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claran

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modular-s2orc

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2021-11-03T15:17:27.644Z

2015-12-29T00:00:00.000Z

240487757

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SYNTHESIS OF ETHYLENE WITH ACREMONIUM SP. 502 PHYTOPATHOGENIC FUNGI The ability of Acremonium sp. 502 phytopathogenic fungi to produce ethylene was studied.It was found that Acremonium sp. 502 is able tosynthesize ethylene. The highest level of biosynthesis was recorded after 5 weeks of cultivationthat coincides with the terms of the highest levelof synthesis of cellulases that are involved in thepenetration of the fungus into the plant. A suggestion that ethylene synthesis plays a certainrole in the mechanism of pathogenesis of cucumbers plants is offered. G. V. Tsehmister Representatives of Cucurbitaceae family are among the most common types of vegetables grown in Ukraine. The problem of their destruction with fungi in Ukraine has not been investigated enough. Besides, pathogenic fungi lead to considerable losses in agriculture. Representatives of Acremonium Link genus, mostly lead saprotrophic way of nutrition, but in certain circumstances they can change specialization and cause diseases of plants of Cucurbitaceae family, manifesting themselves as facultative parasites. Fungi of Acremonium genus were isolated from diseased plants of melons in Spain, Italy and the US (California and Texas) [13-18; 20]. In 2011, S.P. Nadkernychnyi separated strain of Acremonium sp. 502 from cucumber plants were grown under conditions of closed ground and had disease symptoms. We have confirmed its pathogenicity on cucumber plants of Koroliok variety. It was revealed that it is localized in the root system, root neck and hypocotyl and the disease starts to manifest itself in the phase of true leaves. [12] Phytopathogenic microorganisms produce a number of secondary metabolites that negatively affect the growth and development of plants. We know that the ability to synthesize phytohormones often play an important role in the pathogenesis and correlates with virulence and specialization of pathogen. In case of obligate parasitism the use of host plant regulatory systems by pathogen is an important combination factor. Phytohormones were detected in spores, culture liquid and mycelium of many pathogenic fungi. It is believed that the intensive production of hormones is associated with the implementation of phytopathogenic features such as tissue necrosis and their hydrolysis into simple compounds [2], it is also known that hypersynthesis of phytohormones unbalances hormonal system of plants and is the cause of many diseases [7]. The determination of fungi ability to produce phytohormonal substances enables to determine the mechanism of their influence on plant organism. One of the components of fungi pathogenesis is ethylene production [4]. Ethylene is a natural plant growth regulator, one of the main phytohormones. In the plant it controls a wide range of physiological processes, ripening of fruit and aging of tissues, seed germination, growth of cells by stretching, as well as it participates in plants response to various stressors. Typical processes that are activated by ethylene are accelerating leaves aging [9], stunting of stem growth in length, its thickening and horizontal growth [1]. At the same time ethylene can reduce polar auxin transportation, inhibit cell division, accelerate fruit falling, cause flowers aging, stun roots growth [19]. It is known that fungi of Fusarium, Penicillium, Verticillium, Mucor, Saccharomyces genera are able to synthesize ethylene [9]. In view of the above, the objective of our study was to investigate the ability of Acremonium sp. 502 to produce ethylene as one of the mechanisms of influence on plants. Materials and methods. Pathogenic strain of fungi of Acremonium genus, was studied, which was isolated from diseased cucumber plants that were grown in the conditions of closed ground. Strain virulence was preapproved on cucumbers of Koroliok variety. The culture of fungus was kept on slant wort-agar (4 Balling degrees). The description of morphological and cultural characteristics was presented previously by us [10]. To study the synthesis of ethylene Acremonium sp. 502 was superficially cultured in glass vials on Chapek synthetic medium at the temperature of 26-28°C. 10 ml of medium were poured on into 30 ml vials. Acremonium sp. 502 seeding material was obtained by flushing conidia and fungus hyphae pieces from slant wort-agar. Sowing was carried with spore suspension (T = 1 x 10 6 CFU) in the amount of 5% from the volume of culture medium. Measurement of ethylene was performed every 7 days over 7 weeks. At this vials were sealed for a day. The composition of air in the gas phase was analyzed on gas chromatograph . The composition of air in vials without fungus inoculums served as a control. Fungus mycelium was separated through filter paper, washed several times with distilled water and dried to constant weight at 105°C. The calculation of ethylene in the sample was done according to the proposed method [6]. As the criterion for assessing of observed changes probability standard deviation was calculated [5]. Thus, we found that pathogenic to cucumber plants Acremonium sp. 502 fungus is able to produce ethylene. The highest level of biosynthesis was recorded after 5 weeks of cultivation, coinciding with the terms of the highest level of cellulases synthesis that are involved in the penetration of the fungus into the plant. We can therefore assume that ethylene synthesis plays a certain role in the mechanism of pathogenesis of cucumber plants by influencing the activity of cellulases and accelerating leaves aging.

v3-fos

2019-04-07T13:04:19.585Z

2015-03-01T00:00:00.000Z

100899287

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EFFECT OF WATER AVAILABILITY ON SOIL MICROBIAL BIOMASS IN SECONDARY FOREST IN EASTERN AMAZONIA Soil microbial biomass (SMB) plays an important role in nutrient cycling in agroecosystems, and is limited by several factors, such as soil water availability. This study assessed the effects of soil water availability on microbial biomass and its variation over time in the Latossolo Amarelo concrecionário of a secondary forest in eastern Amazonia. The fumigation-extraction method was used to estimate the soil microbial biomass carbon and nitrogen content (SMBC and SMBN). An adaptation of the fumigation-incubation method was used to determine basal respiration (CO 2 -SMB). The metabolic quotient ( q CO 2 ) and ratio of microbial carbon:organic carbon (C MIC :C ORG ) were calculated based on those results. Soil moisture was generally significantly lower during the dry season and in the control plots. Irrigation raised soil moisture to levels close to those observed during the rainy season, but had no significant effect on SMB. The variables did not vary on a seasonal basis, except for the microbial C/N ratio that suggested the occurrence of seasonal shifts in the structure of the microbial community. INTRODUCTION Secondary forests play a major ecological role in the Amazon region, for playing relevant ecological roles, including the fixation of atmospheric C, conservation of biodiversity, connection between forest remnants, and maintenance of the hydrological regime. In addition, secondary forests play a key role in crop rotation, in traditional shifting cultivation systems of the region (Vliet et al., 2013), by increasing soil biomass and nutrients to meet the nutritional demands of crops (Schroth and Lehmann, 2003;Denich et al., 2004). Therefore, studies on nutrient cycling in the secondary forests of Amazonia are extremely important from both an ecological and agronomic standpoint. Soil microbial biomass (SMB) is an important parameter of nutrient cycling in ecosystems, because it is the fraction of soil organic matter (SOM) that is most rapidly decomposed. The dynamics of SMB are determined by biotic and abiotic factors that vary in space and over time in both natural and man-modified ecosystems (Jenkinson and Ladd, 1981;Wardle and Hungria, 1994). Variation in SMB over time is closely associated with changes in water availability in the soil (Patel et al., 2010), and its study contributes to the understanding of release and mineralization patterns (Wardle, 1998) of nutrients that will consequently be available for plants (Singh et al., 1989;Luizão et al., 1992). The patterns in temporal variation of SMB in temperate climate are already well understood; they are closely associated with seasonal changes of temperature and hydrological regime (Wardle, 1998). In tropical soils, where temperatures do not vary greatly, water regime plays a major role in the dynamics of soil microbiota (Lodge et al., 1994). However, few studies have been conducted in tropical environments that describe the variation of SMB in relation to water availability in tropical soils. The effects of abiotic factors, such as water, on the functioning of ecosystems can be assessed by observational or manipulative experiments (Sala and Jackson, 2000). Observational experiments on the effects of soil moisture usually consist of the evaluation of variables over periods of differing precipitation rates (e.g., dry and rainy periods), whereas manipulative experiments involve an increase or reduction in the input of water into the ecosystem, by irrigation or absence of precipitation, respectively (Meir et al., 2009). Our study was developed in the context of an experiment involving the manipulation of soil water availability in a secondary forest in eastern Amazonia, resulting in alterations in several processes of the ecosystem, including the flow of greenhouse gases (Vasconcelos et al., 2004), input and decomposition of leaf litter (Vasconcelos et al., 2007;, net primary productivity (Vasconcelos et al., 2012), and leaf gas exchanges (Fortini et al., 2003). The objective of this study was to assess the response of microbiological variables and their temporal variation to changes in soil water availability resulting from seasonality of rainfall and irrigation during the dry season. Experimental area and sampling The experimental area consisted of a 15-year-old secondary forest colonizing an area abandoned after multiple cycles of slash-and-burn agriculture. Palavras-chave: capoeira, irrigação, fumigação-extração, C-microbiano, N-microbiano, relação C/N microbiana. located at the Fish Farming Station of the Rural Federal University of Amazonia (Universidade Federal Rural da Amazonia), alongside the BR 316, km 63, in the region of Apeú, Castanhal, in the watershed of the River Praquiquara, Baixo Guamá (1° 19' S, 47° 57' W). The climate is classified as AM3 (Köppen classification), with annual rainfall between 2,000 and 2,500 mm. The rainy season lasts from December to May, and the dry season from June to November. The relative air humidity varies between 78 and 90 %. The soil was characterized as phase I Latossolo Amarelo distrófico (concretionary, laterite) (Oxisol), and the vegetation classified as secondary broadleaf forest. The chemical composition of the soil is shown in table 1. In September 1999, eight 20 × 20 m plots were established, spaced at least 10 m apart. Four plots were randomly assigned to receive irrigation treatments during the dry season, and the others the control treatment (no irrigation). In August 2001, during the dry season, micro-sprinkler irrigation was initiated, i.e., 5 mm of water was applied daily, corresponding to the evapotranspiration rate of the regional native forests (Jipp et al., 1998). Soil sampling was performed at a depth of 0-10 cm in November 2000 (dry season), April 2001 (rainy season), and October 2001 (dry season), after three months of irrigation treatment. Six soil cores were combined to provide one composite sample for each experimental plot. Figure 1 show the rainfall events that occurred in the 30 days before sampling. Sample preparation and laboratory procedures The samples were stored in plastic bags and maintained at a temperature of 4 °C until analysis. They were ground, sieved (< 2-mm), and mixed. All plant and animal residues were removed. The moisture of the samples collected in the dry seasons, in November 2000 and October 2001, was standardized to 60 % of the maximum water retention capacity. To facilitate sieving, the samples collected in April 2001 were air-dried for one night, for being very wet. The samples had a relative moisture content of approximately 60 % of the maximum water retention capacity. Soil gravimetric moisture was determined as proposed by Embrapa (1997). Organic carbon, soil microbial biomass carbon and soil microbial biomass nitrogen Organic carbon (C ORG ) was determined by a colorimetric method based on the oxidation of organic matter by a sulfochromic solution and heating, and adapted from Baker (1976). To determine soil microbial biomass carbon ( S M B C ) a n d n i t r o g e n ( S M B N ) , w e u s e d SMB fumigation-extraction (Brookes et al., 1985;Vance et al., 1987;Tate et al., 1988). The extraction was performed using K 2 SO 4 (0.5 mol L -1 ) on non fumigated samples and fumigated samples in alcohol-free chloroform. M i c r o b i a l C w a s d e t e r m i n e d u s i n g t h e colorimetric method (Anderson and Ingram, 1993) and microbial nitrogen determined by the Kjeldahl method (Embrapa, 1997). The K EC used was 0.26 (Feigl et al., 1995) and K EN was 0.54 (Brookes et al., 1985;Joergensen and Müeller, 1996). Microbial biomass basal respiration Microbial biomass basal respiration (CO 2 -SMB) was estimated using an adaptation of the fumigation-incubation method (Jenkinson and Powlson, 1976), which consisted of the incubation of soil samples for 10 days in a container of NaOH, to capture CO 2 released by SMB. During the experimental period, the samples were standardized to 75 % of the maximum water retention capacity without humidity correction. Calculated indices and ratios The C MIC :C ORG ratio was determined using the results of the SMBC and soil organic carbon analyses. The metabolic quotient (qCO 2 ) (Santruckova and Straskraba, 1991), also called microbial biomass respiratory quotient (MBRQ) according to Gama-Rodrigues et al. (1997), was calculated using the microbial biomass basal respiration and microbial biomass carbon data. Statistical analysis The effects of irrigation, sampling period, and interactions between irrigation and sampling period were tested using analysis of variance of repeated measurements considering a two-factor design. The differences between means were tested by Tukey's test at a significance of 5 %. Statistical analyses were performed using SigmaStat software, v. 2.0 (Jandel Scientific, 1994). RESULTS The results showed a significant effect of the interaction between irrigation and sampling period on soil gravimetric moisture ( Table 2). During the dry season (November 2000), the gravimetric moisture contents were approximately half the values measured in the rainy period (April 2001) ( Figure 2). During the irrigation period, gravimetric moisture was significantly higher in the irrigated plots than the control plots, approximately 80 % of the values measured in the rainy period ( Figure 2). There was no interaction effect between sampling period and treatment, though sampling period did have a significant effect on the following variables: C ORG , SMBC, SMBN, CO 2 -SMB, qCO 2, C MIC :C ORG, and C:N MIC ( Table 2). The SMBC values varied between 469 and 924 µg g -1 of C and were significantly higher in November 2000 (dry season) than in the other sampling periods (Table 3). The SMBN varied between 34 and 63 µg g -1 of N, and was significantly higher in November 2000 and April 2001. The microbial C:N ratio varied between 9 and 19, and was significantly higher in October 2001 (dry season) and lower in April 2001 (rainy season). The values of soil microbial biomass basal respiration (CO 2 -SMB) were significantly different among all sampling periods ( Table 3). The metabolic quotient (qCO 2 ), which also increased with time, was significantly higher in April and October 2001 (Table 3). The values of soil organic C differed between the two dry periods; however, they were statistically similar to those in the rainy season. The C MIC :C ORG ratio was significantly higher in November 2000 than in the other sampling periods. (Table 3). DISCUSSION The values for SMBC and SMBN agreed with results of earlier studies conducted in the same region (Rangel-Vasconcelos et al., 2005;Sotta et al., 2008;Lopes et al., 2011;Melo et al., 2012). Bittencourt et al. (2006) found a higher SMBN content in Amazonian soils under secondary vegetation in the rainy season. Other studies in tropical forests indicated that seasonal variation in soil moisture is associated with seasonal variation in soil microbial C and N (Singh et al., 1989;Luizão et al., 1992;Srivastava, 1992). However, in this study no clear pattern of soil microbial mass variation associated with seasonal changes in soil moisture was observed. The high concentrations of SMBC and SMBN in the first soil sampling, during the dry season, may have been the result of nutrient release from dead SMB and/or the decomposition of leaf litter, in addition to rainfall in the days before sampling. Prolonged dry periods followed by rainfall cause osmotic stress in microbial cells and promote cell lysis, resulting in the release of a pulse of nutrients (Lodge et al., 1994;Wardle, 1998;Yang et al., 2008), that become available to the soil microbiota and plants (Singh et al., 1989;Srivastava, 1992). In fact, pulses of soil CO 2 efflux, possibly associated with the increase in microbial activity, were observed in the same experimental area in response to rainfall during prolonged dry periods (Vasconcelos et al., 2004). In addition, in the dry season, there is an accumulation of leaf litter as a combined consequence of increased litter production and reduced decomposition rate (Vasconcelos et al., 2007;. Therefore, rainfall in the dry season favors the release of nutrients from the accumulated leaf litter, which benefits the soil microbial biomass. The occurrence and amount of daily precipitation during the 30 days preceding the two samplings performed in the dry season (November 2000 andOctober 2001) are shown in figure 1. In the first soil sampling, pluvial precipitation was 77 mm during the 30 preceding days, 17 mm of which fell in the week immediately before sampling, characterizing a period of possible nutrient pulses (Lodge et al., 1994). Prior to the October 2001 sampling, precipitation was 24 mm in the preceding 30 days (no rain fell in the 15 days before sampling). The values of basal respiration were higher than those found by previous studies conducted in Amazonia: 0.81 μg g -1 h -1 of C-CO 2 (Luizão et al., 1992), 0.35 to 0.70 μg g -1 h -1 of C-CO 2 (Gama-Rodrigues et al., 1994), and 0.85 to 1.11 µg g -1 h -1 C-CO 2 (Melo et al., 2012). This may have been a result of soil-climatic differences, as well as differences between the methods used to determine basal respiration. In general, there was an increase in carbon mineralization during the observation period, rather than the expected increase in the amount of SMBC. Over time, qCO 2 lost efficiency, since carbon was mineralized at a faster rate than it was fixed, indicating that the greater the amount of carbon in SMB, the lower its mineralization rate and vice-versa (Santruckova and Straskaba, 1991;Gama-Rodrigues et al., 1997). Contrary to what was expected, the high respiration rate in the third sampling (October 2001) indicated an increase in microbial activity, although this increase was not stimulated by soil water availability. Although no consistent pattern of seasonality was observed for SMBC and SMBN during the experiment, the microbial C/N ratio tended to vary on a seasonal basis, suggesting an alteration in the microbial community composition with the changes between dry and rainy seasons. Bacterial microbiota is richer in proteins, and therefore in N content, than fungal microbiota (resulting in a lower C/N ratio) (Anderson and Domsch, 1980;Brady, 1989;Tate, 2000). Ross (1987) characterized the C and N contents of soil microorganisms and observed a C:N ratio of 22 for Penicillium novae-zelandiae, but a C:N ratio for Pseudomonas sp. of 3.3. Cornejo et al. (1994) studied the effects of irrigation on the microbial community structure in a tropical forest and observed a higher density of bacteria and a lower density of fungi in an irrigated plot. On the other hand, Holland et al. (2013) found a higher density of fungi in soils under grape cultivation with periods of irrigation. Repeated cycles of soil drying and wetting can favor the growth of microbial populations adapted to these conditions. Marschner et al. (2002) demonstrated that bacteria and fungi are able to tolerate sudden changes in soil matric potential, and grow rapidly with the availability of nutrients from the labile fraction of soil organic matter or dead microorganisms. However, fungi can respond better to fluctuating conditions of soil moisture and survive periods of drought, for being more resistant to osmotic stress (Wardle, 2002;Coleman et al., 2004). The C ORG content increased over the observation period, possibly as a result of the increase in phytomass, production of net organic matter, and secondary vegetation succession (Vieira, 1996;Rangel-Vasconcelos et al., 2005). The values of C MIC :C ORG indicate the extent to which soil C ORG is in the form of SMB, and the extent to which C ORG will return to the atmosphere in the form of CO 2 . Usually, SMB represents between 1 and 5 % of total C ORG in the soil (Powlson and Jenkinson, 1981;Balota et al., 1998). According to Anderson and Domsch (1989), the C MIC :C ORG ratio varies between 0.3 and 7 %. Basante et al. (2001) observed values between 3 and 11 % in the native forests of Amazonia, whereas Rangel- Vasconcelos et al. (2005) reported values between 2 and 18 % in secondary vegetation of various ages. CONCLUSIONS The increase in soil water availability resulting from irrigation during the dry season and under the experimental conditions of this study had no effect on soil microbial biomass. Carbon, nitrogen, and microbial activity indicated no seasonality in relation to the dry and rainy seasons. T h e m i c r o b i a l C / N r a t i o i n d i c a t e d n o seasonality; however, the results indicated changes in the microbial community according to the sampling period.

v3-fos

2016-05-12T22:15:10.714Z

2015-02-17T00:00:00.000Z

5845527

s2

Morphological, physicochemical, and antioxidant profile of noncommercial banana cultivars Banana cultivars––Luvhele (MusaABB), Mabonde (MusaAAA), and Muomva-red (Musa balbisiana) ––were characterized for morphological, physicochemical, and antioxidant properties. All three cultivars varied significantly (P < 0.05) in their morphology, pH, titratable acidity and total soluble solids with no significant difference in their ash content. Individual cultivars showed variations in flour starch granule when observed using a scanning electron microscope. Characterization of cultivars for total polyphenols (TPs) and antioxidant activity upon pretreatment with ascorbic, citric, and lactic acid shows that the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay of samples varied significantly as Muomva-red cultivar (1.02 ± 0.01 mg GA/g) expressed the highest DPPH activity at lactic acid concentration of 20 g/L. Total polyphenol content was also highest for Muomva-red [1091.76 ± 122.81 mg GAE/100 g (d.w.)]. The high amount of TPs present in these cultivars make them suitable source of bio-nutrients with great medicinal and health functions. Introduction Banana is an edible fruit grown in tropical and subtropical regions of the world at latitude 20°close to the equator, with characteristic seasonal variation in rainfall and temperature (Pua 2007). Globally, there exist more than a 100 common names for which the fruit from the genus Musa is associated, with over 1000 cultivars and landraces emanating from more than fifty Musa species (Heslop-Harrison and Schwarzacher 2007; Arvanitoyannis and Mavromatis 2009). The name banana is said to originate from the coastal part of West Africa, Guinea or Sierra Leone, and was accepted in the New World as a term used for describing the fruit's peel. The oldest record of edible banana has been traced to originate from India (600 B.C.). Banana, apart from apple, is the highest consumed fruit in Europe (Arvanitoyannis and Mavromatis 2009) and it can either be classified as commercial or noncommercial cultivars. The noncommercial cultivars also referred to as indigenous varieties are so-called due to the fact that they are rarely cultivated for export or trade, but are grown in household gardens by small-scale growers mostly for consumption (Anyasi et al. 2013). Basically, two major noncommercial cultivars are grown in Limpopo Province of South Africa: Mabonde and Luvhele cultivars (Fig. 1). Muomva-red, another noncommercial variety cultivated in Limpopo Province, is also grown in other parts of the world. Variations exist among these noncommercial varieties in their morphological features of length, width, peel and pulp colour, weight, and overall shape as well as their antioxidant properties. These properties are factors that are used in the classification of these fruits to their corresponding groups and subgroups. Commercial cultivars are known to be larger in size, weight, length, and overall shape and have higher consumer acceptability when compared to the noncommercial cultivars. Although the fruit's final shape and size are representatives of the cultivars, they are also affected by environmental and genetic interactions (Robinson and Sauco 2010). Accordingly, the commercial varieties have been grouped into various cultivars using these parameters due majorly to their acceptability as well as nutritional content. However, the noncommercial cultivars are rarely known or classified even though different cultivars exist. Current trends show an increase in research on the utilization of unripe banana products for use by consumers due to the presence of polyphenols (Aurore et al. 2009), free and bound phenols such as anthocyanins in fruit pulp (Bennett et al. 2010), and moderate antioxidant capacity in flour of unripe green banana (Menezes et al. 2011;Sarawong et al. 2014). Nutritional differences have also been observed in the mineral and bioactive profiles of these fruits. Literatures studied show that some of these noncommercial cultivars contain higher antioxidant nutrients when compared to the commercial cultivars (Faller and Fialho 2010;Fu et al. 2011). However, there is scarce information on variation in morphological, physicochemical, and antioxidant properties that exist among these noncommercial banana varieties cultivated in South Africa and other tropical and subtropical countries. This research therefore seeks to comparatively profile the morphological, physicochemical, and antioxidant properties of three noncommercial banana cultivars. Plant materials and treatments A total of 30 banana fingers, each selected randomly from different parts of six fruit bunches of three noncommer- cial banana cultivars: Luvhele, Mabonde and Muomvared and obtained at the unripe green stage 2 of ripeness (Aurore et al. 2009) from household banana farms in Thulamela Municipality, Vhembe District of South Africa, were used for this research. The fingers were randomly collected from two bunches of each of the individual banana fruits. Characterization of all three cultivars was done by determining the morphological parameters of finger length and girth, physicochemical properties, total polyphenol, as well as antioxidant properties of the fruits. Noncommercial cultivars were also compared with information from Musa Germplasm Information System (MGIS) as well as from other literatures (Daniells et al. 2001;Aurore et al. 2009;Anyasi et al. 2013). The cultivar names, their species, subspecies, accession names, and numbers obtained from the MGIS database and other literatures are shown in Table 1. To obtain flour used for the determination of phenolic and antioxidant properties, pulp of noncommercial cultivars was cut to 4 mm size and pretreated with organic acids; ascorbic, citric, and lactic acid at concentrations of 10, 15, and 20 g/L for 10 min. The mixture containing the pretreatment and sliced pulp was allowed to drain for 2 min. Fruit pulp was then conventionally dried in an air oven dryer (Prolab instruments, South Africa) at a temperature of 70°C for 12 h. Dried pulp was later milled (Retsch ZM 200 miller, Haan, Germany) at 16,000 rpm for 30 sec. Banana flour obtained from milled pulp was then used to determine the total polyphenol and antioxidant activity of banana cultivars. Determination of fruit length and girth Fruit length and girth were determined from a selected banana bunch using the protocols of Dadzie and Orchard (1997). From two bunches of noncommercial banana cultivars, 10 randomly selected individual fingers from different hands, top to bottom of banana bunch, were used to determine fruit length and girth. Fruit length determination involves measuring the outer and inner curve of individual fruits with a tape from the distal end of the fruit to the point at the proximal end where the fruit pulp is judged to terminate. Fruit girth: distal end, widest midpoint, and proximal end were determined by measuring individual fruit with a tape at the widest midpoint, distal, and proximal end of each individual fruit finger (Dadzie and Orchard 1997). Total soluble solids and titratable acidity The total soluble solids (TSS) of fruit cultivars were determined using the methods of Dadzie and Orchard (1997). Approximately 30 g of banana pulp tissue was homoge-nized in 90 mL of distilled water for 2 min and filtered using Whatman No.1 filter paper. A single drop of the filtrate was placed on the prism of a refractometer and readings for percentage TSS were taken. Recorded values were multiplied by three due to the dilution factor of the pulp, which is three times the amount of distilled water. For the determination of total titratable acidity (TTA), from 100 mL of filtered banana pulp, 10 mL was pipette into a conical flask, and diluted to about 80 mL with distilled water. About 0.3 mL phenolphthalein was then added to the solution titrated to a faint pink end-point with 0.1 N NaOH. TTA was expressed as percentage malic acid. pH and ash content The pH was determined using the protocols of Dadzie and Orchard (1997). Approximately 30 g of banana pulp tissue was homogenized with 90 mL of distilled water for 2 min and filtered using Whatman No 1 filter paper. Readings were recorded by inserting the pH electrode in the filtrate on stabilization of sample filtrate. Ash content of fruit sample was determined using method of Horwitz (2000). Empty crucibles were placed in a muffle furnace at 600°C for an hour, cooled in desiccators, and weighed. About 1 g of each sample of banana flour was placed in the crucible and ignited over a burner with the help of a blowpipe until it was charred. The principle was placed in a muffle furnace at 600°C for 2-4 h. The appearance of gray white ash indicates complete oxidation of all organic matter in the sample. Scanning electron microscopy (SEM) Imaging of unripe fruit flour was conducted using a Leo 1430VP SEM. Prior to imaging, flour samples were mounted on a stub with double-sided carbon tape. Samples were then coated with a thin layer of gold in order to make flour surface electrically conducting. SEM micrographs revealed the surface structure of banana flour of all three cultivars. Beam conditions during surface analysis were 7 kV and approximately 1.5 nA, with a spot size of 150 and a magnification of 1000 kX. Total polyphenol Total polyphenols of flour obtained from the three noncommercial banana cultivars was determined using the Folin-Ciocalteu colorimetric methods of Prabhu and Barrett (2009) with slight modifications. The method is based on the reduction of MoO 4+ to MoO 3+ that is detected by color change from yellow to blue; measured at 760 nm. Approximately 0.2 g of milled oven dried fruit pulp was weighed and 2 mL of acetone was added. The mixture was incubated for 1 h at room temperature, shaking occasionally and centrifuged at 6000 rpm for 5 min at 4°C. To 9 lL of centrifuged sample in a microplate, 109 lL of Folin-Ciocalteu solution was added. About 180 lL of 7.5% Na 2 CO 3 was added to the mixture, covered with aluminium foil and incubated at 50°C for 5 min. Absorbance was read at 760 nm, using an UV spectrophotometer microplate reader (Zenyth 200rt Biochrom, UK). Gallic acid was used as the standard phenol compound and acetone used as the extraction solvent. The results were expressed as equivalents of gallic acid (mg GAE/ 100 g d.w.) from the calibration curve. Determination of 1,1-diphenyl-2picrylhydrazyl (DPPH) scavenging activity The ability of the banana flour to scavenge the unstable free radical 1,1-diphenyl-2-picrylhydrazyl was determined using the methods of Ribeiro et al. (2008). This capacity to scavenge the stable DPPH free radical can be used in expressing the measure of antioxidant activity in fruits (Musa et al. 2013). Methanol was used as the extraction solvent and gallic acid used as standard with result of analysis measured in mg GA/g (d.w.). To 0.2 g of milled banana flour, 2 mL of methanol was added to the sample, incubated for 30 min at room temperature and centrifuged at 6000 rpm for 10 min at 4°C. Dilution of different concentrations of 10, 20, 30, 40, and 50 mg/mL of the sample was used to determine the IC 50 of the sample. Final values of IC 50 were obtained by plotting the percentage disappearance of DPPH as a function of the sample concentration. To 250 lL of DPPH solution, 28 lL of sample mixture was added in a microplate, covered with aluminum foil and incubated for 1 h at room temperature. Absorbance was read at 517 nm using an UV spectrophotometer microplate reader (Zenyth 200rt Biochrom, UK). Statistical Analysis All measurements were conducted in triplicate and results presented as mean values AE standard deviation (SD). Statistical analysis was conducted using the one-way analysis of variance (ANOVA) and means of results for each experiment was compared using the Tukey Honest Significant Difference (HSD) Test (P < 0.05 confidence levels). Inner curve lenght (cm) SPSS 21 for windows (SPSS Inc., Chicago, IL) statistical software package was used to conduct the statistical analysis. Results and Discussion Fruit morphology Differences that exist in fruit morphology are due mostly to variations in cultivars. Banana cultivars differ in their fruit girth measurements with different cultivars showing different sizes and shapes in those parts of the banana peel ( Fig. 2A). The outer curve length of Mabonde had the lowest length in almost all fingers when compared statistically (P < 0.05) to Luvhele and Muomva-red cultivars. Muomva-red banana outer curve length was highest for fingers 1, 2, 3, 4, 5, 7, and 9 when compared to other cultivars and differed significantly (P < 0.05) from Luvhele and Mabonde. The outer curve length of Luvhele was significantly the smallest except for finger 6 when compared to other cultivars. Cultivar Muomva-red is thus said to have significantly longer banana fingers when compared to the other noncommercial cultivars used in this study. Results of finger length of individual commercial banana fingers show that the commercial variety such as Williams and Grand Nain were longer in length: 17-24 cm (Daniells et al. 2001) when compared to the noncommercial cultivars used in this study. Results from the inner curve length showed that Mabonde banana had the highest inner curve length with finger 3 (15.47 AE 0.25 cm) and varied significantly (P < 0.05) from other noncommercial cultivars. There was a marked significant difference in the inner curve length of Mabonde banana cultivar in all fingers of the three cultivars. The inner curve length of both Luvhele and Muomva-red were significantly different (P < 0.05) for all fingers except for fingers 2 and 9 (Fig. 2B). Similar results were reported by Belayneh et al. (2014) on the fruit length of some noncommercial cooking bananas. The distal end, widest midpoint, and proximal end of banana fruits are parameters that make up the girth and morphological structure of the fruit. These morphological properties could be used for characterization and differentiation of one banana cultivar from another (Dadzie and Orchard 1997). Among all three cultivars analyzed, there were significant differences in banana distal end with Mabonde and Muomva-red varying significantly in very few fingers. Mabonde banana cultivar had the widest midpoint for all cultivars, varying significantly (P < 0.05) from other cultivars ( Table 2). The widest midpoint of Muomva-red was significantly higher than that of Luvhele for all fingers measured. The proximal end varied significantly in all three cultivars apart from finger 1 in which there was no significant difference in the measured proximal end. Results obtained on fruit girth for all cultivars agree with results obtained by Belayneh et al. (2014) on the fruit girth of some cooking bananas. Physicochemical properties of fruit cultivars The total titratable acidity (TTA) of cultivars analyzed showed that there was no significant difference in the TTA of Luvhele (1.61 AE 0.13) and Muomva-red (1.65 AE 0.15). Cultivar Mabonde showed marked significant difference in its TTA when compared to other noncommercial cultivars (Table 3). Similar results for TTA were reported by Belayneh et al. (2014), while lower TTA values were recorded for unripe Robusta banana cultivar after 6 days of storage upon pretreatment of the individual banana fingers with ethrel solution (Kulkarni et al. 2011). According to Sadler and Murphy (2010), TTA is used in the determination of total acid concentration present in food products. In fruits, acidity decreases with concomitant rise in maturity, thus TTA and sugar content of fruits acts as an important parameter in the determination of both flavour and overall maturity of the fruit. Total soluble solids (TSS) varied significantly (P < 0.05) in all noncommercial banana cultivars. According to Dadzie and Orchard (1997), TSS can be used in the screening of different banana hybrids. Generally, the amount of TSS in a fruit is directly proportional to the degree of fruit ripeness as TSS is said to increase with fruit ripeness. Thus, TSS can also serve as a useful index in the determination of fruit maturity and ripeness. Cultivar red exhibited a significantly higher TSS (2.92 AE 0.14) when compared to other fruit cultivars. Cultivar Mabonde had the least soluble sugar content (1.49 AE 0.05) when compared with all three cultivars. Similar results were recorded for TSS of green Cavendish banana flour (Alkarkhi et al. 2011) and unripe banana (Kulkarni et al. 2011). The low values recorded for the TSS of all cultivars are attributed to the fact that the available starch present in the unripe cultivars are yet to be converted into soluble sugars through enzymatic degradation (Zhang et al. 2005). Degradation and consequent reduction in starch proceed rapidly during the onset of ripening thus leading to an overall increase in TSS of fruits. pH of all analyzed samples varied significantly (P < 0.05) in all fruit cultivars. Cultivar Luvhele was the least acidic in all cultivars (6.12 AE 0.03) when compared to Mabonde (5.46 AE 0.02) and Muomva-red (5.36 AE 0.02) on fresh weight basis. Muomva-red was more acidic when compared to the other cultivars, which agrees with values obtained by Kulkarni et al. (2011) for unripe banana cultivars. Alkarkhi et al. (2011) also report similar results for the pH of unripe green banana flour. Conversely, the pH values of all unripe cultivars were higher than values obtained in ripe cultivars as reported by Arvanitoyannis and Mavromatis (2009) due to the associated increase in organic acids present in fruits as ripening increases. The pH of food measures the amount of hydronium ions (H 3 O + ) present in a food produce. Many food quality criteria have been found to correlate better with pH than with acid concentration (Sadler and Murphy 2010). Cultivar Luvhele had a percentage ash content of 1.13 AE 0.05, cultivar Mabonde 1.22 AE 0.58 and Red 1.33 AE 0.11 (f.w.). The result also agrees with that of Nwokocha and Williams (2009) on white and yellow plantain. Results also showed that there was no significant difference (P < 0.05) in the percentage ash of all unripe banana cultivars analyzed. Ash content in fruits and vegetables are affected by agro-climatic conditions such as cultivation practices, nature of soil, and climatic conditions. Ash content is used to determine the total mineral present in a food produce. A high percentage ash value equals a high total mineral value in the fruit sample. Mineral availability in fruits and vegetables are influenced positively or negatively by these agro-climatic conditions (Forster et al. 2002). The absence of marked differences in the nature of soil and climatic conditions are factors that explain the lack of difference in the ash content of all cultivars examined (Bugaud et al. 2006). All three banana cultivars were obtained at the same location with the same soil and climatic conditions applied during cultivation. SEM analysis of fruit flour Surface morphology obtained from SEM micrographs of all three cultivars showed marked variations in structure, shape, and size of starch granules of the different banana flour. Electron micrographs suggest that granules were considerably irregular in their structures among cultivars. Observed shapes include polygonal for Luvhele, oval for Mabonde and elongated for Muomva-red unripe banana flour (Fig. 3). Oval shape of granules obtained from unripe Mabonde flour is in agreement with the work of Utrilla-Coello et al. (2014) which showed starch granules exhibiting regular shapes with oval appearance. Drying temperature of 70°C for 12 h during flour production, as well as differences in cultivars (Sivak and Preiss 1998;Jackson 2003) account for the variations in granule morphology among the different cultivars. Irregular shapes and size of granules as observed in micrographs could be attributed to high heat treatment leading to irreversible swelling, puncturing, and gelatinization of banana flour. Pretreatment with GRAS organic acids of individual cultivars, however, showed no difference in shape and structure of granules obtained from flour as all cultivars retained their shape and size (Fig. 4). Adhesion between granules was also observed in flour samples of all cultivars irrespective of pretreatment concentration. This result is in agreement with Wang and Copeland (2012) on effect of alkali treatment on structure and function of pea starch granules. Presence of adhesion between granules can be attributed to the occurrence of lipid and protein molecules in the granules of unripe flour samples (Perez et al. 2009). Total phenolics of unripe banana cultivars Fruit samples with different pretreatment showed various total polyphenol content (TPC) at varying levels of pretreatment concentration. Cultivar Mabonde had a high coefficient of determination (r 2 = 0.919) for all organic acid treatment with ascorbic acid treatment recording the highest relationship between treatment and cultivars (Fig. 5A) for fruit TPC. Weak relationship (r 2 = 0.1734) was observed between cultivar Luvhele and citric acid treatment (Fig. 5B) and very weak relationship (r 2 = 0.0026) between cultivar Muomva-red with lactic acid treatment (Fig. 5C). There was significant increase in TPC yield in all cultivars as the concentration of pretreatment increased. Cultivar Muomva-red showed the highest amount of TPC in all pretreatment and at different levels of concentration except for concentration levels of citric acid 20 g/L and lactic acid 15 g/L ( Fig. 5B and C). Cultivar Luvhele showed a significantly higher TPC yield of 707.87 AE 12.62 mg GAE/100 g and 841.59 AE 38.39 mg GAE/100 g at such concentration levels. Also fruits treated with citric acid had a higher TPC yield in all cultivars with pretreatment concentration of 15 mg/L having the highest significant yield (P < 0.05) of TPC for Muomvared. Generally, there were significant differences in yield of TPC in all cultivars at all treatments except for pretreatment concentration of citric acid 20 g/L where there was no significant difference in TPC across all cultivars. Results also show that TPC was highest for Muomva-red with a value of 1091.76 AE 122.81 mg GAE/100 g (d.w.). Significant variations that exist in quantity and quality of total polyphenols in plant foods have been attributed to diverse inherent and external conditions such as genetic composition, plant cultivar, soil composition, state of plant maturity, and postharvest practices (Jaffery et al. 2003;Faller and Fialho 2010). The TPC values obtained from the noncommercial cultivars were higher than those obtained by Sarawong et al. (2014) with TPC of native banana flour at 220.30 AE 0.59 mg GAE/100 g (db) and Fu et al. (2011) who reported values of 57.13 AE 3.64 mg GAE/100 g (general banana), 29.07 AE 2.06 mg GAE/100 g (cooking banana), and 25.55 AE 0.60 mg GAE/100 g (royal banana). Similarly, Menezes et al. (2011) showed lower total polyphenols of 50.65 AE 0.80 mg GAE/100 g (d.w.) in flour of unripe banana. Anusuya et al. (2013) also recorded polyphenol content (110.45 mg GAE/100 g, 94.03 and 79.92 mg GAE/100 g) from banana pseudo stem flour when compared to results obtained from this present study. The high TPC content in the noncommer- cial banana cultivars, especially Muomva-red could be attributed to the colour of the banana cultivars which has a red peel when unripe. This could also be seen in almost all the pretreatment concentration where Muomva-red significantly exhibited the highest amount of TPC. High temperature processing leads to alteration of the molecular compounds resulting in polymerization and alteration of the molecular structure of phenolic com-pounds thus leading to a reduced extractability (Altan et al. 2009;Brennan et al. 2011;Nayak et al. 2011;Sharma et al. 2012). The high phenolic contents in all fruit cultivars analyzed agree with Sarawong et al. (2014) who argue that increase in phenolics could be due to the disruption of cell walls by all extrusion conditions thus resulting in higher TPC content. Accordingly, the TPC of fruits and plant produce is generally dependent on part of the plant and the solvent used for extraction. Apart from varietal difference, high TPC yield in all cultivars could also be as a result of pretreatment with organic acid used in the preparation of unripe banana flour. Sulaiman et al. (2011) stated that significant differences observed in the phenolic content among different cultivars can be attributed to the breakdown of phenols influenced by varietal differences. Antioxidant properties of unripe banana cultivars Antioxidant capacity of all noncommercial banana cultivars was determined by DPPH assay. Results of analysis showed that the antioxidant capacity varied significantly in all cultivars and at different organic acid pretreatment concentration (Table 4). Mabonde cultivar recorded the lowest DPPH activity in all cultivars and varied significantly in pretreatments. Mabonde banana cultivar showed a low significant scavenging activity for citric acid pretreatment and at a concentration of 10 and 15 g/L. The DPPH scavenging activity also differed significantly (P < 0.05) for Muomva-red and was highest (1.02 AE 0.01 mg GA/g with lactic acid concentration of 20 g/L). There was a significant difference in the antioxidant activities of cultivars as a result of organic acid pretreatment, with cultivars treated with lactic and ascorbic acids showing significantly higher antioxidant activity in all cultivars. IC 50 is the amount of antioxidant necessary to decrease the initial DPPH absorbance by 50%. The lower the IC 50 obtained during DPPH assay the better the scavenging property and ability to break the free radical chain reaction (Frankel 1991;Lim et al. 2007). The DPPH assay measures the ability of the extract to donate free hydrogen ions to the radical. During this assay, there is a reduction of the purple chromogen radical to pale yellow hydrazine by antioxidant compounds. This reduction activity of the purple chromogen radical corresponds to a decrease in optical density at long wavelengths (Musa et al. 2013). Generally, lactic acid pretreatment showed the highest DPPH activity (P < 0.05) in all cultivars at pretreatment concentration of 20 g/L. DPPH scavenging activities of noncommercial cultivars were also significantly higher when compared to the results obtained by Moyo et al. (2013) on the antioxidant capacity of nonconventional leaves consumed in South Africa. The high DPPH scavenging activity of the noncommercial cultivars agrees with results obtained by Sarawong et al. (2014). Similar results obtained for DPPH scavenging activity were reported by Sulaiman et al. (2011) on eight Malaysian bananas using hexane, chloroform, and 80% methanol (v/v) as extraction solvent. The antioxidant capacity of banana cultivars as comparable to TPC is significantly affected by the mode of sample preparation as well as the solvent used for extraction. Accordingly, the distinctive properties of the structure and composition of individual plant materials make their behavior unpredictable when in combination with various solvents (Gonz alez- Montelongo et al. 2010). Similarly, the overall antioxidant properties of food produce can be enhanced by the formation of products synthesized by Maillard reaction. Products formed from Maillard reaction are due mainly to intense heat treatment or prolonged storage, thus resulting in a strong antioxidant activity. Conversely, heat treatment and drying process can also result in a corresponding decrease of naturally occurring antioxidants (Nicoli et al. 1999;Toor and Savage 2006;Sulaiman et al. 2011). Conclusion Significantly marked morphological differences exist among all the three noncommercial banana cultivars. Morphological profiles of different cultivars examined can be used as parameters for distinguishing these noncommercial banana varieties. The variations that exist in total polyphenol content (TPC) are other useful parameters needed for nutritional classification and differentiation. Among all cultivars examined, Muomva-red had the longest finger, highest amount of TPC, and the highest antioxidant activity. Compared to works done by other authors, high amount of total polyphenols (TPs) obtained in cultivars used in this study make them a suitable source of bio-nutrients with great medical and health function in the body. Recorded TPs in all samples were higher when compared to results obtained from other banana cultivars from different parts of the world. Thus, further processing, use and application of these noncommercial cultivars otherwise underutilized will be highly beneficial due to the presence of antioxidants.

v3-fos

2015-09-18T23:22:04.000Z

2015-07-01T00:00:00.000Z

6035237

s2

Impact of Data Processing and Antenna Frequency on Spatial Structure Modelling of GPR Data Over the last few years high-resolution geophysical techniques, in particular ground-penetrating radar (GPR), have been used in agricultural applications for assessing soil water content variation in a non-invasive way. However, the wide use of GPR is greatly limited by the data processing complexity. In this paper, a quantitative analysis of GPR data is proposed. The data were collected with 250, 600 and 1600 MHz antennas in a gravelly soil located in south-eastern Italy. The objectives were: (1) to investigate the impact of data processing on radar signals; (2) to select a quick, efficient and error-effective data processing for detecting subsurface features; (3) to examine the response of GPR as a function of operating frequency, by using statistical and geostatistical techniques. Six data processing sequences with an increasing level of complexity were applied. The results showed that the type and range of spatial structures of GPR data did not depend on data processing at a given frequency. It was also evident that the noise tended to decrease with the complexity of processing, then the most error-effective procedure was selected. The results highlight the critical importance of the antenna frequency and of the spatial scale of soil/subsoil processes being investigated. Introduction The assessment of soil water content (SWC) variation on both spatial and temporal scales is fundamental in many research areas and applications, such as land use planning, irrigation management, ecological and hydrological modelling. A great deal of research has gone into the development of novel SWC techniques capable of providing measurement of a physical variable that is a surrogate for SWC across a wide range of spatial scales. Reviews of soil moisture measurement techniques are given by Robinson et al. [1] and Vereecken et al. [2], with the aim to identify several emerging methods and technologies from geophysics. Geophysical methods provide a low cost and noninvasive way of gathering a large amount of information on various physical soil properties. In agriculture, the use of these methods was largely motivated by the need for reliable, quick and easy measurements of soil parameters at field and landscape spatial extents. Ground Penetrating Radar (GPR) is one of the geophysical techniques, currently used in agricultural research and application to monitor shallow soil water content [3][4][5][6]. A GPR system consists of an arrangement of antennas that can emit and receive electromagnetic pulses in the radar frequency range of 1 MHz to a few GHz. These pulses propagate through low-loss materials until they are reflected or diffracted by interfaces and objects that exhibit contrasts in the electric permittivity (ε) [7]. A review of the numerous laboratory studies, as provided in Knight [8], shows that the dominant factors controlling ε of a material are: (1) the volume fractions and dielectric constants of the individual components; (2) the geometrical arrangement or distribution of the components; and (3) the physical and/or chemical interactions between components. Conversely, given the large contrast between ε of water (81) and that of air (1) and of commonly occurring solid components (5)(6)(7)(8)(9)(10)(11)(12), it is often assumed that variation in ε is primarily due to variation in water content. However, the development and acceptance of GPR as a soil water content sensor is still limited by the cumbersomeness of its application in field conditions, due to the complexity of data acquisition and processing. In particular, processing procedures are necessary to compensate or minimize undesirable effects and enhance the radar images of subsoil. Filtering processes are very time consuming, requiring automation and a well-designed, conceptually rigorous filter. Several publications have described the use of 2-D and 3-D GPR images and, the quality of processed GPR image was generally evaluated visually and qualitatively according to the subjective ability of the operator to detect different interfaces or reflections. Moreover, the application of signal processing may alter drastically some characteristics of the the raw signal data. In this work the selected characteristic being investigated is the amplitude of signals reflected by any surface. The hypothesis is based on the facts that the resulting amplitude of a radar wave depends not only on intrinsic attenuation, mostly controlled by electrical conductivity, but also on reflection coefficient between layers with different dielectric properties. The amplitude will then provide information directly related to subsurface changes of the dielectric properties affecting the hydraulic properties [9][10][11]. In particular, enveloped amplitude is associated with the reflection strength of the signal, so that a large value of amplitude may indicate major changes in subsurface layers and/or lower attenuation through the layer of soil crossed by radar waves. Generally, the amplitude values were inversely proportional to water content. Data processing may affect the spatial dependence of GPR data. In particular, some researchers have studied how the geostatistical structure of surface GPR reflection data was affected by different data gains and migration for a single radar section [11]. They observed that the sedimentary materials and their geometrical arrangement affected spatial structures of radar data but geostatistical models of radar reflection amplitudes were a function of data processing and signal frequency. Another researcher [12] used geostatistical tools in order to characterize spatial variations of the data and their resolution, and to filter out the spatial components identified as noise. In this study, a series of data processing sequences was applied to the recorded data and after each processing sequence, and the amplitude envelope of radar signal was calculated. It is important to note that all GPR data used in this study are displayed in "time slice" (or depth slice) maps [13], a way of displaying the data in horizontal maps of recorded reflection amplitudes. The radar processing was kept to a minimum, in order to preserve the original amplitude values. GPR response, for each applied data processing sequence, was described and the approaches were compared through variography analysis and traditional statistical analysis. The objectives were: (1) to investigate the effects of data processing on radar signal; (2) to select a quick and error-effective data processing for detecting subsurface features. The data analysis was aimed to quantify measurement error in the data submitted to different processing procedures. Finally, because the spatial structures of GPR data can exhibit marked dependence on antenna frequency, a comparison among different frequencies (250, 600 and 1600 MHz) was performed using variography techniques aimed (3) to estimate measurement error and to establish an objective criterion for selecting the most suitable antenna frequency to investigate a complex system such as the agricultural soil. Description of the Field Site and Mapping Protocol The experiment was conducted at the "Maria Elisa Venezian Scarascia" farm, one of the experimental farms of the Italian Agricultural Research Council (CRA), located in Rutigliano-Bari (40°59′48.25″ N, 17°02′02.06″ E), in south-eastern Italy ( Figure 1). The test plot selected for this study was bare soil of 40 m × 20 m size (red rectangle in Figure 1). The study area is located in the Murgia Plateau, characterized by homogeneous sequence of calcareous and dolomitic rocks. Limestones have quite low porosity but are usually fractured and affected by karst dissolution. The soil is classified as fine, mixed, superactive, thermic Typic Haploxeralfs, according to the Soil Taxonomy [14], and as Cutanic Luvisol (Hypereutric, Profondic, Clayic, Chromic), according to the WRB [15]. Soil texture is mainly clayey with the clay content ranging from 30% to 60% by weight increasing in depth and with high content of gravel (15% by weight). The coarse soil mineral fraction increases in volume in the neighbourhood of the outcropping bedrock. The soil depth, as it was also revealed by the pedological survey [16], is rarely deeper than 0.60-1 m, owing to the occurrence of shallow bedrock (Figure 1b). Since high contents of clay may strongly attenuate the signal, a preliminary ERT survey was carried out which allowed us to estimate the resistivity of soil (values between 25 and 60 Ωm) and radar energy attenuation (values between 6 and 14 dB/m). These values show that Ground Penetrating Radar can be effectively and reliably used in this study area. Additional CMP measurements, carried out through a bistatic GPR system with central frequency of 450 MHz, were used to determine velocity profiles [17]. The bare plot was surveyed with two different monostatic GPR equipments along transects N-S and E-W oriented and about 1 m apart using the common-offset method: one, a Noggin 250 MHz (Sensors & Software Inc., Mississauga, ON, Canada), operates with a central frequency of 250 MHz and spans a 3 dB bandwidth from 125 to 375 MHz and with shielding front to back >20 dB and the other, a RIS 2 k-MF Multifrequency Array Radar-System (manufactured by IDS Ing, Pisa, Italy), with two central frequencies of 600 MHz (bandwidth from 300 to 900 MHz) and 1600 MHz (bandwidth from 800 to 2400 MHz). The 250 MHz GPR system acquired the data using a time window of 100 ns with a temporal sampling interval of 0.2 ns and spacing between the traces of 0.05 m collected after 16 stacked radargrams with a performance factor of 172 dB. The 600 and 1600 MHz GPR system worked with a time window of 60 ns and a temporal sampling interval of 0.05 ns; successive traces were collected every 0.024 m with a performance factor of 172 dB. The coordinates of the initial and final positions of GPR transects were recorded using a differential global positioning system with planimetric centimeter accuracy. Detailed discussions of the fundamental principles of GPR can be found in the publications by Daniels et al. [19] and Davis and Annan [7]. GPR Data Processing All GPR data were processed with ReflexW Software [20] and six different data processing sequences were applied to all radar sections ( Figure 2): 1° procedure: Time zero correction; 2° procedure: Time zero correction and dewow filtering; 3° procedure: Time zero correction, dewow filtering and band-pass frequency filter; 4° procedure: Time zero correction, dewow filtering, band-pass frequency filter and running average on a defined number of traces covering 0.5 m; 5° procedure: Time zero correction, dewow filtering, band-pass frequency filter and running average covering 1 m; 6° procedure: Time zero correction, dewow filtering, band-pass frequency filter and migration. Detailed descriptions of the above procedures can be found in the historical publications by Reynolds [21] and Cassidy [22]. The signal processing parameters applied to the data are summarized in Table 1. The computer time varied for each procedure, increasing with the complexity of processing. No amplitude gain functions were applied to the data because they are more likely to destruct relative amplitude information [23]. After each procedure, the instantaneous amplitude or envelope of data was calculated using a quadrature filter (Hilbert transformation). This filter is traditionally used to transform a real-value signal to an analytic signal, i.e., a signal that has no negative frequency components. In practice, quadrature filters give an estimation of the energy distribution of the traces [24]. It is a measure of the reflectivity strength and is proportional to the square root of the total energy of the received signal at a given time instant. Envelope can then give an overview of the distribution of the different types of reflectors present in the subsoil and then permits the construction of a structural model of subsoil. One of the most impressive ways of displaying GPR data is in horizontal maps that allow easy visualization of location, depth, size and shape of the radar anomalies buried in the ground. The maps can be created at various time levels within a data set to show radar information at a specified time (depth) across a surveyed site. Therefore, enveloped amplitude maps (time slices) were built averaging the amplitude (or the square amplitude) of the radar signal, expressed in digital number (DN), within overlapping time windows of width Δt. Typically Δt must be of the order of the dominant period of the antennas (4 ns, 2 ns and 1 ns for 250, 600 and 1600 MHz antennas, respectively), because GPR reflections are normally taken over a time window of a microwave pulse length. The total time interval was of 20 ns for 250 and 600 MHz because this time was comparable with the depth of soil (at 0-0.30 m depth), and of 5.5 ns for 1600 MHz because of the attenuation of radar signal. For visual representation and interpretation of radar sections, reported in order to support the interpretation of the relationship observed by statistical and geostatistical analysis, standard GPR data processing was performed including: time zero correction, dewow, automatic gain control (AGC), background removal and bandpass filter. Geostatistical Methodology The multivariate dataset, consisting of GPR time/depth slices after processing (Figure 2), was interpolated using multiGaussian cokriging [25]. This approach is based on a multiGaussian model which requires a prior Gaussian transformation of each attribute into a Gaussian shaped variable with zero mean and unit variance. Such a transformation procedure, known as Gaussian anamorphosis, consists in determining a mathematical function which transforms a variable with a Gaussian distribution into a new variable with any distribution [26,27]. Gaussian anamorphosis was performed by using an expansion in Hermite polynomials restricted to a finite number of terms [27]. As for variogram fitting, a Linear Model of Coregionalization (LMC) was applied. LMC, developed by Journel and Huijbregts [28], considers all the studied variables as the result of the same independent physical processes, acting at Ns spatial scales, u. The n(n + 1)/2 simple and cross-semivariograms (γ) of the n variables are modelled by a linear combination of Ns semivariograms standardized to unit sill gu(h), which are assumed to be the same for all variables at a given spatial scale u. Using the matrix notation, the LMC can be written as: is a symmetric matrix of order n × n, whose diagonal and out of-diagonal elements represent simple and cross-semivariograms, respectively for lag h; B u = [b u ij] is called coregionalization matrix and it is a symmetric positive semi-definite matrix of order n × n with real elements b u ij, which represent the sills of the direct (if i = j) and (cross-) variograms ij (for i ≠ j) at a specific spatial scale u among the selected Ns scales. The model is authorized if the mathematical functions g u (h) are mathematically authorized semivariogram models under the constrain of positive semi-definiteness of each B u , being assumed to represent a variance-covariance matrix at the spatial scale u. Fitting of LMC is performed by weighed least-squares approximation under the constraint of positive semi-definiteness of the B u , using an iterative approach developed by Rivoirard [29]. Goodness of fitting was evaluated using cross-validation and in particular by calculating mean error and mean squared standardized error, which have to be close to 0 and 1, respectively. In LMC, total variance can be decomposed into spatially structured variance at different spatial scales and spatially unstructured variance (nugget). The nugget effect represents unexplained spatially dependent variation (microvariability at distances closer than the smallest sampling lag) or purely random variance (like measurement or sampling error). The proportion of total variation can warn us of large measurement errors or of the need to sample more densely, or both [30] and for this reason, it can be used as indicator of map quality. The GPR data were interpolated with block cokriging in order to reduce the variability, using a regular 5 × 5 discretization of each 0.5 m × 0.5 m block; finally the estimates were back-transformed to amplitude of the radar signal. Geostatistical procedures were separately applied to the data of each antenna frequency, by using the software package ISATIS ® , release 2014 [31]. After interpolation, to eliminate the noise due to variable energy of the transmitted signal, the estimated amplitudes were normalised by using, as reference, the first time slice mainly related to the radar waves in air. Comparison between the Maps In order to make the comparison among the maps more objective and to choose the most efficient GPR processing in a less subjective way, the spatial association between paired GPR maps was calculated using confusion matrix and k statistic. The efficiency of data processing was evaluated in terms of maps quality and computer time consuming. The maps were preventively classified in ten isofrequency classes of radar signal amplitude and the classes were then compared in pairs by using a two-enter table (confusion matrix). Each cell of the matrix gave the absolute frequency of the occurrence of the two corresponding classes [32]. The overall consistency, which is a measure of the spatial association between two maps, was computed as the proportion of the total number of observations along the main diagonal of the contingency matrix. Weighted kappa coefficient, introduced by Cohen [33], measures the inter-classification agreement and equals 0 when the agreement is due to chance and +1 when there is perfect agreement. When kappa is negative, it is assumed no agreement. Besides kappa coefficient, its confidence limits [34] were computed. The approach was implemented with the FREQ procedure of the SAS/STAT software package [35]. First Visual Interpretation From the visual inspection of GPR radar sections and CMP data, three main layers were synthetically disclosed: a first layer at time ranging between about 3 ns to about 5 ns (0.09-0.15 m depth, considering a velocity of 0.06 mns −1 ), visible only in the radar sections acquired with 1600 MHz frequency (an example is reported Figure 3), a second layer between 10 ns and 14 ns (0.3-0.4 m depth with a velocity of 0.06 mns −1 ) and a third layer between 20 ns and 22 ns (0.6-0.66 m depth with an average velocity of 0.1 mns −1 ), detectable from all the radar sections acquired with 250 and 600 MHz frequencies and from CMP data (example of CMP data is reported in Figure 4). For convenience these reflections are referred to as the "first", "second" and "third" reflection, respectively. The "first" and "second" reflected layers may be related to interfaces in the soil, probably due to shallow ploughing or soil compaction caused by tractor passage and/or tillage. Conversely, the "third" reflected layer was ascribed to the soil-bedrock interface because of its wide amplitude, denoting a strong electromagnetic contrast, and on the basis of pre-existing pedological profile (Figure 1b). The bedrock reflection was generally characterized by marked roughness (more or less evident in different parts of the site) and many anomalies of various types (hyperbolic signals) were observed in the overlying soil layer. The radar sections of the different antennas showed varying features in terms of resolution, and it was preferred not to select only one antenna because all antennas jointly captured the scale-dependent variation of soil/subsoil. Moreover, the propagation velocity was equal to 0.06 mns −1 up to 10 ns and for longer times the average velocity was 0.1 mns −1 , assuming a subsoil model with horizontal stratification and constant lateral velocity (Figure 4b). Statistical Analysis of Data All the time slices showed a sensible attenuation of signal at about 20 ns (about 0.90 m depth) for 250 and 600 MHz frequencies and at about 5.5 ns (about 0.15 m) for 1600 MHz frequency; therefore, all GPR data coming from the longer travel times (deeper depths) will not be treated from now onwards. Statistical analysis highlighted that GPR data at any frequency and for all procedures showed clear departure from normal distribution, however the addition of further steps in the processing (after the third procedure) caused a more symmetric distribution. In addition, Person's correlations allowed you to disclose the main reflections observed visually in the radar sections and to evaluate the strength of spatial association between the data at the different depths as function of the processing procedure. The GPR amplitudes at the different times were strongly correlated within an interval of 10-14 ns, for 250 MHz antenna (mainly evident in the data processed with the third procedure, as reported in Figure 5). This time interval may be related to the "second" reflection, which was not detectable visually in the radar sections but only in CMP data. The correlation coefficient showed a discontinuity in the range 10-12 ns for the 600 MHz antenna at the corresponding depth range of 0.3-0.36 m, not evident in the radar sections, which may be due to agricultural tillage. On the contrary, the correlations between the time slices corresponding to the 1600 MHz antenna indicated the presence of a discontinuity at 3.5-4 ns (corresponding at 0.1-0.12 m depth), not detectable in the other antennas, which may be due to the presence of organic residuals in the first ten centimeters. Furthermore, from the statistical analysis, the third procedure seems to have achieved a good trade-off between the quality of the signal at any travel time (the data distribution was quite normal and with few outliers, as a consequence of the band-pass filter, and the correlation coefficients increased so improving the signal in depth) and the computer processing time demand (the computer time for the fourth procedure was longer) for any frequency. Visual Interpretation of the Estimated Maps All GPR data at any time interval for each antenna were correlated so to justify the application of a multivariate approach. The data, being generally skewed, were previously transformed into standard Gaussian variables and a linear isotropic model of coregionalization (LMC) was fitted to the all experimental direct and cross-variograms separately for each processing procedure and each antenna. Independently of the kind of processing, the basic structures were identical for any antenna, showing that the procedures did not alter the intrinsic spatial structures. Geostatistical modelling of = 12 m). The goodness of LMC fitting was satisfactory because mean error was quite close to 0 and the mean square standardised error was almost 1 for all data sets. As regards the first procedure at 250 MHz frequency, the visual inspection of the estimated normalized amplitude maps at the different times showed that the processing did not modify sensibly the signal at any depth, preserving the raw information. However, the signal in depth was not usable because of the high attenuation (Figure 6a). For both 600 and 1600 MHz antenna, the maps were very noisy and did not showed well defined spatial structures (Figure 6b,c). For the second processing, the maps at 250 MHz frequency and corresponding to the longer time slices (16-18 ns) appeared well structured and more associated with the ones observed in the shallower depths. Therefore, the addition of a further step in the GPR processing improved the signal, recovering more information from the deeper depths. This characteristic was not valid for the other frequencies, suggesting that no improvement was obtained with the addition of dewow, as expected for the higher sampling frequencies used. As for the third procedure, all the maps of the estimated amplitude at different times (Figure 7) displayed some consistency up to the deeper depth. A tendency for higher values of amplitude was detectable along the north-eastern and south-western sides of the plot. An improvement was also visible for the maps of the other frequencies (Figures 8 and 9) which looked more variable because of their finer spatial resolution. They showed a similar spatial pattern after 10 ns for 600 MHz frequency and after 5.5 ns for the 1600 MHz frequency, which consisted of two blocks along the longitudinal axis, with the eastern side characterized by higher values of amplitude. This type of processing then seems to reach a good trade-off between the quality of signal at any travel time/depth and the complexity of signal processing. No significant improvement was observed in the maps obtained with the application of running average (fourth and fifth processing) even if the maps looked slightly more smoothed. Finally, the maps obtained with the sixth procedure were quite similar to the previous maps, though the quality has worsened for some of them, in particular for the deeper maps (Figure 10), because of the presence of quite evident artifacts. This was probably due to the assumption of a subsoil model with horizontal stratification and the use of an average velocity for all radar sections of a given frequency. In a complex environment, as the one studied, this assumption is probably violated, therefore the migration can cause errors and does not improve the quality of maps. Quantification of Measurement Error Since the best processing should be selected on its capability of filtering spatially uncorrelated error (white noise of GPR signal, nugget effect in geostatistics), the proportion of the total variance associated with nugget component was calculated ( Figure 11). It is evident that the error has a tendency to decrease with the complexity of processing, with the exception of the last procedure, confirming in objective way most of the previous considerations. As for 250 MHz frequency, the main differences were between the first two processing procedures and the third one (about 16%), while the successive steps did not reduce sensibly the measurement error, with the exception of the last one that caused an increase. As explained previously, a reason, is that migration relies on the knowledge of velocity; therefore, an uncertain estimation may cause error in prediction of signal. An improvement in the results may be achieved by using an accurate subsurface velocity model obtainable with CMP semblance technique, which requires a longer time of signal acquisition and processing. For the other antenna frequencies (Figure 11), the nugget proportion was lower than the one at 250 MHz frequency, due to the finer footprint. The third procedure did not reduce greatly the measurement error (13%), as much as for the fourth and fifth procedures (about 40%-45% for 600 MHz antenna and about 35%-37% for 1600 MHz antenna). These last procedures (for all frequencies) showed the lowest values of nugget proportion, but no significant improvement was observed in the maps. The sixth procedure, also in this case, caused an increase of nugget proportion compared with the ones of the 3-5 procedures, and the quality of maps was then worse. Therefore, also in the light of the previous considerations, the third processing could be considered as optimal in terms of map quality and complexity of signal processing. Comparison among the Different Processing Procedures: Results For each frequency, all maps corresponding to the different types of processing were compared with the one of the third processing procedure, assumed as reference. In the interest of conciseness, only the results at 250 MHz frequency will be presented. As regards the shallower depth, the maps corresponding to the first two procedures generally showed lower overall accuracy and weighted kappa coefficient, which is indicative of poor spatial association with the reference map. The use of these procedures then changed the signal structure in the shallower layers, though at a visual inspection the maps appeared very similar. Moderate association (about 50%) was obtained with the fourth procedure, indicating no actual improvement. Moreover, the poor agreement (about 20%) with the procedure after migration confirmed the result obtained through a visual comparison of the maps. On the contrary, the overall accuracy for the maps corresponding to 10 ns (Table 2) was higher for the first two procedures and the kappa coefficients were significantly greater than 0 and indicative of a moderate spatial agreement. The use of additional steps of processing (from the fourth to sixth procedure) reduced the overall accuracy. Finally, the results related to the deeper time slices indicated very weak agreement, demonstrating the modification of signal in depth with the use of these additional steps. In conclusion, the traditional statistical analysis allowed you to objectively state that the spatial association with the map of the third procedure decreased with the complexity of processing. However, geostatistics with nugget effect estimation provided a tool of testing the quality of maps and then a criterion for selecting the optimal processing. Comparison among the Different Antenna Frequencies: Results In order to investigate the response of GPR as a function of operating frequency, we restricted the geostatistical analysis only to the data processed according to the third procedure. The estimated structures (type, range, sill) of GPR data are expected to depend on the antenna frequency, since the radar signal wavelength affects spatial resolution, which is determined by the area illuminated by a GPR antenna, often referred to as the Fresnel zone or antenna footprint [7] or support in geostatistics. The LMCs (reported in Section 3.3) revealed the presence of three basic structures at different spatial scales in the horizontal plane. The cumulative values of the eigenvalues associated with each spatial structure showed the main components of variation to be the nugget effect (0.58, 0.47 and 0.3 for 250, 600 and 1600 MHz frequencies respectively) and the structure at short range (0.25, 0.34 and 0.62 for 250, 600 and 1600 MHz frequencies respectively). For 250 MHz frequency the highest proportion of variation was associated with nugget effect, because of a wider support. At the lowest frequency, the maps looked smoother and more continuous than at higher frequency, because they were less sensitive to small scale features and produced a LMC with longer ranges. This result was confirmed by the visual interpretation. It can be observed that the shorter range (3 m, 1 m and 0.5 m for 250, 600 and 1600 MHz frequencies, respectively) was quite close to the wavelength calculated for propagation velocity of 0.06 m/ns (2.4 m, 1 m and about 0.4 m for 250, 600 and 1600 MHz frequencies, respectively), which is directly proportional to the measurement resolution. The short-range structure was probably more sensitive to the type of equipment used, whereas the longer range was more influenced by the material properties. Some researchers obtained similar results using different antenna frequency but they asserted that they depend not only on the material and its intrinsic structure but also on data processing and signal frequency [10]. The longer-scale structure may then be related to soil texture and hydraulic properties. This issue should be further investigated by assessing and modelling the relationships of GPR data with textural and hydraulic properties [36]. Conclusions The approach presented in this paper aims to investigate the effects of data processing on radar signal and to select a data processing procedure which is quick and efficient in terms of both computer time and quality of maps. Geostatistical analysis applied to three different frequencies, stressed the critical importance of the scale of survey and the need to utilise a proper equipment to capture the scale-dependent variability of soil/subsoil. The processing procedure, based on the instantaneous amplitude or envelope, was evaluated with statistical and geostatistical techniques. Statistical analysis allowed you to detect the major reflections of the radar signal, and other useful information not evident at visual interpretation of radar sections. Geostatistical analysis provided also a criterion to select the most efficient processing on the basis of its capability of reducing spatially uncorrelated error. The selected error-effective procedure included time zero correction, dewow filter, bandpass filter and envelope. The main limitation of the proposed procedure consisted in the extremely heterogeneous subsurface conditions of study site because the received signal was the result of multiple interactions. Further studies should be conducted to improve the interpretation of such type of data. In particular the obtained results indicated that geostatistical tools, like geostatistical component filtering [25], could be applied to reduce noise and systematic variation and produce more reliable maps of amplitude. Signal frequency exhibited a drastic influence on the spatial structures of geostatistical modelling, because of its relation to spatial resolution. The pattern of spatial structures estimated by geostatistics can be related to both radar equipment (short range) and soil properties (long range). Disclosing which soil physical properties are mainly responsible for the observed spatial structures of GPR signal remains an interesting and challenging research topic for future investigations.

v3-fos

2019-04-01T13:02:39.297Z

2015-09-01T00:00:00.000Z

89481797

s2

Sweet Cherry Skin Has a Less Negative Osmotic Potential than the Flesh The skin is the primary load-bearing structure in a sweet cherry fruit (Prunus aviumL.). Failure of the skin in rain cracking is considered to be related to water uptake. Little is known of the skin’s water potential, its osmotic potential (CP ), and turgor. The objective here was to quantify CP S relative to the osmotic potential of the flesh (CP ). Spatial resolution was achieved by monitoring plasmolysis in epidermal cells in tissue sections, incubated in selected osmotica using a light microscope method. Decreasing the osmotic potential [CP (more negative)] of the incubation medium increased the proportion (percent) of plasmolyzed epidermal cells. The pattern of increasing plasmolysis was sigmoidal with increasing osmolyte concentration. The value of CP for 50% of cells plasmolyzed, depended to some extent on the osmolyte used. The value ofCP became slightly less negative for the osmolytes tested in the order: 1) mannitol, 2) sucrose, and 3) artificial cherry juice (a solution comprising the five major osmolytes of sweet cherry juice in the appropriate proportions and concentrations). There was little difference in the value ofCP at 50% plasmolysis between the cultivars Hedelfinger, Sam, and Sweetheart. In all three cultivars, the value of CP F (measured for expressed juice using an osmometer) wasmarkedly more negative than that ofCP S (measured for 50% plasmolysis). Incubating skin segments in juice from the same fruit resulted in the plasmolysis of most (85.7% to 96.4%) of the epidermal cells. As fruit development progressed from stage II [27 day after full bloom (DAFB)] to the fully mature stage III (97 DAFB), plasmolysis occurred for increasingly more negative values of CP. Moreover, the difference between the osmotic potential values recorded for the flesh CP F and for the skin CP S increased. Plasmolysis of epidermal cells was accompanied by a marked swelling of their walls. The results indicate a marked difference in the osmotic potential of flesh (CP F trended more negative) and skin cells (CP S trended less negative). The water potential (Y) of the sweet cherry fruit and its two components, osmotic potential (YP) and turgor (YP) (whereY = YP + YP), are likely to be important factors affecting fruit cracking. First, fruit Y affects the rate of water uptake through the skin surface. Here, the rate is related to the difference between the value ofY for an adhering water droplet (probably very close to zero) and that of the epidermis (Beyer and Knoche, 2002). Second, for vascular flow through the xylem of the pedicel, the flow rate will be related to the difference between the value of YP in the apoplast of the spur and the average value ofYP in the apoplast of the fruit. Third, according to the critical turgor pressure concept, rain cracking is thought to be a function of fruit turgor in grapes [Vitis vinifera L. (Considine and Kriedemann, 1972)] and also in sweet cherries (Measham et al., 2009). Here fruit (tissue) turgor is generated by the stress in the fruit skin, which envelopes the semifluid parenchyma of the flesh (Considine and Brown, 1981). Despite its possible role in fruit cracking, little is known about the values taken by Y and its components within the various tissues of a developing sweet cherry fruit. Till now, for sweet cherries, it is the situation prevailing in the large parenchyma cells of the flesh of the outer mesocarp that has received most attention with data now being available for the osmotic potential (YP) and turgor of these cells (YP) (Knoche et al., 2014; Schumann et al., 2014). The turgor of these cells is surprisingly low (YP zero) compared with their highly negative osmotic potentials (YP), implying highly negative values for their water potentials (Y). The thin [<100 mm (Glenn and Poovaiah, 1989)] skin of a sweet cherry fruit forms its structural ‘‘backbone’’ (Br€uggenwirth et al., 2014) holding the flesh under compression, the skin under tension, like a football (Grimm et al., 2012). However, similar information relating to the epidermal cells that (with the hypodermis) make up the fruit’s skin is not yet available and is difficult to obtain. The desired values for the epidermis are the skin’s water potential (Y) with its components YP and turgor (YP). These values are especially difficult to obtain because 1) the skin is quite thin and 2) the size of the epidermal cells is much smaller than the parenchyma cells of the flesh. This makes direct determination of cell turgor using a pressure probe difficult (Steudle, 1993). The only published information of which we are aware is based on osmometric measurements of juice extracted from 1 ± 0.2-mm-thick skin tissue samples (Moing et al., 2004). Based on their dataset, the value of YP for the outer 1-mm layer of the fruit was slightly less negative than that of the flesh beneath. However, the thin (<100 mm) skin can have comprised only 10% or less of their 1-mm sample (Glenn and Poovaiah, 1989), so their result must have been dominated (90% or more) by the parenchyma cells immediately underlying the hypodermis and epidermis. Hence one might reasonably expect the true difference between YP and YP to be markedly greater than that reported by them. Nevertheless, direct evidence for our assertion is lacking. The objective of this study was to measure the osmotic potential of the epidermal cells of sweet cherry fruit (YP) relative to those of the flesh cells (YP). Because of the need for Received for publication 5 May 2015. Accepted for publication 12 June 2015. This research was funded in part by a grant from the Deutsche Forschungsge- ADDITIONAL INDEX WORDS. Prunus avium, epidermis, exocarp, mesocarp, turgor, water potential ABSTRACT. The skin is the primary load-bearing structure in a sweet cherry fruit (Prunus avium L.). Failure of the skin in rain cracking is considered to be related to water uptake. Little is known of the skin's water potential, its osmotic potential (C P S ), and turgor. The objective here was to quantify C P S relative to the osmotic potential of the flesh (C P F ). Spatial resolution was achieved by monitoring plasmolysis in epidermal cells in tissue sections, incubated in selected osmotica using a light microscope method. Decreasing the osmotic potential [C P (more negative)] of the incubation medium increased the proportion (percent) of plasmolyzed epidermal cells. The pattern of increasing plasmolysis was sigmoidal with increasing osmolyte concentration. The value of C P for 50% of cells plasmolyzed, depended to some extent on the osmolyte used. The value of C P became slightly less negative for the osmolytes tested in the order: 1) mannitol, 2) sucrose, and 3) artificial cherry juice (a solution comprising the five major osmolytes of sweet cherry juice in the appropriate proportions and concentrations). There was little difference in the value of C P at 50% plasmolysis between the cultivars Hedelfinger, Sam, and Sweetheart. In all three cultivars, the value of C P F (measured for expressed juice using an osmometer) was markedly more negative than that of C P S (measured for 50% plasmolysis). Incubating skin segments in juice from the same fruit resulted in the plasmolysis of most (85.7% to 96.4%) of the epidermal cells. As fruit development progressed from stage II [27 day after full bloom (DAFB)] to the fully mature stage III (97 DAFB), plasmolysis occurred for increasingly more negative values of C P . Moreover, the difference between the osmotic potential values recorded for the flesh C P F and for the skin C P S increased. Plasmolysis of epidermal cells was accompanied by a marked swelling of their walls. The results indicate a marked difference in the osmotic potential of flesh (C P F trended more negative) and skin cells (C P S trended less negative). The water potential (Y) of the sweet cherry fruit and its two components, osmotic potential (Y P ) and turgor (Y P ) (where Y = Y P + Y P ), are likely to be important factors affecting fruit cracking. First, fruit Y affects the rate of water uptake through the skin surface. Here, the rate is related to the difference between the value of Y for an adhering water droplet (probably very close to zero) and that of the epidermis (Beyer and Knoche, 2002). Second, for vascular flow through the xylem of the pedicel, the flow rate will be related to the difference between the value of Y P in the apoplast of the spur and the average value of Y P in the apoplast of the fruit. Third, according to the critical turgor pressure concept, rain cracking is thought to be a function of fruit turgor in grapes [Vitis vinifera L. (Considine and Kriedemann, 1972)] and also in sweet cherries (Measham et al., 2009). Here fruit (tissue) turgor is generated by the stress in the fruit skin, which envelopes the semifluid parenchyma of the flesh (Considine and Brown, 1981). Despite its possible role in fruit cracking, little is known about the values taken by Y and its components within the various tissues of a developing sweet cherry fruit. Till now, for sweet cherries, it is the situation prevailing in the large parenchyma cells of the flesh of the outer mesocarp that has received most attention with data now being available for the osmotic potential (Y P F ) and turgor of these cells (Y P F ) Schumann et al., 2014). The turgor of these cells is surprisingly low (Y P F %zero) compared with their highly negative osmotic potentials (Y P F ), implying highly negative values for their water potentials (Y F ). The thin [<100 mm (Glenn and Poovaiah, 1989)] skin of a sweet cherry fruit forms its structural ''backbone'' (Br€ uggenwirth et al., 2014) holding the flesh under compression, the skin under tension, like a football (Grimm et al., 2012). However, similar information relating to the epidermal cells that (with the hypodermis) make up the fruit's skin is not yet available and is difficult to obtain. The desired values for the epidermis are the skin's water potential (Y S ) with its components Y P S and turgor (Y P S ). These values are especially difficult to obtain because 1) the skin is quite thin and 2) the size of the epidermal cells is much smaller than the parenchyma cells of the flesh. This makes direct determination of cell turgor using a pressure probe difficult (Steudle, 1993). The only published information of which we are aware is based on osmometric measurements of juice extracted from 1 ± 0.2-mm-thick skin tissue samples (Moing et al., 2004). Based on their dataset, the value of Y P S for the outer 1-mm layer of the fruit was slightly less negative than that of the flesh beneath. However, the thin (<100 mm) skin can have comprised only %10% or less of their 1-mm sample (Glenn and Poovaiah, 1989), so their result must have been dominated (90% or more) by the parenchyma cells immediately underlying the hypodermis and epidermis. Hence one might reasonably expect the true difference between Y P S and Y P F to be markedly greater than that reported by them. Nevertheless, direct evidence for our assertion is lacking. The objective of this study was to measure the osmotic potential of the epidermal cells of sweet cherry fruit (Y P S ) relative to those of the flesh cells (Y P F ). Because of the need for high spatial resolution, we used microscopy and a plasmolysis technique to limit our analyses just to the epidermal cell layer. The same values for the flesh were measured conventionally using osmometry. Materials and Methods PLANT MATERIAL. Fruit of the sweet cherry cultivars Adriana, D€ onissens Gelbe, Flamengo Srim, Fr€ uhe Rote Mecklenburger, Hedelfinger, Rainier, Sam, Staccato, and Sweetheart, all grafted on Gisela 5 rootstocks (Prunus cerasus L. · Prunus canescens Bois), were sampled at commercial maturity as indexed by color, size, and taste. In addition, 'Regina' fruit was sampled during development from 27 d to maturity at 97 DAFB. Trees were grown in a greenhouse and an experimental orchard of the Horticultural Research Station of the Leibniz University in Ruthe (lat. 52°14#N, long. 9°49#E). Fruit were selected for freedom from defects and for uniformity, and transferred to the laboratory. Unless otherwise specified fruit was processed within 48 h of sampling. GENERAL EXPERIMENTAL PROCEDURE. Sections of the fruit skin comprising epidermal and hypodermal cells were prepared using a razor blade, blotted with tissue paper or rinsed by dipping briefly (5 s) in deionized water. A section was immediately transferred to a microscope slide, covered with a cover slip, and incubated for 30 min in an aqueous solution containing one of a broad range of concentrations of an osmolyte. Unless otherwise specified, sucrose was used as the standard osmolyte. After 30 min, the slide was transferred to the stage of a microscope and viewed in incident transmitted light at ·40 magnification using a fluorescence microscope (BX-60; Olympus Europa, Hamburg, Germany). Occasionally, differential interference contrast was used to enhance contrast. Unless otherwise specified, cells were observed only in sections that appeared healthy as indexed by the absence of coagulated protoplasts. Calibrated digital images were taken (DP 71, Olympus Europa) and the percentage of plasmolyzed cells was determined. In some experiments, swelling of the cell walls was quantified by determining cell wall thickness using image analysis (Cell-P, Olympus Europa). The osmolarity of incubation media and of juice extracted from the same fruit as that used in the plasmolysis and cell wall swelling assays was quantified by vapor pressure osmometry (VAPRO Ò 5520 and 5560; Wescor, Logan, UT). EXPERIMENTS. The effects of osmolyte type and concentration on the percentage plasmolysis were investigated in 'Adriana' sweet cherry using sucrose, mannitol, or a mix of osmolytes that mimicked the composition of extracted sweet cherry juice. We refer to this solution as artificial cherry juice. Where solubility permitted, osmolarities ranged from 0 to 5 MPa (equivalent to 0 to 2000 mmolÁkg -1 ). For the artificial juice, a 1000 mmolÁkg -1 solution was composed of glucose (431.8 mM), fructose (393.5 mM), sorbitol (76.9 mM), malic acid (13.8 mM), and potassium malate (56.3 mM) (Herrmann, 2001). These five osmolytes accounted for 98% of the osmolarity recorded for sweet cherry juice. The pH of the artificial juice (pH 3.4) was close to that of natural sweet cherry juice (pH 3.6). The percentage of plasmolyzed cells was quantified as described above on sections excised from five fruit per osmolyte type. Differences in the osmolarity of the skin and flesh were investigated in 'Hedelfinger', 'Sam', and 'Sweetheart' fruit. The osmolarity of the epidermis was assessed by quantifying the percentage of plasmolysis of fruit skin sections after 30 min of incubation in sucrose solutions and that of the flesh by water vapor pressure osmometry of expressed juice. Sections were prepared from 10 fruits per cultivar. Plasmolysis was also determined following incubation of skin sections in juice extracted from flesh of the same fruit in the immediate vicinity of the position where the skin section was excised. Plasmolysis was assessed after 30 min. Segments incubated in silicone oil (AK 10; Wacker Chemie, Munich, Germany) served as controls and were inspected immediately after excision. To broaden the database, this experiment was also conducted with 'D€ onissens Gelbe', 'Flamengo Srim', 'Fr€ uhe Rote Mecklenburger', 'Hedelfinger', 'Rainier', 'Sam', and 'Staccato'. Two sections per fruit from a total of 10 fruit per cultivar were assessed. The effect of development on the osmolarity of epidermal cells as indexed by plasmolysis was studied in developing 'Regina' (27 to 97 DAFB). Sections of the fruit skin were prepared, incubated in sucrose solutions (30 min), photographed, and the percentage of plasmolyzed cells and thickness of cell walls were quantified as described above. This procedure allowed direct comparisons between cell wall thickness and the extent of plasmolysis on an individual skin segment basis. The number of replicates was 10. Data for the osmolarity of the flesh of the fruit (obtained from the same batch of fruit) were taken from Schumann et al. (2014). The time course of cell wall swelling was established using skin sections from 'Sam' sweet cherry (n = 5). Because cells burst when incubated in water for extended periods of time, incubation was ended after 3 h. By this time, the majority of protoplasts were still intact and alive so that swelling could be established on an individual segment basis. Potential relationships between the swelling of cell walls and the initial cell wall thickness were identified in 'Sam' sweet cherry by quantifying cell wall thickness within 5 min of excision of skin segments and then repeating the measurement on the same segment at the same position 3 h later. Fruit used in this experiment was held at 2°C for 5 to 6 d. Whether cell wall swelling was affected by the health (or otherwise) of neighboring epidermal cells was studied in 'Sweetheart'. In these experiments, a polyethylene glycol solution was used that was osmotically buffered at an osmolarity of 200 mmolÁkg -1 . A 6-h incubation period was selected. Under these conditions, some protoplasts collapsed as indexed by their coagulated cytoplasm and epidermal sections contained both healthy and collapsed cells. Cell wall thickness was measured between two healthy epidermal cells, between a living cell and a dead cell and between two dead cells. Three measurements were made per pair of cells and section on a total of 20 fruit. DATA ANALYSES. Results in tables and figures are presented as means ± SE. Where error bars are not visible, they are smaller than the symbols. Analyses of variance (PROC GLM) and regression analyses (PROC REG, PROC NLIN) were carried out using SAS (version 9.1.3; SAS Institute, Cary, NC). Percentage plasmolysis data were arcsine transformed before analysis of variance. The osmolarity at 50% plasmolysis was estimated as the point of inflection of a logistic regression model. At this osmolarity, the turgor pressure is zero (the point of insipient plasmolysis) and the osmolarity of the symplast is assumed to be equal to that of the incubation solution. Results Plasmolysis occurred when skin sections were incubated in hypertonic sucrose solutions. Vacuoles and protoplasts of some epidermal cells shrank, so that the cell membranes separated and receded from the cell walls ( Fig. 1A-C). As the osmotic potential of the incubation medium fell (became more negative with increasing concentration of osmolytes) so the percentage of plasmolyzed epidermal cells increased. The profile of the plasmolysis increase was sigmoidal with falling osmotic potential ( Fig. 2A). When different osmolytes were compared, the osmotic potential associated with 50% plasmolysis changed. Thus, the osmotic potential with mannitol as osmolyte was less negative (-0.8 ± 0.1 MPa), than with sucrose (-1.2 ± 0.0 MPa), than with artificial sweet cherry juice (-1.4 ± 0.0 MPa). It was also observed that juice extracted from the flesh had an osmotic potential of -1.9 ± 0.0 MPa. This value is significantly more negative than the osmotic potentials of any of the pure osmolytes or of the artificial juice for the 50% plasmolysis condition in the epidermis. When skin segments of 'Hedelfinger', 'Sam', and 'Sweetheart' were incubated in solutions of different osmolarities, the relationships observed between percentage plasmolysis and osmotic potential were qualitatively and quantitatively similar (Fig. 2B). In all cultivars examined, the osmotic potential of the flesh was markedly more negative than that of the epidermal cells of the same individual fruit (Table 1). Across all cultivars and fruit, the average difference was 1.1 MPa [range 0.8 to 1.5 MPa (Table 1)]. It is interesting to note that in the comparison of osmolytes ( Fig. 2A) and the comparison of cultivars (Fig. 2B), a marked difference (up to 1 MPa and more) existed between the least and the most negative osmotic potentials of bathing solutions causing plasmolysis in a fraction of the population of epidermal cells (insets in Fig. 2A and B). Thus, the distribution of osmotic potentials of epidermal cells must have been broad. When skin segments of selected sweet cherry cultivars were incubated in juice extracted from the same batch of fruit, epidermal plasmolysis was the general result ( Fig. 1D and E). In six out of seven cultivars, between 85.7% and 96.4% of epidermal cells were plasmolyzed. This indicates that the skin has a less negative osmotic potential than the flesh of the same fruit (Table 2). 'Flamengo Srim' differed from all other cultivars examined in that 1) the percentage of epidermal cells plasmolyzed in juice from the same fruit was lower but still significantly higher than in the control and 2) a significant proportion of epidermal cells exhibited granulated cytoplasm and showed no indication of plasmolysis. Plasmolysis depended on the stage of development (Fig. 3). As fruit developed from stage II (27 DAFB) to full maturity at stage III (97 DAFB), plasmolysis occurred at increasingly more negative osmotic potentials. A comparison of fruit of different developmental stages revealed that 1) osmotic potentials of flesh and skin decreased steadily (became increasingly negative) with the largest change occurring around the stage II/III transition, 2) skin osmotic potentials were always less negative than those of the flesh, and 3) the osmotic potential difference between flesh and skin increased steadily with development ( Fig. 4A-C). Plasmolysis of epidermal cells was accompanied by a marked swelling of their cell walls ( Fig. 1B and C). The time-course experiment established that swelling approached an asymptote within %3 h (Fig. 5A). Furthermore, the thickness of the swollen cell walls (after 3 h) was linearly related to their initial thickness (Fig. 5B). Because the slope of the linear regression was close to unity (slope 0.88 ± 0.09; r 2 = 0.85, P < 0.001), the amount of cell wall swelling was essentially independent of initial cell wall thickness. The increase in cell wall thickness averaged %0.64 ± 0.15 mm. The extent of cell wall swelling depended on the health of the cells bordering a particular cell wall. Cell wall swelling was greatest for a cell wall between two nonliving cells and least when both bordering cells were healthy and fully turgid. The extent of cell wall swelling was intermediate for a cell bordering a healthy cell on one side and a nonliving one on the other ( Fig. 1F; Table 3). Cell wall swelling depended on the osmotic potential of the incubation medium and on the developmental stage of the fruit (Fig. 3). Before 55 DAFB, cell wall thickness was nearly independent of the osmotic potential of the bathing solution indicating the absence of significant swelling. As fruit development progressed to 55 DAFB during stage III and beyond, cell wall thickness increased under more negative osmotic potential conditions in a manner consistent with swelling. When cell wall thickness is considered as a function of the percentage of plasmolyzed cells, for stages <55 DAFB, cell wall thickness was essentially independent of the extent of plasmolysis ( Fig. 6A and B). However, beyond 55 DAFB, cell wall thickness increased markedly and approached an asymptote as cells began to plasmolyze and turgor pressures most likely approached zero (Fig. 6B). Discussion Our data clearly establish that 1) for ripening sweet cherries, most individual fruit, of most cultivars, the value of Y P S (skin) is less negative than that of Y P F (flesh) with the result that almost all epidermal cells suffer plasmolysis when exposed to juice from their own fruit [this includes even those few individual epidermal cells lying close to the extreme (negative) end of the osmotic potential demographic]; 2) the difference, DY P, (where DY P = Y P S -Y P F ) between skin and flesh increases during stage III averaging 1.1 MPa across cultivars at maturity; and 3) plasmolysis is accompanied by marked swelling of the cell walls. EVIDENCE FOR A LESS NEGATIVE OSMOTIC POTENTIAL IN SKIN THAN FLESH. The less negative osmotic potential of skin than flesh is consistent with earlier data by Moing et al. (2004) who reported a difference in Y P of 0.5 MPa (range in DY P = 0.4 to 0.7 MPa). The DY P values in our study were 2-to 3-fold larger than those of Moing et al. (2004) probably because of our much higher spatial resolution achievable by a microscope assessment of epidermal plasmolysis, whereas Moing et al. (2004) quantified juice osmolarity from 1.0 ± 0.2-mm thick tissue blocks, where only the outer %100 mm (%10%) was epidermal (Glenn and Poovaiah, 1989) and most of the rest (%90%) was parenchymatous. Hence, in their samples, the juice from the true skin (having a less negative Y P S ) was mixed with that from some adhering parenchyma cells beneath (having a more negative Y P F ), probably resulting in an underestimate of the magnitude of DY P, the difference in osmotic potential between skin and flesh. The conclusion that osmotic potential differs between skin and flesh is based on different techniques (i.e., plasmolysis for the skin vs. osmometry of the flesh). Unfortunately, it was technically impossible to observe plasmolysis in a parenchyma cell of the flesh (cells far too large) or, conversely, to use 3.1 ± 0.0 ab 6.7 ± 1.9 a 91.5 ± 1.8 ab a vapor pressure osmometer to record the osmotic potential of an epidermal cell (cells far too small). The large, thin-walled parenchyma cells prevent critical assessment of the onset of plasmolysis in the narrow focal plane of the microscope. Moreover, the soft flesh tissues of a mature fruit are difficult to handle in thin sections that require the absence of mechanical stress and minimal juice leakage. Nevertheless, we believe a methodological artifact is an unlikely explanation for the DY P value we infer between skin and flesh. Furthermore, the observation that epidermal cells consistently plasmolyze when incubated in juice from the very same fruit would seem to offer conclusive and direct evidence for the existence of a major difference in Y P between skin and flesh. Lastly, the significant value for DY P we report here is not a singular event at one point in the course of development, but is present throughout stage III during which time it increases continuously (Fig. 4C). METHODOLOGICAL ARTIFACTS. It is important to understand potential methodological limitations of this study as they affect the estimates both of Y P S and of Y P F and, hence, of DY P . Y P S : The excision of a skin strip for microscopy allows its relaxation (release of elastic strain) and this may have altered the water potential. Relaxation is rapid [halftime of %2.7 min (Grimm et al., 2012)] and this is accompanied by a decrease in planar area of the sample and consequently in the planar area of each epidermal cell. Cell wall strain release allows each epidermal cell to assume a more spherical shape and hence a decrease in turgor and thus a more negative water potential than before. If the skin sample is now bathed in an excess volume of an external solution, epidermal water potential will soon come into balance with this and the result will be a generally too-negative estimate of epidermal water potential. This suggests, the true difference in osmotic potential between flesh and skin is actually greater than that quantified above-in other words our estimate is conservative and the true difference will more likely be greater than (not less than) that we suggest. Y P F : Juice extraction by crushing the flesh tissue will destroy any in vivo compartmentation and hence will create a potential for autolysis. What is the likelihood that enzyme reactions will significantly alter (decrease) juice osmotic potential? We have already seen that the predominant juice osmolytes are the hexose monomers glucose and fructose (Herrmann, 2001) and these are less likely to be affected with any rapidity. Sucrose is a possible candidate for rapid enzymatic cleavage but this moiety occurs at a too-low concentration for its possible autolytic cleavage to markedly decrease juice osmotic potential (Herrmann, 2001). We suggest that the net effect of any methodological artifacts will more likely decrease the measured osmotic potential difference (DY P ) we report, than increase it. Hence, our inferences are satisfactorily conservative. ARE SKIN AND FLESH AT WATER POTENTIAL EQUILIBRIUM? A central question is whether skin and flesh are at water potential equilibrium or could their water potentials differ significantly? A water potential equilibrium implies the absence of a net movement of water and/or of osmolytes between flesh and skin. Under equilibrium conditions, Y F = Y S and, hence, where the much more negative value taken by Y P F than by Y P S requires Y P F to be correspondingly much higher (i.e., much more positive) than Y P S . Rearranging the above equation shows that under equilibrium conditions, DY P and DY P must be numerically equal. This would seem to imply that Y P F must greatly exceed Y P S and that it would do so by an average of 1.1 MPa (Table 1). However, recorded values of Y P F of mature fruit are always very much less than 1.1 MPa, indeed they usually take values of less than 10% of this value [%0.1 MPa Schumann et al., 2014)]. This would seem to imply a highly negative value for Y P S , which inference is difficult to accept as being real. Thus, a high value for Y P F can be excluded as a mechanism for balancing the very negative value of Y P F . In grape berries, high values for Y P F as a consequence of similarly very negative values for Y P F have been shown to be prevented by the accumulation of osmolytes in the apoplast (Wada et al., 2008(Wada et al., , 2009). Here, the high levels of apoplastic osmolytes roughly balance the high symplastic ones, thereby preventing the development of a high Y P F . If this situation was also to occur in sweet cherry, then Eq. [1] must be modified by relaxing its implicit assumption that the apoplasts of the flesh and skin are essentially composed of pure water. This means we must include the opportunity for an apoplastic osmolyte in the notation for the flesh tissue (Y P Fa ), not just a symplastic one (Y P Fs ) and likewise in the skin (Y P Sa and Y P Ss ). ½3 This shows that the essential absence of Y P Fs (obtained by measurement) requires Y P Fs to be about equal to Y P Fa (Schumann et al., 2014). The value of Y P Ss needs not be similar to Y P Sa or the epidermal cells would not be turgid. Next, because Y P Fs is much more negative than Y P Ss (i.e., a major result presented in this study) Y P Fa must be much more negative than Y P Sa . Such a difference in the osmotic concentrations of the two adjoining apoplasts (i.e., that of the flesh and that of the skin) requires postulation of a sharp gradient in osmolyte concentration along the planar interface between the apoplasts of the flesh and skin tissues. This gradient will likely generate a powerful concentrative driving force for the diffusion of osmolytes from flesh apoplast to skin apoplast. Similarly, it will generate a powerful water potential driving force for the diffusion of water in the reverse direction, from skin apoplast to flesh apoplast. Although plausible, or even inevitable, direct evidences for these counter-directional diffusions of osmolyte and water are lacking. of the skin as determined by plasmolysis (Y P S ) and of juice extracted from the flesh (Y P F ) of developing 'Regina' sweet cherry fruit. Data for Y P F were obtained by vapor pressure osmometry from the fruit of the same batch (redrawn from Fig. 2A in Schumann et al., 2014) for comparison. Data for Y P S represent osmolarities of the incubation solutions yielding 50% plasmolysis (Fig. 3). (C) Gradient in osmotic potential (DY P ) between skin and flesh. The DY P value was calculated by subtracting Y P F from Y P S . X-axis scale in days after full bloom (DAFB). If a large difference in osmolyte concentration is to be sustained between the apoplasts of the flesh and skin (DY P ), two possibilities remain. The first is that there is a diffusion barrier in the apoplast (equivalent to an endodermis) at the flesh-skin tissue interface that significantly impedes the counter-directional diffusions of osmolytes and water. The other possibility is that a standing difference (Y F < Y S ) is maintained actively by a continuous supply of carbohydrates to the flesh from the tree via the pedicel phloem, and/or on the other by a strong metabolic sink for carbohydrates in the skin. Although there does not seem to be a vascular system within the thin (100 mm) skin layer, there is a prolific but fine network of ventral and dorsal vascular bundles in the outer flesh (mesocarp). The distribution of the vascular system is consistent with this hypothesis, but it would also require that the skin be very active metabolically so as to ''use up'' the major proportion of the centrifugally diffusing osmolytes (predominantly sugars). Returning to the idea of an apoplastic barrier, the swollen cell walls of the skin could represent a significant diffusion resistance that slows progress toward osmotic and thus water potential equilibrium. Unlike the internal cuticle of tomato [Solanum lycopersicum L. (Matas et al., 2011)], there is no evidence for an internal transport barrier in sweet cherry fruit that could hydraulically ''isolate'' skin from flesh. Based on the above arguments, the existence of a gradient in Y between skin and flesh is a more likely explanation for the DY P , but definitive evidence is lacking. It is important to note that in both cases, the consequences would be similar, i. e., a driving force for diffusion of osmolytes from flesh to skin and for flow of water in the reverse direction from skin to flesh. The broad range of osmotic potentials of bathing solutions (up to 1 MPa and more) causing plasmolysis of fractions of epidermal cells of the very same fruit deserves some further comments (Fig. 2). Based on the arguments presented above, this range reflects the range of Y P S of the epidermal cells indicating considerable heterogeneity within the population of cells in a given fruit. Assuming water potential equilibrium within the epidermis, we would expect a correspondingly broad range in their Y P S . Unfortunately, due to the small size of epidermal cells, direct evidence for this inference using pressure probe techniques is difficult to obtain. SWELLING OF CELL WALLS ON PLASMOLYSIS. Swelling of cell walls is commonly observed in ripening fruit that has a ''soft and melting'' texture, but not in fruit having a ''crisp and fracturable'' texture (Redgwell et al., 1997). Our observations in sweet cherry are consistent with this conclusion. According to Redgwell et al. (1997) swelling is thought to result from pectin solubilization and a subsequent increase in volume when water moves into voids of the cellulose-hemicellulose network formed on pectin solubilization and extraction. We observed increased swelling beyond 55 DAFB as compared with before 55 DAFB, which is in agreement with increased activity of pectinases during stage III of fruit development [Fig. 3 (Brummell, 2006;Kondo and Danjo, 2001)]. Unfortunately, little is known about the physical properties of swollen cell walls and we are not aware of published information quantifying the ''osmotic'' potentials and pressures exerted by the swelling and hydrating cell walls. Our data suggest 1) that the swelling pressure is probably low, whereas 2) the volume increase due to swelling is quite significant [+ 0.64 mm in width ( Fig. 5A)]. First, cell wall swelling occurred only when living cells plasmolyzed and hence, turgor was absent. Second, cell wall swelling was greatest in cell walls separating two dead cells, intermediate for cell walls separating a dead from a living cell, and least when cell walls separated two living and turgescent cells. Apparently, in turgescent cells, the turgor balanced the swelling of cell walls. Because the turgor of cells of the outer mesocarp is lower than 0.1 MPa at maturity Schumann et al., 2014), the pressure developed by swollen cell walls is expected to be of the same order of magnitude or slightly lower. This is of a similar magnitude to the swelling pressure reported for the starch biopolymer in potato [Solanum tuberosum L. (Jarvis et al., 1992)]. The volume increase of the apoplast due to cell wall swelling indicates significant water uptake, the forces driving this uptake are not yet known. Conclusion Our results provide direct and conclusive evidence for the existence of a marked difference (1.1 MPa) in osmotic potential between the sweet cherry fruit skin and its flesh. The most likely explanation is a standing gradient in osmotic potentials where osmolytes accumulate in the flesh due to vascular import while the skin lags behind. From a practical point of view, the less negative osmotic potential of the skin has some interesting consequences: First, a less negative osmotic potential of the skin will prevent the development of significant turgor in the skin, where the bursting of epidermal cells would weaken the tissue as a load-bearing structure (Br€ uggenwirth et al., 2014). Second, a more negative osmotic potential in the flesh in the absence of significant turgor Schumann et al., 2014) would essentially ''dehydrate'' the skin thereby making the skin flaccid and hence more extensible as indexed by a decreased modulus of elasticity (Br€ uggenwirth, personal communication). Finally, as a further consequence, the skin may serve as a transient buffer that allows uptake into the cells and enhanced water transport into the flesh (driven by the skinflesh osmotic potential difference). All three effects would tend to render a fruit less susceptible to cracking.

v3-fos

2018-04-01T10:50:48.609Z

2015-07-01T00:00:00.000Z

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A Hard Nut to Crack: Reducing Chemical Migration in Food-Contact Materials When we buy food, we’re often buying packaging, too. From cherries to Cheez-It® crackers, modern foods are processed, transported, stored, and sold in specialized materials that account, on average, for half the cost of the item, according to Joseph Hotchkiss, a professor in Michigan State University’s School of Packaging. Consumer-level food packaging serves a wide range of functions, such as providing product information, preventing spoilage, and protecting food during the journey from production to retail to pantry, fridge, or freezer. That’s why food producers lavish so much time and money on it. But what happens when these valuable and painstakingly engineered containers leach chemicals and other compounds into the food and drink they’re designed to protect? Such contamination is nearly ubiquitous; it happens every day, everywhere packaged food is found, with all common types of packaging, including glass, metal, paper, and plastic.1,2,3,4 Many manufacturers are eager to alleviate the problem of chemical migration from food packaging, but progress in identifying viable alternative materials has been incremental at best. Even as awareness of the issue grows, large-scale solutions that are scientifically and financially viable remain out of reach. The challenges in reaching them are many. Yet some of the world’s leading health authorities and largest food producers are working toward fixes (and in cases already deploying them), despite the absence of scientific consensus or regulatory requirements around most food-packaging chemicals of concern. The Winding Path of Chemical Replacement Due primarily to consumer demand, health concerns represent the largest force driving innovation within the food-packaging industry today, Hotchkiss says. "I believe the safety issues will continue to grow, and those who can assure consumers that they are concerned about it and are doing what they can to address it will be rewarded in the marketplace," he says. "Those that don't will be punished in the marketplace." People around the world are familiar with bisphenol A (BPA) and concerns about its migration into food and drink from plastic bottles, metal cans, and other consumer products. To date U.S. and European authorities have concluded, based on the available evidence, that the levels of BPA that currently occur in foods are safe for all consumers. 5,6 Other scientists suggest the experimental evidence for BPA's adverse health effects is strong enough to warrant removing the chemical from food-use applications as a precaution. 7,8 In recent years U.S. manufacturers voluntarily abandoned the use of BPA in baby bottles, sippy cups, and infant-formula packaging, and the U.S. Food and Drug Administration (FDA) formally ended its authorizations of these uses thereafter. 9 Beyond our borders, several other countries have banned BPA from some infant products, including Canada, the European Union, South Africa, China, Malaysia, Argentina, Brazil, and Ecuador. 10 France went even further with its recently implemented ban of BPA from all packaging, containers, and utensils that come into contact with food. 11 The BPA debate illuminates many of the challenges involved in stemming chemical migration. As France recognized with its ban, prohibitions for baby products alone don't address the fact that BPA exists in countless consumer products and food-packaging materials to which infants and expectant mothers, 12 among other susceptible populations, may still be exposed-such as metal beverage and food cans, which are often lined with BPA-based epoxy resins. 13 Plastic bottles Depending on the type of plastic, bottles may leach catalysts or stabilizers. Foil retort pouches Some leaching may occur with the adhesives used to seal pouches. Polypropylene inner layers also may leach stabilizers. Glass jars Glass itself is mostly inert but can be a source of naturally present metals at low levels. Jar lids may be equipped with BPA-based epoxy liners and/or gaskets that leach plasticizers. Metal cans Epoxy linings can leach BPA. Paperboard boxes with polyethylene liner bags Recycled paperboard may be contaminated with chemicals from papers not originally intended for food-contact uses (e.g., newsprint, thermal receipts). Polyethylene liner bags may leach stabilizers. Liquid paperboard Label inks have caused problems in the past. Inner polyethylene layer may leach stabilizers. Greaseproof wrappers Poly-and perfluorinated compounds are used to make some packaging greaseproof. Ceramic kitchenware The glazes used in artisanal pottery and older massproduced ceramics may leach toxic metals. Offset migration Offset migration occurs when the printed outer surface of food packaging transfers chemicals to the inner food-contact surface. Packaging Pathways These are just a few of the potential routes of chemical migration from food packaging itself. However, foods and beverages also can be contaminated prior to packaging, through handling and storage of either finished products or their ingredients. A 177 BPA is just one of many known or suspected endocrine disruptors commonly found in food packaging that can migrate into food and drink. 14,15 Furthermore, endocrine disruptors from plastics are far from the only class of potentially harmful chemicals that can leach into food or drink from food packaging; depending on factors including temperature, storage time, and physicochemical properties, a wide variety of compounds-including components of coatings and films, adhesives and glues, and inks and pigments-can migrate from packaging materials. 16,17 For these reasons, Laura Vandenberg, an assistant professor of environmental health at the University of Massachusetts Amherst, believes most existing bans on BPA do little to ensure food safety. "This was a very empty victory, I think, to focus on BPA and baby bottles," she says. Alternative Plastics Sure enough, in some applications BPA was replaced with other bisphenols, including BPS and BPF, which laboratory experiments indicate have estrogenic effects at least as pronounced as those of BPA. 18 In others, including baby bottles, polycarbonates were replaced by alternative plastics with migration issues of their own. 19 Chemists are now on the hunt for effective alternatives to BPA. To date no one has identified any drop-in fixes that will work in all the same applications, for the same or a lesser cost, with an established lack of estrogenic activity (now known in the marketplace as "EA-free"). But partial solutions are beginning to appear. One of the most widely available is a polymer called Tritan that can replace traditional polycarbonate in clear, hard plastics used for water and baby bottles. According to its manufacturer, Eastman Chemical Company, Tritan is free of estrogenic activity within the human body. 20 Not everyone agrees. In 2011 a pair of affiliated firms called PlastiPure and Certi -Chem published a study showing the potential for endocrine disruption in Tritan. 21 This sparked a lawsuit from Eastman, which it later won. 22 At the core of the case was the question of how best to detect and define estrogenic activity; the two sides used different tests that each insisted was accurate. 23 Tritan is still used widely in hard-plastic bottles sold by Nalgene, CamelBak, Nathan, and other brands, while PlastiPure and Certi-Chem continue to support the development of other alternative plastics and products, including food packaging, says chief economic officer Mike Usey. In addition to testing and consulting, the sister companies will soon expand into product development, Usey says. "We've had so much interest in the last year and a half from consumers for safer products, and a lack of traction with manufacturers, that we've decided to spin off a product company." But full-scale solutions remain at least a few iterations away, says John Warner of the Warner Babcock Institute for Green Chemistry. "Something like reinventing plastic isn't going to happen in a day, a month, or a year," he says. "This isn't a matchmaking game. It's not like the solutions are out there, if only the companies could be matched up with those solutions. I really feel we are inventions away from success." Much of Warner's personal research centers on developing biobased plastics (i.e., derived from renewable biomass sources) that are safer, cheaper, and as effective as traditional fossil-fuel plastics for food packaging. However, plant-based plastics still may contain some of the same harmful additives and manufacturing by-products (known as non-intentionally added substances) that can migrate into food and drink. These plastics do offer one distinct advantage, Warner says: "Because bioplastics are new, they have less of an incumbent history, so designers, inventors, and developers can create a better formulation of additives that have less impact on human health and the environment." In other words, although it doesn't guarantee success, there may be more opportunity for creativity and innovation around bioplastics than with traditional plastics that are more entrenched in industry, he speculates. A Silver-Bullet Lining? Beyond reusable hard plastic bottles, the most prominent source of BPA in food-contact materials is the ubiquitous metal can. The BPA-based epoxy resin linings of cans serve a dual purpose by protecting the container from acidic or otherwise corrosive elements in foods as well as protecting food and drink from the can's metallic taste. Within this sector of the food-packaging industry, researchers have worked for years to identify a replacement for standard BPAcontaining epoxies that performs just as well across the same range of food and beverage types. 24 Such a coating must be physically stable and resistant to all manner of foods and beverages, and, in the case of food cans, must maintain its performance at elevated temperatures while foods are being sterilized after sealing. No replacement has yet emerged. But efforts now under way could pay dividends in the not-too-distant future. Valspar Corporation, a Minneapolisbased manufacturer that bills itself as the number-one global supplier of coatings for metal packaging, is motivated to develop an EA-free can lining for use with a wide variety of foods. And staff toxicologist Mark Maier, who's leading the company's efforts, thinks he's found it. He says Valspar has developed a replacement coating that several academic laboratories have shown to be EA-free. But even if testing validates Valspar's invention, that doesn't guarantee economic viability. "The supply chain challenge may be bigger than the safety challenge," he says. "It doesn't matter how good your technology is-if it costs too much, nobody's going to buy it." Daniel Schmidt, an associate professor in the Department of Plastics Engineering at the University of Massachusetts Lowell, is leading another group in search of a new can lining. Schmidt's lab has already made an epoxy from 2,2,4,4-tetramethyl-1,3-cyclobutanediol (CBDO), the same monomer that is at the heart of Tritan, 25 and is working to scale it up. Funding to date has come from the university and its Toxics Use Reduction Institute, but Schmidt says a private company has recently agreed to provide support for continued research into applications that meet its needs, primarily in the beverage sector. As to whether Schmidt's design will ultimately show any estrogenic activity, which CertiChem's tests on Tritan suggest it could, he admits there's some uncertainty. "We do need to do more to ensure that everything is okay in all respects," he says. "One of the main reasons we chose CBDO was for its structure, which bears little or no resemblance to known endocrine disruptors. This doesn't guarantee success, but it's a good place to start." Other large corporations including Dow Chemical have also alluded to their own efforts to develop safer drop-in can-lining solutions. 26 And a number of natural and organic food brands, including Muir Glen, Eden Foods, Wild Planet, and Amy's Kitchen, have already touted a transition to BPA-free can linings-but details are spotty as to what alternatives they've embraced or what level of endocrine disruption or migration the replacements represent. Amy's, for example, gives no information on its website as to what alternative formulation it is using, although it does say that low levels of BPA are still migrating into its food. 27 In 1999 Eden Foods switched its linings for low-acid foods to oleoresin, a mixture of oil and resin extracted from plants such as pine and balsam fir, but high-acid foods like tomatoes are still canned with liners formulated with BPA, or bottled in jars with lids containing BPA. 28 Pressure up the Supply Chain Nestlé Corporation, the world's largest food producer with thousands of brands selling nearly any prepackaged food one can imagine, must manage the entire spectrum of foodpackaging materials and their potential risks. It therefore has a considerable incentive to ensure the safety of its packaging. The Swiss company's food-packaging safety program got its start after a huge 2005 recall caused by the discovery that traces of isopropyl thioxanthone, a chemical used to cure packaging inks, was migrating through liquid paperboard cartons into ready-to-drink baby formula sold by the company. 29 "Nestlé got burned and said, 'That will never happen again,'" says Stephen Klump, the company's head of packaging quality and safety. "That was a big wake-up call for the industry." Eventually Nestlé published guidance for inks that prohibits more than 50 acrylates, solvents, photoinitiators, and pigments. 30 These prohibitions are based on health risks (recognized by Nestlé or perceived by the public), migration potential, and, in some cases, negative impacts on taste, smell, or color. The company also has a policy against food contact with BPA, phthalates, and recycled paperboard, which can contain harmful chemicals derived from sources not originally intended for use in food packaging-such as newspaper ink or BPA-/BPS-containing thermal receipts that are added to recycling bins. In addition, Klump says Nestlé aims to phase out BPA from all its can linings and polycarbonate plastics by the end of 2015, but he did not specify which alternatives the company is embracing. In February of this year, Nestlé announced it is developing guidance on packaging adhesives in order to clarify its position on additional substances of concern. 31 The company asks suppliers to formally declare compliance with its guidances as part of their contract, but does not enforce them; Klump says it can be hard to verify total compliance. Nevertheless, through these directives, Nestlé can use its sheer size to spur innovation within the food-packaging industry, and companies selling safer inks and adhesives can tout their compliance with Nestlé's guidance as a benchmark, as SPGPrints has done with its new line of lowmigration ultraviolet inkjet inks. 32 Other large food producers hold similar sway, says Jane Muncke, managing director and chief scientific officer of the Switzerlandbased Food Packaging Forum, a nonprofit foundation formed in 2012 to communicate information about food packaging and health. "They have such big buying power they'll just switch suppliers if they're not happy with the product." In this sense, the onus is often on packaging suppliers to make their products safer, which many are trying to do. A number of manufacturers have introduced new barrier films for dry foods such as pasta, cereal, and rice, among them Clondalkin Flexible Packaging, 33 Innovia Films, 34 Smurfit-Kappa, 35 Imerys Kaolin, 36 BASF, 37 MM Karton, 38 and Sappi Fine Paper Europe. 39 These barriers are intended to prevent label inks and their constituent chemicals from migrating from the exterior of the package into the food, as well as stop mineral oils and other harmful substances present within recycled paper packages. 40 Migration of mineral oils has become a significant concern for some European consumers following a European Food Safety Authority probe into the issue. 41,42 Incremental Changes While it's clear that a number of packaging manufacturers are eager to switch to alternative packaging whether required to or not, progress to date has been incremental at best. "The unqualified success may be out there, and I really do hope that these companies are developing them, but for the most part what I have seen are just-barely-studied alternatives," says Vandenberg. Many researchers and innovators in the field who believe they're on the right track have yet to see their eureka moment, if indeed it's coming. Still, change is happening. Consumer demand in Europe contributed to the development and rollout of the world's first PVCand plasticizer-free glass-jar lid by German packaging manufacturer Pano, says Rolf Rohrkasse, manager of product and material development for the company. Since 2011 Pano has sold 450 million of its BLUESEAL® lids in Europe, but it has yet to break into the U.S. market. However, Pano is in discussions with Coca-Cola, Unilever, and Nestlé, among others, to expand its global reach. The caps still contain a plastic seal-a polyolefin-based elastomer called Provalin®. 43 While migration is not eliminated, Pano claims that migration levels are significantly lower compared with polyvinyl chloride (PVC) and its many additives. 44 (The rubbery gaskets on almost all glass-jar lids available today contain PVC, which can leach a host of chemicals, including phthalate plasticizers, directly into foods. 45 This is particularly true for fatty and oily foods. 46 ) However, like relying on dry-food barriers to reduce migration rather than eliminating the harmful chemicals in the first place, Muncke sees Pano's lids as only a small step in the right direction. "It's kind of a half-solution," she says. "It doesn't solve the whole issue." Some nongovernmental organizations are taking steps to get specific chemicals removed from food packaging. 47 Within the last year the Natural Resources Defense Council (NRDC) has teamed with citizens' groups in Safety Testing As migration concerns drive chemists, food producers, and packaging manufactures to seek out and market new chemicals and materials, the threshold for deeming a substance "safe" is likely to become more hotly contested. Although traditional toxicity tests can be used to evaluate some outcomes of concern, endocrine disruption poses a particular challenge due to the fact that such chemicals may produce effects in experimental models at very low doses. 50 Environmental Health Sciences, have developed an endocrine-disruption detection system known as TiPED that is designed to help chemists formulate safer chemicals. 51 TiPED involves a series of tests with ascending sensitivities: computational assessments, high-throughput cellular assays, cell process assays, live-animal testing with fish and amphibians, and, ultimately, mammalian testing. Meanwhile, a European program known as LIFE-EDESIA-designed to identify three to five EA-free alternatives each for bisphenols, phthalates, and parabens-has developed a simpler in silico and in vitro tiered structure that foregoes any animal testing. 52 And Nestlé has promoted its own computational screening method, while Valspar employs four or five assays in a tiered system that staff toxicologist Mark Maier says is essentially the same as TiPED, except it stops shy of animal testing. "The trick is when do you stop [searching for effects]," Maier says. "It just depends on who's talking." Environmental Health Perspectives • volume 123 | number 7 | July 2015 A 179 petitioning the FDA to withdraw its decadesold approvals of a handful of chemicals, including perchlorate, an endocrine disruptor used to produce rubber gaskets and to reduce static charge in plastic dry-food packaging, and long-chain perfluorocarboxylates, used to greaseproof paper and paperboard. 48 The latter have been largely abandoned by U.S. manufacturers but increasingly are employed in India and China and are still legal to import and use, says Tom Neltner, an independent consultant. Maricel Maffini, a consultant and former senior scientist with the NRDC, is concerned that the development of safer alternatives is being hampered by a lack of regulatory incentives and oversight. "There is no regulatory pressure for innovation," she says. "And when [manufacturers] do take the initiative to go for an alternative, we don't know the safety profile of that alternative, we don't know the exposure, we don't know if it gets metabolized when it gets into the environment. So there are still a lot of systemic improvements that we need." Schmidt points out that even if consumer packaging is totally free of harmful substances, there are still many opportunities during processing and handling for foods and beverages to become contaminated, even before they are packaged. As an illustration, he points to a study of phthalates in olive oil, which found contamination in every sample tested, but no significant difference in the degree of contamination between oils packaged in glass, plastic, or metal. 49 "Packaging is important," he says, "but the issue is even bigger still. Make the packaging perfect, and you've still got [contamination] coming from further up the supply chain." Muncke, for one, is prepared to concede that a true food-packaging panacea may not be anywhere around the next bendespecially when one takes into account the environmental impacts of producing and discarding so much packaging, and the carbon footprint of the global food system. "If you want to preserve food by using packaging, then you have to make compromises," she says. "There is no packaging that is perfect." Nate Seltenrich covers science and the environment from Petaluma, CA. His work has appeared in High Country News, Sierra, Yale Environment 360, Earth Island Journal, and other regional and national publications.

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2019-04-01T13:09:12.856Z

2015-08-01T00:00:00.000Z

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CIRRIG: Weather-based Irrigation Management Program for Container Nurseries A goal of irrigation best management practices in container nurseries is to conserve water while maintaining optimal plant growth and quality. A web-based, container irrigation management program (CIRRIG) was developed to automatically provide daily irrigation run times for sprinkler-irrigated crops in container nurseries. The program estimates evapotranspiration rates based on weather uploaded from a weather station located on-site and plant production conditions monitored in each zone and adjusts irrigation run times based on irrigation application rate, the plant’s irrigation-capturing ability (for sprinkler irrigation), desired leaching fraction, and irrigation system uniformity. For this project we interfaced CIRRIG output with a programmable logic controller (PLC) to automatically irrigate a sprinkler-irrigated crop at a container nursery in Florida. Sweet viburnum (Viburnum odoratissimum) in 10-inch-diameter containers were grown by the nursery for 24 weeks in adjacent irrigation zones, one controlled automatically using CIRRIG and the other by the nursery’s traditional practice of manually turning on and off irrigation. Water use was monitored with flowmeters and plant growth by measuring plant size and shoot dry weight periodically throughout the trial. Plant growthwas not different (P < 0.05) because of irrigation practice. CIRRIG reduced water use during the study period by 21% (42 vs. 53 inches) compared with the nursery’s irrigation practice. An assessment of the watersaving benefits ofmaking daily adjustments to irrigation run times based onweather including rain indicated savings of 25% and 40% compared with biweekly adjustments with andwithout automatic rain cutoff, respectively. This trial demonstrated that CIRRIG coupled with an on-site weather station and a computer-controlled irrigation system can be used to manage irrigation while conserving water in a container nursery. SUMMARY. A goal of irrigation best management practices in container nurseries is to conserve water while maintaining optimal plant growth and quality. A web-based, container irrigation management program (CIRRIG) was developed to automatically provide daily irrigation run times for sprinkler-irrigated crops in container nurseries. The program estimates evapotranspiration rates based on weather uploaded from a weather station located on-site and plant production conditions monitored in each zone and adjusts irrigation run times based on irrigation application rate, the plant's irrigation-capturing ability (for sprinkler irrigation), desired leaching fraction, and irrigation system uniformity. For this project we interfaced CIRRIG output with a programmable logic controller (PLC) to automatically irrigate a sprinkler-irrigated crop at a container nursery in Florida. Sweet viburnum (Viburnum odoratissimum) in 10-inch-diameter containers were grown by the nursery for 24 weeks in adjacent irrigation zones, one controlled automatically using CIRRIG and the other by the nursery's traditional practice of manually turning on and off irrigation. Water use was monitored with flowmeters and plant growth by measuring plant size and shoot dry weight periodically throughout the trial. Plant growth was not different (P < 0.05) because of irrigation practice. CIRRIG reduced water use during the study period by 21% (42 vs. 53 inches) compared with the nursery's irrigation practice. An assessment of the watersaving benefits of making daily adjustments to irrigation run times based on weather including rain indicated savings of 25% and 40% compared with biweekly adjustments with and without automatic rain cutoff, respectively. This trial demonstrated that CIRRIG coupled with an on-site weather station and a computer-controlled irrigation system can be used to manage irrigation while conserving water in a container nursery. D etermining irrigation run times that minimize water use while sustaining optimal production is a difficult task for container nursery managers. This is particularly true for production in small containers as conditions in the irrigated area that affect evapotranspiration (ET) rates (e.g., plant growth, pruning, spacing) can change rapidly compared with microirrigated plants grown in large containers. Furthermore, the capture of sprinkler irrigation water can be greatly affected by the plant canopy so that crops with similar ET rates may require different irrigation rates (Million and Yeager, 2015). The dynamic natures of ET and irrigation capture provide great challenges for ET-based irrigation scheduling. Several systems are being used to implement ET-based irrigation in container nurseries. One approach is to directly monitor substrate moisture content with sensors (Kohanbash et al., 2013;van Iersel et al., 2013). Sensors can be programmed to open solenoid valves when substrate moisture falls below critical values and then to close valves once favorable substrate moisture thresholds are reached. Sensor-based irrigation has the distinct advantage of directly monitoring the water status of the container substrate. A disadvantage of sensor-based irrigation is the cost for purchasing, installing, and maintaining a sensor network in the nursery. However, as the technology improves, this approach will become less expensive and easier to use and manage (Belayneh et al., 2013). Another means for implementing ET-based irrigation, and one used in this research, is a software approach whereby substrate water loss is estimated with ET functions. A general function used to estimate ET for a wide range of agricultural crops entails multiplying potential ET (ETo) by a crop coefficient to determine actual ET (Schuch and Burger, 1997). Because crop coefficients depend on rapidly changing growing conditions, continuous functions have been developed to estimate actual crop ET rates (ETc) from ETo (Beeson, 2005(Beeson, , 2010Grant et al., 2012;Irmak, 2005;Pardossi et al., 2008). For example, Beeson (2010) developed ET functions that use projected plant area and a growth index to estimate ETc from ETo. Million et al. (2011), using a partial cover function developed by Ritchie (1972) for field crops, estimated ETc as a function of ETo and leaf area index. Unlike field crop situations, partial cover in container nurseries can result in significant temperature increases when solar radiation not intercepted by the plant canopy heats nonradiating black container sidewalls and ground-cloth surfaces. To account for the heating effect, Million et al. (2011) used a biased maximum daily temperature in their ETo calculation. Another source of variation when estimating the irrigation requirement using an indirect approach is the influence that the plant canopy can have on the capture of sprinkler irrigation water. We define the capture factor (CF) as the amount of sprinkler irrigation water captured by the container with a plant relative to that amount of water that would be captured without a plant. CF >1 indicates that the plant canopy is augmenting irrigation capture so that irrigation amounts can be reduced accordingly. Similarly, CF <1 indicates that the plant canopy is reducing irrigation capture and irrigation amounts would need to be increased accordingly. Functions for estimating CF based on plant size, container size and spacing, and the plant species' watercapturing ability were reported by Million and Yeager (2015). Using a software approach for indirectly estimating the irrigation requirement for sprinkler-irrigated container crops, daily irrigation run times can be output by estimating ETc and CF and then calculating the irrigation run time that will deliver the required amount of water to the container, taking into account any rain received. The advantage of a software approach is that no hardware is required except a weather station. A disadvantage of the software approach compared with a sensor-based system is that irrigation run times are indirectly estimated and therefore a degree of uncertainty always exists. Both approaches require labor to either monitor sensor function (sensor approach) or to monitor plant conditions in the irrigated area that affect ET and CF estimation (software approach). The purpose of this study was to evaluate an irrigation management program, CIRRIG, for implementing ET-based irrigation in a container nursery. In the first section, we describe CIRRIG and discuss the required inputs to be monitored by nursery staff. In the second section, we describe how the program was used to automatically control an irrigation valve in a nursery and compare water savings of the program with the nursery's traditional irrigation practice. Using results from the trial, we also evaluated the benefit of adopting a weather-based irrigation management program that adjusts run times daily compared with a periodically adjusted irrigation management practice. 2015) is an irrigation management program that was developed at the University of Florida for container nurseries in the humid southeastern states; its applicability in other regions and other climates has not been tested. All CIRRIG programs and user account data currently reside on a dedicated server located at the Department of Environmental Horticulture in Gainesville, FL. CIRRIG uses weather data acquired from a weather station on-site and plant production conditions to automatically output daily irrigation run times for each irrigation zone created by the user. Hourly weather data are uploaded to the user's account on the server using a Java agent residing on a host computer connected to the weather station. Hourly weather data uploaded to the server includes temperature maximum, temperature minimum, solar radiation, and rain. CIRRIG allows the user to view historical hourly and daily weather data. Required plant production-related inputs for each zone include percent plant cover, container diameter, container spacing, plant height and width, irrigation-capturing ability of plant, and irrigation application rate ( Fig. 1). Additional options for adjusting irrigation include irrigation distribution of uniformity [DU (Burt et al., 1997)] and a target leaching fraction. Because CIRRIG estimates daily container ET on 24-h day basis, we partition daily estimated ET rates into hourly ET rates based on the hourly distribution of solar radiation. CIRRIG then balances estimated hourly ET water loss and hourly rain input to arrive at a net water deficit for calculating irrigation demand. Materials and methods The user selects the irrigation schedule for each zone. Schedule options include daily irrigation, odd day irrigation, and fixed day irrigation (particular days of the week). If a nondaily schedule is selected, the water deficit in the container substrate is carried over each day that irrigation is not scheduled. At a user-selected time, typically just before irrigation, CIRRIG calculates the irrigation run time based on the past 24-h weather and the latest plant production-related inputs for each zone. Besides irrigation run time, displayed output also includes supportive information including estimated ET, CF, irrigation rate, and weather data. Historical zone output can also be viewed for a user-selected period. Output can be viewed on a computer or mobile device or exported in a comma-delimited (*.csv) file for automation. For automation, the user assigns an external reference (e.g., valve number) to each zone and this reference is output in the *.csv file to enable interfacing with the nursery's own computer-controlled irrigation system. We developed a Java program for the client side to manage the daily download of the *.csv file. The nursery will then need to develop its own program to incorporate the *.csv file data into the database associated with the nursery's computer-controlled irrigation system. For our own research purposes, we developed a Java program that directly acquired irrigation run times from CIRRIG and set timer values on our PLC thereby eliminating the need to download a *.csv file. FIELD DEMONSTRATION TRIAL. CIRRIG technology was evaluated at Salmon's Wholesale Nursery (SWN), a 70-acre container nursery located in Dunnellon, FL (lat. 82.5°W, long. 29.0°N). Two adjacent independently controlled irrigation zones in the nursery were selected to compare water use and plant growth of container shrubs produced with either the nursery's traditional irrigation practice or with CIRRIG. The irrigation system used at SWN was manually turned on and off by nursery staff. Once turned on, the system automatically cycled (typically one to three cycles per zone) through all irrigation zones until turned off. Because they did not use traditional time clocks to set the irrigation schedule, start times and run times varied daily. Several tasks were required to implement CIRRIG at the nursery. A weather station (Vantage Pro2 Plus Ò ; Davis Instruments Ò , Hayward, CA) was installed 50 ft from a shed that housed the nursery's irrigation electrical hardware. The station was attached to a tripod so that the fanaspirated radiation shield was 5 ft above the ground. Cabling was run from the weather station to the data-logging console located inside the shed. The data logger recorded hourly weather data that was automatically downloaded via a USB connection to a laptop computer using proprietary software (Weatherlink Ò , Davis Instruments Ò ). Weatherlink was configured to automatically export hourly weather data to a text file that was uploaded hourly to the CIR-RIG server via a Java program residing on the laptop. Weather data logged at the top of the hour was downloaded to the computer 5 min past the hour and uploaded to CIR-RIG at 10 min past the hour. A second task was to setup an internet connection in the remotely located irrigation shed. For this, we used an USB cellular modem (UML290 4G USB; Pantech Mobile, Atlanta, GA) connected to a wireless router (BR95; Cradlepoint, Boise, ID). A static IP address was acquired (Verizon Wireless, Wallingford, CT) to enable a constant internet connection. Another task was to install a PLC to serve as a computer-controlled irrigation system that could interface with CIRRIG. A PLC (D0-06DA; Automation Direct, Cumming, GA) with an Ethernet communications module was installed in the irrigation shed. The PLC was configured for the static IP address to allow constant connectivity. A graphical user interface (GUI) for controlling the PLC remotely was developed to allow us to manage the acquisition of CIRRIG output by the PLC as well as monitor real-time PLC activity. The GUI also had functions for manually controlling irrigation or for setting default irrigation run times if internet connection was lost. PLC irrigation history was archived in a text file that could be monitored remotely. To complete the PLC system, wires to the solenoid valve controlling one of the two irrigated zones were rerouted to the PLC leaving the other test area on the nursery's system. Because we could not use the nursery's 24-V AC circuit, we installed a 24-V AC transformer to supply electrical current for the PLC-controlled circuit. The two irrigation zones selected for testing were each 85 · 225 ft. Water from 3-inch supply lines were distributed through 1 1 /4-inch pipes to 34 sprinklers per zone on risers 5 ft tall. Sprinklers (Xcel-Wobbler highangle; Senninger Ò Irrigation, Clermont, FL) were fitted with #9 (grey) nozzles rated at 2.5 gal/min at 20 psi. Sprinklers were arranged in four rows 25 ft apart in an offset pattern. The irrigation application rate was 0.43 inch/h and DU was >90%. Flow meters (Omniä T 2 ; Sensus Ò , Raleigh, NC) were installed in each of the 3inch-diameter pipes supplying the two test zones to monitor and compare water amounts applied to each of the two irrigation zones during the trial. The relationship between flowmeter readings in gallons to irrigation depth in inches was determined by collecting irrigation water in twenty 4-inch-diameter containers over a 10-d period. Based on these tests, the average ratio of inches of irrigation to gallons of water flow was used to convert flowmeter readings to depth of irrigation water. On 4 Dec. 2013, 1280 rooted cuttings of sweet viburnum were transplanted by nursery staff into 10-inch-diameter containers (Classic 1000; Nursery Supplies Ò , Kissimmee, FL) filled with a substrate mix comprised by volume of 5.5 pine bark:4.5 Florida sedge peat:1 sand and amended with gypsum at 3 lb/yard 3 and a 0N-0P-5.0K fertilizer with minor elements at 2 lb/yard 3 (Reliable Peat, Leesburg, FL). An 18N-2.2P-7.5K, resin-coated, controlledrelease fertilizer (Osmocote Ò Pro 18-5-9 with micronutrients, 11-12 month release at 70°F; Everris, Dublin, OH) was placed underneath each transplanted cutting at the time of planting at the rate of 16 g per container. Containers were placed outdoors on black, woven, polypropylene ground-cloth in an offset pattern with no spacing between adjacent containers. Plants were equally distributed across the two test irrigation zones. Additional species planted and placed in the two test irrigation zones included japanese privet (Ligustrum japonicum), yellow anisetree (Illicium parviflorum), jack frost privet (Ligustrum japonicum), and chinese fringe flower (Loropetalum chinense var. rubrum). From planting until the initiation of the trial, SWN staff controlled all irrigation. Plants were covered with cold protection cloth as needed by nursery staff during the same time period. On 4 Mar. 2014, 80 plants in each test zone were grouped into five blocks of 16 plants. The 16 plants within each block were randomly assigned to one of four harvest dates in 2014: 7 Mar., 5 May, 30 June, and 28 Aug. On harvest dates, biomass of plant shoots was determined by cutting plants at the substrate surface and drying shoots in a forced-air dryer at 70°C for 48 h. Plant size measurements of the final harvest plants were taken biweekly throughout the trial. Plant height was measured from the substrate surface to the uppermost foliage while plant width was the average of two perpendicular measurements, one being the widest. All production activities (except irrigation management in the CIRRIG zone) were conducted by nursery staff. Nursery staff applied preemergence herbicide (OH2 Ò ; Everris, Dublin, OH) to all plants on 28 April Controlled-release 21N-1.7P-6.7K fertilizer (Nursery Mix 21-4-8; Everris, Dublin, OH) was surface-applied at 45 g per container on 8 May. Plants were pruned on 6 June by removing 9-10 inches of plant height and 2-3 inches of plant width. On 10 July containers were spaced 12 inches apart by nursery staff. The irrigation trial began on 11 Mar. 2014. Flowmeter readings of each test zone were taken on 11 Mar. and at least once per week throughout the 6-month trial. Irrigation of the CIRRIG zone was managed by University of Florida staff. Irrigation in the CIRRIG zone was typically scheduled for 0630 HR while irrigation in the SWN zone was typically scheduled for 1630 HR or later. About once every 2-3 weeks, plant production inputs required for CIR-RIG were monitored. Percent plant cover (PPC) was determined throughout production by taking digital images 4 ft above the canopy of each block of plants and estimating PPC using image processing freeware (GNU Image Manipulation Program, 2015). With GIMP, dense green foliage was selected using an oval selection tool then deleted turning selected pixels to white. The result was a processed image with white areas where dense foliage was deleted (Fig. 2). The percentage of white pixels to total pixels as determined by GIMP's histogram tool provided an estimate of PPC. The average PPC of the five images was input into CIRRIG. Percent plant canopy cover was determined in the same manner throughout production as plants grew in size and/or plants were pruned or spaced. The effect of irrigation on plant size index and shoot dry weight at each measurement date was evaluated using a paired t test with n = 20 and a confidence level of 5% (SAS version 9.2; SAS Institute, Cary, NC). Cumulative water use by the two irrigation practices as measured by flowmeters could not be compared statistically. We report both a cumulative depth of irrigation water applied as well as amounts applied between successive flowmeter reading dates. An additional analysis of the potential benefits of using a weatherbased irrigation program vs. a fixedrate program was made by dividing the trial into twenty-four 2-week intervals and comparing the depth of water applied with CIRRIG to two hypothetical fixed-rate irrigation schedules. One fixed-rate schedule took the maximum daily irrigation amount applied during a given 2-week interval (according to CIRRIG) and applied this amount each day during the same 2-week interval. A second fixed-rate schedule was the same as the first except no irrigation was applied if 0.5 inch or more rain fell on the previous day. Results and discussion Plant size index (Fig. 3) and shoot dry weight (Fig. 4) were not different (P > 0.05) because of irrigation schedule. Plant height and width were 10 and 9 inches, respectively, at the start of the experiment and 27 and 24 inches, respectively, at the end of the trial. Plants grew well throughout the trial with only the June pruning resulting in a decrease in plant size and shoot biomass. Cumulative irrigation water applied during the trial was 42.1 and 53.0 inches for the CIRRIG and SWN schedules, respectively. The 21% reduction in cumulative water use when irrigation was automatically controlled using CIRRIG was relatively constant throughout production (Fig. 5). Higher water use by SWN may be due in part to reduced efficiency of getting irrigation water into the container in the afternoon when evaporative losses were likely higher compared with CIRRIG's early morning application when evaporative losses would be expected to be lower (Playan et al., 2005). The linear shape of the cumulative water use plots indicate that irrigation amounts did not change significantly during the trial despite increased plant growth. The reason for this was that CF increased in proportion to Fig. 2. An image processing program was used to estimate percent plant cover (PPC), a required input of irrigation management program. A selection tool was used to delete dense foliage and the percentage of white pixels to total pixels in the image using a histogram provided an estimate of PPC, 65% in this example. increases in ET. For example, ETc and CF measured on 1 April were 0.23 inch and 1.2, respectively, and when measured on 12 July were 0.51 inch and 2.5, respectively. In this case, ETc increased 2.2· but at the same time CF increased 2.1·. A similar increase in CF was reported for sweet viburnum produced in 6.3-inchdiameter containers (Million et al., 2010). A comparison of the irrigation water applied by the two irrigation programs on an interval basis shows that SWN staff had a remarkable ability to increase and decrease irrigation in a similar pattern as CIRRIG (Fig. 6). We attributed this in part to SWN's irrigation system which required staff to manually turn on and off the irrigation system each day so that irrigation run times were determined on a day-to-day basis based on apparent need. For intervals where CIRRIG reduced irrigation, SWN also reduced irrigation, often to the same extent. For intervals where irrigation amounts peaked for SWN, CIRRIG also peaked but often at much reduced amount. For example, two peaks in May when little rain occurred (Fig. 7) indicated that CIRRIG applied 25% to 60% less water. Compared with peak interval irrigation water use in May and June, peak interval irrigation in July and August decreased, which was because of increased rainfall and decreased solar radiation levels that coincided with afternoon cloud formation typical of that time of the year. The nursery's method of manually turning on and off irrigation is in contrast to many growers who set irrigation time clocks to apply a fixed amount of water. In many situations, irrigation run times are not changed for weeks or months at a time. To get some perspective of how CIRRIG in the present trial might compare with a fixed-rate schedule, we compared CIRRIG water use to the water use that would have occurred with two fixed-rate programs, one that accounted for rain (FIXED-RAIN) and one that did not (FIXED). Because the fixed rate of irrigation for both FIXED and FIXED-RAIN was the maximum daily amount applied during each 2-week interval, the daily fixed rate of irrigation was changed once every 2 weeks. The benefit of using CIRRIG, which made daily adjustments in irrigation based on the past day's weather, is depicted in Fig. 8. CIRRIG reduced cumulative irrigation water applied 40% (37 vs. 62 inches) when compared with FIXED and 25% (37 vs. 50 inches) when compared with FIXED-RAIN. FIXED-RAIN reduced cumulative irrigation water applied 20% (50 vs. 62 inches) compared with FIXED providing evidence that turning off irrigation after significant rain is an important conservation practice. Compared with using a traditional rain sensor to cutoff irrigation, CIRRIG had the added advantage of objectively evaluating rain quantity and timing relative to any water deficit. During the trial, total rainfall was 27.8 inches. According to CIRRIG, only 8.5 inches or 31% of total rain directly reduced irrigation during the trial. Interestingly, for the 21 d that it rained ‡0.5 inch, only 17% (3.8 vs. 21.8 inches) of rain effectively replaced the irrigation requirement. The low percentage was due in part to the fact that many of the rain events of ‡0.5 inch occurred toward the end of the season when CF >2 and ET rarely exceeded 0.8 inches. Also, 7 of the 21 d received >1.0 inch and on those days ET rates were low because of low solar radiation levels. For the 34 d that it rained <0.5 inch, 79% (4.7 vs. 6.0 inches) of rain directly reduced irrigation. The take-home message is that accounting for rain in a sprinkler irrigation program requires an objective accounting of when the rain fell and what amount actually got into the container relative to the container substrate's water deficit to estimate the rain's effectiveness for decreasing the irrigation requirement. This trial demonstrated that CIRRIG can be used to effectively guide irrigation. Although the scope of this trial was limited, it provides evidence that when automated, daily adjustments to irrigation based on estimated ET and effective rain can conserve irrigation water without reducing plant growth. CIRRIG requires the user to monitor and input plant production conditions in the irrigation zone and as such requires active involvement by nursery staff to make it work. CIRRIG is being used to automatically irrigate %160 zones of sprinkler-irrigated container production in a container nursery in VA. Staff at the nursery adopted several measures, which have helped them implement CIRRIG. For monitoring PPC, the staff visually compares the existing plant cover to a palette of images representing a range of PPC values and selects the one that best represents the existing condition. Another example is that pruning schedules and pruning specifications (e.g., plant height and width) are forwarded to the irrigation manager to input into CIRRIG. Similarly, spacing schedules are forwarded to the irrigation manager to input into CIRRIG. To save time, the effects of pruning and spacing on PPC can be estimated and input at the same time. Finally, leaching fraction testing (Stanley, 2012) can be conducted periodically throughout the nursery to ensure CIRRIG is performing effectively. Saunders Brothers (Piney River, VA) estimated that 10 h/week are devoted to day-to-day management of CIRRIG (J. Stanley, personal communication). The cost for adopting CIRRIG technology at a nursery will depend on the cost of the two main components: CIRRIG and the computercontrolled irrigation system. The rights to CIRRIG are owned by the University of Florida and we have just begun investigating opportunities to offer the program to interested growers. We envision that future users will enter into an annual subscription agreement with UF that includes upfront training costs to ensure the nursery is using CIRRIG properly. The CIRRIG subscription would cover technical support as well as maintenance and program improvements. We estimate an annual subscription will cost %$10,000, which will vary depending on nursery size and number of consulting trips needed. The second component is the installation of a computer-controlled irrigation system and this cost will vary tremendously based upon nursery size, complexity, pumping challenges, local network upgrades, and other features (e.g., fertilizer or chlorine injection). The irrigation control contractor that installed the PLC system at Saunders Brothers nursery indicated that a 32-valve control box with control software and network connectivity would cost %$8000 to $10,000 (R. Illig, personal communication). Additional costs for installation, programming, technical support are unknown but will certainly be higher during the first few years as the ''kinks'' get worked out. We believe several conditions are needed to make a CIRRIG work effectively at any given nursery. The staff must be dedicated to the idea of weather-based irrigation. Automated CIRRIG technology is not a ''set and forget it'' system like a traditional irrigation controller that is set manually. Staff must be dedicated to managing CIRRIG inputs and outputs on a daily basis as well as ensuring irrigation effectiveness by diligent monitoring of plants in the field. It is also important that the nursery have good IT support to troubleshoot networking and computer problems that can often arise during power outages or following irrigation system malfunctions. On the production side, CIRRIG will be more effective if plants within irrigated areas have similar irrigation requirements; a nursery with diverse plantings within the same zone will not likely benefit greatly from CIRRIG. CIR-RIG assumes the irrigation system provides relatively uniform delivery of water over the irrigated area and that application rates are consistent from one day to another. A container nursery that meets the above conditions and that is dedicated to irrigating efficiently should find that CIRRIG is a viable tool for implementing weather-based irrigation at the nursery.

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Phenotypic and Transcriptomic Analyses of Autotetraploid and Diploid Mulberry (Morus alba L.) Autopolyploid plants and their organs are often larger than their diploid counterparts, which makes them attractive to plant breeders. Mulberry (Morus alba L.) is an important commercial woody plant in many tropical and subtropical areas. In this study, we obtained a series of autotetraploid mulberry plants resulting from a colchicine treatment. To evaluate the effects of genome duplications in mulberry, we compared the phenotypes and transcriptomes of autotetraploid and diploid mulberry trees. In the autotetraploids, the height, breast-height diameter, leaf size, and fruit size were larger than those of diploids. Transcriptome data revealed that of 21,229 expressed genes only 609 (2.87%) were differentially expressed between diploids and autotetraploids. Among them, 30 genes were associated with the biosynthesis and signal transduction of plant hormones, including cytokinin, gibberellins, ethylene, and auxin. In addition, 41 differentially expressed genes were involved in photosynthesis. These results enhance our understanding of the variations that occur in mulberry autotetraploids and will benefit future breeding work. Introduction Mulberry (Morus alba L.) [1] is a woody plant native to China that is commercially valuable. The most important use of mulberry is as the sole food source of the domesticated silkworm (Bombyx mori L.). However, mulberry is also used in animal fodder, pharmaceuticals, food production, and landscaping [2,3]. Polyploidy is a heritable change in which the entire chromosome set is multiplied, and it plays an important role in plant evolution [4]. Two forms of polyploidy are often considered: allopolyploidy, which originates from interspecies hybrids, and autopolyploidy, which originates from intraspecies genome duplication events. Polyploidy is particularly widespread in the flowering plants (angiosperms), including many major crops [5]. Polyploid plants are often larger and have larger organs than their diploid relatives, including higher yield, larger leaves, larger fruit, more robustness, and some other agronomic characters [6][7][8], which makes polyploids quite appealing for agricultural breeding. How are the larger plants regulated by the polyploidization? The most naïve hypothesis was that increase in gene copy number increased the amount of protein, which in turn increased the cell volume [9]. It was found that the ploidy-dependent increase in cell volume is genetically regulated in the experiment of investigating a wide range in cell size by tetraploidizing various mutants and transgenics of Arabidopsis thaliana [10]. Early research reported that polyploidization increased the chloroplast number and photosynthesis per cell, which may be due to increasing size of cells [11]. However, the mechanism behind the ploidy-related regulation of cell size, cell proliferation and expansion remains largely unclear. In recent years, plant breeders have worked with polyploids in mulberry and several artificially generated polyploids with "larger" mulberry characteristics have been reported [12][13][14]. Hence, we sought to investigate the physiological and molecular mechanisms for the enlargement phenomenon in mulberry polyploids. Transcriptome-wide gene expression analysis has been demonstrated in many bred and natural polyploid plants. Research on transcriptional analyses of autotetraploids and their related diploids show a significant divergence in species-specific traits even though a great deal of common characteristics also exist [5,15]. For example, only ~1%-3% of genes are significantly differentially expressed between the autotetraploids and diploids of Arabidopsis (Arabidopsis thaliana L.), rice (Olyza Sativa L.), and Chinese woad (Isatis indigotica Fort.) [16][17][18], whereas ~10% of potato (Solanum phureja L.), birch (Betula platyphylla Suk.), and Paulownia (Paulownia fortune Hemsl.) genes are significantly differentially expressed [8,19,20]. In addition, RNA profiling using different tissues may partly cause transcriptome divergence. Among woody plants, the biosynthesis and signal transduction of indole-3-acetic acid and ethylene have been altered by a genome duplication event in birch [8], whereas differentially expressed transcripts are enriched in the energy metabolism pathway and in genetic information storage in Paulownia [20]. Hence, it is necessary to investigate the changing expression in key genes after polyploidization in the mulberry. RNA-Seq is a powerful tool for detailed transcriptomic studies that is cost-efficient and yields a far greater amount of information than traditional sequencing technology [21]. In this study, we obtained a series of mulberry autotetraploids using a colchicine treatment and compared differences between the transcriptomes of diploid and autotetraploid mulberry plants using RNA-Seq technology. The results improved our understanding of the genetic regulation associated with mulberry autopolyploidization. Detection of Mulberry Autotetraploids A series of mulberry autotetraploids were generated with the colchicine treatment. We investigated the ploidy of two-month old mulberry seedlings using a flow cytometry analysis and chromosome counts. The DNA content of the control diploids had a main flow cytometry peak at channel 100 ( Figure 1A). In contrast, the DNA content of the autotetraploids showed a main peak at channel 200 ( Figure 1B). The chromosome count of diploids was 2n = 2x = 28, whereas that of the autotetraploids was 2n = 4x = 56 ( Figure 1C,D). Of all 247 novel saplings generated with the colchicine treatment, about 49% saplings were autotetraploids while the rest of them were chimeras with part of polyploidization cell. Phenotypic Changes versus Ploidy in Mulberry Diploid and autotetraploid mulberry trees at adult stage were characterized morphologically. The height, breast-height diameter, leaf area and cross-section, inflorescence length, fruit length and diameter of autotetraploid trees were larger than those of the diploid trees. The mean value of leaf area and leaf cross-section of autotetraploids were 99.28 cm 2 and 2.88 μm, respectively, which were ~40% larger and ~19% thicker compared with those of diploids ( Figure 2; Table 1). As shown in the leaf cross-section in Figure 2C,D, it appears that the cell size of spongy tissue and palisade tissue were larger in the autotetraploid leave, but the cell number did not reveal an apparent difference. Further, this was simply a direct observation, and with no statistical significance. The fruits of autotetraploids were larger than those of diploids at different fruit stages while the fruit maturation period had no significant differences. At the black fruit stage, the mean weight of autotetraploid fruit was 8.57 g, which was ~70% heavier than diploid fruit. And the mean value of fruit length and diameter were 5.70 and 2.22 mm, respectively, which were ~40% longer and ~55% greater compared with those of diploids ( Figure 2; Table 1). The height and breast-height diameter of autotetraploid trees were also greater than those of diploids (Table 1). Gene Expression in Diploids and Autotetraploids To compare differences between the transcriptomes of diploid and autotetraploid, cDNA libraries were generated from leaves of adult stage plants, and then Illumina paired-end sequencing was performed. A total of ~12 M, 50-bp, single-ended RNA-Seq reads were generated from each sample. There were three biological replicates from three separate trees each for diploid and autotetraploid. Each of the reads was mapped to the mulberry genome sequence, which contains 29,338 predicted genes [22]. All of the samples showed similar match results, with ~70% of reads matching the genome sequence and ~64% being unique matches. Additionally, ~62% of reads matched the predicted genes of the genome, with ~58% being unique matches. Approximately 20,000 predicted genes were covered, constituting ~68% of the total predicted genes of the genome ( Table 2). The number of detected genes was saturated when the sequencing counts surpassed 5 million ( Figure S1), and ~50% of the matched genes had >70% coverage by reads in all of the samples ( Figure S2). Differential Gene Expression between Diploids and Autotetraploids Among all of the samplings, 21,229 genes were detected. To compare the gene expression differences between diploids and autotetraploids, a reads per kilobase of exon per million reads mapped (RPKM) value for each sample was calculated. We considered genes with ≥2-fold changes in expression (log2(RPKMautotetraploid/RPKMdiploid) ≥ 1) and probability ≥ 0.8 to be regulated genes. Based on this criterion, 609 (240 up-regulated and 369 down-regulated) differentially expressed genes were identified between diploids and autotetraploids ( Figure 3; Tables S1 and S2). Among all of the expressed genes, only 2.87% of genes were differentially expressed. This indicated that gene expression was not highly altered after polyploidization in mulberry, which corroborates reports in other plant species [16,17]. To validate the gene expression data obtained through RNA-Seq, 10 genes were randomly selected from the differentially expressed genes for a quantitative real-time PCR (qPCR) analysis ( Figure 4). The expression patterns of the 10 genes obtained through qPCR were largely consistent with the RNA-Seq data. The qPCR analysis confirmed that the RNA-Seq approach provides reliable differential gene expression data for ploidy analysis in mulberry. Functional Classifications of Differentially Expressed Genes To further analyze the differentially expressed genes, we functionally classified these genes using the public gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Using the GO database, we characterized 432 differentially expressed genes were grouped into 44 GO terms based on sequence homology, which fell into three main groups-molecular function, biological process, and cellular component ( Figure 5). For the biological processes, besides the large categories "cellular process" and "metabolic process", genes were mostly enriched in "response to stimulus", "single-organism process", and "biological regulation". In addition, the processes "localization", "developmental process", "multi-organism process", and "signaling" differed significantly between autotetraploids and diploids. Using the KEGG database, we annotated 350 differentially expressed genes to 100 KEGG pathways, using a blastx search with an E-value threshold of 1.0 × 10 −5 . The top 20 enriched KEGG pathways are shown in Figure 6. Besides the most highly represented group "metabolic pathways", the "biosynthesis of secondary metabolites", "plant-pathogen interaction", and "plant hormone signal transduction" pathways were also highly enriched ( Figure 6). Additionally, the pathways "flavone and flavonol biosynthesis", "limonene and pinene degradation", and "stilbenoid, diarylheptanoid and gingerol biosynthesis" differed significantly between autotetraploids and diploids. Differentially Expressed Genes Related to Plant Hormones Plant hormones are a category of important regulatory factors affecting plant growth and development. Among the differentially expressed genes between autotetraploids and diploids, a series of genes involved in the biosynthesis and signal transduction of plant hormones was found. Of the 609 regulated genes, 30 (4.9%) genes were associated with plant hormones (Table 3). Among them, six genes were related to cytokinin biosynthesis and signal transduction, including two adenylate isopentenyltransferases (Morus025042 and Morus010031), three two-component response regulator ARRs (Morus001125, Morus023955, and Morus023956), and one cytokinin dehydrogenase (Morus018596). The two adenylate isopentenyltransferase genes-important enzymes catalyzed during cytokinin biosynthesis [23]-were up-regulated in autotetraploids compared with the diploids of mulberry. Meanwhile, the three two-component response regulator ARR genes-which act as negative regulators of cytokinin signal transduction [24]-were down-regulated in the autotetraploids (Table 3). There were five regulated genes associated with gibberellin (GA) signal transduction, including one GA receptor (Morus027556) and four DELLA proteins (Morus013990, Morus004260, Morus025266, and Morus025269). The GA receptor GID1-a hormone-sensitive lipase regulated by GA perception [25]-was up-regulated in autotetraploids compared with in diploids. DELLA proteins are key regulators of GA signaling, acting as negative regulators of GA responses [25]. Among the four regulated DELLA proteins in this study, three were down-regulated in the autotetraploids (Table 3). Nine regulated genes were involved in auxin biosynthesis and signal transduction, including auxin-induced protein, auxin-binding protein, auxin-repressed protein, and auxin hydrolase genes. Among the nine regulated genes, three were up-regulated whereas six were down-regulated in autotetraploids compared with diploids (Table 3). Of the 10 regulated genes related to ethylene biosynthesis and signal transduction, two were 1-aminocyclopropane-1-carboxylate oxidase (ACO) genes (Morus004820 and Morus012808) and eight were ethylene response factor genes. ACOs are important enzymes during ethylene biosynthesis [26]. Several ethylene response factors play crucial roles during ethylene signal transduction [26]. In this study, among the 10 regulated genes related to ethylene, nine genes were down-regulated, whereas only one was up-regulated in autotetraploids compared with diploids (Table 3). These results suggested that plant hormones may play important roles in the phenotypic changes of autotetraploids compared with diploids. Differentially Expressed Genes Related to Photosynthesis Photosynthesis is another important factor affecting plant growth. Among the regulated genes in the autotetraploids, 41 (6.9%) were involved in photosynthesis (Table 4). There were 14 genes expressed specifically in chloroplasts, including a phospholipase, thylakoid protein, aminotransferase, phosphate translocator, and polyphenol oxidase. Additionally, there were 20 cytochrome genes, including cytochrome b, cytochrome c, phytochrome interacting factor, and a series of cytochrome p450s. Seven other genes were associated with photosynthesis, including homogentisate phytyltransferase, photosystem I reaction center subunit XI, NADPH, and quinone oxidoreductase. Among these, the photosystem I reaction center subunit XI gene was up-regulated significantly in autotetraploids, with a log2(RPKMautotetraploid/RPKMdiploid) value of 7.68. Another gene that encodes a homogentisate phytyltransferase-a key enzyme of the biosynthesis of the strong antioxidant tocopherol during photosynthesis [27]-was also up-regulated significantly in autotetraploids, with a log2(RPKMautotetraploid/ RPKMdiploid) value of 9.45. Table 4. Differentially expressed genes involved in photosynthesis. The fold change of each regulated gene is reported as log2, and the cutoff log2(RPKMautotetraploid/RPKMdiploid) ≥ 1 and probability ≥ 0.8. Discussion Polyploidy has played an important role in the evolution of angiosperms and was involved in the speciation of many important crops [28]. Autopolyploidy is usually associated with increased plant, organ, and cell sizes, so polyploids generated through plant breeding have been used as tools to increase crop yields [29][30][31]. By targeting relevant genes through transcriptomic techniques, the genetic basis of autopolyploidism has been investigated [19,20]. RNA-Seq is a newly developed high-throughput sequencing technology that provides a powerful and cost-efficient research platform for transcriptional profile analyses [32]. In this study, the transcriptomes of autopolyploid and diploid mulberry were investigated using Illumina RNA-Seq technology with mulberry genome sequences as the reference [22]. We obtained 609 transcripts that are differentially expressed between autopolyploids and diploids, accounting for ~2.87% of the total genome sequences. Transcriptomic analyses have been performed in the generated autopolyploids of several plants, including Arabidopsis, potato, rice, Chinese woad, Rangpur lime (Citrus limonia Osbeck), birch, and Paulownia [8,[16][17][18]33]. The percentage of differentially expressed genes in generated autopolyploids varies from 1.08% in Rangpur lime [33] to 12.6% in birch [8]. In herbaceous plants, leaflet and root tip tissues of potato has ~10% differentially expressed genes among different ploidies, whereas seedlings of Arabidopsis and pollen of rice have 1.63% and 2.59% between autotetraploid and diploid, respectively [16,17,19]. Compared to the other woody plants studied, mature leaves of mulberry had a higher percentage (2.87%) of differentially expressed genes than the 1.08% in mature leaves of Rangpur lime, but much lower than the 12.6% in shoot tips of birch or the 9.49% in young leaves from Paulownia [8,20,33]. Different tissues source for RNA profiling maybe another reason for transcriptome divergence. The percentages of differentially expressed genes between autopolyploids and diploids show species-specific and tissue-specific features. Among the genes demonstrating expression changes between autopolyploids and diploids, plant hormone-related genes are an important category [8,16]. Ethylene-and auxin-related processes are controlled by highly regulated genes in autopolyploid Arabidopsis seedlings [16], and the biosynthesis and signal transduction of the auxin and ethylene pathways are altered after genome duplication in birch [8]. In Rangpur lime, GA-and auxin-related GO categories are over-expressed in autotetraploid plants [33]. In this study, among the 609 regulated genes in the autotetraploids, 30 (4.9%) genes were associated with plant hormones (Table 3). Cytokinin, GAs, and auxin-all of which are plant hormones that promote plant development and growth [34]-were significantly affected in autotetraploid mulberry compared with in diploid mulberry (Table 3). There were six regulated genes related to cytokinin biosynthesis and signal transduction, and five regulated genes associated with GA signal transduction in the autotetraploids (Table 3). Among them, two adenylate isopentenyltransferase genes-important enzymes catalyzed [23] during cytokinin biosynthesis-were up-regulated, whereas three two-component response regulator ARR genes -which are negative regulators of cytokinin signal transduction [24]-were down-regulated in autotetraploid mulberry (Table 3). In the GA signal transduction pathway, a GA receptor, the GID1 gene, was up-regulated, whereas three genes encoding negative regulators of DELLA proteins [25] were down-regulated in the autotetraploids ( Table 3). Levels of ethylene-a plant hormone suppressing development and growth [34]-were significantly altered in autotetraploid mulberry (Table 3). Of the 10 regulated genes related to ethylene, most of them were down-regulated in the autotetraploids (Table 3). In summary, plant hormones-especially cytokinin, gibberellin, and ethylene-may play important roles in the phenotypic changes of autotetraploids. The rate of photosynthesis and chloroplast numbers both increase in association with ploidy increases [11,35,36]. In addition, photosynthesis-related genes are up-regulated in polyploidy plants [18,37]. In Arabidopsis, photosynthesis-and chlorophyll-related GO categories are enriched in autotetraploids compared with diploids [16]. In the present work, a series of differentially expressed genes that are involved in photosynthesis-including genes specifically expressed in chloroplasts, cytochrome genes, and photosystem-related genes-were up-regulated in autotetraploid mulberry (Table 4). Several important genes were substantially up-regulated in autotetraploids, such as the photosystem I reaction center subunit XI gene and the homogentisate phytyltransferase gene (Table 4). RNA for Illumina sequencing in this study were from mature leaves, the main tissues for photosynthesis, which may be another reason for so many different expressed genes related to photosynthesis. In brief, photosynthesis may be an important factor affecting phenotypic changes in autotetraploid plants. The mechanism of polyploidization regulating larger organs has been studied for several years. Previous studies have reported that polyploidization increased the cell size, chloroplast number and photosynthesis per cell [10,11]. In this study, we investigated the leaf cross-section of diploid and autotetraploid mulberry. We seemed to observe the cell size increased in autotetraploid compared with diploid but the cell number did not reveal an apparent difference (Figure 2). On the transcriptome level, we found that gene expression of two important hormones, cytokinin and GAs-promoting plant growth and affecting cell size [34]-were positively regulated in autotetraploid mulberry (Table 3). Moreover, a series of photosynthesis related genes, including several chloroplast specifically expressed genes, were up-regulated in autotetraploid mulberry (Table 4). It could be speculated further that mulberry autotetraploid could increase level of cytokinin and GAs, which thereby increased the cell size and photosynthesis, ultimately resulted in larger organs. Further research on the mechanism of larger organs regulated by polyploidization is needed. Plant Materials Seeds of diploid mulberry (M. atropurpurea) were soaked in 0.1% colchicine for 48 h in the dark to induce autotetraploidy, and seeds soaked in distilled water under the same conditions acted as controls. The seeds were sown in a greenhouse after colchicine treatment, and 247 novel saplings and 39 control diploid saplings were transplanted into plastic pots in a greenhouse. Ploidy Measurement The DNA content of the leaves of two-month old seedlings was evaluated by flow cytometry using the methods of Galbraith et al. [38] with some modifications. Three biological replicates performed of each sample. Briefly, ~0.5-1 cm 2 young intact leaves were chopped in 1 mL of ice-cold extraction buffer (50 mM MgCl2, 50 mM citric acid, 5 mM HEPES, 0.1% Triton X-100, and 1% PVP-40) using a new razor blade. The crude suspension was filtered through a 42-μm nylon filter to remove cell debris and then added to a propidium iodide staining solution to a final concentration of 50 μg/mL. After 1 h of incubation at room temperature, the fluorescence intensity was measured using a FACSAriaII (BD Biosciences, San Jose, CA, USA) flow cytometer with excited blue light at 488 nm and 5 × 10 8 J/s. The percentages of the cells that showed varied DNA contents were determined using BDFACSDiva software, and the DNA content of diploid leaves in the control group were measured in parallel. Mitotic chromosomes were counted in young leaf bud of two-month old seedlings. Three biological replicates performed of each sample. Buds were collected and treated with a fixative buffer (concentrated hydrochloric acid/45% acetic acid/ethanol at 2:1:1 (v/v/v)) for 5 min. After flushing with water two or three times and immersing in water for 10 min, leaf buds were placed onto microscope slides and stained with one drop of carbolfuchsin and one drop of 45% acetic acid. Then, leaf buds were mashed with tweezers, the cytoplasmic residue was cleared, and the remains covered with glass. The chromosomes were visualized under a microscope (AxioScope A1, Carl Zeiss MicroImaging, Göttingen, Germany) using 1000× magnification. Approximately 10 metaphase cells were assessed for each leaf bud. Phenotype Measurement A series trees of autotetraploid (YY56) and diploid (TL) were adjacent planted in a plantation with the same parcel and climate condition. Ten adult stage (three-year old) mulberry trees of each cultivar were used in phenotype measurement. Leaf cross-sections were evaluated using scanning electron microscopy. An area ~1 cm 2 from the center of mature leaves was fixed for 24 h in 2.5% glutaraldehyde fixation solution containing 2.5% glutaraldehyde and 0.1 M phosphate-buffered saline (PBS, pH 7.4) and dehydrated using a graded series of alcohol-isoamyl acetate concentrations, each for 15 min. The samples were dried using a critical point dryer (HCP-2, Hitachi, Tokyo, Japan), mounted on scanning electron microscopy stubs, sputter-coated with gold using an ion coater (Eiko IB-5, Hitachi), and observed under a scanning electron microscope (Philips XL30, Philips Electron Optics, FEI UK Ltd., Cambridge, UK). At least five leaf samples from each individual tree were viewed and measured at 20 kV using 2000× magnification. Leaf areas were measured from at least 10 healthy and fully expanded leaves collected at random from each tree. Height and breast-height diameters were measured from ten trees. The lengths, weights and diameters of the fruits were measured using a Vernier caliper, and the fruit maturation period were measured from end of the flowering to black fruit stage. At least 10 healthy fruits were selected at random from each tree and measured. SIGMASTAT from SPSS [39] was used to analyze the morphological data. Student's t test were used to detect differences between autotetraploid and diploid at the usual probability level p = 0.05. RNA Extraction, Illumina Sequencing, and Data Processing For Illumina sequencing, three biological replicates from six independent adult stage trees of autotetraploid and diploid were used. Total RNA from each sample was extracted using the RNAiso Plus reagent (Takara BIO Inc., Otsu, Japan) and further purified using RNeasy Plant Mini kit reagents (Qiagen, Valencia, CA, USA). RNA quality was verified using a 2100 Bioanalyzer RNA Nanochip (Agilent, Santa Clara, CA, USA). All samples had an RNA integrity value of >7.5. RNA was then quantified using a NanoDrop ND-1000 Spectrophotometer (Nano-Drop, Wilmington, DE, USA). Total RNA (10 μg) was prepared for the cDNA library for each pool. Illumina sequencing was performed at the Beijing Genomics Institute, Shenzhen, China using the HiSeq 2000 platform (Illumina, San Diego, CA, USA). First, poly-T oligo-attached magnetic beads (Illumina) were used to isolate poly(A) mRNA from total RNA. The purified mRNA was then fragmented into 200-to 700-nt pieces. The first strand of cDNA was synthesized using random hexamer primers, followed by synthesis of the second strand using SuperScript Double-Stranded cDNA Synthesis kit reagents (Invitrogen, Camarillo, CA, USA). The synthesized cDNA was subjected to end repair and phosphorylation using T4 DNA and Klenow DNA polymerases and T4 polynucleotide kinase, respectively. The repaired cDNA fragments were 3ʹ-adenylated using the Exo-Klenow fragment, and the Illumina paired-end adapters were ligated to the ends of these 3ʹ-adenylated cDNA fragments. To select templates for downstream enrichment, the products of the ligation reaction were purified by electrophoresis in a Tris-acetate-EDTA (2% w/v) agarose gel. cDNA fragments (200 ± 25 bp) were excised from the gel. Fifteen rounds of PCR were performed to enrich the purified cDNA templates using PCR primers PE 1.0 and PE 2.0 (Illumina) with Phusion DNA polymerase. Finally, after validation on an Agilent Technologies 2100 Bioanalyzer using Agilent DNA 1000 Chip kit reagents, the cDNA library was constructed by 50-bp, single-end RNA sequencing (RNA-Seq) in a PE flow cell using the Illumina Genome Analyzer HiSeq 2000. Sequencing quality was evaluated and the data were summarized using the Illumina/Solexa pipeline software. Library saturation was also analyzed. For the raw data, adaptor sequences were eliminated, and distinct clean reads were identified. Subsequently, clean reads and distinct clean reads were classified based on their copy numbers within the library, and the percentages of total clean and distinct reads were calculated. The raw data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database [40] under submission number GSE70428. Annotation and Analysis of Sequence Data For annotation, all sequences were mapped to the mulberry genome [22]. The expression levels of each gene were estimated using the frequency of clean reads and then normalized to RPKM [41]. Differential expression genes analysis was used the NOIseq metheod [42]. Fold changes were assessed using the log2 ratio after expression abundances were normalized to RPKM. Differential expression genes were cutoff log2(RPKMautotetraploid/RPKMdiploid) ≥ 1 and probability ≥ 0.8. Sequences were characterized using the GO database (http://www.geneontology.org/) [43]. Pathway assignments were determined with the KEGG pathway database [44] using the blastx algorithm with an E-value threshold of 1.0 × 10 −5 . The differential gene expression analysis data have been submitted to the GEO database under submission number GSE70428. qPCR and Statistical Analysis Mature leaves of adult stage mulberry trees were used in qPCR. Primers were designed using Primer 5.0 software [45]. Mulberry MaACT3 (GenBank accession number: HQ163775) gene was used as the reference gene. Expression levels for all of the candidate genes were computed based on the stable expression level of the reference gene. qPCR was performed in 96-well plates on a Roche LightCycler 480 system using SYBR-GREEN1 fluorescent reagents (Takara, Otsu, Shiga, Japan). Reactions were each carried out in 20 μL containing 0.4 μM (final concentration) of each primer (Table S3). The qPCR thermal profile consisted of 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s, 58 °C for 10 s, and 72 °C for 10 s. Dissociation curves were obtained from a thermal melting profile generated under a final PCR cycle of 95 °C for 5 s followed by a constant increase in temperature from 65 to 97 °C. Threshold values were empirically determined based on the observed linear amplification phase of all of the primer sets. Sample cycle threshold (Ct) values were standardized for each template based on the reference gene control primer reaction, and the 2 −ΔΔCt method was used to analyze relative changes in gene expression. Three biological replicates were used to ensure statistical credibility. SIGMASTAT from SPSS was used to analyze the qPCR data. Student's t tests were used to detect differences between autotetraploid and diploid at the usual probability level p = 0.05. Conclusions In this study, phenotypic and transcriptomic changes between autotetraploid and diploid mulberry plants were compared. Larger plants and organs were observed in the autotetraploids. Only a few changes in gene transcriptional levels, including genes associated with plant hormones and photosynthesis, occurred in the autotetraploids. Further molecular mechanism-based studies of genome duplication events are still needed.

v3-fos

2019-04-04T13:07:27.716Z

2015-03-18T00:00:00.000Z

54973487

s2

A comparative study of nutrients and mineral composition of Carallia brachiata (Lour.) Merill The proximate composition and mineral constituents of Carallia brachiata leaf and fruit powder were evaluated for their nutritional values and mineral compositions by using standard techniques. In proximate analysis, ash, carbohydrate, proteins, fiber, fat, moisture, total energy content (dry basis) was assayed while mineral analysis were carried out by using atomic absorption spectrophotometer. The species showed variable results in proximate analysis of both the parts; however, the fruit of C. brachiata have revealed higher percentage of carbohydrate (65.74%) and energy values (310.25Kcal/100g). The leaf showed higher percentage of proteins (13.59%) and crude fibers (18.87%). From the results is clear that both the parts of C. brachiata are rich in micronutrients like Cu, Zn & Fe. The proximate and nutrient analysis of the species can help us to determine the health benefits achieved from their use in marginal communities. Introduction Carallia brachiata (Lour.) Merill (Family-Rhizophoraceae) is commonly known as karalli. It is a large evergreen ornamental tree. It has many medicinal uses. Bark of Carallia brachiata is traditionally used in wound healing, treating itch, oral ulcer, inflammation of throat and stomatitis [1]. The ethyl acetate and methanol extracts of bark exhibited anti-inflammatory and wound healing activities [2]. The leaves and bark are used medicinally against itch and septic poisoning. The fruit extracts have medicinal importance to treat ulcers [2]. The occurrence of trees is reported in semi-evergreen forest and also along the coastal areas in mangrove habitat [3]. The subcanopy tree is geographically distributed in Indo-Malasia and Australia [4]. In developing countries, numerous types of edible wild plants are exploited as sources of food; to provide an adequate level of nutrition to the inhabitants, where poverty and climate change are causing havoc to the rural people. In this context, this analysis was carried out to evaluate the nutritional value and mineral composition of Carallia brachiata with hope that it would be incorporated into food basket of the country. The aim of analysis is the preliminary assessment of nutritional value and mineral composition of the plant-based diets. Attention has been drawn to this undervalued natural resource specifically, to make a bigger contribution against malnutrition. Collection of Material: Fruits as well as leaves were collected from coastal area of Ratnagiri district. Sample Preparation: The leaves and fruits were air-dried and ground to a fine powder. Powder is stored in air-tight containers prior to further analysis. Proximate analysis: The moisture and ash content was determined by gravimetric method. The crude fiber was calculated by acid-base digestion. Crude protein was determined by Macro-Kjeldahl method. Crude fat content was determined gravimetrically following Soxhlet extraction with ether according to Official "Association of Official Analytical Chemists" (AOAC) method [5]. Available carbohydrate was estimated "by difference" using the formula, TCH (%) =100-% (CP+A+CF+M). The energy value were estimated by calculation method using following formula, Energy value (g/100g) = [4x crude protein] + [4 x carbohydrate] + [9 x crude fat]. www.ssjournals.com Mineral Analysis: Acid digestion was carried out by the method followed by Toth [6]. The mineral elements like Cu, Zn, Co, Fe, Ca, Mg, Mn etc. were analyzed by Atomic Absorption Spectrophotometer (AAS). Proximate composition: The results of proximate composition of leaf and fruit powder of C. brachiata are shown in Table 1. The ash content, which is an index of mineral contents; it is found in the leaf powder of C. brachiata 4.87% Dry Weight (DW) was more than the fruit (3.42%). It is apparent that leaves of C. brachiata are good source of crude fibers, while fruits are good source of carbohydrates. The crude protein content of both leaf and fruit was 13.59% and 10.9% respectively. Plants foods that provide more than 12% of their calorific value from protein are a good source of protein. The crude lipid contents of leaf and fruit of C. brachiata were less than the range (8.3-27% DW) [7]. The estimated carbohydrate content in fruit of C. brachiata (65.74%) was found to be higher than that of leaves (59.79%). The crude fiber content in leaves was found 18.87%, which was more than the fruit fiber content (12.82%). The fiber RDA values for children, adults, pregnant and breast-feeding mothers are 19-25%, 21-38%, 28% and 29% respectively. The total energy content of leaf was estimated to be 306.21±0.015 Kcal/100g (DW) and for fruit it is 310.25±0.017 Kcal/100g (DW), which is an indication that it could be an important source of dietary calorie. Calorific content of C. brachiata could be attributed to high carbohydrate and protein contents. Fig. 1 and 2 represent the results of mineral compositions of C. brachiata leaf and fruit. Mineral composition Nutritional significance of elements in both leaf as well as fruit has adequate level of all the essential minerals. There is a range of minerals needed regularly by our bodies. Iron is needed by our blood and helps produce the red colouring. Iron is higher in the leaf of C. brachiata (117.84 mg/100g) than the fruit (54.66 mg/100g). This deficiency is very common around the world and women, children and old people are much more likely to suffer from iron deficiency. Iron is essential trace element for haemoglobin formation, normal functioning of central nervous system and in the oxidation of carbohydrates, proteins and fats [8]. Calcium is found nearly same amount in both the parts of C. brachiata. Calcium is very important for bones and teeth but also affects many other things within our bodies. Zinc has now been recognized as very important especially for the growth of children. Zinc is used in our bodies in chemicals called enzymes that control how our bodies work. Zinc is found to be higher in fruits as well as in leaves of C. brachiata. Several coastal seeds and nuts are important sources of zinc and that are often eaten by children along seashores in all tropical countries as a good source of zinc. Sodium is important for fluid distribution, blood pressure, cellular work and electrical activity. Potassium is essential for the ability of skeletal and smooth muscles to contract. Because of this, an adequate intake of potassium is important for regular digestive and muscular functioning. In C. brachiata sodium and potassium were found to be higher in fruits than the leaves ( Table 2). According to Food and Agricultural Organization (FAO), food balance data, it has been calculated that about 20% of the world's population could be at risk of deficiency of essential minerals. In the leaves and fruits of C. brachiata, the range of copper is less than 5mg, which is recommended by World Health Organization (WHO). Conclusion The results of the proximate and mineral assessment showed that fruit is good source of potassium, moisture and carbohydrate whereas leaf is good source of zinc, fiber, lipid and ash. The results suggest that the plant fruits if consumed in sufficient amount could contribute greatly towards meeting human nutritional requirement for normal growth. Fruits of C. brachiata are recommended for continues used for nutritional purposes, considering the amount and diversity of nutrients it contains. Biochemical analysis alone cannot be the exclusive criteria for judging the nutritional significance of plant parts. Thus, it becomes necessary to consider other aspects like antinutritional/toxicological factors. Also, the awareness about their use and rational sustainable harvesting from the area is the need of the present scenario for meeting demand as ethnic food with nutritional security in rural areas.

v3-fos

2019-04-07T13:09:57.989Z

2015-12-15T00:00:00.000Z

102230997

s2

Effect Of Chromium Nicotinate On Oxidative Stability, Chemical Composition And Meat Quality Of Growing-Finishing Pigs The effect of different organic sources of Cr on growth, feed efficiency and carcass value is known but there is a lack of information between chromium nicotinate (CrNic) and pork quality. Therefore, purpose of this research was to investigate the effects of CrNic on chemical composition, quality and oxidative stability of pork meat. In the study, pigs of Large White breed (40 pcs) were used. The pigs were divided into two groups, namely the control and the experimental of 20 pcs with equal number of barrows and gilts. The pigs were fed the same diet which consisted of three feed mixtures applied at the different growth phases, from 30 - 45 kg OS-03, 45 - 70 kg OS-04 and 70 - 100 kg OS-05. The pigs were allowed ad libitum access to feed and water. The diet of experimental group was supplemented with 0.75 mg.kg -1 CrNic in the form of chromium-inactivated yeast Saccharomyces cerevisiae . The fattening period in pigs lasted from 30 to 100 kg. The chromium supplementation led to a significantly higher content of chromium in longissimus thoracis muscle (LT) of experimental pigs. In addition, the results showed a statistically significant difference ( p ≤0.05) in retention of chromium in the LT, monounsaturated and omega-3 polyunsaturated fatty acids content in experimental group compared with control. Moreover, there was highly significant ( p £0.05) difference in essential fatty acids, as well as in oxidative stability in 7 days, among the groups. The highly significant differences were also observed among sexes, namely in total water, protein and intramuscular fat contents, colour CIE b* in both times, and oxidative stability. However, physical-technological parameters (pH, drip loss, shear force and meat colour) were not affected when pigs were fed the supplement. On the whole, the positive effect of chromium nicotinate in most of investigated parameters may be beneficial not only for pork industry but also for consumers. Normal 0 21 false false false CS JA X-NONE However, most grains and feedstuffs are deficient in Cr and must be supplemented with a bioavailable source of Cr (Bunting, 1999). Animals cannot utilize glucose when chromium is deficient in feed (Tang et al., 2001). It is generally accepted that organic sources of Cr like chromium picolinate and chromium nicotinate are utilized more efficiently than inorganic Cr sources Supplemental chromium nanoparticle (CrNano) has shown beneficial effects on carcass characteristics and pork quality in finishing pigs (Wang and Xu, 2004). Research with four organic sources (Cr-tripicolinate, Cr-propionate, Cr-methionine, Cr yeast) in concentration 5000 μg.kg -1 of Cr was reported (Lindemann et al., 2008). The effects of the forms of Cr fed on the meat quality and the carcass measurements were minimal. Selenium-yeast combined with chromium-yeast has positive effect on performance and carcass composition of finishing lambs Animals and diets The experiment was carried out in an Experimental Centre near the Department of Animal Husbandry at the Slovak University of Agriculture in Nitra. In the study, 40 pigs of Large White breed were used. The genotype of all pigs on the marker RYR-1 (malignant hyperthermia syndrome) was analysed by the DNA test. All the experimental animals were detected as homozygous dominant (NN). The pigs were divided into a control group and an experimental group (each of 20 animals) with equal number of barrows (S 1 ) and gilts (S 2 ). Both, control (G 1 = Cont) and experimental group (G 2 = CrNic) of pigs were fed the same diet which consisted of three feed mixtures applied at the different growth phases, from 30 -45 kg OS-03, 45 -70 kg OS-04 and 70 -100 kg OS-05 (Table 1). The diet of experimental group was supplemented with 0.75 mg.kg -1 nicotinate (CrNic) in the form of chromium-inactivated yeast Saccharomyces cerevisiae fermented on the substrate which was from natural resources with a higher content of chromium during the whole fattening period. The pigs were housed in an environmentally controlled finishing barn with two pigs in each pen. They were allowed ad libitum access to feed and water. The fattening period in pigs lasted from 30 to 100 kg. The growth performance of pigs was controlled by weighing with an accuracy of 0.5 kg. The weighing was realised in two-week intervals (30 -90 kg) and one-week intervals (90 -100 kg). Slaughter and sample collections The slaughtering and the carcass dissection of pigs were carried out in the slaughterhouse of Experimental Livestock Centre near the Department of Animal Husbandry. The pigs were slaughtered at an average live weight of 102.5 kg and the dissection of carcasses was done according to standard practices STN 466164. Carcasses were chilled at 3 -4 °C overnight. The samples (100 g) for chemical analysis and determination of some meat quality traits were taken from LTon right half-carcass 24 hours post mortem. The place of sampling was above the last thoracic vertebra. After that, the samples were labelled and stored frozen at -19°C ±0.5 °C for 14 days until analysis. Chemical analysis The chemical composition of pork in LT was determined from samples of muscle homogenate (50 g) using the FT IR method (Nicolet 6700). The analysis of infrared spectra of muscle homogenate was done by the method of molecular spectroscopy. The principle of the method was the absorption of infrared radiation by the transition the sample, in which were changes in rotation vibrational energy states of molecules in response to changes in dipole moment of the molecule. The analytical output was an infrared spectrum which was a graphical display of functional dependence of the energy, usually expressed in percentage of the transmittance (T) or in units of absorbance (A) on the wavelength of the incident radiation. The transmittance (throughput) was defined as the ratio of the intensity of the radiation that passed through the sample (I) and the intensity of the radiation emitted by the source (Io). The absorbance was defined as the common logarithm of 1/T. The energy dependence on the wavelength was logarithmic; the wave number was used, which was defined as the reciprocal of the wavelength, and thus the energy dependence of the wave number will be a linear function. Individual groups of fatty acids (g.100g -1 FAME, Fatty Acid Methyl Ester) were determined from the muscle homogenate of LT in the Laboratory of gas chromatography at Faculty of Natural Sciences (Comenius University, Bratislava, Slovakia). Preparation of fatty acid methyl esters Small amount (4 -5 g) of muscle tissue was sampled and homogenized by grinding. From the obtained homogeneous mixture, 1 g sample was collected. After that, 4 mL of mixture for the extraction was used, chloroform: methanol (2:1) and the sample was shaken for 1h. After extraction, 2 mL of saline solution (0.9% NaCl) was added and shakedagain for 10 minutes. After few minutes, it was taken approximately 2 mL of lower layer which was subsequently centrifuged. From the adjusted sample, it was collected 1 mL for the transesterification. Discovery Ag-Ion SPE preseparation columns were developed for the separation of methyl esters according to the degree of saturation of fatty acids using a method of Kramer et al., (2008). Meat quality measurements The physical characteristics of meat quality were measured in the laboratory conditions of the Experimental Centre near the Department of Animal Husbandry, SUA in Nitra. The meat colour was determined on the cut of the LTabove the last thoracic vertebra 24 h post mortem using a spectrophotometer CM-2600d. Commission Internationale de IʼEclaire (CIE) L*, a*, and b* values were determined using the CIE Lab space with a D65 illuminate. For determination of drip loss, the methodology described by Honikel (1998) was used. In time 24 h to 48 h post mortem, a sample (approximately 50 g) was taken from the LT, placed in vacuum plastic bagsand hung in the refrigerator at 4 -6 °C. After 7 day-storage at temperature 4 ±1 °C, the Warner-Bratzler shear force was analysed. The samples were heated to temperature of 71 ±1 °C for 30 minutes and then cut for chips in 1x1 cm across fibers. Shear force was determined using the device Chatillon. TBA method Procedure for the sample preparation and determination of MDA was done according to the method of Marcinčák et al., (2009). A ground sample (1.5 g) was weighed in a 50 ml centrifuge tube and 1 mL EDTA (complex-forming agent) was added immediately. After gentle agitation, 5 mL 0.8% BHT was added, and the tube was gently shaken again. Just before homogenization, 8 mL 5% TCA was added to the tube and homogenization was carried out for 30 s at maximum speed. After homogenization, the sample stood for 10 minutes and then it was centrifuged for 5 min (3500 rpm, 4 °C). After centrifugation, the top hexane layer was discarded and the bottom layer was filtered through Whatman filter paper No. 4 into a 10 mL volumetric flask and diluted to volume with 5% TCA. After that, a 1 mL of TBA was added to the tube of 4 mL sample. The samples and MDA standards were incubated in a water bath for 90 min at 70 °C. After cooling in an ice bath, samples were incubated at room temperature for 30 min and extinction of samples was measured by UVspectrophotometer at a wavelength of 532 nm. Preparation of calibration curve From the stored MDA solution, 1 mL was pipetted to 25 mL volumetric flask and added 0.1 mol -1 HCl. The resulting MDA working solution with a concentration of 0.1748 g.mL -1 was used to preparation of the calibration curve. Statistical analysis The parameters of meat quality were statistically evaluated by statistical methods described by Grofík and Fľak (1990) and by statistical package Statistix, Version 8 and 9 (Anonymous, 2001). At first, the basic statistical characteristics, means (ӯ) and standard deviations (SD) of analysed traits were computed. The differences of analysed traits between studied groups (G i ), sex (S j ), their interactions (GS) and pens (P k ) were evaluated by twofactor analysis of variance (AOV) with repeated measurements/animals on pens factor. The colour of meat was evaluated by three-factor AOV, with these same factors and factor time (24 hours and 7 days). The linear regression method was used for describing the dependence of oxidative stability of LT muscle after Cr-supplementation on time of storage. Chemical parameters Supplementation of the diet with 0.75 mg.kg -1 chromium nicotinate resulted in a significantly higher content of chromium in the LT of experimental pigs than that of control pigs (0.199 vs. 0.153 mg) as shown in Table 2. The percentage of total water content in LT muscle was the same in both control pigs and pigs fed chromium. The percentage of total protein content was lower in the control group compared with the experimental, but the effect was According to study of Štefanka et al., (2013), addition of selenium with chromium nicotinate has reduced cholesterol content in the pork muscles. In our experiment, the differences in the fatty acids content in intramuscular fat of LT (g.100 g -1 FAME) are presented in Table 2. There are highly significant differences caused by sex in total water, protein and intramuscular fat content. The content of monounsaturated fatty acids was significantly lower in the control (51.63 g) compared with the experimental group (53.61 g). Also, the content of the essential fatty acids in the control group was significantly lower than that of experimental group (6.65 g vs. 7.80 g), see Tables 2 and 3. On the other hand, the chromium supplementation resulted in the significant increase (p ≤0.05) of omega 3-polyunsaturated fatty acids in experimental pigs compared with the control ones (0.47 vs. 0.42 g). Lien et al., (2001) suggest that the carcass of the pigs that received the chromium picolinate supplemented diet (400 μg/kg) contained less oleic acid (C18:1) and total unsaturated fatty acids (p ≤0.05). The total saturated fatty acid content in chromium fed group was higher than that in control. Physical and technological quality of pork The results for the physical and technological quality of pork are presented in Table 2 and Table 3 115 Note: ӯmean; SDstandard deviation; G 1control group; G 2experimental group; G 1 S 1 , G 1 S 2 , G 2 S 1 , G 2 S 2subgroups. y y y y In our study, an effect of chromium supplementation on some meat colour parameters 7 days post mortem was determined. The values of meat colour were not significantly different between experimental and control group. The differences between sexes in CIE b* in 24 hours and 7 days were significant. The means of meat colour CIE L*, a* and b* in LT 24 hours and 7 days by groups, sex and time are presented in Table 6. It was found out that the differences between analysed groups, sexes and time were not significant for CIE L*. The results showed that there was a highly significant difference between Time and interaction Group x Time in parameter CIE a*. Highly significant difference in CIE b* was caused by differences of Sex and Time. Also, there was a significant interaction in Group x Sex. Oxidative stability The effect of dietary chromium supplementation on the antioxidative stability of LT muscle is presented in Tables 2, 3 and 5. It was showed a highly significant difference between groups in 7 th day (0.161 mg.kg -1 in G 2 group vs. 0.314 mg.kg -1 in G 1 group). The means and standard deviations of the oxidative stability of LT for total observations in the 1 st , 3 rd , 5 th and 7 th day, for groups and sex and also for subgroups G i S j are presented in Table 5. The linear regression parameter estimates, the corresponded analyses of variance and significance of differences between elevations and slopes of oxidative stability of LT muscle are presented in Table 4. The dependence of oxidative stability on time of storage (x = t = 1, 3, 5, and 7 days) was highly significant. The deviations from linearity, i. e. nonlinearity (NonLin) were not significant. The dependence of oxidative stability had a pure linearity form. The comparison between groups and sexes showed significant or highly significant differences between elevations andslopes (Table 4). There was a similar situation by comparison of sexes (S j ) in analysed groups (G i ). Figure 1 and 2 illustrate the linear functions. The coefficients of determination R 2 were highly significant. The slope in the G i = Cont was two times higher thanslope in the G 2 = CrNic group (b 1 = 0.0478 vs. 0.0229 MDA). The reverse situation was observed by comparison of sexes. Recent research indicates that there are two mechanisms for chromium to affect the pork quality. It is an effect on carbohydrate metabolism or effect on stress. The investigation has shown that Cr-propionate and Cr-picolinate increases insulin sensitivity (Amoikon et al., 1995;Matthews et al., 2001). We can assume that Note: nnumber; ӯmean; SDstandard deviation; G 1control group, G 2experimental group; S 1 -barrows,S 2gilts; G 1 S 1 , G 1 S 2 , G 2 S 1 , G 2 S 2subgroups. Figure 2 Linear regressions of oxidative stability of longissimus thoracis muscle after Cr-supplementation for subgroups G i S j , (G 1 = Contr, G 2 = CrNic, S 1 = barrows, S 2 = gilts). These findings would be indicated that the Cr may affect glycolytic potential in muscles and subsequently impact the pork quality. The glycolytic potential of muscle tissue also plays an important role in the preslaughter stress. Some research has mentioned that Cr may partially mitigate the effect of short-term stress (National Research Council, 1997). CONCLUSION According to the results obtained in vivo experiment, it can be concluded that the supplementation of organic chromium as chromium nicotinate (0.75 mg.kg -1 ) in the pig diet resulted in a higher retention of chromium in LT. The dietary addition of organic chromium to growing and finishing diets for pigs caused a higher content of monounsaturated fatty acids and essential fatty acids in intramuscular fat of LT. However, the feeding with the supplementation increased the content of polyunsaturated and omega 3 fatty acids in the experimental group of pigs. On the other hand, the chromium nicotinate have no effect on the chemical composition of meat and meat quality traits, except with some colour parameters after 7-days storage. The Cr-addition had significantly positive impact on the oxidative stability of pork during its storage. It could be demonstrated that Cr consistently affects pork quality, so that may be beneficial from pork industry and consumer point of view. However, more research is needed to investigate the consistency in which the chromium nicotinate may improve the pork quality.

v3-fos

2016-05-12T22:15:10.714Z

2015-11-01T00:00:00.000Z

15084788

s2

Worse Comes to Worst: Bananas and Panama Disease—When Plant and Pathogen Clones Meet This article deals with: Bananas: their origin and global rollout; genetic diversity of Fusarium oxysporum f.sp. cubense, the causal agent of Panama Disease; Panama Disease: history repeats itself; tropical race 4, a single pathogen clone, threatens global banana production; strategies for sustainable Panama Disease management. Unfortunately, it is not well known which VCGs (the so-called Foc race 1 strains) caused the Panama disease epidemic in "Gros Michel" and, hence, their geographical dissemination is still unclear (I. Buddenhagen and M. Dita, personal communications). The current epidemic in Cavendish bananas, however, is caused by VCG01213 [5], colloquially called Tropical Race 4 (TR4). Panama Disease: History Repeats Itself Large railway projects in Central America in the late 1800s facilitated industrial banana production and trade [10], which was entirely based on "Gros Michel" bananas [8]. The vegetative compatibility groups (VCG) in F. oxysporum f. sp. cubense resulted in 12,978 DArTseq markers that divide Foc into two distinct clades-clade 1 and clade 2. VCG01216 is considered the same as VCG01213 [13]. The labels for race 1 isolates are based on personal communications with I. Buddenhagen and M. Dita. Although VCG01213 contains all TR4 isolates that cause the current Panama disease epidemic in Cavendish bananas, VCG0120-which has also been considered as race 4 [5]-and VCG0124 [36] have also been recovered from symptomatic Cavendish plants. doi:10.1371/journal.ppat.1005197.g001 unparalleled vulnerability of "Gros Michel" to race 1 strains drove aggressive land-claiming policies in order to continue banana production. However, this did not stop the epidemic as Panama disease was easily entering these new areas through infected planting material. Hence, by the 1960s, the epidemic reached a tipping point with the total collapse of "Gros Michel" [9]. Fortunately, there was a remedy: Cavendish bananas-maintained as interesting specimens in botanical gardens in the United Kingdom and in the United Fruit Company collection in Honduras-were identified as resistant substitutes for "Gros Michel." A new clone was "born" that, along with the new tissue culture techniques, helped save and globalize banana production [5,8,9]. However, in the late 1960s, Panama disease emerged in Cavendish bananas in Taiwan, but TR4 was only identified as its cause in 1994 [9,24,25]. Surprisingly, this initial outbreak did not awaken the banana industry and awareness levels remained low, despite the lack of any Cavendish replacement that met market demands and the susceptibility of many local banana cultivars to TR4 [5] (see also http://panamadisease.org/en/news/26). Thus, TR4 threatens not only the export trade but also regional food provision and local economies. Tropical Race 4, a Single Pathogen Clone, Threatens Global Banana Production Ever since TR4 destroyed the Cavendish-based banana industry in Taiwan, its trail in Southeast Asia seems unstoppable with incursions and expansions in the Chinese provinces of Guangdong, Fujian, Guangxi, and Yunnan as well as on the island of Hainan. Since the 1990s, TR4 has also wiped out Cavendish plantations in Indonesia and Malaysia; between 1997 and 1999, it significantly reduced the banana industry near Darwin in the Northern Territory of Australia. It was first observed in the early 2000s in a newly planted Cavendish banana farm in Davao (on island of Mindanao, Philippines), where it currently threatens the entire banana export trade [26]. Since 2013, incursions outside Southeast Asia were reported in Jordan [27], Pakistan, and Lebanon [28], informally announced in Mozambique and Oman, and just recently noted in the Tully region of Northern Queensland, Australia. By now, TR4 may have affected up to approximately 100,000 hectares, and it is likely that it will disseminate further-either through infected plant material, contaminated soil, tools, or footwear, or due to flooding and inappropriate sanitation measures [5,29]. Clearly, the current expansion of the Panama disease epidemic is particularly destructive due to the massive monoculture of susceptible Cavendish bananas. Foc is a haploid asexual pathogen [8] and is therefore expected to have a predominantly clonal population structure [13,14,[19][20][21][22]. Comparison of re-sequencing data of TR4 isolates from Jordan, Lebanon, Pakistan, and the Philippines-with the publicly available reference genome sequence of Foc TR4 strain II-5 (http://www.broadinstitute.org/)-indeed shows a very low level of single nucleotide polymorphisms (SNPs) (about 0.01%). This, together with a highly similar set of DArTseq markers, suggests that the temporal and spatial dispersal of TR4 is due to a single clone (Fig 2). This finding underscores the need for global awareness and quarantine campaigns in order to protect banana production from another pandemic that particularly affects vulnerable, small-holder farmers. Strategies for Sustainable Panama Disease Management Any disease management eventually fails in a highly susceptible monoculture. Managing Panama disease with its soil-borne nature, long latency period, and persistence once established is, therefore, impossible without drastic strategy changes. Evidently, exclusion is the primary measure to protect banana production, which requires accurate diagnosis based not only on visual inspection, as this overlooks important aspects of its genetic diversity and epidemiology. New molecular-based diagnostics rapidly detect TR4 in (pre)symptomatic plants [30], soil, and water and, hence, can be used for surveillance and containment, which are key to avoiding an encounter of TR4 with Cavendish monocultures. Additionally, a thorough understanding of Foc epidemiology and pathology is urgently required, as this facilitates developing effective methods to destroy infected plants and (biological) soil treatments, thus reducing the inoculum quantity. Furthermore, we showed that high-throughput genome analyses unveil Foc population diversity (Figs 1 and 2), rather than lengthy and cumbersome VCG analyses, which enables resistance deployment strategies. Finally, effective disease management cannot be achieved without adequate disease resistance levels. "Cavendish"-based somaclones [31] do not satisfy local or international industry demands (apart from the epidemiological risks), as this germplasm is, at most, only partially resistant to TR4 [32]. Instead, the substantial genetic diversity for TR4 resistance in (wild) banana germplasm, such as accessions of Musa acuminata ssp. malaccensis [4], can be exploited in breeding programs and/or along with various transformation techniques [33][34][35] to develop a new generation of banana cultivars in conformity with consumer preferences. Developing new banana cultivars, however, requires major investments in research and development and the recognition of the banana as a global staple and cash crop (rather than an orphan crop) that supports the livelihoods of millions of smallholder farmers. Until new, commercially viable, and resistant banana cultivars reach markets, any potential disease management option needs to be scrutinized, thereby lengthening the commercial lifespan of contemporary banana accessions. The current TR4 epidemic and inherent global attention should be the wake-up call for these much needed strategy changes. Supporting Information S1 Limited genetic diversity between multiple Foc TR4 isolates from distinct geographical locations revealed by hierarchical clustering, based on 4,298 DArTseq markers. Countries of origin for each of the TR4 isolates are indicated by different colors. (C) Phylogenetic analysis of selected Foc TR4 isolates (highlighted in bold in panel B) and related F. oxysporum species, based on whole-genome re-sequencing data. Phylogenetic tree analysis was performed using REALPHY [37], applying the PhyML algorithm for tree constructing (Foc II5 reference genome). The F. oxysporum f.sp. lycopersici and the F. oxysporum f. sp. cubense II5 genomes, as well as Foc race 4 and race 1 genomes, are publicly available at GenBank (http://www.ncbi.nlm.nih.gov/ genome/genomes/707). Robustness of the grouping was assessed by 500 bootstrap replicates, and thick branches indicate maximum support. doi:10.1371/journal.ppat.1005197.g002

v3-fos

2018-04-03T01:49:35.549Z

2015-02-04T00:00:00.000Z

9700358

s2

Analysis of the Thinopyrum elongatum Transcriptome under Water Deficit Stress The transcriptome of Thinopyrum elongatum under water deficit stress was analyzed using RNA-Seq technology. The results showed that genes involved in processes of amplification of stress signaling, reductions in oxidative damage, creation of protectants, and roots development were expressed differently, which played an important role in the response to water deficit. The Th. elongatum transcriptome research highlights the activation of a large set of water deficit-related genes in this species and provides a valuable resource for future functional analysis of candidate genes in the water deficit stress response. Introduction Water deficit is responsible for the greatest crop losses worldwide and is expected to worsen, heightening international interest in drought tolerance in crops [1]. Plant adaptation to water deficit is the result of many different physiological and molecular mechanisms that interact in a complex manner. Previous studies have shown that the plant response to water deficit stress involves numerous genes, which activate a series of physiological and biochemical processes to counteract the effects of the water-limited environment, including (1) the synthesis and accumulation of various osmoprotectants, (2) maintaining intracellular ion homeostasis via the expression of transporters, and (3) scavenging of reactive oxygen species (ROS) generated as a secondary effect of water deficit by detoxification enzymes [2,3]. In addition to these physiological and biochemical effects, regulatory systems that link the sensing and signaling of environmental stress in plants also play important roles in the response to water deficit [4,5]. The components that control and modulate stress-adaptive pathways mainly include transcription factors and protein kinases [2,6]. Many transcription factors belonging to different transcription factor families, such as bZIP, AP2/ERF, MYB, NAC, WRKY, and zinc finger, are important regulators of the plant response to abiotic stress, and their activity can improve stress tolerance in transgenic plants [2,3]. Moreover, these protein kinases, including calmodulin-dependent protein kinases (CDPKs), mitogenactivated protein kinases (MAPKs), receptor protein kinases (RPKs), and ribosomal protein kinases, participate in signal transduction processes in abiotic stress signaling and function as hubs in abiotic stress signaling. Thinopyrum elongatum (syn. Lophopyrum elongatum or Agropyron elongatum), a perennial species in the tribe Triticeae and the genus Elytrigia, shares an ancestor with common wheat [7][8][9]. This species is easily crossed with common wheat, which makes it a good source for genetic improvement of wheat. In the past several decades, a number of genes from Th. elongatum have been introduced into common wheat to improve yield and provide resistance to wheat streak mosaic virus, barley yellow dwarf virus [10], stripe rust [11,12], leaf rust [13], Fusarium head blight, and so on [14]. In addition to harboring pathogen resistance genes, Th. elongatum has also been used to improve tolerance to abiotic stresses, such as drought, waterlogging, and salinity, through the introduction of its chromosomes into wheat [7,[15][16][17]. However, the genome of Th. elongatum has not yet been published, which seriously limits the identification, characterization, and development of valuable genes in this species. In this study, we performed large-scale transcriptome sequencing of Th. elongatum under water deficit stress using ion torrent sequencing technology. We then compared the global expression profiles of Th. elongatum shoot and root tissues under control and water deficit stress conditions and identified a number of differentially expressed genes in response to water deficit stress. Gene annotation analysis of these genes provided novel insights into the response of Th. elongatum to water deficit stress, which should greatly facilitate wheat improvement in the future. Plant Material and Water Deficit Treatment. Thinopyrum elongatum (PI 531718, 2 = 14) seeds were kindly provided by GRIN (http://www.ars-grin.gov/), ARS, US Department of Agriculture. The seeds were pregerminated on wet filter paper in the dark at 25 ∘ C as described by Placido et al. [7]. When the coleoptiles were approximately 1 cm long, uniform seedlings were selected and transplanted to plastic pots filled with a mixture of surface soil collected from a field and washed sand. At 16 weeks after transplanting, each pot was supplied with 50 mL of water and 50 mL of half-strength Hoagland solution twice weekly (irrigated four times per week). Then, 16 weeks later, the plants were randomly divided into two groups, including the control group (watered normally as described above) and the water deficit group (supplied with only 50 mL of half-strength Hoagland solution twice weekly). After eight weeks of treatment, plant materials were collected from both the control and water deficit groups. All seedlings were grown in a greenhouse from March 2013 to September 2013 in Harbin, China. The greenhouse temperature was between 26 ∘ C and 30 ∘ C, the humidity ranged from 40% to 80%, and the light period was 06:00 to 18:00, as supplied by metal halide lamp 1 kW bulbs (Philips Lighting). The root and shoot tissue samples were separated, cleaned quickly, frozen in liquid nitrogen, and stored at −80 ∘ C for RNA isolation. 2.2. Total RNA Extraction, RNA-Seq Library Construction, and Sequencing. Frozen plant samples were ground in liquid nitrogen and total RNA was extracted using One Step RNA Reagent (Bio Basic Inc., Canada) as per the manufacturer's protocol and purified using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA). The integrity of the RNA was assessed by formaldehyde agarose gel electrophoresis. Total RNA was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and a Bioanalyzer 2100 (Agilent Technologies, CA). RNA integrity number (RIN) values were greater than 8.0 for all samples. Ribosomal RNA depletion was carried out using a RiboMinus RNA plant kit for RNA-Seq (Life Technologies, CA). The whole-transcriptome cDNA library was prepared using an Ion Total RNA-Seq kit v2 (Life Technologies Corporation, CA). Double-stranded cDNA was ligated to barcoded adapters and sequenced by BGI-Shenzhen Ltd. (Shenzhen, China) using an Ion PI Chip (ion torrent, Life Technologies, CA). Processing of raw data, removal of adapter sequences, base-calling, and quality value calculations were performed using Torrent Suite Software 4.0 (ion torrent, Life Technologies, CA). Quality reads were obtained by trimming the raw reads at a minimum PHRED score of = 20. RNA-Seq Data Processing, De Novo Assembly, and Annotation. RNA-Seq reads were first processed with FASTX toolkit to remove low-quality sequences with parameters "− 33-20-70". The resulting high-quality cleaned reads were assembled de novo into contigs using Trinity with the parameters "min kmer cov 2" [25]. To remove the redundancy of Trinity-generated contigs, the reads were further assembled de novo using iAssembler with minimum percent identity (− ) set to 97 [26]. Blast searches of the resulting unique transcripts were performed against combined databases harboring Arabidopsis, rice, maize, and Brachypodium distachyon protein sequences with a cutoff -value of 1 − 5 [27]. Gene ontology (GO) terms were assigned to the assembled transcripts based on the GO terms annotated to their corresponding homologs in the combined database, and the GO annotation results were explored using WEGO [28]. Annotations from MapMan were also retrieved based on homology search results [29]. Plant transcription factors (TF) and protein kinases were identified and classified into different families (or groups) using the iTAK pipeline (http:// bioinfo.bti.cornell.edu/tool/itak/) [30]. Gene Expression Quantification and Differential Expression Analysis. High-quality cleaned RNA-Seq reads were aligned to the assembled Th. elongatum transcripts using the Bowtie program, allowing one mismatch [31]. Following the alignments, raw counts for each transcript and in each sample were derived and normalized to reads per kilobase of exon model per million mapped reads (RPKM). Differentially expressed genes (fold changes ≥ 2 or fold changes ≤ 0.5 and adjusted value ≤ 0.001) between normal and water deficit stress conditions were identified with the edgeR package [32]. GO terms enriched in the set of differentially expressed genes affected by water deficit stress were identified using the topGO package [33]. Sequencing and De Novo Assembly of the Thinopyrum elongatum Transcriptome. To obtain a global view of water deficit stress-induced changes in Th. elongatum at the transcriptome level, we performed whole genome transcriptome sequencing of shoots and roots collected from control and water deficitstressed plants using the ion proton platform [34]. After removing low-quality, adaptor, and barcode sequences, a total of 39,273,796 reads were obtained. All raw and processed data were submitted to the NCBI database (Accession numbers: SRX729803 and SRX729805-07). De novo assembly of these high-quality cleaned reads generated 169,990 unique transcripts with an average length of 550.5 bp; the longest International Journal of Genomics transcript was 10,851 bp long. The length distribution of the assembled Th. elongatum unique transcripts is shown in Figure 1. Annotation of Thinopyrum elongatum Unique Transcript Sequences. The assembled Th. elongatum unique transcripts were annotated by Blast analysis against the combined databases, including Arabidopsis, rice, maize, and Brachypodium distachyon protein sequences, revealing a total of 81,061 (47.9%) unique transcripts with significant hits. Consistent with previous reports [6], the results show that the percentage of genes that could be annotated was positively correlated with the length of the genes, as shown in Figure 1. The short transcripts were annotated to fewer targets, and the longer transcripts generated more hits. Among these unique transcripts, 59,704 (35.1%) were assigned to at least one GO term in three main categories, that is, biological process, molecular function, and cellular component. We further classified these unique transcripts into different functional categories, as shown in Figure 2. The result shows that metabolic process (GO:0008152) was the most abundant group in the biological process category, followed by biological regulation (GO:0065007), while response to stimulus (GO:0050896) and response to stress (GO:0006950) were also common, which is consistent with the transcriptome data collected from Th. elongatum plants under water deficit stress. In the molecular function category, the most abundant groups included binding (GO:0005488), catalytic activity (GO:0003824), oxidoreductase activity (GO:0016491), transferase activity (GO:0016740), transporter activity (GO:0005215), and transcription regulator activity (GO:0030528). There were also transcripts classified into specific groups, such as antioxidant activity (GO:0016209), indicating that antioxidants play an important role in trapping free radicals to protect Th. elongatum from water deficit damage. To investigate the transcriptional regulation process in detail, we used the iTAK pipeline to mine for transcription factors and protein kinases. In total, we identified 2,988 transcription factors classified into 77 different families and 3,154 protein kinases classified into 85 different families from among the Th. elongatum transcripts, shown as Figure 3. These TFs belong to many families that play important roles in the plant response to abiotic stress, such as C2H2, C3H, WRKY, MYB, SNF2, bZIP, bHLH, NAC, AUX/IAA, AP2-EREBP, CCAAT, and MADS. The protein kinases were classified into legume lectin domain kinase, leucine rich repeat kinase, DUF26 kinase, S domain kinase, GmPK6/AtMRK1 family, CDPK, SnRK, MAPK family, and so on. These protein kinases broadly participate in the regulation of gene expression, while protein kinases involved in the plant response to abiotic stress, especially the CDPK and MAPK families [35,36], were also highly abundant in our transcriptome dataset. To estimate possible differences in transcript sequences between Th. elongatum and wheat, a BLASTN search was performed against the wheat transcript sequences from IWGSC. The results show that 46.63% (79,265/169,990) of unique transcripts from Th. elongatum had significant matches with wheat transcripts, most with high identity percentages, as shown in Figure 4. The remaining transcripts from Th. elongatum (53.37%) without significant matches in wheat represent Th. elongatum-specific genes, which could be beneficial for wheat improvement. Differentially Expressed Genes under Water Deficit Stress. Using the edgeR Bioconductor package, we identified 1,300 and 3,604 differentially expressed transcripts from Th. elongatum shoot and root tissue, respectively, while 122 transcripts were differentially expressed in both tissues, shown as Figure 5. Among these transcripts, 2,690 were induced by water deficit stress in root tissue, while 914 were repressed in roots. There were almost three times as many upregulated transcripts as downregulated transcripts in roots. However, in shoots, 700 transcripts were induced while 600 were repressed. GO terms were assigned to all 4,782 differentially expressed transcripts, and enrichment analysis for GO annotation was performed using the topGO package; the results are shown in Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/265791. As expected, GO terms in the biological process category were highly enriched, including GO:0050896 (response to stimulus), GO:0009628 (response to abiotic stimulus), GO:0006950 (response to stress), GO:0009651 (response to salt stress), and GO:0006970 (response to osmotic stress), which is consistent with previous reports in other plants. Meanwhile, GO terms in the molecular function category, such as GO:0005507 (copper ion binding), GO:0016491 (oxidoreductase), GO:0016209 (antioxidant), GO:0004784 (superoxide dismutase), GO:0022857 (transmembrane transporter), and GO:0005215 (transporter), were also highly enriched under water deficit stress in Th. elongatum, indicating that the antioxidant and transport systems play important role in protecting plants from damage due to environmental stress. We further annotated the functions of differentially expressed transcripts using MapMan. The results show that shoots and roots have different ways of responding to water deficit stress, as shown in Figure 6. In shoots, differentially expressed transcripts were more enriched in the category photorespiration. However, compared to shoot tissue, some transcripts were more abundant in roots, including those in the categories across cell wall, lipids, antioxidant (including ascorbate, glutathione, and OPP), and sucrose metabolism. Meanwhile, transcripts in the categories abiotic stress (20.2) and ascorbate and glutathione (21.2) were highly enriched, which is consistent with the GO annotation results. We analyzed the expression of transcripts in these two categories based on transcriptome data, revealing that most of these transcripts were induced under water deficit stress, as shown in Figure 7. Discussion In plants, roots are often able to continue growing under water deficit stress in order to seek deeper water resources, while shoot elongation is completely inhibited due to a decline in photosynthesis. In this study, RNA-Seq technology was utilized to compare the shoot and root transcriptomes of Th. elongatum under water deficit stress to those grown under control conditions. We identified a total of 4,782 differentially expressed transcripts. Among these water deficit-responsive genes, 3,604 were detected in roots, while 1,300 were detected in shoots and only a few (122) were expressed in both roots International Journal of Genomics 5 100 99 98 97 96 95 94 93 92 91 90 88 89 86 85 87 84 83 82 81 80 78 77 79 76 75 74 73 72 71 2000 4000 6000 8000 10000 12000 Identities (%) Number of transcripts and shoots, which is consistent with previous reports in maize and cotton [37,38]. Placido et al. [7] examined the role of the brassinosteroid gene regulatory network in root development, finding that the drought tolerance of common wheat was improved through introgressing an alien chromosome segment from Th. elongatum. In the current study, we identified three differentially expressed transcripts involved in brassinosteroid metabolism and the brassinosteroidmediated signaling pathway in Th. elongatum (ThUN007188, ThUN021506, and ThUN006380), which is consistent with the transcripts detected in wheat by Placido et al. [7]. We also found that GO categories GO:0044036 (cell wall macromolecule metabolic process, nine of 50 differentially expressed transcripts) and GO:0048364 (root development, six of 45 differentially expressed transcripts) were enriched in root tissues, which is consistent with the fact that brassinosteroids promote cell wall loosening, root elongation, and root development to mitigate the effect of water deficit stress on plant growth. Our transcriptome data suggest that TFs play important roles in the response of Th. elongatum to water deficit stress, since we identified 47 differentially expressed TF genes. In plants, the bHLH TF directly regulates the GA and JA signaling pathways to induce the initiation of trichomes [39], which act as barriers to protect plants from water loss. Indeed, in this study, we identified 12 differentially expressed bHLH TF genes, including 10 expressed in shoots and three expressed in roots (with one expressed in both tissues). Moreover, nine transcripts involved in JA and GA metabolism were also differentially expressed in shoots compared to two in roots. These results suggest that the modulation of the GA and JA signaling pathways by bHLH in shoots helps protect Th. elongatum from water deficit stress. In addition to bHLH, other TFs reported to be involved in plant responses to abiotic stress, such as AP2/ERBP (9) [40,41], MYB (8) [42][43][44] (6), and WRKY [45] (6), were also differentially expressed under water deficit stress, confirming that they play crucial roles in regulating transcription processes under water deficit stress. ROS are produced in plant tissues due to the partial reduction of oxygen, for example, in the photosynthetic and respiratory electron chains, and their levels increase dramatically under environmental stress [46]. However, as ROS can cause cellular damage, they are scavenged (and generated) by oxidoreductases. Therefore, the homeostasis of ROS is determined by interactions between the ROS-producing and ROS-scavenging pathways in plants. ROS play important roles as signaling molecules that modulate many pathways [47], for example, MAPK cascades [48], and influence the activity of TFs under water deficit stress. Based on the transcriptome data, we identified 28 differentially expressed transcripts that are related to ROS metabolism, most of which were induced by water deficit stress, as shown in Figure 7. These transcripts were mainly classified into two highly represented GO categories, "oxidoreductase activity" and "antioxidant activity, " which were identified by topGO analysis. These genes encode enzymes including oxidoreductase, ascorbate, tocopherol, and glutathione, all of which scavenge harmful ROS to protect Th. elongatum from cellular damage [20,49]. However, the detailed mechanisms of ROS metabolism are unknown. Additional studies are needed to fully elucidate the complex interactions of ROS metabolism under water deficit stress. Conclusions In the present study, thousands of transcripts were identified from Th. elongatum shoots and roots that were differentially expressed in response to water deficit stress, with three times as many transcripts in roots as in shoots. These transcripts are mainly involved in the response to stress, signal transduction, transcriptional regulation, ROS metabolism, and so on, especially the regulation of root development under water deficit. All of these genes help Th. elongatum adapt to water stress, and their introduction into cultivated wheat may improve the water deficit tolerance of this crop in the future.

v3-fos

2016-05-04T20:20:58.661Z

2015-03-19T00:00:00.000Z

19012418

s2

Automated integrative high-throughput phenotyping of plant shoots: a case study of the cold-tolerance of pea (Pisum sativum L.) Background Recently emerging approaches to high-throughput plant phenotyping have discovered their importance as tools in unravelling the complex questions of plant growth, development and response to the environment, both in basic and applied science. High-throughput methods have been also used to study plant responses to various types of biotic and abiotic stresses (drought, heat, salinity, nutrient-starving, UV light) but only rarely to cold tolerance. Results We present here an experimental procedure of integrative high-throughput in-house phenotyping of plant shoots employing automated simultaneous analyses of shoot biomass and photosystem II efficiency to study the cold tolerance of pea (Pisum sativum L.). For this purpose, we developed new software for automatic RGB image analysis, evaluated various parameters of chlorophyll fluorescence obtained from kinetic chlorophyll fluorescence imaging, and performed an experiment in which the growth and photosynthetic activity of two different pea cultivars were followed during cold acclimation. The data obtained from the automated RGB imaging were validated through correlation of pixel based shoot area with measurement of the shoot fresh weight. Further, data obtained from automated chlorophyll fluorescence imaging analysis were compared with chlorophyll fluorescence parameters measured by a non-imaging chlorophyll fluorometer. In both cases, high correlation was obtained, confirming the reliability of the procedure described. Conclusions This study of the response of two pea cultivars to cold stress confirmed that our procedure may have important application, not only for selection of cold-sensitive/tolerant varieties of pea, but also for studies of plant cold-response strategies in general. The approach, provides a very broad tool for the morphological and physiological selection of parameters which correspond to shoot growth and the efficiency of photosystem II, and is thus applicable in studies of various plant species and crops. Introduction In plants, acclimation to cold, causes reduced growth, increase in antioxidant content, reduced water content, and changes in gene regulation, hormone balance, membrane composition, osmotic regulation, and photosynthetic function [1]. The adaptability and productivity of legumes (chickpea, faba bean, lentil, and pea) are limited by abiotic stresses in general [2], and their high sensitivity to chilling and freezing temperatures is well described [3]. Since cold tolerance is an important agronomical problem in Central and Northern Europe and geographically similar regions, we aimed to develop a routine measuring procedure for automated integrative highthroughput screening for selection of potentially cold tolerant cultivars. Pea (Pisum sativum L.) was chosen as a model crop because its tolerance to cold stress is one of the limiting factors in autumn sowings which allows for the enhanced productivity of pea plants. Overwintering plants have developed adaptive responses to seasonal weather changes. For example, overwintering evergreens have developed so-called sustained non-photochemical quenching (reviewed, e.g., by Verhoeven [4]) as a protection mechanism against absorbed light which is in excess with respect to the capacity of the carbon photosynthetic reactions and which is decreased during winter. These plants sense the upcoming cold period through the perception of environmental impulses, mainly temperature and day length. However, the sustained non-photochemical quenching does not work in modern pea cultivars. For this reason, we chose two modern cultivars and investigated their reaction to cold stress. We employed digital RGB imaging to study shoot growth, and chlorophyll (Chl) fluorescence imaging (CFIM) to analyze various parameters of plant photosystem II (PSII) efficiency. The cultivars used in this study were morphologically similar which facilitated the validation of sensitivity and resolution of our visible imaging analysis. There is a paucity of information on the acclimation of pea plants to cold. An extensive study was published by Markarian et al. [5]. These authors evaluated 26 pea lines based on their winter survival. Further physiological parameters (total dry matter and photosynthetic area) of autumnand spring-sown pea plants were evaluated by Silim et al. [6]. Autumn-sown plants produced similar seed yields to spring sowings when the winter survival was adequate, and autumn sowings matured 2-4 weeks before the springsown crops, depending on the variety and season [6]. The effects of short term acclimation (four days) of pea plants to cold temperatures (5°C) were explored by Yordanov et al. [7] who measured the rate of oxygen production and CO 2 assimilation, and Chl fluorescence parameters in order to evaluate photochemical activity and functional heterogeneity of PSII. They found that cold-acclimated plants showed higher photosynthetic rates and better Chl fluorescence parameters than non-acclimated plants [7]. The effects of short term cold acclimation (three days, 4°C) and subsequent recovery (2 days) of standard pea plants were studied by Chl fluorescence measurements in more detail by Georgieva and Lichtenthaler [8]. The Chl fluorescence parameters reflecting photosynthetic function decreased during cold acclimation but were reversible in the subsequent recovery [8]. A similar study was later carried out with three different pea cultivars by Georgieva and Lichtenthaler [9]. These studies revealed the importance of two potential traits that could be used to distinguish between pea cultivars with different cold-sensitivity: rate of shoot growth and values of Chl fluorescence parameters. Both traits can now be studied by non-invasive high-throughput platforms to provide integrative insight into plant physiology during cold acclimation. The spatio-temporal changes in shoot biomass or leaf area can be assessed using automated RGB imaging and image-analysis software, as has been shown for many species such as cereals, tomatoes, soybean and beans [10][11][12][13]. The Chl fluorescence parameters are routinely analyzed by nonimaging fluorometres (NICF) or the imaging system (CFIM). For physiological studies, kinetic types of CFIM that allow computation of various Chl fluorescence parameters on the whole leaf or shoot are the most valuable. However, the kinetic type CFIM has not been commonly integrated into high-throughput systems [14] and in recent reports only systems measuring a single Chl fluorescence level have been employed [11,15]. The intensity of Chl fluorescence depends on the amount of chlorophylls; thus, a single Chl fluorescence level can be used, e.g., to distinguish between non-stressed and senescent leaves (when amount of Chls is decreased) at late stages of stress. However, this does not provide any information about earlier processes in PSII that are not necessarily linked to later senescence events. In this report, we describe a procedure employing an automated integrative high-throughput platform suitable for studies of the physiological basis of cold-stress adaptation and selection of pea cultivars with cold sensitivity/ tolerance. The platform measures shoot area and Chl fluorescence to provide a complex analysis of plants during cold-acclimation. For this purpose, we developed new software for automatic RGB image analysis and we evaluated various parameters of Chl fluorescence obtained from CFIM. The data from the automated phenotyping platform were validated through estimation of shoot biomass by manual weighing of the shoots and by measurement of Chl fluorescence by a NICF hand operated fluorometer. Despite the complexity of pea shoots, very good correlation between pixel based shoot area and fresh biomass were obtained. Similarly, the Chl fluorescence parameters measured by NICF fully confirmed the reliability of the automated CFIM analysis. Visible imaging used for shoot growth To compare the influence of cold acclimation on biomass production, two putative cold-resistant cultivars of pea Terno and Enduro were selected (labeled as TER and END, respectively). After germination, the seedlings were grown in a growth chamber at 22/20°C (see Materials and methods) and after the development of the first true leaf, the cold stress conditions were established. The seedlings continued growing in 5°C for 21 days and were screened twice per week in the automated platform. The green area of each individual seedling was extracted from particular projections ( Figure 1) and combined to account for the overall shoot biomass. As shown in Figure 2, the total green area of the plants was calculated at 7 time-points. The cultivar TER showed a significantly higher (for p values see Table 1) increase in the total green area compared to the cultivar END ( Figure 3A). Because the green area of the cultivars was different at the beginning of the experiment, the normalized green area (NGA) was calculated, where the green area on the n th (5, 8, … 21) day of measurement was divided by the green area obtained on the 1st measuring day. The TER cultivar showed higher shoot growth which on the 21st day was almost a 3.5 fold increase in the green area, whereas END multiplied its projected area by only about 2.5-times ( Figure 3B). To analyze how the cultivars differed in their growth rates, the relative growth rate (RGR) was used according to Hoffmann and Poorter [16]. We used the following formula: where À lnW 1 and À lnW 2 are the means of the natural logarithms of the plant's green areas and t 1 and t 2 are the times at which the green areas were measured. The TER cultivar relative growth rate was significantly higher (for p values see Table 1) during the whole period of cold acclimation. Moreover, at the beginning of the cold stress, the TER cultivar tended to speed-up its growth, then reached a steady state and finally decreased its RGR by the end of the experiment. The second cultivar, END, was very stable, slightly decreasing its growth rate during the experiment ( Figure 3C). To examine the statistical significance of the differences between obtained TER and END growthrelated parameters, the non-parametric Mann-Whitney U test was performed for each measuring day. The p values obtained for each measuring day are shown in Table 1. It has been reported that cold-treatment affects total shoot biomass production and growth-rate in springsown and overwintering pea cultivars [6,17]. Besides shoot growth cold-treatment affects also growth of the root as showed in work by Bourion et al. [17]. However, the effect on the root is less severe compared to the above ground parts of the plants [17]. Due to this fact and due to the technical set up of our automated platform in this study we focused only on the analyses of cold-treatment effects on shoot growth. We describe here the development of the measuring setup for automated screening of pea cultivars with different coldsensitivity through analysis of the shoot growth by RGB imaging followed by precise image-analysis. A similar approach has been shown for different species and different types of stresses. Considering crop species alone, most of the protocols for automated phenotyping using RGB imaging were designed for cereals, most often to Figure 1 The example images of three optical projections of single END seedling used for calculation of total green area on 8th day of cold acclimation. The green area that was digitally extracted from the images is marked by white border line. screen for drought, or salt tolerant plants [10,15,[18][19][20][21][22][23]. Surprisingly, use of such a method has not been presented so far for any crops studied for cold-acclimation. Although there was no presumed effect of cold-treatment on the reliability of RGB imaging, the complicated morphology of field pea cultivars could potentially affect the accuracy of the automated measurements. For this reason, we tested our method of the green area (or projected area) estimation from automated RGB imaging by its comparison with a method of manual weighing of the shoots. The shoots of both cultivars were harvested on the last measuring day and FW of individual plant shoots was measured. Subsequently, correlations between the green area and FW were calculated using the non-parametric Spearman correlation coefficient. A similar approach has been reported recently by Hairmansis et al. [15] for rice. These authors found a correlation of projected area and FW ranging from 0.96 to 0.97. A more sophisticated calculation was developed by Golzarian et al. [22] who used estimated shoot area as a function of plant area and plant age. This method was applied by Pereyra-Irujo et al. [12] in experiments with soybean, providing a correlation of 0.97 in dry mass. Shoots of cereals and soybean have relatively low spatial-complexity. In contrast, shoots of field pea cultivars TER and END are formed mainly by stem and tiny tendrils (Figures 1, 2) requiring very precise identification by image-analysis software. Despite the challenging pea shoot morphology, Spearman correlation coefficients of 0.91 and 0.96 for TER and END cultivars, respectively, were found in our analysis (p < 0.05; Figure 4). This is fully comparable with the phenotyping protocols designed for other crop species and provides an efficient and reliable tool for the evaluation of pea growth. Chlorophyll fluorescence imaging used for determination of photosynthetic function Further variables used for phenotyping of the two pea cultivars were those obtained from measurements of Chl fluorescence induction (CFIN), which reflects photosynthetic function, mainly of PSII. Based on our knowledge of the parameters that can be determined from CFIN (reviewed in Lazár [24]), we selected the following parameters: i) the maximal quantum yield of PSII photochemistry for a dark- and F V are the minimal, maximal, and variable fluorescence levels, respectively, for a dark-adapted state; ii) the actual quantum yield of PSII photochemistry for a light-adapted state, , where F M ' and F (t) are the maximal and actual (at time t; usually in the steady state) fluorescence levels for a light-adapted state; iii) the quantum yield of constitutive non-light induced (basal or dark) dissipation processes consisting of Chl fluorescence emission and heat dissipation, Φ f, D = F (t)/F M ; and iv) the quantum yield of regulatory light-induced heat dissipation, is the coefficient of photochemical quenching which estimates a fraction of the so-called open PSII reaction centers; and that Φ PSII (= (F M ' -F 0 ')/F M ') is the maximal quantum yield of the PSII photochemistry for a light-adapted state. The F 0 ' in the last two equations is the minimal fluorescence level for a lightadapted state which was estimated from: [24]). The changes in these Chl fluorescence parameters measured during acclimation of TER and END cultivars to 5°C for 21 days are shown in Figure 5. Φ Po is affected very little by the cold acclimation of TER but there is a continual decrease in Φ Po of END ( Figure 5A). Φ P initially decreases more in TER than in END but after 6 days it maintains its value in TER but continues to decrease in END ( Figure 5B). The continual decrease in Φ P in END is mostly caused by a continual decrease in Φ PSII ; q P slightly increasing in the last two measurements in END ( Figure 5B). On the other hand, the initial decrease in Φ P in TER is mostly caused by decrease in Figure 3 Analyses of the growth progress of shoots of TER (red boxesfull line) and END (blue boxesdashed line) pea cultivars. The values derived from the green area on n th days (1, 5, 8,…, 21) are presented as medians (black bars) and quartiles (boxes). For better readability, the boxes are shifted in x-axes to not to overlap, but still represent the values measured on the same days. A) A total green area. B) A normalized green area. C) A relative growth rate. The error bars show minimal and maximal values. q p but the almost unchanged value of Φ P in TER after 6 days is caused by the counter action of q P , which increases, and of Φ PSII , which decreases ( Figure 5B). Therefore, it can be concluded that photosynthesis of the two pea cultivars uses different strategies for cold acclimation. Whereas in END, the number of open reaction centers as well as their maximal photosynthetic quantum yield in light generally decrease with prolonged cold acclimation, in TER, a decrease of the maximal quantum yield of PSII photochemistry in light (Φ PSII ) is compensated by an increase of number of the open PSII reaction centers (q P ) ( Figure 5B). Furthermore, END shows an increased quantum yield of constitutive nonlight induced dissipation processes (Φ f, D ) at the end of the cold acclimation compared to TER ( Figure 5C), whereas the rise of the quantum yield of regulatory light-induced heat dissipation (Φ NPQ ) during the acclimation is faster in TER than in END ( Figure 5D). It is interesting to note that cold-induced changes of the Chl fluorescence parameters for given cultivar and differences (or about the same values) of the parameters between the cultivars ( Figure 5) are not accompanied by expected changes and differences of green areas and growth rates ( Figure 3). Even when the photosynthetic function was decreased by cold treatment (decrease of the Φ Po , Φ P , q P , and Φ PSII parameters; Figure 5A and 5B), the total and normalized green area of both cultivars was still increased ( Figure 3A and 3B). It might show that the grow rate changed (for TER; Figure 3C) or decreased (for END; Figure 3C) with increasing duration of the cold treatment, however, these changes were not statistically significant (data not shown). The uncorrelated behavior of photosynthetic and growth parameters reflects different temperature dependences of photosynthesis and processes hidden behind the plant growth. While photosynthetic function was decreased by treatment of the cultivars at 5°C, probably much lower temperatures would be needed to stop plant growth. Therefore, FCIM data and RGB imaging data carry different and complementary information about acclimation of plants to lower temperatures. To take advantage of the high-throughput capacity of our phenotyping platform, we used a relatively short protocol to measure CFIN. This set up, however, did not allow for determination of photoinactivated centers which might be formed during a joint action of light and cold [25][26][27][28]. Depending on the theory used, the formation of the photoinactivated PSII centers can influence all quantum yields of the light-adapted state (for a review see [24]) used in this work. Therefore, in the next study we aim to modify the CFIN measuring protocol in order to determine the quantum yield of photoinactivated PSII centers as well. Furthermore, we tested the reliability and accuracy of the Chl fluorescence parameters measured by the automated CFIM in a high-throughput set up by comparing the selected parameter (Φ Po ) with the same parameter measured by a hand-operated non-imaging Chl fluorometer. For this purpose the overall Chl fluorescence images were separated into images of the second and third leaves and their Φ Po were evaluated. On the other hand, Φ Po was evaluated from the fast Chl fluorescence rise as measured by the nonimaging Chl fluorometer with a different set of leaves (see Materials and methods). The results of these comparisons are presented in Figure 6A for the second leaves and in Figure 6B for the third leaves, respectively. A representative image of the spatial distribution of Chl fluorescence is presented in Figure 6C. Not surprisingly, the data show that there is no statistically significant difference (at p < 0.05) between Φ Po measured for given leaves by the two different approaches. Moreover, Figure 6C documents another advantage of using the CFIM in automated highthroughput platforms. Although the software is primarily adjusted to calculate the mean value of fluorescence from the total surface of every plant, if needed, the CFIN images can be later separated for subsequent calculation of the Chl fluorescence parameters taken from the individual selected areas which represent individual plant parts ( Figure 6C). To the best of our knowledge, only one study was published reporting on use of CFIM integration into a highthroughput phenotyping platform to analyze cold-or chilling-stress. Using an automated phenotyping platform Jansen et al. [14] evaluated only the F V /F M parameter (Φ Po ) for two different Arabidopsis plants (wild-type and a mutant), and wild-type tobacco plants. Φ Po decreased in the wild-type tobacco plants during the cold treatment, and the same decreasing trends were found with Arabidopsis plants, however, the differences between the wild-type and a mutant were not convincing. Using a CFIM system, Lootens et al. and Devacht et al. [25,29] studied the effect of different cold temperatures on industrial chicory plants. In agreement with our results, the authors found again only a small decrease of Φ Po after 10-day incubation at 4°C and the values of the Φ P and Φ PSII parameters caused by the incubation were similar to those obtained in our study. Mishra et al. [30,31] used CFIM to study the effect of a two-week incubation at 4°C on nine Arabidopsis thaliana accessions differing in cold tolerance. In addition to evaluation of standard Chl fluorescence parameters, like Φ Po , Φ P , and q P , the authors also showed that combinatorial imaging of Chl fluorescence transients combined with classifier and feature selection methods could discriminate between detached leaves from cold sensitive and cold tolerant accessions. Plant material Two morphologically similar field pea (P. sativum subsp. sativum var. sativum) cultivars Terno (TER) and Enduro Figure 5 Changes of CFIN parameters of TER (full symbols) and END (open symbols) pea cultivars measured during the 21 days of cold acclimation. Changes in A) the maximal quantum yield of PSII photochemistry for a dark-adapted state (Φ Po ); B) the maximal and the actual quantum yield of photosystem II photochemistry for a light-adapted state (Φ PSII , Φ P respectively), the coefficient of photochemical quenching (q P ); C) the quantum yield of constitutive non-light induced dissipation processes (Φ f, D ); D) the quantum yield of regulatory light-induced heat dissipation (Φ NPQ ); are shown. The values represent medians from 15 measurements. The error bars represent quartiles. The medians of all the TER and END parameters at the end of measurements were statistically significant (p value < 0.05), except of q p and Φ NPQ . (END) were used in the experiment. TER is pea cultivar, used for spring sowing term with a certain capacity to cold-acclimation, whereas END is a cold-tolerant overwintering cultivar. The END cultivar was obtained from the Selgen a.s. company (Prague, Czech Republic). The TER cultivar was taken from the Czech collection of pea genetic resources kept in Agritec Ltd., Šumperk, Czech Republic. The collection is run according to the general rules of the National Programme for Plant Genetic Resources of the Czech Republic and the passport data are available on http://genbank.vurv.cz/genetic/resources/. Cultivation conditions and experimental setup The TER and END pea cultivars were sown into standardized pots (65 x 65 x 95 mm, Plant-It-Rite, Australia) filled with 100 g of soil (Substrate 2, Klasmann-Deilmann GmbH, Germany) and watered to full water capacity. The seeds were germinated in mini-greenhouses (50 x 32 x 6 cm with clear plastic lid) in a growth chamber with white LED lighting (150 μmol photons of PAR m -2 s -1 ). The conditions were set-up to simulate a long day (16 h day, 8 h night) with temperatures of 22°C during the light period and 20°C in the night. The relative humidity was set to 60%. After the development of the first true leaves, the temperature was decreased to 5°C for the entire experiment, the other parameters remained unchanged. The plants were regularly watered with the same amount of water. Fifteen seedlings from each cultivar were used for the automated phenotyping, and another fifteen plants were used for control measurements of maximal quantum yield of PSII photochemistry through the use of a handoperated non-imaging Chl fluorometer. For measurements in PlantScreen TM phenotyping platform (Photon Systems Instruments, Brno, Czech Republic), the pots with the seedlings were placed in standardized trays; two pots per tray and automatically loaded and measured by the platform. The movement of the trays was performed by a roboticdriven conveyor belt that routinely transferred experimental plants between the growing and measuring areas according to a user-defined protocol. A single measuring round of 8 trays consisted of 20 minutes of dark-adaptation, followed by the measurement of Chl fluorescence and digital RGB imaging from three optical projections. Approximately 16 plants per hour were analyzed, due to the length of the measuring round that is dependent on the length of the dark adaptation and CFIM measurement. In the case of RGB imaging the platform throughput increases to about 60 experimental trays (120 plants) per hour. The data from Chl fluorescence and RGB imaging were stored in a database server, and analyzed either by the software provided by the manufacturer or by the software developed by the authors of this study as described below. RGB software image analysis The plants were automatically loaded into the measuring cabinets of the PlantScreen TM platform where the three RGB imagesthe top, front, and side views - (Figure 1) of each experimental tray containing two plants were taken. To assess the total green area, the green mask of the individual plants has to be found in the image. To this end, we used a combination of automatic thresholding procedures and automatic edge detection techniques. First, the image was converted from the RGB colour space into the HSV colour space. It is much easier to find the green mask in the H channel of the HSV colour space because the S and V channels only contain information on the saturation and brightness of the colour but not the hue itself. The region in the three dimensional RGB space which defines the 'plant green' colour may have a rather complicated shape, however, it is reduced to a line-segment in the one-dimensional H space as the S and V coordinates can be ignored. For thresholding in the H channel, several standard automatic algorithms can be used, e.g., the most popular Otsu method [32] that calculates the optimum threshold separating the foreground and background pixels so that their combined intra-class variance is minimal. In our case, we used an even simpler techniqueforeground (i.e., the plant) was predefined as a particular line segment in the H channel. This was possible due to the standardized image acquisition setting. The thresholding step usually provides very good discrimination between the plant and its background and no further processing is necessary. However, the pea plants possess very thin offshoots (only one or two pixels thick) that may be difficult to find by thresholding alone. If the thresholding routine makes a single-pixel mistake, which often happens due to noise in the image, the entire offshoot is lost, which is undesirable. We solved this problem by exploiting the Canny automatic edge detection algorithm which tracks the contours of the plant image [33]. The thin offshoots were tracked particularly well because the edge detection algorithm focused on such thin structures. The results of the thresholding step were then combined with the edge detection step and the final green mask of the object was found. Finally, a couple of post processing steps were performed (e.g. median filtering and image opening and/or closing) to enhance the quality of the mask. It only took several seconds on a standard PC to find the green mask of a single pea plant. The mask provided information about the projection of the plant surface area onto the three image planes. The projections can be expressed in square millimeters because the RGB camera had been calibrated beforehand. The calibration proceeded as follows. Two bars covered by millimeter paper were placed in the pots instead of the pea plants. The bars were approximately the same height as the plants. Three images (top, front, side) of the two bars were acquired with the same camera setting used for the entire experiment. These images served as the standard for converting the leaf area from pixels to square millimeters. The total green area of the plant is then estimated as A = √(A x 2 + A y 2 + A z 2 ), where A x , A y , and A z are the respective projections onto the three image planes. This procedure is naturally not precise but it gives an estimate which is in good correlation (Figure 4) with the fresh biomass of the above ground plant parts. CFIM and non-imaging Chl fluorescence measurements A standard protocol was used for the measurement of Chl fluorescence quenching using the CFIM part of the Plant-Screen TM platform. The plants underwent 20 -40 minutes of dark adaptation before CFIM measurements. During all signal recordings, short (33.3 μs) red (650 nm) "measuring" flashes were applied and a Chl fluorescence signal was detected a few microseconds before the measuring flash and during the flash, and then the two signals were subtracted. This is a pulse amplitude modulation (PAM) type of measurement. To measure the minimal fluorescence for a darkadapted state, F 0 , only the measuring flashes were applied for an initial 5 seconds. Then, a saturation pulse of 800 ms duration (white light, intensity of 1000 μmol photons of PAR m -2 s -1 ) was applied and the maximal fluorescence for a dark-adapted state, F M , was measured. After the F M measurement, fluorescence was kept relaxed in darkness for 17 seconds. Red actinic light (650 nm, intensity of 100 μmol photons m -2 s -1 ) was then switched on for 70 seconds to drive photosynthesis. It was visually checked so that a steady state fluorescence signal was attained at 70 s of illumination. During the actinic illumination, saturation pulses were applied at 8, 18, 28, 48, and 68 seconds from the beginning of the actinic illumination. The value of the maximal fluorescence measured during the last saturation pulse was taken as the maximal fluorescence signal for the light-adapted state, F M '. The fluorescence signal caused by the actinic illumination measured just before the last saturation pulse was taken as the steady state fluorescence for a light-adapted state, F (t). The four fluorescence levels (F 0 , F M , F (t), F M ') were used for calculation of the minimal fluorescence level for a light-adapted state, F 0 ' , the quantum yields, and the other fluorescence parameters as defined and described in the Results section. A hand-operated FluorPen fluorometer (Photon Systems Instruments, Brno, Czech Republic) was used for control measurements in order to compare the results obtained using automatized CFIM with hand-operated non-imaging Chl fluorescence measurements. Blue light (455 nm) of intensity 1000 μmol photons m -2 s -1 and a duration of 1 second was used by FluorPen for illumination of the sample and a whole fast fluorescence rise (the O-J-I-P curve) was recorded. However, only the minimal and maximal fluorescence levels, F 0 and F M , respectively, for the dark adapted state, were evaluated from the curve using built-in routines. The two fluorescence levels were used for calculation of the maximal quantum yield of PSII photochemistry (see Results). The data for Chl fluorescence measurements are presented as medians and lower and upper quartiles [34]. Conclusion In this proof-of-concept study, the high-throughput method for automated screening of cold-tolerant pea (Pisum sativum L.) cultivars was designed. TER and END cultivars were screened simultaneously in an automated way with throughput of 16 plants per hour for i) growth of the aerial parts by RGB imaging and ii) for the efficiency of photosynthesis by chlorophyll fluorescence imaging. We demonstrated that the presented integrative approach based on analyses of differences in relative growth rate and selected CFIM parameters can provide deeper insight into the physiological base of cold-acclimation. Data from both analytical tools pointed to significant differences in the growth and photosynthesis of TER and END cultivars, and indicated that the two pea cultivars use different strategies for cold acclimation differing in number of open PSII reaction centers, their maximal photosynthetic quantum yield in light and quantum yield of constitutive non-light induced dissipation processes. The reliability of the screening was verified by independent measuring of the fresh weight of the shoots and by Chl fluorescence measurement by hand fluorometer. Since the CFIM analysis is not limited to plant morphology and our image analysis was sensitive enough to detect tiny tendrils of pea, we believe that the described procedure can be easily employed for shoot analyses of other different plant species. Competing interests The authors declare that they have no competing interests. Authors' contributions JFH and DL carried out the visible and fluorescence imaging analyses, data processing and interpretation, participated in the design of the study and drafted the manuscript. TF developed and carried out the software image analysis, data processing, performed the statistical analysis, and drafted the manuscript. AH carried out the visible and fluorescence imaging analyses, data processing and interpretation, participated in the design of the study and drafted the manuscript. MH contributed to the design of the study, and helped to draft the manuscript. LS conceived the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.

v3-fos

2019-04-02T13:05:31.624Z

2015-10-11T00:00:00.000Z

90874258

s2

Growth Inhibition Potentials of Leaf Extracts from Four Selected Euphorbiaceae against Fruit Rot Fungi of African Star Apple ( Chrysophyllum albidum G . Don ) Chrysophyllum albidum G. Don commonly called African star apple and locally called udara (Igbo), agbalumo (Yoruba) belongs to the family Sapotaceae [1]. It features prominently in the compound agro forestry system for fruit, food, cash income and other auxiliary uses including environmental purposes. It is also a tree that is common throughout the Tropical Central, East and West Africa regions for its sweet edible fruit and various ethnomedical uses [2]. Introduction Chrysophyllum albidum G. Don commonly called African star apple and locally called udara (Igbo), agbalumo (Yoruba) belongs to the family Sapotaceae [1]. It features prominently in the compound agro forestry system for fruit, food, cash income and other auxiliary uses including environmental purposes. It is also a tree that is common throughout the Tropical Central, East and West Africa regions for its sweet edible fruit and various ethnomedical uses [2]. C. albidum fruits are widely eaten in Southern Nigeria. The fruit is seasonal (December-March), when ripe. It is flattened seeds or sometimes fewer by abortion. The fruit is ovoid to sub-globose pointed at the apex and up to 6 cm long and 5 cm in diameter. The skin or peel is grey when immature turning orange red, pinkish or light yellow within the pulp having three to five seeds arranged as a star [3]. The fruit has been found to have the highest content of ascorbic acid with 1000 to 3330 µg of ascorbic acid per 100 gm of edible fruit or about 100 times that of oranges and 10 times of that of guava or cashew. It is also an excellent source of vitamins B and D as well as iron [4]. Umoh [5] and Ureigho [6] reported on the proximate composition, minerals and vitamins content of Chrysophyllum albidum. The fruit has immense economic potential, especially following the report that jams that could compete with rasp berry jams and jellies could be made from it and it is eaten especially as snack by both young and old [2]. The fruits contain 90% anacadic acid, which is used industrially in protecting wood and as a source of resin. The fruits can be used in the preparation of wine, soft drink, jams and jellies [3,6]. The seed are used for local games; it is also a source of oil, which used for diverse purposes [7]. The seeds along with those of other Sapotaceae are used as anklets in dancing. It was also discovered in the removal of Ni 2+ ions from synthetic wastewater [8]. The cotyledons are useful in the preparation of medicine for the treatment of infertility problems in both male and female; infertility due to the presence of abnormalities within the uterus and female tubes, abdominal pains in dysmenorrheal, secondary ammenorrhae in women (loss or absence of menstrual cycle). The seed cotyledon has been reported to possess antihyperglycemic and hypolipidemic effects [9]. Fungi have been reported to be associated with post harvest deterioration of agricultural products in Nigeria. However, F. solani, L. theobromae, Rhizopus spp and A. flavus have been reported to be associated with C. albidum [10]. Since most microbial spores are small in size and light, they could settle on the surface of African Star Apple fruits resulting in the range of microbial group isolated from them. Preserving the freshness of these fruits for many days or months is therefore the problem, which most farmers and the traders seek to solve. Control of fruit rot by employing the use of local preservatives (plant extracts) like Afromomum danielli, Afromomum melegueta and chemical disinfectants like (parazone), sodium chloride and sodium benzoate at mild form has been suggested to reduce the losses due to storage moulds [10]. The objective of this study is therefore to isolate and identify fungi associated with C. albidum fruits rot in storage as well as to determine the effects of various concentrations of ethanolic extracts of Phyllanthus amarus, Euphorbia hirta, Euphorbia heterophylla and Acalypha fimbriata on the identified fungi. Materials and Methods Collection of plant materials for the study Mature healthy and rotted C. albidum fruits were purchased at Abraka Main Market, Delta State. Fresh and healthy leaves of Euphorbia hirta, Euphorbia heterophylla Phyllantus amarus and Acalypha fimbriata free from insect and pathogen attack were collected from different areas within Abraka community. Abraka (Ethiope East Local Government Area of Delta State lies within latitude 05° 47˝N and longitude 06° 06˝E of the Equator with an annual rainfall of 3,097.8 mm, annual relative humidity of 83% and annual mean temperature of 30.6°C [11]. The plants were identified using Akobundu and Agyakwa [12]. Isolation and identification of fungi Isolation and identification of fungi from diseased C. albidum fruits was carried out using the method adopted from Ilondu [13]. Sections, 4 mm long, excised from the margins of diseased spot with sterile razor blade were surface-sterilized for 2 min in 2% aqueous solution of commercial bleach (sodium hypochlorite solution), rinsed in two changes of sterile distilled water. The disinfected tissue pieces were blotted between sterile Whatman No. 1 filter paper and aseptically plated on potato dextrose agar (PDA) plates (3 pieces per plate). The plates were then incubated at room temperature (32 ± 2°C) for five days. Any observed mycelial growth was repeatedly transferred to fresh PDA plates until pure cultures of isolates were obtained. The frequency of isolations of the different types of fungi associated with C. albidum fruit rot diseases was determined. The number of times each fungus was encountered was recorded. The percentage frequency of occurrence was calculated with the formula below: Number of times a fungus was encountered 100 × Total fungal isolations 1 Plant sample preparation and extraction procedures The plants were collected into polyethylene bags and taken to the laboratory for processing. The leaves were separately plucked and rinsed in flowing tap water, shade dried on the bench in a ventilated section of the Department of Botany herbarium at ambient temperature (30°C ± 2) for two weeks [14]. Dried leaves were separately ground into powder using an electric blender before extraction. For extraction procedures, one hundred gram of each pulverized sample was put into Soxhlet extractor and three hundred milliliter of absolute ethanol (HPLC grade) was added and extracted for 8hrs for each batch of sample. The extracts were evaporated on a rotary evaporator at 40°C to remove excess alcohol. The solvent free extracts were stored at 4°C till needed. Phytochemical tests One gram of powdered sample was subjected to phytochemical test for alkaloid (Myers reagent), Flavonoids were determined by magnesium rebbon test, Sapoins by chloroform and H 2 SO 4 tests, Tannins, by Ferric salt test, Sterol by Chloroform-acetic anhydride, Terpenes and phenols by following the procedures of Oyewale and Audu [15]. GC-MS analysis was done at National Research Institute for Chemical Technology (NARICT) Zaria, Kaduna state, Nigeria. A SHIMADZU GCMS-QP 2010 Plus system was used. The GC-MS was operated under the following conditions: Column oven temperature: 70°C; Injection temperature: 250°C; Injection mode: split; Pressure: 104.1 kPa; Total flow: 6.2 ml/min; Column flow: 1.59 ml/min; Linear velocity: 46.3 cm/sec; Purge flow: 3.0 mL/min; and Split ratio: 1.0. The generated chromatogram was recorded. The identification of the components was carried out using the peak enrichment technique of reference compounds and computer matching with those of NIST.05 library mass spectrum [14,16]. Effect of extracts on fungal growth Different concentrations (100, 80, 60 40 and 20 mg/ml) were prepared from each of the extracts. One millilitre of each level of concentration was aseptically incorporated into 20 ml of cool molten PDA in sterile test tube. Each medium was homogenized by gentle agitation before dispensing into sterile 9 cm Petri dishes. The control was set up using extract free PDA plates. The plates were allowed to set for 3 hr. The effect of the extracts on fungal growth was determined using the method of Chohan et al. [17]. This was done by inoculating at the Centre of 90 cm Petri plates with a mycelia disc (4 mm) obtained from the colony edge of 7-day old culture of the test fungi. Three replicates of both the control and PDA-extract plates per isolate were incubated at room temperature (28 ± 2°C) and radial growth was measured with a metric ruler daily for seven days. Colony diameter was taken as the means along two directions on two perpendicular lines drawn on the reverse of the plates. The percentage inhibition was calculated by the method of Ayodele et al. [18]. Data analysis Data obtained were subjected to Analysis of Variance (ANOVA) using Statistical Package for Social Science (SPSS) version 17.0 and means were separated according to Duncan's Multiple Range Test (DMRT) at 5% probability level. Results The fungi isolated from the diseased Chrysophyllum albidum fruits were Aspergillus niger and Fusarium solani. A. niger occurred more frequently with 69.6% followed by F. solani with 30.4% (Table 1). The classes of natural products present in the plant investigated are shown in Table 2 The gas chromatography profiles of the plants extracts used in the study were shown in Figures 1-4. The analysis of the extract revealed complex mixture of constituents ranging from 7-14 compounds in the samples (Table 3). 9-Octadecenoic acid (Z)-methyl ester was most abundant among the (7) compounds in E. hirta, Erucic acid was most abundant in E. heterophylla and A. fimbriata. The two fungi were very sensitive to various concentrations of the plant extracts tested since the extracts significantly reduced the mycelia growth of the fungi at all concentrations (Table 4). However, the effectiveness of the plant extracts increased with increased in concentration and this was significantly different (p<0.05) when compared to the control. Similarly, percentage growth inhibition generally increased with increase in concentration of the leaf extracts when compared to the control. Although, the plant extracts could not give complete inhibition at the highest concentration tested, their effectiveness increased with increase concentrations. There was no significant difference in the inhibitory effect of P. amarus on A. niger at the concentrations of 20 and 40 mg/ml, 60 and 80 mg/ml concentrations with E. heterophylla as well as 80 and 100 mg/ml concentrations with A. fimbriata. Similarly, there was no significant difference in the inhibitory effect of A. fimbriata extract on F. solani from 60-100 mg/ml concentrations (Table 5). A. niger was most sensitive to E. heterophylla followed by A. fimbriata, P. amarus and E. hirta respectively. Similarly, F. solani was most sensitive to A. fimbriata followed by E. heterophylla, E. hirta and P. amarus. Discussion The present study showed that two fungi were associated with post harvest fruit rot disease of Chrysophyllum albidum, which include Aspergillus niger and Fusarium solani. These fungi have previously been reported as fruit rot pathogens [13,19,20]. Aspergillus niger has the highest percentage occurrence of 69.6% followed by F. solani which is 30.4%. This was enhanced by the light Phytochemical screening of the plants revealed the presence of saponin, alkaloid, tanin, steroids, Phenols, terpenes, glycosides, and flavonoids. The presence of these secondary metabolites could be responsible for their antifungal activity. Egwin et al. have earlier demonstrated the presence of tannins in Euphorbia hirta and opined that it may account for its antimicrobial activity. Tannins have been reported to be toxic to bacteria, filamentous fungal and yeast [22]. Ogbo and Oyibo [19] reported that the presence of alkaloids, saponins and terpenoids in the extract of Ocimum gratissimum may have accounted for the broad spectrum of activities on the fungal isolate tested. The analysis of the plant extract of the leaves in this study showed a complex mixture of constituents. The total number of compounds identified varied from 7-14 in all the plant samples. It is possible that these compounds identified in the plant extracts were responsible for the observed fungi-toxic effects in the study. Sunderham [23] reported that the toxic action of the plant extract of E. heterophylla is due to the combined action of its constituents this is similar to the observations of Ilondu [14,16]. Erucic acid was the highest constituent found in E. heterophylla (22.22%) and A. fambiata (28.88%) extracts. Antimicrobial activity of Eruca sativa seed oil has been reported to be due to higher concentration of erucic acid present in the oil [24,25]. Varied concentrations of fatty acid including their ethyl and methyl esters were found abundant in all the plant extracts. Several researchers have reported the antifungal activity of fatty acid and their ethyl and methyl esters against pathogenic fungi [14,16,26]. The percentage inhibition of the mycelia growth of the tested fungi was found to increase as concentration of the plant extracts increased. This may be as a result of the presence of the biologically active antimicrobial compounds of the extracts in higher quantity at lower dilutions, this findings is in consonance with the work of Fernadex et al. [27] who suggested that with increasing concentrations the antagonistic property of the extract increased. The above result clearly confirms that the test fungi varied widely in the degree of their susceptibility to the extracts. The extract of Euphorbia heterophylla was the most effective of all the extracts in inhibiting the growth of Aspergillus niger followed by Acalypha fimbriata, Phyllanthus amaraus and Euphorbia hirta. While ethanolic extracts of Acalypha fimbriata was the most effective in inhibiting the growth of Fusarium solani followed by Ephorbia heterophylla, Euphorbia hirta and Phyllanthus amarus. Previous studies have shown that ethanolic leaf extracts of E. hirta, E. heterophylla, A. fimbriata, P. amarus and other species of these genera were capable of inhibiting the growth of bacteria, and fungi [13,23,[28][29][30][31][32]. Conclusion The result of this study is an indication that these Euphorbiaceae could be a potential source of antifungal agents. Knowledge of chemical constituents of non-economic plants is desirable because such information could be valuable in discovering new source of economic materials, which may be precursors for the synthesis of complex chemical substances. Such screening of various natural organic compounds and identification of active agents is the need of the century for the formulation of plant biofungicide and improvement of food security for the timing world population. Values with the same superscript(s) in the same column are not significantly different at P>0.05 by DMRT.

v3-fos

2018-12-20T22:29:44.676Z

2015-03-16T00:00:00.000Z

59402838

s2

Effects of selenium on the growth and photosynthetic characteristics of flue-cured tobacco (Nicotiana tabacum L.) The objective of this study was to investigate the effect of Selenium (Se) supply (0, 3, 6, 12, 24 mg kg−1) on the growth, photosynthetic characteristics, Se accumulation and distribution of flue-cured tobacco (Nicotiana tabacum L.). Results showed that low-dose Se treatments (≤6 mg kg−1) stimulated plant growth but high-dose Se treatments (≥12 mg kg−1) hindered plant growth. Optimal Se dose (6 mg kg−1) stimulated plant growth by reducing MDA content and improving photosynthetic capability. However, excess Se (24 mg kg−1) increased MDA content by 28%, decreased net photosynthetic rate and carboxylation efficiency by 34% and 39%, respectively. The Se concentration in the roots, stems, and leaves of the tobacco plants significantly increased with increasing Se application. A linear correlation (R = 0.95, P < 0.01) was observed between Se level and tobacco plant tissue Se concentration. This correlation indicated that the tobacco plant tissues were not saturated within the concentration range tested. The pattern of total Se concentration in the tobacco plant tissues followed the order root > leaf > stem. The Se concentration in the roots was 3.17 and 7.57 times higher than that in the leaves and stems, respectively, after treatment with 24 mg kg−1 Se. In conclusion, the present study suggested that optimal Se dose (6 mg kg−1) improved the plant growth mainly by enhancing photosynthesis, stomatal conductance, carboxylation efficiency and Rubisco content in the flue-cured tobacco leaves. However, the inhibition of excess Se on tobacco growth might be due to high accumulation of Se in roots and the damage of photosynthesis in leaves. Introduction Selenium (Se) is essential to animals and humans [1]. Recent research has shown that this trace element is also beneficial to plants [2]. However, high Se concentrations may elicit toxic effects on plants [3]. The difference between the deficiency and toxicity of Se, as in other essential trace elements, is narrow [4]. Plant species differ in Se uptake and accumulation in shoots and roots, as well as in tolerance to high Se concentrations in solution or soil [5]. For example, Astragalus bisulcatus and Stanleya pinnata exhibit high tolerance to Se in soil; these plants can hyperaccumulate Se up to 1% of their dry weights (DWs) [6]. By contrast, tobacco and soybean are sensitive to Se; these plants can be affected by low Se concentrations (e.g., 1 mg kg −1 ) in culture media [7]. It's been well reported that the phytotoxicity of Se varies among agricultural crops [5]. Evidence to prove that nonaccumulator plants require Se remains lacking. However, numerous studies have reported that low Se concentrations benefit the growth of these plants. Turakainen et al. [8] showed that appropriate Se concentrations has positive effects also on potato carbohydrate accumulation and possibly on yield formation. Similarly, other studies revealed that Se promotes the growth of ryegrass [3], tea [9], rice [10], and soybean [11]. However, excess Se accumulation (>0.1% plant DW) is generally toxic to plants, except for rare Se-hyperaccumulating plants [4]. Plants subjected to Se stress exhibit different physiological changes, including stunted root growth, reduced biomass, chlorosis, reduced photosynthetic efficiency, and ultimately plant death [4]. Previous studies reported that Se improves the antioxidant capacity of plants [3,12]. Feng et al. [2] have recently discovered that Se elicits protective effects on plants against abiotic stresses. Soil treatment with Se has been highly recommended to produce Se-enriched food for human consumption. Se-enriched products, such as tea [9], rice [10,13], and vegetables [14] have been developed. Furthermore, various studies have associated the consumption of Se-enriched vegetables with reduced risk of developing cancer [14,15]. Broccoli can accumulate Se and convert it into a form that is chemoprotective against cancer [15]. These findings suggest that Se-enriched vegetables benefit human nutrition and health [14]. Therefore, understanding the effect and function of Se on the plant growth is important to the development of Se-enriched agricultural products. Although Se is known to elicit detrimental effects on plants, the effects of Se stress on the photosynthetic characteristics of tobacco have yet to be elucidated. Tobacco (Nicotiana tabacum L.) is an economically important nonfood crop worldwide; flue-cured tobacco accounts for approximately 80% of the world's tobacco production [16]. Therefore, the present study aims to determine the effects of treatments with different Se concentrations (0 mg kg −1 to 24 mg kg −1 ) on the plant growth, gas exchange, chlorophyll concentration, Rubisco content, malondialdehyde (MDA) content, Se accumulation and distribution of flue-cured tobacco. Experimental materials and growth conditions A soil pot experiment was conducted in greenhouse conditions in major rice-growing areas of Anhui province, China. Tobacco (N. tabacum L., cv. Yunyan 87), a popular flue-cured tobacco cultivar in China, was used in this study. The seeds were provided by Chizhou Tobacco Corporation, Anhui province, China. Seeds were surfaced-sterilized with 2% (v/v) NaOCl for 10 min, rinsed with deionized water, and then sown in floating nursery. During the growing season, the plants were placed in a greenhouse with a daytime temperature of 25°C to 33°C, a nighttime temperature of 15°C to 23°C, and a relative humidity (RH) of 60% to 85%. The soil was paddy soil, collected from the tobacco field in Chizhou, Anhui province, China. Main physical and chemical properties were as follows: pH water 2.5:1 5.4, organic matter 17.6 g kg −1 , available N 157.7 mg kg −1 , available P 16.6 mg kg −1 , available K 184.7 mg kg −1 , and total Se 0.16 mg kg −1 . Experimental design Five levels of Se (sodium selenite, Na 2 SeO 3 ) treatment, i.e. 0 (CK), 3, 6, 12, and 24 mg kg −1 Se were performed in the experiment. Each pot (35 cm in diameter, 28 cm in height) was filled with 20 kg of air-dried and 2 mm-sieved soil. Therefore, each pot was added with 0, 60, 120, 240, or 480 mg of Na 2 SeO 3 to produce the five treatments. The fertilizers for each pot were as follows: flue-cured tobacco special fertilizer (N:P:K = 9:13.5:22.5) 45.45 g, KNO 3 10.10 g, K 2 SO 4 3.28 g, Ca(H 2 PO 4 ) 2 14.61 g. All the fertilizers and Na 2 SeO 3 were mixed thoroughly and applied as basal dressing at 10 days before tobacco seedlings transplanting. Tobacco seedlings (approximately 12 cm in height) were transplanted into the pots on April 9, 2011, with one plant for each pot. The pots were arranged at 1.2 m inter-row spacing and 0.5 m intrarow spacing. Each treatment was replicated for four times, and each replicate included four plants. Gas exchange measurements At 40 d after the treatments, the gas exchange on the newly expanded leaves was measured from 8:30 to 12:00 using a Li-6400 portable photosynthesis system (Li-Cor, Inc., Lincoln, NB, USA) as previously described [17]. During the measurements, leaf temperature was maintained at 25 ±1°C with a photosynthetic photon flux intensity of 1200 μmol m −2 s −1 . Ambient CO 2 concentration in the cuvette (C a-c ) was adjusted to Ca (380 μmol CO 2 mol −1 ), and RH was maintained at 50% ±5%. After 10 min, C a-c was controlled across the series of 1000, 800, 600, 400, 200, 100, and 50 μmol CO 2 mol −1 . Carboxylation efficiency (CE) was calculated as the initial slope of the A/Ci response curves. Data were recorded after equilibration to a steady state. Leaf chlorophyll concentration determination At 40 d after the treatments, the relative chlorophyll concentrations in the newly expanded leaves of the labeled leaf segments were determined using a SPAD-502 Chlorophyll Meter (Minolta, Mahwah, NJ, USA). MDA content determination At 40 d after the treatments, the MDA contents in the newly expanded leaves of the labeled leaf segments were measured as previously described [18]. The absorbance at 450, 532, and 600 nm was obtained with an ultraviolet spectrophotometer (UV-755B, Shanghai Precision and Scientific Instrument Co., Ltd., China). The concentration of MDA was calculated using the following formula: C = 6.45(D 532 − D 600 ) -0.56D 450 . Rubisco measurements After the gas exchange measurements, the Rubisco contents in the newly expanded leaves were measured according to the method of Li et al. [19]. Briefly, about 0.50 g newly expanded leaves were ground with a cooled extraction buffer containing 50 mmol l −1 Tris-HCl (pH 8.0), 5 mmol l −1 β-mercaptoethanol and 12.5% (v/v) glycerol at 0-4°C. The homogenate was centrifuged at 1500 g for 15 min at 4°C. The supernatant solution was mixed with a dissolving solution containing 2% (w/v) SDS, 4% (v/v) β-mercaptoethanol and 10% (v/v) glycerol. Then the mixture was boiled in water for 5 min for gel electrophoresis. An electrophoretic buffer system was used for SDS-PAGE with a 12.5% (w/v) stacking gel and a 4% (w/v) separating gel. Afterwards, the gels were washed with deionized water several times then dyed in 0.25% Coomassie Blue for 12 h and detained. Large subunits and relevant small subunits were transferred to a 10 ml cuvette with 2 ml of formamide and washed in a 50°C water bath for 8 h. The washed solutions were measured at 595 nm using background gel as a blank and bovine serum albumin (BSA) as the protein standard. Determination of plant biomass After the above measurements were completed, the plants were harvested and analyzed for root, stem, and leaf fresh weights. All samples were oven-dried at 105°C for 30 min and then at 60°C until constant weight were reached. Total Se analysis The roots, stems, and leaves of the plants were milled into powder using a mixer mill (Shanghai Bilon Instrument Co., China). The total Se concentrations in the samples were determined as previously described [20,21]. The dried plant powders (0.5 g) were digested overnight with 10.0 ml of HNO 3 :HClO 4 (9:1) in a polypropylene sample tube at room temperature. The digested solution was heated on an electrical hot plate at 60°C for 2 h and then at 100°C for 1 h. The tube containing the solution was added with 10.0 ml of HNO 3 :HClO 4 (9:1) and then stored at 170°C for 2 h until a white fume formed. After cooling to room temperature, the tube was added with 5 ml HCl (1:1) and then heated until the solution became colorless within at least 3 h. After cooling, the solution was filtered and diluted to 50 ml with deionized water. The total Se concentration in the solution was analyzed through inductively coupled plasma-mass spectrometry [X Series ICP-MS (Thermo Electron Corporation, United States)]. Four replicate determinations were performed for each material. Statistical analysis All measurements were carried out on replicate samples collected from four individual plants. Statistical analyses were performed using one-way ANOVA with SPSS statistical software. Data were presented as mean and SE. The results were verified via Duncan's multiple-range test. Effects of Se treatment on plant growth Tobacco plants were grown in soil with fertilizers containing five Se concentrations (0, 3, 6, 12, and 24 mg kg −1 ) under greenhouse conditions. As shown in Tab. 1, lowdose Se treatments (≤6 mg kg −1 ) enhanced the growth of tobacco plants. Treatment with 6 mg kg −1 Se significantly enhanced root, stem, leaf, and whole-plant DWs by 11%, 29%, 18% and 19%, respectively, compared with CK. In contrast to low-dose Se treatments, high-dose Se treatments (≥12 mg kg −1 ) reduced the growth of tobacco plants. For instance, treatment with 24 mg kg −1 Se decreased root, stem, leaf, and whole-plant DWs by 18%, 10%, 19%, and 16%, respectively, compared with CK. However, no significant difference in root/shoot ratio was detected between the CK-and Se-treated tobacco plants. Effects of Se treatment on gas exchange Tab. 2 presents the changes in the gas exchange parameters of newly expanded tobacco leaves after 40 d of Se treatment. Compared with CK, low-dose Se treatments (≤6 mg kg −1 ) significantly increased the net photosynthetic rate (Pn) in the leaves, whereas high-dose Se treatments (≥12 mg kg −1 ) significantly decreased this parameter. The Pn values of the tobacco leaves under 3, 6, 12, and 24 mg kg −1 Se treatments were 1. Effects of Se treatment on leaf chlorophyll concentration and Rubisco content As shown in Fig. 1, the chlorophyll concentration (SPAD value) slightly increased in the newly expanded leaves under low-dose Se treatments (≤6 mg kg −1 ). However, the chlorophyll concentration declined with increasing Se concentration. Compared with CK treatment, 24 mg kg −1 Se treatment significantly decreased the chlorophyll concentration in the leaves. Similarly, compared with CK treatment, low-dose Se treatments (≤6 mg kg −1 ) increased the Rubisco content, whereas 24 mg kg −1 Se treatment decreased this parameter by 18% (Fig. 2). Effects of Se treatment on MDA content Compared with CK treatment, low-dose Se treatments (≤6 mg kg −1 ) significantly reduced the MDA contents in the tobacco leaves, whereas high-dose Se treatments (≥12 mg kg −1 ) increased this parameter (Fig. 3). The MDA contents in the leaves under 3 and 6 mg kg −1 Se treatments were only 86% and 82% those in the leaves under CK treatment, respectively. The MDA contents in the leaves under 12 and 24 mg kg −1 Se treatments were 1.05 and 1.28 times those in the leaves under CK treatment, respectively. Se concentration and accumulation in tobacco plant tissues The Se concentration in the different tobacco plant parts significantly increased (P < 0.01) with increasing Se application in soil (Tab. 3). For example, the Se concentrations in the roots, stems, and leaves under 24 mg kg −1 Se treatment were 6.59, 5.91 and 5.43 times higher than those in the same plant parts under 3 mg kg −1 Se treatment, respectively. Under the same Se treatment, the Se concentration in the roots was evidently higher than those in the stems and leaves. In general, the pattern of total Se concentration in tobacco plant tissues followed the order root > leaf > stem. Under the same treatment of 24 mg kg −1 Se, the Se concentration in the roots (31.36 mg kg −1 ) was 3.17 and 7.57 times higher than those in leaves and stems, respectively (Tab. 3). The accumulation of Se in the tobacco roots, stems, and leaves significantly increased with increasing Se application (Tab. 4). For example, the roots, stems, and leaves under 24 mg kg −1 Se treatment accumulated 254.64, 43.60, and 179.30 μg plant −1 of Se; these values were 6.28-, 5.51-, and 4.81-fold higher than those accumulated by the roots, stems, and leaves under 3 mg kg −1 Se treatment, respectively. Under high-dose Se treatments (24 mg kg −1 ), the roots accumulated up to 254.64 μg plant −1 of Se, which was 1.42 and 5.84 times higher than those accumulated by the leaves and stems, respectively. Relationship between Se concentration and tobacco plants The relationship of the Se concentrations in the different treatments with those in the different tobacco plant parts (root, stem and leaf) was analyzed and compared after 40 d of treatment (Fig. 4). The Se concentrations in the roots, stems, and leaves significantly correlated with those in the different treatments. The amounts of absorbed Se in the roots, stems, and leaves were closely related to the Se concentrations in the different treatments. Discussion Research has revealed that Se exerts a dual effect on the growth of different plant species [1,5,22]. At low doses, Se stimulates plant growth; at high doses, this element hinders plant growth [2]. In the present study, the exposure of plants to 6 mg kg −1 Se increased the yield of roots, shoots and leaves by 11%, 18% and 29%, respectively (Tab. 1). These findings indicate that the growth of flue-cured tobacco plant increased at low-dose Se treatments (≤6 mg kg −1 ), which agreed with earlier observations of Yao et al. [22], who found that treatment with 1 mg kg −1 to 3 mg kg −1 Se promotes biomass accumulation in wheat seedlings. However, several reports have provided evidence that high Se addition levels decrease the biomass of non-accumulator plants like wheat [5] and rice [23]. Our results showed that selenite inhibited tobacco plant growth at concentrations up to 12 mg kg −1 . The decrease in the root, stem, and leaf DWs was much more apparent at concentrations up to 24 mg kg −1 (Tab. 1). The improvement of plant growth at low-dose Se treatments (Tab. 1) may be due to the significant increase in photosynthesis of tobacco plants (Tab. 2). In the present study, low-dose Se treatments (≤6 mg kg −1 ) increased the photosynthetic rate, stomatal conductance and carboxylation efficiency in tobacco leaves (Tab. 2). A similar pattern was observed in rice [23] and sorghum leaves [11]. Wang et al. [23] revealed that in rice seedlings, low doses of Se enhanced photosynthesis. In addition, in sorghum, Se application significantly increased the photosynthetic rate and stomatal conductance [11]. In contrast, at high Se supply levels, the photosynthetic rate in tobacco leaves decreased significantly. High-dose Se treatments (24 mg kg −1 ) reduced the Pn, CE and Rubisco content by 34%, 39%, and 18%, respectively (Tab. 2 and Fig. 2). This fact suggests that, growth inhibition of tobacco plants under high-dose Se treatments may result from impaired photosynthesis. The values are presented as mean and SE (n = 4). Different letters indicate a significant difference in the same column at P < 0.05. Excess Se was toxic to tobacco plants, leading to reduction of chlorophyll concentration (SPAD value; Fig. 1) that may cause photosynthesis suppression. In our experiment, the chlorophyll concentration in the leaves decreased significantly under 24 mg kg −1 Se treatments (Fig. 1). Similarly, Chen et al. [24] reported that Chlorella vulgaris has lower total chlorophyll content under high Se treatments than under low Se treatments, suggesting that Se affects chlorophyll synthesis or chlorophyllase activity. A decrease in pigment concentrations could also decrease photosynthetic functioning and elicit negative effects on the levels [24]. However, to acquire detailed regulatory mechanisms underlying these effects, further studies should focus on the nature of PSII photochemistry and photosynthetic apparatus under excess Se. The inhibition of photosynthesis in tobacco plants under high Se application may be closely related to the increassed MDA levels. As a product of lipid peroxidation, MDA is an indicator of oxidative damage [25]. Reports have shown that proper doses of Se can reduce MDA accumulation in various plants [2,26]. In the present study, Se application of 6 mg kg −1 significantly decreased the MDA content in tobacco leaves; however, 24 mg kg −1 Se remarkably increased this parameter (Fig. 3). Similarly, Cartes et al. [26] found that low-dose Se treatments (≤6.0 mg kg −1 ) reduce the MDA content in ryegrass, and vice versa. The MDA content reflected the extent of lipid peroxidation and indirectly reflected the degree of cell damage. Therefore, our results suggested that the optimal Se dose (≤6 mg kg −1 ) enhanced antioxidant capacity and reduced lipid peroxidation in flue-cured tobacco leaves, whereas excess Se accelerated lipid peroxidation. Plant growth responses were closely related with the concentrations of Se in the plant tissues (Tab. 3). The Se concentrations in the different parts of tobacco plant increased as those in the different treatments increased (Tab. 3). A linear correlation (R = 0.95, P < 0.01) was found between soil and tobacco plant tissue Se concentrations (Fig. 4), indicating that the tobacco plant tissues were not saturated within the tested concentration range. Moreover, Se concentration was much more higher in the tobacco roots than in the leaves and stems (Tab. 3). Therefore, a significantly higher amount of Se was accumulated in the roots than in the leaves and stems (Tab. 4). The pattern of total Se concentration in tobacco plant tissues (root > leaf > stem) was similar to that previously observed in ryegrass [27], wherein Se was principally accumulated in the roots. Previous studies have demonstrated that hyperaccumulators were characterized by a high leaf Se concentration, and a higher shoot:root Se concentration ratio [28,29]. Valdez et al. [30] reported that as a Se hyperaccumulator, the pattern of total Se concentration in Astragalus bisulcatus follows the order root < leaf < stem. Under 24 mg kg −1 Se, the Se concentration in the roots (31.36 mg kg −1 ) was 3.17 and 7.57 times higher than those in leaves and stems, respectively (Tab. 3). It suggested that high accumulation of Se in roots (31 mg kg −1 ) caused tobacco plant toxicity. Generally, most cultivated plants contain less than 25 mg Se kg −1 DWs and are considered to be non-accumulators [31]. More recently, it was revealed that most plant species growing on seleniferous soils contain <10 mg Se kg −1 DWs, and experience toxicity at levels above ~100 mg Se kg −1 DWs [32]. Therefore, from our results it can be suggested that the flue-cured tobacco had a low tolerance to high Se levels, an should be classified as a Se non-accumulator. Conclusions The present results showed that Se stimulated tobacco plant growth at low-dose application (≤6 mg kg −1 ) but inhibited the plant growth at high-dose (≥12 mg kg −1 ), and the optimal dose of Se application was 6 mg kg −1 . Optimal Se dose (6 mg kg −1 ) improved the plant growth mainly by enhancing photosynthesis, stomatal conductance, carboxylation efficiency and Rubisco content in the flue-cured tobacco leaves. However, the inhibition of excess Se on tobacco growth might be due to high accumulation of Se in roots and the damage of photosynthesis in leaves.

v3-fos

2019-04-06T13:04:11.187Z

2015-01-01T00:00:00.000Z

97805828

s2

Comparison of Yield, Nutritive Value, and In Vitro Digestibility of Monocrop and Intercropped Corn-Soybean Silages Cut at Two Maturity Stages Limited information on nutrient composition and in vitro digestibility of corn-soybean intercropped silage is available. The objective of this study was to compare corn (Zea mays L.) or soybean (Glycine max L. Yesilsoy) monocrop silage with corn-soybean intercropped silages in term of yield, nutritive value, and in vitro digestibility. Intercropping was as follows: 1 row corn to 1 row soybean (1M1S), 1 row corn to 2 rows soybean (1M2S) and 2 rows corn to 1 row soybean (2M1S). The crops were harvested when the corn reached 3/5 or 1/4 milk line. The silage samples were analysed for pH, dry matter (DM), crude protein (CP), ether extract (EE), neutral (NDF) and acid detergent fibre (ADF), calcium, potassium, magnesium and phosphorus. Also, in vitro true (IVTD) and in vitro NDF (IVNFD) digestibilities were determined in the silages samples. The DM, EE, calculated non structural carbohydrate values were higher in silage harvested at 1/4 than 3/5 milk line. All intercropped silages had higher CP values (1M1S, 8.3%; 1M2S, 10.1%; 2M1S, 8.0%) than the monocrop corn (SM, 6.8%) silage. The NDF and ADF levels were higher for 1M1S, 2M1S and SM compared with 1M2S and monocrop soybean (SS) silage. In vitro true DM digestibility of all silages increased with maturity stage; it was higher for the 1M2S than other silages. It is concluded that corn-soybean intercropped silage has better nutrient composition and digestibility than SM or SS silage. Introduction Because of high yield in a single harvest, simplicity for ensilaging and high energy value (net energy for lactation=1.45 Mcal/kg) corn silage is a major forage source for dairy cows throughout the world (National Research Council, 2001;Guadarrama-Estradal et al., 2007). Comparing with legume silage (Anil et al., 2000), it is poor in protein content (8.8%) (National Research Council, 2001). On the other hand, legume material is highly difficult to ensile because of its high buffering capacity and low level of water soluble carbohydrate (Maasdorp and Titterton, 1997). Therefore, protein-rich legume and high-energy corn silage can be ensiled to form better nutrient composition (Anil et al., 2000). As a cultivation system, intercropping involves the planting of two or more crop species on the same field (Kipkemoi et al., 2010;Costa et al., 2012). Intercropping has several advantages such as higher total yield and improved soil conservation, better utilisation of water and light (Akinlade et al., 2003;Costa et al., 2012). Moreover, intercropping corn with legumes for silage is a feasible strategy to improve crude protein (CP) level (Prasad and Brook, 2005;Costa et al., 2012;Contreras-Govea et al., 2009;Zhu et al., 2011). Compared with the ensilage of monocrop corn (SM), intercrops of corn and legumes for silage have higher CP, fibre and lactic acid concentrations (Contreras-Govea et al., 2009;Zhu et al., 2011). Because of optimum nutritive composition, predicting the best harvest time of intercropping is an important issue in intercropped silage (Salawu et al., 2001). Salawu et al. (2001) reported that level of potentially digestible nutrients is an appropriate measure of forage quality. However, limited information on nutrient composition and in vitro digestibility of corn-soybean intercrop silage is available. Therefore, the objective of this study was to evaluate the yield, nutrient composition and in vitro digestibility of corn intercropped with soybean in comparison with SM or monocrop soybean (SS) silages. Materials and methods Crop production and silage preparation The crops were produced during the second crop growing season in summer 2010 at the Eastern Mediterranean Agricultural Research Institute (36°51'18" latitude N, 35°20'49" longitude E) in Adana, Turkey. The crop production was a split plot design in a randomised complete block design replicated four times. The main plots represented intercropped and monocrop silages, whereas, maturity was assigned to the sub plots. It was determined that soil contained 7.7 pH, 20% lime, 2% organic matter, 28% sand particle, 31% clay, and 41% silt. Fields were fertilised according to the soil test results and were cultivated before planting. Corn (Zea mays L. Pioneer 31Y43) and soybean (Glycine max L. Yesilsoy) were seeded as monocrop [SM and soybean (SS)] or intercropped as follows: 1 row corn to 1 row soybean (1M1S), 1 row corn to 2 rows soybean (1M2S) and 2 rows corn to 1 row soybean (2M1S). The SM and SS were spaced at 70 cm¥15 cm and 70 cm¥5 cm with population of about 95,240 and 285,710 plants per hectare, respectively. The crops were planted at the end of June and harvested (FX40 New Holland) when the corn reached 3/5 kernel milk line (in early September) or 1/4 kernel milk line (mid September) of maturity according to Afuakwa and Crookston (1984). Mean, maximum and minimum daily air temperatures were 28, 33, and 22°C respectively, and precipitation was 776 mm during the crop production. Botanical composition of the intercrops was estimated by hand-clipping six different areas (1.0 m¥0.5 m) per field before each harvest, separated by hand into individual species. All crops were chopped with a conventional harvester. Yields for each of the crops were estimated by averaging the weights of individual wagonloads from measured areas of the field. The mixed forages were packed in cylindrical plastic tubes with a volume of 3.5 L per treatment and compacted by hand, to exclude as much air as possible, and then tied by a string to ensure air-tightness. The top lid was provided with a CO2-lock, enabling escape of fermentation gasses. The material was left to incubate in a room for 60 days at room temperature (~25°C) before samples were taken for nutrient composition analysis. In addition, in vitro degradabilities were determined. Determination of nutrient composition The pH of the silages was determined (PT-10P; Sartorius, Göttingen, Germany) on the aqueous extract of silage. Silage samples were dried at 60°C for 48 h and ground to pass through a 1 mm screen. The ground samples were ashed at 550°C for 4 h in a muffle furnace (Nabertherm, Lilienthal, Germany). The CP content was determined as N x 6.25 using the Kjeltec 2300 instrument (Tecator; Foss, Hillerød, Denmark). Ether extract (EE) was analysed by a standard ether extraction method (AOAC, 2000). Neutral detergent fibre (NDF) and acid detergent fibre (ADF) were determined with Van Soest et al. (1991) procedures. Ca, Mg, and K were analysed by atomic absorption spectrophotometry (AOAC, 2000). Phosphorus was analysed by colorimetry (AOAC, 2000). All analyses were done in triplicate. In addition, non-structural carbohydrates (NSC) were calculated as 100-(CP+EE+NDF+ash). In vitro digestibility The ground samples were exposed to in vitro digestion using ANKOM DAISY II incubator (Ankom Technology Corporation, Fairport, NY, USA) using the method outlined by Goering and Van Soest (1970). Briefly, approximately 0.50 g of each silage was weighed into separate Ankom F57 filter bags. The bags were placed in digestion jars. Two buffer solutions were warmed to 39°C before setting up each in vitro digestion. The solutions were mixed in a 5:1 ratio, and 1800 mL of the mixed buffer solution was added to digestion jars. Digestion jars were sealed and placed in the preheated incubator for 20 min, allowing the temperatures of the incubator and vessels to equilibrate to 39°C. Rumen liquor was collected from two ruminally cannulated infertile Holstein heifers (approximately 350-400 kg live weight) 2 h after morning feeding. The heifers were fed a corn silage-based diet at maintenance level [dry matter (DM) basis; 2.1 Mcal/kg metabolisable energy, 10% CP, 32% NDF). The rumen liquor was strained through four layers of cheesecloth before mixing with the buffer solution. After 48 h, the bags were removed from the digestion jars, rinsed and, then placed in an ANKOM 200/220 Fibre Analyzer (Ankom Technology Corporation). Digesta samples were exposed to NDF extraction. In vitro true DM digestiblity and in vitro NDF digestibility (IVNFD) were calculated as described by Thomas et al. (2001). Statistical analysis Data were analysed using the PROC MIXED procedure of SAS (2000). The statistical model included maturity, silages and the maturity x silages interaction. Excepting K and ash values in nutrient compostion of monocrop and intercropped silages, nonsignificant interactions were found for all variables. Therefore the interactions were removed from the model. Kenward-Rogers adjustment was used for calculation of denominator degrees of freedom. Pre-planned contrasts were used to compare yield, nutritive value, and in vitro digestibility of silages. The contrast were as follows: SM vs SS (C1), SM vs intercropped silages (C2), SS vs intercropped silages (C3), 1M1S vs 1M2S (C4), 1M1S vs 2M1S (C5), 1M2S vs 2M1S (C6). All results are reported as least squares means. In statistical analyses, P≤0.05 was taken as the level of significance and P≤0.10 was considered to indicate a tendency. Results and discussion In the intercropped silages, the proportion of corn declined while soybean proportion increased (Table 1). The lowest percentage of corn (51.8%) was observed in 1M2S silage at 1/4 milk line. On the other hand, the highest percentage of corn (82.2%) was observed in 2M1S silage at 3/5 milk line. Soybean proportion was lowest (17.8%) for 2M1S silage and increased as the maturity progressed. The maturity stage did not (P>0.05) affect fresh forage, and DM yields ranged from 29.9 Comparison of corn-soybean silages to 46.9 t/ha and 5.9 to 12.0 t/ha (Table 2). Monocrop corn had a higher forage yield (average of harvesting times: 44.8 t/ha) than SS (average of harvesting times: 31.2 kg/ha; contrast C1; P<0.01) and of all intercropped silages (average of harvesting time: 39.3 kg/ha; contrast C2; P<0.05). Similar response was observed in DM yield except for comparison SM with intercropped silages (contrast C2; P<0.10). Fresh forage and DM yields were higher in SM silages, followed by three intercropped silages and the SS silage. Several researchers have reported variable results of intercropping systems. Geren et al. (2008) indicated that intercropped corn with cowpea (Vigna unguiculata) and bean (Phaseolus vulgaris) produced higher DM yield than SM. On the other hand, Maasdorp and Titterton (1997) reported that because of tall and leafy structure, corn in row intercropping had a marked depressing effect on legume growth. Competition and unequal use of environmental or underground resources, such as light and water, seem to account for problems experienced on intercropped communities. These imbalances may have negative effects (for example reduced leaves or leaf area index) on crop yield (Chui and Shibles, 1984;Esmail, 1991). Nutrient value of silages is given in Table 3. The maturity stage did not affect pH and CP content of silages. Silages harvested at 1/4 milk line had higher DM, EE, and NSC (P<0.01 for all). Also, there was a trend for increased P in association with maturity (P<0.10). On the other hand, NDF, ADF, ash, Ca, K, and Mg contents were decreased with maturity (P<0.05). There were significant differences between monocrop silages (SM and SS) and intercrop silages in pH (P<0.01), SM having the lowest pH (3.8). The DM contents differed (P<0.01) between the silages and the 1M2S silage had the highest DM value (27.1%). The highest CP (11.3%) and EE (2.2%) contents were determined in the SS. When compared to SM, the inclusion of soybean as an intercrop increased CP and EE contents (P<0.01), whereas decreased NDF (P<0.01), ADF (P<0.05), and ash (P<0.01) contents. Also, Ca, K, and Mg in the intercrop silage were higher (P<0.01) than SM. The P concentration was similar (P>0.10) between all silages with the one exception being monocrop SM and SS (P<0.10). The high pH of SS and 1M2S silages may be due to the buffering effect of soybean (Mugweni et al., 2000). Legumes may result in relatively high pH values in the silages. The pH values achieved in this study seem to suggest that when the soybean is mixed with corn, which has high levels of fermentable carbohydrates (Bal et al., 1997), the buffering effect is reduced and desirable pH levels are achieved. Also, these findings confirm the technical feasibility of intercropped corn-soybean silages. The DM contents of the silages were between 20 and 29%. McDonald et al. (1987) suggested that optimum DM range of ideal corn silage is between 28 and 32%. Especially, the silages harvested at 1/4 milk line presented higher DM levels than the silages harvested at 3/5 milk line. Costa et al. (2012) reported that the DM level was related to the fermentation conditions of the material and to the levels of loss in the systems. One of the main objectives of intercropped silage is to obtain a complementary effect of the desirable nutrient characteristics of two or more crops. In the present study it was deter-Serbester et al. mined that average protein value of intercropped silages was higher (29%) than SM silage. Legumes are rich in protein. Anil et al. (2000) reported that intercropping corn with a variety of protein-rich forages could increase silage CP level by 3-5% and improve N digestibility, indicating a potential to reduce the requirement for purchased protein supplements. In addition, previous researches have shown that CP concentration increases when the proportion of corn decreases or legumes increase in the mixture (Maasdorp and Titterton, 1997;Contreras-Govea et al., 2009). However, it should be noted that CP levels of corn and soybean grown as monocrop were low because of second crop production and high enviromental temperature during growing months. The NDF contents of the silages varied from 39 to 54% and decreased with the percentage of soybean increased in the mixture. The presence of leguminous plants in the ensiled mass affected NDF and ADF levels in the present study. There is usually lower concentration of fibres in the DM of legumes in relation to grasses (Costa et al., 2012). In addition, NDF level is related to the maturity stage of the forage sources, because of levels of cell wall components, chiefly the cellulose, hemicellulose, and lignin (Mugweni et al., 2000). However, such an effect had not been observed in other experiments and found no effect of intercropping on the NDF and ADF level (Costa et al., 2012). When comparing SM and intercropped silages, it is evident how these last had higher content of Ca and Mg. Paulson et al. (2008) reported that legumes had more total macro and micro minerals and ash than grasses. For example, legumes contain 2 to 3 times Ca than in grasses (Paulson et al., 2008). This result is in agreement with the current study. Table 4 summarises the in vitro true digestibility (IVTD) of all silages increased (P<0.01) with maturity and value for the 1M2S was the highest (68.1%) at 1/4 milk line. However, NDF digestibility of the silages was decreased (P<0.01) by the stage of maturity. At the 3/5 milk line stage, NDF digestibility of silages was 42.8% while that value was 35.4 at 1/4 milk line stage. There was a trend for increased IVTD in association with intercropping (P<0.10). Also, increased proportion of soybean in the intercrop silage increased (P<0.05) IVTD. No significant difference in NDF digestibility was observed between corn and intercropped silage. The lowest NDF digestibility (35.5%) was observed in SS. In addition, intercropped silages had higher NDF digestibility (P<0.01) than monocrop SS. True DM of the silages were improved with maturity. This effect may result in increased soybean ration in mixture. Murphy et al. (1984) reported a more adequate protein for rumen bacteria when sheep are fed corn-fababean silage rather than corn silage. On the other hand, no significant differences in these parameters were observed between SM and intercropped silages. Zhu et al. (2011) reported that in vitro DM and NDF digestibility were similar among silages made from the vine peas, corn, and mixtures. Conclusions Intercropped corn with soybean increased CP, and decreased NDF and ADF concentrations in silages. The optimum time for harvesting intercropped silage could be at 1/4 kernel milk line stage of corn. However, for high yield, SM silage is recommended. Finally, among all intercropped silages the 1M2S (1 row corn to 2 rows soybean) was preferable according to nutrient composition than other intercropped silage. Therefore it may be an alternative to SM silage in nutrition of ruminants. In vivo studies are needed for further confirmation.

v3-fos

2016-06-18T00:05:21.955Z

2015-07-07T00:00:00.000Z

9428268

s2

Genome-wide digital transcript analysis of putative fruitlet abscission related genes regulated by ethephon in litchi The high level of physiological fruitlet abscission in litchi (Litchi chinensis Sonn.) causes severe yield loss. Cell separation occurs at the fruit abscission zone (FAZ) and can be triggered by ethylene. However, a deep knowledge of the molecular events occurring in the FAZ is still unknown. Here, genome-wide digital transcript abundance (DTA) analysis of putative fruit abscission related genes regulated by ethephon in litchi were studied. More than 81 million high quality reads from seven ethephon treated and untreated control libraries were obtained by high-throughput sequencing. Through DTA profile analysis in combination with Gene Ontology and KEGG pathway enrichment analyses, a total of 2730 statistically significant candidate genes were involved in the ethephon-promoted litchi fruitlet abscission. Of these, there were 1867 early-responsive genes whose expressions were up- or down-regulated from 0 to 1 d after treatment. The most affected genes included those related to ethylene biosynthesis and signaling, auxin transport and signaling, transcription factors (TFs), protein ubiquitination, ROS response, calcium signal transduction, and cell wall modification. These genes could be clustered into four groups and 13 subgroups according to their similar expression patterns. qRT-PCR displayed the expression pattern of 41 selected candidate genes, which proved the accuracy of our DTA data. Ethephon treatment significantly increased fruit abscission and ethylene production of fruitlet. The possible molecular events to control the ethephon-promoted litchi fruitlet abscission were prompted out. The increased ethylene evolution in fruitlet would suppress the synthesis and polar transport of auxin and trigger abscission signaling. To the best of our knowledge, it is the first time to monitor the gene expression profile occurring in the FAZ-enriched pedicel during litchi fruit abscission induced by ethephon on the genome-wide level. This study will contribute to a better understanding for the molecular regulatory mechanism of fruit abscission in litchi. Introduction Fruit abscission, occurring during fruit development, is characterized through a high coordination of biochemical events that take place in a group of specialized cells located between the pedicel and fruitlet, known as abscission zones (AZs, Bonghi et al., 2000;Sun et al., 2009). In agricultural production, shedding of fruit is a major limiting factor of yield. Over the last few decades, it is widely believed that abscission involves in multiple changes in cell structure, metabolism and gene expression, and divides into four major steps (Patterson, 2001;Estornell et al., 2013): (i) the ontogeny of AZ, (ii) the acquisition of competences to respond to abscission signals, (iii) the onset of the cell separation, (iv) the differentiation of a protective layer. It is well achieved that plant hormones are deeply involved in abscission, and ethylene operates as an efficient accelerator for organ abscission. Although there is no clear and sufficient evidence for a direct link between the ethylene perception and the onset of abscission, it is well supported that the development of this process is concomitant with an increase in the production of ethylene (Zhu et al., 2008). In fact, application of ethephon, an ethylene-releasing compound, effectively hastens the abscission of fruit in apple (Yuan, 2007;Kolarič et al., 2011), sweet orange (John-Karuppiah and Burns, 2010), sweet cherry (Smith and Whiting, 2010), and olive (Zahra, 2014). However, aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis, blocked the fruit abscission promoted by auxins in apple (Zhu et al., 2008), while 1-Methylcyclopropene (1-MCP), an inhibitor of ethylene perception, did not affect the abscission-promoted effect of ethephon in orange (John-Karuppiah and Burns, 2010). In this regard, understanding the regulatory effects of ethylene on abscission is important for the fruit industry. Up to date, gene expression and enzymatic studies on organ abscission have shown that ethylene either facilitates the efficacy of ethylene signaling pathways (Li and Yuan, 2008;John-Karuppiah and Burns, 2010), or activates the synthesis and secretion of several cell wall and middle lamella hydrolytic enzymes associated with the separation of cells at the AZ, such as cellulase (Abeles and Leather, 1971;MacDonald et al., 2011) and polygalacturonase (Taylor et al., 1993). A transcriptome analysis could be one of the most powerful tools to understand complicated transcriptional regulation during plant organ shedding. In order to provide a new insight into the molecular basis of ethylene-mediated abscission, there are few cases of transcriptome analyses performed using ethylenetreated pedicels as materials. In the study of ethylene-promoted citrus leaf abscission, Agustí et al. (2008Agustí et al. ( , 2009) discovered the preferential accumulation gene families in laminar AZ after ethylene treatment, such as cell wall modification, lipid transport, protein biosynthesis and degradation, transcription factors (TFs), stress and pathogen-related genes and some special genes involved in signaling events. In tomato, Wang et al. (2013) compared the transcriptome difference between the AZ and neighboring portion (the basal and apical) of pedicel in a time course after ethylene treatment, proposing a possible regulatory scheme involving in tomato flower abscission. However, the comparative analysis of the transcriptome profiles involved in ethylene-promoted fruit abscission using AZ as materials is lacking, although it has long been observed that application of exogenous ethylene accelerates fruit abscission. Litchi (Litchi chinensis Sonn.), an important economic fruit crop in subtropical area, has been challenged by massive fruit drop, one of the major factors causing a low yield (Yuan and Huang, 1988;Mitra et al., 2003). For example, a medium size tree may produce about 60,000 female flowers but, typically, less than 5% of flowers develop into mature fruits (Stern et al., 1995). Yuan and Huang (1988) reported that there were three to four waves of physiological fruit drop throughout fruit development in 70∼90 days depending on cultivars. Wave I, wave II, and wave III of abscission occurred around 1 week, 3 weeks, and 6-7 weeks after full bloom, respectively, but wave IV was specific to cultivars with aborted seeds and occurred 2-3 weeks before harvest. Previously, few studies focus on the molecular regulation mechanism of litchi fruit abscission. Through the application of ethephon, ethylene has been proved to have an unequivocal promotive effect on litchi fruitlet abscission and increase the expression of LcPG1 encoding a pectin-degrading enzyme (Peng et al., 2013). On the other hand, there was circumstantial evidence that there had a higher fruit abscission rate and ACO (1-aminocyclopropane-1carboxylic acid oxidase) gene expression level in fruits treated by NAA (naphthalene acetic) spraying, suggesting a potential role in fruit abscission . Nevertheless, the comprehensive transcriptome-wide expression profiling analysis under ethylene-induced abscission has not yet been documented in litchi. In this experiment, we performed a genome-wide digital transcript analysis on fruit abscission zone (FAZ) enriched pedicel at 0, 1, 2, 3 d time points of ethephon treatment. Our results showed that a total of 6167 ethylene-regulated genes were preferentially expressed in ethephon-treated FAZ-enriched tissues. Among them, 2730 candidate genes were considered to be involved in ethylene-promoted fruit abscission process by further Gene Ontology (GO) and KEGG pathway enrichment analyses. It was demonstrated that a range of functional categories such as plant hormone synthesis and signaling, carbohydrate metabolism, TFs and cell wall modification, were highly regulated by ethylene. These results will provide a new insight of the ethylene regulatory fruit abscission molecular mechanism in litchi. Plant Materials and Treatment Nine 9-year-old litchi trees (L. chinensis Sonn. cv. Feizixiao) were randomly selected in an orchard located at South China Agricultural University in 2012 (Guangzhou, China), and blocked into three biological replicates of three trees each. At 25 d after anthesis, 20 fruit-bearing shoots (about 5∼8 mm in diameter) located in different directions from each tree were tagged. Ten of them were dipped in 250 mg L −1 ethephon solution (containing 0.05% Tween-80 surfactant) for 1 min, while the remaining 10 shoots dipped in water were used as control. Three out of ten treated shoots were used to monitor fruit abscission dynamic and the others were used for sampling. Samples were conducted at 0, 1, 2, and 3 d after treatment. Fruitlet and FAZ-enriched pedicels were collected immediately after the samples were taken back to laboratory on ice. FAZenriched pedicels were excised by cutting around 2 mm at each side of the abscission fracture plane (Supplementary Figure 1). After separation, all tissues were quickly frozen in liquid nitrogen and stored at −80 • C for future analysis. Determination of Fruit Abscission and Ethylene Production Rate of Fruit Cumulative fruit abscission rate (CFAR) was calculated according to our previous method (Kuang et al., 2012). Ethylene production was measured according to the method described by Yan et al. (2011) with some modifications. Two fruit from each treatment on each tree were collected and enclosed in a 30 mL airtight syringe equipped with a rubber piston for 2 h at 25 • C. Air within the syringe was forced into an airtight container filled with saturated salt water with a needled inserted to allow replacement. After all the samples were collected, 1 mL air sample was then withdrawn from the headspace of the container with a syringe and injected into a GC-17A gas chromatograph (Shimadzu, Kyoto, Japan) fitted with a flame ionization detector and an activated alumina column (200 cm × 0.3 cm). The injector temperature was 120 • C; the column temperature was kept at 60 • C and the detector temperature at 60 • C. Helium was used as carrier gas at a flow rate of 30 mL min −1 . The ethylene production rate was expressed as microliters of C 2 H 4 kg −1 h −1 . Digital Transcript Abundance Library Preparation and Illumina Sequencing Total RNA from FAZ-enriched pedicel was isolated using Column Plant RNAout 2.0 kit (TIANDZ, Inc, China). The quantity and quality of RNA samples were evaluated using 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Equal amounts of total RNA from three biological replicates were pooled to construct seven libraries named CK0, CK1, CK2, CK3, ETH1, ETH2, and ETH3. For example, CK1 and ETH1 were the libraries from pedicels harvested at 1 d after water and ETH treatment, respectively. After RNA extraction, mRNA purification by Oligo (dT), fragmentation, cDNA synthesis by random hexamer primers, size selection and PCR amplification were performed by BGI-Shenzhen as described previously . Data Analysis for Digital Transcript Abundance Profiles High-quality reads used for further downstream processing were filtered through the standard Illumina pipeline to remove the low-quality reads and those containing adaptor/primer contaminations. All clean reads were mapped back to the litchi genome (http://litchidb.genomics.cn, unpublished) using SOAPaligner (Version 2.21) allowing up to two nucleotide mismatches with the parameters of "-m 0 -x 1000 -s 28l 32 -v 2 -r 2, " which are specified on http://soap.genomics. org.cn/soapaligner.html. Clean reads mapped to reference, from multiple genes, were filtered and unambiguous clean reads were remained. For gene expression analysis, the number of unambiguous clean reads for each gene was calculated and normalized to RPKM (Reads Per Kilo base per Million reads) (Mortazavi et al., 2008). Six paired-libraries including CK0 vs. CK1, CK0 vs. CK2, CK0 vs. CK3, CK1 vs. ETH1, CK2 vs. ETH2, and CK3 vs. ETH3 were used to analyze the differential gene expression, according to the method described in Audic and Claverie (1997). Two filter criteria were used to identify differentially expressed genes (DEGs): a four-fold change in transcript levels and a FDR (False Discovery Rate) value ≤ 0.001 among every comparison. In order to eliminate the control background noise, take day 1 treatment data for example, we excluded the DEGs identified in CK0 vs. CK1 library from the DEGs identified in CK1 vs. ETH1 library, and the obtained data was recorded as ETH1/CK1. The other two time points were done as the same way, and the result was recorded as ETH2/CK2 and ETH3/CK3, respectively. Then the union of DEGs among ETH1/CK1, ETH2/CK2, and ETH3/CK3 was defined as ethephon-responsive genes. Finally, all ethephon-responsive genes were mapped to terms in GO and KEGG databases for functional and pathway-enrichment analysis. And those genes significantly enriched in GO term analysis (FDR ≤ 0.05) or enriched in KEGG pathway (Q-value ≤ 0.05) were screened to be the candidate genes involved in the fruit abscission process. Heat maps showing expression profiles were generated using the MultiExperiment Viewer (MeV, v4.9). Quantitative Real-Time PCR To validate the accuracy of our DTA profiles results, 41 randomly selected DEGs were evaluated by quantitative real-time PCR (qRT-PCR) after the ethephon treatment in litchi FAZ. The RNA samples for DTA analysis were also used for qRT-PCR. Genespecific primer sequences were designed using Primer Premier 5.0 and listed in Supplementary Data Excel File 1. Purified total RNA (2 µg) from each sample was reverse-transcribed to synthesize cDNA by ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). Then, the cDNA was amplified using SYBR Green-PCR master kit (THUNDERBIRD SYBR qPCR Mix, TOYOBO) and LightCycler 480_II Real-Time PCR System (Roche). The PCR amplifications included the following condition: 95 • C for 1 min, followed by 40 cycles of 95 • C for 5 s, 55 • C for 30 s and 72 • C for 30 s. Dissociation curves were run to determine the specificity of the amplification reactions. The data were normalized using cycle threshold (Ct) value corresponding to two litchi reference genes, EF-1α and GAPDH (Zhong et al., 2011). The relative expressive level of the target genes were calculated using the Ct method. Duplicates from three biological replicates were used. Changes in Fruit Abscission Rate and Ethylene Production CFAR and ethylene production in fruitlet were compared between the control and ethephon (ETH) treatment (Figure 1). The CFARs showed similar trends (Figure 1A), which gradually increased in the first day and had no visible difference. Two days after treatment, the CFAR in ETH-treated fruitlet was significantly higher than that in the control. Consequently, 100% of the fruitlet abscised by 4 d after ETH treatment, compared with a ∼50% loss in the control, indicating that ETH treatment significantly accelerated fruitlet drop. In addition, a clear impact on ethylene production was also observed in ETH-treated fruitlet. Within 3 days of observation, ethylene production in the control fruitlet remained more or less flat and kept below 8 µl kg −1 h −1 . While ethylene production in the ETH-treated fruitlet increased rapidly and continuously, and achieved nearly a ten-fold higher level at day 3 than the control ( Figure 1B). The increase in ethylene production suggested that ethephon application probably accelerates the fruit drop following the induction of ethylene production in fruitlet. Digital Transcript Abundance Profile Analysis To explore the transcriptional changes of litchi FAZ-enriched pedicel in response to ethephon treatment, seven digital transcript abundance (DTA) tag profiles of the control (CK0, CK1, CK2, and CK3) and ETH-treated samples (ETH1, ETH2, and ETH3) were sequenced ( Table 1). After quality filtering, nearly 82 million clean reads were generated from the above libraries (10-12 million reads for each library). The tag sequences of the seven libraries were mapped to the litchi genome, and 87.11, 86.93, 86.69, 87.20, 87.53, 87.37, and 86.52% of all clean reads were obtained, respectively. These results showed that both the throughput and sequencing quality were high enough for further analysis. Transcirptome Responses during Fruit Abscission After comparing three paired-libraries (CK1 vs. ETH1, CK2 vs. ETH2, and CK3 vs. ETH3), a total of 6167 genes were found to be significantly up-or down-regulated which named as ethephon-responsive genes (Supplementary Data Excel File 2). Based on GO enrichment analysis, 3249 genes (53%) were divided into the three principal GO organization categories (Supplementary Figure 2A): biological process (2437 genes), cellular component (1459 genes) and molecular function (2825 genes). The enriched GO terms included carbohydrate metabolic process, photosynthesis, extracellular region, cell wall, thylakoid, catalytic activity and oxygen binding. KEGG pathway enrichment analyses showed that a total of 3420 genes were assigned to the 118 related pathways, and the top 20 enriched pathways including plant hormone signal transduction (ko04075) and starch and sucrose metabolism (ko00500) were illustrated in Supplementary Figure 2B. Analysis of the Candidate Genes Involved in the Fruit Abscission Process A total of 2471 and 2344 genes were selected in GO (FDR ≤ 0.05) and KEGG enrichment analyses (Q-value ≤ 0.05) with a significant level, respectively. After eliminating duplicated, 2730 genes were identified as the candidate genes involved in the fruit abscission process regulated by ethephon (Supplementary Data Excel File 3). The up-and down-regulated genes accounted for 37.44% (1022 genes) and 62.56% (1708 genes) of them, respectively. Based on their patterns of expression, these candidate genes could be classified into four groups, which consisted of genes with similar temporal patterns of expression kinetics (Figure 3). Group I included 1867 early-responsive genes whose expression were up-or down-regulated early at 1 d after treatment; Group II had 148 middle-responsive genes whose expression were not induced or suppressed until 2 d after treatment; Group III contained 258 late-responsive genes that were not regulated until 3 d after treatment; Group IV consisted of 457 constant-responsive genes that up-or down-regulated early and whose expression was maintained constant during the treatment. By hierarchical cluster analysis, each group could be subsequently divided into two to six clusters, for example, Group I included cluster 1A, 1B, 1C, 1D, 1E, and 1F which had 172, 261, 55, 776, 434, and 169 genes, respectively. In total, 723 up-regulated and 1601 down-regulated genes were found at 1 d after treatment, and 299 up-regulated and 107 down-regulated genes were found at 2 d or 3 d after treatment. These results showed that the majority (85.13%) of those candidate genes made a quick response to the ETH treatment in 24 h when no significant difference on fruit abscission rate was found between the control and the ETH treatment. Except 101 genes encoding proteins of unknown functions, the other 2629 genes had unambiguous annotations. Those genes related to plant hormones, cell wall metabolism TFs, carbohydrate metabolism, ROS and calcium signaling were further analyzed as follows. Genes Related to Plant Hormone Biosynthesis and Signaling Pathway A total of 195 candidate genes were found related to plant hormone biosynthesis and signaling pathway (Figure 4, Supplementary Data Excel File 4). Of these, 124, 16, 17, and 38 genes belonged to the Group I, Group II, Group III, and Group IV, respectively. Sixty and fifty-five genes were related to auxin and ethylene, among them, 47 auxin-related genes were down-regulated and 39 ethylene-related genes were up-regulated. These genes should be closely associated with fruitlet abscission, including those encoding auxin efflux carrier component (PIN), auxin influx carrier (AUX1), AUX/IAA protein, auxin response factor (ARF), SAUR family protein, GH3 protein, 1-aminocyclopropane-1-carboxylate oxidase (ACO), 1-aminocyclopropane-1-carboxylate synthase (ACS), AP2/ERF transcription factor and ethylene receptor (ETR), et cetera. Twenty-eight gibberellins (GA) related and 18 cytokinin-related genes were found, most of them were down-regulated, such as genes encoding gibberellin 20 oxidase (GA20ox), gibberellin receptor GID1, cytokinin hydroxylase (CYP735A), histidine kinase (AHK) and two-component response regulator (AARA), etc. . . Eight of eighteen abscisic acid (ABA) related genes were upregulated including those encoding zeaxanthin epoxidase (ZEP), abscisic acid receptor (PYR/PYL) and protein phosphatase 2C (PP2C), and the repressed genes mainly encoded 9cis-epoxycarotenoid dioxygenase (NCED), carotenoid cleavage dioxygenase (CCD), and abscisic acid 8 ′ -hydroxylase (CYP707). Moreover, six salicylic acid (SA) related genes encoded with salicylic acid-binding protein (SBP) and regulatory protein NPR1 were highly up-regulated, and five jasmonic acid (JA) related genes mainly encoded allene oxide cyclise (AOC) and jasmonate ZIM domain-containing protein (JAZ) were decreased. These results suggested that seven classes of plant hormones were involved in the process of fruitlet abscission induced by the ETH treatment. The most important hormones were ethylene and auxin, followed by GA, cytokinin and ABA. JA and SA ranked the last according to the number of DEGs. Genes Encoding for Transcription Factors Except for 58 genes putatively encoding TFs related to plant hormones, there were 127 candidate TFs belonged to diverse families including ABI3/VP1, bHLH, BLH1, bZIP, GRAS, HD-ZIP, HSF, KNOX, LBD, MADS-box, MYB, NAC, WRKY, Trihelix, and zinc finger (Figure 5, Supplementary Data Excel File 5). Of these, 51 and 76 genes were up-and down-regulated, respectively. And 78, 9, 14, and 26 belonged to Group I, Group II, Group III and Group IV, respectively. Remarkably, 71 out of 114 TFs in Group I and Group IV were down-regulated, indicating that most TFs were repressed at 1 d after the ETH treatment. There were more than 10 members in those families including bHLH, MYB, WRKY, NAC, LBD, and HD-ZIP. Most members of bHLH and HD-ZIP families were down-regulated, while 13 of 15 members in the family of WRKY were up-regulated, and all members in the families of BLH1, MADS-box, KNOX, and Trihelix were down-regulated. Moreover, 33 of the 51 up-regulated TFs were induced at 1 d after treatment, and the largest TF family was the WRKY (10 genes), followed by MYB (6 genes) and NAC (5 genes), implying that those genes from these TF families could be related with triggering the transcriptional chain reaction during ETH-induced abscission. Genes Related to Cell Wall Biosynthesis, Degradation, Loosening, and Modification A total of 208 genes including 56, 104, 21, and 27 genes related to cell wall biosynthesis, degradation, loosing and modification were found, respectively (Supplementary Data Excel File 6). Of these, 72 and 136 genes were up-and down-regulated, respectively. Of the up-regulated genes, there were 51 cell wall degradation, loosing and modification related transcripts FIGURE 3 | Ethephon-responsive genes expression pattern obtained by kinetics-based clustering analysis. Group I, cluster of genes with early and transient changes; group II, clusters of genes modified in their expression until 2 d after treatment; group III, cluster of genes with expression kinetics exhibiting late changes; group IV, cluster of genes with persistent changes during the whole abscission process. The + and − signs in bracket represent up-and down-regulated of genes, respectively, while 0 represents no change. The numbers on the right of bracket indicate the total numbers of genes in each cluster. All of these changes were based on a four-fold change criterion (log2 ratio) indicated by blue dotted line. Gray dotted line indicates the gene-expression levels and the average values of gene-expression level in clusters is shown by the red solid lines. (Figure 6). Among them, 4 genes were related to callose degradation, like endo-1,3-β-glucosidases (ENGs) and β-1,3glucanases (BGN13s); 5 genes were involved in cellulose degradation, such as endo-1,4-β-D-glucanases (CELs) and βglucosidases (BGLUs); 11 genes like endo-1,4-β-mannosidase (MAN), xyloglucan endotransglucosylase/hydrolases (XTHs) and β-D-xylosidases (BXLs) were related to hemicellulose degradation; 13 genes such as polygalacturonases (PGs) and pectate lyase (PLs) were associated with pectin degradation; 12 expansins (EXPs) related to cell wall loosening and 6 pectinesterase/pectinesterase inhibitors (PE/PEIs) genes were involved in cell wall modification. Of the down-regulated genes, there were 35 cell wall biosynthesis-related genes (Figure 6), including those encoding cellulose synthase, extensin, glycinerich cell wall structural protein, UDP-glucuronate 4-epimerase and xyloglucan glycosyltransferase. There were not surprised that those genes encoding enzymes mentioned above may be involved in the process of fruitlet abscission induced by ethephon. However, 101 down-regulated genes related to cell wall degradation, loosening and modification, and 21 up-regulated genes related to cell wall biosynthesis were also found in our study. Genes Related to Photosynthesis, Carbohydrate, and Energy Metabolism The expression of over 93% (103 out of 110 genes) genes involved in photosynthesis pathway were strongly decreased, especially repressed exclusively at 1 day after the ETH treatment, and most of them belonged to Group I (Supplementary Data Excel File 7). The affected genes function in chlorophyll biosynthesis, thylakoid formation, chlorophyll a/b binding protein, light harvesting (PSI and PSII), electron transport and carbon fixation. These results suggested repressed expression of those encoding for chloroplast and light harvesting associated proteins by ethephon might lead to the process of chloroplast malfunction and photosynthesis inhibition. Not surprisingly, the down-regulated photosynthesis related genes are linked with changes in the expression of genes in carbohydrate metabolism. A total of 137 candidate genes were found in this section (58 up-regulated and 79 down-regulated, Supplementary Data Excel File 7). Overall, 24 out of 32 genes involved in glycolysis and all members of the TCA cycle (7 genes), such as alcohol dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase and ATP-citrate synthase, were strongly downregulated within Group I, indicating that the degradation of sugar was inhibited during the early ETH-induced abscission. However, an increased expression of seven genes associated with fructose and mannose degradation and four genes involved in galactose hydrolysis were also found during the late induction of abscission. These results suggested that sugar degradation was inhibited during the early induction but increased during the late induction. Moreover, our data also showed that 13 out of 17 genes encoding ATPase were repressed after the ETH treatment, especially down-regulated exclusively at 1 day post-treatment ( Supplementary Data Excel File 7). These results indicated that ethephon might lead to the interdiction of ATP synthesis and the high energy supply. Genes Related to ROS Response, Calcium Signal Transduction, and Protein Ubiquitination A total of 88 transcripts belonging to the reactive oxygen species (ROS) response were preferentially regulated in the ETH-treated FAZ-enriched pedicels (Supplementary Data Excel File 8). Of these, 39 genes were up-regulated and the other 49 genes were down-regulated. Among the up-regulated genes (Figure 6), we found eight respiratory burst oxidase homolog proteins (Rboh) and one peroxisomal-(S)-2-hydroxy-acid oxidase (GLO) involved in ROS production, indicating a high ROS content might be produced in FAZ. Not surprisingly, 27 genes putatively encoding ROS scavenging enzymes of diverse families showed increased expression, and the most abundant transcripts encoding glutathione S-transferase (GST) and peroxidase (POD). However, the down-regulated ROS scavenging genes were also found, indicating that a competing activity occurred in the regulation of fruit abscission. In addition, 20 genes encoding the key enzymes related to ascorbate synthesis and metabolism, including L-ascorbate oxidase (AO) and inositol oxygenase (MIOX), showed an immediate decreased regulation at the 1 day post-treatment. As an important signaling molecular, 52 transcripts related to calcium transport and perception displayed altered changes (Figure 6, Supplementary Data Excel File 8). Among them, 19 and 33 genes were up-and down-regulated respectively, and 46 of them belonged to Group I, indicating that most genes had instantaneous response to the ETH treatment. For example, two calcium influx transporters, CNGC (13 out of 18 genes) and ANX (2 genes), showed strongly decreased expression during the early induction. In contrast, 4 out of 6 transcripts encoding calcium efflux carrier proteins (PMCAs) were increased. These results indicated that a role regulating calcium influx and maintaining calcium level in the FAZ may be associated with the beginning of abscission process. Moreover, two calcium responsive genes, CML (6 out of 10 genes) and CDPK (3 genes), were also repressed during the early induction. A total of 40 genes involved in protein ubiquitination were found (Figure 6, Supplementary Data Excel File 8), and 32 of them belonged to Group I and IV, indicating that most genes had a quick response to the ETH treatment. Among them, 26 genes were up-regulated, including those encoding 26S proteasome, cullin, E3 ubiquitin-protein ligase, EIN3-binding f-box protein, U-box domain-containing protein and ubiquitin-conjugating enzyme E2. However, 14 down-regulated genes involved in protein ubiquitination were also found. Discussion Ethephon (2-chloroethylphosphonic acid) is a synthetic plant growth regulator discovered several decades ago, which acts by releasing ethylene when it penetrates plant tissues (Royer et al., 2006). The application of exogenous ethylene inducing fruit abscission has been observed in apple (Yuan, 2007;Kolarič et al., 2011), sweet orange (John-Karuppiah and Burns, 2010), sweet cherry (Smith and Whiting, 2010) and olive (Zahra, 2014). The data herein presented strengthen the direct observation that ethephon aggrandizes both the fruitlet abscission and ethylene FIGURE 6 | Expression profiling of genes related to cell wall modification, ROS response, calcium signaling transduction, and protein ubiquitination in the FAZ-enriched pedicel after ethylene treatment. Up-regulated genes involved in cell wall degradation, cell wall loosening and ROS response, and down-regulated genes related to cell wall biosynthesis were showed. All genes involved in calcium signaling transduction and protein ubiquitination were exhibited. Gene expression levels were indicated with color bars. Additional information was presented in Supplementary Data Excel File 4. Frontiers in Plant Science | www.frontiersin.org evolution in litchi. It is speculated that the mechanism of fruitlet abscission after treatment with exogenous ethephon may relate to the action of ethylene. However, a deep knowledge of the molecular events occurring in FAZ during fruit abscission induced by ethephon is still unknown. Although our previous study cloned a LcPG1 gene and found its expression in FAZ was paralleled with the alteration of fruitlet abscission in litchi, induced by the ethephon treatment and inhibited by spraying 2,4-dichlorophenoxyacetic acid (2,4-D) (Peng et al., 2013). So this work mainly focused on genome-wide mining putative fruit abscission related genes regulated by ethylene in litchi. Litchi fruit growth could be divided into two stages (Li et al., 2003). The first Stage constitutes about two thirds of the whole fruit growth cycle, which is the phase mainly characterized by the growth of pericarp and seedcoat; and the second Stage is the phase mainly characterized by the growth of embryo and the rapid aril growth. In the case of "Feizixiao" litchi used in our study, it needs 70-75 days from female flowering to a mature fruit, which can fluctuate some depending on the female opening date (Li et al., 2004). Fruit weight is about 25-30 g at maturity, but it is approximately 1.0 g when fruit develops at 25 days post anthesis (DPA) which is the time of the ETH treatment in this study. There were three to four waves of physiological fruit drop throughout fruit development in 70-90 days depending on cultivars (Yuan and Huang, 1988). Wave I, wave II, and wave III of abscission occurred around 1 week, 3 weeks and 6 weeks after anthesis, respectively. For a normal inflorescence of "Feizixiao" litchi, there are about 500-800 female flowers. Only 10% of them may set fruit successfully at 1 week after anthesis (wave I), and 30-50% of the surviving fruitlet will drop during wave II, after which no abscission will occur for the next 2-3 weeks. This study focuses on fruitlet abscission occurred at the second wave. The first 3 days after treatment might be coincidence with the peak of fruit drop wave II. It is not surprising that ∼50% of the control fruitlet abscised between 25 and 28 DPA. Moreover, after 28 DPA, the remaining 50% of the fruitlet in the control treatment did not abscise over the next 2-3 weeks. ETH treatment, however, induced abscission of 100% of the fruitlet by 28 DPA. Thus, the ETH treatment largely magnified the second physiological fruit drop, which inducing a significantly higher rate of fruitlet abscission. This is exactly the biological effect expected for ETH. Moreover, ethylene production in fruit between the control and the ETH treatment had the substantial difference. Ethylene production in the control fruitlet remained more or less flat and kept below 8 µl·kg −1 ·h −1 in the period of treatment, while which in ETH-treated fruitlet increased rapidly at 1 d after treatment and achieved nearly a ten-fold higher at 3 d when compared with the control. When the pH is above 4.0, ethephon slowly decomposes to release ethylene. The increase in ethylene production was the results of endogenous synthesis and ethephon release, which probably accelerates the fruit drop. In addition, the CFAR had no difference with the control in the first day after the ETH treatment, after then, the CFAR was sharply increased and significantly higher than that of the control. There were two obvious stages during the fruitlet abscission induced by ethephon: the early induction (0-1 d after treatment) that might induce acquisition of ethylene sensitivity and abscission competence, and the late induction (1-3 d after treatment) that might lead to the execution of fruitlet abscission and formation of the defense layer. A total of 6167 significantly DEGs were screened as ethyleneresponsive genes and 2730 of them were identified as candidate genes involved in the fruitlet abscission process. Over 85% of the candidate genes displayed a significant transient change during the early ethylene-induction. It is generally accepted that ethylene operates as an activator, while auxin act as retardants (Roberts et al., 2002). In agreement with this supposition, 115 out of 195 candidate hormone related genes were involved in biosynthesis and signaling pathway of ethylene and auxin. These evidences were supported by the high expression levels of ethylene signal pathway related genes such as ETR2, EBF, EIN3/EIL and a class of ERF TFs, as previously demonstrated by John-Karuppiah and Burns (2010) in sweet orange fruit and leave abscission zone; and the repressed expression of transcript levels for auxin polar transport carriers (PIN and AUX1) and auxin responsive genes (TIR1, Aux/IAA, ARF, and SAUR). A decline in the abundance of auxin efflux carrier might be responsible for fruitlet abscission induced by shading and NAA in apple (Zhu et al., 2011) and mature-fruit abscission in melon (Corbacho et al., 2013). Moreover, Zhu et al. (2011) found that genes involved with cytokinin and gibberellic acid (GA) signaling pathways were down-regulated by shading and NAA in apple fruitlet FAZ. Similarly, a strongly decreased expression of large number of genes related to cytokinin and GA biosynthesis and signaling, such as CYP735A, AHK, AHP, GA20ox, and GID1, were also found in our study, probably implying that the metabolism of the cytokinin and GA in FAZ were affected in the early ethephon-promoted abscission process. On the other hand, it has been proposed that ABA and JA might be correlated with the abscission activation in citrus fruitlet (Gomez-Cadenas et al., 2000) or leaves (Agustí et al., 2009). They exhibited exactly the opposite results on mature fruit abscission, six of eight DEGs involved in ABA biosynthesis were up-regulated in melon (Corbacho et al., 2013), and six out of the seven DEGs were down-regulated in olive (Gil-Amado and Gomez-Jimenez, 2013). Our result was quite different from them, half of the eight DEGs involved in ABA biosynthesis showed increased transcript abundance during the ethephon-promoted fruitlet abscission in this study. All together, the mentioned above plant hormones were involved in the process of fruitlet abscission induced by the ETH treatment and the most important hormones were ethylene and auxin. TFs are concerned as major switches of regulatory cascades during development, and the changes in the expression of such genes may affect various biological processes (Riechmann et al., 2000). A total of 185 different TF genes transcript such as AP2/ERF, Aux/IAA, bHLH, MYB, WRKY, NAC, LBD, and HD-ZIP, were affected by the ETH treatment. Those thought to be directly involved in ethylene and auxin signal transduction, such as AP2/ERF, Aux/IAA, and ARF, were already discussed before. The expression of most genes belonging to the family of bHLH was sharply down-regulation, and it was consist with the reports in the flower abscission zone in tomato after flower removal (Meir et al., 2010). Other up-regulated TFs in our work such as NAC and WRKY, were previously reported to be involved in mature fruit abscission in melon and olive (Corbacho et al., 2013;Gil-Amado and Gomez-Jimenez, 2013). Many reports have shown that numerous characterized NAC and WRKY genes are involved in response to environmental stimuli and play various roles in response to biotic and abiotic stress (He et al., 2005;Jensen et al., 2010;Zhao et al., 2012). Thus, these up-regulated NAC and WRKY genes might be similar to ethylene-or stressinduced TFs found in other species (Yang et al., 2009;Jensen et al., 2010) and putatively involved in the downstream of ethylene signaling. Concerning MYB, Corbacho et al. (2013) reported that MYB was the most represent up-regulated TFs during the late mature-fruit abscission in melon, while only 7 members of the 15 affected MYB genes were induced in litchi. All these differential expression of genes encoding TFs belonging to different families might act as early regulators of the abscission induction, but the exact roles of these regulatory factors responding to ethylene induction remain to be further investigated. ROS are versatile molecules related to a wide range of cellular processes, including programmed cell death, development, and hormonal signaling (Kwak et al., 2006). Previously, some reports supported a link between ROS and abscission. In tobacco, Henry et al. (1974) found that POD activity was increased during the ethylene-induced pedicel abscission. In tomato, delayed abscission of flowers and fruits was related to the increase of ROS-scavenging enzymes (Djanaguiraman et al., 2004). In pepper, Sakamoto et al. (2008) reported that H 2 O 2 was involved in stress-induced petioles abscission, indicating that H 2 O 2 acts downstream abscission signaling from ethylene. In ethylenetreated citrus leaves, a set of transcripts belonging to the ROS scavenging machinery have been reported to be over-represented in petioles rather than the laminar abscission zone (Agustí et al., 2008(Agustí et al., , 2009. Results in this study showed that a number of Rboh (role in ROS production) and genes encoding ROS scavenging enzymes were induced, suggesting the burst of ROS caused by ethephon treatment. Calcium has been considered as an important intracellular messenger in plants and is essential for the maintenance of structural integrity of biomembrane and cell wall (Poovaiah and Rasmussen, 1973), and is also required for a variety of ethylene-dependent abscission processes (Raz and Fluhr, 1992). Xu et al. (2009) reported that direct application of calcium on tomato pedicel explants under ethylene would accelerate abscission but there are a number of reports describing that calcium delayed organ abscission (Poovaiah and Leopold, 1973;Beyer and Quebedeaux, 1974;Iwahori and Van Steveninck, 1988). In fact, Poovaiah and Rasmussen (1973) showed that ethephon treatment for bean leaf explants decreased calcium level in the AZ just prior to separation. The role of this element in organ abscission is still controversial. But how does the calcium signaling communicate with the fruit abscission? Our results showed that ethephon treatment up-regulated genes encoding calcium efflux carrier proteins (PMCA), down-regulated different genes encoding calcium influx carrier proteins such as cyclic nucleotide-gated channel (CNGC) genes and calcium responsive genes (CML and CDPK) in the litchi FAZ-enriched pedicel. These molecular results strongly suggested that regulating calcium influx and maintaining calcium level in the FAZ might be associated with the onset of ethephon-promoted litchi fruitlet abscission process. It is speculated that ethylene might lead to a high extracellular calcium level in FAZ, and result in the deposition of calcium on cell wall and the deficiency on the cell inside. Ubiquitylation-dependent proteolysis is a major event during both the induction and execution of cell death (Estelle, 2001), and could be triggered by H 2 O 2 in tobacco (Vandenabeele et al., 2003). In ethylene-treated citrus leaves, a group of transcripts involved in the ubiquitin/proteasome system have been reported to be induced in both laminar abscission zone and petiolar cortical tissue (Agustí et al., 2008(Agustí et al., , 2009). The up-regulation of both ubiquitin-conjugating enzymes (E2) and ubiquitin-protein ligases (E3) after the ETH treatment in litchi FAZ-enriched pedicel, suggested that a similar proteolytic mechanism might be involved in ethephon-induced abscission. Also, two 26S proteasome components and a large number of proteins with Fbox and U-box domain like potential E3 ligases were induced by ethephon. In Arabidopsis, abscission of floral organs is arrested with suppressed expression of a F-box protein (González-Carranza et al., 2007). These observations strongly suggested that a general proteasome-related mechanism might play a role in ethephon-induced abscission. Zhu et al. (2011) found both shading and NAA treatment for apple tree resulted in a large number of photosynthesisrelated genes were down-regulated in the FAZ. For instance, 90 out of the 94 DEGs were repressed in shading-treated FAZ. In our study, 103 out of the 110 DEGs involved in photosynthesis pathway were strongly decreased. These results also indicated that photosynthesis was one of the GO terms enriched in FAZ. The structural features of AZ cells were described by Sexton and Roberts (1982) as densely protoplasmic, with small intercellular spaces, containing large deposits of starch, and with a high density of branched plasmodesmata. Then, AZ cells should not contain photosynthetically active chloroplasts. However, handdissected AZ-enriched young and green fruitlet pedicle (see Supplementary Figure 1) used in our study should contain photosynthetically active chloroplasts, which is why so many down-regulated of photosynthesis-related genes were found. In order to uncover the abscission-associated metabolism of AZ cells we should take a laser capture microdissection (LCM) approach to obtain AZ-specific cells sample for performing an accurate study of the abscission events in the future. Thus, the roles of these genes on fruit abscission need to be further evaluated. Some evidences supported a strong connection between the carbohydrate amounts available for the fruit and their probability of abscission (Yuan and Huang, 1988;Iglesias et al., 2003;Zhou et al., 2008). One of the reasons of abscission may be due to a lack of carbohydrate. Not surprisingly, ETH treatment also affected the carbohydrate and energy metabolism. Affected genes within these groups include those associated with glycolysis, TCA cycle and ATPase. These results indicated that ethephon might lead to the carbohydrate stress and the interdiction of ATP synthesis during the early abscission induction. Moreover, an increased ROS production may be linked to the inhibition of ATPase, as a consequence to the mitochondrial damage (Roy et al., 2008), and the release of cytochrome c from mitochondria, which have been implicated as regulator of programmed cell death (Tiwari et al., 2002). After the early induction, there was a lot of fruitlet dropped in paralleled with a continuous highly release of ethylene yield during the late induction (1-3 d after ETH treatment). Ethylene may act as the signal generated within the fruit, FAZ, or released from ethephon, through diffusion, triggering abscission event. It was supported by the abundant of several transcripts (ACS, ACO, ERF) involved in ethylene biosynthesis and transductive pathway. Several up-regulated genes were linked to ABA biosynthesis and signaling transduction, such as ZEP and PP2C. It is proposed that ABA might be corrected with the late stage of abscission process. Similarly, ethylene appeared to be a positive regulator of SA action during the abscission induction, since the expression level of SBP and NPR1 genes were highly up-regulated, as found during melon mature-fruit abscission (Corbacho et al., 2013). It assumed that SA might be as an endogenous signaling molecule for stress response or the formation of protective layer after AZ cell separation. Homologs of LBD and WRKY TFs were also highly expressed at 1-3 d after ETH treatment, showing similar change to that reported in tomato flower abscission zone . The LBD family played a possible role in lateral meristem initiation (Shuai et al., 2002), while the WRKY family hold central positions mediating the regulation of disease resistance (Robatzek and Somssich, 2001). These TFs might have functions in stress defense and the formation of protective layers after fruitlet abscission. For fruit to be shed, cell separation must occur in FAZ, and the abscission is paralleled with intercellular space increase and middle lamella lysis in the FAZ which is the result of the degradation and/or remodeling of cell wall (Lee et al., 2008;Bowling and Vaughn, 2011). There were 43 cell wall degradation and loosing related transcripts up-regulated and 29 cell wall biosynthesis related genes down-regulated, and most FIGURE 7 | The possible molecular events to control the ethephon-promoted litchi fruitlet abscission based on expression data obtained from DTA analysis. Gene expression levels were indicated with color bars: red (up-regulated) and green (down-regulated). of them had significantly differential expression throughout the whole ethephon treatment, indicating that these genes might be involved in the process of fruitlet abscission induced by ethephon. Several researchers have already reported that the expression of genes encoding for cell wall-hydrolyzing enzymes was associated with abscission and also regulated by ethylene (Lashbrook et al., 1994;Kalaitzis et al., 1997;Roberts and Gonzalez-Carranza, 2009). However, 92 down-regulated genes related to cell wall degradation and loosing, and 15 up-regulated genes related to cell wall biosynthesis were also found in our study, which might be involved in the new cell wall synthesis and reconstruction for the formation of protective layers after fruitlet abscission. This study only focus on the gene expression profile occurring in the FAZ-enriched pedicel during litchi fruit abscission induced by ethephon. In conclusion, a total of 2730 candidate genes were involved in the process of litchi fruit abscission induced by ethephon treatment. A preliminary molecular regulatory scheme was herein prompted out for litchi fruitlet abscission induced by ethephon based on our results (Figure 7). At the early beginning, the ethylene evolution in fruitlet was greatly increased by ETH treatment, which would suppress the synthesis and polar transport of auxin and trigger abscission signaling. At the same time, FAZ might perceive the abscission signals, and then, 1867 early-responsive genes were up-or down-regulated from 0 to 1 d after ETH treatment. The most affected genes included those related to ethylene biosynthesis and signaling, auxin transport and signaling, TFs, protein ubiquitination, ROS response, calcium signal transduction and etc. . . Then, a lot of genes related to cell wall degradation, TFs, ethylene, ABA and JA biosynthesis and signaling cascade were upregulated. At the last, cell separation happened and the fruitlet abscission was enhanced. To the best of our knowledge, this study provides the first global monitoring of gene expression changes occurring in FAZ-enriched pedicel during litchi fruit abscission. Author Contributions JL was responsible for the overall concept and experimental design, and revising and finalizing the manuscript. CL carried out ethephon treatment, DTA data integration and analysis, performed qRT-PCR experiments, and drafted the manuscript. YW was responsible for bioinformatics analysis. PY and WM performed the ethephon treatment and sample collection. All the authors read and approved the final manuscript.

v3-fos

2018-04-03T03:12:01.134Z

2015-08-27T00:00:00.000Z

978088

s2

Effects of ascorbic acid on α-l-arabinofuranosidase and α-l-arabinopyranosidase activities from Bifidobacterium longum RD47 and its application to whole cell bioconversion of ginsenoside Bifidobacterium longum RD47 was cultured in 24 kinds of modified MRS broths containing various ingredients to select the most promising source that induces microbial enzymes. Among the various ingredients, ascorbic acid significantly enhanced α-l-arabinofuranosidase and α-l-arabinopyranosidase activities in Bifidobacterium longum RD47. Addition of 2 % ascorbic acid (w/v) to MRS showed the maximum enzyme activities. Both whole cell and disrupted cell homogenates showed efficient ρ-nitrophenyl-β-d-glucopyranoside and ρ-nitrophenyl-β-d-glucofuranoside hydrolysis activities. The initially enhanced α-l-arabinopyranosidase and α-l-arabinofuranosidase activities by ascorbic acid were maintained over the cell disruption process. The optimal pH of α-l-arabinofuranosidase and α-l-arabinopyranosidase was 5.0 and 7.0, respectively. Both enzymes showed the maximum activities at 40.0 °C. Under the controlled condition using Bifidobacterium longum RD47, ginsenoside Rb2, and Rc were converted to ginsenoside Rd. Introduction Panax ginseng (Panax ginseng C. A. Meyer) has been widely regarded as an important oriental plant medicine in East Asia since cultivation started around 11 BC (Jia and Zhao 2009). Ginseng contains various phytochemicals such as polyacetylenes, polyphenolic compounds, and ginsenosides (saponins). Among these phytochemicals, ginsenosides generally exhibit the pharmacological and nutraceutical effects of ginseng (Nagai et al. 1972;Karikura et al. 1991). As reported by Tawab et al. (2003), the conversion of ginsenosides into deglycosylated form is crucial for its in vivo biological activity. Various methods (e.g., chemical treatment, mild acid hydrolysis, and alkaline cleavage) have been developed as ways to convert ginsenosides into deglycosylated form (Han et al. 1982;Bae et al. 2003;Ko et al. 2003). However, these methods produce significant amount of by-products (e.g., epimerization, hydration, and hydroxylation) (Chen et al. 1987;Elyakov et al. 1993;. In order to resolve these problems, numerous studies in both academia and industry use probiotic enzymes to transform ginsenosides into aglycones (e.g., Hasegawa et al. 1997;Bae et al. 2000;Ko et al. 2000;Ko et al. 2003;Bae et al. 2004). For example, several Korean food and pharmaceutical conglomerates have applied for patents to achieve ginseng market dominance over the last several years (Table 1). Nowadays, the size of the Korean ginseng market is estimated to be $1.14 billion (Baeg and So 2013). In order to convert ginsenoside Rc and/or Rb2 into Rd, a-L-arabinofuranosidase (Abf) and a-L-arabinopyranosidase (Abp) have been cloned in Escherichia coli An et al. 2012). However, from a marketing and food safety point of view, using genetically modified organism and E. coli has practical limitations for use in the food industry. Several studies have shown that the addition of specific nutrients can considerably change microbial enzyme activities; in this case, the induced enzyme can be applied to ginsenoside conversion (Crociani et al. 1994;Degnan and Macfarlane 1995;Salyers et al. 1977;Tzortzis et al. 2003;Hsu et al. 2005;Ku et al. 2011). Herein, we aim to show the optimal condition (i.e., the concentration of ascorbic acid and ginseng extract, temperature, cell disruption step, and pH) to improve Abf and Abp activities in Bifidobacterium longum RD47 (BL47). The induced Abf and Abp were applied to convert ginsenoside Rb2 and Rc into Rd. Materials Panax ginseng roots were purchased from a local grocery store in Korea. Chiro-inositol and pinitol were provided by Amiocogen Co., Ltd. (Korea). Acetonotrile, methanol, and water were purchased from J. T. Baker Ò (USA). Ginsenoside standard Rb2, Rc, and Rd were purchased from BTGin Co., Ltd. (Korea). Yeast extract, proteose peptone, beef extract and deMan, Rogosa, Sharp (MRS) media were purchased from Becton, Dickinson and Company (BD) (USA). Glucose-free MRS was formulated according to the manual of microbiological culture media (Difco TM and BBL TM Manual 2009;Ku et al. 2009Ku et al. , 2011. The glucosefree MRS contained 10 g proteose peptone, 10 g beef extract, 5 g yeast extract, 1 g polysorbate 80, 2 g ammonium citrate, 5 g sodium acetate, 0.1 g magnesium sulfate, 0.05 g manganese sulfate, and 2 g dipotassium phosphate in 1 l of distilled water. The pH of the broth was 6.5 ± 0.2 at 25°C and 2 % of agar was added if needed. Cell growth condition In order to select a promising nutrient for the enzyme induction, various modified MRS broths containing different carbon sources were designed ( Table 2). The pH of all broths was adjusted to 7.0 via the addition of sodium hydroxide, and all the broths were sterilized using 0.2 lm A method of preparation for fermented red ginseng using conversion by enzyme mixture and fermentation by lactic acid bacterium and the products containing fermented red ginseng manufactured thereof as effective factor 13 June 2014 Daesang Corp. A novel strain of kimchi lactic acid bacteria having ginsenoside Rg3 enrichment activity and methods for preparing fermented ginseng using the strain syringe Ersatz-Membranfilter (BRAND Ò , Germany). After two successive transfers in the MRS broth, 1 % (v/v) of activated BL 47 was inoculated into each modified MRS broth and grown anaerobically at 37°C for 18 h. The viable cell counts were determined by plating on MRS containing 2 % agar (BD, USA) under anaerobic conditions. Cell growth rates were measured optically using a spectrophotometer at 600 nm (Model Benchmark, Bio-Rad, Japan) ( Table 2). Enzyme assay Enzyme activities were measured for three different samples: whole, lysed, and disrupted cells. Whole cell suspension was prepared as previously described (Park et al. 2012). Cell lysis step was carried out to extract microbial enzyme from the whole cell using lysis solution as described in Ku et al. (2011). Disrupted cell suspension was prepared by the cell sonicator set at 45 amplifications for 3 min at 4°C. For the enzyme reaction, 5 mM of qnitrophenyl-b-D-glucopyranoside (pNPP), and q-nitrophenyl-b-D-glucofuranoside (pNPF) (Sigma, St. Louis, Mo., U.S.A.) were used. The released pNP was measured at 405 nm (Model Benchmark, Bio-Rad, Japan) after enzyme reaction at 37°C. Enzyme activity was evaluated using the following equation: Determination of the optimal enzyme condition (ascorbic acid concentration, ginseng extracts concentration, pH, cell disruption time, and temperature) To determine the optimal concentration of ascorbic acid, BL47 was anaerobically grown in MRS with 0-5 % (w/v) of ascorbic acid at 37°C for 18 h. After determination of the optimal ascorbic acid concentration, 0-55 % (v/v) of ginseng extracts were added to the modified MRS broth containing 2 % ascorbic acid (w/v). The activated BL47 was inoculated to each media and anaerobically grown at 37°C. One ml (5 9 10 8 CFU/ml) of the cell suspension was harvested, washed twice in PBS, and then re-suspended in 4 ml of PBS at 37°C. During the cell sonication process, enzyme activity was evaluated at 30 s interval as described above. The degree of cell disruption was measured by the optical density at 600 nm. The optimal pH and temperature of Abf and Abp were determined by the aforementioned method (Ku et al. 2011). Treatment of ginseng extracts using disrupted cell suspension Ginsenosides were extracted from the ginseng root using the method described in our previous study (Kim et al. 2008). The disrupted cell suspensions from 5 9 10 8 CFU/ ml were mixed with the ginseng extracts at the ratio of 19:1 (v/v) and incubated at 37°C. The cell-ginseng extract suspensions were collected after 3, 6, 9, and 12 days and evaluate the bioconversion of ginsenosides through TLC analysis (Park et al. 2012). Addition of ginseng extracts to MRS broth supplemented with ascorbic acid and ginsenosides conversion Five to 55 % (v/v) of ginseng extracts were added to MRS ? 2 % ascorbic acid (w/v) broth. The initial pH of all broths was adjusted to 7.0 by adding sodium hydroxide. Activated BL 47 was anaerobically cultured at 37°C for 7 days without shaking. The whole cell and ginseng extract suspensions were collected after 2, 3, 4, 5, 6, and 7 days to evaluate the bioconversion of ginsenosides using the TLC analysis (Park et al. 2012). The changed Abf and Abp activities by the concentration of ginseng extracts were determined using our previous method (Ku et al. 2011). Statistical analysis For the statistical evaluation of cell growth rates and changed enzyme activities, the analysis of variance (ANOVA) was applied using the program Minitab Ò 16, and the Tukey's test was applied for the post hoc comparison. Significant differences were considered at p \ 0.05. Fig. 1 Relative enzyme activities of Bifidobacterium longum Rd47 cultured in the various broths (n = 3). Cell lysis solution was treated to samples. Error bars standard deviation. White bars aarabinofuranosidase, black bars a-arabinopyranosidase Fig. 2 The effect of ascorbic acid on the production of a-Larabinofuranosidase and a-Larabinopyranosidase from Bifidobacterium longum Rd47 (n = 3). Cell lysis solution was treated to samples. Error bars represent standard deviation. White bars aarabinofuranosidase, black bars a-arabinopyranosidase, black circles population growth Results and discussion Induction of Abf and Abp using ascorbic acid In our previous work, various carbon, nitrogen, and ion sources were added to the microbial culture broth and determined the optimal aand b-galactosidases production from BL47 (Han et al. 2014). In this study, we aimed to investigate which sources were effective inducers for the Abf and Abp and whether any of the sources could promote the growth of BL47 (Table 2). As sole carbon sources, L-arabinose, lactose, and xylose (#1, 2 and 8) were ineffective for the growth of BL47. Fructose and maltose showed a slight enhancement (#4 and 6). BL47 showed the best growth when sucrose (#7) was added. There were no statistically significant growth differences between commercial MRS (control) and modified MRS containing glucose (#5) (p \ 0.05), which demonstrates that commercial MRS and lab-made MRS have a similar effect on cell growth (Fig. 1). Several studies have shown that the addition of certain organic acids to culture media can improve the cell viability by neutralizing hydrogen peroxide and reducing redox potential (Brewer et al. 1977;Collins and Hall 1984;Ku et al. 2011). Interestingly, the addition of some organic acids to culture media modified the morphology of Bifidobacterium spp. by ion chelation during fermentation (Kojima et al. 1968(Kojima et al. , 1970Ku et al. 2009). In this work, the addition of 2 % (w/v) lactic acid (#21) and citric acid (#24) to commercial MRS showed a decrease in cell viability (p \ 0.05). However, the degrees of the BL 47 growth were not significantly affected by the presence of ascorbic acid (p [ 0.05), as compared to those cultured in normal MRS (Fig. 1). The microscopic morphology of BL47 was not changed by ascorbic acid (data not shown). Both Abf and Abp activities of BL47 were outstandingly increased by adding 2 % of ascorbic acid (w/v) (p \ 0.05). For further examination of the role of ascorbic acid, 0-4 % (w/v) of ascorbic acid was added to commercial MRS (Fig. 2). As a result, the degree of enzyme activity increased as the concentration of ascorbic acid increased up to 2 %. Conversely, BL 47 cultured in media containing 4 % of ascorbic acid showed significantly decreased enzyme activity (p \ 0.05) and decreased growth (p \ 0.05). These results suggested that ascorbic acid can enhance Abf and Abp activity and were optimal at 2 %. Because majority of probiotic bacteria are anaerobic microorganisms, it is common in the food industry to add ascorbic acid into commercial probiotic products in order to scavenge oxygen. Several studies have demonstrated the effects of ascorbic acid on lactic acid bacteria (Dave and Shah 1997;Talwalkar and Kailasapathy 2004;Santiesteban-López et al. 2013;Shu et al. 2013). These studies focused on evaluating changed cell growth rates to determine best conditions for the yield of lactic acid bacteria. The concentrations of ascorbic acid used in their experiments were relatively lower (\0.1 % w/v) than in our experiment (2 % w/v). The present study reports a newly observed enhancement of Abf and Abp activities from the genus Bifidobacterium by ascorbic acid. Optimal pH, temperature, and disruption conditions for Abf and Abp Some microbial enzymes produced from lactic acid bacteria showed high enzyme activity in the acidic conditions (Ku et al. 2011). The optimal pHs of Abp in B. breve K-110 and B. longum H-1 were 5.8 and 6.8, respectively (Shin et al. 2003;Lee et al. 2011). The optimal pHs of Abf activity in B. breve K-110 and B. longum H-1 were 4.5 and Fig. 3 The effect of cell disruption (Sonication) on a-Larabinofuranosidase and a-Larabinopyranosidase activities from Bifidobacterium longum Rd47 (n = 3). Error bars represent standard deviation. White bars aarabinofuranosidase, black bars a-arabinopyranosidase, black circles degree of cell disruption 4.7, respectively. The maximum Abf and Abp activities of the BL47 cultured in MRS were observed at pH 6.0 and 5.0, respectively, whereas those cultured in MRS broth containing 2 % of ascorbic acid (w/v) were observed at pH 7.0 and 5.0 (p \ 0.05), respectively. The maximum Abf and Abp activities were observed at 40°C for BL47 within our experimental range of 4-70°C and about 60 % of both Abf and Abp activities were detected after incubation at 60°C for 10 min compared to the maximum activity (data not shown). During the cell Fig. 4 The effect of ginseng extracts on the production of a-L-arabinofuranosidase and a-Larabinopyranosidase from Bifidobacterium longum Rd47 (n = 3). Enzyme activities were evaluated without disruption step. Error bars standard deviation. White bars aarabinofuranosidase, black bars a-arabinopyranosidase, black circles population growth sonication process, the degree of cell disruption was increased while the optical density of cell suspension was gradually decreased at 600 nm (Fig. 3). There was no statistically significant loss of the Abf and Abp activities (p [ 0.05) during this process. The whole cell suspension efficiently hydrolyzed q-nitrophenyl-b-D-glucopyranoside and q-nitrophenyl-b-D-glucofuranoside. These results suggest that both enzymes apparently have high resistance to physical disruption. Cell disruption process is essential to use cytosolic enzymes for the bioconversion Yan et al. 2008;Noh and Oh 2009;Yoo et al. 2011). However, these processes are time consuming and labor intensive. In our previous work (Ku et al. 2011;Park et al. 2012), we reported successful hydrolysis of glycosides using whole cell without cell disruption process. This minimal microbial process may lead to cost reduction, an important practical application in the food industry. Bioconversion of ginsenosides The use of microbial crude enzymes can reduce food processing cost, as compared to the use of purified enzymes (Singh et al. 2013). Based on the presently determined optimal conditions [ascorbic acid concentration: 2 % (w/ v); temperature: 40°C; pH: 5], the disrupted BL 47 homogenates (5 9 10 8 CFU/ml) and the whole cell suspension of BL 47 (5 9 10 8 CFU/ml) were applied to the hydrolysis of natural substrates. Ginsenosides Rb2 and Rc are differentiated from Rd by the presence of a-L-glucopyranoside and a-L-glucofuranoside, respectively; therefore, aglycone of ginsenosides Rb2 and Rc is the same as that of ginsenoside Rd (Shin et al. 2003). The whole cell suspension converted both Rb2 and Rc into Rd; however, the disrupted cell extracts only converted Rb2 to Rd. The effect of BL47 enzymes on the experimental ginsenosides was slightly different from its effects on pNP substrates. This difference may be caused by the direct application of crude enzyme homogenates (i.e., the whole cell and disrupted cell suspensions) into the bioconversion step without protein purification. Similar results were reported with microorganisms Flavobacterium johnsoniae and Cladosporium cladosporioides (Hong et al. 2012;Wu et al. 2012). When BL47 was cultured in modified MRS containing 2 % ascorbic acid and various levels of ginseng extracts, both Abf and Abp showed maximum activities when the ginseng extracts were 10 % (v/v) without significant changes in growth rate (p [ 0.05) (Fig. 4). However, the enzyme activities were gradually decreased by adding additional ginseng extract to the broths. During the processing step to make ginseng extracts, a high level of carbohydrates can be extracted from ginseng because more than 60 % of the ginseng root consists of carbohydrates (Van et al. 2009;Choi et al. 2014). Several studies report that the addition of a high concentration of carbon sources to culture media decreased the induced enzyme activity (van der Veen et al. 1994;Gielkens et al. 1999;Gueimonde et al. 2007;Hetta et al. 2014). Sánchez and Hardisson (1980) hypothesized that this enzyme inhibition may be the result of catabolite repression and inactivation, or the reduced usability of the inducer. The TLC profile of transformed ginsenosides by using the whole cell suspension of BL47 showed a transformation of ginsenoside Rc and Re to Rd. We also detected ginsenoside F2 and unknown faint bands (Figs. 5, 6). This study revealed that the addition of 2 % of ascorbic acid to MRS media caused a significant increase in Abf and Abp activities of BL47. We also showed the optimal conditions for the induced enzymes. Based on our results, we applied the whole cell and disrupted cell homogenates to the bioconversion of ginsenosides in order to use this process in industrial applications. The bioconversion using whole and living cell in media containing ginseng extracts is not perfectly completed; however, the potential for reducing production cost has been approved. Our protocol is more practical for the bioconversion of ginsenosides than conventional methods, which usually include numerous procedures (such as enzyme purification, cell disruption, gene work). Further work in the molecular level should be conducted in order to investigate the effect of ascorbic acid on BL 47.

v3-fos

2018-12-17T19:32:38.554Z

2015-05-30T00:00:00.000Z

59056888

s2

Oxidative Stress on Buccal Mucosa Wound in Rats and Rule of Topical Application of Ethanolic Extracts of Mauli Banana (Musa Acuminata) Stem The present study was undertaken to evaluate the effect of a topical application of ethanolic extracts of Mauli banana stem on buccal mucosa wounds in rats (Rattus novergicus). Three sets of experiments with 3 groups male rats each consisted of 9 animals were used for studying wound oxidative stress status. Group 1 (P0) as the negative control was left to heal spontaneously; group 2 (P1) as the postive control were treated with alocair topically; and group 3 (P2) as treatment group were treated with ethanloic extracts of Mauli banana stem topically, respectively, 24h after wound creation for 3 days. The oxidative stress status was evaluated by monitoring the SOD, CAT activity, MDA and CC levels. The effect of ethanolic extracts of Mauli banana stem on oxidative stress status revealed significant increased in SOD activity, decreased in MDA levels, and no significant change both in CAT activity and CC levels compared to negative control. These results showed that The ethanolic extracts of Mauli banana stem might affect the oxidative stress status during wound healing process. South Kalimantan Indonesia is a tropical country that has a variety of fruits [1]. In this province, medicines based on herbal origin have been the basis of treatment and cure for various diseases. A large number of plants are used by folklore traditions in Indonesia. In this day, the use of traditional plants for wound healing has received much attention [2]. It is because these herbal drugs have real effectiveness wit low side effects and cost [3]. It is well known that conditions such as wound increase oxidative stress. Hence, if there is a compound has antioxidant content and activity, it can be an excellent therapeutic agent for enhancing the wound healing process. Antioxidants are capable of promoting rapid reepithelialization of acute wounds and burns and have antimicrobial properties [4]. Mauli banana (Musa acuminata) is a famous and delicious banana that grows plenty throughout Kalimantan Selatan [5]. It is well known that all the parts this plant are beneficial to in the medical aspects and ornamental uses [6,7]. The beneficial medicinal effects of these plants materials typically result from the com-binations of antioxidant content in this parts of this plants [8]. Our previous study showed Mauli banana stem contained bioactive compounds such as ascorbic acid, b-carotene, lycopene, tannin, saponin, and flavonoid. Mauli banana stem have antioxidant activities and/or free radical scavenging activity [5]. Since ROS was involved in wound healing, the presence of those antioxidants might be important in the successful treatment of wounds. Thus, in this present study we investigated the effect of Mauli bananas stem ethanolic extracts on oxidative stress status during buccal mucosa woundhealing. Research Design This research was a true experimental study with post test-only with control design to test the effect of Mauli banana stem ethanol extract on buccal mucosal wound healing in rats (Rattus novergicus). Plant Materials The Then the small pieces of Mauli banana stem were pounded and blenderized into a dry powder. The dry powder then was used for maceration. First, the dry powder put into an Erlenmeyer flask containing 500 ml of 70% ethanol. Then the mixture was allowed stand at room temperature for five days. This process was performed up to three times. The resulting solution then filtered and concentrated in an evaporator at 40 o C. The dilution is then performed to obtain the ethanol extract of banana stem mauli 25%. Dilution is done by mixing the extract with distilled water in the ratio of 25:75. Experimental animals The experimental animals used were 48 2-3 months old male rats (Rattus novergicus) weighing 200-250 gram. The animals were obtained from the Abadi Jaya farm at Yogyakarta, with the provision of being in healthy condition (active and well-formed). The rats were kept in housing cages with four animals per cage. Standardized food and water were performed under a light/dark cycle of 12 h. The cages kept in a room that had a constant temperature of 25±1°C. In order to prevent the animals from coming in contact with their feces and/or urine, a husk was added to the cages. Excision Wound The surgical procedures were performed under general anesthesia, by inhalation administration of diethyl ether. After anesthesia, the buccal mucosa was antiseptically cleaned with povidone iodine. Then a 10 mm length and 1 mm depth surgical mucosal wound was made in the buccal mucosa of all animals with a disposable scalpel. Treatment Protocol The animals were randomly divided into three groups. P0 was left to heal spontaneously, P1 was treated with Alocair (Aloe vera) topically and P2 was treated with 25% of Mauli banana stem. The treatment applied to the wound site on the two times daily for three days. At the end of treatment, rats will be anesthetized using diethyl ether. Then the scars tissue were taken with 5 mm length, 3 mm wide, and 3 mm depth from the side of the wound. The sample tissues were immediately fixed in phosphate buffer solution pH 7. Then the sample was ground to form a liquid. Subsequently the solution was taken and centrifuged at 3000 rpm for 45 minutes. The top layer of 500 mL were taken for further biochemical analysis. Biochemical Analysis Hydrogen peroxide measurement were made by FOX2 methods with slight modification [9]. SOD activity was measured by Misra and Fridovich method. MDA level of the homogenate was measured by the Buege and Aust method [10]. Carbonyl compound level in homogenate are estimated according by the DNPH method with slight modification [11]. Statistical Analysis Statistical analysis was performed using SPSS for Windows version 16.0. Data were checked for normality (Shapiro-Wilk normality test) and homogeneity of variance (Levene's test). Then, data was divided into two assumptions. The normally and homogeneity distributed data were run with the parametric one-way analysis of variance (ANOVA) and followed by post hoc Tuckey HSD test. The non-normally and/or nonhomogeneity distributed data were run with the nonparametric Kruskal-Wallis Test and followed by Mann-Whitney U test. P-values <0.05 were considered statistically significant. In this wound model, the SOD and catalase activity were determined. The results are presented in figure 1A and 1B. Figure 1A and 1B shows the SOD and CAT activities in wound model homogenate. Compared with the P0, the SOD activity of P1 and P2 increased significantly (p<0.05). In other hand, the CAT activity of the P1 groups is increased compared with P0 groups but not statistically significant. The CAT activity of P2 groups also increased if compared with P0 groups, but decreased if compared with P1 groups. Wound healing is a natural process of regenerating dermal and epidermal tissue, and may be classified into three phases. The three phases of wound healing process are inflammation, proliferation and remodeling phase [12]. ROS and oxidative reactions play a signifi-cant role in those whole steps of wound healing, providing signaling and defense against microorganism [13]. Overproduction of these ROS results in oxidative stress is thereby causing cytotoxicity and delayed wound healing. Therefore, elimination of oxidant could be an important strategy in healing of acute and chronic wounds [14]. Hence, estimation of antioxidants like SOD and CAT in wound healing tissue is also relevant because this antioxidant hasten the process of wound healing by destroying these ROS. Preventive antioxidants such as SOD and CAT are the first line of defense gainst ROS. SOD catalyzes radical superoxide dismutation producing hydrogen peroxide, whereas CAT remove it [15]. In our test, we found that in Mauli banana stem extracts groups, SOD activities increased significantly in wound tissue. Nonetheless, CAT activities had no change. These results show that Mauli banana stem extracts might have different effects on different antioxidant enzymes in wound tissue. Furthermore, the wound site also might absorb these extracts differently, and the metabolic enzymes in wound side were different as well. We hypothesize that the change of enzyme activities is related to the components or metabolites of Mauli banana stem extracts, which could affect enzymatic activities or enzyme contents. Further studies are needed to confirm this hypothesis. To determined the impact of ROS production in the wound site, MDA and carbonyl compound were estimated. The estimated levels of MDA and carbonyl compound have been presented in figure 2A and 2B. Figure 2A suggested that the lipid peroxidation level in the negative control group (P0) was higher as shown by high MDA content. However, treatment with standard as well as ethanolic extracts of Mauli banana stem significantly reduced the level of MDA in comparison to the negative control group. The results also suggested that standard treatment with alocair is more efficient than Mauli banana stem extracts. Figure 2B suggested that the carbonyl compound level in the negative control group (P0) was higher compared with the other two groups (P1 and P2). However, treatment with standard as well as ethanolic extracts of Mauli banana stem not significantly reduced the level of carbonyl compound in comparison to the negative control group. The result of this study in MDA level suggests that the significant decreased might be correlated with alteration in antioxidant profile. The increasing activities of antioxidant enzymes will reduce the lipid peroxides (MDA) in treated rats both with alocair or Mauli ba-nanas stem extracts. On the other hand, this result study revealed that carbonyl compound levels had no change in the wound site. The reason maybe same with the reason as mentioned earlier in antioxidant activities parts in this discussion. From this analysis, it can be concluded that the application of ethanolic extracts of Mauli banana stem extracts topically has different effects on oxidative stress status in buccal mucosa wounds healing process. The extracts significantly increased the SOD activity and decreased the MDA levels. On the other hand, the extracts were not significantly effect on CAT activity and CC levels. Further controlled clinical studies should observed and its modest efficacy. Authors are thankful to Iskandar, dr. from Research Unit Mutiara Bunda Mother and Child Hospital, Martapura South Kalimantan Indonesian, for his support and encouragement during the process of writing this research.

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2018-04-03T03:42:30.271Z

2015-11-25T00:00:00.000Z

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Detection of Capripoxvirus DNA Using a Field‐Ready Nucleic Acid Extraction and Real‐Time PCR Platform Summary Capripoxviruses, comprising sheep pox virus, goat pox virus and lumpy skin disease virus cause serious diseases of domesticated ruminants, notifiable to The World Organization for Animal Health. This report describes the evaluation of a mobile diagnostic system (Enigma Field Laboratory) that performs automated sequential steps for nucleic acid extraction and real‐time PCR to detect capripoxvirus DNA within laboratory and endemic field settings. To prepare stable reagents that could be deployed into field settings, lyophilized reagents were used that employed an established diagnostic PCR assay. These stabilized reagents demonstrated an analytical sensitivity that was equivalent, or greater than the established laboratory‐based PCR test which utilizes wet reagents, and the limit of detection for the complete assay pipeline was approximately one log10 more sensitive than the laboratory‐based PCR assay. Concordant results were generated when the mobile PCR system was compared to the laboratory‐based PCR using samples collected from Africa, Asia and Europe (n = 10) and experimental studies (n = 9) representing clinical cases of sheep pox, goat pox and lumpy skin disease. Furthermore, this mobile assay reported positive results in situ using specimens that were collected from a dairy cow in Morogoro, Tanzania, which was exhibiting clinical signs of lumpy skin disease. These data support the use of mobile PCR systems for the rapid and sensitive detection of capripoxvirus DNA in endemic field settings. Summary Capripoxviruses, comprising sheep pox virus, goat pox virus and lumpy skin disease virus cause serious diseases of domesticated ruminants, notifiable to The World Organization for Animal Health. This report describes the evaluation of a mobile diagnostic system (Enigma Field Laboratory) that performs automated sequential steps for nucleic acid extraction and real-time PCR to detect capripoxvirus DNA within laboratory and endemic field settings. To prepare stable reagents that could be deployed into field settings, lyophilized reagents were used that employed an established diagnostic PCR assay. These stabilized reagents demonstrated an analytical sensitivity that was equivalent, or greater than the established laboratory-based PCR test which utilizes wet reagents, and the limit of detection for the complete assay pipeline was approximately one log 10 more sensitive than the laboratory-based PCR assay. Concordant results were generated when the mobile PCR system was compared to the laboratory-based PCR using samples collected from Africa, Asia and Europe (n = 10) and experimental studies (n = 9) representing clinical cases of sheep pox, goat pox and lumpy skin disease. Furthermore, this mobile assay reported positive results in situ using specimens that were collected from a dairy cow in Morogoro, Tanzania, which was exhibiting clinical signs of lumpy skin disease. These data support the use of mobile PCR systems for the rapid and sensitive detection of capripoxvirus DNA in endemic field settings. Capripoxviruses (CaPVs) cause serious pox diseases of domesticated ruminants (Carn, 1993). Comprising sheep pox virus (SPPV), goat pox virus (GTPV) and lumpy skin disease virus (LSDV), they are large, complex, doublestranded DNA viruses within the genus Capripoxvirus, subfamily Chordopoxvirinae, family Poxviridae (Buller et al., 2005). SPPV and GTPV are normally restricted to Asia and North Africa, although clinical cases of sheep pox have also been detected in Europe (Mangana et al., 2008), and recently in Bulgaria and Greece (during 2013). Lumpy skin disease (LSD) occurs across Africa, and in recent years, LSDV has also been found in several countries of the Middle East , including Turkey where more than 236 outbreaks have occurred since 2013 (ProMed 20130831.1915595). The World Organization for Animal Health (OIE) classifies CaPVs as notifiable disease agents, and molecular diagnostic tests play an important role in monitoring the spread of these viruses in susceptible livestock. A range of conventional agarose-gel-based polymerase chain reaction (PCR) assays (Ireland and Binepal, 1998;Heine et al., 1999;Markoulatos et al., 2000;Tuppurainen et al., 2005;Zheng et al., 2007), or real-time PCR assays (Balinsky et al., 2008;Bowden et al., 2008;Stubbs et al., 2012) are used in diagnostic laboratories. However, poorly equipped laboratories often face difficulties accessing these molecular techniques (particularly real-time PCR) that are reliant upon expensive and relatively fragile equipment. In particular, the ability to perform nucleic acid-based tests such as PCR in field settings has proven to be a challenging goal largely due to the reliance upon pre-processing of samples (nucleic acid extraction), the lack of stable reagents that are suitable for use in environments where it is not possible to maintain a cold chain (King et al., 2008) and the cost of the field equipment. The Enigma Field Laboratory (FL) is a hardware platform which undertakes nucleic acid extraction, PCR thermocycling and analysis of data without user intervention, which has been applied for the detection of other notifiable diseases such as foot-and-mouth disease (Madi et al., 2012). The aim of this study was to optimize and evaluate a mobile PCR platform for the simple detection of CaPV DNA. This study utilized the real-time PCR primers, probes, master mixes and cycling conditions that have been previously described (Bowden et al., 2008). Pilot studies were undertaken to assess the performance of newly developed lyophilized PCR reagents that were prepared and assembled into assay cartridges by Enigma Diagnostics (Salisbury, UK). A decimal dilution series (Neat to 10 À10 ) of DNA prepared from an LSDV isolate (Israel LSD-07 POX-V1-07-08, isolated from naturally infected cattle in 2007) was prepared in nuclease-free water containing carrier RNA (1 lg ml À1 ). In these experiments, 5 ll of each dilution was mixed with 20 ll of nuclease-free water and this was used to re-suspend the lyophilized reagent pellets prepared by Enigma Diagnostics. This 25 ll suspension was then transferred into a 96-well PCR plate. For the conventional wet reagents, 2 ll of DNA was added to 18 ll of diagnostic assay mastermix (Bowden et al., 2008) prior to transfer into 96-well PCR plate. This initial laboratory validation of lyophilized reagents was carried out on the Mx3005P quantitative PCR machine (Stratagene). Parallel testing demonstrated an improved analytical sensitivity of one log 10 for the lyophilized reagents when compared to the reference test (Fig. 1a). This one log 10 increase in analytical sensitivity was maintained when the new lyophilized assay was applied to a decimal dilution series (10 À1 -10 À10 ) of Israel LSD-07 POX-V1-07-08 virus prepared in 10% w/v homogenized cattle skin suspensions and run in full, including DNA extraction on the Enigma FL (Fig. 1b). For the above comparison, one aliquot per dilution was extracted on the MagNA Pure LC Robot (Roche) using the Total Nucleic Acid Isolation kit (Roche) following manufacturer guidelines, followed by real-time PCR performed using wet reagents assayed on the Mx3005P quantitative PCR machine (Stratagene) (reference test). The extraction and real-time PCR for the second aliquot (0.5 ml) was performed in a complete automated cycle on the Enigma FL. The suitability of this assay to detect CaPV DNA in clinical samples was evaluated using archived field and experimental samples held at the OIE Reference Laboratory for LSDV and GPV/SPV (The Pirbright Institute, UK) and the National Veterinary Reference Laboratory in Tanzania (Tanzania Veterinary Laboratories Agency-TVLA). Fourteen clinical samples (Table 1), comprising nine from two experimentally infected animals where cattle had been infected with LSDV Neethling strain (VN83 and VN84), and five from field samples submitted to The Pirbright Institute were used. Five additional skin scrapings from the TVLA archive were analysed within East African laboratory settings. For each sample, one duplicate was extracted and assayed using the established reference test (Bowden et al., 2008) whilst for the second duplicate the extraction and real-time PCR was performed on the Enigma FL. There was complete concordance between positive results (n = 19) and negative results (n = 3) generated on the Enigma FL and the standard laboratory pipelines (Table 1). Opportunistic testing of samples collected from a Holstein-Friesian cross-dairy cow on a small holder farm in Morogoro, Tanzania, displaying clinical signs of LSD (Fig. 2) was also undertaken. Two samples comprising EDTA blood and skin scrapings were tested; skin scrapings were processed using a field-based tissue processing kit (Svanodip â Ag extraction kit; prior to being added into the Engima FL sample loading chamber, whilst EDTA blood was added directly to the sample chamber). Both specimens were positive for CaPV DNA using the Enigma FL. It should be noted that the current diagnostic reference test (Bowden et al., 2008) utilizes a conservative cut-off C T value of <37 to define a positive result. However, for this study, all C T values are reported because amplification of CaPV in animals with late infection may generate weak values that would be missed with a cut-off of 37. Suitable negative controls were also included in the data set to confirm the absences of any false amplification. These data show that a mobile PCR platform can rapidly detect CaPVs from suspect cases, within 60 minutes of sample collection, offering a sensitive molecular technology that can be deployed into field settings. Furthermore, the comparative data for the lyophilized reagents demonstrate that it is possible to generate a stabilized assay with equivalent (or better) performance compared with the wet-assay format. This current assay format represents the results of a collaborative research project that was undertaken to highlight the potential of these technologies for diagnostic use. Future validation to include a greater sample data set would increase confidence in the test and could be combined with optimization of the DNA extraction steps to bring its performance in line with that of a standard laboratory extraction robot. Further availability of this particular assay (via commercial sources), as well as other tests that might also exploit this format, will be dependent upon demand and interest from customers. Conflict of Interest James Wood and Paul Martin are employees of Enigma Diagnostics. All laboratory work and evaluation of the equipment was undertaken by staff from The Pirbright Institute, Sokoine University of Agriculture and TVLA, and no financial support was provided from Enigma Diagnostics to The Pirbright Institute to conduct this study.

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2016-06-18T00:22:32.514Z

2015-12-10T00:00:00.000Z

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Ectopic Expression in Arabidopsis thaliana of an NB-ARC Encoding Putative Disease Resistance Gene from Wild Chinese Vitis pseudoreticulata Enhances Resistance to Phytopathogenic Fungi and Bacteria Plant resistance proteins mediate pathogen recognition and activate innate immune responses to restrict pathogen proliferation. One common feature of these proteins is an NB-ARC domain. In this study, we characterized a gene encoding a protein with an NB-ARC domain from wild Chinese grapevine Vitis pseudoreticulata accession “Baihe-35-1,” which was identified in a transcriptome analysis of the leaves following inoculation with Erysiphe necator (Schw.), a causal agent of powdery mildew. Transcript levels of this gene, designated VpCN (GenBank accession number KT265084), increased strongly after challenge of grapevine leaves with E. necator. The deduced amino acid sequence was predicted to contain an NB-ARC domain in the C-terminus and an RxCC-like domain similar to CC domain of Rx protein in the N-terminus. Ectopic expression of VpCN in Arabidopsis thaliana resulted in either a wild-type phenotype or a dwarf phenotype. The phenotypically normal transgenic A. thaliana showed enhance resistance to A. thaliana powdery mildew Golovinomyces cichoracearum, as well as to a virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Moreover, promoter::GUS (β-glucuronidase) analysis revealed that powdery mildew infection induced the promoter activity of VpCN in grapevine leaves. Finally, a promoter deletion analysis showed that TC rich repeat elements likely play an important role in the response to E. necator infection. Taken together, our results suggest that VpCN contribute to powdery mildew disease resistant in grapevine. INTRODUCTION Plants have evolved multiple mechanisms to protect themselves against pathogens (Jones and Dangl, 2006). The first line of defense is microbe-associated molecular pattern (MAMP)triggered immunity (MTI) following MAMP perception by membrane-resident pattern recognition receptors (Maekawa et al., 2011). MTI is thought to limit the growth of invasive pathogens. The second line of defense is plant innate immunity, which is activated by the specific recognition of pathogenderived effectors by intracellular host resistance (R) proteins, and is termed effector-triggered immunity (ETI) (Chisholm et al., 2006). ETI typically leads to a hypersensitive response (HR) and gives rise to a faster and stronger defensive response than MTI-triggered immunity (Cesari et al., 2013). Understanding the function of R proteins, and the mechanisms by which they recognize pathogen effectors, can potentially lead to the development of a long-term strategy for the control and prevention of pathogen invasion. Over the past few decades, numerous R genes have been cloned from model plants and important crops (Pan et al., 2000b;Collier and Moffett, 2009;Sekine et al., 2012). Most R proteins contain a nucleotide binding (NB) domain and a Cterminal leucine-rich repeat (LRR) domain, and belong to the so-called NB-LRR protein family (Ooijen et al., 2008). The most conserved domain in NB-LRR proteins is an NB domain that is found in proteins such as human Apaf-1, plant R proteins and Caenorhabditis elegans Ced-4 (ARC), and as such is referred to as the NB-ARC domain (Ooijen et al., 2008;van der Biezen and Jones, 1998). As a consequence of determining its threedimensional structure, Albrecht and Takken (2006) proposed that the NB-ARC domain can be further divided into three subdomains (NB, ARC1, and ARC2). Several conserved motifs have been identified thoughtout the NB-ARC domain in R proteins, such as Walker B, GxP, hhGRExE, Walker A or P-loop, MHD, and RNBS-A-D (Meyers et al., 1999;Pan et al., 2000a;Ooijen et al., 2008). Crystal structure analysis of the NB-ARC domain has led to the suggestion that it may function as a molecular switch to regulate signaling pathways through conformational changes (Riedl et al., 2005;Takken et al., 2006). It has also been shown that the nucleotide binding of the NB-ARC domain in the R proteins, I-2, and Mi-1, requires a P loop, since a P-loop mutant abolished the binding capacity (Tameling et al., 2010). Likewise, the oligomerization of an NB-ARC-LRR protein in the presence of its elicitor requires an intact P-loop in the NB-ARC domain (Mestre and Baulcombe, 2006). Plant NB-LRR proteins can be divided into two distinct classes: the TNL and the CNL type, based on the domains present at their N terminus. Those that possess a Toll and human interleukin-1 receptor (TIR) domain are referred to as TIR-NB-ARC-LRR or TNL proteins, while those carrying a predicted coiled-coil (CC) domain are classified as CC-NB-ARC-LRR, or CNL proteins (Pan et al., 2000a;Lukasik-Shreepaathy et al., 2012). The potato (Solanum tuberosum) Rx protein is a typical CC-NB-ARC-LRR protein mediates resistance to potato virus X (PVX) (Kohm et al., 1993;Bendahmane et al., 1999), the CC domain of RX protein has a four bundle structure and forms a heterodimer with RanGAP2 WPP domain (Hao et al., 2013). The N-termini of the CC and TIR domains are thought to mediate downstream immune responses. It has been reported that in CNL proteins, the CC domain of NRG1 is capable of independently inducing defense responses (Collier et al., 2011), and in TIR proteins the TIR domain plays a crucial role in the cell death signaling pathway (Zhang et al., 2004;Weaver et al., 2006). The identification and functional characterization of NB-ARC domain R proteins is of considerable interest in developing novel sources of disease resistance in crop plants that are threatened by phytopathogens. For example, Erysiphe necator is a fungus that causes powdery mildew (PM) disease in grapevine worldwide, resulting in serious losses in both grape yield and quality. The most economically important cultivated grapevine is V. vinifera, which is highly susceptible to PM (Gadoury et al., 2012). To combat the pathogen, fungicides are widely used, which causes environmental and financial pressure on grape growers and reduces wine quality. Thus, developing new grape cultivars with enhanced disease resistance mechanisms is of considerable interest. The wild Chinese Vitis, "Baihe-35-1, " is an accession of wild Chinese V. pseudoreticulata W. T. Wang that possesses high resistance to multiple fungi, and particularly to E. necator (Wang et al., 1995;Lin et al., 2006;Yu et al., 2011). To elucidate the resistance mechanisms involved in the defense response to fungal infection in this species, we previously performed an RNAseq based transcriptome analysis V. pseudoreticulata "Baihe-35-1" that had been inoculated with E. necator (Weng et al., 2014). Among the pathogen induced genes, one was predicted to encode an NB-ARC domain protein. In this current study, we report the isolation of the full length cDNA of this gene, which we designated VpCN, and its functional characterization following ectopic expression in Arabidopsis thaliana. Conclusions regarding its role in conferring Chinese Wild V. pseudoreticulata "Baihe-35-1" with disease resistance to powdery mildew are presented. Plant Materials and Growth Conditions Grapevines (Chinese wild V. pseudoreticulata accession Baihe-35-1 and V. vinifera cv. "Red globe") were maintained in the grape germplasm resources orchard, Northwest A&F University, Yangling Shaanxi, China. A. thaliana (ecotype type, Columbia-0) was grown in a growth chamber under the following conditions: 22 • C, 50% humidity, a 16/8 h day/night intensity of 125 µmolm −2 s −1 provided by cool white fluorescent bulbs. Cloning and Sequence Analysis Total RNA was extracted from grapevine as previously described (Zhang et al., 2003). First strand cDNA was synthesized from 1 µg of total RNA with the PrimerScript ™ II 1st Strand cDNA Synthesis kit (TaKaRa Bio Inc., Dalian, China), according to the manufacturer's instructions. LA Taq (Takara Bio. Inc.) was used to amplify the ORF sequence of VpCN. The PCR products were cloned into the T-easy vector (Promega, USA), sequenced (Beijing Genomics Institute, Beijing, China) and submitted to GenBank (accession number KT265084). The VpCN cDNA sequence was analyzed using BLAST (http:// Ncbi.nlm.Nih.gov/blast) in the NCBI database. Grapevine DNA extraction was conducted as previously described (Yu et al., 2013), primers for amplify promoter sequence were designed according to acquired sequence from Grape Genome Database (12×; http://www.genoscope.cns.fr), after cloning into the Teasy vector and sequencing, the promoter sequence was analyzed using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/ plantcare/html/) (Lescot et al., 2002). The deduced amino acid sequence of VpCN was aligned with closely related proteins and a phylogenetic tree was generated using neighbor joining algorithm with 1000 bootstrapping with the ClustalW tool in the MegAlign program (Version 5.07, DNASTAR Inc.) ( Figure 1D). A structural model of the NB-ARC domain of VpCN was constructed using the structure of PDB 4m9x.1.C (Huang et al., 2013) in SWWISS-MODEL ( Figure 1C). Real time PCR was conducted using SYBR @ Premix EX Taq ™ II (Tli RNaseH Plus) (Takara Bio. Inc.) in a 20 µl volume reaction following the manufacturer's instructions using the CFX96TM real-time system (Bio-Rad, Hercules, CA, USA). The amplification cycles were as follows: initial denaturation at 94 • C for 30 s, 40 cycles at 95 • C 5 s, 60 • C for 30 s. For melting curve analysis: 40 cycles at 95 • C for 15 s followed by a constant increase from 60-95 • C. The grapevine Actin 1 (GenBank Accession number AY680701) was used as reference gene. Construction of Vectors for Ectopic Expression and A. thaliana Transformation To generate 35S:VpCN, the open reading frame (ORF) region of VpCN was cloned into the binary vector, pCAMBIA 2300 (CAMBIA company), downstream of the CaMV 35S promoter. The construct was introduced into Agrobacterium tumefaciens, strain GV3101, via electroporation, and the transformed A. tumefaciens was used to transform A. thaliana using the floral dip method (Clough and Bent, 1998). Transgenic plants were screened on MS (Murshige and Skoog, 1962) medium containing 60 mg/mL kanamycin, PCR amplification was performed to identify transgenic plants with gene specific primers. Construction of VpCN Promoter:: GUS Gene Fusion Vectors and A. tumefaciens Mediated Transient Expression Assays To generate the VpCN promoter:GUS vector, the VpCN promoter was cloned into the T-easy vector, digested with BamHI and PstI, and finally cloned into the binary vector pC0380GUS. 35S:GUS was used as a positive control (Xu et al., 2010). Four pVpCN promoter fragments with different 5 ′ deletions were amplified (Supplement Table 1). All the constructs were introduced into A. tumefaciens strain GV3101 via electroporation. The A. tumefaciens mediated transient expression assays were performed as previously described (Guan et al., 2011). A. tumefaciens GV3101 lines harboring the different constructs were grown in liquid Yeast Extract Phosphate (YEP) (Smith and Goodman, 1975) medium (supplemented with 100 µgml −1 kanamycin, 60 µgml −1 gentamycin, and 30 µgml −1 rifampicin) to an OD 600 of 0.6, and harvested by centrifugation at 5000 ×g for 10 min, before being resuspended in filtration solution (10 mM 2-(N-morpholino) ethanesulfonic acid (MES), pH 5.7, 10 mM MgCl 2 and 15 µM acetosyringone) and adjusted to an OD 600 of 0.6 for infiltration of young grapevine leaves using a vacuum infiltration method (Santos-Rosa et al., 2008). After infiltration, the leaves were kept in a chamber at 16/8 h day/night cycle at 23 • C with 70% humidity for 48 h, before inoculation with E. necator (Guan et al., 2011;Yu et al., 2013). A. thaliana powdery mildew G. cichoracearum was maintained on highly susceptible pad4 A. thaliana mutant plants. The infection was conducted as previously described (Tang and Innes, 2002). The susceptibility or resistance phenotypes were scored 8 days after infection (Nie et al., 2011). Analyses of pathogenesisrelated 1 (PR1) gene expression were performed using qRT-PCR using the same PCR program as for the VpCN analysis. The A. thaliana tubulin gene (GenBank Accession number NM_179953) was used as a reference. Rosett leaves from 4 week old Arabidopsis were harvested at 0, 12, 24, 36, and 48 h after inoculation. P. st DC3000 cells grown in King's B medium (supplemented with 100 µgml −1 kanamycin and 30 µgml −1 rifampicin) to an OD 600 of 0.6, harvested by centrifugation for 5000 × g for 10 min and re-suspended in 10 mM MgSO 4, adjusted to optical density at OD 600 of 0.02. The bacterial suspension containing 0.025% Silwet-77, and the mixture were hand infiltrated into the abaxial side of the A. thaliana leaves using a needless 1 ml syringe (Fan et al., 2008). P. st DC3000 bacterial growth were assessed 3 and 5 days after infection as described (Ahn et al., 2007). Trypan Blue Staining For trypan blue staining, A. thaliana leaves were collected 12 hpi (hours post-inoculation) and boiled in alcoholic lactophenol trypan blue solution (20 mL of ethanol, 10 mL of phenol, 10 mL of water, 10 mL of lactic acid [83%], and 30 mg of trypan blue). Stained leaves were cleared in chloral hydrate (2.5 g dissolved in 1 mL of water) for 3 h, before placing under a coverslip in 50% glycerol (Koch and Slusarenko, 1990;Frye and Innes, 1998). Peroxide Assay Peroxide (H 2 O 2 ) was assayed using a hydrogen peroxide kit, according to the manufacturer's instructions (Nanjing Bio Ins., Nanjing, China). Quantification of dead cells was performed 12 hpi by staining leaf discs (0.5 mm in diameter) with 0.2% Evans blue (Sigma) for 30 min, followed by several washes with water to remove excess stain (Mino et al., 2002;Ahn et al., 2007). One (Bent et al., 1994), gi46395604 (Bevan et al., 1998), gi46395938 (Theologis et al., 2000), gi75318159 (Ori et al., 1997), gi325511400 (Theologis et al., 2000) The third to fifth fully expanded young grapevine leaves beneath the apex were selected for samples. The experiment encompass three independent biological replicates, for each biological replicate three leaves haversted from three plant and three technical replicates were performed. Data represent means of three biological replicates ±SE, asterisksin indicate statistical significance in comparison with control (Student'st-test, significance levels of *P < 0.05, **P < 0.01 are indicated). Frontiers in Plant Science | www.frontiersin.org milliliter of 50% methanol supplemented with 1% SDS was added and the samples were incubated at 50 • C for 1 h. Absorbance at OD 600 was determined by ultraviolet spectrophotometry after a 10-fold dilution of the extracts (Ahn et al., 2007). The nitro blue terazolium (NBT) staining was performed as described (Kim et al., 2011). Callose Accumulation To observe callose accumulation, leaves (3 dpi) were immersed in destaining solution (10 ml phenol, 10 ml glycerin, 10 ml lactic acid, 10 ml H 2 O, and 80 ml ethanol) and kept in an oven at 60 • C for 1 h to remove chlorophyll. The samples were washed to remove the destaining solution, and stained with 0.1% aniline. The fluorescence of callose was detected using an epifluorescence microscope (E800, Nikon) with a V-2A filter (Reuber et al., 1998;Ahn et al., 2007). For quantitative determination of callose, A. thaliana, leaves (3 dpi) were immersed in ethanol for 2-3 days to remove the chlorophyll, before centrifugation at 5000 × g for 10 min. The supernatant was discarded and the pellet resuspended in 0.4 ml DMSO. One hundred microliter of the supernatant was supplemented with loading mixture [400 µl 0.1% (w/v) aniline blue, 590 mL 1 M glycine/NaOH (pH 9.5), 210 mL 1 M HCl] and 200 µl 1 M NaOH. The control samples were not supplemented with aniline. The samples were incubated in a water bath 50 • C for 20 min and cooled to room temperature before detection with a fluorescence spectrophotometer (F-4600, Hitachi, Tokyo, Japan) under 393 nm excitation, 479 nm emission and a voltage of 400 v. The fluorescence of the samples was determined by subtracting the fluorescence value of the control from those of the samples (Kohler et al., 2000). GUS Staining, Histochemical and Fluorometric Assays for Determining GUS Activity A histochemical β-glucuronidase (GUS) assay of leaves was carried out as previously described (Jefferson, 1987). Briefly, leaves were immersed in GUS staining solution at 37 • C for 24 h, before washing in 70% ethanol at 37 • C and viewing macroscopically (Guan et al., 2011;Yu et al., 2013). GUS fluorescence was determined quantitatively according to Jefferson (1987). Protein concentrations in grapevine extracts was normalized by dilution with extraction buffer according to Bradford (1976 VpCN Expression during Powdery Mildew Infection To identify potential resistance mechanisms and resistance related genes in the response of wild Chinese V. pseudoreticulata to powdery mildew, we previously performed a transcriptome analysis of the "Baihe-35-1" using RNA-seq (Weng et al., 2014). We observed that the expression of VpCN (GenBank accession number KT265084) was strongly induced by inoculation with E. necator. To verify this, we performed quantitative real-time PCR (qPCR) analysis of VpCN expression in V. pseudoreticulata leaves that had been inoculated with E. necator, and observed 4.2-fold greater VpCN transcript levels than in leaves prior to inoculation. Subsequently, VpCN expression decreased but remained at a higher level than in mock inoculated plants (Figure 1E). Cloning and Sequence Analysis of VpCN To investigate the putative role of VpCN in providing resistance to pathogens, we first designed primers based on a cDNA sequence obtained from the Grape Genome Database (12×; http://www.genoscope.cns.fr), and isolated and designated the gene VpCN (GenBank accession number KT265084). The VpCN gene is located on chromosome 15 (Figure 1A), has an ORF of 1773 bp (Supplement Figure 1) and is predicted to encode a protein of 590 amino acids with a molecular mass of 67,390 Da and a theoretical pI value of 5.45. The amino acid sequence was further predicted to contain a RxCC-like domain in the Nterminus from residue 6-119, a Ran GTPase-acting protein 2 (RanGAP2) interaction site in the RxCC-like domain and an NB-ARC domain spanning residues 129-414. The NB-ARC subdomains, NB, ARC1, and ARC2 were all present. Furthermore, several conserved motifs, such as a P-loop, RNBS A-D, and a GLPL ( Figure 1B) were detected. In addition to a RxCC-like domain and an NB-ARC domain, we also found an AAA domain and a PLN03210 domain in the predicted amino acid sequence (picture not shown). A structure-based multiple amino acids sequence alignment was performed to compare the NB-ARC domain of VpCN with those of other closely related plant R proteins, including RPS2 (gi30173240) (Bent et al., 1994) and I-2 (gi75318159) (Ori et al., 1997). The amino acids sequence identity between the VpCN and the A. thaliana RPS2 NB-ARC domain was shown to be 33%, while the VpCN and I-2 NB-ARC domains had a 29%, sequence identity, concentrated on the conserved motifs of the NB-ARC subdomains ( Figure 1B). Ectopic Expression VpCN in A. thaliana Enhance Resistance to Powdery Mildew We next transformed the VpCN in A. thaliana under the control of the constitutive 35S promoter (Figure 2A). A total of 42 independent transgenic T1 lines were obtained and the presence of the transgene confirmed by PCR using VpCN specific primers. The T2 progeny segregated so that 39 lines displayed wild type morphology while three lines exhibited a dwarfed phenotype and morphological abnormalities, such as small yellow leaves, stunted growth, and chlorotic tissue ( Figure 2B). These dwarf lines eventually died. The lines with a wild type phenotype were challenged with G. cichoracearum, and three transgenic lines with higher resistance were chosen for the generation of homozygous T3 generation lines. The transgenic lines displayed few visible white powdery areas on their leaves at 8 dpi, whereas the wildtype (Col-0) exhibited abundant powdery mildew development (Figures 2C,D). To determine whether the enhanced resistance to G. cichoracearum in the transgenic lines was related to an increase in the expression of a known defense gene, we evaluated PR1 (Pathogenesis Related 1) (Friedrich et al., 1996) transcript levels at 0, 12, 24, 36, and 48 hpi. Three transgenic plants displayed higher PR1 transcript abundance after pathogen inoculation than wild type plants, reaching a maximum level at 12 hpi. The PR1 transcript levels of transgenic plants were ∼4-5fold higher after inoculation than in wild type at all time points (Figure 2E). Ectopic Expression of VpCN Results In Enhanced Protection Against The Bacterial Pathogen, P. st DC3000 Since amino acid sequence of VpCN was predicted to contain a PLN03210 domain, which has been shown to be correlated with resistance to Pseudomonas syringae pv. glycinea race 6 (Kim et al., 2009), we hypothesized that it might function in providing resistance to bacterial infection. To test this, transgenic and control plants were challenged with the bacterial P. st DC3000 pathogen by leaf infiltration (Figure 3A). Most infiltrated wild type leaves exhibited water-soaking at 1 dpi, turned yellow and finally wilted at 5 dpi. In contrast, the transgenic plants infected with the pathogen showed fewer symptoms (Figure 3B), and when the growth of P. st DC3000 in the inoculated plants was quantified, it was found that the bacterial number in the transgenic plants was significantly lower than in the wild type plants (Figure 3F). To observe the effect of VpCN expression on cell death, trypan blue staining was performed of leaves and we observed that cell death was more widespread in the transgenic lines than the wild type plants (Figure 3C). Additionally, cell death quantification by Evans blue staining followed by spectrophotometric analysis, showed a 5-6 fold higher level cell death in the transgenic plants ( Figure 3H). Nitroblue tetrazolium (NBT) staining for the superoxide anion also showed higher accumulation in the transgenic plants (Figure 3D), as did quantitative measurements of H 2 O 2 ( Figure 3G). Finally, the accumulation of the (1,3)-β-glucan polymer callose, which is known to be involved in plant defense responses (Brown et al., 1998), was visualized by aniline blue staining of wild type and transgenic plants after treated with P. st DC3000 (Figure 3E). Greater accumulation of callose was observed in the transgenic plants than in wild, and when callose levels were quantified, it was confirmed that the transgenic lines contained significantly (P < 0.05) more callose (Figure 3I). Isolation and Analysis of the VpCN Promoter Sequence A 1440 bp upstream sequence was cloned using wild Chinese V. pseudoreticulata "Baihe-35-1" genomic DNA by PCR, regulatory cis-acting elements predicted showed that several putative regulatory elements involved in the activation of defense-related genes, including 72 predicted TATA boxes, 32 CAAT boxes, and two TC-repeat elements, which are known to be involved in defense and stress responses, a TCA element, which is involved in salicylic acid (SA) responses, a TGACG motif, which is associate with methyl jasmonate-response, an HSE element, which is involved in heat stress responses and two TATC elements, which are related to gibberellin responses ( Figure 4A). Additional predicted cis-regulatory elements included light response elements (TCCC-motif, MRE, I-box, GT1-motif, GAGmotif, GA-motif, G-box, CATT motif, Box-I, AT1-motif, and Box-4), as well as others cis-elements (5UTR Py-rich stretch, circadian element and, TATC box). Several of the predicted ciselements are known to be involved in responses to environmental stresses, further suggesting that the VpCN promoter may play a role in defense responses. Promoter::GUS (Glucuronidase) Assays To test the activity of the VpCN promoter, the 1440-bp promoter fragment was fused to a reporter gene encoding β-glucuronidase (GUS), generating the construct pCVpCNGUS. As a positive control, a CaMV35S::GUS (PC35SGUS) construct was used and a construct with no promoter was used as a negative control (pC0380GUS) (Xu et al., 2010; Figure 4B). All the constructs were expressed transiently in grapevine leaves, which were subsequently subjected to GUS staining. Leaves transformed with the PC35SGUS construct showed strong GUS activity, while no activity was detected in wild type (WT) and very little in PC0380GUS. pCVpCNGUS transformed leaves showed GUS activity but at a lower level than leaves transformed with PC35SGUS (Figure 4C), and when leaves were infected with E. necator 2 dpi prior to GUS staining, the infected leaves exhibited stronger GUS activity than mock-inoculated control leaves. To further determine the location of the pathogenresponsive cis-regulatory region, we generated four promoter deletion fragments and fused them to GUS (−1360, −700, −400, and −240 bp) (Figure 5A). When the GUS activity was quantified fluorescently, the highest levels were measured in grapevines containing the −1440 bp fragment, where it was induced 1.57fold after treatment with E. necator compared to mock controls. Leaves transformed with −1360, −700, and, −400 promoter fragments exhibited a relative low level of GUS activity; however, they showed increased GUS activity after being challenged with E. necator ( Figure 5B). Since the leaves transformed with the −240 bp fragment showed no significant difference in GUS activity before and after treatment with E. necator (Figure 5C), the −400 bp promoter fragment was deduced to be the minimal promoter region required for the response to E. necator infection. DISCUSSION We previously reported the leaf transcriptome of wild Chinese grape (V. Pseudoreticulata, "Baihe-35-1") that had been inoculated with E. necator, and showed that expression of a unigene corresponding to VpCN was strongly induced by the infection (Weng et al., 2014). Here, we isolated the ORF sequence of VpCN and ectopically expressed it in A. thaliana. This resulted in enhanced disease resistance to the pathogens G. cichoracearum and P. st DC3000. The deduced amino acid sequence of the corresponding protein is predicted to contain an RxCC-like and an NB-ARC domain. Most currently known R proteins have a NB-ARC domain and the CC domain is thought to initiate signaling (Radirdan et al., 2008). Given the rapid and strong up-regulation of VpCN transcript accumulation in wild Chinese Vitis after treatment with E. necator, we suggest that VpCN may play a role in the early defense signaling pathways in pathogen recognition. In addition to these two domains, the deduced amino acid sequence also contained a PLN03210 domain, which is thought to contribute to the identification of resistance signaling components and to convey resistance to P. syringae (Kim et al., 2009), suggesting that VpCN may also be associated with bacterial disease resistance. Several studies have already demonstrated that overexpression of an R-gene can cause growth retardation, spontaneous cell death, and constitutive defense activation (Tao et al., 2000;Bendahmane et al., 2002;Stokes et al., 2002;Mohr et al., 2010;Nandety et al., 2013) due an over activation of the ETI system. In this study, three independent transgenic lines exhibited dwarfism and stunted growth, as well as other morphological defects, although since these plants eventually died, we were unable to investigate whether they also exhibited enhanced resistance to G. Cichoracearum. In agree with these results we suggest that VpCN ectopic expression may active ETI system and cause constitutive defense in three transgenic plants and cause growth retardation, spontaneous cell death. Further studies will investigate whether the three dwarf and lethal phenotypes is caused by toxic effects of high level of VpCN expression or the co-suppression between VpCN and Arabidopsis endogenous genes with VpCN-homologous sequences. There have been several reports suggesting that overexpression of R genes enhances disease resistance due to constitutive SA accumulation, PR gene expression and active defense responses (Keller et al., 1999;Tang et al., 1999;Kim et al., 2001;Shirano et al., 2002;Stokes et al., 2002). In this study, ectopic expression of VpCN in A. thaliana enhanced disease resistance to G. cichoracearum, and when the PR1 transcript levels was assessed, a 4-5 fold increase in expression was observed in 12 hpi in transgenic plants compared to WT, and these levels remained higher over the time course. These results suggest that ectopic expression of VpCN in A. thaliana activate defense responses after pathogen inoculation. The production of reactive oxygen species (ROS), mainly in the form of a superoxide burst and H 2 O 2 accumulation, is thought to enhance plant defense responses and to be essential for the establishment of plant immunity (Alvarez et al., 1998;Grant and Loake, 2000;Punja, 2004;Choi and Hwang, 2011;Kim and Hwang, 2014). In agreement with these results, we found that higher levels of O − 2 anions and H 2 O 2 in the transgenic plants than in WT after challenging with P. st DC3000. This suggests that ectopic expression of VpCN triggers an oxidative burst to induce plant immunity to P. st DC3000; however, further studies are needed to investigate how oxidative burst and H 2 O 2 accumulation is mediated by VpCN. High concentrations of ROS can result in HR-like cell death (Kovtun et al., 2000; The various deletion fragments of the VpCN promoter fused to GUS and relative GUS activity driven in the transiently transformed grapevine leaves. The dark bars indicate the average GUS activity for deletion constructs in transiently transformed grapevine leaves treated with E. necator, the gray bars indicate the mock treatment (sterile water). Numbers adjacent to the bars indicate the fold difference in GUS activity leaves harboring the various constructs challenged with E. necator relative to the mock samples. The mean GUS activity (±SD) is averaged from three independent experiments (n = 3), the errors bars indicate the stand deviation. Significant difference between treatment and mock conditions was analyzed using one sided paired t-test (**and * meaning P < 0.0.1 or P < 0.05, respectively). Wang et al., 2007;Zhang et al., 2012), and over-expression of a TIR-NB-LRR gene from wild north American grapevine in V. vinifera wine grape cultivars was reported to lead to HR-like cell death after inoculation with E. necator (Feechan et al., 2013). Moreover, over-expression of a RPP1A truncation in A. thaliana induced elicitor-independent HR-like cell death (Weaver et al., 2006). In this study, an increase in ROS (O − 2 and H 2 O 2 ) accumulation followed by H 2 O 2 induced HR-like cell death was observed after ectopic expression of VpCN in A. thaliana, when the transgenic plants were inoculated with P. st DC3000. Callose-containing cell-wall appositions, called papillae, provide a physical barrier that slows pathogen invasion at the site of pathogen attack (Luna et al., 2011). Callose deposition is typically triggered by conserved pathogen-associated molecular patterns (PAMPs) and contributes to the innate immunity (Brown et al., 1998;Luna et al., 2011). Ellinger et al. (2013) demonstrated that over-expression of PMR4 in transgenic plants promoted early callose accumulation at attempted fungal penetration sites, which provided complete resistance to G. cichoracearum, and the non-adapted PM agent, B. graminis. In this study, transgenic plants displayed more callose deposition than WT plants in response to treatment with P. st DC3000, suggesting that callose deposition may contribute to the enhanced disease resistance to the pathogen displayed by the transgenic plants. To elucidate the molecular basis of VpCN transcript induction after inoculation with E. necator, the VpCN promoter was isolated and its activation investigated using A. tumefaciens-mediated transient expression of VpCN in V. vinifera leaves. Bioinformatic analysis of the promoter sequence revealed two TC-rich repeats ( ′ 5-ATTCTCTAAC-3 ′ ), which are thought to be involved in defense and stress responses (Diaz-De-Leon et al., 1993). We hypothesized that these might be involved in the response to E. necator, and generated four promoter deletion constructs to test this idea. Plants harboring a −1360, −700, or −400 bp region of the promoter sequence, all of which contain two or one TC rich repeat elements (Supplement Figure 2), showed increased GUS activity after challenge with E. necator. However, plants containing only a −240 bp region sequence, which has no TC-rich repeat elements (Supplement Figure 2), showed no significant change in GUS activity after inoculation with E. necator. Thus, we propose that the TC-rich repeat elements may play a role in the VpCN promoter activity in response to E. necator infection. This study suggests that VpCN is a disease resistance gene, and we will investigate that whether the VpCN is interact with AVR protein (effector) from Erysiphe necator. Further functional studies to the VpCN with other proteins and downstream defense signaling involved in the powdery mildew disease resistance will be helpful in understanding the molecular mechanisms of powdery mildew disease resistance in Chinese wild V. pseudoreticulata. AUTHOR CONTRIBUTIONS XW and ZW designed the experiments. ZW, LY, RW, and ZL performed the experiments. XW, ZW, and CL analyzed the results and wrote the manuscript. All authors read and approved the final manuscript.

v3-fos

2019-03-20T13:05:12.225Z

2015-09-18T00:00:00.000Z

84029648

s2

Suppressive soil against Sclerotinia sclerotiorum as a source of potential biocontrol agents: selection and evaluation of Clonostachys rosea BAFC1646 The fungal diversity structures of soils that are suppressive and non-suppressive to Sclerotinia sclerotiorum were characterised and screened for fungal strains antagonistic to the S. sclerotiorum pathogen. Soil suppressiveness was associated with a particular fungal diversity structure. Principal component analysis showed that antagonism by fungal species in suppressive soils was associated with the occurrence of Fusarium oxysporum, Fusarium solani, Talaromyces flavus var. flavus and Clonostachys rosea f. rosea. In particular, C. rosea f. rosea occurred exclusively in suppressive soil samples, suggesting that this morpho-species plays an important role in suppression of S. sclerotiorum diseases. One strain of C. rosea f. rosea (BAFC1646) was selected for further experiments. Dual-culture assays confirmed the antagonistic behaviour of C. rosea f. rosea BAFC1646 against three different S. sclerotiorum strains. Antifungal activity was corroborated by diffusion assays with metabolite extracts. Greenhouse assays with soybean plants showed that the selected C. rosea f. rosea strain reduced the percentage of dead plants when co-inoculated with S. sclerotiorum. In addition, inclusion of C. rosea f. rosea alone increased shoot lengths significantly. In this work, we established the involvement of fungal species in soil suppressiveness and in further assays confirmed that C. rosea f. rosea BAFC1646 exhibits a bioprotective effect against S. sclerotiorum in soybean plants. Introduction The fungal plant pathogen Sclerotinia sclerotiorum (Lib.) de Bary affects many economically important crops worldwide (Boland & Hall, 1994). This fungus survives in soil in the form of sclerotia, which germinate myceliogenically or carpogenically depending on environmental conditions (Bardin & Huang, 2001). Recurrent fungicide application has led to fungicide resistance in S. sclerotiorum populations (Gossen, Rimmer, & Holley, 2001;Kuang, Hou, Wang, & Zhou, 2011). Consequently, the approach of biological control has become an attractive potential means of reducing the incidence of Sclerotinia diseases (e.g. Sclerotinia wilt and stem temperate, with an annual precipitation of~900 mm and an annual average temperature of about 17°C (Gómez, 2008). Isolation and identification of fungi Active saprotrophic fungal strains were isolated from suppressive and non-suppressive soil parcels by a simple soil particle washing method wherein fungal hyphae are isolated from soil samples in an automated washing machine. Each soil sample was placed in a separate sterile box containing a 0.2 mm sieve. They were washed vigorously for about 2 min and in 35 steps with sterile water. The washing action was achieved by passing sterile air through the system. This method removes nearly all fungal spores from soil particles (Parkinson, 1994). Soil particles were cultured on malt extract agar (MEA) with streptomycin (0.5% v/v) and chlorotetracycline (0.25% v/v) at 25°C in the dark. Most of the isolated fungi were identified based on culture characteristics and spore morphology (Domsch, Gams, & Anderson, 1980). S. sclerotiorum strain BAFC225 (Buenos Aires Fungal Collection, Universidad de Buenos Aires) isolated from a sclerotium found in a non-suppressive soil sample was used for antagonism and biocontrol evaluation assays. Characterisation of fungal diversity structure The diversity of culturable and morphologically identifiable species in the two soil types studied was characterised according to the following parameters: Fr (frequency of occurrence of each species) = No. of species occurrences × 100/ total no. of inoculated soil particles Ar (relative abundance of each species) = No. of species isolates × 100/total no. of isolates obtained SR (species richness) = No. of species found at each site × 100/total no. of inoculated soil particles The Shannon-Wiener Diversity Index (H) was used to calculate the diversity of filamentous fungi in each site as follows (Krebs, 1994): where s is the number of isolates of the ith species and pi is the proportion of the total sample belonging to the ith species. This function incorporates two components of species diversity: the number of species and the proportion of individuals of each species (Donnison, Griffith, Hedger, Hobbs, & Bardgett, 2000). The obtained frequencies of occurrence of each species (Fr) were used to examine trends in species diversity structure in the two soils by principal component analysis (PCA), a classical method of data analysis for synthesis of information (Kenkel & Booth, 1992). Characterisation and selection of antagonistic soil fungi Three different in vitro assay methods were used to evaluate the antagonistic ability of each fungal isolate. Their effects on pathogen growth and sclerotia formation in dual culture and volatile and non-volatile metabolite production were evaluated in 90 mm Petri dishes at 25°C in the dark, as detailed below. Dual culture All of the soil fungal isolates obtained were tested against the pathogen S. sclerotiorum BAFC225 on MEA in a dual culture (Whipps, 1987). For each confrontation, two plugs (diameter, 4 mm) were used, one from the potential antagonist (i.e. the target isolate) and the other from S. sclerotiorum. The plugs, excised from the edge of an actively growing MEA culture of each colony, were planted 4.5 cm apart in 90 mm diameter Petri dishes (Whipps, 1987) as shown in Figure 1. Each dual-culture plate was grown in parallel with two controls plates: a pathogen alone plate and a target isolate alone plate. The interaction observed for each isolate was characterised according to the following four parameters: type of interaction [types (TIs) defined in Table 1], index of dual-culture growth inhibition, effect on S. sclerotiorum sclerotia formation and inhibition halo width (Ih) (Whipps, 1987). Production of sclerotia was determined in four colony zones after 6 days and after 13 days in dual culture (Figure 1). To assess effects on sclerotium formation capacity, the number of sclerotia encountered in dual culture was compared with a dual-culture system based only on BAFC225 (Jackson, Whipps, & Lynch, 1991). Each condition was examined in triplicate for each fungal isolate evaluated. The parameters considered in the dual cultures are summarised in Table 2. Production of non-volatile metabolites Plugs (4 mm diameter) retrieved from isolate colonies were placed on a 6 cm diameter cellophane membrane (Sterlitech) in MEA-containing Petri dishes. After three days, the membrane and the fungus were removed. Then, a single S. sclerotiorum BAFC225 colony plug (4 mm diameter) was placed in the centre of each MEA plate. The control treatment consisted of the same steps but without isolate inoculation of the cellophane membrane, as described by Whipps (1987). Incubation was carried out at 25°C; S. sclerotiorum colony diameters were measured periodically. Growth inhibition was measured by the Index of Growth Inhibition in the cellophane membrane assay (IGIc). It was calculated as the diameter of the pathogen colony on medium with membranes pre-inoculated with a potential antagonist compared with the diameter of the control colony (Table 2). IGIc values were determined on the day that the pathogen reached the Petri dish edge in the control treatment. Each treatment condition was examined in triplicate for each fungal isolate evaluated. To confirm that the fungal isolates did not penetrate the membrane, the experiment included a control condition in which a membrane was treated with the antagonist, with subsequent removal of the membrane, but without Sclerotinia slcerotiorum inoculation. Production of volatile metabolites Modifications of compartmentalised cultures were used to establish each fungal isolate's capacity to produce inhibitory volatile metabolites (Dennis & Webster, 1971). Two MEA-containing Petri dishes (90 mm diameter) were employed: one was inoculated with a pathogen plug and the other with the isolate being evaluated. Then, the bottoms of the two Petri dishes were joined and sealed hermetically with Parafilm®. In the control condition, only the pathogen plug was inoculated. The incubation was performed at 25°C and the colony diameter was recorded Figure 1. Dual cultures of some fungal isolates from soybean soils and Sclerotinia sclerotiorum BAFC225, 13 days after confrontation. (a) Illustration of areas assessed to determine antagonist effects on sclerotial production in dual cultures. X corresponds to the location of inoculum of each strain evaluated (modified from Jackson et al., 1991). Some of the observed patterns of sclerotia formation are shown in panels b-f as follows: (b) scerotia in zones 1, 2, 3 and 4 (in dual culture with Fusarium semitectum); (c) scerotia in zones 2, 3 and 4 (F. oxysporum); (d) scerotia in zones 3 and 4 (F. equiseti); (e) scerotia only in zone 4 (vs. moniliaceous sterile mycelium 5); and (f) absence of scerotia formation (Trichoderma koningii). periodically. Growth inhibition was evaluated through the Index of Growth Inhibition in the volatile metabolites assay (IGIv), with the IGIv value being determined when the control colony reached the Petri dish edge ( Table 2). The experiment included three replicates of each of the evaluated isolates. Analysis of antagonism The antagonistic capacity of each soil was evaluated through the behaviour of the isolates obtained from each one and analysed using a matrix of antagonism and PCA. The parameters considered in the matrix of antagonism were the aforementioned parameters for each in vitro assay (summarised in Table 2). Different isolates of the same species in each soil sample were grouped as a function of their morphotype and their antagonistic behaviour and were considered to be members of the same taxon (strain) in the PCA. Also percentage (%) of antagonistic isolates was determined for each soil as: Ant ðpercentage of antagonistic isolates‚ %Þ ¼ No of antagonistic isolates of a species  100⁄Total isolates evaluated Selection of an antagonistic strain The PCA results (based on frequency of occurrence of each species and antagonistic behaviour) were then used to associate observed soil suppressiveness with particular fungal taxa and to select the most promising antagonistic strains. The species whose presence and antagonistic activity showed the best separation between suppressive and non-suppressive soils were selected for further evaluation in subsequent anti-S. sclerotiorum antagonism assays. Table 1. Types of interaction considered in dual-culture confrontation assays (modified from Whipps, 1987). Type of interaction Summary of types of interaction between colonies in confrontation TI1 Colonies meet and form a straight line in center of the Petri dish, with both ceasing growth and a mixing zone of mycelia in the interaction zone (Iz), with Iz ≤ 2 mm. TI1+ Like TI1, but Iz > 2 mm. TI2a Antagonist colony growth surrounds pathogen colony with Iz ≤ 2 mm. TI2b Pathogen colony growth surrounds antagonist colony with contact between the hyphae, Iz ≤ 2 mm. TI2a+ Antagonist colony growth surrounds pathogen with subsequent growth over the pathogen with Iz > 2 mm. TI2b+ Pathogen colony growth surrounds antagonist colony with subsequent growth over the pathogen with Iz > 2 mm. TI3a Antagonist colony growth surrounds pathogen colony without contact between the hyphae and with a growth inhibition halo (Ih) ≤ 2 mm. TI3b Pathogen colony growth surrounds fungal antagonist without contact between the hyphae and with Ih ≤ 2 mm. TI4 Mutual inhibition with Ih < 2 mm, straight line interaction between the colonies. TI5 Mutual inhibition with Ih > 2 mm. Identification and characterisation of the selected antagonistic strain Identification A highly promising strain of fungus was identified with the aid of DNA barcodes in polymerase chain reaction (PCR) experiments, and it was incorporated in the Buenos Aires Fungal Collection (Universidad de Buenos Aires) as BAFC1646. The strain was cultured on 20% (w/v) malt extract broth (MEB) at 25°C for one week. Genomic DNA was extracted from the harvested mycelium (≈80 mg dried weight) using UltraClean® Microbial DNA Isolation Kit (MO BIO Laboratories, Inc. Carlsbad, CA). The rDNA ITS region was amplified by PCR with specific primers, namely ITS1 and ITS4 (White, Bruns, Lee, & Taylor, 1990). PCR amplification was performed in a Table 2. Indices of antagonism determined in in vitro assays and considered in the PCA of antagonism of suppressive and non-suppressive soils. Hydrolytic enzyme production. Enzyme activities were measured qualitatively by means of the halo generated by the degradative activity of the produced enzymes. The enzyme activities evaluated included cellulolytic, xylanolytic, pectinolytic, amylolytic, lipolytic and proteolytic activities. The culture media used included carboxymethylcellulose (CMC-Sigma, St. Louis, MO), oat xylan (Sigma,, St. Louis, MO), apple pectin (Sigma, St. Louis, MO), soluble starch (Sigma, St. Louis, MO), Tween20 (Sorbitan Monolaurate) and gelatin as substrates. Cellulases and xylanases were revealed with Congo Red dye (Pointing, 1999), pectinases with Ruthenium Red (Hankin & Anagnostakis, 1977) and amylases with I 2 -KI (Gessner, 1980). Lipases were detected by the presence of a precipitate around the fungal colonies caused by the formation of lauric acid calcium salt crystals (Abdel-Raheem & Shearer, 2002). Proteolytic activity was evidenced by visualisation of a precipitate, which results in a more opaque agar and an enhanced clear zone around the colonies (Hankin & Anagnostakis, 1977). Three replicates were used for each enzyme assay. Antagonism of C. rosea f. rosea BAFC1646 In vitro assays The antagonistic behaviour of C. rosea f. rosea strain BAFC1646 was evaluated against three S. sclerotiorum strains (BAFC225, BAFC2232 and BAFC217) in dualculture assays on MEA and potato dextrose agar (PDA; Whipps, 1987). A 4 mm colony plug was used for BAFC1646 inoculation. After two days, the S. sclerotiorum strain colony plugs were inoculated at a distance of 4.5 cm (Whipps, 1987). Three replicates were used for each confrontation. Control dishes were inoculated only with the pathogen strain in each of the media being assessed. All cultures were incubated at 25°C in the dark. The width of the inhibition halo was determined and the percentage of radial growth inhibition (RGI %) was calculated as: where rc is the radius of the control S. sclerotiorum colony and rd is the radius of S. sclerotiorum in a dual-culture colony (Whipps, 1987). Antifungal activity MEB (100 ml) was inoculated with a 4 mm colony plug excised from the edge of an actively growing MEA culture of BAFC1646. After seven days of incubation, the culture (mycelium plus spores) was used to inoculate 1 L of MEB (in a 4-L Erlenmeyer flask). Incubation was performed at 25°C for 21 days (stationary growth phase) under static conditions. This procedure was done in duplicate. Amberlite XAD-16 (150 g L −1 ) was then added to 2 L of filtered broth. The suspension was filtered 18 h later. The Amberlite was washed with distilled water and then eluted with MeOH (2 L). The MeOH eluate was evaporated to dryness and subjected to vacuum chromatography on RP-C18 using water and mixtures of water and MeOH of decreasing polarity (90: (Hadacek & Greger, 2000). Dried extract samples (100 µg) were used to impregnate filter paper discs (4 mm diameter) placed on MEA in the centre of a Petri dish confronting four S. sclerotiorum colonies at 25°C in the dark. The pathogen was inoculated at four equidistant locations as 4 mm diameter colony plugs excised from the edge of an actively growing MEA culture. The distance between the impregnated filter papers and the colony plugs was 2.5 cm. Filter paper with each solvent mixture, but without organic extract, was employed in control treatments. Each mixture and control sample was evaluated in triplicate. The RGI (%) was evaluated after 4 days and observed over a period of 10 days. Bioprotective capacity of BAFC1646 in soybean plants A glass-house experiment was conducted using G. max (soybean). Pathogen infection through mycelium and myceliogenic germination of the sclerotia was employed to simulate the type of infection observed in the field (in non-suppressive soil parcels). Soybean seedlings were sown in 200 ml of steam-pasteurised soil inoculated with the antagonistic strain added at a concentration of 1.2 × 10 6 colony-forming units per gram of soil (cfu g −1 ; determined by the method of soil dilution plate). The antagonist inoculum was incorporated into the soil as a mass of boiled and autoclaved rice previously inoculated with mycelium plugs to full colonisation (100 g substrate/ 10 days). The amount (i.e. mass) of this substrate was incorporated to reach the above-mentioned concentration (1.2 × 10 6 cfu per g of soil). The seedlings were planted in 200 ml plastic pots (one seedling per pot). After three days, the plants and soil were transferred to 600 ml pots by breakage of the 200 ml pots and the addition of 300 ml of soil colonised by S. sclerotiorum BAFC225 containing mycelia and sclerotia at the bottom and around the sides of the pot (final concentration: 15%, w/v of pathogen inoculum). The pathogen-infected soil was prepared in sterile polypropylene bags containing rice:bran:water (20:20:100; v/v/v) as substrate, and was inoculated with 5 mm pathogen plugs (six per 350 g of substrate) and then incubated in the dark for 20 days at 24-28°C (Rodríguez, Cabrera, Gozzo, Eberlin, & Godeas, 2011). A completely randomised design was employed. Four treatments with five replicates each were used: S. sclerotiorum only, C. rosea f. rosea only, S. sclerotiorum + C. rosea f. rosea together and control without any fungus inoculated (i.e. plants received only substrate). Four replicates per treatment were used in the assay repetition. The percentage of alive plants, shoot lengths and the dry weights of roots and shoots was assessed. Harvested plants were dried in an oven at 80°C until at a constant weight. Identification and characterisation of the fungal diversity structure A total of 146 isolates belonging to 30 different morpho-species were isolated from suppressive and non-suppressive soybean soils (Table 3). Fusarium oxysporum, Clonostachys rosea f. catenulata, Humicola grisea, Talaromyces helicus var. helicus, Trichoderma harzianum and Trichoderma koningii were common in both soils. The five species with the highest frequency of occurrence in suppressive soils were F. oxysporum, H. grisea, T. harzianum, T. koningii and C. rosea f. rosea, with the latter species being recovered exclusively from suppressive soil. T. koningii, F. oxysporum, H. grisea and Phoma exigua were the most frequent species found in non-suppressive soils. Among the species found to occur exclusively in nonsuppressive soils, Trichoderma viride and P. exigua were noteworthy for their high frequency. Greater SR and H values were observed for non-suppressive soils (SR = 0.33 and H = 3.77) than for suppressive soils (SR = 0.24 and H = 2.38), indicating that there was greater species richness and diversity (i.e. according to the Shannon-Wiener index) in the non-suppressive soils. PCA based on frequency of occurrence (Fr) revealed distinctive patterns differentiating samples from suppressive versus non-suppressive soils. As shown in Figure 2a, there was separation along the ordination axes as a function of components 1 and 5, which showed the best separation between samples. Component 1 segregated suppressive soil samples due to the presence of Fusarium solani, Fusarium dimerum and Fusarium equiseti, whereas component 5 segregated suppressive soil samples due to the presence of C. rosea f. rosea, T. harzianum and Fusarium semitectum. Discriminant analysis showed that 92% of the samples were well grouped. Characterisation and selection of antagonistic fungal strains It was determined that 71% of the isolates from suppressive soil samples were antagonistic against S. sclerotiorum. Among the isolates with antagonistic effects, 82% showed evidence of volatile or non-volatile metabolites inhibiting the pathogen. Antagonistic isolates exhibited TI5 and TI3b types of interaction (Table 1) or another TI combined with observed growth inhibition in antifungal metabolite assays. Antagonism against S. sclerotiorum was observed for 100% of the F. oxysporum, T. harzianum and C. rosea f. rosea isolates, which represent three of the five species most frequently isolated from suppressive soils. Antagonistic activity against S. sclerotiorum was observed in only 51% of strains isolated from non-suppressive soils. Isolates of T. koningii, F. oxysporum and H. griseathe species most frequently isolated from non-suppressive soilsexhibited antagonistic activity in 69%, 71% and 0% of trials, respectively (Table 3). About three quarters (76%) of the antagonistic isolates from non-suppressive soils showed growth inhibition indicative of the presence of inhibitory metabolites. That is, they exhibited inhibition types TI5 or TI3b, or another TI combined with observed growth inhibition in antifungal metabolite assays (see Table 1). The PCA of antagonistic behaviour showed that the suppressive and nonsuppressive soil samples were best segregated by representation of component 3 in the functions of components 4 and 5 (Figure 2b and 2c). Component 4 segregated the samples mainly according to the antagonism of F. oxysporum (strains 1 and 5) and F. solani, whereas component 5 segregated suppressive soil samples characterised mainly by the antagonistic behaviour of T. flavus var. flavus and C. rosea f. rosea (strain 2). Identification and characterisation of C. rosea BAFC1646 The strain BAFC1646 was identified as being C. rosea f. rosea based on its molecular barcode and morphological characteristics. Alignment of the generated sequence in Sequencher software and comparisons with deposited sequences in BLAST searches indicated that the ITS sequenced from the strain (Query coverage 97%) was 99% similar to AF358235 from strain CBS 710.86, the ex-neotype strain of C. rosea (Schroers, 2001;Schroers, Samuels, Seifert, & Gams, 1999). The GenBank accession number of the newly generated nucleotide sequence is KF765504. The BAFC1646 strain did not show P-solubilisation ability as evidenced by the absence of halos in NBRIP medium. Enzyme activity assays indicated that this fungal strain produces cellulases, amylases, lipases, and xylanses, but not pectinases, and no evidence of proteolytic activity was observed. Production of IAA was detected. Antagonism of C. rosea f. rosea BAFC1646 In vitro assays All three S. sclerotiorum strains tested were inhibited by C. rosea f. rosea BAFC1646 (Figure 3a and 3b). Inhibition halos were observed with all three S. sclerotiorum strains on MEA and against S. sclerotiorum strain BAFC225 on PDA (classified as TI3b, Table 1). The antagonistic C. rosea f. rosea strain BAFC1646 stopped growth of S. sclerotiorum strains BAFC2232 and BAFC217 on PDA upon colony contact with a width ≤2 mm (classified as TI2b, Table 1). As shown in Table 4, inhibition of S. sclerotiorum radial growth was more effective on PDA [RGI (%) range 47-54%] than on MEA (43-50%) in dual-culture assays with all three pathogen strains. On MEA, BAFC225 exhibited significantly more growth inhibition than the other two strains. Meanwhile, on PDA, both BAFC225 and BAFC2232 exhibited significantly more growth inhibition than BAFC217 (Table 4). C. rosea f. rosea BAFC1646 produced inhibition halos with all three S. sclerotiorum strains on MEA, but only with BAFC225 on PDA. The inhibition halos observed for the three S. sclerotiorum strains on MEA did not differ significantly from one another (Table 4). Changes in the growth of pathogen colony hyphae in contact with inhibition halos were observed under an optical microscope; the observed changes included increased branching, shortening of branches and collapsed cytoplasm (Figure 4). 44.4 ± 0.9 b 2.5 ± 0.3 a 53.9 ± 2.1 a 0.0 ± 0.0 Note: Values are means for each treatment with standard errors. Different letters indicate significant differences between treatments (ANOVA Tukey test p < .05). Ih on PDA data were not been analysed due to 0 counts for both BAFC217 and BAFC2232. Antifungal activity Organic extracts of C. rosea f. rosea strain BAFC1646 (eluted with 10:90 and 0:100 of H 2 O-MeOH in the C18 fractionation) produced significant inhibition zones (halo > 5 mm) in diffusion assays. The pathogen showed morphological abnormalities of the mycelium, similar to those observed in the dual-culture assays, as well as melanisation of the colony in the zone of contact with crude extract (Figure 3c and 3d). The RGI (%) values for the pathogen in contact with the extract were >20%. sclerotiorum co-inoculation condition survived (symptoms of disease was not observed in surviving plants). Plants in the C. rosea f. rosea BAFC1646 only condition had significantly longer shoots than plants in the other conditions (P < .05), however shoot and root dry weight did not differ significantly between the treatment groups ( Figure 5). Values are the mean from two assays. Re-isolation of S. sclerotiorum from the roots and stems of plants exhibiting symptoms of disease allowed us to confirm that it was the causal agent of disease. Discussion This study focused on the role of fungi in suppressive soils in inhibiting the growth of the phytopathogenic fungus S. sclerotiorum. Knowledge of the fungal species that inhabit suppressive and non-suppressive soils is important for understanding soil suppressiveness. It is also essential for isolating and identifying the specific microorganisms responsible for effective biocontrol (Borneman & Becker, 2007). Here, we found that F. oxysporum, C. rosea f. catenulata, H. grisea, T. helicus var. helicus, T. harzianum and T. koningii were present in both suppressive and non-suppressive soils, but with different frequencies. We observed higher frequencies of F. oxysporum, H. grisea, T. helicus var. helicus and T. harzianum in suppressive soils, but higher frequencies of C. rosea f. catenulata and T. koningii in non-suppressive soils. These results support Dix and Webster' s (1995) hypothesis that primary differences in community composition are related more to variations in species frequencies than fundamental composition. The soil washing method used had the benefit of ensuring that we were sampling active fungi (Parkinson, 1994). Our diversity analyses showed that suppressive soils tended to have a less diverse fungal structure than non-suppressive soils (see SR and H data), which indicates that antagonism against S. sclerotiorum is attributable to particular species. Indeed, some taxa (e.g. C. rosea f. rosea and F. solani, both with 100% of isolates being antagonistic) were found exclusively in suppressive soils, and some (e.g. F. oxysporum, T. harzianum and T. koningii) were present in both suppressive and non-suppressive soils, but were found in higher proportions in antagonistic isolates of suppressive soils. Some authors have suggested that changes in frequency mask changes in relative levels of various subpopulations (Hunter et al., 2006). Our experimental results were confirmed by PCA. The PCA results indicated that antagonism differences between suppressive and non-suppressive soils could be attributed to C. rosea f. rosea and F. solani in suppressive soils. In fact, C. rosea f. rosea, among the five most frequently encountered morpho-species, was the only taxon that was observed exclusively in suppressive soils. All recovered isolates of this species displayed antagonistic activity against the pathogen. Our results support the notion that naturally occurring soil micro-organisms are important for both natural and induced disease suppressiveness. The suppressiveness of soils may be characterised by high microbial population diversity (Ghorbani, Wilcockson, Koocheki, & Leifert, 2008;Weller et al., 2002). Although the present study did not address this question directly, our findings are consistent with the hypothesis that suppressive soils should have a high frequency and inoculum density of specific antagonistic fungal taxa (Bonanomi, Antignani, Capodilupo, & Scala, 2010). In fact, when we evaluated antagonism, we found that 71% and 51% of all the isolates obtained from suppressive and non-suppressive soils, respectively, showed some kind of antagonism. All isolates of three of the five species most frequently found in suppressive soils (F. oxysporum, T. harzianum and C. rosea f. rosea) showed antagonistic abilities. Most of the antagonistic isolates from suppressive soil samples produced antifungal metabolites and inhibited growth of the pathogen. A low proportion of the isolates overgrew pathogen colonies. This is the case of Trichoderma spp. Talaromyces flavus var. flavus and C. rosea f. catenulata. Relationships among organisms are critical aspects of the fungal diversity structure (Dix & Webster, 1995). In this regard, our results establish that soybean soil suppressiveness was associated with the antagonistic activity of certain fungal strains, with the inhibitory effect due to antifungal metabolites. Other studies have shown that both soil microbial community composition and antifungal metabolite production can be major determinants of soil fungistasis (de Boer et al., 2003;Garbeva et al., 2011;Vey, Hoagland, & Butt, 2001). de Boer et al. (2003) consider that although the nutrient status of the soil can exert a role in the production of antifungal compounds, it is the microbial community composition and the interactions between their members that determine the quality and quantity of these antifungal compounds. The ordination analysis (i.e. PCA) based on the antagonistic behaviour of different species and/or species-specific mechanisms indicated that F. oxysporum, F. solani, T. flavus and C. rosea f. rosea were the most likely sources of suppressiveness. This conclusion is supported by prior work (Rodríguez, Cabrera, & Godeas, 2006) in which a strain of F. oxysporum was found to protect soybean plants against S. sclerotiorum through the production of antifungal secondary metabolites. It is noteworthy that the occurrence frequencies of F. solani and C. rosea f. rosea were determinant components in the separation between suppressive and non-suppressive soils and that C. rosea f. rosea, in particular, was present at high frequencies in suppressive samples. Indeed, C. rosea f. rosea meets the desirable biocontrol agent criterion of being ecologically adapted to the environment of its target pathogen . However, C. rosea is used already in commercially available formulations. In this context, our findings suggest that the BAFC1646 strain of C. rosea may be a promising biological control agent. The findings from our in vitro dual-culture assays, an established method for initial evaluation of a microbial antagonist (Gromadzka et al., 2009;Whipps, 1987), provide strong evidence in support of C. rosea f. rosea BAFC1646 as a biocontrol agent. Although other strains of C. rosea have been shown to inhibit phytopathogens (Mejía et al., 2008;Whipps, 1987;Zazzerini & Tosi, 1985), it is noteworthy that our BAFC1646 strain showed an unusually high RGI (%) in all the conditions evaluated and against all strains of S. sclerotiorum evaluated. Interestingly, the power of inhibition exerted varied among the three S. sclerotiorum strains tested. Inhibition in sclerotia formation and alteration of mycelial morphology were observed in S. sclerotiorum colonies interacting with C. rosea f. rosea BAFC1646. Similar modifications of hyphae morphology have been described previously when phytopathogenic fungi were confronted with a range of micro-organisms. Their effects have been related to antifungal metabolites (Aryantha & Guest, 2006;Rodríguez et al., 2011;Zazzerini & Tosi, 1985). The antifungal activity of C. rosea f. rosea BAFC1646 was confirmed in diffusion assays with metabolite extracts associated with the stationary growth phase. Metabolite extracts changed the morphology of the mycelium of the pathogen in dual cultures. These changes corresponded with melanisation of pathogen structures in diffusion assays. The purification and complete structural elucidation of the metabolite(s) involved is being undertaken currently and the results will be published elsewhere. In our greenhouse assays, C. rosea f. rosea reduced S. sclerotiorum disease in soybean plants. In addition, in the absence of the pathogen, it significantly increased the shoot lengths of soybean plants. Using similar conditions, Harman, Petzoldt, Comis, and Jie Chen (2004) and Rodríguez et al. (2011) showed growth promotion of Zea mays plants and Lactuca sativa plants with T. harzianum and C. rosea inoculation, respectively. Thus, C. rosea f. rosea could be used as both a biocontrol agent and a plant growth-promoting agent. Multi-faceted mechanisms can be at work in microbial-plant interactions (Whipps, 2001). Here, our C. rosea f. rosea strain showed growth promotion and various hydrolytic enzyme activities in addition to its antifungal activity. These multiple effects could have implications for improving plant growth, facilitating access to nutrient sources otherwise not available to the plant (Whipps, 2001). The observed production of IAA by C. rosea f. rosea could be involved directly or indirectly in the promotion of soybean plant growth. IAA production by saprotrophs and endophytes has been reported to be involved in promoting growth in various plants and thus could be responsible, at least in part, for the presently observed plant growth promotion (Yuan, Zhang, & Lin, 2010;Zhang et al., 2012). Saprophytic ability and IAA production have been described as common attributes of fungal biocontrol agents (Altomare, Norvell, Björkman, & Harman, 1999;Shoresh, Harman, & Mastouri, 2010). A few consistent patterns relating plant and soil community composition have been detected; however, they have not been studied in detail (Singh et al., 2013). Soil microbial populations are immersed in a system of interactions that affect plant fitness and soil quality (Barea, Pozo, Azcon, & Azcon-Aguilar, 2005). Here, we uncovered characteristic differences between soils that are or are not suppressive against S. sclerotiorum through a two-pronged approach. On the one hand, we isolated active fungi, identified the isolated fungi and determined their frequencies of occurrence. On the other hand, we evaluated the antagonistic activity of the isolates against S. sclerotiorum, and identified C. rosea f. rosea strain BAFC1646 as a promising biocontrol agent. Our characterisations of fungal diversity structures allowed us to elucidate the differential behaviour of suppressive versus non-suppressive soils. Those findings led us to select suppression-associated fungal species for further analysis. The present results indicate that C. rosea f. rosea has a bioprotective activity that protects soybean plants from S. sclerotiorum diseases and further suggest that this activity may be mediated through antifungal metabolite production. Disclosure statement No potential conflict of interest was reported by the authors.

v3-fos

2016-03-22T00:56:01.885Z

2015-09-30T00:00:00.000Z

18382388

s2

Construction of Specific Primers for Rapid Detection of South African Exportable Vegetable Macergens Macergens are bacteria causing great damages to the parenchymatous tissues of vegetable both on the field and in transit. To effectively and rapidly investigate the diversity and distribution of these macergens, four specific primers were designed by retrieving 16S rDNA sequences of pectolytic bacteria from GenBank through the National Center for Biotechnology Information (NCBI). These were aligned using ClusterW via BioEdit and primers were designed using Primer3Plus platform. The size and primer location of each species and PCR product size were accurately defined. For specificity enhancement, DNA template of known macergens (Pectobacterium chrysanthermi) and fresh healthy vegetable were used. These primers yielded expected size of approximately 1100 bp product only when tested with known macergens and no amplicon with fresh healthy vegetable was detected. Rapid detection of macergens in rotten vegetable samples was then carried out using these primers. Nucleotide sequences of macergens identified were deposited into the GenBank and were assigned accession numbers. Hence, with these specific primers, macergens can be identified with minimal quantities of the vegetable tissues using molecular techniques, for future use of the quarantine section of the Agricultural Department of the country for quick and rapid detection of macergens before exportation. Introduction Several bacteria species classified to different genera that can macerate parenchymatous tissues of a wide range of plants termed macergens, can occur in growing plants and on the harvested crop either in storage or transit [1]. They cause greater losses in the production and economy of the affected plant depending on the severity of the attack [2]. The different tissue maceration enzymes produced by these macergens result in rapid tissue degradation in plants [3]. The macergens include pectolytic strains of bacteria belonging to mainly six genera namely Erwinia, Xanthomonas, Pseudomonas, Clostridium, Cytophaga, and Bacillus [4]. The activities of these macergens are tightly detrimental to agricultural efficiency and plant production, leading to greater economic losses [5]. For a complete diet, fruit and vegetables (leafy and fleshy vegetables) are highly essential, however, fruits and vegetables are being threatened by macergens both on the farm, transit and in storage, reducing their quality, yields, shelf-life and consumer satisfaction. If mistakenly eaten, it can result in food poisoning and allergens [6]. In order to guide against this, early detection of these macergens needs to be considered. Although conventional methods have been in use, they are laborious and time consuming. No diagnostic primer is yet available to discriminate macergens [7]. Polymerase Chain Reaction (PCR) assay is the most sensitive of all the existing rapid methods, to detect microbial pathogens in many specimens [8]. This involves several critical steps such as Deoxy Ribonucleic Acid (DNA) extraction, PCR amplification and the detection of amplicons through electrophoresis study. Hence, needs for rapid and accurate detection of these become imperative. Rapid detection of macergens in vegetables is becoming more critical and the development of rapid and sensitive methods is of great interest for human safety. However, molecular techniques can be used to confirm the identity and the nature of the macergens, thus the major aim of this article is to design specific primers for rapid, accurate detection and identification of macergens. Primer Design All database searching was done through the website of the National Center for Biotechnology Information (NCBI) at http://www.ncbi.nlm.nih.gov/. Sequences retrieved were as follows: These pectolytic macergens 16S rDNA nucleotide sequences from NCBI were saved as FASTA files. FASTA files were copied into BioEdit files in the program BioEdit Sequence Alignments Editor, Version 7.0.9.0 [9]. Multiple Sequence alignments (MSA) were performed using the ClustalW 2.0 algorithm [10]. Stringency was varied to achieve an alignment with a small number of gaps and mismatches. Altering the stringency was also done to yield as many regions with a high degree of sequence similarity as possible. MSA's were consolidated based on obvious discrepancies (i.e., the presence of a pectolytic bacterium) and a lack of sequence similarity to the consensus. The lack of sequence similarity was measured subjectively and on a percent similarity basis when needed. Consolidated trials were then aligned with each other and sequences with low similarity were discarded. They were then opened in BioEdit to determine the highly conserved regions where primers can be designed for macergens. The primers were designed using the Primer3Plus interface (http://frodo.wi.mit.edu/) and the best primers were selected using criteria for good primer design [11]. Before proceeding to empirical testing, the finally selected primer sequences were checked for potential hairpins structure, self-dimer, cross-dimer, and cross-homology, and tested for binding affinities to the priming sites (delta G values) using Gene infinity Platform. Their specificity was determined through in silico PCR in Gene Infinity platform. NCBI Blast was also used to see if the primers were able to give the target macergens. Finally, the best primers were synthesized by Integrated DNA Technology at Inqaba Biotechnical Industrial (Pty) Ltd, Pretoria, South Africa. Primer Development Primers were tested with a gradient PCR machine from 47 to 59 °C to test for varying annealing temperatures. Concentrations of MgCl2 were varied from 1.0 to 4.0 mM and 10 ng of DNA was used per reaction tube. Reaction volumes of 50 μL consisted of 5 μL 10 × Buffer, 10 mM dNTPs, 20 μg/mL BSA, 5 U/μL Taq polymerase, 10 μM forward and reverse primers and enough nanopure water to fill reaction volumes to 50 μL. The PCR began with a 94 °C hot start for 10 min. The PCR cycles consisted of a 94 °C melting temperature for 30 s/cycle, a 47-59 °C annealing temperature for 30 s/cycle, and a 72 °C polymerase elongation step for 1 min/cycle. The PCR ended with a 72 °C elongation for 10 min and a holding period at 4 °C for infinite time. Samples were loaded into a 1.6% agarose gel stained with EtBr (Ethidium Bromide), 1 kb DNA ladders were loaded in 5 μL volumes, while 7 μL of the sample was loaded with 2 μL of loading dye. The gel was allowed to run for 2 h at 60 V. Test results were visualized with a ChemiDoc ™ MP System (Bio-Rad Laboratories, Hercules, CA, USA). Primers were empirically checked for specificity, by using them to amplify a known macergens DNA template of Pectobacterium chrysanthemi (31 ng/μL) serving as positive control and fresh healthy vegetable DNA template as negative control. This was done in order to know if they really amplified the target region of pectolytic gene and also to eliminate any possible contamination in the PCR assay. Detection of Macergens from Vegetable Samples The designed primers were used for detection of macergens after the specificity test. Extraction of Metagenomic DNA from Vegetables DNA was extracted from the twenty-six rotten South African vegetables using ZR Fungal/Bacterial DNA MiniPrep ™ (Zymo Research) according to the manufacturer instructions. PCR Amplification The average amount of the DNA used as template for PCR was 1 ng per reaction using the previously described conditions in this study. These were repeated at least twice, unless the result was not clear enough, hence were repeated three times. PCR amplicons were analyzed by electrophoresis on 1% (w/v) agarose gel as above to confirm the expected size of the amplicons and visualized using ChemiDoc Image Analyzer while remaining PCR products were purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel GmbH & Co., KG Düren, Germany). DNA Sequencing The sequencing of the purified PCR products were done at Inqaba Biotechnical Industrial (Pty) Ltd, Pretoria, South Africa with PRISM ™ Ready Reaction Dye Terminator Cycle Sequencing Kit using the dideoxy chain termination method and electrophoresed with a model ABI PRISM ® 3500XL DNA Sequencer (Applied Biosystems, Foster City, CA, USA) by following manufacturer's instructions. Sequence Analysis ChromasLite version 2.33 software was used for the analysis of Chromatograms, (sense and antisense) resulting from sequencing reaction for good quality sequence assurance [12]. The resulting chromatograms were edited using BioEdit Sequence Alignment Editor [9]. After which, the resulting consensus 16S rDNA sequences obtained were Blast in the NCBI database (www.ncbi.nlm.nih.gov) with the Basic Alignment Search Tool (BLASTn) for homology in order to identify the probable organism in question [13]. These sequences were deposited in the GenBank. Phylogenetic Analysis The phylogenetic analyses based on the 16S rDNA gene for pectolytic bacteria were further used to characterize the macergens in order to establish relationship among them. The partial 16S rDNA sequences obtained for the macergens were utilized in the search of reference nucleotide sequence available in NCBI GenBank database using BlastN algorithm [13]. MAFFT version 7.0 was employed in the multiple alignment of nucleotide sequences [14], while trees were drawn based on three major techniques using MEGA 6 [15]. These techniques include: distance based (Neigbour-Joining (NJ) with cluster-based algorithm) used in calculating pairwise distance between sequences and group sequences that are most similar and character based method (Maximum Likelihood) for comparing set of data against set of models of evolution to select the best model for the variation pattern of the sequences [16]. Results and Discussions The 16S rDNA gene of the pectolytic bacteria were best target genes for primer development because they are highly conserved regions of the bacteria and most reliable. They are present in all target organisms as single copy per genome and are improbable to undergo horizontal gene transfer. The significance of the alignment containing several pectolytic different bacterial species was that the developed primers would have a better chance of amplifying macergens community DNA as a whole. This means that our primers may encompass a broader range of species to be recognized by PCR analysis. These species could be bacteria that we had not considered during our development process. The primer sets were developed around bacterial species that can macerate plant tissues so that they could be used to amplify community DNA extracted from plant. Four primers sets were successfully developed, from the 16S sequences of the pectolytic bacteria downloaded for better performance. The designed primers tested in the Gene Infinity Platform for binding affinities to the priming sites (delta G values), showed that they did not have potential hairpin structures, self-dimer, cross-dimer and cross-homology. All the forward primer sets sequence are good due to their legitimate G/C clamp at the 3' end, its moderate melting temperature, and its location past the 5' end of the coding sequence. These sequences are moderate in length, which facilitate specific binding to the target gene. The in silico PCR performed in the Gene Infinity platform revealed an excellent specificity of designed primers. Further primer specificity, in NCBI's Primer-BLAST also resulted in the target macergens. These are in line with the primer properties proposed by Innis and Gelfand [11] which resulted into an excellent results. Thus, generated macergens-specific PCR primers from 16S rDNA sequences of pectolytic bacteria with their properties and the locations were depicted in the Table 1. In Figure 1, the sensitivities of Polymerase Chain Reaction (PCR) assay of the primers revealed that, 1000-1200 base pairs product were obtained only when macergens specific primers were used to amplify the DNA of positive control in which the vegetable were exposed to P. chrysanthemi and negative control that were not exposed to any macergens or microorganisms (DNA template of fresh healthy vegetable). The result obtained from the use of these specific group primers, on vegetable DNA samples revealed their ability to amplify 16S rDNA product of the correct size exclusively from DNA of these vegetables. These are depicted in Figure 2. Hence, there is clarity in the specificity of designed primers because they did not bind to the DNA template that is devoid of the target gene in question. As a result of this, they were able to detect macergens from the vegetable samples. With the use of these designed primers, fourteen macergens were detected in sixteen vegetables out of twenty-six samples examined. Enterobacter sp., Lelliottia sp and Klebsiella sp. were detected by all the primer sets. The most abundant out of all the macergens detected is Citrobacter sp. detected by Primer Sets 1, 2, and 4 ( Figure 3). The sequences of the macergens detected were deposited in the GenBank. In addition, the macergens detected by the primer pairs from the samples with their accession numbers are shown in Tables 2-4, respectively. Identification of bacteria has often been difficult using traditional methods, but it has become easier with 16S rDNA sequencing [17]. Although this has insufficient discriminating power in some genera, phylogenetic analysis allows us to exclude other species and genera. This can be used to eliminate the hypothetical cause of diseases in the quarantine section. The 16S rDNA constitutes a real step forward towards accurate identification with 85.8% of species level identification, as compare to the traditional methods that are slow and unreliable [18]. Furthermore, Figures 4 and 5 depicted the analysis on phylogenetic relationships of thirty-one sequences of macergens detected alongside with twenty-seven 16S rDNA sequences of the most closely related taxa retrieved from GenBank. Table 3. (c) Ethidium bromide-stained gels of PCR amplification products obtained from different rotten vegetable samples using M180F + M1190R. Lane 2,5,6,7,8,9,10,13,14,15,16,19,22,23,24,25 and 26: No amplification; Lane 1, 3, 4, 11, 12, 17, 18, 20 and 21: Amplicon size ranges from 1000 to 1100 bp. These macergens detected are represented in Table 4. Figure 4. Neighbor Joining method of phylogenetic tree based on partial 16S rDNA gene sequence, showing the phylogenetic relationships between macergens and the most closely related strains from the GenBank. Numbers at the nodes indicate the levels of bootstrap support based on 1000 resampled data sets. Only values greater than 50% are shown. The scale bar indicates 0.5 base substitution per site. Pantoea species were set as the out-group. Sequences obtained in this study are denoted with a triangle . This relationship was based on two methods of phylogenetic tree namely: distance and likelihood methods. This was done in order to establish the proven resolution and statistical significance of the various treeing algorithm according to [19,20]. The distance based method inferred the evolutionary relationship using Neighbor Joining (NJ) clustered-based algorithm. The concatenated NJ showed the optimal of 46.60977 branch length with 207 position in the final dataset. Based on the cluster algorithm, NJ tree revealed the percentage of evolutionary relationship with the macergens based on the degree of differences between the sequences. The concatenated NJ showed that M32 and M112 have very high homology of 100% with Enterobacter ludwigii and Enterobacter sp., respectively. Equally, M35, M40 and M118 also shared 100% homology in NJ with Rahnella genomosp. In NJ, M20, M21, M31, M33, M37, M39 and M115 are closely related to Kluyvera intermedia, Citrobacter murliniae, Cronobacter malonaticus, Leclercia adecarboxylata, Tatumella terrea and Enterobacter sp., respectively, with 99% bootstrap value. Based on this distance tree, M114 and 23 have 99% homology with Rautella sp., Yesina murmii and Pectobacterium sp., which is a well-known primary macergens [21]. Equally, M28 and M36 also possessed 99% similarities with Klebsiella michiganensis and uncultured Pectobacterium sp. also a primary macergens [22]. In addition, M25 exhibited 99% evolutionary relationship in NJ with Citrobacter youngae and Enterobacter youngae. In NJ, M27 expressed 94% homology with Citobacter freundii, while M111 has 91% homology with Rahnella genomosp. These high bootstrap value expressed by the afore-mentioned macergens is beyond 70% borderline of degree of relatedness proposed by Wayne et al. [23]. In addition to this, similarities expressed by these marcergens with the reference taxa belonging to different species, is due to their high similarity value, which result in DNA reassociation values that fall below the 70% threshold values [24]. This showed high genetic relatedness that is increasingly reliable because they cannot be wiped out overnight according to Konstantinidis and Stackebrandt [19]. In NJ, M22, M24, M26, M29, M30, M38, M113, M117, M119 and M120 form distinct clades with bootstrap value less than 50% but are closer to Enterobacter sp. M34 and M116 also have very low bootstrap values that are less than 50% but have closest relative to be Citrobacter murliniae and Cronobacter malonaticus, respectively. These macergens did not cluster with any strains as a result of peculiarity of their nucleotide signature pattern [25]. This indicates that M22, M24, M26, M29, M30, M34, M38, M113, M116, M117, M119 and M120 are novel macergens based on their distinctness [19]. The maximum likelihood method was based on Kimura-2-parameter model [26]. This showed the relatedness of macergens based on the discrete character shared with the reference taxa. The tree with the highest log likelihood of −9764.8523 was shown with 205 final data position. This tree showed that M35, M40, and M118 are closely related to Rahnella genomosp with 99% bootstrap value. M111 also has a moderately similarity of 74% bootstrap value with Rahnella genomosp. M20 and M112 also have high homology of 96% with Kluyvera intermedia and Enterobacter sp. There is high relatedness of 98% bootstrap value between M21 and Citrobacter murliniae. Enterobacter ludwigii is closely related to M32 with 94% bootstrap value. Pantoea agglomerans and Klebsiella sp. are moderately similar to M39 with 89%, while Proteus mirabilis have 88% homology with M39. Klebsiella michiganensis and uncultured Pectobacterium sp. have 93% homology with M28 and M36. In addition, M33 expressed 92% similarities with Raoultella terrigena and Lerclercia adecarboxylata. Moderate relatedness of 81% was seen in M27 with Citrobacter freundi, 85% in M115 with Enterobacter sp. and 87% in M25 with Enterobacter cloacae and Citrobacter youngae. High levels of similarities of 95% were also expressed in M23 and M114 with Yersinia murmii, Raoultella sp. and Pectobacterium sp., respectively, and 97% in M37 with Tatumella terrea. All these results are still in accordance with the distance based method with the exception of M31 that clustered with 99% homology in NJ. This clustered with Cronobacter malonaticus in ML with a bootstrap value that is less than 50%. Hence, the relationship between Cronobacter malonaticus and M31 has been wiped out [19]. It is not reliable because their DNA reassociation is above the threshold level based on result depicted by ML tree [24]. ML tree also shows that some macergens did not align with any of the reference taxa based on their uniqueness, including M22, M24, M26, M29, M30, M31, M34, M38, M113, M116, M117, M119 and M120. These were classified as novel macergens with unique nucleotide signature pattern [27,28]. From the phylogenetic point of view, with the use of different algorithms, the trees inferred well-supported phylograms of macergens with high resolution of the inner branches. They all revealed that macergens are heterogeneous as they cut across different species. This is in line with [29]. Thus, it is not surprising that novel strains that do not cluster with the current known members of the previous macergens have emerged. The four oligonucleotide primer (M101F + M1208R, M182F + M1190R, M180F + M1190R, and M57F + M296R) designed in this study enhanced specificity for DNA from macergens which provides a simple method for identifying macergens. Conclusions The four primers designed were able to produce amplicons of expected sizes upon PCR analysis; they were optimal for heterogeneity of macergens. The high degree of similarity between the sequences chosen, through many rounds of search and refinement, implies that the primers are specific for pectolytic gene. Since these primers were designed around bacterial species, we can conclude that they must be specific to the certain amount of bacteria necessary. This method offers advantages over classical methods of detection, in the sense that the entire assay is fast, reliable, cost effective and no taxonomist is required before the identification is complete. This can be employed in analyzing and monitoring plant materials for macergens invasion in a quarantine section of the agricultural sector of a country before importation and exportation of these plants.

v3-fos

2016-06-17T23:51:31.955Z

2015-01-26T00:00:00.000Z

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GIGANTEA – an emerging story GIGANTEA (GI) is a plant specific nuclear protein and functions in diverse physiological processes such as flowering time regulation, light signaling, hypocotyl elongation, control of circadian rhythm, sucrose signaling, starch accumulation, chlorophyll accumulation, transpiration, herbicide tolerance, cold tolerance, drought tolerance, and miRNA processing. It has been five decades since its discovery but the biochemical function of GI and its different domains are still unclear. Although it is known that both GI transcript and GI protein are clock controlled, the regulation of its abundance and functions at the molecular level are still some of the unexplored areas of intensive research. Since GI has many important pleotropic functions as described above scattered through literature, it is worthwhile and about time to encapsulate the available information in a concise review. Therefore, in this review, we are making an attempt to summarize (i) the various interconnected roles that GI possibly plays in the fine-tuning of plant development, and (ii) the known mutations of GI that have been instrumental in understanding its role in distinct physiological processes. INTRODUCTION GIGANTEA (GI), the unique plant specific nuclear protein, although identified way back (Rédei, 1962) as a late flowering mutant (gi) in Arabidopsis thaliana (At), its precise biochemical roles are far from being understood (de Montaigu et al., 2010). The genomic organization of GI was evident after it was fine-mapped to chromosome 1 and subsequently, the GI cDNA was isolated (Fowler et al., 1999). The genomic locus of GI of At consists of 14 exons and encodes for a protein of 1173 amino acids (Fowler et al., 1999;Park et al., 1999). GI expression is ubiquitous and is detected throughout various stages of plant development indicative of its involvement in several functions summarized in Figure 1 (Fowler et al., 1999;Park et al., 1999). It is interesting to note the ubiquitous expression of GI that reflect upon its pleiotropic roles in multitude of responses ranging from breaking of seed dormancy, hypocotyl elongation, initiating the circadian rhythm in seeds to the setting of fruits in the adult plant. Many of the above listed responses integrate information from the light input and external temperature, making it an interesting but complicated area of plant science. Experiments aimed at understanding the abundance of the transcript and the protein are typically carried out in controlled cabinets, where the subjective time of the diurnal cycle are referred as the Zeitgeber time (ZT). Both the GI transcript and GI protein are under the control of diurnal regulation. Under long day (LD) growth cycle of 16 h light and 8 h dark (16 hL/8 hD), the GI mRNA peaks at ZT 10 and shows a trough at ZT 0, while under short day (SD) cycle of 8 hL/16 hD, GI transcript level peaks at ZT 8 (Fowler et al., 1999). The GI protein abundance also follows a similar pattern to its transcript accumulation (David et al., 2006). The regulation of GI is important for the control of circadian clock and several genes such as FLAVIN-BINDING, KELCH REPEAT, F BOX 1 (FKF1), a blue light photoreceptor, and CYCLING DOF FACTORs (CDFs), which are involved in the transcription of a flowering time regulator CONSTANS (CO; Fornara et al., 2009). In addition, the diurnal regulation of the protein might also play an important role in the diurnal control of stomatal opening (Ando et al., 2013). In order to assign a function to GI, it was of interest to enumerate its precise sub-cellular localization. Therefore, N-terminal GFP fusion of GI was constructed and transiently transfected in onion epidermal cells. The fluorescence microscopy of the fusion protein for the first time demonstrated that GI is predominantly localized to the nuclei and forms nuclear bodies (Huq et al., 2000). Later, the GI protein was also found to be localized in the nucleus of different cell types of transgenic At plants over-expressing GI:GFP (Mizoguchi et al., 2005). Four clusters of basic amino acids resembling the nuclear localization signal (NLS) in the GI sequence explained its nuclear abundance (Huq et al., 2000). GI has been shown to form nuclear bodies of diverse numbers, size, and shape (Kim et al., 2013c). To understand the molecular composition of GI nuclear bodies, attempts were made to evaluate the co-localization of GI with marker proteins of known sub-nuclear compartments such as heterochromatin bundles, nucleoli, spliceosome, and Cajal bodies. This piece of work demonstrated that GI did not localize to any of the above known nuclear compartments (Kim et al., 2013c). This suggested that GI might not have role in processes such as biogenesis of rRNA and snRNP, pre-mRNA splicing, and protein degradation. Since these co-localization studies were carried out in Arabidopsis mesophyll protoplasts using a transient over-expression method, it does not mimic the exact physiological environment. Furthermore, the association and dissociation rate of proteins to nuclear bodies has been shown to be affected by specific post-translational modifications. The spatio-temporal mis-localization of proteins can also affect its post-translational modifications. With so many complexities involved, stable transgenic lines expressing fluorescent tagged marker proteins and GI under their native promoters would be an impressive feat to achieve in order to understand the molecular composition of the GI complexes. Understanding the molecular composition of GI nuclear bodies (NBs) at different diurnal time-points would be a valuable asset. The formation of GI nuclear bodies is light dependent since, the sequestration of GI into NBs is facilitated by EARLY FLOWER-ING 4 (ELF4) during the day, thus inhibiting the CO transcription. Likewise, EARLY FLOWERING 3 (ELF3) promotes the interaction of GI and CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) to form NBs which degrade GI in planta (Yu et al., 2008). The dynamic association of GI with heterogenous nuclear bodies during the light to dark transition needs to be evaluated. In other words, the question still remains, if GI associates and dissociates in a light dependent manner on a core complex within the nuclei based on its differential post-translational modification status. Although studies showed the presence of GI predominantly in the nuclei, in silico analysis predicted the presence of 11 transmembrane domains in AtGI which argues in favor of a possible membrane localization (Fowler et al., 1999). Furthermore, membrane localized GI possibly has a role in the regulation of ion channels during salt stress and stomatal opening as seen in phototropins (Stoelzle et al., 2003). Purified recombinant GI from Escherichia coli when subjected to electron microscopic study, revealed a tetrameric arrangement in vitro. However, its quaternary structure in vivo is still unclear (Black et al., 2011). This multimeric organization of a protein would not only offer more epitopes for interactions with diverse regulators but also would offer additional layers of control on its stability. ALLELES OF GIGANTEA WITH DISTINCT PHENOTYPES The gi mutants were described as late flowering mutants for the first time (Rédei, 1962). There are several gi mutants described in literature such as gi-1, gi-2, gi-3, gi-4, gi-5, gi-6, gi-11, gi-12, gi-100, gi-200, gi-201, gi-596, and gi-611 (summarized in Table 1; Figure 2). Some of the gi mutants were shown to influence the activity of the circadian clock, while others alter diverse responses (Park et al., 1999). The gi-1 allele, lacking the C-terminal part of GI, was responsible for shortening the period of the clock, while the gi-2 allele, lacking both the C-terminal and the central region of GI, lengthened the period. While the gi-1 mutation shortened the period of CAB2 expression, the gi-2 mutation lengthened the period of CAB2 expression (Park et al., 1999). This suggests that the central region of the protein or the terminal half of the protein most probably fine-tunes the period length of the circadian clock. The gi-2 mutant at higher temperature of about 28 • C showed longer hypocotyl and flowered earlier in comparison to the plants grown at temperatures of 18 and 22 • C (Araki and Komeda, 1993). Even though higher temperature were shown to regulate flowering (at 18, 22, 28 • C) and hypocotyl elongation (at 22, 28 • C) in gi-2 mutant, it was almost equally sensitive toward vernalization as in WT. Vernalization is the exposure of plants to prolonged cold temperature that leads to earlier flowering cue in Arabidopsis. This implies that probably GI regulates flowering using a vernalization-independent pathway (Martinez-Zapater and Somerville, 1990;Koornneef et al., 1991;Araki and Komeda, 1993). The alleles of GI are the result of random mutagenesis or T-DNA insertion which have aided in understanding its various functions. Alleles such as gi-1, gi-2, gi-3, and gi-6 introduce premature stop codon whereas gi-4 and gi-5 most probably alter the C-terminus of the protein due to frame-shift mutations (Fowler et al., 1999). No GI expression was detected in the gi-11 and gi-201 alleles carrying T-DNA insertion (Richardson et al., 1998;Martin-Tryon et al., 2007). The gi-100 mutation, originally identified in a red light screen, also contained a T-DNA insertion, but produced a truncated transcript of about 2 kb due to the absence of the 3 end of GI (Huq et al., 2000). The transcript level in gi-1, gi-2, and gi-3 is lower compared to that of gi-4, gi-5, gi-6, and gi-100, which show similar or higher levels compared to their respective WT (Fowler et al., 1999;Huq et al., 2000). The role of GI in blue light dependent hypocotyl elongation was revealed using the gi-200 allele, consisting of a substitution of the serine 932 . Various deletions in GI sequences and its phenotypes are summarized in Table 1. After analyzing the data depicted in Table 1, it is evident that any deletion in GI mostly causes defects in the flowering time, circadian clock, and control of hypocotyl elongation. In the gi-4 mutant, improper splicing leads to a loss of 90 amino acids from the C-terminus causing late flowering. This deletion also causes the over-expression of its own transcript suggesting that the C-terminal 90 amino acids are required for its auto-regulation and flowering time (Fowler et al., 1999). The abundance of the gi-4 transcript could be due to increased stability or decreased decay which needs to be verified. Since GI stimulates CO transcription, this C-terminal domain of GI might be acting as an enhancer of CO transcription or involved in the recruitment of activators to the CO promoter. The seeds of Wassilewskija (Ws) ecotype expressing CAB:LUC were mutagenized and screened for altered period length. Two novel alleles gi-596 and gi-611 were identified in this screen . In the gi-596 allele, mutation caused by the substitution of the serine residue at 191 position to phenylalanine (S191F) did not affect the flowering-time although the period length of the circadian clock is lengthened and longer hypocotyl was observed under both red and blue light conditions. This suggests that the Frontiers in Plant Science | Plant Genetics and Genomics serine 191 residue might have an important role in photoreceptor signaling. On the contrary, the mutation in gi-611 allele was mapped to the lysine 281. This allele showed significantly early flowering in SDs suggesting that this lysine in WT is involved in decelerating the flowering time . Since the Ws ecotype is a natural null for the high light sensor Phytochrome D, the phenotype observed could be a combinatorial effect of the lack of this photoreceptor and the respective mutations in GI allele (Aukerman et al., 1997). It would be interesting to evaluate if these alleles in Col background would show the similar light dependent effect to rule out the involvement and interaction of PHYD in this process. Both the positions, Lys281 and Ser191 are conserved in the Col-0 and Ler-0 ecotypes and thus, the role of these residues could be confirmed by the expression of the respective GI alleles containing the substitutions in these ecotypes to determine the importance of these mutated residues. TRANSCRIPTIONAL REGULATION OF GIGANTEA Defects in the circadian clock components have been found to affect the GI transcription. CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), a core component of the circadian clock, reduces the GI expression by binding to CCA1-binding site on GI promoter (Lu et al., 2012). GI transcript, thus accumulates toward the middle of the day, when CCA1 expression is repressed by TIMING OF CAB EXPRESSION 1 (TOC1). The rhythmicity of GI transcript level is lost in elf3 mutant in continuous light (LL) suggesting that ELF3 might also regulate the GI mRNA abundance (Fowler et al., 1999). Since CCA1 and ELF3 have been proposed to physically interact to control flowering time and hypocotyl elongation, it would be interesting to investigate the coordinated involvement of these two proteins in the regulation of GI transcription. Clock proteins, such as, LIGHT-REGULATED WD 1 and 2 (LWD1 and LWD2) also affect the GI expression pattern, since in lwd1lwd2 double mutant GI transcript is most abundant at ZT 6 instead of ZT 10 (Wu et al., 2008). The two proteins being very similar (∼90% identity) possess functional redundancy, evident from single mutants being phenotypically similar to WT. Another clock associated gene, TIME FOR COFFEE (TIC) is also known to regulate the rhythmicity of GI in Arabidopsis. In tic mutants, GI transcript level is lower and the peak is shifted ∼4 h earlier than in WT plants (Hall et al., 2003). Since in both the lwd1lwd2 and tic mutants the GI expression is shifted to ZT6, it suggests that the activities of both the proteins might be required for the repression of the GI transcription in the morning. pseudo-response regulators (PRRs), namely, PRR5, PRR7, and PRR9 also have been proposed to regulate GI expression and therefore, flowering time via the CO-FT module (Nakamichi et al., 2007;Kawamura et al., 2008). Epistatic analysis and mutant combinations between LWD1/2, PRRs, and TIC would be beneficial to explain the additive roles of these genes products and the genetic hierarchy of the genes regulating the inhibition of GI expression. The expression of GI at the wrong time of the diurnal cycle is known to cause flowering time defects in At (Fornara et al., 2009). These mutants might behave as late flowering due to the untimely expression of GI. Although a lot is known from the transcript analysis, the work at the protein level is far from being understood due to the unavailability of a GI anti-serum that could detect the endogenous GI protein. The detailed post-translational regulation of GI is explained in the Section "Post-Translational Regulation of GIGANTEA." Several studies have demonstrated that light quality and quantity influence GI transcription, although systematic studies involving changes in the light fluence and wavelength to evaluate GI expression is yet to be carried out. In Arabidopsis, upon transition to night, GI mRNA level decreases with a half-life of about 1 h irrespective of the photoperiod (Fowler et al., 1999). A significant light dependent down-regulation is also detected in the legume Medicago truncatula suggesting a similar mechanism might coordinate light sensing with transcriptional activity (Paltiel et al., 2006). GI mRNA accumulation pattern in both Arabidopsis and M. truncatula showed a secondary peak at ZT 2 under SDs as well as LDs (Paltiel et al., 2006). This peak could be the result of an acute response to light. A similar peak of GI mRNA at ZT 2 has also been documented in plants grown under blue light. The role of blue light in the regulation of this early secondary peak of GI needs to be thoroughly examined using mutants that are affected in blue light signaling. This would clarify if the peak at ZT 2 is due to photoreceptors or secondary signaling components involved in blue light signaling. The peak expression of GI is delayed by approximately 4 h in plants grown in low red:far-red (R:FR) light conditions in comparison to plants grown in white light condition (Wollenberg et al., 2008). This indicated that the photoreceptors and their activity are fine-tuning the timing and quantity of the GI transcript. The accumulation of the GI protein in the morning around ZT 3-4 and its consequence in plant development has not been studied yet, that needs to be evaluated in depth. Besides light, temperature too has been found to regulate GI expression. Warmer temperature of 28 • C up-regulates GI mRNA www.frontiersin.org level as compared to the cooler temperatures of 12 • C at dawn (Paltiel et al., 2006). The night time repression of GI transcription was shown to be temperature dependent and regulated by evening complex (EC) night time repressor constituted of ELF3, ELF4, and LUX ARRHYTHMO (LUX; Nusinow et al., 2011;Mizuno et al., 2014). The EC night time repressor was revealed to bind to the GI promoter through LUX binding site (LBS). GIGANTEA has been proposed to regulate its own expression, since the mutants, gi-1 and gi-2, have lower expression of the GI alleles, approximately 40 and 20% of the WT transcript, respectively (Fowler et al., 1999;Park et al., 1999). But this autoregulatory role of GI transcription is contradictory, since, gi-4 and gi-6 lines show ∼30% higher expression of GI compared to its WT (Fowler et al., 1999). This effect could be either due to the difference in the ecotypes or differential regulation of the transcript stability. Another question worth investigating would be the abundance of the mutant proteins produced in each mutant, which would require a functional GI antiserum. The positive or negative auto-regulatory role suggests that mutations at different residues in the coding sequence can influence the abundance of transcriptional enhancers or repressors, affecting GI expression. POST-TRANSLATIONAL REGULATION OF GIGANTEA Over-expression of GI leads to the constitutive accumulation of the GI transcript throughout the photoperiod. Despite its constant expression level, GI protein follows a cyclic pattern of accumulation in both LDs and SDs. This is suggestive of the degradation of the GI protein (David et al., 2006). GI was found to be ubiquitinated upon dusk, a pre-requisite for its degradation via the 26S proteasome mechanism (David et al., 2006). In the dark phase, nuclear GI abundance has been shown to be regulated by the E3 ubiquitin ligase activity of COP1 and ELF3 (Yu et al., 2008). The interaction between COP1 and GI is ELF3 dependent, where ELF3 serves as an adaptor protein (Yu et al., 2008). The shuttling of COP1 between the nucleus and the cytoplasm is regulated by light (von Arnim and Deng, 1994). COP1 being nuclear localized in the night phase makes it competent for COP1-ELF3 mediated degradation of GI through 26S proteasome. Upon heat shock GI is SUMOylated (López-Torrejón et al., 2013). It has been proposed that SUMOylation prevents the degradation of GI, thus enhancing its abundance. GI accumulation has been correlated with earlier flowering under heat stress. The identification of SUMOylation and ubiquitination sites in GI that alter its stability and degradation could be of pivotal importance in manipulating flowering time of crop plants. Current knowledge on the transcriptional and post-translational regulation of GI is presented schematically in Figure 3. ROLES OF GIGANTEA GIGANTEA plays multiple roles throughout plant development. Its functions in processes such as light signaling, circadian clock regulation, flowering time control, chlorophyll accumulation, sugar metabolism, and stress tolerance have been discussed below. LIGHT SIGNALING Photoreceptors such as phytochromes, cryptochromes, UV-light receptor, and phototropins help plants to sense variations in the light quality, quantity, and direction. The red and far-red light photoreceptors, phytochromes, are encoded by a multigene family, PhyA-E in Arabidopsis. While PhyA is the far-red light receptor, PhyB-E function as red light receptors with PhyB playing a predominant role. They mediate very-low-fluence responses (VLFRs), low-fluence responses (LFRs), and the high-irradiance responses (HIRs), with reference to the photon flux density (Casal et al., 1998). Like phyB-9 mutant, gi-100 also shows elongated hypocotyl when grown under saturated red light (Huq et al., 2000). Neither the genes nor the proteins abundance of PhyA and PhyB are influenced in gi-100 (Huq et al., 2000). Therefore, GI was suggested to function downstream of PhyA and PhyB. Mutation in GI leads to decreased VLFR under FR light suggesting its role in PhyA signaling (Oliverio et al., 2007). The gi mutants showed reduced seed germination and cotyledon unfolding in FR light conditions. These phenotypes are rescued by over-expression of GI. This suggested that GI might have a positive role specifically in PhyA mediated VLFR. GI also has a role in regulating flowering in low R:FR ratio which might be attributed to PhyA signaling (Wollenberg et al., 2008). Both PhyA and PhyB form NBs like GI. It would be interesting to determine if Phys and GI are present in the same sub-nuclear complexes and the localization of GI in the NBs alters the Phy-mediated functions. The gi mutants showed longer hypocotyl in comparison to WT under blue light . Earlier, it had been suggested that GI may be either a positive regulator of TOC1 or act parallel to it for the regulation of hypocotyl elongation. Since only gi not toc1 mutants show the longer hypocotyl in blue light, it can be inferred that GI does not regulate TOC1 for hypocotyl elongation . CIRCADIAN CLOCK CONTROL The circadian clock controls many processes depending on the length of the day and night cycle in an organism. In plants, the rhythmic expressions of various genes are influenced by the circadian clock, thereby regulating functions such as elongation of hypocotyl, petioles and inflorescence stem, movement of cotyledon and leaf, and flowering time. CCA1, LATE ELONGATED HYPOCOTYL (LHY), and TOC1 are the core components of circadian oscillator in plants (Somers, 1999). In 2005, the clock was proposed to be an interlocking network of proteins working in a feedback loop (Locke et al., 2005). According to the new model of clock, while the morning elements LHY and CCA1 repress TOC1 transcription, the evening element TOC1 downregulates LHY/CCA1 accumulation, differing with the earlier observations (Alabadí et al., 2001;Gendron et al., 2012;Huang et al., 2012). To understand the circadian rhythm in Arabidopsis, the ESPRESSO Quantitative Trait Loci (QTL) was generated from the cross between Ler and Cvi ecotypes (Swarup et al., 1999). Ler and Cvi ecotypes were suggested to comprise of an even distribution of alleles involved in the shortening and lengthening of period, since the progeny of their cross generated lines which had period length both longer and shorter than the parents. GI was identified as one of the genes that could be responsible for regulating the rhythms of cotyledon movement (Park et al., 1999;Swarup et al., 1999). The gi mutants have diverse circadian periods than WT concluding that GI has a role in period length regulation. Mutation in GI affects CHLOROPHYLL A/B-BINDING PROTEIN 2 (CAB2) gene expression which is also under the control of circadian clock (Park et al., 1999). Soon after a day of imbibition of seeds, GI is required for initiating the rhythmicity of the circadian clock (Salomé et al., 2008). Mutations in the GI locus affect the CCA1 and LHY gene expression in both LDs and SDs conditions (Fowler et al., 1999). A recent study proposed that both the nuclear and cytosolic GI are required to positively and negatively regulate LHY expression, respectively, that fine-tunes the clock function (Kim et al., 2013b). Over-expression or mutations of CCA1 and LHY disrupted the GI expression (Fowler et al., 1999). Accordingly, the double mutant of LHY and CCA1 showed early abundance of GI transcript (Mizoguchi et al., 2002(Mizoguchi et al., , 2005. It suggests that GI operates in a feedback loop as a component to maintain the rhythmicity and period length of the clock. The established LHY/CCA1-TOC1 module of the clock could not explain the experimental data like the time difference of about 12 h between LHY/CCA1 abundance in morning and TOC1 accumulation in evening (Alabadí et al., 2001;Locke et al., 2005). It was therefore proposed that LHY/CCA1-TOC1 module comprises of other components. One of the components was predicted to be GI, whose expression followed the same pattern as predicted by the in silico analysis and was subsequently experimentally confirmed . Further work suggested that GI alone would not be able to regulate the observed time difference (Kawamura et al., 2008). TOC1 in turn is regulated by GI along with ZEITLUPE (ZTL), an F-box protein (Kim et al., 2011). ZTL is a blue light photoreceptor which is stabilized by its interaction with GI and Heat Shock Protein 90 (HSP90). Together the ZTL-GI complex control TOC1 level (Kim et al., 2007). Temperature compensation is an important characteristic of the circadian clock to maintain the rhythm over a range of environmental temperature. GI was recognized as a candidate regulating temperature compensation effect, especially at higher temperatures (Edwards et al., 2005;Gould et al., 2006). Since fluctuations in the temperature regulate the abundance of GI transcript, it could be plausible that GI and temperature sensing mechanism crosstalk and feedback each other. Arabidopsis thaliana dawn and dusk, GI regulates the clock rhythm along with ELF4 . GI was also required for iron-deficiency induced long circadian clock rhythm (Chen et al., 2013). Reduced depolymerization of actin filament caused the period of the circadian clock to shorten, as evident from the shortened period of GI expression (Tóth et al., 2012). Since GI expression is under the control of the circadian clock, GI accumulation pattern has been exploited to screen for novel clock mutants (Onai et al., 2004). Many components that mediate between GI and the clock are still to be unraveled. The role of GI in the regulation of the clock documented till date is summarized in Figure 4. PHOTOPERIODIC FLOWERING-TIME REGULATION GIGANTEA is a major mediator between the circadian clock and the master regulator of photoperiodic flowering time control, CO. GI upregulates CO transcription, thereby accelerating time required to flower. Koornneef et al. (1998) showed that a novel mutant, gi-3, is epistatic to CO and FLOWERING LOCUS T (FT) way back. Mutation in GI led to a decrease in the accumulation of CO mRNA without affecting its cycling phase compared to its WT that led to delayed flowering (Suárez-López et al., 2001). Mutants in the GI locus or over-expressors of GI did not discriminate daylength for flowering. Accordingly, gi mutants were later flowering and over-expressors were earlier in both LDs and SDs (Rédei, 1962;Araki and Komeda, 1993;Mizoguchi et al., 2005). During dawn, CO transcription is repressed by the combinatorial activity of the DOF transcription repressors bound to the CO promoter. In LDs, the expression of FKF1 and GI coincide at ZT10. Therefore, toward the middle of the day the accumulation of GI along with FKF1 forms a complex competent to degrade the DOF factors. This elevates the CO transcription, thereby leading to FT expression (Imaizumi et al., 2003(Imaizumi et al., , 2005Sawa et al., 2007). While in SDs, since FKF1 accumulates 3 h after GI peaks, it does not allow the formation of the degradation complex, therefore leading to a low abundance of CO transcript. This photoperiod FIGURE 4 | Circadian clock control by GI. GI and the central clock components work in a feedback loop. GI along with ELF4 positively regulates the clock while GI and ZTL form a complex to degrade TOC1 in evening. GI and HSP90 regulate the stability of ZTL, thereby influencing clock. www.frontiersin.org pathway where GI regulates FT expression in a CO-dependent manner is schematically depicted in Figure 5. GI regulates the abundance of FKF1, which is involved in the proteasomal degradation of proteins (Fornara et al., 2009). Post-transcriptionally, GI also controls the sub-cellular level of CYCLING DOF FAC-TOR 2 (CDF2; Fornara et al., 2009). FKF1 belongs to a family of F-Box proteins containing two other candidates -LOV KELCH Protein 2 (LKP2) and ZTL. The blue light dependent interaction between GI and FKF1 is mediated by the LOV (Light, Oxygen, or Voltage) domain of FKF1 and the amino-terminal of GI in vivo (Sawa et al., 2007). The gi-100 mutant is later flowering than the F-Box triple mutant fkf1 ztl-4 lkp2-1. This might be due to the presence of GI in fkf1 ztl-4 lkp2-1, which downregulates the abundance of CDF transcripts, or the presence of an additional layer of control by GI bypassing the triple F-Box module. There are at least two independent mechanisms through which GI regulates FT expression independent of CO. While the first mechanism involves microRNA, the second mechanism is through the binding of GI to the FT promoter. The microRNA based control involves miRNA172, which is positively regulated in the presence of GI. The miR172 inhibits the expression of TARGET OF EAT1 (TOE1), an APETALA 2 (AP2)-related transcriptional repressor of FT (Jung et al., 2007). In the recent past, expression of GI specifically in the mesophyll or vascular tissue was carried out. This rescued the late-flowering phenotype of gi-2 under both day length conditions and two different temperatures of 16 and 23 • C (Sawa and Kay, 2011). The expression of GI in mesophyll and vascular tissue was done using tissue specific promoters LIGHT-HARVESTING COMPLEX B2.1 (pLhCB2.1) and SUCROSE TRANSPORTER 2 (pSUC2), respectively. While expression pattern of GI under the control of pLhCB2.1 is altered and peaked at ZT 0, GI expressed under the phloem specific promoter led to the over-expression of the transcript with peak at ZT 10. The FT transcript level was up-regulated without an increase in CO mRNA in both day-length conditions. GI was shown to binds to the FT transcriptional repressors such as SHORT VEGETATIVE PHASE (SVP), TEMPRANILLO 1 (TEM1), and TEMPRANILLO 2 (TEM2), and their specific target regions within the FT promoter in the mesophyll, thereby relieving the repression and promoting FT transcription (Sawa and Kay, 2011). The degradation of the FT transcriptional repressors or the unavailability of their binding sites on the FT promoters due to the presence of GI could lead to the abundance of the FT transcript. FT expressed in the vascular tissue normally triggers flowering. Since GI expressed in mesophyll accelerated flowering, elevating the FT level in vasculature, the signal from mesophyll GI most likely induces CO transcription in vasculature. Alternatively, the GI could be transported to the vascular tissues and induce the photoperiod module which needs to be investigated. Expression of 35S::GI:GFP in gi-3 plants complemented the late flowering phenotype of gi-3. On the contrary, expressing the 35S::GFP:GI in gi-3 caused later flowering compared to the background lines indicating that the N-terminal fusion of GI might be either non-functional or might not be imported into the nucleus. In the transgenic line expressing C-terminal fusion, the fusion protein was localized to the nucleus and formed NBs (Mizoguchi et al., 2005). In an independent study, transgenic plants expressing glucocorticoid receptor (GR) fusion of GI flowered with ∼20 leaves less when treated with dexamethasone, compared to its untreated control which flowered with ∼55 leaves under LDs (Günl et al., 2009). In 15 day old seedlings, the induction of flowering time genes like CO and FT took place ∼28 h after dexamethasone treatment causing early flowering. This indicates that cytoplasmic retention of GI most probably delays time to flower. Mutation in GI is epistatic to mutation in ELF4 and together regulate CO expression . Recent studies showed that ELF4 sequesters GI into nuclear bodies, thereby preventing GI to associate with the CO promoter (Kim et al., 2013b). It would be interesting to know the nature of the GI nuclear bodies and the components there in, using biochemical approach followed by mass spectrometric analysis. GIGANTEA interacts with N-terminal tetracopeptide domains of SPINDLY (SPY), a plant O-linked β-N-acetylglucosamine transferase, and antagonizes its activity, thereby, promoting flowering (Tseng et al., 2004). Acetylglucosamine transferases have role in the addition of acetylglucosamine residues to proteins, which often competes with phosphorylation. This suggests that sugar modification may function as an important event in flowering time regulation. The known pathways through which GI regulates flowering are summarized in the Figure 6. PLEOTROPIC FUNCTIONS OF GIGANTEA Besides flowering time, circadian clock, and light signaling regulation, GI has been implicated in other processes such as, sucrose signaling (Dalchau et al., 2011), starch accumulation (Eimert et al., 1995), and stress tolerance (Kurepa et al., 1998a;Fowler and Thomashow, 2002;Kim et al., 2013a;Riboni et al., 2013). The control of cotyledon movement, transpiration, and hypocotyl elongation responses have been shown to be attributed to the concerted activity of SPY and GI (Sothern et al., 2002;Tseng et al., 2004). The precise nature of this interaction is still unclear. However, GI functions antagonistically to SPY. The interaction of GI with SPY and ELF4 independently regulates hypocotyl length, where mutation in ELF4 and SPY are epistatic to gi-2. GIGANTEA has been demonstrated to play a role between sucrose signaling and the circadian clock while grown in DD (Dalchau et al., 2011). Plants entrained in LD when shifted to DD, maintained the rhythmic GI expression exclusively in the presence of sucrose suggesting light independent control of GI rhythmicity. Although contradictory evidence on role of sucrose on GI expression has been reported, sucrose seems to affect the GI expression through SENSITIVE TO FREEZING6 (SFR6) locus (Knight et al., 2008;Usadel et al., 2008). More precise experiments are required FIGURE 6 | Flowering time regulation by GI. GI regulates flowering time through many pathways mostly up-regulating FT. The mechanism of flowering time control by GI along with ELF4 and SPY is unknown. The miR172 processed by GI inhibits TOE1/2 that up-regulates FT transcription. GI also degrades inhibitors of FT transcription like SVP, TEM1 and 2. GI-FKF1 complex tunes CO transcription, which in turn controls FT accumulation. to unravel this mechanism. In the leaves of Arabidopsis, starch accumulation is elevated in the gi mutants (Eimert et al., 1995). On the contrary, presence of multiple copies of GI led to starch accumulation in the progeny of a cross between A. thaliana and A. arenosa, suggesting the antagonistic role of GI in these plants (Ni et al., 2009). The gi-3 mutants showed higher tolerance capacity to redox cycling agent, paraquat, and H 2 O 2 (Kurepa et al., 1998a). Tolerance against paraquat is counteracted by the exogenously applied polyamines such as spermidine, spermine, and putrescine (Kurepa et al., 1998b). Paraquat treatment upregulated endogenous levels of putrescine in gi-3 and WT. Since exogenous application of polyamines is effective for the resistance, the mechanism of the transporters during this stress needs attention. Oxidative stress due to herbicide imazethapyr has been shown to increase GI abundance and cause earlier flowering by ∼4 days (Qian et al., 2014). The mechanism behind higher tolerance to oxidative stress mediated by GI is still unclear. Kurepa et al. (1998a) showed that gi mutants, gi-3, gi-4, gi-5, and gi-6, have more chlorophyll accumulation in comparison to WT in presence of paraquat. Even treatment with nitric oxide (NO) reduces the GI mRNA abundance and increases the chlorophyll content (He et al., 2004). In both the cases above, lower abundance of functional GI can be correlated to higher accumulation of chlorophyll. The role of GI in regulating the chlorophyll content needs to be studied in mutants and over-expressors of GI. Chlorophyll accumulation in allotetraploid, obtained by a cross between A. thaliana and A. arenosa, is higher than the WT individuals (Ni et al., 2009). The starch and chlorophyll accumulation in allotetraploids is exactly opposite in comparison to that seen in A. thaliana. The reverse trend might be due to post-transcriptional silencing posed by the presence of multiple homologous sequences of GI transcript, essentially a co-suppression phenomenon. Dynamin, a GTPase having role in vesicle recycling during endocytosis, was found to interact with TAP tagged GI in rice (Abe et al., 2008). Although mutation in dynamin gene did not have any effect on the flowering time, it showed aerial rosette phenotype in Arabidopsis. In Arabidopsis, GI has been found to be involved in setting of fruits (Brock et al., 2007). No significant association of the GI haplogroup was detected with days to flower, petiole length, and inflorescence height. A significant association was observed between one haplogroup with fruit set, producing 14% more fruit than other haplogroups. Such studies in the crop plants could help in increasing the yield. GIGANTEA mRNA levels increases about five-to eightfold in the cold treated Arabidopsis plants suggesting that GI is a cold-responsive gene (Fowler and Thomashow, 2002). The flowering time of gi mutants was further delayed when exposed to low temperature compared to WT (Cao et al., 2005). C-repeat Binding proteins (CBFs) have been known to regulate various genes responsive to cold and are implicated in cold stress tolerance. On the contrary, Cao et al. (2005), it was revealed that GI regulates cold acclimation through CBF-independent pathway. The ability to tolerate and acclimatize toward cold is reduced in gi mutants suggesting the protective role of GI in cold tolerance. www.frontiersin.org Recently, the role of GI under salt stress was documented (Kim et al., 2013a). Although, salt stress did not affect the GI expression, it affected the GI protein stability in pGI::GI-HA transgenics (Cao et al., 2005;Kim et al., 2013a). It seems plausible that there is a mechanism at the post-translational level that regulates GI abundance. GI also regulated the activities of the proteins involved in the salt stress tolerance. It interacts with Salt Overly Sensitive 2 (SOS2) directly and inhibits the activity of SOS1, a Na + /H + antiporter. Therefore, GI is a negative regulator of salt tolerance and is degraded during salt stress. According to a recent model, plants under salt stress would flower later than when grown in normal growth conditions reasoned for the degradation of GI (Park et al., 2013). In At and other plants, the tolerance to higher salinity, enhanced cold, and sustained drought were manifested by the increase of sub-cellular level of abscisic acid (ABA). Recent reports indicated that GI has role in ABA-dependent drought escape tolerance. It suggests that the GI regulation of salt and cold stress tolerance could very likely be ABA-mediated (Riboni et al., 2013). Drought stress up-regulates GI transcription and in turn, increases the abundance of miR172E variant (Han et al., 2013). WRKY DNA binding protein 44 (WRKY44) was found to be suppressed by GI in drought stress and interact with TOE1. GI-miR172-WRKY44 were proposed to be in the same pathway possibly associated with drought stress tolerance. On the same line of thinking, the light dependent GI-mediated stomatal opening response could be ABA mediated (Ando et al., 2013). GI also has a role in wall in-growth deposition in phloem parenchyma transfer cells in A. thaliana in response to high light and cold stress (Edwards et al., 2010;Chinnappa et al., 2013). ROLE OF GIGANTEA HOMOLOGS GIGANTEA homologs in prokaryotes, fungi, mosses, or animals have not been reported as yet (Holm et al., 2010). GI homolog has been shown to be absent in the green unicellular alga Ostreococcus tauri (Corellou et al., 2009). Evolution of GI has been correlated with the evolution of higher plants from liverwort onward, although being absent in mosses. The evolution of GI can be proposed to have taken place alongside the origin of land plants. The role of GI in light signaling, circadian clock control, and flowering time regulation seems to be conserved across the plant kingdom, as inferred from the various studies to understand the role of GI homologs in Arabidopsis. GI homologs from the non-flowering and flowering plants have been summarized below. The GI-FKF1 interaction and function has been recently shown to be conserved in the liverwort Marchantia polymorpha (Kubota et al., 2014). The LOV domain of FKF1, which has been found to be required for the interaction with GI, contains a conserved cysteine residue in AtGI and MpGI important for its blue light dependent functions (Sawa et al., 2007). GI-FKF1 module has been proposed to be important for the transition of plants from water to land and the evolution of vascular system. The Marchantia polymorpha ortholog of GI, MpGI, has been shown to partially rescue the late flowering phenotype of Arabidopsis gi mutant suggesting the functional conservation of GI across the plant kingdom. The FKF1 homologs have been shown to be present in A. thaliana (AtFKF1, AtZTL, and AtLKP2), Oryza sativa (OsFKF1, OsZTL1, and OsZTL2), Glycine max (GmFKF1, GmZTL1, and GmZTL2), Triticum aestivum (TaFKF1 and TaZTL), Allium cepa (AcFKF1 and AcZTL), Mesembryanthemum crystallinum (McFKF1 and McZTL), and Selaginella moellendorffii (SmFKF; Kubota et al., 2014). The GI counterparts in the above mentioned species are also conserved. This shows that GI-FKF1 module has been conserved since the primitive time and thus might be have been important in shaping the development of higher plant. This light perceiving module needs to be studied in detail to understand the evolution of various functions and residues along with putative domains required to carry out these functions in plants. The conserved interaction of GI with FKF1 has been shown to be conserved in soybean. Norway Spruce (Picea abies) GIGANTEA ortholog of Picea abies, PaGI and AtGI share 58% identity and 72% similarity. Natural variations in GI have been correlated to clinal variations in the different populations of close relative of the Scandinavian Norway spruce (Chen et al., 2014). Over-expression of PaGI in WT Arabidopsis did not show any phenotypical changes (Karlgren et al., 2013). However, when PaGI was over-expressed in gi-2 mutant, it partially rescued the late flowering phenotype and flowered at the same time as WT plants suggesting that PaGI and AtGI are functionally conserved to large extent. The strength of the over-expression has neither been verified at the gene expression level nor the protein accumulation level and therefore needs to be confirmed. Barley (Hordeum vulgare) GIGANTEA homolog in Barley was identified using BLAST searches and later confirmed by Southern hybridization analyses. Only one homolog was detected in barley. Barley GI (HvGI) has ∼94 and ∼79% similarity with OsGI and AtGI, respectively (Dunford et al., 2005). Barley, being a long-day plant, its GI expression followed the pattern documented for AtGI. Characteristically, in SDs, the peak of expression was noticed about 6-9 h after dawn whereas, in LDs, the peak is shifted to 15 h after dawn (Dunford et al., 2005). The mutation in HvELF3 (mat-a.8), a 4 bp deletion causing frame shift and premature stop codon, was found in the barley cultivar Mari (Zakhrabekova et al., 2012). This mutation led to the up-regulation of HvGI transcription and was found to be the reason for early flowering phenotype in this cultivar. Although, post-translational interaction between AtELF3 and AtGI is known, no evidence is there in Arabidopsis suggesting the transcriptional regulation of GI by ELF3. Duckweed (Lemna gibba) The AtGI homolog of L. gibba, LgGIH1, a LD plant, plays a pivotal role in its circadian clock control, since the LgGIH1 knockdown resulted in the arrhythmic gene expression phenotype in plants (Serikawa et al., 2008). Earlier reports suggested that AtGI and LgGIH1 followed similar expression pattern in both LD and LL conditions (Miwa et al., 2006). The function of GI and ELF3 homologs are shown to be conserved between Arabidopsis and L. gibba. Maize (Zea mays) Maize is a SD plant, which has two diurnally regulated GI homologs called gigantea of Z. mays 1a and 1b (gi1 and 2) due to tetraploidy events and genome evolution expressed in leaves (Gaut and Doebley, 1997;Swigonová et al., 2004;Miller et al., 2008;Hayes et al., 2010;Khan et al., 2010;Schnable et al., 2011). Among the two homologs, the gigantea1 transcript was highly expressed. Mutation in gi1 caused early flowering in LD but had lesser effects in SD. The gi1 mutation also increases plant height and alters the timing of the vegetative phase (Bendix et al., 2013). The early flowering phenotype of gi1 mutant was due to the conserved pathway involving the up-regulation of CO-like flowering regulatory gene called CO of Z. mays1 (conz1) and FT-like floral activator gene named Z. mays centroradialis8 (zcn8). Purple False Brome (Brachypodium distachyon) GIGANTEA ortholog of B. distachyon (BdGI) is rhythmically regulated by the circadian clock and up-regulated by both cold and dark (Hong et al., 2010). BdGI was identified by BLAST search followed by Southern hybridization analysis. The BdGI transcript level was found to be oscillating in both SD and LD conditions, like AtGI. While the lowest transcript level in both SD and LD was at ZT 0, the peak in SD was at ZT 8 and in LD was at ZT 12. BdGI shares 65% identity with AtGI. BdGI, like AtGI, is a nuclear localizing protein and interacts with COP1 and ZTL proteins as evident from the yeast two-hybrid assays. BdGI complements the late flowering phenotype of Arabidopsis gi-2 mutant suggesting the conserved function of GI in monocots and dicots. While PhyC does not show a pronounced effect in the LD model Arabidopsis, it causes late flowering in this temperate grass (Woods et al., 2014). In phyC mutants, GI expression is almost undetectable. The low GI expression could explain the lower abundance of the homologs of CO and FT. The delayed flowering phenotype suggests that the photoperiodic flowering pathway through GI is conserved in grasses as in Arabidopsis. Rice (Oryza sativa) Rice and Arabidopsis GI share 67% similarity and the NLS are quite conserved between OsGI and AtGI (Hayama et al., 2002). GI expression pattern was similar in both rice and At (Hayama et al., 2002) and similarly, OsGI acts as a positive regulator of Hd1 (CO homolog of rice; Hayama et al., 2003). It controls the rhythm of nearly 27000 genes in rice (Izawa et al., 2011). When gi mutants are grown in field conditions, sucrose, and starch content increases, chlorophyll content decreases, stomatal conductance increases, panicle, and spikelet number increases and fertility was reduced. OsGI was shown to be involved in ETR2 (ethylene receptor)-dependent late flowering phenotype and starch accumulation thus, regulating the developmental transition based on the availability of energy (Wuriyanghan et al., 2009). Tulip (Liriodendron tulipifera) GIGANTEA ortholog was shown to be closer to eudicot GI sequence than the monocot sequences (Liang et al., 2010). Wheat (Triticum aestivum L.) Wheat is a LD plant and has been shown to have an ortholog of AtGI, referred as TaGI1 (Zhao et al., 2005). TaGI1 has ∼81 and 63% identity with OsGI and AtGI, respectively. The TaGI1 follow rhythmic pattern of expression similar to that of Arabidopsis and over-expression of TaGI1 complements late flowering phenotype of gi-2 mutant Arabidopsis. TaGI was also associated with "earliness phenotype" of wheat which helps in its adaptation and increase in yield in varied environmental conditions (Rousset et al., 2011). Common Ice Plant (Mesembryanthemum crystallinum) A crassulacean acid metabolism plant, Mesembryanthemum crystallinum, also showed a rhythmic expression of the orthologs of GI, McGI (Boxall et al., 2005). The ortholog was identified using BLAST search and later isolated and sequenced. McGI expression peaks at ZT 9 similar to AtGI. Morning Glory (Pharbitis nil) PnGI protein shares 70 and 67% identity with AtGI and OsGI protein, respectively (Higuchi et al., 2011). PnGI mRNA is also circadian regulated like the other GI orthologs. Over-expression of PnGI led to altered period length affecting the expression pattern of downstream genes. Pharbitis nil is a SD plant, and like OsGI, PnGI inhibits the expression of PnFT (FT homolog of morning glory). Pea (Pisum sativum) LATE BLOOMER 1 (LATE1) is the AtGI ortholog in pea, a LD plant, and follows a rhythmic pattern of expression as seen in Arabidopsis (Hecht et al., 2007). LATE 1 was shown to be regulating the pea homologs of Arabidopsis circadian clock genes. Apart from its role in flowering time and circadian clock regulation, LATE1 has been implicated in Phy-B dependent seed de-etiolation in red light. LATE1 was found to regulate circadian clock gene expression in constant light and dark . In LD and SD, LATE1 was shown to control a mobile signal that regulates the flowering time. Radish (Raphanus sativa) In another instance, expression of antisense AtGI gene, under the constitutive 35S promoter, led to delayed bolting in LDs, proving that GI has an important role in photoperiodic flowering time control in this plant (Curtis et al., 2002). The bolting and flowering time was delayed by 17 and 18 days, respectively, with respect to WT plants. Soybean (Glycine max) Glycine max, a SD plant, has two GI orthologs -GmGIa and GmGIb (Watanabe et al., 2011). Both the GmGI sequences have nearly 70-91% identity to eudicot and monocot genes. Like OsGI, GmGI regulated GmFT paralogs. GmGI has been shown to have role in soybean seed maturity. GmGI loss of function leads to early flowering as in the model SD rice plant. Interestingly, a recent study in soybean suggested that there are three AtGI homologs in the soybean genome unlike previously suggested two orthologs GmGIa and GmGIb (Li et al., 2013). The third form is a result of alternative splice form of GmGIa, resulting in GmGIα and GmGIβ. The GI orthologs were diurnally regulated and differentially expressed in different tissues adding up to a more complex www.frontiersin.org regulation. GmGI proteins have the conserved NLS and localize to nucleus. GmGI proteins have been shown to interact with orthologs of FKF1 in soybean suggesting that function most likely is conserved. Tomato (Solanum lycopersicum) Tomato is a day neutral plant. GI was shown to be up-regulated and inhibit tomato seed germination thereby promoting seed dormancy under FR condition in the presence of functional PhyA (Auge et al., 2009). On the contrary, in Arabidopsis, loss of function of GI led to elevated dormancy (Penfield and Hall, 2009). In other members of the Solanaceae such as potato and tobacco, photoperiodic control of GI was also shown to be operational (Rutitzky et al., 2009). The conserved diurnal regulation of GI in different plants described above suggests the prevalence of an important transcriptional machinery as well as the GI promoter. The availability of GI antiserum would help to understand the regulation of GI in these crop plants. The localization and the stability of GI in most of these plants are still to be addressed. While few of the interaction with proteins such as orthologs of ELF3, COP1, ZTL, and FKF1 are shown to be conserved, the function of these complexes needs to be disclosed in various species. CONCLUSION AND PERSPECTIVE GIGANTEA seems to be a very important plant protein involved in various processes, from developmental regulation to metabolic flux. Despite its pivotal roles, it is surprising that GI null mutants are not lethal. Being a large protein, it might satisfy to function in several pathways summarized, yet to be fully understood. It would be a great challenge to understand and connect the functional roles of GI at different developmental stages. Although GI is a multifunctional protein, the role of its various functional domains are still in darkness. A functional antiserum against a conserved domain of GI that would detect the endogenous level of protein across species and in multiple mutational background would be very useful. The lack of such an antiserum possesses a serious bottleneck delaying the understanding of its abundance, regulation at the protein level and regulatory functions like the GI-FKF1 module across the plant kingdom. Despite this problem, several elegant experiments have been published where researchers have attempted to understand its role using transgenic plants expressing tagged versions of GI. Although time-consuming, these are the impressive feats that place GI mechanistically in a network of photoperiod control pathway. The role of GI in flowering time regulation, circadian clock control, and light signaling is still being pursued. But less-known functions such as sucrose signaling, chlorophyll accumulation, oxidative stress resistance demand more attention. More functions of GI are beginning to be documented. Recently, the emerging role of GI in salt tolerance has been demonstrated, which indicates that we are still not saturated in understanding the various functions GI. It would be interesting to understand how GI regulates so many functions before going into the complex cross talk between them it can fine tune. The lower plant moss Physcomitrella patens does not have a GI ortholog but still carries out most of the developmental aspects except flowering. It is very interesting to note that they do have CO-like genes, therefore the evolution of GI function is still an interesting area and demands further attention (Zobell et al., 2005).

v3-fos

2018-04-03T01:04:14.240Z

2015-05-26T00:00:00.000Z

204962248

s2

Cofactor composition and function of a H2-sensing regulatory hydrogenase as revealed by Mössbauer and EPR spectroscopy† †Electronic supplementary information (ESI) available: Tables with the simulation parameters and details of the Mössbauer, and EPR spectra (Tables S1–S4). additional EPR and Mössba A regulatory hydrogenase is characterised by Mössbauer, EPR and FTIR yielding insight into structure and function of this dihydrogen sensor. Introduction Hydrogenases, the biocatalysts carrying out the reversible oxidation of H 2 in nature, are complex metalloenzymes, which harbor metal cofactors with a fascinating coordination chemistry. [1][2][3][4] According to their active site metal content, they are grouped into [NiFe]-, [FeFe]-and [Fe]-hydrogenases. 4 The enzyme investigated in this study belongs to the class of [NiFe]hydrogenases, whose basic module consists of two subunits. The large subunit harbors the heterobimetallic [NiFe] center, whereas the small subunit carries up to three iron-sulfur clusters, which serve as an electron relay during catalysis. [4][5][6][7][8] [NiFe]-hydrogenases are classied into ve phylogenetically distinct groups on the basis of their structure and cellular function. 9,10 Although most [NiFe]-hydrogenases are strongly inhibited by molecular oxygen, at least four of the subfamilies contain members that retain catalytic activity in the presence of O 2 , 10,11 which is in particular a most interesting property with respect to any potential application for photocatalytic hydrogen production. 4 Both the degree and the biophysical/biochemical origin of this O 2 -tolerance differ between the different subgroups. In selected representatives of group 1, O 2 -tolerance is crucially linked to the presence of a novel [4Fe-3S] cluster with unprecedented redox properties. 7,8,[12][13][14][15][16] H 2 sensors belong to the group 2 of hydrogenases and are virtually resistant towards O 2 and CO. This property has been primarily associated with a narrow intramolecular hydrophobic gas channel that impedes O 2 diffusion to the [NiFe] site. [17][18][19] The facultatively lithoautotrophic b-proteobacterium Ralstonia eutropha (Re) H16 harbors four different O 2 -tolerant [NiFe] hydrogenases: a membrane-bound hydrogenase (MBH), a NAD + -reducing soluble hydrogenase (SH), an actinobacterialtype hydrogenase (AH) and a regulatory hydrogenase (RH). 10,11,[19][20][21][22][23][24][25][26] The RH is a group 2b protein and acts as H 2 sensor in a signal transduction pathway, which ensures that the energy-generating hydrogenases, MBH and SH, are only synthesized if their substrate H 2 , is available. 9,24 The H 2 -sensing unit is composed of two RH heterodimers that are tightly connected to a histidine protein kinase homotetramer (Fig. 1). 22 A single RH heterodimer consists of the large, active sitecontaining subunit, HoxC, and the small subunit, HoxB, that harbors three iron-sulfur clusters (FeS) of so far unknown structure. Amino acid sequence analysis predicts the presence of three [4Fe-4S] clusters. 24 This is in contrast to prototypical group 1 hydrogenases, which usually possess two [4Fe-4S] clusters and one [3Fe-4S] cluster, 2,4,17,27 but is similar to the group 1 subclass of [NiFeSe]-hydrogenases. 28,29 Though the molecular structure of regulatory hydrogenases is unknown, a number of biochemical and biophysical investigations by EPR, 20,21 FTIR, 30 Resonance Raman 31 and X-ray Absorption Spectroscopy (XAS) 22,26 have provided valuable information on the cofactor structure, composition and function of the RH from R. eutropha. Due to the high complexity of the system, in vitro studies were mainly carried out on a truncated version of the protein (designated as RH stop ), which allows for both isolation by affinity chromatography and formation of a single heterodimer (HoxBC), the tertiary structure of which is similar to that of classical hydrogenases. 5,22 The as-isolated (oxidized) RH stop exhibits no EPR signals related to paramagnetic Ni or FeS clusters. In the most oxidized state, the [NiFe] active site resides in the EPR-silent, catalytically competent Ni-SI state, 20 which contrasts the case of "standard" [NiFe]-hydrogenases that exhibit a superposition of paramagnetic states, known as Ni-A and Ni-B, reecting oxidative modications at the active site. [32][33][34] The absence of the Ni-A and Ni-B states in RH, both of which require reductive reactivation, is fully consistent with the sensory character of the protein that needs to react immediately in the presence of H 2 . 22,24,35,36 Incubation of RH with H 2 in absence of articial electron acceptors affords the paramagnetic Ni-C state characterized by a hydride ligand in the bridging position between the Ni III and Fe II ions. 20 This state appears to be a thermodynamic "bottleneck". Even prolonged incubation with H 2 does not detectably yield the otherwise occurring most reduced Ni-R state(s), typically observed in standard hydrogenases. Ni-SI and Ni-C are therefore the only intermediates to accumulate in signicant amounts in RH. 20 The hydride ligand in Ni-C is photolabile at cryogenic temperatures due to its presumed translocation as a proton. This yields two well-dened paramagnetic forms, termed Ni-L 1 and Ni-L 2 , where the Ni ion resides formally in the Ni 1+ state. 28,37,38 Paramagnetic states of the FeS clusters in RH so far have not been amenable to characterization by EPR spectroscopy. Therefore, the identity, electronic properties, and their possible role in the O 2 -tolerance of this enzyme have remained elusive. This prompted us to employ Mössbauer spectroscopy, which has proven to be a powerful tool for the characterization of the electronic structure of iron-containing cofactors in general and in [Fe]-, 1 [FeFe]-, 39,40 [NiFe]-13,41-44 and [NiFeSe]-hydrogenase, in particular. 45,46 The study was complemented by EPR and FTIR spectroscopy. The Mössbauer spectra of RH can be explained by the presence of three [4Fe-4S] clusters, at least two of them with distinct redox properties. Strong reducing agents were employed to ascertain complete reduction of all three FeS clusters, which for the rst time also afforded detectable EPR signals of the reduced FeS centers. Moreover, the low-spin Fe II site in the [NiFe] center could be unambiguously identied, based on its unique properties which differ from those of other [NiFe] enzymes studied so far. The information obtained by Mössbauer spectroscopy was complemented by a 57 Fe HYS-CORE characterization of the [NiFe] center in the Ni-C state. Overall, the present study contains a comprehensive characterization of the metallocofactors in RH and sheds light onto their structural and electronic properties, which were so far enigmatic. On the basis of the present results the mechanism of the regulatory hydrogenase is revisited and discussed with regard to its physiological functionsensing of H 2 . Reagents All chemicals were of analytical grade and used without further purication. 57 Fe was purchased as metallic iron from Chemotrade Handelsgesellscha, Düsseldorf (Germany) and subsequently dissolved in HCl to yield FeCl 3 for preparing the fermentation media. Protein preparation For the investigation of the RH by Mössbauer, EPR and FTIR spectroscopy, we have employed the so-called RH stop protein, which represents a simplied version of the dimeric wild-type RH, comprising only one functional moiety of the dimer (Fig. 1). 22,26 Because of the replacement of a 50 amino acid extension of the RH small subunit HoxB with a Strep-tagII affinity peptide, formation of the double-dimeric RH is prevented and instead the heterodimeric RH stop protein can be obtained and further puried by affinity chromatography. The RH stop derivative was puried from the transconjugant strain R. eutropha HF574 (pGE567), 19 which was cultivated under hydrogenase-depressing conditions in the presence of 57 FeCl 3 as the sole source of iron. The RH was then puried by Strep-Tactin affinity chromatography as described previously. 19 The elution fractions were pooled and the RH protein was subsequently concentrated to a nal concentration of ca. 400 mM, suitable for spectroscopic analysis. Reduction with H 2 was performed by ushing the sample with hydrated H 2 gas (99.99%, Air Liquide) for 60 minutes in an anaerobic glovebox (Coy). From these preparations, samples were obtained for parallel EPR, IR and Mössbauer experiments, respectively. Reduction with reducing agents was carried out anaerobically through addition of either Ti 3+ citrate or sodium dithionite (Sigma-Aldrich, Germany) to a nal concentration of 10 mM. Sodium dithionite was freshly prepared in 100 mM Na 2 CO 3 / NaHCO 3 buffer (pH 10). Ti 3+ citrate was prepared by dissolving the necessary amount of TiCl 3 (anhydrous, Sigma-Aldrich, Germany) in a solution containing ten-fold excess of trisodium citrate. The color of the solution turned immediately from violet to dark orange (nal pH was 7.5). The Ti 3+ stock concentration was 15 mM. In a rst set of experiments, 1.5 equivalents of Ti 3+ were added anaerobically to an RH sample and were allowed to react for 15 min, before dividing the sample in two aliquots and separate freezing for parallel examination by Mössbauer and EPR spectroscopy. In a second set of experiments, four more equivalents of Ti 3+ were added to the previous Mössbauer sample to ensure complete reduction. No signicant changes were observed in the spectra recorded with this sample, and it was therefore further used for eldand temperature-dependent Mössbauer spectroscopic studies. Mössbauer spectroscopy Mössbauer spectra were recorded on an alternating constant acceleration spectrometer. The minimum experimental line width was 0.24 mm s À1 (full-width at half maximum). The sample temperature was maintained constant either in an Oxford Variox or an Oxford Mössbauer-Spectromag cryostat. The latter is a split-pair superconducting magnet system for applying elds of up to 8 Tesla to the samples that can be kept at temperatures in the range 1.5-250 K. The eld at the sample is perpendicular to the g beam. The 57 Co/Rh source was positioned at room temperature inside the gap of the magnet in a reentrant bore tube at a distance of about 85 mm from the sample. The eld is zero at this position. All isomer shis are quoted relative to the centroid of the spectrum of metallic airon at 300 K. Mössbauer spectra were simulated employing the usual spin Hamiltonian formalism. 47 Theoretical details are described in the ESI. † Electron paramagnetic resonance (EPR) spectroscopy Continuous wave (cw) X-Band measurements were carried out using an X-band Bruker ESP 300E instrument (9.4 GHz, TE 012 resonator) equipped with a helium ow cryostat (Oxford Instruments, ESR910) and an ITC 503 temperature controller. CW S-band (2-4 GHz, loop gap resonator) and CW Q-Band (34 GHz, TE 011 resonator) measurements were carried out using a Bruker ESP 300 spectrometer equipped with an Oxford Instruments helium ow cryostat. Pulse Q-Band experiments were carried out on a Bruker ELEXSYS E-580 FT EPR Q-band spectrometer equipped with a SuperQ-FT microwave bridge, a home-built slightly overcoupled cylindrical TE 011 resonator, with a construction described by Reijerse et al., 48 and an Oxford CF935 Helium ow cryostat. The solid-state microwave amplier in this bridge produces a power of 3 W at the resonator. Pulsed X-band EPR measurements were performed on a Bruker ELEXSYS E-580 Xband spectrometer with a SuperX-FT microwave bridge and a CF935 Oxford ow cryostat in the temperature range 10-20 K. An over-coupled Bruker ER 4118X-MD4-W1 dielectric ring ENDOR resonator was used for these experiments. The MW pulses were amplied by using an Applied Systems Engineering Traveling Wave Tube (TWT) amplier (1 kW). Q-band and Xband HYSCORE spectra were recorded using the standard Bruker data acquisition soware. W-band measurements were carried out on a Bruker ELEXSYS E-680 spectrometer, using the commercial W-band ENDOR probehead (Bruker). Spin quantitation was carried out by using as a standard 1.0 mM Cu 2+ chloride in water (1 mM Cu 2+ , 2 M NaClO 4 , 10 mM HCl), measured under non-saturating conditions. Simulations of the cw and pulse pseudo-modulated spectra, and HYSCORE spectra were done using home-written simulation programs implemented in the Kazan soware (Dr Alexey Silakov, The Pennsylvania State University, and Prof. Boris Epel, University of Chicago) that employs MATLAB (Mathworks) as an interface. Details are described in the ESI † or in previous publications of our group. 49 Fourier transform infrared (FTIR) spectroscopy Measurements were performed on a Bruker IFS 66v/s FTIR spectrometer with 2 cm À1 resolution. The detector was a photovoltaic mercury cadmium telluride (MCT) element. Room temperature measurements were done with a liquid cell that consists of two CaF 2 windows (3.5 cm diameter) separated by a 0.1 mm Teon spacer. The temperature was regulated by a thermostatic bath. The soware for data recording consisted of the OPUS package (Bruker Optics). Analysis and further processing were performed with home-built routines written in MATLAB version 6.5 (Mathworks). Results The as-isolated, oxidized state of RH stop The FTIR spectrum of the as-isolated RH stop (Fig. 2) 26,50 exhibits three characteristic signals from the diatomic ligands at the Fe in the [NiFe] center that can be assigned to the Ni-SI state, with a strong band at 1943 cm À1 (corresponding to the stretching vibration of CO) and two weaker bands at 2081 cm À1 , 2072 cm À1 (corresponding to the vibrationally coupled CN À stretches). 2,51 A small band at 1961 cm À1 coincides with the CO stretch of the Ni-C state, the concentration of which however, is presumably too low for the detection of the corresponding Ni-related EPR signals. Also the concentration of another minor component, causing a weak band at 1932 cm À1 appears to be undetectably low by EPR. The EPR spectrum of the as-isolated enzyme shows only faint signals at g ¼ 4.23 and g av $ 2.01, which we assign to adventitiously bound high-spin Fe III (S ¼ 5/2) and [3Fe-4S] 1+ clusters (S ¼ 1/2), respectively. Since in particular the [3Fe-4S] 1+ clusters are present to only substoichiometric amounts (their spin quantication is in agreement with the abundance of only 0.3 clusters per protein dimer, as estimated below from the Mössbauer spectra), both EPR-active species appear to be oxidation-derived degradation products. This is a common observation for hydrogenases, because of the partial accessibility of O 2 to the FeS clusters (the distal FeS cluster is particularly exposed to the protein surface). 52 The Mössbauer spectra of the as-isolated 57 Fe labeled RH stop recorded at various magnetic elds are shown in Fig. 3. In these spectra all Fe containing species are detected irrespective of their redox and spin states. The dominant contribution (corresponding to $79% of the total iron content, blue lines) originates from a diamagnetic species with intermediately strong isomer shi and quadrupole coupling constants ( Table 1). The values are typical of iron in the high spin state with quasitetrahedral sulfur coordination and mixed oxidation state (+2.5). Together with the diamagnetic behavior the features are characteristic for valence-delocalized [4Fe-4S] 2+ clusters with four essentially indistinguishable Fe 2.5+ sites and total spin S ¼ 0. 53 Other FeS clusters with Fe 2.5+ sites would be either paramagnetic or have additional iron sites with different spectroscopically detectable oxidation states. Similar signals have been found in all [NiFe] hydrogenases studied by Mössbauer spectroscopy to date (Table S1 †). [41][42][43][44][45][46] A well-resolved shoulder observed in the zero-eld spectra at ca. À0.8 mm s À1 can be ascribed to another, rather particular subspectrum with unusually low isomer shi and large quadrupole splitting (d ¼ 0.10 mm s À1 , DE Q ¼ 1.60 mm s À1 , green lines), accounting for $7% of the total Fe content. Because the isomer shi is clearly below the range known for physiological FeS sites (and their degradation products in proteins) 54-56 this subspectrum can be associated with the low-spin Fe II ion of the [NiFe] active site. As expected for the Ni-SI state, the Mössbauer spectrum reveals diamagnetic behavior, as can be seen, e.g., from the eld dependence of the low-energy line, yielding a shoulder throughout the series of spectra shown in Fig. 3. Fig. 2 FTIR spectra of the RH from R. eutropha in its as-isolated (green trace), H 2 -reduced (red trace) and Ti 3+ citrate-reduced (blue trace) states, which demonstrate the presence of 82% Ni-SI, 100% Ni-C and 85% Ni-C state, respectively. In the Ti 3+ -citrate treated sample the band at 1948 cm À1 , may be correlated with a substoichiometric accumulation of Ni-R, an assignment that is however precluded, because the expected bands of the corresponding CN ligands of Ni-R are absent. The absence of any additional Ni signals in EPR, suggest that the 1948 cm À1 band corresponds either to a different conformation of Ni-C or to an EPR-silent state. Moreover, two weak paramagnetic components are present in the spectra, but they are of only minor intensity ($7% of the total iron content each, Tables 1 and S1 †). These can be attributed to the adventitiously bound high-spin Fe III and a contamination with [3Fe-4S] + clusters, respectively, which combined with their correspondingly weak EPR signals at g ¼ 4.3 and g av $2.01, suggest them to be oxidative degradation products (vide supra). In summary, the relative intensities of the Mössbauer components correspond to a model with 13 genuine iron sites in RH stop , arising from 12 Fe in three [4Fe-4S] clusters and one iron in the [NiFe] active site (one Fe of 13 would correspond to 7.7% relative intensity, we observed $7% for the [NiFe] species). In practice minor decomposition of some cubanes can be inferred from the presence of nuisance Fe III and [3Fe-4S] clusters, which changes the percentages slightly, revealing ca. 0.3 [3Fe-4S] + clusters per heterodimer (see ESI †). The H 2 -reduced state of the RH stop FTIR. The FTIR spectrum of the H 2 -reduced RH stop exhibits three bands that are characteristic of the Ni-C state, 19 with a CO stretching vibration at 1961 cm À1 and two CN-related stretches at 2083 and 2071 cm À1 , respectively. This spectrum demonstrates that exposure to H 2 leads to a well-dened and homogeneous reduction of the [NiFe] center to the Ni-C state (Fig. 2). Mössbauer. The Mössbauer spectra of the H 2 -reduced RH stop recorded at different magnetic elds and temperatures are shown in Fig. 4 The results provide profound evidence for the reduction of tetranuclear iron-sulfur cofactors in the regulatory hydrogenase, a conclusion that has been drawn previously on the basis of UV/VIS and XAS spectroscopy, albeit invoking a different FeS cofactor composition. 19 The intensity ratio of the Mössbauer subspectra demonstrates that maximally two of the three clusters in HoxB can be reduced by molecular H 2 . Unique, model-free simulations of the complex magnetic Mössbauer spectra shown in Fig. 4 are virtually impossible in all details because of the large number of overlapping subspectra, which, even with the variety of eld conditions and temperatures, cannot be fully disentangled. We were therefore aiming for 'generic' solutions, which allowed us to characterize the general types and numbers of different FeS clusters, and iron in the catalytic site. To this end, a set of Mössbauer parameters could be obtained ( 43,45,46,53 These yielded the nice global simulation of the spectra shown in Fig. 4, but we cannot readily exclude ambiguities for each and every value of magnetic hyperne coupling constants or individual isomer shis and quadrupole splittings. However, the general assignments to oxidized and reduced [4Fe-4S] clusters are clear and the result excludes major deviations from the typical properties of cubane clusters. The subspectrum of low-spin Fe II in the catalytic center in the Ni-C state shows a similar low isomer shi (0.07 mm s À1 ) as found above for the oxidized Ni-SI state (0.10 mm s À1 ). This observation is in agreement with the active site iron persisting in its low-spin Fe II state upon reduction of the RH with H 2 , which is supported by the diamagnetic behavior of the component in the magnetic Mössbauer spectra, revealing very small spin density on the Fe of the active site in the Ni-C state (vide infra). However, the quadrupole splitting (0.69 mm s À1 ) of the Fe II ion in Ni-C is much lower than that in Ni-SI (1.60 mm s À1 ), revealing a signicant change in the ligand environment. a Obtained from the simulations of the spectra at different applied elds perpendicular to the g beam at 4.2 K. Isomer shis (d) and quadrupole splittings (DE Q ) that are shown in parenthesis correspond to 160 K (these parameters were also used to simulate the spectrum at 80 K). h is the asymmetry parameter, A i are the hyperne tensor components and G is the line width. g ¼ 2.0 was used for all the species. For the Fe III high spin species S ¼ 5/2, D ¼ 1.0 cm À1 and E/D ¼ 0.33 were assumed. The slow relaxation limit was assumed at 4.2 K and the fast relaxation limit was used at 160 and 80 K. Errors are estimated as follows: A minor contribution from [3Fe-4S] clusters, as seen above by EPR for the oxidized protein, could not be unambiguously derived from the Mössbauer spectra of the reduced RH. Typical subspectra for the corresponding 1+ and 0 oxidation states of cubane trinuclear FeS centers with spins S ¼ 1/2 and S ¼ 2, respectively, were introduced in the simulations, but these did not signicantly affect the quality of the ts or the distribution of the native FeS clusters (Fig. S2 †), and therefore have not been further considered in our analysis. Presumably, magnetic broadening or heterogeneity in structure and environment of the non-physiological 3Fe clusters hampered their detection in the complex spectra of H 2 -reduced RH stop . Eventually, also reconstitution of [4Fe-4S] clusters from [3Fe-4S] species under reductive conditions in the presence of adventitious Fe II ions cannot be completely ruled out as a rationale for the apparent absence of such signals in the spectra. 58 Reduction of RH stop with strong reducing agents FTIR. The FTIR spectrum of the Ti 3+ citrate-reduced sample (Fig. 2) shows the typical Ni-C state signals. 26,50 Even under these strongly reducing conditions (E m ¼À510 mV at pH 7.5), the oneelectron more reduced Ni-R state was not generated in detectable amounts, which is in agreement with previous observations. 23 The Ni-R state, which is believed to be the direct outcome of the reaction of the Ni-SI state with H 2 , 59-62 was accumulated neither with H 2 (physiological) nor with more potent chemically reducing agents (non-physiological). Thus, Ni-C represents the only tractable reduced state of RH stop , which might be rationalized by the explicit redox equilibria of the RH cofactors (discussed below). A small band at 1948 cm À1 , may indicate a substoichiometric generation of the Ni-R state. Mössbauer. The Mössbauer spectrum of the Ti 3+ citratereduced RH stop protein recorded at 4.2 K with 1 T applied magnetic eld is shown in Fig. 5 (top), and a series of eldand temperature-dependent Mössbauer spectra is displayed in Fig. S7. † Employing the same approach and starting parameters as for the simulation of the spectra of the H 2 -reduced RH stop (Tables 2 and S2 †), analysis of the spectra of the Ti 3+ citratereduced samples reveals almost full reduction of all three [4Fe-4S] clusters to the 1+ state ($82% of the Fe in the sample, 2.67 clusters per protein). Similar results were obtained with samples reduced with sodium dithionite at pH 10 (data not shown). It should be noted that super-reduction of the cubanes to the [4Fe-4S] 0 state, which would result in high isomer shi values and large quadrupole splittings exhibited by typical Fe II S 4 sites, was not observed. [63][64][65][66] The diamagnetic Mössbauer spectrum of the low-spin Fe II ion in the [NiFe] center was identied reasonably well again, in particular in the spectra recorded at low temperature and small elds ( Fig. 5 and S7 †). In detail, the parameters could be taken the same as those determined for the H 2 -reduced samples (d ¼ 0.10 mm s À1 , DE Q ¼ 0.69 mm s À1 ), which is supported by the FTIR spectra, demonstrating that the majority of the [NiFe] sites remains in the Ni-C state. Non-cluster-related high-spin Fe II was also present as an impurity and amounted to $8%. EPR spectra of reduced RH stop 'Unsplit' Ni-C signal of the H 2 -reduced RH stop . The X-band cw spectrum of the H 2 -reduced RH stop shows at 5 K a single S ¼ 1/2 signal with principal g-values (2.199, 2.140, 2.015) that are characteristic of the [NiFe] center in the Ni-C state (Fig. S2 †), and which have been explained formally by adopting a conguration of a low-spin Fe II (S ¼ 0) and a Ni III (S ¼ 1/2) center, with the singly occupied orbital exhibiting mainly d z 2 character. 20,21 Remarkably, no indications from FeS resonances were found in the cw EPR spectra, although the majority of clusters were in the reduced state with spin S ¼ 1/2 (see below). In addition, line broadening in the Ni-C spectra due to 57 Fe hyperne coupling Fig. S4 † discloses a 'splitting' in the g y component of Ni-C. Because this splitting originates neither from 57 Fe hyperne interactions (spectrum of the 56 Fe sample is identical) nor from magnetic interactions with the proximal [4Fe-4S] 1+ cluster (splitting is eld-independent), it is rather consistent with a conformational heterogeneity of Ni-C, which appeared to slightly vary between different sample preparations. This scenario is supported also by spectra obtained at higher microwave frequencies (e.g., W-Band, Fig. S4 †). 57 Fe hyperne coupling in the Ni-C related EPR signal of H 2reduced RH stop . The weak magnetic hyperne coupling of the low-spin Fe II site (S ¼ 0) in the Ni-C state, which is caused by (covalent) spin density delocalization from the Ni III ion, could not be resolved by Mössbauer spectroscopy. Such weak interactions can be probed and studied by advanced pulsed EPR techniques. 32,67 Indeed, 2-and 3-pulse ESEEM experiments at Qand X-band frequencies showed intense modulations, originating from the electron spin interaction with the 57 Fe nuclear spin (I ¼ 1/2), which were absent in the 56 Fe-labeled samples. Analyses of the orientation-selective HYSCORE spectra recorded at Q-band ( Fig. 6 and S5 †) and X-band (Fig. S6 †) frequencies yielded principal values A Fe ¼ (5.0, 1.1, À0.5) (AE0.5) MHz for the 57 Fe hyperne coupling tensor. Additional signals due to 1 H and weakly coupled 14 N nuclei from the protein backbone were also detected, in particular in the X-band HYSCORE spectra ( Fig. S6 †), but not further analyzed. 'Split' Ni-C EPR signal in the chemically reduced RH stop . Incubation of the RH stop with either Ti 3+ citrate or dithionite resulted in the same Ni-C EPR spectrum without any discernable differences in the overall signal intensity. This is in contrast to canonical hydrogenases, in which the Ni-C signal diminishes upon prolonged activation with H 2 as well as electrochemically at reduction potentials similar to those of the two strong reductants. 4,51 The spectra of the Ti 3+ citrate-and dithionite-treated RH stop recorded at 40 K are shown in Fig. S8. † Lowering the temperature to #10 K led to the appearance of a 'split signal' (see also Fig. 7). This 'splitting' is indicative of magnetic interactions between the [NiFe] site and another paramagnetic species, which on the basis of its spatial localization (distance # 14Å) has to be the reduced proximal [4Fe-4S] cluster in the 1+ state. Similar 'interaction' spectra have been reported for all [NiFe]hydrogenases studied so far, with the exception of the RH-type proteins. 20 Hence, the 'magnetic ngerprint' found here is the rst direct evidence of such a spin-spin interaction between adjacent metallocenters in a regulatory hydrogenase. The Mössbauer results suggest a certain heterogeneity with respect to the degree of reduction of the FeS clusters of the chemically reduced samples of RH stop , a fact that is reected also in a mixture of 'split' and 'unsplit' Ni-C signals contributing to the EPR spectra. It is difficult to ascertain the precise amount of 'unsplit' Ni-C species in the spectra of the chemically reduced protein. However, by using the Ni-C signal of H 2reduced RH stop as a reference for an almost pure 'unsplit' Ni-C spectrum, an upper threshold of less than 30% can be estimated for the fraction of magnetically uncoupled Ni-C in the dithionite-reduced sample ( Fig. 7 and S8 †). This estimation is in full agreement with the corresponding Mössbauer results on samples prepared under identical conditions, which demonstrated that 2.7 clusters (of three) are in the 1+ reduced state. With this presumption, the X-and Q-band Ni-C-related EPR spectra in the dithionite-reduced RH stop could be well simulated to obtain a reasonable solution of the magnitude and orientation dependence of the electron spin-spin interaction between the participating paramagnetic centers. The spectral features can be reproduced by a simple three-spin model accounting for magnetic interaction between the [NiFe] site in the Ni-C state and the proximal [4Fe-4S] 1+ cluster as well as between the proximal and the medial [4Fe-4S] 1+ cluster. The interaction between the [NiFe] site and the medial cluster was assumed to be negligible and therefore not considered. The g-values of the [4Fe-4S] 1+ clusters were chosen similar to those of other typical tetranuclear clusters, Fig. 6 Q-band 57 Fe HYSCORE spectra of the H 2 -reduced forms of 57 Fe-enriched RH stop at magnetic fields corresponding to two principal g-components of the Ni-C spectrum. Experimental conditions: mw frequency ¼ 33.90 GHz, T ¼ 20 K, p/2 ¼ 40 ns, s ¼ 300 ns, shot repetition time ¼ 1 ms. Simulation parameters are given in Table 3. Fig. 7 X-band CW EPR spectra of the dithionite-reduced RH stop at two different temperatures, 5 K and 80 K, respectively. The experimental signals (black lines) and the simulations (red lines) correspond to the spin-coupled Ni-C sites since the contribution of the uncoupled Ni-C signal ('unsplit'), estimated to be <30% in the spectra, has been subtracted from the spectra. The part of the simulated spectrum that corresponds to the [4Fe-4S] 1+ cluster signals is depicted with a dashed line (this was experimentally not detectable presumably due to g-strain effects and relaxation enhancement caused by spin-spin interactions). Because of the fast electronic relaxation of the FeS cluster signals at 80 K, their signals are not detectable and thus the 'fingerprint' of their magnetic interaction on the [NiFe] is absent from the NiC-related EPR signals. Experimental conditions: mw frequency 9.47 GHz, modulation amplitude 0.7 mT, mw power 0.2 mW. constrained, however, to reproduce the respective Q-band EPR spectra (Fig. 8, Table S2 †). The interaction between the [NiFe] center and the proximal cluster was described by an anisotropic J tensor (H electron spinÀspin coupling ¼ ÀS 1 $J$S 2 ) with an isotropic component J iso ¼ 31.7  10 À4 cm À1 (95 MHz) and an anisotropic component J dip ¼ [À60, 110, À50] MHz. The rst is the trace of J and accounts for the through-bonds interaction of the paramagnetic centers, whereas the second part is traceless and accounts for through-space dipole interaction. Using the point-dipole approximation, J dip is consistent with the [NiFe] site residing at a distance of r ¼ 9.8Å from the proximal [4Fe-4S] 1+ cluster with spherical polar coordinates (q, 4) ¼ (80 , 80 ) relative to the magnetic axes given by the two g-matrices, which were taken collinear. 68 The interaction between the two [4Fe-4S] 1+ centers was in good approximation considered to be isotropic with J iso ¼ 43.4  10 À4 cm À1 (130 MHz). The signals of the [4Fe-4S] 1+ clusters are not clearly detectable (presumably due to g-strain effects), which is similar to the case of standard O 2 -sensitive group 1 hydrogenases, but unlike [NiFeSe] hydrogenases. [68][69][70] EPR spectra of the FeS clusters. Whereas the 'split' Ni-C EPR signal in chemically reduced RH stop samples is readily observable by using conventional cw spectroscopy with eld modulation, the signals of the reduced [4Fe-4S] 1+ clusters are hardly detectable. Even measurements with dispersion-detection at temperatures as low as 2 K and high microwave power (providing rapid-passage conditions) were unsuccessful. Probably spin-spin interactions between the clusters, g-tensor anisotropy and g-strain effects broaden the signals beyond recognition. However, eld-swept 2-pulse echo-detected Q-band spectra of H 2 -and dithionite-reduced RH stop showed for the rst time two distinct signals that can be assigned to Ni-C and the [4Fe-4S] 1+ clusters (Fig. 8), respectively. The assignment is based on the typical low g-values of FeS clusters in conjunction with fast spin relaxation (the broad quasi-absorption signals are detectable only at lower temperatures, Fig. S3 †). The intensity of the FeS subspectrum is signicantly higher for the dithionitereduced samples than that for the H 2 -reduced samples, given relative to the resolved Ni-C contribution, appearing at higher g values. This is in agreement with the Mössbauer results that demonstrated reduction of almost all three [4Fe-4S] clusters with dithionite. The region of the spectra in which the FeS clusters are expected could not be explored in the Ti 3+ -reduced samples, because the strong signal from residual reductant overlaps with the [4Fe-4S] 1+ signals (Fig. S8 †), hindering their detection and analysis. 21,24 Discussion The RH active site The low-spin Fe II site in the catalytic center of [NiFe] hydrogenases has not been discerned in most Mössbauer spectroscopic studies to date, because its spectrum is usually masked by the iron-sulfur cluster signals that impede site-resolution and characterization. In this study, however, we obtained wellresolved signals attributed to the corresponding low-spin Fe II center in RH stop . This site exhibits a low isomer shi characteristic of low-spin Fe II centers, because shielding of the 57 Fe nucleus is less effective compared to the case of high-spin Fe II centers. 71,72 Furthermore, the quadrupole splitting parameter varies between the two different redox states. Upon transition from the oxidized Ni-SI to the one-electron more reduced Ni-C state, the quadrupole splitting DE Q decreases from 1.60 mm s À1 to 0.69 mm s À1 , reecting signicant changes in the ligand environment of the Fe II ion, whereas the isomer shi remains almost invariant. Only minor differences in the isomer shi of the Fe II ion were observed for the Ni-SI and Ni-C states, which suggest that there is no marked effect on the average bond lengths or the strength of the p-/s-bond interaction with the diatomic Fe ligands. The quadrupole splitting, however, was signicantly different for the two redox states, which might result from a change in the direct coordination environment of the Fe II . In fact, a vacant bridging position between the two heterometals has been proposed for the Ni-SI state, whereas in the Ni-C state, the presence of a bridging hydride between the Fe and Ni ions has been demonstrated. 20,37 The observed decrease in the quadrupole splitting for the RH can therefore be correlated with the change in the Fe coordination number from Ni-SI (5-coordinate) to Ni-C (6-coordinate), the latter of which reects a more symmetric environment. This signicant difference of the quadrupole splitting between the Ni-SI and Ni-C states might not be a unique feature of H 2 -sensors and is expected to be observed also in other [NiFe]-hydrogenases. The unprecedented resolution of the low-spin Fe II site (at least) in the as-isolated RH is presumably due to the almost stoichiometric presence of the Ni-SI state and the concomitant absence X-and Q-band 57 Fe HYSCORE experiments were carried out to characterize the electron spin density at the Fe center, from which detailed information about the electronic structure of the active site can be obtained. The anisotropic hyperne tensor has an isotropic component of 1.9 MHz, which is two-fold larger than the 57 Fe isotropic hyperne coupling constants determined for the Ni-B state of the D. vulgaris hydrogenase (A iso ¼ 0.8 MHz) 33 and the Ni-A state of the D. gigas enzyme (A iso ¼ 1.0 MHz). 73 Because the isotropic hyperne constant is proportional to the electron spin density at the nucleus, all data indicate a higher (although very small) electron spin density at the Fe center in the Ni-C state compared to the Ni-A and Ni-B states. Furthermore, the data are consistent with a low-spin Fe II center in all states, with most of the spin density located at the Ni ion, as has been previously shown by 61 Ni enrichment and subsequent determination of the respective 61 Ni hyperne tensor in the standard [NiFe] hydrogenase from D. vulgaris. 4,74,75 These results are in full agreement with density functional theory calculations of the 61 Ni hyperne coupling constants. 76 In the present as well as in previous studies of RH, the fully reduced Ni-R state could not be generated in signicant amounts, neither by incubating the enzyme with H 2 nor with strong reducing agents. In the FTIR spectra of the chemically reduced RH, a small intensity band at 1948 cm À1 is present and could represent the CO stretching vibration characteristic of the reduced Ni-R state. However, there is no evidence for the corresponding bands of the CN ligands, which for all reported Ni-R forms are typically shied at least by 10 cm À1 to lower wavenumbers with respect to those of Ni-C. 51 Nevertheless, we cannot exclude that Ni-R accumulated in small amounts, but the CN bands have been too low in intensity to be observed. The FeS clusters Amino acid sequence analysis of the RH small subunit HoxB revealed 12 conserved cysteine residues to be the likely ligands of three predicted tetranuclear clusters. 24 In previous studies, however, the iron-sulfur cofactors could not be successfully detected by EPR under any conditions. 20,21,23,50 The unprecedented O 2 -tolerance (or rather insensitivity) of the RH protein and the limited spectroscopic accessibility of the iron-sulfur clusters invoked a series of proposals for the chemical nature and role of these metallocofactors in the function and O 2resistance of H 2 -sensing hydrogenases. In addition, previous XAS results suggested the presence of [2Fe-2S] clusters and an unusual [4Fe-3S-3O] cluster in the RH wild-type protein. 23,26 However, these assumptions are not in line with our Mössbauer and EPR results, which neither showed signals attributable to [2Fe-2S] clusters 55,56,77 nor to a [4Fe-4S] cluster with unusual oxygen ligation at one or more of the Fe sites. In the latter case, the more ionic oxygen ligands are expected to cause a greater electric eld gradient asymmetry around that Fe site and result in a large quadrupole splitting, which, however, was not observed in our study. The Mössbauer and EPR results obtained for the RH stop protein in combination with the presence of twelve conserved cysteine residues arranged in characteristic FeS cluster binding motifs are consistent with the presence of three [4Fe-4S] clusters in H 2 -sensing hydrogenases and afford for the rst time the characterization of their electronic properties, which were found to be similar to those of classical tetranuclear cubanes. Without a crystal structure of the RH, however, unusual structures of any of the tetranuclear cubanes cannot be readily excluded. In the as-isolated RH stop , all three clusters reside in their oxidized, diamagnetic [4Fe-4S] 2+ form. Upon reduction with H 2 , approximately two (1.8) of the clusters are reduced to the 1+ state, whereas stronger (non-physiological) reducing agents, i.e. Ti 3+ citrate and sodium dithionite, afford almost complete reduction of all three clusters (2.7). The expected EPR signals of the reduced [4Fe-4S] 1+ clusters in both the H 2 and dithionite/ Ti 3+ citrate-treated samples are hardly detectable, which may be a result of g-strain effects and the inter-cluster spin-spin interactions, as has been previously observed for group 1 hydrogenases. 43,68 On the basis of the amino acid sequence, two of the three [4Fe-4S] clusters in the RH are coordinated by four cysteinederived thiolate ligands, while the 'distal' cluster is coordinated by one His and three Cys residues. Thus, at least with respect to the rst coordination sphere, the proximal and the distal clusters are expected to have similar electronic properties as their counterparts in the mostly O 2 -sensitive [NiFe]-hydrogenases. However, compared to other [NiFe]-hydrogenases, including the O 2tolerant membrane-bound enzymes, it is rather unusual that H 2 incubation does not lead to detectable accumulation of the reduced state of the proximal [4Fe-4S] cluster. On the basis of our observations, the cluster remains largely oxidized under steady state conditions, which is also supported by the absence of a magnetic splitting in the Ni-C-derived EPR signal generated upon H 2 incubation. The geometric arrangement of the clusters relative to the active site dictates that the proximal cluster is the rst FeS cofactor to become reduced upon H 2 oxidation. Thus, the present results may support a fast discharging of the proximal Interaction between the [NiFe] active site and the FeS clusters Although the FeS clusters of reduced RH stop are essentially 'silent' in the cw EPR spectra, a spin coupling model of three spins S i ¼ 1/2 including the Ni center, the proximal and the medial clusters (both in their reduced 1+ forms) could be successfully employed to simulate the spectra of Ni-C in chemically (dithionite) reduced RH stop . In order to limit the number of parameters, the fourth paramagnetic species, the distal [4Fe-4S] cluster, was not included into the simulations. In most hydrogenases studied so far, the long-range spinspin interaction between the reduced proximal cluster and the [NiFe] center can be considered, in good approximation, to be isotropic. In the case of RH stop , however, the spin coupling tensor, J, is quite anisotropic as reected in the Ni-C-related EPR spectra. The largest component of the anisotropic magnetic interaction tensor is 110 MHz and directed along the y-axis of the g-tensor of the Ni-C center, which is consistent with the absence of resolved splittings in the g x and g z components in the rhombic Ni-C spectrum. The isotropic component caused by the through-bond exchange interaction is found to be 95 MHz (assuming a traceless tensor for the anisotropic magnetic dipolar interaction). 78 This value is comparable to the exchange coupling constant of J iso ¼ 120 MHz (40  10 À4 cm À1 ) used for modeling the magnetic interaction between the paramagnetic active site in the Ni-C state and the proximal [4Fe-4S] 1+ cluster in the standard hydrogenase of D. gigas. 68 Recently, the exchange coupling constant between the active site in the Ni-B state and the superoxidized [4Fe-3S] 3+ proximal cluster of the O 2 -tolerant hydrogenase I from Aquifex (A.) aeolicus was also found to lie in this range (J iso ¼ 107 MHz, 36  10 À4 cm À1 ). 13 The isotropic exchange coupling constant between the medial and the proximal clusters in RH stop was determined to be at 130 MHz, which is noticeably larger compared to the corresponding value found in the case of A. aeolicus Hydrogenase I (21 MHz, 7  10 À4 cm À1 ). 13 This difference might provide an additional rationale for the difficulty to detect signals from the reduced [4Fe-4S] clusters in RH preparations due to a correspondingly increased sensitivity of the spectra for strain (distributions of different local electronic changes/interactions) and relaxation enhancement effects. Structure and function of H 2 -sensor hydrogenases As described above, the RH harbors three [4Fe-4S] clusters and is in this respect structurally similar to the group 1 [NiFeSe] enzymes, which contain the same iron-sulfur cluster complement. 28,29 The presence of a tetranuclear FeS cofactor in the medial position, which is usually occupied by a "high-potential" [3Fe-4S] center in most other hydrogenases, is consistent with the cytoplasmic localization of RH. In functional terms, the RH protein deviates from most other [NiFe(Se)] hydrogenases in some or all three of the following features: (i) it does not form the oxidized, inactive Ni-A and Ni-B states (ii) it hardly accumulates the most reduced Ni-R form upon reduction with H 2 , and (iii) H 2 does not lead to detectable population of the proximal [4Fe-4S] cluster in the 1+ state. The rst property is most likely related to the RH-specic narrow hydrophobic gas channel that restricts access of molecular oxygen to the active site, 18,19 which apparently renders H 2 sensors O 2 -insensitive rather than O 2 -tolerant. The second and third features are presumably relevant for the catalytic function underlying the H 2 -sensing mechanism of regulatory hydrogenases. Accumulation of the Ni-C state upon incubation of the RH with H 2 and the apparent inability of RH to generate detectable amounts of the Ni-R state under steady-state conditions can be explained by one (or both) of the following thermodynamic considerations. First, the redox potential of the proton-coupled Ni-C to Ni-R transition might be more negative compared to that of standard hydrogenases. Second, the proximal [4Fe-4S] cluster may have an unusual low potential, which is supported by its difficult reducibility through H 2 . Assuming a redox potential that is appreciably more negative than that of the proton-coupled Ni-SI to Ni-C transition, 38 the RH would be trapped in the Ni-C state under steady state conditions. The low-potential Ni-C to Ni-R transition may be promoted by alterations in the rst and second coordination sphere of the RH active site, which might also result in lowering of the pK a of the cysteine thiol-based proton present in the Ni-R state (e.g., due to a more hydrophobic environment). 61 One characteristic of the second coordination sphere of RH proteins is the presence of a glutamine residue instead of a histidine that is conserved in all other [NiFe]-hydrogenases. This glutamine residue might play a role in the redox properties of the active site but seems not to be involved in H 2 sensing. 22 The low redox potential of the proximal [4Fe-4S] cluster might also be caused by the immediate protein environment. In fact, a number of glycine residues in the vicinity of the proximal cluster, which are conserved only in sensory hydrogenases (Fig. S9 †), are situated at the interface of the small and large subunits. It is difficult to predict how the presence of these conserved glycine residues may change the reduction potential, but presumably these may alter the local electrostatic environment. Remarkably, a low redox potential of the proximal cluster would impede particularly the Ni-C to Ni-SI transition and thereby slowing down the catalytic activity of the enzyme. This is in-line with the low H 2 turnover rate observed for H 2 sensors, which, in turn, prevents energy loss and saves H 2 for the energyconverting hydrogenases. 36 Summary and conclusions The present study provides new insight into the nature and role of the metal cofactors in H 2 -sensing regulatory hydrogenases. Mössbauer spectroscopy allowed the identication of the lowspin Fe in the [NiFe] active site. As the Mössbauer parameters are sensitive to changes in the ligand eld coordination and symmetry around the active site Fe ion, they can be used to probe structural and electronic changes otherwise inaccessible by other techniques. Only, the quadrupole splitting, but not the isomer shi, is markedly dependent on the redox state (Ni-SI and Ni-C) of the catalytic site. It is considerably smaller in the Ni-C state compared to that of the Ni-SI state, which correlates well with the iron being 6-and 5-fold coordinated respectively. Mössbauer and EPR spectroscopy allowed for the rst time the identication and characterization of three [4Fe-4S] clusters as constituents of the electron transfer relay in the RH. Their electronic properties are similar to those of conventional tetranuclear FeS centers in low-potential ferredoxins. The extent of their reduction is dependent on the chemical reductant employed, i.e. non-physiological, strong reducing agents led to reduction of essentially all three [4Fe-4S] clusters, while H 2 afforded the reduction of only the medial and distal clusters. Upon H 2 treatment, a magnetic 'splitting' in the EPR signal of the active site-related Ni-C state, which is typical for most [NiFe]hydrogenases, was not observed for the RH. The splitting was detectable, however, when the enzyme was treated with dithionite and identied the proximal cluster as the last cofactor being reduced. This observation supports a low redox potential of the proximal cluster that promotes rapid transfer of electrons to the adjacent [4Fe-4S] 2+ centers, which, on the basis of our data, are supposed to have higher redox potentials. Concomitantly with the absence of the 'split' Ni-C EPR signal, also the Ni-R state as the most reduced catalytic intermediate is not accumulated upon "physiological" treatment of the RH with H 2 . Even in the presence of strong reducing agents, solely the Ni-C state is observed. Stabilization of the Ni-C state is presumably accomplished through appropriate adjustment of the redox potential of both the Ni-C-to-Ni-R transition (and the concomitant proton transfer) and the proximal [4Fe-4S] cluster. The potential of the latter seems to be too low to support an efficient redox transition from Ni-C to Ni-SI, which coincides with the low H 2 turnover rates of H 2 -sensing regulatory hydrogenases. In summary, the present spectroscopic study unravels the structural basics that have been evolved to convert an energy-generating into a cytoplasmic, H 2 -sensing hydrogenase.

v3-fos

2016-06-18T00:11:59.910Z

2015-09-15T00:00:00.000Z

18128008

s2

Proteomic analysis of middle and late stages of bread wheat (Triticum aestivum L.) grain development Proteomic approaches were applied in four grain developmental stages of the Chinese bread wheat Yunong 201 and its ethyl methanesulfonate (EMS) mutant line Yunong 3114. 2-DE and tandem MALDI-TOF/TOF-MS analyzed proteome characteristics during middle and late grain development of the Chinese bread wheat Yunong 201 and its EMS mutant line Yunong 3114 with larger grain sizes. We identified 130 differentially accumulated protein spots representing 88 unique proteins, and four main expression patterns displayed a dynamic description of middle and late grain formation. Those identified protein species participated in eight biochemical processes: stress/defense, carbohydrate metabolism, protein synthesis/assembly/degradation, storage proteins, energy production and transportation, photosynthesis, transcription/translation, signal transduction. Comparative proteomic characterization demonstrated 12 protein spots that co-accumulated in the two wheat cultivars with different expression patterns, and six cultivar-specific protein spots including serpin, small heat shock protein, β-amylase, α-amylase inhibitor, dimeric α-amylase inhibitor precursor, and cold regulated protein. These cultivar-specific protein spots possibly resulted in differential yield-related traits of the two wheat cultivars. Our results provide valuable information for dissection of molecular and genetics basis of yield-related traits in bread wheat and the proteomic characterization in this study could also provide insights in the biology of middle and late grain development. Introduction Hexaploid wheat (Triticum aestivum, 2n = 6× = 42, AABBDD) is one of the most important cereals that provides a large proportion of essential nutrients in the human diet. The major constituents of wheat grain are starch (70-80% dry weight) and proteins (10-15% dry weight; Tasleem-Tahir et al., 2012). Of the total wheat grain proteins, the major protein (80%) reserves are the prolamins, which are a mixture of monomeric gliadins and polymeric glutenins located in the starchy endosperm. In contrast to the gliadins and glutenins, the other major protein families of the wheat endosperm, are the non-prolamins, including albumins and globulins (Vensel et al., 2005a). Wheat grain development is divided into two main stages: (1) grain enlargement, and (2) grain filling and desiccation/maturation. Grain enlargement involves early and rapid division of the zygote and triploid nucleus. Cell division is followed by the influx of water, which drives cell extension. This stage occurs at approximately 3-20 days postanthesis (dpa). During the grain filling stage, cell division slows and then ceases and beginning at around 10 dpa until maturity, storage products are accumulated, at which point the endosperm serves its function as a carbohydrate store (Nadaud et al., 2010). In recent years, different approaches including transcriptomics, proteomics, and metabolomics have been used to understand the diversity and development of grain. However, the expression profiles of accumulated proteins are often poorly correlated with their corresponding mRNAs; e.g., in Arabidopsis (Ruuska et al., 2002), rice (Zhang et al., 2010), and wheat (Dong et al., 2012;Ma et al., 2014). Two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) proteomic approaches have been broadly applied to investigate the dynamic expression profiles of proteins during grain development in different plant species, including Arabidopsis (Ruuska et al., 2002;Li et al., 2007), soybean , maize (Méchin et al., 2007), and rice (Thelen, 2009;Zhang et al., 2012). Further, a significant study on the proteomics of the wheat grain developmental period has been carried out. Proteomic studies on the response to heat stress during grain filling in 10 wheat cultivars indicated that primarily changes in both the amount and activities of enzymes involved in photosynthesis and antioxidant activities contributed to relatively higher heat tolerance (Wang et al., 2015). Identification of proteins in the first 2 weeks of grain development stages showed that a total of 10 clusters of genes were examined in bread wheat (Nadaud et al., 2010). The proteomes of hard and soft near-isogenic wheat lines at four grain developmental stages revealed that kernel hardness is related to the amplification of a stress response during endosperm development (Lesage et al., 2012). Proteome characterization of four grain developmental phases in wheat cultivars Jimai 20 and Zhoumai 16 indicated that differences in seed storage proteins were related to different flour quality performance from these wheat cultivars (Guo et al., 2012). Ethyl methanesulfonate (EMS) mutants have been widely used as an important method to develop new germplasms in wheat breeding programs due to its high mutant frequency (Henry et al., 2014). The key reasons for this include their highly beneficial mutations, excellent phenotypic characteristics, and novel gene traits. The EMS mutation technique has also reached a mature stage, in which damage in plants is reduced and abundant plant mutations are generated by controlling EMS use. Additionally, EMS mutants have been employed as basic materials in some studies (Botticella et al., 2011;Bonchev et al., 2012;Henry et al., 2014). For example, six EMS-mutagenized lines were validated to improve lodging resistance in Tef (Eragrostis tef ; Zhu et al., 2012). Stay-green and fast-senescing EMS mutated wheat lines with similar anthesis were characterized to investigate the impact on yield and nitrogen partitioning (Derkx et al., 2012). A combination of 2-DE and EMS mutants in wheat proteomic studies were rarely applied. The Chinese winter wheat cultivar Yunong 201, developed by Agronomy College of Henan Agricultural University, was released as a high-quality noodle wheat cultivar by Henan province in 2006. An elite M 2 line was screened from a large EMS-mutagenized population because of its different plant architecture, larger kernel size, and higher grain weight. This line was self-crossed four times into Yunong 3114. Compared with Yunong 201, Yunong 3114 showed relatively larger kernel size, higher thousand grain weight and higher yield per plot. Therefore, comparison of proteomics of mid and late grain developmental stages of the bread wheat Yunong 201 and Yunong 3114 could provide valuable information for dissection of molecular and genetics basis of yield-related traits in bread wheat, and the proteomic characterization could also provide insights in the biology of middle and late grain development. Plant Materials A Chinese winter wheat cultivar Yunong 201 (released no. Yushenmai 2006006) was treated by 0.8% EMS (ethyl methanesulfonate) in 2007. An elite M 2 line was screened from the EMS mutated population containing 2000 lines due to its differential plant architecture, larger kernel size and higher grain weight, which was self-crossed four times into an M 6 line Yunong 3114. Yunong 201 and Yunong 3114 were planted at the Zhengzhou Scientific Research and Education Center of Henan Agricultural University (longitude 113.6 • E; latitude 34.9 • N) during the 2013-2014 cropping seasons under nonstressed natural soil conditions. Differently developmental seeds of Yunong 201 and Yunong 3114 were collected during the postanthesis period based on thermal times that corresponded to the cumulative average daily temperatures as shown in Table 1, and grain size and weight of each sample were investigated (Figure 1). Sampled grains were stored at −80 • C prior to analysis. Protein Preparation For two-dimensional gel electrophoresis (2-DE), protein samples with three biological replicates were prepared according to the method of Gao et al. (2009). Grain samples of 500 mg were extracted in the mid-ear region of each spike, and were ground into a powder in liquid nitrogen with a mortar and pestle. Ten volumes of cold extraction buffer containing 100 mM Tris-HCl (pH 8.8), 10 mM fresh dithiothreitol (DTT), and 10% sodium dodecyl sulfate (SDS) were added, and further ground for 1 h on ice. After centrifuging at 10,000 g for 10 min at 4 • C, the supernatants were collected to new tubes, and an equal volume of phenol was added, following which samples were shaken gently for 30 min, and centrifuged at 14,000 g for 10 min at 25 • C. Below the top phenol phase, the samples were collected to new tubes, and then the above cold extraction buffer was added again to extract once more. The phase of phenol was acquired again, and samples were precipitated with five-fold volumes of cold ammonium acetate/methanol at −20 • C for 2 h. After centrifugation at 14,000 g for 15 min at 4 • C, the supernatants were discarded and the pellets were washed three times in ice-cold acetone containing 5 mM DTT. The pellets were vacuum-dried and resuspended in lysis buffer containing 8 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), and 20 mM DTT at 25 • C for 2 h according to method of Li et al. (2013). The suspension was centrifuged at 14,000 g for 40 min at 25 • C to remove insoluble materials. Concentrations of total protein were determined by the Bradford assay (Bio-Rad) based on a bovine serum albumin standard (Li et al., 2013). Detailed standard curves with seven different concentrations of BSA (0-100 µg) resuspended in lysis buffer and water in triplicate were shown in Additional file 2 (Data sheets 11, 12). 2-DE and Images Analysis For 2-DE, 800 µg of protein samples were loaded onto an ReadyStripTM IPG Strip (24 cm, pH 4-7, BIO-RAD, USA) and hydrated passively with 450 µL of protein solution containing 0.5% (v/v) immobilized pH gradient (IPG) buffer (pH 4-7) for 12-18 h at 20 • C using a PROTEAN IEF Cell (BIO-RAD, USA). The first-dimension isoelectric focusing (IEF) was performed with six steps: 250 V for 130 min, 250 V for 90 min, 500 V for 90 min, 1000 V for 2 h, 9000 V for 5 h, and 9000 V for 10 h with a total of 99 kVh and a constant 500 V for the last 12 h. After IEF, the strips were incubated for 15 min in "equilibration buffer I" consisting of 6 M urea, 2% (w/v) SDS, 1.5 M Tris-HCl (pH 8.8), 20% (v/v) glycerol, 0.01% (w/v) bromophenol blue, and 2% (w/v) DTT and then in "buffer II" consisting 6 M urea, 2% (w/v) SDS, 1.5 M Tris-HCl (pH 8.8), 20% (v/v) glycerol, 0.01% (w/v) bromophenol blue, and 2.5% (w/v) iodoacetamide for 15 min. For second-dimension electrophoresis, the strips were transferred to 12% vertical sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gels. All seed samples were run in triplicate to obtain statistically reliable results. After electrophoresis, gels were fixed in 40% (v/v) methanol and 10% (v/v) acetic acid for 40 min. To visualize the gels, they were stained with staining solution consisting of 0.12% (v/v) Coomassie brilliant blue (CBB) G-250, 20% (v/v) alcohol, 10% (v/v) phosphoric acid, and 10% (w/v) ammonium sulfate, and then destained in double-distilled water . The 2-DE images were scanned at 300 dpi with a UMAX Power Look 2, 100XL scanner (Maximum Tech, Taiwan, China), and quantitative intensity analysis was performed using PDQuest software (version 8.0.1, Bio-Rad, USA). First, the 2-DE gel of 21 dpa seed samples of Yunong 210 and Yunong 3114 was selected as the reference gel. All gels of other stages were matched to the reference gel. Automatic groups formed, and single spots that differed between replicates were manually checked and corrected when necessary. The spots that existed in three independent sample sets were selected. Image quantitative analysis revealed significant differences in protein spot abundance by Student's t-test (abundance variation at least two-fold, P < 0.05). Two-dimensional Gel Excision, Tryptic Digestion, and Desalting Protein extracts were separated on preparative gels and 130 proteins of interest were recovered from the gels for identification. Proteins (800 µg) from samples were resolved on separate preparative polyacrylamide gels and were visualized by staining with a modified silver staining method that was compatible with subsequent mass spectrometric analysis (Yan et al., 2000). Protein spots of interest were cut from the preparative gels, destained for 20 min in 30 mM potassium ferricyanide/100 mM sodium thiosulfate (1:1 v/v) and washed with Milli-Q water until the gels were destained. The spots were incubated in 0.2 M NH 4 HCO 3 for 20 min and then lyophilized. Each spot was digested overnight in 12.5 ng/µl trypsin in 25 mM NH 4 HCO 3 . The peptides were extracted three times with 60% acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA). The extracts were pooled and dried completely by a vacuum centrifuge. MALDI-TOF/TOF Analysis MS and MS/MS data for protein identification were obtained using a MALDI-TOF-TOF instrument (4800 proteomics analyzer; Applied Biosystems). Instrument parameters were set using the 4000 Series Explorer software (Applied Biosystems). The MS spectra were recorded in reflector mode and a mass range from 800 to 4000 and a focus mass of 2000. MS was used using a CalMix5 standard to calibrate the instrument (ABI 4700 Calibration Mixture). For one main MS spectrum 25 sub-spectra with 125 shots per sub-spectrum were accumulated using a random search pattern. For MS calibration, autolysis peaks of trypsin ([M+H] + 842.5100 and 2, 211.1046) were used as internal calibrates, and up to 10 of the most intense ion signals were selected as precursors for MS/MS acquisition, excluding the trypsin autolysis peaks and the matrix ion signals. In MS/MS positive ion mode, for one main MS spectrum 50 sub-spectra with 50 shots per sub-spectrum were accumulated using a random search pattern. Collision energy was 2 kV, the collision gas was air, and the default calibration was set using the Glu1-Fibrino-peptide B ([M+H] + 1570.6696) spotted onto Cal 7 positions of the MALDI target. Combined peptide mass fingerprinting (PMF) and MS/MS queries were performed using the MASCOT search engine 2.2 (Matrix Science, Ltd.) that was embedded into the GPS-Explorer Software 3.6 (Applied Biosystems) on the NCBI database with the following parameter settings: 100 ppm mass accuracy, with trypsin cleavage and one missed cleavage allowed, carbamido methylation set as a fixed modification, and oxidation of methionine allowed as a variable modification. Additionally, the MS/MS fragment tolerance was set to 0.4 Da. A GPS explorer protein confidence index ≥95% was used for further manual validation. Comparison of Grain Size of Yunong 201 and Yunong 3114 Kernel sizes and weights of the grain in both Yunong 201 and Yunong 3114 increased gradually from 21 to 35 dpa, and then decreased from 35 to 42 dpa (Figures 1E,F). Compared with Yunong 201, Yunong 3114 possessed longer kernel length and higher grain weight at the four stages of development (detailed data in Figures 1A-D) but there was no obvious difference on grain width between the two cultivars. Identification, and Classification of Differentially Accumulated Proteins during Grain Development Yunong 201 and Yunong 3114 had similar proteomic profiles at the four stages according to the 2-DE protein maps that were extracted from both samples (Figures 2, 3). There were more than 1000 gel spots detected over the gel, and 173 spots were detected that displayed altered abundance, which were then analyzed by mass spectrometry. Finally, 130 spots out of the 173 spots, representing 88 unique proteins, were successfully identified based on BLASTp analyses of NCBI databases (Additional file 1: Data sheets 1-10; Table 2). Protein Expression Profiles during Grain Development The expression profiles of the 130 protein spots were investigated by hierarchical cluster analysis ( Figure 5). Four main expression patterns (A-D) were presented and clearly reflected two distinct grain development phases: grain filling (21, 28), and desiccation/maturation (28-42), as shown in Figure 5. Expression pattern A included 54 protein spots in Yunong 201, and 48 spots in Yunong 3114 that exhibited up-regulation during the four grain developmental stages, which contained many stress/defense-related proteins, such as α-amylase inhibitor 0.19 (protein spots 10 and 19-1), CM 17 protein precursor (protein spots 32 and 33), disease resistance protein RPP13 (protein spot 70), vicilin-like antimicrobial peptides 2-2 (protein spot 71), and 1-Cys peroxiredoxin (protein spot 83), which all accumulated significantly at the desiccation/maturation developmental stages in Yunong 201 and Yunong 3114. Almost all of the storage proteins including globulin 3 (protein spots 5 and 15), globulin-3A (protein spots 22, 25, and 130), globulin 3B (protein spot 23), and gamma gliadin (protein spot 78) displayed this pattern in both Yunong 201 and Yunong 3114. Besides, the same responses were seen for glutaminyl-tRNA synthetase (i.e., protein spot 101), serpin-Z1C (i.e., protein spot 104), which is involved in protein synthesis/assembly/degradation (Fernando et al., 2015;Kodera et al., 2015) and 27 K protein (Kimoto et al., 2009;i.e., protein spot 76-2), which is involved in transcription/translation. Expression pattern B included the largest proportion of identified proteins whose expression was down-regulated during the mid and late grain developmental stages. Moreover, 71 protein spots in Yunong 201 and Yunong 3114 belonged to this expression group, respectively. All of the proteins associated with glycolysis and most of the proteins involved in starch metabolism, photosynthesis, and energy production and transportation/signal transduction displayed this expression pattern in two samples; for example, ATP synthase CF1 beta subunit (protein spot 107), 2,3-bisphosphoglycerateindependent phosphoglycerate mutase (protein spots 142, 143, and 144), adenosine kinase 2 (protein spots 89 and 90), granule bound starch synthase (protein spot 138), and the 23 kDa oxygen evolving protein of photosystem II (protein spot 166). Expression pattern C showed both down-and up-regulated expression trends, including protein spots 49, 67, and 111, which were seen in the 14-3-3-like protein B, serpin-N3.2, and β-amylase in Yunong 201. There were five protein spots (i.e., 103, 104, 105, 106, and 111) that showed this pattern of expression in Yunong 3114. Unlike expression pattern C, expression pattern D displayed both an up-and down-regulated expression trend. Only protein spot 60 (chitinase 2) belonged to this pattern in Yunong 201, as did protein spots 121 (cerpin-Z2B), 122 (cerpin-Z1C), and protein spot 99 (serpin-N3.2) in Yunong 3114. Protein spots 168 (unnamed) and 167 (unnamed) displayed higher expression levels in Yunong 201 than did Yunong 3114, and protein spot 168 accumulated a single pattern E (not shown in Figure 5) that remained constant during the four grain developmental stage of both cultivars. In addition, protein spot 167 also belonged to this pattern in Yunong 3114, while it displayed expression pattern B in Yunong 201. Comparative Proteomic Characterization in Yunong 201 and Yunong 3114 during Grain Development A total of 12 protein spots with different expression patterns co-accumulated in both samples (Figure 5), including stress/defense, protein synthesis/assembly/degradation, signal transduction, starch metabolism, photosynthesis, and the presence of two unnamed proteins. For example, protein spot 67 was identified as serpin-N3.2 that displayed expression pattern C in Yunong 201, and pattern B in Yunong 3114. Protein spot 7 (Nucleoside diphosphate kinase 1) accumulated steadily at the four developmental stages in Yunong 201, and showed pattern E, although it displayed a down-regulated trend in Yunong 3114. Serpin-Z2B, Serpin-Z1C (protein spots 121 and 122) showed expression pattern A in Yunong 201, but displayed expression pattern D in Yunong 3114. In addition, protein spots 103 (ATP synthase subunit beta, mitochondrial), 104 (UTP-glucose-1-phosphate uridylyltransferase) and 105 (ATP synthase beta subunit) showed expression pattern C in Yunong 201, and showed a down-regulated trend in expression in Jimai 20 during the four developmental stages. Protein spots with two-fold changes or greater in abundance at particular times between the two cultivars were considered as cultivar-different proteins (Guo et al., 2012). Altogether six protein spots displayed cultivar-different proteins during the four developmental stages, which involved three groups: stress/defense, starch metabolism, protein synthesis/assembly/degradation. Among them, protein spot 99 (serpin-N3.2) was only identified in Yunong 3114; however, this protein spot was absent in Yunong 201. Meanwhile, protein spot 169 (i.e., small heat shock protein Hsp 23.5) was not detected in Yunong 3114, which was only identified in Yunong 201. Discussion In this study, a Chinese winter wheat cultivar Yunong 201 and its EMS mutant line Yunong 3114 were selected to study the proteomic expression differences during mid and late stages of grain development. Proteomic expression profiles during four grain development stages and cultivar-variable proteins of the Yunong 201 and Yunong 3114 were investigated by 2-DE and MALDI-TOF/TOF-MS. Proteomic characterization in this study could provide insights in the biology of middle and late grain development. Analysis of Cultivar-Different Proteins in Developmental Seeds of Yunong 201 and Yunong 3114 Up to date, a considerable work has been carried out on wheat grain proteomics through different wheat cultivars (Majoul et al., 2004;Kim et al., 2010), for instance, grain storage proteins (Mamone et al., 2009;Dupont et al., 2011), endosperm and endosperm amyloplasts (Vensel et al., 2005b;Dupont, 2008), and kernel peripheral and aleurone layers (Tasleem-Tahir et al., 2011;Nadaud et al., 2015). Those studies provided the important information on biochemical processes of wheat grain development, however, few studies on proteomic of EMSmutagenized cultivars were conducted in bread wheat. Due to the significant differences on kernel size, thousand grain weight and higher yield per plot of Yunong 201 and Yunong 3114, proteomics analysis of Yunong 201 and Yunong 3114 could provide valuable information for further understanding function of candidate cultivar-different proteins (e.g., serpin for spot 99, small heat shock protein for spot 169, β-amylase for spot 111, α-amylase inhibitor for spot 11, dimeric α-amylase inhibitor precursor for spot 12, and cold regulated protein for spot 35) which were possibly associated with yield-related traits in bread wheat. Heightened stress interrupts normal protein functions. Small heat shock proteins (sHSPs) are produced in seeds during maturation and under various stress conditions, which can form large multimeric structures and display a wide range of cellular functions, as well as being able to act as molecular chaperones. These sHSPs do this by forming stable complexes with folding intermediates of their protein substrates (Omar et al., 2012;Wu et al., 2014). In our study, the abundance of a sHSPs (spot 169) was up-regulated in Yunong 201, but was absent in Yunong 3114. In addition, stress-related cold regulated protein (spot 35) had a higher abundance in Yunong 201 than in Yunong 3114 during the four grain developmental stages. α-Amylase inhibitors are high molecular weight macromolecules that are particularly abundant in certain cereals and leguminosae, which specifically involved in the degradation of α-1,4-linked sugar polymers, such as starch and glycogen, into oligosaccharides (Franco et al., 2000). α-Amylase inhibitors play important roles in protecting starch and protein reserves in the endosperm against degradation, particularly that caused by biotic stresses like insect attack (Franco et al., 2002). In our study, α-amylase inhibitors (spots 11 and 12) accumulated gradually from 21 to 42 dpa in Yunong 201, however, displayed down-regulated trends during β-Amylase is a starch-degrading enzyme that hydrolytically cleaves α-1,4-D-glucosidic bonds to liberate β-maltose from the non-reducing ends of a variety of polyglucans that are synthesized during grain development, and is one of the major proteins in the starchy endosperm (Yin et al., 2002;Vinje et al., 2011). They can only contribute to starch granule hydrolysis by degrading solubilized intermediates that are released from the granules by α-amylase (Sun and Henson, 1991). The identified protein spot 111 (β-amylase), which displayed pattern C, accumulated at a higher level of abundance in Yunong 201 than in Yunong 3114. This is possibly one of the important reasons to result in differences of grain size and weight between Yunong 201 and Yunong 3114. Analysis of Protein Spots during the Developmental Stage of Yunong 201 and Yunong 3114 A total of 173 identified protein spots showed more than a two-fold difference in abundance in Yunong 201 and Yunong 3114 at the four stages by means of the classic 2-DE method in this study. Of them, 130 were successfully identified by MALDI-TOF/TOF analysis. The identified protein spots had specific functions in stress/defense, carbohydrate metabolism, protein synthesis/assembly/degradation, storage proteins, energy production and transportation, photosynthesis, transcription/translation, signal transduction, and unknown functional groups. Protein spots with unknown functions were those not identified by interrogating three database including NCBI Triticum, NCBI Viridiplantae, and the Universal Protein Resource (UniProt), probably owing to the lack of a genomic sequence of bread wheat until now. A vast majority of the 130 protein spots had similar proteomic profiles at four stages of development in Yunong 201 and Yunong 3114. However, there were still 12 protein spots exhibiting differential expression patterns and six protein spots exhibiting cultivar-differential expression. It suggests that these six protein spots abovementioned possibly contributed to difference of yield-related traits between Yunong 201 and Yunong 3114. Therefore, the identified protein spots showing differential expression could be used for further digging genes related to yield-related traits in bread wheat and characterization of these protein could also provide new insights into the biology of middle and late grain development in bread wheat. Stress/Defense Plants responsiveness to stress entails a complex mechanism and is involved in a large number of enzymes. Chitinase has been proven to play important physiological roles including defense from attack morphological changes, and digestion (Suzuki et al., 2014). Plants with over-expressed Chitinase genes showed stronger disease resistance in different crops (Cletus et al., 2013). In our study, the proteins for spots 60, 61, and 62 were identified as Chitinase 2, which gradually accumulated at four stages of development in Yunong 201 and Yunong 3114, suggesting a vital role of Chitinase in the response to different stress/defense challenges during the mid and late grain developmental periods. The R-proteins recognize pathogenic effectors and activate an efficient defense system that includes a "hypersensitive response (HR)" of programmed cell death or apoptosis at the infection site (Jones and Dangl, 2006). Disease resistance protein RPP13 (i.e., spot 70) is an R-protein, and our proteomic analysis showed that the abundance of RPP13 increased during the grain phase of development, which probably suggested a protective role from pathogens during the grain phase of development during mid and late developmental stages. Vicilin-like antimicrobial peptide 2-3 is a processing product of the 7S globulin precursor that is found in Macadamia integrifolia kernels that display antimicrobial activity (Marcus et al., 1999), the abundance of which (i.e., spots 71 and 100) also gradually increased, which was consistent with RPP13, and might indicate positive roles in antipathogen defense. 1-Cys peroxiredoxin (1-cysPrx) is a novel antioxidant enzyme that reduces phospholipid hydroperoxides, playing an important role in cellular defense mechanisms against oxidant stress (Manevich et al., 2002). The identified protein spots 83 and 84, (1-Cys peroxiredoxin) showed an enhanced trend in expression at the grain phases in Yunong 201 and Yunong 3114. Serpins are likely to participate in a range of biochemical pathways in distinct cell types, tissues and organs in plants to protect cells from oxidative stress, and are highly expressed during seed maturation and occur in tissues during all development stages (Roberts and Hejgaard, 2008). Four types of serpins including serpin-N3.2 (i.e., spots 40, 67, and 99), serpin 1 (i.e., spots 42 and 116), serpin-Z2B (spot 121), and serpin-Z1C (spots 122 and 124) were identified in this study, and spots 121 and 122 showed up-regulated trends in Yunong 201 but down-regulated trends in Yunong 3114. Moreover, spot 67 showed down-regulated trends in Yunong 3114, while it presented a C expression pattern in Yunong 201. In addition, spots 40 and 42 displayed down-regulated patterns of expression during the mid and late grain developmental stages in both cultivars. However, the abundance of spot 124 was increased, which was because of EMS mutagenesis that contributed to four types of serpins displaying differential trends in expression in the two wheat cultivars. Therefore, proteomic studies in this study could also be used in practical applications such as breeding for an enhanced stress tolerance as suggested by Kosova et al. (2014). Carbohydrate Metabolism Glycolysis provides energy and intermediates for the synthesis of metabolites, of which, we identified nine protein spots representing six types of proteins. These were referred to fructokinase-2 (spot 87), phosphoglycerate kinase, cytosolic (spot 123), enolase (spot 115), cytosolic 3-phosphoglycerate kinase (spot 127), UTP-glucose-1-phosphate uridylyl transferase (spot 104 and 106), and 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (spot 142, 143, and 144), with the exception of the abundance of spot 104 (UTP-glucose-1phosphate uridylyl transferase), which decreased in Yunong 201, and showed an expression pattern C in Yunong 3114. The remaining spots all showed down-regulated patterns of expression from the 21 to 42 dpa. Moreover, 21 dpa belonged to the late stage of wheat filling, which has enhanced glycolysis that is a significant source of energy for grain filling and accumulation of dry matter. Thus, this coincided with the synthesis stage of starch. During the filling stage, and due to the sharp accumulation of starch, ATPases are activated to provide more energy demands for an organism. Our research suggested that the reduced abundance of energy production from 21 dpa, as indicated by the down-regulated trend of the ATP synthase CF1 beta subunit (spot 107), ATP synthase subunit alpha, mitochondrial (spot 132 and 135), adenosine kinase 2 (spot 89 and 90), and the ATP synthase CF1 alpha subunit (spot 154). However, spot 103 (ATP synthase subunit beta, mitochondrial), and spot 105 (ATP synthase beta subunit) displayed an enhanced trend at the grain mature period in Yunong 3114. Starch Synthesis and Storage Proteins Starch is the major energy reserve for a large variety of higher green plants, such as cereals, legumes, and tubers (Miao et al., 2015). The biosynthesis of starch is the major determinant of overall yield in cereal grains (Emes et al., 2003). In all plant tissues capable of starch biosynthesis, adenosine diphosphate glucose (ADPGlc) pyrophosphorylase (AGPase, EC 2.7.7.27) is the enzyme that is responsible for the production of ADPGlc, the soluble precursor and substrate for starch synthases. The AGPase reaction is the first committed step in the biosynthesis of stored starch in amyloplasts (Tetlow et al., 2004). Our results demonstrated that ADP glucose pyrophosphorylase (spot 54), ADP-glucose pyrophosphorylase large subunit (spots 92 and 153), and small subunit ADP glucose pyrophosphorylase (spot 141) were all down-regulated from the filling stage to trace levels at the desiccation phase, which matched the increase in starch content and grain weight during the mid and late grain developmental stages. Glycogen synthase catalyzes the formation and elongation of the α-1,4-glucose backbone using ADP-glucose, the second and key step of glycogen biosynthesis. Elongation and branching of amylopectin is a complex process and it requires an array of enzymes including starch synthases (SS), starch branching enzymes (SBE), and debranching enzymes (DBE). However, synthesis of amylose is brought about solely by the enzyme granule-bound starch synthase I (GBSSI) or waxy protein (Ahuja et al., 2014). In our study, the abundance of granule bound starch synthase (spot 138) was decreased from the 21 dpa, which belonged to the late stage of grain filling, which was consistent with the filling phase, and the most vital stage of starch accumulation. We identified one β-amylase that was involved in seven spots (i.e., 109, 111, 114, 119, 125, 128, and 133), except for spot 111. The remaining six spots showed a down-regulated trends. There were three types of globulin that were identified at the four grain stages, including the globulin 3 (spot 5 and 15), globulin-3A (spot 22, 24 and 25), and globulin 3B (spot 23). Further, with the exception of gamma gliadin (spot 78) and avenin-like seed protein (spot 74), they accumulated gradually at the mid and late developmental periods in both samples. In general, biosynthesis of seed storage protein is dependent of amino acid synthesis and the transport of nitrogen metabolism (Hernández-Sebastià et al., 2005). We identified one alanine aminotransferase 2 (i.e., spot 151) and one glutaminyl-tRNA synthetase (i.e., spot 101). However, they displayed the opposite expression trend for the two wheat cultivars. Other Functional Proteins 14-3-3 Proteins function as homodimers or heterodimers and bind a large number of differentially phosphorylated substrates to regulate a wide array of cellular signaling and physiological processes (Lozano-Durán and Robatzek, 2015). We identified one 14-3-3 protein (i.e., spots 46, 47, and 48), and one 14-3-3-like protein B (i.e., spots 49 and 50), among them, 14-3-3 proteins were down-regulated in two samples, while the abundance of protein spot 50 increased in Yunong 201 and Yunong 3114. Nevertheless, the other 14-3-3-like protein B (i.e., spot 49) displayed a steady expression trend in Yunong 201, but was gradually down-regulated at the four stages of development in Yunong 3114. The cytosolic NDPK1 is the main nucleoside diphosphate kinase (NDPK) isoform in plants, which operates in the context of homeostasis of cellular nucleoside triphosphate (NTP) pools that accounts for more than 70% of total NDPK activity in plants (Prabu et al., 2012). In our study, the abundance of nucleoside diphosphate kinase 1 (spot 7) was gradually reduced in Yunong 3114; however, it showed a steady expression trend during the four developmental grain stages in Yunong 201. Thus, nucleoside diphosphate kinase 1 might play a different role in signal regulation in the two wheat cultivars. Conclusions In our study, 2-DE and tandem MALDI-TOF/TOF-MS were implemented to characterize protein accumulation at the middle and late stages of grain development in Yunong 201 and Yunong 3114, which differ by grain weight and size. Totals of 130 differentially accumulated protein spots representing 88 unique proteins were identified and they showed four main expression patterns in Yunong 201 and Yunong 3114. Moreover, six cultivardifferent protein spots were examined. These included cultivardifferent protein spot 111 (β-amylase), which accumulated at much higher abundance in Yunong 201 than in Yunong 3114. This difference was possibly related to the difference in grain size and weight between the two wheat cultivars. In addition, the absence or down-regulation of three protein spots identified as 11, 12, and 169 in Yunong 3114 were all related to stress/defense, the results possibly revealed that Yunong 201 and Yunong 3114 possessed differential adaptation to abiotic stress. Our results could provide valuable information for dissection of molecular and genetics basis of yield-related traits in bread wheat as well as new insights into the biology of late grain development. Author Contributions FC and DC designed the project. NZ and WH performed the experiments. NZ and FC wrote the paper. NZ performed the analyzed the data. All authors read and approved the manuscript.

v3-fos

2019-04-08T13:08:34.490Z

2015-07-16T00:00:00.000Z

99650148

s2

Whey protein isolate modified by transglutaminase aggregation and emulsion gel properties Whey protein isolate and commercial soybean salad oil were used to produce the WPI emulsion dispersions. The properties of TG-catalyzed emulsion gelation produced from WPI emulsion dispersions were investigated by the amount of TG, temperature, pH and reaction time. Specifically, the texture properties (hardness and springiness), water-holding capacity and rheological properties (G' and G") were assessed. The result of Orthogonal tests showed WPI emulsion can form better hardness and springiness gel when the ratio of TG and WPI was 20U/g, pH 7.5, treatment temperature and time were 50°C and 3 h, respectively. The microstructure of TG emulsion gels was more compact, gel pore is smaller, distribution more uniform, the oil droplets size smaller compared with untreated emulsion gels. Compared to the control of rheological properties, G' and G" were significantly increased and G' > G", results showed that the gel was solid state, and TG speeded up the process of gelation. particles and the interaction with the gel matrix and oil droplets. Therefore, the research about emulsion gel to improve the textural properties of the food is necessary. In the present work, there are few papers published concerning with whey protein emulsion gel. To be competitive in food ingredient markets, the functionality of whey proteins must be continuously improved and designed for specific end uses. Chemical and physical methods are commonly used. Food proteins can have their functionality altered by temperature and other chemical means. Specific functional attributes could be obtained by enzymatic polymerization of proteins. Here the enzymatic reaction could be controlled by time to enhance the functionality to the desired level [5]. The enzyme transglutaminase (EC 2.3.2.13) catalyzes an acyl transfer reaction between the γcarboxyamide group of peptide-bound, glutamine residues as acyl donors and primary amines as receptors [6]; when transglutaminase acts on protein molecules, intra-and inter-molecular ε-(γglutamyl) lysine crosslinks are formed by the enzyme reaction [7]. The inter-molecular cross-linking, which increases the weight average molecular weight of the protein, may improve the gelation and emulsifying properties [8]. Many studies have been carried out the TG-induced polymerization of modified whey protein to obtained different functional properties of gels, such as high hardness [9], the changes temperature of the gel point [10], the reduction of syneresis cracking of gel catalyzed by TG [11], but the properties of whey protein emulsified gel catalyzed by transglutaminase rarely reported. The effect of the amount of TG, pH, temperature, heating time on emulsion gel were observed by texture analysis, water-holding capacity and rheology, and provide the reference about theoretical studies to improve whey protein gel emulsion properties. Materials The whey protein isolate powder used in this study was a commercial product provided by Columbia company (USA), with an approximate composition of >90% protein, <5.1% moisture, <1.5% minerals as per experiment's data. Soybean salad oil (JIUSAN Oil Industry Group Co. Ltd. Harbin, China). Ca 2+ -independent microbial transglutaminase (TG-B) was provided by Yiming Biological Products Co. Ltd. (Jiangsu, China). The enzyme (composed of lactose, maltodextrin and transglutaminase) presented mean enzymatic activity of 100 U/g, as per manufacturer's data. The enzyme was used in the original form without further purification. All other chemicals were of analytical grade. WPI emulsion dispersions preparation Preparation of 8% (w/v) whey protein isolate emulsion dispersions. WPI solutions were prepared by dispersing the WPI powder in distilled water adjusted to 7.0 pH using either 1.0 M HCl or 1.0 M NaOH, followed by mechanically stirring at room temperature for 120 min, then left overnight at 4℃ to equilibrate and ensure completely hydration. Commercial soybean salad oil was added to whey protein isolate solution, and the ratio of oil is 15% (v/v). This system was stirred and premixed for 15 min at 60℃. Emulsions were obtained using an YQ-3 high speed homogenizer (Scientific research machinery work, Jiangsu, China) at 10000 rpm for 120 s. Emulsions were cooled and stored at 4℃ until further analyses. All samples were prepared in triplicate. The effects of TG on the characteristics of emulsion gel The basic parameters were setting for the temperature of 45℃, cross-linking of time 3 h, pH of 7.5, TG-B for 30 U/g. The sample was heated at 90℃ for 30min, and then gel was rapidly cool to room temperature in ice-water bath. Finally, left overnight at 4℃ before the analysis. Single factor experimental design. In order to investigate the effects of TG on emulsion gel characteristics, texture properties and water-holding capacity were determined. The amount of TG (0, Texture analysis of emulsion gel The texture of emulsion gel was determined according to the method of Xin Gu [12] with minor modification by TA-XT plus Texture Analyzer (Stable Micro System, England). The gel (25 mm in height and 50 mm in diameter) was equilibrated at room temperature for 30min before measured. Using P/0.5 cylindrical probe, which moves downward twice during measuring. setting forward speed: 10 mm/s, measuring speed: 10 mm/s; retreating speed: 10 mm/s, compression depth: 10 mm. Three measurements were conducted for each replication, and there were three replications in all treatments. Means and standard deviations were calculated from nine data. Water-holding capacity The water-holding capacity (WHC) of emulsion gel was determined according to the method of Vale'rie Leung Sok Line [13] with minor modification (centrifugal machine, Medicine centrifuge works, Beijing). The WPI emulsion gel was weighed and placed into 50 mL centrifugal tubes, and centrifuged at 9000 rpm for 20 min, then measured the weight of overflow water. Values are means of three replications with duplicate measurements. Water-holding Capacity equation is as follows: Where W t (g) is the weight of total moisture of gel and W r (g) is the weight of overflow water after centrifuging. Scanning electron microscopy The gel was spiced into squares of 3×3×3 mm 3 , and were determined according to the method of Chin K B [14]. Accelerating voltage 5 KV, observing and photographing the sample particle morphology with Japan JME-100CXII TEM. Rheology The Rheology of emulsion gel was determined according to the method of Li [15] with slight changes by MALI038384 Rotational rheometer (Malvern Instruments Ltd. England). Rheological measurements carried out using parallel-plate with 20 mm diameter and 0.3 mm gap. In order to prevent evaporation of moisture during the test, the sample was sealed around paraffin oil. The gelation conditions were the same as those previously described in Section 2.3. Heating temperature from 30℃ to 85℃ by 3℃/min constant heating rate and maintain 10 min. Oscillation frequency of 1 Hz, the stress amplitude of 0.122, which is the linear viscoelastic region, as measured by the initial trial. The parameter G' (storage modulus) and G" (loss modulus) were measured. Statistical analysis Each experiment was repeated three times. The data are the average of three times, standard deviation of the error term; Analysis of variance was used (p<0.05) and when the effect of the factors was significant. Data were statistically analyzed using SPSS13.0 software. The effects of the amount of TG on texture and water-holding capacity The gel strength increased significantly at TG dosage (0-20 U/g).The emulsion gel strength reached maximum with the amount of enzyme 20 U/g; over 20 U/g, the gel strength decreased with the amount of TG increasing (figure 1). Figure 2 showed that amount of TG was 0-15 U/g, the gel springiness significantly enhanced; and then decreased when the dosage was over 15 U/g. The α-lactalbumin structure has 8 glutamine residues and 12 lysine residues; while β-lactoglobulin has 16 glutamine residues and 15 lysine residues in its protein chain [16]. However, not all of these residues are available for enzymatic reaction with transglutaminase, due to the globular structure of whey proteins. The rate of crosslinking by transglutaminase is dependent on the macromolecular structure of each protein substrate; reactive glutamine residues, i.e., reside in flexible regions of the polypeptide chain or in regions with reverse turns [17]. Nieuwenhuizen et al. [18] investigated the accessibility of the lysine and glutamine residues of a-lactalbumin to the microbial transglutaminase reaction and showed that a maximum of five lysines and five glutamines can be modified by transglutaminase depending on the temperature, pH, and the presence or absence of calcium (Ca 2+ ). Transglutaminase concentrations above 20 U/g of protein may be higher than the quantities required for the reaction with the available residues of α-lactalbumin and β-lactoglobulin, saturation of the protein substrate may occur, without altering, even decreasing the hardness and springiness of the gel at transglutaminase concentrations higher than 20 U/g of protein, this probable due to excessive crosslinks. Similar results were observed by Han Cui-ping et al. [19]. The polymerization of whey proteins, the gel strength reached maximum at the amount of 10 U/g of TG, over 10 U/g the strength decreased. The water-holding rate was high at 94-98% at the of 5-30 U/g, Figure 3 showed when TG dosage was 0-5 U/g, the water-holding capacity of gel was significantly increased; subsequently slowly improved with 5-15U /g; enzyme dosage is 15 U/g, the WHC was maximum.As a consequence, when TG dosage was a range of 5-20 U/g, emulsion gel strength, springiness and WHC were at optimum values. The effects of pH on texture and WHC of emulsion gel Emulsion gel strength increased with increasing pH.The gel strength significantly increased at pH 5.0-7.5, and reached maximum when the pH is 7.5, whereas when the pH continues increasing, gel hardness declined ( figure 4).Tang Chuan-he [20] also confirmed the optimum pH of TG catalyzed β- lactoglobulin was 7.5, however the pH over 7.5, the catalysis of TG was weakened, TG was almost inactivation, when pH was over 8.5. Figure 5 described the effect of pH on gel springiness of TG-WPI emulsion gels. Like hardness, gel elasticity increases with increasing pH. Gel elasticity increases slowly at pH values 5.0-6.5, but at pH 6.5-7.5, the elasticity increases significantly and reached maximum at pH 7.5, the treatment of pH exceeded 7.5 was not found to significantly increase. As shown in figure 6, WHC of emulsion gel significantly increased at pH 5.0-7.0. When the pH was 7.0-8.0, the WHC was not significant increased. Overall, the emulsion gel strength, elasticity and water-holding capacity are at optimum values at pH 7.0-7.5. The effects of temperature on texture and WHC of emulsion gel Emulsion gel strength increased with increasing temperature. Specially at 35-45℃, the strength is significantly increased. Each of enzymes itself has optimal temperature and pH. The optimal temperature of TG is at the range of 45-55℃, when the temperature is higher 55℃, the TG is passivated even inactivated. Therefore, as the temperature continues to rise, the emulsion gel strength is slightly decreased (figure 7). As shown in figure 8, Emulsion gel elasticity increases with increasing temperature, the gel elasticity is significantly improved at 35-45℃; however, the elasticity has no significant change at 45-55℃; as the temperature continues to rise, the elastic gel is significantly reduced. The effect of temperature on WHC is the same like to hardness. When the temperature is 45℃, WHC of emulsion gel is maximum (figure 9). According to above analysis, the optimum values of gel strength, elasticity, WHC are at the range of 45-55℃. The effects of heating-time on texture and WHC of emulsion gel The emulsion gel strength showed no significant change as heating-time extended, gel strength was maximized when heated 3-4 h ( figure 10). As shown in figure 11, gel elasticity significantly increased when the reaction 2-3 h; at the time of 5-7 h, the emulsified gel elasticity decreased. As reaction time extended, the WHC increased firstly, then decreased, when the reaction was at 3-4 h, WHC reached the maximum ( figure 12).This could be related that with heating-time extending, whey protein occured so excessive crosslinks that it is not prone to aggregate and form gel. Another reason maybe the mechanism of TG catalysis, the TG enzyme can catalyze and decomposite glutamine at the present of H 2 O, thereby as the reaction extended, TG probably promotes reverse reaction and parts of aggregation are resolved. Result of orthogonal design According to single factor experimental results, L 9 (3 4 ) orthogonal test was carried out by optimum levels of the amount of enzyme added (A), pH (B), temperature (C), reaction time (h) in table 2 and table 3. The characteristics of gel were evaluated by lining-up score method to determine the optimum process conditions of the emulsion gel of whey protein isolate. Lining-up score method [21]: For the No. i (i = 1,2, ......, 9) test, Xmax represents the best, in the first place, marked 10 scores; Xmin means the worst, in the last, marked 1 score; for the remaining number of other indicators points, according to proportion of their degree of difference between the outstanding value of the index, namely: ΔX = (X max -X min ) / 9; A (score) = (X max -X i ); final score = 10-A; Then the sum of each number test score for all indicators was comprehensive scores in table 4. TG has the potential to improve gelation through the formation of covalent intra-and intermolecular bonds. Covalent bonds, such as disulfide and ε-(γ-Glu)-Lys bonds, can restrict the flow of protein chains, thereby enhancing the elasticity and hardness of the network [22]. Orthogonal test results in table 5 showed primary and secondary relationship of the four factors on the impact of the emulsion were A> B> D> C, that is the amount of TG impacts the most, and temperature the least. Optimal conditions established by the test results for the main parameters A 2 B 2 C 3 D 2 , namely transglutaminase catalyzed emulsion gel processing as follows: enzyme dosage 20 U/g, pH 7.5, temperature 50℃, reaction time 3 h. By variance analysis in table 6, the main factor was enzyme dosage, consistent with the range analysis in table 5. Be verified experimentally, the measured value of the gel strength of 211.927±2.433 g; gel elasticity was 2.018± 0.234, WHC was 98.357± 5.903%. 3.6. The microstructure of TG emulsion gels Electron micrographs were taken using SEM in order to get insight into the structure of emulsion gel. The optimum conditions were tested rather than the full experimental design. Scanning electron microscopy (SEM) showed that there were differences in the microstructure of the control and experimental gel (figures 13(a) and 13(b)). The differences were mainly associated with the compactness of the protein matrix and the size of the void spaces containing the aqueous portion of the gel. Compared to the control ( figure 13(a)), the network structure is more compact, led to emulsion gels with higher density and less porosity, more uniform and smoothly distribution, smaller particle size of droplets. Rheology Rheological properties of gels can be described by the storage modulus (G ') and loss modulus (G' '). When the G ' value is much greater than G' ' values, indicating the status of the sample exhibits solidlike behaviour with high elasticity; the performance characteristics similar to liquid (G' '>G '), on the contrary, has a high viscosity [23]. The control WPI emulsion gel ( figure 14(a)) the maximum G 'values and G "values were 12670.6 Pa and 7840.8 Pa. TG catalyzed WPI forming emulsion gel ( figure 14(b)), G ' value and G " values were increased by 334.85% and 338.81%, which implied that TG-WPI emulsion gels were more compact than WPI emulsion gels, this agreed with the micrographs (figure 13). The gel point was about at 70℃ (figure 14a), probable due to the denatured temperature of whey protein was over 65℃. Although not the true definition, the gel point represented quite accurately the transition point from what was perceived to be a liquid to a more solid-like material. Gel point temperatures were lower for samples that had more extensive cross-linking [24]. The control of G ' increased rapidly at 60℃( figure 14(b)), the gel point decreased by 10℃ compared to figure 14(a), which showed the gel point temperature was decreased, indicating that TG enhanced the ability of the emulsion solutions forming gel. Conclusion This study demonstrates that the optimum condition of forming emulsion gels catalyzed TG, enzyme dosage 20 U/g, pH 7.5, temperature 50℃, reaction time 3 h. Emulsion gels with high G ' and G " and good water-holding capacity were obtained. SEM show TG leads to changes in the structure of emulsion gels from a particulate to a mixed-type gel, where both fine-stranded and random aggregates are found. TG-WPI emulsion results in emulsion gels of unique properties that can be tailored to specific food applications.

v3-fos

2016-05-15T04:30:48.489Z

2015-03-01T00:00:00.000Z

17280498

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A Review of Recent Developments in Buffalo Reproduction — A Review The buffalo is an important livestock resource in several countries of South Asia and the Mediterranean regions. However, reproductive efficiency is compromised due to known problems of biological and management origins, such as lack of animal selection and poor nutrition. Under optimal conditions puberty is attained at 15 to 18 months in river buffalo, 21 to 24 months in swamp buffalo and is influenced by genotype, nutrition, management and climate. However, under field conditions these values deteriorate up to a significant extant. To improve reproductive efficiency, several protocols of oestrus and ovulation synchronization have been adopted from their use in commercial cattle production. These protocols yield encouraging pregnancy rates of (30% to 50%), which are comparable to those achieved in buffaloes bred at natural oestrus. The use of sexed semen in buffalo heifers also showed promising pregnancy rates (50%) when compared with conventional non-sexed semen. Assisted reproductive technologies have been transferred and adapted to buffalo but the efficiency of these technologies are low. However, these latest technologies offer the opportunity to accelerate the genetic gain in the buffalo industry after improving the technology and reducing its cost. Most buffaloes are kept under the small holder farming system in developing countries. Hence, future research should focus on simple, adoptable and impact- oriented approaches which identify the factors determining low fertility and oestrus behaviour in this species. Furthermore, role of kisspeptin needs to be explored in buffalo. INTRODUCTION Buffalo has a significant role in the agricultural economy of many developing countries by providing milk, meat and draught power. The world population of buffalo is estimated to be 199 million (FAOSTAT, 2012) with more than 96% of the population located in Asia including 16.4% of Pakistan's contribution. In recent decades, buffalo farming has expanded widely in the Mediterranean and Latin America as well as, in Central/Northern Europe where several herds were introduced. Dairy buffaloes have been used for milk production in India, Pakistan, some other South Asian countries, the Middle East and Italy; while dairy characteristics are being induced in the local population of Indo-Chinese Region and South America through crossbreeding with Pakistani Nili Ravi and Indian Murrah buffaloes. The milk yield increased from 700 to 2,000 kg/year in China through crossbreeding (Yang et al., 2007). The buffalos can utilize poorer quality roughages, adapt to harsher environments and are more resistant to several bovine tropical diseases. Despite these merits, buffalo have relatively poor reproductive efficiency irrespective of their location throughout the world. Buffalo exhibit many of the known reproductive disorders including delayed onset of puberty, poor oestrus expression, longer postpartum ovarian quiescence, and most importantly lowered conception rates particularly when bred artificially (Gordon, 1996). However, higher fertility could be achieved through better feeding and management (Perera et al., 1987;Usmani et al., 1990;Qureshi et al., 2007). It appears that because buffalo are located mostly in developing countries with meager resources, there is limited quality research in the area of basic physiology, health, management, nutrition and applied reproduction. The objective of this review is to examine the major recent developments in buffalo reproduction. We discuss the impact of the various techniques as well as bottlenecks and possible future developments which will lead to improve reproductive performance in this species. PUBERTY Buffalo usually attain puberty when they reach about 60% of their adult body weight (250 to 400) kg, but the age at which they attain puberty can be highly variable, ranging from 18 to 46 months (Jainudeen and Hafez, 1993). The factors that influence this are genotype, nutrition, management and climate. It could be attained under optimized conditions at 15 to 18 months in river buffalo and 21 to 24 months in swamp buffalo (Borghese, 2005). The delay in puberty, consequently delays conception and results in low reproductive efficiency and lengthening of the non-productive life. A major cause of delayed puberty may be poor feeding and management under field conditions. OESTROUS CYCLE In order to enhance reproductive efficiency of buffalo, a thorough understanding of the regulatory mechanisms involved in the oestrus cycle is required. The duration of the oestrous cycle in buffalo is similar to that in cattle, ranging from 17 to 26 days with a mean of around 21 days (Jainudeen and Hafez, 1993). However, there is a greater variability of the oestrous cycle length in buffalo compared to cattle, with a greater incidence of both abnormally short and long oestrous cycles. This may be attributed to various factors including adverse environmental conditions, nutrition and irregularities in secretion of ovarian steroid hormones (Kaur and Arora, 1982;Nanda et al., 2003). In buffaloes, ovarian follicular dynamics during the oestrous cycle is similar to that in cattle. Studies from India (Taneja et al., 1996), Brazil (Baruselli et al., 1997) and Pakistan (Warriach and Ahmad, 2007; Figure 1) have shown clearly that the majority of buffalo have two waves of follicular activity during their oestrous cycle. More investigations on the effect of follicle stimulating hormone and nutrition on number of follicular waves need to be studied in buffaloes. Studies on oestrous behavior and endocrinology in buffalo (Roy and Prakash, 2009;Singh et al., 2000) indicate considerable variations in reproductive endocrine activity without external signs of oestrus (silent heat) are common. The low intensity of oestrus in buffaloes may be due to low circulating concentrations of 17-β oestradiol in comparison with dairy cattle (Seren et al., 1995). Furthermore, tying up the animals as per normal husbandry practices in many developing countries restricts the ability of buffalo farmers to observe heat signs (Warriach et al., 2009). Buffalos also tend to show heat signs during the night when farmers are not observing their animals (Unpublished data). Season is another extrinsic factors that influences the characteristics of oestrous behaviour. In the tropics, high ambient temperature reduces sexual activity during the day (Jainudeen, 1977) and shortens the oestrous period (Gill et al., 1973) with the incidence of silent oestrous more common during the hot summer season. These adverse effects of heat stress make oestrous detection much more difficult in buffalo. Oestrous detection could be significantly improved through the introduction of a teaser bull or an androgenzied female (Chohan et al., 1992). The interval between standing oestrous and ovulation, which is very important for artificial insemination, was 30 hours in buffaloes (Warriach et al., 2008). Under field conditions, the am-pm rule of insemination originally developed for cattle (Trimberger, 1948) is generally followed in buffaloes. To follow this rule, the buffaloes should be bred 12 h after the detection of standing oestrus. However, onset of heat signs instead of onset of standing oestrus has been erroneously considered as the land mark with buffaloes often being inseminated, earlier than required. This early breeding is potentially responsible for lowered fertility, and can be explained by the fact, there is an interval of about 8 to 10 h between onset of heat signs and onset of standing oestrus. This indicates buffaloes should be inseminated 12 h after the detection of standing oestrus (detection by bull/teaser) or alternatively 18 to 24 h after the onset of heat signs. In order to confirm this approach, investigations are required on the timing of insemination in relation to standing oestrus and pregnancy rate. SYNCHRONIZATION OF OESTROUS CYCLE Various studies using protocols for synchronization based on progesterone and gonadotropin releasing hormone (GnRH) administration together with prostaglandin to induce luteolysis during breeding season have yielded quite promising conception rates ranging from 30% to 50% (Table 1). However, some buffaloes do not respond to treatment, especially during the low breeding season. There could be several reasons for this, but among the most likely is the animal's follicular status at the beginning of treatment. The ideal time of treatment can be established by determining ovarian activity by ultrasound (De Rensis and López-Gatius, 2007). The presence of the dominant follicle and an active corpus luteum (CL) indicate the success of synchronization. Protocols for buffaloes with limited follicular and luteal activity, remain to be refined, but they most likely will be devised around the strategic timing of administration of reagents currently used in synchronization protocols while ensuring that the supply of dietary energy and protein are not lacking. SEXED FROZEN SEMEN Semen sexing has been successfully used for producing living offspring in bovine species (Seidel et al., 1999). In buffalo, a difference in DNA content between X and Y sperm was found, and based on this difference it has been further demonstrated that processing buffalo semen was feasible (Lu et al., 2007). In a recent study, promising pregnancy rates (50%) were achieved when inseminating a dose of sexed semen containing 4 million spermatozoa (Gaviraghi et al., 2013). In order to expand use of this technology, there is a need to further refine this protocol for buffalo breeds of commercial significance such as the Nili-Ravi buffalo of Pakistan. ASSISTED REPRODUCTIVE TECHNOLOGIES The first successful embryo transfer in buffalo was performed in the United States of America (Drost et al., 1983). Subsequent successful transfers have been reported from many other countries. However, the success rate is much lower in buffaloes, due to their inherently low fertility and poor superovulatory response (Misra et al., 1990). The average yield of transferable embryos is less than one per superovulated donor. The buffalo ovary has a smaller population of recruitable follicles at any given time; an average of 12,000 primary follicles has been reported (Danell, 1987), compared to the average in the ovary of the cow, which has an average of 133,000 (Erickson, 1966). This technique comprises a series of carefully integrated sequential steps including donor selection, donor treatment, recipient selection, insemination of the donor, embryo recovery, embryo handling and evaluation, embryo transfer, and recipient care. The technology has to be refined to account for the lower, less responsive follicle population in the buffalo. Assisted reproductive technologies have been introduced to overcome the inherent reproductive problems, fast propagation of superior germplasm, and to reduce the generation intervals. These technologies provide an excellent source of embryos for carrying on basic research in developmental physiology, farm animal breeding, and for commercial application of the emerging bio techniques like cloning and transgenesis. During the past two decades, considerable advances have been made in our understanding of buffalo reproductive physiology, however, previous reviews (Palta and Chauhan, 1998;Gasparrini, Chohan, 1998 PGF2α (cloprostenol) 53 Rao andRao, 1983 PRID 41 2002) and more recent studies suggest that the rate of transferable embryo yield remains at a plateau (Manjunatha et al., 2009). Results have been quite variable between laboratories and are most likely related to differences in embryo physiology, metabolism, and culture requirements among buffalo breeds. Further studies are also needed to improve the cryopreservation of in vitro embryo production embryos. FUTURE RESEARCH The hypothalamo-pituitary-gonadal axis is the regulatory system for reproduction in mammals. A newly discovered neural peptide, kisspeptin, has opened a new era in reproductive neuroendocrinology. As shown in a variety of mammals, kisspeptin is a potent endogenous secretagogue of GnRH, and the kisspeptin neuronal system governs both the pulsatile GnRH secretion that drives folliculogenesis, spermatogenesis and steroidogenesis, and the GnRH surge that triggers ovulation in females (Okamura et al., 2013). Role of kisspeptin needs to be explored in buffalo. CONCLUSIONS Buffaloes are an important livestock resource for many countries. Most buffaloes are kept under the small holder farming system in developing countries. Future research should focus on simple, adoptable and impact oriented approaches which identify the factors limiting fertility and oestrus behaviour in this commercially significant species. Despite the inherited problems in buffalo slow progress has been made in the application of assisted reproductive techniques. Artificial insemination is practiced commercially; embryo transfer, in vitro embryo production, and nucleus transfer remain in the realm of experimentation. If their costs are reduced these latest techniques offer the opportunity to accelerate the genetic gain in the buffalo industry with the proviso that they are used in conjunction with efficient national progeny testing and sire evaluation programs.

v3-fos

2017-06-27T20:00:48.925Z

2015-11-24T00:00:00.000Z

7949118

s2

The opioid effects of gluten exorphins: asymptomatic celiac disease Gluten-containing cereals are a main food staple present in the daily human diet, including wheat, barley, and rye. Gluten intake is associated with the development of celiac disease (CD) and related disorders such as diabetes mellitus type I, depression, and schizophrenia. However, until now, there is no consent about the possible deleterious effects of gluten intake because of often failing symptoms even in persons with proven CD. Asymptomatic CD (ACD) is present in the majority of affected patients and is characterized by the absence of classical gluten-intolerance signs, such as diarrhea, bloating, and abdominal pain. Nevertheless, these individuals very often develop diseases that can be related with gluten intake. Gluten can be degraded into several morphine-like substances, named gluten exorphins. These compounds have proven opioid effects and could mask the deleterious effects of gluten protein on gastrointestinal lining and function. Here we describe a putative mechanism, explaining how gluten could “mask” its own toxicity by exorphins that are produced through gluten protein digestion. Background Gluten is the main structural protein complex of wheat consisting of glutenins and gliadins. Glutenins are polymers of individual proteins and are the fraction of wheat proteins that are soluble in dilute acids. Prolamins are the alcohol-soluble proteins of cereal grains that are specifically named gliadins in wheat [1], which can be further degraded to a collection of opioid-like polypeptides called exorphins in the gastrointestinal tract [2]. Gliadin epitopes from wheat gluten and related prolamins from other gluten-containing cereal grains, including rye and barley, can trigger celiac disease (CD) in genetically susceptible people [3], and accumulating data provide evidence for the deleterious effects of gluten intake on general human health. Nevertheless, until now, there is no consent about the possible detrimental health effects of gluten intake because of often failing gastrointestinal symptoms even in individuals with proven CD. By describing our "silent opioid hypothesis," we hope to shine light on this highly conflictive scientific item. Our review process and literature search was based on the use of the following key words: gluten, gliadin, celiac disease, asymptomatic celiac disease, gluten and transglutaminase, gluten and exorphins, gluten and intolerance, gluten and DPP IV, gluten and substance P, DPP IV, gluten and neoantigens, celiac disease and epidemiology, and gluten-free diet. Literature inclusion criteria included in vitro, in vivo, and human trial studies; indexed publications; full-text papers; and research methodology. Papers were excluded when not indexed and when methodology did not reach minimal criteria, and papers older than 2005 were excluded when more actual publications were available. Asymptomatic celiac disease CD normally presents itself with a number of typical signs and symptoms of malabsorption: diarrhea, muscle wasting, and weight loss. Other gastrointestinal (GI) symptoms like abdominal pain, bloating, and flatulence are also common. Curiously, a large group of patients that have been diagnosed with CD through screening for CD-specific antibodies and duodenal biopsy [3,4] lack these classical symptoms, a condition that is also referred to as "asymptomatic CD" (ACD). Many disorders are present in patients with ACD, including diabetes mellitus type I [5,6], severe hypoglycemia in diabetes mellitus type I [7], psoriasis [8], sleep apnea in children [9], neoplasia [10], atopic dermatitis [11], depression [8], subclinical synovitis in children [12], autism [13], schizophrenia [14], and irritable bowel syndrome (IBS) [8], suggesting that gluten intake is related to the development of these conditions. ACD is present in a large group of diagnosed celiac patients [15,16]. A study based on the data of the National Health and Nutrition Examination Survey showed that only 17 % of patients with serologically diagnosed CD suffer from the classical celiac symptoms [17]. A human study in 2089 elderly individuals looking for possible persistence of anti-gliadin antibody (AGA) positivity showed that 54 % of the AGA-positive patients suffered from intestinal inflammation, but only a small number of them complained about gastrointestinal symptoms [18]. The rate of elderly people suffering from mild inflammation in the gut mucosa and being AGAnegative is, according to a recent Swedish-populationbased study, only 3.8 % [19], again showing that gluten can cause inflammatory injury in the gut, without suffering any gastrointestinal symptoms. The presence of possible ACD is further recognized by the National Institute for Health and Care Excellence (UK) [20]. According to the guidance for CD screening issued in 2009, it is recommended to screen for CD when patients suffer from diabetes mellitus type I, IBS, thyroid hormone disturbances, Addison's disease, epilepsy, lymphoma, rickets, repetitive miscarriage, Sjögren's disease, and Turner disease. The following question arises: why do patients with ACD, with proven inflammatory signs, not suffer from pain, bloating, and other typical symptoms? Could it be that substances present in gluten with opioid effects mask the deleterious effects, functioning as masking compounds of gastrointestinal symptoms, converting the causal factor of CD, gluten, into a silent killer? CD is characterized by the presence of serum antibodies against tissue transglutaminase The most reliable way to diagnose CD is through small intestinal biopsy and measurement of the presence of serum antibodies against tissue transglutaminase (tTG), the main endomysial auto-antigen in CD [21][22][23]. Tissue TG deamidates glutamine residues from the gliadin peptide into glutamic acid, leading to enhanced immunogenicity of the resulting modified peptides. In addition, tTG can, in the absence of any other protein substrate, crosslink with gliadin, producing a tTG-gliadin complex, which can be considered a neo-antigen with possible immune toxicity [24,25]. Both symptomatic and asymptomatic CD are associated with certain immune systemrelated genetic polymorphisms, of which the HLA-DQ2 and HLA-DQ8 polymorphisms are expressed in the majority of CD patients [3]. However, many more genes, all related to a more pro-inflammatory activity of the immune system, are also linked with increased CD susceptibility [26]. A recent study by Sironi et al. [27] showed that several interleukin/interleukin receptor genes, involved in the pathogenesis of CD, have been subjected to pathogen-driven selective pressure. Particularly, CD alleles of IL18-RAP, IL18R1, IL23, IL18R1, and the intergenic region between IL2 and IL21 display higher frequencies in populations exposed to high microbial/viral loads, suggesting that these variants protected humans against pathogens. Since CD occurred after the increase of hygiene management and the incorporation of cereals into the human diet, it can be assumed that individuals bearing these genotypes are better protected against pathogens but at the same time are more susceptible for autoimmune diseases in general and CD specifically [28]. Although the above-described events explain the development of the typical inflammatory symptoms of CD and even the flattening of the gut lining through this immune response, they do not explain the phenomenon that many patients suffer from ACD at the level of the intestine and often, in parallel, suffer from extra-gastrointestinal disorders [8]. Gliadin is degraded to a collection of polypeptides called exorphins in the gastrointestinal tract Breakdown of gliadin from wheat is achieved through hydrolysation by intestinal pepsin, leucine aminopeptidase, and elastase, resulting in the release of immune-reactive and opioid-like peptides, including gliadinomorphin-7 (Tyr-Pro-Gln-Pro-Gln-Pro-Phe) from α-gliadin [2]. Further breakdown of these peptides, which are rich in proline, depends on the enzyme dipeptidyl peptidase IV (DPP IV), capable of cleaving N-terminal dipeptides with proline at the second (penultimate) position [29][30][31]. The remaining tripeptide (in the case of gliadinomorphin-7) with proline in the center is slowly hydrolyzed and acts as a selective competitive inhibitor for DPP IV [32][33][34]. Whereas total breakdown of gliadin into isolated amino acids prevents the presence of the gluten epitopes which are known to provoke a pro-inflammatory response of the immune system in genetically susceptible people [35,36], a possible DPP IV deficiency/inactivity could result in the incomplete breakdown of gluten, and thereby increase the presence of immune-reactive and opioid-like peptides, also known as gluten exorphins [36][37][38][39]. Gluten is not the only source of exorphins. Dairy products and certain vegetables such as soy and spinach also contain proteins, which can be converted in bioactive exorphins [40]. Gliadin from gluten and casein from dairy products show surprisingly high substrate specificity for DPP IV when compared with other endogenous DPP IV substrates. For example, DPP IV shows higher affinity for gliadin and casein than for substance P (SP) [41] and glucagon-like peptide (GLP) [42]. Gliadin is highly specific for DPP IV [36], which is further evidenced by its binding affinity with human DPP IV. By using an enzyme-linked immunosorbent assay, it was shown that binding of gliadin and casein to DPP IV inhibited DPP IV binding to anti-DPP IV by 52 and 44 %, respectively [43]. The fact that gliadin has a high affinity for DPP IV might explain why so many patients with proven CD are asymptomatic. Inhibition of DPP IV by gliadin can result in increased levels of non-metabolized gliadin molecules with opioid activity that can inhibit the typical abdominal pain associated with classical CD (Fig. 1). Opioid pathways could be responsible for the development of ACD It is surprising that a large group of patients, positive for the presence of CD antibodies and with proven histological CD, do not suffer from any gastrointestinal symptoms. If it is the opioid effects of gluten itself masking the classical symptoms of CD, then symptoms should be provoked when patients are given naloxone, a natural antagonist of morphine. Opioid effects on intestinal transit time Gastric emptying and intestinal transit are influenced by endogenous and exogenous opioid substances. It is known for long that morphine increases gastrointestinal transit time in humans and that this can be reversed by naloxone [44]. Early research showed that gluten exorphins induced a significant increase in transit time, and this effect was abolished when naloxone was administered [45]. A more recent study supports these early findings. In a single-center study, Urgesi et al. [46] observed that patients suffering from CD show a significantly longer small bowel transit time. In the discussion, the authors mention different pathways explaining their findings but do not mention the possible effect of opioids on intestinal transit time. Gluten-derived exorphins mimic endogenous opioid activity Stimulation of insulin production after meal intake is considered an endogenous opioid activity. Early research in rodents showed that oral administration of gluten exorphin A5 stimulated insulin production after food intake. The postprandial increase of insulin release by gluten exorphin was completely abolished by the opioid antagonist naloxone, implying that gluten exorphins maintain bioavailability for the peripheral nervous system within the gastrointestinal tract and pancreatic tissues [47]. Elevated circulating prolactin levels were Fig. 1 The development of symptomatic and asymptomatic CD and NCGS. Incomplete gluten breakdown results in inhibition of DPP IV and the possible increase of SP, leading to intestinal and extra-intestinal gluten-induced disorders. Gluten-derived DPP IV inhibition also increases the presence of GIP and GLP in the gut, leading to improved glucose homeostasis observed in individuals diagnosed with CD [48]. A short gluten-free diet period lowered prolactin levels in these patients, suggesting that gluten (or glutenderived substances), similar to endogenous opioids, directly affects prolactin secretion. This was further evidenced by Fanciulli et al. [49]. In rats, by using an opioid antagonist unable to cross the blood-brain barrier (naloxone methobromide), intracerebroventricular (ICV)-injected gluten exorphins stimulated prolactin release through activation of opioid receptors probably also outside the brain. Gluten exorphins influence behavior and pain perception A recent review of the literature concluded that foodderived exorphins are bioactive and affect behavioral traits such as spontaneous behavior, memory, and pain perception in rodents. The highest behavioral influence was measured for casein and spinach-derived exorphins (respectively, B-casomorphin and rubiscolin) [50]. Only one of the reviewed studies described the effects of gliadin exorphins in this context. Takahashi et al. [51] showed that ICV-administered gliadin exorphin A5 induced antinociceptive effects and orally delivered gliadin exorphin A5 modified learning and anxiety behavior during several laboratory stressors in mice, thus indicating that orally delivered exorphins can influence both the peripheral and central nervous system and suggesting that gluten exorphins possess opioid activity that could potentially mask symptoms in ACD patients. Besides explaining the lack of intestinal symptoms through gluten exorphin opioid activity in individuals suffering from ACD, DPP IV inhibition by gluten intake can have many other consequences on human health. DPP IV inhibition is known to have anti-diabetic effects but at the same time could be responsible for the presence of extra-intestinal symptoms and disorders in ACD and the occurrence of intestinal and extra-intestinal symptoms and disorders in CD and non-celiac gluten sensitivity (NCGS) patients (described below). DPP IV blockage by gliadin peptides improves glucose homeostasis Casein is not the only protein competing with gluten as a substrate for DPP IV (a nice overview of natural DPP IV substrates is provided by Gorrel et al. [52]). Other N-terminal dipeptides with proline at the second position, like the incretins, GLP and glucose-dependent insulinotropic polypeptide (GIP), both important regulators of glucose metabolism and essential to gut function, compete with gluten as substrates for DPP IV [52][53][54][55][56][57]. Carbohydrate intake increases the secretion of both incretins, which are normally rapidly broken down by DPP IV [58]. In a recent review [58], it is described how the inhibiting effect of gliadin on DPP IV increases the presence of GLP and GIP, by using whole wheat as a natural DPP IV inhibitor. DPP IV inhibition by gliadin could explain the "health promoting" effects of whole wheat intake, as the suppression of GLP and GIP breakdown has anti-diabetic effects [58] (Fig. 1). Contrasting data were found in a randomized, controlled, and openlabeled study [59]. Two days of DPP IV inhibition increased GLP and GIP levels but did not affect glucose values, transit time, or gastric emptying in healthy subjects, suggesting that short exposure to DPP IV inhibition does not affect any function related with DPP IV. The latter makes sense when observing the toxic effects of gluten as a natural DPP IV inhibitor. When patients suffering from NCGS followed a gluten-free diet and were re-challenged with gluten intake, it took approximately 7 days before new symptoms were provoked [60]. Longer use of gluten and synthetic DPP IV inhibitors have been shown to influence gastric emptying and transit time significantly. It is even so that deceleration and slowing of transit time are considered the most important mechanisms by which DPP IV inhibitors influence glucose homeostasis [56,61,62]. DPP IV blockage by gliadin peptides induces intestinal and extra-intestinal disorders Breakdown of SP is also dependent on DPP IV activity. SP has neurological, immunological, and endocrinological functions and influences pain sensitivity, gut peristaltic, inflammation, and social interaction [63]. Increased concentrations of SP in the gut can produce abdominal pain with diarrhea [64] and angioedema with swelling and abdominal pain [65]. High SP levels in the gut can even produce pancreatitis together with abdominal pain, diarrhea, and vomiting [66]. A recent doubleblind placebo-controlled human trial showed that the intake of a small amount of gluten (4.375 g/day for 1 week) significantly increased intestinal and extra-intestinal symptoms in individuals with self-reported gluten sensitivity [67]. Typical intestinal symptoms such as bloating and abdominal pain were increased after 1 week of gluten intake, as were extra-intestinal symptoms, such as depression and apthous stomatitis. Because in patients suffering from NCGS no known intestinal lesions or other biomarkers such as antibodies against gluten/gliadin/self-antigens seem to be present [68], their symptoms have to be explained by different pathways. NCGS presents itself with intestinal symptoms like diarrhea, abdominal discomfort, and flatulence, while headache, lethargy, attentiondeficit/hyperactivity disorder, ataxia, or oral ulceration appears as an extra-intestinal symptom [3,67]. Most, if not all, of these symptoms can be explained by increased levels of SP [64,69,70], suggesting that, in NCGS patients, gliadin blocks DPP IV activity and thereby inhibits SP breakdown. Thus, interventions targeting SP release in this group of patients could be a possible strategy to alleviate their intestinal and extra-intestinal symptoms [67] (Fig. 1). DPP IV inhibition increases the development of angioedema DPP IV inhibition by synthetic inhibitors, such as sitagliptin, is known to increase the possibility of developing angioedema [71]. CD produces the same symptoms as angioedema, and both disorders are so similar that, in general, it is advised to screen people with hereditary angioedema for CD [72]. Skin disorders, CD, and angioedema seem to be associated as seen in patients suffering from gluten-induced chronic urticaria [73] of which approximately 40 % also experience angioedema [74]. Even guidelines for the management of urticaria are similar as for angioedema [74,75], suggesting that both disorders have the same etiology, which could be CD and/or increased levels of SP through DPP IV inhibition. A study by Ramsay et al. [76] indicated that patients with gastrointestinal disorders (CD, morbus Crohn, colitis) suffer from mast-cell-induced inflammation. Interestingly enough, mast-cell inflammation can be induced by SP [77]. DPP IV inhibition by gluten would also explain the relationship between gluten intake and skin disorders [78,79]. Many skin diseases, including acne vulgaris, are associated with higher serum levels of SP [69], which can be induced by blockage of DPP IV [65]. Another gluten intake-related disorder, major depression, is also related with DPP IV inhibition; low serum DPP IV is an important marker for depression [80], and gluten could function as an inhibitor of DPP IV. Gliadin peptides can cause anatomical changes at the level of the brain Recent research in humans has shown that gluten intake can even cause anatomical changes at brain level, although neurological symptoms are absent. Anatomical MRI shows silent neurological changes, including bilateral decrease in cortical gray matter and caudate nuclei volumes in celiac patients compared to controls [81]. Negative correlations were found between the duration of the disease and the volumes of the affected regions. Similar neurological changes were observed in a retrospective examination of the brain by MRI of patients suffering from biopsy-proven CD who were referred for a neurological opinion by their gastroenterologist [82]. Patients were divided into subgroups based on their primary neurological complaint (balance disturbance, headache, and sensory loss). The outcome was that CD patients suffer from a significant loss of cerebellar volume compared with healthy controls. Affected area were brain regions below and above the tentorium cerebelli. These changes were the highest in the headache subgroup and unexpected for the patient's age. The headache group had an average loss of white matter in these regions two times more than the subgroup with balance disturbance and six times more than the subgroup suffering from sensory loss. One possible explanation for the loss of white matter in people suffering from CD is the presence of gluten-induced autoimmune vasculitis [83]. Another more recent hypothesis explaining the loss of white matter caused by reactions against gluten is related with the complementary immune system. The complement protein C1Q is known to bind and help eliminate complexes of immune globulins bound to antigens [84]. C1Q further is considered a "punishment factor" of the central neurological system, produced by strong synapsis and marking weak synapsis for possible phagocytosis by neighboring glia cells [85]. The process of synapsis breakdown should be considered normal in early life with the purpose of remodeling the central nervous system during neurological development [86]. Increased expression of C1Q has been observed in patients suffering from Alzheimer [87], autism [88], and schizophrenia [89]. Severance et al. [84] showed that C1Q binds preferentially to immune globulins coupled with casein and gluten antigens. Their results suggest that the increased expression of C1Q increased synaptic breakdown and could be responsible for schizophrenia onset. We speculate that the increased presence of gliadin peptides, induced by DPP IV inhibition, could be responsible for stimulating C1Q expression and, thereby increases disease susceptibility for these neurodevelopmental and neurodegenerative disorders. Conclusions The precise pathway leading to the development of ACD still needs to be discovered. However, the putative mechanism presented in this review could explain this intruding phenomenon. The incomplete breakdown of the gluten protein, resulting in the presence of gliadin peptides with opioid effects, makes it plausible to suggest that the opioid effects of gluten exorphins could be responsible for the absence of classical gastrointestinal symptoms of individuals suffering from gluten-intake-associated diseases. Moreover, the partial digestion of gluten, leading to DPP IV inhibition, could also account for the presence of extraintestinal symptoms and disorders in ACD and the occurrence of intestinal and extra-intestinal symptoms and disorders in CD and NCGS patients. If so, then individuals suffering from any of these conditions should be recognized in time and engage in a gluten-free lifestyle to prevent gluten-induced symptoms and disorders.

v3-fos

2016-05-12T22:15:10.714Z

2015-04-30T00:00:00.000Z

13938978

s2

Ensemble Learning for Spatial Interpolation of Soil Potassium Content Based on Environmental Information One important method to obtain the continuous surfaces of soil properties from point samples is spatial interpolation. In this paper, we propose a method that combines ensemble learning with ancillary environmental information for improved interpolation of soil properties (hereafter, EL-SP). First, we calculated the trend value for soil potassium contents at the Qinghai Lake region in China based on measured values. Then, based on soil types, geology types, land use types, and slope data, the remaining residual was simulated with the ensemble learning model. Next, the EL-SP method was applied to interpolate soil potassium contents at the study site. To evaluate the utility of the EL-SP method, we compared its performance with other interpolation methods including universal kriging, inverse distance weighting, ordinary kriging, and ordinary kriging combined geographic information. Results show that EL-SP had a lower mean absolute error and root mean square error than the data produced by the other models tested in this paper. Notably, the EL-SP maps can describe more locally detailed information and more accurate spatial patterns for soil potassium content than the other methods because of the combined use of different types of environmental information; these maps are capable of showing abrupt boundary information for soil potassium content. Furthermore, the EL-SP method not only reduces prediction errors, but it also compliments other environmental information, which makes the spatial interpolation of soil potassium content more reasonable and useful. Introduction High quality soil property maps based on spatial patterns of soil variability are needed for agricultural planning, risk assessments, and decision making in regards to environmental management and conservation. However, such maps are usually not readily available and they are often difficult and expensive to acquire, especially for mountainous and high altitude regions. Furthermore, sampling points for soil potassium content are typically sparse and the available data may be insufficient to characterize the highly variable soil potassium content and its spatial patterns. Therefore, it would be worthwhile to develop methods that can estimate soil potassium content in areas where soil potassium content has not been measured. Spatial interpolation, which is method that can be used to construct continuous data from variables measured at point locations, is a promising technique for soil potassium content studies [1]. Three main limitations are typically encountered when using spatial interpolation techniques to estimate soil properties. These limitations include small numbers of available soil samples, nonlinearity of the relationships between environmental variables and soil properties, and the optimal spatial interpolation model for a given study region is often not known. When sampling points are sparse or poorly correlated in space, it can be difficult to interpolate the distributions of soil properties accurately without secondary environmental variables [2][3][4][5]. Some studies have indicated that important relationships exist between soil properties and different types of geographical information, including terrain attributes derived from digital elevation models (DEM) [6][7][8][9], and the distributions of land use types, rock types, soil types, etc., [10], and other soil attributes related to target variables [11]. Many interpolation methods can be used to process soil potassium content data. Non-geostatistical methods such as inverse distance weighting (IDW) with an interpolator assume that each input point has a local influence that diminishes with distance and no additional assumptions are required for the data [12]. The IDW technique is commonly applied because of its relative simplicity and availability. However, predictions from IDW are usually associated with large errors. Geostatistical methods like kriging assume that the distances and directions between sample points contain spatially correlated data that can be used to explain variation in the surface [13]. The kriging interpolation model provides the best linear unbiased estimates, accurate descriptions of the spatial structure of data, and valuable information about the estimation error distributions [14]; while it is usually better than IDW, kriging is based on a model whose assumptions (e.g., stationary hypothesis) may not be met in practice. The interpolation methods discussed so far often need data that meet certain conditions or are parameter-specific, and the performances of the methods can be influenced by many factors [4]. These influencing factors are problematic in that no consistent findings have been obtained on them to date, which makes it challenging to select an optimal method for spatial interpolation. Therefore, it is often difficult to select an appropriate spatial interpolation method for a given study area. In recent years, some machine learning methods have been applied to the fields of data mining and spatial interpolation, and these studies have demonstrated the utility of such methods in terms of data accuracy and processing efficiency. e.g., random forest(RF), support vector machine (SVM), and some other learning methods [8,[15][16][17][18][19][20][21][22][23][24][25]. Furthermore, SVM has been applied to rainfall data in a study by Gilardi [26]. Notably, ensemble learning has not been applied yet to the spatial interpolation of soil properties. The specific objectives of this study are (1) to describe spatial distributions of soil properties accurately based on soil types, geology types, land use types, and slope by applying ensemble learning with ancillary environmental information for improved interpolation of soil properties (hereafter, EL-SP) and (2) to compare the performance of the EL-SP method with IDW, universal kriging (UK), ordinary kriging (OK), and a method that combines OK with different types of geographic information. To accomplish these objectives, we applied the EL-SP method along with the other interpolation methods to soil potassium content data we collected from around Qinghai Lake in China during September 2013. The prediction patterns of the interpolation methods were analyzed based on their prediction maps. EL-SP As a modified versions of regression kriging [9,27], each observation z(xi,yj) of one specific soil potassium content at grid (i,j) can be expressed as: where t(xi,yj) is the trend value of z(xi,yj) in the grid (i,j) and r(xi,l,k,yj,l,k) is the residual in the lth type of kth environmental information computed by subtracting the trend value of t(xi,yj) from the measured value of soil potassium content. We assumed that t(xi,yj) and r(xi,l,k,yj,l,k) are independent of each other and the variation of r(xi,l,k,yj,l,k) is homogeneous over the overall study area. The residuals of the relevant types of environmental information were then used to interpolate the surface of residuals in the whole study area by ensemble learning. The interpolated values of residuals were finally summed to the soil potassium content trend as the final interpolated values of EL-SP interpolation. The framework of EL-SP (Fig 1), and the processes used were as follows. 1. With the measured soil potassium content values, we calculated the trend and residuals of soil potassium content. 2. According to the spatial distribution of the soil potassium content trend value, we mapped the overall distribution of soil potassium content t (xi,yj) for each grid (i,j). 3. According to the related residuals, we used OK combined with soil type (OK-Soil), OK combined with geology type (OK-Geology), OK combined with land use type (OK-Landuse), and OK combined with slope type (OK-Slope) for the remaining residual interpolation. 4. Ensemble learning was then used to integrate all the residual surfaces (OK-Soil, OK-Geology, OK-Landuse, and OK-Slope) to obtain the soil potassium content r(xi,l,k,yj,l,k). 5. Finally, we added up the trend surface and residual surface for the EL-SP simulation results. Typically, an ensemble is constructed in two steps. First, a number of base learners are produced (e.g., OK-Landuse, OK-Soil, OK-Geology, and OK-Slope). Then, the base learners are combined for use, where the most popular combination schemes are used in majority voting for classification and weighted averaging for regressions. The pseudo-code of ensemble learning is as below: is the interpolation model set (e.g., OK-Landuse, OK-Soil and OK-Geology) Base learner h; Number of learning rounds n. Process: for t = 1,. . .,n; //Measure the interpolation error of h t The final interpolation function: Parameter specification and secondary variable selection The specification of parameters was based on the requirements of the interpolation methods and data characteristics. The interpolation methods were performed using the geostatistics analysis and 3D analyst modules of ArcGIS 10.1. Different parameters for IDW, UK, OK, and OK-Geo (OK with combined geographic information) were compared, and the optimal parameters with the smallest root mean square error (RMSE) values were determined. For the OK and its combined methods, the spherical, exponential, Gaussian, and linear models were fitted to the experimental variogram, and the number of the closest samples chosen varied from 5 to 30 with five-step intervals. The IDW was estimated with powers of 1, 2, 3, and 4. In order to analyze which secondary variables were significant for soil potassium content, the analysis of variance (ANOVA) procedure was used to test for the significance of geographic type effects on the variances of soil potassium content (Table 1). Table 1 shows that land use types, soil types, geology types, and slopes were the four most strongly correlated variables with soil potassium content based on the results of the ANOVA analysis; these variables were all significant at the 0.01 level, and they were used as secondary variables in EL-SP and OK-Geo. The OK and IDW methods do not need secondary variables. Study area The study area (36°32 0 56@-37°48 0 40@N, 99°51 0 29@-101°12 0 82@E) is located in the northwest region of the Qinghai Province in China (Fig 2A). This region covers an area of 8753.73 km 2 , and a large portion of this area is covered with water (4473.96 km 2 ). The region comprises three geomorphic counties including Gangcha, Haiyan, and Gonghe counties. The study area is part of the Qinghai Lake basin, and the elevation is varies from 2008 to 4616 m. According to 1:1000, 000 scale soil maps reported by the National Soil Census Office, there are 9 soil types in this region, and these soil types include alpine meadow soil, semi-fixed sandy soil, meadow marsh soil, alpine shrub meadow soil, chestnut soil, and leached chernozem soil, etc. (Fig 2B). According to the 1:500,000 geologic map of the Qinghai Province from the Qinghai Provincial Bureau of Geology and Mineral Resources, the regional geology types include valley plain, alluvial terrace, lake beach, and denudation high terrace, etc. (Fig 2C). Regional land use types were classified into water body, croplands, grasslands, swamp meadow land, shrub land, and unused lands, etc. ( Fig 2D). According to the correlation between slope and soil potassium content, the slopes were classified into five groups including 0°-5°, 5°-8°, 8°-15°, 15-25°, and >25° (Fig 2A). Dataset We collected a total of 193 topsoil samples (0-30 cm) from the Qinghai Lake region in September 2013(S1 Dataset). The sampling activities were approved orally by members of the Environmental Monitoring Center of the Qinghai Province, who were project participants. At the sampling sites, we recorded the locations of the soil samples, the elevation, soil types, geology types, and land use types. Each sample was air-dried and passed through a 2 mm sieve prior to determining the soil potassium contents used in this study. A large amount of environmental information can be used as secondary variables to improve the performance of spatial interpolation methods as discussed by Li and Heap [4] and Shi et al [5]. After preliminary analyses, soil types, geology types, land use types, and slope data were considered as important secondary information in this study. Soil types, geology types, and land use types have been used in earlier work to improve the performance of spatial interpolators of soil properties [5], so the inclusion of this type of environmental information was expected to improve the prediction accuracy. Slopes are likely to have some influence on the transfer of soil potassium content from high slope regions to low slope regions, so slope was also considered as an important secondary variable in this study with the potential to improve the overall prediction accuracy. Since the relationships between the soil potassium content and the secondary information variables were nonlinear, ANOVA analysis was used measure the correlations: the soil potassium content displayed a significant correlation with land use types (f = 7.342, p-value = 0.000), soil types (f = 6.781, p-value = 0.000), geology types (f = 7.512, p-value = 0.000), and slope types (f = 5.250, p-value = 0.000). The dataset of environmental information was generated in ArcGIS 10.1, and where necessary, the data were resampled to a 30 m resolution. However, the limited soil potassium content sample sizes and uneven distribution of sample points meant that there were some geographic areas without enough sample data for modeling; hence, we tried to use the environmental variables to improve the spatial interpolation accuracy in such areas. The soil potassium content samples were divided into two groups. One was the training subset with 150 samples, and the other was the test subset with 43 samples. We used the training subset to estimate values of the test subset, and then, we compared the predicted and measured values for every data point of the test subset. The mean error (ME), mean absolute error (MAE), and RMSE were calculated using the predicted and measured values at each validation sample site for the test subset. Accuracy of EL-SP In order to assess the accuracy of EL-SP for interpolating soil potassium contents, we compared the performance of the proposed EL-SP method to the UK, OK, IDW, and OK-Geo techniques. The ME, MAE, and RMSE values, which were calculated using the predicted and measured values, are shown in Table 2; these values reflect the interpolation quality of the different methods. We found that the interpolators that were combined with environmental information, i.e., EL-SP and OK-Geo, were the most accurate methods. Additionally, the OK and IDW methods outperformed the UK method. The combination of environmental information with OK considerably improved the prediction accuracy, although the resulting values were less accurate than those from EL-SP. The higher accuracy of EL-SP is thought to be related to its good ability to discern boundaries among the different geographic features, which in turn led to more accurate descriptions of the spatial variation characteristics of soil potassium content in different geographic types. Maps of EL-SP The spatial predictions of soil potassium content for the five methods (i.e., UK, OK, IDW, OK-Geo, and EL-SP) are illustrated (Fig 3). The spatial distribution patterns of the two most accurate methods (i.e., EL-SP and OK-Geo) were similar and they captured the major spatial distribution patterns and trends of soil potassium content; but weak "bull's eyes" patterns were evident with OK-Geo. The ranges of results were somewhat narrower in the predictions. The predictions of UK ( Fig 3A) produced a map with linear tracks, sharp transitions, and banding patterns. The predictions of OK ( Fig 3B) produced a map similar to that of OK-Geo and EL-SP, but with the sharp transitions and evident "bull's eyes" patterns. The predictions of IDW (Fig 3C) reproduced the major patterns, but it failed to predict changes in local variation and displayed strong "bull's eyes" patterns at sample points with either high or low values. In OK-Geo ( Fig 3D) and EL-SP (Fig 3E), the combined environmental information helped to eliminate the linear tracks, sharp transitions, and banding pattern effects that were apparent in the OK and UK maps. Overall, these results suggest that interpolators that combine data with additional environmental information can describe the local variation more accurately. Additionally, such techniques show great promise for improving the interpolation performance in difficult to study regions. Although the proposed EL-SP method was found to produce more accurate results than the other traditional spatial interpolation methods that were tested, the search for optimized statistical spatial interpolation methods is still in its early stages. Other combined approaches that use machine learning methods with existing spatial interpolation techniques may also yield valuable results. In particular, learning methods such as distribution learning, Hausdorff distance learning, and visual-textual joint relevance learning, etc. [18][19][20][21][22][23]25], which have shown good performance in the fields of data mining and image retrieval, may have great potential when used in spatial interpolation applications, especially in combination with other methods. Thus, future studies should aim to investigate the potential of other combination techniques and test their performances under different field scenarios. Conclusions Large spatial variations in soil potassium content within different geographic regions can make it difficult to assess soil properties accurately when there are few data points available. We proposed the use of an ensemble learning model for soil potassium content interpolation, and the method uses valuable information from secondary environment variables for making predictions. Compared with more traditional UK, OK, IDW and OK-Geo interpolation techniques, the proposed EL-SP method not only reduced prediction errors at the study site, but it also produced spatial interpolation maps of soil potassium content that were more reasonable. Kriging interpolation models usually perform better than IDW and are excellent at least in theoretical analyses [4]. However, in this study the kriging interpolation accuracy was similar (e.g., OK) or worse (e.g., UK) than that of the the IDW method. Other research has found that IDW can be perform better than kriging models when data are isotropic and there are no correlations between the primary variables and the secondary variables [28]. However, the correlations between the primary variables and secondary variables were strong in this study, which suggests that kriging may not always be the optimal model to use for spatial interpolation under a wider range of conditions than previously suspected. In this study, the interpolation accuracy of the methods that used secondary environmental variables (e.g., OK-Geo and EL-SP) was better than that of the methods that did not use such ancillary information (e.g., UK, IDW, and OK). Thus, interpolation models when combined with appropriate secondary environment variables can effectively improve the simulation accuracy. limitation of EL-SP is that it has a smoothing effect and that the surface variation is smaller than the ensemble object values. If the ensemble results for an object are smaller than the measured value, the EL-SP results will be lower than the measured value. The current research method used 'tandem' ensemble interpolation models, incorporating a global interpolation model for the entire study area, although a simple global model cannot explain the spatial instability of soil properties. In future, we will use 'parallel' ensemble interpolation models, based on the different regional characteristics of the study area and with consideration of the problems of simulation scale, to select the appropriate interpolation model integration.

v3-fos

2016-06-02T01:40:34.545Z

2015-09-06T00:00:00.000Z

18948278

s2

Rapid detection of contagious ecthyma by loop-mediated isothermal amplification and epidemiology in Jilin Province China The aim of this experiment was to develop a loop-mediated isothermal amplification (LAMP) assay and to research the recent epidemiology of contagious ecthyma in Jilin Province, China, using the assay. A LAMP assay targeting a highly conserved region of the F1L gene was developed to detect contagious ecthyma virus (CEV). Three hundred and sixty-five cases from 64 flocks in 9 different areas of Jilin Province, China, from 2011 to 2014 were tested using the LAMP assay. The results showed that the sensitivity of the LAMP assay was 100 copies of the standard plasmid, which is 100-fold higher than the sensitivity of PCR. No cross-reactivity was observed with capripoxvirus, fowlpox virus, foot-and-mouth disease virus serotype O, foot-and-mouth disease virus serotype Asia I and bluetongue virus. The average positive rate was 19.73% (72/365), and the positive rate was highest in lambs aged 1–6 months. Our results demonstrated that CEV infection was very widespread in the flocks of Jilin Province and that the LAMP assay allows for easy, rapid, accurate and sensitive detection of CEV infection. Contagious ecthyma (CE), also known as Orf, is a debilitating disease of sheep and goats caused by the contagious ecthyma virus (CEV). CEV is a dsDNA virus belonging to the Parapoxvirus genus of the Chordopoxvirinae subfamily and Poxviridae family. CEV is the cause of a papular dermatitis in sheep and goats known as CE and is zoonotic, affecting humans [10]. The disease is characterized by inflammatory, proliferative and scabby lesions on the lips, nostrils and muzzle, and its frequency of occurrence and severity are particularly high in lambs. CE has a worldwide distribution, and it affects the majority of small ruminants and some wild animals. In an outbreak, up to 10% mortality in lambs and 93% mortality in kids have been recorded [7]. This disease impacts the economic well-being of farmers and causes economic losses. Loop-mediated isothermal amplification (LAMP) is a highly specific, efficient and rapid nucleic acid amplification method that amplifies DNA under isothermal conditions [11]. Gene amplification products form a ladderlike pattern on an agarose gel and show a visual color change when the fluorescent dsDNA intercalating dye SYBR Green I is used. LAMP may be used more easily and rapidly than PCR in clinical medicine and has been successfully developed to diagnose many diseases [1,6,8,9,13,17]. The outer primers (5′ GACCCCGAGCTCATGGT 3′ for F1L-F3 and 5′ GCCGCGTCTTCACCTGTA 3′ for F1L-B3), inner primers (5′ CCTCCTTGATGATCGC-GTCGTGACGTCTCGCTAGACGCCTA 3′ for F1L-FIP and 5′ GAGGTGTTCACGCTGGAGAAGCAGTACTC-GGGGTAGACCAC 3′ for F1L-BIP) and the loop primer (5′ GAGCTTCTTCATGCCGCC 3′) of the F1L gene were designed using the PrimerExplorer 4.0 software based on a conserved region of the F1L gene identified by sequence alignment. The primers were synthesized by Takara. Viral genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. A standard plasmid, pGEM-Teasy-F1L, was constructed by insertion of a F1L gene fragment generated using the F1L-F3 and F1L-B3 primers into the pGEM-T-easy plasmid (Promega, Madison, WI, U.S.A.). The sequence of the pGEM-T-easy-F1L plasmid was verified by sequencing at SinoGenoMax. The standard plasmid DNA was extracted using an AxyPrep Plasmid Miniprep Kit (Axygen BioScience, Union City, CA, U.S.A.). The concentration of plasmid was measured using an Epoch Multi-Volume Spectrophotometer System (BioTek, Winooski, VT, U.S.A.), and the copy number was then calculated. The LAMP assay was performed in a reaction mixture of The specificity of the LAMP assay was determined by applying the DNA from CEV, capripoxvirus (CPV), fowlpox virus (FPV), foot-and-mouth disease virus serotype O (FM-DV-O), foot-and-mouth disease virus serotype Asia I (FM-DV-Asia I) and bluetongue virus (BTV). These viruses were maintained in our laboratory. To evaluate the sensitivity of the LAMP assay, the CEV standard plasmid (1-10 8 copies) was used as template in PCR reactions. The PCRs were performed in a 25 µl reaction mixture containing 1×PCR buffer, 0.2 mM each dNTP, 0.2 µM F1L-F3 and F1L-B3 primers, 1 µl extracted DNA and 0.1 U Taq DNA polymerase (Takara, Otsu, Japan). The PCR reactions were performed using the following protocol: 95°C for 10 min; 35 cycles denaturation at 94°C for 30 sec, 30 sec of annealing at 55°C and 30 sec of primer extension at 72°C; and then a final extension at 72°C for 10 min. The LAMP products were visualized with the naked eye after the addition of SYBR Green I. Both the LAMP and PCR products were separated on 2% agarose gel and visualized on a UV transilluminator. Positive results were obtained for the detection of CEV genome DNA and the standard plasmid with the LAMP method established in this study. After the LAMP reaction was completed, we observed a color change from orange to green with a positive LAMP reaction with the addition of SYBR Green I (1000×, 1 µl, Invitrogen, Thermo Fisher Scientific) to the reaction mixture (Fig. 1A). This color change was particularly significant against a black background. The positive LAMP reaction generated a laddering pattern with a set of different sized bands consisting of several invertedrepeat structures, as determined by 2% agarose gel electrophoresis (Fig. 1B). Positive amplification was detected in 45 min with both genomic DNA and the standard plasmid when the reaction temperature was higher than 61°C. The gel electrophoresis bands were clearly visible when the reaction temperature was higher than 63°C, and no difference was observed when the reaction temperature varied from 63 to 69°C (Fig. 2A). The results indicate that the Mg 2+ concentration must be at least 6 mM to yield a positive reaction, and no additional change was observed up to 14 mM Mg 2+ (Fig. 2B). Positive reactions were obtained using ratios of outer, loop and inner primers ranging from 1:2:4 to 1:2:14, and no significant difference was observed ( Fig. 2C and 2D). Positive amplification was detected within as little as 30 min, and product formation reached the maximal level after 45 min (Fig. 2E). The good conditions for the LAMP assay were determined to be 65°C for 45 min with 8.0 mM MgSO 4 , 0.8 M betaine, 1.4 mM dNTPs, 0.2 µM each outer primer, 0.8 µM each inner primer, 0.4 µM loop primer and 8 U Bst polymerase. All viruses were detected with this LAMP assay, and only DNA from the CEV was amplified; the results confirmed the specificity of this assay. Both SYBR Green I staining and agarose gel electrophoresis yielded the same result. The detection limits for the LAMP assay and PCR were 10 2 and 10 4 copies of the standard plasmid, respectively. No amplified products were detected in the negative controls. Therefore, the sensitivity of the LAMP was 100-fold higher than that of PCR (Fig. 3). The fluorescent green and orange colored products were visualized after SYBR Green I staining, and the sensitivities were consistent with the gel electrophoresis results. The results showed that the LAMP assay has the potential to replace PCR because of its simplicity, rapidity, specificity, sensitivity and cost-effectiveness without the need for specialized equipment. Jilin Province is the main sheep-breeding region in China, and CEV has been reported in the literature in China [3,5,18]. CE is an important zoonotic disease, and detailed and updated information on epidemiology in sheep would not only help in updating the knowledge of the scientific community but also would be useful for policy makers in formulating appropriate measures for control and eradication of the disease. Here, three hundred and 65 cases (including the skins of the back, udder, limbs and tail) from 64 flocks in 9 different areas of Jilin Province, China, from 2011 to 2014 were tested using the LAMP assay. The results showed that the average positive rate was 19.73% (72/365), and the positive rate was highest in lambs aged 1-6 months ( Table 1). Conventional PCR assays were also used to evaluate the three hundred and 65 cases, and the positive rate for conventional PCR was 19.18% (70/365). The results showed that the LAMP assay is more sensitive than PCR for the detection of clinical samples. The CEV antibodies of 2104 serum samples collected from the flocks mentioned above were examined by indirect ELISA. The results showed that 812 serum samples were positive, and the average positive rate was 38.59% (812/2104). The epidemiology of CEV has not been reported in the literature in Jilin Province, China, and our results demonstrated that CEV infection was very widespread in the flocks of Jilin Province. According to the sites of pathological changes, the disease can be divided into the lip type, breast type, vulva type and mixed type, and among these types, the lip type was most common. None of the animals had been inoculated with a CEV vaccine, so the feeding density and differences in environment were considered to be the main causes of infection. Currently, PCR is an important technique for disease diagnosis, A conventional PCR assay based on amplification of part of the CEV gene (B2L) has been developed [14]. Moreover, Real-time PCR has also proved to be accurate and effective in the quantification of Orf viral DNA [2,4,12], However, these assays require complex operations and high-precision instruments. LAMP is especially useful in resource-limited situations. Reliable detection of CEV is fundamental for the exclusion of other rash-causing illnesses (e.g, capripox, foot-and-mouth disease, staphylococcal infection and bluetongue). To date, a LAMP assay based B2L for the detection of CEV has been reported [15]. In this study, the sensitivity of the LAMP assay was 100 copies, which was less sensitive than assays previously reported [14,16]; however, the assay in this study can be completed within 45 min, which can save 15 min of time and the established LAMP assay in this study had good specificity.

v3-fos

2018-04-03T04:18:19.161Z

2015-12-15T00:00:00.000Z

15189948

s2

Ramie Leaf Extracts Suppresses Adipogenic Differentiation in 3T3-L1 Cells and Pig Preadipocytes The present study was carried out to evaluate the anti-obesity effect of different concentrations of extracts of hot air-dried ramie leaf (HR) and freeze-dried ramie leaf (FR) in 3T3-L1 cells and pig preadipocytes. To analyze the effect on cell proliferation, cells were treated with 25 μg/mL or 100 μg/mL HR or FR extract for 2 days. Cell differentiation was evaluated by measuring glycerol-3-phosphate dehydrogenase and lipoprotein lipase (LPL) activities and intracellular triglyceride content. Treatment with either HR or FR extracts inhibited the proliferation of 3T3-L1 cells and pig preadipocytes in a dose-dependent manner. HR extract treatment inhibited the differentiation of both cell types more effectively than FR treatment. The extent of triglyceride accumulation decreased significantly in both cells following either HR or FR treatment. Furthermore, LPL activity significantly decreased after treatment with HR or FR extract. These results indicated that HR and FR extracts may inhibit proliferation and differentiation of 3T3-L1 cells and pig preadipocytes. Further studies are needed to explore the anti-obesity effect of HR and FR extracts. INTRODUCTION Obesity is considered a public health problem and is caused when energy intake exceeds energy expenditure. Obesity is closely linked to metabolic diseases such as diabetes, hypertension, heart disease, and cancer (Cao, 2007). Adipogenesis is a sequence of events that includes adipose tissue proliferation and differentiation, and lipid accumulation (Fernyhough et al., 2007). Obesity causes normal or excessive fat accumulation and adipocyte differentiation (González-Castejón et al., 2011). Many studies have shown that plant-derived foods have the potential to reduce obesity (Boeing et al., 2012). Furthermore, understanding the anti-obesity mechanism of plant-derived products will help prevent obesity. In vitro and in vivo evidence suggests that natural compounds inhibit obesity by inhibiting adipocyte differentiation and altering lipid metabolism (Yun, 2010). Ramie (Boehmeria nivea L.) belongs to the family Urticales and is widely cultivated in Asian countries such as Korea, India, and China. Ramie is used as a traditional herbal medicine and has been shown to have antioxidant, anti-hepatitis B virus, anti-inflammatory, and antifungal effects (Lee et al., 2009;Xu et al., 2011;Wei et al., 2014). Ramie is a rich source of amino acids, vitamins, minerals, dietary fibers, and flavonoids (Lee et al., 2009). Recent studies have shown that the physiochemical composition and the antioxidant properties of ramie vary between hotair-dried ramie leaf (HR) and freeze-dried ramie leaf (FR) (Kim et al., 2014a, b). However, the effect of preparation method on its anti-adipogenic effect has not yet been evaluated. In the present study, we used 3T3-L1 cells, clonal line of preadipocyte that was capable of differentiating adipocyte and porcine preadipocytes (Green and Kehinde, 1975). Pig has been used as an ideal model due to its similar metabolic features, cardiovascular systems, and organ sizes with humans (Houpt et al., 1979;Spurlock and Gabler, 2008;Litten-Brown et al., 2010). It may be very helpful to understand the effect of HR and FR extracts on adipogenesis using these model. Preparation of ramie leaf extract Ramie leaf was purchased at a local market in Gwangju, Korea. It was rinsed in water and blanched in boiling water for 1 minute. Afterwards, the blanched portion was drained of water. HR was prepared by placing in a 60°C hot-air oven for 40 h. FR was dehydrated at -70°C for 72 h. HR and FR powders were added with 80% ethanol solution. Using a heating mantle, solutions were heated at 65°C for 9 h with constant stirring and then filtered through filter paper. Samples were concentrated on a rotary evaporator and stored at -70°C, before analysis. 3T3-L1 cell culture conditions 3T3-L1 murine preadipocytes were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured as described previously (Chen et al., 1997). For the proliferation experiments, cells (1×10 5 ) were plated in 6-well plates and media were changed every 2 days. To induce cell differentiation, cells (3×10 4 ) were plated in 6well plates and were grown to confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS). Media was changed every 2 days until 14 days. On day 1, confluent cells were changed to differentiation medium, which consisted of 10 μg/mL insulin, 1 μM dexamethasone and 0.5 μM 3-isobutyl-1-methylxanthine (IBMX) in DMEM supplemented with 10% FBS. From day 2, media was replaced with 10 μg/mL insulin in DMEM supplemented with 10% FBS. Stock solutions of Ramie leaf were prepared in dimethyl sulfoxide (DMSO) and diluted with medium prior to experimental procedures. Pig preadipocyte isolation Pig preadipocytes were obtained from newborn male pigs. The back fat tissue taken from the pigs were treated with collagenase treatment. The isolated tissue was digested with collagenase for 40 minutes at 37°C. After removal of undigested tissue using nylon mesh, tissue was centrifuged at 1,500×g, 10 minutes. The cell pellet was placed into a Krebs-Ringer Bicarbonate buffer, centrifuged at 1,796×g for 10 mins, and cells were collected. Determination of cell viability Cells were treated with DMSO (control) or various concentrations of each Ramie leaf extract for 2 days. Cells were washed with phosphate-buffered saline (PBS) and cells were collected cells using trypsin. After centrifugation, cell proliferation was determined using a hemocytometer and the trypan blue exclusion assay. Measurement of cell differentiation Glycerol-3-phosphate dehydrogenase (GPDH) activity was measured according to the method of Wise and Green (1979). After removing the culture media, differentiated cells were washed with PBS and lysed in a pH 7.4 buffer consisting of 0.25 M sucrose, 5 mM Tris base, 1 mM ethylenediaminetetraacetic acid, and 1 mM dithiothreitol. After centrifugation, GPDH activity in the supernatant was measured. Quantification of triglyceride content Cellular triglyceride content was isolated from the supernatants of differentiated cells by homogenization. Supernatant (5 μL) was mixed with free glycerol assay reagent (160 μL) in a 96-well plate. After incubation for 5 mins at 37°C, the absorbance of the solution was measured at 540 nm using a microplate reader. The absorbance was measured again after adding triacylglycerol reagent (40 μL). Measurement of lipoprotein lipase expression Lipoprotein lipase (LPL) activity was measured by detecting liberated 3H triolein substrate emulsion according to previously reported method (Nilsson-Ehle and Schotz, 1976). LPL activity was expressed as units of fatty acid released per gram of tissue. Statistical analysis All data are expressed are means±standard error. All results were compared by one-way analysis of variance using the statistical package of social science (SPSS) program. Group means were considered significantly different at p<0.05. Ramie leaf extract inhibited the viability of 3T3-L1 cells and pig preadipocytes Initially we examined the effect of ramie leaf extracts on viability of 3T3-L1 cells and pig preadipocytes. As shown in Figure 1A, the viability of 3T3-L1 cells did not show any difference between HR and FR extract at 24 h (data not shown), but examined the significance of 100 μg/mL HR or FR extract after 48 h. Treating pig preadipocytes with 25 μg/mL or 100 μg/mL HR or FR extract significantly inhibited their viability after 48 h ( Figure 1B). These results indicated that HR and FR extracts reduced the viability of pig preadipocytes at lower concentrations than that required for 3T3-L1 cells. Ramie leaf extract reduced differentiation of 3T3-L1 cells and pig preadipocytes We analyzed the effect of ramie leaf extract on the differentiation of 3T3-L1 cells and pig preadipocytes. Compared to DMSO treatment (control), the treatment of 3T3-L1 cells with 100 μg/mL HR or FR extract for 48 h resulted in 36% and 34% inhibition, respectively, of GPDH activity. The two drying methods did not affect 3T3-L1 cell differentiation following treatment for 48 h (Figure 2A). The HR extract inhibited the differentiation of pig preadipocytes at 100 μg/mL. However, the FR extract suppressed the differentiation in a concentration-dependent manner ( Figure 2B). These data indicate that the HR extract is superior to FR extract in inhibiting the differentiation of pig preadipocytes. Effect of ramie leaf extract on lipid accumulation in pig preadipocytes We measured the effect of ramie leaf extract on lipid accumulation in pig preadipocytes. Figure 3 shows the proliferation of pig preadipocytes after 2 days of cultivation. After 4 days of cultivation, cells had reached confluency. At 6 days, compared to the control, HR and FR extract treatment at a concentration of 100 μg/mL inhibited lipid accumulation in pig preadipocytes. This finding indicates that HR and FR extract inhibited adipocyte differentiation. Ramie leaf extract inhibited triglyceride accumulation in 3T3-L1 cells and pig preadipocytes To explore the effect of ramie leaf extract on intracellular triglyceride accumulation, 3T3-L1 cells and pig preadipocytes were exposed to HR and FR extract for 6 days. HR and FR extract at a concentration of 25 µg/mL decreased triglyceride levels by 11.6% and 9.6%, respectively. HR and FR extract at a concentration of 100 µg/mL reduced triglyceride levels by 29.9% and 26.6%, respectively ( Figure 4A). Triglyceride accumulation in pig preadipocytes was inhibited by 15% following treatment with HR extract at 25 μg/mL; 100 μg/mL HR inhibited triglyceride accumulation by 44.5%. FR extract also decreased triglyceride content in pig preadipocytes, but no significant changes were observed at 25 μg/mL ( Figure 4B). These results demonstrate that both ramie leaf extracts inhibited the accumulation of triglycerides in 3T3-L1 and preadipocytes and that HR extract was more effective than FR extract in inhibiting triglyceride accumulation. Ramie leaf extract inhibited lipoprotein lipase activity in 3T3-L1 cells and pig preadipocytes Because LPL is a key factor in the development of obesity (Wang and Eckel, 2009), we evaluated the effect of HR and FR extracts on LPL activity in 3T3-L1 cells and pig preadipocytes. As shown in Figure 5A, compared to the control, treatment with 100 µg/mL HR or FR significantly inhibited LPL activity in 3T3-L1 cells. In pig preadipocytes, treatment with HR extract decreased LPL activity at both concentrations 25 μg/mL and 100 μg/mL ( Figure 5B). HR Figure 3. Micrographs showing differentiation of pig preadipocytes treated with air-dried ramie leaf (HR) or freeze-dried ramie leaf (FR) extracts. Pig preadipocytes treated with HR or FR extracts (100 μg/mL) were incubated for 6 days. Each experiment was repeated at least triple. Figure 2. Effects of hot air-dried ramie leaf (HR) or freeze-dried ramie leaf (FR) extracts on differentiation of 3T3-L1 cells (A) and pig preadipocytes (B). Cell differentiation was determined by glycerol-3-phosphate dehydrogenase (GPDH) activity. Reported values are means±standard error. a-c Values with different superscripts are significantly different at p<0.05 according to Tukey's test. # Significantly difference compared with freeze drying at p<0.05 according to Student's t-test. Each experiment was repeated at least triple. extract treatment showed more sensitive than FR extract treatment on inhibition of LPL activity at 25 µg/mL concentration. These findings imply that HR and FR extract inhibited LPL activity in both 3T3-L1 cells and pig preadipocytes. DISCUSSION Obesity is caused by the growth and expansion of adipose tissue (de Ferranti and Mozaffarian, 2008). Adipocyte differentiation and fat accumulation are closely linked to obesity (Fève, 2005). In the present study, we evaluated the effect of HR or FR extract treatment on the proliferation and differentiation of 3T3-L1 cells and pig preadipocytes at concentrations of 25 and 100 μg/mL. The 3T3-L1 cell line and primary preadipocytes are useful models for studying the differentiation of preadipocytes into adipocytes (Kim and Moustaid-Moussa, 2000;Fève, 2005). Hot-air and freeze drying are common methods for preserving food and feed (Ratti, 2001). Food preparation methods can affect the nutritional and functional qualities of the food. We have investigated to compare the physicochemical composition and antioxidative properties between HR and FR (Kim et al., 2014a, b). There were no significant differences in moisture, crude protein, crude fat, crude ash, and carbohydrate content depending on the drying methods, but the dietary fiber content was significantly higher in FR than in HR. In addition, the contents of vitamin A, E, and C, total minerals, total organic acids and total free sugars in HR were significantly higher than those in the FR. Phenolic compounds such as flavonoids and chlorogenic acid are considered to be the major contributors to the antioxidant ability of plants (Ong et al., 2013). Total polyphenolics, flavonoids, and chlorogenic acid contents in HR and FR used in this experiment were found to be 132.50±2.76 and 138.00±2.64 tannic acid equivalent mg/g dry matter (DM), 119.00±1.15 and 124.25±0.58 rutin equivalent mg/g DM, and 15.24±0.65 and 15.78±0.53 mg/g, respectively. Several studies have shown that the antioxidant capacities of natural products depend on the drying method used (Kim et al., 2007;Ferreira and Luthria, 2010;Gümüşay et al., 2015). Our previous study revealed that both HR and FR extracts had a high 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and high total flavonoid contents (Kim et al., 2014b), but that there was no difference between them (Chen et al., 1997). Oxidative stress is caused by an imbalance between pro-oxidants and antioxidants and it plays a critical role in the pathology of various diseases (Furukawa et al., 2004). Low levels of oxidative stress allow cells to maintain a normal redox status, but high levels damage cell composition and function (Yu, 1994). Obesity increases oxidative stress and induces lipid peroxidation (Vincent and Taylor, 2006). Excessive levels of lipid in the blood can induce elevated oxidative levels in vivo and in vitro (Furukawa et al., 2004;Vincent and Taylor, 2006). In this study, we observed that HR and FR extract treatment inhibits the proliferation and differentiation of 3T3-L1 cells and pig preadipocytes in a dose-dependent manner (Figure 1 and 2). Furukawa et al. (2004) demonstrated that reactive oxidative species was produced during differentiation of 3T3-L1 cells into adipocytes (Furukawa et al., 2004). Therefore, inhibition of proliferation and differentiation by HR and FR extracts which contain high levels of antioxidants may have the potential to prevent obesity. In this study, HR and FR extract treatment decreased lipid accumulation in 3T3-L1 cells and pig preadipocytes ( Figure 4A, 4B). A dose of 100 μg/mL HR extract significantly inhibited triglyceride accumulation in pig preadipocytes. It inhibited the proliferation and differentiation of 3T3-L1 cells in a similar manner. These results indicated that HR and FR extract-mediated decrease of proliferation and differentiation might affect the accumulation of lipid in 3T3-L1 cells and pig preadipocytes. Further studies are needed to assess the underlying mechanism. To our knowledge, this study is the first to demonstrate that after HR and FR extract reduce differentiation of and lipid accumulation in 3T3-L1 cells and pig preadipocytes. LPL has an important role in lipoprotein metabolism, the hydrolysis of triacylglycerols into free fatty acids, and the formation of monoglyderides from plasma lipoproteins (Wang and Eckel, 2009). LPL acts as a gatekeeper enzyme that determines the amount of free fatty acid that enters cells. Semenkovich et al. (1989) reported that differentiated 3T3-L1 cells had increased levels of the LPL gene and enzyme activity. The results of the present study revealed that HR and FR extract treatment decreases the expression of LPL in 3T3-L1 cells and pig preadipocytes ( Figure 5A, 5B). These indicate that the inhibition of LPL activity following HR and FR extract treatment was, at least in part, caused by the inhibition of differentiation of both cells. Interestingly, at a low dose of HR extract (25 μg/mL), the LPL activity at the cellular level decreased compared to FR treatment, suggesting that HR may have an inhibitory effect on LPL activity in pig preadipocytes. CONCLUSION In summary, treatment with HR and FR extract inhibits the proliferation and differentiation of, lipid accumulation, and LPL activity in 3T3-L1 cells and pig preadipocytes. Therefore, we suggest that extracts of natural products such as HR and FR extract may have potential to reduce obesity. Further studies are needed to clarify and compare the mechanism of these two extracts in adipocytes.

v3-fos

2019-04-06T13:07:55.640Z

2015-02-16T00:00:00.000Z

35028908

s2

Investigation and Evaluation on Heavy Metal Contaminations of Green Salads and Potato Fried in Different Restaurants and Fresh Vegetables in Some Egyptian Governorates The content of Cd, Pb, Al, and As in fresh vegetables, green salads and potato fried purchased from the Egyptian market and restaurant were determined using atomic absorption spectrometry (AAS).The results of this study showed that there was wide variation in the concentration of these metals in vegetables collected from different sites. The highest Cd level in watercress (8.33 mg/Kg) was found in Cairo. Vegetables grown in Cairo and Alexandria had cadmium many folds higher than those of other towns. Lead concentration in potato of Alexandria is more than permitted level. Aluminium (Al) content was high concentration in all vegetable samples and arsenic (As) concentration mostly was appeared in potato. Analytical results indicated that the concentration of Cd, Pb, and As in green salad samples were so few. The highest concentration of Al was detected in salad collected from popular restaurant 8. The results show that the heavy metal content in unprocessed potato appears to be much higher than that in processed potato. The highest levels of Cd, Pb, and Al were detected in popular restaurant 9. Cd and As contents of international chain restaurant 1 were found in high level (2.958 and 0.95mg/Kg). Introduction Food safety is a major public concern worldwide. During the last decades, the increasing demand for food safety has stimulated research regarding the risk associated with consumption of foodstuffs contaminated by pesticides, heavy metals and/or toxins (1).Vegetables constitute an important part of the human diet since they contain carbohydrates, proteins, as well as vitamins, minerals and trace elements (2,3).Vegetables also act as buffering agents for acidic substances obtained during the digestion process. However, these plants may contain both essential and toxic elements, such as heavy metals, at a wide range of concentrations (4).Publicity regarding the high level of heavy metals in the environment has created apprehension and fear in the public as to the presence of heavy metal residues in their daily food. The public is confused and alarmed about their food safety. Keeping in mind the potential toxicity and persistent nature of heavy metals, and the frequent consumption of vegetables and fruits, it is necessary to analyze these food items to ensure the levels of these contaminants meet agreed international requirements (5). Heavy metals are easily accumulated in human vital organs and threaten human health. Vegetables are part of human diet to take up a lot of essential nutrients and certain trace elements in a short period. In this situation, safety of vegetables is very important (6, 7, 8, and 9). Heavy metals are one of a range of important types of contaminants that can be found on the surface and in the tissue of fresh vegetables. Heavy metals, such as cadmium, copper, lead; chromium and mercury are important environmental pollutants, particularly in areas under irrigated with waste water. Several investigations of water, soil and vegetables pollution by waste water are available (10, 11, 12, 13, 14, 15, and 16). Strictly speaking, heavy metals are defined as those with higher density than 5 mg mL -1 (17) but the collective term now includes arsenic, cadmium, chromium, copper, lead, nickel, molybdenum, vanadium and zinc. Some interest also exists in aluminium, cobalt, strontium and other rare metals. Physiologic roles are known for iron (haemmoeties of heamoglobin and cytochromes), copper (amine oxidases, dopamine hydrolase and collagen synthesis), manganese (superoxide dismutase), zinc (protein synthesis, stabilization of DNA and RNA) with low requirements of chromium (glucose homeostasis). Other heavy metal ions are not believed to be essential to health even in trace amounts. Vegetables, especially those of leafy vegetables grown in heavy metals contaminated soils, accumulate higher amounts of metals than those grown in uncontaminated soils because of the fact that they absorb these metals through their leaves (18).Research findings show that at least 20 million hectares of land in North and South Africa, South America, Middle East, Southern Europe, South West America, Mexico and a significant part of Central and East Asia is irrigated by raw sewage, mainly for cultivation of vegetables. Consequently, this usage ends to soil contamination and heavy metals accumulation both in soil and crops (19). Heavy metal accumulation in soils is of concern in agricultural production due to the adverse effects on food quality (safety and marketability), crop growth (due to phytotoxicity) (20, and 21). Metals such as lead, mercury, cadmium and copper are cumulative poisons. These metals cause environmental hazards and are reported to be exceptionally toxic. On consumption of food in the diet, the trace metal contents of food are directly taken into the body (22, and 23).These metals enter the human body mainly through two routes namely: inhalation and ingestion, and with ingestion being the main route of exposure to these elements in human population. Heavy metals may enter the human body through inhalation of dust, direct ingestion of soil and consumption of food plants grown in metal contaminated soil (24, and 25).Heavy metals intake by human populations through the food chain has been reported in many countries with this problem receiving increasing attention from the public as well as governmental agencies, particularly in developing countries (26, and 27).The allowable Limit of Heavy Metals, as safe values for copper, lead, and cadmium in fruit and vegetables recommended by the WHO / FAO are 40, 0.3, and 0.2 mg/kg, respectively. Vegetables take up metals by absorbing them from contaminated soils, as well as from deposits on different parts of the vegetables exposed to the air from polluted environments (28). It has been reported that nearly half of the mean ingestion of lead, cadmium and mercury through food is due to plant origin (fruit, vegetables and cereals). Metal contamination soils may be widespread in urban areas due to past industrial activity and the use of fossil fuels (29).Additional sources of heavy metals for plants are: rainfall in atmospheric polluted areas, traffic density, use of oil or fossil fuels, for heating, atmospheric dusts, plant protection agents and fertilizers which could be adsorbed through leaf blades (30,31,32,33).The content of essential elements in plants is conditional, the content being affected by the characteristics of the soil and the ability of plants to selectively accumulate some metals (34). Prolong consumption of unsafe concentrations of heavy metals through foodstuffs may lead to the chronic accumulation of heavy metals in the kidney and liver of humans causing disruption of numerous biochemical processes, leading to cardiovascular, nervous, kidney and bone diseases (35).The main source of human exposure to Pb and Cd is food, which is believed to provide about 80-90% of daily doses (36,37). Pb and Cd toxicity is well documented and is recognized as a major environmental health risk throughout the world. Pb affects humans and animals of all ages, but the effects of lead are most serious in young children. Cadmium is a toxic and carcinogenic element. The International Agency for Research on Cancer has identified Cd as a known human carcinogen. Pb and Cd poisoning results from the interaction of the metal with biological electron-donor groups, such as the sulfhydryl groups, which interferes with a multitude of enzymatic processes. Clinical manifestations of Pb toxicity include symptoms referable to the central nervous system, the peripheral nervous system, the hematopoietic system, the renal system, and the gastrointestinal systems. Cd is a cumulative nephrotoxicant that is absorbed into the body from dietary sources and cigarette smoking (37,36).Potentially toxic metals are also present in commercially produced foodstuffs. Results of (38) indicated that the cadmium concentrations in shoots and roots varied both with different Cd levels and type of vegetable. Generally Cd accumulation in various plant parts in vegetable crops was increased with the increasing cadmium concentrations in the growth medium. Cd increased more sharply in roots than shoots. Celery contained higher Cd in the edible parts than other vegetable species. Permissible level of consumption of Cadmium, for human is 70µday -1 . The effect of environmental pollution on contamination of foods and on their safety for human consumption is a serious global public issue and widely addressed (39,40). Some of these elements are toxic to humans even at a very low level. Excessive content of Pb and Cd metals in food is associated with etiology of a number of diseases especially with cardiovascular, kidney, nervous as well as bone diseases (41,42,43,17). They have also been implicated in causing carcinogenesis, mutagenesis and teratogenesis (44,45).Lead is a toxic element that can be harmful to plants, although plants usually show ability to accumulate large amounts of lead without visible changes in their appearance or yield. In many plants, Pb accumulation can exceed several hundred times the threshold of maximum level permissible for human (46). The introduction of Pb into the food chain may affect human health, and thus, studies concerning Pb accumulation in vegetables have increasing importance (47). Although a maximum Pb limit for human health has been established for edible parts of crops (0.2 mg/kg) (48), soil Pb thresholds for producing safe vegetables are not available. Further investigations Zhang and Zhou (49) showed that the Al-based coagulants at the tested concentrations had a poisonous effect on the germination of vegetable seeds. While aluminum can be toxic at higher levels, it is considerably less toxic than either mercury or lead. In fact, aluminum is found at easily measurable levels in various biological fluids and tissues. However, at high levels aluminum has the potential to cause a number of health problems such as anaemia and other blood disorders, colic, fatigue, dental caries, dementia dialactica, kidney and liver dysfunctions, neuromuscular disorders, osteomalacia and Parkinson's disease (50). Arsenic is regarded as human carcinogen from extremely low levels of exposure, having no possible beneficial metabolic functions for humans. Its low level exposure cause nausea and vomiting decreased production of RBCs and WBCs, abdominal pain and its long term exposure causes darkening of skin and appearance of small corns on palm soles. Other affect includes anorexia, fever, fluid loss, goiter, hair loss, headache, herpes, impaired healing, jaundice, keratosis, kidney and liver damage, muscle spasms, pallor, peripheral neuritis, sore throat, weakness and interferes with the uptake of folic acid (50). There were positive curvilinear or linear relationships between the inhibitory rate of seed germination and the concentration of Al in the acidic and neutral conditions except for the toxic effects of PAC (polyaluminum-chloride) on Brassica chinensisin the neutral condition (49). Moreover there were obvious differences in root elongation of Brassica chinensis exposed to AlCl 3 in various pH conditions. The aim of the present study was to evaluate the safety of selected fresh vegetables in different regions of Egypt, green salads, and potatoes fried available in Egyptian restaurants in the regions of Cairo and Giza in 2014, as determined by the content ofPb, As, Al, and Cd. Materials Various kinds of fresh vegetables (33 samples) were randomly collected from the main farmlands around the city (Cairo, Giza, Alexandria, menoufia, and kalyopia). Green salads (12 samples) and potato fried were collected from International chain, local, and popular restaurants in February and March during the year 2014 submitted to a laboratory for analysis. All reagents which used in these methods were obtained from Merck-Darmstadt. Determination of Mineral Contents The content of metals in vegetables and green salads samples was determined according to a procedure advised by the Regional Center for Food and Feed. The samples were brought in plastic bags to Laboratory. Vegetables samples were cleaned peeled (if necessary) and washed to obtain edible parts prior to analysis. Green salads and potato fried samples were homogenized. All samples were weighed (1g±0.01) in Pyrex tubes, add 10 ml of concentrated nitric acid, and allow standing overnight. Heat carefully on a hot plate unit the production of red NO 2 fames has ceased. Cool the beaker and a small amount (2-4 mL) of 70 % HClO 4 . Heat again and allow evaporating to a small volume transfer the sample to a 50 mL flask and diluting to volume with distilled water (51).The samples were analyzed by Contraa 700-P, atomic absorption spectrophotometer-Analytical Jena. Statistical Analyses Analysis using the Dunks test was carried out to examine the statistical significance of differences in the mean concentration of metals between groups of vegetables using SPSS, version 10. A probability level of p<0.05 was considered statistically significant. Results Heavy metal content obtained on tomatoes, cucumber, lettuce, watercress, and potatoes collected from different agricultural locations (Cairo, north and south Giza, Alexandria, Menoufia, Kalopia, and Sohag) in Egypt are given as mean values and standard Error in Table 1. Results of monitoring studies carried out in Egypt in 2014 showed that the heavy metal content of vegetable samples were regionally dependent. The highest Cd level in watercress (8.33 mg/Kg) was found in Cairo and higher in potatoes (4.41 mg/Kg) from Kalyopia while the lowest was determined in the cucumber (0.0000198 mg/Kg) from north Giza. Cd content of potatoes were found partly high levels in Cairo, north Giza, and Alexandria (1.3, 0.33, and 0.95 mg/Kg) compared with other agricultural location. The content of Cd in lettuce from Cairo, Alexandria, Menoufia, and south Giza was 1.06, 1.69, 0.81, and 1.31 mg/Kg, respectively. The highest Pb content was found in potatoes (61.1mg/Kg) from Alexandria while the lowest were found in Tomatoes (0.0000062, 0.000006, 0.000004, and 0.000069) from north Giza, Alexandria, Menoufia, and Kalyopia, respectively. On the other hand, the Pb content of lettuce, potatoes were high (0.57, 0.51, and 0.543 mg/Kg) in Cairo, Menoufia, and Kalyopia independent of region. Al contents varied in agricultural locations among the vegetable samples. The highest levels were 4178.67 and 1820.85 mg/Kg in potatoes from north Giza, and lettuce from Alexandria while the lowest Al levels were found in cucumber from Cairo, Alexandria, and tomatoes from Kalypia (3.369, 3.547, 3.34 mg/Kg, respectively). As content of vegetable samples were found at low levels. As content of vegetable samples were changed between 0.00042 (in cucumber from North Giza) to 0.319 (in potato from North Giza) mg/Kg. Concentration of some heavy elements found in green salad collected from international chain restaurant, local restaurant, and popular restaurants were summarized in Table 2. Cd, Pb, and As contents of green salad collected from different restaurants were found at low levels. Al content of salad samples in all restaurant categories ranged from 1.603 to 914.11mg/Kg. The highest concentration of Al was detected in salad collected from popular restaurant 8 (914.11 mg/Kg) while international chain restaurant (2), local restaurants (3,6), and popular restaurant (10,11) had the lowest concentration (1.603,3.998, 4.03, 3.93, and 4.32mg/Kg). The concentrations of heavy metals (Cd, Pb, Al, and As) in potatoes fried from three level restaurants (international chain, local, and popular) analyzed are reported in Table 3. The highest levels of Cd, Pb, and Al were detected in popular restaurant 9. Cd and As contents of international chain restaurant 1 were found in high level (2.958 and 0.95mg/Kg) compared with other restaurants, While Pb contents was found in low level (0.033 mg/Kg). As content in potatoes fried from popular restaurant 7 and 8 were non-detectable amounts. At the same form, potatoes fried contained Al at the low concentration. Al values showed a notable increase from 56.88 mg/Kg in popular restaurant 7 to 2305.87 mg/Kg in popular restaurant 9. Pb was detected at lowest levels in potatoes fried from local restaurant 6 (0.0019 mg/Kg) and Cd was lowest in potatoes fried from local restaurant 4 (0.419 mg/Kg). Discussion The toxicity of the metals in agricultural products depends upon relative level of exposure of crops to the contaminated soils as well as the deposition of toxic elements in the polluted air by sedimentation. Different metals show the toxicity at different concentrations and can be potentially toxic at sufficiently high concentrations. However, certain metals exhibit toxic effects even at relatively low concentrations. Metal toxicity depends on the element, its chemical form and its oxidation state. The results in Table 1 show a high degree of contamination in vegetables, when compared with the permissible levels given by the FAO and WHO (36). Our data showed that the overall toxic metal accumulation was greater in leafy vegetables lettuce, watercress and tubers such as potato. Heavy metal content has been reported in various common vegetables of many towns in Egypt. Our data showed that large concentrations of cadmium (toxic trace element) in lettuce (Cairo, Alexandria, menofia, and South Giza), watercress (Cairo and Alexandria) and potatoes (Cairo, Alexandria, and Kalyopia). The above data reveal that cadmium content in same vegetable differs from town to town. According to above data, cadmium content in vegetables of Cairo and Alexandria are many folds higher than those of other towns. It might be because the metal uptake in vegetables is influenced by several factors such as metal concentrations in agricultural soils, soil pH, physico-chemical characteristics of the soil, soil classification, etc. Moreover, vegetable consumption (per person per day) is not same for the residents of different regions. Abdel-Shafyet al., (52) reported the levels of the metals in the surface layer of soil irrigated with the Nile water and ground water in Qatta village Egypt were arranged in the following ascending orders Fe > Zn >Mn>Pb> Cu > Cd and Fe >Mn> Zn = Cu >Pb> Cd, respectively. The level of heavy metals in different parts of potato plants irrigated with Nile and ground water for over four successive seasons in Qatta village. In the period of continuous soil irrigation with both the Nile and ground water, most of metals, namely cadmium and lead, are accumulated in plants. The level of heavy metals in soil does not depend essentially on the sources of irrigation, but first of all on the metal constituents of the fungicide applied. Okwulehie and Ogoke (53) reported that cadmium in the range of 0.7-0.94 ppm for certain mushrooms in Nigeria. Banerjee et al., (54) recorded cadmium levels in some fruits and vegetables in India to be in the range of 0.03 to 7.32 µg/g dry weight. Cadmium content of tomato and cucumber analyzed in Egypt was below the WHO standards limit (0.2 mg/kg) (35).The high contamination levels found in some vegetables may be related to pollutants in irrigation water, farm soil or pollution from highway traffic (55).In a study Bahemuka and Mubofu (4) reported that the cadmium content in vegetables of Tanzania ranged between 0.1 and 0.6 mg/kg dry weight. Studies have shown that metals such as iron, copper, cadmium, chromium, lead, mercury and nickel have the ability to produce reactive oxygen species. The result of this is lipid peroxidation, DNA damage and altered calcium homeostasis (56,57). Elsokkary and Sharaf (58) reported that the bioaccumulation ratio of Cd in the plants followed the order: Chard>Spinach> Lettuce> Parsley>Rocket> Coriander. The main cause for concern in terms of contamination of vegetables in Alexandria and Menoufia by heavy metals relates to Lead (Pb). Although a maximum Pb limit for human health has been established for edible parts of crops in China is 0.2 mg/kg (48) but this limit by WHO standards is 0.3 mg/kg (35). Data showed that in potato of Alexandria, lead concentration is more than permitted level, so they are not suitable for consumption. Lead is a toxic element that can be harmful to plants, although plants usually show ability to accumulate large amounts of lead without visible changes in their appearance or yield. In many plants, Pb accumulation can exceed several hundred times the threshold of maximum level permissible for human (46). The introduction of Pb into the food chain may affect human health and thus, studies concerning Pb accumulation in vegetables have increasing importance (47,3). On the whole, some vegetables that were studied in this study were contaminated by lead and they were toxic to consumer. Zhuanget al., (59) have found higher contents of Cd and Pb concentrations than the maximum permissible levels in vegetables collected from six sampling sites around Dabaoshan mine located at Shaoguan city, Guangdong, southern China. It was found that most of the vegetable types marketed in Kathmandu were grown along the bank of rivers, roadsides and highways irrigated with wastewater as well as from vegetable farms with possible use of fertilizers and pesticides in excessive amount (source: personal communication with vegetable grosser). Continuous irrigation of agricultural land with sewage and wastewater may cause heavy metal accumulation in the soil and vegetables (60).Besides, pesticides and fertilizers are known to be the main sources of heavy metal pollution in agricultural areas (61).Moreover, the transportation and marketing systems of vegetables play a significant role in elevating the contaminant levels of heavy metals which may pose a threat to the quality of the vegetables with consequences for the health of the consumers of locally produced foodstuffs (62,63).It is concluded that atmospheric deposition from urban and agricultural areas may play an important role in the enrichment of agricultural produce from Cd and/ or Pb (64). Abdel-Sabour and Rabie (65) also studied ten sample of vegetable plant species, plants grown on soils (irrigated with industrial wastewater of Shebin EI-Qanater and Mostorod collectors) and (irrigated with polluted water of EI-Shaboura canal), whereas its soils have been subjected to prolonged domestic and industrial wastewater irrigation (about 35 years). Results revealed that vegetable plants species varied in their affinity to accumulate metals in their edible parts. Irrigation with different wastewater significantly increased the concentration of Pb and Cd in vegetable plants especially the leafy species. Rashad and Shalaby, (66) measured the levels of heavy metals at different sites with different distances and directions from two dumpsites in Alexandria for vegetation. The concentrations of metals in leaves and roots of tomatoes, carrots and potatoes plants were higher in plants grown at the site close to Abis (the municipal solid waste) dumpsite and decreased with increasing distance. The only difference between Abis and El-Montaza is that the recorded levels of heavy metals at Abis area were higher than obtained at El-Montaza area. This could be due to the other pollution sources found at Abis area, including the cement company and other petroleum companies near Abis area which is a serious source of their metals. A market basket survey showed that Aluminium (Al) content was high concentration in all vegetable samples. Al content in some common vegetables in North Giza which show that potato had the highest amount while the lettuce had lowest (Table 1). In Alexandria the highest variation in Al level was observed in lettuce and tomato and lowest in cucumber. However the Kalyopia vegetables (cucumber and watercress) contain much higher amount of Al compared to Sohag. It can be seen that total Al in vegetable samples ranged from 3.34 to 4178.67 mg/kg, in green salad was 1.603 to 914.11 mg/kg, and in processed potato ranged from 56.88to 2305.87 mg/kg. Similar high concentrations of Al in black tea leaves were from 685 to 1200 mg/kg. In the other vegetable samples the concentration of Al is much higher, ranging from 70 to 400 mg/kg. The highest concentration of about 400 mg/kg of Al was found in lamb's lettuce and in garden lettuce was reported in the literature (67).Müller et al., (68) also found higher Al concentrations in various sorts of lettuce (200-1000 mg/kg). Since Al-tolerant plants can accumulate Al from soil solution, higher concentrations of Al may be found in vegetables (up to 80 mg/kg) (68,69), in different kinds of lettuce (up to 1000 mg/kg), in herbs and spices(up to 300 and 1000 mg/kg Al, respectively) and in black tea leaves (from 600 to 1200 mg/kg of Al) (68). Al enters drinking waters mainly due to the use of Al 2 (SO 4 ) 3 that is applied as a coagulant to clarify turbidity (70).Another possible source of the entry of Al into foodstuffs is the use of Al containing food additives, which in cheese processing may increase Al concentrations up to 70 mg/kg (68). Lokeshappaet al., (71) analyzed the Aluminium in selected vegetables available in Powai area, Mumbai, India. The content of Aluminium in spinach and coriander was in high concentration and in moderate concentrations in tulsi, potato and ladyfinger. Cadmium was present in small concentrations in all samples except coriander and carrot. It was found that the overall toxic metal accumulation was greater in leafy vegetables viz. spinach, coriander and tubers such as potato. Data in Table 1 showed that arsenic (As) concentration mostly was appeared in potato and in fallow order by town: North Giza >Kalyopia> Alexandria. Several countries, including the UK and Australia, currently use a 1 ppm limit for arsenic in food and this is often cited as a "safe" level for rice. Most of the values exceeded the maximal permissible limit of food As standard 1.0 mg/kg dry weight (72), but this limit by WHO standards is 0.43 mg/kg (35). As reported in the literatures, the total arsenic contents in vegetable products were < 0.004 to 0.303 mg/kg fresh weight (73,74,75,76). The average arsenic concentration in the vegetables collected from some arsenic prone areas of Bangladesh was 0.28 mg/kg fresh weight, which was higher than that of the United Kingdom, 0.003 mg/kg fresh weight (77), and Croatia, 0.0004 mg/kg fresh weight (78). Concerning plants growing on contaminated soils of Egypt, several investigators studied the accumulation of heavy metals in these plants. ElSabbagh (79) found that Cd, Cu, Pb and Zn were accumulated in vegetable plants growing on contaminated soils in industrial area. Vegetables Cd ranged from 0.03-0.34 mg/100g. The cultivated vegetables revealed that it absorbs soilPb with varying concentrations from < 0.4-2.5 mg/100g, green onion ranks the first order as it contains 2.5 mg/100g followed by Radish. Abdel-Maksoud (80) reported that using wastewater for irrigation lead to the accumulation of Fe, Cd and Pb in vegetable crops such as Spinach, Potato, Tomato and Celery growing on polluted soil at Giza Governorate. Heavy metal contents of leaves Ficusretusa showed that their concentrations increased with the increase in the concentration of these metals in the soil: plants grown at AbouQuir had the highest concentrations (81). According to the results of this study, the heavy metal (Cd, Pb, and As) contents of green salad in washing samples are so small that one must draw the conclusion that migration of heavy metal from the washing into the green salad can be ignored. However Al was high in some restaurant categories as well as in vegetable samples ( Table 2). Washing treatments significantly (P ≤ 0.05) reduced concentrations of heavy metal contents in the leaves of Ficusretusa (81).El-Desoky and Ghallab (82) found that Contents of Pb in the unwashed plant samples (6.00-40.18 ppm) are quite high, compared with the washed samples (0.34 to 29.53 ppm) which were collected from the area around the superphosphate factory. Amounts of cadmium accumulate on plant surface from dusts carried from the factory chemnies. The results show ( Table 3) that heavy metal is widely contained in processed potato (potatoes fried). Furthermore, the heavy metal content in unprocessed potato appears to be much higher than that in processed potato. These results suggest that the presence of heavy metal from processed potato due to the containers used in the manufacturing, processes of heat, type of oil, and oil contamination. These differences in restaurant categories may be probably due to raw material, process conditions and analytical procedures. Harmankayaet al., (83) found that Cd and Pb contents of all chips (from several markets in Konya in Turkey) were at the low levels. Fried potatoes from the Egyptian market content of the studied metals were found to be ranged from 0.054 to 0.10 of Cd, 0.065 to 0.159 of Pb (64). Heavy metal contents in fried potatoes were found to be ranged from 0.054 to 0.10 of Cd and 0.065 to 0.159 mg/kg of Pb. The levels of Cd in all samples are higher than the permissible limits (84). In conclusion, it is well known that environmental pollution is a product of urbanization and technology and other attendant factors of population density, industrialization and mechanization that serve to provide the necessities of the population. Aluminium (Al) content was high concentration in all vegetable samples. Data showed that potato of Alexandria, lead concentration is more than permitted level. Among the restaurant categories Cd, Pb, and As contents of green salad were found at low levels. The highest levels of Cd, Pb, and Al were detected in popular restaurant 9. Cd and As contents of international chain restaurant 1 were found in high level. If these vegetables are to be used for a short period of time or in small doses, washing can reduce the surface contamination to a great extant. Hence, it is suggested that consumers wash the vegetable prior to use. This result will therefore help the government, individuals and communities to take necessary measure in controlling heavy metal pollution and to minimize exposure of people living town of Egypt. Next study will be on the Egyptian soil and water.

v3-fos

2019-04-08T13:07:43.679Z

2015-08-30T00:00:00.000Z

99661478

s2

Determination of Quercetin a Biomarker in Hepatoprotective Polyherbal Formulation through High Performance Thin Layer Chromatography Methods: Polyherbal hepatoprotective formulation was developed by using five bioactive fractionated extracts of three plants namely Butea monosperma, Bauhinia variegate and O. gratissimum. All three plants contain quercetin. Chromatographic separation was performed on aluminium foil plates coated with 200 μm silica gel 60F254 Linear ascending development with toluene:ethyl acetate:formic acid, 5:4:0.1 (v/v/v) was performed at room temperature (25 ± 2°C) in a twin-trough glass chamber saturated with mobile phase vapor. Compact bands (Rf=0.38) were obtained for quercetin. Spectro densitometric scanning was performed in fluorescence mode at 380 nm. The method was validated for precision, recovery, specificity, detection and quantification limits. Introduction Nature still obliges as the man's primary source for the cure of his ailments. Research in preventive medicine showed the importance of functional nutrition in reducing the risk factor of certain chronic diseases. Innate defense system of the human body may be insufficient for the damage caused by continued oxidative stress [1]. Quercetin and other flavonoids, have the structure to act as powerful antioxidants, and have often proven so in vitro. Quercetin, being a major constituent of the flavonoid intake, could be a key in fighting several chronic degenerative diseases [2]. Growing scientific evidence has shown adverse side effects, like liver damage and mutagenesis, of synthetic antioxidant [3]. Therefore, recently there has been an upsurge of interest in natural products as antioxidants, as they inhibit the free radical reactions and protect human body from various diseases, such as cancer and diabetes. Recent studies showed that a number of plant products including polyphenolic substances (e.g., gallocatechins, delphinidin, cyanidin, gallic acid, ellagic acid, pelargonidin and sitosterol) and various plants or herbal extracts exert potent antioxidant actions, which are very well known for their healing powers [4]. Stem bark powder is used to apply on injury caused due to axe. Stem juice is applied on goitre of human being. Paste of stem bark is applied in case of body swellings. Bark is acrid, bitter, appetizer, aphrodisiac and laxative, anthelmintic, useful in fractures of the bones, diseases of theanus, dysentery, piles, hydrocele, cures ulcers and tumors. Bark is useful in biliousness, dysmenorrhea, liver disorder, gonorrhea and it also purifies the blood. The ash of young branch is prescribed in combination with other drugs in case of scorpion sting [5]. O. gratissimum is associated with chemo-preventive, anticarcinogenic, free radical scavenging, radio protective and numerous others pharmacological use [10]. O. gratissimumis used to treat different diseases, e.g., upper respiratory tract infections, diarrhea, headache, ophthalmic, skin diseases, pneumonia, and also as a treatment for cough, fever and conjunctivitis [11,12]. Earlier reports have shown the smooth muscle contracting lipid soluble principles, and antimutagenic activity in organic solvent extracts of O. gratissimum leaves [12,13]. This medicinal plant has also potential role as antibacterial [14,15], antifungal [16,17,18], antimicrobial [19,20] and anthelmintic [21]. The aqueous leaf extract and seed oil showed anti-proliferative and chemo-preventive activity on HeLa cells. Nangia-Makker et al. reported that, aqueous extract of O. gratissimum leaves inhibits tumor growth and angiogenesis by affecting tumor cell proliferation, migration, morphogenesis, stromal apoptosis and induction of inducible cyclooxygenase (COX-2) [22]. Ursolic acid was determined in dichloromethane and ethyl acetate fractions of methanolic extract of O. gratissimum in previously published report [23]. A limited number of study have been used for the determination of quercetin in Butea monosperma bark [24] and Ocimum gratissimum leaf and Bauhinia vareigata bark [25]. The quercetin concentrations were also determined by UV spectropho-tometry [26], liquid chromatography coupled with different types of detectors [27][28][29][30][31]. Even though these analytical procedures are suitable for the detection of quercetin in samples originating from plants, they have limitations with respect to their applications in the determination of quercetin in plant samples. The reported colorimetric method lacks sensitivity and is tedious and time-consuming. Even though high performance liquid chromatography (HPLC) is a method of choice, it is limited by extensive sample clean-up and requires expensive solvents and longer periods of column stabilization. In comparison to HPLC, HPTLC is a versatile analytical technique that requires less expensive instrumentation and expertise. The present study was carried out to develop a rapid, sensitive and accurate analytical method for estimation of quercetin in bioactive fractions of plant extracts and its pharmaceutical dosage form (hepatoprotective tablet formulation) for the routine analysis of a large number of plant extract samples and their formulations. Reagents and standards Quercetin was purchased from Yucca enterprises, Wadala, Mumbai and methanol AR grade from S.d. fine-Chem Ltd., Mumbai. Plant materials Polyherbal hepatoprotective tablet was prepared by using fractions obtained from alcoholic extracts of Butea monosperma, Bauhinia variegata stem bark and O. gratissimum leaves ( Figure 1). All these ingredients were collected from Maliba Pharmacy College campus and were authenticated by Prof. Minoo H. Parabia, Department of Bioscience, Veer Narmad South Gujarat University, Surat. Voucher specimen (No: MPC/13032010/01, 02 and 03) has been deposited in the Department of Bioscience. Extraction and fractionation procedures: The dried and powdered material of each plant (500 g) was extracted with methanol at room temperature for three weeks with shaking and stirring. Combined methanolic extracts were evaporated to dryness under reduced pressure below 40°C and then dissolved in distilled water and subjected to solvent-solvent fractionation. Bauhinia variegate L.: Methanolic extract was fractionated with hexane, ethyl acetate (EtOAc) and n-butanol (n-ButOH) in the order of their increasing polarity to obtain respective fractions [18]. Ocimum gratissimum L.: Alcoholic extract was fractionated with hexane, dichloromethane (DCM) and ethyl acetate (EtOAc) in the order of their increasing polarity to obtain respective fractions [33]. Each fraction was concentrated to dryness under reduced pressure and below ( Establishment of qualitative and quantitative phytoprofile of fractionated extracts Qualitative phytochemical analysis: Each fraction was subjected to various qualitative chemical tests using reported methods to determine the presence or absence of metabolites viz., alkaloids, tannins, flavonoid, steroid, terpernoids and phenolic compounds, etc. [34]. Chemical test for flavonoids: Chemical tests were performed for flavonoids according to Macdonald et al. [35]. Quantitative phytochemical analysis Determination of total phenols: Each sample was mixed with 1 mL Folin-Ciocalteu reagent and 0.8 mL of 7.5% Na 2 CO 3 . The resultant mixture of was measured at 765 nm after 2 hr at room temperature. The mean of three readings was used and the total phenolic content was expressed in milligram of gallic acid equivalents/1 g extract. The coefficient of determination was found to be r 2 =0.992 [36]. sample and standard solution were measured at 415 nm. The mean of three readings was used and the total flavonoid content was expressed in milligram of quercetin equivalents/1 g extract. The coefficient of determination was r 2 =0.99020 [37]. Sample preparation Preparation of standard solutions of quercetin: Stock solution of quercetin was prepared by dissolving 50 mg quercetin in 100 mL of methanol (500 µg/mL). Standard solutions of concentration 0.5, 1.0, 1.5, 2.0 and 2.5 in µg/mL were prepared by dilution of the stock solution with methanol. Samples preparation from each plant extracts fractions: Accurately weighed 100 mg of each, acetone fraction of Butea monosperma, ethyl acetate and n-butanol fractions of Bauhinia variegata and dichloromethane and ethyl acetate fractions of Ocimum gratissimum was transferred to separate 10 mL volumetric flask and dissolved in 10 mL of methanol. These solutions were sonicated for 10 minutes and filtered through Whatman No. 1 filter paper to get solution containing 10 mg/mL each. Sample preparation from polyherbal tablet: Polyherbal tablets equivalent to about 100 mg of mixture of fractionated extracts of Butea monosperma, Bauhinia variegata and Ocimum gratissimum was weighed and transferred to 10 mL volumetric flask containing 10 mL methanol to get solution containing 10 mg/mL. The resulting solution was centrifuged at 3000 rpm for 5 min and supernatant was analyzed for quercetin content [38]. Instrumentation and chromatographic conditions HPTLC was performed on 15 cm × 10 cm aluminum backed plates coated with silica gel 60F254 (Merck, Mumbai, India). Standard solution of quercetin and sample solution were applied to the plates as bands 6.0 mm wide, 9.2 mm apart, and 15.0 mm from the bottomedge of the same chromatographic plate by use of a Camag (Muttenz, Switzerland) Linomat V sample applicator equipped with a 100 μL Hamilton (USA) syringe. Ascending development to a distance of 80 mm was performed at room temperature (28 ± 2°C), with toluene: ethyl acetate: formic acid, 5:4:0.2 (v/v/v), as mobile phase, in a Camag glass twin-trough chamber previously saturated with mobile phase vapour for 20 min. After development, the plates were dried with a hair dryer and then scanned at 380 nm with a Camag TLC Scanner with WINCAT software, using the deuterium lamp. The method was validated according to the ICH guidelines [11]. Calibration curve of quercetin Different volumes of stock solution (500 µg/mL) were spotted on the TLC plate to obtain concentration 0.5, 1.0, 1.5, 2.0 and 2.5 µg /spot of quercetin, respectively. The data of peak areas plotted against the corresponding concentration. Method Validation The proposed method was validated as per ICH guidelines [39]. Samples were prepared as per the earlier adopted procedure given in the experiment. Linearity and range Linearity is expressed in terms of correlation coefficient of linear regression analysis. The linearity response was determined by analyzing 5 independent levels of calibration curve in the range of 0.5, 1.0, 1.5, 2.0 and 2.5 µg /spot of quercetin respectively. The calibration curve of absorbance vs. concentration was plotted and correlation coefficient and regression line equations were determined. Precision Result of precision should be expressed as relative standard deviation (% R.S.D) or coefficient of variance (% C.V.). Repeatability Standard solutions were applied by Linomat 5 automatic sample applicator. Sample was spotted seven times for repeatability studies. The peak area obtained with each solution was measured and % C.V. was calculated. Intraday precision Mixed solution containing (1.0-2.0 µg/spot) of quercetin was analyzed three times on the same day and % C.V. was calculated. Interday precision Mixed solution containing (1.0-2.0 µg/spot) of quercetin was analyzed on three different days and % C.V. was calculated. Accuracy It was determined by calculating the recovery of quercetin by standard addition method. Recovery studies The accuracy of the method was established by performing recovery experiments at three different levels using the standard addition method. In 1 µl (1 µg/mL) of samples, known amounts of quercetin (0.5, 1.0 and 1.5 µg/spot) standard were added by spiking. The values of percent recovery and average value of percent recovery for quercetin were calculated. Limits of detection and limit of quantization The LOD and LOQ were estimated from the set of 5 calibration curves. The LOD and LOQ may be calculated as Specificity The specificity of the method was ascertained by analyzing the standard drug and extract. The spot for quercetin in the sample was confirmed by comparing the R f values and spectra of the spot with that of the standard. The peak purity of the quercetin was assessed by comparing the spectra at three different levels, viz. peak start, and peak apex and peak end positions of the spot. Optimization of mobile phase Various ratios of solvents were tried as a mobile phase and optimum mobile phase was selected was toluene:ethyl acetate:formic acid, (5:4:0.1 v/v/v). This mobile phase allowed good resolution, dense, compact and well-separated spots at R f value 0.38. Wavelength 380 nm was used for quantification of the drug. Since there is only one peak seen, is shown in Figure 3. Quantification by HPTLC Method development In HPTLC chromatogram, all tracks for standard quercetin at wave length 380 nm were shown in Figure 2. The R f value of standard quercetin was found to be 0.38 and peak area was 9726 (Figure 3). Method validation Linearity and range: The linearity was determined for both drugs at five different concentration levels. The linearity of quercetin was in the range of 0.5-2.5 µg/spot and calibration curves are shown in Figure 3. Correlation co-efficient for calibration curve of quercetin was 0.9843. The regression line equation for quercetin is as follows: y=9076x+6315 (Figure 4). Precision Repeatability: The data for repeatability are shown in Table 1. The % C.V for repeatability was found to be 0.5 (24341 ± 125) ( Table 1) Intra-day precision: The data for intra-day precision for quercetin are shown in Table 2. The % C.V of quercetin was found to be in range of 0.69%-0.97% (Table 2). Inter-day precision: The data for inter-day precision for quercetin are shown in Table 3. The % C.V of quercetin was found to be in range of 0.77%-1.50% (Table 3). Accuracy: Accuracy of the method was confirmed by recovery at three level of standard addition. Percentage recovery for quercetin was found to be in range of 97.33%-99.11%. The results are shown in (Table 4). Limits of detection (LOD) and limit of quantitation (LOQ): Limit of detection and quantitation were determined by equation LOD=3.3 × (SD/s) and LOQ=10 × (SD/s) LOD and LOQ results are shown in (Table 5). Estimation of Quercetin in Fractionated Extracts of Butea monosperma, Bauhinia variegata and Ocimum gratissimum and Polyherbal Formulation The peak purity was assessed by comparing the spectra at peak start, peak apex and peak end positions of the spot. Good correlation (R 2 =0.9843) was obtained between the standard and the samples in the range of 0.5-2.5 µg/spot. (Table 6). Acetone fraction of Butea monosperma showed eight peaks; the fourth peak R f value (0.39) was coinciding with standard R f value ( Figure 5). The concentration of quercetin was found to be 0.395 (µg/10 mg). Ethyl acetate fraction of Bauhinia variegata showed eight peaks; the fourth peak R f value (0.38) was coinciding with standard R f value of quercetin ( Figure 6). The concentration of quercetin was found to be 0.174 (µg/10 mg). n-butanol fraction of Bauhinia variegata showed six peaks, the third peak R f value (0.38) was coinciding with standard R f value (Figure 7). The concentration of quercetin was found to be 0.138 (µg/10 mg). Dichloromethane fraction of Ocimum gratissimum showed nine peaks, the third peak R f value (0.38) was coinciding with standard R f value (Figure 8). The concentration of quercetin was found to be 0.322 (µg/10 mg). Ethyl acetate fraction of Ocimum gratissimum showed seven peaks; the third peak R f value (0.39) was coinciding with standard R f value (Figure 9). The concentration of quercetin in ethyl acetate fraction of Ocimum gratissimum was found to be 0.673 (µg/10 mg) [39,40]. Polyherbal tablet of formulation showed eighteen peaks, the R f value (0.38) of seventh peak was coinciding with standard R f value. The HPTLC densitogram is shown in Figure 10. The concentration of quercetin was found to be 0.113 (µg/10 mg) [38]. Summary of validation parameters The detailed summary of validation parameters is described in (Table 7)

v3-fos

2019-04-15T13:03:52.264Z

2015-12-18T00:00:00.000Z

114530868

s2

Thermal Performance Testing of Cryogenic Multilayer Insulation with Silk Net Spacers Early comprehensive testing of cryogenic multilayer insulation focused on the use of silk netting as a spacer material. Silk netting was used for multiple test campaigns that were designed to provide baseline thermal performance estimates for cryogenic insulation systems. As more focus was put on larger systems, the cost of silk netting became a deterrent and most aerospace insulation firms were using Dacron (or polyester) netting spacers by the early 1970s. In the midst of the switch away from silk netting there was no attempt to understand the difference between silk and polyester netting, though it was widely believed that the silk netting provided slightly better performance. Without any better reference for thermal performance data, the silk netting performance correlations continued to be used. In order to attempt to quantify the difference between the silk netting and polyester netting, a brief test program was developed. The silk netting material was obtained from Lockheed Martin and was tested on the Cryostat-100 instrument in three different configurations, 20 layers with both single and double netting and 10 layers with single netting only. The data show agreement within 15 - 30% with the historical silk netting based correlations and show a substantial performance improvement when compared to previous testing performed using polyester netting and aluminum foil/fiberglass paper multilayer insulation. Additionally, the data further reinforce a recently observed trend that the heat flux is not directly proportional to the number of layers installed on a system. Introduction Early in the development of multilayer insulation (MLI) systems, multiple spacer materials and reflector materials were tested. Different vendors settled on different configurations for manufacturing including using aluminum foil, single aluminized mylar, and double aluminized mylar for reflectors and Dacron tufts, polyester netting, silk netting, and fiberglass paper for spacers [1][2][3][4]. For "In-space" applications, eventually double aluminized mylar was settled upon as a consensus reflector, however no consensus in spacer material was developed [5][6][7][8]. In the meantime, Lockheed was awarded a contract by NASA for a series of tests to standardize the expected performance of MLI to be used for engineering design. In performing this testing, they used fiberglass paper and silk netting as different spacer materials [8]. As NASA's exploration research continued through the 1980s and 1990s, silk netting was abandoned in favor of the less expensive polyester netting (often referred to as Dacron). Fox, Kiefel, et.al. reported that in 1993, the cost of silk netting was seventeen times higher than polyester netting [9]. While the authors were unable to verify that data point using current prices, they were able to verify a large discrepancy of more than five times the cost, however, the grade of silk netting was not confirmed to be suitable for use in high performance MLI systems. NASA completed multiple test campaigns using a combination of double aluminized mylar and polyester netting [8][9][10][11]. The data from these tests always had a significantly higher heat load than was predicted from the correlations previously developed by Lockheed. Lockheed continued to use and perform testing on silk netting due to its established (and presumed slightly better) performance, as the extra cost for small dewars used on science missions was not as significant as the performance benefit [12]. This situation left the aerospace industry in the quandary of having tank-applied MLI data for double aluminized mylar and polyester netting, yet relying on a basic foundational data set built upon double aluminized mylar and silk netting calorimeter testing. There were no comparable data sets between the two material sets and no clear way to assess if the equations developed using silk netting were appropriate for use with polyester netting. At the time there were no means of achieving the desired comparison in a cost effective manner. Experimentation In order to measure the thermal performance of various insulation systems, NASA Kennedy Space Center's Cryogenic Test Laboratory uses liquid nitrogen boil-off calorimetry for a variety of its instruments. This test program used the Cryostat-100 calorimeter, which is well documented elsewhere [13][14][15]. Cryostat-100 testing yields absolute thermal performance of the multilayer insulation systems in terms of heat load (Q). The heat load can then be normalized to the logarithmic mean surface area and converted to heat flux (q) in accordance with ASTM C1774, Annex A1. [16] The objectives of the testing were to measure and compare the thermal performance of silk netting to previously tested polyester netting. Based on these comparisons, the relative performance between the silk netting, polyester netting, and analytical models can be ascertained. The approach to carrying out the tests was straight forward. The test blankets were built out of previously tested double aluminized mylar sheets (care was taken to ensure no degradation due to previous handling had occured), which were interleaved between the silk netting. Each layer was individually overlapped in order to minimize the effect of seam performance on the test data. A laboratory standard evacuation and bakeout process, including multiple purge cycles with gaseous nitrogen, was performed on all test coupons. In order to maximize utilization of the test time, multiple different warm boundary temperatures (WBT) were used (293 K, 305 K, and 325 K) for better comparison and parameterization of the warm boundary temperature on the first test only. Both high vacuum [~10 -6 torr (~10 -4 Pa)] and degraded vacuum [~10 -3 torr (~10 -1 Pa)] tests were run. In one case no vacuum [760 torr (101 kPa)] tests were also run. While the high vacuum test is mainly of interest for comparisons with the various performance models discussed above, the other vacuum levels serve as points of interest for comparisons with other types or applications of thermal insulation systems. All tests were conducted in cold vacuum pressure (CVP) conditions with a residual gas of nitrogen. The complete test matrix is shown in Table 1. The first test had 20 layers (n) of double aluminized mylar, each separated by two layers of silk netting. The second and third test had 20 and 10 layers of double aluminized mylar, respectively, that were each separated by a single layer of silk netting. This sequence provided the effect of lowering the layer density (z) from 0.85 layers/mm to 1.3 layers/mm. Type E thermocouples were placed on selected layers throughout the blankets to provide temperature profiles throughout the thickness of the MLI system. Further details on the preparation steps and nomenclature are found in ASTM C740. [17] Results and Discussion The silk netting results are given in Table 2 and The thermocouple data for the layers showed fairly typical temperature profiles through the blanket. The temperature profiles for A177 are shown in Figure 2. As would be expected with radiation heat transfer, the temperature gradient is much steeper closer to the cold boundary. All three warm boundary temperature tests are shown for comparison. The profile gives a curved shape, somewhere between a 2 nd and 4 th order polynomial. The silk netting results are compared to polyester netting in both heat load and mass. Table 3 shows that the silk netting when used as a single spacer weighed slightly less than the comparable coupons made with polyester netting [18]. In Table 4, the thermal performance is compared to previous testing performed on Cryostat-100 using many different materials. The silk netting is dramatically better than other material types, with heat fluxes as low as half of that for normal polyester netting. Part of this difference can be attributed to slightly different layer densities and thicknesses; however, the silk netting is still dramatically better across the board, even when accounting for the minor differences. Further comparisons of the current data with previous silk netting testing by Lockheed [19] and Stochl [20] are shown in Figure 3. The test data compare well with the previous test data. The current data are slightly better performing than the Stochl data due to the lower layer density as expected. The current data also projects in line with the hallow dot, which was also tested at 290 K. This excellent agreement builds confidence in the calorimeter data. Also of note is the increasing trend shown on the right side of Figure 3 which shows a slightly increasing q*N product (heat flux multiplied by number of layers) with increasing number of layers. This effect was noted by Fesmire and Johnson [17] and is further reinforced here: the heat flux is not inversely proportional with number of layers in a real system as is assumed by most analytical models. The data from this test series is plotted against the previously published data for comparison in Figure 4. Both the LB-MLI and the silk netting show much less propensity for increasing q*N with increasing number of layers, at least on the scale of the previously reported data. Table 5 below. This work was completed in 1974 and since then a number of tests and analyses have produced variants of coefficients for different materials and layups of the blankets. The heat flux for a silk net spacer system with double aluminized Mylar is lower than that for a system utilizing Dacron netting. The comparison between the predicted and measured values is quite good (except for the 10 layer case, which is different by 40 %). In some cases, the measured data is lower than the predicted value. When modelling insulation systems utilizing these equations derived from calorimeter tests, a number of considerations related to layer density control, end effects and attachment techniques can be found in the work of Nast, Frank, and Feller [21]. Conclusions Silk netting was tested on the absolute cylindrical boiloff calorimeter, Cryostat-100, at the NASA Kennedy Space Center between warm boundary temperatures of 293 K, 305 K, or 325 K and a cold boundary temperature of 78 K. The silk netting material was provided by Lockheed Martin from remnants of legacy flight programs. The test results show a dramatically lower heat load with a minimally lower mass than using polyester netting or fiberglass paper but with the same general trend of q*N increasing when increasing number of layers as other MLI systems. The results of this series of test showed that the double silk net did better than single net as expected. Equation 4-14 from reference 7 under predicted the performance by 0-10% for the double net (A177). For the single net the equation over predicted by 15-40% (A178/A179). The mass difference between polyester netting and silk netting is minimal. The agreement of the current test data with previous testing further strengthens the use of the benchmark thermal performance data from Cryostat-100 along with the recently noticed trends from it. Due to the cost and availability of silk in general, silk netting may be prohibitive for most applications and spacer materials of polyester netting and fiberglass paper will still be used. However, these test data confirm that there is a significant difference between the silk netting and polyester netting. Silk netting should still be considered in cases where optimum levels of performance are needed and the benefits outweigh the costs.

v3-fos

2016-06-17T02:08:34.719Z

2015-12-15T00:00:00.000Z

2013788

s2

Targeted Analysis Reveals an Important Role of JAK-STAT-SOCS Genes for Milk Production Traits in Australian Dairy Cattle The Janus kinase and signal transducer and activator of transcription (JAK-STAT) pathway genes along with suppressors of cytokine signalling (SOCS) family genes play a crucial role in controlling cytokine signals in the mammary gland and thus mammary gland development. Mammary gene expression studies showed differential expression patterns for all the JAK-STAT pathway genes. Gene expression studies using qRT-PCR revealed differential expression of SOCS2, SOCS4, and SOCS5 genes across the lactation cycle in dairy cows. Using genotypes from 1,546 Australian Holstein-Friesian bulls, a statistical model for an association analysis based on SNPs within 500 kb of JAK-STAT pathway genes, and SOCS genes alone was constructed. The analysis suggested that these genes and pathways make a significant contribution to the Australian milk production traits. There were 24 SNPs close to SOCS1, SOCS3, SOCS5, SOCS7, and CISH genes that were significantly associated with Australian Profit Ranking (APR), Australian Selection Index (ASI), and protein yield (PY). This study supports the view that there may be some merit in choosing SNPs around functionally relevant genes for the selection and genetic improvement schemes for dairy production traits. INTRODUCTION Hormones and cytokines play an essential role in the growth and differentiation of the mammary gland. This is reflected in differential expression of genes during mammary gland development, and across different stages of lactation (Alluwaimi and Cullor, 2002). The cellular responses to cytokine signals are controlled by complex networks of intracellular signaling pathways which, despite the diversity of cytokines and growth factors, are highly conserved. The Janus kinase and signal transducer and activator of transcription (JAK-STAT) pathways are particularly important during lactation. Four different JAK kinases, namely JAK1, JAK2, JAK3, and TYK2, and seven different STAT members, namely STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6, have been described (Imada and Leonard, 2000). Various members of these gene families are key regulators of alveolar proliferation and differentiation in the mammary gland. Gene deletion analysis in mice confirms the mandatory requirement of the JAK-STAT signaling pathway in mammary gland development and lactation (Hennighausen et al., 1997;Yamaji et al., 2013). A key regulatory feature of this pathway is a family of genes that encode a group of negative inhibitors named suppressors of cytokine signalling (SOCS; Linossi et al., 2013;Linossi and Nicholson, 2015). The SOCS family is required for the attenuation of cytokine signals in mammary epithelial cells, and acts to limit proliferation through a negative feedback mechanism. The SOCS family is comprised of eight members named SOCS1 to SOCS7 and CISH (cytokine-inducible SH2), and are characterized by a central Src-family homology 2 (SH2) domain and C-terminal SOCS box (Jegalian and Wu, 2002). SOCS1 and SOCS2 are required for the attenuation of the prolactin receptor signaling pathway during pregnancy and lactogenesis (Ramanathan et al., 2008;Riley et al., 2010;Wei et al., 2013). SOCS3 has been credited with control of the regulation of involution of mammary tissue through programmed cell death and tissue remodeling, initiated after the termination of lactation (Sutherland et al., 2007). Differential expression of SOCS3 relative to controls correlates with failed lactation in prolactin receptor knockout and galanin knockout mice (Naylor et al., 2005). The present study examined gene expression of five SOCS family members across the lactation cycle in dairy cows. Additionally, we report the design and validation of a dairy trait association model using a targeted JAK-STAT pathway candidate gene approach, primarily focusing on the SOCS family genes. Animal Selection and Collection of Mammary Tissue Five multiparous Holstein-Friesian cows entering their third or fourth lactation were evaluated for milk production and had an Australian selection index (ASI) value in the top 25% of the Australian herd, and a previous lactation production range of 5,300-8,800 L per lactation. Mammary tissue biopsy samples were collected as described (Sheehy et al., 2009) serially from the five animals at three different time points either 5 days following termination of milking from the previous lactation (involution sample), approximately 20 days (8-23 days, average 17.8 days) prior to calving (pregnancy sample), and approximately 30 days (30-35 days, average 33.2 days) following calving (lactation sample). At the time of tissue sample collection, each animal was evaluated for body condition score. No pathogens were observed in any milk samples (as determined by culture) at the time of biopsy and the absence of negative energy balance was determined by blood metabolite analysis. All work with animals was conducted in accordance with the guidelines of the Animal Research Act, NSW, Australia, and was approved by the Animal Ethics Committee of the University of Sydney. RNA Extraction and Expression Studies using qRT-PCR Approximately 100 mg of mammary tissue from each biopsy was treated with 1 ml of Tri-reagent (Sigma-Aldrich Pty. Ltd., NSW, Australia) and the RNA extracted was quantified by spectrophotometry. Approximately 100 µg of purified RNA was further purified by RNeasy column including an on-column DNase1 treatment (QIAgen, Doncaster, VIC, Australia). Single stranded cDNA was synthesized from 2 µg of RNA according to the manufacturer's protocol using SuperScript III (Invitrogen Aust. Pty, Melbourne, VIC, Australia). Oligonucleotide PCR primers were manufactured by a commercial manufacturer (Sigma-Aldrich Pty. Ltd., NSW, Australia). The details of the primers for the five members of SOCS genes and the house keeping control gene used for relative quantification are summarized in Table 1. PCR was performed in the presence of Sybr Green and monitored for real time analyses using a Rotorgene 6000 instrument (QIAgen, Doncaster, VIC, Australia), over 35 cycles of 95 • C, 30 s, 60 • C 30 s, 72 • C 1 min. Each gene was analyzed for 15 different samples (n = 5 in each stage of lactation cycle) in triplicate and relative quantification was measured against a standard housekeeping gene, namely Large Ribosomal Protein (RPLP0). Statistical analysis to compare expression levels across the three stages of lactation was undertaken using a balanced ANOVA, incorporating cow as a blocking term, with statistical significance between the comparisons determined by a protected Fisher least significance difference (GenStat; VSN International Ltd., Hemel Hempstead, UK). Selection of SNPs Genotyping data from 1,546 Australian Holstein-Friesian bulls was available for analysis as previously described (Khatkar et al., 2007(Khatkar et al., , 2008. The SNPs within 500 kb of all SOCS genes, and genes associated with the JAK-STAT pathway, were collated for analysis. Each set of SNPs was analyzed for association against the range of dairy traits, as quantified by the Australian Breeding Value (ABV) for these milk production traits, obtained from the Australian Dairy Herd Improvement Scheme 1 . The traits included were 1 http://adhis.com.au/ the Australian Profit Ranking (APR), ASI, protein yield (PY), protein percentage (PP), milk yield (MY), fat yield (FY), and fat percentage (FP), and were analyzed for all 1,546 bulls. The set of SNPs in the gene region was considered as a genotype, and this was conducted by concatenating the number of copies of the minor allele (e.g., "0-2-1" for a set with three SNPs). Then the associations between the genes (as assessed by constructed genotypes) and the traits were evaluated through fitting general linear models in GenStat, with the significance of each gene being assessed with an F-test. A backward elimination procedure was used to arrive at a minimum number of significant genes in the model, starting from a full model where all eight SOCS family genes, and 21 genes from JAK-STAT pathway genes were included. Genes were removed from the model using a threshold of P > 0.05. At the conclusion of the elimination procedure, the P-values, the contribution to total variation (R 2 ) and the adjusted contribution (adjusted R 2 ) for all genes was obtained. The procedure was repeated using SOCS genes only. A simulation model was then developed to validate the results obtained for JAK-STAT-SOCS genes. The loci used for this model were selected randomly from the dataset. Following the same procedure, SNPs were selected within a distance of 500 kb of each locus. Subsequently, each set of SNPs was analyzed and the least significant were removed. One thousand permutations of this procedure were performed with SNPs selected at random from different locations each time. For comparison with results from the JAK-STAT-SOCS results, the distributions of P-values, the contribution to total trait variation and the adjusted variation were plotted for the 1000 permutations. This permutation analysis was conducted with a user-written procedure in R. 2 SOCS Gene Expression qRT-PCR was used to analyze SOCS1-SOCS5 gene expression across the three stages of the lactation cycle. When considering the relative levels of gene expression compared to the control gene (RPLPO), i.e., the 'normalized ratio, ' expression of SOCS2 was notably greater, followed by SOCS5 (Figure 1). Overall levels of SOCS1, SOCS2, and SOCS3 were much lower. The patterns of expression were either, increased in lactation compared to the other stages (SOCS2, SOCS4, SOCS5), decreasing from pregnancy to involution (SOCS1), or increasing from pregnancy to involution (SOCS3). Statistically, there was no difference seen between animals (n = 5) at each stage (all P > 0.05), but a difference was seen between lactation stages for SOCS2 (P = 0.057, suggestive), SOCS4 (P = 0.007) and SOCS5 (P < 0.0001) based on ANOVAs. Further analysis based on Fisher's protected LSD revealed that SOCS2, SOCS4, and SOCS5 were differentially expressed during lactation compared to the other two stages, but not when levels during pregnancy were compared to expression levels during involution. Analysis of Genotyping Data A total of 98 SNPs were identified from the neighborhoods of eight SOCS genes and 21 JAK-STAT pathway genes, and were then collated into a group of SNPs representing all 29 genes. A preliminary analysis of SNPs around each gene revealed a significant association with each of the seven dairy traits tested. Subsequently, a general linear model was fitted to calculate the total variation explained by all SNPs across all the genes. After removing less significant predictors using a backward elimination procedure, the final gene-SNP combinations found 2 https://www.r-project.org/ to be significantly associated with each dairy trait were collated (Tables 2 and 3). When all genes from JAK-STAT pathways, including SOCS genes, were considered, the optimized set accounted for 28.1% of the total adjusted variation for APR. The range for the other six traits was from 20.8% for ASI down to 10.3% for FP (Table 4). A model was also developed based only on SNPs around eight members of the SOCS family genes. Here, there was a more conservative removal of SNPs during elimination. The final combinations accounted for 23.7% of the adjusted total variation for APR ( Table 4). The range for the remaining traits was from 20.8% for PY to 6.2% for FP. Notably, the total adjusted variation explained for PY was equivalent for both the JAK-STAT pathway genes together and the SOCS genes alone. The effects of various SNP combinations around the eight members of SOCS genes for each dairy trait were estimated. In general, there was a greater number of combinations with a positive rather than a negative effect, especially for PY, FY, FP, and MY. In contrast, there was a greater number of SNP combinations with a negative rather than positive effect on PP. Simulation Model A simulation model was developed to mimic and validate the results of the SOCS association model. With 1000 permutations for each trait, this resulted in three or fewer significantly associated loci for any of the traits, i.e., the probability of randomly identifying these loci was 0.003. An example of the comparative analysis between the simulation and SOCS model for APR is depicted in Figure 2. This shows the results of the observed data, as indicated by the red arrows, in comparison to the distribution of random SNP-gene associations. For all measures, the observed values are at the extreme end of the distributions, confirming the true statistical significance of these associations. DISCUSSION Genes that are functionally relevant to any biological process are often differentially regulated, both temporally and between physiological states. Members of the JAK-STAT pathways, and SOCS genes in particular as negative regulators, play an important role in mammary gland growth and development as mediators of hormone and cytokine signals (Chen et al., 2000;Alluwaimi and Cullor, 2002;Jegalian and Wu, 2002). In the present study, gene expression analysis identified that SOCS2, SOCS4, and SOCS5 genes were differentially expressed in mammary tissue across the lactation cycle in dairy cows. Focusing on the JAK-STAT pathways, a model was developed for JAK-STAT pathway genes, and separately for SOCS genes, to test association with dairy traits. The model revealed significant associations with genetic variants linked to some JAK-STAT pathway members, and specific members of the SOCS family. Further, the simulation study by randomly selecting genes as trait predictors indicated that the genes detected were indeed strongly associated, given the extremes they had on the distributions on all the metrics (Figure 2). Gene expression analysis presented here showed that there is a relatively high level of expression of SOCS2, SOCS4, and SOCS5 in lactation when compared to the other two stages, whereas by comparison, SOCS1 and SOCS3 expression levels were 10-fold lower in relative terms, and showed a pattern in which expression during lactation was intermediate between that seen in the other two stages. The patterns of SOCS1 and SOCS2 expression are consistent with those measured in a study of photoperiod effects on dairy cattle (Wall et al., 2005;Dahl, 2008). In that study, the cows showed the highest expression of SOCS1 during pregnancy and the lowest expression during involution, and SOCS2 expression was highest during lactation. The pattern of SOCS3 expression was different between the studies, but in our study there was a very low level of SOCS3 expression and more variable. However, prolactin levels are raised during peak lactation and lower levels of SOCS3 expression may be expected, as noted in another study by those same authors (Wall et al., 2006). Other research has suggested a primary role of SOCS3 in apoptosis and tissue remodeling during involution (Sutherland et al., 2007), and SOCS3 activity may also reflect leukocyte infiltration and activation during this stage. Expression of SOCS mRNA levels in the liver of dairy cattle are also elevated during lactation, consistent with insulin effects (Winkelman et al., 2008;Cummins et al., 2012). A similar pattern was seen in the present study in the mammary tissues of cows during lactation when compared to pregnancy and involution. Although SOCS3 expression was not influenced by genotype (Cummins et al., 2012), variation in SOCS2 expression could potentially affect milk production traits. There have been several studies that have used SNP selection methods based on co-expression or other functional parameters to assess genetic contributions to production traits in cattle [see e.g., (Reverter and Chan, 2008;Moser et al., 2009Moser et al., , 2010Raven et al., 2014;Widmann et al., 2015)]. Here, eight candidate genes from the SOCS family and 21 regulatory JAK-STAT pathway candidates were selected for analysis based on the generic function of these genes and their potential for involvement in biological processes relevant to the lactation cycle. JAK-STAT pathways act directly in providing intracellular signals that coordinate gene transcription in response to a wide range of cytokines and hormones. Some of these pathways have specific roles in mammary development and the lactation cycle, while others provide signals that are common in cellular responses. The strategy employed here was to capture family members and regulators without specifically selecting genes from within the family based on a priori knowledge. Some emphasis was placed on SOCS genes on the basis that negative regulators of intracellular pathways have the potential to have high impact. That is, in a network of interactions, negative regulators of key pathways are often represented as substantial nodes. Using this strategy, significant associations were identified, initially for SNP combinations from both the JAK-STAT members and SOCS family genes together, and subsequently for SOCS family genes alone. Interestingly, the genes neighboring the SNP combinations that were selected from the backward elimination procedure did not align with those that may have been expected, either from the SOCS expression data, or known involvement in lactation studies. Specifically, SOCS2 and STAT5 genes were not represented in the model. This is in contrast to the model developed by Raven et al. (2014), who analyzed SNPs associated with genes from three annotated pathways related to lactation. This most likely reflects the larger number of genes and SNPs included in the latter study. The analysis suggests that the association with milk production traits contributed by the JAK-STAT pathway was significantly influenced by the inclusion of SOCS genes. The range of observed variation in the trait attributed to the optimized model for all JAK-STAT related SNPs was between 10 and 28%, and for SOCS genes alone was 6-24%. Biologically, this suggests that the JAK-STAT-SOCS genes play a significant role in determining performance in milk production and composition. The utility of SOCS gene-related SNPs in the model, may relate to the significant biological effect exerted by negative regulators of important functional pathways. The absence of SOCS2 in the model is likely to be explained by SNP allele frequencies, but could also reflect functional redundancy or cross talk in the JAK-STAT pathways. To summarize this study, gene expression analyses supported an active role for members of the SOCS family of genes during the lactation cycle. SNPs linked to the SOCS and JAK-STAT pathway genes were useful in developing a model that accounted for significant variation in a number of important dairy traits. This study supports the view that there may be some merit in utilizing the SNPs around these genes for selection and genetic improvement schemes for dairy production traits. FUNDING This work was funded in part by a scholarship to Sondur J. Arun from the Cooperative Research Centre for Innovative Dairy Products.

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A Cross-Sectional Study of Pesticide Use and Knowledge of Smallholder Potato Farmers in Uganda In response to increased pest and disease problems, potato farmers use pesticides, which could raise environmental and health concerns. This study sought to promote proper and safe pesticide-handling practices by providing data needed to guide pesticide regulation policy and training for extension staff and farmers. A household survey was conducted in three major potato-growing agroecological zones of Uganda. Two hundred and four potato farmers were interviewed about the type and source of pesticides they use in potato cultivation, the frequency of applications, the use of protective clothing, and cases of pesticide poisoning. The types of pesticides used in potato were fungicides (72%), insecticides (62%), and herbicides (3%). Overall, use of personal protective equipment was low, that is, gumboots (73%), gloves (7%), face masks (16%), and long sleeve shirts (42%). Forty-three percent of farmers who applied pesticides reported having experienced skin itching, 25% skin burning sensation, 43% coughing, 60% a runny nose, 27% teary eyes, and 42% dizziness. An IPM approach involving only moderately to slightly hazardous pesticides when pest and disease incidence has reached economic injury levels and by considering all safety measures during application and storage would be environmentally recommendable and result in reduced health risks. Background Potato (Solanum tuberosum L.) is an important food security and cash crop for smallholder farmers in midelevation and highland areas of Uganda with an annual production of 0.8 million tonnes, produced on approximately 112,000 ha [1]. It ranks 5th among the food crops grown in Uganda after sweet potato (Ipomoea batatas (L.) Lam.), maize (Zea mays L.), cassava (Manihot esculenta Crantz), and banana (Musa spp.). Most (71%) of the potato produced is for sale as ware potato in local markets with limited formal and informal cross border trade to neighbouring countries of Rwanda and Democratic Republic of Congo [2]. Pests and diseases are among the most important constraints to potato production in Uganda. If not adequately controlled, yield losses from fungal and bacterial diseases alone can reach up to 100% [3]. Yield losses from insect pests in Uganda have not been quantified although their severity and damage is feared to become important with global warming [4,5]. The absence of environmentally friendly approaches for management of potato pests and diseases has left farmers with no option other than use of chemical pesticides on a routine basis. Farmers get exposed to toxic pesticides by eating while spraying, entering into freshly sprayed fields, inhalation, and direct contact of the skin with any form (liquid, powder, or aerosol) of pesticides [6]. The Food and Agriculture Organization (FAO) Code of Conduct for Pesticide Use is most of the time not adhered to in many developing countries [7][8][9][10]. Misuse of pesticides can lead to illness which reduces the availability of family farm labour and increases the resistance of pests to pesticides due to low pesticide rates and the frequent use of the same active ingredients [11]. In Uganda, the impact of pesticides on human health, environment, and farm productivity among potato farmers has never been estimated. However, isolated cases of farm workers using pesticides to commit suicide do occur. Ngowi et al. [12] observed that it is a challenge to estimate all costs to human health (medical expenses, recuperation costs, transport costs, and labour losses) and the environment (ecosystem degradation) resulting from pesticide use. Indiscriminate use of pesticides, however, raises a number of environmental and health concerns including soil and water pollution and human and livestock diseases among others. For instance, high pesticide residue levels have been reported in water bodies and foods. Evidence of pesticide poisoning, unsafe pesticide-handling practices, and inadequate use of personal protective equipment has been reported among farmers of horticultural crops in Uganda [13] and coffee (Coffea arabica L.) in Jamaica [14]. In 2002, 103 cases of pesticide poisoning leading to four deaths were registered in Poland [15]. However, there are barely any statistics in Uganda for cases of agricultural pesticide poisoning since most farmers are rural and do not seek treatment from hospitals. Even if treatment was sought, it is more likely that health care providers are not adequately trained to make proper diagnosis of pesticide-related illnesses as has been observed in Ghana [9], Ivory Coast [10], Tanzania [16], and South Africa [17]. Some programs such as the Pesticides Initiative Programme that promotes safe pesticide use especially in fresh export produce do exist in Uganda but no such program is known to exist for nonexport produce like potato [18]. The lack of knowledge or training in safe pesticide-handling practices, however, exposes both the environment and potato farmers to the negative effects of pesticides. There is a need to set up policies and programs to promote the safe use of pesticides. Adherence to the international food safety standards will increase not only market avenues of potato but also household income. Integrated Pest Management (IPM) strategies for potato pests ought to be promoted in Uganda to reduce the overall use of pesticides. This study sought to (i) identify the types of pesticides used in potato farming systems in Uganda, (ii) document the self-reported symptoms of pesticide poisoning, and (iii) describe pesticide-handling practices among potato farming households. Study Area. Six subcounties (Muko, Nyarusiza, Kapchesombe, Wanale, Kibalinga, and Kakabara) in six major potato-growing districts of Uganda (Kabale, Kisoro, Kapchorwa, Mbale, Mubende, and Kyegegwa), respectively, were purposely selected for this study. District selection was based on representation of the three most important potatogrowing agroecological zones of Uganda, that is, southwestern highlands (Kabale and Kisoro), eastern highlands (Mbale and Kapchwora), and Lake Albert Crescent (Mubende and Kyegegwa) districts. One subcounty in each district that was observed by the agricultural extension officers to grow most of the amount of potato was purposively selected. Verbal informed consent was sought from the respondents prior to the beginning of the interview. Respondents were informed of their right to refuse participation and to withdraw from the study at any given time. The confidentiality of the collected information was also assured. Sampling Procedures. Farm household selection was random and involved stopping at regular intervals (1-5 km) along main roads traversing each subcounty. Respondents were household heads or any adult household member who had grown potatoes in the previous cropping season and was present at home at the time of the study. Two hundred and four potato farmers (34 per district and subcounty) verbally consented to be interviewed. A structured questionnaire was used to interview farmers. The questionnaire was written in English and administered in English and local languages (Luganda, Kupsabiny, Lumasaaba, Rutooro, Rukiga, and Rufumbira) by agriculture extension officers and research assistants under the supervision of the first author. The interviews covered the following themes: (1) the type and source of pesticides used in potato farming, (2) frequency of pesticide application in a cropping season, (3) the use of protective gear when applying pesticides, (4) any cases of pesticide poisoning experienced by potato farmers, and (5) individual knowledge on the negative effects of pesticide use on the environment among others. Data for this household baseline survey were collected between August and September 2013. Statistical Analysis. Raw data were coded, entered, and analyzed using the statistical program SAS V.9.2 for Windows (SAS, Cary, NC, USA) [19]. For each agroecological zone, a chi-square test was used to test whether the obtained data and their differences were significant or whether variables were related to each other. The significance levels were set at ≤ 0.01, ≤ 0.05, and ≤ 0.1. The results were then presented in tables separately for each agroecological zone, from which inferences were drawn. Sociodemographic Profile. Of the 68 respondents that were interviewed per agroecological zone, the number of females and males was not significantly different at ≤ 0.1 for all the three agroecological zones (Table 1). Respondents were mainly between the ages of 31-64 years, followed by the youth (18-30 years). Most of the respondents had attended school for 1-7 years with the Lake Albert agroecological zone having the largest proportion of farmers (72%) in this category. Pesticide Groups Used by Potato Farmers. All farmers in the southwestern highlands used insecticides and fungicides on potato followed by farmers in the eastern highlands ( Table 2). Pesticides were significantly least used in the Lake Albert Crescent with only 16% and 12% of the farmers using fungicides and insecticides, respectively. Generally, herbicides were used by very few farmers (3%) and no farmer in the southwestern highlands used herbicides. The use of both fungicides and insecticides by a large percentage of farmers indicates that fungal diseases specifically late blight and insect pests are perceived to be equally important. 1.01 ns 6.28 * * 3.77 * * * * , * * , and * indicate statistical significance at ≤ 0.01, ≤ 0.05, and ≤ 0.1, respectively. ns: not statistically different at ≤ 0.1. = number of respondents. SWH: southwestern highlands; EH: eastern highlands; LAC = Lake Albert Crescent. Active Ingredients and Toxicity Classes of Pesticides Used by Potato Farmers. The classification of pesticide active ingredients in this study followed the WHO Recommended Classification of Pesticides by Hazard and Guidelines to Classification 2009 [20]. Most (54.9%) of the fungicides used belonged to the WHO class U (unlikely to present acute hazard in normal use) while 28.9% of the insecticides belonged to the WHO class II (moderately hazardous) ( Table 3). Only one highly hazardous (Class 1b) insecticide was used by very few (0.5%) farmers. Due to the lack of formal seed potato suppliers, farmers often save potatoes from the previous own harvest for use as seed in the next cropping season. To control the potato tuber moth Phthorimaea operculella (Zeller) during storage, farmers used malathion in southwestern highlands. Some farmers (2.5%) did not know the name of the fungicide they used since it was sold to them in unlabelled polythene bags. Nearly equal number of farmers used fungicides (75.1%) and insecticides (76.5%). However, herbicide use was very low among potato farmers (5.4%). Highly hazardous pesticides have been reportedly used in many low-and middle-income countries like Peru and Ecuador [8], Philippines [21,22], Cambodia [23], and Kenya [24]. In Uganda, moderately hazardous pesticides like lambda-cyhalothrin, dimethoate, chlorpyrifos, and cypermethrin have been used in cowpea (Vigna unguiculata L. Walp.) [25]. Jensen et al. [23] urged that farmers often think that broad spectrum pesticides are more effective at controlling pests and diseases and therefore the widespread use of highly and moderately hazardous pesticides. Frequency of Pesticide Application. The number of pesticide applications per season of three months was highest in the eastern highlands for fungicides (5.3 ± 0.4) and insecticides (4.2 ± 0.3) but lowest in Lake Albert Crescent for both fungicides (2.2 ± 0.3) and insecticides (1.4 ± 0.3) ( Table 4). Some farmers applied fungicides up to 18 times and insecticides up to 12 times per cropping season. Frequencies of pesticide application of twice a week have been reported in other crops like tomato (Lycopersicon esculentum Mill.) in Uganda [13]. Other countries in Africa reporting heavy use of pesticides include Ghana where tomato farmers sprayed up to 12 times per season [9] and Tanzania where vegetable farmers sprayed up to 16 times per cropping season [12]. Spray frequencies observed in this study are relatively low and may be economical [3]. An Integrated Pest Management approach that has been specifically developed to control economically important potato pests in Uganda involving pesticide applications only when pest and disease incidence has reached economic injury levels would be more sustainable and economically friendly to the environment and hence would also reduce health risks of farmers and consumers. Calendar spraying has also been reported to reduce pests' natural enemies and increase the pest burden [26]. In a related study, we also noted that potato farmers lack general knowledge on the existence of other pest management strategies like the use of intercropping, early planting, early harvesting, use of trapping devices, sanitation, crop rotation, biopesticides, and biological control agents in an Integrated Pest Management approach [27]. IPM for both insect and disease management has to be region specific. IPM for disease (bacterial wilt, viruses, and late blight) management also involves a combination of a number of approaches including use of resistant varieties, clean seed, fungicides, cultural practices (planting at high altitude, crop rotation), and farmer education [28]. In the Andean region of Peru, for instance, IPM for insect management involving the use of plastic barriers, attract-and-kill, and one application of a low-toxic insecticide has been shown to be effective in preventing Andean potato weevils (Premnotrypes spp.) infestations, managing potato tuber moths (Phthorimaea operculella (Zeller) and Symmetrischema tangolias (Gyen)), and controlling flea beetles (Epitrix spp.) [29]. In the Republic of Yemen, P. operculella was the only economically important potato pest which could be controlled by using healthy uninfested seed and biological control both under field and storage conditions [30,31]. There is therefore a need to bring to the attention of farmers the existence of more environmentally friendly pest management methods that can increase profit margins. Sources of Pesticides and Pesticide Information. Most farmers received information about which pesticide to use from other farmers (45%) and only 2% of the farmers received information directly from agricultural extension officers (Table 5). When it came to the doses of pesticides to use, farmers in the southwestern highlands and eastern highlands relied mostly on their own previous experience and reading instructions on the pesticide label (38% and 55%, resp.) while in Lake Albert Crescent, most farmers (50%) relied on pesticide retailers. On average, agroinput shops were the primary source of pesticides in the three agroecological zones (60%), followed by general household merchandise shops (40%). Other farmers (1%) represented a minor role as source of pesticides. Pesticides were dispensed in quantities of 0.8 ± 0.1 to 8.2 ± 3.5 Kg or litres and it was common to find small quantities of 3.08 ns 1.67 ns 2.12 ns * * * , * * , and * indicate statistical significance at ≤ 0.01, ≤ 0.05, and ≤ 0.1, respectively. ns: not statistically different at ≤ 0.1. SWH: southwestern highlands; EH: eastern highlands; LAC: Lake Albert Crescent. The sample size ( ) for each percentage is indicated in parenthesis. fungicides in unlabelled plastic polythene bags. All farmers used knapsack sprayers to apply pesticides. Knowledge of Pesticide Toxicity Labels. Less than half of the respondents could read the pesticide labelling across the three agroecological zones; almost all respondents (91%) were not able to explain the toxicity label ( Table 6). The relatively low level of education by the majority of the farmers (i.e., <7 years of school) may explain the inability of farmers to read pesticide labels which are often written in English. For the few farmers who knew how to read but did not read the pesticide label, it could be due to reluctance or ignorance of its presence. It should be noted that there is no legislative control in Uganda requiring sellers and users of pesticides to be formally trained. This weakness on the part of the pesticide regulatory bodies may explain the presence and use of highly hazardous (Class 1b) insecticides such as dichlorvos pesticides on the market and the reluctance to use personal protective equipment during pesticide application reported in Table 8. About a third of the farmers mentioned as negative effects of pesticides symptoms of illness, reduced soil fertility, reduction of beneficial insects, pollution of water sources, and also crop biodiversity loss relating this specifically to the disappearance of the red-fruited nightshade (Solanum villosum Miller) in the southwestern highlands. Nearly half (49%) of the farmers in the southwestern agroecological zone applied pesticides before disease symptoms or insect pests occurred. The number of farmers who routinely applied pesticides was lowest in the eastern highlands. There was significant chronic exposure to pesticides among potato farmers (76% and 58% of the farmers in the southwestern and eastern highlands, resp.) of more than 10 years. Majority of farmers (91% in the eastern highlands, 89% in the southwestern highlands, and 67% in Lake Albert Crescent) perceived that the use of pesticides in potato farming has increased in the last 10 years. This trend in pesticide use could be due to increased disease and pest incidence as a result of increased potato production and climate change. It is also possible that the protection against crop loss reaped from calendar spraying has led to high frequencies of pesticide applications in potato [32]. Nearly two-thirds of the farmers applied pesticides in mixtures. It was common for farmers to combine a contact and systemic fungicide plus an insecticide within a single tank mixture to reduce costs for pesticide applications. Reducing costs associated with spraying was also the main reason for combining more than one pesticide among potato farmers in Ecuador [33] and vegetable farmers in Tanzania [12]. Although mixing pesticides can increase efficacy against pests and diseases compared to single applications of each pesticide, care should be taken to ensure that the pesticides being combined are compatible with no antagonism and cannot cause plant toxicity [34]. Farmers' Reports of Pesticide Poisoning Symptoms. Several farmers reported having felt sick after application of pesticides (Table 7). A runny nose was the most common reported symptom by 54%, 72%, and 40% of the farmers in the southwestern highlands, eastern highlands, and Lake Albert Crescent. Skin burning and eye irritation were less common. Headache, dizziness, itchy skin, cough, dry throat, blurring of vision, general body weakness, and sneezing are some of the most common mild poisoning symptoms usually experienced by pesticide sprayers [10,12,23,35]. Contact with pesticides has been reported to cause higher risk of cancers, neuropsychological impairments, accidental mortality, leukaemia, and even death [15,22,36,37]. Though no cases of deaths were reported in this study, pesticide self-poisoning accounts for about one-third of the world's suicides [38]. During 2002 in Uganda, pesticides accounted for 46% of selfpoisoning episodes that received hospital admissions [39]. It should be noted however that even fungicides like mancozeb which are unlikely to cause acute hazard in normal use can lead to long-term risk for cancer development and endocrine disruption [40]. Use of Personal Protective Equipment as Reported by Farmers. Use of personal protective equipment while applying pesticides was very low despite the high risk and frequency of exposure. Boots were the protective equipment worn by majority of the farmers (66%, 83%, and 65% in the southwestern highlands, eastern highlands, and Lake Albert Crescent, resp.), and practically no farmer used a hat, an overall, or goggles (Table 8). Very few farmers used gloves when handling pesticides. Handkerchiefs were often used instead of face and nose masks which likely give a much lower level of protection. The low investment in protective clothing during pesticide handling could be explained by the lack of knowledge on the pesticide toxicity plus the high levels of poverty which makes farmers unable to buy protective clothing. Relatively very few farmers sought medical treatment after getting signs of pesticide poisoning and the cost of medication was relatively low (≤2$US, data not shown), not considering overall costs which would include consultation fees, cost of diagnosis, travel to and from the health centres, cost of time spent in the health centre, and costs out of productive work, among others. Farmers often believe that pesticide-related symptoms are normal and therefore do not seek medical treatment as was the case in Tanzania [12], Indonesia [41], and Ivory Coast [10]. Conclusions, Recommendations, and Policy Implications This baseline study gives an insight into the range of pesticides used in the management of potato pests and diseases in Uganda, pesticide-handling practices, and symptoms of occupational pesticide poisoning. The protection against loss reaped from calendar spraying has led to high frequencies of pesticides applications in potato cultivation. Many farmers in the study areas are not adequately informed about the hazards associated with pesticide use and do not strictly use protective measures to guard them and the environment from hazards of pesticide exposure. The improper use of highly and moderately hazardous pesticides by farmers often resulted in pesticide poisoning among farmers. However, the affected farmers rarely sought medical treatment. More indepth studies on the impact of pesticide exposure on the livelihoods are recommended. Information gathered in this study will help to guide or improve future pesticide regulation and health interventions. 8 BioMed Research International The lack of knowledge of pesticide use and handling calls for investments in farmer training by governmental extension organizations, NGOs, pesticides policy and regulatory bodies, food safety standard regulatory organization(s), and the Ministry of Agriculture. An Integrated Pest Management approach would be the most effective way of reducing pesticide use in potato production while protecting the environment, increasing the productivity of potato, promoting natural enemy population build-up, and reducing the development of pesticide resistance and human health related risks. This baseline survey is the first step towards the development of an IPM system conducive to Ugandan potato farming systems. CIP: International Potato Center CSI-CC: Crop Systems Intensification and Climate Change DCE: Disciplinary Center of Excellence FAO: The Food and Agriculture Organization IPM: Integrated Pest Management NGO: Nongovernmental Organization WHO: The World Health Organization.

v3-fos

2019-04-08T13:12:45.253Z

2015-07-08T00:00:00.000Z

99148758

s2

Statistical Methodology for Cadmium (Cd(II)) Removal from Wastewater by Different Plant Biomasses Bior emediation & Biodegradation The combined effects of metal ion concentration (X), hydrogen ion concentration (pH) and biomass dose (BD), on the biosorption of Cadmium Cd(II) were investigated. Two different plant biomasses; rice straw (Oryza sativa) and dragon tree leaves (Dracaena draca) were studied. The optimum conditions were found at (X)=10 ppm, (pH)=7 and (BD)=0.5 g. Under these conditions, desirability values of 0.996 and 0.997 for rice straw and dragon tree leaves were obtained, showing that the calculated model may represent the experimental model and give the desired conditions. The samples before and after biosorption experiments were characterized by Energy Dispersive X-Ray Spectroscopy. Introduction The availability of water resources are becoming increasingly scarce; the consumption and exploitation of water resources, along with exponential increase in population have caused water pollution [1]. Toxic metals of particular concern in treatment of industrial wastewaters include: mercury, lead, cadmium, zinc, copper, nickel, and chromium [2]. So this study focuses on Cadmium (Cd(II)) that is attracting wide attention of environmentalists as one of the most toxic heavy metals. Currently methods that are being used to remove heavy metal ions include chemical precipitation, ion-exchange, adsorption, membrane filtration, electrochemical technologies. These methods are usually inadequate and expensive [3]. Biosorption is an emerging technology that is used to sequester toxic heavy metals and is particularly useful for the removal of contaminants from industrial effluents [4]. The biosorbent term refers to material derived from microbial biomass, seaweed or plants that exhibit adsorptive property [5]. Many biosorbents have been used in biosorption processes such as bacteria, fungi, algae [6] and agricultural wastes such as rice husk [7], Pequi Fruit Skin [8], Psidium guajava leaves powder [9], sugarcane bagasse, maize corncob, Jatropha oil cake [10] and cork waste [11]. The utilization of agricultural waste materials is increasingly becoming important concern because these wastes represent unused resources and, in many cases, present disposal problems [6]. So the use of natural biomaterials, especially crop wastes as biosorbents, is a promising alternative due to their relative abundance and their low commercial value [12]. Nearly 3 Million tons of rice straw is burned annually in the field of Egypt every year causing "Black cloud" [13]. However, no available literatures about using waste of ornamental plants as natural biosorbent. In this work, the Central Composite Design (CCD), which is a type of Response Surface Methodology (RSM), was employed for Optimization the biosorption of Cd(II) using two different dried plant biomasses: rice straw (Oryza sativa) and dragon tree leaves (Dracaena draca); a common ornamental plant in Egyptian gardens. Samples before and after biosorption of Cd(II) were characterized using Energy Dispersive X-Ray Spectroscopy. Biosorbent preparation Plant biomasses were dried, then were washed with tap water to remove any dust or foreign particles attached to them and finally rinsed with deionized water. The washed biomasses were dried at 60 o C for 48 hours and grounded to powder then sieved through a siever; mesh size ≤ 0.5 mm for biosorption experiments. Reagents and equipments Cadmium standard solution with initial concentration 1000 ppm was used to prepare experimental concentration of 10 and 100 ppm using deionized water. pH adjustment of the solutions was made by HNO 3 and NaOH utilizing a pH/mV hand-held meter (Crison pH meter, PH 25). Biosorption experiments Response surface methods are used to examine the relationship between response variable (RF%) and the studied factors (X, pH and BD). RSM is applied to optimize the studied factors that produce the best response and model a relationship between the factors and the response [14]. All data were analyzed using MINITAB ® 16 software. A2 3 full factorial central composite design with two coded levels was performed. For statistical calculation, the variables were coded according to Eq. (1): Where x i is the dimensionless coded value of the variable Xi, X0 the middle value of X i , and ∆X the step change. Batch experiments were conducted with the following conditions: 0.5 g of each biomass and 100 ml of Cd(II) solution with an agitation speed 300 rpm (round per minute) at room temperature. The influence of three factors i.e., initial metal ion concentration (X), hydrogen ion concentration (pH) of the solution, biomass dose (BD) have been investigated. The range and the levels of the variables investigated in this research are given in Table 1. Then samples were collected after 2 hours to reach equilibrium in biosorption. Control samples were prior to batch biosorption experiment to determine initial metal concentration and all samples were conducted in triplicate. The metal ions contents in all the samples prior to and after batch biosorption experiments were analyzed by Varian Inductively Coupled Plasma (ICP-AES). Removal efficiency (RF%) of biosorbent was calculated using the following equation Where: C i = Initial concentration of metal in solution, before the sorption analysis (mg/l), C f = Final concentration of metal in solution, after the sorption analysis (mg/l). Characterization of biosorbents Energy Dispersive X-Ray Spectroscopy (EDAX): EDAX spectra can be collected from a specific point on the sample, giving an analysis of a few cubic microns of material. Each biosorbent was characterized by EDAX before and after Cd(II) biosorption. Biosorption experiments Batch experiments were conducted as tabulated in Table 2, '+1' for the higher level and '−1' for the lower level of the studied factors. Removal efficiency percentage (RF%) were calculated according to Eq.(1). Regression coefficients (Coef) and the associated standard errors (SE Coef) of results are shown in Table 3. Results revealed that all the studied factors together with their interactions were significant at 95% confidence limits (P>0.05). The response variable (Cd(II) removal %) was fitted by the following equation: At X=10 ppm, pH=7 and BD=0.5 g, the highest percentage of Cd(II) removal by rice straw was 82.60% while that for dragon tree leaves was 79.60% (Table 2). It worth noting that the effect of all the studied main factors (X, pH, BD) was identical for both biosorbents. As such, our results demonstrated that the factor (X) had the largest effect on biosorption process by rice straw and dragon tree leaves (Table 3). Results also showed that Cd(II) biosorption was favored at low metal concentration values (X=10 ppm). This is in line with [15,16]. In the current work, the biosorption percentage was decreased as the metal ion concentration from 10 to 100 ppm. This is may be because the biomass surface area available for metal biosorption was higher the ratio of active adsorption sites to the initial Cd(II) ions is larger, resulting in higher removal efficiency [17]. This is in agreement with many researchers [6,11]. The second important main factor in the biosorption process was pH. Results indicated that as the pH value increases, Cd(II) biosorption increases by both biosorbents (Table 3). At lower pH values, the H 3 O + ions compete with the metal ions for the active sites on the biosorbent [18]. In our work, the optimum higher pH value for Cd(II) biosorption was 7. The hydrolysis of Cd(II) ions occur beyond pH=7 as reported in [10]. In this account, the third main factor in the biosorption process was BD. Results indicated that as (BD) increases, Cd(II) biosorption increases by both biosorbents (Table 3). An increase in the biomass dosage generally increases the amount of solute biosorbed, due to the increased surface area of the biosorbent, which in turn increases the number of binding sites [19][20][21]. Data obtained from the response surface plots of both biosorbents are illustrated in Figures 1-3. These plots are used to visualize the relationship between response (%RF) and the level of each studied factors. Every one of them is mapped against two experimental factors while the third is fixed at two different levels [22]. Figure 1 illustrated the removal efficiency of Cd(II) by both biosorbents over (pH) and (BD). At constant metal ion concentration (100 ppm, 10 ppm), a remarkable increase in Cd(II) removal was attained as pH increases till reaching its maximum at pH=7 for both biosorbents. However, a slight increase in Cd(II) removal was observed as (BD) increases till reaching its maximum at BD=0.5 g. Figure 2 illustrated the removal efficiency of Cd(II) by both biosorbents over (X) and (BD). When keeping pH constant (7, 2) for both biosorbents, a remarkable increase in Cd(II) removal was attained as (X) decreases till reaching its maximum at X=10 ppm for both biosorbents. However, a slight increase in Cd(II) removal was observed as BD increases till reaching its maximum at BD=0.5 g for both biosorbents. a slight increase in Cd(II) removal was observed as pH increases till reaching its maximum at pH=7. Analysis of variance (ANOVA - Table 4 ) showed the sum of squares used to estimate the factors' effect and the F-ratios defined as the ratio of the respective mean-square-effect and the mean-square-error. The significance of the present biosorption models as assessed by F-values and P-values indicated that the studied factors and their interactions (X.pH.BD) except (X.pH, X.BD, pH.BD) are statistically significant in the case of rice straw and the studied factors and their interactions except (X.BD and X.pH.BD) are statistically significant in the case of dragon tree leaves. Characterization of biosorbents The results of EDAX (Figure 4) showed that raw biosorbents did not contain any Cd(II) ions on their surfaces and these ions appeared only after batch biosorption experiments. Response optimization After Response Surface Methodology was carried out, Minitab's Response Optimizer was used to get the optimized factors and responses. The goal for the studied factors (X, pH, BD) was to maximize them as listed in Table 5. All results had relatively high desirability scores of rice straw and dracaena draca were 0.961 and 0.970, respectively as listed in Table 5 because the predicted response of them were 82.30 and 79.39 which were quite close to the targets of each one of 82.60 and 79.60, respectively and optimization plot was shown in Figure 5. Desirability is an objective function that ranges from zero outside of the limits to one at the goal [23,24]. The composite desirability (D) of 0.99650 combined the individual desirabilities and it is high as it is closer to 1 and the best removal percentage of Cd(II) obtained at X=10 ppm, pH=7 and BD=0.5 g for each biosorbent where the vertical lines on the graph represent the current factor settings, the horizontal datch lines represent the current response values Conclusion It may be concluded that: • The most significant effect for Cd(II) biosorption by rice straw and dragon tree leaves was ascribed to (X). • Main factors exert more effect than interaction factors by both biosorbents. • Ion exchange and complexation processes are the mechanisms of biosorption that occurred in rice straw and dragon tree leaves, respectively. • EDAX confirmed biosorption process by the changes occurred on the surfaces of both biosorbents. • Desirability values (0.996 and 0.997) indicated the calculated model can represent the experimental model and give the desired conditions for both biosorbents.

v3-fos

2018-04-03T03:30:48.198Z

2015-10-01T00:00:00.000Z

5848178

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Water Properties Influencing the Abundance and Diversity of Denitrifiers on Eichhornia crassipes Roots: A Comparative Study from Different Effluents around Dianchi Lake, China To evaluate effects of environmental conditions on the abundance and communities of three denitrifying genes coding for nitrite (nirK, nirS) reductase and nitrous oxide (nosZ) reductase on the roots of Eichhornia crassipes from 11 rivers flowing into the northern part of Dianchi Lake. The results showed that the abundance and community composition of denitrifying genes on E. crassipes root varied with different rivers. The nirK gene copies abundance was always greater than that of nirS gene on the roots of E. crassipes, suggesting that the surface of E. crassipes roots growth in Dianchi Lake was more suitable for the growth of nirK-type denitrifying bacteria. The DGGE results showed significant differences in diversity of denitrifying genes on the roots of E. crassipes among the 11 rivers. Using redundancy analysis (RDA), the correlations of denitrifying microbial community compositions with environmental factors revealed that water temperature (T), dissolved oxygen (DO), and pH were relatively important environmental factors to modifying the community structure of the denitrifying genes attached to the root of E. crassipes. The results indicated that the specific environmental conditions related to different source of rivers would have a stronger impact on the development of denitrifier communities on E. crassipes roots. Introduction Phytoremediation technology using floating macrophytes (Eichhornia crassipes) performed very well in remediation eutrophic water body since E. crassipes is capable of assimilating large amount of nutrients efficiently [1][2][3]. During 2010-2012, large-scale confined growth of E. crassipes was used to remove pollutants (mainly N and P) from Dianchi Lake as well as the rivers connecting to the lake. Dianchi Lake is the sixth largest freshwater lake in China. There are more than 31 rivers, which carried wastewater discharged from different types of sewage treatment plants (STPs), agriculture, and domestic source, flowing into the lake. The macrophytes significantly improved water quality in both inflow rivers and Dianchi Lake [4]. To evaluate the contributions of water hyacinth to the removal of nitrogen from the lake, both assimilation and stimulated denitrification by the macrophyte are important since N-15 tracing experiment in labs indicated that the values of N-15 at.% excess of N 2 -N production were significantly ( < 0.05) higher with the growth of E. crassipes than that without [5,6]. The presence of E. crassipes roots has positive effect on stimulating the activity, abundance, and diversity of denitrifiers. Studies have reported that plant rhizosphere enhanced bacterial abundance, activity, and diversity [7]. Previous studies also suggested that the root system of floating macrophyte could support the attachment of microorganism and enhance the growth and activity of bacteria for removing organic matter and nutrients [8]. Environmental conditions are also critically important in mediating the activity, abundance, and diversity of bacteria [9,10]. The abundance and diversity of denitrifiers on E. crassipes roots grown in the rivers receiving wastewater with different water properties may vary with the variation of environmental factors. The abundance of the functional genes and community compositions of denitrifiers can be affected by many factors, such as water temperature, pH, DO, and nutrient concentrations. In the rivers with different sources of condensed pollutants and diverse physiochemical properties, the abundance and diversity of denitrifiers on the E. crassipes roots may be modified in various patterns. Hence, in the present study, we investigated the abundance and diversity of denitrifying bacteria on E. crassipes roots in 11 rivers with different pollution sources in the north side of Dianchi Lake. It put an emphasis on understanding the interactions between the changes of environmental factors and the abundance and diversity of denitrifying bacteria attached to E. crassipes roots. It was expected that the results would shed some insight on how environmental factors and cultivation of E. crassipes mediate denitrification process in different eutrophic rivers flowing into the Dianchi Lake. Water temperature, pH, and dissolved oxygen (DO) were measured in situ using portable meter (YSI ProPlus, USA). One-liter water samples were collected from each site with three replicates at 0-0.5 m of the water column of the eleven rivers using cylinder sampler in September 25, 2012. Total nitrogen (TN), ammonium (NH 4 + ), nitrate (NO 3 − ), nitrite (NO 2 − ), and total soluble nitrogen (TSN) were analyzed using a SEAL AutoAnalyzer 3 (SEAL Analytical Co., Hampshire, UK). Mixed root samples were collected randomly with three replicates at each sampling site using sterile scissors and forceps and then stored in an ice box and taken back to the laboratory. Fresh roots (2 g) of E. crassipes were transferred into 200 mL sterile water. Bacteria attached to E. crassipes roots were detached by vigorous shaking for 30 min (18.3 Hz, Thermomixer Eppendorf) and filtered through a 0.45 m sterile filter. The resultant filtrates were filtered through 0.22 m Millipore membrane filters using a vacuum air pump and the membranes stored at −80 ∘ C for DNA extraction [5]. DNA Extraction. All the abovementioned membranes were cut into pieces with sterile scissors and used immediately for DNA extraction, which was performed using an E.Z.N.A. Water DNA Kit (OMEGA Bio-Tek Inc., Doraville, GA, USA) by following the manufacturer's instructions. The extracted DNA was stored in a −20 ∘ C freezer [5]. Real-Time Polymerase Chain Reaction (qPCR) Assay. Real-time quantitative PCR was performed to estimate the denitrifying bacteria abundance using the primers listed in Table 1. Real-time polymerase chain reaction (qPCR) was performed on ABI 7500 real-time System (Life Technologies, USA). Amplification was performed in triplicate in a total volume of 20 L reaction mixtures by using SYBR Premix Ex TaqTM (TIiRNaseH Plus) qPCR Kit as described by the suppliers (Takara Bio, Dalian, China). For each assay, three different PCR conditions were performed separately or 58 ∘ C (nirK-F1aCu/nirK-R3Cu). The qPCR amplification was performed as follows: initial denaturation at 95 ∘ C for 2 min, followed by 35 cycles consisting of denaturation step at 95 ∘ C for 5 s, varying annealing temperature for 30 s, and elongation at 72 ∘ C for 30 s. The data were collected during the 72 ∘ C for 30 s step. Data was analyzed using the ABI 7500 software (Version 2.0.6, Life Technologies, USA). The parameter Ct (threshold cycle) was determined as the cycle number at which a statistically significant increase in the reporter fluorescence was detected. The standard curves for real-time PCR assays were developed as previously described [5]. PCR Amplification Denaturing Gradient Gel Electrophoresis (DGGE) Analysis. For denaturing gradient gel electrophoresis (DGGE) analysis, the PCR was performed in reaction mixtures including 1 L of template DNA, 5 L of 10 × PCR buffer, 1 L of dNTPs (10 mM each), 1 L of each primer (20 M) ( Table 1), and 2 U of Taq polymerase (Takara Bio, Dalian, China) and adjusted to a final volume of 50 L with sterile deionized water. The reaction was performed in a Bio-Rad C1000 thermal cycler (Bio-Rad, USA) using different cycling conditions. The nirK gene (F1aCu/R3Cu-GC) PCR program was carried out with an initial denaturation at 94 ∘ C for 3 min, followed by 32 cycles of 94 ∘ C for 30 s, 58 ∘ C for 30 s, and 72 ∘ C for 45 s, followed by 72 ∘ C for 10 min, and ended at 10 ∘ C. The touchdown PCR amplification of nirS (Cd3Af/R3cd-GC) and nosZ (nosZ-F/nosZ1622R-GC) was performed as follows: 94 ∘ C for 2 min, followed by 10 cycles, 94 ∘ C for 30 s, and 57 ∘ C for 30 s in the initial cycle and at decreasing temperatures by 0.5 ∘ C/cycle until a temperature of 52 ∘ C was reached in the subsequent cycles. The extension step was performed at 72 ∘ C for 1 min. After the touchdown program, 30 cycles at 94 ∘ C for 30 s, 53 ∘ C for 30 s, and 72 ∘ C for 1 min, followed by 72 ∘ C for 10 min, and ended at 10 ∘ C. The amplified products were pooled and resolved on DGGE gels using a Dcode system (Bio-Rad Laboratories, Hercules, USA). The purified PCR products (30 L) of nirS, nirK, and nosZ containing approximately equal amounts of PCR amplicons were loaded onto the 1 mm-thich 6% (w/v) polyacrylamide (37.5 : 1, acrylamide : bisacrylamide) gels with denaturing gradients of 50-75% for 15 h (nirS), 50-70% for 12 h (nirK), and 50-70% for 15 h (nosZ) (100% denaturant contains 7 mol/L urea and 40% (v/v) formamide). The gels were run in 1 × TAE (40 mM Tris-acetate and 1 mM EDTA) at 100 V and 60 ∘ C. The gel was silver-stained using protocol [11]. Polaroid pictures of the DGGE gels were scanned using an Epson Perfection V700 Photo scanner (Seiko Epson Corporation, Nagano, Japan). DGGE profiles were digitized after average background subtraction for the entire gel using Quantity One software (Version 4.5, Bio-Rad, USA) as previously described [5]. Digitized information from the DGGE banding profiles was used to calculate the diversity indices such as richness ( ), which was determined from the number of bands in each lane, and Shannon-Wiener Index ( ), which was calculated from = − ∑ × ln [12], where is the importance probability of the bands in a gel lane, calculated as = / , where is the intensity of a band and is the sum of intensities of all bands. Data Analysis. Three replicates were used in all parameter analyses. Data presented as mean values ± SD. One way analysis of variance (ANOVA) followed by ---test was performed to check for quantitative differences between samples; < 0.005 was considered to be statistically significant. All statistical analyses were done using SPSS software. The relative intensity of a specific band was transformed according to the sum of intensities of all bands in a pattern [13]. Redundancy analysis (RDA) for community ordination was conducted using CANOCO (version 4.5, Centre for Biometry, Wageningen, Netherlands) for Windows using relative band intensity data obtained from the Quantity One analysis [14]. Eight environmental parameters, including water temperature, pH, DO, ammonia, nitrate, nitrite, total nitrogen, and total soluble nitrogen, were selected to perform RDA-based variance inflation factor (VIF) analysis with 499 unrestricted permutations to statistically evaluate the significance of the first canonical axis and of all canonical axes together. Statistical significance was kept at < 0.05 for all analyses. Water Properties. The corresponding environmental parameters (Table 2) of the eleven rivers represented their own properties of different pollution sources. The water from STP sites was characterized by relatively high concentrations of nitrate (4.79-12 mg L −1 ) and low concentrations of ammonia nitrogen (0.06-1.98 mg L −1 ) and organic matter, which had contrary properties comparing to those rivers receiving raw sewage from industrial, domestic, and agricultural sources. The XB and YA rivers had similar characteristics to those rivers receiving water from STP but lower dissolved oxygen and higher ammonia nitrogen. Quantification of Denitrifying Genes (nirK, nirS, nosZ). The results showed that the abundance of nirK, nirS, and nosZ gene copies per gram fresh root ranged from 4.13 × 10 7 to 6.11 × 10 8 , 1.45 × 10 8 to 1.99 × 10 8 , and 2.20 × 10 8 to 2.20 × 10 10 , respectively ( Figure 2). The nirK and nirS abundance on the roots of E. crassipes in YA river and XB river were significantly higher than those in other rivers ( < 0.05). The highest abundance of nosZ was observed on the root sample in JJ river. The lowest abundance of nirK and nosZ type denitrifiers were determined on the root sample from DG river. The nirK, nirS, and nosZ copy abundance varied between sites indicated that different pollution source would influence the abundance of denitrifiers in rivers. The highest abundance ratio (125.34) of nosZ/(nirK + nirS) occurred in JJ river, followed by GPG (39.75), while the lowest ratio was in XYL river (1.43), and the ratios in other rivers were similar, ranging from 3.26 to 8.38. However, the nirK/nirS ratio in all samples ranged from 1.70 to 6.60. To explain the relationship between environmental factors and the abundance of nirK, nirS, and nosZ, the gene copy numbers of three denitrifiers and eight parameters were explored by redundancy analysis (Figure 3). The gray circle area implies a positive correlation and the white circle area implies a negative correlation. The larger the circle area, the greater the impact corresponding to the changes in environmental factors that would have influenced the denitrifiers. Denitrifier lines at the end in the gray circle had positive regression coefficients for that environmental variable with the corresponding -value larger than 2.0. The results showed that the temperature, pH, and nitrate circle areas were larger than other environmental factors, which indicated that temperature, pH, and nitrate circle greatly affected the nirS, nirK, and nosZ abundance than other factors. The abundance of nirK and nirS was positively correlated with water temperature, nitrate, and nitrite concentrations and was negatively correlated with the other factors (pH, DO, DTN, TN, and ammonium). The abundance of nosZ was negatively correlated with water temperature and was positively correlated with the pH and DO, while there were no significant correlations with other factors. each gene type to illustrate resolution. However, all the three replicates of the profiles were digitized and were used in statistics analysis. of E. crassipes (Table 3). The significant differences among them were observed statistically ( < 0.05). With respect to the richness and diversity of denitrifier communities in all sites, similar trends emerged with low richness and diversity of nirK and nirS genes in XBX and DG rivers, which mainly received effluent from STPs. Next trends were in the H, JJ, and XBH rivers with relatively higher richness and diversity of nirK and nirS genes, which were less impacted by the effluent from STPs. The highest richness and diversity of nirK showed in the XB and YA rivers, which received wastewater after flowing through a wetland incubation on the water way. The richness and diversity of nosZ, which was mainly impacted by temperature, gave a similar trend for all rivers due to the fact that temperature did not vary too much in all sites. 6 International Journal of Genomics Relationship between Environment Matrices and Denitrifier Diversity. To determine to what extent the eight environmental properties affected the three types of denitrifier community compositions, nirK, nirS, and nosZ DGGE fingerprints were evaluated by redundancy analysis ( Table 4). The first axis explained 26.9% of the nirK-type denitrifier diversity, and the second axis explained 21.8% of the diversity. For nirS-type denitrifier, the first two canonical axes explained 29.5% and 11.5% of the variation, respectively. For nosZ-type denitrifier, 37.8% and 13.7% of the variation were explained by the first two canonical axes (Table 4). Of the parameters, total N, DO, pH, and water temperature appeared to be the relatively important environmental factors for denitrifiers (Table 5). For nirK-type denitrifier, water temperature, DO, and total N explained 46% variations of microbial communities, leaving 54% of the variation unexplained. Variation partitioning analysis showed that water temperature, DO, and total N separately explained 19% ( = 0.020), 13% ( = 0.054), and 14% ( = 0.240) of the variation, respectively. For nirS-type denitrifier, water temperature (18%, = 0.066), pH (10%, = 0.304), and DO (8%, = 0.038) explained 36% variations of microbial communities, leaving 64% of the variation unexplained. Compared to nirS, the total N rather than DO was relatively important for nosZ-type denitrifier ( Table 5). The relationships of microbial patterns to environmental variables were summarized in RDA ordination plots ( Figures 4(b), 5(b), and 6(b)). The RDA charts (Figure 4(b)) of nirK gene showed four rivers (PLJ, XYL, DG, and CF) grouped into one type, while other four rivers (XBH, XBX, H, and YA) clustered together. Other rivers were located independently; they did not belong to either group. For nirS gene, PLJ, JJ, and CF rivers were similar and grouped into one type, while other three rivers (XBH, XYL, and XBX) clustered together. Other rivers were not similar and did not belong to either group ( Figure 5(b)). According to the RDA chart ( Figure 6(b)) of nosZ gene, DG, H, GPG, and XBH rivers were located independently and did not cluster with any group. However, PLJ, XYL, JJ, and CF rivers clustered into one group, while other three rivers (XB, XBX, and YA) clustered together. Discussion The activity of denitrifying microorganisms leads to significant net removal of dissolved nitrogen from the water, Partial RDAs based on Monte Carlo permutation ( = 499) kept only the significant water properties in the models. For each partial model, the other significant water properties were used as covariables. and values were estimated using Monte Carlo permutations. Sum of all eigenvalues for both partial RDAs was 1.000. resulting in considerable improvement of water quality in aquatic ecosystem [15]. Denitrifiers play an important role in buffering of the excessive load of nitrogen from upstream to downstream [16]. In aquatic ecosystems, mats of macrophytes are important sites for microbial mediated biogeochemical processes, as accrual of biomass and increases in mat density reduce the degree of external factors to influence internal processes [17]. The suspended root system of E. crassipes could provide a large surface area, approximately 2.5 to 8.0 m 2 kg −1 on a dry weight basis, for microbial attachment [5,18]. Releasing of oxygen and dissolved organic carbon from roots of E. crassipes would support an appropriate microenvironment for nitrification and/or denitrification [19,20]. Process of denitrification is driven by the denitrifying microorganisms under the influence of environmental conditions. Water properties in different rivers in the present study were shown altering the abundance and diversity of denitrifiers on E. crassipes roots. Differential Characteristics of Different Rivers Impact the Abundance of Denitrifier on the Roots. The abundance of nirK, nirS, and nosZ denitrifiers on the root of macrophytes varied with the variation of environmental parameters in different rivers, which seemed depending on the nitrogen concentrations, water temperature [21], water turbulence, and pretreatment of wastewater using wetland. The abundance of nirK, nirS, and nosZ denitrifiers on root samples from XBX, DG, CF, and PLJ rivers was relatively stable and low. These rivers were larger than other rivers around Dianchi Lake [22], which were important sites receiving effluent from the STPs. The fast-flowing water and irregular discharge of effluent of these rivers [22] may prevent development of the stable environment properties from microbial attachment and propagate. Contrarily, the abundance of denitrifiers genes on roots samples in XB and YA rivers (Xiaba and Yaoan rivers) was higher than that in other rivers. The XB river received both the wastewater from industrial and residential areas and the tidal water from Dianchi Lake, when water level increased in rainy seasons (May to October) [23]. The water merged at Wujia wetland, and then part of it was pumped into YA river after 45 days of retention in Wujia wetland. This implies that a combination of wetland and growth of water hyacinth may further promote denitrification processes in eutrophic water. The abundance of nirK gene was always greater than that of nirS gene on the roots of E. crassipes, suggesting that the fresh water of Dianchi lake was more suitable for the growth of nirK-type denitrifying bacteria. This was different from the previous reports [24]. The nirS gene of cytochrome cd1 type has also been found more often in anoxic locations, where DO levels were consistently low. In contrast, nirK genes of copper containing type have been found where diurnal DO swings are greater [24,25]. This finding was of coincidence with that E. crassipes releases oxygen from roots, which facilitates the creation of aerobic microsites on the roots [26]. Even though the nirK and nirS are functionally equivalent, denitrifying bacteria harboring either nitrite reductase seems to be likely not under the same community assembly rules [27]. Philippot et al. [28] suggested that the existence of the two types of nitrite reductase (nir-gene) was due to differential niche preferences. This speculation was consistent with previously identified habitat preferences of nirS-gene and nirK-gene bearing organisms [29]. Moreover, nirK and nirS sequences may come from different sources. Jones and Hallin [27] found that most nirK sequences were derived from soil but that most nirS sequences were prominently derived from marine and estuarine environment. Bacteria suspended in water and attached to the root of E. crassipes may originate from many different sources. Autochthonous bacterioplankton populations that developed in the water column were likely to be mixed with allochthonous populations from forest soils, urbanized land, farm fields, and wetlands as well as hyporheic sediments in the rivers. This mixed origination, impacted by varied environmental parameters, seemed to be the main cause of the discrepancy of denitrifiers found in the eleven rivers. This, however, did not necessarily indicate that nirKtype denitrifiers contributed more or less in denitrification than nirS-type ones; rather it may only imply that the root of E. crassipes could provide a broad support for different kinds of microorganisms. Relationship between Environmental Factors and Community Compositions of Denitrifying Genes on E. crassipes Root at Different Rivers. Dianchi Lake together with surrounding rivers comprised a plateau water catchment to provide ecological services and fresh water supply for more than seven million people in the area. Its geochemical characteristics have made the water pH relatively high (7.56-8.03) and its geophysical characteristics have made the water temperature moderate with winter months (December to March next year) around 12 ∘ C. Its heavy load of organic matters have made the DO level relatively low (0.20-3.80 mg L −1 ) in the 11 rivers investigated. These environmental properties dominated the community assembly processes of the genetic makeup of the denitrifiers in the rivers. Nevertheless, specific environmental conditions in different rivers favored the variation in richness and diversity of different denitrifying genes. The DGGE profiles for denitrification genes encoding nitrite and nitrous oxide reductase (nirK, nirS, and nosZ) on the root of E. crassipes growing in 11 rivers around Dianchi Lake supported our hypothesis of profound differences in community composition, although a complex picture of denitrifier community similarity emerged depending on which functional denitrification gene was evaluated. The correlations of denitrifying microbial community compositions with abiotic environmental factors, using redundancy analysis (RDA), confirmed that water temperature (Temp), dissolved oxygen (DO), and pH appeared to be the most important factors to alter the denitrifier community structures significantly by serving as essential conditions for the growth of microorganisms on the roots of E. crassipes ( Table 5). The results of this study indicated that the development of denitrifier communities on roots corresponded to different origins of rivers. The physiochemical characteristics of water from the river inlet varied with water origin and pollution sources [22,23,30], resulting in the variation in DO, pH, pollutant species and concentrations, and organic carbons in rivers. These environmental factors, including DO, carbon content, water temperature, and pH, influenced denitrification rates in rivers [31] and as a consequence they might also affect the denitrifier community composition [32,33]. Braker et al. [34] found that the change of temperature resulted in gradually changed denitrification activity but also in abundance mutative of nitrate reducers and in different denitrifier community compositions. There are some indications that temperature and pH may directly or indirectly influence the abundance and communities composition of denitrifiers [35,36]. The excess O 2 resulted in reduced denitrifying bacterial growth and a smaller bacterial density versus nitrate reducing bacteria ration [37], which indicated that the development of the denitrifying bacteria was influenced by the DO concentration. Many investigators had found that the pH, temperature, and DO generally affect diversity and richness of denitrifier community [32,38], and microbial community assembly was more dependent on local-scale environmental factors [39]. On the other hand, different microorganisms may have their physiological constraints for growth and reproduction within narrow pH ranges, specific DO, and nutrient availability, which affect the community structures directly [40,41]. Activities of microorganisms could change the environmental properties that differed in the concentrations of enzymes and nutrients or DO, the form and amount of dissolved carbon present, and pH [42] hence to affect denitrifier community structure. Previous studies have found that growth of E. crassipes could regulate water at neutralize pH significantly [43], as a result of increase in the rate of denitrification in aquatic ecosystems [44]. Conclusions The variation in abundance of denitrifier communities on E. crassipes roots, grown in rivers flowing into Dianchi Lake, corresponded to different water properties of rivers. The ratio of nirK/nirS gene copies abundance was always greater than 1, indicating that the surface of E. crassipes roots was more suitable for the growth of nirK-type denitrifying bacteria. The temperature of water, nitrate concentration, and pH greatly affected the nirS, nirK, and nosZ abundance than other factors. Meanwhile, the temperature of water, DO, and pH appeared to be the most important factors to alter the community structures of denitrifiers on the roots of E. crassipes. As process of denitrification is driven by denitrifies under the influence of environmental conditions, a variation of denitrification capability in different rivers would be expected.

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2018-11-09T11:04:33.082Z

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Genetic Diversity of Mexican Avocado in Nuevo Leon, Mexico Mexico is considered the center of origin of avocado, and in the mountains of Sierra Madre Oriental in Nuevo Leon the remains of an avocado have been found, which are evidence of the place or origin of Persea americana var. drymifolia, or the Mexican avocado. Wild creoles have been found growing with native vegetation and have contrasting characteristics with cultivated varieties (improved creoles) and creoles (seedling) found in orchards. All this diversity represents a valuable source of genes and genetic combinations that can be used in Persea breeding programs. Therefore, we performed studies in the north and south areas of Nuevo Leon, Mexico, in order to determine the genetic diversity of this crop and implement actions for the characterization and conservation of this genetic resource, which has been disappearing due to changes in soil use, the introduction of improved varieties and the destruction of their habitat. Introduction Mexico is considered the center of origin of avocado, and in the mountains of Sierra Madre Oriental in Nuevo Leon the remains of an avocado have been found, which are evidence of the place or origin of Persea americana var. drymifolia, or the Mexican avocado. Wild creoles have been found growing with native vegetation and have contrasting characteristics with cultivated varieties (improved creoles) and creoles (seedling) found in orchards. All this diversity represents a valuable source of genes and genetic combinations that can be used in Persea breeding programs. Therefore, we performed studies in the north and south areas of Nuevo Leon, Mexico, in order to determine the genetic diversity of this crop and implement actions for the characterization and conservation of this genetic resource, which has been disappearing due to changes in soil use, the introduction of improved varieties and the destruction of their habitat. An analysis of 42 Mexican avocado samples based on the fruits traits as: fruit weight, fruit length, fruit diameter, seed weight, seed length, seed diameter, length seed cavity, the ratio of fruit length/fruit diameter and the ratio of seed weight/fruit weight, was carried out. The results of the analysis showed that the classification of genetic diversity with these fruit traits was not well represented. The characterization of the samples with AFLP molecular markers, allowed the differentiation of five genotypes in the cluster analysis and the separation of those with the same local name, thus demonstrating genetic differences within Mexican avocado genotypes. When the molecular data were analysed with the morphological data, 11 genotypes were differentiated and the separation of genotypes with the same local name was possible. At present, we are working with 10 genotypes to determine genomic regions that help us to identify and differentiate genotypes. The research results have identified about 37 improved creoles, 19 creoles (seedling) and nine wild creoles (seedling), all of them with different traits. To preserve this diversity, we are implementing a local germplasm bank together with Mexican avocado growers in order to start a breeding program. Origin of Mexican avocado (Persea americana var. drymifolia) Mexico has a wide variety of types of avocado-there are at least 20 different species related to the avocado [1]. The avocado is part of the Lauraceae family, considered by many botanists to be among the most primitive of the dicotyledonous plant; the centre of origin of the avocado is located in the highlands of central and east-central Mexico and the Guatemalan highlands [2]. In Mexico, three races are recognized: Mexican, Guatemalan and West Indian, which were classified as botanical varieties [3]; the possible centre of origin of these races is shown in Figure 1 [4]. Persea americana var. drymifolia, or the Mexican avocado, is the oldest variety used as food [2]; seeds of avocados were found in cave deposits (El Riego, Purrón and Coxcotlán) in the Tehuacan Valley, Puebla in Mexico, the oldest cotyledon from the Coxcotlán cave deposit is dated to at least 7,000 B.C. [4]. In 1966, the geographical distribution of the P. americana var. drymifolia in Mexico was determined in the mountain forests of the eastern and south-eastern and in the Pacific zone ( Figure 2) [5]. The findings of Mexican primitive avocados in the Sierra Madre Oriental of Nuevo Leon show that this area is part of the centre of origin of Persea americana var. drymifolia [6]. The Mexican avocado is grown in different intensive agricultural systems and both in orchards and backyard gardens, these forms of production are important centres of experimentation, plant introduction, empirical improvement and shelters of unique genetic diversity that contain genes that have not been studied and could have potential use in breeding programmes. Persea americana var. drymifolia trees are characterized by their resistance to cold and high oil content, a very distinctive characteristic is its strongly aromatic leaves (anise scented) in almost all individuals; usually, they grow at altitudes greater than 2,000 m. The leaves are dark green with light green or reddish young shoots; the fruit has a thin, smooth and soft skin, the seed can be adhered or loose, the cotyledons are smooth or slightly rough, and it is common to find fibre in the flesh, although this is not found in most cultivated species [1]. Given that the avocado is an open-pollinated species, it contains great genetic variability with almost unlimited possibilities for utilization [7]; a wide diversity of germplasm allows the advancement of botanical and agronomic knowledge and the development of new cultivars [6]. The germplasm of Mexico has been the basis of breeding programmes in other countries, so it is necessary to focus on the exploration, classification and preservation of the germplasm of the genus Persea to implement breeding programs. The generation of improvement varieties involves searching for genes that may exist in some cultivated varieties or wild plants; if these sources of genes do not exist, the possibility of the transferring of its characteristics will be lost forever. In Nuevo Leon, Mexico, it is still possible to find different wild plants of the Mexican avocado (wild creoles) growing among natural vegetation, and their morphological traits are contrasting with cultivated varieties. Cultivated varieties consist of local selections of plants that have been grown for several years and which the growers have selected based on their production and quality (fruit size, mainly); these cultivars are grafted trees with genotypes of interest and are called 'improved creoles'. Native plants are those from avocado seeds that have not been grafted (seedlings) and are called 'creoles'. The genetic diversity of wild plants, improved creoles and creoles, represents a valuable source of genes and genetic combinations that can be used in Persea breeding programmes. The main problems in identifying the genetic variability of Mexican avocados in Nuevo Leon, Mexico, are, among other factors, the diversity of local names that are used for the improved creoles, and the use of the same rootstock for grafting different varieties, resulting in a tree with branches belonging to different cultivars ( Figure 3). Since 2005, in order to study this genetic diversity, we performed studies of the morphological and molecular characterization of Mexican avocado varieties. First review of Persea americana var. drymifolia in Nuevo Leon, Mexico In order to identify the genotypes of Mexican avocados (improved creoles, creoles and wild creoles), from June 2005 to November 2006, surveys were conducted in the municipalities of Dr. Gonzalez and Sabinas Hidalgo, located north of Nuevo Leon, Mexico, and in the municipalities of Aramberri, General Zaragoza and Rayones, located south of the state. A total of 30 improved creoles and 19 creoles and wild creoles, were identified. We collected five fruit of each of the trees that had fruit at the time of the visit (26 improve creoles and 12 creoles). The improved creoles collected in Dr. Gonzalez were Huevo de Toro Blanco, Larralde, Verde Perez and Perales, whereby the fruits of these cultivars mature in green color; and Anita, Floreño, Huevo de Toro, El Cuervo, Negro Santos Normal, Negro Santos Especial, Rodriguez and Rosita, the fruits of these improved creoles mature in black color (Figure 4), also were collected four creoles ( Figure 5). In Aramberri, we collected the improved creoles: Mantequilla, Pagua, Platano Delgado, Maria Elena, Fuerte, Leonor, Pato and De la Peluqueria ( Figure 6), as well as five creoles ( Figure 5)-in the case of Mantequilla, the skin of the ripe fruit is yellow. In the General Zaragoza municipality, only two creoles were identified ( Figure 5 and Figure 8). In Sabinas Hidalgo, improved creoles were identified, such as Anita, Floreño, Negro Santos and Pepe ( Figure 7). In Rayones, the improved creoles were Verde Fuerte and Pagua (Figure 7), as well as two creoles ( Figure 5). Four improved creoles had no fruits: Lampazos, Blanco and Sanjuanero of Dr. Gonzalez municipality, and Israel of Aramberri municipality. Fourteen improved creoles were reported in Sabinas Hidalgo. In the study of evaluation of native avocados in the northern region of Nuevo Leon [8], six genotypes corresponded with those we found. In this same report, 10 improved creoles that were not documented by us were reported in Sabinas Hidalgo: Blanquito, Fosa, Cuervo, Pera, Sabroso, Chapeño, Pecoso, Pila, Salazareño, Especial. Negro Santos and Especial genotypes were reported in Bustamante [8]this area was not considered in our study. El Salto is a wild creole collected in the municipality of General Zaragoza and it was named this way because the trees were located in Parque Recreativo El Salto, located in this municipality. The fruit has a length of 2 cm and, according to researchers at Red Aguacate-SNICS-SAGARPA in Mexico, it could be a primitive Mexican avocado genotype-this fruit's characteristics are similar to those reported by Storey, who mentioned the discovery of a primitive form of avocado fruit which is about 2 cm length, in the pine and oak forests of Nuevo Leon, Mexico [4]; this vegetation type coincides with the area where we located this genotype ( Figure 8). The harvest of Mexican avocados in Nuevo Leon, Mexico, is performed during the period from June to December, although there are genotypes with fruit production throughout the year. The collections of the samples were conducted both in gardens and in areas of wild vegetation. The identification of the samples was performed according to the local name provided by the growers-those samples of non-grafted trees were identified as creoles and samples collected in areas of wild vegetation were identified as wild creoles. Eleven improved creoles corresponding to 26 samples, seven creoles and nine wild creoles were identified ( Table 1). The Platano Grueso improved creole is more commercially accepted by the characteristics of size and appearance of the fruit; this cultivar characteristically presents an early harvest starting cycle, in mid-June, which limits its production from concentrating in a relatively short period of time [9]. The tendency of some producers is the replacement of treetops to grafts of this cultivar. Some other cultivars are preserved due to its adaptive characteristics, palatability and harvest out of season, allowing marketing for the best price. The study of the evaluation of creole avocados in the southern region of the state of Nuevo Leon reported 23 improved varieties [9], of which 13 coincide with those that have been identified in our studies. Due to the lack of definition in the branching pattern of trees by grafting different varieties in the same plant (Figure 3), it was decided only to perform a morphological evaluation of the fruits. For the molecular analysis of the samples and the morphological analysis of their fruits, 10 leaves and five ripe fruits per tree were collected. The characterization of the fruits was performed according to the morphological descriptors for avocado fruit given by the International Plant Genetic Resources Institute [10]. The characteristics were: fruit weight (g), fruit length (cm), fruit diameter (cm), seed weight (g), seed length (cm), seed diameter (cm), seed cavity length (cm), seed cavity diameter (cm), ratio of the fruit length/fruit diameter, and ratio of the seed weight/fruit weight. In order to determine the variables with greater weight on the morphological characterization and the behaviour of each variable, the mean, standard deviation and coefficient of variation were estimated. The data analysis was performed by principal components analysis and based on this; a cluster analysis was performed using the UPGMA method (un-weighted pair groups method with arithmetic averages) with the Gower distance (1-S) [11]. Statistical analyses were performed using the software InfoStat/Pv.2006p.3 [12]. In the fruit's morphological characterization, the traits with higher coefficients of variation were: seed weight (39.42%) and fruit weight (38.42%); the seed cavity length and fruit length showed coefficients of 24.82% and 20.34%, respectively. Values greater than 50% suggest that the characteristics have the highest variability within a species, while values below 20% indicate little variability in the species [11], so these characteristics were considered as classificatory. Figure 9. Comparison of the sizes and shapes of fruits of Persea americana Mill. var. drymifolia from Aramberri and General Zaragoza, Nuevo Leon, Mexico [13]. The sample identification is according to Table 1. As to the seed weight and fruit weight, the highest averages were 69.76 g and 251.40 g, respectively, corresponding to P26 (Platano Grueso); the lowest values for this variable were 7.08 g and 10.53 g, respectively, corresponding to P57 (El Salto). In the case of seed cavity length, the highest average corresponded to P21 (Platano Delgado) with 11.46 cm, the lowest average was 2.60 cm and corresponded to P57 (El Salto). For fruit length, the highest average value was 12.76 cm and corresponded to P4 (Platano Grueso), the lowest value corresponded to El Salto and was 2.88 cm; the fruit traits of this wild creole contrast sharply with the others samples collected (Figure 9). The relationship between the traits evaluated and the similarity between the samples was performed by principal component (PC) analysis with standardized data. The PC1 and PC2 represented 75.8% of the total variance, values greater than 60-70% explaining a reasonable percentage of the total variability of the samples [14]. The traits with the greatest influence in PC1 (59.7%) were fruit weight, seed weight and fruit length, while in PC2 (16.1%) they were seed diameter and seed cavity length. The cophenetic correlation was 0.984; PC analysis results coincided with the results of analysis of variation coefficients; of the samples organized in six clusters, P57 had the highest distance, and P45 and P48 (wild creoles) formed a cluster. P53, P56 and P11 formed another cluster-the first two corresponding to wild creoles while P11 corresponds to Platano Delgado. The samples P25 (Leonor) and P21 (Platano Delgado) were separated from the rest of the samples, while the other samples were grouped into a cluster. The dendrogram was performed with the following variables: seed weight, fruit weight, seed cavity length and fruit length; with the addition of the ratio of fruit length/fruit diameter and the ratio of seed weight/fruit weight, the cophenetic correlation increased to 0.888. Four clusters were formed at a distance of 0.23; two clusters coincide with those formed by the principal components analysis. Cluster I was formed with P57, cluster II with P21. Cluster IV was formed by five samples identified as wild creoles (P45, P47, P48, P53 and P56), sample P16 (creole), P1 (Huevo de Paloma), P25 (Leonor) and P32 (Chino). Cluster III was formed with the remaining samples ( Figure 10). Although the morphological differences between fruits are evident, only the sample P57 was identified. Linkage distances within the dendrogram reflected the differences and inconsistencies between presumably identical genotypes; we expected that the samples identified with the same name would be grouped together, which did not happen. These results agree with those reported by [15], who assert that fruit size is a trait that does not help in the differentiation of wild and cultivated avocado plants, because trees produce fruits of different sizes. Avocado groups are not well represented when the morphological characteristics of fruits are used [16]. Traditional plant identification is performed by phenotypic characterization-this is slow and limited, and the expression of the quantitative characteristics is subject to environmental influences. Molecular markers allow the identification, classification and use of genetic diversity present in the genomes of plants; differences or similarities at the DNA level in the individuals are observed directly. The AFLP (amplified fragment length polymorphism) is a highly efficient molecular marker [17]-in avocados they have been used to study the genetic diversity of germplasm [16,18,19]. The molecular characterization of the samples was performed using AFLP molecular markers; AFLP generation was performed using the IRDye Fluorescent AFLP Kit for Large Plant Genome Analysis (LI-COR® Biosciences); for the construction of the binary data matrix, it was assumed that bands with the same molecular weight are identical, assigning the number 1 to the presence of bands and the number 0 to the absence of bands [13]. Similarity indices were determined by the Gower coefficient (1-S). A cluster analysis of the molecular data was performed using the UPGMA method using standardized variables through the Info/Gen 2006p.1v software [20]; the cophenetic correlation coefficient was calculated. With the cluster analysis of 683 AFLP markers (Figure 11), two clusters were defined (distance 0.14). The samples P9 (Verde), P44 and P39 (Creoles), P38 and P29 (Platano), P4 (Platano Grueso), P3 (Pagua) and P23 (Calabo), were not clustered. The separation of these samples shows that they are different genotypes, but in those samples that were not clustered with samples with the same local name, the question is whether there is genetic variation among the improved creoles. Cluster "I" was composed of samples P48, P46 and P45, identified as wild creoles, while cluster "II" was composed of 32 samples. By decreasing the distance to 0.09 in the dendrogram, samples identified as Platano (P30, P15, P13, P12) and Platano Delgado (P11), were grouped; while P56, P50 (both wild creoles) and P32 (Chino) samples were separated. The clustering at very short distances (0.015) is between P6 (Campeon), P7 (Platano Delgado) and P1 (Huevo de Paloma), and between P53 (wild creole) and P33 (Maria Elena), which shows that they are closely related. The clustering at distances greater than samples identified with the same name calls into question the genetic identity of improved creoles. To perform the comparative analysis between cluster groups formed in the morphological and molecular analysis, a mixed data matrix was constructed with morphological data and AFLP binary data; a cluster analysis was performed by UPGMA with the Gower distance (1 S) through the software Info/Gen 2006p.1v [20]. As in previous cases, the cophenetic correlation coefficient was calculated. (Figure 12). The separation of P9, P44, P3 and P4 coincided with that obtained in the molecular data analysis. The separation of P48, P57, P45, P56 and P50 identified as wild creole is a difference between the analysis of mixed data and analysis of molecular data. With increasing distance in the dendrogram, the P57 and P45 (wild creoles) samples were clustered; these samples are characterized by the lowest values of fruit length and seed length. When the cluster distance was increased in the dendrogram, the influence of the morphological variables in the formation of the groups was evident, so the group consisting of P48, P57 and P45 was characterized by the lowest seed weight values. When analysing in detail the scheme of clustering, we observed that P26 and P27 were grouped at a shorter distance-an important feature of these samples is that they showed the highest fruit weight and that they correspond to the improved creoles Platano Grueso and Platano, respectively. As such, we can conclude that they present similarities. The results of this study demonstrated that AFLP molecular markers allow the estimation of the genetic diversity of the Mexican race of Persea americana Mill. when molecular data are combined with morphological data. When only molecular data generated by AFLP are used, the differentiation between genotypes was ambiguous; we demonstrated the effectiveness and facility of AFLP used to characterize avocado accessions based on the race origin [19]; however, there is no reference about its usefulness in differentiating between varieties. In vitro culture callus We have worked with in vitro culture techniques to conserve the germplasm of different avocado genotypes. The selection of the explant to be used in the conservation of plant tissue depends on the resources and the goal of the project; in avocados, different explants have been used to obtain different types of morphogenesis [21]. The avocado morphogenetic capacity under in vitro conditions is linked to the use of materials with juvenile characteristics [22]. The avocado cotyledon callus was kept for over 15 years without apparent differentiation conditions [23]-these tissues remained as callus divisions through a series of small segments and were transferred to fresh media at intervals of one-to-three months. The callus tissue is essential for obtaining somatic embryos by indirect embryogenesis [24,25]. Avocado somatic embryos were regenerated from calluses [26][27][28][29]. We used the developed young leaves of a Mexican avocado tree (six years old) as explants to establish an in vitro culture callus. The avocado leaves were washed with a commercial detergent and running water for 30 minutes. Subsequently, petiole were cut and the leaves were placed in an antioxidant solution, 400 mg L -1 of ascorbic acid, 150 mg L -1 of citric acid and 30 g L -1 of sucrose with 1 g L -1 systemic fungicide metalaxyl-M (Ridomil, Syngenta Agro), for 2 min at a vacuum pressure of-20 bar; they were placed in alcohol 70% v / v for 30 s followed by three rinses with sterile water, then placed in a solution of 2.4% NaOCl with two drops of Tween 20 for each 100 ml for 15 min, followed by three rinses with sterile distilled water; the leaves were kept in sterile antioxidant solution above without systemic fungicide, until their establishment in vitro. Explants of 1 cm 2 were obtained and placed in glass vials with 20 ml of DCR medium [30] pH 7.5, supplemented with vitamins and growth regulators, 200 mg L -1 of inositol, 50 mg L -1 of glutamine, 500 mg L -1 of casein hydrolyzate, 0.3 mg L -1 of 6-benzylaminopurine, 0.01 mg L -1 of naphthaleneacetic acid, 0.1 mg L -1 of thiamine, 0.5 mg L -1 of nicotinic acid, 0.5 mg L -1 of pyridoxine, 2 mg L -1 of glycine, 10 mg L -1 of ascorbic acid, 10 mg L -1 of citric acid, 1.5 mg L -1 of 2,4-D, 0.3 mg L -1 of kinetin, 20 g L -1 of sucrose and 4.5 g L -1 of Phytagel. The glass vials with the explants were placed in a controlled temperature of 20 ° C ± 2 ° C in complete darkness for callus formation. Callus formation from the leaves was completed out in 30 days ( Figure 13). Using the same culture medium, two subcultures of each callus were performed with a period of 30 to 45 days between them. AFLP markers were generated and 341 polymorphic bands were used to calculate the polymorphic information content (PIC) and the genetic distance to measure genetic stability of the avocado callus. We analysed molecularly 94 samples, the mother plant, 31 calluses formed from the mother plant explants, 31 calluses from the first subculture, and 31 calluses from the second subculture-the results of this research are in press. Present investigations Actually, we are working with the fruit gene expression of genotypes with contrasting characteristics (Figure 14). Using the technique of differential display [31], we obtained differential fragments of the genome of the fruit ( Figure 15); these amplified fragments were sequenced in order to identify regions of the genome to help us in the identification and differentiation of genotypes (improved creoles, creoles and wild creoles). We are analysing the sequences obtained with data from the NBCI and hope that these results can be used in the future in the genetic characterization of genotypes as well as in avocado breeding programs. In order to preserve all the genetic variability of the Mexican avocado that has been detected in Nuevo Leon, Mexico, a local germplasm bank is being implemented together with growers where they can safeguard copies of improved creoles, creoles and wild creoles. The intention is to have plant material for them and to start in a short period with the creation of a breeding program for the Mexican avocado. Conclusion The genetic diversity of Persea americana var. drymifolia, found in Nuevo Leon, Mexico, is broad and can be used for incorporation into breeding programme cultivars, as well as for use as rootstocks and inter-stocks; wild varieties are a genetic patrimony that can provide innovative sources of advantageous use for producers. Identifying the characteristics of agronomic interest (genes) boosts the Mexican avocado crop through its alternate use (processed fruit, oil extraction, medicinal use, use as a condiment, animal feed, etc.). The classification and conservation of plant germplasm should be seen as an activity to preserve this heritage of diversity-it requires a major effort to preserve the genotypes of cultivated plants, but especially wild and native plants that are threatened with the destruction of their natural habitat and being replaced by improved varieties. Author details Adriana

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Somatic embryogenesis in ferns: a new experimental system Somatic embryogenesis has never been reported in ferns. The study showed that it is much easier to evoke the acquisition and expression of embryogenic competence in ferns than in spermatophytes. We discovered that the tree fern Cyathea delgadii offers an effective model for the reproducible and rapid formation of somatic embryos on hormone-free medium. Our study provides cyto-morphological evidence for the single cell origin and development of somatic embryos. Somatic embryogenesis (SE) in both primary and secondary explants was induced on half-strength micro- and macro-nutrients Murashige and Skoog medium without the application of exogenous plant growth regulators, in darkness. The early stage of SE was characterized by sequential perpendicular cell divisions of an individual epidermal cell of etiolated stipe explant. These resulted in the formation of a linear pro-embryo. Later their development resembled that of the zygotic embryo. We defined three morphogenetic stages of fern somatic embryo development: linear, early and late embryonic leaf stage. The transition from somatic embryo to juvenile sporophyte was quick and proceeded without interruption caused by dormancy. Following 9 weeks of culture the efficiency of somatic embryogenesis reached 12–13 embryos per responding explant. Spontaneous formation of somatic embryos and callus production, which improved the effectiveness of the process sevenfold in 10-month-long culture, occurred without subculturing. The tendency for C. delgadii to propagate by SE in vitro makes this species an excellent model for studies relating to asexual embryogenesis and the endogenous hormonal regulation of that process and opens new avenues of experimentation. Introduction Somatic embryogenesis is the developmental pathway by which plant somatic cells develop into somatic embryos. Apart from the lack of syngamy, these resemble zygotic embryos. SE is currently used to propagate hundreds of species of seed plant in vitro, and forms the basis of fundamental studies that help us understand how a single somatic cell develops to form an entire plant (Vogel 2005). The process serves as a model system for the study of molecular, biochemical and physiological events that occur during both the induction and the development of the embryo. Although SE was discovered more than half a century ago (Steward et al. 1958), our entire knowledge of this process is based on seed plants. It has not previously been reported for monilophytes. Moreover, it has been reported only twice for cryptogams, and in each case these were lycopods, i.e. Lycopodiella inundata (L.) Holub (Atmane et al. 2000) and Huperzia selago (L.) Bernh. Ex Schrank and Mart. [Lycopodium selago L.] (Szypuła et al. 2005). Since ferns and spermatophytes are sister lineages (Pryer et al. 2001), ferns are useful subjects for developmental and morphological research, comparative evolution, and functional genomics Renzaglia 2008, 2009;Der et al. 2011;Tomescu 2011;Vasco et al. 2013). They can also help to improve our understanding of SE. In terms of zygotic embryogenesis, ferns have been relatively under-investigated compared to spermatophytes. Extensive studies of the sexual reproduction of ferns were conducted at the turn of the twentieth century and a considerable amount of information regarding the embryology of this plant group was collected, collated and published by Wardlaw (1955). He demonstrated the pattern of development that occurred during the early embryology of various fern species and how the two-celled early embryo of leptosporangiate ferns underwent six additional regular divisions to create an eight-celled embryo. The proliferation of zygotic initial cells eventually led to a four-part embryo, the four quadrants forming the root, leaf, foot, and shoot apex, respectively, that would later produce additional vegetative organs. Subsequently, morphological, cytological, and biochemical studies conducted on fern zygotic embryos at various stages of development were summarized by DeMaggo (1977). Since that time, progress has not been as rapid or as extensive in its scope, and there still remains a dearth of embryological information relating to those processes that follow on from cell and organ differentiation. By contrast, there is a wealth of information available relating to the developmental anatomy and morphology of shoot, leaf and root of the fern sporophyte (White and Turner 1995). Currently, researchers aim for a better understanding of the evolution of land plants, and alternation of generations, as observed in ferns (Niklas and Kutschera 2009;Ligrone et al. 2012). Renzaglia (2008, 2009) elucidated the development of the embryo and gametophyte placenta for the fern model Ceratopteris richardii Brongn. and significantly broadened our knowledge of the development of the fern zygotic embryo. It was also shown that auxin is involved in the initial zygotic cell division of Marsilea vestita Hook. & Grev. and organization of body plan in fern sporophytes (Poli 2005). In addition to the sexual life cycle, some fern species undergo asexual development resulting in the formation of sporophytes from gametophyte somatic cells (apogamy) (Raghavan 1989). Either obligate or induced apogamy is considered equivalent to organogenesis of sporophytes (Raghavan 1989;Fernández et al. 1996;Gabancho et al. 2010). An apogamous system of reproduction occurs both in nature and in vitro, and it has been regularly studied ever since its discovery by Farlow (1874) up to the present time (Cordle et al. 2011). This method of reproduction became established in fern lineages that experienced frequent reticulate evolution in combination with polyploidy and has been recognized for approximately 10 % of extant ferns (Liu et al. 2012). However, apogamy has been only sporadically reported for tree fern species (Stokey 1918;Parajuli and Joshi 2014). Propagation of tree fern species is a difficult challenge and thus, few tree ferns are propagated by commercial nurseries. Some of the species can be propagated by spores, but they cannot be propagated vegetatively as they do not produce offsets from their 'trunk' (Large and Braggins 2004). However, for commercial purposes, the erect 'trunk' of tree ferns (e.g. Dicksonia antarctica Labill.) can be cut, transported and replanted, and can still continue to grow, as long as the crown remains intact (FPA Biodiversity Program 2012). Over the last few years, the propagation of tree fern species from spores has become the priority area for ex situ conservation studies (Simabukuro et al. 1998a, b;Arens 2001;Hiendlmeyer and Randi 2007;Rechenmacher et al. 2010;Ranil et al. 2011;Martíez et al. 2014). Conversely, methods of in vitro culture have been exploited for the propagation of tree ferns by gametophyte multiplication and sporophyte regeneration. Media used for growth and proliferation of gametophytes were either supplemented with plant growth regulators (PGRs) (Bonomo et al. 2013;Das et al. 2013) or not Rybczyński 1995, 2007;Kuriyama et al. 2004;Khare et al. 2005;Moura et al. 2012). The application of biotechnology methods for tree ferns was summarized by Rybczyński and Mikuła (2011). Their list of 20 species has recently been extended by at least 3 new taxa, namely: Alsophila odonelliana (Alston) Lehnert, Cyathea gigantea (Wall. ex. Hook.) and C. cunninghamii Hook. f. (Moura et al. 2012;Bonomo et al. 2013;Das et al. 2013). Gametophytes cultured in vitro also provide sufficient plant material for cryo-studies and the long-term preservation of tree ferns in liquid nitrogen (Mikuła et al. 2011a). Further methods are required for the efficient, quick and effective propagation of tree fern species in vitro. The present work relates to Cyathea delgadii Sternb., a species of evergreen, non-seasonal tree fern (10 m tall) native to the gallery, montane, cloud, and rain forests of the Caribbean, Central and South America, including valuable ecoregions of the Atlantic Forest. It grows at an elevation of 100-2,730 m above sea level (Oliveira-Filho and Ratter 1995). Cyathea delgadii is a member of a large complex centered on Cyathea fulva (M. Martens & Galeotti) Fée (Arens 2001). It produces spores all the year round (some 300 million spores per frond), but their viability, like the spores of most species belonging to family Cyatheaceae, diminishes after a few weeks of storage at room temperature or after a 2-year period of storage at 4°C (Simabukuro et al. 1998a). This species is cultivated as a garden ornamental plant (Hiendlmeyer and Randi 2007). Our study focuses on the induction and description of SE in the fern C. delgadii, belonging to a group of plants (Monilophyta) for which this phenomenon has not yet been reported. Emphasis is focused on cyto-morphological evidence for SE induction, embryo development, and the efficiency of this process during short-and long-term culture. Plant material Laminae of C. delgadii fronds were collected from a plant growing in the greenhouse of the PAS Botanical Garden-CBDC, Warsaw, Poland. They were dried at room temperature for 5 days to liberate spores. The released spores were surface sterilized by wrapping sporangia in Whatman no. 1 filter paper and immersing the package in 70 % (v/v) ethanol for 30 s and in 5 % (v/v) commercial bleach (Domestos) for 20 min and then washing the package three times in sterile distilled water. After disinfection, spores were blotted onto medium containing half-strength MS micro-and macronutrients (Murashige and Skoog 1962) with full complement of vitamins (1/2MS), 2 % (w/v) sucrose and 0.7 % (w/v) plant agar. The pH of the medium was adjusted to 5.8 before autoclaving at 120°C for 20 min. The spores germinated at 22 ± 1°C under a 16/8 h photoperiod, at a light intensity of 3.5 lE m -2 s -1 . The young gametophytes were transferred separately onto fresh 1/2MS medium and subcultured only once. They were maintained in subculture until they reached maturity. After spontaneous syngamy, zygotic embryos and young sporophytes were produced after 1 year of gametophyte culture. Somatic embryogenesis induction and the assessment of its efficiency Stipes of zygotic embryo-derived sporophytes (Fig. 1a) developed under 16/8 h photoperiod were used for the initiation of primary SE. Secondary SE was induced on intact somatic embryos that had reached the first crozier stage and on the stipes of somatic embryo-derived sporophytes ( Fig. 1b) that had developed 2 or 3 fronds, growing in darkness. The plant material was cultured on 1/2MS agar medium supplemented with 2 % (w/v) sucrose and 0.7 % (w/v) plant agar. The cultures were kept in a climatic chamber at ?22 ± 2°C, in constant darkness, and at a relative humidity of 35-55 %. The percentage of responding explants and the number of somatic embryos per responding explant was calculated after 9 weeks of culture. Subsequent evaluations were carried out every month for almost 1 year of culture. The somatic embryo production capacity index (SEPCI) was calculated by multiplying the proliferation percentage by Sporophyte acclimatization and transfer to soil For acclimatization, sporophytes cultured under 16/8 h photoperiod conditions were used. Sporophytes with 4-6 fronds were transferred to pots of peat and perlite (3:1) substrate, and kept in mini greenhouses. The potting mixture was autoclaved at 121°C for 20 min. The mini greenhouses were lightly ventilated daily, and the sporophytes periodically misted for 4-6 weeks. The plantlets were then transferred to a greenhouse. Microscopic preparation Stipes of somatic embryo-derived sporophytes were fixed in 2 % glutaraldehyde in 0. , followed by mixtures of absolute ethanol and propylene oxide (3:1; 1:1; 1:3), and finally, propylene oxide. Explants were then embedded in Epon-Spurr epoxy resin mixture. Twomicrometre-thick sections were cut using an LKB ultramicrotome (Sweden) and stained for several seconds with aqueous 0.1 % toluidine blue solution. They were examined using a Vanox epifluorescence microscope (Olympus, Japan) equipped with a computer image analysis system (cellSens Standard ver. 1.7). To detect the natural red autofluorescence of chlorophyll, a blue-violet light (BV filter: 400-440 nm) was used. For examination of the first few divisions of epidermal cells, plant material was fixed in FAA (5 parts formaldehyde:5 parts glacial acetic acid:90 parts ethanol) overnight and then dehydrated in graded ethanol solutions (70, 80 and 100 %). Next, explants were cleared in methyl salicylate as described by Young et al. (1979) and examined under a Vanox microscope (Olympus, Japan) using blue-violet light (BV filter: 400-440 nm). Statistical analysis Results were analyzed by means of a one-way ANOVA analysis of variance and Fisher's least significant difference (LSD) procedure using Statgraphics Plus software. Significance was set at the 0.05 level. The results were expressed as the mean ± standard deviation based on three independent experiments, each consisting of at least 30 explants. Results Production of zygotic embryo-derived sporophytes as a source of primary explants Propagated gametophytes of C. delgadii achieved maturity within a year of in vitro culture on 1/2MS medium. Further details of sex organ formation for the species were provided by Rybczyński and Mikuła (2011). Following fertilization, the early development of the zygotic embryo was confined to the archegonium. Within 4-5 weeks, the main organographic regions had been determined. Subsequently, the first leaf elongated and the second developed (Fig. 1a). The stipes of the first young fronds, grown at a photoperiod of 16/8 h, were used for the following experiments (Fig. 1a). h Numerous somatic embryos with first leaf after 6 weeks growth. i Partly green, differentiated lamina of the first leaf of juvenile sporophyte. j Somatic embryo-derived young sporophyte showing extended lamina of primary frond and primordium of second leaf, as well as two roots, following development in the presence of light. C cortex, Ep epidermal cells, L first leaf, R root, SA shoot apex, T trichomes, Vc axial cylinder (color figure online) Cyto-morphological evidence for somatic embryogenesis induction and embryo development Within 2 weeks following culture initiation, divisions of epidermal cells of stipe explants began (Fig. 2a). The first division of the epidermal cell was perpendicular to the polar axis of the explant and led to the formation of two, almost equal or unequal daughter cells. The next few divisions (divisions 8-10) were also perpendicular to the stipe axis (Fig. 2b). Within 3 weeks of culture, these Plant Cell Rep (2015) 34:783-794 787 divisions resulted in the formation of linear somatic proembryos. Dividing epidermal cells were widely distributed along the explants (Fig. 3a). Within 3 weeks following culture initiation, numerous epidermal cells of stipe explants were present, resulting from several anticlinal, periclinal and inclined cell divisions. These irregular divisions led to the formation of four separate and unequally sized segments of the pro-embryo (Fig. 3b). During the early stage of somatic embryo development, trichomes developed on two of four of the visible segments of the pro-embryo (Fig. 3c). Further development of somatic embryos focused on the differentiation of the embryonic leaf (Fig. 3d). Later, the emerging lamina primordium of the first frond and primordium of the shoot apex were visible (Fig. 3e). Histological analysis revealed that the somatic embryos originated directly from epidermal cells of stipe explants (Fig. 3f, g). The structure of the stipe was maintained for the entire duration of the initial culture on 1/2MS medium. The single-layered epidermis, cortex and closely arranged axial cylinder cells of explants were clearly distinguishable, even after 6 weeks of culture (Fig. 3g). The majority of the epidermal and cortex cells were rich in amyloplasts, which stained intensely with toluidine blue. The axial cylinder appeared to be structurally intact. During the first leaf stage, the somatic embryos showed a normally developed vascular system and the first leaf, shoot apex and the primordium of the second leaf were also visible (Fig. 3g). On some explants, all the somatic embryos produced during the first 6 weeks of culture were almost at the same stage of development (Fig. 3h). Somatic embryos which developed in darkness were opaque (Fig. 3h) or somewhat translucent at the base (Fig. 3d), but apically, were white, yellow or greenish in color. At transition from embryo to juvenile sporophyte, the lamina of the embryonic leaf became pale green during the next week of culture, and the whole leaf elongated (Fig. 3i). The next step of sporophyte development was the formation of the second frond and the elongation of the root. On returning to light, the frond lamina of the etiolated sporophyte quickly became dark green and regained its typical shape (Fig. 3j). The efficiency of somatic embryogenesis in short-and long-term culture When stipes of the first frond of zygotic embryos were cultured on 1/2MS agar medium containing 2 % sucrose, primary somatic embryos were formed at a frequency of 19.3 % over a 9 week period (Fig. 4). When SE was induced in stipes of somatic embryo-derived juvenile sporophytes, 85.71 % of explants formed new somatic embryos. In intact somatic embryos, the percentage of responding explants was lower (71.43 %), but the difference was statistically insignificant. The average number of somatic embryos produced was similar for each type of initial explant studied, ranging from 11.97 to 13.57 per stipe (Fig. 4). Somatic embryo-derived stipe explants were able to produce somatic embryos within 11 months of maintenance on 1/2MS medium supplemented with 2 % sucrose without any subculture. The cultures were kept constantly in darkness. The frequency of explants producing somatic embryos ranged between 61.0 and 86.4 % ( Table 1). The efficiency of C. delgadii SE in long-term culture differed significantly (Table 1). After 2 months, each stipe produced 13.2 somatic embryos. The embryogenic potential increased gradually, reaching 84.3 and 80.1 somatic embryos per responding explant by the end of months 9 and 10, respectively. The efficiency of SE decreased to 55.2 embryos per stipe after 11 months of continuous culture. The highest SEPCI index was based on 10-monthold cultures. The progress of an 11-month-old culture maintained in darkness is shown in Fig. 5. The fronds of somatic embryoderived sporophytes gradually elongated, and the mass of tissue was seen to increase (Fig. 5a-c). After 3 months of culture, mass-produced young sporophytes (Fig. 5d) developed two fronds and one or two roots (see Fig. 1b). After 5 months, the fronds possessed long stipes and most of their laminae remained as croziers (Fig. 5e). A few of these commenced further development (Fig. 5e). After 7 months of culture, the first symptoms of sporophyte aging were observed. Some fronds turned brown (Fig. 5f). During 11 months of extended culture, some fronds died (Fig. 5c). The aging fronds spontaneously produced new somatic embryos directly on their laminae and stipes (Fig. 5f, g). These somatic embryos were capable of further development without any subculture (Fig. 5g). Alternative ways of embryo formation were also investigated. When cultures were maintained in darkness for at least 5 months, brown tissue was formed at the base of sporophytes (Fig. 5e). During the next 3 months, somatic embryos arose from this tissue (Fig. 5h). Moreover, the laminae and stipes of aging fronds developed embryogenic callus tissue (Fig. 5i, j). The resultant somatic embryos were also able to develop into sporophytes (Fig. 5k). Under 16/8 h photoperiod conditions, all etiolated sporophytes turned green and developed normally to form plantlets (Fig. 5l). Sporophytes began to produce spores after 6 months of growing under ex vitro conditions. The process of SE for C. delgadii is shown schematically in Fig. 6. It commences with spores, passes through the gametophyte stage, the induction of zygotic and somatic embryogenesis, the growth of mature plantlets and their acclimatization to ex vitro conditions, and ends with a second generation of spores (Fig. 6). Plant regeneration and acclimatization After 3 weeks of culture of initial explants, embryos were formed (Fig. 3a). Most of these had the capacity for further development. Under conditions of constant darkness, embryos developed a few roots and fronds, but the latter remained at the crozier stage for several months (Fig. 5d, e). When a 3-week-old initial culture was subjected to photoperiod conditions, the embryos expanded sequentially and developed into sporophytes without any additional subculture (Fig. 5l). The sporophytes grew quickly and, within 8 months, their acclimatization to ex vitro conditions was complete. Following acclimatization, 23 of the 25 tested sporophytes survived and were transferred to a greenhouse. During the next 6 months, the sporophytes grew to maturity. They produced spores that were able to germinate and form fertile gametophytes (Fig. 6). Discussion In this paper, we report on a novel SE experimental system for ferns. Our aim was to pioneer a new line of research into this process. This we accomplished by investigating a species of the Monilophyta clade. We discovered that epidermal cells of C. delgadii stipe explants have the potential to initiate somatic embryo development equal to that of many spermatophytes. However, it is much easier to evoke the acquisition and expression of embryogenic capacity of somatic cells in ferns than in spermatophytes. This study has enabled us to develop an effective, reproducible and rapid method of propagating C. delgadii sporophytes. Furthermore, the method of cultivation presented here is particularly attractive in that the use of exogenous PGRs is not required. Somatic versus zygotic embryogenesis in ferns Species belonging to Cyatheaceae, like most leptosporangiate ferns, exhibit a type of embryology in which the first zygotic division is transverse to the polar axis of the gametophyte and longitudinal to the axis of the archegonium (Johnson and Renzaglia 2009). According to Wardlaw (1955) this first division is slightly asymmetric in Pteris serratula L.f. and Marsilea. The following two divisions give rise to a 4-celled zygotic embryo. Contrary to reports of typical leptosporangiate fern zygotic 5 Progress of somatic embryo production in Cyathea delgadii during long-term culture. General view of a culture maintained in darkness for: a 3 months, b 5 months, c and 11 months. d Numerous young sporophytes with roots and croziers (arrows), after 3 months. e Typical fronds with long stipes and croziers (arrows), with brown tissue at their bases; a few laminae that have developed further (rectangle and small picture), after 5 months. f Spontaneous production of somatic embryos on aging fronds (arrows), after 7 months. g Further development of spontaneously induced somatic embryos. h Somatic embryos derived from the brown tissue (arrows), after 8 months. i Yellow and green callus on surface of lamina. j Yellow and green callus on surface of stipes with differentiated somatic embryos (arrows). k Young sporophytes (asterisks) formed by secondary, spontaneous somatic embryogenesis. l Image of sporophytes after 2 week-long light exposure. R root, S stipe (color figure online) embryogenesis, somatic embryo formation in C. delgadii was initiated by several cell divisions perpendicular to the polar axis of the stipe. These divisions led to the formation of a linear somatic pro-embryo. Despite these differences between somatic and zygotic embryogenesis in the way the first division occurs, complete C. delgadii somatic embryos were produced. This may strengthen the hypothesis that the initial division may not be important in the fate determination of the embryonic cell in pteridophyte groups (Johnson and Renzaglia 2009). Variations in the plane of zygotic division in leptosporangiate ferns (particularly in angle shift) may be regulated by its proximity to an auxinsecreting meristematic region of the gametophyte, gravity or gametophyte habit (Stone 1958;Jayasekera and Bell 1959). Thus, we speculate that the pattern of early SE is also determined by the impact of the polar gradient of endogenous hormones as the latter pass from the blade to the base of the frond. In zygotic embryogenesis of leptosporangiate ferns, following the first division of the zygote, the epibasal cell becomes located towards the apex of the prothallus, and after it divides (transversely to the first division), the daughter cells independently give rise to the shoot apex and first leaf. Division of the hypobasal cell leads to the formation of the first root and foot (Wardlaw 1955;Johnson and Renzaglia 2008). Our studies showed that despite the linear course of somatic pro-embryo formation, its further development resulted in division of the embryo body into four clearly visible segments and subsequently into two regions which can be distinguished based on the presence or absence of trichomes. We speculate that the region devoid of trichomes can give rise to foot and embryonic root, whereas the region with trichomes can develop into the shoot apex and embryonic leaf. It seems that despite the lack of an archegonium and adjacent gametophyte tissue able to supply nutrients and growth-regulating substances (e.g. auxins) to the developing zygotic embryo (DeMaggo 1977), the polarity of C. delgadii somatic embryos was established very early. This forms the subject of a future investigation. Developmental differences between embryo-derived and apogamous sporophyte The development of C. delgadii somatic embryo and juvenile sporophyte was similar to that observed during the zygotic embryogenesis of other leptosporangiate ferns (Wardlaw 1955). Likewise, the first embryonic leaf differed from subsequently formed leaves, and an embryonic root was formed at the base of the first leaf. Development of an apogamous sporophyte differs from the development of an embryo-derived sporophyte in certain details. It was shown that the young apogamous sporophytes of Asplenium auritum Sw., A. monodon Liebm., Phegopteris connectilis (Michx.) Watt. and Pteris multifida Poir. did not form roots in parallel to the first leaf (Kawakami et al. 1995;Soare et al. 2007;Gabancho et al. 2010). Moreover, the first sporophyte leaf possessed stomata, multicellular glandular hairs, and scales (Gabancho et al. 2010). Contrary to the first leaf of the apogamous sporophyte, the juvenile, embryonic leaf of C. delgadii did not develop stomata. C. delgadii somatic embryogenesis versus somatic embryogenesis of seed plants and lycopods Propagation of spermatophytes in in vitro culture by SE is a complicated process requiring the proper combination of Fig. 6 Schematic diagram representing the course of the newly described somatic embryogenesis process for C. delgadii: commencing with spores, passing through the gametophyte stage, followed by induction of zygotic and somatic embryogenesis and finally, the production of mature plantlets initially grown in vitro and later under ex vitro conditions. SE somatic embryogenesis, initial explants are shown in red (color figure online) Plant Cell Rep (2015) 34:783-794 791 various factors such as: type of plant material, PGRs, sugars, light, mineral salts, etc. (Gaj 2004). For the majority of seed plant species, exogenous PGRs are amongst the most important factors involved in the induction and maintenance of SE (Raemakers et al. 1995;Fehér et al. 2003). Our studies showed that SE in both primary and secondary explants of the fern C. delgadii can be induced without the application of PGRs. Moreover, spontaneous formation of somatic embryos and callus production, which improved the effectiveness of the process several fold in 10-month-long culture, occurred without subculturing. This suggests that the juvenile, etiolated stipe explant that was used for culture initiation and a starvation play a crucial role in SE induction in C. delgadii. We do not know of any other species for which the process of SE is so highly effective, yet demands so little effort. It is possible that the low margin requirement in the culture may be specific for cryptogamic plants. In the lycopod Lycopodiella inundata, the nodular callus and somatic embryo production were established on a MS medium with half-strength mineral salt content, but supplemented with PGRs (Atmane et al. 2000). For a second species of lycopod, Huperzia selago, both the induction of callus and the formation of nodular structures on its surface and their subsequent development into somatic embryos occurred on MS hormone-free medium containing an identical mineral salt concentration to that used for C. delgadii (Szypuła et al. 2005). For some species, non-hormonal factors, for example, osmotic shock, drought, mechanical wounding of tissues, macro-salts, heavy metal ions and heat or cold shock, can also be used to induce or enhance SE efficiency (Smith and Krikorian 1989;Choi et al. 1998a;Patnaik et al. 2005;You et al. 2007;Mikuła et al. 2011b). Our study showed that in the case of C. delgadii, none of the aforementioned inducers (even mechanical damage) are essential to regain cell totipotency and to acquire the competence necessary to convert somatic cells to embryogenic cells. Our success was probably due to the use of a specific type of explant, i.e. a piece of very young etiolated sporophyte, and maintaining the initial culture in constant darkness. It would appear that endogenous hormone levels in cells and tissues of this species are a major factor in determining cellular response. Choi and Soh (1996) showed that direct somatic embryo formation from ginseng zygotic embryos grown on regulator-free medium was related to the excision of zygotic embryos and polar endogenous auxin accumulation. Conversely, the origin of somatic embryos as multiple or single-state forms, depends on the degree of maturity of the plant material used for experiments (Maheswaran and Williams 1985;Choi et al. 1998b). We have produced somatic embryos of single-cell origin from the maturing epidermis of juvenile sporophyte stipes of C. delgadii. According to Maheswaran and Williams (1985), this type of tissue can contain some immature epidermal cells and these single cells are capable of embryogenic response. Embryo development It is well known in pteridophyte embryology that the patterns of zygotic embryo development are defined by the plane of zygotic division and the direction in which the first leaf and shoot apical meristem grow with respect to the gametophyte (Wardlaw 1955;Johnson and Renzaglia 2009). However, in comparison to spermatophytes, the studies of fern embryo development are still incomplete and the developmental stages have not yet been given names. We demonstrated how the pivotal phases of C. delgadii somatic embryo formation have morphological counterparts in fern zygotic embryogenesis. Leptosporangiate fern embryos have no suspensor, whereas this is present both in zygotic and most of somatic embryos of seed plants (Young 1995). Instead, during the first stage of fern embryo formation, the foot plays a crucial absorptive and nutritional role in the growth of the embryo (Johnson and Renzaglia 2008). However, using current methods for plant body investigations, we were not able to recognize a foot in the somatic embryos of C. delgadii. In future, we intend to use transmission electron microscopy to provide an anatomical framework for studies of somatic embryogenesis. Another important issue is the designation of morphogenetic stages of the fern embryo, in which the basic structure of the embryo is established. In angiosperms, the development of both zygotic and somatic embryos occurs via the globular, heart, torpedo and cotyledonary stages (Young 1995;Filonova et al. 2000). The sequence of gymnosperm embryo development can be divided into three phases: proembryogeny (up to suspensor elongation), early embryogeny (up to the root meristem establishment) and late embryogeny (including establishment of the root and shoot meristems) (Filonova et al. 2000). At present, we are able only to define three different morphogenetic stages representing three major events in the development of somatic embryo in ferns: 1. linear stage: from the first cell division until the formation of several-celled pro-embryo (Figs. 2, 3a, b); 2. early embryonic leaf stage: until the emergence of the first leaf (Fig. 3c, d); 3. late embryonic leaf stage: until the emergence of the second leaf primordium (Fig. 3e, g, h). Although the earliest stage of somatic embryo development in C. delgadii, comparable to globular stage was not observed, the subsequent developmental patterns appeared to correspond to those observed during the zygotic embryogenesis of leptosporangiate ferns (Wardlaw 1955;Johnson and Renzaglia 2008). The fern zygotic embryo grows and develops its first leaf without any interruption caused by dormancy until it emerges from the gametophytic tissue and becomes established as a freeliving sporophyte (Wardlaw 1955). Similarly, the development of the somatic embryo into a juvenile sporophyte is rapid and proceeds directly under the light conditions stated for the tree fern C. delgadii. Conclusions Ferns, as a group, are the closest living relatives of spermatophytes, and are of interest not only because of their ornamental value, but also for their usefulness as models for evolutionary, morphological and developmental studies. The phylogenetic position of ferns and their amenability to apogamic reproduction both in vivo and in vitro make them valuable models for studying how asexual plant embryogenesis evolved. The simple, effective and hormone-free system of SE induction described for C. delgadii can help broaden our fundamental knowledge of this process. Looking to the future, it also provides an excellent model for the study of endogenous hormonal regulation of SE. This novel method for the in vitro reproduction of tree ferns may also be valuable for the rapid and mass propagation of these plants both for conservation and commercial purposes.

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Stabilization of Organic Matter in the Raised-Bed Soils of Tidal Swamplands is Influenced by The Types and The Amounts of Organic Matter Application Farmers in tidal swamplands annually added organic matter (OM) onto the raised beds to maintain organi c matter contents and thereby maintain soil productiv ity of the raised beds. This experiment aimed to st udy the influence of the types and the amounts of OM on the s abilization of organic matter in the raised-bed soils. Four types of OM: rice straw, eceng gondok ( Eichornia crassipes), purun tikus ( Eleocharis dulcis) and mixed rice straw-eceng gondok were added to a 27-y ear raised bed soil with 4 different rates: 0, 0.5, 1 0 and 2.0 of maximum sorption capacity (Q max), and the OM stabilization was quantified after 10 weeks of OM addition. Results of this study showed with the ex ception of rice straw, OM addition to soil resulted in increases in the mineralization of soil OM thereby inducing priming effect. Addition of rice straw at rate of 0.5 of Qmax resulted in stabilization of 46% added OM, while o nly 30% and 37% of added OM was stabilized when OM was added to soils at rates of 1.0 and 2.0 Qmax, respectively. This study showed that the stabilization of OM in raised bed soils were influe nc d by the chemical composition of OM and the amou nt of added OM. Key-words: adsorption, green-house gas emission, carbon retention, ligand exchange INTRODUCTION Farmers of tidal swamplands generally halved their land; some areas of the land were left in flooded condition and planted with rice, while other part of the land were elevated through the deposition of muddy siltclay minerals from surrounding areas. In the raised-beds, the farmers then cultivated crops, cassava, fruits and perennial crops. Crop productivity in the raised beds varies with the age raised beds. Reduction in the crop productivity may due to changing organic matter contents in the raised-beds with increasing the age of raised-beds. Study conducted by Rasmadi (2003) showed that the Correspondence: A.R. Saidy, Department of Soil, Faculty of Agriculture, Lambung Mangkurat University, Banjarbaru 70714, Indonesia. Phone: +62 511 4777540, fax: +62 511 4772254, email: [email protected] content of organic carbon and nitrogen decreased with progressing the age of raisedbeds. Farmers annually deposed organic matter that has decayed and mixed with muddy siltclay minerals from sunken-beds and then piled them onto the raised-bed. The purpose of mixed silt-clay-OM deposition onto the raised-bed is to elevate raised-bed surface. This process may also improve the stabilization of organic matter through the interaction between iron and aluminium oxides that may be contained in the silt-clay minerals and organic carbon present in soils. Stabilization of OM in soils is controlled by several factors: chemical composition of OM, presence of soil minerals such as phyllosilicate clays and oxide and the accessibility of microorganisms for OM in soils (Baldock et al., 2004;Saidy et al., 2012a;2013b). Lignin,with its aromatic ring structures, is recognised to be more resistant to decomposition than carbohydrates, and together with alkyl carbon areconsidered to account for a biochemically stable component of SOC (von Lützow et al., 2006). However, information on the stabilization of OM in the raised bed soils with different types of OM is not available. The objective of this study was to quantify the effect of types and the amount of OM addition on the amount of OM stabilized in the raised-bed soils through incubation study in the laboratory. METHODS Soils used for the study were sampled from the Village of Karang Indah, Mandastana District, Kuala Barito Regency, South Kalimantan Province. A-raised-bed experiencing 27 years (made in 1987) has been selected for this study based on the results of interviews with farmers and field surveys.Soil samples were collected from a depth of 0-30 cm at several different points. Once cleaned for plant debris, soil samples were then homogenized, stored in plastic bags and stored at 4 o C. Samples were then airdried for incubation experiment. Four types of OM: rice straw, eceng gondok (Eichornia crassipes), purun tikus (Eleocharis dulcis) and mixed rice straweceng gondok were collected from same area of soil sampling. All OM were oven-dried at 50 o C and then ground to <2 mm. Chemical analyses were conducted for all OM to determine contents of organic C, total N, hot water soluble C, cellulose, hemicellulose and lignin (Bremer and Malvaney, 1982;Chesson, 1991;Nelson dan Sommers, 1996). The chemical composition of OM used in this study is described in Table 1. Air dried-raised bed soils were mixed homogenously with each OM (for each treatment) in the PVC tube (radius 1.95 cm). The mixtures were then compacted to a give a depth of 2.0 cm to obtain a bulk density (BD) of compacted soils similar to the BD measured in the field. Distilled water was then added to obtain 70% water-filled pore space (WFPS). The PVC tubes were then transferred into 1 L Mason jars containing 5 mL deionised water in a 20 mL plastic vial to maintain humidity. The jars were sealed with air-tight lids with rubber septa to allow sampling of gas from the jars and incubated in the dark at room temperature for 10 weeks. For each treatment, three replicate samples were prepared and incubated. Organic C stabilization was measured by determining the headspace CO 2 concentrations within each jar, using a Servomex 1450 infra-red gas analyser (Servomex, UK). Carbon dioxide was measured repeatedly for each sample over the duration of the experiment. Analysis of variance was performed on the data of C mineralization to quantify the effect the types and the amounts OM addition on OM stabilization. All statistical analyses were performed using GENSTAT 12 th Edition (Payne, 2008). RESULTS AND DISCUSSION Analysis of variance revealed that the types and the amount of OM added to soils influenced significantly OM stabilization. Based the types of OM, carbon mineralization increased in the order of rice straw < mixed rice straw-eceng gondok < eceng gondok < purun tikus (Fig. 1). The lowest carbon Fig.1.Carbon mineralization of OM applied to the raised after reduced by carbon mineralization from soils. The l of the mean (n = 3). The same letters above the bars indicate the treatment effect is not different based on the Duncan's Multiple Range Test (DMRT ) at 5%. Notation of 0.5 Q Q max indicates the amount of OM applied to soils is equivalent to 0.5, 1.0, and 2.0 times the soil maximum capacity soil to adsorb OM. To determine the amount of carbon mineralized only from OM, carbon mineralized from the soil and OM is re by the amount of carbon mineralized only from thesoils (Fig.1). Fig.1 shows that with the exception of rice straw, the amount of carbon mineralization of OM after deducting the carbon mineralization of the soil is higher than the amount of carbon added to the soils. This was observed at all levels of the addition of organic matter (Fig. 1). The higher carbon mineralization of eceng gondok, purun tikus, and mixed rice straw and eceng gondok was attributed to the mineralization of organic /index.php/ijwem matter RESULTS AND DISCUSSION Analysis of variance revealed that the dded to soils influenced significantly OM stabilization. Based the types of OM, carbon mineralization increased in the order of rice straw < mixed eceng gondok < eceng gondok < purun tikus (Fig. 1). The lowest carbon mineralization was observed application was attributed to the fact that rice straw had the highest lignin content compared to other OM (Table 1). Effect of lignin content on OM decomposition were also reported by Yuwono (2008) and Aprianis (2011) who found an inverse between lignin content and decomposition rate of OM. Fig.1.Carbon mineralization of OM applied to the raised-bed soil with different types and amount after reduced by carbon mineralization from soils. The line above the bars are the standard deviation of the mean (n = 3). The same letters above the bars indicate the treatment effect is not different based on the Duncan's Multiple Range Test (DMRT ) at 5%. Notation of 0.5 Q the amount of OM applied to soils is equivalent to 0.5, 1.0, and 2.0 times the soil maximum capacity soil to adsorb OM. To determine the amount of carbon mineralized only from OM, carbon mineralized from the soil and OM is reduced by the amount of carbon mineralized only from thesoils (Fig.1). Fig.1 shows that with the exception of rice straw, the amount of carbon mineralization of OM after deducting the carbon mineralization of the soil is higher ed to the soils. This was observed at all levels of the addition of organic matter (Fig. 1). The higher carbon mineralization of eceng gondok, purun tikus, and mixed rice straw and eceng gondok was attributed to the mineralization of organic carbon present in raised addition of OM to soils. Mineralization of natural OM in soils due to the addition of fresh or new OM is known as priming effect (Fontaine et al., 2004;Fontaine et al., 2007). The addition of fresh OM will activate soil microorganisms that were previously dormant, which in turn improve the processes mediated by microorganisms such as the decomposition of natural organic matter (Blagodatskaya and Kuzyakov, 2008). Therefore, the addition of eceng gondok, purun tikus and mixed r gondok will reduce the content of OM in the 22 mineralization was observed with rice straw application was attributed to the fact that rice straw had the highest lignin content compared to other OM (Table 1). Effect of lignin content on OM decomposition were also reported by Yuwono (2008) and Aprianis (2011) who found an inverse relationship between lignin content and decomposition bed soil with different types and amount ine above the bars are the standard deviation of the mean (n = 3). The same letters above the bars indicate the treatment effect is not different based on the Duncan's Multiple Range Test (DMRT ) at 5%. Notation of 0.5 Q max , 1.0 Q max and 2.0 the amount of OM applied to soils is equivalent to 0.5, 1.0, and 2.0 times the soil in raised-bed soils after the addition of OM to soils. Mineralization of natural OM in soils due to the addition of fresh or new OM is known as priming effect (Fontaine et al., 2004;Fontaine et al., 2007). The addition of fresh OM will activate soil oorganisms that were previously dormant, which in turn improve the processes mediated by microorganisms such as the decomposition of natural organic matter (Blagodatskaya and Kuzyakov, 2008). Therefore, the addition of eceng gondok, purun tikus and mixed rice straw-eceng gondok will reduce the content of OM in the raised-bed soils, while rice straw addition resulted in an improvement of OM content in the raised-bed soils through OM stabilization. Fig. 1 also shows that 54 % of the OM added to the soilswas decomposed and returned to the atmosphere as CO 2 at level addition of 0.5 Q max . A portion of added OM that does not decompose (46 % of rice straw added to soil) was stabilized at raised-bed soils which in turn will increase the OM content of soils. At the level of the addition of OM 1.0 and 2.0 Q max, only 30 % and 37 % of rice straw added to the soilswas stabilized by mineral soils, respectively. Thus, the amount of rice straw to be applied to the raised-bed soil to generate increased soil OM is equivalent to 0.5 Q max (half of the maximum capacity of the soil adsorb OM). CONCLUSION Results obtained in this study revealed that among types of OM (rice straw, eceng gondok, purun tikus, and mixed rice straweceng gondok) were applied to the raised bed soils, rice straw produces the highest stabilization of OM. The stabilization of rice straw was related to the chemical composition of rice straw containing compounds are relatively difficult to be decomposed. Results of the study also show that the addition of OM to the raised-bed soils, with the exception of rice straw, resulted in priming effect. Therefore, rice straw is recommended for use as a source of OM to increase OM content of the raised-bed soils through the stabilization of OM.

v3-fos

2017-06-20T18:17:13.520Z

2015-04-01T00:00:00.000Z

14099178

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Effects of L-tryptophan, Fructan, and Casein on Reducing Ammonia, Hydrogen Sulfide, and Skatole in Fermented Swine Manure The effects of daily dietary Bacillus subtilis (Bs), and adding L-tryptophan, fructan, or casein to fecal fermentation broths were investigated as means to reduce the production of noxious gas during manure fermentation caused by ammonia, hydrogen sulfide (H2S), and 3-methylindole (skatole). Eighty swine (50.0±0.5 kg) were equally apportioned to an experimental group given Bs in daily feed, or a control group without Bs. After 6 weeks, fresh manure was collected from both groups for fermentation studies using a 3×3 orthogonal array, in which tryptophan, casein, and fructan were added at various concentrations. After fermentation, the ammonia, H2S, L-tryptophan, skatole, and microflora were measured. In both groups, L-tryptophan was the principle additive increasing skatole production, with significant correlation (r = 0.9992). L-tryptophan had no effect on the production of ammonia, H2S, or skatole in animals fed Bs. In both groups, fructan was the principle additive that reduced H2S production (r = 0.9981). Fructan and Bs significantly interacted in H2S production (p = 0.014). Casein was the principle additive affecting the concentration of ammonia, only in the control group. Casein and Bs significantly interacted in ammonia production (p = 0.039). The predominant bacteria were Bacillus spp. CWBI B1434 (26%) in the control group, and Streptococcus alactolyticus AF201899 (36%) in the experimental group. In summary, daily dietary Bs reduced ammonia production during fecal fermentation. Lessening L-tryptophan and increasing fructan in the fermentation broth reduced skatole and H2S. INTRODUCTION Environmental contamination caused by swine manure has provoked much public concern. More than 160 noxious substances have been discovered in swine waste (Le et al., 2005). Ammonia and hydrogen sulfide (H 2 S) are routinely measured, and 3-methylindole (skatole), a byproduct of tryptophan degradation, also increases fecal odor. Deposition of skatole in adipose tissues can taint the meat, with harmful consequences for the beef and pork industries. In efforts to reduce the production of ammonia, H 2 S, and skatole, dietary additives have been investigated. These have included probiotics (Wutzke et al., 2010), fructan (Xu et al., 2002), and enzymes or amino acids (Vhile et al., 2012). Castration and vaccination have also been used to control the production of noxious gases. Among the probiotics, effective microorganisms, Bacillus subtilis (Bs), and lactic acid bacteria reduce the production of ammonia by modifying the intestinal microflora (Jeon et al., 1996). Dietary fructan has been shown to selectively promote Bifidobacterium spp. in the gut, thereby reducing the production of skatole (Li, 2009), and there is evidence for the prebiotic effects of inulin-derived fructans in humans (Salazar et al., 2014). Amino acid-balanced diets (such as those containing tryptophan) were shown to reduce the protein ratio in daily feedings, which led to less ammonia production in swine manure (Powers et al., 2007). The purpose of the above methods was to reduce the production of foul-smelling gas from within the bodies of live swine. To our knowledge, there have been no investigations to reduce the unpleasant odor of fecal matter once outside the body, i.e., conducted in vitro. The present study evaluated the effects of externally applied tryptophan, fructan, or casein on manure fermentation, and noted the interactions among these additives and dietary Bs. Our results should provide a solid reference for studies aimed at reducing the odor of fecal waste outside the live swine body. MATERIALS AND METHODS The Animal Welfare Committee of Shandong Academy of Agricultural Sciences reviewed and approved the study protocol. Experimental design The 80 pigs were randomly divided into an experimental group and a control group (n = 40, each), constituting four replicates in each group and ten pigs in each replicate. Both groups were raised on a concrete floor. Pigs in the experimental group (Bs + ) were given a cornsoybean basal diet supplemented with 0.2% Bs. The control group (Bs -) received the same diet, but without the bacteria. The basal diet was prepared in accordance with the China feeding standard for swine (NY/T65-2004). After 6 weeks, fresh manure was quickly collected, before feeding in the morning, from 2 randomly selected pigs in each replicate for subsequent studies (Yokoyama and Carlsonet, 1974). In brief, 280 g of each fecal sample was first suspended in 2.8 L anaerobic mineral buffer (5.0 g/L NaHCO 3 , 0.9 g/L NaCl, 0.9 g/L (NH 4 ) 2 SO 4 , 0.45 g/L KH 2 PO 4 , 0.45 g/L K 2 HPO 4 ·3H 2 O, 0.03 g/L CaC1 2 ·2H 2 O, 0.02 g/L MgCl 2 , 0.01 g/L MnSO 4 ·4H 2 O, 0.01 g/L CoCl 2 ·6H 2 O, 0.01 g/L FeSO 4 ·7H 2 O, 0.1 g/L cysteine). The suspensions were subsequently transferred to sterile plastic bags filled with CO 2 , thoroughly shaken for 5 min, and filtered through 6 layers of gauze to remove small particles. The filtrate from each suspension was distributed into 27 aliquots with 100 mL in each flask, and divided into 9 groups based on the amounts of L-tryptophan, fructan, or casein added to each flask (Table 1). The broths were incubated anaerobically for 24 h at 38°C. A 20-mL sample was taken from each flask to measure the gas composition. One milliliter of clear fermented broth was separately taken from both the Bs + and Bssamples (the blank, containing no added L-tryptophan, fructan, or casein, broth 1 in Table 1), mixed, and stored at -20°C for microflora isolation and identification. Measurement of substances in the noxious gas The fermented broth samples were measured after filtration through a 0.45-μm organic membrane. Ammonia was measured by the Nessler reagent colorimetric method. H 2 S was quantified via sulfate-iodometric titration. Ltryptophan and skatole were measured using highperformance liquid chromatography, with reference to Li (2009), and conducted at the Center for Food Quality Supervision and Testing Ministry of Agriculture (Jinan, China). Isolation and identification of fecal bacteria in the fermentation broth Genomic DNA of fecal bacteria was purified using an OMEGA Stool DNA kit (Omega Bio-Tec, Norcross, GA, USA). Using V3-340F (5'-FAM-TCCTACGGGAGGCA GCAGT-3') and V3-532R (5'-TCCTACGGGAGGCAGC AGT-3') as universal primers, the V3 region of 16S rDNA was amplified through fluorescence-based polymerase chain reaction-single-strand conformation polymorphismfragment length polymorphism (F-PCR-SSCP-FLP) methods, as described by Zhang et al. (2000). The 195-bp PCR products were analyzed with an Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA, 96-capillary array), in which the amount of each specific bacteria was quantified by the area size of their corresponding peaks. The 16S rDNA clone library (target fragment size 1.6 kb) was constructed by amplifying the 16S rDNA region of each bacteria using the V3 region fluorescent-labeled primers. The PCR products were purified and sequenced at the Biotechnology Research Center, Shandong Academy of Agricultural Sciences. All the sequences were BLASTed in the NCBI database to identify the bacterial species, and quantified using Mega6 software (http://www.megasoftware.net). Statistical analyses The amounts of ammonia, H 2 S, and skatole of the Bs + and Bsgroups were compared using one-way analysis of variance and Student's t-test. A probability (p) value less than 0.05 was the standard for significance. The generalized linear model procedure (PROC GLM) was used in the 3×3 orthogonal experimental design, to analyze the average k values and the residual R values. Duncan's method was used for multi-comparisons (p = 0.05). SAS (V9.1) was used for statistical analyses. Data are presented as means± standard error of the mean. Effect of different additives on ammonia, H 2 S, and skatole production The amounts of ammonia, H 2 S, tryptophan, and skatole produced in the fermentation broths prepared from fecal samples from pigs given Bs (Bs + group) were significantly lower than that of pigs of the control group (Bs -). This suggests that the addition of Bs in the diet was associated with reduced ammonia, H 2 S, tryptophan, and skatole production in the fermentation broths ( Table 2). Effect of different added substances on the residual Rvalue of noxious gas Casein showed the largest R-value to the ammonia in the fermentation broth in the Bs + group, and L-tryptophan resulted in the largest R in the Bsgroup (Table 3). This indicated that the main experimental additives affecting the Bs + and Bsgroups were different. In the production of H 2 S, fructan resulted in the largest R-value in both groups, which indicated that fructan was the main substance that affected the production of H 2 S. In the result regarding tryptophan, Ltryptophan showed biggest R-value in both groups, indicating that external added tryptophan was the main cause for the tryptophan in the fermentation broth. For skatole, external L-tryptophan showed the biggest R-value, which indicated that external L-tryptophan was the main affecting element. Regarding the effect of adding casein to the fermentation broth on ammonia production, there was no correlation (r = -0.1551) between Bs + group (k1>k2>k3) and the Bsgroup (k1>k3>k2; Table 4). This shows that the effects of casein at different concentrations were different in the Bs+ group and the Bsgroup. Fructan at different concentrations showed similar effects on H 2 S, since the two effects (both k1>k3>k2) were highly correlated (r = 0.9981); k1 was higher than k3 in both groups (p<0.05), which indicated that the 1.5% added fructan reduced the H 2 S levels in both the Bs + and Bsgroups. Tryptophan showed similar effects on skatole (both k2>k3>k1, r = 0.9992), and k1 was lower than k3 in both groups (p<0.05). This indicated that the 0.1% added tryptophan increased the concentrations of skatole in both the Bs + and Bsgroups. Correlation between externally added substances and Bacillus subtilis In the Bs + group, there were no associated changes between externally added L-tryptophan and ammonia, H 2 S, tryptophan, or skatole production (p>0.05; Table 5). There was significant correlation between casein and Bs in the production of ammonia (p = 0.0395), as well as between fructan and Bs on the production of H 2 S (p = 0.0141). DISCUSSION The ammonia, H 2 S, and skatole produced during pig raising not only adversely affect the growth of pigs, but also contaminate the environment and harm human health. In this study, we found that incorporating Bs in the pigs' daily feed significantly reduced the concentrations of ammonia, H 2 S, and skatole in the manure fermentation broth. Moreover, adding external L-tryptophan increased the production of skatole in the manure fermentation broths. Fructan inhibited the production of H 2 S. The effect of casein to the ammonia production depended on the presence of Bs. These discoveries provide an important reference for the control of noxious gases. Effects of Bacillus subtilis on the production of ammonia, H 2 S and skatole in the fermented broth Bs is routinely added to the daily swine diet to reduce the production of noxious gas. In the present study, ammonia production from fermentation broths prepared from the fecal matter of pigs given Bs was also reduced compared with that of pigs of the control group, which is consistent with the results of Kim et al. (2005). This may be because Bs secretes proteases that degrade proteins which otherwise transition to ammonia (Zhang et al., 2009). It is also possible that Bs promotes the presence of Lactobacillus amylovorus in the microflora (Su et al., 2006), which produces amylase (Eom et al., 2009) that facilitate the breakdown of ammonia. Consistent with the discovery of Lee et al. (2009), in the present study dietary Bs was also associated with less H 2 S production from the fermentation broths, compared with the control group. This reduction may be caused by metabolites of Bs that contain catalase and oxidase, which decrease the production of H 2 S (Yumoto et al., 2004). In the present study, dietary Bs was also associated with less skatole produced by the fermentation broths, compared with the control group. It was reported previously that bacteria could affect the pathway of skatole formation by way of tryptophan metabolism (Li, 2009;Vhile et al., 2012), although the microorganisms tested were not Bs. The identity of the microorganisms responsible for the conversion of tryptophan to skatole has not been established. However, bacteria known to facilitate the degradation of skatole are: lactic acid bacteria 11201 (Yokoyama et al., 1983), Clostridium drakei and C. scatologenes (Whitehead et al., 2008), C. disporicum (Li, 2009), and C. scatologenes ATCC 25775 (Doerner et al., 2009). The predominant bacteria discovered in the manure broth in this study were Streptococcus alactolyticus AF201899 and Lactobacillus amylovorus. No reports regarding the function of Streptococcus alactolyticus AF201899 have been published previously. Rinkinen et al. (2004) demonstrated that S. alactolyticus was the predominant lactic acid bacteria in the empty intestine and feces. S. alactolyticus could secret β-glycoside hydrolase, αgalactosidase, amylase, urease, acidic galactose, and galactosidase. It has also been reported that lactic acid in the daily diet could reduce skatole production in the gut (Nowak and Libudzisz, 2009). Investigated as a probiotic, L. amylovorus secreted lactic acid and amylase (Eom et al., 2009) and promoted the growth of lactobacilli and bifidobacteria when galactooligosaccharides and Bifidobacterium were added to an in vitro model of the large intestine (Martinez et al., 2013). L. amylovorus was not found to degrade skatole directly. Skatole could be degraded in vitro by Streptococcus 6020 (Li, 2011). The effect of S. alactolyticus AF201899, L. amylovorus, and Bacillus MA001 need further investigation. In this study, the effect of Bs in reducing ammonia, H 2 S, and skatole occurred in the swine gut, in the in vitro manure fermentation process, or both. Suárez-Estrella et al. (2013) reported that Bs could reduce changes in bacterial species during vegetable anaerobic composting, but there are no reports on the amount of ammonia, H 2 S, or skatole. In the present study, some isolated bacteria could not be grown in culture, which is consistent with the general knowledge that there are many undiscovered bacteria in the swine intestine. Further investigations are needed to elucidate the effect of Bs on the production of ammonia, H 2 S, and skatole. Effect of added L-tryptophan, casein, and fructan on ammonia, H 2 S and skatole production In the present study, tryptophan as an additive was the main variant that affected ammonia production in the Bs + group, but not the Bsgroup. No significant interaction was observed between tryptophan and Bs, indicating that added tryptophan might affect the nitrogen level in feces through other bacteria. Pierce et al. (1931) discovered that yeast might enhance nitrogen and indole production; added tryptophan, cysteine, and phenylalanine inhibited ammonia production in feces. There has been no report regarding whether added tryptophan could affect the production of H 2 S in swine manure. In our study, added tryptophan was not the main element affecting the production of H 2 S. We found that added tryptophan was the main variant affecting the amount of tryptophan and skatole produced in the fermented manure broth. This is consistent with the previous discovery that skatole in swine blood could be increased by injecting tryptophan into the swine appendix (Jensen, 2006). However, it was also found that addition of tryptophan into the daily diet had no significant effect on the level of skatole (Wesoly and Weiler, 2012). The differences might be caused by how the tryptophan was acquired by the swine or how it interacted with the manure. Wesoly and Weiler (2012) indicated that the insignificance of dietary tryptophan might be because tryptophan might be absorbed by the small intestine, instead of being utilized by bacteria in the colon. In the present study, added Ltryptophan showed no significant effect on skatole in either the Bs + or Bsgroup (same k value, r = 0.9992), indicating that the fermentation of Bs in the intestine might not be simulated by the fermentation of tryptophan in vitro. In this study, casein was the main factor affecting ammonia production in the control group, but was not significant for H 2 S production, which is consistent with the reports of Powers et al. (2007). They showed that a diet balanced in amino acids with low crude proteins helped to reduce the ammonia level in feces, but exhibited no effect on H 2 S. We found that the combination of dietary Bs with casein added to the fermentation broth was significantly associated with a reduction in ammonia (p = 0.0395), but not H 2 S (p = 0.9505) produced from the fermentation broth. This was in accord with the various effects of different concentrations of casein on ammonia production in both the Bs + and Bsgroups (r = -0.1551). The effect of casein on ammonia production may be related to the protease secreted by Bs. Casein was not the main additive to affect skatole production. This is consistent with the conclusion of Lin et al. (1992) that daily diet had no effect on the production of skatole. Lundstrma et al. (1994) reported that an increase in the concentration of skatole did not correlate with an increase of protein. Casein or dietary proteins could result in changes in the microbiota in the intestine and therefore affect the fermentations . Fructan, as a probiotic, modifies the microflora by increasing the amount of beneficial bacteria (Salazar et al., 2014). In the present study, the addition of fructan to fermentation broths was associated with a reduction in H 2 S, which correlated with the addition of dietary Bs. This is similar to a previous study (Zhao et al., 2013), which reported that a 1% addition of fructan had no effect on fertility, but lactic acid bacteria dramatically increased, with a significant decrease in E. coli (p<0.001); the amount of ammonia, H 2 S, and organosulfur significantly decreased (p<0.05). Fructan had similar effects on H 2 S in both the Bs + and Bsgroups of the present study (r = 0.9981), indicating that Bs did not affect the amount of H 2 S. However, fructan did not significantly affect skatole, which is consistent with the conclusion drawn by Xu et al. (2002). They observed that 0.5% to 1% fructan increased the amount of indole, but not skatole, perhaps because the pH change resulted in a difference in the microbiota. In our research, although the microbiota of the Bs + and Bsgroups differed, we regret that the pH was not measured. In this study, Bs was added to the diet, and not directly to the fecal fermentation broths. In fact, Bs can also be added directly to the broths. The production of crude proteins in fecal broth depended on the amount of casein added. Further study is required to determine the optimal means of introducing Bs to fermentations broths, and its interactions with different protein, tryptophan, and fructan concentrations. CONCLUSION Adding L-tryptophan to fecal fermentation broths increased production of skatole. Adding fructan to the broths reduced the production of H 2 S. The effect of casein on ammonia depended on the addition of Bs in the daily diet. Daily dietary Bs reduced the production of ammonia, H 2 S, and skatole in fecal fermentation broths.

v3-fos

2016-12-22T08:44:57.161Z

2015-02-01T00:00:00.000Z

17314058

s2

Using Near Infrared Reflectance Spectroscopy ( NIRS ) to predict the protein and energy digestibility of lupin kernel meals when fed to rainbow trout , Oncorhynchus mykiss This study examined the potential of using near infrared spectroscopy (NIRS) to predict the nutrient composition, energy density and the digestible protein and digestible energy values of lupin kernel meals when fed to rainbow trout. A series of 136 lupin kernel meals were assessed for their protein and energy digestibilities using the diet-substitution approach in a series of 10 experiments over a 6-year period from 2002 to 2008. Two reference diets were also included in each experiment. Minimal variance in the digestibility parameters of both reference diets was observed among the experiments ensuring that there was a high degree of robustness in the across-experiment evaluations. The same lupin kernel meal samples were also scanned using a diode array near infrared spectrophotometer (DA-NIRS). The spectra obtained by the DA-NIRS were chemometrically calibrated against both the chemical composition and the digestible value data using multivariate analysis software. The cross validation tests used in this study provide a valid indication of the potential to predict the nutrient composition, energy value and digestible protein and energy values of the lupin kernel meals as used in diets for rainbow trout. That the standard errors of cross validation (SECV) of the parameters investigated were generally commensurate with the cross trial variation seen in the reference sample indicating robust calibrations for the two target parameters of digestible protein and digestible energy. Therefore this study demonstrates that within one raw material type that not only does significant variability in the digestible value of the raw materials exist, but that it is possible to use NIRS technology to provide rapid estimates of the digestible value of those raw materials in near real-time. Introduction Lupin meals are one of many raw materials that have consistently been shown to provide sound nutritional value in the diets of a range of aquaculture species (De la Higuera et al., 1988;Burel et al., 1998;Glencross et al., 2005;2011b;Smith et al., 2008). This raw material has also shown interesting functional properties in extruded feeds in addition to its nutritional attributes adding extra value above their inherent nutritional values (Glencross et al., 2011a;. However, like all raw materials, the composition of lupin meals can vary considerably depending on a range of genetic, environmental and processing factors (and of course the interaction between these) (Longnecker et al., 1998;Glencross et al., 2007a;2008a;. Importantly, this variability in composition has also been noted to extend to the digestible value of lupin meals (Glencross et al., 2008b). This digestible value of lupin meals and indeed, that of most plant proteins is usually a direct reflection of their digestible protein and/or energy content (Burel et al., 1998;Glencross et al., 2008bGlencross et al., , 2011a. Accordingly any variability in these digestible value parameters (protein and energy) of these meals should translate to variability in their economic value. In lupin meals, an increase in protein concentration is typically reciprocated by a decrease in the levels of non-starch polysaccharides (NSP) (Glencross et al., 2007b;2008b). However, high levels of some types of NSP have been implicated in lower nutritional value of lupin meals (Glencross, 2009). In particular the level of lignin has been implicated as a negative factor in protein digestibility via both multivariate analysis and empirical means (Glencross et al., 2008b;2012). Furthermore, because lupin meals are largely devoid of starch it is recognised that the nutritional value of these raw materials is largely dependent on their protein and lipid components (Glencross et al., 2007b;2008b). This lends the application of these raw materials to the development of rapid analysis assessments of nutritional value such as the use of near infrared spectroscopy (NIRS). This is particularly pertinent given that virtually all modern aquaculture diets are formulated on a digestible nutrient and energy basis (Glencross et al., 2011a). Therefore an improved assessment of the nutritional value of these raw materials, and on a near-real-time basis, will provide significant advancements in the responsiveness and cost savings in diet formulation by the aquaculture feed industry. This study reports on the evaluation of the digestibility of a large number of meals of narrowleaf lupins, Lupinus angustifolius when fed to rainbow trout (Oncorhynchys mykiss). Part of this dataset is that used in Glencross et al., (2008b), but it is further expanded by an additional 60 samples to provide the critical mass of data to enable potential NIRS calibrations to be developed. The variability in this data set (digestible nutrient/energy values and chemical composition) was studied using diode array near infrared spectroscopy (DA-NIRS). Based on this DA-NIRS analysis of each lupin meal this study reports on this potential of DA-NIRS to predict nutrient composition, energy value and protein and energy digestibility of narrow-leaf lupin kernel meals. Ingredient and diet development Over a six-year period (2002 -2008), separate batches of seed of Lupinus angustifolius were collected from the Department of Agriculture's (Western Australia) germ plasm and breeding lines. This seed in many cases constituted the same genotype over several seasons, often from the same site. Samples of the seed were then split using a small disc-mill and aspirated to separate hulls from kernels. A final manual clean of the kernels to remove any remaining hull material was also undertaken on each sample to ensure 100% purity of each kernel preparation. Each kernel sample was then milled using a RetschTM ZM200 rotor mill (Retsch Pty Ltd, North Ryde, NSW, Australia) with a 750 m screen to create consistent particle sized kernel flour. Additional meals were created based on whole-seed and blended seed and kernel meals and were also included in the sample set. In addition to the lupin meals, each of the other ingredients used in this study was thoroughly ground such that they each passed through a 750 m screen. The experiment design was based on a strategy that allowed for the diet-substitution digestibility method to be used (Glencross et al., 2007a). To achieve this a basal diet was formulated and prepared to include approximately 500 g kg -1 DM protein, 210 g kg -1 DM fat and an inert marker (yttrium oxide at 1 g kg -1 ) ( Table 1). Each test ingredient being studied was added at 30% inclusion to a 70% sub-sample of the basal mash (see Table 1). The diets were then processed by addition of water (about 30% of mash dry weight) to the combined mash during mixing to form a dough, which was subsequently screw pressed using a pasta maker through a 4 mm diameter die. The resultant moist pellets were then oven dried at 70C for approximately 12 h before being allowed to cool to ambient temperature in the oven. The basal diet for each experiment was prepared in a similar manner, but without the addition of any test ingredient. In addition a reference lupin kernel meal was included in every digestibility study to allow for cross-comparison across all studies. The basal diet and an example test diet formulations and their composition are presented in Table 1. Fish handling and faecal collection These digestibility studies constituted ten separate experiments. Each experiment had two common diets, which included the reference diet and a reference lupin kernel meal (cv. Myallie 2002 season from Coorow). For each experiment hatchery-reared rainbow trout (Oncorhynchus mykiss, Pemberton heat-tolerant strain, Western Australia) were transferred from grow-out ponds to experimental tanks (200 L) before being introduced to the experimental diets. Freshwater (salinity < 1 PSU) of 16.0  0.1C (mean  S.D. across each of the ten experiments) at a flow rate of about 4 L min -1 was supplied to each of the tanks. For each experiment the tanks were stocked with 15-20 trout of 254  62.5 g (mean  S.D.; n = 10 experiments). Treatments were randomly assigned amongst 48 tanks within each experiment, with each treatment having three replicates. Fish were manually fed the diets once daily to apparent satiety as determined over three separate feeding events between 1500 and 1600h each day. The fish were allowed to acclimatise to the allocated dietary treatment for seven days before faecal collection commenced consistent with earlier studies by this group (Glencross et al., 2005;2007b;2008a;. Faeces were collected using manual stripping techniques based on those reported by Glencross et al. (2005;2007b). Stripped faeces were collected during 0800 to 1000h over a four-day period, with each fish only being stripped twice and not on consecutive days. Faecal samples collected from different days were pooled within tank, and kept frozen at -20C before being freeze-dried in preparation for analysis. Chemical and digestibility analysis All chemical analyses were carried out by official NATA (National Association of Testing Authorities) accredited analytical service providers (Chemistry Centre (WA), East Perth, WA, Australia and Animal Health laboratories, Department of Agriculture and Food Western Australia, South Perth, WA, Australia) to AOAC (2005) standards. In this regard each of the diet and faecal samples were analysed for dry matter, yttrium, ash, phosphorus, nitrogen and gross energy content. The dry matter of each sample was calculated by gravimetric analysis following oven drying at 105ºC for 24 h. Total yttrium concentrations were determined after mixed acid digestion using inductively coupled plasma atomic emission spectrophotometry (ICP-AES). Protein levels were determined based on the measurement of total nitrogen content of each sample using a LECO auto-analyser, and based on N x 6.25. The amino acid composition of samples was determined by an acid hydrolysis prior to separation via HPLC. The acid hydrolysis destroyed tryptophan making it unable to be determined using this method. Total lipid content of the diets was determined gravimetrically following extraction of the lipids using the chloroform:methanol (2:1). The gross ash content of each sample was determined gravimetrically following the loss of mass after combustion of a sample in a muffle furnace at 550C for 12 h. Crude dietary fibre was determined by digesting the defatted sample with multiple washes of acetone and ethanol. The resulting residue was corrected for undigested protein and ash. To determine neutral-detergent fibre (NDF) content samples were then boiled with buffered NDF solution. The residue was collected on a coarse sintered glass crucible. The acid-detergent fibre (ADF) was determined following a sample being reacted in 0.5M acid detergent solution and the residue is collected on a coarse sintered glass crucible. The lignin content was determined by reacting the ADF residue with cold 72% sulphuric acid. The sample was then ashed and the residue measured gravimetrically. Gross energy content of each sample was determined by adiabatic bomb calorimetry. Differences in the ratios of the parameters of dry matter, protein, amino acids or gross energy relative to yttrium content, in the feed and faeces in each sample were calculated to determine the apparent digestibility coefficient (ADCdiet) for each of the nutritional parameters examined in each sample of each diet based on the following formula as reviewed in Glencross et al. (2007a): where Ydiet and Yfaeces represent the yttrium content of the diet and faeces respectively, and Parameterdiet and Parameterfaeces represent the nutritional parameter of concern (dry matter, protein or energy) content of the diet and faeces respectively. The digestibility values for each of the test ingredients in the test diets examined in this study were calculated according to the formulae: Where Nutr.ADingredient is the digestibility of a given nutrient from the test ingredient included in the test diet at 30%. ADtest is the apparent digestibility of the test diet. ADbasal is the apparent digestibility of the basal diet, which makes up 70% of the test diet. NutrIngredient, Nutrtest and Nutrbasal are the level of the nutrient of interest in the ingredient, test diet and basal diet respectively (as reviewed by Glencross et al., 2007a). All raw material inclusion levels were corrected for dry matter contribution and the effects that this may have had on the actual ratio of reference diet to test ingredient. Based on this assessment digestibilities determined greater than 100% were not corrected because they were considered potentially indicative of interactive effects between the diet and test ingredient and therefore should be stipulated as determined. However, for reasons of practicality, the total levels of digestible nutrients/energy were only calculated assuming a maximum digestibility of 100% or a minimum of 0%. NIRS scanning and chemometrics A Diode Array Near Infrared Spectrometer (DA7200, Perten Instruments, Huddinge, Sweden) was used to scan each of the 135 lupin meals samples. These samples were scanned in reflectance mode using the rotating 75mm sample cup. The spectra from all of the samples were collected across the full wave length range (950 to 1650nm) of the instrument as absorbance at a resolution of 2nm using 9 scans per sample (DA7200 Operation Manual, 2007). The scans were collected in groups of 3 with the sample cup repacked between each group. There were processed by the DA7200 to provide a single spectra for analysis. These spectra were then combined with the nutrient composition, energy value and digestibility data which was copied in to the UNSCRAMBLER ® multivariate analysis software package ready for calibration model development. The raw lupin meal spectra obtained is shown in Figure 1. Initially all the primary spectra were examined visually to eliminate anomalous scans before being copied into the UNSCRAMBLER ® multivariate analysis software (Workman and Weyer, 2008). The reference data was then incorporated to form the calibration data set. Cross validation was then used to evaluate the relationship between the spectra and the digestibility, nutrient and energy values. The UNSCRAMBLER ® was then used to develop a model that provided a regression based on the whole spectra after specific mathematical treatments of the data. The calibration was evaluated by the statistical measurements of the standard error of cross validation (SECV) and the correlation coefficient (R 2 ) (Workman & Weyer, 2008). An optimisation program was used to determine the math pre-treatments and wave number ranges to use with the data that gave the lowest standard error of cross validation (Workman & Weyer, 2008). Cross validation tests were run on the suggested combinations after taking into consideration any Wave Number ranges known to be appropriate for the parameter under consideration. Validation tests were then re-run after excluding outliers (samples the software flags as either bad reference results or extremely unusual spectrally) (Esbensen, 2004). This process was continued until a balance was struck that included the following elements; a) the standard error of cross validation (SECV) is similar to the standard error of the reference method, b) the number of outliers (poor prediction samples) remaining is small enough or their residual vales are low enough to still be able to meet the objectives of the calibration, and c) the correlation coefficient (R 2 ) is sufficiently close to a perfect correlation of 1.0 to indicate probable future robustness and to meet the objectives of the calibration (Esbensen, 2004). Provided the SECV value is in the order of the reference method standard error values of R 2 of 0.6 or even lower can be acceptable but values of over 0.8 are desirable (Workman & Weyer, 2008). Also for calibration robustness the standard deviation of the total population used in the calibration model should be at least 1.5x (preferably 2 times or more) the SECV value (Workman & Weyer, 2008). Statistical analysis All values are means unless otherwise specified. Figures were constructed and Multivariate chemometric analysis was undertaken using the UNSCRAMBLER ® software. Results and Discussion Variability exists in all ingredients. This variability can be managed through a variety of means, either by the ingredient supplier, or by the feed manufacturer. Examples of this include the large-scale blending by commodity handlers of grains of different protein levels to produce a more homogenous product, or the analysis of batch variation by feed manufacturers to allow precise customisation of each diet according to each batch of ingredients supplied (Jiang, 2001;Glencross et al., 2008a). In addition to these ingredient management strategies an improved understanding of the level of variability in the chemical composition of the ingredient and how that variability contributes to changes in nutritional value is a key step to maximising the potential value of the ingredient (Glencross et al., 2008a;. In this study a series of 136 lupin (L. angustifolius) meal samples were collected over a six-year period and examined in a series of digestibility assays with rainbow trout. This work builds on earlier datasets already published by these authors (Glencross et al., 2008a;, by adding an additional 60 samples and also further analysing all 136 of the samples using NIRS technology. Data variance Over a series of ten independent experiments both the basal and reference diets had minimal variability in their digestibility parameters among experiments (Table 2). Dry matter diet digestibilities were different for both diets, but had a similar coefficient of variance of 1.9% and 2.3%. Coefficients of variance (CV) for diet protein digestibility were low at 1.2% and 1.3%, and the means identical at 0.905. Diet energy digestibilities were different for both diets (0.899 and 0.812), but also had low CV's of 1.4 and 1.9%. These data are consistent with other similar such data published on lupin meal digestibility in rainbow trout (Glencross et al., 2005;2007b;2008b;2011b). The variability of the ingredient apparent digestibility coefficients for the reference were typically greater than that observed of the diet digestibilities (Table 2). This is to be expected because of the inherent nature in which the ingredient digestibility values are calculated depending on the diet digestibility values of two separate diets and the ingredient composition values. As such this parameter incorporates the errors of all three of these assessments (as reviewed by Glencross et al., 2007a). However, the resultant data still proved to be very robust and from it energy digestibility was the most consistent of the ingredient parameters evaluated, with a CV of 4.0%. Digestibilities for the dry matter had the highest variability with a CV of 9.8%. There was substantial variability in the composition of the lupin kernel meals used in this study. As a summary of Table 3; the mean ± S.D., protein (N x 6.25) concentration in lupin meals, across all 136 samples was 445 ± 66 g kg -1 on a dry basis (range 27.7 to 61.3 g kg -1 ). Total lipid was 79 ± 14 g kg -1 (range 50 to 171 g kg -1 ) and ash 32 ± 7 g kg -1 . Carbohydrates, measured by difference between dry matter minus protein, lipid and ash, were 426 ± 50 g kg -1 on a dry basis (range 254 to 539 g kg -1 ). Mean gross energy was 20.6 ± 0.6 MJ kg -1 DM (range 18.7 to 23.0 MJ kg -1 DM). Dietary crude fibre was 309 ± 4.6 g kg -1 on a dry basis (range 175 to 434 g kg -1 ), acid-detergent fibre was 101 ± 52 g kg -1 on a dry basis (range 52 to 262 g kg -1 ), neutral-detergent fibre was 64 ± 44 g kg -1 on a dry basis (range 30 to 200 g kg -1 ) and lignin was 6.2 ± 3.5 g kg -1 on a dry basis (range 2.2 to 21.7 g kg -1 ) ( Table 3). These composition values are similar, but a little more variable than that of most lupin studies published (De la Higuera et al., 1988;Burel et al., 1998;Glencross et al., 2005;2007b;2008b;2011b). Substantial variability in ingredient digestible protein/energy parameters was measured across all experimental ingredients (Table 3). This variability was compounded by the combined variability in ingredient composition and ingredient digestibility. The ingredient digestible protein had a coefficient of variation of 15.3%, with a range in digestible protein levels of 244 to 595 g kg -1 on a dry basis ( Table 3). The ingredient digestible energy levels had a coefficient of variation of 14.9%, with a range in ingredient digestible energy of 7.7 to 20.5 MJ kg -1 on a dry basis (Table 3). These digestible nutrient values are similar, but a little more variable than that of most other lupin studies published (Burel et al., 1998;Glencross et al., 2005;2007b;2008b;2011b). NIRS calibration statistics Calibrations were successfully developed for each of the parameters in this study (Table 3). Although the focus of this was on calibration development for the digestible protein and digestible energy values (Figures 3 and 4; Table 3), calibrations were also successfully developed for a suite of compositional features (Table 3). Among the composition calibrations the number of factors used to derived the calibration varied from 3 (sum of amino acids) to 9 (energy). These digestible protein and digestible energy calibrations appear to be quite unique within the scientific literature. Not only are they the only such calibrations found for a feed grain for these digestible value parameters in fish, they also appear to be relatively unique within broader monogastric research in that they base the development of the calibrations for a single type of feed grain. This is in contrast to similar such work done with pigs which used a range of cereal varieties (barely, wheat, sorghum, triticale and maize) (van Barneveld et al., 1999). These earlier pig studies also had problems with sample analysis by different laboratories and the lack of inter-experimental reference ingredients, issues that have both been addressed in the present study. It has been suggested that an acceptable calibration should have an accuracy >1.5 times the value reported as the standard error of the reference method used for that parameter, a value referred to as the RPD (Workman & Weyer, 2008). Based on this assessment the digestible energy calibration had a RPD of 1.50 and the digestible protein a RPD of 1.18. Therefore this would suggest that the digestible energy calibration is acceptable, but that the digestible protein calibration still needs further refinement, despite having a R^2 > 0.80. This refinement could potentially be achieved by either fortifying the dataset further with additional samples and digestibility data, or by removing those data points from the calibration that weaken the calibration R^2. Conclusions The cross validation tests used in this study provide a valid indication of the potential to predict the nutrient composition, energy value and digestible protein or digestible energy values of the lupin kernel meals as used in the rainbow trout feeding trials. Overall the standard errors of cross validation (SECV) of the parameters investigated were generally commensurate with the cross trial variation seen in the reference sample (cv Myallie) and the RPD values at or close to values indicating robust calibrations for the two target parameters of digestible protein and digestible energy. This study therefore demonstrates that there is great potential to use NIRS to predict both the composition and digestible values of kernel meal samples of narrow-leaf lupins either by scanning the kernel meal before diet preparation.

v3-fos

2016-03-22T00:56:01.885Z

2015-01-30T00:00:00.000Z

17077288

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Volatile Compounds of Raspberry Fruit: From Analytical Methods to Biological Role and Sensory Impact Volatile compounds play a key role in the formation of the well-recognized and widely appreciated raspberry aroma. Studies on the isolation and identification of volatile compounds in raspberry fruit (Rubus idaeus L.) are reviewed with a focus on aroma-related compounds. A table is drawn up containing a comprehensive list of the volatile compounds identified so far in raspberry along with main references and quantitative data where available. Two additional tables report the glycosidic bond and enantiomeric distributions of the volatile compounds investigated up to now in raspberry fruit. Studies on the development and evolution of volatile compounds during fruit formation, ripening and senescence, and genetic and environmental influences are also reviewed. Recent investigations showing the potential role of raspberry volatile compounds in cultivar differentiation and fruit resistance to mold disease are reported as well. Finally a summary of research done so far and our vision for future research lines are reported. Introduction Raspberry (Rubus idaeus L.) is a member of the Rosaceae family producing a red fruit with a sweet but tart flavor. Some cultivars with recessive genes giving an extremely low concentrations of anthocyanin produce yellow berries [1], but it is the European red fruited cultivars that are most widely grown and economically most important. Although it is called a berry, the fruit produced by the raspberry is, in botanical terminology, a collection of numerous drupelets around a central core. The drupelets typically separate from the core when pickled. This commodity is of continuously increasing economic importance, as witnessed by nearly 50 raspberry breeding programs around the world [2] and the ongoing raspberry sequencing project [3]. Red raspberries contain high amounts of polyphenols and antioxidants, and have a unique phytochemical profile rich in ellagitannins and anthocyanins that distinguishes them from other berries and fruits [4] and has positive implications for human health and the prevention of chronic diseases [5], although these fruits are mostly recognized and appreciated for their characteristic flavor. Volatile compounds play a key role in the formation of the flavor of food products and nearly 300 volatile compounds have so far been reported in raspberry. Volatile organic compounds (VOCs) are organic molecules with appreciable vapor pressure at ordinary room temperature. They are usually small molecules with a molecular weight lower than 300 Dalton. People often associate scents with volatiles that can be perceived by the human nose and have a pleasant smell [6] and flavor. It should be mentioned that volatile compounds in plants have various ecological and productive impacts: they attract pollinating insects, advertise that fruit are ripe and ready for seed dispersal, modulate systemic acquired resistance to pests and diseases, and also seem to alleviate abiotic stress [7]. Despite the economic and nutraceutical importance of raspberry, there has been little mention in the literature over last ten years of the volatile compounds in this fruit. A series of studies carried out by Firmenich in the '60s and '70s defined the basic methodologies and listed the compounds isolated and identified in raspberries. Of these, 4-(4-hydroxyphenyl)butan-2-one was recognized as the key compound in defining typical raspberry flavor and was therefore named "raspberry ketone". In the following decades only a few investigations on volatile compounds in raspberry were carried out and very little is known about the real impact the different volatile compounds have on human sensory perception or about their role in pest defense. Volatile Compounds in Raspberry Raspberry volatiles are important for the perception of sensory quality (odor, flavor) and for mold resistance [13], and some are claimed to have nutraceutical properties [28,29], although the nutritional role of volatile compounds is still very controversial. Fruit volatile compounds are influenced by numerous factors including cultivar variation, climate, soil, ripeness, and many other variables [11,19,30]. Early studies focused on isolation and identification of these volatile compounds in particular those most likely related to raspberry aroma, later moving attention to the factors affecting volatile composition and their possible biological roles. Since terms such as aroma and flavor are extensively used in the literature reviewed, sometimes to indicate volatile compounds in general, before we begin we would like to draw attention to the significance of these terms. The term "aroma" refers to an odor, or to a compound responsible of an odor, with a pleasant or unpleasant connotation [31]. Odor is a sensation perceived by the olfactory receptors in sniffing certain volatile substances (ortho-nasal route). The term "flavor" refers to "a perception resulting from stimulating a combination of the taste buds, the olfactory organs, and chemesthetic receptors within the oral cavity" [32], in other words it is the combination of taste and smell features (through the retro-nasal route). The volatile compounds released by raspberry are free forms of different metabolites, most of them often in the form of glycoside bound to sugars. The presence of glycosidically-bound volatile compounds in plants is well established [33]; they are able to release free volatile compounds by enzymatic or chemical cleavage during plant maturation, industrial pretreatments or processing and may be considered potential aroma precursors [34]. Pyysalo compared the volatile compounds of a cultivated raspberry (Rubus idaeus cv. Ottawa) with a hybrid obtained by crossing raspberry (Rubus idaeus, L.) with arctic bramble (Rubus arcticus, L.) [12]. The volatile compounds were isolated from the press juice of the berries in a continuous vacuum evaporator, and separation, identification and quantification were than performed in three stages. The carbonyl compounds were determined as 2,4-dinitrophenylhydrazones, while the volatile acids and the neutral components were determined separately in a combined gas chromatograph-mass spectrometer using glass capillary columns coated with a polar phase (FFAP) [12]. More than 70 compounds were reported in the volatile fraction of the hybrid and 30 in the cultivated raspberry (the latter are reported in Table 1): identification was carried out from the recorded MS spectra. Honkanen and collaborators obtained an extract containing neutral components and free fatty acids from press extracted berry juice of wild and cultivated (Rubus idaeus cv. Ottawa, Preussen) raspberries [11] using a pentane/ethyl ether extraction. A total of 75 volatile compounds were identified and quantified by GC-MS systems equipped with polar capillary columns coated with FFAP (Table 1). Compound identification was achieved by comparing the acquired MS spectra with those of the reference compounds. A few years later, Guichard isolated volatile compounds in frozen raspberry (Rubus idaeus cv. Lloyd George) using three different extraction methods [9]: vacuum distillation, liquid-liquid extraction and a sorbent trapping method (Chromosorb 105). A total of 126 components were then identified by GC-MS ( Table 1). The three methods were also qualitatively compared in terms of rapidity, fraction recovery and reproducibility [20]. Vacuum distillation allows a more efficient, preferential extraction of alcohols, including terpene alcohols. Liquid-liquid extraction is less reproducible but allows isolation of compounds of different classes and gives a better recovery of ionones. The sorbent trapping method uses a stream of N2 to strip the volatile compounds, which are then trapped on Chromosorb 105: this method is rapid and reproducible with a preferential recovery of monoterpenes [20]. In a later work, Guichard compared this trapping method [21] with the method used by Rapp and Knipser, originally optimized for wine aroma extraction [39], which also uses a stream of N2 but volatile components are trapped in a solvent (trichlorofluoromethane) [39]. Reproducibility was worse with Rapp and Knipser's method than with sorbent trapping, especially for the terpene fraction [21]. Robertson and co-workers trapped the volatile compounds flushed by a zero-air gas flow over flowers or intact berries of Rubus idaeus cv. Glen Prosen in Haysep Q or Tenax TA tubes [19]. The trapped volatiles were than analyzed by automated thermal desorption-gas chromatography-mass spectrometry equipped with a medium polarity column (DB 1701): 61 chemical compounds were identified [19] (Table 1) by comparing retention time and acquired mass spectra with those reported in MS-libraries or published reports. Malowicki and co-workers used the stir bar sorptive extraction (SBSE) to trap the volatile components from the juice of frozen raspberries (Rubus idaeus cv. Meeker, Chilliwack, Tulameen, Yellow Meeker, Willamette) and analyzed them by GC-MS [22,23]. Separation was carried out using a polar column (ZB-FFAP). They were able to identify 29 volatile compounds (confirmed with authentic standards) ( Table 1) showing quantitative differences between cultivars and between different growing sites for the same cultivar [22]. Aprea and co-workers studied the volatile compounds released by the mashed fruits and juices of two raspberry cultivars (Rubus idaeus cv. Tulamen, Polka) by means of two rapid, solvent-free headspace methods: dynamic headspace by Proton-Transfer Reaction Mass Spectrometry (PTR-MS) and semi-static headspace solid phase micro-extraction (SPME) [24]. The GC-MS analysis of SPME trapped volatiles resulted in identification of 45 compounds (28 of which confirmed by authentic reference) while PTR-MS, a direct injection mass spectrometer [40], allowed the monitoring of hundreds of mass spectrometric signals, 29 of which were tentatively identified and 4 had established identities ( Table 1). The same SPME-GC-MS procedure with separation carried out in a polar fused-silica capillary column (HP-Innowax) was used to compare the volatile profiling of different raspberry varieties (Rubus idaeus cv. Anne, Autumn Bliss, Caroline, Heritage, Himbo Top, Josephine, Opal, Pokusa, Polana, Polesie, 2 Polka accessions, Popiel, Tulameen) [13]: the 45 compounds identified are reported in Table 1. Vrhovsek et al., quantified 39 volatile compounds (Table 1) in a solvent extract of 5 raspberry varieties (Rubus idaeus cv. Autumn Treasure, Glen Ample, Himbo Top, Rubyfall and Sugana) using GC-triple quadrupole MS/MS [16]. GC separation was performed on a polar (VF-WAXms) capillary column. The five varieties investigated differed in both qualitative and quantitative volatile compound composition. A total of 276 volatile compounds are reported in Table 1 (note that three entries refer to undetermined isomers of reported compounds). Data are collected from the 20 papers dealing with raspberry fruit volatile compounds reviewed in this section. Of the 276 volatile compounds reported in raspberry, 141 have been confirmed by comparison with authentic standards (references in bold in Table 1). Figure 1 shows distribution of the 276 volatile compounds according to chemical class. The largest class of compounds is constituted by the 56 monoterpenes reported (we use the term terpene to indicate both terpenes and terpenoids). Of these, terpinen-4-ol, geraniol, linalool, limonene, nerol, p-cymene, terpinolene, α-and β-phellandrene, γ-terpinene and α-and β-pinene are the most frequently reported. Terpenes derive from the common building unit isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP) [41]. In plants, two parallel pathways lead to the formation of both IDP and DMADP: the mevalonate (MVA) pathway, which is active in the cytosol, and the methylerythritol 4-phosphate (MEP) pathway, which is active in the plastid. It is generally acknowledged that monoterpenes are synthesized in the plastids whereas sesquiterpenes are produced in the cytosol [42], with some exceptions [43]. A total of 38 volatile acids are reported in Table 1. A characteristic of raspberry volatile composition is the relatively high amounts of some of these volatile acids. Acetic acid is reported to be between 20 ppb and 135 ppm, the wide variability mainly attributed to variety differences [15]. Hexanoic and octanoic acids are reported at concentrations up to 19.3 ppm and 600 ppb, respectively. Concentrations of volatile compounds observed by various authors in raspberry fruits can vary several-fold (Table 1). These differences are generally attributed to particular characteristics of raspberry cultivars or non-homogeneity of the fruit ripening stage. In one of the most recent works [16], the concentrations of the different volatile compounds were, in most cases, reported to be two orders of magnitude lower than previous findings. Direct comparison of the quantities reported by different authors is not feasible because of the different extraction methods and quantification procedures used. Most papers do not report recoveries of the extraction method used, and when carrying out quantitative procedures the matrix effect is most of the time not taken into account when calibration curves are built. Glycosidically-Bound Compounds A large number of volatile compounds, several of them with odor activity, are glycosylated and accumulate as non-volatile and flavorless glyco-conjugates in plant tissues [48]. These glycosidically-bound compounds are present in several fruits [48] and their occurrence is typically two to eight times greater than that of their free forms [49,50]. Pabst and collaborators studied glycosidically-bound volatiles in raspberry fruit (Rubus idaeus cv. Heritage) after enzymatic hydrolysis [51]. In total, 57 bound aglycons originating from fatty acid, phenylpropanoid, and terpene metabolisms were separated by GC on a Chrompack fused silica CP-Wax-58-CB WCOT capillary column and identified by MS using reference standards. Terpenes and C13-norisoprenoids were the largest classes with 14 and 12 compounds each respectively, followed by alcohols with 11 compounds, and acids with nine compounds. The other 11 compounds were seven phenols, one furane, one ketone and three lactones ( Table 2). More recently, Vrhovsek et al. quantified the amounts of 24 glycosidically-bound compounds in five raspberry varieties (Rubus idaeus cv. Autumn Treasure, Glen Ample, Himbo Top, Rubyfall and Sugana) [16]. This group developed a selective GC/MS/MS method for quantitative metabolite profiling of volatile compounds in apple, grapes and raspberries [16]. The volatile compounds were extracted from frozen powder of the fruits according to the solid phase extraction method reported in previous works [49,52]. Compound separation was carried out on a polar (VF-WAXms) capillary column. The compounds belonging to several classes, alcohols (4), aldehydes (4), terpenes (4), C13-norisoprenoids (7), one sesquiterpene, one acid, one ester, one lactone and one ketone, are reported in Table 2 with quantitative information. Several of the compounds identified are present in bound form with concentrations 2 to 40 times higher than their free forms ( Table 2). In the cultivar Autumn Treasure the bound form of benzyl alcohol is 44.7 times that of the free form. The amount of α-ionol present as glycol-conjugate is 40.7 and 27.5 times that of the free form in Himbo Top and Rubyfall varieties, respectively. β-damascenone was found only in bound form in the varieties investigated. a The identity of compounds has been confirmed by comparison with authentic standards but for 3-hydroxy-5,6-epoxi-β-ionone. b Quantitative data obtained after solvent extraction of mashed fruit by GC-triple quadrupole methods using authentic standards. "X" refers to identified but not quantified compounds. Enantiomeric Distribution Natural volatile molecules are generally found with one enantiomer predominating, attributable to stereoselectively controlled biogenetic formation mechanisms [53]. It is also known that certain enantiomeric chemicals have different sensory properties in terms of both odor quality and intensity [54]. Therefore, knowing the enantiomeric distribution of chiral compounds may help in understanding aroma perception. Furthermore, enantioselectivity and isotope discrimination during biosynthesis have been recognized as important indicators of authenticity of the natural product [55] and as such represent a useful method for differentiating natural raspberry products from those adulterated with synthetic aromas [56]. Nitz and co-workers determined the enantiomeric distribution of seven γ-lactones in deep-frozen raspberry (unknown variety) by multi-dimensional GC with achiral-chiral column combinations [57]. They found γ-octalactone and γ-hexalactone predominating with 65 and 30 ppb, respectively, and a prevalence of (S) enantiomers. The other γ-lactones had concentrations of 5 ppb or less with a prevalence of (S) enantiomers for hepta-, nona-and undeca-lactones, while a racemic distribution was observed for the deca-, and dodeca-lactones. Werkhoff and co-workers found that 99.9% of α-ionone is present in raspberry as (R) enantiomer [58]. The same result was obtained by Casabianca and Graff, who studied the enantiomeric distribution of α-ionone and δ-decalactone in three raspberry cultivars (Rubus idaeus cv. Mecker, Heritage and Williamette) and commercial raspberry products (tea, syrup and juice) [56]. One enantiomer was predominant in raspberry fruit while commercial product prepared with synthetic flavors displayed a racemic distribution of the two enantiomers. The (R) enantiomer of α-ionone was found to be more than 98%. In contrast, the (S) form was more than 98% in δ-decalactone. The results for α-ionone were corroborated by Sewenig et al. [59] in different raspberry varieties (Rubus idaeus cv. Rucami, Schönemann-Meyer, Meeker, Rumiloba, Glen Ample and Tulameen). Development during Ripening Fruit ripening is a highly coordinated, genetically programmed, irreversible phenomenon involving a series of physiological, biochemical, and sensory changes that lead to the development of a soft, edible ripe fruit with desirable attributes [60]. During ripening the odor and flavor of the fruits develop through the production of several volatile and non-volatile compounds (sugars, acids) and/or degradation of bitter principles (flavonoids, tannins, and related compounds) [61]. Guichard followed the evolution of different volatile compounds in two raspberry varieties (Rubus idaeus cv. Lloyd George and Rose de Côte d'Or) during ripening [62]. Four stages of ripening were identified: green-pink, pink, ripe and over-ripe. In both varieties all the terpenes and sesquiterpenes measured (α-pinene, β-pinene, myrcene, α-phellandrene, p-cimene, β-phellandrene, γ-terpinene, caryophyllene and humulene) greatly increased during ripening. The chromatographic peak areas varied from 10 to 1000 for the different terpenes and sesquiterpenes. Esters (isopentyl-, pentenyl-, (Z)-3-hexenyl-and methyl-acetate) also increased 10-100 fold during ripening. Geraniol was at its highest at the ripening stage in the cultivar Lloyd George but continued to increase up to the over-ripe stage in the cultivar Rose de Côte d'Or. Dihydro-β-ionone was at its highest at the ripe stage then decreased. α-Ionone increased slightly during ripening in both varieties, while β-ionone increased slightly only in Lloyd George and not at all in Rose de Côte d'Or. Robertson and co-workers included the flowering stages in their study of the evolution of volatile compounds in raspberry [19]. They sampled volatile compounds at six sequential stages of inflorescence development in the raspberry (Rubus idaeus) cultivar Glen Prosen: green buds, flowers, old flowers/early green fruit, green fruit, pink fruit, mature red fruit. During raspberry ripening, the saturated aldehydes from six to 10 atom carbons increase steadily, as did several monoterpenes such as α-pinene, camphene, α-phellandrene and limonene, in agreement with previous observations [62]. However, the two terpenes (E)-and (Z)-ocimene and the ester (Z)-3-hexenyl acetate greatly decreased. The terpene α-copaene and the sesquiterpene β-caryophyllene reached a maximum at the green stage then decreased considerably during berry ripening. The two ionones α-and β-only appeared during the last two stages of ripening as did the three esters methyl acetate, propyl acetate and ethyl hexanoate [19]. Postharvest Development Boschetti et al. measured the volatile compounds in raspberry [25] by direct injection method. They used a Proton Transfer Reaction Mass Spectrometer (PTR-MS) to monitor the emission of volatile organic compounds during postharvest aging of six different kinds of berry including the raspberry cultivar Tulameen. Using PTR-MS it was possible to monitor VOC emission from individual or small quantities of intact berries in real time and at high sensitivity without the need for any treatment or accumulation method [25]. Raspberries were monitored for three consecutive days before they started to decay at the end of the third day. The highest emissions recorded on the first day were methanol, acetaldehyde (4-5 ppm) and ethanol (1 ppm). Methanol, a major volatile associated with aging, reached a concentration of 40 ppm. Masses related to esters were constant and below 10 ppb over the three days [25]. It was suggested that simultaneous monitoring of the emissions of a large number of volatiles in real time and at high sensitivity can be used to describe fruit products and processing [25]. Fast Processes Aprea et al. monitored real time release of VOCs during mashing of the fruit [24] to simulate what happens during chewing or what could be the consequence of fruit damage during handling. In general, volatile emission increases after crushing of the fruits as the physical barriers trapping these secondary plant metabolites are disrupted. As well as the preformed plant metabolites, several other compounds of neo-formation (mainly oxidation product) are released. Figure 2, taken from Aprea et al. [24], reports the development over time of selected compounds monitored during raspberry crushing. At 10 min from the beginning of the experiment (indicated by the arrow) a sudden increase in the volatile compounds emitted occurs. Methanol, acetate ester, and acetic acid signals increase 4-to 5-fold in less than 1 min. (Z)-3-Hexenol increases 13-fold, while the peak in the C6-VOC signal after 4 min represents a 150-fold increase. These latter compounds together with C5-VOC are typical wounding products emitted by leaves and fruits, which originate from the lipoxygenase and hydroperoxide lyase pathways and are responsible for the typical green notes of fruits and leaves when crushed [63]. The experiment revealed that compounds produced by plant metabolism and accumulated in fruit tissue or constantly released, such as acetate esters, and compounds that are a direct consequence of tissue damage, such as C6-VOCs, have different release patterns. Since these compounds are also produced during food consumption they could affect sensory perception of the berries. The study mentioned in this section [24] was carried out using instruments coupled with a quadrupole mass analyzer which provides only the nominal mass of the observed spectrometric peaks, and therefore several interferences cannot be excluded (a version of the instrument coupled with time of flight mass spectrometry which improves the capacity of compounds identification is currently available [64]). Odor-Active Compounds Few of the many volatile compounds reported in raspberry (Tables 1 and 2) are recognized as important for the aroma of this fruit. One of the first compounds to be identified as having an impact on the character of raspberry is 4-(4-hydroxyphenyl)butan-2-one [38], which for this reason was named raspberry ketone. This compound is synthetized in Rubus idaeus by condensation of p-coumaroyl-CoA with malonyl-CoA and successive reduction [65]. Borejsza-Wysocki and co-workers measured the content of raspberry ketone in six raspberry cultivars (Rubus idaeus cv. Camby, Meeker, ORUS 576-47, ORUS 2078, Royalty and Willamette) and subjected them to organoleptic evaluation [66]. The 11 judges scored the varieties on a 0-100 scale for intensity of "raspberry" flavor and aroma. The highest flavor score (56.2) was obtained for the Willamette variety, which had the highest raspberry ketone content of the six raspberry cultivars investigated [66]. In their study, Larsen and co-workers identified raspberry ketone and α-and β-ionone as the most important aromas in the 10 different raspberry varieties investigated (Rubus idaeus cv. Camenzind, Chilcotin, Glen Prosen, Glen Moy, Glen Clova, Meeker, Rutrago, Skeena, Vaten and Zenith) [15]. They confirmed that pure raspberry aroma was highly dependent on raspberry ketone content, while α-and β-ionone were found to be important for the overall aroma. α-Ionone in raspberry is known to be present in the (R)-enantiomeric form [33,34,67], which is reported to produce a violet-like, fruity, raspberry-type, floral odor [68], while β-ionone is described as "fragrant" and "floral" [69]. Klesk and co-workers investigated odor-active compounds in red raspberry cultivar Meeker by Aroma Extract Dilution Analysis (AEDA) [10]. This technique involves the flavor extract being sequentially diluted and each dilution analyzed by GC-O by a small number of judges. The flavor dilution (FD) of an odorant corresponds to the maximum dilution at which that odorant can be perceived by at least one of the judges [70]. Although FD factors do not conclusively establish that one sample contains more of a given aroma compound than another, it gives an indication of the compounds that may contribute to the overall aroma of a product. Klesk and co-workers identified 75 odor-active volatiles (see Table 1) in the Meeker raspberry cultivar from two locations in the United States (Oregon and Washington) [10]. Compound identifications were confirmed by injection of authentic standards. The most intense compounds found in both samples included strawberry furanone, hexanal, β-ionone, (E)-β-ocimene, 1-octanol, β-pinene, (FD 2048), β-damascenone (FD 512), acetic acid, (Z)-3-hexenal, methional (FD 256), (Z)-3-hexenol, and linalool (FD 128). Differences between the fruit from the two locations were found for other compounds [10]. As the authors themselves recognize, defining FD is only a first step towards measuring the true odor impact of these compounds [70], which would require chemical quantification of these potent odorants and generation of their OAVs to be carried out. Wild Raspberries In 1980 Honkanen et al. carried out qualitative and quantitative comparisons of the volatile compounds in Finnish wild raspberries and in two cultivated varieties (Ottawa and Preussen) using GC-MS [11]. Volatile compounds were isolated from pentane/ethyl ether in raspberry juice extract and GC separation was performed in a polar (FFAP) capillary column. A total of 75 molecules were identified (Table 1) with the aid of authentic standards. As with the cultivated varieties, volatile acids (especially acetic and hexanoic) in the wild varieties were found to be present in high concentrations (24 ppm). The authors reported the presence in wild raspberries of two acids, 3-methyl-2-butenoic and 3-methyl-3-butenoic, which have not been found in any other cultivated raspberry. A few terpenes and sesquiterpenes have been found to be specific to wild raspberries, such as (Z)-sabinol, menthol, and α-elemene. The alcohol fraction in wild raspberries was reported to be about twice that of the cultivated varieties (24 and 10%-15%, respectively). The two trans enantiomers, 2-hexen-l-ol and 3-hexen-l-ol, not found until now in cultivated berries, were also reported to be present in wild berries. Several volatile phenolic compounds were identified in the wild berries, such as 2-methoxy-4-vinylphenol, 2-methoxy-5-vinylphenol, 3,4-dimethoxybenzaldehyde, and 4-vinylsyringol, none of which has been reported in any cultivated variety. The amount of raspberry ketone (4-(4-hydroxyphenyl)butan-2-one), one of the most important compounds impacting on raspberry flavor [38], was found to be 3 times higher in wild berries than in cultivated varieties, although the amount of α-and β-ionone was 1.5-2 times lower. With the exception of ionones, the amounts of individual volatile compounds were generally 3-4 times higher in wild raspberries than in the cultivated varieties. The higher amounts of volatile compounds and the presence of several compounds only in wild raspberry species may contribute to their distinctive aroma. The authors also suggest that increased berry size as a result of breeding programs, hybridization and/or fertilization leads to a deterioration in the aroma of the berries [11]. Differences among Cultivars Terpenes, terpenoids and nor-isoprenoid volatile compounds are the major compounds that have been examined for the differentiation of raspberry cultivars [15,22] as they are highly related to raspberry odor and flavor [15]. Larsen and co-workers reported relatively small variations in raspberry ketone and ionones in the 10 cultivars they compared (Rubus idaeus cv. Camenzind, Chilcotin, Glen Prosen, Glen Moy, Glen Clova, Meeker, Rutrago, Skeena, Vaten and Zenith) [15]. Greater differences between the varieties were observed in the concentrations of linalool, geraniol, benzyl alcohol, acetoin, acetic acid, and hexanoic acid. The high variations in the three latter compounds were ascribed to differing enzymatic activity influenced by both variety and different degrees of ripeness [15]. Malowicki [13]. Volatile compounds were separated in a polar fused-silica capillary column (HP-Innowax). All fruits were harvested in the same experimental field using the same agronomic practices. Crop season strongly influenced the total volatile emissions. In 2007 the raspberries had higher amounts of volatile compounds (two fold for many varieties), which was attributed to the colder temperatures (and higher thermic excursions) recorded over the 2007 season in the experimental fields, located in Vigolo Vattaro (Trento, Italy) [13]. Similar effects due to temperature excursions were reported in previous works [30,71]. Nonetheless, the assembled data set allowed raspberry varieties to be clustered in groups of similar volatile patterns. In general, Polka and Popiel were characterized by low amounts of volatile compounds, while Caroline, Heritage, Himbo-top, and Josephine were much richer. Levels of terpene alcohols and C13-norisoprenoid compounds were found to be higher in Anne, Polana, Polesie, Polka-I, Polka-P, and Popiel, while monoterpenes and sesquiterpenes were higher in Autumn Bliss, Caroline, Heritage, Himbo Top, Josephine, Opal, Pokusa, and Tulameen. Tulameen was further differentiated for the amounts of C6 compounds (aldehydes and alcohols) and their esters [13]. In a subsequent study, advanced chemometric methods were used to classify the same 14 cultivars using both GC data and PTR-MS measurements [72]. Specifically, random forest (RF), penalized discriminant analysis (PDA), discriminant partial least Squares (dPLS) and support vector machine (SVM) were used for cultivar classification, and random forest-recursive feature elimination (RF-RFE) was used for feature selection [72]. These analyses revealed 2-heptanone, 2-heptanol, (E)-caryophyllene, and dehydro-β-ionone to be the most useful compounds for raspberry cultivar classification. Thus, not only terpenes and derivative compounds, as suggested in previous works [15,22], but also other classes of compounds may contribute to the characterization of raspberry cultivars. These cultivar differences are then reflected in the diverse aroma and possible defense mechanisms (see Section 2.8) of the selected raspberry varieties. Environmental and Seasonal Effects The raspberry fruit produces an array of volatile compounds with significant variations in their contents influenced by numerous factors including genotype, climate, soil, ripeness, and many other variables [2,10,11,13,19,22,30] that impact on odor and flavor. Mold Resistance Raspberries are delicate fruits that soften and deteriorate rapidly after harvest. They are also highly susceptible to fungal diseases, particularly gray mold caused by Botrytis cinerea especially during postharvest storage [73]. Plants possess a range of preformed or inducible defense mechanisms, many of them involving secondary metabolites [74]. In fact, several volatile compounds are recognized for their inhibitory activity against pathogens, in particular B. cinerea in the case of raspberry [75,76]. Other studies have demonstrated that the same volatile compounds can have an opposite effect on pathogen development. For example, (E)-2-hexenal stimulates both B. cinerea spore germination and mycelial growth when present at low concentrations [77]. It was recently demonstrated that key strawberry aroma compounds stimulate B. cinerea conidial germination and some typical wound volatiles stimulate pathogen conidial germination or mycelial growth [78]. Thus, along with other resistance factors and defense mechanisms [79,80], volatile compounds seem to play a central role in mediating plant/pathogen interactions. Aprea et al. compared susceptibility to B. cinerea and volatile profiles in 14 raspberry cultivars [13] and found nine compounds to be negatively correlated with raspberry B. cinerea susceptibility: α-pinene, β-phellandrene, p-cymene, 2-heptanol, 4-terpineol, (E)-β-caryophyllene, β-damascenone, dehydro-β-ionone, and caryophyllene oxide. The authors suggested that quantification of these compounds in raspberry could be used as an indicator of fruit resistance to B. cinerea [13]. A subsequent study confirmed the importance of dehydro-β-ionone, 4-terpineol, p-cymene, (E)-β-caryophyllene for predicting raspberry susceptibility to B. cinerea [72]. Conclusions and Perspectives Little literature on raspberry volatile compounds has appeared in the last 10 years and most of what there is concerning isolation and identification was concentrated during the period of Firmenich's pioneering work. Later, the availability of more powerful analytical techniques allowed raspberry volatile composition to be studied in greater detail but only a few investigations looked at their roles in sensory perception and the ecological and physiological implications. Aside from their nutraceutical properties, one of most important traits of raspberries in terms of human consumption is their pleasant aroma. Only a small fraction of the volatile compounds identified in raspberry fruits contribute to the aroma, so that distinguishing odor-active compounds from other volatile compounds is an important step in aroma research. The association between volatile compounds and aroma/flavor perception in a complex matrix, such as fruit, is not straightforward. For example, multiple volatiles are responsible for aroma/flavor sensations, combinations of volatiles yield flavors differing from those expected of individual compounds, and perception of volatiles differs in different matrices [81]. Moreover, the final sensory evaluation can even be influenced by psychological and multisensory factors. The only "instrument" which can discriminate between odor active compounds and other volatile compounds is the human nose. Therefore, the primary measure of the sensory attributes of flavor and aroma is descriptive sensory analysis, typically with trained sensory panels [81]. For all the above-mentioned reasons we think that the relationship between volatile compounds and odor and flavor in raspberry is worth further investigation using appropriate methodologies. Furthermore, there is little literature on raspberry characterization by sensory descriptive methods. In our opinion this issue should be better addressed in future research and breeding programs. For example, it would be desirable to include sensory traits and volatile compounds in research on quantitative trait loci, as has been done for apple [82][83][84]. Other aspects of raspberry research, only partially addressed in the literature and deserving more attention, relate to plant communication and plant-pathogen interaction mechanisms mediated by endogenous volatile compounds, as studied in other fruit (e.g., strawberry [78]). These studies will contribute to a better understanding of some of the natural defense mechanisms activated by plants with the aim of helping agronomists to manipulate and manage them in order to reduce the use of pesticides. The identity, biochemical pathways and release of volatile compounds in raspberry has been widely investigated, but more studies are needed to better understand the various biological roles played by the different volatile compounds in raspberry.

v3-fos

2019-04-01T13:16:17.896Z

2015-01-01T00:00:00.000Z

89286932

s2

INVESTIGATION OF APRICOT REPRODUCTIVE STRUCTURES, CREATION AND PROPAGATION OF NEW FORMS SUMMARY One of the main problems of modern fruit agriculture is creation of new productive apricot cultivars by hybridization method with suitable selection of parent couples with functional gametes. Analysis of male generative sphere One of the main problems of modern fruit agriculture is creation of new productive apricot cultivars by hybridization method with suitable selection of parent couples with functional gametes. Analysis of male generative sphere development in 25 apricot cultivars demonstrated that they have different quality of pollen grains. The least amount of morphologically proper pollen grains was noticed in cultivars Dionys, Harcot, Mamai thus they should be used as maternal forms. The greatest pollen grains viability is typical for cultivars Ananasny Tsiurupinsky, Krasny Vympel, Magistr and Holovous that are recommended as paternal forms. In the process of parent forms selection flower structure should be considered since flowers with short pistils have undeveloped embryo sacs with undifferentiated egg apparatus. In the flowers with long pistils two ovules are usually, initiated one of which gradually degenerates and the other develops successfully. Under the pollen grains placement on the stigma proper ones enlarge rapidly, pollen tube develops and mitosis of generative cell occurs in it resulted in the formation of two sperms. Pollen tube grows and comes to the embryo sac, pours its content and double fertilization occurs. Using of embryoculture and in vitro cultivation of hybrid embryos on the modified Murashige and Skoog, Gamborg & Eveleigh, Monnier and Quoirin & Lepoivre media gave us possibility to obtain seedlings of 12 breeding combinations. Such complex of investigation methods allows creating new apricot forms for their further use in breeding and propagation processes. INTRODUCTION Apricot (Prunus armeniaca L., SYN. Armeniaca vulgaris Lam.) is one of the most popular and promising stone fruit. Its fruits have high taste qualities due to the presence of sugars, organic acids, pectin, vitamins, anthocyanins, minerals, etc. The apricot fruits are used fresh, in confectionery production and medicine. As a food product apricot has been used over the millennia. It is grown and cultivated in many warm temperate countries of Europe, Central Asia, the Caucasus, Ukraine, Moldova and in the southern regions of Russia. Modern scientist distinguish between three and six possible places of the apricot origin, most likely it`s from the Tian Shan region of China (Dalby, 2003;Folta, 2009). During the cultivation of apricot many of its cultivars have been created, but there is still a need in new genotypes and forms with a complex of economicallyvaluable traits that would be adapted to the particular growing conditions and would have exceeded the available cultivars (Fideghelli and Strada, 2008;. Therefore, the aim of our study was to identify the best ways of getting new apricot genotypes for the subsequent breeding process. RESULTS AND DISCUSSION Many cultivars of apricot are susceptible to late-winter and early-spring air temperature fluctuations, which are specific to the southern regions and their generative buds are damaged with spring frosts. It remains relevant to create cultivars with the slow development of generative buds and their long period of dormancy (Sholokhov, 1961;Gorina, 2014, etc.). Cytoembryological studies of apricot by S.I. Elmanov, A.M. Sholokhov and E.A. Yablonsky (1969) and our observations have demonstrated that the mechanism of incompatibility blocking is a multi-stage process. It can occur in the time of pollen grains` germination, growth of pollen tubes in the tissue of the pistil, fertilization and embryogenesis. Frost resistance and winter-hardiness of apricot cultivars are greatly depended upon the genotype and development of generative buds. Analysis of the male generative sphere development in 25 apricot cultivars has demonstrated that they possess pollen of different quality. The smallest number of morphologically normal pollen grains is formed in cultivars Dionys, Harcot, Mamai, therefore it is advisable to use them as a parent form. The highest vitality of pollen was in cultivars Krasny Vympel, Magistr and Holovousy, which are recommended as paternal forms. For example, cultivar Ananasny Tsyurupinsky has over 90% of morphologically normal pollen grains. More than 50% of them are fertile and on 15% sucrose solution via 6 hours after sowing they sprouted and gave long (more than 10 diameters of the pollen grain) pollen tubes. When selecting the original forms the structure of flower should be also taken into account, because flowers with short pistils have undeveloped embryo sacks, the egg apparatus is undifferentiated. In flowers with long pistils two ovules are usually placed, one of which gradually degenerates, and the other develops normally. When applied on the stigma of the pistil normally developed pollen grains rapidly swell, pollen tube develops and division of the generative cell resulted in two sperms formation occurs in it. The sprouting pollen tube reaches the embryo sac, pours out its contents and double fertilization takes place. It should be noted, that apricot is peculiar to the protandry phenomenon that should be considered in hybridization. From many available cultivars in our collection compatible, high yield cultivars with high quality of fruits and frostresistance that could be used as parental forms have been selected. For example, cultivar Krymsky Amur with large high quality fruits is well compatible with frost-resistant cultivar Harcot that is also resistant to the Sharka virus, but sensitive to the spring frosts. As the result of their crossing, interesting hybrids could be obtained. Development of hybrid embryos in vitro could pass the different ways -via callusogenesis and via direct organogenesis (Figures 1 and 2). Crossing between cultivar Shalah with large high-quality fruits and frostresistant cultivar Veecot could produce the hybrids with large fruits of new colours and taste resistant to stress environmental factors (Figures 3 and 4). The results of our investigation and data presented by other researchers (Mitrofanova et al., 2000;Pintea, 2014) illustrate possibilities for increasing effectiveness of new apricot cultivars breeding due to the use of embryoculture method along with hybridization. CONCLUSIONS Thus, on the base of our investigations it could be concluded that complex approach is necessary to increase the effectiveness of the breeding process aimed in obtaining new cultivars with high-quality fruits, resistant to stressful environmental factors. It includes biological and phenological studies, investigations of generative sphere and strict selection of the parent forms according to their characteristics, as well as the use of in vitro culture method for growing up immature embryos and their virus free, as well as for propagation of obtained valuable genotypes.

v3-fos

2018-04-03T03:04:17.111Z

2015-10-27T00:00:00.000Z

16385862

s2

Developmental Changes in Composition and Morphology of Cuticular Waxes on Leaves and Spikes of Glossy and Glaucous Wheat (Triticum aestivum L.) The glossy varieties (A14 and Jing 2001) and glaucous varieties (Fanmai 5 and Shanken 99) of wheat (Triticum aestivum L.) were selected for evaluation of developmental changes in the composition and morphology of cuticular waxes on leaves and spikes. The results provide us with two different wax development patterns between leaf and spike. The general accumulation trend of the total wax load on leaf and spike surfaces is first to increase and then decrease during the development growth period, but these changes were caused by different compound classes between leaf and spike. Developmental changes of leaf waxes were mainly the result of variations in composition of alcohols and alkanes. In addition, diketones were the third important contributor to the leaf wax changes in the glaucous varieties. Alkanes and diketones were the two major compound classes that caused the developmental changes of spike waxes. For leaf waxes, β- and OH-β-diketones were first detected in flag leaves from 200-day-old plants, and the amounts of β- and OH-β-diketones were significantly higher in glaucous varieties compared with glossy varieties. In spike waxes, β-diketone existed in all varieties, but OH-β-diketone was detectable only in the glaucous varieties. Unexpectedly, the glaucous variety Fanmai 5 yielded large amounts of OH-β-diketone. There was a significant shift in the chain length distribution of alkanes between early stage leaf and flag leaf. Unlike C28 alcohol being the dominant chain length in leaf waxes, the dominant alcohol chain length of spikes was C24 or C26 depending on varieties. Epicuticular wax crystals on wheat leaf and glume were comprised of platelets and tubules, and the crystal morphology changed constantly throughout plant growth, especially the abaxial leaf crystals. Moreover, our results suggested that platelets and tubules on glume surfaces could be formed rapidly within a few days. Introduction observations allow one to infer that the wax crystals of the wheat leaf surface may show developmental differences. Wheat is one of the most important staple crops in the world. Although the morphology and component of cuticular waxes have been extensively studied in wheat [17,18,19,20,32,33], it remains unclear how the cuticular wax component and crystal structure of wheat leaves and spikes change with the increasing wheat plant age. Thus, gas chromatography with flame ionization detection (GC-FID), gas chromatography and mass spectrometry (GC-MS) and SEM were applied to investigate the changes in the cuticular wax composition and the crystal morphology of leaves and spikes in parallel, in order to comprehensively understand the wax developmental patterns in wheat leaf and spike. Leaf and spike sampling To analyze developmental changes in the wax load, composition and morphology during the leaf developmental cycle, leaves were sampled at 50, 100, 200 and 230 days after seed germination. In general, different growth stages of wheat plants at 50, 100, 200 and 230 days represent seedling stage, wintering stage, heading stage and filling stage, respectively. Leaves at 50 and 100 days were young and not completely unfolded. Therefore, the first leaves were sampled from different plants per replicate. At 200 and 230 days, flag leaves were collected. Spikes were harvested randomly from three individual plants at 1, 3, 5, 7, 9 and 15 days after heading (DAH). Leaf and spike samples were divided into two groups: one group was used for GC-MS and GC-FID analysis, and the other was used for wax morphology analysis. Cuticular wax extraction Leaf and spike samples collected at different development stages were immediately immersed in chloroform after tissues were photographed, and shaken for 1 min at room temperature to extract cuticular waxes. The wax-extracted spikes were dried at 50°C for seven days and weighed. A known amount of n-tetracosane (C 24 alkane) was added as an internal standard. The resulting extracts were filtered and dried completely under a gentle stream of nitrogen gas. The samples containing the internal standard were re-dissolved in chloroform, transferred to a GC autosampler vial, and dried again under a stream of nitrogen gas. Subsequently, the extracted samples were treated with 100 μl bis-N,N-(trimethylsilyl) trifluoroacetamide (BSTFA, Sigma) and 100 μl pyridine (Fluka) for 1 h at 70°C to transform hydroxyl containing compounds into their corresponding trimethylsilyl derivatives. Then, the surplus BSTFA was quickly evaporated under nitrogen gas flow and re-dissolved in 500 μl of chloroform for chemical analysis. Chemical analysis of cuticular waxes Chemical composition of the wax extract was analyzed by a capillary gas chromatograph equipped with a Rxi-5ms column (30 m length, i.d 0.25 mm, film thickness 0.25 μm; Restek, USA) and attached to a mass spectrometer (GCMS-QP2010, Shimadzu, Japan) using helium as the carrier gas. GC was carried out with temperature-programmed on-column injection and oven temperature set at 50°C for 2 min, raised by 20°C min -1 to 200°C, held for 2 min at 200°C, raised by 2°C min -1 to 320°C, and held for 15 min at 320°C. Individual wax components were identified by comparison of their mass spectra with those of authentic standards and literature data. The quantitative compositions of the mixtures were studied using GC-FID (GC-2010 Plus, Shimadzu, Japan; column 60-m Rtx-1, 0.32-mm i.d., df 0.25 μm; Restek, USA). GC was carried out under the conditions as described above but with nitrogen as the carrier gas. Quantification was based on the FID peak areas and the internal standard (n-tetracosane), which was added to the wax samples before GC-FID. The total amount of leaf wax components was expressed per unit of leaf surface area, and the total amount of spike wax components was expressed in μg of wax per g of spike (dry weight). The total leaf blade surface areas were calculated with ImageJ software (http://rsb.info.nih.gov/ij/) by measuring the apparent leaf blade areas in digital images and multiplying by 2. All quantitative data are given as mean values and standard deviations (SD). SEM To examine the differences in wax morphology during various stages of leaf and glume development, fresh leaf blades of four wheat varieties were collected at 50, 100, 200, and 230 days of plant growth, respectively. Glumes were sampled at 1, 3, 5, 7, 9 and 15 DAH. The samples were air-dried for seven days in a desiccator at room temperature and then carefully dissected. 3-5 mm completely dried pieces were attached with double adhesive tape to the aluminium stubs and sputter-coated with gold particles using 90-s bursts from a sputter coater. Coated surfaces were investigated using a Hitachi S4800 SEM at an accelerating voltage of 10 kV and a working distance of 8.4 mm. Changes in the amount and individual composition of leaf surface wax during leaf development The total wax from leaves of the four wheat varieties, including both glossy species (A14 and Jing 2001) and glaucous species (Fanmai 5 and Shanken 99), was extracted with chloroform ( Fig 1). The chemical composition of cuticular wax was analyzed at four plant age stages (50, 100, 200 and 230 days). The total leaf wax load of the four wheat varieties increased steadily during initial leaf development between 50 and 200 days, and then decreased from 200 to 230 days, except for Shanken 99, in which the total wax load increased continuously throughout the entire sampling period (Fig 2 and S1 Table). Hence, it can be concluded that a general trend of the total wax load is initially to increase and then decrease during leaf development. For example, the total wax load on Jing 2001 leaves was 324.9, 370.2, 389.4 and 367.4 μg dm -2 at 50, 100, 200 and 230 days, respectively (S1 Table). Chain length distribution of leaf cuticular waxes composition The chain length distributions of individual wax constituents were further analyzed by GC-FID and GC-MS. The major alcohols constituent on leaves of the four wheat varieties consisted of even numbers of carbon chain length from C 20 to C 32 , with C 28 being the dominant chain length. The alcohols chain length remained constant throughout the growth period. The odd number carbon chain length of alkanes ranged from C 23 to C 33 (Fig 3). Interestingly, a shift in the dominant alkanes chain length was observed during leaf development. The dominant alkanes chain length was C 27 from 50 to 100 days, but C 29 became the dominant chain length from 200 to 230 days. The aldehydes chain length contained even carbon number chain lengths between C 22 and C 28 , with a maximum of C 28 . The aldehydes chain length distribution did not change during leaf ontogenesis (Fig 3). Additionally, there was a significant change in the chain length distribution of fatty acids constituent. At 50 days, the chain length distribution ranged from C 20 to C 24 or C 20 to C 26 , without a dominant chain length. However, the chain length distribution became increasingly longer ranging from C 20 to C 28 at 100 days, with C 28 forming the dominant chain length, and then remained constant throughout the remaining period of leaf development. A single carbon chain length, C 31 , was detected for βand OH-βdiketones. The carbon chain length of esters contained C 44 to C 46 , with C 44 being the major chain length (Fig 3). Our studies suggested that the individual wax constituent of the four wheat varieties shared nearly identical changes in chain length distribution during leaf development. Morphological changes in leaf surface wax crystals during leaf development To gain insight into the dynamic development of epicuticular wax crystals, we investigated the wax crystal micromorphology on both the adaxial and abaxial sides of leaf blades by SEM. Leaf blades were obtained from four wheat varieties at four plant ages (50, 100, 200 and 230 days). Based on the classification and terminology of plant epicuticular waxes presented by Barthlott et al [31], we identified two forms of wax crystals on the wheat leaf surface: platelets and tubules (S1-S4 Figs). At 50 days, both the adaxial and abaxial leaf sides of all four wheat varieties were covered with platelet-shaped wax crystals. Some platelets were connected to their neighboring crystals and formed a dense network. The size of the wax platelets was between 0.3 and 0.7 μm in length and between 0.3 and 0.5 μm in height. In general, the wax platelets had irregular margins, which were slightly curved and arranged in widely varying angles towards each other (S1-S4 Figs). SEM showed no significant differences in the wax crystal morphology of leaf blades with different varieties, or between the adaxial and abaxial leaf sides during initial leaf development at 50 days. The crystal shapes at 100 days were similar to that of 50 days, with platelet-shaped structures depositing on both the adaxial and abaxial leaf sides (S1-S4 Figs). Nevertheless, the platelets at 100 days were distinguished by a significantly longer length of 0.6-1.0 μm and a silimar height of 0.3-0.5 μm, compared to those at 50 days. Additionally, the adaxial and abaxial sides of the leaf blades at 100 days showed a crystal structure with more sparse arrangements of plate-shaped wax, suggesting a decrease in the total number of crystalloids present per unit area compared to that at 50 days (S1- S4 Figs). Strikingly, dramatic changes in wax morphology occurred at 200 days. Leaves from glossy species (A14 and Jing 2001) showed significantly different structures with deposits of plate-shaped wax on the adaxial leaf surface and the smooth wax film coverages, without any crystalline structures present, on the abaxial leaf surface (S1 and S2 Figs). In contrast, the epicuticular wax of glaucous species (Fanmai 5 and Shanken 99) formed platelets on adaxial sides of leaf blades and tubule-shaped structures on the abaxial sides of leaf blades. The tubules were approximately 0.1-0.3 μm in diameter and 5-20 μm in length (S3 and S4 Figs). Consequently, it was concluded that the crystals morphology of the abaxial leaf surface changed drastically from 100 to 200 days. The abaxial surface may show a smooth wax film or form tubule-shaped structures at 200 days, depending on the wheat variety. During further leaf development, the cuticular wax layer shared very similar crystals structure between 200 and 230 days (S1-S4 Figs). Taken together, these results clearly show that as plant age increased, epicuticular wax crystals display morphology changes specifically on the abaxial side of the wheat leaf blade. Developmental changes in the amount and individual constituent on the spikes of wheat Besides functioning as inflorescence and protectors of the growing grains, spikes play a major role in the production of assimilates for grain filling [34]. To this end, there is no information available on changes of wheat spike wax constituents with different growth stages. Therefore, we investigated the changes in wax amount and composition on spikes of four wheat varieties at six stages (1,3,5,7,9 and 15 DAH). The total wax load on the spikes of all four wheat varieties increased steadily from 1 to 9 DAH and reached a maximum at 9 DAH, and then decreased from 9 to 15 DAH (Fig 4). For instance, the total wax load on glossy variety Jing 2001 spikes was 250.7, 345.0, 525.6, 685.2, 886.0 and 788.3 μg g -1 at 1, 3, 5, 7, 9 and 15 DAH, respectively (S2 Table). For all four wheat varieties, the spikes wax fraction consisted of the five compound classes, including βand OH-β-diketones, alcohols, alkanes, aldehyde and fatty acids. The wax amount of alkanes, diketones and alcohols constituents continuously increased from 1 to 9 DAH, and then decreased or changed slightly until 15 DAH depending on varieties (Fig 4). During the investigated period of 15 DAH, the wax amount of aldehydes and fatty acids constituents showed a slight fluctuation (Fig 4). These tendencies of changes in spike waxes were shown almost similarly in all four varieties. Studies presented here demonstrated that the total wax amount of wheat spike surface firstly increased and then decreased during spike development. βand OH-β-diketones mainly consisted of a single carbon chain length of C 31 . Interestingly, OH-β-diketone was not detected in glossy varieties (Fig 5A and 5B), and a small amount of OH-β-diketone was detected in Shanken 99 (Fig 5D), while Fanmai 5 yielded high amount of OH-β-diketone (Fig 5C), suggesting that the amount of OH-β-diketone strictly depended on varieties. Generally, βand OH-β-diketones were the major constituent on spikes of glaucous species, with relative portions ranging from 41.0 to 69.6% (S2 Table). The odd number carbon chain length of alkanes ranged from C 23 to C 31 or C 23 to C 33 depending on wheat varieties, with a relatively sharp maximum at both C 29 and C 31 . Unlike C 28 being the dominant chain length in alcohols from leaves, the alcohols constituent of spikes consisted of even numbers of carbon chain length from C 22 to C 30 or C 22 to C 32 , with C 24 or C 26 being the dominant chain length. The fatty acids constituent consisted of even numbers of carbon chain length C 16 and C 18 with a maximum for C 16 . The aldehyde contained a single even carbon number chain length of C 22 (Fig 5). Additionally, unlike the chain length changes of leaf surface wax, there were no significant changes in the chain length distributions of all the five wax components on spikes of the four wheat varieties during spike development (Fig 5). In conclusion, our results disclose that the wax amounts and components on the spikes of wheat exhibit the significant developmental changes with increasing spike age. Morphological changes in wax crystal on the glumes of wheat Because the major surface of a spike was covered by glumes, to characterize the morphological development of the spike epicuticular wax crystals, native glume surfaces of four wheat varieties were investigated by SEM at the same six stages. Similarly to the leaves, the glumes of wheat were also covered with two forms of crystalloids: platelets as well as tubules. Unexpectedly, the platelets were only present on glossy species, whereas tubules were present on all four varieties (Fig 6). The glume wax crystals developed differently between glossy and glaucous varieties. For glossy species, at 1 DAH, the glume surfaces were covered by a relatively smooth film without any crystal structures present, typically rendering their surfaces glossy. This trend continued from 1 to 3 DAH. At 5 DAH, glumes of A14 still appeared smooth. In contrast, a small amount of tubules began to deposit on the glumes of Jing 2001. At 7 DAH, tubules were recognized for the first time on A14 glumes, and Jing 2001 glumes formed a more dense covering of tubules (Fig 6). Strikingly, at 9 DAH, both platelets and tubules can be found on glumes of A14 and Jing 2001. Notwithstanding, during spike development, platelets disappeared with only tubules present on the glume surfaces of glossy species at 15 DAH, suggesting a very short window to view the plate-shaped wax crystals on glume (Fig 6). For glaucous species, a small amount of tubules began to deposit at 1 DAH, and tubule-shaped wax crystals became considerably great density during the glume development. This trend continued until 15 DAH. At 15 DAH, the glumes of all four wheat varieties showed the tubule-shaped wax structure, and the tubules were parallel to the plane of the glume surface, with a diameter of 0.1-0.3 μm and a length of 5-20 μm (Fig 6). Compared to glossy species, glaucous species displayed a dense array of tubules from 1 to 15 DAH (Fig 6). Consequently, it was concluded that tubule-shaped wax crystals first appeared, and platelets started to form, finally, platelets disappeared with only tubules present on the glume surface of glossy species. In contrast, only tubules were present on glaucous species throughout the entire period of 15 DAH (Fig 6). Taken together, these results again demonstrate that the development of epicuticular wax crystals is a dynamic process and that platelets and tubules on wheat glume surfaces can be formed rapidly within a few days. Discussion Despite several reports on wheat wax content and morphology [17,18,19,20,32,33], this is the first detailed and precise characterization of the developmental changes in cuticular wax composition and morphology on the leaves and spikes during wheat plant growth. Wheat leaves vary in size according to growth stage. We found that the total amounts of cuticular wax on leaf surfaces increased continuously from 50 to 200 days, matching the time period of leaf expansion, and then decreased until the end of leaf development, except for Shanken 99, in which wax levels gradually increased throughout the analyzed leaf development stages (Fig 2). We speculate that Shanken 99 leaves continue to expand at 230 days and that the total wax amounts may decrease during further leaf development. Therefore, it can be concluded that wax biosynthesis is in full progress during early leaf development. However, when leaf expansion is complete, wax accumulation ceases. The wax amount of all four wheat varieties constantly changed during the investigated period of 230 days (Fig 2 and S1 Table). This result indicates that any amount of wax given for wheat in previous report is only representative of a certain plant age [17,18,26,32,35]. Additionally, it should be noted that alcohols are always the major components of cuticular waxes mixture throughout leaf development. When βand OH-β-diketones presented, the alcohol content decreased correspondingly. In the present study, we also investigated the chain length changes in individual wax constituents of leaves. Generally, these results are in accordance with observations on other wheat cultivars [17,35]. Interestingly, there was a significant change in the alkane chain length distribution, in which the dominant chain length of alkanes shifted from C 27 to C 29 during leaf development. Likewise, the chain length for fatty acids became increasingly longer from 50 to 100 days, and then remained almost constant in chain length distribution between 100 and 230 days (Fig 3). Similar to developmental changes of leaf surfaces wax, the amount and composition of spike surfaces wax showed the dynamic changes. The total spike wax load first increased, and then decreased during spike development. The amount of alkanes, diketones and alcohols continuously changed throughout the entire period of spike growth. Compared to leaves, alkanes and diketones were major factors that caused the waxes changes in spike growth. There was no significant change in the chain length distributions of all the five wax components of spikes. The prevailing consensus of opinion regarding the diversity of wax crystals on plant surfaces is that the various morphologies result from self-assembly processes, based mainly on the chemical composition of the waxes [32]. Our results showed that epicuticular wax crystals patterns on wheat leaf surface consisted of platelets and tubules, and the crystals morphology and size changed constantly during leaf development. Between 50 and 100 days of plant, both the adaxial and abaxial sides of leaf blades were covered by platelet-shaped wax crystals (S1-S4 Figs). Similar to our results, the cuticular wax on both sides of the blades from 8-week-old T. aestivum plants formed platelets [32]. Interestingly, from 200 to 230 days, the adaxial wax layer was characterized by a homogeneous coverage of platelet-shaped crystals, but the abaxial side of flag leaf blade changed to a smooth wax film in a glossy varieties or tubules in a glaucous varieties. Previous studies have shown that the adaxial wax bloom in wheat leaves consisted mainly of deposits of thin wax plates, and fibrillar waxes predominated on the abaxial surface of flag leaves [18,20,36]. For spikes, this is no literature report on the developmental changes in crystal morphology on wheat glumes. Our results showed that epicuticular wax of glumes was composed of platelets and tubules. Interestingly, for glossy species, tubules first formed, and then platelets began to appear, finally, platelets disappeared with only tubules depositing. Nevertheless, for glaucous species, only tubules appeared during the entire sampling period of 15 DAH. In particular, platelets and tubules on glume surfaces can be formed rapidly within a few days. Based on these results of the chemical composition and the crystals morphology, it is reasonable that the wax chemical composition changes in different stages correspondingly cause the wax crystals changes. Epicuticular wax layer that covers leaves, fruits and stems, gives the plant surface a glaucous or gray appearance [22,29,30]. It has been known that platelet crystals are positively correlated with alcohols on the wheat leaf surface and the recrystallization of the pure octacosan-1-ol constituent of wheat waxes formed upright platelets [32]. Prior studies have demonstrated that tubules are dominated by secondary alcohol (nonacosan-10-ol) or β-diketone [37][38][39]. Barthlott et al also proposed the term 'β-diketone tubules' [40]. The β-diketone tubules mainly contribute to the glaucous phenotype in wheat [17,18,19,41]. In this study, glaucous and glossy wheat varieties were used for wax analysis. Our results showed that the abaxial leaf surface of glossy varieties displayed a smooth wax film between 200 and 230 days (S1 and S2 Figs), while glaucous varieties were covered with wax tubules (S3 and S4 Figs). The wax chemical data indicated that βand OH-β-diketones could be detected in both glossy and glaucous leaves. This result suggested that the amount of βand OH-β-diketones must reach a certain threshold before tubules could be observed. For example, when the amounts of βand OH-β-diketones ranged from 24.1 to 26.1 μg dm -2 , tubules were absent on the abaxial surface of A14. When the amounts of βand OH-β-diketones ranged from 48.0 to 52.1 μg dm -2 , tubule-shaped wax crystalloids are present on the abaxial surface of the Fanmai 5 leaf, suggesting that 48.0 μg dm -2 of βand OH-β-diketones is a critical threshold value for the occurrence of tubules, at least in wheat leaf. Our results also showed that β-diketone could be detected in the spikes of both glossy and glaucous varieties, but OH-β-diketone could be detected only in the spikes of glaucous varieties. Consequently, it was concluded that βand OH-β-diketones could be synthesized by different enzymes and these varieties were good materials for further research on the biosynthesis of OH-β-diketone. Conclusions Our results clearly indicated that the cuticular wax on wheat leaves and spikes represented a highly dynamic system, and the individual wax component and wax crystal gradually changed during the investigated season. The general accumulation trend of the total wax amount of wheat leaf and spike surfaces is first to increase and then decrease during plant development, but these changes were caused by different compound classes between leaf and spike. Alcohols are the predominant wax constituent on leaves, but the relative percentages decrease continuously throughout the entire period of leaf growth. βand OH-β-diketones are abundant in spikes, particularly in glaucous varieties. There was a significant shift in the chain length distribution of alkanes between early stage leaf and flag leaf. Unlike C 28 alcohol being the dominant chain length in leaf waxes, the dominant alcohol chain length of spikes became C 24 or C 26 depending on varieties. Epicuticular wax crystals on wheat leaf and glume surfaces consist of platelets and tubules, and the crystal structures change constantly throughout plant growth. In particular, platelets and tubules on glume surfaces could be formed rapidly within a few days.

v3-fos

2016-05-12T22:15:10.714Z

2015-08-05T00:00:00.000Z

10757062

s2

Genome-wide identification of sweet orange (Citrus sinensis) histone modification gene families and their expression analysis during the fruit development and fruit-blue mold infection process In eukaryotes, histone acetylation and methylation have been known to be involved in regulating diverse developmental processes and plant defense. These histone modification events are controlled by a series of histone modification gene families. To date, there is no study regarding genome-wide characterization of histone modification related genes in citrus species. Based on the two recent sequenced sweet orange genome databases, a total of 136 CsHMs (Citrus sinensis histone modification genes), including 47 CsHMTs (histone methyltransferase genes), 23 CsHDMs (histone demethylase genes), 50 CsHATs (histone acetyltransferase genes), and 16 CsHDACs (histone deacetylase genes) were identified. These genes were categorized to 11 gene families. A comprehensive analysis of these 11 gene families was performed with chromosome locations, phylogenetic comparison, gene structures, and conserved domain compositions of proteins. In order to gain an insight into the potential roles of these genes in citrus fruit development, 42 CsHMs with high mRNA abundance in fruit tissues were selected to further analyze their expression profiles at six stages of fruit development. Interestingly, a numbers of genes were expressed highly in flesh of ripening fruit and some of them showed the increasing expression levels along with the fruit development. Furthermore, we analyzed the expression patterns of all 136 CsHMs response to the infection of blue mold (Penicillium digitatum), which is the most devastating pathogen in citrus post-harvest process. The results indicated that 20 of them showed the strong alterations of their expression levels during the fruit-pathogen infection. In conclusion, this study presents a comprehensive analysis of the histone modification gene families in sweet orange and further elucidates their behaviors during the fruit development and the blue mold infection responses. Introduction In eukaryotes, the dynamics of chromatin structure regulate DNA accessibility and DNA-templated processes, and affect various biological processes (Ho and Crabtree, 2010). Nucleosome is the basic unit of chromatin and it compacts DNA by nearly sevenfold with ∼146 bp of DNA wrapped around a histone octamer. The histone octamer is composed by two copies of H2A, H2B, H3 and H4 histone proteins. The histone tails are modified by dynamic post-translational modifications (PTMs) including methylation/demethylation, acetylation/deacetylation, and so on (Patel and Wang, 2013). Various histone modifications, which are termed as the "histone code, " collectively build up an enriched and complicated pattern of chromatin structure and powerful function modulations (Strahl and Allis, 2000). It is well reviewed that a series of gene families involved in the establishment of histone methylation/demethylation and acetylation/deacetylation (Berr et al., 2011). Methylation of histone lysine residues is an important epigenetic regulation mechanism which can activate or silence gene expression. It is known that histone lysine methylation modifications (except the methylation of H3K79) are catalyzed by a series of histone methyltransferases (HMTs), which were mainly encoded by a family of SET DOMAIN GROUP genes (SDGs; Feng et al., 2002). The SDG family is divided into different classes according to the sequence similarities with the suppressor of variegation 3-9 [SU(VAR)3-9], enhancer of zeste [E(z)], trithorax (TRX), and absent, small, or homeotic disks 1 (ASH1). The functions of histone lysine methylation in plant biological processes are involved in floral organ development, flowering transition, shoot and root branching, endodormancy release, carotenoid biosynthesis, hormone regulation, thigmomorphogenesis, and fungal pathogens resistance (Dong et al., 2008;Cazzonelli et al., 2009Cazzonelli et al., , 2014Berr et al., 2010a,b;Sui et al., 2012;Sun et al., 2012;Kim et al., 2013a;Saito et al., 2015). Moreover, histone methylation also occurs at arginine residues and histone arginine methylation is involved in many cellular processes including transcription, RNA processing and transport, signaling, subcellular transport and so on (Pahlich et al., 2006). Histone arginine methylation is controlled by a conserved protein family named protein arginine methyltransferases (PRMTs). Plant PRMT genes are involved in the regulations of several essential developmental processes, including vegetative growth, circadian cycle, flowering process, and response to ABA and high salinity (Ahmad and Cao, 2012). On the other hand, histone methylation can be directly erased through the action of histone demethylases (HDMs). So far, two types of HDMs have been identified: lysine-specific demethylase 1 (LSD1) and jumonji C (JmjC) domain containing proteins. LSD1 is an amine oxidase, which removes monoand di-methyl groups from H3K4 residue. Arabidopsis has four genes encoding LSD1 [LDL1, LDL2, LDL3 and FLOWERING LOCUS D (FLD)] which can regulate the flowering time with partial redundancies (Jiang et al., 2007). The other type of HDMs is JmjC-domain protein family (JMJ famlily) which has been assigned to distinct groups including JmjC-domainonly group, JHDM1/FBX/KDM2, JMJD1/JHDM2/KDM3, JMJD2/KDM4, JARID/KDM5, and JMJD3/KDM6. Studies on JMJs in plant have uncovered their important roles in chromatin regulation and plant development, including flowering time, floral organ development, female gametophyte development, BR signaling and circadian regulation (Chen et al., 2011). Histone acetylation and deacetylation underlie a mechanism for reversibly modulating chromatin structure and transcriptional regulation (Tian et al., 2005). The homeostatic balance of histone acetylation is maintained by two types of antagonistic proteins: histone acetylases (HATs) and histone deacetylases (HDACs). So far, plant HATs have been distinctly divided into four groups including: (1) HAG group contains GCN5-, ELP3-, and HAT1-like histone acetylases; (2) HAM group featured by a MOZ-YBF2 (MYST) domain; (3) HAC group is similar to p300/CREB-binding protein (CBP) coactivator family in animals; (4) HAF group is related to mammalian TAF II 250 (TATA binding protein-associated factors; Pandey et al., 2002). Genes encoding HATs have been widely reported in the regulations of developmental transitions, responses to environmental signals and integrations of stress hormone signals (Tian et al., 2005;Sheldon et al., 2006;Chen and Tian, 2007). Plant HDACs have been classified into three families including RPD3/HDA1 superfamily (HDA), Silent Information Regulator 2 (SRT) and HD2 (HDT) families (Hollender and Liu, 2008). Currently, studies have revealed key roles of plant HDACs in regulating plant vegetative and reproductive development, stress responses, gene silencing, as well as cell death and cycle (Ma et al., 2013;Wang et al., 2014). Overall, these gene families involved in histone modifications cooperatively alter the chromatin structures and performances of nucleosomes in order to specifically control gene expression. Moreover, in spite of involvements of these genes in developmental regulations, a growing body of studies has been revealed their crucial roles in abiotic stresses and plant immunity (Berr et al., 2010a;Kim et al., 2010;Luo et al., 2012). Citrus is an important and widely grown fruit crop with richness of nutritional components such as carotenoids and vitamin C. Its fruit development and ripening process shows a single sigmoid curve including two stages of slow growth with a period of rapid growth in between (Bain, 1958). After the fruit ripening, most of citrus fruits have been proceeded to the postharvest storage. Blue mold (Penicillium digitatum) is the most devastating pathogen in citrus fruit post-harvest process and responsible for nearly 90% of production losses during fruit postharvest handling (Macarisin et al., 2007). Although HM genes had been investigated during the fruit development process in tomato (Aiese Cigliano et al., 2013) and grape (Aquea et al., 2011), as well as the plant-pathogen response in Arabidopsis (Alvarez et al., 2010;Berr et al., 2010a), little is known regarding the functions of HMs in citrus. Given the critical roles of plant HMs in regulations of fruit development and pathogen responses, it is expected that they are also involved in citrus fruit development and fruitblue mold infection. In this study, 136 CsHMs, belonging to 11 families were identified in sweet orange. Then, genomic organization, phylogenetic relationship, domain architecture, and gene structure of these genes were comprehensively analyzed. Additionally, expression profiles of CsHMs were analyzed in six stages of fruit development and four periods of blue mold infection. Such a comprehensive analysis of these CsHMs will provide fundamental to understanding their diverse roles in citrus development and be useful for future functional genomic studies on regulations of histone modifications in citrus. Identification of CsHM Families The HMM files containing conserved domain of each HM families (HMTs: SDG-PF00856, PRMT-PF05185; HDMs: HDMA-PF04433, JMJ-PF02373; HATs: HAG-PF00583, HAM-PF01853, HAC-PF08214, HAF-PF09247; HDACs: HDA-PF00850, SRT-PF02146) were downloaded from Pfam protein database 1 . These HMM files were used as a query to search the two sweet orange genome databases 2 [Orange genome Annotation Project (Xu et al., 2013); Sweet Orange Genome Project 2010 3 (Wu et al., 2014)] using HMMER 3.0 software (HMMER 3.0 4 ) with the default parameters. In order to obtain the complete catalog of CsHMs, the output results from two genomes were combined and filtrated the redundant sequences. For CsHDTs, AtHDT1 (At3g44750), AtHDT2 (At5g22650), AtHDT3 (At5g03740), and AtHDT4 (At2g27840) from Arabidopsis thaliana were used to perform a Blastp algorithm from sweet orange genome 2 and two sequences named as CsHDT1, CsHDT2 were obtained. The final ID numbers and DNA sequences of CsHMs are listed in Supplementary Table S1 and Data Sheet 1, respectively. The ID numbers with red fonts were the additional predicted genes from the second sweet orange genome (Wu et al., 2014). Genomic Organization of CsHMs To determine the physical location of HMs, the MapChart software (Voorrips, 2002) was applied to locate the CsHMs on sweet orange chromosomes according to their positions given in the genome database (Xu et al., 2013). Analysis of Domain Compositions and Gene Structures To investigate the domain compositions of CsHMs, the complete amino acid sequences of these genes were subjected to SMART website, including outlier homologs and PFAM domains. The genomic DNA sequences and corresponding CDS sequences of CsHMs were submitted to Gene Structure Display Server (GSDS 5 ) website to visualize the gene structures. Phylogenetic Analysis The HM protein sequences from Arabidopsis, rice and maize were collected from ChromDB database 6 . Each HM family including citrus HMs was aligned with ClustalW program. The generated files were subjected to phylogenic analysis by using MEGA 5.05 program 7 with Neighbor-Joining method. The phylogenic trees were constructed with the following settings: pairwise deletion for sequences analysis, poisson model for substitution, and bootstrap test of 1000 replicates for internal branch reliability. For SDG and HAG families, the conserved domain sequences of SET and AT1 identified in citrus together with the domain sequences from Arabidopsis, rice and maize were used for tree constructions, respectively. Plant Materials and Blue Mold Infection To analyze the expression patterns of HMs during the fruit development, fruit samples were collected from the adult plants of sweet orange (Citrus sinensis [L.] Osbeck), cultivated at the Institute of Citrus Research located in Guilin, Guangxi Province, China. Fruit samples with three independent repeats were collected from different position and orientation of six different trees. The fruit samples were continuously collected from July to December in 2011 as six fruit developmental stages, which were 90, 120, 150, 180, 210, and 240 days after flowering (daf), respectively. The peel and flesh tissues were separated from sampled fruits, and then immediately frozen in liquid nitrogen and kept at −80 • C until further analysis. Sweet orange fruits were used as the materials for the investigation of fruit-blue mold infection. Mature fruits were treated with 2% NaClO for 2 min and washed with distilled water for three times. A uniform lesion (5 mm wide, 3 mm deep) was made at the equator of the fruit using a sterile nail. An aliquot of 20 μL suspension of P. digitatum at 1 × 10 6 spore mL −1 was inoculated into each wound site. After the inoculation, fruits were incubated in a storage chamber with 95% relative humidity at 25 • C temperature for 6, 24, and 48 h to collect the samples. An aliquot of 20 μL double distilled water was inoculated into the fruits as the control (CT). 10 mm of peel around the wound was collected and immediately frozen in liquid nitrogen and kept at −80 • C for RNA extraction. Each of the inoculation experiment was performed with the three replications. Expression Analysis of CsHMs To investigate the expression patterns of all CsHMs in different citrus tissues, the normalized RPKM (reads per kilobase per million mapped reads) values of these genes were extracted from the dataset of the Citrus sinensis Annotation Project (CAP) and visualized by the heat maps with transformed log 10 values using MeV 4.7 software (Saeed et al., 2006). In order to gain an insight of their roles in citrus fruit development, genes whose RPKM values were higher than 5.0 in fruit tissues were selected to further analyze their expression profiles during the six stages of fruit development using real-time PCR. Total RNA was extracted from the peel and flesh samples of citrus fruits according to the previous description (Liu et al., 2006). First strand cDNA was synthesized from 1.5 μg of total RNA using the ReverAid first strand cDNA synthesis KIT (Fermentas). Real-time PCR primer pairs were designed by Primer Express software (Applied Biosystems, Foster City, CA, USA) and their sequences were listed in Supplementary Table S2. The primers were tested to ensure amplification of single discrete bands with no primerdimers. The primers were diluted in Power SYBR R Green PCR Master Mix (Applied Biosystems) and the amplification mixture volume was 10 μL per reaction. Reaction conditions were an initial incubation for 2 min at 50 and 95 • C for 1 min, and then followed by 40 cycles of 95 • C/15 s and 60 • C/1 min. Reactions were run on a 7900 HT Fast Real-Time PCR System with 384-Well Block Module (Applied Biosystems). The β-actin gene was used as an endogenous control and comparative Ct method (2 − Ct ) was adopted to calculate the expression data . The expression levels of 90 daf flesh or peel were used as the calibrator for the relative expression analysis. Expression analysis of all CsHMs response to blue mold infection was performed with real-time PCR. The expression levels of control were used as the calibrator for the analysis. The heat maps and hierarchical clustering of gene expression data were visualized in MeV 4.7 software. Genes with fold change (log 2 value) higher than 1.0 or lower than −1.0 were selected and their expression profiles were shown in Supplementary Figure S2. SPSS software was applied to the statistical analysis of these data in the present study. In addition, the 16 CsHDACs have nine CsHDAs, five CsSRTs, and two CsHDTs ( Table 1). All of gene IDs was listed in Supplementary Table S1. CsHMTs/CsHDMs The chromosomal locations of CsHMs were demonstrated on sweet orange chromosome available at CAP 8 . The members of CsSDG family were widely distributed in eight chromosomes with no distribution in the ninth chromosome (Figure 1). The largest number of CsSDGs was located on chromosome 5 (seven CsSDGs). However, eight genes including CsSDG6,21,22,34,37,38,39, and 40 were not determined because the physical map of sweet orange was incomplete. CsPRMTs were distributed at chromosomes 4, 5, 7, and 9 ( Figure 1). As regard CsHDMAs, CsHDMA2 was located in chromosome, while CsHDMA1 and CsHDMA3 were closely located in chromosome 3, suggesting the occurrence of tandem duplication. CsJMJs were widely distributed at chromosomes 2, 3, 5, 6, 7, and 8 and six of CsJMJs were located in chromosome 5. CsHATs/CsHDACs As shown in Figure 1, lots of CsHAGs displayed the close locations from each other. This might imply that the tandem duplication events occurred in this gene family. The citrus GCN5 (CsHAG25) and ELP3 gene (CsHAG20) were located in chromosome 5, while citrus HAT1 gene (CsHAG38) was in chromosome 4. Members of CsHDAs were distributed at chromosomes 1, 4, 5, 6, 7, and 8. Five CsSRTs named as CsSRT1-5 were located at chromosomes 1, 2, and 4. Two CsHDTs named as CsHDT1 and CsHDT2 were located at chromosomes 7 and 6, respectively. Phylogenetic Analysis, Conserved Domains, and Exon/Intron Organizations of CsHMs To explore the phylogenetic relationships among CsHM proteins and group them within the established classes, the predicted amino acid sequences of each HM family from various species were aligned and phylogenetic trees were further constructed. Furthermore, the gene structures of all CsHMs and the domain compositions of their coding proteins were analyzed. CsHMTs All of the 40 CsSDGs were divided into seven classes according to the classification criteria of SDG family in Arabidopsis (Springer et al., 2003;Figure 2 (Figure 4) were categorized to two classes (Supplementary Figure S1) according to the previous study (Aiese Cigliano et al., 2013). CsPRMT2 to 5 proteins were clustered to class I, and CsPRMT1, 6, 7 proteins belonged to class II. CsHDMs The Phylogenetic tree of HDMAs was clustered to two main clades (Supplementary Figure S1) and all of three CsHDMAs were characterized by conserved N-terminal SWIRM (PF04433) domain and C-terminal Amino_oxidase (PF01593) domain. JMJ family was grouped into five classes based on sequence similarities, including JMJ-only (class I), KDM3 (class II), KDM4 (class III), KDM5 (class IV), and JMJD6 (class V) groups (Figure 3; Lu et al., 2008). JMJ-only class included four citrus members (CsJMJ9 and CsJMJ18-20) which only had JmjC domain and were not clustered to other groups. However, amino acid analysis of Arabidopsis and rice JMJ-only members indicated that they could be active demethylases (Lu et al., 2008). KDM3 class had six citrus members (CsJMJ10-13, CsJMJ16, and CsJMJ17), featured by a JmjC domain at the C-terminal with Ring finger domains (SM000184) ahead of it (Figure 5). The CsJMJs of KDM4 class fell into two main subclasses corresponding to the domain composition. Subclass I was characterized by four tandem repeats of ZnF_C2H2 domain (SM000355), while subclass II contained a zf-C5HC2 domain (PF02928) at the C-terminal. KDM5 group was also divided into two main subclasses which contain one (CsJMJ3) and three citrus members (CsJMJ1, 2, and 8), respectively. Additionally, the class of JMJD6 had two citrus members which had JmjC and N-terminal FBOX domain. CsHDACs As shown in Supplementary Figure S1, CsHDAs were divided into three classes according to the previous study in Arabidopsis, rice and maize (Alinsug et al., 2009). All of them contained conserved Hist_deacetyl domain (PF00850) and an additional STYKc (SM00221) domain was presented in CsHDA4, 5, 6, and 8. Five of CsSRTs are characterized by an SIR2 domain (PF02146) and CsSRT4 and 5 had an additional DUF domain (PF02714) at the C-terminal. In addition, the phylogenetic tree of HDTs showed that CsHDT1 was close to AtHDT1 and AtHDT2, while CsHDT2 was most closely related to AtHDT3 (Supplementary Figure S1). Expression Patterns of CsHMs in Different Tissues The expression patterns of all CsHMs in different tissues (callus, leaf, flower, and fruit) revealed by RNA-seq data of the CAP Figure 7. Hierarchical cluster analysis was performed based on the expression data of each CsHM gene family corresponding to the four different tissues. According to the hierarchical clustering results, we classified genes of each family to different expression pattern groups (I-IV). As shown in Figure 7, the group I genes of CsSDGs showed a low expression level in fruit tissues, while the members of group III were expressed highly in leaf. The members of CsPRMTs presented a high expression level among these four tissues. For CsJMJs, genes in group I were expressed lowly in fruit, while group II genes showed a relative high expression level in callus and fruit. The CsHAGs mainly grouped to four expression patterns and the high expression in callus was prevalent among the group III members. Moreover, the genes belonged to CsHDAs group I expressed highly in fruit and the group II members showed high expression in callus. Above all, the various expression profiles of CsHMs among the four tissues indicated that these genes might take part in different biological processes in sweet orange. Dynamic Expression Patterns of CsHMs in Different Fruit Developmental Stages In order to gain insights into the biological roles of CsHMs in citrus fruit development, genes with RPKM values higher than 5.0 in fruit tissues according to RNA-seq data were selected to further analyze their expression profiles during the six fruit developmental stages using real-time PCR. The real-time PCR primers of each CsHMs were listed in Supplementary Table S2. CsHMTs/CsHDMs As shown in Figure 8, most of selected CsSDGs were expressed highly in flesh of citrus fruit at the mature stage (240 daf). Notably, the increasing expression levels of CsSDG6,7,18,23,and 40 in flesh were strongly correlated with the fruit development process (Figure 8). For CsPRMTs, the mRNA abundance of CsPRMT1, 2, and 4 were expressed highly in flesh at the 240 daf stage. As regard CsJMJs, all members of KDM5 class (CsJMJ1, 2, 3, and 8) showed the increasing expression patterns in flesh along with the fruit development. Additionally, the expression levels of CsJMJ14 also increased during fruit development in flesh. However, the expression profiles of these selected genes showed more complicated in peel during the fruit development. high expression level at the mature stage (240 daf) of fruit. As regard CsHDAs, the expression profiles of CsHDA5, 6, 7, and 8 presented an increasing trend during the fruit development. CsHATs/CsHDACs Notably, the expression level of CsHDA7 showed the strong positive correlation with the citrus fruit development. In peel, the selected CsHATs genes showed a similar expression pattern with high expression levels at 120 daf stage of fruit development. Expression Profiles of CsHMs Response to Blue Mold Infection A growing body of studies had been revealed the crucial roles of HMs in various abiotic stresses and plant immunity. Blue mold is considered as the most devastating pathogen in citrus fruit post-harvest process and causes lots of rotting losses. In order to determine the CsHMs responding to fruit-blue mold infection, the expression profiles of all CsHMs were detected among the four periods of infection using real-time PCR. The results were visualized by the heat maps and hierarchical clustering was further analyzed in each CsHM family (Figure 10). A numbers of CsHMs were up (yellow color) or down (blue color) regulated by the blue mold infection. According to these data, 20 genes including five CsSDGs, one CsHDMA, four CsJMJs, seven CsHAGs, one CsHDA, and two CsSRTs were selected to present their expression patterns in Supplementary Figure S2 for their expression fold change higher than 2.0 compared with the control. The expression levels of CsSDG6, 7, and 11 were inhibited at 6 h after infection (hai) and recovered at 24 and 48 hai. On the contrast, CsSDG37 was up-regulated by the infection. Three CsJMJs (CsJMJ1, 4, and 14) were strongly down-regulated, while CsJMJ11 were induced by the infection. Five CsHAGs including CsHAG2,7,14,15, and 44 were up-regulated, while the expression levels of CsHAG29 and 31 were strongly inhibited at 6 and 24 hai and then recovered at 48 hai. For CsHDAs, CsHDA3 was selected out and showed the increased expression levels under the infection. Additionally, two CsSRTs (CsSRT3 and 4) presented different responses to infection. CsSRT1 was downregulated, while CsSRT4 was highly up-regulated at the 6 h and then slightly induced at the 24 and 48 h by the infection. Discussion Chromatin based gene regulation affects various processes such as root growth, flowering time, floral organogenesis, gametophyte, and embryo development as well as plant response to pathogens and environmental changes (Alvarez et al., 2010;Deal and Henikoff, 2011;Gu et al., 2014;Kim et al., 2015). A series of gene families have been proved to be involved in establishment of histone modifications which can determine chromatin state to regulate biological processes (Berr et al., 2011). Here, genes involved in histone methylation/demethylation and acetylation/deacetylation have been genome-widely characterized in sweet orange based on the sequenced genome (Xu et al., 2013;Wu et al., 2014). Eleven gene families (SDGs, PRMTs; HDMAs, JMJs; HAGs, HAM, HACs, HAFs; HDAs, SRTs, and HDTs) containing 136 CsHMs were identified in sweet orange genome. The gene numbers of these families in sweet orange are close to the Arabidopsis. For example, CsSDGs and CsJMJs have 40 and 20 members respectively (Table 1) and the corresponding AtSDG and AtJMJ families contain 41 and 21 members (ChromDB database). However, much more HAG genes (45 members) were identified in sweet orange genome compared with in Arabidopsis (three members), rice (3) and maize (4; ChromDB database). But if we used the AT1 domain as a query to apply the Blast program, 33 HAG members were obtained in Arabidopsis (Aiese Cigliano et al., 2013), which was close to the number of CsHAGs. Moreover, 26 predicted HAG proteins were identified in tomato genome based on the similar searching methods (Aiese Cigliano et al., 2013). Recently, an increasing number of HMs have been identified and unraveled their pivotal roles in regulations of essential processes (Berr et al., 2011). The involvements of HMs in citrus fruit development have not yet been described. However, numbers of tomato HMs had been characterized their potential roles in tomato ripening process (Aiese Cigliano et al., 2013). A study on grape SDG family has revealed that several VvSDGs were increasing their expression levels during the grape berry development (Aquea et al., 2011). In our study, we also revealed that a number of CsHMs showed the increasing expression patterns during the citrus fruit development (Figures 8 and 9). Regarding histone methylation in plants, SDG family controls the methylation of histone lysine residues, which are involved in various biological processes such as flowering transition, hormone regulation, and carotenoid biosynthesis (Cazzonelli et al., 2009;Sun et al., 2012;Kim et al., 2013a). Generally, histone H3K9 and H3K27 methylation are two repressive marks, whereas H3K4 and H3K36 methylation activates gene expression (Berger, 2007). A number of SDGs has been characterized their catalytic functions in Arabidopsis and rice . AtSDG9, 23, 31, 33 and OsSDG714, belonging to Class V (Figure 2), FIGURE 6 | Domain composition and gene structure of sweet orange CsHAGs, CsHAM, CsHACs, CsHAFs, CsHDAs, CsSRTs, and CsHDTs. Exon/intron structures of these genes are placed on the right side of the domain composition. Exon(s) and intron(s) were represented by green boxes and black lines, respectively. The blue box represented UTR region of gene upstream and/or downstream. are responsible for H3K9 methylation, while AtSDG1, 5, 10 (Class I), 15 and 34 (Class IV) catalyze H3K27 methylation. For the activation marks, AtSDG27, 30 (Class III) and 4 (Class II) act on the H3K4 residues and AtSDG4, 8 and 26 (Class II) catalyze the H3K36 methylation. Although the enzymatic activity and specificity of citrus SDGs are not known, genetic data suggest that they may catalyze the same lysine residues and act the similar repression/activation functions with Arabidopsis. During citrus fruit development, CsSDG7 showed the increasing expression levels and CsSDG13 presented the high expression level at the mature stage (Figure 8). Moreover, CsSDG7 and CsSDG13 are homologous to AtSDG8 and AtSDG27 which catalyze the activation marks H3K36 and H3K4 methylation respectively, indicating that CsSDG7 and CsSDG13 could have similar functions and activate genes expression during the fruit development. Furthermore, AtSDG8 encoding a HMT can affect the carotenoid biosynthesis via regulating the H3K4 tri/dimethylation on CRTISO (CAROTENOID ISOMERASE) which controls the carotenoid isomerization in carotenoid biosynthetic pathway (Cazzonelli et al., 2009). Citrus fruits accumulated nearly 115 kinds of carotenoids and the total carotenoids content increased rapidly during the fruit development (Rouseff et al., 1996;Kato et al., 2004). Previous study revealed that the ' Anliu' sweet orange used in this study showed a rapid increase of total carotenoids in flesh after the green stage (150 daf), which was attributed to the increased accumulation of β-cryptoxanthin and violaxanthin (from an undetectable level to 2.28 μg/g and from 0.99 to 4.63 μg/g, respectively; Liu et al., 2007). In this study, CsSDG7, being homolog to AtSDG8 (Figure 2), showed the increasing expression levels in flesh during the fruit development process (Figure 8). This implied that CsSDG7 could also be involved in citrus fruit carotenoid accumulations during the Figure S3). For histone arginine methylation, atprmt4a and atprmt4b double mutant, atprmt5/skb1, and atprmt10 display the late flowering phenotype by increasing the FLC expression in Arabidopsis, indicating these AtPRMTs are required in the Arabidopsis flowering transition process (Pei et al., 2007;Niu et al., 2008;Schmitz et al., 2008). In our study, CsPRMT1 and CsPRMT2, being homolog to AtPRMT5 and AtPRMT4 respectively, showed the high expression levels in flesh of fruits at 240 daf stage. Based on the similarity and expression profiles, we expected that these two CsPRMTs could also be required in citrus fruit ripening process. Regarding histone demethylases, Arabidopsis JMJ14 catalyzes histone demethylation at H3K4 residues and represses flowering . A recent study has revealed that two novel NAC transcription factors NAC050 and NAC052 interacted with the FYRC domain of AtJMJ14 to regulate gene expression and flowering time (Ning et al., 2015). AtJMJ15 is an H3K4me3 demethylase and AtJMJ15 overexpression resulted in an obvious early flowering phenotype (Yang et al., 2012). CsJMJ1 with a FYRC domain (Figure 5) is clustered with AtJMJ14 and AtJMJ15 (Figure 3), suggesting CsJMJ1 is a predicted H3K4 demethylase with repression of gene expression. Moreover, the expression levels of CsJMJ1 and the other members of KDM5 class (CsJMJ2,3,8) presented an increasing trend during the citrus fruit development (Figure 8) and we expect that these genes could be functional during citrus fruit development. For histone acetylation/deacetylation, one HAF gene (SlHAF1) was identified in tomato genome and it has the strongest expression in tomato fruit at 10 days after breaking, suggesting an important role in tomato maturation (Aiese Cigliano et al., 2013). Similarly, two identified CsHAFs (CsHAF1 and 2) also showed a high expression level in flesh at the mature stage (240 daf) of citrus fruit development (Figure 9), implying that they could have the similar functions with SlHAF1 in tomato. The best studied HDACs in Arabidopsis belonged to RPD3 (Class I), including AtHDA6, 19, 7, 9 and pseudogenes AtHDA10 and 17 (Supplementary Figure S1). AtHDA19 was a member of AP2-TPL-HDA19 repressor complex which negatively regulated multiple floral organ identity genes in Arabidopsis (Krogan et al., 2012). AtHDA9 repressed Arabidopsis flowering by removing H3K9Ac and H3K27Ac on the flowering promoting gene AGAMOUS-LIKE19 (AGL19; Kim et al., 2013b). AtHDA9 and AtHDA6 worked redundantly in the repression of embryonic properties (Tanaka et al., 2008) and AtHDA7 was required for female gametophyte development and embryogenesis . Moreover, a study on tomato revealed five SlHDAs (SlHDA1, 3, 5, 6, and 7) could have the potential roles in fruit development and ripening process by their high expression levels in tomato fruit development (Aiese Cigliano et al., 2013). In this study, CsHDA5 and 6, being homolog to AtHDA9 and 19 respectively, also showed the increasing expression levels in flesh during the citrus fruit development (Figure 9), suggesting that these two genes could also have deacetylation functions with the repression on genes involved in fruit development. A few studies have shown that the HM genes play vital roles in plant immunity (Alvarez et al., 2010). In Arabidopsis, HMT FIGURE 8 | Expression profiles of selected CsHMTs and CsHDMs (RPKM > 5 in fruit tissue based on RNA-seq data) in peel and flesh during six fruit developmental stages (90-240 daf-days after flowering) using real-time PCR. Data were mean ± SD of three separate measurements. Capital letters indicated significant differences at P < 0.01. FIGURE 9 | Expression profiles of selected CsHATs and CsHDACs (RPKM > 5 in fruit tissue based on RNA-seq data) in peel and flesh during six fruit developmental stages (90-240 daf-days after flowering) using real-time PCR. Data were mean ± SD of three separate measurements. Capital letters indicated significant differences at P < 0.01. gene SDG8 was confirmed to be crucial in plant defense against fungal pathogens by regulating genes within JA (jasmonic acid) and/or ethylene signaling pathway (Berr et al., 2010a). ATX1 (AtSDG27), a TRX (TRITHORAX) member involved in H3K4 trimethylation, activated WRKY70 and SA-sensitive genes and reinforced basal resistance to Pseudomonas syringae (Alvarez-Venegas et al., 2007). In our study, CsSDG7 and CsSDG13, being homologous to AtSDG8 and AtSDG27 which catalyzed FIGURE 10 | Expression profiles of all CsHMs in response to blue mold (Penicillium digitatum) infection of citrus fruit at different periods (Control, 6, 24, and 48 h) using real-time PCR. Transcripts were normalized to Actin gene expression and the expression level of control was used as the calibrator for relative expression analysis. Hierarchical clustering of each CsHM family was performed using MeV 4.7 software. Genes with log2 value higher than 1.0 (up-regulated) or lower than −1.0 (down-regulated) were marked with yellow box or blue box, respectively. the H3K36 and H3K4 methylation respectively (both of them activated gene expression), were down-regulated under the blue mold infection (Figure 10). For HDAs, AtHDA6 was involved in regulating tolerance to necrotrophic fungi by repressing the JA and ethylene signaling in Arabidopsis (Zhu et al., 2011). AtHDA19 activated the resistance against Alternaria brassicicola and was also involved in JA and ethylene signaling of pathogen response in Arabidopsis (Zhou et al., 2005;Choi et al., 2012). In citrus, CsHDA4, being homologous to AtHDA6, was upregulated responding to blue mold infection, while CsHDA6, clustering with AtHDA19, was suppressed (Figure 10). Overall, 20 genes including five CsSDGs, one CsHDMA, four CsJMJs, seven CsHAGs, one CsHDA, and two CsSRTs exhibited the strong alterations of their expression levels under the infection (Supplementary Figure S2). The expression change of these genes implied that they could be involved in fruit-blue mold infection process. Conclusion This study provided the first insight into the CsHMs in citrus and their expression patterns during the citrus fruit development as well as response to fruit-blue mold infection. These CsHM genes were further characterized from the perspectives of genomic organization, phylogenetic relationship, domain composition, and gene structure. Additional expression analysis of these genes was measured in six different fruit developmental stages and four periods of blue mold infection. From the results, we obtained a numbers of genes with the increasing expression profiles during the fruit development and 20 strongly blue mold responsive genes. The comprehensive characterizations of CsHMs presented in our study will be useful for future research to unravel the mechanisms of histone modification regulations in citrus. Acknowledgments We thank Dr. Yizhong He, from the Key Laboratory of Horticultural Plant Biology of Ministry of Education, Huazhong Agricultural University, for providing the peel samples of blue mold infection. We thank Prof. Zuoxiong Liu, from the College of Foreign Languages of Huazhong Agricultural University, for reading the manuscript. We also thank Dr. Chengquan Yang, from the Key Laboratory of Horticultural Plant Biology of Ministry of Education, for reading the manuscript. This project was supported by the Ministry of Science and Technology of China (2011CB100600) and the National Natural Science Foundation of China (31330066 and 31221062). Supplementary Material The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2015.00607 TABLE S1 | List of the histone modification genes (CsHMs) in sweet orange.

v3-fos

2019-04-06T13:08:17.418Z

2015-01-01T00:00:00.000Z

97623462

s2

Understanding the destructuration of starch in water–ionic liquid mixtures The destructuration of native maize starch in mixtures of water and ionic liquids (ILs) containing acetate anions was studied in dynamic heating conditions, combining calorimetry, rheology, microscopy and chromatographic techniques. A phase diagram of starch in water – IL solutions was established. The phase transitions undergone by starch include the typical endothermic gelatinization phenomenon for IL – water ratios lower than 0.5, while for mixtures with a higher ionic liquid content, a complex exothermic phenomenon combining mild degradation and solubilization takes place. This results in an optimum destructuration temperature as low as 40 – 50 °C for an IL – water ratio close to 0.7. In addition, speci fi c macromolecular chain breaking reactions appear to take place, depending on the nature of the cations present, resulting in di ff erent macromolecular structures of the recovered starch. These results suggest the possibility of using solvent media design for a controlled modi fi cation of starch macromolecular characteristics. Introduction As renewable resources become essential for industry, the creative design and application of innovative technology for the optimization of such resources is a research topic raising huge interest in the past decade. 1 Among all polysaccharides investigated as potential alternatives to conventional oil based plastics, starch has attracted a large amount of attention. 2 Starch is one of the most abundant biopolymers in nature, and considering its low cost, renewability and biodegradability, it can be considered as a raw material for the fabrication of biologically degradable materials. Starch is composed of two different glucose polymers: amylose, a predominantly linear macromolecule formed from α(1→4) linkages with a molar mass of ∼10 5 -10 6 g mol −1 , and amylopectin, a massive multiply branched polymer containing both α(1→4) and α(1→6) linkages with a molar mass of ∼10 7 -10 9 g mol −1 . Starch is synthesized in the form of densely packed granules, containing both amorphous and crystalline regions. 3 Given its granular structure, starch shows low solubility in any conventional solvent despite being highly hydro-philic. However, when suspended and heated in excess water, starch undergoes an order-disorder transition called gelatinization. During this phenomenon, starch granules swell and amylose progressively leaches out of the granules, and the semi-crystalline structure is disrupted. Although some starch molecules are readily solubilized in water, some granule remnants may still be present even after gelatinization has occurred. Thus, starch insolubility represents a problem when trying to obtain homogeneous amorphous materials. In recent years, the performance of ionic liquids (ILs) as solvents for biopolymers has generated lots of interest. ILs are room-temperature molten salts; since they present high thermal stability and are not volatile, it has been reported that they offer an alternative to common organic solvents. For this reason and because they are easily recyclable, and even some of them are biodegradable, 4 they have been classified as 'green solvents.' For these reasons, they have attracted enormous attention over the past decade, becoming a very important area of research. 5 Over the last few years, the use of ILs to dissolve and process starch has been reported. [6][7][8][9][10] While the first reports focusing on ionic liquids containing chlorine anions showed strong depolymerisation of starch, thus limiting potential applications, 9 more recent works on acetate based ionic liquids are more promising, 8 despite the fact that no clear evaluation of starch degradation in such systems has been communicated by the authors. In the presence of chlorine anions, the macromolecular degradation of starch has been related to the acidic hydrolysis of glycosidic bonds. 6,9,[11][12][13] According to Mateyawa et al. 8 the presence of acetate based ionic liquids is likely useful for avoiding such phenomena. The same authors also reported an exothermic transition when starch was heated in pure and concentrated 1-ethyl-3methylimidazolium acetate (EMIMAc)-water solutions and it was proposed that this exothermic transition was due to starch dissolution, without gelatinization. Stevenson et al. 9 reported no enthalpic transition when analysing recovered starch, previously treated with 1-butyl-3-methylimidazolium chloride, suggesting that after being heated in ILs, the starch is destructured and no further gelatinization can be observed when reheating in water. As recalled by Brennecke et al. 14 one of the major advantages of ILs is that they offer the possibility of being tailored by modifying the chemical structure of the cation and anion moieties. Consequently, in the present work, we focus on the influence of the cation by comparing the thermal destructuration of starch mixed with water and two acetate based ionic liquids: EMIMAc and cholinium acetate. The latter presents the advantage of very low toxicity, choline being an essential nutrient and thus biocompatible. Materials Regular corn starch (Maritena 100) was purchased from Tate & Lyle (Paris, France) with an initial moisture content of 12%. EMIMAc was produced by BASF and supplied by Sigma-Aldrich. Before use, both materials were dried with P 2 O 5 under vacuum at room temperature for one week. After this time, the starch moisture was lower than 3%. Cholinium acetate (CholAc) was synthesized by a metathesis reaction. 15 Equivalent amounts (0.06 mol each) of cholinium chloride and potassium acetate (both purchased from Aldrich) were dissolved in absolute ethanol, and mixed and stirred for 1 hour at room temperature. A white precipitate of potassium was formed and removed by filtration. The ethanol was evaporated on a rotary evaporator. The CholAc thus produced was freeze-dried prior to use. After purification and freeze-drying, the melting point of CholAc was 83°C. For analysis, the starch was suspended in aqueous solutions of varying IL concentrations (from 0% w/w to 100% w/w IL). Since different concentrations of starch were also studied, a phase diagram was prepared. ILs are known to be highly hygroscopic, thus the sample preparation was carried out in a glove box under dry gas purge. Methodology 2.2.1. Micro differential scanning calorimetry (µDSC). Mixtures of 20% w/w starch in different solvents were prepared. The solvent composition varied from 0% IL ( pure water), to 100% EMIMAc or 95% CholAc (due to its high melting point, 5% water was added to CholAc; the melting point of CholAc 95% was 53°C). Appropriate amounts of IL and water were weighed and thoroughly mixed before the addition of the starch. A reference cell was prepared by adding the same water content as in the sample cell. The sample was stirred (50 rpm at room temperature) for 1 h before being heated from 20°C to 120°C and cooled from 120°C to 20°C in the µDSC (µDSC7evo, Setaram, Caluire, France) at a heating/cooling rate of 1°C min −1 . The onset (T o ), peak (T p ) and conclusion temperatures (T c ), and the enthalpy of the transition (ΔH) were determined using Calisto software (Calisto v1.32 DB v1.33). All mixtures were analysed in duplicates. 2.2.2. Macromolecular characterization of samples. High Performance Size Exclusion Chromatography coupled with Multi-Angle Laser Light Scattering (HPSEC-MALLS) was used for characterization of the samples. The starch was suspended in the IL-water solutions and stirred for 1 h. These suspensions were then heated in an oil bath (Ministat 240, Huber, Offenburg, Germany), which was used to mimic the dynamic heating applied by the µDSC: from 20°C to 120°C at 1°C min −1 . After this thermal treatment, the samples were pretreated with DMSO, precipitated with ethanol, dried and solubilized by microwave heating under pressure, as previously described by Rolland-Sabaté et al. 16 Each suspension of the sample in water at a concentration of 0.5 g L −1 was heated for 40 s (maximal internal temperature reached: 152°C) at 900 W. The starch solutions were then filtered through 5 µm Durapore TM membranes (Waters, Bedford, MA, USA). Concentrations of the carbohydrate were determined by the sulphuric acid-orcinol colorimetric method described by Planchot et al. 17 Sample recoveries were calculated from the ratio of the initial mass and the mass after filtration. Solutions were immediately injected into the HPSEC-MALLS-system. The equipment and the method used were the same as that described previously. 18 The SEC column was a Shodex® KW-802.5 (8 mm ID × 30 cm) with a KW-G guard column (6 mm ID × 5 cm) both from Showa Denko K. K. (Tokyo, Japan). They were maintained at 30°C. The two on-line detectors were a Dawn® Heleos® MALLS system fitted with a K5 flow cell and a GaAs laser, (λ = 658 nm), supplied by Wyatt Technology Corporation (Santa Barbara, CA, USA) and a RID-10A refractometer from Shimadzu (Kyoto, Japan). The eluent (Millipore water containing 0.2 g L −1 of sodium azide) was carefully degassed and filtered on-line through Durapore GV (0.1 µm) membranes from Millipore (Millipore, Bedford, MA, USA), and eluted at 0.5 mL min −1 . Sample recovery rates were calculated from the ratio of the mass eluted from the column (integration of the refractometric signal) and the injected mass. These last values were determined using the sulfuric acid-orcinol colorimetric method. 17 M n , M w , the dispersity (M w /M n ), and the radius of gyration, R G (nm), were established using ASTRA® software from WTC (version 6.1 for PC), as previously described by Rolland-Sabaté et al. 16,18 A value of 0.146 mL g −1 was used as the refractive index increment (dn/dc) for glucans and the normalization of photodiodes was achieved using a low molar mass pullulan standard (P20). 2.2.3. Rapid Visco Analyser (RVA). Viscosity properties of the starch in different solutions were studied with a Rapid Visco Analyser (RVA-3, Newport Scientific Pty. Ltd., Australia). Starch suspensions (7.5% w/w) were prepared by weighing the solvent in a canister and adding starch slowly while stirring. The slurry was heated from 20°C to 95°C while being stirred at 960 rpm for the first 10 s and then at 160 rpm until the assay was completed. The heating rate was 10°C min −1 . It was held at 95°C for 10 min, and finally cooled to 20°C at a cooling rate of 6.7°C min −1 . The pasting temperature (T p ), peak viscosity (PV), final viscosity (FV), breakdown (BD) and setback (SB) were obtained from the pasting curve. Samples were assessed in duplicate. 2.2.4. Microscopy. The microstructure of the starch suspended in different IL solutions before and after the enthalpic transitions was analysed with a light microscope, LEICA DMRD. Light, polarized light and differential interference contrast images were obtained. Suspensions of the starch were prepared in the same way as the samples analysed by µDSC. The appropriate amount of IL, water (when required) and starch were weighed. These mixtures were stirred for 1 h and then heated in an oil bath (Ministat 240, Huber, Offenburg, Germany) at 1°C min −1 until the corresponding temperature was reached. Samples were then removed from the oil bath and cooled in an ice bath. The samples were immediately observed under the microscope. 2.2.5. Statistical analysis. The data obtained were statistically treated by variance analysis, while means were compared by the Fisher LSD test at a significance level of 0.05 (INFOSTAT statistical software, Facultad de Ciencias Agropecuarias, Universidad Nacional de Cordoba, Argentina). Differential scanning calorimetry (µDSC) The thermal behaviour of the starch mixed with aqueous IL solutions of different concentrations was monitored by µDSC ( Fig. 1). Increasing concentrations of the ILs were used from the bottom to the top of the figure. When the IL concentration was low, the starch underwent a typical gelatinization, represented by an endothermic transition (a second endothermic transition can be observed at around 100°C, and is ascribed to the melting of amylose-lipid complexes). The fact that the gelatinization shifts to higher temperatures with increasing IL concentration is consistent with the previously described effect due to the presence of different salts in aqueous solution. 19,20 The effect of the salts on starch gelatinization has been found to follow the Hofmeister series, with kosmotropes (structure making, salting-out) delaying gelatinization and chaotropes (structure breaking, salting-in) accelerating it. Acetate is a wellknown kosmotrope, so it was expected to produce a shift of gelatinization toward higher temperatures. Nevertheless, a further increase in IL concentration (up to 50% for EMIMAc and 60% for CholAc) led to a decrease in the gelatinization temperature. This trend will be discussed below (see Macromolecular characteristics). If we now consider high IL concentrations (from the top to the bottom, Fig. 1) an exothermic transition is present. The highest concentration of CholAc studied was 95%, but these results were not included in the figure since the exothermic peak was not complete at 120°C, which is the upper limit for the µDSC; the peak onset temperature is presented in Table 1. This exotherm starts at lower temperatures when water is added and the heat released (ΔH) is also decreased. There is a critical concentration (depending on the IL used) at which both transitions (exo-and endothermic) take place: 70% CholAc and 60% EMIMAc. In the former case, both transitions can be observed, but in the latter both phenomena seem to happen at very close temperatures thus probably cancelling one another. The same behaviour was also observed by Mateyawa et al. 8 working with EMIMAc and by Koganti et al. 21 using N-methyl morpholine N-oxide (NMMO). These authors attributed the exothermic transition to starch dissolution in these solvents. Enthalpy values for the exotherm of normal corn starch in NMMO were 17.5 J g −1 (no enthalpy change was observed when increasing NMMO concentration from 70 to 78%), whereas Mateyawa et al. 8 did not provide any ΔH value. In the present study, ΔH of the exothermic transition ranged between 17.3 J g −1 (70% EMIMAc) and 180.7 J g −1 (100% EMIMAc) ( Table 1). Moreover, when heated at low rates (0.1°C min −1 ), two peaks were clearly observed by µDSC (data presented in the ESI, Fig. S1 †); this finding indicates that more than a single phenomenon would be responsible for the exothermic transition. The same trends were observed at different starchsolvent ratios (data not shown). Macromolecular characterization of treated starches In order to understand the phenomena underlying the exothermic transition, the macromolecular properties of starch treated with ILs were studied using the HPSEC-MALLS system. To this end, the starch was treated with different IL-water solutions, recovered and pre-treated with DMSO. The DMSO pre-treatment recoveries were between 95% and 100% for all samples. DMSO pre-treatment is known to remove the polysaccharide oligomers with degree of polymerization (DP) lower than 12. Thus, this high recovery percentage shows that the heating of samples in different IL solutions does not induce the apparition of sugars with DPs smaller than 12. The solubilisation recovery rates and the elution recoveries of the starches were higher than 90%. The high sample recovery values obtained here indicate that the fractionation response was quantitative for all the samples. Overall, this solubilisation procedure was thus considered to enable the structural characterization of these samples. Fig. 2a presents chromatograms for starch heated in pure water and in EMIMAc solutions. When considering starch heated in pure water, two peaks were observed for the differential refractive index signal (corresponding to the concentration of the chains). The first and bigger one ( peak I, 5.8 mL) corresponds to amylopectin population, while the second, and smaller, to amylose ( peak II, 6.6 mL). When analysing the starch-EMIMAc 100% chromatogram, two peaks are also observed; nevertheless, some important features can be highlighted: (1) the first peak started to elute at higher volumes, indicating a lower size for these molecules as the elution volume is inversely proportional to the molecular size, (2) the second peak is bigger than the first one, and (3) no evident shift in peak II is observed. In addition the molar mass is clearly lower for each fraction of starch-EMIMAc 100% compared to starch-pure water solutions. Overall, these features indicate that amylopectin is depolymerized when heated in EMIMAc, which explains the shift of the amylopectin peak (which accounts for a smaller size), while there is co-elution of the depolymerisation products and amylose, thus explaining the increased area of the second peak. Finally, no evidence of amylose depolymerisation is found (no shift of the peak II). For the samples treated with EMIMAc 70%, amylopectin also eluted at higher volumes, although the overall profile and molar mass distribution are more similar to that of pure water. For EMIMAc 50%, no shift of the amylopectin peak was observed, but the area of peak II is bigger than for starch treated with water, indicating the presence of amylopectin depolymerisation products. Nevertheless, since mild depolymerisation occurs under these conditions ( Table 2) the detector response to amylopectin is still high. For the starch heated in 95% CholAc, only one peak could be clearly detected, while the amylopectin fraction is represented by a shoulder (Fig. 2b) and the molar mass is smaller for each elution volume. This accounts for the depolymerisation of amylopectin by CholAc as well. When water was added to CholAc, a shift of amylopectin elution toward higher volumes is still present, but again the overall behaviour is more similar to that of pure water. Table 2 shows M w and R G values obtained by integrating the signals for the whole population of molecules present in the sample. A progressive and linear reduction in M w is observed when the EMIMAc concentration is increased, with a reduction of 29%, 48% and 80% for EMIMAc 50%, EMIMAc 70% and EMIMAc 100%, respectively, compared to starch heated in pure water. Interestingly, when treated with CholAc, the reduction in M w is non-linear, and reductions are 25%, 24%, 27% and 81% for CholAc 60%, CholAc 70%, CholAc 80% and CholAc 95%, respectively. The same trend is observed for R G . This indicates that both ILs have a different response in the presence of water, with rather small quantities of water (20%) significantly reducing the depolymerisation caused by CholAc. The dispersity (M w /M n ) decrease for the samples treated with ILs (from 7.56 to 7.10 and 4.48 for EMIMAc 100% and CholAc 95%, respectively) is linked to the reduction of the overall peak broadness, and explained by the reduction of the amylopectin molar mass. Although the two acetate based ionic liquids tested do not completely avoid starch depolymerisation, the reductions of the molar masses found in this study are very different to those obtained after treating starches with halide based imidazolium IL, where reductions of 1-3 orders of magnitude can be observed. 6,9,12 From Table 2 it can be observed that a slight depolymerisation takes place when treating starch with EMIMAc 50% and CholAc 60%, even though no exothermic transition was observed by µDSC. This finding may explain why the gelatinization shifts to lower temperatures and ΔH decreases when the starch is heated in µDSC with these IL solutions, since this mild depolymerisation may facilitate starch swelling, shifting gelatinization toward lower temperatures. Moreover, the significantly lower molar masses observed for the amylopectin populations ( peak I, Fig. 2) in EMIMAc 100% and particularly in CholAc 95% treated samples compared to starch-pure water solutions account for a less dense structure (as these fractions exhibit the same size because elution volume is proportional to size in HPSEC). One can deduce that the original amylopectin population is linearized after heating in the ILs, and further in CholAc. To summarize, it is clear that the starch is depolymerized when heated in IL and that the depolymerization pattern varies according to the cation nature, not only to anion characteristics. Though at present it is not possible to propose a mechanistic explanation for this differential behavior, these results suggest the possibility of tailoring the ionic liquid for a controlled modification of the macromolecular characteristics of starch through mild depolymerisation during destructuration. The possible interaction between the starch and the IL during heating that could lead to the formation of new mole-cular species was monitored by FTIR and NMR. No significant changes were found between starch heated in water or the ILs. These results are presented in the ESI (Fig. S2 and S3 †). It is also possible that the mechanism involves not only starch and the IL, but also water molecules. For future studies, a possibly fruitful approach for trying to understand the interactions between these three components and their influence on the destructuration mechanism would be the use of molecular simulation. A recent paper showed the particular interest in this tool for understanding the interactions in the case of cellulose dissolution in IL-water and IL-DMSO mixtures. 22 It would also be interesting to study the destructuration of starch in IL-DMSO mixtures, since these simulations show that the co-solvent nature plays an important role in cellulose dissolution by IL. 22 (Fig. 3a), and differential interference contrast and polarized-light microscopy ( Fig. 3b and c). Fig. 3a presents images of the samples before the enthalpic transition (endothermic or exothermic depending on the water content) for the starch suspensions heated in EMIMAc solutions where a well-defined granular structure can be observed (similar images were obtained for CholAc, data not shown). Images of the starch suspensions after the enthalpic transition when heated in EMIMAc and CholAc solutions are presented in Fig. 3(b and c). From these images, it can be observed that, when heated in pure water, starch granules are gelatinized: swelled and deformed with no remaining crystallinity. However, when EMIMAc is added (30 and 50%), gelatinization is not complete, since some granules are still birefringent as a result of their crystallinity. When the amount of EMIMAc reached 70%, however, no polarization was evident after heating, nor the presence of granular remnants; the same was observed for EMIMAc 100%. The absence of granular remnants may indicate that starch is not only depolymerized in concentrated EMIMAc solutions, but is also solubilized. Both phenomena, leading to an overall starch destructuration, may account for the exothermic transitions observed by µDSC. Fig. 3c shows the same behaviour when CholAc was used, but 80% of CholAc was necessary to achieve complete destructuration, since at a lower concentration (70%) granular remnants were present. When EMIMAc 100% and CholAc 95% were used, a few gas bubbles were observed under the microscope. These gas bubbles may indicate the formation of volatile products, but could not be identified. These results are supported by images obtained with an Environmental Scanning Electron Microscope (ESEM) for starch heated in pure water, pure EMIMAc and CholAc 95%. In these images, the destructuration/solubilisation process is evidenced (Fig. S4 †). Rapid Visco Analyser (RVA) RVA is an empirical study commonly performed on starch slurries to follow the viscosity behaviour as the sample is heated. While heated, the starch granules start to retain solvent and swell, which results in a concomitant increase in viscosity, i.e. viscosity onset temperature. The viscosity of the suspension increases to the point where the number of swollen intact starch granules is maximal. The peak viscosity (PV) is indicative of the solvent-binding capacity. When the temperature increases and the granule absorbs as much solvent as to achieve its rupture point, the viscosity decreases to a minimum. This decrease in viscosity is called breakdown (BD). When the starch suspension cools, the amylose retrogrades (re-crystallizes), resulting in an increase in viscosity named setback (SB), until a gel is formed at the end of the test. In this study, the viscosity onset temperature correlated with the µDSC onset temperature, although the former was higher than the latter (Tables 1 and 3). It has been established that the viscosity onset temperature is higher than the gelatinization onset temperature, 23 since different techniques detect starch transitions in different ways, giving slight differences in the determined parameters. Fig. 4 presents the viscosity profiles of starch in EMIMAc (Fig. 4a) and CholAc (Fig. 4b) solutions. The ILs alone were also analysed: their viscosity was near zero and no change in viscosity was observed during the heating and cooling pro- cesses. From Fig. 1 it can be seen that the starch heated in concentrated EMIMAc solutions (100% and 70%) undergoes an exothermic transition, related to starch depolymerisation/solubilisation; when the amount of EMIMAc is 50% or lower, an endothermic transitionascribed to gelatinizationtakes place. Fig. 4a shows that when EMIMAc 100% is used, the viscosity increases as depolymerisation/solubilisation take place. During the cooling stage, the viscosity increases significantly, probably as a consequence of the interaction between the products of depolymerisation, which are smaller and less branched than amylopectin, favouring their association, and also as a consequence of amylose retrogradation. As water is added (EMIMAc 70%), the viscosity onset temperature and peak viscosity are lower (in agreement with µDSC results, Table 1). The decrease in the viscosity onset temperature is related to the lower viscosity of the solvent when compared to pure EMIMAc, and its diffusion into the granule would be faster facilitating depolymerisation. However, starch depolymerisation is lower in EMIMAc 70%, explaining the lower viscosity value during heating when compared to pure EMIMAc (Table 3). On increasing the water content to 50% (EMIMAc 50%), solvent diffusion into the granules increases and the amount of water is enough to gelatinize the starch. In addition, a slight depolymerisation is also observed with EMIMAc 50% (Table 2). Both phenomena may explain the higher viscosity shown by this sample (Table 3). When EMIMAc 30% is used, the pasting behaviour is closer to that of starch in pure water, although the overall viscosity is higher. These results are in good agreement with Mateyawa et al. 8 Fig. 4b shows that the starch in CholAc solutions has a different behaviour than in EMIMAc solutions. When heated in the concentrated CholAc solution (CholAc 95%), two peaks are present: at the beginning of the test, CholAc is in the solid stateexplaining the high viscosity of the sample at this point but as the temperature increases, it melts; a second increase in the viscosity is observed during the cooling period. The exothermic peak (observed by µDSC) for this sample starts at around 97°C (Table 1); this may explain the absence of a viscosity peak during heating. However, some depolymerisation may have occurred during the heating at 95°C, forming smaller and more linear molecules from amylopectin, explaining the slight increase in viscosity during cooling. CholAc 70% could not be analysed since the viscosity exceeded the RVA limit (10 000 cP). For CholAc 50% and 30%, an increase in the viscosity was observed between 85 and 90°C (Table 3), and no viscosity breakdown was found. The viscosity increase started late during heating, while the maximum temperature reached by the RVA is 95°C. This temperature may not be sufficient to completely disrupt the starch granular structure, although granules may swell and some amylose may leach out, resulting in a viscosity increase. Phase diagram In summary, Fig. 5 shows the corn starch phase diagrams when treated in different IL solutions. When the IL concentration is high, a complete loss of granular structure is observed (results are supported by microscopy images) and this destructuration is accompanied by starch depolymerisation (HPSEC-MALLS results); the degree of depolymerisation depends on the water amount. When the water content is sufficiently high, gelatinization occurs, instead of destructuration/solubilisation. Under these conditions, granular remnants are still observed after heating. When EMIMAc 60% is used, a partial gelatinization followed by partial destructuration/solubilisation takes place, and the same is true for CholAc 70%. Conclusions The results presented in this study show that two different phenomena take place when starch is heated in EMIMAc and CholAc solutions: when the concentration of both ILs is low enough, gelatinization is the dominant phenomenon, whereas when the concentration is higher, depolymerisation and dissolution of the starch take place. The effect of both ILs on gelatinization corresponds to that of stabilizing salts. EMIMAc and CholAc have been shown to be appropriate solvents for starch destructuration when mixed with the correct amount of water (30% water for EMIMAc and 20% water for CholAc). At these concentrations, destructuration (depolymerisation and dissolution) starts at temperatures as low as 36°C and 68°C, respectively and, after heating at 120°C, the average molar mass of starch is reduced by 27% and 48% when heated in CholAc 80% and EMIMAc 70%, respectively. This suggests that specific starch chain breaking reactions may occur depending on the cation present in the IL, which could open the possibility of solvent media design for a controlled modification of the macromolecular characteristics of the starch.

v3-fos

2018-04-03T03:46:03.094Z

2015-04-10T00:00:00.000Z

18004582

s2

Maternal fucosyltransferase 2 status affects the gut bifidobacterial communities of breastfed infants Background Individuals with inactive alleles of the fucosyltransferase 2 gene (FUT2; termed the ‘secretor’ gene) are common in many populations. Some members of the genus Bifidobacterium, common infant gut commensals, are known to consume 2′-fucosylated glycans found in the breast milk of secretor mothers. We investigated the effects of maternal secretor status on the developing infant microbiota with a special emphasis on bifidobacterial species abundance. Results On average, bifidobacteria were established earlier and more often in infants fed by secretor mothers than in infants fed by non-secretor mothers. In secretor-fed infants, the relative abundance of the Bifidobacterium longum group was most strongly correlated with high percentages of the order Bifidobacteriales. Conversely, in non-secretor-fed infants, Bifidobacterium breve was positively correlated with Bifidobacteriales, while the B. longum group was negatively correlated. A higher percentage of bifidobacteria isolated from secretor-fed infants consumed 2′-fucosyllactose. Infant feces with high levels of bifidobacteria had lower milk oligosaccharide levels in the feces and higher amounts of lactate. Furthermore, feces containing different bifidobacterial species possessed differing amounts of oligosaccharides, suggesting differential consumption in situ. Conclusions Infants fed by non-secretor mothers are delayed in the establishment of a bifidobacteria-laden microbiota. This delay may be due to difficulties in the infant acquiring a species of bifidobacteria able to consume the specific milk oligosaccharides delivered by the mother. This work provides mechanistic insight into how milk glycans enrich specific beneficial bacterial populations in infants and reveals clues for enhancing enrichment of bifidobacterial populations in at risk populations - such as premature infants. Electronic supplementary material The online version of this article (doi:10.1186/s40168-015-0071-z) contains supplementary material, which is available to authorized users. Background The establishment of the intestinal microbiota after birth is an important event in the life of a newborn [1]. Bifidobacterium species are among the early colonizers of breastfed infants [2], with evidence that they are uniquely beneficial to the newborn infant in various ways [3][4][5][6][7]. Non-digestible sugars in breast milk known as human milk oligosaccharides (HMOs) are protective to infants [8] and function as a prebiotic in the establishment of bifidobacteria. Select members of the genus Bifidobacterium commonly found in breastfed infants are able to utilize HMOs as carbon sources, including fucosylated oligosaccharides [9][10][11][12][13]. The relationship between mothers, infants, and bifidobacterial species appears to have coevolved over mammalian history [14], perhaps to aid the infant in avoiding infection. Bifidobacteria have also been shown to reduce inflammation and gut permeability [7,[15][16][17]. A recent analysis of breastfed infants in Bangladesh revealed that higher bifidobacterial populations correlate with improved responses to both oral and parenteral vaccines early in infancy [5]. Bifidobacteria are not alone in their ability to consume HMOs, as members of the genus Bacteroides are known to consume some types of HMOs [18]. These two groups are both involved in the production of short-chain fatty acids and lactate, which alter the pH of the environment, modulate the microbiota, and have other systemic properties [19]. HMOs can be bound to other compounds in milk as glycoconjugates, which may play a similar role to free HMOs [20]. Together, free HMOs and their related glycoconjugates have been referred to as human milk glycans (HMGs) [6]. Among the genes that build HMGs in the mammary gland is the fucosyltransferase 2 (FUT2) gene, which catalyzes the transfer of fucose residues by an α1,2-linkage to glycans found in human milk. Known as the 'secretor' gene because of its role in the expression of ABO blood types in various secreted body fluids (tears, saliva, breast milk, and so on), this gene has well-known mutations that inactivate transferase activity which occur in most populations across the world, including in about 20% of the population of the United States [21]. FUT2 seems to be under balancing selection [22,23], as there are both advantages and disadvantages to possessing an active copy of the gene. For example, non-secretors are resistant to rotavirus [24], norovirus [25], and Helicobacter pylori [26] infections, while secretors have lower risk in developing type 1 diabetes [27] and Crohn's disease [28]. Breastfeeding mothers who are secretors also confer resistance to diarrheal disease on their children [29]. Morrow et al. found that there were differences in survival between premature infants of differing secretor statuses [30], although how much the mother's genotype plays into this outcome is unknown. The amount of fucosylation in breast milk is also known to change over the course of lactation [31], which may affect the protection conferred to an infant over time. There are phenotypic differences in the milk glycans from secretors and non-secretors [32] and in their ability to deflect pathogen binding to the epithelium [33]. HMOs containing α1,2-fucosyl linkages have been shown to promote the growth of bifidobacteria due to prebiotic action [34]. Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum possess glycosyl hydrolase family 95 (GH95) fucosidases that act on 2′-fucosylated HMOs [12,35]. In other bifidobacteria such as Bifidobacterium breve, GH29 fucosidases enable the consumption of 2′-fucosylated HMOs [36]. Regardless, there is little known about how actual breast milk from mothers of different secretor statuses affects the resulting gut community of a breastfed infant. This study examines the differences in infant gut microbial populations that arise from these compositional differences in HMGs. Maternal secretor status A subset of 44 infant/mother dyads from the existing UC Davis Foods For Health Institute Lactation Study were selected for an analysis of the effects of a mother's secretor status on her infant's gut microbiota over four time points ranging from 7 to 120 days of life. To determine each mother's secretor status, several specific 2′-fucosylated HMO 'markers' were quantitated in the earliest milk available from each mother (Additional file 1: Table S1). Of the 107 milk samples, 35 were found to be from 12 non-secretor donors (33%) and 72 were from 32 secretor donors (67%). Aside from the levels of secretor status marker oligosaccharides, milk determined to be from non-secretor mothers showed other significant differences in glycan composition when compared to secretor milk over all four time points (Table 1). Although the total oligosaccharide abundance and the relative amount of total (including 2′ and 3′) fucosylation were comparable among averaged data from the two phenotypes, the relative abundances of non-fucosylated neutral and sialylated structures differed. In non-secretors, the average amount of sialylation was 23.4% ± 5.7%, which is significantly higher than the amounts of sialylation found in secretors, which averaged 18.2% ± 4.8% (p < 0.0001). Conversely, non-secretor milk had lower relative amounts of non-fucosylated neutral structures than secretor milk, with 21.3% ± 8.8% and 25.4% ± 7.3%, respectively (p = 0.023). To rule out some potential external factors that could confound a microbiota comparison between the two secretor phenotypes, maternal and infant demographics and clinical characteristics were compared between the two groups. No obvious differences between secretor and non-secretor groups were found (Tables 2 and 3). All infants consumed breast milk throughout the study duration; however, based on parental reports, some infants occasionally consumed limited amounts of supplemental infant formula and/or solid foods (see Additional file 2: Table S2 for details). Additional file 2: Table S2 also indicates from which infants samples were available for each of the four time points. To validate the phenotypic designation (secretor or non-secretor) assigned to each mother, genotypic information about secretor status was also generated for each mother. Mothers determined to be homozygote nonsecretors by genotype were, in all cases, also determined to be non-secretors in phenotype as described above. In two cases (mothers 1036 and 1041), the genotypic data showed either homozygote secretor or heterozygote (respectively), but the phenotype indicated non-secretor. In all other cases, the homozygotic and heterozygotic secretors were determined to have a secretor phenotype. In the two aberrant cases, later time points revealed secretor levels of 2′-fucosylation in these two mothers, suggesting that secretor phenotype might change over the course of lactation in some mothers. Fecal bifidobacterial levels To investigate if the grouping by mother's secretor status produced differences in the gut microbiota of the infant, we used 16S rRNA gene amplicon sequencing to probe the fecal microbiota of the infants. In general, the most common bacterial groups found in all the infants were Bifidobacteriales, Lactobacillales (mostly Streptococcus), Bacteroides, Enterobacteriaceae, and Clostridiaceae. As shown in Figure 1, there is a general trend towards increasing amounts of Bacteroidales and Bifidobacteriales and decreasing amounts of Lactobacillales (mostly Streptococcus) and Enterobacteriales over time. Differences were seen between the five C-section-born infants and the 39 vaginally born infants, with C-section infants having much lower levels of bifidobacteria and Bacteroides, although the number of C-section infants was too low to draw any robust conclusions (Additional file 3: Figure S1). When grouped by the mothers' secretor status, differences were seen in the aggregated infants' gut microbiota ( Figure 2). Specifically, secretor-fed infants generally had higher relative amounts of Bifidobacterium (p < 0.05) and Bacteroides and lower levels of enterobacteria, clostridia, and streptococci (p < 0.05). To verify the higher levels of bifidobacteria found in secretor-fed infants, we probed the samples with bifidobacterial-specific quantitative PCR (qPCR). The aggregated secretor-fed infant samples were, on average, found to have significantly higher absolute levels of bifidobacteria (10 9.0 /g feces versus 10 7.7 /g feces, p < 0.001) (Figure 3). The distribution of the qPCR data was bimodal, with a large group of samples with low levels of bifidobacteria (<10 7.6 /g feces, average 10 6.1 ) and a separate group with high amounts of bifidobacteria (>10 8.9 /g feces, average 10 10.3 ), with a striking lack of values falling in between the two ranges ( Figure 4). These two groupings of samples were labeled 'Low-Bif qPCR ' and 'High-Bif qPCR ' samples, respectively. Values are frequencies per total sample size, n = 44. a Genotyping data missing for one subject phenotypically described as a secretor. Much of the bifidobacterial abundance difference between the two secretor status milk phenotypes appears to come from variation in the time at which each infant transitions from possessing a Low-Bif qPCR ' gut community to possessing a High-Bif qPCR gut community. Bifidobacteria were found to be established (High-Bif qPCR ) earlier in secretorfed infants (60% of infants versus 37.5% at day 6) and more often (80% versus 50% by day 120) ( Figure 5). To examine the dependence of bifidobacterial abundance in stool on secretor status phenotype in milk, a contingency analysis was performed on the 105 available matching milk and stool pairs ( Figure 6). Of 35 milks from non-secretors, 20 of the matched infant stool samples were Low-Bif qPCR (57.1%). Of the 70 milks from secretor women, 23 of the matched infant stool samples were Low-Bif qPCR (32.9%). A Pearson chi-square test was significant (p = 0.0171), indicating that mother's secretor status and infant bifidobacteria levels are dependent variables. A Fisher's exact test yielded p = 0.015, suggesting the probability that bifidobacteria levels will be High-Bif qPCR is greater for infants who are receiving milk from secretor mothers. We then tested whether differences in the types and amounts of oligosaccharides present in the milk other than 2′-fucosylation leads to differences in the amount of bacteria present. Figure 7 shows that stool samples with Low-Bif qPCR counts (N = 43) and those with High-Bif qPCR counts (N = 62) were from infants that received matching milk samples of mostly comparable glycan composition. On average the milk received by Low-Bif qPCR infants was significantly higher in α(1-3/4)-fucosylated oligosaccharides than milk received by the High-Bif qPCR infants (14.2% ± 9.2% versus 10.1 ± 7.9%, respectively; p = 0.021), but this may be mostly due to the trade-off between 2′-and 3′fucosylation related to secretor status, which was shown above to correlate with an increase in bifidobacterial Figure 2 Comparison of relative levels of gut microbiota in secretor-fed infants and non-secretor-fed infants. Asterisks indicate significant differences (p < 0.05) in the relative levels of various gut microbes using a Wilcoxon rank sum test. The color boxplots show the quartiles above and below the median; the dark line near the center of the box denotes the median. The whiskers extend to the first and fourth quartiles, and the black dots show outliers. N = non-secretor, S = secretor. Fecal glycoprofiles To test whether bifidobacteria in general were a driver of oligosaccharide consumption in the infant gut, we measured types and amounts of oligosaccharides in the feces as a proxy for (lack of ) consumption. Of 107 stool samples, glycans were detected in 103, of which bifidobacterial abundance was also measured in 102 samples. Figure 8 shows a breakdown of how fecal glycoprofiles differed between samples that were High-Bif qPCR and Low-Bif qPCR . The absolute abundance of fecal glycans (residual milk glycans present in infant stool) were significantly higher in Low-Bif qPCR stool samples (N = 42) than in High-Bif qPCR (N = 60). Significantly higher amounts of non-fucosylated neutral (p < 0.0001), fucosylated (p = 0.009), and sialylated species (p = 0.045) were left behind in the Low-Bif qPCR fecal samples, as determined in ion counts per 100 μg of stool. The two groups showed no significant differences in glycan composition in terms Figure 6 Contingency plot of secretor status by bifidobacterial content. Pearson chi-square test was significant (p = 0.0171), indicating that mother's secretor status and infant bifidobacteria levels are dependent variables. A Fisher's exact test yielded p = 0.015. of relative abundance (percent of total HMO signal) between the three glycan types. Fecal bifidobacterial isolates Noting the previous differences in fecal glycoprofiles, we hypothesized that secretor mothers would enrich their infants in bifidobacteria that are able to consume 2′-fucosylated HMOs. To test whether there were functional differences in the ability of bifidobacteria from infants with mothers of differing secretor statuses to consume a secretor-type oligosaccharide (2′-fucosyllactose), we obtained over 400 isolates from the 107 infant fecal samples in this study for the purpose of growing them in vitro with only 2′-fucosyllactose as a carbon source. Of these isolates, 382 were identified by the matrix-assisted laser desorption ionization (MALDI) Biotyper (Bruker, Fremont, CA, USA) as belonging to the genus Bifidobacterium. Bifidobacteria were successfully isolated from 73 of the 107 samples. Figure 9 shows a breakdown of these isolates by species and mother's secretor type over each of the four time points. The most commonly isolated species were B. breve and members of the B. longum group. As MALDI may not reliably distinguish between members of this group, we do not include subspecies designations in the description of the isolates. Notably, the B. longum group increased in proportional representation over time in the secretor-fed, but not in the non-secretor-fed infants. This data may however be skewed by isolation bias. Of the 382 bifidobacteria isolates, 97 were chosen as a subset of 'unique' isolates to study further. The 'unique' subset included only one isolate of each bifidobacterial species obtained from each sample, selected at random from among isolates of the same species. Multiple isolates of the same species from the same fecal sample are likely from a clonal population in the infant gut, thus our designation of these isolates as the 'unique' subset [37]. This subsetting was necessary due to limitations in the amount of 2′-fucosyllactose growth substrate available. Each unique isolate was grown on 2′-fucosyllactose (2FL) to test its ability to consume this prototypical secretor-type sugar. Using the cutoff maximum optical densities (ODs) shown in the example growth curves in Additional file 4: Figure S2, we classified each isolate as a high, medium, or low grower. Notably, a higher percentage of isolates from secretor-fed infants grew to medium and high ODs than from non-secretor-fed infants ( Figure 10). In addition, of the two isolates obtained from non-secretor-fed infants that could grow on 2FL, one was a Bifidobacterium dentium strain, a species adapted to oral niches [38,39]. Fecal bifidobacterial (sub)species profiles As different species and subspecies of bifidobacteria have differences in their glycan consumption capabilities, we investigated whether secretor-and non-secretor-fed infants differed in their bifidobacterial content at a taxonomic resolution higher than achievable by our marker gene sequencing method (with current read lengths). At first glance, it appeared as if there were no species-level differences in the bifidobacterial populations of secretorfed and non-secretor-fed infants ( Figure 11). However, a difference was noted between secretor-fed infants and non-secretor-fed infants in which bifidobacterial species were present in high relative abundances when compared to the microbiota as a whole. Specifically, there was a difference in the correlation of the relative abundance of various bifidobacterial species (from the bifidobacteriaterminal restriction fragment length polymorphism (Bif-TRFLP) data) with absolute (qPCR) and relative (amplicon sequencing) bifidobacterial abundance ( Figure 12, top and bottom). Both the B. longum group and B. breve are positively correlated with bifidobacterial abundance in secretorfed infants, while only B. breve holds that distinction in non-secretor-fed infants (B. bifidum is shown as correlated, but is only present in one non-secretor-fed sample for which marker gene sequencing data is present, and at a level of only 3% of total bifidobacteria). Interestingly, the B. longum group is strongly anti-correlated with bifidobacterial abundance in non-secretor-fed infants. B. longum subsp. infantis was not found in a single high-bifidobacteria sample from non-secretor-fed infants (data not shown). Phrased differently, distinct sets of bifidobacterial species seem to be able to dominate the community in infants fed by mothers of different secretor statuses. To test whether the presence of different bifidobacterial species led to differences in oligosaccharide content of the feces (and thus putative oligosaccharide consumption), we compared the fecal glycome between samples possessing different dominant species of bifidobacteria ( Figure 13). Of the 60 stool samples with high-bifidobacterial abundances, the species identified were longum subsp. infantis and B. longum subsp. longum were of roughly equal abundance and, thus, were designated more generally as 'B. longum group'. Species groups with N < 4 were omitted from statistical analysis. As expected, glycan presence was not significantly different between samples with different major bifidobacterial species in Low-Bif qPCR stool, as bifidobacteria were not likely the major glycan consumer in samples where they were not abundant (data not shown). In High-Bif qPCR stool, there were significant differences in the percentage of fucosylated species (ANOVA p = 0.0001) and the percentage of non-fucosylated neutral species (ANOVA p < 0.0001). By a pairwise comparison, samples high in B. longum subsp. infantis were shown to have a higher percentage of nonfucosylated neutral oligosaccharides left behind in the feces. Of the oligosaccharides not ostensibly consumed by B. longum subsp. infantis, 52.3% ± 5.8% were nonfucosylated neutral species on average. The relative abundance of non-fucosylated neutral species was considerably lower in samples high in all the other oligosaccharide types, all with averages ≤30%. The percentage of fucosylated oligosaccharides among the residual fecal glycans was highest in infants with B. longum subsp. longum as their dominant species of bifidobacteria, with an average of 77.3% ± 3.7%. This value was significantly higher than the fucosylated oligosaccharide percentages leftover in samples dominated by B. breve (57.3% ± 3.7%, p = 0.003) and B. longum subsp. infantis (42.7% ± 6.3%, p = 0.0001). Relative Abundance (% of total HMO Signal) Figure 8 Differences in fecal HMOs between samples that were either High-Bif qPCR or Low-Bif qPCR . The p values are from a twotailed unpaired t-test. * = Significant at 95% confidence level. HMO = human milk oligosaccharide. Broader infant bacterial communities As we had observed the impact of mother's secretor status on the amount and type of bifidobacteria in the infant's feces, we wished to investigate the impact of secretor status on the rest of the infant's gut community structures. We first classified infant gut communities in a less-supervised manner, which would independently capture important differences in community structures while requiring fewer a priori choices on our part. Using the QIIME 1.8.0 implementation of the BIO-ENV function of the 'vegan' R package ('BEST'), a subset of the most abundant bacterial species were tested for their impact on community UniFrac distances [40]. Results indicated that genera Bifidobacterium and Bacteroides were the top two contributors to differences among the given inputs (rho statistic = 0.586 when only those two factors are considered, see Additional file 5: Table S3). A principle coordinate analysis of the marker gene sequencing results revealed three main clusters of samples, (Additional file 6: Figure S3, middle plot) with one outlier. The three clusters were respectively distinguished by high bifidobacterial content (BI), high Bacteroides (BA), and high levels of a number of other taxa including streptococci, enterobacteria, and clostridial species (OT) (see Additional file 6: Figures S3, Additional file 7: Figure S4, Additional file 8: Figure S5, Additional file 9: Figure S6). The one outlier sample that fell into its own category (high enterococci) was from a C-section infant (Additional file 6: Figure S3). Infants often moved between groups over time, and by day 120, few infants remained in the OT group (Additional file 6: Figure S3). Notably, a higher abundance of non-secretor-fed infants fell into the OT area of the plot (Additional file 6: Figure S3). The infant stools were thus divided into groups of Bacteroides (BA PCoA , N = 24), bifidobacteria (BI PCoA , N = 38), or OT PCoA (N = 39) dominated samples, based on the PCoA groupings described previously. ANOSIM was used to test the The OD achieved by each strain during growth on 2′-fucosyllactose (2FL) was compared with the OD obtained in the absence of sugar source as a negative control and lactose as a positive control. This difference in OD (ΔOD) was used as a parameter to evaluate the strain's ability to grow on the 2FL. Table S4). To investigate the impacts of having a microbiota dominated by taxa other than bifidobacterial species, we compared fecal glycomes across these three groups using ANOVA (Figure 14). There were significant differences in total oligosaccharide abundance (p = 0.0003) among the three groups, as well as differences in the absolute and relative abundances of fucosylated (p = 0.033 absolute, p = 0.027 relative) and nonfucosylated neutral (p < 0.0001 absolute, p = 0.010 relative) oligosaccharide types. The OT PCoA group differed the most from the BI PCoA and BA PCoA groups in terms of absolute abundance of non-fucosylated neutral oligosaccharides (Tukey-Kramer honestly significant difference (HSD) p < 0.0001 and p = 0.0002, respectively), having significantly higher oligosaccharide amounts in the stool, with an average of 1.51e08 ± 1.45e07 counts in the OT PCoA group, versus 5.20e07 ± 1.47e07 counts and 5.12e07 ± 1.90e07 counts in the BI PCoA and BA PCoA groups, respectively. The relative abundance of nonfucosylated neutral species differed most in the BA PCoA group (accounting for 16.0% ± 3.9% of the total), being significantly lower than those in the BI PCoA group (28.5% ± 3.0%, p = 0.035) and OT PCoA group (30.8% ± 2.9%, p = 0.009). Additionally, the BA PCoA group also had a higher percentage of fucosylated oligosaccharides than the other two groups, with 71.3% ± 4.2% fucosylation, versus 57.9% ± 3.3% in the BI PCoA group (p = 0.035) and 58.44% ± 3.2% in the OT PCoA group (p = 0.044). The absolute abundance of fucosylation in the OT PCoA group (2.57e08 ± 2.52e07 ion counts) was significantly higher than that of the BI PCoA group (1.64e08 ± 2.56e07 ion counts, p = 0.029). Absolute and relative amounts of sialylation were similar across all three groups. Fecal lactate Bifidobacteria produce two main metabolic end products: lactate and acetate [41]. As a measure of metabolic output from oligosaccharide consumption, we investigated whether the concentration of lactate varied with microbiota composition. Lactate was chosen due to its lower volatility than acetate. Figure 15 shows a summary of the differences in fecal lactate that correlate with microbiota differences. The results are from a subset of the samples, as only 87 samples had sufficient sample quantity for the analysis (updated group sizes are included with the tabulated result). Lactate concentration did not appear to be normally distributed nor did the Bartlett test indicate that most groupings were homoscedastic. Accordingly, log transformations were performed prior to statistical analysis, and the results are tabulated as the median in parts per thousand (ppt) by mass along with the interquartile range. When comparing groups based off of the bifidobacterialspecific qPCR data (high/low), the High-Bif qPCR group was found to have higher levels of lactate (p = 0.0006). In the groups based off of PCoA clustering (BA PCoA , BI PCoA , OT PCoA ), lactate was found to have significant differences by ANOVA. Pairwise comparison of the lactate results found no significant differences between BI PCoA and BA P-CoA groups, but found that the OT PCoA group was lower than both the BI PCoA group and the BA PCoA group with p < 0.0001 in each case. Discussion While breastfeeding is overwhelmingly recommended as the best source of nutrition for a newborn, it is clear that not all breast milk is the same. Maternal diet, gestational age of the infant, and lactation stages have been known to influence the lipid and protein content of milk [42,43]. Conversely, term milk glycan compositions appear relatively stable throughout lactation, perhaps highlighting their importance to the developing infant [44]. Decreasing milk glycan concentrations over the course of lactation have been observed, for which the increased volume consumed by infants at later stages of nursing may compensate [45]. Milk glycan composition from mothers who deliver prematurely was recently shown to be more variable than mothers delivering term infants [46]. Milk composition is also affected by the mother's glycosyltransferase genotype [47]. In this study, the milk of secretors and non-secretors had modest differences besides the amount of 2′-fucosylation. Secretors had higher absolute amounts of sialylated sugars and higher relative amounts of undecorated sugars. Nonsecretors had higher relative amounts of sialylated sugars. These factors may also play a part in the shaping of the secretor-fed infants' microbiota. The potentially confounding factor of the infant's own glycosylation system, the timeline and levels of its expression, and its potential influences on the microbiota were not explored in this study and remain to be elucidated. It is well established that differences in gut glycan content affect the gut microbiota. Secretor status has been shown to affect both the gut microbiota and metabolite profiles in adults [48,49]. Secretor status also specifically affects the species composition and absolute abundance of gut bifidobacterial populations in adults, with secretors having higher bifidobacterial abundance [28,50]. Numerous studies have also associated intake of milk glycans with the initial colonization of the infant gastrointestinal tract [34,51,52]. Our data provide insight into what types of milk inputs are most likely to lead to a high-bifidobacteria microbiota in the context of our cohort (healthy infants in a developed nation). The strongest corollaries of input milk matched with a High-Bif qPCR stool were high absolute amounts of non-fucosylated neutral HMOs, high absolute amounts of α(1-2)-fucosylated HMOs, and low relative amounts of α(1-3/4)-fucosylated HMOs, perhaps reflecting competitiveness with 2′-fucosylation. This may be due to adaptations of infant-type bifidobacteria to efficiently and selectively consume fucosylated and undecorated oligosaccharides [11,12,52,53]. Our data also provide insight into the metabolism of input milk glycans by different types of microbiota. BI PCoA feces possessed fewer oligosaccharides of all types, suggesting that these populations are capable of metabolizing greater amounts of fucosylated, sialylated, and undecorated sugars than communities low in bifidobacteria. Some environmental pressures (pH, carbon source availability, and so on) that select for bifidobacteria likely select for other taxa as well. It is important to note that relative abundances (such as provided by most marker gene sequencing workflows) are a zero-sum game; when one taxa's relative abundance increases, it registers as a concurrent decrease in other taxa. For some communities, this type of data may not adequately describe the underlying ecological interactions. This is illustrated in our study by the correlation difference between the relative and absolute bifidobacterial abundances with Bacteroidales in secretor-fed infants (−0.3 with relative abundance; 0.2 with absolute abundance). The relative abundance data seem to indicate that Bacteroides is antagonistic to (negatively correlated with) bifidobacteria, while the absolute abundance (qPCR) data show that they are mildly positively correlated with each other. It may be that bifidobacteria and Bacteroides populations respond to some environmental conditions in the same way but not to the same magnitude. The presence of HMGs that both genera can consume may be an example of this [54]. Both genera were enriched in secretor-fed infants, but the amplitude of the response as shown by the correlation matrix, however, appears to differ. This fact would be disguised by relative abundance data alone. Bifidobacteria and Bacteroides were implicated here as the major HMO consumers in infants, which agrees with previous in vitro work [55]. Bacteroides-dominated communities had a lower percentage of undecorated HMOs remaining, suggesting that they consume undecorated sugars preferentially to at least some decorated sugars. Our data show that fucosylated sugars remain at a significantly higher level (relative to other sugars) by Bacteroides-dominated feces, implying that they are not a preferred substrate of members of that taxa. Bifidobacteria-dominated feces have lower absolute amounts of fucosylated oligosaccharides than Bacteroides-and OTdominated communities. This suggests that fucosylated oligosaccharides might enrich bifidobacteria more than Bacteroides, supporting the difference shown in the correlation matrix between absolute and relative abundance of bifidobacteria and Bacteroides in secretor-fed infants. However, it may be that only some bifidobacterial species would possess this advantage, due to differences in abilities of various species to consume fucosylated substrates. In aggregate, the bifidobacterial species distribution did not differ greatly between secretor-fed infants and non-secretor-fed infants. As the species of bifidobacteria present within an infant are dependent on environmental exposure, we tentatively conclude that this subset of geographically co-located infants was exposed to similar sets of bifidobacterial species. However, there was a large difference in which species of bifidobacteria thrived in each group of infants (defined as a species whose presence tended to lead to domination of the microbiota by bifidobacteria). A broader range of bifidobacterial species were positively correlated with the amounts of overall bifidobacteria within secretor-fed infants than non-secretor-fed infants, suggesting that the presence of 2′-fucosylated sugars allows a broader array of bifidobacterial species to colonize the gut environment. According to our fecal isolate data, bifidobacteria that can grow on 2′-fucosylated substrates are relatively uncommon, but represent a larger proportion of total bifidobacteria in secretor-fed infants, suggesting that this substrate is related to the competitive fitness of these strains. Previous work supports the idea that fucosidases are a differentiating factor in the ability to grow on 2′-fucosylated sugars, although further study is needed to definitively identify which class(es) of fucosidase(s) is/are necessary [10]. That B. longum subsp. infantis thrives in secretor-fed infants is no surprise. It possesses both classes of fucosidases (GH95 and GH29) and was shown here to grow on 2′-fucosyllactose in vitro [10]. Feces dominated by this subspecies also had lower percentages of fucosylated oligosaccharides remaining. However, why B. longum subsp. infantis failed to dominate in any non-secretor-fed infant is somewhat perplexing. It may be that B. longum subsp. infantis specializes in consuming 2′-fucosylated oligosaccharides to gain an advantage over other species. B. breve on the other hand seems to be an oligosaccharide generalist, as it was dominant in examples of infants fed by both types of milk. B. breve strains are known to be variable in their capacity to consume 2′fucosylated oligosaccharides [36]. The fact that B. breve and not B. longum subsp. infantis seems to thrive in non-secretors may account for the observation of Avershina et al. that B. breve abundance separated the 10-day-old infants in their study into two groups; one group where it accounted for <15%, whereas in the other it accounted for >75% of the bifidobacterial load. B. longum group members were dominant among all infants in that study [56]. The B. breve-dominated group may correspond to a non-secretor-fed infant minority in their study. As they did not track the secretor status of the mothers in their study, this remains an open question. It is also important to note that their cohort was located in Norway and that the bacterial exposure pattern may differ from that of our cohort. The differing levels of competitiveness of B. breve and B longum subsp. infantis in infants fed by mothers of differing secretor status may also account for the observation that a three-probiotic mixture containing both B. breve and B longum subsp. infantis was more effective at promoting high levels of bifidobacteria in breastfed premature infants than a probiotic containing B. breve alone [57]. The bimodal distribution of bifidobacterial abundance shown by qPCR is intriguing from an ecological perspective. A remarkably similar bimodal distribution with comparable ranges was found by Mikami et al., suggesting that this phenomena may be widespread [58]. According to this distribution, a useful cutoff value for defining the population level of bifidobacteria in an infant as high or low would be around 10 8 /g of feces. Using this cutoff, our data also agree with the growing consensus that the establishment of bifidobacteria can happen in many infants in the first week of life [59,60]. The ecological phenomena of alternative stable community states may help explain this bimodal distribution in the infant gut. Simply stated, alternative stable state theory posits that a change in an ecosystem or in environmental conditions can result in a drastic shift in the composition of a community once some threshold or breakpoint is reached (review of concept here [61]). Some bifidobacteria are known to produce bacteriocins, which could contribute environmental pressure to maintaining alternate stable community states, once a threshold number of bifidobacteria producing these bacteriocins is reached [62]. A more likely mechanism by which these alternative stable states might be formed in this environment involves the production of lactate and short-chain fatty acids (SCFA) and their influence on environmental pH. Perhaps once a certain threshold of bifidobacteria/gram feces is reached the amount of SCFA and lactate produced alters the pH of the gut lumen, overcoming the buffering capacity of the luminal contents. Due to the lower pH, a shift in the community structure could occur as non-acid-tolerant members of the community die off. It is known that pH is a major driver in the composition of soil microbial communities [63][64][65][66]. The same physiological constraints that select for microorganisms able to grow at a low pH in soil likely apply in gut communities as well. One of these constraints may be effect of environmental pH on intracellular pH homeostasis and proton motive force [67]. Bacteroides in particular is known to be sensitive to low-pH conditions, and the sensitivity is increased in the presence of SCFAs [68][69][70][71]. At least some enterobacteria are known to be pH sensitive as well, which may indicate that a low-pH gut is protective against infectious disease [70]. A decrease in the abundance of other microbes due to pH changes, especially a decrease in competitors for HMG substrates, such as Bacteroides [54,72], would allow bifidobacteria to thrive from the reduced competition for nutrients and space. A survey of breastfed and formula-fed infants showed that fecal pH and SCFAs were lower in breastfed infants (pH mean 5.8) than formula-fed infants (pH mean 7.1), but that lactic acid was higher, suggesting that lactic acid might be a driver of pH [73]. Lactic acid has a lower pKa than the common SCFAs and would be hypothesized to have a greater effect on pH. B. longum subsp. infantis grown on HMOs was found to produce higher molar amounts of acetate and lactate [74]. Breastfed infants are known to have higher levels of bifidobacteria than formula-fed infants and also have different types of bifidobacteria, which may be important to the SCFA profile [75,76]. Both Bacteroides and bifidobacteria are known to produce lactate, which may explain the difference we observed between the amount of lactate in feces dominated by those groups as opposed to other species [77]. Although our data showed that both bifidobacteriaand Bacteroides-dominated fecal communities were higher in lactic acid than communities dominated by other species, our methods did not test the flux of lactate production and utilization by both the microbiota and the host. Nevertheless, lactate levels may be an interesting biomarker of the composition of the microbial community of the infant gut. There are numerous ways in which the composition of the gut microbiota impacts health. In the first 2 years of life, the infant gut microbiome progresses through a series of age-associated taxonomic changes. Infants and their gut microbiota are sensitive to disruptions during the early days and weeks of life [1,78,79]. Indeed, recent evidence suggests infants suppress the immune system early in life to aid in developing a healthy microbiota [80]. Like any new ecosystem, the microbial community in the gut has unfilled niches and has not developed mature levels of colonization resistance [81]. The establishment of the gut microbiota can impact an individual's lifelong health, and an early intervention that makes beneficial changes could have lifelong positive effects [82,83]. Early establishment of bifidobacteria is thought to be beneficial in numerous ways. Recently, domination by bifidobacteria was shown to be associated with improved immune response to vaccines [5]. Other benefits include protection from pathogens and development of the neonatal immune system [3,5,84,85]. For these reasons, our finding of delayed bifidobacterial colonization of non-secretor-fed infants (despite a higher incidence of exclusive breastfeeding in non-secretors) has important implications. Understanding the mechanism behind these differences will prove crucial to potentially compensating for this problem, perhaps through carefully chosen prebiotics and/or probiotics. It may be that the deficit in bifidobacteria that nonsecretor-fed infants experience is due to the likelihood of acquiring a species of bifidobacteria with metabolic abilities appropriate for the type of milk being consumed. As colonization by bifidobacteria is thought to be dependent on stochastic exposure to environmental strains, fewer appropriate potentially colonizing species may mean a lower likelihood of obtaining an appropriate one, whatever the mechanism of acquisition. As modern hygiene standards and other cultural practices may lead to a reduction in the exposure levels to different bifidobacterial species, the phenomenon of the low bifidobacteria infant might be an artefact of developed countries. Indeed, recent studies of developing areas of the world have revealed widespread domination of bifidobacteria in infants [5,86,87]. More surveys of the absolute abundance of infant bifidobacteria in developing and undeveloped areas of the world are needed, along with the measurement of relevant exposure-related metadata. Conclusion In conclusion, our work reveals important functional differences in the microbiota of infants fed by mothers of differing secretor. This knowledge will be useful to those selecting bifidobacterial species for probiotic interventions in breastfed infants [88]. As a mother's secretor status can be determined relatively easily, it could be used as a marker to target clinical interventions administering probiotics to infants to match the set of glycans the mother provides. This work provides context and insights for future hypothesis testing related to the in vivo competition between bifidobacteria and other members of the microbiota, as well as among bifidobacterial species. Further work is necessary to determine if these apparent differences in bifidobacterial populations between secretor phenotypes are indeed a developed-world phenomenon, and if the 'hygiene hypothesis' mechanism we propose here plays a role. Subjects Milk samples were obtained from 44 healthy women enrolled in the Foods for Health Institute Lactation Study at UC Davis. Subjects were enrolled at approximately 34 weeks of gestation and asked to fill out detailed health history questionnaires regarding demographics, anthropometrics, pregnancy history, current and prior health history, dietary habits and restrictions, physical activity level, as well as medication and supplementation intake history. Subjects reported the mode of delivery of their infants (C-section versus vaginal), infant sex, weight, length, and gestational age at birth, and filled out questionnaires regarding the health of themselves and their infants, as well as their diet throughout the study. Subjects received lactation support and training on proper sample collection from the study's lactation consultant. The UC Davis Institutional Review Board approved all aspects of the study, and informed consent was obtained from all subjects. This trial was registered on clinicaltrials.gov (ClinicalTrials.gov Identifier: NCT01817127). Breast milk samples Subjects were instructed to write on all sample tubes the time, date of collection, time of last meal prior to collection, and contents of the meals. Milk samples were collected in the morning on day 6, 21, 71, and/or 120 postpartum using a modified published method [89] involving milk collection by the subject from one breast using a Harmony Manual Breastpump (Medela Inc., McHenry, IL, USA) 2 to 4 h after feeding her infant. Subjects fully pumped one breast into a bottle, inverted six times, transferred 12 ml into a 15-ml polypropylene tube, and subsequently froze the sample in their kitchen freezers (−20°C). Samples were picked up biweekly, transported to the lab on dry ice, and stored at −80°C until processing. Infant stool samples Infant fecal samples were collected from the 44 breastfedterm infants born to women in the study at 6, 21, 71, and/or 120 days of life. Only one of the infants enrolled in this study had received antibiotic treatment at 89 days postpartum. All of the infants consumed breast milk, and several infants also consumed infant formula or solid food throughout the study duration. Parents were prompted to fill out detailed labels on each stool sample vial regarding infant intake of solids, infant formula, medications, and supplements. Parents transferred their infant fecal samples into sterile plastic tubes and were instructed to immediately store the samples in −20°C until transported by study personnel. Fecal samples were transported to the laboratory on ice packs and stored at −80°C before processing. Infant metadata statistics Differences in demographics and characteristics between secretor and non-secretor women were analyzed by the Pearson chi-squared test. Differences in clinical characteristics between secretor and nonsecretor women were analyzed non-parametrically for unequal sample sizes using Mann-Whitney U test. Alpha was set at 0.05. Oligosaccharide extraction from milk and stool Glycans were extracted from 50 μl of breast milk that was aliquoted onto two 96-well plates. Milk was defatted via centrifugation; the skimmed milk was collected and subjected to an ethanol precipitation for the removal of proteins. Following protein precipitation, the liquid fraction containing the oligosaccharides was dried completely using centrifugal evaporation. The oligosaccharides were reconstituted in 50 μl of water and reduced to their alditol forms with 1 M NaBH 4 . This is done in order to eliminate alpha and beta anomers on the reducing end of the sugars. Following reduction, the oligosaccharide mixture was desalted and enriched by solid-phase extraction on graphitized carbon-packed 96-well plates. Samples were desalted with six column volumes (approximately 1.2 ml) of deionized water and eluted with 20% acetonitrile in water, followed by 40% acetonitrile and 0.01% trifluoroacetic acid in water. Eluent was dried completely, reconstituted with 50 μl of deionized water, and diluted 50 fold for liquid chromatographymass spectrometry (LC-MS) analysis. Glycans were extracted from 50 mg of homogenized stool. Stool was diluted to 100 mg/ml with deionized water. Diluted stool samples were then homogenized by rocking the vials overnight. The solid components of the stool were then separated by centrifugation, then 100 μl of the oligosaccharide-rich supernatant was aliquoted onto two 96-well plates. Two times the sample volume (approximately 300 μl) of ethanol was added to each well. Proteins were precipitated at −80°C for 1.5 h, and then centrifuged at 3,220 rcf for 30 min at 4°C. The supernatant containing the HMOs was then collected and dried completely. Samples were reconstituted in 100 μl of deionized water and reduced with 100 μl of 2 M NaBH 4 (1 M final concentration in 200 μl solution total). Reduction was performed in a 65°C water bath for 1.5 h. Samples were immediately transferred to C8 packed 96-well plates for removal of residual proteins and peptides by solid-phase extraction. The C8 flow-through containing the oligosaccharides was then desalted and enriched by solid-phase extraction on graphitized carbon 96-well plates. Samples were desalted and enriched following the same protocol for milk as described above. Eluted glycans were then dried down, reconstituted in 100 μl of deionized water, and diluted tenfold for LC-MS analysis. Glycoprofiling by nano-LC chip TOF (time of flight) mass spectrometry Both milk and fecal HMOs were analyzed using an Agilent nano-LC Chip time-of-flight mass spectrometer (Agilent, Santa Clara, CA, USA), as described previously [32,90]. Briefly, all chromatography was done on a nano-scale microfluidic chip, equipped with a trapping column for sample enrichment and an analytical column for separation, both packed with porous graphitized carbon (PGC). Directly from the 96-well plate, 1 μl of HMO sample was injected and loaded onto the enrichment column and subsequently separated on the analytical column with a gradient optimized for glycan separation, using 3% acetonitrile and 0.1% formic acid in water as aqueous solvent A and 90% acetonitrile and 0.1% formic acid in water as organic solvent B (gradient also described previously). LC-MS data was deconvoluted using Agilent's MassHunter Qualitative Analysis software, version B.03.01 (Agilent, Santa Clara, CA, USA). Oligosaccharides were identified by matching retention time and exact mass to a fully annotated, inhouse HMO library [90,91]. Oligosaccharide quantitation and statistics Total oligosaccharide abundance was determined for each sample by summing the signal of all identified HMO peaks (in ion counts). Oligosaccharides were then grouped by glycan class, designated as either fucosylated, sialylated, or non-fucosylated neutral (containing neither fucose nor sialic acid residues). Total absolute amounts of fucosylation and sialylation were determined by summing the abundance (peak volume in ion counts) of all HMOs containing either fucose or sialic acid residues, respectively. Relative amounts for each glycan class were determined by normalizing absolute abundance of each class to the total HMO signal and were expressed as percentages. Two-tailed, unpaired t-test, with an α of 0.05, was used to compare glycan expression in milk between secretor and non-secretor mothers, as well as comparing milk and fecal HMOs received by infants with low amounts of bifidobacteria versus those with high amounts of bifidobacteria, as defined in the Bifidobacterium-specific qPCR section below. ANOVA was used to compare fecal glycan expression between infants whose dominant bacterial species were categorized as Bacteroides, bifidobacteria, or 'other', by PCoA grouping (explained below) followed by a pairwise comparison of means between each of the aforementioned groups using Tukey-Kramer HSD test, with an α of 0.05. Bifidobacterial isolations To isolate bifidobacteria, 100 mg of each fecal sample was aseptically transferred to a sterile tube, diluted tenfold with sterile phosphate buffered saline (PBS), and homogenized by vortex. Serial dilutions were prepared in PBS and inoculated on modified Bifidobacterium selective iodoacetate mupirocin (BSIM) agar. Modified BSIM agar was prepared by supplementing de Man Rogosa Sharpe (MRS) media with 13 g/l agar, 500 mg/l of L-cysteine-HCL, 20 mg/l of nalidixic acid, 50 mg/ml mupirocin, 50 mg/ml kanamycin, 50 mg/ml polymixin B sulfate, 100 mg/ml Iodoacetate, and 100 mg/ml 2,3,5-triphenyltetrazolium chloride. The plates were inoculated for 48 h at 37°C in an anaerobic chamber with an atmosphere containing approximately 5% carbon dioxide, 3% hydrogen, and the remainder nitrogen. Up to ten resulting colonies from each sample with the appropriate colony appearance were streaked onto BSIM plates for purity for two passages. The resulting pure strains were grown in MRS broth supplemented with 0.05% L-cysteine and stored at −80°C in 50% glycerol. MALDI-TOF Biotyper MS identification of isolates Glycerol stocks of each isolate were streaked on MRS plates and incubated at 37°C for 48 h in an anaerobic chamber. A colony from each plate was added to 300 μl nuclease-free water in a microcentrifuge tube and homogenized by vortex. Next, 900 μl of 98% ethanol was added to the tube, pulse vortexed, and centrifuged for 2 min at maximum speed. The supernatant was removed and the tubes were centrifuged again for 2 min. All liquid was removed from the pellets, and the samples were left at room temperature to allow the ethanol to evaporate. Subsequently, 25 μl of formic acid was then added to each tube and homogenized by vortex, followed by the addition of 25 μl of acetonitrile. Samples were then centrifuged for 2 min, and 1 μl of extract was placed on a MALDI target plate, left to dry at room temperature, covered with an α-Cyano-4-hydroxycinnamic acid (HCCA) matrix, and air dried. The MALDI target plate was then analyzed by a MALDI Biotyper (Bruker, Fremont, CA, USA), and the best database match for each isolate was recorded. Bacterial in vitro growth on 2′-fucosyllactose Unique bifidobacterial isolates (one isolate of each identified species from each infant fecal sample) were tested for growth on modified MRS (mMRS) with 3% filter-sterilized 2′-fucosyllactose as the sole carbon source, using mMRS with 3% lactose as a sole carbon source as a positive control and a no-sugar mMRS as a negative control. mMRS contains 10 g/l bacto-peptone, 5 g/l yeast extract, 2 g/l dipotassium phosphate, 5 g/l sodium acetate, 2 g/l ammonium citrate, 200 mg/l magnesium sulfate, 50 mg/l manganese sulfate, 5 g/l beef extract, 500 mg/l cysteine-HCL, and 1,000 g/l Tween 80. B. infantis ATCC 15697 and B. animalis UCD316 were included as positive and negative growth controls, respectively. Isolates were streaked from glycerol stock onto reinforced clostridial media (RCM) plates and incubated at 37°C in an anaerobic chamber for 48 h. A resulting colony was inoculated in 1 ml RCM broth at 37°C in an anaerobic chamber for 16 h. Five microliters of each resulting overnight culture was used to inoculate 100 μl of mMRS medium supplemented with either 3% (w/v) of 2′-fucosyllactose, 3% (w/v) of lactose, or mMRS without added sugar. The cultures were grown in 96-well microtiter plates in triplicate and covered with 20 μl of sterile mineral oil to avoid evaporation. The incubations were carried out at 37°C in an anaerobic chamber. Cell growth was monitored by OD at 600 nm every 30 min preceded by 30 s of shaking at a variable speed for a total of 96 h. The OD obtained for each of the technical triplicates from each strain grown on each substrate was averaged together and compared to the OD obtained in the absence of sugar source. This difference in OD (ΔOD) was used as a parameter to evaluate the strain's ability to grow on the different substrates. 2′-fucosyllactose was produced as described previously [92]. Fecal DNA extraction DNA was extracted from 150 mg of stool sample using the ZR Fecal DNA MiniPrep kit (ZYMO, Irvine, CA, USA) in accordance with the manufacturer's instructions, which included a bead-beating step using a FastPrep-24 Instrument (MP Biomedicals, Santa Ana, CA, USA) for 2 min at 25°C at a speed of 6.5 m/s. Breast milk and saliva DNA extraction DNA was extracted from breast milk for the secretor genotyping assay using the Qiagen DNeasy Blood and Tissue kit (Qiagen, Venlo, Netherlands) with a modified protocol for extracting DNA from animal saliva obtained from the Qiagen website. Briefly, 2 ml of breast milk or saliva was spun in a microcentrifuge at 15,000 rpm for 30 min to pellet human cells. Cells were washed once in PBS and re-pelleted. The pellet was re-suspended in 180 μl of PBS and incubated with 25 μl of proteinase K and 200 μl of buffer AL for 10 min at 56°C. Two hundred microliters of ethanol was added to the sample and mixed by vortexing. The entire sample was loaded onto a spin column, and purification proceeded as per the manufacturer's recommended protocol from that point. DNA was eluted in 30 μl of buffer EB for increased concentration. Determination of secretor genotype Genomic DNA purified from each mother's breast milk or saliva was amplified with primers FUT2-F (5′-CC TGGCAGAACTACCACCTG) and FUT2-R (5′-GG CTGCCTCTGGCTTAAAGA), which produces a 608bp amplicon. Each reaction contained 25 μl of 2X GoTaq Green master mix (Promega, Madison, WI, USA), 5 μl of DNA, 1 μl of each primer (10 μM), 6 μl of MgCl 2 (25 mM), and 12 μl of nuclease-free water. Cycling conditions were 95°C for 2 min followed by 35 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min. A final elongation was allowed at 73°C for 5 min, after which the products were kept at 4°C overnight. Successful amplification was confirmed by gel electrophoresis, and the PCR products were purified using the QIAquick PCR purification kit (Qiagen) according to the manufacturer's instructions. Samples with low amplicon concentrations were attempted again with 50 cycles of PCR, which was successful in amplifying difficult samples. The amplicons were then digested with BfaI, which cuts the DNA of individuals containing the mutated non-secretor rs601338 SNP (G → A, Trp → Ter) allele of the FUT2 gene, the predominant non-secretor mutation in the U.S. The resulting digests were electrophoresed on a 2% agarose gel for approximately 2 h at 80 V, and the resulting bands were visualized using GelGreen dye (Biotum, Hayward, CA, USA) under UV light. Individuals possessing a secretor allele produce a 608-bp band on the gel after digestion, while a non-secretor allele produces two bands with sizes of 516 and 92 bp. In this way, it is possible to easily distinguish both homozygote genotypes from each other and from heterozygotes. Determination of secretor phenotype The mother's secretor status phenotype in milk was determined by quantitating fucosylated glycan markers that have been previously described and assessed for sensitivity and specificity [32]. Secretor status was determined once per mother using milk from the earliest available time point, as the influence of early milk is thought to be most influential in establishing microbiota [6]. Thus, by our definition, 'secretor' and 'non-secretor' might be thought of as 'early secretor' or 'early non-secretor' since the phenotypes were defined at an early time point. Among these markers are α(1-2)-fucosylated structures, including 2′-fucosyllactose (2′FL, m/z 491.19), lactodifucotetraose (LDFT, m/z 637.25), lacto-N-fucopentaose I (LNFP I, mz/ 856.33), isomeric fucosylated lacto-N-hexaose (IFLNH I, m/z 1221.45), difucosyllacto-N-hexaose a (DFLNH a, m/z 1367.51), and difucosyllacto-N-hexaose c (DFLNH c, m/z 1367.51). Cutoff values for the relative amounts of each marker were used to distinguish secretor women from non-secretor women, as described previously [32]. Bif-TRFLP The method of Lewis et al. was used to perform the Bifidobacterium-specific terminal restriction fragment length polymorphism assay [93]. Briefly, DNA from feces was amplified in triplicate by PCR using primers NBIF389 (5′-[HEX]-GCCTTCGGGTTGTAAAC) and NBIF1018 REV (GAC-CATGCACCACCTGTG). DNA was purified using the Qiagen QIAquick PCR purification kit and then cut with restriction enzymes AluI and HaeIII. The resulting fragments were analyzed on an ABI 3100 genetic analyzer, and sizes were compared against the published database for species identification. Bifidobacterium longum/infantis ratio (BLIR) A PCR-based assay, BLIR, was developed in order to determine which subspecies of B. longum were present in each sample and to gain an estimate of their relative abundance to each other. Three primers (FWD_ BL_BI (5-[HEX]-AAAACGTCCATCCATCACA), REV _BL (5-ACGACCAGGTTCCACTTGAT), and REV_BI (5-CGCCTCAGTTCTTTAATGT)) were designed to target a conserved portion of the genome (between Blon_0424 and Blon_0425) shared by both subspecies using multiple genome sequences of each subspecies. FWD_BL_BI is complementary to a sequence in both subspecies, while REV_BL and REV_BI are complementary to nearby sequences in only B. longum subsp. longum and B. longum subsp. infantis, respectively. FWD_BL_BI and REV_BL amplify a fragment of the B. longum spp. longum genome 145 bp in length, while FWD_BL_BI and REV_BI amplify a fragment of the B. longum subsp. infantis genome 114 bp in length. DNA from each sample was amplified by PCR using 0.5 μl of 10 μM stock of each of the above primers, 12.5 μl GoTaq Green Master Mix (Promega), 1 μl of 25 mM MgCl 2, 1 μl of template DNA, and 9 μl of nuclease free water. Cycling conditions were 95°C for 2 min, 30 cycles of 95°C for 1 min, 54°C for 1 min, and 72°C for 30 seconds, followed by a 72°C extension for 5 min. PCR products were purified from the mixture using the QIAquick PCR purification kit (Qiagen), and diluted 1:10. 1.5 μl of the dilutions were analyzed by capillary electrophoresis on an ABI 3100 genetic analyzer (Applied Biosystems, Carlsbad, CA, USA). The HEX fluorophore (Abcam, Cambridge, UK) on the common primer allowed detection and differentiation of amplicon sizes and a rough quantitation of the abundance of each amplicon based on peak area when the samples were analyzed with PeakScanner 2.0 software (Applied Biosystems, Carlsbad, CA). A positive control was included with each PCR run to ensure potential amplification of both B. longum subsp. longum and B. longum subsp. infantis products. Bifidobacterium qPCR Levels of Bifidobacterium were measured by qPCR using the methods of Penders et al. and performed on a 7500 Fast Real-Time PCR System (Applied Biosystems) [94]. All reactions were carried out in triplicate with a nontemplate control and compared to a standard curve with known quantities of bifidobacterial DNA. Sequence analysis The QIIME software package (version 1.7.0) was used to analyze the results of the Illumina sequencing run. Illumina V4 16S rRNA gene sequences (Illumina) were demultiplexed and quality filtered using the QIIME 1.7.0 software package with default settings unless otherwise specified [96]. Reads were truncated after a maximum number of three consecutive low quality scores. The minimum number of consecutive high-quality base calls to include a read (per single end read) as a fraction of the input read length was 0.75. The minimum acceptable Phred quality score was set at 20. Similar sequences were clustered into operational taxonomic units (OTUs) using open reference OTU picking with UCLUST software [97]. Taxonomy was assigned to each OTU with the Ribosomal Database Project (RDP) classifier [98] and the RDP taxonomic nomenclature [99]. OTU representatives were aligned against the Greengenes core set [100] with PyNAST software [101]. PCoA plots were generated using the default beta diversity analysis parameters. Lactate concentrations Lactate concentration was measured in a modified version of the procedure developed by Ford et al. [102]. Shortly, lactate was extracted from solid feces by agitation in a 12-fold excess of pH 5.5 phosphate buffer for 3 h at 4°C. Proteins were removed by ethanol precipitation, and 100 μl of each extract was collected for analysis. After spiking the samples with stable isotope standards, 0.2 M aqueous 3-Ethyl-1-[3-(dimethylamino)propyl]carbodiimide was used to link an excess of 2-phenyl-2-ethanamine to the fatty acids via a peptide bond. The reaction was run for 20 min at room temperature and quenched by an ice bath followed by immediate C18 solid-phase extraction. The extracted phenylethylamine adducts were dried in a vacuum and reconstituted in water. The aqueous derivatives were analyzed on an Agilent 6490 QQQ LC/MS system (Agilent), and the response was gauged by characteristic transitions due to the Y and B fragmentation of the peptide bonds. Quantitation was achieved by comparison of the derivatized analytes to internal stable isotope standards. Lactate was quantified by comparison to 3,3,3-D 3 -lactate. During statistical analysis, the Bartlett test was used to test for homoscedasticity, and when needed, data was log transformed to meet the assumptions required to conduct a parametric test. For pairwise comparisons, two-tailed Student's t-tests were used. Results were determined to be significant for p < 0.05.

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2017-06-24T13:39:01.333Z

2015-01-05T00:00:00.000Z

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Ethnoecology of the palm Brahea dulcis (Kunth) Mart. in central Mexico Background There have been few studies on the sustainable use of non-timber forest products in arid and semi-arid zones. The palm Brahea dulcis has been one of the most important resources in semi-arid Mesoamerica, since pre-Hispanic times. Currently, some populations grow within protected natural areas, representing both a challenge and an opportunity for local development. This ethnoecological study of B. dulcis in central Mexico aimed to evaluate their uses, harvesting context, and potential for exploitation, in order to give practical advice on their best use and management. Methods Ethnographic and ecological information was obtained in Barranca de Metztitlán Biosphere Reserve and Valle del Mezquital, Mexico. We studied the population structure and density; additionally, we evaluated the rate of leaf production, leaf renewal rate, percent survival of new leaves, the development of reproductive structures and performed a one-year defoliation experiment (involving a control and four treatments including a mix of semiannual and annual frequency of harvest and removal of two new leaves and/or two mature leaves). Results Twenty uses of the palm were recorded in the study area. Religious/symbolic and handicraft uses are highlighted. The population density of this species was the highest reported for the genus (1244 ± 231.7 ind/ha). The leaf production rate was the highest reported for arborescent palms of the Americas (11.83 ± 0.036 leaves/individual/year). The sexual reproductive cycle was 2.3 years long. A one-year defoliation experiment did not show statistically significant differences. Recommendations include: 1) implement management focused on increasing the abundance and quality of this useful resource in Metztitlán; 2) employ a strategy of focusing on ethnicity and gender in promoting their exploitation; 3) learn from theoretical frameworks of other non timber forest product studies. Conclusions We propose that Brahea dulcis is the palm with the highest potential for sustainable use in the arid and semi-arid zones of Mexico. The challenge to improving management includes simplifying the legal protection framework, promoting uses and developing a market strategy. Collaborations to share experiences with peasant farmers from Guerrero is recommended. We further recommend the development of a governmental strategy to enhance and reassess this important resource. Electronic supplementary material The online version of this article (doi:10.1186/1746-4269-11-1) contains supplementary material, which is available to authorized users. Background The palm is the archetypical non-timber forest product (NTFP); since the dawn of humanity it has served many purposes [1]. There are numerous genera and species of palms typical of arid and semi-arid areas (A-SA), including Brahea, Copernicia, Phoenix, and Washingtonia. However, there are fewer studies of sustainable management of A-SA palms than of palms in humid areas [2]. In our opinion, it is very important that A-SA resources be studied more extensively for a variety of reasons, including: a) A-SA make up about 41% of the land area of the earth and these zones are increasing in area [3]; b) they are the predominant zones in some countries and regions; and c) some 33% of the world's population lives in these ecosystems [3]. Much of Mexico's land area is A-SA, where genera such as Brahea and Washingtonia evolved. 10 of 13 Brahea species are endemic to Mexico, as is W. robusta. Extensive areas of Mexico, located particularly in poor rural regions, are dominated by Brahea dulcis, where it clearly has great cultural, religious and economic importance currently and in the past [4][5][6]. For example, it is heavily traded in several major traditional markets, namely Tehuacán (Puebla), Sahuayo (Michoacán), and Tlapehuala and Chilapa (Guerrero) [7]. The use and commercial importance of this NTFP is so large that in the Chilapa market alone, which may be where the most of the species is sold, it is estimated that an average of 400 tonnes are sold per month (Cati Illsley, pers. comm., Sept. 2012). Brahea dulcis, "palma sombrero" or "soyate", has been one of the most important NTFP in the arid and semiarid zones of Mesoamerica since pre-Hispanic times. Virtually all parts of the plant have been heavily used as NTFP. In pre-Hispanic times, tribute was paid to the Aztec empire with baskets and mats made of this species [6]. In Colonial Mexico, the palm hats made by the Franciscan friars became a major industry in the nineteenth and early twentieth century [4]: in 1877, 46,392 hats were produced in the state of Guerrero [8]. From ancient times, and still today, palm blades have been used to weave a basic piece of furnishing, the petate, a thin mat used as a floor covering and sleeping mat. The petate is part of the material culture of some indigenous groups (v.g. Nahuas). Even today, the palm is used for religious offerings, and to make a variety of utilitarian items; humans have put almost every part of these palm plants to some use. The young leaves are used to make mats, hats, and brooms, while the older leaves are used in flower arrangements, and the dried leaves are used as fuel [4][5][6]8,9]. The roots are used to make dish scrubbers, and the flowers and fruits are edible [6]. Some of the populations of this useful species grow within protected natural areas (PNA), specifically biosphere reserves: Barranca de Metztitlán, Tehuacán-Cuicatlán, and Sierra Gorda. The inclusion of useful species such as B. dulcis in PNA represents both a challenge and an opportunity within and outside of PNA, where the sustainable use of NTFP could be a key contribution to local development. There are important local ethnobotanical studies of B. dulcis [5,6,8,10], and some ecological [11] and genetic studies [12,13], but currently it is not clear how their ecological features can help or impede their harvesting. Of the few studies of B. dulcis focusing on management, Illsley et al. [6] evaluated the impact of harvesting leaves, while [14,15] developed management plans for certain areas of the state of Guerrero, based on population studies and particularly on knowledge from peasant farmers. The aim of the present paper was to conduct an ethnoecological study of B. dulcis in the Barranca de Metztitlán biosphere reserves (RBBM in Spanish) and adjacent areas in Hidalgo state in central Mexico. The study aimed to answer the following questions: Which types of products are and have been produced currently and in the past? Which populations have the most potential for exploitation? What is the rate of production of new leaves and how does it vary through the year? What is the growth rate of the petioles? How long do the leaves last? How long does each stage of leaf development last (emergence, new, mature, senescent)? What frequency and intensity of harvesting young leaves will maximize production of new leaves? How long does sexual reproduction take from flower production to ripe fruit? It was hypothesized that this NTFP is used in a broad variety of ways, and that the rate of new leaf production per individual would be low due to the arid conditions, but that the potential harvest per unit area as a whole would be significant due to the high densities of populations in the RBBM. Meeting the study objectives enabled us to suggest better ways to manage and use this NTFP in the semiarid regions of central Mexico, ideas that can be applied to other regions as well. Methods The study species Brahea dulcis, B. nitida André and B. salvadorensis H. Wendl. ex Becc. are the only species of the genus whose range extends beyond the borders of Mexico into Central America. In Mexico, B. dulcis is prevalent in the Balsas Basin, extending to southern Oaxaca, the high part of the Papaloapan Basin, and along the Sierra Madre Oriental as far as southern Tamaulipas [16]. The species grows most abundantly between 1200 and 2200 m.a.s.l. in the understory of oak and pine-oak forests, low deciduous forests, and in disturbed areas where it may form almost monospecific palm groves that are apparently secondary [16,17]. This palm grows up to 8 m tall, with sometimes solitary, erect stems and frequently has caespitose growth; in adults, the crown has 10-15 flabellate leaves, petioles with armed margins, inflorescences of sessile flowers hanging in modified racemes, and monocarpic, monosperm drupe fruits [18]. The palms are hermaphroditic plants with bisexual flowers [17]. They reproduce by both sexual and asexual means [6], as the suckers are fire-resistant [19]. They interact with ants and bees [9], which may serve as pollinators. The fruits are dispersed by birds, small mammals, coyotes, and humans. There is evidence that B. dulcis can hybridize with B. nitida when the two species grow sympatrically [13]. According to [4,6] there are two types of B. dulcis palmars: a) the low-growing or "manchonera" palms about 1.5 m tall, which most often reproduce asexually, increasing the density of ramets; and b) the tall "soyacahuitera" palms six or more meters tall, in which sexual reproduction is more common. These two different morphologies are the result of management practices employed by the local people (intensive leaf harvesting, burning, and grazing), which keeps the palm trees short; moreover continual leaf harvesting increases plant population density [6]. A manchonera can grow to be a soyacahuitera under human management. Study area Our study area was located within the A-SA regions of Hidalgo state in central Mexico, particularly the Barranca de Metztitlán Biosphere Reserve (RBBM) and adjacent areas of Valle del Mezquital. The RBBM is a 2,090,512-hectare PNA characterized by a high richness of cactus species, especially endemic Mexican cacti. The RBBM includes tropical arid scrub, tropical deciduous forest, submontane scrub, pine forest, pasture, and riparian woodland ecosytems [20]. The RBBM is located in the Huasteca Karst subprovince of the Sierra Madre Oriental province, where regional tectonic movements and erosion caused by the Amajac and Metztitlán Rivers carved deep canyons. The climate is generally hot dry and semi-dry due to the rain shadow effect of the Sierra Madre Oriental. Average annual precipitation is 500 mm, mostly in summer and less in winter. The rainy season lasts from June to October, but can be shorter in some parts of the region (Figure 1). The average annual temperature ranges from 18 to 22°C [20]. In the Valle del Mezquital the towns of Naxthe, Taxhie and Ixmiquilpan were studied. The predominant local culture in this semi-arid region is hñä hñü. Interviews In March 2008, RBBM staff were interviewed about their opinions on the general status of the palm. They informed us that Tlaxco is currently the main local palm handcraft town. We then visited Tlaxco and Metztitlán for five days during Holy Week to conduct open interviews. Although at the beginning the people were somewhat reserved, all the respondents consented to be interviewed, especially when they learned that we were interested in talking about the palm and that we were from the university. We asked for general ethnographic information including: 1) the home towns of harvesters and handcrafters currently and in the past; 2) the handcrafters' opinion about the main problems and challenges relating to the use of this NTFP; 3) understanding the local commercial network; 4) finding out the types of palm products produced in the region. In this first exploratory trip we conducted open interviews with some twelve handcrafters in Tlaxco (all women, because there were no male handcrafters in the town). Additionally, we spoke with the main middleman in Tlaxco, to find out where he goes to sell the products and how they are marketed. Finally, we also spoke with one handcrafter and seller of palm goods in Metztitlán to learn their perspective. After two weeks, we returned to interview four sellers at the Ixmiquilpan market, to ask about where they found the handcrafters whose work they sold. Between August 2008 and April 2009 we conducted 25 semi-structured interviews and carried out participant observation in Naxthé (2), Taxhie (2, in Alfajayucan municipality) and Tlaxco (21 in Metztitlán municipality). These places were chosen based on information obtained from sellers in Ixmiquilpan. At the beginning, the research was explained to the informants. Interviews were conducted with handcrafters to find out: 1) the type of products that they can make; 2) how long it takes them to weave each type of product; and 3) how many palm blades they require for each product. By means of participant observation, the second author worked closely with the handcrafters and learned how to make the crafted articles. Before the research was conducted, we asked permits from the authorities of the Biosphere Reserve and from Communal authorities ("Delegado"). Additionally, we explained the purpose of our study to each person interviewed and asked for his/her verbal consent. Potential for exploitation of palm populations In June, 2008, we made a reconnaissance visit, guided by RBBM staff. They were interested in evaluating which populations have the greatest potential for exploitation, and suggested five sites for study: La Rivera, Tlaxco, La Yerbabuena, Zotola (Metztitlán municipality) and San José Zoquital (Atotonilco El Grande municipality). Five palm populations were of the manchonera type, and are now harvested almost exclusively for Palm Sunday, and the dry leaves for fuel. Each population's potential for exploitation was estimated by assessing its density and population structure. The distance between sites ranges between 4.3 to 50 Km ( Figure 1). The general characteristics of the sites are listed in Table 1. At each site, a sampling area of 0.1 ha was chosen, made up of ten 10 × 10 m randomly selected quadrants. The size of the quadrants was chosen because we observed many individuals generally distributed in clusters. The total number of B. dulcis individuals in each quadrant was counted, the heights of the stipes were measured, and they were categorized. If two palms close together were joined at the root, it was counted as one individual. Height was measured from the ground to the apical meristem. In individuals with prostrate or curved stems, the convex side of the stipe was measured. To characterize the population structure, individuals were subdivided into five classes based on the recommendations of Balslev et al. [21] for studying palms, although we made some changes; our classes were defined as: 1) seedlings (S): lanceolate leaves, stems of some centimeters, sucker absent; 2) saplings (I): leaves partially divided, similar size to seedlings, sucker absent; 3) juveniles (J): flabellate leaves, stipe to one meter, can present suckers; 4) subadult (SA): flabellate leaves, stipe generally at least one meter, can present suckers; 5) adults (A): flabellate leaves, stems generally at least 1.5 m, can present suckers, reproductive. The population density by site was calculated for the total number of individuals and for each size category. For each individual, we also recorded whether the palm had harvestable leaves; that is, leaves large enough to be useful. Total density was calculated, and the population structure was graphed, distinguishing between individuals that were useful for supplying handcraft materials and those that were not. Rate of leaf production, leaf renewal rate and percent survival of new leaves Since harvesting the leaves can influence the production of new leaves, leaf production was measured every two weeks at San José Zoquital, the site where leaves are harvested the least by the local residents (Table 1). Every two weeks, from July 2008 to July 2009, 42 individuals were observed. These palms were in the useful range of 0.6 to 1.6 m tall; harvesters can reach their leaves from the ground. In order for the sample to be statistically independent, palms grouped in patches were excluded, since it is known that individuals located close to each other may be genetically identical [13]. At the first sampling, each individual was labeled and located geographically, its total height was measured and the number of green leaves, inflorescences and infructescences counted. Following this, at each fortnightly sampling, new leaves were marked (with paint on the petiole), using a different color each time. Leaves, inflorescences and infructescences were each marked individually in order to identify and record their stage of development. The following data were recorded every two weeks for each of the 42 selected individuals throughout the oneyear sampling period: a) the number of new leaves per individualthe average yielded information on leaf production rate per individual and variation over time; b) the length of the new leaf petiole, to evaluate the average rate of growth (this was only measured in 179 leaves on 15 individuals); c) the first new leaf produced by each individual during the observation periodthese were marked and followed in order to measure the average annual leaf survival rate. To measure leaf turnover, all the leaves of each individual were classified as new, green or dry. A new leaf (NL) was defined as one that is budding out from the apical meristem, with petiole visible and leaf blade at most partially opened (the part closest to the petiole no more than 15 cm wide). A green leaf (GL) was a leaf with a blade more than 15 cm wide and bright green in color. A dry leaf (DL) was brown in color and no longer borne erect. Defoliation experiment Due to differences in harvest regime between populations, we decided to conduct defoliation experiment at the La Yerbabuena site, which is the most heavily harvested site ( Table 1), so that the resulting potential management recommendations would be applicable to a realistic harvesting scenario. The defoliation experiment was conducted for a period of one year (July 2008 to July 2009) on 100 individuals, to which a total of four different treatments and a control were applied. A random block experimental design was applied, using 20 blocks throughout the site. Each block consisted of five individuals, one randomly assigned to each treatment and the control. The treatments involved a combination of different harvesting frequencies (semiannual or annual) and intensities (two new leaves and/or two mature leaves) ( Table 2). The experimental treatment was determined based on what had been done in other studies with mature leaves. Although in this case the useful resource is the new leaves, in some treatments (T3 and T5, Table 2) we decided to harvest both new and mature leaves since harvesting mature leaves can potentially stimulate physiological responses in the plant that can increase the production of new leaves. The 100 individuals in the experiment had heights ranging from 0.6 to 1.6 m high, this being the range of plants that are usually harvested. Each individual was labeled and georeferenced (GPS), and the newest, secondnewest and third-newest leaves were marked with a dab of colored paint on the petiole in order to differentiate between treatments. Data recorded for each plant at the beginning and end of the experiment were total plant height (ground to base of the petiole of the youngest leaf ), total number of leaves, and the number of inflorescences and green and dry infructescences. The numbers of new and green leaves were recorded before the experimental treatments were applied. In January 2009, the numbers of leaves produced over the previous six months of the experimental period were counted for individuals in the T2 and T4 groups, and leaves were then harvested according to the respective treatment. Development of reproductive structures The time from emergence of inflorescences to mature fruit production was recorded on the 42 study individuals. Eighteen of them had inflorescences or infructescences during the study period. The reproductive structures were marked as described in the section "Rate of leaf production, leaf renewal rate and percent survival of new leaves" and classified into developmental stages. The inflorescences were classified into five stages: emerging, pubescent, bud, flower and dry flower ( Figure 2). Emerging inflorescences did not yet show rachillae, and were dark red. Pubescent inflorescences had smoothtextured, pearlescent cream-colored rachillae. The bud stage showed immature green petals. The flower stage corresponded to full anthesis, with the colorful whorls visible. In the dry flower stage, the floral whorls were dark brown. The infructescences were classified into four categories according to the dominant color of the fruit; green, yellow, black, or dry ( Figure 2). These data were used to estimate the total duration of flower and fruit production, and the duration of each stage. It should be noted that the size of the sample was different for each stage, as it depended on flower and fruit production in the sample of plants. Data analysis The ethnobotanical information was summarized in a table. The population structure was compared between localities using the chi-square statistic and a posteriori adjusted residual analysis described by Habermann [22]. The potential for use by site was evaluated on the basis of density of useful individuals. The leaf production rate per individual (mean ± 1 s.e.) was calculated for each two-week period and averaged for the 42 individuals to give the fortnightly leaf production rate per individual and its variation across fortnightly periods. In addition, the rate (by month) was related to average monthly rainfall of the current and previous month [23], available for the period 1991-2006 using a simple linear regression model. To explore which variables could explain leaf production per plant, we used a generalized linear model (GLM) that had the leaf production rate of each individual as the response variable, and plant height, height squared, the number of suckers and number of reproductive structures as explanatory variables. In these models, the parameters and random variables are necessarily linear, while the mathematical variables may not be [24]. Since the response variable was leaf counts, we used a Poisson distribution. A chi-square function was used to evaluate the statistical significance of the variables. The GLIM statistical package (Numerical Algorithms Group, UK) was used. Average growth rate of the leaf petioles per individual was calculated by averaging the growth values of each petiole (for example, the average growth in the first twoweek period of the 6 to 17 leaves produced by each plant) even though these were produced in different periods. The average leaf growth rate for the 15 individuals observed was also averaged and graphed. The percentage Ethnobotany According to our ethnographic information, people have been harvesting only the leaves for at least eight decades in the region. They take them to the markets, or directly to the craftspeople, but there are no useful palms in the towns where the craftspeople live (Tlaxco, Naxthé, Taxhie). Ixmiquilpan, Actopan and Atotonilco are the main palm markets in the region; Metztitlán is a smaller, seasonal market which is most active in Holy Week. Ixmiquilpan is the most important market for trade of food, fibers such as ixtle, and palm, and has been so since pre-Hispanic times. There is a considerable geographic division between craftspeople and harvesters. As a result, transporting the material is an essential part of the process, in the past on donkeys and now in trucks. In Tlaxco, Naxthé, Taxhié, the most common handcrafted palm products are petates (18 of 25), followed by brooms (12/25), fans (4/25), hats (4/25) and miniature implements (3/25). Petates require 15 to 40 leaves and one to seven days to make but are not the most profitable product; a hat takes the same time to make but sells for a higher price (Table 3). Twenty different types of products are crafted in the region as a whole (Table 4); the most common uses for the palm were found to be utilitarian and religious/symbolic objects. Utilitarian items include hats, bags, and brooms (Table 4, Figure 3). They also used to include "capotes" or raincapes that currently are in disuse (replaced by plastic); photographs of capotes were collected in the ethnographic work of [10, pp 65]. Religious uses include the branch or "ramo" used on Palm Sunday in many towns (Table 4) and the burial headpiece reported for the first time. In Taxhie, the dead are buried with a woven palm leaf headpiece shaped like a simple crown bearing a small cross. A village tradition says that once a man who died in Taxhie met St. Peter and was asked why he arrived with his head uncovered. St. Peter explained that this is a requirement for entering heaven. The man was told that he could return to earth and tell people that they had to be buried with a headpiece. The role of protection attributed to the palm also includes the power of calming storms when the leaves are placed or burned behind the main door of a house (in Hidalgo and Mixteca Oaxaqueña). Many of these uses require leaves. There are two ways to process the new leaf to make the final product: either the new leaf is cut, boiled, dried in the sun and woven, or it is only cut and woven. The results are different. Boiled and dried, the leaf is more flexible and easier to weave, and the final product is lighter in color. In the latter case, the leaf is stiffer, darker in color and more readily torn or broken. The majority of products are made with new leaves (not expanded yet). Dead leaves are currently used as fuel and until a few decades ago, were used for thatching traditional roofs of houses. Variation among populations in potential for exploitation of palm The differences in population structures of B. dulcis between the five study sites were statistically significant (χ 2 = 269.3, P <0.05, df = 16; Figure 4). Populations consistently tended to have low seedling densities and a very low density of individuals > 2.01 m (Figure 4), but the population as a whole showed significant densities in the RBBM (1244 ± 231. 7 ind/ha), with a strong variation between sites (450 to 1690 ind/ha, Table 5). In descending order of total density, the sites are Zotola > Tlaxco > La Yerbabuena > Atotonilco > La Rivera (Table 5). Ordered by density of useful individuals, however, the sites are Yerbabuena > Atotonilco > La Rivera > Zotola > Tlaxco. The total density of the densest population (Zotola) was 3.76 times that of the least dense population (La Rivera). The density of useful individuals (defined as those bearing leaves useful to craftsperson) differed even more across populations. By this measure, La Yerbabuena was 5.1 times denser than Tlaxco ( Table 5). Rate of leaf production, leaf growth rate and percent survival of new leaves One palm produced on average (±1 s.e.) 11.83 ± 0.036 leaves per year, with a notable variation among individuals (ranging from 5 to 19). Leaf production was positively correlated with plant height (R 2 = 0.3805, P <0.05). Trunk height, the only significant variable in the GLM, explains 30% of the total variation in leaf production. In the rainy season (June to October) leaf production was 0. Pulido and Coronel-Ortega Journal of Ethnobiology and Ethnomedicine 2015, 11:1 Page 8 of 16 http://www.ethnobiomed.com/content/11/1/1 rainfall in the same month (R 2 = 0.3328, P = 0.050) and the previous month (R 2 = 0.5105, P = 0.013). The length of the leaf life cycle was 24 fortnights, during which time the leaf grew from the new to the green stage and in some cases to the dry leaf stage. The petiole showed its highest growth rate (9 cm) during the second fortnight and then began to decrease with a negative geometric distribution (Figure 6). A leaf lasted on the plant an average of 20.18 fortnights, and the GL stage was twice as long as NL and DL ( Table 6). The 21 leaves marked for the study of leaf survival had an annual survival rate of 76% (Figure 7). The first leaf deaths occurred in the 17th fortnight, and continued gradually decreasing. Reproductive structure development stages and duration Throughout the year, it was always possible to find at least one individual in nearly every stage of inflorescence and infructescence; in general the reproductive cycle is not associated with particular times of the year, and there is little synchronization among individuals (Table 7). There was however, synchronization within individuals; their reproductive structures moved through the stages almost simultaneously (Additional file 1). Emergence of inflorescence was the only stage restricted to a particular season of the year: emergence was only observed from April to June, as the season changed from dry to the beginning of the rainy season. However, the duration of a single stage often varied greatly between different individuals, especially the stages of flowering, dry flower, yellow fruit and black fruit (Tables 7 and Additional file 1). For example, the flower stage varied from 3 to 10 fortnights among different individuals. Based on the data (Tables 7 and Additional file 1), which is limited for some stages, the best estimate for the Pulido and Coronel-Ortega Journal of Ethnobiology and Ethnomedicine 2015, 11:1 Page 9 of 16 http://www.ethnobiomed.com/content/11/1/1 average length of inflorescence is 36.9 fortnights, and for infructescence (except the dry stage) 18.26 fortnights. The total reproductive cycle from the emergence of the inflorescence to mature fruit production takes at least 55.2 fortnights; that is, 2.3 years. Discussion Ethnobotany: use and disuse of B. dulcis Although some significant ethnobotanical research of this species has been undertaken, such as the work of [5] in Huizltepec (Guerrero), [10] in Michoacán and Pulido and Coronel-Ortega Journal of Ethnobiology and Ethnomedicine 2015, 11:1 Page 10 of 16 http://www.ethnobiomed.com/content/11/1/1 [6,8] in La Montaña de Guerrero, as well as the present study, no thorough ethnobotanical studies at the national scale have yet been published. Our study reports new uses including miniature implements, the burial headpiece, leaf strips for tying bundles of vegetables, and leaves to wrap zacahuiles (a type of tamal). While the species is and has been extensively used currently and in the past, certain uses have greatly decreased or discontinued. These include the use of the leaves to make capotes (rain capes), petates (sleeping mats), and the substitution of leaves used for thatching and handcrafts by synthetic materials (it was once common in the study region for house roofs to be thatched with the leaves of this palm). The substitution phenomena is common to many NTFP, caused by resource scarcity and the lower prices of some synthetic materials [25]. In the RBBM, substitution and decreased use of B. dulcis is due to difficulty in obtaining the raw material (whether legally or illegally) and probably to emigration, cultural change and/or low prices of palm products. The problem of obtaining the raw material is due to the difficulty of meeting the requirements of current regulations. For example, when the RBBM was established, the people of the hñä hñü village of El Palmar, which owes its name to the high abundance of B. dulcis, submitted an official letter complaining of legal impediments to the use of the palm, which have not been resolved to date [26]. In spite of the trend of decreased use, maintaining use is very important because it not only produces useful objects, but is also part of cultural identity, as well promoting commerce. Additionally, objects made of NTFP help the carbon sink, and offer a better alternative than plastic to fight climate change. Density and population structure The density of B. dulcis is an order of magnitude greater than that reported for other species of Brahea, corroborating its value as a NTFP and supporting the hypothesis that the potential for its exploitation per unit area in the RBBM is high. Extrapolating our results, B. dulcis showed an average density of 1244 ± 231.7 ind/ha (Table 5) compared to 278 ± 66.9 ind/ha (unpublished data) recorded for B. armata, a species endemic to the Baja California Peninsula, at six desert canyon sites. For B. aculeata, endemic to Sonora, the average density was 121.7 ± 36.3 ind/ ha in tropical deciduous forest [27]; the variation between species is extensive. The densities of B. dulcis found in the RBBM (1244 ± 231.7 ind./ha) are very close to those obtained by [6] for B. dulcis at 18 sites in the Chilapa region (Guerrero, Mexico), where a density of 1157.9 ± 174.6 genets/ha (calculated from 6, Table 2) was reported, ranging from 350 to 3233 genets/ha across sites. Clustered palms were counted as one genetic individual or genet in both studies, making comparison possible. Current density and population structure of B. dulcis in the RBBM vary significantly between sites, meaning that the potential for exploitation varies greatly from one locality to another. Thus, while Zotola has the highest density, many individuals are juveniles with prostrate stems and are therefore not suitable for harvest. The area is heavily eroded and grazed (the latter particularly in the past). Tlaxco had the next-densest population, but it was dominated by juveniles, which are not useful, and by seedlings and saplings, which are not yet useful. Curiously Tlaxco had a scarcity of adults, but seeds appear to be originated by individuals present in numerous ravines which were not sampled. La Yerbabuena, Atotonilco and La Rivera had the lowest density of total individuals, but the highest density of useful individuals. Adults made up a significantly higher proportion at La Rivera and Atotonilco, contributing to management potential. Additionally, the three populations share the characteristic that leaves are harvested at least twice per year, contributing to higher levels of use and management. More precise measurement of the intensity of management of B. dulcis could be an important aspect of future research, by means of appropriate indexes such as that of [28]. Ramírez-Rodríguez et al. [13], building on the system described and studied by Illsley et al. [6], recently found that management increased genetic diversity (heterodicity), increased the density of genets and ramets in the population, and increased the number of genotypes in B. dulcis. Thus the highest density was observed at the La Yerbabuena site, followed by Atotonilco, which could be in part a result of more intensive management at these places compared with the others. Leaf longevity and production rate Our results are consistent with leaf longevity reported for the species in the state of Guerrero, where [15] notes that it varies from 8 to 14 months, and is directly proportional to the height of the individual. Small farmers in Guerrero know that the leaves take eight months to complete their life cycle [15], similar to our data. Illsley et al. [6] report an annual rate of 20 leaves per year, higher than our results; these differences may be due primarily to the fact that these authors selected tall palms, while the present study included relatively short individuals. When these results are compared to those recorded for other palms, it can be seen that B. dulcis annual leaf production is the highest reported for arborescent palms of the Americas (Table 8), while leaf longevity is low. This suggests that the strategy of B. dulcis is to produce many leaves, but with each leaf having a relatively short life. This makes it ideal from the point of view of sustainable NTFP use, since although removing leaves for handicraft uses removes photosynthetic production for an average of 20.18 fortnights, which has an impact on the energy balance of the plant, at the same time the characteristics of the palm's life cyclehigh leaf productivity and short leaf life cyclesmean that humans have less effect on the plant while they are taking advantage of its life cycle. The findings for B. dulcis are consistent with the trends described by [29] for palms of the world, in which he found a positive relationship between leaf production rate and the number of leaves in the crown. He also reported a negative correlation between the number of leaves produced per year and leaf longevity. This suggests, furthermore, that the more dry and seasonal the climate, the more leaves there tend to be in the crowns of the palms. He also found that the number of leaves per tree is higher in dry than in wet habitats. Petiole growth pattern was similar to that reported for Euterpe edulis [30]. Optimal harvest This study is the first to have the stated goal of optimizing new leaf production. Our results are consistent with the results reported by [15], who compared leaf production between harvested and unharvested plots during a twoyear period. This failure of the plant to show a response to the experimental treatments we applied is largely due to the fact that few leaves were harvested, and these at a low frequency compared to the high rates of production. Differences would probably have been observed if the treatments had involved harvesting more leaves more often, and the observation period been longer. Our results are consistent with the life strategies described by [29], where palms in desert and xeric shrubland had more leaves in [31,32]. However, when evaluating the effects of leaf harvesting, it must be taken into account that effects are not limited to potential plant mortality but may also include changes in growth, such as decreased leaf length or decreased reproductive activity [33,34]. Examining these aspects in B. dulcis could be subjects of future studies. The insignificant effect that harvesting new and mature B. dulcis leaves has on individuals and populations is ideal from the perspective of NTFP exploitation, since harvesting of the resource does not appear to have significant implications, at least in the short term. Development of reproductive structures B. dulcis has a biannual reproductive cycle, as do Borassus aethiopum [35] and Oenocarpus bataua [36]. Perhaps high leaf production rates in B. dulcis can be related to slow reproduction. In spite of the reproductive activity of B. dulcis at the study sites, no recruitment of new individuals through sexual reproduction was observed during the study period. That is, no seeds were observed to germinate or seedlings to establish. This could be due to the high rate of solar radiation received at the site, trampling by livestock (sheep, goats and donkeys), and also partly because of seed depredation by weevils. In addition, there were cases of abortion, particularly in the transition from the flower to dry flower stage, and from dry flower to green fruit (Additional file 1). In the field, ants were often observed in the inflorescences and on the infructescences, and bees in the inflorescences, and fox feces containing seeds of this palm species were observed. Recommendations for sustainable use 1. As a first step, we propose apply in RBBM the traditional palm management practices used in Guerrero, including removing suckers and controlling grazing [15]. We consider it a mistake to invest money and effort in planting seeds at these locations (which is the simplistic option recommended by many managers), since this species regenerates easily if it is managed by proper sucker removal and controlled pasturing. Additional management might be required, but these two measures should be tried first, and the results evaluated. 2. Given that the few indigenous populations (Hñä hñü groups) living in the RBBM are the landholders and managers of these palm forests, and that the craftspeople are mostly women, the RBBM should promote the exploitation of this NTFP with a focus on ethnicity and gender, taking advantage of the need to promote sustainable activities in buffer zones. 3. Promotion of the use of any NTFP in PNA should take into account the lessons learned from comparative studies of NTFP around the world [37][38][39]. These studies show that a successful NTFP requires at the very least: a) assessing which NTFP have economic potential, and not naively assuming that the most biologically abundant NTFP has the greatest potential; b) understanding that in Latin America NTFPs are a complementary strategy to local economies; c) recognizing that although the key to success of an NTFP is in the economic sphere, biological limits and the maintenance of cultural values must be respected. In light of the above, it is recommended that RBBM officials design actions to: a) improve the quality of craftsmanship and product diversification; b) seek more and better fair trade markets for these products; c) provide a Figure 1 in the original paper). ***see Figure 6 in original paper. practical legal framework for the use of NTFP, rather than promoting conservation policies based on not managing resources. Conclusions We suggest that B. dulcis is the most important palm with the highest potential for sustainable use in A-SA zones in Mexico because of its wide distribution across Mexico (13 states); the remarkable versatility of its parts, nearly all of which have a use; and the evident preponderance of this palm in the major traditional markets of Mexico. No other palm species in A-SA zones has as wide a geographic distribution or such wide use: for example other species of the genera Brahea and Washingtonia are typical of dry environments but are distributed in much smaller geographic areas, and are used only locally. For example, palmars (palm forests) composed of two Brahea and two Washingtonia species cover an area of 8533 ha in Baja California [40], while palmars of B. dulcis cover vast areas of unknown size in 13 states. Additionally, as was shown, the ecological behavior of B. dulcis includes the highest densities reported for the genus, and the highest rate of new leaf production per individual in spite of the arid conditions where it grows. In spite of its huge potential, B. dulcis is not well managed in central Mexico because of legal framework, substitution and disuse. In the context of rural Mexico, it would be very complicated to comply with all legal requirements, as they set forth a set of procedures that would be difficult to follow. A reform of the official standards on palm harvesting in Mexico is therefore strongly recommended. Instead of requiring a series of bureaucratic procedures for obtaining the raw material, the procedures should be streamlined and simplified, and there should be an incentive to have agencies in place that could act as a communication bridge between rural residents and government agencies.

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The K-segments of wheat dehydrin WZY2 are essential for its protective functions under temperature stress Dehydrins (DHNs), group 2 of late embryogenesis abundant (LEA) proteins, are up-regulated in most plants during cold, drought, heat, or salinity stress. All DHNs contain at least one K-segment, which is believed to play a significant role in DHN function by forming an amphipathic helix. In previous studies, wzy2, an YSK2-type DHN gene, was isolated from the Zhengyin 1 cultivar of Triticum aestivum under cold and drought stress treatment conditions. Four WZY2 truncated derivatives were constructed to knock out the K-, Y- or S-segment, which potentially affect the function of the protein. In vivo assays of Escherichia coli viability enhancement, in vitro lactate dehydrogenase (LDH) activity protection and ex vivo protein aggregation prevention assays revealed that WZY2 acted as a protectant and improved stress tolerance during temperature variation. The results also showed that unlike the truncated derivative without K-segments, the derivative containing two K-segments had remarkable effects that were similar to those of full-length WZY2, indicating that the K-segment is the major functional component of WZY2. Moreover, compared with the other segments, the first K-segment might be the most critical contributor to WZY2 functionality. In general, this work highlights the behavior of DHNs in relieving cold stress ex vivo and the contribution of the K-segment to DHN function. Introduction Abiotic stress such as cold, drought, heat, or salinity can cause plant dehydration and even death. As a typical response to such stress, most plants up-regulate the expression of dehydrins (DHNs), which are group 2 of late embryogenesis abundant (LEA) proteins; these proteins are believed to participate in plant environmental stress tolerance (Close, 1997). Most DHNs have one to three repeats of the Y-segment (T/VDEYGNP) near their N-terminus. Following the Y-segment, the S-segment is a tract of five to seven serine residues. The unique characteristic of DHNs is their lysine-rich consensus domain, EKKGIMDKIKEKLPG, which is referred to as the K-segment (Close, 1996) and plays an active role in protecting cellular macromolecules and lipidmembranes (Koag et al., 2009;Drira et al., 2013). It is possible that K-segments do form an amphipathic helix (Allagulova et al., 2003;Hanin et al., 2011). Five types of DHNs have been identified based on the existence of these three segments: Y m SK n , K n , K n S, SK n , and Y m K n (Close, 1996). The -segment, which is also noteworthy, is interspersed throughout DHNs, shows lower conservation and is frequently repeated (Close, 1997). In addition, the DHNs are shown to lack cysteine and tryptophan residues and to be rich in charged and polar amino acids. These properties confer them with highly hydrophilic and boiling-resistant features, similar to other intrinsically disordered proteins (IDPs; Tompa, 2002;Dyson and Wright, 2005). Some DHNs have been reported to be components of the freezing stress response, including WCOR410 (Triticum aestivum; Tsvetanov et al., 2000), COR15 (Arabidopsis thaliana; Baker et al., 1994), and CAS15 (Medicago sativa; Monroy et al., 1993). Furthermore, it was demonstrated that WCOR410 could improve freezing tolerance of transgenic strawberries (Houde et al., 2004). The α-helical conformation of the K-segment is essential for the binding of DHNs to anionic phospholipid vesicles, as confirmed by lipid vesicle-binding assays of three K-segment deletion derivatives of the maize DHN1 (YSK 2 ; Koag et al., 2009). The K-segments of wheat DHN-5 (YSK 2 ) protect Escherichia coli exposed to various stresses by preventing protein aggregation, and DHN-5 also acts as an antibacterial and antifungal factor during biotic stress (Drira et al., 2015). Moreover, DHN-5 was found to protect lactate dehydrogenase (LDH), β-glucosidase, and glucose oxidase from cold and heat damage in vitro (Brini et al., 2010;Drira et al., 2013). Interestingly, truncated forms of DHN-5 with one or two K-segments also showed the same function, whereas YS-truncated derivatives had no effect in these experiments (Drira et al., 2013). Two variants of YSK 2type VvcDHN1a have been reported: spliced DHN1a_s (YSK 2 ) and unspliced DHN1a_u (YS). Only DHN1a_s was reported to be involved in resistance to cold and drought as well as the growth of Botrytis cinerea (Rosales et al., 2014). There are two forms of DHN in Jatropha curcas seeds, JcDHN_1 (Y 3 SK 2 ) and JcDHN_2 (Y 2 SK 2 ); the transcript level change of JcDHN_2 was 8-fold that of JcDHN_1 at its maximum value, a time when the water content of the seed changed dramatically from 42% for mature seeds to 12% for desiccated seeds (Omar et al., 2013). In addition, LEA proteins might function as molecular chaperones (Wise and Tunnacliffe, 2004) to help non-natural proteins resist aggregation in vitro (Goyal et al., 2005). In previous studies, we cloned the full-length cDNA of the DHN wzy2 (accession no. EU395844, YSK 2 ) from the wheat cultivar Zhengyin 1. wzy2 expression varies depending on genotype, stress type, and stress duration (Huang et al., 2009;Zhu et al., 2012). Furthermore, quantitative real-time PCR analysis of wzy2 showed that this gene could be induced by low temperature, anoxia, indoleacetic acid, methyl jasmonate, abscisic acid, and gibberellin treatments (Zhu et al., 2014). To further understand the function of the K-segment of DHNs, we generated four truncated WZY2 constructs; each construct retained different conservative segments of the protein. Our data provide evidence that the K-segment plays a significant role in WZY2 function. This segment is critical for maintaining bacterial growth, enhancing LDH activity, and preventing protein aggregation during temperature stress. Construction, Expression, and Purification of WZY2 and its Truncated Derivatives Full-length and truncated wzy2 cDNAs were amplified with specific primers (Supplementary Table S1). All the forward primers contained an EcoR I site (underlined), and the reverse primers contained a Hind III site (underlined). PCR reactions to achieve the desired Y-, S-, and K-segment deletions were performed following the protocol for overlap extension PCR (Heckman and Pease, 2007) using TransStart FastPfu DNA Polymerase (Transgen, P.R. China). The open reading frame (ORF) of wzy2 was amplified from the plasmid wzy2-pMD19-T with the wt_F/wt_R primers (Junyi et al., 2012). The PCR products were digested with EcoR I and Hind III and ligated into the expression vector pET28a (Novagen, USA), which had been digested with the same enzymes. The recombinant plasmid was transformed into E. coli strain BL21 (DE3) according to Novagen's protocol. The plasmid wzy2-pET28a, containing the full-length wzy2 gene cDNA, was used as the template for the generation of the truncated derivatives. To obtain recombinant proteins without the specified segments (i.e., K1, K2, YS, and K1K2), eight primers were designed and employed in the PCR protocol. Some primers included the nucleotide sequence that spanned the region targeted for deletion (Figure 1). The first K-segment deletion variant ( K1) was generated with primers wt_F/wt_R; the amplified product mixture of wt_F/k1b and k1a/wt_R (amplified from wzy2-pET28a) at a 1:1 ratio as the template for this reaction. To remove the second K-segment (K2) to obtain K2, primers wt_F/k2 were used. To generate the variant K1K2 lacking both K-segments, PCR was performed with primers wt_F/k2. The K1 PCR amplified fragments were used as the template. The strategy to generate variant YS, with deleted Y-and S-segments, was based on wzy2-pET28a. Three pairs of primers (i.e., k4a/k4b, k4c/k4d, and k4e/wt_R) were utilized to generate three small products from this plasmid, and the mutant protein YS was created by splicing these pieces together. All PCR products were inserted into the pET28a vector following digestion with EcoR I and Hind III, and the vector was transformed into E. coli BL21 (DE3). All recombinant constructs were confirmed by DNA sequencing. Escherichia coli BL21 (DE3) strains carrying recombinant plasmids WZY2, K1, K2, YS, or K1K2 or the control vector pET28a were grown in Luria-Bertani (LB) medium with 50 μg/mL kanamycin at 37 • C until an OD600 = 1.0 was reached. Protein expression was induced by 1 mM isopropyl β-Dthiogalactoside (IPTG) for 3 h. After the induction period, the bacteria were harvested by centrifugation at 6,000 × g for 10 min at 4 • C, and the pelleted cells were suspended with phosphatebuffered saline (PBS; pH 7.0). The E. coli cells suspended in PBS were placed in liquid nitrogen for 5 min and then successively incubated in a 90 • C water bath for 30 min with manual agitation every 10 min. The upper clear supernatant was transferred to a fresh tube after centrifugation at 12,000 × g for 15 min at 4 • C, and a second centrifugation step was required. Soluble proteins were purified with a gel filtration system with Sephacryl S-100 High Resolution medium (GE Healthcare, USA) and verified by gel electrophoresis. E. coli Stress Tolerance Assay To evaluate temperature stress tolerance, 1 mL of culture medium from each IPTG-induced sample was taken, and the samples were adjusted to equal cell concentrations. The samples were incubated at different temperatures (0, 37, and 50 • C) for 20 min before being diluted at an appropriate ratio for spotting onto LB basal plates. Cell survival was measured as the ratio of colony-forming units (CFUs) between the treatment (0 or 50 • C) and control (37 • C). The experiment was repeated three independent times in triplicate for each sample. LDH Protection Analysis Lactate dehydrogenase activity was used as a marker to evaluate the protective function of DHN during cold and heat stresses. The assay was based on that described by Drira et al. (2013), with minor modifications. LDH (EC1.1.1.27, from rabbit muscle, Sigma-Aldrich, USA) was dissolved in 10 mM sodium phosphate (pH 7.4) at a final concentration of 10 μg/mL. Purified WZY2, its truncated variants, HIS (from the control vector), and bovine serum albumin (BSA, Sigma-Aldrich, USA) were all prepared at 20 μg/mL with 10 mM sodium phosphate (pH 7.4). The LDH assay reaction buffer contained 2 mM NADH, 10 mM pyruvic acid, and 10 mM sodium phosphate (pH7.4). Equal volumes of LDH enzyme solution and protein solution were mixed for temperature stress treatments. For the cold stress test, we incubated LDH and the protein mixture on ice for 0, 2, 4, 6, 8, 10, 12, or 24 h and transferred 70 μL of the mixture into 630 μL of reaction buffer at the end of the incubation. After thorough mixing, the samples were incubated at 25 • C for 30 s, and the absorbance at 340 nm was measured at 30s intervals for 6 min using a microplate reader (SpectraMax M2, Molecular Devices, USA). Triplicate reactions were set up for each treatment. The LDH reaction data were analyzed with ancillary software (SoftMax Pro, Molecular Devices, USA). A high temperature stress test was also performed by incubating the LDH and protein sample mixtures in a 45 • C water bath and adding 70-630 μL of the reaction buffer at 0, 10, 20, and 30 min. The LDH activity was then measured as in the cold stress test. Plant Materials and Growth Conditions The winter wheat (T. aestivum L.) cultivar Zhengyin 1, from which wzy2 was first isolated, was used in this study. Seeds were obtained from the College of Life Science of Northwest A&F University in Yangling, China. The plants were grown in a commercial soil mix under 200 μE m −2 s −1 light at 18/25 • C (night/day) temperature with a 16-h photoperiod. Wheat Leaf Blade Epidermal Cell Transient Expression Assay The vector pTF486, containing cauliflower mosaic virus (CaMV) 35S promoter-driven enhanced GFP (eGFP), was used in this study (Yu et al., 2008). To create a C-terminal GFPtagged fusion protein, the ORF of wzy2 was amplified using primers GFP_F and GFP_R1 (Supplementary Table S1) and then inserted into the pTF486 vector at the Nco I site (underlined) to generate the recombinant plasmid P35S::WZY2::GFP. The same set of primers was used to generate the constructs P35S:: K1::GFP and P35S:: YS::GFP. P35S:: K2::GFP and P35S:: K1K2::GFP, which were created using primers GFP_F and GFP_R2 (Supplementary Table S1). All the generated recombinants were confirmed by DNA sequencing and prepared for transient expression in wheat leaf blade epidermal cells. The desired plasmids were introduced into the host cells using a gene gun. Young leaves of Zhengyin 1 at the third leaf stage were selected and disinfected with 70% ethyl alcohol for 10 s, followed by rinsing three times with sterile water. These disinfected leaves were then snipped to 3-cm-long pieces and grown on Murashige and Skoog (MS) solid medium at 25 • C for 4 h in the dark. Five constructs and a control vector were used for transfection via the PDS-1000/He gene gun system (Bio-Rad, USA), per the manufacturer's protocol. After 24 h of incubation, the wheat leaf tissues were kept at 4 • C for 72 h and then rewarmed at 25 • C for 24 h. GFP fluorescence in the transfected wheat leaf blade epidermal cells was examined periodically (at 4 • C at 0, 12, 24, 48, and 72 h, and at 25 • C at 12 h and 24 h) during the incubation using a Leica DM5000 fluorescence microscope system (Germany). Statistical Analysis All data were analyzed with a t-test or two-way ANOVA using GraphPad Prism version 5.0 (USA). Significant differences were considered at a P-value less than 0.05. Sequence Analysis of WZY2 and its Truncated Derivatives To determine whether the K-segment is necessary for WZY2, we constructed a series of truncated recombinant WZY2 proteins (Figure 1 and Supplementary Figure S1). Full-length WZY2 contains a Y-segment (Y), an S-segment (S), and two K-segments (K1, K2). The derivative proteins K1 and K2 had either the first K-segment (K1) or the second K-segment (K2) deleted. YS lacked the Y plus S elements but retained both K-segments. For K1K2, the K1-and K2-segments were removed, but the Y plus S segments were retained, in contrast to YS. DNA sequencing was used to verify that the mutants were only deleted for the selected regions. Dehydrins are considered IDPs (Hughes and Graether, 2011), which do not have a unique, well-defined protein structure (Hughes et al., 2013). Using the disordered region prediction tool PONDR-Fit 1 , the intrinsically disordered characteristics of these full-length, and truncated proteins were calculated (Dunker et al., 2002); the scores were all higher than 0.5, indicating that all five proteins are disordered. There were three low-score fragments in the amino acid sequence of WZY2: 20-50, 70-90, and 125-150, corresponding to the regions between the Y-and S-segments, K1-segment, and K2-segment, respectively (Supplementary Figure S2). A consistent conclusion could be inferred from the curves of the WZY2 truncated derivatives. Moreover, the curve obtained for YS was similar to that of WZY2, except for a slight decrease in the first low-score region (Supplementary Figure S2). The 3D structures of WZY2 and its truncated derivatives were predicted using Phyre2 2 . As expected, WZY2 was predicted to contain the random coil and two α-helices related to the K-segments. All nine amino acid residues involved in the helicesprotein interaction were polar amino acids, and seven of them were lysines (Figure 2). YS also showed two α-helices and seven polar amino acids, including five lysines that involved in interactions between helices and protein (Figure 2). K1 and K2 contained one α-helix each, with only two residues ( K2) in the interaction (Figure 2). K1K2 was predicted to adopt a loosely folded structure with a random order (Figure 2 To investigate whether WZY2 and its truncated derivatives enhance stress tolerance in vivo, the cell viability of E. coli transformed with WZY2, K1, K2, YS, K1K2, or the control vector was measured under freezing and thermal conditions. These cultures were treated at 0, 37, or 50 • C, as described earlier in Section "Materials and Methods, " and survival ratios were calculated. A temperature of 37 • C was chosen as the standard condition, and the control vector was used as a negative control. The bacterial cells overexpressing WZY2 and YS showed the best viability under cold stress, followed by K1 and K2. The growth of E. coli with K1K2 or the control vector was ∼10% lower than that of the others (Figure 3A and Supplementary Table S2). The survival rates of the cells overexpressing WZY2, YS, K1, K2, and K1K2 were up to 2.8-, 2.8-, 2.5-, 2.5-, and 1.8-fold, respectively, compared with the controls after heat treatment ( Figure 3B). In both stress treatments, YS, similar to WZY2, showed a better effect on improving viability than did K1K2. These data confirmed that the K-segments of WZY2 are crucial for the temperature tolerance of E. coli. WZY2 and its Truncated Derivatives Protected LDH Enzyme Activity Under Freezing and Thermal Stress Conditions Because LDH loses its activity during freezing and thermal conditions, we selected this enzyme to evaluate the protective effect of WZY2 and its truncated derivatives. The time-dependent loss of LDH activity was measured as described previously in a method that employed BSA and PBS buffer as the positive and negative controls, respectively. WZY2 and its truncated derivatives were purified by a gel filtration system and verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Supplementary Figure S3). LDH activity before treatment was regarded as 100%. After incubation in an ice-bath for 24 h, HIS did not show any protective effect on LDH, with rates of activity loss that were similar to the buffer condition ( Figure 4A). In contrast, LDH activity increased nearly 20% in the presence of WZY2, which was higher than that with BSA (12%; Figure 4A). YS exhibited a similar protective effect to that of BSA, and the activity rates reached 102% up to the end of the freezing stress ( Figure 4A). When LDH was incubated on ice in the presence of K1, K2, or K1K2, the activities showed the same trend, decreasing to ∼45-50%. After 4 h of incubation with K1 or K2, LDH still retained more than 80% of its activity, and this retention was much longer than that observed for K1K2 (2 h; Figure 4A). The effects of WZY2 and its truncated derivatives with regard to protecting LDH activity during heat stress were also evaluated. In the first 10 min, LDH activity with HIS and buffer decreased nearly 50%, with only 30% remaining after 30 min ( Figure 4B). In contrast, LDH inactivation was dramatically reduced in the presence of WZY2, with 87% of the activity being FIGURE 2 | Three-dimensional structure prediction of WZY2 and its truncated derivative polypeptides. The K1-segment is shown in yellow, and the K2-segment is in blue. The amino acid residues involved in the helices-protein interaction are shown in yellow (in the K1-segment), in green (with the K1-segment), in blue (in the K2-segment), and in orange (with the K2-segment). FIGURE 3 | The cell viability of Escherichia coli transformed with WZY2, K1, K2, YS, and K1K2 or the control vector when subjected to temperature stress. E. coli cells were exposed to 0 • C (A) or 50 • C (B) for 20 min, and CFUs were calculated. The cell survival ratio was derived from a comparison of CFUs at 37 • C. Three independent assays were performed, and SE are included. Values are the mean ± SD (n = 3). Significant differences in the survival ratio are indicated as * P < 0.05 or * * P < 0.01, which were evaluated with a t-test. retained after 30 min ( Figure 4B); WZY2 also protected LDH activity more effectively than BSA. The protective effects of the truncated derivatives on LDH activity resulted in similar rates of enzyme activity; among the truncated derivatives, YS and K1K2 showed the highest and lowest protection efficiency, respectively ( Figure 4B). The protective effects of WZY2 and YS were similar to each other, and these proteins offered greater protection than the other truncated forms; K1, K2, and K1K2 were also similar to each other in terms of their protective effects and offered a moderate level of protection. WZY2 and its Truncated Derivatives Prevented Protein Aggregation Under Cold and Rewarming Conditions The WZY2 (or K2, YS, K1K2)::GFP fusion protein was overexpressed in Zhengyin 1 leaf epidermal cells ( K1::GFP could not be observed), and GFP fluorescence was examined by fluorescence microscopy. All fusion proteins and GFP were observed in the nucleus and cytoplasm prior to low temperature stress. In dehydrated cells, some green fluorescence spots (white arrow in Figure 5) were found near the plasma membrane where the fusion proteins aggregated. The pattern of aggregation varied during cold and rewarming treatments. Control GFP diffused throughout the entire cell, and this pattern did not change with an increase or decrease in temperature. The accumulated WZY2::GFP and YS::GFP proteins disappeared on the third day of cold stress (Supplementary Figure S4). K2::GFP was found to diffuse from the spots after 48 h of cold treatment, and the aggregation dissipated within 24 h of rewarming (Supplementary Figure S4). The distribution of K1K2::GFP showed no change compared to controls during the entire treatment, similar to GFP (Supplementary Figure S4). The merged image of the GFP channel and bright field for WZY2::GFP showed that the fusion proteins were located adjacent to the space between the epidermal cells and mesophyll cells before cold stress. No fusion proteins were found in this area after 48 h of cold treatment, even though plasmolysis was occurring (Figure 5). To maintain the consistency of the experiment, each fusion protein was observed in one cell, and images were captured under the same conditions. Discussion Dehydrins are a group of intrinsically disordered proteins that respond to abiotic and biotic stresses (Graether and Boddington, 2014; Rosales et al., 2014). The importance of DHNs has been demonstrated by genetic and protein evidence in vitro; however, an ex vivo, or in vivo protective mechanism has not been elucidated (Graether and Boddington, 2014). The wheat DHN WZY2 is induced by drought, low temperature, and other stresses (Zhu et al., 2014). The data presented in the present paper indicate that WZY2 exerts its function as a protectant. It increases E. coli viability, protects LDH activity and inhibits protein aggregation during temperature variation. The K-segment is a major functional component of this DHN. The proteins with two K-segments (WZY2 and YS) had the most significant impact on improving cold and heat stress tolerance in all the experimental systems employed in this study. YS performed in a manner similar to WZY2 with regard to maintaining bacterial growth, enhancing LDH activity and preventing protein aggregation. The recombinant proteins that contained one K-segment (i.e., K1 and K2) had complex effects, and K1 and K2 had similar effects that were smaller than those of WZY2 and YS both in vitro and in E. coli cells. K1::GFP could not be observed in wheat leaf epidermal cells under fluorescence microscopy. Although the recombinant protein with no K-segment ( K1K2) exhibited higher LDH activity protection efficiency than the controls, it resulted in almost identical consequences as the controls in other assays. In our study, K1, K2, Y plus S, or K1 plus K2 were removed from full-length WZY2 to generate the truncated derivatives K1, K2, YS, and K1K2, respectively. DNA sequence analysis verified that except for the removed component(s), the remainder of the proteins remained intact in the constructs. Since no additional amino acids was removed, K1K2 differed from the recombinant protein YS (from TaDHN5; Drira et al., 2013) in protecting LDH activity under freezing and thermal stresses. Group 1 and group 3 LEA proteins have been shown to function as molecular chaperones that protect citrate synthase and LDH from aggregation due to water stress (Goyal et al., 2005). LEA proteins are not classical chaperones but are more likely unstructured proteins, such as α-synuclein (Kumar et al., 2014;Manda et al., 2014) and EhPDI (Mares et al., 2014). Chaperones will translocate to specific locations to carry out their functions (Vaseva et al., 2012), and DHNs behave in the same way. To provide the most realistic intracellular microenvironment for WZY2, wheat Zhengyin 1 leaf tissues were used, and single cells were selected to measure each recombinant protein. This work tested the functions of protective agents in ex vivo experimental systems for the first time and further evaluated the results of other experiments in vitro (LDH activity) and in vivo (bacterial viability). Previous studies on dynamic changes in DHN protein localization in cells were performed using immunohistochemical methods. Immuno-gold labeling of LTI29 (A. thaliana) indicated that this protein changed location from the cytoplasm to the plasma membrane in cold acclimated (Puhakainen et al., 2004). Comparing immunomicroscopy images of wheat Irkutskaya ozimaya seedlings grown at 4 and 22 • C, it was clear that the density of DHNs increased significantly in the rough endoplasmic reticulum, mitochondria, and intercellular space at low temperatures (Romanenko et al., 2010). One paper reported dynamic DHN movement in protoplasts. Actinidia chinensis DHN1 is normally located in the nucleus and then moves to the cytoplasm near the plasma membrane in response to osmotic stress (Qiu et al., 2001). Based on available evidence, DHNs move toward the plasma membrane and intracellular membrane systems during cold acclimation, a result that is consistent with DHNs' binding gain of membrane stability in vitro (Koag et al., 2003(Koag et al., , 2009Rahman et al., 2010Rahman et al., , 2011aRahman, 2012). However, our results conflict with this notion. Indeed, WZY2, YS, and K2 left areas near the plasma membrane under cold stress. This result indicated that unlike the DHNs mentioned above, the primary function of WZY2 is to protect biomacromolecules and prevent protein aggregation. Analysis of the circular dichroism (CD) spectra of DHN1 (YSK 2 ) and its truncated proteins with anionic lipid vesicles showed that wild-type DHN1 and K2 (the deletion mutant lacking the second K-segment) adopt more α-helical structures than K1 (the deletion mutant lacking the first K-segment; Koag et al., 2009). The comparison of the CD spectra of mutant proteins with K-segment deletions in the presence of SDS showed that K2, but not K1, was nearly equivalent to normal DHN1 (Koag et al., 2009). It should be noted that we could not observe the K1 recombinant in wheat leaf epidermal cells. Our hypothesis is that K1::GFP may be an unstable protein in wheat leaf epidermal cells or that the first K-segment may be the most critical segment in this wheat expression system, with its deletion leading to reduced or no WZY2 expression. This speculation requires further investigation. The procedure for monitoring dynamic changes in the localization of GFP-tagged DHNs in our system was rapid and easy to perform and showed the natural movements of the DHNs. However, not all stresses were suitable, such as the high temperature stress, because the leaf tissue could not survive for 48 h at high temperatures. It would be worthwhile to apply this method to the study of osmotic, salinity, and heavy-metal ion stresses. The DHNs showed different behaviors in different stress situations with regard to factors such as the pathway and strength of dynamic movement. If the movement of DHNs is predictable, these proteins will be potential candidates for the experimental systems employed in this study. The data represented in this paper strongly suggest that WZY2 is essential for the maintenance of the cell survival rate, the protection of LDH enzyme activity, and the prevention of protein aggregation under temperature stress. The results illustrate that K-segments play a significant role in WZY2 function, especially the first K-segment. Further studies are needed to determine which proteins regulated by WZY2, how WZY2 protects proteins from aggregating ex vivo and in vivo, and the contribution of the K1-segment to the function of WZY2. Author Contributions WY proposed the ideas and designed the experiments. WY, HL, HL, and YX performed the experiments. WY and YZ analyzed the data. WY drafted the manuscript and revised it. JY and LZ ensured that all work was appropriately investigated. All authors approved the final manuscript. Supplementary Material The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls. 2015.00406/abstract Figure S1 | Comparison of the amino acid sequences of full-length WZY2 and the deletion mutants. The primary amino acid sequences and Y-, S-, and K-segments are shown. Hyphens indicate the deleted positions. Figure S2 | Disordered regions predicted with PONDR-Fit software for WZY2 and its truncated derivative polypeptides. Green lines represent the confidence scores of the disorder prediction. Predictions were evaluated with a standard, such that scores of 0.0-0.5 indicated order and 0.5-1.0 indicated disorder.

v3-fos

2018-12-14T20:25:43.674Z

2015-02-01T00:00:00.000Z

56100152

s2

OF ORGANOPHOSPHOROUS PESTICIDE RESDUES IN SAMPLES OF BANANA , PAPAYA , AND BELL PEPPER The objective of this study was to monitor 11 organophosphorus pesticides in samples of papaya, bell pepper, and banana, commercialized in the metropolitan area of Vitória (ES, Brazil). The pesticides were determined by an optimized and validated method using high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). All three samples exhibited a matrix effect for most of the pesticides, mainly with signal suppression, and therefore the calibration curves were produced in matrices. Linearity revealed coefficients of determination (r2) greater than 0.9895 for all pesticides and recovery results ranged from between 76% and 118% with standard deviation no greater than 16%. Precision showed relative standard deviation values lower than 19% and HorRat values lower than 0.7, considering all pesticides. Limits of quantification were less than 0.01 mg/kg for all pesticides. Regarding analysis of the samples (50 of each), none of the pesticides exceeded the maximum residue limit determined by Brazilian legislation. INTRODUCTION With large, fertile lands and favorable weather for agriculture, Brazil is one of the world's main producers and suppliers of food, reaching third place in the global ranking of fruit production (41.5 million tons) in 2010, with significant banana and papaya crops. 1 During the same year, the country was one of the largest exporters of fruits and vegetables of Latin America, thus contributing to the growth of Brazilian agribusiness, which was corroborated in 2012, when the export revenues raised to 910 million dollars. 2 Among several fruits and vegetables commercialized in Brazil, papaya, banana, and bell pepper achieved great prominence in the country's economy. Carica papaya, commonly known as papaya, is produced in large scale in Brazil, which holds the second place in global production, only behind India. 3 Moreover, the exports of this fruit accounted for 23 thousand tons, causing it to be the fifth most exported product in January/2013. 4,5 Similarly, banana (Musa spp.) is one of the most produced and consumed fruits worldwide, mainly in Brazil, which has a crop area of 71,253 hectares and exports accounting for 82 thousand tons in 2013. 4 It is a fruit known for its nutrients that contribute for elevated nutritional and energetic levels. 6 Bell pepper (Capsicuum annuum), a vitamin C source and rich in minerals, stands among the top ten vegetables consumed in Brazil and around the world. In 2011 the Brazilian production was approximately 365.7 million tons and its sales accounted for near 1.5 billion reais. 7 Fruits and vegetables are crucial for a healthy diet, due to the presence of significant amounts of nutrients and minerals. However, at the same time they may contain hazardous substances, such as pesticides. 8 Agricultural procedures with them still the most common way in order to achieve food production in adequate quantities, as they are an efficient tool against pests that can jeopardize production and lead to food shortage. 9 Between 2007 and 2012, the amount of pesticides used in crops was 346.6 thousand tons, making Brazil the world's leader in pesticide commerce in 2010 and the second largest market for pesticides in 2012, the first being the United States. 10,11 Amid the classes of most toxic compounds and with greatest incidence in food in Brazil stand the insecticides, among which the organophosphorus compounds are prominent, accounting for more than 36% of the global market. 12 With the rising of the organophosphorus, which are less persistent in the environment, organochlorine pesticides, though less hazardous, were substituted for being more resistant in the nature. 13 In particular, organophosphorus are highly neurotoxic, presenting inhibitory function of cholinesterase, which controls the nervous system, leading to an elevation in levels of the neurotransmitter acetylcholine at nerve endings and causing neurobehavioral losses in humans. 14,15 Besides, pesticides are connected to other chronic health issues, such as cancer and adverse reproductive effects, dermatitis, respiratory problems, and can also be mutagenic and teratogenic. 16,17 In Brazil there are several monitoring programs focusing on evaluating food quality and on implementing controlling actions for pesticides residues, as an endeavor to minimize the exposure of the population to these contaminants. 18,19 The Program for Analysis of Pesticides Residues in Food (PARA), coordinated by the National Sanitary Surveillance Agency (ANVISA), and the National Program for Residues and Contaminants Control (PNCRC), ran by the Department of Agriculture, Livestock, and Supply (MAPA), are responsible for the monitoring of pesticides residues and for instituting maximum residue limits (MRLs) in food. In recent years, pesticides residues were found in about 65% of analyzed samples, revealing bell pepper to be the vegetable with largest percentage of irregular samples, 90%, and papaya with 20% of unsatisfactory samples. [20][21][22] It is relevant to keep in sight that, in the same year, the most abundant chemical group found in crops was the organophosphorus one, with 38% nonconformities. [20][21][22] In spite of the existence of the above-mentioned programs, they only monitor small quantities of samples at each region, and that is why they do not present the thorough reality of the incidence of pesticides in food. Therefore, the objective of this paper is to validate a method using sample preparation by QuEChERS method 23 and high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) to monitor organophosphorus residues in samples of banana, papaya, and bell pepper from ten different market from at Vitória and Vila Velha cities (Espírito Santo, Brazil), during a three-month period. Samples From September to November, 2013, 50 samples of Cavendish bananas, 50 of papaya, and 50 of green bell pepper were purchased from markets and open markets at the metropolitan area of Vitória (ES, Brazil). Samples were collected every fifteen days in polyethylene bags to store 1.0 kg of each sample (randomly chosen) and conducted immediately to the laboratory for the analyses. Extraction Samples of banana, papaya, and bell pepper were extracted according to the QuEChERS method described by AOAC Official Method 2007.01. 24 Each unit of the studied food, from its respective sample, was cut in four pieces. Two were discarded and the remaining ones were homogenized in a food processor. Part of the ground sample was stored under refrigeration for retest. A 15 g portion was inserted into a 50 mL Falcon tube and then 15 mL of acetonitrile with 1% acetic acid, 6 g of anhydrous MgSO 4 , and 1.5 g of NaOAc were added to the system. Next the system was homogenized in an automatic agitator for 1 min and then centrifuged (Laborline Omega P.I.C microprocessor system centrifuge) at 3000 rpm for 1 min. An aliquot of 6 mL of the supernatant was transferred to a 15 mL Falcon tube that previously contained 150 mg of anhydrous MgSO 4 and 50 mg of PSA. Afterwards the system was once again homogenized in an automatic agitator and centrifuged at 3000 rpm for 1 min. The final extract was filtered through a membrane (0.45 µm pore, 13 mm, non-sterile) to a vial and finally injected into the chromatography. Extractions were conducted in duplicates and the samples were stored at low temperatures (-10 ºC to -30 ºC range) prior to the assays. Chromatographic conditions (HPLC-MS/MS) The analyses were conducted in an Agilent Technologies 1200 Series chromatography, with automatic sampler, quaternary pump, degassing system, and reverse phase C18 column (4.6 mm x 150 mm, 5 µm i.d., Agilent Eclipse XDB), kept at 35 ºC, following AOAC Offical Method 2007.1 with modifications. 24 The compounds were separated using as mobile phase Milli-Q water with 0.1% (v/v) formic acid (phase A) and methanol with 0.1% (v/v) formic acid (phase B). The elution gradient started at 40% B, staying at this level for 2 min, followed by linear growth up to 70% B in 5 min and up to 90% B in 8 min, and then kept constant for another 5 min. Reequilibration time was 2 min. Injected sample volume was 20 µL and the flow rate was constant at 0.8 mL min -1 . The chromatography was coupled to a mass spectrometer with triple quadrupole (API3200, Applied Biosystems), operating in positive (+5500 V) ionization mode. Ion source temperature was kept at 600 ºC and nitrogen was used as collision gas. Data were collected by multiple reaction monitoring (MRM) and processed by the Analyst ™ 5.0 software. HPLC-MS/MS parameters optimization Due to its sensitivity and selectivity for trace analysis in complex matrixes, liquid chromatography is a widespread technique to determine pesticides in large scale using MRM. As a way to obtain maximum sensitivity to identify and quantify the target compounds, the optimization of all mass spectrometer parameters was performed for each analyte in a 0.8 µg mL -1 acetonitrile solution with 0.1% (v/v) acetic acid. Optimization of the parameters is displayed in Table 1. Method validation Method performance was evaluated according to the reference document DOQ-CGCRE-008, by the National Institute of Metrology, Normalization, and Industrial Quality. 25 Evaluated analytical parameters were: selectivity (matrix effect), linearity, precision (repeatability and intermediate precision), accuracy (recovery), and limits of detection (LOD) and quantification (LOQ). The validation process was developed using spiked samples and pesticide-free samples as blank (with previously confirmed absence of pesticides in samples of banana, papaya, and bell pepper). Selectivity and matrix effect Selectivity assesses the studied substance in presence of other interfering compounds in the sample. Matrix effect is observed by the increase or decrease of the detector response of an analyte in the matrix extract when compared to the same substance analyzed in an organic solvent. This evaluation was done by comparing detector responses (peak areas), analyzing a standard solution of pesticides in solvent (acetonitrile 0.1% (v/v) acetic acid) and in extracts of each matrix (banana, papaya, and bell pepper) at three spike levels (0.05, 0.025, and 0.0125 mg L -1 ), with seven repetitions each. The effect was evaluated by the statistical test ANOVA. Groups with p<0.05 were considered statistically different. Matrix effect was quantified by comparison of the slope of the curve done in matrix extract and in solvent, estimated by the following equation. 19 Matrix effect (%) = 1 100 slope of the curve in matrix slope of the curve in solvelt Linearity corresponds to the ability of a method to present results directly proportional to the concentration of the analyte within a determined range. Linearity was assessed by coefficient of determination (r²), obtained by linear regression. In order to determine the linear range, the statistical method of least squares was applied and points with average residuals smaller than 15% were approved. For the smallest concentration, however, this criterion was raised up to 20%. After evaluation of the linearity, the analytical curve was plotted with at least 5 points by external standard. Precision Precision was tested by repeatability and intermediate precision parameters in three concentration levels (0.05, 0.025, and 0.0125 mg L -1 ) added to each matrix extract. For the repeatability assays, seven consecutive repetitions in each concentration level for each matrix were performed. For intermediate precision, 21 tests in three different days (7 per day) were done. In order to validate the acceptability of precision, HorRat values were calculated by the following expression: HorRat = RSD/PRSD, where RSD is the relative experimental standard deviation and PRSD is the predicted relative experimental standard deviation, given by Horwitz equation: PRSD = 2 (1-0.5logC) , where C is the concentration. Precision was considered adequate when the HorRat value remained below 2.0. Limits of detection and quantification LOD reveals the smallest concentration of the studied substance that can be detected, but not necessarily quantified. LOQ, on the other hand, is the lowest concentration by which the analyte can be measured with a certain confidence level. Determination of LOD and LOQ was done by the signal-to-noise ratio method, using proportions of 3:1 and 6:1, respectively, obtained from seven sample blank injections (pesticide-free matrixes). Practical quantification limit (PQL) was defined as the lowest concentration of the linear working range. Recovery Recovery is defined as the proportion of the quantity of the target substance present in or added to the samples from which it is extracted and able to be quantified. Recovery of pesticides was estimated by analysis of spiked samples in three different concentration levels, with seven repetitions each. Each test utilized different concentrations according to the matrix's correspondent linear range: banana (0.009, 0.025, and 0.01 mg kg -1 ), papaya (0.009, 0.125, and 0.025 mg kg -1 ), and bell pepper (0.00625, 0.0125, and 0.1 mg kg -1 ). Recovery was determined by the arithmetic mean of the obtained values from the following equation: where C1 is the concentration of the analyte in the spiked sample, C2 is the concentration of the analyte in the non-spiked sample, and C3 is the concentration of the analyte added to the spiked sample. Statistical analysis All results are displayed as arithmetic mean ± standard deviation. Data were analyzed by ANOVA, followed by a post-hoc Tukey test and significance was accepted when p<0.05. Analyses were conducted by Statistica™ 6.0 Statsoft, Inc. RESULTS AND DISCUSSION All 11 studied pesticides (azinphos-methyl, mevinphos, sulprofos, demeton-S-methyl sulfone, diazinon, fensulfothion, fenthion, etoprophos, coumaphos, dichlorvos, and tetrachlorvinphos) belong to the class of organophosphorus compounds, the major group of pest controllers used in agricultural production. They are organic compounds that contain phosphorous and generally present an ester in their structure. 26,27 The pesticides selected for this study are not authorized for banana, papaya, and bell pepper crops; 20 consequently, the employed analytical methodology has minimum required performance limit (MRPL) of 0.01 mg kg -1 . 20 But the fact that these pesticides are still authorized for other crops makes their improper use persistent, bringing along serious consequences for the environment and for human health, as they are classified as highly or moderately toxic. Matrix effect It is known that matrix interferents can increase or decrease the signal of the analyte in chromatography tandem mass spectrometry, generating the so-called matrix effect. Both matrix components and pesticide structure can affect the analyte signal. Therefore, as an alternative, the calibration curve in matrix extracts was used, as it is recommended when matrix effect occurs. 28 Results revealed that more than 78% of the analytes presented matrix effect in all different concentrations for all samples, except for demeton-S-methyl sulfone, which did not display matrix effect in banana extracts, and for fensulfothion, in bell pepper. It was also observed that the lowest concentration, 12.5 mg L -1 , did not present significant difference when comparing the curves in solvents and in matrixes in the majority of the analyses. Furthermore, from the three examined matrixes, papaya showed more intense matrix effect than banana and bell pepper. Curbelo et al. analyzed matrix effect in banana leaves and proved that all tested analytes (seven organophosphorus and one tiadiazinone) exhibited relevant matrix effect when assessed in samples. 6 In other study, matrix effect was evaluated by comparison of the detector response for standards of seven pesticides prepared in solvent with those prepared in matrixes of banana and mango, and the effect was also observed. 29 As matrix effect was confirmed for most analytes, it was quantified by the comparison of the slope of the curves done in matrixes of banana, papaya, and bell pepper and the slope of the curve done in pure solvent for each pesticide. Negative or positive values from the equation mean matrix-induced suppression or enhancement of the signal, respectively. Depending on the results, distinct matrix effects can be observed: it was considered low signal suppression when the result was between -20% and 0%; low signal increase when between 0% and 20%; moderated effect when between -50% and -20% or 20% and 50%; and strong signal suppression or signal enhancement when the values were below -50% or above 50%, respectively. 19 In this study, most of the pesticides displayed a decrease in signal, that is, from the 31 samples of papaya, banana, and bell pepper, 26 had negative matrix effect results. In a HPLC-MS system, signal suppression occurs more often, since the matrix components decrease the efficiency of spray formation and/or decrease the amount of generated ions. 30 Three cases revealed mainly strong signal suppression, but papaya was significantly stronger when compared to banana and bell pepper. Signal suppression or signal enhancement responses for the three matrixes altogether were: low in 16.13% of the samples; moderated in 19.35%; and strong in the 64.52% remaining. These results are shown in Table 2. Linearity As matrix effect was noticed in the majority of the analyzed matrixes, calibration cures were done in matrixes of banana, papaya, and bell pepper for the eleven pesticides in concentrations ranging from 0.2 to 0.00625 mg kg -1 , mainly due to matrix effect. Results exhibited that the coefficient of determination for all pesticides were above 0.9903, except for fenthion, which had r² of 0.9895 in papaya samples (Table 3). Residuals found in the three samples remained below 19.7% for the PQL and below 14.9% for the remaining points, which is under the limits established by ANVISA (lower than 15% for regular points and lower than 20% for the lowermost concentration). The coefficient of determination of the curve done in banana samples were between the 0.9920 and 0.9992 range, which agrees with studies done in Spain, which certified that the detector response for the banana matrix was linear for the tested organophosphorus pesticides tested with coefficients above 0.992. 6,31 For papaya samples, on the other hand, r² varied between 0.9895 and 0.9994. In a study done by Navickiene et al., linearity was also determined by papaya sample blank and the results revealed linear response with r² ranging from 0.962 to 0.999. 28 For bell pepper samples, coefficients of determination were between 0.9940 and 0.9998. As the matrix effect was significant, quantification was developed using the calibration curves obtained by the samples of banana, papaya, and bell pepper. Precision After the conclusion of the 90 analyses, it was observed that all organophosphorus in the three studied samples and in the three concentrations presented RSD below 20%, corroborating other authors who assessed precision in banana and papaya samples. 28,32,33 Values of RSD (%) in the repeatability studies varied from 2.8 to 18.4 for banana; from 3.1 to 13.8 for papaya; and from 4.2 to 14.3 Accuracy Method accuracy was estimated by recovery assays that were conducted for the pesticides at three spike levels. For the recovery done in the banana matrix, the concentrations were 0.1, 0.025, and 0.009 mg kg -1 ; for papaya, 0.025, 0.0125, and 0.009 mg kg -1 ; and for bell pepper, 0.1, 0.0125, and 0.00625 mg kg -1 , with seven replicates for each level in the three samples. These results are displayed in Table 2. All tested pesticides exhibited recoveries between 76 and 118% with RSD between 0.7 and 16%. Carneiro et al., who used a QuEChERS method along with UHPLC-MS/MS in order to quantify 128 pesticides in bananas, also reached good recovery results (70-120%), with RSD below 20% for most tested analytes. 32 Navickiene et al. applied a method based on the dispersion of solid phase matrix and GC-MS to determine seven pesticides in papaya and mango, obtaining average recovery rates from 80 to 146%, with RSD between 1.0 and 28%. 29 Limits of detection and quantification LOD and LOQ were evaluated by the injection of banana, papaya, and bell pepper matrix blanks and of the lowermost concentration of the calibration curve, respecting the linear working range of each analyte. LODs of all three matrixes were between 0.02 and 2.14 µg kg -1 ; 0.03 and 1.18 µg kg -1 ; and 0.11 and 2.89 µg kg -1 , respectively (Table 3). For the LOQs, on the other hand, the results ranged between 0.05 and 4.28 µg kg -1 for banana; 0.05 and 2.36 µg kg -1 for papaya; and 0.22 and 5.79 µg kg -1 for bell pepper. Sample monitoring Method development was used for monitoring and analyzing pesticides residues in samples of banana, papaya, and bell pepper that were bought during three months at ten markets and open markets. All the 150 samples were extracted by QuEChERS and then analyzed by HPLC-MS/MS. This extraction method, along with chromatography and spectrometry techniques, has been widely used for the analysis of pesticide residues in food, presenting itself as an excellent option for cleaning up complex samples. Peak identification was verified by comparison of retention times, by formation of precursor ion and product ion, and by injection of the spiked sample. No residue of the assessed pesticides was found in the studied samples. Only dichlorvos was detected in a single bell pepper sample, at the first fortnight of October, but in a level below the LOQ. The presence of pesticides in food is so worrying that ANVISA created PARA in 2001 as a way to monitor pesticide residues in several foods in Brazil. In 2010, this program tested 18 crops, including papaya and bell pepper, in 26 states. In Espírito Santo State, 6 papaya samples and 6 bell pepper samples were analyzed. Only one papaya sample was irregular, while all bell pepper samples presented nonconformities. The major pesticide class found in the analyses was the organophosphorus one, which present high toxicity and is not allowed for these crops, causing occupational risk to rural workers and other harmful effects to consumers. 21 The report of the activities of the 2011/2022 biennium released by PARA monitored 67 foods in Espírito Santo and 22 irregularities were found, among which the ones related to papaya and bell pepper crops. From the eight analyzed samples of each food, one was illegal for papaya and 5 for bell pepper. In the same way as the previous year, the organophosphorus class was the major chemical group found in the study, accounting for 38% of the irregularities. Another governmental agency that works with pesticide residue monitoring is MAPA, which coordinates the PNCRC of products with vegetal origin. During the 2011/2012 crop year, PNCRC evaluated 23 crops, including banana, papaya, and bell pepper, revealing 100%, 91.3%, and 37.3% conformity rates, respectively. Papaya was the only analyzed sample from Espírito Santo, with 90.81% conformity, as 9 were irregular from the 98 tested. 34 24 samples of papaya were also monitored in the state, during the 2012/2013 crop year, and 7 presented nonconformities. 35 After the comparison between the data of this study with those of PARA and PNCRC, it can be concluded that the results were analogous. PARA did not find any residues of the pesticides analyzed by this study when testing the same crops. Likewise PNCRC revealed 100% conformity for all three samples regarding the organophosphorus pesticides analyzed by both studies (azinphos methyl, coumaphos, demeton, diazinon, dichlorvos, etoprophos, fenthion, mevinphos, and sulprofos). Therefore it can be noticed that the producers from Espírito Santo either are not using these pesticides or are utilizing them correctly; nevertheless, it is important to keep in sight that the pesticides assessed by this study are not allowed by Brazilian legislation for these crops. CONCLUSION The developed and validated multiresidue method for the simultaneous determination of 11 organophosphorus in samples of banana, papaya, and bell pepper was efficient for this application. Significant matrix effect with signal suppression for most compounds was observed; thus, the analytical curves were done in the matrixes. None of the pesticides in the monitored samples exceeded the allowed limit, which indicates that these forbidden compounds are not being used by their producers in Espírito Santo.

v3-fos

2019-04-04T13:05:21.109Z

2015-07-09T00:00:00.000Z

93828602

s2

Extraction and Characterization of Fibres from the Stalk and Spikelets of Empty Fruit Bunch 1Department of Mechanical Engineering, Higher Technical Teachers’ Training College, The University of Bamenda, P.O. Box 34, Bambili, Cameroon 2Department of Mechanical Engineering, Ecole Normale Superieur d’ Enseignement Technique (ENSET), University of Douala, Douala, Cameroon 3Department of Chemistry, Faculty of Science, University of Buea, Buea, Cameroon 4School of Engineering, University of Technology, Kingston, Jamaica Introduction The sustainable exploitation and development of resources and materials is one approach to ensuring sustainable technologies. Biobased industries are receiving increased interest as one response to sustainability issues associated with the exploitation of nonrenewable resources. The effectiveness of this response, however, depends on the judicious use of biomass as feedstock for chemical industries, biomaterials, and bioenergy. The cultivation of crops for biomass has gained commercial status, but there is stiff competition on arable land for food production due to a rapidly increasing world population. This has led to increased interest in agricultural waste, which is abundant and cheap, as a renewable feedstock [1]. The cultivation and processing of important cash crops such as cocoa, cotton, rice, and oil palm generate large quantities of agricultural waste which presents disposal problems but is increasingly regarded as a resource for biobased industries. Palm oil is produced in many tropical countries, notably in Africa and Asia, and it is the largest global source of edible oil. It constitutes 38.5 million tons or 25% of world's total oil and fat production [2]. Cameroon, a West African country, produces 3.5 tons of oil/ha/year [3]. Waste from oil palm cultivation and palm oil processing include empty 2 Journal of Applied Chemistry fruit bunches (EFB), shell, palm kernel, sludge, palm oil mill effluent (POME), fronds, and trunk [4]. EFB is a highly fibrous, mineral rich material [5,6] and is one of the most important solid waste fractions from the oil mill [7]. The EFB is 23% of the weight of a fresh fruit bunch and an oil mill with a capacity of 60 t of fruit bunches per hour produces more than 54,000 t of empty fruit bunches per year [8]. EFB has low commercial value and poses a disposal problem due to its bulky nature. Conventionally, it is burnt, disposed of in landfills, or composted to organic fertilizer [9]. The burning of EFB is, however, no longer recommended as it causes air pollution. There is therefore the need to optimize the use of EFB so as to solve these problems and enhance its judicious us as feedstock for valuable products in the chemical, energy, and biomaterials sectors. Current uses of EFB from large and medium-scale industrial units include recycling for nutrients and moisture control in farms, fuel, mushroom cultivation, biogas production, cellulose, and fibre extraction. Fibres from EFB have found applications in the production of composites, textiles, cellulose, and paper [10]. Interest in biofibres has grown as they are increasingly viewed as a green alternative to the more conventional fibre reinforcements such as glass fibres [11]. The composition and fibrous character of EFB determines its various applications such as recycling for nutrients and moisture control in farms, fuel, mushroom cultivation substrate, biogas production, cellulose, and fibre extraction. In addition to its high mineral content, EFB is also rich in holocellulose (82.4% of extractives-free organic fraction) and lignin (17.6% of extractives-free organic fraction) [10]. Lignin, cellulose, and hemicellulose contents are variable, but comparable to soft and hardwoods, indicating a potential for pulping and use as raw material in biorefineries [10]. The pretreatment of EFB fibres using merely water, acid, and alkali increases sugar production and high removal of hemicellulose and/or lignin in the fibres [12]. Palm oil production is carried out in two types of facilities, distinguished by the scale of operations and processes/technologies used [13,14]. Generally, processing units handling up to 2 tons of fresh fruit bunches per hour are considered to be small-scale, while large-scale mills are able to process more than 10 tons per hour. Large plantations operate modern industrial mills where the full fruit bunches are steam-sterilized before the nuts are extracted mechanically using a hammer mill. The oil is subsequently pressed out, separated and purified [15]. The global palm oil market is dominated by these operators, but artisanal producers in West and Central Africa still contribute a significant proportion of the production [13,14]. Smallholders who process their own fruit operate small, usually manuallyintensive mills, where the nuts are extracted from bunches before cooking. In some traditional practices, the fruitcontaining spikelets are separated from the stalk before the fruit is extracted. The spikelet is subsequently used as fuel while the stalk is scattered in gardens or simply discarded. The full fruit bunches do not undergo steam treatment as is the case in modern mills [16]. Data available on the chemical composition and other fibre characteristics make no difference between the spikelet and the stalk [17][18][19]. The fresh fruit observed growing directly on the spikelet supports the structural development as spikelets and not separate rachises [20]. However, the observation on artisanal oil production in some parts of Cameroon where the spikelet is separated from stalk before fruit extraction has prompted a more systematic characterization of EFB spikelet and stalk. The fibres of spikelet are higher in dry weight than those of the stalk from EFB of the oil palm. This alternative is expected to lead to reductions in steam requirements for sterilization and thus presents the potential for more sustainable operations. It might be more labour-intensive, but Nkongho et al. [13] make a case for more labour-intensive artisanal processing methods in a context of high unemployment. This paper presents results of investigations of the physical characteristics, chemical composition, tensile strength, microstructure, and response to alkali treatment of fibrous biomass from EFB stalk and spikelet from 2 types of palm oil production facilities in Cameroon. Materials and Methods Empty fruit bunches were obtained from an industrial operator, Cameroon Development corporation (C.D.C.) (Mondoni and Idenua Mills), and a small-scale operator in Muyuka, all located in Fako Division of the South West Region of Cameroon. The empty fruit bunches were separated manually into stalk and spikelet and then shredded. The stalks were defibred in one pass through a screw-type shredder fitted with and 7.5 horsepower motor while the spikelets required three passes. Proximate composition of fibres was determined and the fibres were then cleaned with mild dilute alkali (2 wt%) before characterization for length, diameter, and tensile strength. The fibres were further subjected to alkali delignification and then characterized for morphology and chemical composition. Techniques used included standard wet chemical methods, scanning electronic microscopy (SEM) with Energy dispersive X-ray (EDX), XRD, and tensile testing. Shredding, Cleaning, and Strong Alkali Treatment of EFB Fibres. Fibre yield was determined on stalk and spikelet from 10 bunches. Samples for strong alkali treatment were oven dried at 100 ∘ C for 24 hrs and ground in a domestic blender. The ground fibre was sieved and fibres of particle size less than 35 mesh were subjected to pretreatment with 5% NaOH with boiling for 2 hrs, followed by treatment with the 10% NaOH at 20 ∘ C for 16 hrs prior to strong alkali treatment. This consisted of treating samples with 24% KOH-2% H 3 BO 3 (boric acid) at 20 ∘ C for 2 hrs. The slurry obtained after KOH treatment was filtered and the residues collected and washed several times with distilled water in order to remove impurities. The product was oven dried at 50 ∘ C for 8 hrs and samples taken for XRD, FT-IR, and TGA analyses. Galacturonic Acid Content. The galacturonic acid content was determined by colorimetry using a modified mhydroxydiphenyl sulphuric acid method [21]. Standards were Journal of Applied Chemistry prepared using galacturonic acid at concentrations of 29-100 microgram per millilitre. This determination was done in triplicates. Determination of Tensile Strength. Tensile strength test was conducted in the mechanical laboratory of ENSET Douala, Cameroon, using a modified fibre traction device designed by Rodrigue (2008) [22] for testing filaments of raffia. Proximate Analysis. Basic cations Ca, Mg, and K are extracted by dry ashing in a muffle furnace at 500 ∘ C, diluted using a dilute acid mix of HCl/HNO 3 , and analyzed using the atomic absorption spectrophotometer [23] and reported as a %. Total N was determined from a wet acid digest [24] by colorimetric analysis [25]. All determinations were carried out on extractives-free samples. Scanning Electron Microscopy (SEM). The surface morphology of samples was examined by a Scanning Electron Microscope (SEM) (JEOL JSM-5600 or TESCAN VEGA III XMU, Q150 TL). Prior to examination, samples were prepared by mounting about 0.5 mg of powder onto a 5 mm × 5 mm double sided carbon tape, on an aluminum stub. The powder was then sputter-coated for 40 seconds with carbon or gold. 2.6. X-Ray Diffraction. The XRD diffractograms of the precursors and the decomposition products were recorded on a Bruker D8 Advance X-ray diffractometer using a Cu K radiation source ( = 0.15406 nm, 40 kV, and 40 mA). Scans were taken over the 2 range from 10 ∘ to 100 ∘ in steps of 0.01 ∘ at room temperature in open quartz sample holders. FTIR. FT-IR spectra were recorded from 4000 to 400 cm −1 on a PerkinElmer Spectrum Two universal attenuated total reflectance Fourier transform infrared (UATR-FT-IR) spectrometer. Results and Discussion 3.1. Physical Properties. The percentages of fibres in the stalk and spikelet of EFB dry weight were in the ranges of 20-32% and 68-80%, respectively. Figures 1(a) and 1(b) represent the stalk and the spikelet of EFB after manual separation using a cutlass and their fibres, while Figures 1(c) and 1(d) show that spikelets yield more fibre per EFB than stalk. The dry weight results of the stalk and spikelet of 10 different EFBs are presented in Figure 2. The quantities of fibres from the histogram show that the stalk and spikelet were 20 to 32% and 68 to 80% dry weight, respectively. The result indicates that there are more fibres in the spikelet than in the stalk from EFB of the oil palm tree. The diameter of EFB fibres (Table 1) showed important variability along the length. The fibres from the stalk were bigger than those from the spikelet. The length of stalk and spikelet varied from 10 mm to 105 mm and 80 mm to 368 mm giving an average The mild alkali treatment with dilute NaOH was intended to remove mostly oils, some lignin, and a limited amount of acidic hemicelluloses from the fibre surface. Hemicelluloses are a highly heterogeneous group of structural polysaccharides which are found very closely associated with cellulose and lignin in the plant cell wall, thus making their extraction difficult. Several studies have found that the use of alkali (NaOH, LiOH, or KOH) together with boric acid additions significantly enhances the release of hemicelluloses [27,28]. The enhanced release is attributed to the ability of the hydroxyl and borate ions to form plyanions with the hemicelluloses, as well as the ability of the alkali to cause swelling of the crystalline cellulose. The polyanions may be carboxylates, borate complexes of some hemicelluloses and alcoholates. The KOH/Boric acid mixture represents a relatively milder but more effective extractant for releasing hemicelluloses within the cell walls at high alkali concentrations and without mercerization. Galacturonic Acid Content. Galacturonic acid content was higher in stalk than the spikelet of EFB crude extract. Also more galacturonic acid was released at higher sodium hydroxide concentration. Galacturonic acid content of the different EFB extract is presented in the histogram in Figure 3. The EFB crude extracts were coded using the following pretreatment conditions as indicated on Table 2. The higher galacturonic acid content correlates with higher cation content; notably, K + and is also a reflection of differences in content of noncellulosic polysaccharides. This difference might explain the differences in response of the fibres to alkali treatments as well as mechanical properties of the fibres. The results also suggest differences in water absorptivity. Ramadevi et al. [29] found that alkalization of abaca fibres which leads to a reduction in hemicelluloses content resulted in a reduction in water adsorption. Mechanical Properties. Fibres from spikelets of EFB showed higher strength than those of the stalk of EFB while the extension at failure of the fibre from the stalk was higher than that of the fibre from the spikelet (Table 3). Fibres from spikelets of EFB have higher strength than those of the stalk of EFB while the extension at failure of the fibre from the stalk is more than that of the fibre from the spikelet. Chemical Composition. The results showed some differences in chemical composition of fibres from stalk and spikelet with the most significant differences recorded in the ash and potassium content (Table 4). Sodium levels are considerably higher in EFB from the plantation closer to the sea (Bakingili), reflecting the importance of soils/environment. The difference in ash content may be due to different cultivation and production area of oil palm in Fako Division. FTIR Analyses. FTIR spectra of fibres dewaxed with acetone (Figures 4(a) and 4(b)) and after delignification (Figure 4(c)) do not show significant differences between spikelet and stalk. Absorption peaks at 3600 cm −1 -3000 cm −1 (strong and broad) for both stalk and spikelet fibres are assigned to the hydroxyl group, present in cellulose, lignin, and hemicelluloses [30]. The absorption bands located at [34], whilst bands at 1566 cm −1 and 1251 cm −1 are attributed to the aromatic skeletal structure of lignin [35]. The Glycosidic C-O-C band occurred at 1022 cm −1 . FTIR of delignified fibres (Figure 4(c)) showed almost complete elimination of the intensity of the bands at 1718 cm −1 and the absence of the band at 1556 cm −1 , indicating significant elimination of hemicelluloses and lignin [36]. There is however evidence of residual ligin (band at 1251 cm −1 ) and hemicellolose (band at 1718 cm −1 ) in both samples. 3.6. Morphological Analysis. SEM micrographs of fibres cleaned with dilute alkali and after KOH-Boric acid delignification treatments are shown in Figures 5, 6, and 7. Figure 5 shows the SEM of fracture and cross-sectional area of fibres from stalk and spikelets. Figure 5(a) reveals the arrangement of fibrils in helical spirals. These micrographs also reveal the widespread occurrence of silica bodies that have previously been reported by other workers [37]. Figures 5(b), 5(c), and 5(d) show important differences in the density and porosity across sections of spikelet ( Figure 5(b)) and stalk (Figures 5(c) and 5(d)). Spikelet fibres appear more compact than stalk fibres. SEM of alkali-delignified fibres reveal remnants of fibril bundles in spiklet fibres and a more extensive degradation of the fibril bundle structure in stalk fibres. SEM micrograph of delignified stalk reveals sheets which are most probably highly crystalline cellulose sheets obtained by chain folding [38]. This difference in response suggests that spikelet fibrous biomass is more recalcitrant to pretreatments than stalk fibrous biomass. SEM/EDX Analysis. SEM of the surfaces of spikelet fibres treated with hot water (100 ∘ C) only and 2% NaOH at 25 ∘ C are shown in Figure 7. The micrographs reveal silica bodies spread uniformly over the fibre surface. Figure 5(b) shows that sodium hydroxide treatment removes most of the silica bodies from the surface. EDX spectra are presented in Figures 8(a), 8(b), 8(c) and 8(d) for selected stalk and spikelet fibres subjected to different treatments. These reveal that the mineral content is largely due to the presence of potassium, silicon, calcium magnesium, and aluminum and that the enrichment in these elements is closely associated with the silica bodies. Journal of Applied Chemistry X-Ray Diffraction (XRD) . The X-ray diffraction of the stalk and spikelet is shown in Figure 9. XRD data was collected for four different samples of treated stalk and spikelet fibres. Both stalk and spikelet-derived material shows a very broad peak at about 17 ∘ and a relatively strong and sharp peak at 22.1 ∘ . The broad peak at 2 angle 17 ∘ is probably due to amorphous cellulose or the overlapping of the (101) and reflections [39], while the peak at 2 of 22.1 ∘ is assigned to the strong (200) reflection of cellulose [36,[40][41][42]. The diffractogram of spikelet-derived cellulose shows additional sharp peaks, including one at 2 reflection angle of 35 ∘ . The latter has been assigned to the (040) reflections of cellulose. This peak appears to be present in the diffractogram of the stalk-derived cellulose, but it is considerably broadened and weaker. This suggests a higher level of transformation of the stalk biomass following the strong alkali treatment. Conclusion Fibres from stalk and spikelet of EFB were pretreated and studied using different techniques. The dry weight percentages of fibre from stalk and spikelet of EFB and their diameters were in the ranges 20-32%, 68-80% and 200-715 m, 150-650 m, respectively. The diameters varied along the length of the fibre. Stalk fibres were richer in potassium, which is the major plant nutrient. The SEM clearly showed silica bodies found in craters on the surface of the fibres. The cross section area showed irregular porosity and significant differences between the porosities of fibres from stalk and spikelet. Spikelet fibres were shown to be stronger and the results from SEM and XRD characterization of alkali-treated material show that spikelet fibres undergo less transformation than stalk fibres. The ease of transformation of biomass to bioenergy using sustainable pretreatment technologies is a key step in biorefineries. Based on composition and response to treatment, the stalk fibres show more promise as feedstock for nutrient recycling or bioenergy due to their significantly higher potassium contents and lower recalcitrance. The spikelet fibres look more interesting for use as reinforcing fibres due to their higher strengths and recalcitrance. These results suggest that there is potential for improving sustainability through the adoption of more appropriate processing of full fruit bunches (FFBs) for the extraction of palm nuts for palm oil production where spikelet and stalk are separated prior to fruit extraction.

v3-fos

2016-05-04T20:20:58.661Z

2015-11-02T00:00:00.000Z

1106472

s2

In Vitro Fermentation of caprine milk oligosaccharides by bifidobacteria isolated from breast-fed infants This study was conducted to investigate the catabolism and fermentation of caprine milk oligosaccharides (CMO) by selected bifidobacteria isolated from 4 breast-fed infants. Seventeen bifidobacterial isolates consisting of 3 different species (Bifidobacterium breve, Bifidobacterium longum subsp. longum and Bifidobacterium bifidum) were investigated. A CMO-enriched fraction (CMOF) (50% oligosaccharides, 10% galacto-oligosaccharides (GOS), 20% lactose, 10% glucose and 10% galactose) from caprine cheese whey was added to a growth medium as a sole source of fermentable carbohydrate. The inclusion of the CMOF was associated with increased bifidobacterial growth for all strains compared to glucose, lactose, GOS, inulin, oligofructose, 3'-sialyl-lactose and 6'-sialyl-lactose. Only one B. bifidum strain (AGR2166) was able to utilize the sialyl-CMO, 3'-sialyl-lactose and 6'-sialyl-lactose, as carbohydrate sources. The inclusion of CMOF increased the production of acetic and lactic acid (P < 0.001) after 36 h of anaerobic fermentation at 37°C, when compared to other fermentable substrates. Two B. bifidum strains (AGR2166 and AGR2168) utilised CMO, contained in the CMOF, to a greater extent than B. breve or B. longum subsp longum isolates, and this increased CMO utilization was associated with enhanced sialidase activity. CMOF stimulated bifidobacterial growth when compared to other tested fermentable carbohydrates and also increased the consumption of mono- and disaccharides, such as galactose and lactose present in the CMOF. These findings indicate that the dietary consumption of CMO may stimulate the growth and metabolism of intestinal Bifidobacteria spp. including B. bifidum typically found in the large intestine of breast-fed infants. Introduction Evidence suggests that the microbial community of the human gastrointestinal tract (GIT) has a core function in maintaining host health by preventing the colonisation of pathogens, 1 degrading dietary compounds, producing metabolites able to be utilised by the host (e.g. short chain fatty acids, SCFA) 2 and maintaining mucosal immunity. 3 Particularly important in early life, the composition of the GIT microbiota influences the development and maturation of the foetal/neonatal GIT and consequently the overall health of the infant. During the perinatal period, the infant GIT is colonised by a relatively simple microbial community, initially derived from vaginal microbiota and maternal faeces. 4 The type of feeding (breast versus formula feeding), local environment, and antibiotic treatment may also play an important role in determining and maintaining the microbiota composition of the infant GIT. 5,6 The GIT microbiota of breast-fed infants has long been thought to be dominated by bifidobacteria, compared to that of adults and formula-fed infants. 7 However, there are conflicting reports regarding differences in the relative abundance of these bacteria between breast-and formula-fed infants. [8][9][10] Moreover, although some studies have demonstrated a specific diversity of bifidobacterial species present in formula-fed infants is more like the adult bifidobacterial diversity, 11,12 culture or culture-independent methods, able to provide information on the identity and relative abundance of bifidobacteria to the species level, have not been used to compare the faecal microbiota of breast-fed vs. formula-fed infants. While the mechanisms for these differences in microbial colonisation and establishment are not fully understood, it is possible that the numbers of bifidobacteria species in breast-fed infants may be enhanced by the oligosaccharides in human milk (which collectively form the third largest solid component of milk (5 to 23 g l ¡1 ) 13 after fat and protein). Milk oligosaccharides (sugar polymers, typically 3 to 10 units) have been studied extensively because of their marked influence on the GIT microbiota (i.e. prebiotic activity). Human milk contains a variety of oligosaccharides able to stimulate the growth of specific commensal GIT microbiota, 14 as well as stimulate the development of the immune system 15 and prevent adhesion of pathogens to epithelial tissues. 16 Infant-type bifidobacterial species such as B. longum subsp longum, B. longum subsp infantis, B. bifidum and B. breve, for example, contain enzymes specifically involved in the metabolism of human milk oligosaccharides (HMO). 17 Modern infant formulas are increasingly supplemented with a mixture of plant derived oligosaccharides, such as fructo-oligosaccharides (FOS) and inulin (DP D 10 to 60), and lactose derived oligosaccharides, such as galacto-oligosaccharides (GOS). 18 These substrates elicit non-specific bifidobacterial growth (the "bifidogenic effect"), 19 and lack the complexity and diversity of HMO, so are unlikely to successfully mimic the structure specific effects of HMO. Fucosylated and sialylated HMO, for example, are complex oligosaccharides known to act as bacterial adhesin analogs and/or to mimic the receptors used by enteric pathogens to adhere to the surface of host epithelial cells. 16 Sialyloligosaccharides support the growth of breast-fed, neonate specific commensal bacteria, such as Bifidobacterium longum subsp. infantis ATCC15697. 21 It has been suggested that these acidic oligosaccharides, in addition to eliciting a more specific bifidogenic effect, may combat influenza infections 22 and ulcers caused by Helicobacter pylori. 23 Other activities reported include regulation of inflammation by reducing adhesion of human leukocytes on activated endothelial cells, and promotion of commensal enteric bacterial proliferation. 24,25 The predominant forms of sialyloligosaccharides found in human milk, 3 0 -and 6 0 -galactosyl-lactose are also the most prevalent oligosaccharides in caprine colostrum, milk and whey. Therefore, the supplementation of infant formula with caprine milk oligosaccharides (CMO) is likely to stimulate the growth and metabolism of bifidobacterial strains typically found in breast-fed infants, and also to mimic the structure specific effects of HMO with associated infant health benefits. Understanding whether bifidobacterial strains from breast-fed infants are able to utilize CMO as a fermentable substrate is an important factor in the potential use of CMO as an infant formula supplement. To explore the effects of CMO on the growth and metabolism of specific postnatal bifidobacteria, this study aimed to investigate the ability of bifidobacterial strains, isolated from exclusively breast-fed neonates, to ferment a caprine milk oligosaccharide enriched fraction (CMOF) (especially sialyloligosaccharides), prepared from caprine cheese whey, using a recently published method, 26 and to produce SCFA (compared with other prebiotics or milk sugars). Results Genetic characterization of bifidobacterial strains A total of 17 bifidobacterial strains were isolated from faecal samples of 4 exclusively breast-fed infants. The strains were positively identified by amplification and sequencing of a 498 bp region of the 16S rRNA gene corresponding to the V2 to V3 variable regions as B. bifidum (n D 4), B. longum (n D 6) and B. breve (n = 7) (Fig. 1). For the 6 B. longum strains, digestion of a partial 16S rRNA gene amplicon with Sau3AI was consistent with B. longum subsp longum. RAPD sub-typing of the 17 bifidobacterial strains was undertaken to provide an indicative level of genetic heterogeneity that could be explored with further phenotypic assays. The dendrogram separated the 17 strains into 4 main clusters, broadly corresponding to the 3 different Bifidobacteria species (Fig. 1). A clear distinction between B. longum subsp longum strains isolated from infant 2 (AGR2170 to AGR2174) and infant 3 (AGR2176) was discernible by RAPD analysis. A single strain representative of each fermentation profile was selected for quantifying SCFA production (Fig. 3, B. bifidum AGR2166 (a), AGR2168 (b); B. longum AGR2173 (c), AGR2176 (d); B. breve AGR2177 (e), AGR2175 (f)). Only acetic and lactic acid were produced after 16 h and 36 h incubation for all the strains tested (Fig. 3). Acetate was shown to be present at time 0, due to the presence of sodium acetate (0.5%) in the basal media. The same media batch was used to investigate the growth of the different bacterial strains/carbohydrates for comparison purposes. All strains (except AGR2168), produced higher concentrations (P < 0.001) of acetic and lactic acid in medium supplemented with CMOF at 36 h post-inoculation compared to the medium supplemented with the combo preparation (Fig. 3). AGR2168 only grew in the medium supplemented with CMOF, thus comparisons with the combo preparation were not possible. Formate was measured over the 36 h fermentation period but was produced at concentrations that were too low to quantify using our HP-LC methods. 27 Production of acetic and lactic acid by all the strains tested was associated with a decrease in overall pH of the culture over 36 h with the CMOF (Fig. 4). The drop in pH from 6.5 to 4.2 -4.9 was correlated with the final OD of the culture (rD -0.95; P D 0.001), and the concentration of acetic (rD -0.85; P=0.02) and lactic acid (rD -0.83; P D 0.03) produced. The concentrations of galactose, glucose, lactose, and GOS in a media supplemented with 1% CMOF were determined after 36 h of bifidobacterial incubation and compared to the concentrations immediately after inoculation (Fig. 6). These data indicate that lactose was depleted by all bifidobacterial strains tested. The degradation of lactose, GOS, and CMOF were likely to have increased the overall concentrations of glucose and galactose in the media, and were not fully utilised by the end of the 36 h incubation. B. bifidum AGR2166 and AGR2168 had lower levels of glucose and higher levels of galactose in their media compared to pre-incubated media and to the other strains after CMOF fermentation. AGR2166 also had higher levels of lactose, compared to other strains, which may have been associated with oligosaccharide catabolism by this strain. The GOS concentration in the B. longum AGR2173 and B. breve AGR2175 media after incubation did not differ from uninoculated media; however, 50% of the GOS present in the pre-incubated media was fermented by the other strains. Identification of genes encoding for exo-a-sialidase and associated sialidase activity Sialidase activity of the bifidobacterial isolates on CMOF was assessed using molecular methods to demonstrate the presence of sialidase-encoding genes and by measuring sialidase activity using a fluorogenic substrate. The presence of 3 reported sialidase genes (BBPR_1793 and BBPR_1794 from B. bifidum PRL2010 and HMPREF92 28_0182 from B. breve ACS-071-V-Sch8b), were confirmed by PCR amplification and DNA sequencing in the B. bifidum and B. breve strains from this study (Fig. 1). Sialidase enzyme activity was also analyzed in 6 selected isolates (highlighted in Fig. 1); 2 B. breve, 2 B. bifidum and 2 B. longum. Only the B. bifidum isolates (AGR2166 and AGR2168) had cellular sialidase activity when grown in the presence of CMOF ( Fig. 7), although B. bifidum AGR2168 had only limited ability to ferment the CMOF sialyloligosaccharides, 3 0 -and 6 0 -sialyl-lactose as a sole source of carbon (Fig. 2). Sialidase activity from B. bifidum was mainly cell-associated, although approximately 12% residual activity was also detected in the culture supernatant of these 2 strains (Fig. 7A). B. bifidum cellular sialidase activity in AGR2166 and AGR2168 was induced by all 4 substrates examined, and was significantly enhanced (P 0.01) in bacterial cell preparations taken from cultures grown in the presence of 3'and 6 0 -sialyl-lactose when compared to CMOF and combo. Despite the presence of 3 0 and 6 0 -sialyl-lactose in CMOF, there was no increase in sialidase activity of AGR2166 and AGR2168 grown in the presence of CMOF compared to the combo (Fig. 7B). Similar sialidase activity was observed from the culture supernatant taken from AGR2166 grown in the presence of 6'sialyl-lactose and CMOF, and this sialidase activity was higher than combo and 3 0 -sialyl-lactose (P < 0.001). Despite the presence of a sialidase-encoding gene in the B. breve isolates examined, no sialidase activity was observed. However, in contrast to the sialidase proteins from B. bifidum that have signal peptide cleavage sites and transmembrane helices, no such structural characteristics were associated with the sialidase from B. breve, suggesting a potential intracellular localization. Intracellular sialidase activity was not determined. Discussion This study investigated the in vitro effects of a CMOF on the growth of selected bifidobacteria isolated from 4 exclusively breast-fed infants. The CMOF (containing high concentrations (46%) of sialyloligosaccharides), supported enhanced growth of selected bifidobacteria strains isolated from breast-fed infants, and stimulated the in vitro production of lactate and SCFA, such as acetate. These results confirm the hypothesis, bifidobacteria isolated from the faeces of breast-fed infants are able to ferment CMOF, increasing bifidobacteria growth and metabolism. In a recent study, CMO was shown to increase the growth of human faecal Bifidobacterium spp in anaerobic batch culture, 28 although the specificity of bifidobacterial CMO consumption was not investigated. The B. breve, B. longum and B. bifidum strains isolated in this work, together with B. longum subsp infantis and B. adolescentis, are among the most prevalent species found in infants independent of their feeding regime. 12,29 Although only a small number of bifidobacterial strains were selected from each infant, previous work suggests that the infant microbiota in the GIT is heterogeneous but is dominated by 3-5 different bifidobacterial species. 12,19 The RAPD analysis undertaken in this study broadly agrees with the well-recognized level of clonal heterogeneity demonstrated among the B. breve, B. longum and B. bifidum strains as determined by genetic fingerprinting methods such as ribosomal intergenic spacer analysis (RISA) and RAPD. 12,29 Among the bifidobacterial species tested, B. bifidum (AGR2166) was shown to utilize both 3 0 -and 6 0 -sialyl-lactose as a sole carbon source to support growth (Fig. 2) with associated depletion of these same oligosaccharide isomers present in CMOF (Fig. 5). Enhanced depletion of 3 0 -and 6 0 -sialyl-lactose from CMOF by B. bifidum (AGR2166) was likely through cellassociated sialidase expression after induction with the same oligosaccharides (Fig. 7). B. bifidum (AGR2168), in contrast, displayed intermediate growth on 3 0 -sialyl-lactose (Fig. 2b) with partial (20%) utilization of 3 0 -and 6 0 -sialyl-lactose from CMOF ( Fig. 5) with cell-associated sialidase expression (Fig. 7). These data largely agree with previous work 30 that suggests a surface or intracellular location for the sialidase enzyme on B. bifidum. The residual activity of sialidase found in the B. bifidum (AGR2166 and AGR2168) culture supernatant therefore may be associated with cell wall debris and/or released enzyme present within the culture supernatant. B. longum and B. breve strains were unable to utilize 3 0 -and 6 0 -sialyl-lactose as a growth substrate when included as the only carbon source (Fig. 2c to 2f) and no expression of cell-associated sialidase was observed (Fig. 7). Limited depletion (5-15%) of these oligosaccharides by B. longum and B. breve strains was detected (Fig. 5) however, when grown in a CMOF enriched media. This partial depletion may have occurred through incomplete catalysis of 3 0 -and 6 0 -sialyl-lactose without fermentation of any resulting breakdown products or that these sugars were selectively adsorbed to the bacterial cells that were present in the media, which were then removed prior to analysis. 31 Contrasting oligosaccharide depletion observed between strains of the same species was in accordance with inter-strain heterogeneity shown in the RAPD analyses. B. breve AGR2175 and AGR2177, for example, both isolated from the same infant (3) and with similar growth profiles (Fig. 2), showed different oligosaccharides catabolism profiles (Fig. 5) which might indicate differential regulation or expression of enzymes involved in carbohydrate metabolism. Augmented microbial biomass associated with enhanced growth and fermentation of CMOF increased microbial fermentation end products such as acetate and lactate. These data agree with a previous study, where CMO was shown to increase the production of acetate, lactate and propionate in anaerobic batch culture inoculated with human faeces. 28 Formate may also be produced as an end-product of bifidobacterial fermentation with the inclusion of fructose or OF as the main carbohydrate source 27 but was produced at levels that were too low to detect using HP-LC. An absolute measurement of CMO utilization in this study was impossible due to the high concentrations (50%) of lactose, GOS, glucose and galactose in the CMOF. However, when used as a sole carbohydrate source, lactose, GOS, glucose and galactose did not support enhanced bacterial growth when compared to the CMOF. It is likely that the CMO component of the overall CMOF, is not only a fermentable substrate, but also stimulates the utilization of other simpler carbohydrates. The GlcNAc-containing oligosaccharides (6 0 -N-acetyL-glucosaminyl-lactose and lacto-N-hexaose), for example, have been reported as a growth factor stimulating lactose utilization by B. bifidum. 38 The mechanism through which these oligosaccharides are used remains to be identified, but studies on the utilization of HMO may provide some clues. 14,39 Certain bifidobacterial strains such as B. bifidum NCIMB41171 have the ability to synthesize long chain carbohydrates (such as GOS) from lactose and galactose using the transglycosylic activity of b-galactosidase. 40 Thus, it is difficult to precisely determine how much lactose was degraded to glucose and galactose through the hydrolytic activity of b-galactosidase, and how much GOS, if any, was produced by transglycosylic activity of b-galactosidase. 41 Lactose, the core of all HMO and CMO, and the main structure of galactosyl-lactose, is likely to be degraded to galactose and glucose in a catabolic reaction that requires b-galactosidase activity. B. bifidum, for example, contains both extracellular and intracellular b-galactosidases. 43 B. breve 41 and B. longum subsp longum, 43 on the other hand, have been reported to contain only intracellular b-galactosidases, and the high utilization of lactose by these strains may indicate that lactose is likely to be actively transported into the cells by a yet unidentified transporter. Although GOS is also hydrolysed to glucose and galactose by b-galactosidase, different strains have been shown to have differential consumption of selected GOS with different DPs. 44 The infant isolates (B. longum subsp infantis and B. breve) are able to more efficiently consume the GOS species with DP from 3 to 8, while B. adolescentis and B. longum subsp. longum exhibited differential consumption of selected DP. 44 These contrasting HMO and CMO utilisations suggest that niche specific adaptation abilities exist among various bifidobacterial species and strains, and with other components of the microbiota of the GIT. These complex effects cannot be reproduced by simple carbohydrate structures most often used as prebiotics. The ability of the bifidobacterial strains to utilize the different carbohydrates present in CMOF and/or stimulate the consumption of other carbohydrate sources is important to determine the effects of these milk components in the GIT microbiota. Although more than 8% of the identified genes from bifidobacterial genomes are predicted to be involved in carbohydrate metabolism, the ability to metabolise certain complex milk oligosaccharides is species and strain specific. 45,46 Analysis of the genes involved in carbohydrate utilization indicate that B. bifidum (JCM1254 and JCM7004) contain genes that encode specialized enzymes associated with the extracellular deglycosylation of milk oligosaccharides, including extracellular a-fucosidases, 47 b-galactosidases, b-N-acetylglucosaminidases 48 and a-sialidases, 30 which efficiently remove monosaccharides from complex milk oligosaccharides. B. bifidum and B. longum 49 also contain a membrane enzyme, lacto-N-biosidase, responsible for the cleavage of the bifidogenic HMO lacto-n-tetraose to lacto-n-biose 50 and lactose. The mono-and disaccharides released by this endoglycosidase (especially lacto-n-biose), are internalised by family 1 solute binding proteins, and metabolised. Family 1 solute binding proteins are part of a gene cluster conserved across all infant GIT-associated bifidobacteria, including B. bifidum, B. infantis, B. longum and B. breve isolates. 39,51 The same enzyme degradation mechanism may be responsible for the utilization of lacto-N-hexaose present in CMO. None of the selected bifidobacterial strains were able to utilize inulin as the sole carbohydrate source, but all except the B. bifidum strains were able to utilize oligofructose. The DP (oligofructose DP 2-10; HP inulin DP 11-60) is likely to influence the ability of bifidobacterial strains to utilize FOS as the sole carbon source. 52 However, bifidobacteria present in breast-fed infants may also be selectively stimulated by milk oligosaccharides instead of plant derived oligosaccharides. Previous studies confirmed the poor growth of B. bifidum strains on inulin type fructans, 53,54 but strain differences in b-fructofuranosidase production levels have been reported. 55 After weaning, with the introduction of plant derived foods, B. bifidum strains are likely to benefit indirectly from the fermentation of inulin type fructans by other members of the GIT microbiota through the lowering of the GIT pH, or the increased availability of monosaccharides as substrates. 53 In conclusion, faecal bifidobacteria species isolated from breast-fed infants are heterogeneous at the genetic level based on RAPD profiles, but also at the level of substrate utilization through the differing depletion of certain carbohydrate components of the CMO preparation including some oligosaccharides. CMOF was able to stimulate the growth of bifidobacteria commonly found in the GIT of breast-fed infants. CMO contained in the CMOF may also have stimulated the consumption of lactose, glucose, galactose and GOS. Comparing the selected strains, B. bifidum were better able to ferment CMOF, especially the sialyloligosaccharides, which may indicate that in vivo, this strain may benefit from CMO consumption. Defining and linking the utilization of specific oligosaccharide structures, such as the sialyloligosaccharides, to cultured bacteria will provide a scientific path for targeting infant health by establishing protective microbial communities, beneficial to their hosts and potentially applicable to different stages of human life and health states. Material and Methods Isolation of bifidobacteria Collection of faecal samples from healthy breast-fed infants was approved by the local human ethics committee (Massey University, Palmerston North, New Zealand). Bifidobacteria were isolated from faeces obtained from freshly soiled diaper/nappy of 4 exclusively breast-fed neonates on modified TPY agar (MTPY). 56 The plates were incubated anaerobically (93% CO 2 , 7% H 2 ) at 37 C for 48 h. Bacterial colonies were then individually picked, subcultured on fresh MTPY agar plates to obtain single colonies and again on anaerobic De Man-Rogosa-Sharpe (MRS, Oxoid, CM0361) agar slopes (pH 6.5 -7.0) supplemented with Lcysteine-hydrochloride (0.5 g l ¡1 ), at 37 C for 48 h, for storage at ¡80 C until further analysis. Bifidobacterial characterization The identity of the bifidobacterial isolates was confirmed using PCR and 16S rRNA gene sequencing using bif 164 (5-GGG TGG TAA TGC CGG ATG-3) and bif 662 (5-CCA CCG TTA CAC CGG GAA -3) primers. 57 Sequencing of the PCR product (15 ng) was performed using the bif 164 or bif 662 primers (3.2 nM) and 16S rRNA gene sequences were compared with known bacterial sequences available from GenBank database using BLAST. Utilization of CMOF by bifidobacteria The utilization of CMO (10 g l ¡1 , final concentration 1% [w/v]), GOS (2 g l ¡1 ), lactose (4 g l ¡1 ), glucose (2 g l ¡1 ) and galactose (2 g l ¡1 ) present in the CMOF was assessed in 6 bifidobacterial strains (2 B. bifidum, 2 B. longum, and 2 B. breve strains) selected on the basis of contrasting growth profiles. Each strain was incubated in triplicate, and culture supernatants (50 ml of each tube) taken immediately after inoculation, and at stationary phase (36 h), were analyzed by LC-MS and HPLC to evaluate carbohydrate depletion. The depletion of specific oligosaccharides present in CMOF was analyzed by the intensity of their specific masses using LC-MS data and reported as percentage of depletion compared to the concentrations in the control. The HPLC and LC-MS methods used were those described previously. 26 Bifidobacterial exo-a-sialidase genes The presence of sialidase genes described for B. longum and B. bifidum 46,64 and a putative B. breve (NC_017218.1) sialidase gene were evaluated in the 17 bifidobacterial strains. PCR primers were designed to amplify the DNA sequence encoding the glycosyl hydrolase (GH) family 33 domain of the sialidaseencoding genes ( Table 1) from B. longum, B. bifidum and B. breve. Sialidase assay The sialidase activity of selected bifidobacteria was determined using the fluorescent substrate 4-methylumbelliferyl-a-D-N-acetylneuraminic acid (4MU-Neu5Ac) (Sigma, M8639) as described previously. 30 Briefly, bifidobacterial strains were grown anaerobically in 5 ml of a semi-synthetic medium 63 supplemented with 10 g l ¡1 CMO (contained in CMOF), combo, or of 3'-syalyl-lactose or 6'-syalyl-lactose as the sole carbohydrate source for 24 h at 37 C. Six separate cultures of each of 2 B. bifidum, 2 B. longum, and 2 B. breve strains were examined, with each biological replicate assessed in triplicate. Enzyme activities from washed bacterial cells and culture supernatant were determined by comparing absolute fluorescence units (Afu) minus the blank, as described previously. 65 Statistical analysis Bacterial growth, SCFA profiles and sialidase production at 36 h of fermentation for each substrate were tested for normality and homogeneity of variances and compared by one-way analysis of variance (ANOVA) using GenStat (15 th edition SPS). Differences were considered significant at P 0.05. To identify the correlation between bacterial growth, pH and SCFA production, Pearson's Rank correlation factors and P values were calculated Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

v3-fos

2016-05-12T22:15:10.714Z

2015-12-10T00:00:00.000Z

4862812

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Role of Maternal Dietary Peanut Exposure in Development of Food Allergy and Oral Tolerance Background The impact of maternal ingestion of peanut during pregnancy and lactation on an offspring’s risk for peanut allergy is under debate. Objective To investigate the influence of maternal dietary peanut exposure and breast milk on an offspring’s allergy risk. Methods Preconceptionally peanut-exposed C3H/HeJ females were either fed or not fed peanut during pregnancy and lactation. The offsprings’ responses to peanut sensitization or oral tolerance induction by feeding antigen prior to immunization were assessed. We also assessed the impact of immune murine milk on tolerance induction pre- or post-weaning. For antigen uptake studies, mice were gavaged with fluorescent peanut in the presence or absence of immune murine milk; Peyer’s patches were harvested for immunostaining. Results Preconceptional peanut exposure resulted in the production of varying levels of maternal antibodies in serum (and breast milk), which were transferred to the offspring. Despite this, maternal peanut exposure either preconceptionally or during pregnancy and lactation, when compared to no maternal exposure, had no impact on peanut allergy. When offspring were fed peanut directly, dose-dependent tolerance induction, unaltered by maternal feeding of peanut, was seen. Although peanut uptake into the gut-associated lymphoid tissues was enhanced by immune milk as compared to naïve milk, tolerance induction was not affected by the co-administration of immune milk either pre- or post-weaning. Conclusion Maternal peanut exposure during pregnancy and lactation has no impact on the development of peanut allergy in the offspring. Tolerance to peanut can be induced early, even pre-weaning, by giving moderate amounts of peanut directly to the infant, and this is neither enhanced nor impaired by concurrent exposure to immune milk. Introduction Peanut allergy is an example of a defect in the development of oral tolerance, which refers to the normal suppression of systemic immune responses to antigens first encountered by the oral route. The prevalence of peanut allergy has dramatically increased in children within the last decade, affecting over 1% of school-aged children in the United States, United Kingdom, Canada, and Australia. [1][2][3][4] The majority of initial reactions to peanut and tree nuts occur on the first known ingestion [5], suggesting that sensitization likely occurred through another route, such as inhalation, epicutaneous, in utero, or through breast milk. Because maternal, rather than paternal, atopic status has a greater effect on the atopic outcome of the progeny [6], perinatal factors including maternal diet and immune status may play a critical role in the development of peanut allergy. Although dietary antigens, such as peanut, egg, and cow's milk proteins, have been detected in breast milk [7][8][9][10] in concentrations sufficient to cause symptoms in individuals already sensitized [10], observational studies provide mixed evidence about the potential role of exposure to peanut through breast milk in the initial sensitization phase. [11][12] All the studies possess potential recall bias and no prospective controlled human clinical trials are available. Furthermore, infant diet, which is typically linked to the maternal diet, may be a confounding factor. In fact, the recent randomized LEAP trial of oral peanut exposure in infancy showed early exposure to be highly efficacious in prevention of peanut allergy in high-risk individuals. [13] However, the role of maternal diet including or excluding peanut in the development of peanut allergy remains unclear. A murine model of peanut allergy can be a useful tool for initial investigation of interventions for peanut allergy. [14] The advantage of using an animal study is the ability to strictly control dietary and environmental exposure. Previous animal studies on maternal exposure showed a protective effect of ovalbumin (OVA) transfer through murine milk in offspring [15][16][17][18], which was reportedly dependent on TGF-ß in milk and induction of T regulatory cells in one study [15] or vertical transfer of IgG-OVA complexes in another study. [17] We have previously shown that ante-and perinatal feeding of low dose peanut to mothers prevented sensitization and anaphylactic reactions in their offspring during the first peanut exposure. [14] Protection was associated with increased peanut-specific IgG2a, which was related to the coadministration of peanut with a mucosal adjuvant during pregnancy and lactation. While the use of an adjuvant may be useful for therapeutic purposes, our goal was to address the impact of a normal maternal dietary exposure on the risk of allergy in the offspring. Prevention of food allergies by infant feeding practices serves as a simple, inexpensive approach to address the growing incidence of food allergy. We sought to assess the impact of maternal perinatal ingestion of peanut and other food allergens (without sensitization of mothers or co-administration with a mucosal adjuvant) on an offspring's risk of developing an allergy or ability to develop tolerance to food. In addition, the isolated impact of breast milk on tolerance induction was assessed. Because the production of food-specific IgG antibodies is a normal physiologic phenomenon following ingestion of foods and because maternal IgG antibodies have been associated with protection against sensitization in offspring [17], we used mothers with previous exposure and presence of antibodies as biomarkers of exposure. We show that, in mice, maternal perinatal ingestion of peanut has little impact on the development of tolerance or allergy to peanut in their offspring and that the early introduction of peanut directly to the infant, in the presence or absence of breast milk, induces oral tolerance. Animals, antigens and adjuvants Six-week-old female and male C3H/HeJ mice were purchased from the Jackson Laboratory (Bar Harbor, ME). BALB/c female mice (5-8 weeks old) were obtained from Taconic Farms (Hudson, NY). Animals were maintained on peanut-free chow under conventional, specific pathogen-free conditions. At the end of the experiments, animals were sacrificed by cervical dislocation. Freshly ground whole roasted peanut for maternal feeding and oral challenges was prepared as previously described. [19] Defatted crude peanut extract (CPE) was used as an antigen in sensitization and tolerance protocols, intraperitoneal (i.p.) challenges and laboratory assays. CPE labeled with fluorescein (FITC) was used for uptake studies. [14] OVA and betalactoglobulin (BLG) were purchased from Sigma. Cholera toxin (CT) was purchased from List Biological Laboratories Inc (Campbell, CA). Ethics statement Albany Medical College Institutional Animal Care and Use Committee (IACUC) approved the study (protocol #11-10003). Maternal exposure In order to determine the influence of maternal peanut ingestion during pregnancy and lactation on the development of peanut allergy in offspring, female C3H/HeJ mice were gavage-fed with ground peanut (10 mg/mouse) three times a week for four weeks prior to conception. Saphenous venous blood was collected at week 5 to determine CPE-specific IgG1, IgG2a, and IgA levels. Subsequently, they were mated with naïve males. These preconceptionally (PC) peanut-exposed mothers were divided into 2 groups for the period of pregnancy (PG) and lactation (LC): those who were gavage-fed peanut (10 mg) three times a week (PC+PG+LC) and those who were on the normal mouse chow alone, which does not contain peanut (PC). Additional peanut was left in the cages to be ingested ad libitum by mothers as indicated by their group assignment. Naïve mothers not fed peanut during pregnancy and lactation were used as controls (None). These mothers were then used as breeders for mice utilized in the following experiments of sensitization or tolerance induction as described below; alternatively only their milk was used, as shown in Fig 1. (Please see S1A Fig for a detailed protocol on maternal feeding.) These mothers develop IgG, but not IgE antibodies to peanut, and are not clinically reactive to peanut. Offspring of these mothers were weaned between 3.5 and 4 weeks of age. Additional groups included preconceptionally peanut exposed mothers who were fed peanut only during pregnancy and those who were fed peanut only during lactation (not shown). Sensitization, allergen challenge and assessment of anaphylaxis Five-week old C3H/HeJ offspring were sensitized with CPE+CT by gavage (5 mg CPE + 20 μg CT/mouse) at weekly intervals for 6 weeks followed by 2 boosters at 2-week intervals (50 mg CPE + 20 μg CT/mouse), as previously published [20]. (Please see S1B Fig for a detailed protocol on offspring sensitization.) Offspring were bled prior to sensitization at week 5 and postsensitization at week 15 of life, and peanut-specific IgE, IgG1, IgG2a, and IgA were measured by ELISA, as described below. Two weeks after the last booster, an oral challenge with 100 mg peanut i.g. was performed, followed by an intraperitoneal injection of 100 μg CPE after 30 minutes. Anaphylactic signs were evaluated 30 minutes after the challenge dose, according to a previously published scoring system. [19] Rectal temperatures were measured 30 minutes after the peanut challenge with a thermal probe (WPI Instrument, Sarasota, FL). Mast cell protease-1 (MMCP) was measured within 1 hour post-challenge utilizing an ELISA kit (R&D Systems). Splenocytes were harvested for cultures as described below. Tolerance induction To determine the influence of maternal peanut ingestion on the development of oral tolerance in offspring, we performed a classic oral tolerance protocol on offspring. Offspring were weaned between 3.5 and 4 weeks of age, bled, and immediately gavage-fed 1 mg CPE daily for 5 days, followed by two immunizations with 0.1 mg CPE with alum i.p. after 2 weeks at weekly intervals. (Please see S1C Fig for a detailed protocol on tolerance induction.) Offspring that were not fed CPE (fed bicarbonate alone) prior to immunization served as controls. Peanutspecific IgE, IgG1, IgG2a, and IgA were measured by ELISA, as described below. This model is not sufficiently sensitized to assess symptomatic anaphylaxis. Experimental protocols. Grey shaded boxes indicate periods of oral peanut exposure. Mothers were exposed to ground peanut for 4 weeks during either in the pre-conception period only (PC), during preconception, pregnancy and lactation (PC+PG+LC); or were not fed peanut, serving as controls (None). All the offspring underwent oral sensitization to peanut by being gavage-fed crude peanut extract (CPE) with cholera toxin or were tolerized by being gavage-fed CPE without adjuvant. In some experiments, young mice born to naïve mothers were exposed to the milk of naïve (naïve milk) or peanut-exposed (immune milk) mice concurrently with CPE and cholera toxin to induce peanut sensitization or CPE alone to induce oral tolerance. doi:10.1371/journal.pone.0143855.g001 Impact of breast milk on tolerance induction The role of breast milk on tolerance induction was assessed in 11-12-day-old and 3-4 week-old mice (born to naïve mothers) that were subjected to a classic oral tolerance protocol. (Please see S1D Fig for a detailed protocol on the impact of breast milk.) These offspring were fed 1 mg CPE i.g. daily for 5 days, followed by 2 immunizations with CPE in alum after 2 weeks at weekly intervals. Alternatively, CPE was administered in the presence of naïve or immune milk (i.e. milk from peanut-exposed animals). Offspring that were not fed CPE (fed bicarbonate alone) prior to immunization served as controls. Offspring were bled a week after the last immunization. In addition, the impact of breast milk on sensitization was assessed in 3-4 week-old mice that were exposed to OVA in the presence or absence of immune or naïve milk, followed by OVA epicutaneous sensitization [21] and challenge as described in Supplemental Methods. Cell culture and cytokine measurements Splenocytes derived from sensitized offspring were isolated using standard techniques. After the red blood cells were lysed, splenocytes were resuspended in RPMI 1640, supplemented with fetal bovine serum. Cells were cultured in 24-well plates (4×10 6 /well/mL) in the presence or absence of CPE (200 μg/mL). Supernatants were collected after 72 hours and IL-5, IL-10, IL-13, and IFN-γ levels were determined by ELISA, according to the manufacturer's instructions (EBioscience Affymetrix Inc, San Diego, CA). Measurement of antibodies Breast milk was collected using an electric murine breast pump and processed. [22] Serum and milk samples were stored at -80°C until analyzed. CPE-specific IgE in serum was measured by a capture enzyme-linked immunosorbent assay. [23] CPE-specific IgG1, IgG2a, and IgA were measured by applying serum and breast milk dilutions to CPE-coated plates and detected with biotinylated anti-IgG1, IgG2a, and IgA, respectively, and total IgE was measured using an ELISA kit (BD Pharmingen, San Diego, CA). In vivo antigen uptake studies BALB/c mice have been used in our laboratories in the past to assess antigen uptake. They were gavaged with 0.5 mg/ml of FITC-labeled CPE in 0.1M sodium bicarbonate. FITC-CPE was pre-mixed with mouse milk obtained from antigen-exposed (immune, PC+PG+LC group) or non-exposed (naïve) mice. After ten minutes, gavage mice were sacrificed and Peyer's patches (PP) were harvested. PP tissues were processed for immunofluorescence as previously described. [24] Immunofluorescence PP sections were stained as previously described. [24] The anti-mouse antibody CD11c-PE from eBioscience (San Jose, CA) was used for immunostaining dendritic cells in PP tissues. All incubations were done at 37°C for 30 min. Slides were mounted with ProLong Gold antifade reagent (Invitrogen, Grand Island, NY) and images were collected using a Leica SP5 ABOS scanning laser confocal microscope (Leica, Wetzlar, Germany). Images were processed using the Fiji software (http://fiji.sc/). [25] Statistical analysis We performed a one-way ANOVA followed by a Newman Keuls test for all pairwise comparisons if the data were normally distributed. For antibody dilution curves, data were analyzed by a two-way ANOVA with a Bonferroni correction for multiple comparisons. Differences between groups were analyzed by a nonparametric test followed by all pairwise comparisons if the data was not normally distributed, with a Dunn's test for multiple comparisons. Pre-post values were analyzed by Wilcoxon signed rank test. P values <0.05, based on two-tailed tests, are considered statistical significant. All statistical analyses were performed with Prism 5.0 (GraphPad, Inc.). Results Maternal peanut exposure prior to conception results in antibody production in serum and breast milk and transfer of immunoglobulins to offspring To assess the efficacy of antibody production induced by oral exposure to peanut, we measured the antibody levels in maternal serum and breast milk. Three weeks after oral gavage with peanut, more than one-half of the mothers exposed to peanut prior to conception, had detectable levels of CPE-specific serum and breast milk IgG1 and IgG2a, whereas mice that were not exposed to peanut did not (Fig 2A-2D). Specific IgE was undetectable and these mothers were not reactive to peanut upon feeding. Also, feeding peanut throughout lactation did not further boost antibody levels (data not shown). Maternal CPE-specific serum antibody levels were efficiently transferred to the offspring, as evidenced by elevated specific IgG1 and IgG2a levels in pups born to peanut-exposed mothers (Fig 2E and 2F). Pups born to non-exposed mothers had no detectable CPE-specific antibody at weaning. Maternal antibody levels positively correlated with IgG1 and IgG2a antibody levels in offspring at weaning (p<0.001 and p = 0.002, respectively, S2 Fig). Feeding peanut throughout lactation did not further boost antibody levels in offspring (data not shown). Maternal peanut exposure during pregnancy and lactation had no impact on peanut sensitization We then assessed whether maternal feeding of peanut during pregnancy and lactation had an impact on development of peanut sensitization in the offspring. Peanut sensitization was associated with increased serum peanut-specific IgE, IgG1, IgG2a, and IgA levels, which were comparable among the 3 groups of offspring: those born to non-exposed mothers versus those born to mothers exposed to peanut prior to conception or those exposed prior to conception and throughout pregnancy and lactation (Fig 3B-3E). After the oral peanut challenge, the symptom scores were comparable among these 3 groups (Fig 3F), and similar drops in body temperature were observed after i.p. peanut challenge (Fig 3G). MMCP-1 and cytokine levels from splenocyte cultures were likewise comparable between the 3 groups (See S3 Fig). Serum levels of total IgE, specific IgE to Ara h2, and ratios of specific IgE and IgG1 to IgG2a were comparable between the three groups of mice (data not shown). Additional groups of offspring born to mothers exposed to peanut prior to conception, and either exposed to peanut only during pregnancy or only during lactation, had comparable levels of antibodies and challenge responses (data not shown). Experiments were repeated using the milk allergen BLG as an antigen. There were again no significant differences among the groups (S4 Fig). Mothers' preconception serum (A-B) and breast milk (C-D) antibody levels in mothers who were fed peanut preconceptionally (PC) and in those who were not (None). Presensitization antibody levels at 5 weeks of age are shown (E-F) for offspring born to peanut-exposed mothers who either continued to feed peanut throughout pregnancy and lactation (PG+LC) and in those who did not (PC). The dilution curves are not shown due to the small amount of serum available. Specific IgE was undetectable (data not shown). Titles indicate sample dilution. Shown is a mean (with SEM). 5-10 mothers per group and all their offspring (>15/group) were used for each experiment. *, p<0.05; **, p<0.01; ***, p<0.001 compared to None. Oral tolerance to peanut develops independently of maternal peanut exposure Food allergy is thought to be a combined result of failed oral tolerance and an activation of pathways that promote sensitization. We then assessed whether maternal feeding of peanut would impact offsprings' development of oral tolerance. This was done utilizing a classic oral tolerance protocol, in which daily antigen feeding for 5 days induces a suppressed antibody response to immunization with the same antigen. We found that tolerance was induced in young mice by feeding a moderate dose (1 mg) of CPE prior to immunization, as evidenced by lower levels of specific IgE antibodies (Fig 4), IgG1, and IgG2a antibodies were also decreased, but not statistically different between the groups (Please see S5 Fig). We also showed that tolerance development was as effective in mice born to mothers exposed to peanut prior to conception and throughout pregnancy as mice born to non-peanut-exposed mothers (Fig 4). , the lowered body temperature during the intraperitoneal (IP) challenge (G), and after the peanut sensitization were assessed in offspring born to mothers exposed to peanut preconceptionally, who either continued to feed peanut during pregnancy and lactation (PC+PG+LC) and those who did not (PC). Offspring born to mothers never exposed to peanut served as controls (None). Naïve, non-sensitized mice served as controls. Shown is a mean with SEM. >15 mice were used per group. Peanut uptake into Peyer's patch associated dendritic cells It has been reported that breast milk OVA-IgG immune complexes facilitate oral tolerance in neonates due to enhanced uptake across the mucosal barrier through the neonatal Fc receptor. [17] To examine the role of antibodies in uptake of peanut antigens following oral delivery, FITC-CPE was administered orally to 5 week-old mice in the presence of immune or naïve milk. We excised small intestinal tissues containing at least one Peyer's patch and subjected the tissues to cryosectioning and confocal microscopy. In the context of naïve milk, we observed the accumulation of FITC-CPE within a subset of Peyer's patch subepithelial dome dendritic cells (DCs), as evidenced by co-localization of CPE with CD11c + cells (Fig 5). CPE was not visibly detected in the lamina propria. Co-administration of FITC-CPE with immune milk slightly enhanced FITC-CPE uptake in Peyer's patch tissues when compared to administration of CPE with naïve milk, thus suggesting that the presence of antigen-specific IgG or IgA antibodies may facilitate transport of CPE across the follicle-associated epithelium and/or uptake by DCs. Oral tolerance can be induced in early life, and is neither enhanced nor impaired by concurrent exposure to immune breast milk Lastly, because we detected enhanced peanut uptake in Peyer's patches in the presence of peanut antibodies in breast milk, we hypothesized that direct administration of antigen to the or not fed (Unfed). Offspring were either born to mothers exposed to peanut preconceptionally who either continued to feed peanut during pregnancy and lactation (PC+PG+LC) or did not (PC). Offspring born to mothers that were never exposed to peanut served as controls (None). In (C), although antibody levels are similar, the dilution curves are significantly different. Shown is a mean with SEM. Each group has 10-18 mice *, p<0.05, ****, p<0.0001. offspring to induce oral tolerance could be influenced by concurrent immune milk exposure. We fed offspring CPE plus murine milk, either at weaning (Fig 6A-6D) or prior to weaning, at 11 days of age (Fig 6E-6H). We showed that a relatively small dose of fed CPE (1 mg) administered alone, post-weaning, was able to tolerize offspring, but co-administration with immune or naïve murine milk did not have any impact on this tolerance development (Fig 6A and 6B). Because the mice above were tolerized post-weaning, we then assessed whether the pre-weaning period would be more susceptible for tolerance induction by immune milk. This was done by utilizing mice at 11-12 days of life and treating them with two doses of antigen. Again, we showed that that immune milk did not significantly impact tolerance induction (Fig 6E-6H). We also showed that a large dose of CPE was better at inducing tolerance than small or trace quantities of antigen (Fig 6E-6H). We also assessed the impact of fed OVA co-administered with murine milk followed by OVA sensitization, with similar results (Fig 7). Discussion The prevalence of peanut allergy has increased in children. [1][2][3][4] Peanut is commonly known as a cause for fatal and near-fatal anaphylactic reactions [26][27] and rarely outgrown. Because prevention of food allergies by maternal and infant feeding practices could serve as a simple, inexpensive approach to address the growing number of subjects with food allergies, we assessed whether maternal peanut exposure has an impact on peanut sensitization in the offspring. We report that despite vertical transfer of maternal antibodies, maternal peanut exposure and immune status had no significant impact on development of sensitization or tolerance in the offspring. In addition, we show that early introduction of peanut directly to the offspring induces dose-dependent tolerance, which is not further facilitated by co-administration of immune breast milk. These findings suggest that (i) the maternal diet plays little role in the development of peanut allergy and (ii) tolerance can be induced early, even pre-weaning, by introducing moderate amounts of peanut directly to the infant, with or without concurrent exposure to immune breast milk. Our study does not address the question of whether breastfeeding overall has an impact on sensitization or tolerance, because all the offspring were breastfed. Our results differ from several previous animal studies that have shown protection with maternal exposure against sensitization to inhaled or ingested antigens. They have mostly been performed using OVA as an antigen in asthma models [15][16][17][18], as opposed to an oral peanut allergy model. Peanut, as opposed to OVA, activates innate immunity and complement [28][29], which may be related to greater clinically evident allergenicity. However, our studies with two other common food allergens, cow's milk β-lactoglobulin and hen's egg OVA, support our findings. Only one study to date has assessed maternal peanut exposure and using mothers sensitized to peanut showed protection provided by low dose, perinatal maternal peanut exposure against peanut anaphylaxis during first exposure. Protection was associated with increased peanut-specific IgG2a in the offspring, [14] related to co-administration of peanut with a mucosal adjuvant. In the current study, peanut was fed to mothers perinatally without a mucosal adjuvant, which is more relevant to the human population in which peanut ingestion typically occurs as part of a diet without co-administration of an adjuvant. Also, mothers who were allergic to peanut would not be ingesting peanut. Consistently, no enhanced IgG2a responses were detected in the current study, indicating that adjuvant use may explain the different Dose-dependent induction of oral tolerance to peanut in mice prior to or after weaning. Mice post-weaning at 3-4 weeks of age (n = 5/group) (A-D), or pre-weaning at 11-12 days of age (n = 7-9/group (E-H) were fed crude peanut extract (CPE, 1 mg or as otherwise indicated) +/-murine milk (immune, unless otherwise indicated being naive) for 5 days followed by immunization with CPE. Mice immunized without prior feeding of ground peanut (Unfed) were used as untolerized controls. Shown is a mean with SEM. **, p<0.01, ***, p<0.001, ****, p<0.00001 when compared to unfed. outcomes between the studies. Different outcome may also be due to the fact that whereas the current model was IgE-mediated, anaphylaxis during first exposure in the previous model was IgG 1 -mediated. Also, because patients with peanut allergy strictly avoid the allergen, our study determined that the impact of maternal ingestion of peanut in mothers with previous exposure and presence of specific antibodies to peanut are associated with maternal tolerance, not sensitization. In humans, there is likewise great controversy regarding the impact of maternal avoidance diets on allergy prevention. [30][31][32][33] Unlike the previous recommendations, [34] the revised maternal and infant feeding guidelines avoid strong recommendations on whether foods of high allergenic potential should be ingested during lactation. [35] More recently, emerging observational studies have created even more controversy. [11][12][13] Recently, two large US cohorts performed in general populations [12][13] reported that a maternal diet including peanut and tree nuts during or around pregnancy could be associated with a reduced risk to peanut or tree nut Offspring's response to OVA challenge (B-C) and antibody responses to OVA sensitization (D-F). Mice post-weaning at 3-4 weeks of age were fed OVA +/-OVA-immune or naïve milk for 5 days followed by cutaneous sensitization with OVA. Mice immunized without prior OVA feeding (Unfed) were used as controls. X axis for D-F shows the dilution factor for the serum. Each group has 5 mice. Shown is a mean with SEM. **, p<0.01, ***, p<0.001, ****, p<0.00001 when compared to unfed. doi:10.1371/journal.pone.0143855.g007 allergy in offspring. However, when nut allergic mothers were assessed separately, one study reported a trend for an association between maternal consumption of tolerated nuts and increased risk of peanut or tree nut allergy in offspring. [13] This is consistent with findings in a human cohort of high-risk infants with possible or likely cow's milk or egg allergy that reported maternal peanut ingestion during pregnancy associated with increased peanut sensitization. [11] Because controlled clinical trials are lacking, confounding factors such as the infant's diet and other environmental exposures may partly explain the differences in findings between cohorts. The capacity of food antigens in breast milk to sensitize or tolerize is largely unknown. Peanut proteins [7][8] and several other dietary proteins, including cow's milk, hen's egg and wheat allergens, have been detected in human and murine milk. [7,8,15] We anticipated that peanut would also be transmitted in murine milk, although in small quantities. These levels did not seem to play a major role in sensitization or tolerance induction. Due to the fact that levels of dietary antigens present in breast milk are generally very low with many mothers having undetectable levels, [7,8,10] we also assessed the potential of larger doses of peanut co-administered with immune milk in inducing tolerance. We showed that large doses of peanut were better at inducing tolerance to peanut in young mice than small doses or trace amounts and that tolerance induction was not impacted by co-administration with immune milk. This suggests that low levels of dietary antigens present in breast milk may not be sufficient to induce tolerance and that doses larger than trace amounts are required. This was surprising in light of the finding that milk antibodies enhanced peanut uptake in Peyer's patches. While animal studies are not a surrogate for human trials, our results suggest that the impact of maternal dietary antigens may be small compared to other factors, including epicutaneous exposure, which has been associated with sensitization [21,[36][37][38] and early oral exposure, as recently shown in the LEAP study to lead to tolerance induction. [13] Our findings further support the early introduction of foods directly to infants for development of tolerance,and although the exact "window of opportunity" cannot be extrapolated from mouse studies, introduction may be beneficial even pre-weaning without a risk of sensitization. Our finding that breast milk does not appear to facilitate tolerance development in offspring pre-or post-weaning is in disagreement with a recent study, which showed that low quantities of immunologically active peanut allergens were secreted in human breast milk and co-administration of peanut with breast milk was associated with partial tolerance to peanut when administered to young mice prior to weaning. [8] The authors suggested that protection may be transferred via human milk antibodies and a neonatal γ receptor (FcRn), which may bind antibodies from different species. The main difference, compared to our study, was that we administered breast milk from the same species, namely murine milk, to assure that receptors facilitating antibody uptake in the neonatal gut would be appropriate to the species used. During maternal exposure, handling pregnant mothers and gavaging these mice consistently can induce stress related changes in immune responses in the pups. A limitation to this study is that the control group was not gavaged a vehicle as compared to the pregnant mothers who were gavage fed peanut three times a week. However, mothers in each group were handled frequently and additionally stressed on a weekly or twice-a-week basis, due to milking and blood draws. Furthermore, because sensitization may be dependent on presensitization IgG levels, and offsprings' presensitization IgG levels were highly variable, the conclusions might have been different if only offspring with high IgG1 levels would have been taken into account. Due to initial study design not accounting for this, the experiments were not sufficiently powered to perform such analyses. In summary, despite vertical transfer of maternal IgG antibodies, we found that ante-and postnatal maternal peanut exposure does not impact peanut sensitization or oral tolerance induction in offspring, which suggests that no dietary restrictions are needed for pregnant and breastfeeding mothers in order to prevent food allergy. However, there is emerging evidence to suggest that early peanut introduction directly to infants is beneficial in prevention of peanut allergy, providing rationale for current interim feeding guidelines. [39] Furthermore, large doses of peanut were better at inducing tolerance to peanut than small doses and tolerance induction was not impacted by co-administration of immune milk either pre-or post-weaning, suggesting that trace amounts of dietary protein detected in breast milk may not be sufficient to induce significant tolerance. Randomized controlled clinical studies are needed to address the impact of maternal exposure to peanut and the "window of opportunity" for early introduction of foods on allergy risk in humans. Supporting Information S1 Fig. Detailed experimental protocol. (A) To assess the impact of the maternal diet, mothers were pre-conceptionally exposed to crude peanut extract (CPE) (Immune Mothers). During pregnancy and lactation, they were divided into those who continued to feed CPE (PG+LC) and those who did not (None). Non-CPE-exposed mothers that did not feed CPE pre-or postconception served as controls. After weaning, offsprings' responses to peanut sensitization (B) or oral tolerance induction (C) were assessed. (D) To assess the role of murine milk, oral tolerance induction or sensitization to CPE was assessed in young mice pre-or postweaning by feeding CPE alone or with murine milk from immunized mothers. (PDF) Cytokine levels were measured in splenocyte cultures stimulated with peanut for 72 hours. In offspring born to mothers exposed to peanut preconceptionally, who either continued to feed peanut during pregnancy and lactation (PC+PG+LC) and in those who did not (PC). Offspring born to mothers never exposed to peanut served as controls (None). Naïve, non-sensitized mice served as controls. Shown is a mean with SEM. (PDF) S4 Fig. Offspring's response to BLG sensitization based on maternal feeding of BLG. Experimental protocol (A). Mothers' preconception serum (B) and breast milk (C) antibody levels in mothers who were fed BLG preconceptionally (BLG Fed) and in those who were not (Naive). Offspring were sensitized to BLG and serum antibodies (D-F) and symptom scores were measured after oral (PO) feeding (G) and body temperature measured after intraperitoneal (IP) injection of BLG (H), in offspring born to mothers exposed to BLG only preconceptually (PC) and in those who continued to feed BLG during pregnancy and lactation (PC+PG+LC). Offspring born to mothers never exposed to BLG served as controls (None). Shown is a mean with SEM. Each group has 4-12 mice. ÃÃ , p<0.01. (PDF) immunization were assessed in offspring who were either fed PN orally prior to immunization (Fed) or not fed (Unfed). Offspring were either born to mothers exposed to CPE preconceptionally who either continued to feed peanut during pregnancy and lactation (PC+PG+LC) and in those who did not (PC). Offspring born to mothers never exposed to peanut served as controls (None). Shown is a mean with SEM. Each group has 10-18 mice. Ã , p<0.01, ÃÃÃ , p<0.001. 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v3-fos

2016-06-17T06:14:38.038Z

2015-06-29T00:00:00.000Z

17261362

s2

Lysine and novel hydroxylysine lipids in soil bacteria: amino acid membrane lipid response to temperature and pH in Pseudopedobacter saltans Microbial decomposition of organic matter is an essential process in the global carbon cycle. The soil bacteria Pseudopedobacter saltans and Flavobacterium johnsoniae are both able to degrade complex organic molecules, but it is not fully known how their membrane structures are adapted to their environmental niche. The membrane lipids of these species were extracted and analyzed using high performance liquid chromatography-electrospray ionization/ion trap/mass spectrometry (HPLC-ESI/IT/MS) and high resolution accurate mass/mass spectrometry (HRAM/MS). Abundant unknown intact polar lipids (IPLs) from P. saltans were isolated and further characterized using amino acid analysis and two dimensional nuclear magnetic resonance (NMR) spectroscopy. Ornithine IPLs (OLs) with variable (hydroxy) fatty acid composition were observed in both bacterial species. Lysine-containing IPLs (LLs) were also detected in both species and were characterized here for the first time using HPLC-MS. Novel LLs containing hydroxy fatty acids and novel hydroxylysine lipids with variable (hydroxy) fatty acid composition were identified in P. saltans. The confirmation of OL and LL formation in F. johnsoniae and P. saltans and the presence of OlsF putative homologs in P. saltans suggest the OlsF gene coding protein is possibly involved in OL and LL biosynthesis in both species, however, potential pathways of OL and LL hydroxylation in P. saltans are still undetermined. Triplicate cultures of P. saltans were grown at three temperature/pH combinations: 30°C/pH 7, 15°C/pH 7, and 15°C/pH 9. The fractional abundance of total amino acid containing IPLs containing hydroxylated fatty acids was significantly higher at higher temperature, and the fractional abundance of lysine-containing IPLs was significantly higher at lower temperature and higher pH. These results suggest that these amino acid-containing IPLs, including the novel hydroxylysine lipids, could be involved in temperature and pH stress response of soil bacteria. Microbial decomposition of organic matter is an essential process in the global carbon cycle. The soil bacteria Pseudopedobacter saltans and Flavobacterium johnsoniae are both able to degrade complex organic molecules, but it is not fully known how their membrane structures are adapted to their environmental niche. The membrane lipids of these species were extracted and analyzed using high performance liquid chromatography-electrospray ionization/ion trap/mass spectrometry (HPLC-ESI/IT/MS) and high resolution accurate mass/mass spectrometry (HRAM/MS). Abundant unknown intact polar lipids (IPLs) from P. saltans were isolated and further characterized using amino acid analysis and two dimensional nuclear magnetic resonance (NMR) spectroscopy. Ornithine IPLs (OLs) with variable (hydroxy) fatty acid composition were observed in both bacterial species. Lysine-containing IPLs (LLs) were also detected in both species and were characterized here for the first time using HPLC-MS. Novel LLs containing hydroxy fatty acids and novel hydroxylysine lipids with variable (hydroxy) fatty acid composition were identified in P. saltans. The confirmation of OL and LL formation in F. johnsoniae and P. saltans and the presence of OlsF putative homologs in P. saltans suggest the OlsF gene coding protein is possibly involved in OL and LL biosynthesis in both species, however, potential pathways of OL and LL hydroxylation in P. saltans are still undetermined. Triplicate cultures of P. saltans were grown at three temperature/pH combinations: 30 • C/pH 7, 15 • C/pH 7, and 15 • C/pH 9. The fractional abundance of total amino acid containing IPLs containing hydroxylated fatty acids was significantly higher at higher temperature, and the fractional abundance of lysine-containing IPLs was significantly higher at lower temperature and higher pH. These results suggest that these amino acid-containing IPLs, including the novel hydroxylysine lipids, could be involved in temperature and pH stress response of soil bacteria. Introduction Intact polar lipids (IPLs) are useful biomarker molecules because their structures can be specific to microbial taxa or environmental conditions (Sturt et al., 2004;Schubotz et al., 2009). For example, marine phytoplankton from multiple regions of the ocean produce non-phosphorus containing lipids in response to phosphorus scarcity (Van Mooy et al., 2009), which demonstrates the importance of microbial membrane modification. IPLs containing the amino acid ornithine as the polar head group (ornithine lipids, OLs) are common phosphorus-free membrane IPLs among bacteria ( Figure 1A). Approximately 50% of bacterial species whose genomes have been sequenced are predicted to have the capacity to form OLs, but they have not been predicted in eukaryotes or archaea (Lopez-Lara et al., 2003;Geiger et al., 2010;Vences-Guzmán et al., 2012. In various bacteria, OL production increases under phosphorus limitation (Weissenmayer et al., 2002;Gao et al., 2004), and in other microbes the fatty acids of OLs are hydroxylated under thermal or acid stress (Taylor et al., 1998;Rojas-Jimenez et al., 2005;Vences-Guzman et al., 2011). In eight Desulfovibrio strains isolated from intertidal sediments of the North Sea, relative OL content was found to increase at higher growth temperature (Seidel et al., 2013). It has also been suggested that OLs are important for Gram-negative FIGURE 1 | High pressure liquid chromatography-electrospray/ion trap/mass spectrometry (HPLC-ESI/IT/MS) base peak chromatograms of (A) Flavobacterium johnsoniae (low abundance glycine lipids at retention time 6.5 min not shown; ornithine lipid structure included with variable fatty acid hydroxy group), and (B) Pseudopedobacter saltans lipid extracts. PE, phosphatidylethanolamine; OL, ornithine lipid; OL HFA , Ornithine lipid with hydroxylated fatty acid; I, I ′ , II, II ′′ , unknown intact polar lipids. Chromatographic separation was performed on a Lichrosphere diol column (250 mm by 2.1 mm; 5-µm particles; Grace Alltech Associates Inc.). Elution was achieved with hexane-2-propanol-formic acid-14.8 M aqueous NH 3 (79:20:0.12:0.04, v/v/v/v) (A) and 2-propanol-water-formic acid-14.8 M aqueous NH 3 (88:10:0.12:0.04, v/v/v/v) (B) starting at 10% B, followed by a linear increase to 30% B in 10 min, followed by a 20-min hold and a further increase to 65% B at 45 min. The flow rate was 0.2 ml min −1 , and the total run time was 60 min, followed by a 20 min re-equilibration period. bacterial outer membrane stability due to their zwitterionic nature (Freer et al., 1996) and essential to maintain a constant level of extracytoplasmic cytochromes in Rhodobacter capsulatus (Aygun-Sunar et al., 2006). The combination of bacterial specificity, OL production and modification in response to environmental conditions, along with a suggested role in membrane stability makes this class of lipids useful potential biomarker molecules for microbial populations. Besides OLs, other amino acid-containing IPLs have also been identified including glycine lipids (Kawazoe et al., 1991;Batrakov et al., 1999), lysine lipids (Tahara et al., 1976a), glutamine lipid (Zhang et al., 2009), and an ornithine-taurine linked lipid in Gluconobacter cerinus (Tahara et al., 1976b) using various combinations of thin layer chromatography, infrared spectrometry, gas chromatographymass spectrometry, electrospray ionization-mass spectrometry, and 1 H nuclear magnetic resonance (NMR) spectroscopy. None of the lipids from these studies were identified using high performance liquid chromatography/mass spectrometry (HPLC/MS) methods, which are far less laborious than the above mentioned methods, and extremely effective for identifying IPLs in complex bacterial cultures and environmental samples (Sturt et al., 2004;Schubotz et al., 2009). The LC/MS chromatographic and fragmentation behavior of OLs is well known (Hilker et al., 1978(Hilker et al., , 1979Tomer et al., 1983;Cerny et al., 1986;Linscheid et al., 1997;Geiger et al., 2010), but not described for various other amino acid containing IPLs. The characteristic multi-stage MS fragmentation of OLs includes the sequential loss of an H 2 O molecule from the head group, a fatty acid moiety, and the βOH-fatty acid moiety, resulting in a diagnostic m/z 115 cyclic fragment (Hilker et al., 1978;Cerny et al., 1986;Geiger et al., 2010). This fragmentation pattern can be used as an example for identifying other amino acid-containing IPLs via LC/MS multistage fragmentation in bacteria which are important in organic matter recycling. Microbial organic matter decomposition is an important part of the global carbon cycle with contributions to climate change through carbon cycle feedbacks (Bardgett et al., 2008;Singh et al., 2010). The bacterial genus Pedobacter is of great interest to understand organic matter decomposition in soils because many species within the genus are able to degrade heparin, the biomolecule with the greatest known negative charge density (Steyn et al., 1998;Liolios et al., 2011). Pseudopedobacter saltans (Cao et al., 2014), originally classified in the Pedobacter genus (Steyn et al., 1998), has a distinct taxonomic position and its genome has the highest number of heparinase coding genes (Liolios et al., 2011). As P. saltans degrades highly negatively charged molecules like heparin and chondroitin, this species may be involved in the degradation of complex organic molecules in the soil such as other mucopolysaccharides or humic acids. Other microbes with the ability to degrade recalcitrant biomacromolecules are important in organic matter decomposition as well. For example, Flavobacterium johnsoniae, formerly Cytophaga johnsonae, another bacterium found in soil, rapidly digests chitin and many other macromolecules (Stanier, 1947;Larkin, 1989) and has been studied extensively for its gliding motility (McBride, 2001;McBride et al., 2003;Nelson and McBride, 2006). Both P. saltans and F. johnsoniae belong to the Cytophaga-Flavobacterium-Bacteroides (CFB) group (Stanier, 1947;Larkin, 1989;Steyn et al., 1998;Liolios et al., 2011). Despite their important role in soil organic matter decomposition, it is not known how the membrane structural components of P. saltans and F. johnsoniae adapt to their different environmental niches. OLs and glycine lipids have both been identified in F. johnsoniae (Pitta et al., 1989;Kawazoe et al., 1991Kawazoe et al., , 1992Okuyama and Monde, 1996), and amino lipids have been identified in P. saltans (Cao et al., 2014), but the full complement of IPLs have not been described for both species. Amino acid-containing IPLs may be involved in environmental stress response by these consequential species. Here, we study the IPLs, and specifically the amino acid-containing lipids, of P. saltans and F. johnsoniae by HPLC/MS with further structural characterization by NMR spectroscopy. We report the IPL content of P. saltans, and F. johnsoniae including the structural elucidation of novel amino acid-containing lipids and discuss the potential genes and environmental factors that influence their production in P. saltans. Materials and Methods Strains and Culture Conditions F. johnsoniae (DSM 2064 T ) and P. saltans (DSM 12145 T ) were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) in Braunschweig, Germany. F. johnsoniae was grown in liquid CYE medium (g per liter of distilled water): Casitone, 10.0; yeast extract, 5.0; MgSO 4 , 0.1; Tris buffer, 1.2; pH 7.2; 25 • C. P. saltans was grown in DSMZ Medium 948 (g per liter of distilled water): Lab-Lemco powder (Oxoid), 1.0; yeast extract, 2.0; peptone, 5.0; NaCl, 5.0; pH 7.2; 25 • C. Additional P. saltans cultures were grown in triplicate at 30 • C/pH 7, 15 • C/pH 7, and 15 • C/pH 9 in temperature controlled rooms to determine changes in IPL fractional abundance due to different environmental conditions. The temperatures and pH levels were chosen based on temperature growth range reported by Liolios et al. (2011) and initial growth experiments, in which P. saltans cultures did not readily grow at temperatures below 15 • C or below pH 7. The pH was adjusted by addition of HCl or NaOH solutions to achieve the desired values. Biomass from 250 ml cultures was collected by centrifugation at the stationary growth phase and freeze dried for lipid extraction and further analysis. Lipid Extraction Lipids were extracted from freeze dried biomass of each culture by a modified Bligh & Dyer method (Rutters et al., 2002). Biomass was fully submerged and extracted three times in methanol/dichloromethane/phosphate-buffer (MeOH/DCM/Pbuffer, 2/1/0.8, v/v/v) extraction solvent for 10 min in an ultrasonic bath (P-buffer: 8.7 g K 2 HPO 4 L −1 bi-distilled water adjusted to pH 7-8 with 1 N HCl). Extracts were centrifuged for 2 min at 1400 g to separate DCM phase from the MeOH/Pbuffer phase, and the lower DCM layer was pipetted into a separate vial. The MeOH/P-buffer layer was washed twice more with DCM, centrifuged, and the resulting DCM layers were combined with the original DCM layer. DCM was removed under a stream of nitrogen, the residue was dissolved in injection solvent (hexane:2-propanol:H 2 O, 718:271:10, v/v/v), and filtered through a 0.45 µm, 4 mm diameter True ™ Regenerated Cellulose syringe filter (Grace Davison) prior to injection. Extracts were dried down and stored at −80 • C until analysis. HPLC-MS Analysis Intact polar lipids were analyzed by high performance liquid chromatography-electrospray ionization/ion trap mass spectrometry (HPLC-ESI/IT/MS) and HPLC-high resolution accurate mass/mass spectrometry (HRAM/MS) according to Moore et al. (2013). The HPLC-ESI/IT/MS methods closely followed Sturt et al. (2004) with some modifications (Sinninghe Damsté et al., 2011). An Agilent 1200 series HPLC, with thermostatted auto-injector, was coupled to a Thermo Scientific ™ LTQ XL ™ linear ion trap mass spectrometer with Ion Max source and ESI probe (Thermo Fisher Scientific, Waltham, MA). Chromatographic separation was performed on a Lichrosphere diol column (250 mm by 2.1 mm; 5-µm particles; Grace Alltech Associates Inc.). Elution was achieved with hexane-2-propanol-formic acid-14.8 M aqueous NH 3 (79:20:0.12:0.04, v/v/v/v) (A) and 2-propanol-water-formic acid-14.8 M aqueous NH 3 (88:10:0.12:0.04, v/v/v/v) (B) starting at 10% B, followed by a linear increase to 30% B in 10 min, followed by a 20-min hold and a further increase to 65% B at 45 min. The flow rate was 0.2 ml min −1 , and the total run time was 60 min, followed by a 20 min re-equilibration period. Lipid extracts were analyzed by scanning the mass range of m/z 400-2000 in positive-ion mode, followed by data-dependent, dual-stage tandem MS (MS 2 ), in which the four most abundant masses in the mass spectrum were fragmented successively (normalized collision energy, 25; isolation width, 5.0; activation Q, 0.175). Each MS 2 was followed by data-dependent, triple-stage tandem MS (MS 3 ), where the base peak of the MS 2 spectrum was fragmented under identical fragmentation conditions to those described for MS 2 . The performance of HPLC-ESI/IT/MS was monitored by regular injections of platelet-activating factor (PAF) standard (1-O-hexadecyl-2-acetyl-snglycero-3-phosphocholine). HPLC-HRAM/MS analysis was accomplished on a Thermo Scientific ™ Dionex ™ UltiMate ™ 3000 series LC with thermostatted auto-injector coupled to a Thermo Scientific ™ Q Exactive ™ Orbitrap ™ mass spectrometer. Higher-energy Collisional Dissociation (HCD) and product ion scan were used for mass fragmentation of sample masses. The chromatographic conditions and column were the same as those described above for HPLC-ESI/IT/MS. The positive-ion ESI settings were as follows: capillary temperature, 275 • C; sheath gas (N 2 ) pressure, 35 arbitrary units (AU); auxiliary gas (N 2 ) pressure, 10 AU; spray voltage, 4.0 kV; probe heater temperature, 300 • C; S-lens, 50 V. Target lipids were analyzed with a mass range of m/z 400-1000 (resolution, 70,000), followed by data dependent MS 2 (resolution, 17,500), in which the five most abundant masses in the mass spectrum were fragmented successively (normalized collision energy, 35; isolation width, 1.0). IPL fractional abundances were calculated based on HPLC-ESI/IT/MS chromatogram base peak area of each lipid group. Student's t-tests were performed using the GraphPad t-test Calculator (GraphPad Software, Inc. La Jolla, CA) in order to identify statistically significant differences in the fractional abundances of IPLs under different growth conditions; p < 0.05 were considered statistically significant. Fatty Acid Analysis Aliquots of Bligh and Dyer extracts of F. johnsoniae and P. saltans were hydrolyzed with 1.5 N HCl in MeOH by refluxing for 3 h. The hydrolysate was adjusted to pH 4 with 2 N KOH-MeOH (1:1, v/v). Water was added to give a final ratio of 1:1 H 2 O-MeOH and this mixture was extracted three times with DCM. The DCM fractions were collected and dried over sodium sulfate. The extract was methylated with diazomethane (Sinninghe Damsté et al., 2004), followed by silylation in pyridine with N,O bis(trimethylsilyl)trifluoroacetamide (BSTFA) at 60 • C for 20 min. The methylated-silylated extracts were dissolved in ethyl acetate for gas chromatography (GC)-MS analysis (Sinninghe Damsté et al., 2011). GC was performed with a Hewlett-Packard gas chromatograph (HP6890) equipped with an on-column injector and a flame ionization detector. GC-MS was performed on a Finnigan Trace Ultra gas chromatograph interfaced with a Finnigan Trace DSQ mass spectrometer operated at 70 eV with a mass range of m/z 40-800 and a cycle time of 1.7 s (resolution, 1000). Target Lipid Isolation Isolation of target lipids from P. saltans and known OL from F. johnsoniae lipid extracts was accomplished using an Agilent Technologies (Santa Clara, CA) 1100 series LC equipped with an auto-injector, and a fraction collector (Foxy Jr., Isco, Inc., Lincoln, NE). A first isolation was achieved on a semi-preparative LiChrospher diol column (10 × 250 mm, 5 µm; Grace Alltech Associates Inc.) according to Boumann et al. (2009) with the same gradient program as described above for HPLC-ESI/IT/MS, but at a flow rate of 3 ml min −1 . Typical injection volume was 200 µL containing up to 2 mg material per injection. Column effluent was collected in 1 min fractions, which were screened for the presence of target lipids by flow injection analysis cf. Smittenberg et al. (2002) using the ESI/IT/MS (5 µl injection of each fraction, ESI source settings same as described above for HPLC-ESI/IT/MS with a scan range of m/z 400-2000). Fractions containing target lipids were pooled and further purified on a second LiChrospher diol column (4.6 × 250 mm, 5 µm; Grace Alltech Associates Inc.). Typical injection volumes were 65 µl containing up to 0.65 mg of material. Lipids were eluted using the identical gradient program and conditions as described above for HPLC-ESI/IT/MS at a flow rate of 1 ml min −1 , however mobile phases A and B did not contain NH 3 or formic acid. Column effluent was collected in 15 s fractions and screened as described above. Fractions containing target lipids were again combined and elution solvent was removed under a stream of nitrogen. Purity was assessed by HPLC-ESI/IT/MS as described above for IPL analysis. Isolated lipids were stored at −80 • C prior to further analysis. Amino Acid Analysis Approximately 8.4 nmol of each of the purified P. saltans lipids, and 40 nmol of a known ornithine containing lipid purified from F. johnsoniae used as a reference compound, were hydrolyzed in 0.5 ml of 6 M HCl at 110 • C for 18 h to liberate the amino acid head groups from the fatty acids for amino acid analysis. Standardized methods for the analysis of the amino acid lipid hydrolysates follow Spackman et al. (1958), Commission Regulation (EC) no. 152 (2009) phase consisting of number of weak acidic Li-citrate buffers. Stepwise pH, temperature and salt concentration gradients were applied. Spectrophotometer detection occurred after post column derivatization with ninhydrin (135 • C) at 570 or 440 nm. Results and Discussion IPLs of F. johnsoniae The IPLs of F. johnsoniae identified using HPLC-ESI/IT/MS analysis included abundant OLs, moderately abundant hydroxylated fatty acid OLs (OL HFA ) and phosphatidylethanolamines (PEs), and low abundance lyso-PE (PE with one fatty acid), and glycine lipids, which make up 47, 11, 12, 2, and 1%, respectively of the total HPLC-ESI/IT/MS chromatogram base peak area ( Figure 1A). OL HFA were hydroxylated at the α-position of the ester linked fatty acid ( Figure 1A). In addition, two low abundance IPLs with OL-like fragmentation were observed at retention times 27.7 and 28.0 min with apparent protonated molecules ([M+H] + ) at m/z values 639 and 625, respectively, making up 4.6% of HPLC-ESI/IT/MS chromatogram base peak area, which we will refer to as group I. The group I IPLs displayed sequential arbitrary units (AU); auxiliary gas (N 2 ) pressure, 10 AU; spray voltage, 4.0 kV; probe heater temperature, 300 • C; S-lens, 50 V. Target lipids were analyzed with a mass range of m/z 400-1000 (resolution, 70,000), followed by data dependent MS 2 (resolution, 17,500), in which the five most abundant masses in the mass spectrum were fragmented successively (normalized collision energy, 35; isolation width, 1.0). fragmentation loss of an H 2 O molecule, a fatty acid moiety, and a βOH-fatty acid, reminiscent of the fragmentation of OLs, but resulting in MS 3 fragment ions at m/z 129, 130, and 147, instead of the characteristic m/z 115 OL MS 3 fragment. To further elucidate the identity of the group I lipids we analyzed the F. johnsoniae lipid extract by HPLC-HRAM/MS, which revealed the elemental composition of the m/z 129, 130, and 147 fragmentation products of group I (Figure 2, Table 1). During fragmentation the loss of fatty acids and H 2 O from the head group likely resulted in the formation of a ring structure as described for the fragmentation of OL (Zhang et al., 2009), giving the most abundant fragmentation product of m/z 129.1022 with elemental composition C 6 H 13 N 2 O. The loss of the fatty acid moieties without the loss of H 2 O results in the m/z 147.1127 fragmentation product, which has the elemental composition (C 6 H 15 N 2 O 2 ) of protonated lysine, and further loss of NH 3 from this fragment results in the m/z 130.0862 fragment ion (C 6 H 12 NO 2 ). The elemental composition of the m/z 129.1022 fragment also corresponds to protonated lysine minus H 2 O. Elemental compositions of fatty acid losses corresponded to the expected molecular formulas of regular and βOH-fatty acids. Based on this information, we propose that group I IPLs in F. johnsoniae are lysine-containing lipids (LLs) with different fatty acid compositions (Table 1, lysine lipid, with 15C:0 and βOH-17C:0 fatty acids shown in Figure 2). GC/MS analysis showed that the most abundant 15C:0 and βOH-17C:0 fatty acids were iso branched ( Figure 1A) as previously reported (Pitta et al., 1989;Okuyama and Monde, 1996). IPLs of P. saltans HPLC-ESI/IT/MS analysis of the lipid extract of P. saltans showed abundant PE, OL, and OL HFA IPLs ( Figure 1B) making up 8, 10, and 12%, respectively, of HPLC-ESI/IT/MS chromatogram base peak area and four clusters with unknown IPLs, groups I, I ′ , II, and II ′ making up 23, 28, 1.5, and 7% of HPLC-ESI/IT/MS chromatogram base peak area, respectively. OL HFA were hydroxylated on the ester linked fatty acids at the α-position. P. saltans group I produced MS 3 fragmentation products of m/z 129, 130, and 147, identical to the group I LLs identified in F. johnsoniae. The relative retention time of group I IPLs in P. saltans are also similar to the retention time of the LLs in F. johnsoniae (Table 1), and were therefore also identified as LLs. This was confirmed by HPLC-HRAM/MS analysis ( Table 1). P. saltans group I ′ IPLs exhibited the same MS 3 fragmentation products (m/z 129, 130, and 147) as group I LLs, but MS 2 fatty acid fragmentation losses were 16 Th larger than observed for group I, indicating the IPLs in group I ′ are potentially hydroxylated-fatty acid versions of group I LLs (LL HFA ). This was also confirmed by HPLC-HRAM/MS analysis ( Table 1). As with OL HFA , group I ′ LL HFA were hydroxylated on the ester linked fatty acid α-position. The most abundant P. saltans fatty acids were iso branched as previously observed (Steyn et al., 1998;Liolios et al., 2011), and the distribution of fatty acid chain lengths and double bond equivalents were identical between the LLs, LL HFA , OLs, and OL HFA , suggesting a biosynthetic link between these IPL classes ( Table 1). Group I LLs and group I ′ LL HFA were isolated from the P. saltans extract using preparatory HPLC to confirm the lysine head group structure. Approximately 0.6 mg of both LL and LL HFA were individually isolated for NMR analysis. For comparison, roughly 2.0 mg of known OL was also isolated from the F. johnsoniae extract. Results from the 1 H-NMR, COSY, and 13 C-NMR analysis of isolated LLs and LL HFA ( Table 2; Supplementary Materials) revealed many similarities with NMR Retention time, RT; Accuracy, mmu*; OH, hydroxy group attached to fatty acids; Gln, glutamine; I, proposed lysine lipid (LL); I ′ , proposed lysine lipid with hydroxylated fatty acid (LL HFA ); I, proposed hydroxylysine lipid (HLL); II ′ , proposed hydroxylysine lipid with hydroxylated fatty acid (HLL HFA ). *In general the 0.5 mmu (milli mass unit) range represented very high confidence molecular formula assignments and the 1.0 mmu range represented good confidence molecular formula assignments (Kostiainen et al., 1997;Sakayanagi et al., 2006;Calza et al., 2012). 2 | Nuclear magnetic resonance (NMR) proton and carbon chemical shifts, and 2D NMR correlations (correlation spectroscopy: COSY; total correlation spectroscopy: TOCSY) of lysine lipid (LL) and lysine hydroxy fatty acid lipid (LL HFA ). characterization reported for OLs (Okuyama and Monde, 1996;Linscheid et al., 1997;Moore et al., 2013). The fatty acid chain chemical shifts were almost identical between LLs, LL HFA , and OLs, except for the OH group attached to the hydroxylated fatty acids of LL HFA . There were additional 1 H and 13 C signals for the head group of LLs and LL HFA corresponding to the ε-position of the lysine head group, compared to the OL head group, in agreement with the fact that lysine has one more carbon than ornithine. The most compelling 2D-NMR evidence of a lysine head group in the proposed LLs and LL HFA was observed in the TOCSY interactions among the head group positions ( Table 2). The same TOCSY interactions were previously described in the structural determinations of components containing an esterified lysine moiety (Anderson et al., 1995), and peptide-bound lysine (Rabenstein et al., 1997). Interactions observed by adjacent carbon and proton atoms in the HSQC spectra, and spatially close protons in NOESY spectra, further supported the lysine head group assignment of LLs and LL HFA . Amino acid analysis of each of the isolated LL, LL HFA , and OL components confirmed the presence of a lysine head group in the LL and LL HFA and an ornithine head group in the OL ( Table 3). Another pair of unknown IPLs (groups II and II ′ ) observed in the P. saltans lipid extract eluted at 29.2 and 34.9 min, respectively ( Figure 1B). The lipids in both of these groups exhibited identical OL-like fragmentation with ESI/IT/MS MS 3 products at m/z 129 and 145. As observed before for OL HFA and LL HFA compared to OLs and LLs, respectively, the later eluting group II ′ IPLs displayed MS 2 fatty acid fragmentation losses m/z 16 larger than group II, indicating groups II and II ′ contain the same polar head group but group II ′ is potentially hydroxylated on one of the fatty acids chains. HPLC-HRAM/MS analysis confirmed the presence of hydroxylated fatty acids in group II ′ , and revealed that in both groups II and II ′ , fragmentation losses of fatty acids results in the m/z 163.1076 product, which has the exact elemental composition of protonated hydroxylysine, C 6 H 15 N 2 O 3 (Figure 3). Further fragmentation (loss of H 2 O) results in the m/z 145.0972 product (C 6 H 13 N 2 O 2 ). The loss of NH 5 O from the C 6 H 15 N 2 O 3 head group results in the abundant proposed cyclic m/z 128.0707 product with elemental composition C 6 H 10 NO 2 , and the loss of CH 5 NO 2 from the C 6 H 15 N 2 O 3 head group results in the proposed cyclic m/z 100.0761 product (C 5 H 10 NO). These observed elemental compositions are similar to those for LL and LL HFA but all with an additional oxygen (Table 1, Figure 3). We propose that these lipids contain hydroxylysine as the amino acid head group, which would explain the additional oxygen in the elemental composition, the observed fragments, auxiliary gas (N 2 ) pressure, 10 AU; spray voltage, 4.0 kV; probe heater temperature, 300 • C; S-lens, 50 V. Target lipids were analyzed with a mass range of m/z 400-1000 (resolution, 70,000), followed by data dependent MS 2 (resolution, 17,500), in which the five most abundant masses in the mass spectrum were fragmented successively (normalized collision energy, 35; isolation width, 1.0). and higher retention times (due to its more polar nature) compared to LL ( Figure 1B). The fatty acid composition of the proposed hydroxylysine IPL (HLL) and fatty acid hydroxylated hydroxylysine lipids (HLL HFA ) in groups II and II ′ , respectively, are also the same as the OLs and LLs, including fatty acid hydroxylation ( Table 1). The retention time of the amino acid released by acid hydrolysis from the isolated HLL HFA (group II ′ ) was closer to the 5-hydroxylysine standard than any other amino acid standard, but the retention times were still 18 s apart ( Table 3). It is therefore likely that the proposed hydroxylysine head group is hydroxylated in a different position than the hydroxylysine standard resulting in a slightly different retention time. This is in agreement with the study by Morin et al. (1998), which found different hydroxylation positions on lysine resulted in different amino acid analysis retention times. Biosynthetic Pathway of Lysine Lipids The synthesis pathway of LL has not yet been described but it appears to be linked to the OL synthesis. The first described synthetic pathway for OLs involves a N-acyltransferase coded by the OlsB gene transferring a 3-hydroxy acyl group from the constitutive acyl carrier protein (ACP) to the α-amino group of ornithine forming lyso-ornithine, and then an Oacyltransferase (encoded by the OlsA gene) transferring a fatty acyl chain from the acyl carrier protein to the 3-hydroxy group of the lyso-ornithine, forming OL (Weissenmayer et al., 2002;Gao et al., 2004). Vences-Guzmán et al. (2014) described the existence of a second pathway mediated by a bifunctional protein with two acyltransferase domains coded by the OlsF gene in some bacterial species, which have been described to form OL but lack the OlsAB genes. Vences-Guzmán et al. (2014) also described the formation of OL and LL, using MS analysis, in E. coli expressing the OlsF-coding protein from the F. johnsoniae strain UW101, but they did not confirm that LL were indeed produced by F. johnsoniae. However, our study confirms that F. johnsoniae synthesizes OL and LL. LLs have previously been reported in Agrobacterium tumefaciens by Tahara et al. (1976a) and in Mycobacterium phlei by Lerouge et al. (1988). The LL characterized in M. phlei is diacylglycerol based, while the structure of the A. tumefaciens LL is similar to the lysine lipids observed in this study by LC/MS and NMR, i.e., composed of an ester linked fatty acid moiety, and an amide linked β-OH-fatty acid. Homologs of OlsF have been identified in genomes of γ-, δ-, and ε-Proteobacteria and in bacteria belonging to the CFB group (Vences-Guzmán et al., 2014). The genomes of F. johnsoniae and Pedobacter heparinus (both belonging to the CFB group) have been shown to harbor a homolog of OlsF (Vences-Guzmán et al., 2014). We searched for homologs of the OlsF-coding protein in genomes of Pedobacter species and P. saltans, whose lipid composition has been analyzed in our study. We detected putative homologs to OlsF in all Pedobacter genomes available (and in P. saltans) with an identity ≥70% to the P. heparinus putative OlsF (Vences- Guzmán et al., 2014), and an identity ≥40% to the OlsF protein of F. johnsoniae. These putative OlsF proteins in Pedobacter genomes have been previously annotated as hemolysines, as one of the genes of the hemolysin operon gene cluster, an acyltransferase catalyzing the transfer of a fatty-acyl group from acyl-ACP to α-amino groups of two lysine residues of the pro-toxin hemolysin converting it into a mature protein (Stanley et al., 1998). Considering this evidence, we speculate that the OlsF coding protein is involved not only in the synthesis of OL (Vences-Guzmán et al., 2014), but also in the synthesis of LL. Additionally, in our study we report for the first time LLs that are modified by hydroxylation on one of its fatty acid chains and on the lysine head group. P. saltans produces a wide range of amino acid-containing IPLs including OL, OL HFA , LLs, LL HFA , HLL, and HLL HFA . All of the various ornithine and lysine lipids produced by P. saltans have the same fatty acid distribution ( Table 1), suggesting a biosynthetic link. Hydroxylation of OL has been previously described and it is known to be mediated by the OL hydroxylase OlsC on ester-linked fatty acids (Rojas-Jimenez et al., 2005;Vences-Guzman et al., 2011), by the OlsD protein coding gene on the amide-linked fatty acid (González-Silva et al., 2011), or by the OlsE protein coding gene on the ornithine head group moiety (Rojas-Jimenez et al., 2005;Vences-Guzman et al., 2011). We performed protein blast searches for homologs of OlsC, OlsE of Rhizobium tropici (Rojas-Jimenez et al., 2005), and OlsD of Burkholderia cenocepacia but no putative homologs were detected in the currently available genomes of Pedobacter or P. saltans. However, we cannot rule out the presence of other putative OL hydroxylases in Pedobacter species genomes, which are expected to be quite divergent from the already described OL hydroxylases of Rhizobium and Burkholderia species, or that the hydroxylation of OL and LL in Pedobacter and Pseudopedobacter species is mediated by an alternative pathway. Environmental Controls on Lysine IPLs in P. saltans The novel characterization of HLL, and the high abundance of LL HFA and OL HFA in the lipid extract of P. saltans raises the question of their functional significance. Various studies have shown that certain bacteria increase fatty acid hydroxylation of OLs under temperature and pH stress (Taylor et al., 1998;Rojas-Jimenez et al., 2005;Vences-Guzman et al., 2011). The high abundance of LL HFA and OL HFA in P. saltans suggested that these lipids could be associated with in environmental stress response. We tested this hypothesis for LLs and OLs in P. saltans by growing triplicate cultures under three temperature/pH combinations: 30 • C/pH 7, 15 • C/pH 7, 15 • C/pH 9. The ratios of LL and OL abundance, and the degree of fatty acid hydroxylation in P. saltans changed under different temperature and pH conditions (Figure 4). There was a statistically significant increase in the ratio of the sum of LL HFA and OL HFA (LL HFA + OL HFA ) to total LLs and OLs (LL HFA + OL HFA + LL + OL) at higher temperatures and higher pH, with the largest ratio increase due to temperature (p < 0.0001) and a smaller increase due to pH rise (p = 0.0085, Figure 4A). There was also a significant increase in the ratio of HLL to the sum of HLL and LL at higher temperatures (p = 0.0115, Figure 4B). There were FIGURE 4 | Changes in amino acid-containing IPL abundance ratios based on HPLC-ESI/IT/MS chromatogram base peak area of each lipid group in Pseudopedobacter saltans lipid extract under 30 • C/pH 7, 15 • C/pH 7, and 15 • C/pH 9 growth conditions. (A) Ratio of total fatty acid hydroxylated lysine and ornithine lipids (LL HFA + OL HFA ) to total hydroxylated plus total unhydroxylated fatty acid lysine and ornithine lipids (LL HFA + OL HFA + LL + OL); (B) Ratio of total hydroxylysine lipids (HLL) to total HLL plus total LL; (C) Ratio of total LL to total LL plus total OL. Student's t-test statistically significant differences (p < 0.05) of IPL ratios between different growth conditions are represented by letters (a, b, c) over each bar (different letters represent statistically significant differences between bar graph values within each graph, values with the same letter within each graph are not statistically different). significant decreases in the ratio of total LLs vs. the sum of total LLs and OLs at higher temperatures (p < 0.0001) and higher pH (p = 0.0025), with the largest LL/(LL + OL) ratio decrease also due to temperature and a moderate decrease due to pH ( Figure 4C). There appears to be an OL and LL fatty acid hydroxylation stress response to temperature and pH in P. saltans, similar to the fatty acid hydroxylation stress response to temperature and pH observed in various other bacteria, which is proposed to increase membrane hydrogen bonding and stability (Taylor et al., 1998;Rojas-Jimenez et al., 2005;Vences-Guzman et al., 2011). In this case, there is a greater response to temperature than pH for P. saltans. The lysine head group hydroxylation also follows the same trend as fatty acid hydroxylation with more hydroxylation at higher temperature, as also observed in OL head group hydroxylation in R. tropici (Rojas-Jimenez et al., 2005;Vences-Guzman et al., 2011). OLs may also be preferentially produced over LLs at higher temperatures or pH levels in order to provide membrane stability due to the higher water solubility of ornithine than lysine. The various IPL composition changes due to temperature and pH show that the cell membrane of P. saltans is very dynamic in response to changing environmental conditions. Conclusions This is the first study to identify LL fatty acid hydroxylation and LL head group hydroxylation in membrane lipids. Confirmation of OL and LL formation in F. johnsoniae and P. saltans and the presence of OlsF putative homologs in P. saltans suggest the OlsF gene coding protein is possibly involved in OL and LL biosynthesis in both species, however, potential pathways of OL and LL hydroxylation in P. saltans are still undetermined. LL and OL fatty acid hydroxylation and LL vs. OL fractional abundance changes due to temperature and pH in microbial culture experiments were observed, thus expanding knowledge of microbial response to environmental change. The IPLs of related soil bacteria (i.e., Pedobacter sp.) should be characterized as well to investigate stress biomarker potential of environmental conditions such as temperature or pH. Analysis of soil samples is needed as well to reveal if these membrane lipid responses to temperature and pH changes can be observed in the environment.

v3-fos

2016-05-04T20:20:58.661Z

2015-03-18T00:00:00.000Z

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Overexpressing CYP71Z2 Enhances Resistance to Bacterial Blight by Suppressing Auxin Biosynthesis in Rice Background The hormone auxin plays an important role not only in the growth and development of rice, but also in its defense responses. We’ve previously shown that the P450 gene CYP71Z2 enhances disease resistance to pathogens through regulation of phytoalexin biosynthesis in rice, though it remains unclear if auxin is involved in this process or not. Methodology and Principal Findings The expression of CYP71Z2 was induced by Xanthomonas oryzae pv. oryzae (Xoo) inoculation was analyzed by qRT-PCR, with GUS histochemical staining showing that CYP71Z2 expression was limited to roots, blades and nodes. Overexpression of CYP71Z2 in rice durably and stably increased resistance to Xoo, though no significant difference in disease resistance was detected between CYP71Z2-RNA interference (RNAi) rice and wild-type. Moreover, IAA concentration was determined using the HPLC/electrospray ionization/tandem mass spectrometry system. The accumulation of IAA was significantly reduced in CYP71Z2-overexpressing rice regardless of whether plants were inoculated or not, whereas it was unaffected in CYP71Z2-RNAi rice. Furthermore, the expression of genes related to IAA, expansin and SA/JA signaling pathways was suppressed in CYP71Z2-overexpressing rice with or without inoculation. Conclusions and Significance These results suggest that CYP71Z2-mediated resistance to Xoo may be via suppression of IAA signaling in rice. Our studies also provide comprehensive insight into molecular mechanism of resistance to Xoo mediated by IAA in rice. Moreover, an available approach for understanding the P450 gene functions in interaction between rice and pathogens has been provided. Methodology and Principal Findings The expression of CYP71Z2 was induced by Xanthomonas oryzae pv. oryzae (Xoo) inoculation was analyzed by qRT-PCR, with GUS histochemical staining showing that CYP71Z2 expression was limited to roots, blades and nodes. Overexpression of CYP71Z2 in rice durably and stably increased resistance to Xoo, though no significant difference in disease resistance was detected between CYP71Z2-RNA interference (RNAi) rice and wild-type. Moreover, IAA concentration was determined using the HPLC/electrospray ionization/tandem mass spectrometry system. The accumulation of IAA was significantly reduced in CYP71Z2-overexpressing rice regardless of whether plants were inoculated or not, whereas it was unaffected in CYP71Z2-RNAi rice. Furthermore, the expression of genes related to IAA, expansin and SA/JA signaling pathways was suppressed in CYP71Z2-overexpressing rice with or without inoculation. Conclusions and Significance These results suggest that CYP71Z2-mediated resistance to Xoo may be via suppression of IAA signaling in rice. Our studies also provide comprehensive insight into molecular mechanism of resistance to Xoo mediated by IAA in rice. Moreover, an available approach for understanding the P450 gene functions in interaction between rice and pathogens has been provided. Introduction Bacterial blight is an important disease in rice caused by Xoo that results in severe loss of rice yield worldwide [1]. Rice has evolved to utilize a network of sophisticated signaling pathways against invasion by phytopathogens, for example pathogen-associated molecular patterns (PAMPs), systemic acquired resistance (SAR) and hypersensitive response [2][3][4][5]. Plant hormones such as SA, JA and IAA mediate broad-spectrum disease resistance in rice and have been widely studied; the mechanisms of resistance have been elucidated [6,7]. IAA, the major form of auxin in rice, is generally believed to play an important role in plant growth and development [8,9]. However, recent studies demonstrate that IAA acts as a negative regulator in the plant immune response [7,10,11], as exogenous application of IAA or auxin analogs in rice and Arabidopsis significantly promotes disease symptoms. Treatment with IAA and 2,4-dichlorophenoxyacetic acid (2, 4-D; an analog of IAA) in rice resistant to various types of bacterial blight significantly stimulates phytopathogenic Xoo proliferation, resulting in high susceptibility to these compounds [12]. Similarly, treatment of resistant rice plants with IAA enhances the infectivity of Xanthomonas oryzae pv. oryzicola (Xooc) and Magnaporthe oryzae to rice [13]. In addition, exogenous application of 1-naphthalacetic acid (NAA) or 2,4-D on Arabidopsis accelerates the development of disease symptoms during infection by Pseudomonas syringae pv. tomato (Pto) DC3000 or Pseudomonas syringae pv. maculicola [14,15]. On the other hand, many phytopathogens are capable of inducing significant IAA accumulation that weakens the host's native defense barrier, the cell wall [16][17][18][19][20]. This inhibits accumulation of endogenous auxin, leading to high disease resistance rates in rice. The mechanism for this is largely believed to be due to inhibition of expansin gene expression, which induces overexpression of GH3 family genes OsGH3.1, GH3-2 and GH3-8 that enhance broad-spectrum resistance to bacterial Xoo, Xooc and Magnaporthe grisea [12,13,21]. Based on these studies, it can be concluded that the suppression of the auxin signaling pathway partly contributes to disease resistance in rice. The synthesis of IAA is dependent on whether the precursor tryptophan (Trp) is involved, with Trp-dependent and Trp-independent pathways found in both the monocotyledonous model rice and the dicotyledonous model Arabidopsis [20,22,23]. In Trp-dependent pathways, indole 3-acetaldoxime (IAOx) is one of the key intermediate metabolites [22,24]. IAOx is a common precursor of auxin, camalexin and indole glucosinolates biosynthesis, and is a crucial branching point from primary metabolism to secondary metabolism in Arabidopsis [25][26][27][28]. The cytochrome P450 monooxygenase CYP79B2 is responsible for catalyzing the conversion of tryptophan to IAOx in Arabidopsis [24,25,29,30]. To date, many other cytochrome P450 monooxygenase genes involved in IAOx biosynthesis and metabolism have been cloned in Arabidopsis. The overexpression of cyp79B2 in Arabidopsis significantly increases IAA content [31], though less IAA is detected in the cyp79B2/cyp79B3 double mutant [31]. Another cytochrome P450 monooxygenase, CYP71A13, is capable of catalyzing IAOx to indole-3-acetonitrile (IAN) in the Trp-dependent IAA biosynthesis pathway [32]. P450 monooxygenases CYP83A1 and CYP83B1 have similar biochemical functions for IAA biosynthesis, which maintains the endogenous IAA balance in Arabidopsis [33][34][35]. Unfortunately, both IAO X and IAN have not been detected in rice, though indole-3-acetamide (IAM) is present [36,37], thus we hypothesize that Arabidopsis and rice have different IAA biosynthesis pathways. The pathway for IAA biosynthesis is very complex in rice, though a few genes involved in IAA signaling have been cloned, including the YUCCA family, the SMALL AUXIN-UP RNA (SAUR) family, the GH3 family and the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) family [38][39][40][41][42]. The cytochrome P450 family is the largest enzymatic protein family in rice and is largely responsible for both growth and development and the defense response [43][44][45][46][47]. In total, 356 P450 genes and 99 related pseudogenes have been identified in rice (indica and japonica) genomes using sequence information [48]. However, it remains unclear whether P450 genes involved in disease resistance to Xoo are responsible for regulating the IAA signaling pathway in rice. A previous study by our group showed that the cytochrome P450 gene CYP71Z2 contributes to bacterial blight resistance by mediating diterpenoid phytoalexin accumulation in rice [47]. Here we present studies on the role of CYP71Z2 in auxin signaling pathway. Constructs and transformation To construct the CYP71Z2 promoter GUS reporter vector, the predicted 1098 bp DNA fragment upstream of the start codon was amplified from Nipponbare genomic DNA and then inserted into the binary expression vector pBI121. The primers used in this study are shown in S2 Table. The recombinant plasmids were transformed into Agrobacterium tumefaciens strain EHA105 using a freeze-thaw method. Subsequently, the T-DNA region with the predicted CYP71Z2 promoter was introduced into calli derived from mature Nipponbare embryos using the Agrobacterium-mediated method [47]. Plant materials and growth conditions Three seedlings overexpressing CYP71Z2 and different RNAi-expressing rice were chosen to analyze the role of IAA in Xoo resistance. Transgenic seedlings (T5, T6 and T7) were grown in a growth chamber (12 h photoperiod; 28°C; 70% relative humidity; light strength, 30,000 lx), and a slow-release fertilizer was applied. All plants (wild-type and transgenic) were then transplanted into pots under normal growth conditions for Xoo inoculation, IAA quantification, RNA extraction and harvest. Measurement of disease resistance Resistance of rice to the bacterial blight pathogen Philippine Xoo strain PXO99 A was evaluated by the leaf-clipping method at the booting stage. Level of resistance was classified into six groups and measured using the percentage of diseased area (lesion length/leaf length) at 2-3 weeks following inoculation. The six classifications are: 1) Leaves without obvious lesions have high resistance, 2) Leaves with diseased area less than 10% have resistance, 3) Leaves with diseased area 10% but < 20% have modest resistance, 4) Leaves with diseased area 20% and < 50%, have modest susceptibility, 5) Leaves with diseased area 50% and < 75% have susceptibility and 6) Leaves with diseased area 75% have high susceptibility [47]. The bacterial growth rate in rice leaves was determined by counting colony-forming units [47]. Bioinformatic analysis of CYP71Z2 The CYP71Z2 promoter region was predicted using the online Promoter Scan (http://wwwbimas.cit.nih.gov/molbio/proscan/). The cis-acting regulatory DNA elements of the CYP71Z2 promoter were determined by searching in the PLACE (http://www.dna.affrc.go.jp/PLACE/ signalscan.html) database. Phylogenetic analysis among eight species was performed by MEGA. Protein sequence alignment was performed by searching in the non-redundant protein sequences database of the NCBI. Gene expression analyses Leaf samples next to the sites of bacterial infection were collected for RNA extraction at different time points post-inoculation with Xoo PXO99 A . Quantitative real-time PCR (qRT-PCR) was conducted on the Applied Biosystems 7500 real-time PCR system using SYBR Premix Ex Taq TM according to the manufacturer's instructions. For qRT-PCR analysis, three independent biological samples were used, each with three technical replicates. The internal reference gene EF-1a (accession no. AK061464) was used to standardize RNA quantities. Primers used in this study for qRT-PCR are shown in S2 Table. GUS histochemical staining and protein activity Rice tissue, including leaf, root and stem, were put into GUS staining solution for *5 hours at 37°C. The staining solution was removed and 75% alcohol was added to wash off excess stain, as described previously [12]. After complete decolorization, photographs of the stained rice tissue were examined using electron microscopy. IAA quantification To determine the concentration of IAA in rice, at least 1 g of sample was cut from each leaf at different time points post-inoculation and kept frozen at −80°C until use. IAA measurement conditions are described in the methods of [12]. IAA concentration was determined using the HPLC/electrospray ionization/tandem mass spectrometry system and the peaks of the precursor ions 176.3 after purification with a C18-SepPak cartridge. To assess the expression pattern of CYP71Z2, a CYP71Z2 promoter/GUS protein (β-glucuronidase) fusion expression vector was constructed (S1 Fig.). CYP71Z2 promoter/GUS transgenic plants were generated by transforming japonica cultivar Nipponbare. All seven confirmed transgenic lines showed a common pattern of GUS distribution, although differences were observed in GUS activity. GUS histochemical staining showed CYP71Z2 mainly expressed in the leaves, node, lemma, palea and primary roots, indicating tissue-specific expression patterns of CYP71Z2 in rice (Fig. 2). Gene expression patterns were analyzed by qRT-PCR, which showed high expression in the leaves, root node and internodes, consistent with results from GUS histochemical staining (Fig. 3). Expression patterns of CYP71Z2 during incompatible and compatible interactions between rice and bacterial blight were detected by qRT-PCR, with results showing that expression of CYP71Z2 in resistant rice NJH12 was higher than that in susceptible rice R109 (Fig. 4A). In addition, GUS activity in the leaves of CYP71Z2 promoter-driven transgenic rice plants significantly increased after inoculation with Xoo (Fig. 4B). These results suggest that CYP71Z2 is quickly activated in rice upon infection with the bacterial blight pathogen Xoo. Overexpressing CYP71Z2 increases resistance to Xoo Previous studies have shown that the rice P450 gene CYP71Z2 is involved in diterpenoid phytoalexin biosynthesis, contributing to bacterial blight resistance [47]. In this study, we selected six CYP71Z2-overexpressing rice (Acceptor rice is the Nipponbare; T5, T6 and T7) to identify the role of auxin in CYP71Z2-mediated blight resistance at the booting stage. Six homozygous CYP71Z2-overexpressing lines showed resistance to Xoo strain PXO99 A , with the average lesion area ranging from 1.86-4.75%, compared with an average of 47.37% in wild-type Nipponbare (Fig. 5A, B; S1 Table). The expression of CYP71Z2 in all six T7 CYP71Z2-overexpressing plants was higher than that in wild-type, showing increases of approximately 8.16-to 12.35fold ( Fig. 5B; S1 Table). Furthermore, bacterial growth analysis showed that the growth rate of PXO99 A in T7 CYP71Z2-overexpressing line OE51 was lower than that in wild-type after inoculation (Fig. 5C). These results suggest that overexpression of CYP71Z2 confers rice with durable, stable resistance to Xoo. We also examined resistance to Xoo in CYP71Z2-RNAi lines (T5, T6 and T7) at the booting stage. Expression of CYP71Z2 in RNAi lines was significantly reduced, as shown in Fig. 6. Moreover, none of the RNAi lines showed a significant difference in response to PXO99 A infection compared with wild-type ( Fig. 6; S1 Table). These results show that suppression of CYP71Z2 expression does not significantly increase susceptibility to PXO99 A in CYP71Z2-RNAi transgenic lines, suggesting that functional redundancy among the CYP71Z of family may mask the effect of CYP71Z2. CYP71Z2 negatively regulates IAA metabolism The endogenous phytohormone auxin is known to be involved in resistance of rice to bacterial blight [12], though it is unclear if auxin is involved in signaling pathways that contribute to CYP71Z2-mediated blight resistance. To examine this possibility, we measured free IAA concentration of CYP71Z2 transgenic lines at the booting stage. The concentration of endogenous free IAA in the CYP71Z2-overexpressing lines OE11, OE35 and OE51 was 3.21, 2.99 and 3.64 pg/mg fresh leaves, respectively. Compared with 5.07 pg/mg fresh leaves in wild-type, these results suggest that IAA expression is 1.47-to 1.7-fold lower than that of wild-type plants (Fig. 7A), which likely contributes to the resistance to Xoo of these transgenic plants. Moreover, the endogenous free IAA levels in the leaves of CYP71Z2-RNAi lines showed no significantly differences compared to those of wild-type (Fig. 7A), supporting the hypothesis of functional redundancy among CYP71Z family proteins in rice. To further analyze whether decreased IAA in CYP71Z2-overexpressing lines was caused by other genes of the IAA biosynthesis pathway, we quantified the expression of AAO1 (indole-3- acetaldehyde oxidase) and NIT1 (nitrilase) in rice using qRT-PCR. The sequence alignment showed 72% and 78% identity with homologous genes AAO1 and NIT1, respectively, in Arabidopsis [12]. Previous reports indicated that AAO1 and NIT1 function in two Trp-dependent IAA biosynthesis pathways (indole-3-pyruvic acid and indole-3-acetaldoxime) [12,38]. Quantitative analysis showed that expression of AAO1 and NIT1 in OE11, OE35 and OE51 CYP71Z2-overexpressing plants was lower by 3.01 to 4.12-fold and 1.46 to 2.01-fold, respectively, than that in wild-type (Fig. 7B). These results support the notion that CYP71Z2 negatively regulates AAO1 and NIT1 expression to suppress IAA accumulation. In addition, previous reports demonstrated that auxin signaling is also affected by changes in IAA concentration [12]. To evaluate this in our study, we analyzed the expression of auxin signaling-related genes (Aux/IAA families) in CYP71Z2-overexpressing lines by qRT-PCR. These results show that expression of IAA1, IAA4, IAA14 and IAA20 was lower in CYP71Z2overexpressing lines (OE11, OE35 and OE51) than in wild-type, especially with respect to IAA4 and IAA20 (Fig. 7C). IAA biosynthesis is suppressed in CYP71Z2-overexpressing rice after inoculation with Xoo Previous reports demonstrated that auxin signaling is involved in rice-Xoo interactions, with auxin seeming to act as a negative regulator of resistance to Xoo in rice [10][11][12]. To further examine whether auxin signaling takes part in disease resistance to Xoo, we analyzed the IAA concentration in the CYP71Z2-overexpressing rice OE51 after inoculation with Xoo strain PXO99 A . As shown in Fig. 8A, accumulation of IAA was induced at 8, 24 and 72 hours post-inoculation in both OE51 and wild-type. However, IAA accumulation in OE51 was found to be significantly lower than that in wild-type regardless of whether the plants were inoculated or not, with up to a 2.7-fold decrease in accumulation at 8 hours after innoculation (Fig. 8A). These results suggest that overexpression of CYP71Z2 in rice negatively regulates IAA biosynthesis in response to Xoo infection. The expression of auxin signaling and biosynthetic genes was also analyzed by qRT-PCR in the resistant transgenic OE51 and the susceptible wild-type rice following Xoo inoculation. Expression of AAO1, IAA1 and IAA20 was induced in both OE51 and wild-type plants after infection, though expression of AAO1 and IAA20 was significantly decreased in wild-type at 24 h post-inoculation (Fig. 8B, C and D). However, we found that expression of AAO1, IAA1 and IAA20 in OE51 was significantly suppressed compared with wild-type after inoculation (Fig. 8B, C and D), suggesting that overexpression of CYP71Z2 in rice suppresses the expression of genes involved in auxin signaling. Overexpressing CYP71Z2 inhibits expression of expansin genes The plant cell wall is a natural protective barrier for phytopathogens. The loosening cell walls are easier to be infected by pathogenic bacteria. Suppression of expansion genes can prevent plant cell walls from loosening, resulting in enhanced physical protection of plants to phytopathogens [12]. To examine their role in the resistance to Xoo, we determined the expression of six expansin genes in CYP71Z2-overexpressing rice, including three rice α-expansin genes (EXPA1, EXPA5 and EXPA10) and three rice β-expansin genes (EXPB3, EXPB4 and EXPB7). qRT-PCR analysis showed that expression of all six expansin genes was decreased in CYP71Z2overexpressing rice compared with wild-type under normal growth condition (Fig. 9). These results demonstrate that expansin genes may partly contribute to the resistance to Xoo in CYP71Z2-overexpressing rice. The SA/JA pathway is not involved in Xoo resistance of CYP71Z2overexpressing rice Previous studies suggested that SA/JA defense responses are independent of the resistance pathway mediated by auxin in rice [12]. To test whether the increased resistance to Xoo in CYP71Z2-overexpressing rice accompanied by the inactivation of SA-and JA-dependent defense pathways, we detected transcripts of four key genes (PR1a, PR1b, LOX and AOS2) that act in two distinct classes of defense signaling pathways. Relative expression levels analyzed by qRT-PCR showed that three genes had lower expression in OE51 than in wild-type without inoculation, with reductions in AOS2, LOX and PR1a being 1.52-, 2.33-and 2.39-fold, respectively, although PR1b was increased by 1.52-fold in OE51 compared with wild-type (Fig. 10). During Xoo infection, these four genes were largely suppressed in OE51 at most time points (Fig. 10). Gene expression analysis demonstrated that CYP71Z2 may function as a negative regulator of the SA/JA defenses signaling pathways during the incompatible interaction between rice and Xoo, which is consistent with results shown in a previous study [12]. Discussion The mechanisms behind bacterial and fungal disease resistance in rice remain largely unknown, though some GH3 genes and auxin biosynthesis regulators have been implicated in the process. Our previous study showed that the P450 gene CYP71Z2 is involved in resistance to Xoo infection through activation of the phytoalexin biosynthesis pathway. In this study, we've found that overexpression of CYP71Z2 confers transgenic (T5, T6 and T7) rice with durable, stable resistance to bacterial blight, which is accompanied by up-regulation of genes related to IAA biosynthesis and IAA response pathways. Moreover, no significant differences were observed in resistance to Xoo and IAA accumulation between CYP71Z2-RNAi and wild-type lines, suggesting some functional redundancy to compensate for reduced CYP71Z2 expression. These results demonstrate that the cytochrome P450 gene CYP71Z2 is involved in disease resistance to Xoo, potentially through negative regulation of IAA/auxin biosynthesis. The role of auxin in plant disease resistance has been widely studied, with recent evidence demonstrating IAA's role as a negative regulator of plant disease resistance to bacterial and fungal pathogens [49,50]. The role of GH3-like proteins in disease resistance mediated by IAA has also been gradually elucidated over recent years [12,13,21]. In this study, homozygous CYP71Z2-overexpressing rice showed durable resistance to Xoo accompanied by a reduction in IAA accumulation (Fig. 5, 7), consistent with the resistance phenotype of IAA-deficient plants in previous reports [12,13,21]. In addition, the putative indole-3-acetaldehyde oxidase (AAO1) and nitrilase (NIT1), two key genes related to auxin synthesis in rice [12,38], were found to have reduced expression in CYP71Z2-overexpressing rice (Fig. 7). These results further confirm that suppression of auxin biosynthesis contributes to disease resistance of CYP71Z2-overexpressing rice, and overall importance of auxin regulation in response to pathogenic infection. P450 genes have been reported to either act as either positive or negative regulators of auxin homeostasis in Arabidopsis. A plausible explanation for this may be that the substrates catalyzed by different P450 oxidases are different, resulting in changes in IAA production. For example, cyp83B1 mutants show significant overproduction of auxin, whereas CYP83B1overexpressing lines display a loss of apical dominance that is typical of auxin deficiency [33][34][35]. These studies also showed that the CYP83B1 protein is responsible for converting IAOx to 1-aci-nitro-2-indolyl-ethane, which functions to maintain the dynamic balance between IAA and indole glucosinolate metabolism [34,35]. In addition, the cytochrome P450 enzyme CYP79B2 catalyzes the transformation of tryptophan into IAOx, playing a positive role in IAA biosynthesis [29,30]. In this study, we demonstrate that another P450 gene, CYP71Z2, shows similar results when overexpressed as the CYP83B1 mutants, suggesting that CYP71Z2 plays a negative regulatory role in IAA biosynthesis. Unfortunately, substrates for CYP71Z2 and the mechanism behind this role have not yet been identified. IAOx and IAN are two key intermediates of camalexin metabolism and IAA biosynthesis in Arabidopsis, suggesting cross-talk between these two pathways [22,24]. Overexpression of CYP79B2 in Arabidopsis has been shown to increase IAA content and lead to excessive auxin production, which was likely due to CYP79B2 catalyzing the transformation of tryptophan into IAOx. Interestingly, CYP79B2/CYP79B3 double mutants had reduced levels of both IAA and camalexin, suggesting some degree of similar regulation between the two pathways [24,32]. Moreover, CYP71A13 was shown to catalyze the conversion of IAOx to IAN, which also led to reductions in IAN and camalexin upon cyp71A13 mutation [32]. Taken together, these data indicate that cross-talk likely exists between the auxin and camalexin biosynthetic pathways in Arabidopsis. Results from our study showing reduced IAA accumulation in CYP71Z2overexpressing rice (Fig. 7A), in conjunction with previous reports showing that CYP71Z2 accelerates phytoalexin biosynthesis [47], lead us to speculate that cross regulation of IAA and phytoalexin biosynthesis also exists in rice, though this hypothesis requires further study. Many phytopathogens produce IAA for survival and multiplication during the infection process [16][17][18][19][20]. Pathogen-produced IAA leads to induction of the expression of rice expansin genes, resulting in an increase in long-term cell wall flexibility [12,51]. This process makes plant cell walls vulnerable and contributes to pathogen infection and multiplication in rice. In this study, the relative expression of expansin genes in Xoo-resistant, CYP71Z2-overexpressing rice was significantly decreased and correlated with suppression of IAA signaling (Fig. 7-9). These results suggest that the suppression of expansin genes may also contribute to disease resistance in CYP71Z2-overexpressing rice, though the mechanisms behind this remain unclear. As has been shown, auxin biosynthesis is suppressed in resistant rice and is always accompanied by decreases in the expression of auxin-responsive genes. The expression of auxin signaling-related genes was found to be significantly decreased in auxin-deficient, GH3-8overexpressing plants exhibiting resistance to Xoo [12,52,53]. Consistently, the accumulation of auxin signaling-related genes AAO1, IAA1 and IAA20 was inhibited in CYP71Z2-overexpressing rice (Fig. 8). This suggests that suppression of auxin response pathways results from reduced IAA accumulation in CYP71Z2-overexpressing rice. JA and SA signaling pathways play an important role in broad-spectrum and durable disease resistance of rice. More studies are finding that immunity conferred by SA or JA is independent of IAA resistance signaling in plants, with no correlation reported between suppression of auxin signaling and the activation of SA/JA signaling pathways in resistant rice [12,49]. Moreover, plant immunity mediated by SA is often accompanied by inhibition of auxin signaling, including down-regulation of auxin-response genes and IAA-amido synthase genes of the GH3 family [54]. In this study, qRT-PCR analysis showed that the expression of four key genes involved in SA/JA signaling was significantly decreased (Fig. 10), suggesting that SA and JA signaling pathways are inhibited by overexpression of CYP71Z2 in rice. These results demonstrate that activation of SA or JA signaling pathways is not required for disease resistance mediated by IAA in CYP71Z2-overexpressing rice. The P450 family is the largest protein family in rice and plays an important role in the growth, development and defense responses of this plant. The function of some P450 genes in auxin biosynthesis has been studied in Arabidopsis, although so far, similar functionality has not been studied for P450 genes in rice. In this study, the overexpression of CYP71Z2 in rice increased resistance to Xoo PXO99 A with suppression of IAA accumulation and IAA response genes, suggesting that the P450 gene CYP71Z2 takes part in IAA signaling in rice. Moreover, no significant differences in IAA accumulation were detected between CYP71Z2-RNAi rice and wild-type, which could be due to residual CYP71Z2 mRNA or functional redundancy among CYP71Z subfamily proteins. Regardless, these results show that a P450 gene plays a significant role in resistance to pathogen infection in transgenic rice by mediation of the auxin signaling pathway. Table. Gene-specific primers used for qRT-PCR analysis and amplication in this article.

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2018-12-14T20:40:18.263Z

2015-02-26T00:00:00.000Z

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Study of reproductive compatibility and morphological characterization of interspecific hybrids in Sesamum sp In the present study, three wild species of sesame, Sesamum alatum, Sesamum malabaricum and Sesamum radiatum and one wild variety of Sesamum indicum, that is, S. indicum var. yanamalaiensis were crossed with eight cultivated varieties of S. indicum L. in both direct and reciprocal forms. All the wild species exhibited different degrees of cross compatibility with cultivated S. indicum. There was no crossed seed set in the direct and reciprocal crosses involving cultivars of S. indicum (2n = 26) with S. radiatum (2n = 64) and with S. alatum (2n = 26). The crosses involving S. malabaricum and S. indicum var. yanamalaiensis having the same chromosome number (2n = 26) as in the cultivated sesame genotypes were fairly successful in producing high percentage of crossed capsules with well filled seeds. The morphology of four wild species along with the cultivated species of sesame and the interspecific hybrids derived were compared. The wild species utilized in the present study differed significantly from the cultivated in branching pattern, leaf pubescence, flower size, color of corolla and anther, size, shape and color of extra floral nectary, capsule size, and shape, texture and size of the seed. All the successful interspecific hybrids showed predominance of wild characters than cultivated S. indicum. INTRODUCTION Sesame is known to be the most ancient oilseed crop dating back to 3050-3500 B.C. (Bedigian and Harlan, 1986) because of its ease of extraction, great stability, and drought resistance. It is also considered to be important because of its nutritional and antiaging features of high quality vegetable oil with oil content ranging from 50 to 60% (Chayjan, 2010). The sesame oil is highly resistant to oxidative deterioration due to the presence of antioxidants such as sesamin and sesamolin (Erbas et al., 2009) and also has high percentage of unsaturated *Corresponding to author. E-mail: [email protected]. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License fatty acids (Yermanos et al., 1972). Though sesame is having all these benefits, the productivity is limited due to low seed yield (Ashri 1989;Pham et al., 2010), frequent occurrence of diseases (El-Bramawy, 2006) and stress factors (Sarwar et al., 2007). Therefore, breeding efforts have mainly concentrated on increasing the seed yield of sesame. One of the important ways for increasing seed yield is utilization of diverse sources, especially the wild species for the exploitation of heterosis as well as to impart biotic and abiotic stress resistance. Hence an attempt was made to study the crossability between the four wild and cultivated species of sesame and to evaluate the hybrid vigour expression in the interspecific crosses. MATERIALS AND METHODS The experimental materials comprised of three wild species of sesame, Sesamum radiatum (2n = 64), Sesamum alatum (2n = 26), Sesamum malabaricum (2n = 26) and one wild form of Sesamum indicum, that is, S. indicum var. yanamalaiensis (2n = 26) as reported by Devarathinam and Sundaresan (1990) with eight cultivated varieties of S. indicum ( Figure 1). This includes CO 1, PYR 1, SVPR 1, VRI 1, VRI(Sv) 2, TMV 3, TMV 4 and TMV 7. The wild species were collected from the Species Garden maintained at Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore, India. The varietal seeds were obtained from the Department of Oilseeds, TNAU, Coimbatore. The seedlings were raised in earthern pots during Summer, 2010. During flowering, the crossing was effected by utilizing the wild species as both male and female parents. This was done by emasculating the female flower buds (removing the corolla with four stamens) in the previous day evening. The just opened male flowers were collected from the respective parents on the following day morning and pollination was done between 6:30 to 8:30 AM. For this, the 1/3 rd of the corolla was removed to expose the stamen outside, which was then smeared on the stigma of the emasculated flower. A small paper tag was tied at the base of the pollinated flowers for easy identification of crossed capsules at the later stage. The entire crossing block was raised in the glass house to avoid insect pollination. The seeds were collected from the crossed capsules and the F 1 generation was raised with the parents, in two replications with each entry in two rows of 5 m length and spacing of 15 × 30 cm during Kharif, 2010. All the recommended package of practices was adopted. Compatibility relationship Based on the crossing data, it was evident that all the wild species exhibited different degrees of cross compatibility with cultivated S. indicum. The details about direct and reciprocal crosses attempted between the cultivars and the wild species are presented in Table 1. From the data on number of flowers pollinated, number of capsules set and number of hybrid seeds obtained, the crossability between the wild species and cultivated varieties were brought out. The overall cross compatibility relationship is given in Table 2. Between S. indicum (2n = 26) and S. alatum (2n = 26) Though S. indicum and S. alatum are having the same chromosome number, capsule and seed setting was not observed in both direct and reciprocal crosses of S. alatum with eight cultivars of S. indicum. Kedharnath (1954) could obtain only shriveled and non-viable seeds in S. indicum and S. alatum combination and presumed an early abortion of young embryo. Similar attempts were made by Amirtha Devarathnam (1965), Sundaram (1968) and Subramanian (1972) who also failed in producing viable hybrids between these two species. Even though these two species are having the same chromosome status (2n = 26), it is probable that a strong mechanism operates, due to which, hybrid seeds were not obtained. Between S. indicum (2n = 26) and S. radiatum (2n = 64) The direct crosses recorded the capsule setting with the maximum of 6.80% in S. radiatum × TMV 7 and minimum of 1.02% in S. radiatum × VRI 1. There was no seed set in any of the eight direct crosses between S. radiatum and S. indicum. The reciprocal cross between S. indicum and S. radiatum was not successful due to the premature dropping of crossed capsules. Hence, no capsule set and seed set was observed in the reciprocal crosses. Earlier studies involving these species also revealed the failure of these crosses (Dhawan, 1946;Ramanathan, 1950;Amirtha Devarathnam, 1965;Subramanian, 1972;Prabakaran, 1992;Vikas, 2006). The failure was attributed to very early collapse of the hybrid endosperm and the subsequent starvation of proembryo, as observed by Dhawan (1946) through embryological studies. Between S. indicum (2n = 26) and S. malabaricum (2n = 26) The crosses involving S. indicum cultivars and S. malabaricum both having the same somatic chromosome number (2n = 26) were fairly successful in producing good number of crossed capsules with well filled seeds. In the direct and reciprocal crosses effected between eight cultivars of S. indicum with S. malabaricum, successful capsule and seed setting was observed in all the 16 crosses ( Figure 2). Only one crossed seed got germinated in the cross between S. malabaricum as female with S. indicum genotype SVPR 1, but the seedling was died subsequently in the two leaves stage, probably due to the abiotic factors. In the earlier description by John et al. (1950), S. malabaricum was referred as the variety of S. indicum, as S. indicum var. malabaricum, which was highly compatible with other genotypes of S. indicum. However, Prabakaran (1992) referred this as the separate species of sesame as S. malabaricum. He reported that S. malabaricum had possessed distinct morphological features like longer duration, green stem with purple tinge, leathery leaves, purple corolla, highly rough testa as seen against the cultivated sesame. Also, S. malabaricum had shown partial capsule set when crossed with cultivated S. indicum (Prabakaran, 1992). The percentage of capsule setting ranged from 4.6% (S. malabaricum × CO 1) to 7.9% (S. malabaricum × VRI 1) in direct crosses. In reciprocal crosses, it was between 0.9% (TMV 3 × S. malabaricum) and 12.6% (SVPR 1 × S. malabaricum). In direct crosses, the mean number of seeds per capsule was the lowest in S. malabaricum × SVPR 1 (6.7) and highest in S. malabaricum × TMV 7 (25.7). Similarly, the cross SVPR 1 × S. malabaricum recorded the lowest number of seeds per capsule (13.1) and the highest was recorded in TMV 7 × S. malabaricum (55.1) in reciprocal crosses. Between S. indicum (2n = 26) and S. indicum var. yanamalaiensis(2n = 26) The cross-compatibility between S. indicum and S. indicum var. yanamalaiensis both having the same chromosome number of 2n = 26 was confirmed both in direct and reciprocal form (Figure 2). But the capsule set and seed set was not observed in S. indicum var. yanamalaiensis with TMV 3 and TMV 4. The range of capsule setting was from 0 to 52.2% in S. indicum var. yanamalaiensis × VRI(Sv) 2. The seed setting was ranged from 0 to 22.3% (S. indicum var. yanamalaiensis × Paiyur 1) in direct crosses. In the cross S. indicum var. yanamalaiensis × VRI 1, crossed seed was obtained but the seeds were small and shriveled and hence not germinated. In the reciprocal crosses, there was capsule set, but no seed set in TMV 4 × S. indicum var. yanamalaiensis. The range of capsule setting was from 1.0% (TMV 3 × S. indicum var. yanamalaiensis) to 14.1% in SVPR 1 × S. indicum var. yanamalaiensis. The seed setting was ranged from 0 (TMV 4 × S. indicum var. yanamalaiensis) to 47.5% (TMV 7 × S. indicum var. yanamalaiensis). Since the flowering of both parents had not coincided and hence, the pollination was attempted in the later stage of flowering. Due to this, the seed set was not observed in few of the direct and reciprocal crosses between S. indicum var. yanamalaiensis and cultivated varieties. Parents The morphology of four wild species and the cultivated species of sesame was compared and given in Table 3. The wild species utilized in the present study differed significantly from cultivated one in the branching pattern, leaf pubescence, flower size, color of corolla and anther, size, shape and color of extra floral nectary and capsules, texture and size of the seed. S. alatum was profusely branching with completely lobed basal leaves. The corolla color was maroon and glabrous with dark purple corolla lip. The anther was dark purple with purple colored extra floral nectary. The capsules were long and tapering with small and winged seeds. The branches of S. malabaricum were profuse with pubescent leaves. The Kumari and Ganesamurthy 915 corolla was pink and densely hairy with dark pink colored corolla lip. The calyx also had dense hairs with flower having purple anther. The glands were yellow colored and prominent. The capsules were medium sized and hairy. The seeds were also medium sized with rough testa. S. indicum var. yanamalaiensis resembled cultivated S. indicum in most of the traits. It differed from cultivars in branching pattern, corolla and corolla lip color and in the size of yellow glands. The capsules were medium sized sparsely hairy with small black colored seeds with smooth testa as in the cultivated varieties. The wild species S. radiatum differed widely from S. indicum. The stem of S. radiatum was pubescent with more number of branches. The leaves were dark green, pubescent with serrated margins. The corolla was hairy, purple colored with dark purple corolla lip. The calyx was also pubescent with flowers having big, cream colored anther. The glands were dark colored with densely hairy capsules. The seeds were small with rough testa. These above mentioned specific traits were not observed in the cultivated S. indicum genotypes. Inter-specific hybrids The observed morphological characters of the direct and reciprocal crosses of wild with cultivated species are given in Table 4. The hybrids developed from the direct and reciprocal crosses involving S. malabaricum and S. indicum were similar in the expression of qualitative traits. But, the hybrids with S. malabaricum as the female parent had taken comparatively more days to germinate, when compared to their reciprocals. This difference was due to the maternal seed traits of the wild parent. The duration taken for germination of hybrids is much more than their cultivar parent. The hybrids exhibited most of the phenotypic characters of wild parent, indicating the dominant nature of S. malabaricum. The direct crosses resembled the wild parent, S. malabaricum in branching pattern, leaf pubescence, corolla and corolla lip color, flower having calyx with dense hairs, and light purple colored anther. The nature and color of extra floral nectary resembled the wild parent. The capsules were very small with few seeds, which was medium sized black with rough testa. The crossed seeds had expressed dormancy as in the wild parent and many of the crossed seeds had not germinated for more than two months. The reciprocal crosses had also expressed similar traits as in direct crosses between S. malabaricum and S. indicum. The F 1 hybrids involving the eight cultivars of S. indicum with S. indicum var. yanamalaiensis, were evaluated for their morphology and it was found that the 12 hybrids resembled the wild parent in branching pattern, corolla and corolla lip color. From this study, it was found that all the successful interspecific hybrids showed predominance of wild characters than cultivated S. indicum. The wild species viz., S. malabaricum and S. indicum var. yanamalaiensis could be effectively utilized for the transfer of essential traits from wild to cultivated through conventional breeding program.

v3-fos

2016-05-12T22:15:10.714Z

2015-09-23T00:00:00.000Z

29887932

s2

Comparative quantitative trait loci for silique length and seed weight in Brassica napus Silique length (SL) and seed weight (SW) are important yield-associated traits in rapeseed (Brassica napus). Although many quantitative trait loci (QTL) for SL and SW have been identified in B. napus, comparative analysis for those QTL is seldom performed. In the present study, 20 and 21 QTL for SL and SW were identified in doubled haploid (DH) and DH-derived reconstructed F2 populations in rapeseed, explaining 55.1–74.3% and 24.4–62.9% of the phenotypic variation across three years, respectively. Of which, 17 QTL with partially or completely overlapped confidence interval on chromosome A09, were homologous with two overlapped QTL on chromosome C08 by aligning QTL confidence intervals with the reference genomes of Brassica crops. By high density selective genotyping of DH lines with extreme phenotypes, using a Brassica single-nucleotide polymorphism (SNP) array, the QTL on chromosome A09 was narrowed, and aligned into 1.14-Mb region from 30.84 to 31.98 Mb on chromosome R09 of B. rapa and 1.05-Mb region from 27.21 to 28.26 Mb on chromosome A09 of B. napus. The alignment of QTL with Brassica reference genomes revealed homologous QTL on A09 and C08 for SL. The narrowed QTL region provides clues for gene cloning and breeding cultivars by marker-assisted selection. via screening the genomic regions that show high differences of reads between 'Highest' and 'Lowest' bulks. In comparison with the traditional QTL mapping, the NGS-aided strategy provides a simple and effective alternative to rapidly identify QTL of interest by genotyping small number of samples from two sets of individuals with distinct or opposite extreme phenotypes 23,24 . By using the NGS-aided strategy, a few QTL of the interested traits have been successfully identified in yeast 23,[25][26][27] , rice 24,28,29 , Arabidopsis thaliana 30 , sunflower 31 , cucumber 32 , wheat 33 , tomato 34 and chickpea 35 . In this study, the strategies of conventional QTL mapping and high-throughput genotyping were combined to dissect QTL of SL and SW in a doubled haploid (DH) population and its reconstructed F 2 (RC-F 2 ) population of rapeseed. Based on the alignment of SSR markers to the reference genomes of Brassica crops, the genetic region on chromosome A09 where the 17 overlapped QTL of SL and SW on chromosome A09 enriched, was revealed to be homologous with the overlapped QTL for SL on chromosome C08. The major QTL region on chromosome A09 was aligned to a ~1 Mb region on the reference genome of B. rapa and B. napus with high density SNP array. Results Variation in silique length and seed weight. The semi-winter parental line 'SWU07' exhibited higher SL and SW values than the winter parental line 'Express' . Wide variation was detected in both the DH and RC-F 2 populations for SL and SW across the years analyzed (Fig. 1). The field performance of the 233 RC-F 2 lines, with an average SL of 6.13 ± 0.71 cm and an average SW of 3.77 ± 0.38 g, was superior to that of 261 DH lines, which had an average SL of 5.67 ± 0.81 cm and an average SW of 3.47 ± 0.46 g. The normal distribution for SL and SW in both populations suggested that SL and SW were controlled by multiple genes (Fig. 1). The ANOVA results showed significant differences among genotypes, years and genotype-by-year interactions for SL and SW in the two populations (P < 0.01) (Table S1). High broad-sense heritability was detected for SL and SW (average of 83.79% for SL and 79.13% for SW). The significant and positive correlation between SL and SW in both populations (r = 0.49 and 0.34 in the DH and RC-F 2 populations, respectively; P < 0.01) suggested that long silique had the potential to increase SW. QTL analysis. 20 QTL were located on chromosomes A01, A05, A09, and C08 for SL in the DH and RC-F 2 populations across years, totally explaining 55.1-75.7% of the phenotypic variation, whereas 21 QTL were detected for SW on A04, A05, A06, A09, C02, and C05, totally explaining 24.4-62.9% of the phenotypic variation in the DH and RC-F 2 populations across years (Table 1, Fig. 2). Among these QTL, 17 QTL for SL and SW were enriched in a region on chromosome A09 from 80 cM to 107.5 cM with partially or completely overlapped confidence interval. The direction of positive additive effect for these QTL was consistent from parental line 'SWU07' (Fig. 2). No digenic interaction with significant and high effect was found for SL and SW, although nine and ten additive by additive interactions were detected for SL and SW with minor effects, total explaining 2.78% and 4.14% of the phenotypic variation in DH and RC-F 2 populations, respectively. Together the positive correlation between SL and SW and overlapped confidence intervals of QTL, indicated that some common genetic factors might regulate both SL and SW. Given the high degree of synteny between A09 and C08 (http://genomevolution.org/wiki/index.php/ Brassica_oleracea_v._Brassica_rapa), the microsynteny between QTL of SL on A09 and C08 was compared by aligning QTL intervals with the reference genomes of B. rapa and B. oleracea (Fig. 3). The interval of 17 overlapped QTL for SL and SW on chromosome A09, linked with 9 molecular markers (CNU402 ∼ CNU263), was aligned to the regions from 26. Fig. 3; Table S2). The co-localization of QTL suggests homoeologous duplicated QTL for SL on chromosome A09 and C08 in rapeseed. In order to narrow the QTL region in A09, 25 DH lines with extreme performance of SL and SW, were screened by 293 SSR markers to evaluate genetic background. Five and six lines were finally chosen to construct the 'Large' and 'Short' groups, respectively. Thus the two groups shared the same genetic background except for the region of QTL on chromosome A09. The average values for SL and SW in the 'Large' group (SL 7.46 cm and SW 4.18 g) were significantly higher (p < 0.01) than the 'Small' group (SL 3.97 and SW 3.04 g). Each individual from two groups was subjected to high-density selective genotyping using the Brassica 60 K SNP Bead Chip Array. Of the 52,157 SNP markers in the Brassica SNP array, 41,645 (80%) could be detected among 11 DH lines, while only 2751 (6.61%) SNPs were polymorphic between the 'Large' group and 'Small' group, indicating their similar genetic background. The Δ (SNP-index) of each SNP locus was calculated by subtraction of SNP-index of the 'Large' group from the 'Small' group, and the Δ (SNP-index) trends were visualized by means of a sliding window (Fig. 4). A single peak region with a significant (p < 0.01) and high value of Δ (SNP-index), harboring 45 SNP markers (Table S3), was identified (Fig. 4). It corresponded to 1.14-Mb region from 30.84 to 31.98 Mb on chromosome R09 of B. rapa (Fig. 4), and 1.05-Mb region from 27.21 to 28.26 Mb on chromosome A09 of B. napus, which were located into the interval of QTL on A09 detected by conventional QTL mapping. A total of 241 and 225 genes were harbored in these two regions, respectively. Of which, 69.71% (168/241) genes on the chromosome R09 of B. rapa were orthologous to 73.78% (166/225) genes on the chromosome A09 of B. napus, suggesting the homology between two regions. To confirm the narrowed region harboring QTL, ten region-specific SSR (RS-SSR) markers were developed according to the genomic sequence of the regions in reference genome of B. rapa and B. napus. Of which, two RS-SSR markers (CY-04 and CY-10) that exhibited polymorphisms between two parents were successfully mapped to the confidence interval of the QTL on A09 in DH population (Fig. 4), further indicating the consistency between QTL mapping and NGS-aided studies. Discussion QTL mapping is the main approach for genetic dissection of quantitative traits, which provides the start point for map-based cloning of related genes and marker-assisted selection (MAS) in plant breeding. Although QTL mapping for silique traits have been reported [11][12][13][14][15][16][17][18][19] , single population was almost adopted in these studies. In this study, two related populations, DH and its derived RC-F 2 populations were used. The RC-F 2 population holds unique characteristics with normal F 2 population if genotypic selection did not exist in the process of microspore culture, but has advantages over normal F 2 population. For example, it permits the possibility of replicated experiments in multiple years or environments. The major QTL on A09 were repeatedly detected in both populations, indicating the credibility of the QTL on A09. More than 100 QTL have been detected for SL and SW in rapeseed [11][12][13][14][15][16][17][18][19] , but the detailed comparative or functional analyses of these QTL have not been reported. In this study, we assigned QTL regions onto the reference genomes of Brassica crops through BLAST analysis of markers linked to the QTL. This enabled us to detect homeologous duplicated QTL for SW and SL on B. napus chromosomes A09 and C08. In order to test the power of this approach, we compared our result with the study of Yang et al. (2012), who also identified a major QTL region for SL and SW on chromosome A09 in B. napus using different markers with this study 16 . By aligning markers in the flank of the QTL (Na10-B07 and CNU008) to the reference genome of B. rapa, the confidence intervals of the QTL were aligned to the genomic region on chromosome R09 of B. rapa from 30.1 to 32.2 Mb, which partially overlapped with that of QTL detected in the current study (Fig. 3). These findings supported the credibility of QTL on A09 controlling SL and SW. The conventional approach to narrow QTL regions is a laborious process that requires the development of DNA markers and the generation of a large number of advanced-generation progenies. These Figure 3. Comparative analysis of QTL for silique length and seed weight on chromosomes A09 and C08 via alignment of the SSR loci linked with the QTL to chromosomes R09 and O08 of the reference genomes of B. rapa and B. oleracea, respectively. ES-DH was derived from the cross between 'Express' and 'SWU07' in this study, and the NS population was from the study of Yang et al. (2012). Lines beside linkage groups represent QTL for silique length and seed weight, respectively. The light-grey highlighted regions on the linkage groups showed the QTL intervals, and the dark-grey highlighted regions on the genomes exhibited the co-location physical regions of the QTL on A09 and C08. requirements limit its use because they are time consuming and costly, particularly in annual crop species like rapeseed. With the release of reference genomes for some species and advances in NGS technology, several novel strategies for QTL mapping have been proposed with the use of high-throughput genotyping, such as microarray-based genotyping or massively parallel sequencing. In the present study, the process of QTL-seq was followed with slight modification, i.e. a high-density Brassica SNP array, instead of resequencing of bulks in QTL-seq, was used to genotype DH lines with the extreme trait values, and the QTL were successfully anchored to a ~1 Mb region on chromosome R09 of B. rapa and A09 of B. napus. Given the high-throughput nature and low cost of the SNP array, this modified approach will dramatically accelerate the process of QTL fine-mapping in a cost-effective manner. Conclusions We identified 20 and 21 QTL for SL and SW in DH and RC-F 2 populations of B. napus across three years. A significant and positive correlation between SL and SW, and overlapped confidence intervals among partial QTL for SL and SW detected in this study suggested long silique has the potential to increase SW. The major QTL for SL on chromosome A09 and C08 were aligned to the same region of the reference genomes of Brassica crops, suggesting they are homologous QTL. By high density selective genotyping of DH lines with extreme phenotypes, using a Brassica single-nucleotide polymorphism (SNP) array, the region of major QTL on chromosome A09 was aligned to a ~1 Mb region on the reference genome of B. rapa and B. napus. Methods Plant materials and phenotypic evaluation. A B. napus DH population, consisting of 261 lines, was developed from a cross between the European winter cultivar 'Express' (female) and Chinese semi-winter line 'SWU07' (male). Two parental lines showed diversity in SL and SW. Two rounds of random crosses between DH lines were performed to reconstruct 233 RC-F 2 lines. Each DH line was used once each round. The Ten well-developed siliques per plant at the base of the inflorescence were collected, and siliques from ten individuals in the center of each plot were pooled to measure silique length and weight at maturity. Statistical analysis. Analysis of variance (ANOVA) was performed using the GLM procedure of SAS , where σ g 2 , σ ge 2 and σ e 2 are estimates of the variances of genotype, genotype × environment interactions, and error, respectively, n is the number of environments, and r is the number of replications per environment 36 . Pearson's correlation coefficients between traits of interest were calculated using the CORR procedure of SAS 37 . QTL analysis. DNA isolation, development of molecular markers and construction of genetic linkage groups were described in previous study 38 , where 293 SSR markers were arranged into 19 B. napus chromosomes, spanning a genetic distance of 1,188 cM with an average distance of 4.05 cM between adjacent markers. Besides, the sequence of target region was downloaded from the reference genomes (http://brassicadb.org/brad/index.ph), and screened for SSR loci using the software "SSR Locator" 39 . Ten region-specific SSR markers were developed according to the genome sequence of the target QTL region. The genotypes for RC-F 2 lines were deduced from the band patterns of their parental lines. A genotype score of '1' was given to a RC-F 2 line if the SSR marker was present in at least one of the parental lines, while the RC-F 2 line was assigned a score of '0' if the marker was absent in both parents. QTL detection was performed with the composite interval mapping (CIM) procedure of the software WinQTL Cartographer 2.5 40 . A 1,000-permutation test was performed to estimate a significance threshold of the test statistic for a QTL based upon a 5% experiment-wise error rate 41 . The genome-wide digenic interactions were estimated using QTL mapper V2.0 software 42 43 . The alignment criteria were set to allow three mismatches and one gap for a given primer pair. When a marker had multiple amplification loci on the same chromosome, an accurate position for a particular locus was determined manually by referring to the physical positions of its upstream and downstream markers. SNP array analysis. In order to narrow the confidence intervals of major QTL in A09, two pools of DH lines with extreme performance were constructed for SNP array analysis. Briefly, The DH lines with extreme performance of SL and SW were first evaluated by 293 SSR markers for evaluating genetic background. And the samples which shared the same genetic background except for QTL on chromosome A09, were selected to construct the 'Large' group (extremely long siliques and high seed weight) and 'Small' group (extremely short siliques and low seed weight). Each DH lines of the two groups was genotyped with the Brassica 60 K SNP Bead Chip Array (Illumina Inc., CA, USA), together with the parental line 'SWU07' . The single-nucleotide polymorphism (SNP) loci were aligned to the reference genomes of B. rapa (version 1.5) (http://brassicadb.org/brad/index.ph) and B. napus (http://www.genoscope.cns.fr/ brassicanapus/cgi-bin/gbrowse/colza/) The process of chip array analysis was performed in accordance with the method of Takagi et al. (2013) with slight modification 23 . The Δ (SNP-index) of each locus was calculated by subtraction of SNP-index of 'large' group from 'small' group with the formula, Δ (SNP-index) = k/5 − j/6, where k and j are the number of accessions that exhibit a consistant genotype from 'SWU07' in the 'Large' and 'Small' groups, respectively. A sliding window analysis was applied to generate Δ (SNP-index) plots with a window size of 80 SNP and increment of 1 SNP. 1,000 random resamplings were performed to estimate a significance threshold of the test statistic for a QTL based upon a 1% experiment-wise error. Thus the statistical confidence intervals under the null hypothesis of no QTL were determined at a significance level of p = 0.01

v3-fos

2018-01-26T21:25:05.222Z

2015-04-28T00:00:00.000Z

1863222

s2

Analysis of Heterosis and Quantitative Trait Loci for Kernel Shape Related Traits Using Triple Testcross Population in Maize Kernel shape related traits (KSRTs) have been shown to have important influences on grain yield. The previous studies that emphasize kernel length (KL) and kernel width (KW) lack a comprehensive evaluation of characters affecting kernel shape. In this study, materials of the basic generations (B73, Mo17, and B73 × Mo17), 82 intermated B73 × Mo17 (IBM) individuals, and the corresponding triple testcross (TTC) populations were used to evaluate heterosis, investigate correlations, and characterize the quantitative trait loci (QTL) for six KSRTs: KL, KW, length to width ratio (LWR), perimeter length (PL), kernel area (KA), and circularity (CS). The results showed that the mid-parent heterosis (MPH) for most of the KSRTs was moderate. The performance of KL, KW, PL, and KA exhibited significant positive correlation with heterozygosity but their Pearson’s R values were low. Among KSRTs, the strongest significant correlation was found between PL and KA with R values was up to 0.964. In addition, KW, PL, KA, and CS were shown to be significant positive correlation with 100-kernel weight (HKW). 28 QTLs were detected for KSRTs in which nine were augmented additive, 13 were augmented dominant, and six were dominance × additive epistatic. The contribution of a single QTL to total phenotypic variation ranged from 2.1% to 32.9%. Furthermore, 19 additive × additive digenic epistatic interactions were detected for all KSRTs with the highest total R2 for KW (78.8%), and nine dominance × dominance digenic epistatic interactions detected for KL, LWR, and CS with the highest total R2 (55.3%). Among significant digenic interactions, most occurred between genomic regions not mapped with main-effect QTLs. These findings display the complexity of the genetic basis for KSRTs and enhance our understanding on heterosis of KSRTs from the quantitative genetic perspective. Introduction Heterosis was proposed in the early 20 th century to describe the superiority of heterozygous F 1 compared with its homozygous parents in one or more traits [1,2]. Since then, heterosis has been widely applied for improving crops, and it has been particularly effective for maize production [3][4][5]. In general, maize crossbreeding efforts first aimed at improving the inbred lines and subsequently hybridizing these lines. Usually, the methods were focused on improving grain yield, which directly affects corn production and/or characters indirectly acting on corn production, e.g., decreasing plant height [6,7], enhancing resistance to diseases and pests [8][9][10], increasing planting density [11][12][13], or enhancing fertilizer utilization efficiency [14][15][16]. Given the quantitative complexity of these studies, grain yield was usually dissected into several components for further analysis. Considered the morphological relations, the relative components could be divided into two parts, ear related traits (e.g., ear length, ear diameter, row numbers, kernel number per row, and kernel number per ear) and kernel related traits (e.g., kernel length (KL), kernel width (KW), kernel thickness, and kernel weight). Researchers have demonstrated that yield related components always exhibit higher heritability than grain yield [17]. Most previous studies on maize yield related traits focused on ear related traits [18][19][20][21]. In recent years, kernel related traits have garnered more attention with studies attempting to elucidate the genetic basis of grain yield for a variety of reasons. For example, kernel size and weight were characterized as important determinants of grain yield [22,23] and large inbred kernels had the potential to produce better early vigor hybrids and promote flowering time [24]. In addition, several reports revealed that KL and KW had strong influences on kernel weight [25,26]. Therefore, kernel shape related traits (KSRTs) such as KL and KW are likely the major characters affecting grain yield. Analyses based on quantitative trait locus (QTL) mapping have been extensively applied for deciphering the genetic basis of kernel shape in major crops [27][28][29][30][31][32][33][34]. In contrast, the corresponding research progress in maize has been slow and only a few QTLs related to kernel shape have been detected [17,26,35,36]. However, these studies all focused on the relationships of kernel weight with KL and/or KW using different mapping populations, e.g., F 2:3 and recombinant inbred line (RIL). To date, there have been no consistent QTLs related to KL and KW found among previous reports. The discrepancy could be caused by the different evaluation methods, different linkage maps, or different mapping populations used. In addition to KL and KW, other kernel shape characters such as perimeter length (PL), kernel area (KA), and circularity (CS) have not be quantified in previous studies on maize. The accurate estimation of genetic effects facilitates a better understanding of target traits. To precisely detect epistasis, the triple testcross (TTC) design was developed by Kearsey and Jinks [37]. The design has the ability to test epistasis with high efficiency and can produce unbiased estimates of additive and dominance effects if epistasis does not exist. Following the RILbased TTC design, digenic epistatic effects have been evaluated in several studies [38][39][40]. In maize, Frascaroli et al. [41] mapped several QTLs for plant height, seedling weight, grain yield, and number of kernels per plant using a TTC design and identified a few QTLs for these traits with digenic epistasis. In the present study, the software SmartGrain was used for comprehensively and precisely evaluating characters affecting kernel shape: KL, KW, length to width ratio (LWR), PL, KA, and CS [42]. The TTC populations were created to test for additive, dominant, and epistatic effects. The main objectives of this work were to: (1) evaluate the level of heterosis for KSRTs; (2) investigate the correlations between KSRTs,heterozygosity, and yield; (3) estimate the number, genomic position, and the genetic effect of QTLs related to KSRTs for kernel shape; (4) characterize the mode of gene action for these QTLs; and (5) detect any digenic epistatic effects and their contributions to phenotypic values for KSRTs. Plant materials The intermated B73 × Mo17 (IBM) populations derived from the B73 × Mo17 cross were used for creating the TTC genetic populations. Based on the four generations of intermating among the F 2 progenies, the IBM populations have the potential to increase the genetic resolution roughly four-fold [43]. According to the testcross (TC) progeny production scheme [37], three TC populations were obtained as follows: three groups of 82 RILs (female parents) were crossed with B73, Mo17, and their F 1 (B73 × Mo17) and the corresponding progeny populations are referred to as TC(B), TC(M), and TC(F). Therefore, the materials tested included the basic generations consisting of B73, Mo17, and F 1 as well as four RILs, TC(B), TC(M), and TC (F) populations, with 82 genotypes within each population. Field experiments All materials were planted in three blocks in the Luhe Experimental Station of the Jiangsu Academy of Agricultural Science. Each block was arranged with the basic generations and the four populations. To evaluate the population traits statistically, two different field designs were laid out. A randomized complete block design was used for the basic generations and a splitplot design was used for the RIL and TTC populations. In the split-plot design, the four populations were deemed the main plot and the subplot was comprised of the RILs and their three TTC progenies. In all instances, each genotype within each block was planted in a single-row with 8 plants after thinning. Thus, the distance between plants in a row was 0.3 m and the distance between rows was 0.6 m. To reduce the boundary effect of the field, border rows were grown all around the blocks. All materials were subjected to the normal field management until the harvest of mature ears. Traits measurement The whole ears were manually harvested at physiological maturity (R6 stage). After several days of airing, the middle part of each ear was shelled. All kernels in a row were then bulked and 30 kernels were selected randomly per bulk to survey the representative KSRTs of genotypes. To measure the kernel shape accurately, SmartGrain, a high-throughput software for seed shape measurement was employed for the analysis [42]. Following the manual, digital images of thirty kernels per genotype (along with a wire for reference length) were captured by large pixel digital camera. The images were then analyzed and kernel outlines were detected by SmartGrain. The software calculated six KSRTs automatically including KL, KW, LWR, PL, KA, and CS. We corrected misdetected parameters manually. Furthermore, 100-kernel weight (HKW) was evaluated by averaging three replicates of randomly selected 100 kernels for each TTC population. Genetic linkage map The release of B73 reference genomic data (V2) and Mo17 genomic sequencing data facilitates development of novel molecular markers. Based on these genomic sequencing data, 93 insertion/deletion (InDel) and 57 presence/absence variation (PAV) polymorphisms exploited between B73 and Mo17 were used for genotyping 302 IBM RILs in our approach (S1 Table). This was also done with 142 single nucleotide polymorphisms (SNP) called based on the RNAbased sequencing data of IBM RILs deposited in NCBI (SRA054779) [44]. Moreover, 667 public markers with genotyping information were selected randomly to incorporate into the genotyping data. In total, 959 molecular markers were used for the genetic linkage map construction by MSTMap (S2 Table) [45]. The QTL analysis was carried out on the three linear transformations Z 1 , Z 2 , and Z 3 with composite interval mapping [47,48] via the software QTLMapper [49]. To obtain high resolution QTL mapping, the main and interaction effects of important markers were taken as the background genetic variation control. In the mapping range setup, the whole genome range was chosen to search QTLs from the entire genome. Following the manual, the main-effect QTLs were first identified and then the digenic epistasis at all possible marker pairs was analyzed. When the threshold of P 0.001 and R 2 >5% was reached, the putative QTL was declared present [49]. If P 0.001 but 0<R 2 5%, QTLs with LOD>3.0 were also considered significant. For all the main-effect and epistasis QTLs, the detection was based on the mix model approach [49]. Regarding the genetic effects of QTLs, the TTC design revealed the novel definitions given by Melchinger et al. [46] Linkage map construction Linkage map construction was carried out according to the procedure of MSTMap based on the 302 IBM individuals. The complete linkage map was comprised of 77 InDels, 32 PAVs, 61 SNPs, and the remaining 574 other public markers. In total, 744 markers were assigned to 20 linkage groups (LGs). Among these LGs, there were four with 12 markers each and 8 with 14 to 71 markers each (Fig 1). The average size of the 20 LGs was 213.2 centiMorgan (cM), ranging from 42.8 cM to 394.7 cM. The linkage map covered all 10 maize chromosomes and spanned 4263.1 cM of the genome with an average interval distance of 5.7 cM between markers (Fig 1). The minimum distance between markers was 0.2 cM, while the maximum marker interval was 69.1 cM. Among 724 marker intervals, the majority of inter-marker distances were no more than 10 cM (608 10 cM marker intervals including 390 5cM), 108 marker intervals were between 10 and 20 cM, and only 8 marker intervals were above 20 cM length (Fig 2). The maximum number of markers appeared on chromosome 1 (136 markers) and the minimum number appeared on chromosome 8 (44 markers). However, a few large distances still existed between adjacent markers (!20cM); this large gap is likely due to the lack of recombination among the close markers. In addition, the large gap might cause several LGs not linked to each other although they were assigned on the same chromosome. Heterosis and population performance of KSRTs KSRTs of all the populations in our study were evaluated by SmartGrain ( Fig 3A). The image shows that the various outlines of seed traits could be recognized clearly ( Fig 3B) The maximum, minimum, and mean values of four populations across three blocks are shown in Table 2. The performance of all seed traits for RILs (Table 2) was almost at the same level with the MP of the parents (Table 1). This stable mean value demonstrates that there was no interference in the artificial selection during the continuous self-pollination program for creating the RIL population from F 1 . Meanwhile, the maximum and minimum values of RIL traits were more extreme than those of the high value parent and low value parent, respectively. Thus, the RILs displayed extensive variation beyond the parents. When we compare the performance of RILs ( With respect to TC progenies, mean values of TC(M) were significantly higher than TC(B) for KL, KW, PL, and KA. On the contrary, the values were significantly lower for LWR ( Table 2) which is consistent with the observation of parental Mo17 to B73 and confirms the contribution of beneficial alleles donated from Mo17 and B73, respectively, to the former traits and the latter trait. As expected, both of the means of crossed heterozygous progenies from TC (B) and TC(M) populations exceeded significantly any homozygous parent of RIL, B73, and Mo17 populations for most of KSRTs (data not shown). This indicates the prevalence of heterosis. Additionally, only LWR exhibited significant difference between the mean of TC(F) and the mean of TC(B) and TC(M). Furthermore, the mean of TC(F) trended higher than the homozygous parent RIL mean and lower than the heterozygous parent F 1 mean for most of the KSRTs. The CS of progenies did not exceed that of any parent. The phenotypic performance difference seems to be relative with heterozygosity (F 1 >TC(F)>RIL). To further explore the relationship between the heterozygosity and the KSRTs, we investigated the correlations between the heterozygous levels of TC(B) and TC(M) individuals (based on the homozygous proportion of RILs inherited from B73 and Mo17) and their KSRTs (S3 Table). Four out of the six traits exhibited significant positive correlation with heterozygosity (Fig 4). However, the Pearson's R values showed that the correlations between the heterozygosity and the KSRTs were not strong. In the meanwhile, the correlations among KSRTs and between KSRTs and HKW were detected (Table 3). Among KSRTs, KL was not correlated with other KSRTs, whereas KW was statistically significant correlated with the KSRTs except KL. The strongest correlation was found between PL and KA in which the R values was up to 0.964. Four KSRTs were shown to be significant positive correlation with HKW, and significant negative correlation was found between LWR and HKW. The correlation between KL and HKW was not existed according to the corresponding P value. Identification of QTLs affecting kernel shape QTL analysis was carried out based on Z1, Z2, and Z3 of three blocks. The putative QTL was declared significant when P 0.001 and R 2 >5% or when P 0.001 and LOD>3.0 (0<R 2 5%). In total, 28 QTLs for KSRTs were detected in three Z transformations with some QTLs related to different traits located within the same QTL regions ( Table 4). The contribution of a single QTL to total phenotypic variation ranged from 2.1% to 32.9% with 18 QTLs >5% ( Table 4). The majority of LOD values exceeded the 3.0 with a maximum of 9.3. However, the LOD of two QTLs did not achieve the threshold with a minimum of 2.4 (data not shown). Seven QTLs were detected for KL: five in Z2 and one in both Z1 and Z3 (Table 4). These QTLs were mapped on six chromosomes. Despite five dominant QTLs detected in Z2 that were all characterized by overdominance, these QTLs could not be considered as major QTLs due to low R 2 values (3.3%-5.5%). However, these dominant QTLs found in Z2 explained 21.0% of total phenotypic variance. The negative additive QTL detected in Z1 located within the marker region of ufg33-InDel14 on chromosome 1 accounted for 12.3% of the phenotypic variation which indicates that an allele from the longer kernel parent Mo17 contributed to There was only one QTL detected for KW (Table 4): qKW4a was identified in Z3, mapped on chromosome 4, accounted for 15.7% of the phenotypic variation, and displayed an epistasis effect. It is suggested that the dominance × additive epistatic interaction might have an effect on kernel width. Nine QTLs were identified for LWR: six in Z1 and three in Z2 (Table 4). These QTLs were dispersed on chromosomes 1, 2, 3, 5, 8, and 10 (one QTL per chromosome for Z1) as well as on 6 and 9 (for Z2). The QTLs in Z1 exhibited additive effects and QTLs in Z2 displayed overdominant effects. Among additive QTLs, four were positive and two were negative, indicating that alleles from both large LWR parent B73 and small LWR parent Mo17 were beneficial for increasing LWR. The LOD of additive QTLs were all greater than 5.0, and their contribution ranged from 6.0% to 13.4%. Two of three dominant QTLs were located on chromosome 6 and the other was on chromosome 9. They were all classified as positive overdominance, explaining individually between 7.7% and 13.6% of total variation. QTLs detected in Z1 accounted for 43.2% of total phenotypic variation due to additive effects, whereas QTLs identified in Z2 accounted for 29.0% of total phenotypic variation due to dominance effects. Five QTLs were found for PL: one in Z1 and two in both Z2 and Z3 (Table 4). The QTL found in Z1 contributed 28.6% to total phenotypic variation and exhibited a negative additive effect. This indicates that an allele from high PL parent Mo17 contributed to improving PL. Two QTLs identified in Z2 were located on chromosomes 5 and 9, respectively, and both showed positive dominant effects with overdominance classification. The simultaneous fit of two overdominant QTLs accounted for 20.0% of total variation (6.4% and 13.6% each). Moreover, qPL5a flanked with umc1447 and umc1315 shared a genomic region with overdominant qKL5a detected in Z2 for KL. For Z3, two QTLs found in chromosomes 5 and 10, respectively, exhibited opposite epistatic effects and explained 7.3% and 10.7% of total variation each. Likewise, five QTLs were detected for KA: one in Z1 and two in both Z2 and Z3 (Table 4). The mapped genetic position and the orientation of genetic effects of QTLs for KA were almost exactly the same as the five QTLs for PL, except for qAS5a and qPL5a with two mapping positions closely linked. In addition, the contribution to total variation explained by individual QTLs of KA was all lower than those of PL. For CS, only one QTL were found. The QTL detected in Z2 was mapped on chromosome 6. This overdominant QTL accounted for 32.9% of total variation which was the maximum contribution of all individual QTLs detected. Analysis of digenic interaction affecting KSRTs Digenic epistasis for QTLs detected in Z1 and Z2 was determined based on an analysis of marker pairs over the whole genome. The epistatic effects detected in Z1 and Z2 are referred to as additive × additive and dominance × dominance interaction effects, respectively. In Z1, the interaction genetic regions were found for all traits, whereas, only three traits in Z2 (KL, LWR, and CS) were found to possess digenic interaction. Table 5 shows the digenic epistasis detected in Z1 and Z2, excluding those with R 2 ij of zero. Totally, there were 28 pairs of digenic epistasis detected for all traits in Z1 and Z2. The number of the digenic interaction pairs for each KSRT ranged from one for KA to nine for LWR. The majority of interaction effects detected in the KSRTs were determined to be significant. The digenic epistatic effects were all significant for KW, KA, and CS. However, there was no significant epistatic interaction detected for PL. In Table 5, most of the single genetic regions which displayed no or a small contribution to phenotypic variation could account for large proportions of total variation when two genetic regions interacted. For example, no single locus detected in CS could be shown as contributing to phenotypic variation. However, two locus interactions could both explain~30% of total variation. There were also some cases where the percentage of total variation due to digenic interaction was obviously lower than that due to a single locus. For example, two genetic regions detected in Z1 for PL accounted for 25.8% and 1.8% of total variation, respectively, but the contribution to total variation explained by their digenic interaction was 2.8%. In addition, the proportion of total variation explained by epistatic interactions detected in Z1 and Z2 ranged from 1.6% to 31.1%. In Z1, the maximum proportion of variation explained by all additive × additive interactions was 78.8% for KW. This is consistent with the observation that epistatic QTL was detected in Z3 without any significant additive or dominant QTLs detected in Z1 or Z2 for KW. This suggests that epistasis interaction may play an essential role in determining maize KW. In Z2, the highest total R 2 was 55.3% for CS and resulted from a dominance × dominance interaction. The majority of genetic regions detected in digenic epistasis analysis exhibited interaction with one another genetic region. However, some digenic epistatic regions were found to have simultaneous interaction with multiple regions. The genetic regions flanked by umc2176 and umc2039 were discovered to have epistatic interactions with various loci for the three traits KL, LWR, and PL. These involved both additive × additive and dominance × dominance interactions which indicate that kernel shape may be regulated by digenic epistasis at this region. Likewise, additive × additive epistatic interactions were found between the genetic region enclosed by ufg33 and InDel14 and two other loci for both KL and KW. Furthermore, this common interactive region was found to be shared with an additive QTL for KL. Most of loci exhibited main-effect and digenic epistatic effect contributing to the same trait (e.g., psr598-nfd110 for KL and bnlg439-InDel18, umc1114-umc2141, and umc2131-bnlg619 for LWR). We also found that the genetic region of umc1658-bnlg1138 on chromosome 2 mapped with an additive QTL for LWR and contributed to CS via a dominance × dominance interaction with the genetic region on chromosome 5. However, it should be noted that the majority of interactive regions were not mapped with main-effect QTLs. Moreover, no two regions showing digenic interaction were found to both co-locate with main-effect QTLs. In addition, the genetic region harboring an epistatic QTL detected in Z3 was not shown to interact with other genetic regions. Heterosis, mode of gene action for KSRTs, and their utilization in maize breeding Heterosis has been widely used in maize breeding for improving production [3][4][5]. Several key traits attracted breeders have been focused on dissecting their genetic basis [50,51]. Among traits with various heterotic levels, KSRTs are considered as important components involved in grain yield. In maize breeding, high length and low width kernels are the preference of breeders because slim kernels provide growing space for closely packed kernels and long kernels increase the grain yield if ear diameter remains unchanged. Therefore, the long KL type is expected to be the initial kernel shape trait in breeding programs. In present study, the highest MPH was 46.33% for KA which was followed by KL (26.8%). It seems that the KSRTs exhibited modest heterosis levels. For traits showing strong heterosis, their MPH level might be more than 100%. For example, MPH of seedling dry weight and number of kernels per plant were found to exceed 100% and even that of grain yield could reach 239% when maize inbred line B73 was crossed with H99 [41]. It is not surprising that different traits expressed various heterosis levels due to the cross between particular maize inbred lines. According to the interpretation of MPH defined by Melchinger et al. [46], MPH can be expressed as the sum of augmented dominance QTL effects. In the formula, the augmented dominance effect is denoted as the dominance effect minus half the sum of its additive × additive interactions. Thus, the dominance and additive × additive effects both contribute to MPH. For most of the KSRTs, augmented dominance QTLs were detected in Z2, except for KW which only displayed epistasis. The mode of gene action of augmented dominance QTLs were all overdominance, suggesting that overdominance or epistasis might contribute to these QTLs. To improve MPH for these traits, the sum of dominance effects of QTLs should increase to a greater extent than the sum of additive × additive interactions during breeding. The augmented additive QTLs were also identified in Z1 for four out of six KSRTs (excluding KW and CS). The augmented additive effect at a QTL was considered as the net contribution of the QTL to the parental difference in the presence of epistasis [46]. The contribution to phenotypic variation explained by an individual QTL for KL was 12.3%, indicating that selection based on the augmented additive locus should be enhanced in the process of maize breeding. For all QTLs identified, the total variation explained by individual QTLs ranged from 2.1% to 32.9%. If marker-assisted selection (MAS) was used for KSRTs, the QTLs with minor contributions should not be abandoned because the selection for beneficial genotypes with more loci would improve the target phenotype value. However, MAS for epistatic QTLs should be cautious due to the difficulties in the prediction of epistatic effects. In our study, the change in phenotypic values of KSRTs can be attributed to B73 and Mo17, implying that alleles from both parents contribute to KSRTs. For the complex traits, it is not difficult to understand that positive effects for different components might be provided by any parent which leads to the observation that the alleles from the two parents were both in favor of the whole complex trait. This has been observed in other studies. For example, the complex trait grain yield gained favorable alleles from both Zong3 and 87-1 parents [18]. Taken together, the occurrence of additive, dominant, and epistatic QTLs for components of KSRTs indicates the genetic complexity of KSRTs. To gain a better understanding of the genetic basis of complex maize KSRTs for their further utilization, the favorable genes should be isolated and characterized. Comparison of QTLs for KSRTs RIL-based genetic design has been proven providing the highest power for QTL detection than other populations [52]. In our study, 28 QTLs were found for six KSRTs using RIL-based TTC populations. Among these QTLs, 10 QTLs involved in three traits were found in five common genomic regions with each region mapped with two QTLs ( Table 4). The QTL region flanked with umc1447 and umc1315 for KL were identified as a QTL for PL with the same mode of gene action. Furthermore, this region was found to be closely linked with a QTL surrounded by umc1315 and InDel80 for KA. Likewise, four QTL regions for PL were also determined to be QTLs for KA with the same types of gene action. Thus, QTLs for PL seem identical to those for KA. One possible explanation for the high consistency of QTLs mapped for these two traits is that KA has the close correlation with PL (Table 3). Moreover, several main-effect QTL regions for one trait expressed interaction with genomic regions for the same trait or another trait. Therefore, some QTL regions might play pleiotropic roles for different traits. On the other hand, traits that are closely linked might also lead to the co-localization for different traits. To evaluate the consistency of QTLs for KSRTs, we collected the QTLs for KL and KW reported in recent studies [17,36]. The comparison results revealed the poor congruency among these QTLs. No QTLs were located on the same chromosomal bins. The inconsistency of QTLs for the same traits has been reported in previous studies [53,54]. The main cause of the discrepancy is usually attributed to genetic background differences which result from utilizing various mapping populations. Interpretation of digenic interaction In addition to main effect, epitasis was also exhibited large effect on complex traits in maize, especially grain yield [55,56]. The types of digenic interactions including additive × additive, additive × dominance and dominance × dominance have been shown to exist in different proportions for the target traits. Additive × additive is usually more common than the other interactive types. For instance, additive × additive epistatic interaction was the major interactive type among digenic interactions detected in maize grain yield and yield components using F 2:3 populations [18]. In another study on maize kernel related traits, additive × additive interaction made up the highest proportion among all interactive types and displayed the large digenic epistatic effects on KL and kernel thickness [35]. In our present work, the proportion of 67.9% for additive × additive interactions detected in Z1 versus 32.1% for dominance × dominance interactions detected in Z2 illustrates the importance of additive × additive digenic interactions for KSRTs. For KL, the contribution to total variation explained by individual additive × additive interactions was higher than that by dominance × dominance interaction. Moreover, for KW, the epistasis detected in Z3 was all attributed to eight additive × additive interactions with a total R 2 ij of 78.8%. Therefore, additive × additive epistatic interactions between two loci may be the important genetic element for KL and KW. On the other hand, only one additive × additive interaction was detected for KA which had the highest MPH. This result demonstrates that reduced additive × additive interaction can increase the MPH for the trait according to the definition of Melchinger et al. [46]. Furthermore, there was no dominance × dominance digenic interaction found for KW, PL, or KA. This suggests that dominance × dominance digenic interactions might not be important for heterosis of these traits. However, most of the interactive genomic regions were not mapped with main-effect QTLs. The majority of the single loci were not significant for the KSRTs but exhibited significant interactive effects. This finding is in agreement with a previous study which reported that 74.9% of significant epistatic interactions occurred between loci not linked with any QTL and that only a few interactive loci were coupled with main-effect QTLs [18]. These results indicate that some main-effect QTL regions could influence the genetic background of corresponding KSRTs. It should be noted that high-order epistasis might influence on KSRTs even though proper evaluation could not be conducted due to the detection methods of our study. All inferences on epistatic interaction should be cautious when the size of mapping population is limited [57]. Supporting Information S1 Table. The primer list of insertion/deletion (InDel) and presence/absence variations (PAV) markers anchored in the linkage groups (LGs). (XLSX) S2 Table. The genotyping information of intermated B73 × Mo17 (IBM) recombinant inbred lines (RILs) used for genetic likage group construction. "A" and "B" represent the genotype of B73 and Mo17 respectively, and "-" represents the missing genotype. "S1_10096204" represents the single nucleotide polymorphism (SNP) occurred in the position 10096204 on Chromosome 1 of B73 reference genome (V2). The naming format is applicable for other SNPs. (XLSX) S3 Table. The

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Transcription of Biotic Stress Associated Genes in White Clover (Trifolium repens L.) Differs in Response to Cyst and Root-Knot Nematode Infection The transcription of four members of the Kunitz proteinase inhibitor (KPI) gene family of white clover (Trifolium repens L.), designated as Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5, was investigated at both local infection (roots) and systemic (leaf tissue) sites in white clover in response to infection with the clover root knot nematode (CRKN) Meloidogyne trifoliophila and the clover cyst nematode (CCN) Heterodera trifolii. Invasion by the CRKN resulted in a significant decrease in transcript abundance of Tr-KPI4 locally at both 4 days post-infection (dpi) and at 8 dpi, and an increase in transcription of Tr-KPI1 systemically at 8 dpi. In contrast, an increase in transcript abundance of all four Tr-KPI genes locally at 4 and 8 dpi, and an increase of Tr-KPI1, Tr-KPI2, and Tr-KPI5 at 8 dpi systemically was observed in response to infection with the CCN. Challenge of a resistant (R) genotype and a susceptible (S) genotype of white clover with the CCN revealed a significant increase in transcript abundance of all four Tr-KPI genes locally in the R genotype, while an increase in abundance of only Tr-KPI1, Tr-KPI2, and Tr-KPI5 was observed in the S genotype, and only at 4 dpi. The transcript abundance of a member of the1-AMINOCYCLOPROPANE-1-CARBOXYLATE (ACC) SYNTHASE gene family from white clover (Tr-ACS1) was significantly down-regulated locally in response to CRKN infection at 4 and 8 dpi and at 4 dpi, systemically, while abundance increased locally and systemically at 8 dpi in response to CCN challenge. Conversely, the abundance of the jasmonic acid (JA) signalling gene, CORONATINE-INSENSITIVE PROTEIN 1 from white clover (Tr-COI1) increased significantly at 8 dpi locally in response to CRKN infection, but decreased at 8 dpi in response to CCN infection. The significance of this differential regulation of transcription is discussed with respect to differences in infection strategy of the two nematode species. Introduction The mechanisms of plant defence against herbivory and microbial pathogens can be broadly classified as constitutive or induced. The induced defences include the biosynthesis of an array of secondary metabolites and proteins that can act as toxins, anti-feedents or anti-nutrients [1]. Of these, one of the most common inducible defences in plants against herbivory is the synthesis of proteinase inhibitors (PIs). The PI proteins are mostly low molecular mass proteins that occur in all life forms and these proteins are widely distributed through the kingdoms [2]. In all organisms, PIs are classified into super-families based on the class of proteinase inhibited to give serine, aspartic, metallo-and cysteine-proteinase inhibitors [3]. One group of the serine PI superfamily are the Kunitz proteinase inhibitors (KPIs) which, where examined in plants, are widespread and generally exist as multi-gene families [4][5][6][7]. In common with the large PI families in plants, the KPIs have been shown to function as storage proteins and insect pest resistance factors [8][9][10][11], and have been used in transgenic approaches to confer protection against insect pests to the transformants [12][13][14][15][16]. In terms of the other herbivorous pests of plants, parasitic nematodes also cause significant crop losses globally [17], with over 4100 species identified thus far [18]. Of relevance to this study are two groups; the root-knot nematodes (RKN), of the genus Meloidogyne from the family Meloidogynidae, and the cyst nematodes (CN) comprising two genera, Heterodera and Globodera, from the family Heteroderidae. In terms of infection strategy, the two groups share some similarities. An infective and motile juvenile second stage (J2) invades host roots near the tips and establishes a feeding site in the vasculature/inner cortex region before becoming sedentary. However, significant differences also exist between the two groups in terms of migration pathways through the roots and the mechanism of feeding site formation. RKN J2 migrate intercellularly in host roots, while CN J2 move intracellularly. The RKN feeding sites are composed of giant cells while CN feeding sites are syncytia, formed by the fusions of hundreds of syncytial initials. During the infection and parasitism process, extensive changes in the expression of host genes are observed [19][20][21][22][23][24][25] which are, in part, in response to nematode invasion and to stylet secretion proteins, termed effectors [26][27][28]. The major secretory proteins from nematodes include the cysteine proteinases, and so the accumulation of cysteine PIs (cystatins) in transgenic backgrounds has been reported to confer a degree of resistance against CN and RKN in tomato [29,30], Arabidopsis [31,32], rice [33], potato [34,35], alfalfa [36], banana [37] and sweet potato [38]. However, in contrast to the cystatins, there are fewer instances where serine PIs have been shown to exert the same effects in transgenic backgrounds against nematode pests [31,39]. Much less is known too in terms of the interactions of nematode feeding and the KPIs. In transgenic studies, over-expression of the sporamin gene from sweet potato in sugar beet did reduce the growth and development of the sugar beet cyst nematode, H. schachtii [40]. More directly, infection by the soybean cyst nematode, H. glycines, was shown to differentially regulate the expression of a family of KPI genes which was dependent on the genetic background of soybean [41], while the infection of potato with the potato cyst nematode, G. rostochiensis, also influenced the expression of a small KPI gene family [42]. In the pasture legume white clover (Trifolium repens L.), four full-length KPI genes, designated Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5, have been isolated, and these have been shown to display differential transcription during seed germination, in different tissues of the mature plant, and transcription has also been shown to be ontogenetically-regulated [43]. Further, both wounding and insect feeding of leaf tissue by the generalist insect herbivore, Spodoptera litura, differentially regulates the transcription of the KPI gene family both locally at the site of insect damage and in remote tissue, supporting a systemic response [43]. Over-expression of Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5 in Nicotiana tabacum retarded larval growth of feeding S. litura [43]. However, the influence of nematode infection has not been evaluated in terms of any such differential expression. In New Zealand, species of root-knot nematode (RKN) and a species of cyst nematode (CN), H. trifolii are parasites of white clover in pastures [44]. Thus here two different root parasites, the clover CN (CCN), H. trifolii and the clover RKN (CRKN) M. trifoliophila, have been used for a comparative study on changes in transcript abundance of the Tr-KPI gene family using a single white clover genotype of the cv. Huia that is susceptible to both nematodes. In addition, both CN-susceptible and CN-resistant genotypes obtained from a white clover breeding programme were used to examine any genotype-dependent regulation. To expand our study on the significance of the observed transcriptional changes that occur within the Tr-KPI gene family in response to nematode infection, we also examined the transcript abundance of two key genes associated with biotic stress responses, including plant-nematode interactions. The first was 1-AMINOCYCLOPROPANE-1-CARBOXYLATE (ACC) SYNTHASE from white clover (Tr-ACS1), coding for the enzyme that is recognised as the rate-determining step in ethylene (ET) biosynthesis [45], while the second was CORONATINE-INSENSITIVE PROTEIN 1 from white clover (Tr-COI1) that encodes a component of the JA signalling pathway [46]. In total, changes in the transcript abundance of all genes examined were evaluated in terms of the documented differences in infection strategy of the two nematode species. Plant material A single genotype (i.e. arising from a single seed) of the white clover cultivar Grassland 'Huia' (AgResearch Grasslands, Palmerston North, New Zealand) that was susceptible to both CRKN and CCN infection (designated as a double susceptible white clover genotype of cv. Huia) was used in the infection experiments with both CRKN and CCN. In separate experiments, genotypes resistant (17R) or susceptible (23S) to the CCN were used; these genotypes were selected from two breeding lines originating four generations earlier from the same line. These lines were from an AgResearch recurrent selection programme developing resistance to CCN in white clover by selecting germplasm supporting fewer cysts, where the numbers of eggs per cyst were concurrently reduced [47]. To maintain each genotype, plant material was propagated vegetatively through stolon cuttings; the apical part of the stolon was excised just proximal to node 4 and all leaves, except the first emerged leaf, were removed. The cuttings were then placed, by burying the basal two nodes, into pots containing either vermiculite or peat-based potting mix followed by regular watering with half-strength Hoagland's solution [48] until rooting had occurred. These cuttings were then maintained in a temperature-controlled glasshouse (minimum 20°C; venting at 25°C) until sufficient growth had occurred to generate well-established stock plants. Root infection experiments The nematode infection experiments were performed using stolon cuttings (obtained as described) excised from a double-susceptible white clover genotype of cv. Huia or the resistant (17R) and susceptible (23S) genotypes, as appropriate. The stolon cuttings from the stock plants were rooted in peat-based potting mix in 300 x 450 mm trays and maintained in a temperature-controlled glasshouse (minimum 20°C; venting at 25°C) on a plant-heating mat for 20 days. Well-established stolons were then selected and transplanted into 60-mm-diameter, 156-mL capacity plastic cups containing pasteurised sand and were watered with half-strength Hoagland's solution on a regular basis. To infect the plants, a hole was made in the middle of each cup and inoculum was added at a rate of 4000 eggs/3 mL of water for M. trifoliophila and 3000 eggs/3 mL of water for H. trifolii. An identically propagated set of plants that were not infected with nematodes served as the control group. Both whole roots (designated the local tissue) and the first fully expanded (FFE) leaf on the stolon (designated the systemic tissue) were harvested from both infected and control plants after 4 and 8 dpi, immediately frozen in liquid nitrogen and stored at -80°C until RNA isolation. To check infection of the plants, four extra plants of the double-susceptible genotype were infected with both CCN and CRKN and the roots were stained with aniline blue at 4 dpi. To do this, infected roots were washed in running tap water, placed in 1.5% (v/v) NaOCl for 5 min with agitation, rinsed again in running tap water for 30 sec and allowed to stand in tap water for 15 min and blot dried. The roots were then incubated in a boiling solution [0.05% (w/v) aniline blue in 33% (v/v) glycerol, 33% (v/v) lactic acid] for 1 min, cooled to room temperature, briefly rinsed in running tap water and then blot dried. The stained nematodes were visualized using a Zeiss Axiophot photomicroscope. Nucleic acid isolation and cDNA synthesis Total RNA was extracted using the Hot Borate method of [49] and [50]. The RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer V3.6 (Thermo Scientific, USA). Genomic DNA-free RNA samples were prepared using an RNase-free recombinant DNase treatment (Roche). To synthesize the first single strand DNA, the Transcriptor First Strand cDNA synthesis kit (Roche) was used using Oligo (dT) 18 primers. For this, total RNA (1 μg) was combined with Oligo (dT) 18 primer in 0.2 mL capacity tubes and the volume was adjusted to 13 μL with water. The RNA and primers were denatured at 65°C for 10 min and placed on ice immediately. Seven μL of master reaction mixture containing 5X transcriptor RT reaction buffer, protector RNase inhibitor (40 U/μL), 10 mM dNTP-Mix and Transcriptor reverse transcriptase (20 U/μL) was then added. The tubes were placed in the thermocycler and, typically, cDNA synthesis was carried out at 55°C for 30 min, before inactivation of the reverses transcriptase at 85°C for 5 min. Quantitative reverse-transcription PCR (qRT-PCR) For qRT-PCR analysis, specific primers (S1 Table) were designed according to the general requirements of qRT-PCR primers [T m = 60°C (± 1°C), a minimal secondary structure, and an inability to form stable dimers] and based on the cDNA sequences for the representative target genes (S2 Table). The efficiency of all the primers was determined by the standard curve method [51]. qRT-PCR was performed using the LightCycler 1 480 Real-Time PCR (Roche) and system series software 1.7, with three technical replicates of each cDNA sample (20-fold dilution). SYBR green I was used to monitor efficient DNA synthesis. Typically a 10 μL of reaction volume was used that consisted of 5 μL of 2 X LightCycler 1 480 SYBR Green I Master Mix (Roche), 2.5 μL of 20-fold diluted cDNA and 0.5 μL of 10 μM forward and reverse primers. Master mixture and cDNA templates were dispensed into 96-well plates. The PCR was performed as follows; 95°C for 5 min (95°C 10 sec, 60°C 10 sec, 72°C 10 sec) x 40 cycles, 95°C melt. Fluorescence measurements were performed at 72°C for each cycle and continuously during final melting. Relative transcript abundance was determined by comparative quantification to the geometric mean using three biological replicates, where one individual plant represents a single biological replicate. Two or three independent qRT-PCR reactions were performed per replicate and transcript abundance was normalized using two internal reference genes, Tr-β-ACTIN and Tr-GAPDH. The reference genes were selected using BestKeeper [52] and the efficiency of all primers was determined by the LinRegPCR quantitative PCR data analysis program [51] Statistical analysis Statistical analysis was performed using the Statistical Package for the Social Sciences (IBM SPSS Statistics) and any significant differences determined using a pairwise Student's t-test for 4 and 8 dpi separately as the transcript abundance of Tr-KPIs are developmentally regulated in white clover [43]. The Holm-Bonferroni method was also used for the application of a multiple testing comparison for the assessment of multiple gene analysis from the same cDNA sample [53]. Results Infection time-course of root knot and cyst nematodes in white clover roots By 4 dpi, the eggs of both nematode species were hatched and invasion of root tissue of the double-susceptible genotype of cv. Huia was evident (Fig 1). Infection with CRKN eggs had produced discernible swelling at the root tips. In both infections, aniline blue-stained J2 were observed near the inner cortex of the root at 4 dpi suggesting successful egg hatching and J2 invasion. By 8 dpi, gall formation and giant cell formation was visible suggesting active feeding (data not shown). CRKN and CCN infections influence the transcript abundance of the Tr-KPI gene family The transcript levels of the four distinct Tr-KPI genes in response to CRKN and CCN infection of the double-susceptible genotype of cv. Huia were investigated initially. In response to CRKN, the transcript abundance of all four Tr-KPI genes in the infected root tissue (locally) did not show any significant increase when compared with the control (uninfected) tissue (Fig 2A), but a significant increase (p = 0.010) in the abundance of Tr-KPI1 only was observed at 8 dpi in the remote FFE leaf tissue ( Fig 2B). However, a significant decrease in the abundance of the root-specific Tr-KPI4 gene was observed locally at 4 dpi (p = 0.010) and 8 dpi (p = 0.038) (Fig 2A). In contrast to the CRKN response, a significant increase in transcript abundance of all four Tr-KPI genes was observed locally at both 4 dpi (Tr-KPI1, (Fig 3A). The increase in the transcript abundance of Tr-KPI2 in response to CCN infection was also significant using the Holm-Bonferroni multiple testing correction ( Fig 3A). However, for Tr-KPI4, a significant decrease in transcript abundance was observed at 8 dpi when compared with 4 dpi (Fig 3A). In the remote FFE leaf tissue, a significant increase in abundance was also observed for Tr-KPI1 (p = 0.004), Tr-KPI2 (p = 0.018) and Tr-KPI5 (p = 0.023), but only at 8 dpi (Fig 3B). The transcription of the Tr-KPI gene family is differentially regulated in resistant and susceptible lines in response to CCN infection A marked increase in transcript abundance of all members of the KPI gene family was observed in the double-susceptible genotype of cv. Huia in response to CCN infection (Fig 3). To examine this in more detail, two genotypes of white clover from a nematode resistance breeding programme were assessed. Data for cyst numbers on four genotypes derived from the breeding programme is given in S3 Table, increase was only observed at 4 dpi (Tr-KPI2, p = 0.011; Tr-KPI5, p = 0.021). No significant differences for Tr-KPI4 could be discerned at either time-point evaluated (Fig 4B). Effect of nematode infection on the transcript abundance of ET biosynthetic and JA signalling genes For the ET biosynthetic genes examined in response to CRKN infection of the doublesusceptible genotype, a significant decrease in the transcript abundance of Tr-ACS1 was observed locally (in the root tissue) at 4 dpi (p = 0.0005) and 8 dpi (p = 0.012), and at 4 dpi (p = 0.001) systemically (in the FFE tissue) (Fig 5A and 5B). The decrease in Tr-ACS1 transcript abundance at 4 dpi in both the local and systemic tissues was also shown to be significant using the Holm-Bonferroni correction. A significant increase in the transcript abundance of white clover ACC OXIDASE (ACO) genes, Tr-ACO2 (p = 0.005) and Tr-ACO3 (p = 0.037), was observed locally at 8 dpi (Fig 5A), and for Tr-ACO2 systemically at 8 dpi (p = 0.028; Fig 5B) with the increase in transcript abundance of Tr-ACO2 locally at 8 dpi also shown to be significant using the Holm-Bonferroni correction (Fig 5A). In contrast to the decrease in transcript abundance of Tr-ACS1, a significant increase in the abundance of Tr-COI1 was observed both locally (p = 0.019; Fig 5A) and systemically at 8 dpi (p = 0.040; Fig 5B). For CCN infection, a significant increase in Tr-ACS1 transcript abundance was observed both locally (p = 0.040) and systemically (p = 0.018) at 8 dpi (Fig 6A and 6B), while a significant increase in the abundance of Tr-ACO2 was observed at both 4 dpi (p = 0.028) and 8 dpi (p = 0.014), but only locally (Fig 6A). For Tr-ACO3, a significant increase in transcript abundance was observed both in local (p = 0.025) and systemic (p = 0.012) tissue at 8 dpi (Fig 6A and 6B). For Tr-COI1, a significant decrease in transcript abundance was observed locally at 8 dpi (p = 0.032; Fig 6A), but abundance increased significantly at 4 dpi (p = 0.013) in the remote FFE leaf tissue (Fig 6B). Discussion This study has sought to examine the effect of nematode infection strategies on the transcript abundance of the KPI gene family from white clover, as well as any changes in the transcription of key biotic-stress-associated hormone biosynthetic and signalling genes. The interactions of PIs and nematodes has been well studied, largely based on the utilisation of these factors for resistance in transgenic plants. While some serine PIs, including Kunitz proteins, have been tested in this way [31,39,40], the larger focus has been on the cystatins as this gene family is more commonly down-regulated during parasitism [19,21,22,24]. For the KPI genes, more targeted transcriptional studies have identified changes where the magnitude of the response can be dependent upon a resistant or susceptible background [41], but no comparative study of CN and RKN infection of the same species has been undertaken. It is well established that the KPI gene family is differentially regulated by wounding [54,55] in plants including white clover [43] and so a comparative examination of changes in the transcript abundance of the Tr-KPI gene family can contribute to the elucidation of the differences in the infection strategies between the two groups of nematodes. RKN parasitic J2 are known to migrate apoplastically through the root cortex towards the vascular tissue where typically 5-7 parenchyma cells adjacent to the xylem elements are selected as founder cells for the development of the giant cells. The giant cells are formed by synchronous nuclear divisions without cytokinesis to form large, multinucleate cells, after which a hyperplasia of pericycle and cortex cells form the root knots/galls [56]. During the intercellular migration process, little damage to the roots occurs and so a signature of the RKN-host interaction is that many of the defence-associated genes in the host are not induced [19,26]. For white clover, the transcript abundance of the Tr-KPI genes was not increased locally, particularly at 4 dpi. Indeed the transcription of the root-specific Tr-KPI4 gene was repressed. This is broadly in agreement with de Sa et al. [25] who did not detect any changes in the transcript abundance of a KPI gene expressed in the M. javanica-soybean interaction, although transcription across the larger gene family was not examined. For the CN interaction, a different infection strategy is invoked. Here, J2 migrate intracellularly, thus causing cellular damage, until they reach the inner cortex where initial syncytial cells (ISC) are established. Unlike the giant cells, the syncytium develops by the coalescence of surrounding ISC that have originated from cortical cells. Together, this infection strategy suggests that plant responses to cell wounding and cell fusion are invoked, and microarray and transcriptome data highlight an increase in the expression of cell wall dissolution enzymes and an induction of the proteasome components [20,22]. The KPI expression data obtained in this study support a wound response to CCN infection with a marked increase in transcript abundance of all of the Tr-KPI genes locally at both 4 dpi and 8 dpi and Tr-KPI1, Tr-KPI2 and Tr-KPI5 systemically at 8 dpi. These changes are very much in contrast to the changes in the CRKN interaction, but agree with the CN-KPI interactions reported by Rashed et al. [41] and Turra et al. [42]. The white clover genotype used in the CRKN and CCN comparative infection study was confirmed to be susceptible to both species of nematode (data not shown). In the CRKN interaction, no marked changes in transcript abundance of the TR-KPI gene family were observed (with the exception of a local repression of Tr-KPI4) suggesting that the Tr-KPI gene family are part of the defensive array in white clover. In contrast, the up-regulation of the gene family in response to CCN infection may reflect a highly altered cellular homeostasis caused by wounding which, in common with other wound responses, is accompanied by the induction of Tr-KPI genes in the systemic tissue (albeit at 8 dpi). In another study, KPI gene expression was measured during a CN interaction in soybean root and a greater induction was noted in the roots of susceptible plants when compared with those of resistant plants, although this was over 0.5-1 dpi [41]. In the current study, a resistant and susceptible genotype of white clover were compared, and while a more marked induction of Tr-KPI gene expression was observed in the resistant genotype (albeit only at 8 dpi) a significant induction was also observed in the susceptible genotype. These data thus suggest that the KPI gene response may not be a primary determinant in the resistance interaction, but more likely reflects the degree of cellular wounding and the corresponding magnitude of the metabolic changes that occur within the infected plants. Given the difference in cellular homeostasis induced by the CRKN and CCN infection, as presaged by the differences in Tr-KPI transcription, it was also of interest, therefore, to examine the transcription of the key ET biosynthetic and JA signalling genes, the two hormones intimately linked to the regulation of plant responses to both biotic and abiotic stresses, including plant CN and RKN interactions [57][58][59]. In the CRKN interaction, the gene encoding for the rate-determining step in ET biosynthesis, Tr-ACS1 was significantly repressed both locally and systemically at 4 dpi, with the decrease also significant using multiple testing correction thus supporting a more convincing difference. While Tr-ACS1 was also repressed at 8 dpi locally, it was not so systemically, and also there was a local induction of the two ET precursor ACO genes examined at this later time point. Indeed, for Tr-ACO2, this difference was also shown to be significant using multiple testing correction. While we did not measure ET production directly, these changes in transcript abundance suggest a biphasic control of the biosynthetic pathway, with reduction of biosynthesis initially (at 4 dpi) and then a later induction (at 8 dpi). In rice, a decrease in OsACS1 expression has also been observed in response to infection with M. graminicola in both local and systemic tissues [59]. Further, the addition of ET to aerial plant plants, via the application of the synthetic ET precursor 2-chloroethylphosphonic acid (ethephon or ethrel), induced a systemic defence response in the roots, as determined by the induction of the pathogenesis-related genes, PR1a and PR1b. These treated plants showed a reduction in the infection of the RKN, M. graminicola [58]. Further, our data support other approaches where the use of L-α-[2-(2-aminoethyoxy)vinyl]glycine (AVG), an inhibitor of ACC synthase activity, increased the attractiveness of Arabidopsis to the RKN, M. hapla [60]. Likewise in the same study, the ET over-producing mutants, eto1, eto2 and eto3 were deemed to be less attractive to the nematode. In earlier studies with the interaction between M. javanica and tomato roots, an increase in ET production was observed, but at 5 dpi [61]. If the natural ET precursor, ACC or the synthetic precursor compound ethrel were added, the gall number increased while added AVG decreased gall number. These workers proposed that ET served to aid cell expansion and subsequent gall formation. From our results, we can speculate for white clover that the later increase in ACO (after 8 dpi) may catalyse the production of ET that is important in gall formation. In contrast, the earlier suppression of Tr-ACS1 (at 4 dpi) may be important at decreasing ET production and so promote the initial penetration by CRKN. In contrast to the RKN interaction in Arabidopsis, the addition of ACC increased the susceptibility to infection by the beet cyst nematode, H. schachtii, as determined by an increase in attractiveness of infective juveniles for the root exudates and the addition of the ACC synthase inhibitor, AVG, decreased susceptibility [62]. Likewise, studies with ET over-producing mutants of Arabidopsis also resulted in an increase in susceptibility, while ET insensitive mutants displayed a decrease in susceptibility [62]. In the interaction between H. glycines and soybean roots, an increase in ACC content was observed in the roots in response to infection, which was accompanied by the induction of a suite of ACS genes from the multi-gene family [63]. For white clover, the observed local increase in Tr-ACS and Tr-ACO in response to CCN infection may therefore indicate that an increase in ET production is part of an infection strategy invoked by the CCN to increase susceptibility to parasitism. In contrast to the regulation of the ET biosynthesis genes, the transcription of the gene encoding the JA receptor, COI1, displayed the opposite pattern in both interactions. In the CRKN interaction, the transcript abundance of COI1 significantly increased at 8 dpi locally, while in the CCN interaction, the abundance of COI1 decreased at this time point. This result supports the transcriptome study in soybean which suggests that some candidate genes in JA signalling and biosynthesis are down-regulated during well-established compatible CN infections [20]. COI1 is a SCF ubiquitin ligase that binds the JA-isoleucine conjugate and forms a complex with the jasmonate ZIM-domain (JAZ) transcriptional repressor protein. The ligase activity degrades the JAZ repressor and the MYC2 transcription factor is then free to activate a programme of jasmonate-dependent gene expression [46]. In Arabidopsis, RKN infection has been shown to suppress JA-dependent systemic-acquired resistance (SAR) [64], while in rice infection by M. graminicola, a systemic attenuation of both ET and JA biosynthesis is observed [59]. However, JA and ET have been shown to interact locally in a coupled manner in the infection of rice roots with M. graminicola [58]. Here, an ET-induced systemic defence pathway requires a functioning JA signalling pathway, while application of ET activates the transcription of JA biosynthesis and signalling genes. Such results have led to the postulate that RKN nematodes do not target suppression of JA signalling to induce susceptibility but rather ET and SA biosynthesis [65], and the results reported here for white clover support this. In contrast, in the interaction between the RKNs Meloidogyne spp., and tomato roots, JA-signalling does not play a role in defence. Instead, an intact JA signalling pathway is required for susceptibility [66]. The data from our study suggests that while there is an activation of the JA signalling pathway in response to RKN infection, there is also a marked down-regulation of ET biosynthesis so possibly compromising the plant defence response. For the CCN interaction, both the induction of ET biosynthesis and down-regulation of JA signalling occur at 8 dpi, well after the establishment of infection. Thus for the CCN interaction, the synergy between ET biosynthesis and JA signalling may become uncoupled but this occurs later in the infection time course. Thus the role of ET and JA signalling in white clover during the earlier infection stages is consistent with other CN-plant interactions. However, during the later establishment stages, the role of the hormonal cues may alter including any concomitant changes in JA biosynthesis, but this aspect of the interaction was not examined directly in this study. Conclusion CRKN and CCN infection induce distinct transcriptional responses of the Tr-KPI gene family. The differences suggest that (i) KPI expression is not a key determinant of nematode resistance, and (ii) the transcriptional programme reflects differences in cellular homeostasis in response to the two distinct infection strategies. Investigation of both ET biosynthesis and JA signalling genes also reflect the differences in infection strategy and suggest that both nematode groups may uncouple the tight regulation between the two hormones. Supporting Information S1 Table. Mean cyst counts after 5 weeks of infection with CCN on five to eight copies of four genotypes of white clover from the breeding lines C13618 (genotypes R17 and R29) and C13704 (genotypes S23 and S27). (DOCX)

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Association of SSR markers with functional traits from heat stress in diverse tall fescue accessions Background Heat stress is a critical threat to tall fescue in transitional and warm climate zones. Identification of association between molecular markers and heat tolerance-related functional traits would promote the efficient selection of heat tolerant tall fescue cultivars. Association analysis of heat tolerance-related traits was conducted in 100 diverse tall fescue accessions consisting of 93 natural genotypes originating from 33 countries and 7 turf-type commercial cultivars. Results The panel displayed significant genetic variations in growth rate (GR), turfgrass quality (TQ), survival rate (SR), chlorophyll content (CHL) and evapotranspiration rate (ET) in greenhouse and growth chamber trials. Two subpopulations were detected in the panel of accessions by 1010 SSR alleles with 90 SSR markers, but no obvious relative kinship was observed. 97 and 67 marker alleles associated with heat tolerance-related traits were identified in greenhouse trial and growth chamber trial (P < 0.01) using mix linear model, respectively. Due to different experimental conditions of the two trials, 2 SSR marker alleles associated with GR and ET were simultaneously identified at P < 0.01 level in two trials in response to heat stress. Conclusion High-temperature induced great variations of functional traits in tall fescue accessions. And the identified marker alleles associated with functional traits could provide important information about heat tolerance genetic pathways, and be used for molecular assisted breeding to enhance tall fescue performance under heat stress. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0494-5) contains supplementary material, which is available to authorized users. Background Tall fescue (Festuca arundinacea Schreb.) is a major cool-season grass species from the family Poaceae. Native to Northern Europe, North Africa, Middle East, Central Asia, and Siberia, tall fescue is most widely utilized as forage and turfgrass attributed to its adaptability, yield, persistence, and other ecosystem services such as soil improvement, recreation, protection, and carbon sequestration. Tall fescue is a self-incompatible allohexaploid (2n = 6x = 42) out-crossing species containing three genomes (P, G1, and G2) with a genome size of approximately 5.27-5.83 × 10 6 kb [1]. Heat stress limits the growth and development of tall fescue in transitional and warm climatic regions. High summer temperature of 30 to 35°C could constrain growth, reduce turf quality, induce leaf withering, and inhibit photosynthesis [2], which would pose severe effects on global climate change. While effective agronomic measures, including heat acclimation, soil temperature reduction, and growth regulators application, could enhance heat tolerance of tall fescue. Heat tolerant cultivars would be key alternative in alleviation of the negative influences of abiotic stress on plant breeding programs [3]. However, plant heat tolerance is a complex quantitative trait, involving multiple regulatory mechanisms, signal transduction pathways, and metabolic pathways. Therefore, a study on genetic and molecular basis for heat tolerance in plants would be necessary. Detailed study in plant physiological responses to heat stress and identification of molecular markers linked to heat tolerance would enhance the efficiency of traditional breeding programs to developing heat tolerant cultivars. The quantitative inheritances of heat tolerance and interaction between gene expression and environment make challenges to our knowledge of genetic basis of heat tolerant traits of plant. During last two decades, molecular marker has applied to insight into complex traits in plant. Many studies on quantitative trait locus (QTLs) mapping have been conducted to dissect numerous vital agronomical and morphological traits under abiotic stress. The results have improved the efficiency of conventional crop breeding via marker-assisted selection (MAS) in some crop species e.g. rice, maize, barley, soybean, and chickpea [4][5][6][7][8]. However, many linkage mapping based on QTLs studies presented modest and unreliable results due to several factors. First, mappingbased cloning of QTL is time-consuming and costly for construction of populations. Secondly, the restricted number of recombination events per chromosome during mapping population development limits the resolution of genetic map [9]. In addition, QTL mapping could not exploit the extensive genetic variation of natural germplasm resources. On the contrary, association mapping could exploit all recombination events and mutations including historical and evolutionary recombination in natural populations with unobserved ancestry [10]. Association mapping has been widely applied to explore the genetic basis of complex quantitative traits in plant species, and reported under favorable conditions like drought [11][12][13][14]. For example, a candidate gene, ZmDREB2.7 associated with drought stress, was identified to be effective in imparting plant tolerance to drought stress in maize [13]. In turfgrass species, a few studies on association mapping have been carried out involving flowering time, leaf length, submergence tolerance, salinity tolerance, and drought tolerance in perennial ryegrass [15][16][17]. Four single nucleotide polymorphisms from LpLEA3, LpFsSOD, and Cu-ZnSOD have been associated with drought tolerance traits in diverse perennial ryegrass accessions [14]. However, there was limited information on the association between marker genes and heat tolerance of plants [8]. Simple sequence repeats (SSRs) or microsatellites are widely distributed in all eukaryotic genomes. They are powerful tools for dissecting cultivar fingerprinting, genetic diversity assessment, evolutionary study, linkage map construction, and marker assisted breeding [18][19][20]. Alternatively, the SSR markers were developed for allohexaploid tall fescue, an out-crossing species with high intra-specific polymorphism, utilized for genomic mapping, identification of variety, population genetic analysis and diversity evaluation of germplasm [21][22][23][24][25]. Recently, SSR markers have been applied in trait and marker association of plants, such as kernel size and milling quality in wheat [26], oil, starch, and protein concentration in maize [27], submergence tolerance in perennial ryegrass [17]. However, the application of association mapping in detecting links between markers with functional traits such as heat tolerance in tall fescue is undocumented. The objective of this study was to identify marker-trait associations for phenotypic and physiological traits under heat conditions. It was hypothesized that tall fescue accessions had high diversity in high temperature response and the population structure would influence individual functional traits associated with heat tolerance. A set of 100 diverse tall fescue accessions originating from different geographical regions was grown in two heat environmental conditions in the greenhouse and controlled growth chambers. The population structure, relative pairwise kinship, and marker-trait association (MTA) by mixed linear model were statistically analyzed based on SSR markers. Heat stress effects and functional traits variation In tall fescue heat stress imposed leaf yellowing and wilting, limited plant growth, and even death. Turfgrass quality (TQ), survival rate (SR), chlorophyll content (CHL), and growth rate (GR) decreased with prolonged heat stress in both trials, but the severity of decline varied with accession and duration. Significant accession and treatment time effects under heat stress were observed on GR, TQ, CHL, and SR in both trials (Table 1). However, no significant time effect for evapotranspiration rate (ET) in growth chamber trial was detected. There was also no significant interaction effect for functional traits between grass accessions and treatment time. With prolonged heat stress at 1-3 weeks, the mean, maximum, and minimum values decreased in two trials ( Table 2). Under heat stress, the average growth rate decreased from 0.24 g d -1 at initial time to 0.05 g d -1 at 14 WOT, turfgrass quality reduced from 6.55 to 2.56, survival rate decreased from 99.65% to 46.66%, chlorophyll content decreased from 2.35 mg g -1 FW to 1.47 mg g -1 FW, and evapotranspiration rate decreased from 61.55 g d -1 to 10.64 g d -1 , respectively in greenhouse trial. Most of the functional traits decreased except for ET, which increased after one week of stress treatment, and then drastically dropped. In growth chamber trial, all functional traits displayed similar trend, whereby the average GR dropped from 0.11 g d -1 at initial time to 0.03 g d -1 at two WOT, TQ from 7.50 to 3.01, SR from 99.75% to 52.50%, CHLT from 2.03 mg g -1 FW to 1.77 mg g -1 FW, and ET from 22.82 g d -1 to 18.93 g d -1 , respectively. After two weeks, heat stress significantly reduced GR by 79.17% in greenhouse and 72.73% in growth chamber trials compared with their relative controls (the time before heat stress). The decline levels of TQ, SR, and ET were lower than that of GR. Significant correlations between survival rate with evapotranspiration rate, turfgrass quality, and turfgrass quality with evapotranspiration rate were found at two time of heat stress that the values had been standard to relative control in greenhouse trial, and relative values of SR of two weeks under heat stress had significant relationship with chlorophyll content (Table 3). Meanwhile, there were significant correlations between turfgrass quality with GR, CHL, ET and SR in growth chamber trail. There was significant correlation between CHL and SR, ET and GR, however there was no relationship between ET and SR in growth chamber trail (Table 4). High correlations were identified for all functional traits between the two sample times under heat stress in both trials, with the highest correlation for ET (r = 0.86, P < 0.01) in greenhouse trial, and SR (r = 0.768, P < 0.01) in growth chamber trial. Population structure, relative kinship A total of 1010 SSR alleles were amplified from 90 SSR markers by genotyping 100 tall fescue accessions (Additional file 1 Table S1). The allele numbers of SSR marker varied from 3 to 27 alleles per marker with an average of 11.22 alleles per locus. For the co-dominant SSR marker transit to dominant marker in this study, the genetic diversity of the 100 tall fescue accessions was at a relative lower level, in which average of Nei's genetic diversity was 0.255, and average of polymorphism information content was 0.211. According to STRUCTURE analysis results based on Bayesian clustering approach model, a significant population structure was detected among the 100 accessions. The results were consistent with those from the preliminary runs, in which the average probability of the data likelihoods for the population structure in the panel of accessions were increased following the increase of K ( Figure 1A). Therefore, the likely number of subpopulations was identified using the Delta method. The optimal number of groups was determined by the maximum likelihood, and k was set at 2 implying two structural groups (G1 and G2) were identified in the panel ( Figure 1B). The population structure matrix (Q) identified at k = 2 was applied to define the membership probability for assigning accessions to subpopulation when the value was >0.7 (Additional file 2 Table S2) There was no obvious kinship (K) that detected based on 90 SSR markers in the panel of populations ( Figure 3). More than 55.3% of the pair-wise kinship estimates were zero while approximately 89% of estimates were between 0 and 0.05. Less than 5% of estimates were >0.1, indicating that the familial relationships minimum among samples, and would not cause further complexity in association analysis. Association analysis and evaluation of association model Combined with all SSR alleles and three traits including turfgrass quality, growth rate and leaf chlorophyll content in growth chamber trial, associations were performed to detect the effects of Q and K for controlling false associations. Owing to the complexity and population structure in out panel, the simple model that Table 3 Pearson correlations coefficients among functional traits of different time in greenhouse trial *significant at P < 0.05, **significant at P < 0.01. 1 a the value at 7 d of heat stress relative the initial value before heat stress. 2 b the reduction value at 14d of heat stress relative the initial value. Abbreviations: TQ-turf quality, ET-evapotranspiration rate, Chl-chlorophyll content. SR-survival rate, GR-Growth rate. Table 4 Pearson correlations coefficients among functional traits of different time in growth chambers trial *significant at P < 0.05, **significant at P < 0.01. 1 a the value at 7 d of heat stress relative the initial value before heat stress. 2 b the reduction value at 14d of heat stress relative the initial value. Abbreviations: TQ-turf quality, ET-evapotranspiration rate, Chl-chlorophyll content. SR-survival rate, GR-Growth rate overlooked Q and K was not performed. For any trait, the P values from the three models were close to the expected P value ( Figure 4). However, the model of Q showed a different distribution with the other models for turfgrass quality and chlorophyll content. On the other hand, the K and Q + K model displayed similar distribution of P values, and the identified associations (P < 0.01) showed the high similarity in both models (Additional file 3 Table S3). The more stringent model was performed, and the less spurious associations were identified. So the results from Q + K model by MLM would be showed and discussed. Marker allele-trait associations In MLM model with Q and K, a total of 97 SSR alleles were associated with five heat-relative traits at two time points (P < 0.01) in greenhouse trial, while that in growth chamber trial resulted in 67 SSR loci that were strongly associated with the 5 traits (P < 0.01) ( Table 5, and Additional file 4 Table S4). In greenhouse trial, 15 alleles of marker NAF057 that amplified 22 alleles showed the association with ET at two time points by using Q + K model. The similar results also occurred in marker NFA87 associated with GR-1, marker NFA155 related with TQ-1 in growth chamber trial. Moreover, many marker alleles could be associated with a functional trait, and one marker allele was associated with more than one trait. For example, SSR marker alleles (NAF036-194, NAF013-250, NAFG17-136, NAFG023-207, and NAF138-211) were associated with SR and TQ at two sampling times under heat stress in greenhouse trial. Comparing with the same association alleles in two trials, only 2 (Table 6). NFA87-418 that located the linkage group 3B was associated with GR-2, and NFA91-152 was associated with ET-2 in both trials by MLM analysis. Heat responses of tall fescue Heat stress is a major factor that limits growth of coolseason turfgrass on a global scale. Turfgrass survive under high temperature through tolerance or escape mechanisms, which involve many phenotypic and physiological characteristics including growth-restricted, higher photosynthesis rate, stay-green, cell membrane thermal stability, and earliness [28]. High temperature decreased turf quality, caused leaf water deficiency and yellowing, constrained growth, and reduced photosynthesis. So, leaf wilting, turfgrass quality, growth rate, evorpotranspiration rate, and chlorophyll content provided convenient and more efficient measurements for studying turfgrass responding mechanism under unfavorable conditions, which have been intensively applied for screening heat-tolerant germplasm of turfgrass [29][30][31]. In our trials, tall fescue accessions under heat stress exhibited varying degree of negative effects based on ANOVA analysis. Relatively low TQ, high leaf wilting, reduced CHL and severe water loss characteristics presented the damage level of heat stress of tall fescue. Large variations in these functional traits of accessions from different geographic locations and significant correlations between functional traits would provide the potential for selecting heat tolerant accessions and evaluating reliable SSR marker by association analysis between marker and functional traits. However, heat tolerance mechanisms of tall fescue would be different in Figure 4 Quantile-quantile plots of estimated -log10 (P) from association analysis using three models in three traits: a turfgrass quality, b growth rate, c leaf chlorophyll content. The black line is the expected line under the null distribution. The blue line represents the observed P values using GLM with Q model; the red line represents the observed P values using MLM model with K; the yellow line represents the observed P values using MLM model with Q and K. the two trials. In growth chamber trial, heat tolerant accessions maintained relatively high growth rate and good turfgrass quality. Simultaneously, heat-sensitive accessions presented lost water rapidly, curled leaves and even died. Meanwhile, in the greenhouse trial, heat tolerant accessions maintained good turfgrass quality and appearance by restraining growth. Similarly heat-sensitive accessions experienced yellowing of leaves and withering. The probable explanation for variations in tolerance mechanisms under heat stress were due to the different stress conditions including soil properties and temperature [32]. In the greenhouse trial the roots temperature was buffered because of properties of soil. But flasks with roots were directly exposed to heat stress in the chamber trial due to utilizing the nutrition solution, which made the turfgrass in flask to be more sensitive to high temperature than in the greenhouse trial. In addition, both trials displayed highly significant correlations in most of the functional traits. This indicated that heat tolerant traits had mutual influence, and these traits could provide adequate parameters for evaluating the heat tolerance in the field. Tall fescue accessions from different collection areas indicated diversity in phenotypic and physiological characteristics [33]. However the trend and level of heat damage of the accessions were roughly consistent in two time points. Therefore, heat tolerant tall fescue accessions would be effectively selected according the phenotypic traits when heat stress conducted early days. Population structure Tall fescue accessions native to Europe and North Africa, were introduced to North and South America in the late 1800s. They eventually became a prominent forage grass in 1940s in the United States where many commercial cultivars were produced through selective breeding [34]. Tall fescue samples were collected from more than 40 cities representing diverse geographical origins. So in view of the geographical origins, local adaptation, and breeding history of genotypes in association mapping panel, the nonindependent samples would often encompass both population structure and familiar relatedness [35,36]. In our study, the Bayesian clustering approach model based analysis divided the panel of samples into two sub-populations. The most of the accessions from European, North America, and all commercial cultivars were separated into the main subpopulation. The wild accessions from North Africa and Asia were separated into the second subpopulation. The division rule cannot be simple explained geographically due to overlapping of several accessions from the same region (European and Asia) in two groups, which indicated regional breeding objectives, the probable different evolutionary paths and methods of ecological adaptation in morphology and agronomic characteristics of ecogeographic races would be considered [37,38]. Presence of population structure could make some allele frequencies significantly differ between subpopulations, which would lead to spurious association (false positives) of markers with traits [39]. Flint-Garcia et al. [40] presented that 33 to 35% of variation of phenotypic traits about flowering time in a diverse maize panel would be attributed to population structure. Therefore, if subpopulation structure is not taken into account, spurious associations may be identified at other loci that were differentially distributed among subpopulations. Moreover, spurious associations cannot be controlled entirely by GLM model. This is because the Q matrix can only carry a rough dissection of population differentiation. Therefore, a unified mixed-model approach for association mapping that incorporates the pairwise kinship (K matrix) and Q matrix to correct multiple levels of relatedness have been developed. This would be a powerful approach for improving accuracy of association in many cases [40,41]. Kang [42] demonstrated that the distribution of P values ideally should follow a uniform distribution with less deviation from the expected P value. In our panel, SSR marker-trait associations were performed for three traits using the Q, K, and Q + K models, and all the three showed a good fit for P values. However, the models showed the different effects of controlling the population structure for different traits. K model was more superior to the Q model, but similar to the Q + K model. This is consistent with some previous studies [41,43]. The K matrix could capture the relatedness between each possible pair of individual in panel. The Q matrix considers a few axes only [44]. Consequently, no vivid familiar relatedness (from the recent co-ancestry) has been detected in the panel. Therefore a model that would test for complex quantitative traits would be necessary for improving the accuracy of association. LG a mean the locus of linkage groups of genetic linkage map of tall fescue according to Sara et al. [21]. Abbreviations: ET-evapotranspiration rate, GR-Growth rate. Marker allelic effects on functional traits Little is known about the association of SSR loci with heat tolerance related traits in plant species. In our study 97 marker-trait associations (MTAs) in greenhouse trial and 67 MTAs were identified for five heat tolerant traits at two time points (P < 0.01). A total of 13 MTAs in greenhouse trial and 29 MTAs in growth chamber trial were identified in present study for growth rate. High temperature would affect pollen viability, fertilization and seed development leading to yield losses. A large number of MTAs for yield and yield related traits under unfavorable conditions were reported in many crop species [7,8,13]. Furthermore, most of marker NAF057 amplifying 22 alleles were associated with ET in greenhouse trial, which implied the marker may be linked with a crucial gene that is necessary for regulating water loss and transpiration cooling under heat stress [45]. Similar results were observed between survival rate and turfgrass quality in greenhouse trial, suggesting survival rate, turfgrass quality and evapotranspiration rate are vital functional traits reflecting heat tolerance of tall fescue and might be regulated by genetically linked homologous genes. Therefore, these associated markers and identified genotypes with favorable alleles can be deployed after validation for molecular marker breeding to develop heat tolerant in tall fescue. It is interesting to found that many marker alleles presented significant association with single trait, or associations with more than one trait. For instance, 5 associations were associated with SR and TQ in greenhouse trial, which would be considered to be pleiotropic or co-localized MTAs [8]. These co-localized or pleiotropic associations may be beneficial to detect some important genomic regions or genes for heat tolerance related traits. Furthermore, the markers associated with more than one trait may be made effectively use of improving more than one trait by marker assisted selection. For screening heat tolerant accessions by phenotypic and physiological traits, our experimental population is relative small, which influence the power of association analysis. Yan et al. [46] showed that association study with a set of 500 individuals would supplyan 80% probability of detecting a gene that explains 3% or more of the phenotypic variation, and increasing the number of population could be more substantial effect on the power of MTAs than increasing the density of markers in genome-wide association (GWS). More reliable markers could be identified for developing elite heat tolerant tall fescue cultivars through marker assisted selection under various conditions: first if higher density DNA polymorphism databases would have been evenly distributed in all genome chromosomes. And secondly larger mapping populations and phenotypic traits under more sites of heat stress would have been used for association mapping. A large challenge for association analysis for complex quantitative traits in plant is large number of loci identified with small effects in some plant species such as barley, maize, wheat and rice. In our study by MLM analysis, the explained variation (Marker R 2 ) for the identified associations were low to modest, ranging from 7.03% (turfgrass quality)-19.21% (turfgrass quality) in greenhouse trial, and 7.06% (leaf chlorophyll content) -21.86% (growth rate) in growth chamber trial, respectively. The explained variation by marker-trait associations (MTAs) for abiotic stress related traits in association analysis of plant species is changeable. Thudi et al. (2014) used DArT, SNP, and SSR markers to study 300 accessions of chickpea for drought tolerance related root traits, heat tolerance, yield and yield component traits cross 6 environments, and showed that phenotypic variance explained of MTAs ranged from low (4.14%) to very high (96.55%). However, Varshney et al. [47] studying a diverse barley panel at a dry and wet location for drought tolerance related traits, found that explained variation for all of identified MTAs was rather low, ranging from 0.1% to 6.7%. Some other studies on GWA analysis in barley also showed that MTAs contributing large phenotypic variation are highly heritable, and MTAs of explained variation >10% seem hard to be identified for the complex quantitative traits like drought tolerance in association analysis [6,48]. Large effect QTL may be due to the inbreeding nature of some species, while out-crossing plants such as tall fescue and maize may have very large number of genes contributing a very small amount to a quantitative trait [46]. Associations identified in our study were not only small, but also little consistent across environments like barley or wheat. In the two trials of our study, there were only two associated SSR alleles were identified in two trials at low threshold, -Log (P-value) ≥2.0 by MLM analysis, which showed influence of environment on heat tolerance related traits that would be low heritability. The observed differences of marker-trait associations in both trials may be due to the different experimental conditions of heat stress, including temperature, heat intensity, duration, and matrix cultivated, which lead to the variation of phenotypic, physiologic and biochemical characteristics in response to heat stress, and even trigger different genetic pathways and mechanisms of heat tolerance. In greenhouse trial, the temperature of greenhouse often exceeded 45°C in summer, and reached 50°C at the noon, which restrained growth of tall fescue and caused severe thermal damage. Most tall fescue accessions halted growth, withered rapidly, and the leaves yellowed after 2 or 3 day of heat stress. However, the growth chambers controlled the temperature at 35°C moderate high temperature. The extreme high temperature would induce specific membrane damage, expression of HSP [49], and alteration of activity of enzymes, which was not prevalent at moderate heat stress [50]. Simultaneously, immersing grasses into nutrient solution in growth chamber trial made the roots that are more sensitive to heat stress than leaves to be directly exposed to high temperature and severe damage [51,52]. Therefore, many heat sensitive tall fescue accessions presented dehydration wilting and even death in growth chamber trials. Specifically, many factors resulted in the differences of phenotypic and physical traits when tall fescue accession responded to heat stress, which made a few SSR alleles associated with functional traits to be simultaneously identified. The observation that the majority of the SSR alleles associated with heat tolerant-related traits could only be identified in a specific condition of heat stress indicated that tall fescue is very sensitive to variation of high temperature. The similar conditions had also reported in association analysis for drought tolerance related traits of barley, wheat, maize, and chickpea [7,8,13,46]. So identified markers may be not suitable for direct application in markerassisted selection (MAS) programme for developing more stable heat tolerant tall fescue varieties or cultivars. Vast studies including complex crosses and QTLs mapping with well chosen parents on the basis of results obtained in our study for verifying effectiveness of marker alleles are necessary for breeding heat tolerance cultivars by marker assisted selection. Conclusion In summary, we initial focus on association mapping analysis of heat tolerance-related functional traits in tall fescue. Five quantitative traits GR, TQ, SR, CHL and ET showed high diversity and significant mutual correlations in response to heat stress in tall fescue. Two subpopulations were detected in the panel of accessions, but no obvious relative kinship was observed. But for any trait, the K model controlling relative kinship showed the similar distribution of P value and associations with Q + K model that controlling both population structure and relative kinship in our study. So model testing is necessary to reduce the spurious associations. By mixed linear model (Q + K) as the best model for association analysis, 97 associations in greenhouse trial and 67 associations in chamber trial were identified for five heat tolerant traits at two time points (P < 0.01). It is necessary for tall fescue selection breeding because these markers would enhance efficiency of identifying heat tolerant accessions bringing desirable alleles. However, only two SSR alleles associated with GR and ET were identified due to the different environments between two trials. And inadequate samples and limited markers were utilized in our study which might have weakened the reliability and effectiveness of associated SSR markers. Hence, it was necessary to confirm the associated marker locus by genotypes F2 grasses and phenotype F3 progeny, or QTL mapping with a high resolution linkage mapping in the next step. Simultaneously, for identification of more effective markers or genes by association analysis, further research need to focus on selecting candidate genes regulating heat tolerance of tall fescue or developing a large amount of single nucleotide polymorphism for genotyping larger association population. Plant materials and growth conditions 100 diverse accessions of tall fescue were employed in this study, including 93 accessions obtained from the United States Department of Agriculture-Agricultural Research Service (USDA-ARS) and 7 turf-type commercial cultivars obtained from the seed industry ( Table 7). The collection of accessions was based on geographical locations for maximizing genotypic diversity. All accessions were confirmed to be hexaploid by flow cytometry (data not shown). This study was conducted at Wuhan Botanical Garden, Chinese Academy of Science, beginning in 2012. A single seed from each accession was sown in petri dishes with a layer of filter paper soaked in water and kept in dark at 22°C for germination. After one week, the accessions were transplanted into plastic pots (15 cm deep, 11 cm wide) containing a mixture of sand and soil (1:1, v/v) in a greenhouse with temperature ranging from 20°C to 26°C, 1000-1500 μmol photons m -2 s -1 , 14 h photoperiod of natural sunlight, and 76% average relative humidity. Plants were irrigated daily to maintain sufficient water supply conditions, fertilized weekly with halfstrength Hoagland's solution [53], and mowed to 7 cm canopy height once a week. Each accession was propagated through tillers multiple times for genetic uniformity. Heat treatment and experimental design Two trails were conducted. One was processed in the greenhouse in June, 2012, the other in growth chambers repeated in August, September, and October 2012, respectively. Greenhouse trail: All 100 accessions were transferred into a natural greenhouse in June 8 th to July 14 th , 2012 after growing in the controlled greenhouse for 30 d. The maximum temperatures varied from 39°C to 51°C during 21 d of heat treatment. Each accession had three replications with same genotypes, and all plots were arranged in a completely randomized block design. The greenhouse had a photosynthetically active radiation (PAR) of 1000-2000 μmol s -1 m -2 of natural sunlight. Grasses were irrigated daily until water could freely drain from the holes under the plots. Growth chamber Experiment The trial was repeated three times in growth chamber in August, September and October 2012. 100 accessions were transformed into 250 mL Erlenmeyer flask wrapping with aluminum foil, containing half-strength Hoagland's solution and 0.1 μmol magnesium oxide to provide additional oxygen after 30 d growing in controlled greenhouse. Grasses with 7-10 tillers were sealed with parafilm to prevent water escaping from gaps. Before heat treatment, all flasks of grasses were pre-incubated 10 d. Two growth chambers during experimental period were controlled in 14 photoperiod, 70% ± 10% relative humidity, and approximately average 450 μmol photons m -2 s -1 . Every other day all flasks were exchanged layers and half-strength Hoagland solution added. This trail included an unheated control (25/16°C, day/night) and heat stress (38/30°C, day/ night) treatment sustaining 15 d. The heat treatment was subjected in different chambers for each replication. Growth and physiological measurements Many growth and physiological traits were measured before and after heat stress interval 7 d in two trails, including turf grass quality (TQ), survival rate (SR), leaf chlorophyll content (CHL), evapotranspiration rate (ET) and growth rate (GR) . Turf quality was evaluated visually using a scale of 0 (yellow, brown or dead) to 9 (optimum greenness, uniformity, cover) based on density, texture, turf color, and smoothness. Survival rate was also assessed by visual rate using a ratio between survival canopy and total plant. Every 7 d leaves of pots were cut at 7 cm canopy height, were collected, immediately killed at 105°C 30 min, dried at 70°C in an oven for 72 h. Growth rate was calculated as dry weight per growth day. Evapotranspiration rate was measured by weight loss of the plant plot every 24 h and the relative transproation was normalized according to a method described by Hu et al. [54]. Leaf chlorophyll content was measured using the method described by Hiscox and Israelstam [55]. Data was collected from the non-heat and heat treatment across all accessions of tall fescue from two trails to examine the efficiency and consistency. The percentage of reduction of all traits, calculated as [(control value or initial value -heat value)/ control or initial value] × 100, was used to indicated the grass heat tolerance. The main treatment effect, variance analysis (ANOVA) and correlation between growth and physiological traits were performed using SPSS18.0 (IBM Corporation, New York, USA). DNA isolation and SSR analysis Young leaves of each accession were collected for DNA isolation using a cetyltrimethyl ammonium bromide (CTAB) method [56]. A set of 90 published genome-wide SSR markers [21,57] mapped in 22 linkage groups in tall fescue were analyzed in all accessions (Additional file 1 Table S1). All forward primer sequence of markers were labeled with four fluorescent dyes of different colors [FAM (blue), HEX (green), TAMRA (yellow), and ROX (red)]. Each 10 μL PCR reaction in 96 microplates consisted of 1 × supplied Taq-buffer, 2.5 mM MgCl 2 , 200 μM dNTPs, 0.2 mM of each primer pair, 0.5 U of Taq DNA polymerase, and 30 ng of template DNA. PCR reaction was started at 95°C for 10 min; followed by 25 cycles of 50 s at 95°C, 50 s at 68°C with a decrease of 0.6°C in each consequent cycle, 60 s at 72°C; then ran for 15 cycles at 95°C for 50 s, 54°C for 50 s, 72°C for 60 s; and a final extension step 72°C for 10 min. All PCR reactions were used a touch-down program in a 96-well My Cycler thermal cycler (Bio-Rad Inc., Hercules, CA, USA). The PCR amplified fragments were separated by an ABI 3730 DNA Sequence (Applied Biosystems Inc., Foster City, CA, USA). Alleles were scored by GeneMarker 1.5 software (Soft Genetics, LLC, State College, PA, USA) and checked twice manually for accuracy. If more than one fragment were amplified by a primer in accession and appeared differently in other accessions, they were scored as different loci. For allohexaploid genome of F. arundinacea, band scores of SSR loci were entered into a binary matrix as presence (1) or absence (0) following Sara et al. [21]. All confirmed polymorphic alleles were applied for population structure and kinship analysis. Population structure and relative kinship As a result of labeling all SSR markers as dominant in each genotype, no information on marker linkage could be obtained in population structure model. A Bayesian model-based clustering method carried out in STRUC-TURE 2.0.1 software [58] was employed to determine population structure (Q) and division accessions into subpopulation. The basis of the clustering method is that it prevented admixture of correlated allele frequencies, therefore the allocation of individual genotype to K subpopulations is in such a way that Hardy-Weinberg and linkage equilibrium is valid within populations. The structure was run ten times by setting pre-defined k (the number of population groups) ranging from 1 to 15 using admixture models with 10,000 MCMC (Markov Chain Monte Carlo) replications and 10,000 burn-in time for each run. Population based on the maximum likelihood was determined by the probability of data likelihood LnP(D) in the output and an ad hoc statistic △K based on the second-order rate of change in LnP(D) between successive K values [58]. 15 independent runs were operated 100 000 iterations of each run after burnin of 100 000 for a value of K setting from one to five. Then SPAGeDi software [59] was applied to evaluating relative pairwise kinship (K) by 90 SSR markers, and then the pairwise kinship matrix (100 × 100) was produced by the Loiselle coefficient [60]. All negative kinship values between the individuals were assigned to zero, according to Yu et al. [41]. Model testing and association mapping Based on the differences in the regime of heat treatment (density, duration time and matrix cultivated plant), the functional traits of heat tolerant in two trials were used for identifying association with SSR loci, respectively. Turfgrass quality, growth rate and leaf chlorophyll content in growth chamber trial were selected to perform marker-trait associations. Three models were used to access the effects of relative kinship (K) and population structure (Q) for marker-trait associations. The Q model was performed using general linear model (GLM). The K and K + Q models were performed using MLM in TASSEL 2.0.1 software [58]. The quantile-quantile plots of estimated -log 10 (P) were drawn using the observed P values from SSR alleles-trait associations and the expected P values assuming that there was no associations identified between marker and trait. The significant threshold for marker-trait associations was set at P < 0.01.

v3-fos

2016-05-12T22:15:10.714Z

2015-02-23T00:00:00.000Z

10607902

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Biodiversity and γ-Aminobutyric Acid Production by Lactic Acid Bacteria Isolated from Traditional Alpine Raw Cow's Milk Cheeses “Nostrano-cheeses” are traditional alpine cheeses made from raw cow's milk in Trentino-Alto Adige, Italy. This study identified lactic acid bacteria (LAB) developing during maturation of “Nostrano-cheeses” and evaluated their potential to produce γ-aminobutyric acid (GABA), an immunologically active compound and neurotransmitter. Cheese samples were collected on six cheese-making days, in three dairy factories located in different areas of Trentino and at different stages of cheese ripening (24 h, 15 days, and 1, 2, 3, 6, and 8 months). A total of 1,059 LAB isolates were screened using Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and differentiated into 583 clusters. LAB strains from dominant clusters (n = 97) were genetically identified to species level by partial 16S rRNA gene sequencing. LAB species most frequently isolated were Lactobacillus paracasei, Streptococcus thermophilus, and Leuconostoc mesenteroides. The 97 dominant clusters were also characterized for their ability in producing GABA by high-performance liquid chromatography (HPLC). About 71% of the dominant bacteria clusters evolving during cheeses ripening were able to produce GABA. Most GABA producers were Lactobacillus paracasei but other GABA producing species included Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus pentosaceus, and Streptococcus thermophilus. No Enterococcus faecalis or Sc. macedonicus isolates produced GABA. The isolate producing the highest amount of GABA (80.0±2.7 mg/kg) was a Sc. thermophilus. Introduction Traditional alpine raw milk cheeses are commonly produced in alpine regions including the province of Trentino in North-Eastern Italy. Here they are called "Nostrano-cheeses" and are semicooked cheese made by mixing approximately in 1 : 1 ratio the raw cow's milk from two different milking. The first milking is carried to dairy factory the evening before the cheese-making and is stored in large shallow tank for 9-11 hours where a spontaneous creaming occurs. After this overnight stage, the partially skimmed milk under the cream in the tank is manually drained from the cream fat and placed in the cheese-making vat. The whole milk from the morning milking, the second milking, is then added to the skimmed milk. No commercial lactic starters are added and the natural milk microbiota obtained from the overnight skimmed milk initiates the acidification process. The vat milk is coagulated by commercial rennet and, after the manual cutting, the curd is cooked at about 48 ∘ C. After moulding and salting, the ripening is held at about 18 ∘ C for 3 to 8 months. The milk for "Nostrano-cheeses" typically comes from Holstein Friesian and/or Brown Swiss cattle breeds, which are fed differently during the year. The cows are typically fed on hay during the cold season in the valleys and from late June to middle September (summer season) are grazed on high mountain alpine pasture. BioMed Research International It has been reported that the use of commercial starters in raw milk cheeses may modify the characteristics of the cheese microbiota, in particular lowering the microbial biodiversity [1] and it is also well known that mainly LAB microbiota developing during ripening influences the typical organoleptic characteristics of the cheese [2]. Thus, LAB represent a fundamental process factor for the final attributes and quality of artisan dairy products such as alpine cheeses. Several studies have focused on the genotypic and technological characterization of LAB isolated from different traditionally fermented cheeses [3][4][5][6], but little work has so far been done on "Nostrano-cheeses. " In addition to the technological relevance of LAB in cheese, there is currently much research and industry interest in the potential biological activity of dairy LAB, either for use as probiotics in their own wright or as bioactive agents capable of modulating the health functionality of cheese and other dairy products [7]. Raw milk cheeses have already been identified as a useful source of microbial biodiversity and new LAB strains with health promoting properties [8]. Since caseins are rich in glutamate which is released by proteolytic action, the decarboxylation of this amino acid into -aminobutyric acid (GABA) can have an important effect on the formation of eyes in cheese [9]. Besides its technological effect in cheese, GABA has several wellcharacterized physiological functions in mammals including neurotransmission, induction of hypotension, diuretic and tranquilizer effects, and stimulation of immune cells [10][11][12]. Some studies have reported also that GABA derived from the gut may be a neuroactive molecule within the gut-brain axis [13], which is a complex communication highway linking the gut environment with both the central and peripheral nervous systems. Strains of Lb. buchneri [14], Lb. brevis, Lb. paracasei, and Lb. plantarum [15] isolated from traditional cheeses have been shown to produce GABA. GABA-producing LAB have not been isolated and extensively characterised from traditional alpine cheeses produced in Trento, though the presence of GABA in these cheeses has been confirmed and its concentration at the end of ripening reported at between 120 and 1,739 mg/kg [16], which is high compared to other Italian cheese varieties (typically 0.260 to 391 mg/kg) [15]. Therefore, the objective of this study was to analyze the diversity and the successional development of LAB in traditional "Nostrano-cheeses" from the Trento alps during cold and summer seasons and to extensively screen and identify GABA-producing LAB isolates. Cheese Factories and Milk Sampling. Cheeses were sampled in three dairy factories (called B, C, and D according to a previous paper [17]) located throughout the Trentino region and producing traditional alpine cheeses called "Nostranocheeses. " Each factory collected milk from farms within a 15 km radius. Two cheese batches from each dairy factory, one in February and the other in July, were sampled, making a total of six batches subjected to microbiological analyses. All factories processed milk obtained from stabled cows fed with hay during the "cold season" from October to May and high mountain pasture fed cattle in the summer season from June to September. For each of the six batches, at least five cheese samples at different stages of ripening were collected (24 hours, 15 days, 1 month, 2 months, and 3 months and for five batches also 6 and 8 months) making a total of 40 cheese samples per factory. Enumeration and Isolation of Microorganisms. Cheese samples (25 g) were homogenized (2 min at 260 rpm) using a stomacher (laboratory blender stomacher 400, Seward, London, UK) in 225 g peptone water (0.1% mycological peptone (Oxoid, Basingstoke, UK)) and serially diluted. Dilutions were plated and incubated as follows: onto MRS agar acidified to pH 5.5 with 5 mol/L lactic acid, anaerobically, for 2 days, at 30 ∘ C and 45 ∘ C for mesophilic and thermophilic rod-shaped LAB, respectively; onto MRS agar added with vancomycin (8 g/mL; Sigma-Aldrich, Saint Louis, MI,US) [18] and acidified to pH 5.5 with 5 mol/L lactic acid, anaerobically, for 72 h at 30 ∘ C for mesophilic heterofermentative rodshaped LAB; onto M17 agar for 2 days, aerobically, at 30 ∘ C and anaerobically at 45 ∘ C for mesophilic and thermophilic coccoid LAB, respectively; onto KAA aerobically, for two days, at 37 ∘ C for enterococci; onto PCA added with 10 g/L skimmed milk aerobically, for 24 h, incubated at 30 ∘ C for total bacterial count (TBC). All culture media were purchased from Oxoid. At least three colonies were picked from each countable plate; Gram-positive colonies (as determined by KOH method; [19]) and negative to the catalase test (as determined by transferring fresh colonies from agar medium to a glass slide and adding 5% H 2 O 2 ) were isolated. Cell morphology was determined by microscopic observation. Each isolate was purified by subsequent culturing onto M17 or MRS and pure cultures were stored at −80 ∘ C in glycerol (20% v/v) stocks. DNA Extraction and RAPD-PCR. DNA was extracted from overnight broth cultures of isolated strains. Cells were centrifuged at 10,000 ×g for 5 min and the pellets were washed twice in sterile distilled water and suspended in 1 mL of distilled water. Cell lysis was achieved using the Instagene Matrix (Bio-Rad, Hercules, CA, USA) following the manufacturer's instruction. RAPD-PCR was carried out in a total volume of 25 L using primer PC1 [20]. Cluster analysis of DNA patterns was carried out using GelCompar II-BioNumerics software (package version 6.0; Applied Maths, Belgium), exploiting the unweighted pair group method arithmetic averages (UPGMA). Similarity of PCR fingerprinting profiles was calculated based on Pearson product-moment correlation coefficient. The threshold breakpoint value was fixed to 80%; isolates with similarity coefficient higher than 80% were classified into the same cluster, according to Gatti et al. [21]. All the amplicons were analyzed by electrophoresis on 2.5% (w/v) agarose gel (Gibco BRL, Cergy Pontoise, France) at 100 V for 90 minutes in 1X TAE buffer and were revealed by staining with ethidium bromide (0.5 g/L). All amplifications were performed with a T100 Thermal Cycler (Bio-Rad Laboratories). -Aminobutyric Acid (GABA) Production and Quantification. Glutamate decarboxylase (GAD) activity of LAB isolates and the production of GABA were checked using the method of Nomura et al. [26], with some modifications; cultures were centrifuged (9,000 rpm for 15 min at 4 ∘ C), washed twice with sterile PBS, and suspended in sterile 0.85% NaCl solution in order to achieve the 620 nm value of 2.5. Afterward; 100 L of cell suspension was mixed with 900 L of 50 mM sodium acetate buffer (pH 4.7) containing 7.0 mM L-glutamate and 0.1 mM pyridoxal phosphate. The reaction mixture was incubated for 24 h at the same temperature of isolation (30 ∘ C for mesophilic and 45 ∘ C for thermophilic isolates) and filtered through a 0.22 m pore size filter (Minisart, Sartorius Stedim Biotech, Goettingen, Germany). The sample, diluted 10 times with sodium tetraborate 0.1 M (pH adjusted to 10.5) and added to glycine, as internal standard to a final concentration of 10 mg/L, was stored at −20 ∘ C before the analysis. L-Glutamic acid, glycine, and GABA were quantified as o-phthalaldehyde (OPA) adducts modifying the method proposed by Lehtonen [27] in order to notably reduce the time of separation to only 2.7 minutes but without worsening selectivity and accuracy. This was possible in the light of the specifically designed and perfectly known matrix. The measures were performed using an UHPLC Ultimate 3000 (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a fluorescence detector (Ex = 336 nm, Em = 445 nm). Separation was carried out with sodium acetate 0.05 M (pH adjusted to 7.5; eluent A) and methanol (eluent B) using a column Chromolith Performance RP-18e (100 × 4.6 mm; Merck, Darmstadt, Germany) with Guard Cartridge Chromolith RP-18e (10 × 4.6 mm; Merck) at 40 ∘ C. The flow rate was set at 2 mL/min. The analytical gradient for eluent B was as follows: 40% for 30 sec, 25% for 90 sec, 100% for 30 sec and 60% for 15 sec. The sample (10 L), kept at 10 ∘ C by the autosampler, was automatically introduced into the loop, added with 10 L derivatising solution, mixed for 1 min, and injected. The derivatising mix was 4.5 g/L of OPA (Sigma-Aldrich) in sodium tetraborate 0.1 M, corrected to pH 10.5, 10% methanol, and 2% 2-mercaptoethanol (Sigma-Aldrich). The detection limit for GABA was estimated at 0.025 mg/L (3 times the standard deviation of the GABA contents measured repeating 10 times the analysis of a sample at unquantifiable content). Microbial Cell Counts. The microbial populations of Nostrano-cheese samples were estimated on different selective media ( Table 1). The total bacterial counts were in the range of 8-9 log cfu/g from 24 h to 3 months of ripening and decreased after 6 and 8 months by 1 order of magnitude. The thermophilic cocci reached the highest counts after 24 h of ripening (mean values of 8.5 log cfu/g); mesophilic cocci reached the highest counts after 2 mo of ripening (mean values of 8.1 log cfu/g). Enterococci were never dominant and reached their highest count after 15 days of ripening (mean values of 6.2 log cfu/g). The lactobacilli group (counts onto MRS at 45 and 30 ∘ C) was higher after 1 mo of ripening. The growth dynamic of the different microbial groups was different (Table 1); thermophilic cocci counts (onto M17 45 ∘ C) were dominant in the first 24 hours; after 15 days to 3 months of ripening, mesophilic cocci (onto M17 30 ∘ C) and lactobacilli counts (onto MRS and MRS VAN 30 ∘ C) started to increase and were dominant together with thermophilic cocci; finally, at the end of ripening (6 and 8 months) mesophilic lactobacilli and thermophilic cocci maintained the dominance within the alpine cheeses microbiota. On average, three colonies, for each colony morphology, were isolated in pure culture from each medium. For summer season at 6 and 8 months, only two types of cheese were available and sampled in dairy factories C and D. A total of 1,105 isolates were collected. From the total number of isolates, 46 were discarded from further analysis as nonlactic acid bacteria (they were found positive to catalase and negative to KOH tests). The remaining 1,059 strains were characterised for cell morphology; 677 were cocci and 382 were rods (Table 2). Molecular Clustering of LAB Isolates and Species Identification. All putative LAB isolates were analyzed by RAPD-PCR as a first grouping into clusters. The isolates from the same kind of cheese showing a RAPD similarity coefficient of at least 80% were considered as belonging to a single cluster. The RAPD-PCR analysis grouped 1,059 LAB into 583 clusters with 80% similarity index (results not shown). From these clusters, 276 isolates were selected for further analysis because they belonged to the dominant microbial populations as enumerated by plate counts on MRS, MRS VAN at 30 ∘ C, M17 at 30, and 45 ∘ C. The RAPD-PCR analysis of these 276 dominant isolates discriminated 97 different clusters defined at a minimum similarity level of 80% (Figure 1). The 97 clusters were designated using a progressive number followed by the letters B, C, or D to indicate the dairy of origin of the clustered isolates ( Figure 1; clusters 1D to 97C). Species identification was performed by species specific PCRs or partial 16S rRNA gene sequencing. Table 3 shows the results of bacteria identification for each ripening time. The highest diversity within a single species was found for Lb. paracasei and Sc. thermophilus with 35 and 23 different genotypes, respectively. Lb. paracasei was the dominant species (72 isolated on MRS and 16 on M17 agar plates), followed by Sc. thermophilus (50 isolated on M17 agar) and Ln. mesenteroides (27 isolated on MRS and 18 on M17 agar). A different dominant species successional development was observed in cheeses at different ripening time: Sc. thermophilus was always dominant in the first 24 hours and one of the codominant species for up to 2 months of ripening; Lc. lactis species was also found codominant in cheese at 24 h ripening with Sc. thermophilus. Streptococci and enterococci species were recorded in abundance in the first three months but largely disappeared after six months and at the end of ripening (Table 3). We did not find difference in species distribution between cheeses sampled in February and July. The same species were recorded both in cold and in summer season (data not shown). After the genotypic characterization, 97 strains, one representative of each dominant cluster, were processed for the detection of GABA production. GABA Production. Sixty-eight isolates out of the 97 different clusters synthesized GABA (GABA amount > 0.25 mg/kg) after 24 h of incubation at 30 or 45 ∘ C in presence of glutamic acid (Table 4). They grouped 195 of the dominant isolates (71% of the tot) and in particular three (1 Lb. paracasei, 1 Lb. rhamnosus, and 1 Sc. thermophilus) were able to produce GABA concentrations higher than 10 mg/L ( Discussion In Italy the province of Trento has a long dairy history with various dairy biotechnological traditions arising from the geographical challenges of transport and communication between different alpine valleys and a diverse cultural heritage. A wide range of cheeses coexist, each with their own specific biotechnological processes, organoleptic characteristics, and history. A previous review has discussed the importance of preserving this type of traditional artisan cheese, usually made from raw cow's milk, because of their high microbial biodiversity and in particular high species richness of "wild" LAB with diverse metabolic activities and of great potential as dairy starters or even probiotic agents [8]. Previous work has shown that the "Nostrano-cheeses" contain high concentrations of GABA compared to other Italian cheeses [16] and it is known that LAB is responsible for producing GABA in cheese [15]. We, therefore, selected Trento "Nostrano-cheeses" for the screening and isolation of GABA-producing LAB. The successional development of the lactic microbiota of six"Nostrano-cheeses" from 24 hours to 8 months of ripening was characterised. 276 isolates belonging to dominant lactic microbiota were grouped into 97 clusters, identified to the species level, and screened for their GABA production. The milk used to produce these cheeses was the subject of a previous report [17] but in summary, the microbiological characterization was in agreement with microbial counts reported for other traditional Italian cow raw milk cheeses [6,28,29]. M17 and MRS were not perfectly selective, in agree with previous works [6,30] in fact some nontarget isolations were recorded; for example, 2 Lc. lactis isolates amongst 13 were found on MRS agar plates and about 14% of all rodshaped isolates were isolated on M17 agar plates. As commonly found in many raw milk cheeses [28][29][30], the microbial composition of the "Nostrano-cheeses" was dominated by LAB. Lb. paracasei was the most abundant species (31.9% of the isolates), followed by Sc. thermophilus and Ln. mesenteroides (18.1% and 16.3%, resp.). These species were amongst the dominant microbiota at all production stages; in particular, Sc. thermophilus dominated after 24 h until 2 months of ripening, while Lb. paracasei and Ln. mesenteroides reached their highest levels in the cheese after 15 days and remained at high levels until 3 months of ripening with a similar trend observed in microbial counts on MRS which started to decrease after 6 months of ripening, probably the result of microbial autolysis [31,32]. Another 11 different LAB species were found in the cheese samples but none at a relative abundance higher than 5%. All species identified were previously recorded and very common in the dairy environment [4,6,[28][29][30], with the exception of Lb. acidipiscis, which is a species described by Tanasupawat et al. [33] and isolated from fermented fish and has also been isolated more recently from traditional Greek cheeses [34]. RAPD analysis displayed a great genetic diversity amongst the isolates. In fact about 33% of the RADP clusters were singletons (one cluster for one isolate). This genetic biodiversity may reflect a real picture of the high species richness amongst the isolates collected from the cheeses but could also be consequence of the large number of strains analysed in this study. A similar result was found in a previous work, where 206 isolates from spontaneously fermented cheeses were analysed by RAPD PCR [30]. We compared all the clusters recovered from "Nostranocheeses" with those found in the corresponding milk samples and reported in a previous report [17] and no milk RAPD pattern was found amongst the 586 cheese clusters. This may be because the fermentation is not spontaneous but driven by a starter culture from the overnight skimmed milk that, even if natural and not commercial, may inhibit milk microbiota growth and development. It is worth highlighting that some isolates from different dairies grouped within the same cluster. The 9 species occurring in different dairies were Lb. paracasei, Lb. rhamnosus, and Ln. mesenteroides. These few coincident clusters occurred often in different dairy environments and might represent part of an endemic geocentric cheese microbiota, not necessarily coming from milk, but adapted to the cheese-making practice, ripening, and local microclimate and environmental conditions specific to the Trento alps. On the other hand, 34 of the 97 clusters were RAPD-PCR singletons and some species like Lb. coryniformis ssp. torquens, Lb. acidipiscis, Lb. curvatus, and Lb. delbrueckii were peculiar only for one of the three dairy factories. These aspects suggest that each manufacturing facility may also be characterized by a unique microbial population. Considering the recent interest in the gut-brain axis, the potential role of neurotransmitters like GABA in the periphery, and the immunological potential of systemic GABA, we screened the 97 dominant clusters for GABA producing strains [35]. A total of 68 GABA producing strains were identified. Previous studies by Siragusa et al. [15] and more recently by Diana et al. [36] found that sheep milk cheeses contained higher levels of GABA than cow's milk cheeses and consequently had higher numbers of GABAproducing LAB. The raw cow's milk cheeses subject of this current study showed higher amounts of GABA at the end of ripening [16] and a higher percent of GABA producer strains (71%) than these two previous studies where GABA producing strains were less than 14%. This difference may be due to the peculiar traditional environment of production of these Trento cheeses. However, it may also be the result of the cheese production times sampled. We screened the isolates starting at 24 h and followed the cheese LAB microbiota until the end of ripening. It is probable that microbial GABA production follows the same trend as the bacterial growth with higher number of GABA producing strains in the first 3 months followed by a rapid decrease in LAB numbers. Amongst the 68 positive strains, 13 GABA producing strains gave more than 4 mg/kg and belonged mainly to Lb. paracasei species but also to Lc. lactis, Lb. plantarum, Pc. pentosaceus, Lb. rhamnosus, and Sc. thermophilus. The ability to produce GABA has been reported in various LAB, in particular Lactobacillus sp. isolated from fermented food [37]. Lc. lactis and Sc. thermophilus were also found to produce GABA in different Italian cheeses screened by Siragusa et al. [15] and Pc. pentosaceus was isolated as high GABA producing strain from a Thai fermented meat [38]. From our screening, no Ec. faecalis or Sc. macedonicus strain was able to produce GABA (less than 0.25 mg/kg) and, to our knowledge, these species have never previously been identified as GABA producers. GABA is a desired bioactive compound because of its physiological functions such as neurotransmission, induction of hypotension, diuretic and tranquilizer effects, and stimulation of immune cells [10][11][12]. For these beneficial effects, GABA has been introduced in the diet as an oral supplement; the Japanese government defines the foods enriched with GABA as "foods for specified health use" [3]. Fermented milk enriched in GABA produced by lactobacilli may have commercial potential as a health-oriented dairy product. Siragusa et al. [15] were the best GABA-producers during the fermentation of reconstituted skimmed milk. A Lb. casei and a Lc. lactis subsp. lactis were used for the manufacture of a GABA-enriched fermented milk: the first strain hydrolyzed milk protein into glutamic acid and the second converted glutamic acid into GABA, respectively [39]. This current study suggests a real potential of the Sc. thermophilus isolate from cluster 84C to produce GABA in fermented dairy products. A daily intake of fermented milk with an amount of 10 mg of GABA for 12 weeks has been shown to decrease blood pressure by 17.4 Hg in hypertensive patients [39]. Sc. thermophilus belonging to the cluster 84C in this current study produces 80 mg/kg of GABA. 125 mg of milk fermented with this strain could, therefore, be enough to obtain the daily intake necessary for a potential antihypertensive effect observed by Inoue et al. [39]. We are now examining the ability of this Sc. thermophilus strain to produce GABA in fermented milk, either alone or in association with other milk protein hydrolyzing LAB and under simulated gastrointestinal conditions. Conclusions This study describes the diverse lactic microbiota of traditional semihard "Nostrano-cheeses" from the Trento alps in Italy and how this microbiota changes during ripening. We have also characterised the potential of selected LAB isolates to produce GABA under controlled conditions, a molecule newly recognised as a putative food bioactive. We identified one Sc. thermophilus strain as a "high GABA producer" with considerable biotechnological potential for the development of new and attractive dairy products, an important commercial objective for increasing the potential of cheese as multifunctional dairy product.

v3-fos

2019-04-01T13:16:17.713Z

2015-05-01T00:00:00.000Z

89285042

s2

Foliar Application of Fly Ash on Wheat Crop Fly ash is a major particulate type of air pollutant affected the opening and closing of stomata by blocking the stomatal aperture and thereby allowed increased transpiration. Low dusting rate of fly ash increased chlorophyll contents significantly, while high dusting rate of fly ash reduced the chlorophyllase enzyme due to the alkalinity caused by excessive soluble salts on the leaf surface and also due to increase of foliar temperature which retarted chlorophylls or breakdown of chlorophyll to form pheophytin. Due to which photosynthesis in leaves also retarded. In present lower dose of fly ash dust was found beneficial for all plant growth (Length, fresh and dry weight of shoot and root and tillers no. leaf area); yield (Ear length, no. of grains/ear and weight of 100 grains) compared to control .While, both another doses (2.5 and 5.0 g mG2) caused reduction in all above parameters and reduction were higher in 5.0 g mG2 treatments. Similarly, all biochemicals (Photosynthetic pigments, seed protein and seed carbohydrates) were also increased at 1.25 g mG2 treated sets. Lower dose of fly ash was also found beneficial to all leaf epidermal characteristics (No. of adaxial and abaxial surface of stomata, length and width of stomatal aperture and no. and length of trichomes). All these parameters were increased significantly. After that there was gradual decrease in all these parameters at both doses (2.5 and 5.0 g mG2). INTRODUCTION Fly ash is a major particulate type of air pollutant generated by the combustion of coal in coal fired thermal power plants. The Indian coal constitutes about 30-40% fly ash after complete burning (Kumar et al., 2000). About 90 million t fly ash was produced during year 2000 and at present about 100 million t is being produced throughout the country. It will likely to cross about 140 million t by the year 2020 AD. It consist minute, glass like particles of 0.01-100 mm having specific gravity 2.1-2.6 (Davison et al., 1974). Although, fly ash is trapped as fine dust in cyclonic and electrostatic precipitators during combustion of coal, but a considerable amount is escaped and emitted into atmosphere and deposited on soil and vegetation around emission sources. In humid condition fly ash sticks on the leaves or fruits and causes small necrotic dark brown spot on the leaves due to killing of the tissues. Fluckiger et al. (1979) and Krajickova and Mejstrik (1984) reported that fly ash particles affected the opening and closing of stomata by blocking the stomatal aperture and thereby allowed increased transpiration. Dubey et al. (1982) observed that low dusting rate of fly ash increased chlorophyll contents significantly. While high dusting rate of fly ash reduced the chlorophyll contents due to the alkalinity caused by excessive soluble salts on the leaf surface and also due to increase of foliar temperature which retarted chlorophyllase or breakdown of chlorophyll to form pheophytin (Mudd and Kozlowski, 1975). Mishra and Shukla (1986) reported that fly ash deposition on leaves also retarted the photosynthesis. Recently, Raghav (2006) has obtained the similar results on photosynthetic pigments by dusting of fly ash on potato leaves. Therefore, present study was carried out to evaluate the beneficial dose of fly ash that will helpful to increases crop productivity without any loss. MATERIALS AND METHODS Plant culture and treatments: For this experiment, seeds of wheat were surface sterilized (dipped in 0.01% HgCl 2 solution) for 15 min. Sterilized seeds were sown in each autoclaved pots. After germination, seedlings were thinned to maintain single seedling per pot. Each treatment was replicated three times along with a control set. After 15 days of germination, plants were exposed to different of fly ash (1.25, 2.5 and 5.0 g mG 2 ) as foliar application. The fine particles of fly ash were dusted by a plastic duster, which delivered the particles uniformly over the aerial part of plant. Dusting was done twice in a week till 100 days. After each exposure all pots were kept on glass house benches in a randomized block design at 27/23°C day/night temperature. Photosynthetic active radiation was PAR>750 μmol mG 2 secG 1 between 1100 and 1200 h and humidity was 67±5%. The experiment was terminated after 120 days and plants were uprooted carefully. Roots were washed thoroughly under tap water to avoid soil particles and debris. Plant growth, yield, photosynthetic pigments (chl a, chl b, total chl a+b and carotenoids), seed protein (soluble and insoluble) and seed carbohydrate (soluble and insoluble) contents were estimated, the photosynthetic pigments and leaf epidermal characters were examined before maturation of crop. Statistical analysis: The data was analyzed using analysis of variance for single factor (ANOVA) and L.S.D. were calculated at p<0.05 and p<0.01 for significance (Gomez and Gomez, 1984). The standard deviation and percent increase (+) or decrease (-) over control were also calculated. RESULTS Lower dose (1.25 g mG 2 ) of fly ash as foliar application was found beneficial for plant growth and yield of wheat. All parameters were increased significantly (p = 0.05 and p = 0.01). After that there were reductions occurred in all parameters. However, decrease in all parameters was nonsignificant (p = 0.05 and p = 0.01) in 2.5 g mG 2 dose, while there was significant decrease in 5.0 g mG 2 dose as compared to control. Thus all doses of fly ash showed varied responses (Table 1, Fig. 1). Res. J. Environ. Toxicol.,9 (5) The data presented in Table 2 also indicate that the lower dose (1.25 g mG 2 ) of fly ash dust was beneficial for all photosynthetic pigments, carbohydrate and protein contents. The increment in all parameters was statistically significant (p = 0.05 and p = 0.01) compared to control, except carotenoids. But at 2.5 g mG 2 the parameters were statistically similar to control, while at 5.0 g mG 2 dose, there were significant reductions in all above parameters (Fig. 2). Table 3 reveals that 1.25 g mG 2 , dose of fly ash was also found beneficial to all leaf epidermal characters of upper and lower surfaces of wheat. Number, length and width of stomata; length and width of stomatal aperture and number and length of trichomes were increased significantly (p = 0.05), except number and width of stomata and aperture length of abaxial surface compared to control. After that there was gradual and significant decrease in all parameters, except the aperture width and trichome length (in 2.5 g mG 2 ) and trichome number (in 2.5 and 5.0 g mG 2 ). DISCUSSION Although, fly ash is a particulate air pollutants but it contains various utilizable plant nutrient elements such as Ca, Mg, Fe, Cu, Zn, K, Mn, B, S and P along with appreciable amounts of heavy metals (Adriano et al., 1980). The response of plants to micro and macro-nutrients in fly ash may vary from beneficial effects of small concentrations of nutrient element to toxic effects of high concentrations of many elements (Chang et al., 1977). Wheat plant dusted with different doses of fly ash did not show any visible injury. Interestingly, the lower dose (1.25 g mG 2 ) was found beneficial to plant growth, yield, photosynthetic pigments, carbohydrate and protein contents of wheat. It was due to availability of more than 10% water soluble components like S, Ca, Mg especially boron through leaf surface . The absorption of water soluble salts has also been observed by Rohrman (1971). The transport of the elements through intact cuticles and stomata has been reported by Murray (1984). The absorbed elements actually improved the plant growth. Other side photosynthetic pigments were also increased which led to increase the photosynthetic rate. Thus, the cumulative effects caused increment in all the considered parameters of wheat. Present findings also confirm the results of Mishra and Shukla (1986) on maize and Siddiqui and Singh (2005) on wheat at lower dusting rate. However, higher dusting rate of fly ash adversely affected the wheat plant. Actually fly ash formed a thick layer on the surface of leaves and stem. Thick layer interferes with incidence of light and thus retards the photosynthesis (Mishra and Shukla, 1986). Reduction in chlorophyll content at high dusting rate is attributed to the alkalinity caused by excessive soluble salts on the leaf surfaces and also due to increase foliar temperature, which retards chlorophyll synthesis (Tomes, 1963). Reduced photosynthetic pigments perhaps caused less production of food in leaves and insufficient supply of food material to plants, which led to reduction in all the growth parameters. Ultimately, all other parameters like yield, carbohydrate and protein contents were reduced. Similar results have also been observed with high dusting rates of fly ash on maize (Mishra and Shukla, 1986), wheat (Siddiqui and Singh, 2005) and potato (Raghav, 2006). Similar results with cement dust were also found earlier on bean by Darley (1966), at 3.8 g mG 3 and on Vigna mungo by Prasad and Inamdar (1990). All stomatal parameters were increased at 1.25 g mG 2 foliar application, while width of aperture was widened. It was due to the deposition of fly ash particles on the leaf surface of guard cells (Mishra and Shukla, 1986), stimulated the mechanism of regulating the opening and closing of the stomata and prevents them from being closed (Fluckiger et al., 1979;Krajickova and Mejstrik, 1984). While, in heavily dusted leaves (2.5 and 5.0 g mG 2 ) a thick layer of dust was formed, which checked the opening and closing mechanism by plugging the stomata and also caused reduction in their numbers. However, the number and length of trichomes were increased at 1.25 g mG 2 fly ash dust. The stimulation of trichome number and increment of length might be a morphological adaptation of wheat plant against the dust particles to prevent on leaf surface, in order to provide physical defense against toxic gases and particulate matter (Levin, 1973). Raghav (2006) also reported similar results on potato plant. However, at higher dose (5.0 g mG 2 ) the length and number of trichomes were suppressed significantly. It might be due to failure of adaptive response of plant because of high dust fall. Interestingly, the fly ash was found beneficial to wheat at lower dose (1.25 g mG 2 ) of foliar application. ACKNOWLEDGMENT The author is highly thankful to UGC. for providing financial assistance in the form of Project Fellow in UGC Scheme.

v3-fos

2017-08-02T20:49:21.538Z

2015-04-14T00:00:00.000Z

14911502

s2

Allocation of the S-genome chromosomes of Aegilops variabilis Eig. carrying powdery mildew resistance in triticale (× Triticosecale Wittmack) It has been hypothesized that the powdery mildew adult plant resistance (APR) controlled by the Pm13 gene in Aegilops longissima Schweinf. & Muschl. (SlSl) has been evolutionary transferred to Aegilops variabilis Eig. (UUSS). The molecular marker analysis and the visual evaluation of powdery mildew symptoms in Ae. variabilis and the Ae. variabilis × Secale cereale amphiploid forms (2n = 6x = 42, UUSSRR) showed the presence of product that corresponded to Pm13 marker and the lower infection level compared to susceptible model, respectively. This study also describes the transfer of Ae. variabilis Eig. (2n = 4x = 28, UvUvSvSv) chromosomes, carrying powdery mildew resistance, into triticale (× Triticosecale Wittm., 2n = 6x = 42, AABBRR) using Ae. variabilis × S. cereale amphiploid forms. The individual chromosomes of Ae. variabilis, triticale ‘Lamberto’ and hybrids were characterized by genomic and fluorescence in situ hybridization (GISH/FISH). The chromosome configurations of obtained hybrid forms were studied at first metaphase of meiosis of pollen mother cells (PMCs) using GISH. The statistical analysis showed that the way of S-genome chromosome pairing and transmission to subsequent hybrid generations was diploid-like and had no influence on chromosome pairing of triticale chromosomes. The cytogenetic study of hybrid forms were supported by the marker-assisted selection using Pm13 marker and visual evaluation of natural infection by Blumeria graminis, that allowed to select the addition or substitution lines of hybrids carrying chromosome 3Sv which were tolerant to the powdery mildew infection. Introduction Powdery mildew caused by Blumeria graminis (DC.) E.O. Speer f. sp. Tritici Em. Marchal (Bgt) = Erysiphe graminis DC. Ex Merat f. sp. Tritici Em. Marchal is one of the widespread fungal diseases in cereals. This pathogen has recently infected triticale (× Triticosecale Wittm.), man-made, artificial cereal, which was created to combine the characteristics of cold, disease tolerance and adaptation to unfavourable soils and climates with the productivity and nutritional qualities (Woś et al. 2002). At the beginning of the triticale production, the diseases did not appear to be a serious limitation, probably because of lack of the appropriate, triticale-directed pathotypes of fungal pathogens. Moreover, the grown areas of this crop were incidental to cause serious shifts in the pathogen virulence (Ammar et al. 2004). While the harvest area of triticale began to increase, the new hybrid pathotypes carrying virulence genes appeared (Arseniuk 1996). The new, resistant cultivars could eliminate the fungicides accumulation in grain and reduce the crop losses caused by powdery mildew. Two types of resistance to powdery mildew have been identified so far (Flor 1971). First is called monogenic (vertical) or racspecific resistance, which is effective for some isolates of the pathogen, but ineffective for others. Race-specific resistance is expressed in seedlings and involve single major R genes, in a gene-for-gene interaction (Chen and Chełkowski 1999). Race-specific resistance genes are widely used to combat the wheat diseases, yet the resistance is often short-lived, especially when the genes are employed singly in new varieties (Marais et al. 2008). Second type of resistance to powdery mildew is known as an adult plant resistance (APR), also called 'slow mildewing' and 'partial resistance,' which decelerates the infection, growth and reproduction of the pathogen in adult plants. APR to powdery mildew is more durable than race-specific resistance; therefore it is more desirable in breeding programmes. One of the APR genes is Pml3 powdery mildew resistance gene that ensures high tolerance to all known races of this disease in wheat. The Pm13 gene has been transferred from the chromosome 3S 1 of Aegilops longissima Schweinf. & Muschl. (2n=2x=14 chromosomes; S l S l ) into common wheat, Triticum aestivum L. cv. 'Chinese Spring' (Ceoloni et al. 1988). Considering the synteny in the genome construction of related species, which evolved from a common ancestral gene by speciation, Cenci et al. (2003) hypothesized that the Pm13 marker linked with powdery resistant gene has a conservative character. On this basis, it can be assumed that species with S-genome chromatin such as tetraploids (Aegilops variabilis Eig.) and hexaploids (Aegilops vavilovi Zhuk.) could carry the genomic region responsible for powdery mildew resistance. What is more, Ae. longissima is considered as a donor of S-genome (Yu and Jahier 1992;Zhang et al. 1992;Badaeva et al. 1998) of Ae. variabilis (U v U v S v S v ). Ae. variabilis has been used as a donor of desirable genes to wheat through interspecific hybridization such as powdery mildew resistance (Spetsov et al. 1997), leaf rust resistance (Marais et al. 2008) and resistance to nematodes (Coriton et al. 2009). The aims of this study were to: (1) evaluate the presence and the expression of Pm13 gene in Ae. variabilis; (2) to identify the individual chromosomes of Ae. variabilis responsible for powdery mildew resistance and (3) transfer them into triticale. The distant crossing between diploid Aegilops species and hexaploid triticale can be disturbed because of (1) different ploidy level of the parental components and (2) the expression of Ph1 gene located on chromosome 5B in wheat (or triticale), responsible for homologues chromosome pairing during meiosis (Riley and Chapman 1958;Lukaszewski and Kopecký 2010). To avoid the unwanted crossing limitations connected with different chromosome number in parental forms and to circumvent the chromosome pairing system controlled by Ph1 gene, we assumed that using amphiploid forms of Ae. variabilis × Secale cereale (U v U v S v S v RR) in the crosses with triticale (AABBRR) will have a significant impact on F 1 hybrid stability because of R-genome chromosomes, which will be able to pair during prophase I of meiosis and will ensure the functional daughter cells formation and sufficient level of vital pollen grains as a consequence. In this purpose, four subsequent generations (F 1 to BC 2 F 2 ) of (Ae. variabilis × S. cereale) × triticale hybrids were obtained. The chromosome composition during metaphase of mitosis in root apical meristems and chromosome pairing during metaphase I (MI) of meiosis of the pollen mother cells (PMCs) were characterized using fluorescence and genomic in situ hybridization (FISH/GISH). Finally, the Pm13 marker (Cenci et al. 1998) was verified in the Ae. variabilis, parental components and in the hybrid plants and compared with visual evaluation of powdery mildew infection. Plant material Glasshouse experiments were carried out in four subsequent vegetation seasons at Institute of Plant Genetics, Polish Academy of Sciences in Poznań, Poland. Seeds of Aegilops umbellulata Zhuk. (PI 222762; 2n=2x=14; U u U u ) and Ae. longissima (PI 604112; 2n=2x=14; S l S l ) were kindly supplied for the study from the National Small Grains Germplasm Research Facility, National Small Grains Collection (Aberdeen, Idaho, USA). Seeds of Ae. variabilis were received from the collection of Professor M. Feldman (The Weizmann Institute of Science, Israel). The Ae. variabilis × S. cereale amphiploids (U v U v S v S v RR, 2n=6x=42) were obtained by Wojciechowska and Pudelska (1999). The F 1 (Ae. variabilis × S. cereale) × triticale hybrids were obtained by crossing of triticale cv. 'Lamberto' with Ae. variabilis × S. cereale amphiploids as a pollinator. Backcrosses with the triticale as a male parent were used to achieve following generations (BC 1 F 1 and BC 2 F 1 ). Finally, the self-pollinations of BC 2 F 1 hybrids were made to gain BC 2 F 2 plants. The percentage ratio of the total amount of seeds from each plant with the total amount of pollinated flowers of each plant was calculated (Table 1). Chromosome preparation Seeds were germinated on moist filter paper in Petri dishes for 3-4 days. For mitosis metaphase accumulation, the root-tips were collected and stored in ice for 26 h. Afterwards, the plants were placed in the vernalisation chamber for 6 weeks and then located in the glasshouse until harvest. The fixation of the root-tips was made using ethanol and acetic acid (3:1, v/v). The chromosome preparations were made according to Hasterok et al. (2006). The F 1 to BC 2 F 2 hybrids were grown in the nursery and their meiotic behaviour was analysed in PMCs at MI of meiosis. Anthers of the hybrids containing PMCs at MI were fixed in 1:3 (v/v) acetic acid/ethanol and stored at −20°C for a maximum of 2 months. MI of meiosis preparations were made according to Zwierzykowski et al. (2008). The anthers were squashed in 45 % acetic acid, and the slides were stored at 4°C until in situ hybridization. Probe labelling Total genomic DNA was extracted from fresh leaves of Ae. umbellulata (UU), Ae. longissima (S l S l ) and triticale 'Lamberto' (AABBRR) using GeneMATRIX Plant & Funghi DNA Purification Kit (EURx Ltd.). Genomic DNA from Ae. umbellulata and Ae. longissima was labelled by nick translation (using NickTranslation Kit, Roche, Mannheim, Germany) with digoxigenin-11-dUTP (Roche) or tetramethyl-5-dUTP-rhodamine (Roche), respectively. Blocking DNA from triticale was sheared to fragments of 5-10 kb by boiling for 30-45 min and used at a ratio of 1:50 (probe:block). The 5S rDNA probe was amplified from the wheat clone pTa794 (Gerlach and Dyer 1980) by polymerase chain reaction (PCR) with tetramethyl-rhodamine-5-dUTP (Roche) using universal M13 'forward' (5′-CAG GGT TTT CCC AGT CAC GA-3′) and 'reverse' (5′-CGG ATA ACA ATT TCA CAC AGG A-3′) sequencing primers. The thermal cycling programme consist of the following: 94°C for 1 min, 39 cycles of 94°C for 40 s, 55°C for 40 s, and 72°C for 90 s, and 72°C for 5 min. The 25S rDNA probe was made by nick translation of a 2.3-kb ClaI sub-clone of the 25-5.8-18S rDNA coding region of Arabidopsis thaliana (Unfried and Gruendler 1990) with digoxigenin-11-dUTP (Roche). It was used for detection of 25-5.8-18S rDNA loci. The pSc119.2 repetitive DNA sequence, kindly supplied from Dr Kubalaková (Laboratory of Molecular Cytogenetics and Cytometry, I n s t i t u t e o f E x p e r i m e n t a l B o t a n y, O l o m o u c , Czech Republic), was amplified and labelled by PCR with digoxigenin-11-dUTP (Roche) by using universal M13 primers (Vrána et al. 2000). The probe pAs1 (Afa family) was amplified by PCR from the genomic DNA of Ae. tauschii and labelled with digoxigenin-11-dUTP (Roche) according to Nagaki et al. (1995). Digoxigenin detection was made using anti-digoxigenin-fluorescein antibody (Roche). In situ hybridization FISH was carried out to study the mitotic chromosomes of root meristems. On the other hand, GISH was used to examine both the mitotic chromosomes of root meristemes and meiotic chromosomes of PMCs. Four probes were subjected to in situ hybridization on the same chromosome preparations. First FISH was made according to Książczyk et al. (2011) with minor modifications of Kwiatek et al. (2013), using 25S (used for detection of 25-5.8-18S rDNA loci) and 5S rDNA (pTa794). The hybridization mixture (40 μl per slide) contained 90 ng of each probe in the presence of salmon sperm DNA, 50 % formamide, 2×SSC, 10 % dextran sulphate, and was denatured at 75°C for 10 min and stored on ice for 10 min. Chromosomal DNA was denatured in the presence of the hybridization mixture at 75°C for 5 min and allowed to hybridize overnight at 37°C. For detection of the hybridization signals, anti-digoxigenin conjugated with FITC (Roche) was used. After documentation of the FISH sites, the slides were washed according to Heslop-Harrison (2000) (2× 45 min in 4×SSC Tween, 2×5 min in 2×SSC, at room temperature). Second FISH with pSc119.2 and pAs1 (labelled with digoxygenin-11-dUTP and tetramethyl-rhodamine-5-dUTP, respectively) was made with the same conditions after reprobing. After second reprobing, GISH was carried out according to Kwiatek et al. (2012) with modifications. Multicolour GISH was carried out using U-genome probe (from Ae. umbellulata), S l -genome probe (from Ae. longissima) and unlabelled triticale genomic DNA which was used as specific blocker. The GISH mixture (40 μL per slide), containing 50 % formamide, 2×SSC, 10 % dextran sulphate, 90 ng each of the genome probes, and 4.5 μg blocking DNA, was denatured at 75°C for 10 min and stored on ice for 10 min. In case of initial GISH on triticale 'Lamberto' chromosomes, the hybridization mix contained the following: A-genome probe generated from genomic DNA of Triticum monococcum L., R-genome probe (rye, S. cereale L.) and blocking DNA from B-genome (Aegilops speltoides Tausch; 2n=2x=14; SS). The chromosomal DNA denaturation, hybridization and immunodetection conditions were the same as above-mentioned. Mitotic and meiotic (MI) cells were examined with an Olympus XM10 CCD camera attached to an Olympus BX 61 automatic epifluorescence microscope. Image processing was carried out using Olympus Cell-F (version 3.1; Olympus Soft Imaging Solutions GmbH: Münster, Germany) imaging software and PaintShop Pro X5 software (version 15.0.0.183; Corel Corporation, Ottawa, Canada). The identification of particular chromosomes were made by comparing the signal pattern of 5S rDNA, 25S rDNA, pSc119.2 and pAs1 probes according previous study ) and similar cytogenetic analysis (Cuadrado and Jouve 1994;Schneider et al. 2003Schneider et al. , 2005Wiśniewska et al. 2013). Single-factor analysis of variance and Tukey's Honest Significant Difference (HSD) test was used to examine the differences of means of chromosome configurations between plants from respective generations and the differences of means of chromosome configurations between plants from BC 2 F 1 with comparison to their progeny in BC 2 F 2 generation. Evaluation of the powdery mildew infection During the vegetation period, the level of powdery mildew natural infection was evaluated according to COBORU (Cultivated Varieties National Research Centre) recommendations on a 9°scale, where 9 is the most favourable state for agriculture (Fig. 1b,c). The means of powdery mildew expression scores in BC 1 F 1 , BC 2 F 1 , BC 2 F 2 hybrids, Ae. variabilis × S. cereale ampiploids and triticale 'Lamberto' were compared each year to the results of PCR amplification of Pm13 marker using ANOVA calculations and Tukey's HSD test. Pm13 marker analysis and powdery mildew reaction in parental forms The amplification products of 517 bp in size were found in DNA extracts of Ae. longissima (PI 604112), Ae. variabilis and 20 plants of Ae. variabilis × S. cereale, which were used in further crosses with triticale. The bands of all samples gave clear and strong fluorescence after separation (Fig. 1a). The marker for Pm 13 (517 bp) was not identified in rye 'Strzekęcińskie' (used for production of Ae. variabilis × S. cereale ampihiploids, Wojciechowska and Pudelska 1999) and triticale 'Lamberto.' The powdery mildew expression mean scores in Ae. variabilis were made in three subsequent years of experiments and ranged between 8.05 and 8.25 (Table 3). The observations of the infection symptoms conducted on triticale 'Lamberto' showed much lower tolerance to powdery mildew. The mean scores of infection ranged between 2.85 and 2.95 (Table 3). Identification of particular mitotic chromosomes of parental forms The chromosome composition of Ae. variabilis (U v U v S v S v ) and triticale 'Lamberto' (AABBRR), used as parental forms in presented distant crossing were studied (Fig. 2). The analysis were made using 5S rDNA, 25S rDNA (Fig. 2a, d), pSc119.2 and pAs1 probes (Fig. 2b, e) and multicolour GISH with total genomic DNA used as a probe (Fig. 2c, f). Identification of particular chromosomes of A-and B-genome, R-genome, U ugenome and S l -genome was made basing on previous reports of Cuadrado and Jouve (2002), Schneider et al. (2003Schneider et al. ( , 2005 and Badaeva et al. 1996a, b and, respectively and chromosome arms ratio. The rDNA-FISH experiment on chromosomes of triticale 'Lamberto' (2n=6x=42 chromosomes, AABBRR) resulted in 12 signals of 5S rDNA (on chromosomes 1A, 5A, 1B, 5B, 1R and 5R) and 6 signals of 25S rDNA (on chromosomes: 1B, 6B and 1R; Fig. 2a). By contrast, rDNA-FISH on Ae. variabilis (U v U v S v S v ) chromosomes showed 8 signals of 5S rDNA in 1U v , 5U v , 1S v and 5S v chromosomes and 8 signals of 25S rDNA in 1U v , 5U v , 5S v (weak) and 6S v (weak) chromosomes (Fig. 2d). The same locations of rDNA signals appeared on chromosomes of Ae. variabilis × S. cereale amphiploid. The repetitive sequence FISH (seq-FISH) with pSc 119.2 and pAs1 probes resulted in specific patterns on chromosomes of triticale 'Lamberto' and Ae. variabilis. The chromosomes of A-genome of triticale carried only pAs1 signals, mainly on the distant and pericentromeric regions (Fig. 2b). The most distinguishable chromosome was 7A with strong pAs1 signal on the short arm. The pSc 119.2 and pAs1 signal locations on chromosomes of B-genome of triticale were more diversified and appeared also in interstitial regions. R-genome chromosomes of triticale had strong pSc119.2 sites and weak, dispersed pAs1 signals. The locations of pSc119.2 sites on 2R and 3R chromosomes were similar, but the difference of chromosome arms length allowed to distinguish those two. The chromosomes U v -genome of Ae. variabilis (Figs. 2e and 3) carried both the pSc119.2 sites and the pAs1 sites. The strongest pSc119.2 signal was observed in the telomeric region of 3U v chromosome. The pAs1 sites were located both on distal and interstitial chromosomes. The most characteristic pattern was observed on 6U v chromosome. The pSc119.2 and pAs1 probes hybridized also with S v -genome chromosomes (Fig. 3). The pSc119.2 sites were located on the telomeric regions of chromosomes with an exception of long arm of 5S v . The strongest signals were observed on the long arms of 3S v and 7S v chromosomes. The pAs1 sites were mostly dispersed. Distal regions of chromosome 4S v and short arm of chromosome 7S v carried the most visible signals of pAs1. Evaluation of crossing efficiency 106 flowers of triticale 'Lamberto' were pollinated by the pollen of Ae. variabilis × S. cereale forms (Table 1). 19 F 1 seeds were obtained, that indicates 18 % of crossing efficiency (CE). Six F 1 plants were germinated and evaluated using GISH analysis. Backcrossing of 68 flowers of F 1 hybrids with the triticale 'Lamberto' pollen resulted in obtaining of 17 seeds of BC 1 F 1 hybrid generation (CE=25 %). Five BC 1 F 1 plants were chosen on the basis of molecular marker (Pm13) test and cytogenetic analysis of mitotic chromosomes of root meristems for further crossing with triticale. After crossing of 330 flowers with triticale pollen, 25 seeds of BC 2 F 1 generation were obtained. Thereafter, 15 plants were chosen for further hybridizations. 329 flowers of BC 2 F 1 hybrids were selfpollinated, that resulted in 50 seeds of BC 2 F 2 generation. Evaluation of introgression of Ae. variabilis chromatin in triticale hybrids The correct establishing of the introgression of Ae. variabilis chromatin carrying the resistance to powdery mildew was assured by combining the GISH and FISH methods with molecular marker (Pm13) analysis and the results of infection scoring. The chromosome constitution of six F 1 (Ae. variabilis × S. cereale) × triticale hybrids consist of 28 chromosomes of triticale (14 chromosomes of A-and B-genomes and 14 Rgenome chromosomes), seven U v -genome chromosomes and seven S v -genome chromosomes, which were detected by probing with U u -and S l -genomic DNA and blocking with total DNA of triticale (AABBRR) (Table 2, Fig. 4a). FISH experiment with 4 kinds of probes allowed to distinguish chromosomes from each group (group-1 to group-7). Afterwards, five of 17 plants of the BC 1 F 1 generation carried Pm13 marker, which was correlated with the infection scores that ranged from 6 to 8, whereas the another 12 plants were more infected, which was comparable with the infection level of triticale 'Lamberto' (Table 3). In those 5 hybrids (with Pm13 marker) the total number of chromosomes varied from Table 2). The number of U v -genome chromosomes was between 2 and 5, the number of S v chromosomes was 3-4, the number of R-genome chromosomes was 14 in each plant and the A and B-genome chromosomes number varied from 18 to 21 (Fig. 4b). The 12 other plants, without Pm13 marker, had large number of intergeneric translocations. The GISH analysis showed the chromosomes of A-and B-genome with the translocations of S-genome chromosome segments (Fig. 4c). Selected five BC 1 F 1 hybrids (with Pm13 marker) were backcrossed with triticale pollen. The molecular analysis showed that the 3 of 5 BC 1 F 1 plants reproduced 15 descendants (BC 2 F 1 ) with the Pm13 marker (Table 1). The infection scores of those group of hybrids were significantly different in comparison with hybrids without Pm13 marker and triticale 'Lamberto.' The U v -genome chromosomes were not identified in all of 15 plants of BC 2 F 1 generation, but 1 to 3 chromosomes of S vgenome appeared in those plants (Fig. 4d). FISH analysis showed that 3 plants carried 41 chromosomes with one chromosome 3S v and the lack of 3B chromosome pair. Another 4 plants possessed additional chromosome 3S v . The 6 other plants carried substitution pair of 3S v /3B chromosomes. Moreover, one of BC 2 F 1 hybrids had a substitution pair of 3S v /3B chromosomes and one additional chromosome 2S v . The other singular plant carried: a substitution pair of 3S v / 3B chromosomes, an one additional 2S v chromosome and one chromosome 2B (Table 2). In the BC 2 F 2 generation the S v -genome chromosomes were eliminated in 24 plants, however in 26 hybrids 1-2 chromosomes of S v -genome were identified and the range of triticale chromosomes was the same as in the previous generation. FISH experiments allowed to distinguish 9 plants with one, additional chromosome 3S v , 10 plants with a substitution pair of 3S v /3B chromosomes and 7 plants with an additional pair of 3S v chromosomes. Pm13 marker was identified only in plants with introgression of Aegilops chromatin, which was correlated with the powdery mildew infection scores (Table 3). Chromosome pairing behaviour in BC 2 F 1 and BC 2 F 2 of (Ae. variabilis × S. cereale) × triticale hybrids The multicolour GISH allowed to distinguish the S v -genome chromosomes (green) and the triticale chromosomes ( Fig. 5ad). Chromosome configuration means at MI of meiosis in PMCs were examined in selected hybrid plants of BC 2 F 1 with total number of chromosomes amounting 42, that carried a substitution pair of 3S v /3B chromosomes (Table 4) and in BC 2 F 2 hybrids divided in two groups. First group consisted of plants with 42 chromosomes, having a substitution pair of 3S v /3B chromosomes (Table 5), while second group associated the plants with 43 chromosomes having an additional 3S v chromosome ( Table 6). The variance analysis of the chromosome configurations in BC 2 F 1 plants with 42 chromosomes, that carried a substitution pair of 3S v /3B chromosomes showed that the differences between the means of chromosome configurations were not significant (Table 4). The mean of total number of bivalents were 18.02. Bivalents ranged from 9 to 20 per cell. The mean of rod bivalents was nearly two times higher than the mean of ring xx″-number of pairs of triticale chromosomes, 1″xy-one pair of y-genome chromosomes of group-x; 1′xy-a singular group-x chromosome of ygenome; 1″xy/xz-substitution pair of chromosomes. The nomenclature and abbreviation of the genetic stocks of hybrids were described according Raupp et al. 1995 (http://wheat.pw.usda.gov/ggpages/nomenclature.html) bivalents (12.22; 5.80; respectively). Similarly, the mean of rod bivalents of A-, B-and R-genome was considerably higher than ring bivalents of those genomes. Considering the S v -genome bivalents, the mean number of S v /S v rod bivalents and S v /S v ring bivalents was almost equal (0.30 and 0.38, respectively). The mean of S v -genome univalents was 0.72 and the number of univalents ranged between 0 and 2. The mean chromosome configuration for five analysed plants (2n=42 chromosomes) with a substitution pair of 3S v /3B chromosomes was 5.96 I+18.02 II (12.22 rod+5.80 ring). The ANOVA test for BC 2 F 2 hybrids with the same chromosome constitution (20′″+3S v ′) obtained from different BC 1 F 1 plants, carrying a substitution pair of 3S v /3B chromosomes showed that the differences between means of the chromosome configurations of particular hybrids were not statistically significant. The mean (and the range) of bivalents per PMC was 18.7 (9-20) and was similar to the results in BC 2 F 1 hybrids. The same situation appeared considering the means of rod bivalents, ring bivalents and univalents, where mean chromosome configuration for five analysed BC 2 F 2 plants (2n=42 chromosomes) with a substitution pair of 3S v /3B chromosomes was 4.60 I+18.70 II (12.56 rod+6.14 ring). The mean of rod and ring S v -genome bivalents was approximate (0.22 and 0.46; respectively). The comparison of ANOVA results of chromosome configuration between BC 2 F 1 and respective BC 2 F 2 progeny hybrids shows that the differences in means are not significant. Considering the S vgenome univalents, the mean in BC 2 F 2 plants (Table 5) was lower than in BC 2 F 1 plants ( Table 4). Five of six hybrids of BC 2 F 1 (42 chromosomes each), which carried a substitution pair of 3S v /3B chromosomes were evaluated ( Table 2). All of them were the progeny of the most fertile hybrid line no. 4 (Table 1). Chromosome configuration means at MI of meiosis in PMCs were also examined in four BC 2 F 2 hybrid plants (2n=43 chromosomes) carrying additional chromosome 3S v . Fig. 4 Genomic in situ hybridization (GISH) on mitotic chromosomes of (Ae. variabilis × S. cereale) × triticale 'Lamberto' hybrids. On the GISH images, the R-genome is visualized in blue, the A-genome and the B-genome in grey; the U v -genome is visualized in red and the S vgenome in green. a F 1 hybrid with 14 chromosomes of Ae. variabilis (7 chromosomes of U v -genome and 7 chromosomes of S vgenome). b BC 1 F 1 hybrid with 7 chromosomes of Ae. variabilis (4 chromosomes of U v -genome and 3 chromosomes of S v -genome). c BC 1 F 1 hybrid with 2 chromosomes from U v -genome of Ae. variabilis and 21 chromosomes of triticale with introgression of S v -genome chromatin. d BC 2 F 1 hybrid with 3 chromosomes from of S v -genome of Ae. variabilis. Scale bars: 10 μm The mean chromosome configuration for this group was 4.65 I+19.18 II (9.9 rod+9.28 ring). The ANOVA and Tukey's HSD test showed that the differences of chromosomes configuration means between plants with the same chromosome constitution (21′″+3S v ′) obtained from different BC 2 F 1 plants (4/6 and 4/10) were significant. The differences affected the means of A-genome, B-genome and R-genome rod and ring bivalents and also means of univalents of A-and B-genome (Table 6). Discussion Considering the growing tendency in brakeage of triticale resistance to fungal diseases, especially powdery mildew, and from the other hand, the narrow genetic diversity of triticale could lead to the conclusion that it is necessary to utilize the wild Triticeae relatives to enrich the genetic pool of cultivated triticale. The gene order in Poaceae species is generally conserved (Chantret et al. 2008) and the synteny facilitates comparative genomics analyses in grass families (Abrouk et al. 2010). Therefore, it could be expected that the region of chromosome 3S l of A. longissima that is responsible for powdery mildew resistance could be collinear with the same region in the chromosome 3S v of Ae. variabilis (2n = 4x = 28, U v U v S v S v ). Nonetheless, there are discrepant reports concerning the powdery mildew resistance of Ae. variabilis. From the one side, Spetsov and Iliev (1991) obtained a disomic addition line (2n=44) by crossing wheat cv. 'Roussallka' with Ae. variabilis, that manifested a high powdery mildew resistance in seedling and in adult plant stage. From the other side, Cenci et al. (2003) reported that disomic line of wheat cv. 'Chinese Spring' 3S v (K-2) and the derived ditelosomic 3S v S (K-2/SvS) addition lines from Ae. variabilis (Yang et al. 1996) were susceptible, with strong powdery mildew symptoms and abundant sporulation. However, the assumption of a possible synteny between the S-genome chromosomes became meaningful, considering the verification of available powdery mildew STS markers made by Stępień et al. (2001), which showed that Pm13 marker was present in Ae. speltoides (accessions 2056, 2067, d10, d42, d50) that also carry S-genome chromosomes. In presented study, the Ae. variabilis and the Ae. variabilis × S. cereale amphiploids carrying Pm13 marker manifested a low powdery mildew reaction, confirmed by infection scores made on 20 plants each year of the experiment ( Fig. 1c; Table 3). In comparison, triticale 'Lamberto' was much more infected, which was confirmed by Tukey's HSD test (Fig. 1c; Table 3). Moreover, 1402 Polish isolates of B. graminis are reported to be 100 % virulent to triticale 'Lamberto' in three subsequent years of experiment (2008)(2009)(2010) carried out by Czembor et al. (2014). Furthermore, the molecular analysis showed the Pm13 marker was not present in triticale 'Lamberto' (Table 3). The Pm13 marker is located (Cenci et al. 2003). In purpose to identify the particular chromosomes of Ae. variabilis, the FISH experiment with repetitive sequences as probes was carried out. The location of 25S rDNA and 5S rDNA signals in U-and S-genome chromosomes of Ae. variabilis were similar like in the ancestor species, considering chromosomes 1U u 5U u and 5S l and 6S l of Ae. umbellulata and Ae. longissima, respectively (Badaeva et al. 1996b). However, the 25S rDNA signals on 1S l , 3S l and 6U u chromosomes were not present on the homologue chromosomes of Ae. variabilis. There were also some differences in pSc119.2 signals pattern between diploid ancestors (Badaeva et al. 1996a) and Ae. variabilis (Fig. 3). There were no signals in the telomeric regions of long arms of 2U v , 3U v , 5U v and 6U v chromosomes. When comparing pAs1 signals on the U-genome chromosomes, small, dispersed signals were observed on 1U v , 3U v and 5U v chromosomes. Moreover, Badaeva et al. (1996a) did not observed the pAs1 signals on S-genome chromosomes of Ae. longissima, however chromosomes of Ae. variabilis carried weak, scattered landmarks on both arm of each chromosome and strong site on distal region of long arm of 7S v chromosome. The cytogenetic analysis of triticale 'Lamberto' chromosomes revealed also some novel data. The elimination of 25-5.8-18S rDNA was observed in 1A chromosome of triticale, comparing to 1A of wheat. The rDNA aberrations are probably connected with the changes in ploidy level, which commonly appear in hybrids (Shcherban et al. 2008). Knowing the cytogenetic markers distribution on the chromosomes of parental forms (Ae. variabilis × S. cereale amphiploids and triticale 'Lamberto'), and the results of Pm13 molecular marker analysis connected with the evaluation of natural infection by B. graminis, the study of hybrid generations of (Ae. variabilis × S. cereale) × triticale 'Lamberto' were made. As expected, the F 1 hybrids (2n=6x=42, U v S v ABRR) carried 7 chromosomes of U v -, S v -, A-and B-genome and complete set of 14 chromosomes of R-genome. The chromosome composition of F 1 hybrids was anticipated on the basis of related studies, i.e. in the study of Aegilops biuncialis (2n= 4x=28, UUMM) × wheat (2n=6x=42, AABBDD) hybridizations (Schneider et al. 2005), the chromosome set of F 1 hybrids were parallel (ABDUM, 2n=5x=35), with only one difference, that in case of (Ae. variabilis × S. cereale) × triticale hybridizations, R-genome chromosomes can pair and behave Mean 9.9 (7-15) 6.38 (4-9) 3.53 (1-6) 0 9.28 (3-14) 6.13 (2-10) n/a n/a n/a n/s n/a n/a 4/10/3 vs 4/10/4 n/s n/s n/s n/a n/s n/s n/s n/a n/a n/a n/s n/a n/a in diploid manner. The crossing of F 1 hybrids with triticale pollen had an influence on reduction of the Aegilops chromosomes in one group of BC 1 F 1 plants and appearing of S v /AB translocations in the latter group of BC 1 F 1 plants. Marker analysis showed that plants with Aegilops chromosomes carried also Pm13 marker. Moreover, those plants were much more tolerant for B. graminis infection (Table 3). The further backcrossing of selected BC 1 F 1 hybrids with triticale pollen resulted in elimination of Aegilops chromosomes. There was lack of Aegilops chromatin in 9 BC 2 F 1 plants. On the other hand, FISH/GISH analysis allowed to distinguish chromosome(s) 3S v in each of 15 BC 2 F 1 plants and in addition, one chromosome 2 v in 2 plants, where also Pm13 marker was identified. Moreover, the intensity of the level of powdery mildew infection on those plants was lower, when comparing with triticale 'Lamberto' and hybrids without Pm13 marker. Two subsequent backcrosses resulted in the elimination of unneeded Aegilops chromosomes and allow to select the plants with the S-genome chromosomes carrying the resistance. Therefore, the self-fertilization of BC 2 F 1 was carried out to maintain the S-genome chromosome in BC 2 F 2 hybrids. 26 of 50 hybrids had singular or a pair of 3S v chromosomes, that carried Pm13 marker and were more tolerant for B. graminis infection. It cannot be omitted, that the HSD test of the means of infection scores of hybrids with Pm13 marker compared with the mean of infection scores of amphiploids (Ae. variabilis × S. cereale) shows the significant differences (Table 3), that points the tolerance for powdery mildew is a little bit lower in hybrids than in amphiploids, however is much higher than in triticale 'Lamberto' and hybrids without Pm13 marker. It can be supposed that triticale 'Lamberto' carry a virulence factors, that have an influence on Pm13 gene expression. Notwithstanding, the tolerance for powdery mildew was markedly improved in hybrids with Pm13 marker. Afterwards, the genomic in situ hybridization was employed to study the 3S v chromosome(s) behaviour in PMC's of selected BC 2 F 1 and BC 2 F 2 hybrids of (Ae. variabilis × S. cereale) × triticale 'Lamberto.' There were no intergenomic chromosome configurations observed in the plant carrying 2S v and/or 3S v chromosomes, which is opposite to other published studies concerning intergenomic hybridizations between cultivated cereals and Aegilops species. For example, Molnár and Molnár-Láng (2010) reported the intergenomic rod and ring bivalents and trivalent between 2 M, 3 M, 3U and 7 M chromosomes of Ae. biuncialis and wheat (Chinese Spring ph1b) chromosomes. It is assumed, that triticale has the same controlling system of homologue chromosome pairing as wheat, that hampers the pairing of the chromosomes from different genomes. In wheat, homoeologous chromosome pairing and consequent recombination is suppressed by the function of the Ph1 locus, localized on the long arm of chromosome 5B (Riley and Chapman 1958). The Chinese Spring ph1b (CSph1b) mutant genotype (Sears 1977), which lacks the Ph1 locus, has been successfully used for the introgression of alien genetic material into the wheat genome by the induction of homoeologous pairing (Lukaszewski 2000). From this reason the intergenomic bivalent and trivalent appeared in Molnár and Molnár-Láng (2010) study. Considering presented study, FISH experiments showed that the pair of chromosomes 5B was present in all hybrids of each generation and probably is responsible for diploid-like pairing of chromosomes during meiosis, which was confirmed by ANOVA tests (Tables 4, 5 and 6) that demonstrated no differences in means of chromosome configurations between hybrid plants. However, Tukey's HSD test showed the differences in means of bivalent configurations between BC 2 F 2 progeny obtained from 4/6 plant compared with the progeny of 4/10 hybrid (Table 6). It can be supposed that S-genome chromatin has no influence on chromosome pairing of triticale chromosomes. In other words, the way of triticale chromosomes behaviour during first metaphase of meiosis of PMCs seems to be individual regarding to parental form. Furthermore, the way of 3S v chromosome pairing and transmission to next generation is independent, diploid-like. In conclusion, our study showed that molecular cytogenetics and marker-assisted selection combined with evaluation of powdery mildew infection constitute a useful tool for the resistance breeding. Using these methods we have obtained 26 plants carrying 3S v chromosome(s) with the powdery mildew resistance, which can be used in the triticale breeding programmes. On the other hand, these genetic stocks could be used for sequencing the specific region of 3S v chromosome, responsible for powdery mildew tolerance and for comparative studies with the Pm13 gene sequence originated from Ae. longissima.

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High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses Background The aim of this study was to employ high-throughput DNA sequencing to assess the incidence of bacteria with biogenic amine (BA; histamine and tyramine) producing potential from among 10 different cheeses varieties. To facilitate this, a diagnostic approach using degenerate PCR primer pairs that were previously designed to amplify segments of the histidine (hdc) and tyrosine (tdc) decarboxylase gene clusters were employed. In contrast to previous studies in which the decarboxylase genes of specific isolates were studied, in this instance amplifications were performed using total metagenomic DNA extracts. Results Amplicons were initially cloned to facilitate Sanger sequencing of individual gene fragments to ensure that a variety of hdc and tdc genes were present. Once this was established, high throughput DNA sequencing of these amplicons was performed to provide a more in-depth analysis of the histamine- and tyramine-producing bacteria present in the cheeses. High-throughput sequencing resulted in generation of a total of 1,563,764 sequencing reads and revealed that Lactobacillus curvatus, Enterococcus faecium and E. faecalis were the dominant species with tyramine producing potential, while Lb. buchneri was found to be the dominant species harbouring histaminogenic potential. Commonly used cheese starter bacteria, including Streptococcus thermophilus and Lb. delbreueckii, were also identified as having biogenic amine producing potential in the cheese studied. Molecular analysis of bacterial communities was then further complemented with HPLC quantification of histamine and tyramine in the sampled cheeses. Conclusions In this study, high-throughput DNA sequencing successfully identified populations capable of amine production in a variety of cheeses. This approach also gave an insight into the broader hdc and tdc complement within the various cheeses. This approach can be used to detect amine producing communities not only in food matrices but also in the production environment itself. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0596-0) contains supplementary material, which is available to authorized users. Background High-throughput sequencing (HTS) has significantly enhanced our ability to profile complex microbial ecosystems such as those in the sea [1], soil [2], gut [3] and various foods including cheese [4][5][6][7]. While most of these studies rely on amplifying regions of the bacterial 16S rRNA or fungal ITS genes to study the microbial composition of these communities, it is also possible to use HTS to sequence select non-16S based genes [8]. With reference to this, HTS-based methods are currently being explored to improve food safety by targeting specific undesirable populations/genes [9,10], and the potential exists to target genes involved in biogenic amine (BA) formation. BAs are low molecular weight organic bases with biological activity produced, primarily, by decarboxylation of precursor amino acids. BAs are classified according to their chemical structures and can be aromatic (tyramine), heterocyclic (histamine and tryptamine) or aliphatic (putrescine and cadaverine) [11][12][13][14]. In eukaryotes BAs are generally associated with a variety of biological processes including blood pressure regulation, neurotransmission, cellular growth and allergic responses. In prokaryotes, however, BA formation is generally linked with cell survival, particularly in low pH conditions where it serves as a stress response mechanism. Up-regulation of decarboxylase gene expression has previously been shown to occur in the presence of the precursor amino acid and in low pH environments, such as those encountered in fermented foods. The amino acid/amine transporter system also acts to generate energy in the form of proton motive force, thus providing a further competitive advantage under such stress conditions [15,16]. Microbial BA formation is encountered in a variety of fermented foods and beverages including cheese, fish, beer, wine, meat products and fermented vegetables [17]. The most commonly occurring BAs detected in foods include histamine, tyramine, putrescine and cadaverine [18]. The accumulation of histamine and/or tyramine at high levels may produce toxicological effects including hypertension, headaches, palpitations and vomiting in certain individuals, particularly those with reduced mono/di-amine oxidase activity, due to either genetic or pharmacological reasons. The European Food Safety Authority regard histamine and tyramine as the most important BAs from a toxicological viewpoint [19]. Additionally, the presence of di-amines, such as putrescine and cadaverine, can further promote toxicological effects as they act as potentiators of histamine and tyramine toxicity by competing for detoxifying enzymes [20][21][22][23][24]. As the detrimental effects associated with consumption of BAs varies depending on the amine in question and the susceptibility of the individual, it is particularly difficult to set defined limits for BAs in food products [25]. Consequently, regulatory limits describing BA concentrations have yet to be established for the cheese industry. Notably, ripened cheeses are second only to fish as the most commonly implicated source of dietary BAs [19,26,27], which has led to the coining of the term the "cheese reaction" [28]. BAs can be formed by a variety of cheese associated lactic acid bacteria (LAB) including Lactobacillus, Lactococcus, Streptococcus, Leuconostoc and Enterococcus [15,17,18,23]. Several factors are associated with the accumulation of BAs in cheese including low pH, milk processing parameters (raw/pasteurised), the presence of amine forming species (starter or non-starter/contaminating bacteria), availability of precursor amino acids, ripening temperature/time and salt content, among other factors [29]. While the majority of cheese is produced from pasteurised milk, raw milk cheeses are also popular due to their unique flavour characterisitics [27]. High levels of secondary proteolysis as a result of starter and non-starter bacterial action, together with higher microbial load and, in many cases, long ripening times make raw milk cheeses particularly susceptible to BA formation [13,14,27,28,30,31]. The presence of BAs can also be used as an indicator of overall product hygiene in the form of biogenic amine indices [19]. Methods employed to detect BAs in dairy products have been extensively reviewed [15,20,29,32,33]. Essentially, detection is either direct, i.e., detection of the respective amines or indirect, i.e., based on identifying amine forming bacteria. Amine detection methods rely primarily on chromatographic techniques such as thin layer and high performance liquid chromatography (HPLC) [29]. While initial approaches for identifying responsible bacteria were based on differential chromogenic agars and enzymatic methods, more recently, molecular based methods such as DNA hybridisation, polymerase chain reaction (PCR) and quantitative (q)PCR have been used [20,32,34]. PCR based approaches are of particular use for establishing the aminogenic potential of various isolates from food products. In this instance, strains associated with raw materials, production equipment and, in the case of cheese, starter bacteria can be pre-emptively screened for decarboxylase biomarkers leading to a potential reduction of amines in the final product. A review published by Landete et al [20] describes several sets of PCR primers for detecting producers of the major food-associated amines [20]. In this study a range of raw milk, speciality cheeses were screened for the presence of histidine decarboxylase (hdc) and tyrosine decarboxylase (tdc) genes associated with the production of histamine and tyramine, respectively. Previously optimised PCR primer pairs amplifying regions of the Gram-positive hdc and tdc gene clusters were employed and the resultant amplicons were cloned and subjected to Sanger sequencing in order to establish that that there was sufficient heterogeneity among the decarboxylases present to merit a more detailed HTS analysis. HTS revealed the dominant and sub-dominant species with tyramine and histamine producing potential, in these raw milk cheeses. More importantly, the value of employing HTS to survey decarboxylase genes within a microbial population is established. Sample collection Ten speciality cheeses were purchased from a local market. Raw milk cheeses with long ripening times (3 -24 months) were selected and divided into 2 groups (hard and semi-hard). Cheeses originated from several European countries including two Irish artisanal cheeses (A and B), Reblochon, Manchego, Morbier, Tête de Moine, Pecorino Sardo, Ossau-Iraty, Comté and Gorgonzola. Cheeses were vacuum packed and stored at 4°C for 3 days prior to DNA extraction. Table 1 provides a description of the cheeses selected for this study. These particular cheeses were selected due to their potential to accumulate BAs and are not reflective of all cheese within the respective categories. Determination of BA content of cheese BAs were acid extracted, derivatised and quantified, in duplicate, using the method described by Özoğul [35] with modifications for a cheese matrix. Five grams of cheese was weighed into a sterile bag containing 20 ml 0.013 N H 2 SO 4 . The suspension was homogenised in a stomacher (Iul Instruments, Barcelona, Spain) for 10 min. The liquid phase was transferred to a sterile 50 ml tube while the remaining cheese homogenate was subjected to a second acid extraction with 20 ml 0.013 N H 2 SO 4 . The liquid phases were pooled and centrifuged at 5000 g, 4°C for 15 min. After centrifugation, the solution was brought to a final volume of 50 ml with 0.013 N H 2 SO 4 . A 10 ml aliquot was filtered using 0.2 μm cellulose acetate filters (Chromacol, Welwyn Garden, Herts, UK). Extracted BAs were then derivatised by mixing 1 ml of each respective extract with 1 ml 2 N NaOH and 1 ml 2 % benzoyl chloride (Sigma-Aldrich, Wicklow, Ireland) in glass test tubes. The mixture was vortexed and allowed to stand for 15 min prior to the addition of 2 ml saturated NaCl. Two ml of diethyl ether was then added. A plastic pipette was used to transfer the top layer of the extract to a second glass test tube with a further 2 ml diethyl ether added and the resultant top layers pooled. Diethyl ether was evaporated off using a stream of nitrogen at 45°C for 20 min. The BA residue was dissolved by adding 1 ml acetonitrile. BAs were separated using a Luna C18 RF 5 μm, 100 Å column 250 x 4.6 mm (Phenomenex Queens Avenue, Macclesfield, UK) and were eluted at an initial flow rate of 1.6 ml/min for 30 min with Acetonitrile (A) and H 2 O (B), using the following gradients: BAs were quantified using 5 data points on calibration curves against standard solutions of histamine (100-2000 μg/ml), tyramine (5-100 μg/ml), putrescine and cadaverine (Sigma-Aldrich, Dublin, Ireland) (Additional file 1: Table S1). Data was presented as mg of individual BA per kg of cheese. Determination of cheese pH, salt and moisture contents Grated samples of each cheese were analysed for salt content [36], moisture [37] and pH [38] using previously described methods. DNA extraction from selected cheeses Five grams of each cheese was homogenised in 45 ml of a 2 % tri-sodium citrate buffer (VWR, Dublin, Ireland). Cheese homogenate was then subjected to enzymatic PCR detection of hdc and tdc gene fragments using selected primer sets PCR based detection of decarboxylase genes was achieved using primers specific for regions of the Gram-positive and Gram-negative hdc operon, respectively, as well as for the tdc operon. Primers for the hdc operon of Grampositive bacteria comprised of a forward (HDC3 5'-GAT GGTATTGTTTCKTATGA-3') and a reverse primer (HD C4 5' CAAACACCAGCATCTTC-3') targeting a 435 bp fragment of the hdcA gene [18]. Primers targeting the Gram-negative hdc operon comprised of a forward (HIS2-F 5'-AAYTSNTTYGAYTTYGARAARGARGT-3') and a reverse primer (HIS2-R 5'-TANGGNSANCCDATCATYT TRTGNCC-3'), and generated a 531 bp product [40]. The tdc primers, comprised of a forward (TD5 '5-CAAATGG AAGAAGAAGTAGG-3') and a reverse primer (TD2 '5-ACATAGTCAACCATRTTGAA-3'), amplified an 1100 bp fragment of the tdc gene as described by Coton et al [24]. PCR reactions were carried out in triplicate and contained 25 μl BioMix Red Master Mix (Bioline, London, UK), 1 μl of each primer (200 nmol l -1 ), 5 μl DNA template (standardised to 100 ng DNA/reaction) and nuclease free water to a final volume of 50 μl. PCR amplification was carried out using a G-Storm Thermal Cycler (Gene Technologies, Oxfordshire, UK). Amplification consisted of an initial denaturation at 95°C for 10 min followed by 40 cycles of; denaturation at 95°C for 45 s, annealing at 48°C for 1 min and extension at 72°C for 90 s. This was followed by a final elongation step at 72°C for 7 min. PCR amplicons were pooled and cleaned using the AMPure XP magnetic bead-based purification system (Beckman Coulter, Takeley, UK). Cloning of PCR amplicons Cleaned PCR amplicons were subjected to TOPO cloning reactions using the TOPO TA cloning kit (Invitrogen, CA, USA Bioinformatic analysis Following Sanger sequencing, hdc reads were analysed using the NCBI nucleotide database (BlastN; http://blast.ncbi.nlm.nih.gov/). Sanger sequencing of the tdc amplicons did not provide forward and reverse reads of the complete 1100 bp, therefore, only the overlap (approximately 800 bp), aligned using the MegAlign programme was analysed using the BlastN database. Raw Ion PGM reads were quality filtered with the fas-tq_filter script in USEARCH. For both tdc and hdc amplicons, a length cut-off of 170 bp was used. Reads were then clustered into operational taxonomical units (OTUs) and chimeras removed with the 64-bit version of USEARCH [41]. Subsequently OTUs were aligned with MUSCLE [42] and a phylogenetic tree generated within Qiime [43]. Alpha diversity analysis was also implemented within Qiime. For taxonomic assignment OTUs were blasted against the NCBI-Nr database and parsed through MEGAN [44]. Results This study used previously published PCR primers, designed based on alignments of conserved regions of decarboxylase gene clusters from known BA producing isolates [20]. In order to be sure that the variety of decarboxylase genes within the selected cheeses was sufficiently heterogeneous to merit culture-independent HTS analysis, an initial Sanger sequencing-based investigation of cloned PCR amplicons was undertaken. This was then followed by HTS to profile the dominant and subdominant histamine and tyramine producing populations present in the respective cheeses. Sanger sequencing reveals the identity of bacteria with histaminogenic potential The selected hdc primers targeted a 435 bp fragment of the Gram-positive hdcA gene. Six of the 10 cheeses sampled generated PCR amplicons corresponding to the hdc operon (Reblochon, Irish artisanal cheese B, Morbier, Tête de Moine, Pecorino Sardo, Ossau-Iraty). No amplicons were generated, across all cheese varieties, when using the selected Gram-negative hdc primers [20]. The Gram-positive hdc amplicons were cloned via the TOPO TA cloning method and a subset of 46 clones were subjected to Sanger sequencing. Table 2 contains a summary of BLAST output for each cheese sample while Additional file 1: Table S2 contains a complete BLAST analysis of each respective cheese including scores generated, query cover and accession numbers. BLAST output indicated that 35 of the 46 clones sequenced (76.1 %) contained a hdc fragment corresponding to the Lactobacillus buchneri hdc operon. Other hdc sequences identified corresponded to the hdc operon that is conserved across Lactobacillus sakei/Tetragenococcus halophilus/T. muriaticus/Oenococcus oeni/Lactobacillus hilgardii hdc operon (hereafter referred to as the Lb. sakei group of hdc operon; 23.4 %). In the Reblochon and Tête de Moine cheeses, all of the sequenced hdc clones (8 and 8, respectively) corresponded to the Lb. buchneri hdc operon. In the Ossau-Iraty cheese all of the hdc positive clones were identified as corresponding to the hdc operon of the Lb. sakei group. The hdc genes from Lb. buchneri and the Lb. sakei group were identified from among the Irish artisanal cheese B, Morbier and Pecorino Sardo cheeses while clones corresponding to the Lb. sakei group hdc operon were identified from among the Ossau-Iraty cheese. Sanger sequencing reveals the identity of bacteria with tyraminogenic potential PCR amplification, using primers designed based on alignments of tyrosine decarboxylases from known producers [20], detected the presence of an 1100 bp fragment of the tdc gene in 6 of the 10 cheeses tested (Irish artisanal cheese A, Reblochon, Irish artisanal cheese B, Tête de Moine, Pecorino Sardo, Ossau-Iraty). Table 3 depicts a summary of the BLAST output for each positive cheese samples while Additional file 1: Table S3 contains a complete BLAST analysis of samples including top hits, scores generated, query cover and accession numbers. Resultant amplicons were cloned and subjected to Sanger sequencing. In this instance, a subset of 44 clones was sequenced across the six positive cheese types. BLAST analysis revealed the presence of tdc fragments corresponding to several species, including Enterococcus faecalis which also identified across the 6 cheese types. With respect to the Pecorino Sardo cheese, all clones contained tdc genes corresponding to that of and E. faecium. In contrast, tdc genes corresponding to those of enterococci, streptococci and lactobacilli were detected across all other cheese varieties. α-diversity of artisanal cheese microbiota with BA-producing potential as revealed by next generation DNA sequencing Sanger sequencing established that several cheese samples contained multiple microbial sources of decarboxylase genes. As a result it was apparent that the use of a culture-independent HTS-based approach to provide an in-depth insight into the diversity of the populations present was justified. The previously generated PCR amplicons were used for HTS sequencing (n = 6 for grampositive hdc primers and n = 6 for tdc primers). Amplicons were subjected to HTS using the Ion PGM platform, generating 938,971 hdc reads and 624,793 tdc reads, after quality filtering (refer to Additional file 1: Table S4 for the complete list of assigned reads/cheese). Mean read length across both tdc and hdc samples was 245 bp. Operational Taxonomic Unit (OTU) diversity (α-diversity) was calculated for both hdc and tdc samples and is displayed in 33.14 -95.11 % of reads assigned in the tdc samples. The Actinobacteria-assigned tdc reads in Ossau-Iraty corresponded to Actinomycetales at the order level and to Micrococcinaeae at family level but could not be assigned at the genus level. With respect to the hdc samples, Lactobacillales accounted for 13.7 -42.3 % of the reads assigned at the order level. At the genus and species levels, the numbers of reads that could be unambiguously assigned was low in all cases (depicted in Additional file 1: Table S4) and this was particularly evident when analysing the hdc samples. With respect to hdc samples, Lactobacillus accounted for 62.5 % to 100 % of all reads assigned at the genus level. Populations corresponding to Staphylococcus (37.5 % of reads assigned at genus level) were present in Irish artisanal cheese B, while Streptococcus (6.93 % of reads assigned at genus level) was identified in the Pecorino Sardo cheese. At the species level, a small cohort of the Staphylococcus population was identified as S. saprophyticus (5.97 % of reads successfully assigned at species level) while Streptococcus populations were successfully classified as S. thermophilus (6.94 % of reads successfully assigned at species level). Lb. buchneri accounted for the majority of reads assigned (93.06 -100 %) at species level and was detected across all cheeses except for Ossau-Iraty (Fig. 1). With respect to the Ossau-Iraty cheese, no genus or species level assignment was possible. For the tdc samples, reads were assigned primarily to the genus Enterococcus and ranged from 7.67 -99.65 % of reads assigned at genus level. Lactobacillus populations were also present and accounted for 0.35 -92.33 % of reads assigned at genus level. At the species level, E. faecalis accounted for the majority (2.29 -100 %) of reads successfully assigned at species level. Other subdominant populations identified included E. faecium, Lb. curvatus, Lb. brevis and Lb. delbrueckii (Fig. 2). Percentage populations of reads assigned exclusively at genus and species levels are shown in Additional file 1: Table S6. Cheese characterisation BAs were detected, at various concentrations, in all cheeses sampled and were found to range from 13.8 -736.5 mg/kg ( Table 5). The average histamine content of the positive samples was 34.48 mg/kg while the average tyramine concentration was 108.69 mg/kg. In all cases more than one BA was present in the cheeses sampled. Although not as toxicologically important as histamine and tyramine, putrescine and cadaverine levels were also measured to give a total BA concentration in each cheese. As expected, tyramine, generally regarded as the most common BA present in cheese [16,19], was present in 9 cheese samples at concentrations ranging from 4.5 to 323.4 mg/ kg. Histamine was present in 8 cheeses (8.4 -85.1 mg/kg). Cadaverine was detected in all cheese samples at concentrations ranging from 1.2 -267.4 mg/kg, while putrescine was detected in 7 cheeses (3.9 -212.7 mg/kg). The presence or concentration of BAs in the respective cheeses did not appear to be influenced by milk type, source or age. The Morbier cheese contained the highest concentration of total BAs (736.5 mg/kg) while the Comté cheese contained only 13.8 mg/kg total BAs. Histamine was not detected by HPLC in the Manchego and Comté cheeses. Similarly, tyramine was not detected in the Gorgonzola cheese by HPLC. Compositional analyses of the cheeses are presented in Table 6. Salt concentrations ranged from 0.65 -1.99 %, while cheese pH values extended from 5.3 to 7.1. Cheese salt in moisture levels ranged from 2.1 to 6.48. Discussion In this study, a novel, targeted sequencing-based approach was used to screen a range of different cheese varieties for the presence of microbial populations capable of producing the major toxic BAs histamine and tyramine. Initially, Sanger sequencing identified common BA producers (Lb. buchneri, E. faecium and E. faecalis) [23,45] but more importantly provided proof of heterogeneity justifying the use of NGS. The longer read lengths associated with the Sanger approach (up to approximately 800 bp in the case of the tdc amplicon) also allowed, in certain instances, successful identification at genus and species levels. However, the highly conserved nature of decarboxylase genes often reduced the capacity for distinguishing between certain species. This was particularly evident with respect the Lb. sakei/T. halophilus/T. muriaticus/O.oeni/Lb. hilgardii hdc operons and the Lb. curvatus/S. thermophilus and Lb. plantarum/Lb. brevis tdc operons identified. In the aforementioned cases, when conducting a BLAST analysis, the query cover and % identity are identical while the maximum scores differ slightly. This is as a result of single nucleotide changes in the analysed sequences (described in Additional file 1: Tables S2 and S3). In the case of the Lb. curvatus/S. thermophilus tdc operons identified, it likely that both of these cheese associated species are present within the samples tested. With respect to the difficulty differentiating Lb. sakei/T. halophilus/T. muriaticus/O. oeni/Lb. hilgardii hdc operons, it is difficult to predict the exact species present. A further 1,563,764 sequence reads were generated by high-throughput DNA sequencing of amplicons (post quality filtering). HTS allowed for greater population coverage but, in many cases, the short read length led to reduced resolution. Decarboxylases from common BA producers such as E. faecalis, Lb. buchneri, Lb. brevis, and Lb. curvatus were again identified. Subdominant populations, for example Staphylococcus saprophyticus and Lactobacillus delbrueckii, which were not observed via Sanger sequencing, were also present at less than 1 % of total reads. The shorter read lengths (mean read Fig. 1 Phylogenetic assignement, using MEGAN, of hdc reads across 10 speciality cheeses at Phylum, Order, Genus and Species level. Note that no genus or species level assignement was possible for the Ossau-Iraty cheese length of 245 bp) associated with using high-throughput sequencing, meant that, in some cases, the assignment of reads at genus and species levels was challenging ( Figs. 1 and 2). This is particularly relevant with respect to the highly conserved hdc operon. The absence of decarboxylase gene specific databases, as compared to the well annotated 16S rRNA databases, also affected the identification by BLAST analysis. Thus the combination of reduced read length and the lack of specific databases reduced the identification capacity of the HTS-based approach. This issue is particularly noticeable when analysing the microbial composition of the raw sheep milk cheese Ossau-Iraty. With reference to Ossau-Iraty, Sanger sequencing allowed for successful identification of genes assigned to E. faecalis, Lb. curvatus/S. thermophilus (both tdc), and Lb. sakei/T. halophilus/T. muriaticus/O. oeni/Lb. hilgardii (hdc), however the high-throughput approach did not permit assignment of the hdc samples at the genus or species level. In the case of tdc analysis, the identification of E. faecalis-associated tdc was possible. Furthermore, while deep sequencing allowed the identification of tdc genes corresponding to Actinomycetales (0.16 %) (Fig. 2), which were assigned to the Micrococcinaeae, the shorter read length prevented assignment of these decarboxylases at genus or species levels. HPLC results established the presence of various BAs across all cheeses sampled. However, the presence of histamine and/or tyramine did not always correlate with the presence of the corresponding decarboxylase gene fragment. This was most evident in the case of the Morbier cheese, which exhibited the highest total BA concentration in this study. Despite a tyramine concentration of 171.3 mg/kg, no tdc amplicons were generated by PCR. This discrepancy may be attributable to the fact that the primers selected for this study were designed to target Gram-positive LAB and were based on alignments with common (type-strains) species including Lb. sakei, Lb. buchneri, Lactobacillus 30a, O. oeni, C. perfringens and T. muraticus (hdc) and Lb. brevis, C. divergens, C. piscicola, E. faecalis and E. faecium (tdc) [18,24]. Therefore, the primers may not bind to all histamine and tyramine decarboxylase determinants present within the cheeses. Indeed, certain yeast species including strains of Y. lipolytica (tdc), D. hansenii and G. candidum (hdc) are recognised BA producers associated with artisanal cheeses, and may have contributed to the amine content, but would not be detected using the primers employed [13]. In this study, the identification of decarboxylase genes, using HTS, from bacteria commonly used as cheese starter cultures, including Lb. delbrueckii and S. thermophilus was of particular interest [46]. In agreement with previous reports [23,47], S. thermophilus was identified as having histidine decarboxylation capacity in the Pecorino Sardo cheese. The origin of these bacteria, i.e., whether they were added as cheese starters or gained access to the cheese via raw milk or during processing or ripening is not known. This highlights the importance of screening starter and adjunct bacteria for aminogenic potential, using molecular methods that can rapidly detect the presence of decarboxylase genes. S. saprophyticus, not commonly associated with BA formation in cheese, was identified in this study and has previously been associated with BA formation in fermented meat products [48,49]. Of the cheeses selected for this study, both Pecorino-Sardo and Manchego have a well-established association with BAs. In particular, Pecorino Sardo, identified in this study as containing several hdc and tdc positive bacteria (Lb. buchneri, E. faecium, E. faecalis), has previously been shown to contain conditions (microbiota, ripening time, physio-chemical factors) complementary to BA production [30,50]. Manchego has also previously been shown to contain tyrosine decarboxylating microorganisms; however, in this study the Manchego cheese sampled had a low level of total BA concentrations (21.9 mg/kg) and no tdc or hdc positive amplicons were generated [51]. Comté and Gorgonzola have also previously been shown to contain various BAs [52] but in our study BA levels were low and no hdc or tdc amplicons were generated. Interestingly, blue cheeses such as Gorgonzola are often regarded as having optimal conditions for BA production, due to milk processing and proteolytic activity (presence of molds), for BA formation, however, in this study the Gorgonzola sample exhibited among the lowest total BA concentrations [33,53]. Conclusions Ultimately, this study shows, for the first time, that sequencing based technologies (Ion PGM platform) can successfully profile the diversity of histaminogenic and tyraminogenic bacteria present in ripening cheese. A similar approach could also be applied to reduce risk factors associated with BA accumulation. This can be achieved by screening starter cultures, milk and manufacturing/storage facilities with a view to reducing/controlling not only populations associated with BA formation, but potential sources of these populations [13,[54][55][56]. In this way, a pre-emptive approach using existing (refrigeration, preservatives, additives) and/or emerging (microbial modelling, high hydrostatic pressure, irradiation) control measures can be implemented [54,[57][58][59][60]. This method cannot, however, determine the transcriptional activity of the respective genes. In addition, while NGS reads indicate, proportionally, the levels of bacterial populations within the cheese matrix, it does not accurately quantify the numbers of bacteria present. While further optimisation is required, sequencing based approaches have the potential to eventually replace labour intensive culture-based methods which often require primary culturing followed by molecular methods to identify responsible genera. Additional file Additional file 1: Table S1. Standards preparation for HPLC analysis of individual biogenic amines. Table S2. Complete BLAST analysis of clones subjected to Sanger sequencing. Table S3. Table S2: Complete BLAST analysis of tdc clones subjected to Sanger sequencing. Table S4. Total reads assigned for each cheese. Table S5a/b. Microbial composition of bacteria at phylum, order, genus and species levels. Figure S6.

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Loss of floral repressor function adapts rice to higher latitudes in Europe Highlight Allelic variants of floral repressor genes have been artificially selected to reduce sensitivity to photoperiod of rice varieties cultivated in Europe, allowing cultivation of a tropical species at higher latitudes. Introduction Photoperiod (or day length) and temperature are crucial cues that plants use to monitor diurnal and seasonal time (Amasino, 2010). Complex molecular networks have evolved in distinct plant lineages to translate environmental signals into molecular signals that drive reproductive development (Andrés and Coupland, 2012). Rice (Oryza sativa) is a facultative short day (SD) plant in which flowering is accelerated upon exposure to photoperiods falling below a critical threshold, and has been extensively used as model system to understand the genetic and molecular basis of flowering time (Brambilla and Fornara, 2013). Besides its prominent role as a plant model system, amenable to genetic studies, rice is a staple food for large parts of the world population, particularly in Asian countries, where rice was originally domesticated and cultivated. Recent molecular data based on re-sequencing of several hundred accessions of cultivated and wild rice, indicate that the centre of rice domestication can be traced to the middle area of the Pearl River Basin, in southern China Huang et al., 2012). From these tropical regions, rice cultivation has enormously expanded, reaching temperate areas in Asia, up to 55°N (Shrestha et al., 2014). During the 15th century, rice cultivation became established in Europe and Italy is currently the major rice producer. As opposed to tropical Asia, cultivation of rice in temperate regions is constrained by low winter temperatures that restrict the cropping season to late spring and summer, when the photoperiod is long and not inductive. Artificial selection has allowed breeding of rice varieties adapted to higher latitudes, improving germinability and tolerance to low temperatures (Fujino et al., 2008;Suh et al., 2010), and reducing sensitivity to photoperiod (Xue et al., 2008;Fujino et al., 2013;Koo et al., 2013). The genetic architecture of flowering in rice depends on a regulatory network controlling expression of two proteins, encoded by HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1), orthologs of FLOWERING LOCUS T (FT) of Arabidopsis (Kojima et al., 2002;Turck et al., 2008;Tsuji et al., 2013). Upon perception of favourable photoperiods, Hd3a and RFT1 are expressed in the vasculature of leaves and move to the shoot apical meristem to promote the switch from vegetative to reproductive development (Tamaki et al., 2007;Komiya et al., 2008;Komiya et al., 2009). Expression of Hd3a and RFT1 is induced by HEADING DATE 1 (Hd1), a zinc-finger CCT domain transcription factor, and EARLY HEADING DATE 1 (Ehd1), a B-type response regulator (Yano et al., 2000;Izawa et al., 2002;Doi et al., 2004;Itoh et al., 2010). Mutations in either Hd1 or Ehd1 cause delayed flowering under SD (Doi et al., 2004). Conversely, when plants carrying hd1 mutant alleles are grown under long day (LD) conditions flowering is induced earlier compared to the wild type, indicating that Hd1 can either promote or repress flowering, depending on the photoperiod (Yano et al., 2000;Izawa et al., 2002;Ishikawa et al., 2011). The repressive activity of Hd1 can be enhanced by Hd6, which encodes the α subunit of CASEIN KINASE II (CKIIα) (Takahashi et al., 2001). Defective Hd6 alleles are present in some japonica rice cultivars and reduce the capacity of Hd1 to repress flowering under LD (Takahashi et al., 2001;Ogiso et al., 2010;Ebana et al., 2011;Shrestha et al., 2014). Several factors play prominent roles as regulators of flowering under LD, primarily acting upstream of Ehd1, to regulate its expression. Among them, GRAIN YIELD, PLANT HEIGHT AND HEADING DATE 7 (Ghd7) and Ghd8/DAYS TO HEADING 8 (DTH8) are potent repressors of Ehd1 expression and strongly contribute to delay flowering (Xue et al., 2008;Wei et al., 2010;Yan et al., 2011). Ghd7 encodes a CCT domain protein that, when mutated, allows rapid flowering also under LD (Xue et al., 2008). Phosphorylation of Ghd7 is necessary for its repressive activity and is mediated by Hd16/EARLY FLOWERING 1 (EL1), encoding a casein kinase I (CKI) protein (Hori et al., 2013). Loss-of-function alleles of Hd16 have been isolated from Asian varieties capable of flowering early under LD conditions (Hori et al., 2013;Kwon et al., 2014). Ghd8 encodes a homolog of the HEME ACTIVATOR PROTEIN 3 (HAP3) of Arabidopsis. Loss-of-function alleles at this locus also cause early flowering under LD, and have been artificially selected in several varieties to expand rice cultivation to northern areas (Wei et al., 2010;Fujino et al., 2013). CONSTANS-LIKE 4 (OsCOL4) encodes a repressor of Ehd1, active under both SD and LD conditions, and no natural genetic variation at this locus has been reported yet . Cloning of Hd2 showed that it encodes PSEUDO RESPONSE REGULATOR 37 (PRR37), a repressor of Hd3a expression under LD (Koo et al., 2013). Loss-of-function alleles at PRR37 promote early flowering at northern latitudes by derepressing Ehd1 and the florigens (Gao et al., 2014). These data indicate that the floral activation pathway is strongly repressed under long photoperiods and short day lengths activate flowering in part by relieving such repression. Mining of allelic variants of flowering time regulators in rice cultivars adapted to LD conditions has identified extensive natural genetic variation that has been selected by ancient farmers or breeders, as it conferred a selective advantage over the wild type alleles in temperate areas. The levels of genetic diversity among some flowering time genes in rice varieties that can flower in Europe have been partly explored (Naranjo et al., 2014). However, it is not clear which level of genetic diversity exists among LD repressors in European varieties, or how it impacts on phenotypic variation across northern latitudes. This study demonstrates that in several European varieties, expression of both Hd3a and RFT1 is de-repressed regardless of photoperiod and causes a strong decrease of sensitivity to day length. Natural genetic variation at LD repressor loci largely explains such behaviour but the impact of lossof-function alleles varies with latitude. These results indicate that fine tuning of photoperiod sensitivity has been a key strategy allowing expansion of rice cultivation to higher latitudes and provide useful data to geneticists willing to breed novel fast-cycling varieties for temperate environments. Plant material Sixteen temperate japonica varieties cultivated in Italy (referred as the mini-panel in the text) were chosen for detailed genotyping and expression analyses. A set of 247 varieties extracted from the European Rice Germplasm Collection (http://eurigendb.cirad.fr), resulting from the pooling of the working collections of breeders from European public breeding institutions, was used for targeted association mapping (Supplementary Table S1). Two Hd1 mutant lines in the Nipponbare background (NG6019 and NG0430) were obtained from the National Institute of Agrobiological Sciences of Japan (https://tos.nias.affrc.go.jp/; Miyao et al., 2003). The insertions of Tos17 in NG6019 (hd1-1) and NG0430 (hd1-2) were confirmed to be 577 and 1478 bp after the transcription start site, respectively. Genotyping was performed using primers Tos17-For GAGAGCATCATCGGTTACATCTTCTC and hd1-1-Rev CACAGATTCCATCAGCAACAG, or hd1-2-Rev GCCAAATTCCAGAATCCTGA. Heading dates and PS index measurements Heading dates were determined under SD (10L/14D) and LD (16L/8D) conditions in Conviron PGR15 growth chambers or in a walk-in growth chamber. Temperature and relative humidity were set at 28ºC/80% during the day and 24ºC/90% during the night, respectively. Seedlings used to measure heading dates under natural LD (NLD) were sown in a cold greenhouse under natural day length conditions on 5 April 2012 and 11 April 2013, and transplanted on 7 May 2012 and 14 May 2013, respectively, in an irrigated field at the University of Milan. About 20 to 30 seedlings per variety were transplanted and heading dates were scored from each individual. Heading dates of the mini-panel under different day lengths were scored at emergence of the first panicle, just after opening of the leaf sheath. The Photoperiod Sensitivity index (PS) was calculated according to the formula: [(heading date under LD − heading date under SD) / heading date under LD]. For the 247 varieties listed in Supplementary Table S1, the phenotypic data of the CT95-37 RESGEN project 'Constitution, description and dynamic management of rice genetic resources of European vocation' supported by the European Community, DG VI, were used (Feyt et al., 2001). The data were collected from experiments conducted between 1996 and 1998 in Arles (France), Alcala del Rio, in Andalusia (Spain), Thessaloniki (Greece) and Salvaterra de Magos in Estremadura (Portugal). Plants were grown under similar conditions in irrigated fields. The tested cultivars were arbitrarily divided into three groups in order to spread the work load over three years as described in Feyt et al. (2001). The experimental design was an augmented design with a common set of seven control varieties present all years in all trials. The control varieties (Ariete, Baldo, Cigalon, Loto, Koral, Senia and Thaibonnet) represented a broad range of flowering date variation from early to late. Dates of 50% flowering were recorded for all accessions and time to 50% flowering computed based on the trial sowing dates. Data were adjusted for the year effect. The Italy_2012 and Italy_2013 datasets were collected at the northernmost European location (Vercelli, Italy) in 2012 and 2013, respectively. The final data used in association mapping analyses are presented in Supplementary Table S1. DNA extraction, PCR and sequencing Young leaf pieces were pooled in 2 ml tubes containing two clean steel balls. Tubes were rapidly frozen in liquid nitrogen and grinded using a tissue-lyser (Retsch). Genomic DNA from varieties of the minipanel was extracted using a modified CTAB and chloroform:isoamyl alcohol method (Doyle and Doyle, 1987). The DNA pellet was resuspended in ddH 2 O and quantified using a NanoPhotometer Pearl (Implen). About 50-150 ng of gDNA was used in each PCR reaction in a final volume of 13 µl. Primers were designed in order to sequence the coding regions (CDS) of Hd1, PRR37, Ghd7, Ghd8 and COL4. The components were as follows: 1.3 µl of 10X PCR buffer, 2 µl of 5M Betaine or 1 µl of 30% DMSO, 0.2 µl of 10mM dNTPs, 0.25 µl of each primer (1 μg/μl, diluted 1:10), 0.2 µl Taq polymerase, and ddH 2 O to volume. For the amplification of fragments with high GC content, LA Taq and GC Buffers (Takara) were used following manufacturer's instructions. Amplification reactions were run for 40 cycles. The PCR products were incubated with Exonuclease I (Thermo Scientific) and Fast Alkaline Phosphatase (Thermo Scientific) for 15 min at 37°C. Enzymes were heat-deactivated for 15 min at 85°C. Purified fragments were sequenced using specific primers. Candidate gene genotyping Seedlings were grown in a greenhouse for two weeks. The aerial part was collected and grinded using steel balls in 2 ml tubes after freezing with liquid nitrogen. Genomic DNA from all varieties was extracted using the Edwards method (Edwards et al., 1991). A total of 17 different non-functional alleles in five different genes were genotyped using markers listed in Supplementary Table S2: six for hd1, three for prr37, three for ghd7, three for ghd8 and two for el1 (Supplementary Table S1). Primers for dCAPS design were obtained using dCAPS Finder 2.0 (http://helix.wustl.edu/dcaps/dcaps.html) allowing up to two mismatches. PCRs were conducted as described before, and digestions with restriction enzymes were carried out at 37°C overnight. The PCR products were separated by agarose gel electrophoresis, and visualized using a Fluorchem imaging system (Alpha Innotech) with Alpha Ease FC software. In association analyses, all loss-of-function alleles of a given gene were grouped into a single non-functional type (NF) (Supplementary Table S1). Quantification of gene expression and correlation between gene expression and heading dates To quantify gene expression from developmental time courses, leaves were sampled at the indicated day, two hours after lights-on (ZT2). For diurnal time courses, leaves were sampled every three hours from three-week-old plants grown under under SD and six-week-old plants grown under LD. Leaf samples from plants grown under field conditions were collected during floral induction (from the middle of May until the end of July) every two weeks, two hours after dawn. Gene expression data from field-grown plants were collected in 2012 and 2013 with similar results. Data from 2012 are presented. Leaves were collected from at least three independent plants for each RNA preparation. Total RNA was isolated using either TRI reagent (Sigma-Aldrich) or the Spectrum Plant Total RNA Kit (Sigma-Aldrich) following manufacturer's instructions. Total RNA was treated with DNase I using the TURBO DNase kit (Life Technologies), and precipitated with 0.1 volumes of sodium acetate and 2.5 volumes of 100% ethanol. The RNA pellet was washed with 75% ethanol and resuspended in ddH 2 O. RNA was quantified using a NanoPhotometer Pearl (Implen). 2 µg of total RNA were retrotranscribed using SuperScript II Reverse Transcriptase (Invitrogen) with oligo-dT and resuspended in a final volume of 140 μl of cDNA. Quantification of gene expression was carried out in a final volume of 10 µl using a Mastercycler Realplex 2 (Eppendorf). qPCR reactions were carried out using 5 μl of 2X Maxima SYBR Green qPCR Master Mix (Thermo Scientific), 0.25 μl of each primer (1 μg/μl, diluted 1:10), 1.5 μl of ddH 2 O, and 3 μl of cDNA template. Primers used to quantify the mRNA levels of Ubiquitin, Hd3a, RFT1, Hd1, Ehd1, Ghd7 and PRR37 are listed in Supplementary Table S5. Correlations between heading date (days) and gene expression values of Hd3a/Ubq and RFT1/Ubq (log of relative gene expression) were determined by calculating the Pearson's correlation coefficient (R 2 ) using Microsoft Office Excel 2010. Association mapping All accessions had been previously genotyped with SNPs spaced of at least 0.4 Mb in the genome (Courtois et al., 2012). A set of 85 SNPs polymorphic in the studied accession set, hereafter called 'neutral SNP set', was retained for further analysis. All analyses were conducted using Tassel v.4.0 (Bradbury et al., 2007). A Principal Component Analysis was conducted on the neutral SNP set using a correlation matrix. Three axes together explaining 40% of the variation were retained. The matrix constituted of the loadings of the accessions on these three axes was used to control population structure. A kinship matrix was computed using the same dataset to capture more complex familial relationships. Several models were compared to test the significance of each SNP × trait association: a simple linear model without any control of population structure (GLM0), a linear model with control of structure (GLM1), a mixed model with control of kinship (MLM1) and a mixed model involving structure and kinship (MLM2). Only alleles with a frequency above 5% were used in the analyses. Taking into account the number of genes and polymorphisms within genes tested, a threshold of 0.005 was used to declare a test significant. Rice varieties adapted to Europe show reduced sensitivity to photoperiod Artificial selection has expanded rice cultivation from tropical Asia to higher latitudes in Europe, partly by influencing the capacity of plants to flower under long days. To address the molecular mechanisms involved, a working panel composed of 16 temperate japonica varieties cultivated in Italy (minipanel) was selected to be representative of the diversity of heading dates observed under natural field conditions (Fig. 1). The mini-panel and Nipponbare were grown under constant LD (16 hours light) and SD (10 hours light) in growth chambers, and under natural long day conditions (NLD) in Milan (~45°N). All varieties flowered earlier under SD conditions compared to LD conditions, and the response to distinct photoperiods showed large variation (Fig. 1A). Most varieties flowered at similar times under NLD and LD, indicating that photoperiodic flowering was similarly promoted both under constant and varying day lengths. Flowering of Thaibonnet, Balilla and Nipponbare was delayed under LD compared to NLD, indicating that these varieties could discriminate between different long photoperiods and that continuous LD of 16h caused stronger floral repression. Based on heading dates under LD and SD, we calculated a photoperiod sensitivity index (PS). All varieties tested showed reduced PS compared to Nipponbare (Fig. 1B), and some varieties, including Augusto and Sant'Andrea, were almost completely insensitive and flowered at the same time regardless of day length (Fig. 1A). These data indicate that flowering of mini-panel varieties can be promoted by SD but not repressed under LD and NLD compared to Nipponbare. Their capacity to discriminate between different day lengths is therefore impaired. Accessions belonging to the mini-panel carry nonfunctional alleles of long day floral repressor genes Mutations in floral repressors have been associated to northern expansion of rice cultivation (Xue et al., 2008;Fujino et al., 2013;Hori et al., 2013;Koo et al., 2013;Kwon et al., 2014;Shrestha et al., 2014). The type and frequency of lossof-function mutations was assessed at seven loci, encoding genes repressing flowering most effectively under LD and including Hd1, PRR37, Ghd7, Ghd8, OsCOL4, Hd16/EL1 and Hd6/CKIIα (Yano et al., 2000;Takahashi et al., 2001;Xue et al., 2008;Lee et al., 2010;Yan et al., 2011;Hori et al., 2013;Koo et al., 2013). The DNA sequence of all repressors was determined, including the CDS and some introns. However, the upstream regulatory regions were not sequenced, as polymorphisms in the promoters would be difficult to associate to loss-of-function alleles. Sequence data were compared to Nipponbare that bears functional alleles at all repressor loci with the exception of Hd6 that bears a premature stop codon, resulting in a truncated non-functional protein (Takahashi et al., 2001). Several allelic variants were identified (Table 1, Supplementary Fig. S1). Ten out of 16 varieties carry at least one loss-of-function allele at Hd1, Ghd7 or Ghd8. Seven different alleles at the Hd1 locus were detected. The 2 bpdeletion observed in Fragrance, Eolo, Thaibonnet, Gladio, Panda and Augusto (Hd1-V, Hd1-VI), and the 43bp-deletion observed in Lido and Selenio (Hd1-II) were previously isolated from the cultivars Kasalath and HS66 respectively, and were associated to loss-of-function alleles (Yano et al., 2000). Balilla bears the same Hd1 allele of Ginbouzu which was suggested to encode a stronger LD repressor allele than Nipponbare (Yano et al., 2000). Four PRR37 alleles were detected but none of them could be clearly associated to a loss-of-function variant ( Supplementary Fig. S1). Four Ghd7 alleles were identified, one of which showed a deletion of the locus and was previously described as Ghd7-0 in early flowering rice varieties (Xue et al., 2008) (Supplementary Fig. S1). The SNP at position 618 converts tyrosine into a Stop codon (Ghd7-I, Supplementary Fig. S1) and is reported here for the first time. The mutation is located in the CCT motif that is a critical functional domain for this class of transcription factors. The Ghd8 locus was represented by four different alleles ( Supplementary Fig. S1), two of which (Ghd8-II and III) were described as loss-of-function and associated to loss of photoperiod sensitivity (Wei et al., 2010). Three genotypes were detected sequencing the CDS of COL4 , none of which was likely to alter the function of the gene. Genotyping of Hd16 for two loss-of-function mutations previously identified in Japanese varieties (Type 2 and Type 3) (Kwon et al., 2014) showed that no such variants were present within the mini-panel. Mutations in Hd16 seem to have arisen locally and are not widely spread (Kwon et al., 2014). Similarly, no mutations in Hd6 were identified and all varieties carried a functional allele identical to that of Kasalath (Takahashi et al., 2001). Expression of Hd3a and RFT1 is not repressed under long or short days in Italian varieties Induction of Hd3a and RFT1 is correlated to heading dates under controlled SD and NLD (Takahashi et al., 2009;Naranjo et al., 2014;Ogiso-Tanaka et al., 2013). However, their seasonal expression dynamics have not been extensively determined for varieties bearing non-functional allelic variants of LD floral repressors, nor it is clear how accumulation of such mutations influences the flowering behaviour of varieties adapted to northern latitudes. The expression levels of Hd3a and RFT1 were quantified in plants grown under field conditions in Milan, between May and July, when flowering is induced. Expression of Hd3a and RFT1 rapidly decreased in Nipponbare as day length became longer (Fig. 2). Conversely, most Italian varieties showed induction of Hd3a and RFT1 expression, and peak expression was observed 96 days after sowing (DAS) in most of them. Varieties bearing wild type alleles of Hd1, Ghd7 and Ghd8 had higher levels of Hd3a and RFT1 mRNA compared to Nipponbare, indicating that de-repression of florigens is not exclusively caused by mutations in these regulators (Fig. 2B, C). Positive correlations between the amount of Hd3a and RFT1 mRNA and heading dates of the mini-panel grown under NLD were observed, and the highest correlations were determined for RFT1 at 68 DAS (Table 2). These data indicate that Hd3a and RFT1 are not repressed in varieties of the mini-panel and expression of both genes likely contributes to flowering under NLD. It has been previously demonstrated that under SD conditions RFT1 expression was lower compared to Hd3a in wild type plants. Further reduction of its expression by RNAi caused no delay of flowering, indicating that RFT1 is not contributing to flowering promotion under SD (Komiya et al., 2008). In Italian varieties, the correlation between heading dates and RFT1 expression was higher than with Hd3a expression at two time points under continuous SD ( Table 2). Expression of upstream regulators of the florigens was poorly correlated to heading dates (Supplementary Table S4). Therefore, the diurnal mRNA expression profiles of Hd3a and RFT1 were determined under both LD and SD. In addition to Nipponbare, which shows a high PS index, three Italian varieties with a low PS index were selected for this experiment, regardless of genotype. Hd3a was induced in all varieties to levels similar to those observed in Nipponbare (Fig. 3A). Additionally, high levels of RFT1 expression in the three Italian varieties (Fig. 3B) were observed, indicating that RFT1 transcription was strongly de-repressed under SD and exceeded that of Nipponbare. Plants grown under LD showed induction of Hd3a and RFT1 transcription (Fig. 3C, D). The overall expression levels of RFT1 were higher under SD than under LD (Fig. 3B, D). In Italian varieties, de-repression of Hd3a and RFT1 transcription occurs under both long and short photoperiods in the morning, differently from the normal day length-induced peak in the evening in Nipponbare. Induction of RFT1 expression is not only a consequence of activation by the LD pathway but rather the result of its transcriptional de-repression in the background of varieties adapted to northern latitudes. Hd1 represses Ehd1 under natural long days Several varieties could flower under NLD despite bearing wild type alleles of all LD floral repressor genes analysed, indicating the existence of additional factors that contribute to reduce the sensitivity to photoperiod (Fig. 2). Whether these factors could act through upstream transcriptional regulators of Hd3a and RFT1 was tested by quantifying the expression levels of Ehd1 and Hd1. Expression of Ehd1 was transiently induced, similarly to Hd3a and RFT1 ( Fig. 2A), and in many varieties its expression was high regardless of the presence of Ghd7 and/or Ghd8 wild type alleles ( Fig. 2A, E, H). Quantification of Hd1 in the same varieties showed that its expression was strongly reduced and often completely abolished compared to Nipponbare (Fig. 2D). Expression of other LD repressors was not reduced (Fig. S2), supporting the idea that additional factors influence flowering and PS by repressing Hd1 expression and by promoting Ehd1 expression. Whether increased expression of Ehd1 mRNA was caused by reduced expression of Hd1 was tested using hd1 mutant alleles isolated from Nipponbare. Two loss-of-function mutants in which Tos17 retrotransposons were inserted in the first and second exon, respectively, were grown under natural long days in Milan (Fig. 4A). Both mutant alleles showed accelerated heading compared to Nipponbare (Fig. 4B) and transiently expressed Hd3a and RFT1 in leaves (Fig. 4E, F). The expression levels of Ehd1 were also higher compared to those of wild type plants (Fig. 4D), indicating that Hd1 repressed Ehd1 under NLD and that such repression was sufficient to prevent flowering at higher latitudes. Single mutations of LD repressor genes are sufficient to promote flowering at higher latitudes Combinations of hd1, ghd7 and ghd8 mutations seemed to contribute to higher expression of Hd3a under NLD, as shown in the group of varieties bearing several non-functional alleles (Fig. 2I) compared to those bearing a single non-functional allele (Fig. 2F) or only functional alleles (Fig. 2B). Nipponbare (Hd1, Ghd7, Ghd8) and Augusto (hd1, ghd7, ghd8) showed different heading dates under SD, LD and NLD, opposing patterns of Hd3a and RFT1 expression under NLD and very different PS ( Fig. 1 and Fig. 2I, J). To assess if pyramiding of hd1, ghd7 and ghd8 mutations is necessary to promote flowering under LD, Augusto and Nipponbare were crossed, F2 individuals bearing all combinations of hd1, ghd7 and ghd8 mutations were selected and heading dates of F3 families grown under constant LD were scored. Plants bearing only functional LD repressor genes recapitulated the late flowering phenotype of Nipponbare and did not express Hd3a or RFT1, whereas plants bearing at least one mutant repressor flowered earlier ( Fig. 5A) and expressed Hd3a and RFT1 at higher levels ( Fig. 5B, C). The Hd6 mutant allele from Nipponbare segregated in F3 families, but it did not affect heading dates or gene expression levels (data not shown). The homozygous mutant ghd8 from Augusto had the strongest effect to increase Hd3a and RFT1 expression, either in combination with ghd7 or alone (Fig. 5B, C). Taken together, these data suggest that single mutations in Hd1, Ghd7 or Ghd8 or reduction of Hd1 expression are sufficient to de-repress Hd3a and RFT1 expression and to promote flowering at higher latitudes. Loss-of-function alleles of Ghd8, Ghd7 and PRR37 but not of Hd1 are associated with early heading at northernmost sites in Europe To assess how long day repressors impact on heading dates across a latitudinal cline at higher latitudes, a germplasm collection comprising 247 varieties cultivated in Europe was used (Ahmadi et al., 2011;http://eurigendb.cirad.fr). The frequency of hd1, prr37, ghd7, ghd8 and el1 mutant alleles was determined for all varieties (Supplementary Tables S1 and S2). Of these, 145 (58.7%) carried at least one non-functional allele (Fig. 6A), a result very similar to that observed using the mini-panel. Non-functional alleles of Hd1 were the most frequent followed by ghd8, ghd7, prr37 and el1 (Fig. 6A). Extensive pyramiding of loss-of-function mutant alleles was observed, including a variety (Timich 108 from Romania) bearing four mutations (hd1, ghd8, prr37, el1; Fig. 6A, Supplementary Table S1). Selected varieties of the European collection were grown across a latitudinal cline at five locations in Europe, comprising Vercelli (Italy, 45.3°N), Arles (France, 43.6°N), Thessaloniki (Greece, 40.6°N), Salvaterra de Magos (Portugal, 39°N) and Alcala del Rio (Spain, 37.5°N). Flowering time data were recorded and made publically available at http://tropgenedb.cirad.fr and in Supplementary Table S1 (Feyt et al., 2001). For each gene, all loss-of-function alleles were grouped into a single NF and genotypic data were used for targeted association analysis. Results of association statistics are presented in Fig. 6B, following a MLM2. The p-values of association calculated according to other models are reported in Supplementary Table S3. The mean effect of allelic substitutions between wild type and mutant alleles on days to heading were calculated as the phenotypic difference between the two homozygotes, by setting to 0 the effect of the functional allele and computing the effect of the non-functional allele. Depending on the location, different alleles were significantly associated with early heading (Fig. 6B). At the northernmost location, strong associations were detected for ghd8, ghd7 and prr37. Only ghd7 mutations were associated to early heading in Arles. No association was detected between hd1 loss-of-function alleles and early heading at any location. A latitudinal gradient of significance was observed when considering the p-values of association for ghd7 and ghd8 (Supplementary Table S3). Therefore, loss-of-function alleles of flowering time genes are differentially affecting cycle duration, depending on the latitude and mutant alleles of ghd7 and ghd8 are the most effective to accelerate flowering at the northern limits of rice cultivation in Europe, but their effect quickly decreases with latitude. Discussion Rice is largely cultivated in Europe and has a major impact on the economy of several regions. Its adaptation to higher latitudes has required the targeted manipulation of several genes. This study provides a comprehensive characterization of the natural genetic variation of loci encoding LD floral repressor genes and shows how selection of non-functional alleles in European germplasm could be part of the mechanism that allowed rice cultivation at northern sites in Europe. These data can be the basis to design varieties better adapted to such environments. Additionally, the study of Hd1 alleles showing reduced or null expression has allowed for better definition of a regulatory node in the LD pathway, suggesting that Hd1 can repress Ehd1 to reduce expression of the florigens under LD. Genetic variation of Hd3a and RFT1 expression contributes to identification of the existence of novel factors regulating flowering Flowering of rice is promoted upon expression of Hd3a and RFT1. These genes are physically located very close to each other and are likely the product of chromosomal duplication. Yet, their function has diversified during evolution and mutant analysis has helped elucidate the role of each gene under different photoperiods. Plants in which Hd3a expression is reduced by RNAi delay flowering under SD, but flower normally under LD. Conversely, reduction of RFT1 mRNA expression delays flowering under LD, but not under SD (Komiya et al., 2008(Komiya et al., , 2009. Transgenic plants in which both genes are down-regulated produce a strong additive effect and do not flower, clearly indicating that they have a fundamental role for flowering in rice (Komiya et al., 2009). These results account for the little functional genetic variation that has been detected at loci encoding Hd3a and RFT1 in cultivated varieties (Takahashi et al., 2009;Ogiso-Tanaka et al., 2013;Naranjo et al., 2014). Still, genetic diversity of RFT1 is higher than that of Hd3a and functional polymorphisms at the RFT1 locus have been recently identified that disrupt protein function and lead to delayed flowering under LD (Hagiwara et al., 2009;Ogiso-Tanaka et al., 2013). Also several species from the genus Oryza bear mutations in RFT1 that lead to non-functional proteins . These data indicate that RFT1 might not be required under specific environmental conditions, particularly at tropical latitudes where Hd3a is the major factor promoting flowering. For these reasons, genetic variation at Hd3a or RFT1 loci was not assessed in this study, but given the importance of their expression levels to regulate floral induction, detailed expression analyses were performed and expression curves were designed for several varieties. The results indicate that varieties adapted to grow in Europe induce both Hd3a and RFT1 under NLD and such induction correlates with flowering. However, variation in expression patterns could not be exclusively correlated to genotypes at known repressor loci, indicating that the contribution of other regulators, likely including transcriptional activators or as yet unknown genes, has to be taken into consideration. Genetic evidence indicated that reducing Hd1 expression is sufficient to adapt rice flowering to higher latitudes because Hd1 could repress Ehd1 under NLD and consequently, Hd3a and RFT1. This finding highlights the central role of Ehd1 in the LD regulatory network, where repression from Ghd7, Ghd8 and Hd1 converge. It further suggests the existence of Hd1 repressors that might be useful tools to breed varieties adapted to long photoperiods, bypassing the need to introduce mutations in Hd1 or other repressors. European varieties could be useful genetic tools to identify such novel regulators. Seasonal induction of Hd3a and RFT1 was transient in all varieties tested. Induction started between 54 and 68 DAS, reached a maximum 96 DAS in most varieties and then rapidly decreased. This pattern seems to be common to other varieties and locations , and suggests that maintenance of high levels of Hd3a and RFT1 expression in leaves is not required to sustain the flowering process. Upon commitment of shoot apical meristems to reproductive development, Hd3a and RFT1 expression is likely not necessary any longer. Transcriptional dynamics were largely shared by both Hd3a and RFT1, suggesting a common mechanism for their temporary transcriptional induction in varieties adapted to LD. The molecular nature of this system is unknown but one possibility is that an epigenetic mechanism might temporarily allow access of RFT1 and Hd3a chromatin to transcriptional activators. Given the close proximity of these two genes, such mechanism might be operating on both of them. Under NLD, Hd3a and RFT1 expression quickly dropped in Nipponbare, a variety strongly sensitive to day length, but under developmental or diurnal time courses under SD their levels were high and promoted flowering. Correlation of heading dates with RFT1 expression levels was higher than with Hd3a and peak levels of RFT1 transcription in several Italian varieties were higher than those of Nipponbare (peak expression of Hd3a and RFT1 showed a shift of up to six hours compared to Nipponbare, a behaviour that we cannot currently explain but that could be related to defects in diurnal time measurement). These results indicate that RFT1 can be strongly activated also under SD conditions, depending on the genetic background and demonstrate that regulation of its transcription can be very adaptable. Mutations in Ghd7, Ghd8 and PRR37 are strongly associated to early heading varieties at northern latitudes but not at southern latitudes in Europe Association mapping between time to flowering and targeted loci across a latitudinal cline in Europe allowed assigning a relative weight to LD repressors. Ghd8, Ghd7 and PRR37 were detected as major repressors of flowering at the northernmost limits of rice cultivation in Europe, but not at southern locations. These genes have been useful tools for breeders to reduce cycle length and varieties bearing the mutant alleles could spread closer to the northern limits of rice cultivation in Asia (Fujino and Sekiguchi, 2008;Xue et al., 2008;Wei et al., 2010;Ebana et al., 2011;Fujino et al., 2013;Gao et al., 2014). However, whereas strong associations between genotypes and heading were detected for Ghd7, Ghd8 and PRR37 at the northernmost location in Europe, the effect of ghd8 and prr37 was milder in France, where only ghd7 was statistically associated to fast-cycling accessions. At even southern locations, including Greece, Portugal and Spain, no statistically significant association could be detected. These data suggest that the photoperiodic network can finely tune flowering responses and has the capacity to detect small changes in day length. Since seasonal variation of photoperiod is different depending on latitude, it is likely sufficient to modulate flowering to the extent that loss of function alleles that are crucial at ~45°N to promote flowering become less relevant at 40°N or lower latitudes. It is however important to note that statistical thresholds do not abolish the biological value of some alleles. For example, despite not being statistically significant according to the models used in this study, ghd7 mutant alleles should be considered useful tools to accelerate flowering in Greece. High natural genetic variation is associated to the Hd1 locus and contributed to diversification of flowering time in many varieties, including those adapted to northern regions (Yano et al., 2000;Fujino et al., 2008;Takahashi et al., 2009;Fujino et al., 2010;Zhao et al., 2011;Naranjo et al., 2014). Hd1 represses flowering under LD, and mutant alleles confer an advantage to varieties adapted to shortgrowing seasons. Several European varieties bear mutant alleles of hd1 and genetic evidence also showed that such mutations can increase Ehd1 transcription and promote flowering. However, targeted association mapping did not detect any significant association between early heading and mutant alleles at this locus. This is likely the result of population structure of the European collection used in this study (Courtois et al., 2012). European varieties are mostly temperate japonica accessions that have been further distinguished into four sub-groups, American, European 1 and 2, and Admixed, based on SSR and SNP markers (Courtois et al., 2012). The American group comprises non-European accessions that contributed hd1 mutant alleles to Europe. Accessions from this group, which have tropical ancestors in their background, are also consistently later than accessions from the other groups (Courtois et al., 2012). When associations with hd1 mutants were corrected for population structure and kinship, they were not anymore significant. Therefore, the full statistical significance and impact of Hd1 alleles in Europe remains to be determined. This specific case highlights some limitations of association analysis, when population structure is strongly correlated with phenotypic differences. Mutant selection as a tool to expand the cultivated area of rice Field data and analysis of F3 families indicated that single mutations in hd1, ghd7 or ghd8 are sufficient to accelerate flowering under NLD or LD, even if not sufficient to produce very fast cycling accessions. Complex genotypes where up to four repressors were mutated in a single variety were observed when mining European germplasm. Yet, the significance of pyramiding of floral repressors remains to be determined. Varieties bearing combinations of ppr37 and ghd7 mutant alleles have been reported as very early flowering and photoperiod insensitive, possibly indicating that targeting these regulators is sufficient to generate genotypes suitable for cultivation at very high latitudes (Ebana et al., 2011;Kim et al., 2013;Koo et al., 2013). Consistently, among European varieties, Auzgusta and Russo1 (ghd7 prr37) were among the earliest flowering accessions when grown in Vercelli. However, ghd7 and prr37 mutant alleles seem to be underrepresented among European rice varieties, at least compared to hd1 and ghd8 mutant alleles. Increasing their frequency, and particularly that of prr37, might facilitate reduction of cycle duration or allow moving cultivation of specific varieties to even northern latitudes. Supplementary Material Supplementary data can be found at JXB online. Supplementary Fig. S1. Natural genetic variation of repressor genes belonging to the mini-panel compared to Nipponbare. Supplementary Fig. S2. Expression dynamics of Ghd7 (A) and PRR37 (B) in Italian varieties grown under NLD. All varieties bear functional alleles of floral repressor genes. Supplementary Table S1. Allelic variants of Hd1, PRR37, Ghd7, Ghd8 and EL1 and heading dates of 247 European rice varieties grown at 5 locations in Europe. Supplementary Table S2. Strategies and primers used for genotyping mutant alleles of LD repressor genes. Supplementary Table S3. p-values obtained from targeted association mapping analyses using different statistical models. NF: non-functional; GLM: General Linear Model, MLM: Mixed Linear Model. Supplementary Table S4. Correlation between Ehd1 and OsMADS51 gene expression levels and heading dates of Italian varieties grown under continuous SD. Supplementary Table S5. Primers used for quantification of mRNA expression.

v3-fos

2017-06-18T00:55:40.848Z

2015-04-08T00:00:00.000Z

10470912

s2

Microencapsulation of bioactives for food applications † Health issues are an emerging concern to the world population, and therefore the food industry is searching for novel food products containing health-promoting bioactive compounds, with little or no synthetic ingredients. However, there are some challenges in the development of functional foods, particularly in which the direct use of some bioactives is involved. They can show problems of instability, react with other food matrix ingredients or present strong odour and/or flavours. In this context, microencapsulation emerges as a potential approach to overcome these problems and, additionally, to provide controlled or targeted delivery or release. This work intends to contribute to the field of functional food development by performing a comprehensive review on the microencapsulation methods and materials, the bioactives used (extracts and isolated compounds) and the final application development. Although several studies dealing with microencapsulation of bioactives exist, they are mainly focused on the process development and the majority lack proof of concept for final applications. These factors, together with the lack of regulation, in Europe and in the United States, delay the development of new functional foods and, consequently, their market entry. In conclusion, the potential of microencapsulation to protect bioactive compounds ensuring their bioavailability is shown, but further studies are required, considering both its applicability and incentives by regulatory agencies. The increasing interest in functional foods Nowadays, food not only serves to satisfy the primal urge of hunger, but also is a means to promote consumer's health. In this context, the food industry has focused on avoiding the potential harmfulness of synthetic food additives and on developing novel food products containing health-promoting ingredients. Therefore, bioactive natural products are considered as viable and safer substitutes to satisfy the world market demand for new products. 1 "Functional foods" arise as the frontier between nutrition and health, providing a long-term beneficial physiological/ health effect beyond their nutritional properties. 1 The concept of functional food appeared 40 years ago, however the growing interest in this type of product, either from industry (through patents) or academia (through scientific research articles and reviews), was only observed from the second half of the 1990s, indicating an increasing tendency (Fig. 1). The exponential growth of patents and scientific research articles/reviews observed since 2005 was accompanied by the regulation (EC) no. 1924/2006 publication by the European Parliament on nutrition and health claims in foods, which was completed and finalized in 2011 by the European Food Safety Authority (EFSA) regarding beneficial health claims in certain food ingredients. 2,3 In the United States (US) the regulation of functional foods is facilitated, as the food industry itself provides the product definition that will be placed on the market; food companies are only obliged to follow labelling and safety rules implemented by the Food and Drug Administration (FDA). 4 Nowadays, consumers' awareness of health issues is growing together with the increasing incidence of chronic agerelated diseases, such as neurodegenerative diseases, diabetes and cancer, usually correlated with the lifestyle and dietary habits of our societies. 5 Moreover, as the life expectancy is rising, with the consequent increase of health care costs, pharmaceutical and food industries have started to consider functional foods as a new market with huge growth potential. Nowadays, Japan, the United States (US) and the European Union (EU) are the leading markets for functional foods, representing in total 90% of the world market supply for this type of product. 6 In 2006, US and EU markets were valued at 33 billion US$ and at 15 billion US$, respectively, with a tendency to grow. Germany, France, the United Kingdom and the Netherlands are considered the most important countries within the European functional food market. 7 The problems related to the use of free bioactives Despite the known beneficial health effects of natural bioactive matrices and isolated individual compounds, as will be discussed in this section, they show some fragility that has to be considered regarding their direct use or incorporation into foods. The main factors limiting the use of bioactives in food applications are shown in Fig. 2. Bioactive ingredients are generally prone to degradation, both during storage and food processing, as many of them are physically, chemically and/or enzymatically unstable leading to their degradation or transformation with the consequent loss of bioactivity. In many cases the mechanism involved in the degradation of these bioactive molecules is very complex and still unknown. 5,8 Wu et al. 9 reported the reduction of the anthocyanin content in blackberry fruits after six months of canned and jam storage and also after drying treatment. Various types of cereals (wheat, barley and oat) were also tested for the content of biologically active compounds, such as tocopherols, phenolic compounds and microelements, and after hydrothermal processing, the concentration of these molecules severely decreased. 10 Rawson et al. 11 described major losses of bioactive compounds after processing exotic fruits such as mangoes, açaí, pineapple and pitanga, subjecting them to heat treatments, pasteurization and drying, canning and even to storage processing steps. All these processes affect, to a lesser or greater extent, the stability, chemical characteristics, concentration, and even antioxidant activity of a number of compounds such as vitamins and phenolic compounds. Another study that describes the modifications occurring in fruits and vegetables during the processing steps was published by Nicoli She obtained her degree in Biochemistry (1996) at the University of Porto, Master's in Sciences (1999), PhD in Sciences-Chemistry (2003), and habilitation in Sciences-Chemistry (2011) at the University of Minho (Portugal). She is the principal investigator of several financed research projects, and an evaluator of international and national research projects, post-doc and PhD grants. She had supervised several post-doc, PhD and master's students in the BioChemCore group. She has received awards from several different organizations such as Calouste Gulbenkian Foundation. She has published over 260 papers in refereed journals and is a highly cited scientist (top 1%) in Agricultural Sciences (Researcher ID: E-8500-2013; ORCID ID: 0000-0003-4910-4882; SCOPUS ID: 7102135224). Her main research interests are nutraceuticals and functional foods, chemistry of natural matrices/products, and emerging technologies for conservation of food matrices. et al., 12 focusing on the antioxidant activity decrease of the food matrix due to the loss and transformation of the antioxidant compounds, and also due to their interaction with other molecules. The processing steps of a food matrix involve the action of endogenous enzymes, water activity, oxygen pressure and also thermal/mechanical energy, and all of these factors can influence the degradation/transformation of the bioactive molecules leading to the loss of their intended characteristics. Nevertheless, not all the compounds are equally affected; phenolic compounds and vitamins (e.g. vitamins C and E) are more sensitive to blanching and long-term freezing treatments than minerals or dietary fibres. 13 Despite the processing steps, the perishability of food is also a limitation in their intake in the free form. This is because the shelf life determines whether a particular food maintains its characteristics and bioactive properties. For instance, edible mushrooms have a very short shelf life and the postharvest changes, such as browning, cap transformation, texture and weight loss changes, occur immediately, which decrease their bioactive components. 14 The ingested amount of the bioactive compound, its structure and chemical form, its interaction with other molecules, and also the organism itself (mucosal mass, intestinal and gastric behaviour, metabolism and protein bonding) will influence the stability and functionality within the human body, and consequently its bioavailability. 15,16 For instance, phenolic compounds present very low bioavailability due to their poor solubility and stability, especially those with high molecular weight. Furthermore, there are no reports on specific receptors on the small intestinal epithelial cell surface, and thus the transport mechanism involves active diffusion and active efflux, lowering the permeability of such compounds. 17 In the case of anthocyanins, they are very sensitive to pH and temperature changes in the medium. 18 Concerning carotenoid compounds, the nature of the food matrix, the particle size and the processing method, and also their interaction with other food constituents, will affect their bioavailability; moreover, fibre constituents decrease the absorption of carotenoids. The nutritional state of the organism itself will influence the absorption of these molecules (e.g., protein deficiency affects the bioavailability). 19,20 As an example, the interaction of mineral elements with other molecules can decrease their bioavailability, as is the case of calcium where compounds such as oxalates, tannins and dietary fibres decrease its absorption due to precipitation. 21 Also, the gastrointestinal environment and epithelial transport can decrease the bioavailability of natural extracts, as described by Vermaak et al. 22 who investigated the biological activity of green tea and sage extracts under simulated gastrointestinal conditions; the authors observed an accentuated decrease in the antimicrobial activity. Lipophilic compounds have also low solubility, which restricts their incorporation into many food matrices, especially in water-based carriers. The molecular weight, functionality and polarity seriously influence their solubility, physical state, chemical stability and bioavailability. 8,23 It is very difficult to evaluate the bioavailability of these types of compounds, since once metabolized they reach the systemic circulatory system where they can be stored, utilized or excreted. Depending on the concentration and time of these molecules in a particular tissue, or their use in some biological function, the bioavailability can be estimated. 24 For instance, the bioavailability of lycopene, a highly lipophilic carotenoid compound, is influenced dramatically by the intestinal lymphatic uptake. Faisal et al. 25 performed by Balakrishnan et al. 26 in order to increase the solubility and bioavailability of the Coenzyme Q 10 , practically insoluble in aqueous medium, by using oil and surfactant compounds for its oral delivery. Another factor that drives researchers to invest their knowledge into the design of novel food delivery systems is the organoleptic behaviour of some bioactive extracts/compounds. They can present unpleasant taste, odour and/or textures. This is a crucial point for the food industry when developing a new product because the consumer gives importance not only to the price, but also to the taste, smell and appearance. Accordingly, consumers will choose the non-functional counterpart of a similar product, even if it has lower bioactive properties. 16,27 It is known that many people avoid eating fruits and vegetables because most of their compounds such as polyphenols, terpenes and glucosinolates have bitter or astringent taste, making them unappealing to the consumer. 28 To overcome the problems related to the direct use of bioactive extracts/compounds, microencapsulation techniques arise as a potential approach in the food industry to deal with their incorporation, either to impart additional functional properties or to protect the bioactive component itself. The main goal of the present review is to highlight the use of microencapsulation techniques for food applications, as well as to discuss the advantages of microencapsulating bioactive extracts/compounds. Various extracts and compounds that have been encapsulated using different techniques and formulations will be enumerated focusing on the potential for functional food development. A particular emphasis will be given to examples where the final application (incorporation into food matrices) is explored. The advantages of using microencapsulated bioactives Microencapsulation can provide a tool to protect natural extracts and compounds from the action of biotic, abiotic, and biological factors. It emerges as a reliable methodology not only for the food industry, but also for the fields of nutrition and health, where the stability, efficacy and bioavailability of these extracts and compounds are needed. As described previously, there are several factors affecting the bioactives' stability in their free form (Fig. 2), however with microencapsulation technology protection from factors such as light, moisture, heat and oxygen is provided. Also, the organoleptic characteristics of many food products can be masked, but most importantly functional/biological characteristics can be maintained after ingestion together with controlled release in a specific target. The success of a delivery system based on microencapsulation can be measured by the bioactives' behaviour during food processing and storage, and after ingestion. 8 From a practical point of view, microencapsulation techniques protect the core material from the outside environment; it increases the product shelf life by reducing the transfer between the core and the surrounding medium, and by protecting the molecules from reaction with other food constituents, which can decrease their bioavailability. 29 It also increases the solubility, dispersibility and flow of the bioactives. 30 Depending on the applied technology and encapsulated bioactive, the response of the produced delivery system will be different; each compound has specific characteristics that should be considered in the design of a novel microcapsulation process. For instance, phenolic compounds are very powerful antioxidant molecules; however they present problems in their bioavailability because they are transformed, after ingestion, into methylated, glucuronated and sulphated metabolites. 31 Nano-and microparticle based delivery systems appear as a response to overcome these problems, increasing the phytochemical absorption of phenolic compounds in epithelial cells. 17,32 In particular, Davidov-Pardo & McClements 33 showed that the microencapsulation of resveratrol increased its bioavailability. Essential oils have also some organoleptic related problems, most of them presenting an unpleasant taste and odour, with very poor water solubility and high volatility. All these limitations can be overcome by using microencapsulation techniques that increase the effectiveness of their biological functions and decrease the sensory impact on food products. 34 Microencapsulation techniques The microencapsulation concept was primarily developed by the pharmaceutical industrial sector, whose goal was to control and/or modify the release of drug substances. Nowadays, it still represents the major field using microencapsulation (68%) while the food sector accounts for only 13%. 35 The amount of scientific reports and patents regarding microencapsulation for food purposes (Fig. 3) is indicative of the growing interest in this technique regarding the incorporation of bioactive extracts and compounds. Nevertheless, the absence of regulation for novel food ingredients, including the ones derived from using nano-and micro-technologies in their preparation, is still remaining. In the USA the FDA is currently developing a recognition program for nanomaterials to overcome the existing scarcity of information, and also to assess food safety of these new ingredients. 36 The introduction of microencapsulation technologies into the food industry allows the incorporation of flavouring agents in certain types of foods, and also the improvement of their functional and health properties. 30,37 Regarding food science and biotechnology, the incorporation of natural ingredients intends to stabilize, protect and preserve the bioactives into a core, surrounded by a wall, or dispersed in a matrix, made of a material chosen to be suitable for the target delivery system. 34 There have already been reviews on microencapsulation of bioactive compounds and extracts for food applications, 29,30,34,37-40 nevertheless they mainly explored the available techniques for microencapsulation, lacking specificity in the existing examples of microencapsulated bioactive extracts and com-pounds together with the applicability of the performed studies. Fig. 4 shows the logical chain, from the choice of bioactives, materials and microencapsulation process to final applications evidencing the crucial points involved in each step. Microcapsules are particles with diameters ranging from 1 to 1000 micrometers. The most common morphology is of two types: (1) shell type, where the core, the bioactive component itself or a carrier containing it (compounds that facilitate the release) is protected by a membrane; (2) matrix type, where the bioactive component is dispersed in a material's matrix. The encapsulation materials, production process, final morphology and ultimate application are the most important factors to be taken into account when designing a novel delivery system based product. Also, the stability and functional properties of the bioactive component must be taken into account when selecting the microencapsulation technique. Furthermore, to achieve high encapsulation yields it is necessary to ensure process reproducibility, release profile and overcome limiting drawbacks such as microsphere aggregation and adherence. 30 The encapsulation methods and materials most commonly used in food applications are described in Tables 1 and 2, respectively (as also in the ESI †). The definition of categories presented in Table 1 was somehow difficult because the microencapsulation processes can be categorized according to the formation mechanism, the consolidation method, and even according to the specific equipment used. A clear distinction among the described possibilities is not always clear in the published work. Therefore, in this work, efforts were made to define categories according to the microcapsule formation process and a set of general categories are proposed: coacervation, extrusion-based processes, spray-based processes, emulsion-based processes, liposomes, supercritical fluid based processes, ultrasound-based processes and others. 2.2.1. Spray-based process. Spray-based processes are by far the most common methods being divided into spraydrying, electrospray, spray-coagulation (according to internal or external gelation) and spray-freeze drying methods. Spraydrying, the oldest microencapsulation process used by the food industry, is a very straightforward technique. It can be described as flexible, allowing continuous production, making it a cost effective process and consequently the most economical among several encapsulation methods. It can be easily industrialized in terms of equipment and materials, which have a low cost compared with other available techniques. 41 The most commonly used shell materials in this technique are carbohydrates which may limit the encapsulation of some bioactives. 39 It produces high quality microcapsules, with a size less than 40 μm, by atomizing a liquid solution or emulsion through a nozzle to a hot gas chamber giving rise to the prompt formation of a powder. The method's speed and effectiveness ensure the production of microbiologically stable products, with lower costs and specific properties. 37,41 There are several applications dealing with the encapsulation of bioactive compounds and extracts by spray-drying. Examples in the published literature are crude extracts, 42-52 carotenoids, 53,54 enzymes, 55,56 essential oils, 57-62 fatty acids, [63][64][65][66] phenolic compounds (including anthocyanins) 67-87 and vitamins. 88 It is also noticeable (ESI †) that the vast majority of the used shell materials, as was previously reported, are carbo- hydrates and derivatives. However, Medina-Torres et al. 72 encapsulated gallic acid in mucilage obtained directly from Opuntia ficus indica, while Cortés-Rojas et al. 61 encapsulated eugenol in lipid formulations, both affording good results and high encapsulation yields. These results show the constant evolution of this method, and the possibility to overcome constraints related to the limited number of available shell materials, as stated by Gouin et al. 39 Coagulation processes are also commonly used to encapsulate bioactive extracts and compounds for food applications, the most common being those based on alginate beads. [89][90][91][92][93][94] Alginate beads are formed from the polyanionic copolymer derived from the brown marine algae, alginate, which is frequently used as a stabilizer and thickener of many food products. Its coagulation can be promoted by external gelation (e.g. using calcium chloride as the calcium source added to the coagulation solution) or internal gelation (e.g. using calcium carbonate as the calcium source added to the alginate solution). In the first case, gelation occurs mainly at the particle surface and in the second case gelation occurs mainly inside the formed particles. The formed materials, due to their degree of ionic reticulation and functionality, allow the control of water intake and thus the release of the bioactive component. 95 The preparation of such alginate beads is easily performed at the lab-scale, and they have been used to encapsulate a wide variety of compounds (hydrophilic, lipophilic, oils among others), and the controlled release is achieved by pH changes. 39,95 Freeze-drying technology allows the encapsulation of many food constituents, being used on a daily basis to stabilize compounds and increase controlled release. 39 It is mostly used to encapsulate bioactive extracts, 96 phenolic compounds, [97][98][99] vitamin C 100,101 and even essential oils. 102 To the best of our knowledge the use of electrospray technology for food applications is not very common and only one work was found in the reviewed literature. 103 This work refers to the encapsulation of folic acid (vitamin B 9 ), and according to the provided description, it is a very appealing technology since the use of organic solvents and high temperatures is not required. Coacervation. Coacervation is the second most commonly used encapsulation technique for food applications, not only because it provides high encapsulation efficiency, but also due to the triggered controlled release that can be based on the temperature, mechanical or biological mechanisms, providing the needed versatility to support the development of a wide range of food products. 39 It can be divided into complex and simple coacervation; the first is based on the complexation of two oppositely charged polymers that form a strong polymeric shell or matrix. 104 For the complex coacervation, chitosan is the preferable wall material, and alginate is the most commonly used polymer in all the mentioned studies. 92,93,[105][106][107] Chitosan has low toxicity, antimicrobial activity, and biocompatibility, but it is mainly muco-adherence that allows transmucosal absorption and better release of the bioactives. 107 In simple coacervation the initially soluble polymer is precipitated by changing the pH or temperature. 34 Milk proteins 108,109 and pectins with PGPR ( polyglycerol polyricinoleate) 110 are some examples of wall materials used in simple coacervation. 2.2.3. Emulsion based process. Emulsion based processes are also commonly used for food encapsulation applications. It allows the encapsulation of both water and oil soluble food ingredients. 34,37 Emulsion based techniques have been successfully used to encapsulate bioactive compounds including fatty acids, 111,112 vitamins, 113 phenolic compounds, 109,[114][115][116][117] anthocyanins, 110-118 oils 119,120 and bioactive extracts. 106,121 This technique is sometimes coupled with a second one, in most cases a spray-drying based process, which gives rise to a dry powder that can be promptly introduced into a food matrix. 37 In fact, several of the commonly used encapsulation processes start with the first step comprising the preparation of an emulsion. This is the reason why a straightforward divi-sion of the encapsulation techniques is not easy to achieve and some superimposition exists. In this work, and given the importance of spray-based processes, the cases dealing with emulsion coupled with spray techniques were included in the spray-based process category. 2.2.4. Extrusion based process. Extrusion methodologies, unlike the above described methods, are not so usual. They can be divided into electrostatic extrusion and co-extrusion. The extrusion method comprises the passage of the polymer melt with the solubilized bioactive through a nozzle, or the polymer melt and bioactive through concentric nozzles, leading to the formation of particles with high density and encapsulation efficiency. 30,37 This technique is primarily used for the encapsulation of volatiles and unstable flavours. 39 Belščak-Cvitanović et al. 105 and Barbosa-Pereira et al. 122 demonstrated the efficiency of this method for the encapsulation of phenolic compounds. Co-extrusion is used to prepare spherical microbeads with a hydrophobic core, 37 nevertheless it can also be used for the encapsulation of hydrophilic compounds in alginate beads as was done by Piazza & Roversi. 123 2.2.5. Liposomes. Liposome technology has been mostly used in pharmaceutical and cosmetic fields, for targeted delivery of therapeutic agents and inclusion of stabilizers in creams and lotions, respectively. For food applicability they represent a highly valuable resource due to their high encapsulation efficiency, stability and easy production. 39 Foremost, liposomes have been used to stabilize and increase the bioavailability of bioactive molecules. [124][125][126][127] Moreover it is widely used to encapsulate compounds that are poorly soluble in certain solvents. Coimbra et al. 128 demonstrated the efficacy of liposomes for the encapsulation of resveratrol, caffeic acid, carvacrol, among others (compounds poorly soluble in water), while Rasti et al. 129 increased the oxidative stability of polyunsaturated fatty acids by means of their encapsulation in liposomes. 2.2.6. Supercritical fluid based process. Supercritical fluid based processes have many advantages for the encapsulation of sensitive substances such as essential oils or enzymes, always being coupled with other encapsulation techniques. Almeida et al. 62 used a supercritical fluid impregnation technique to encapsulate oregano essential oil into a starch matrix, achieving a homogeneous product in a faster way due to the low viscosity and higher diffusion of supercritical CO 2 . On the other hand, Santos et al. 94 by using rapid extraction of a supercritical solution and Sosa et al. 130 and Visentin et al. 87 by using a supercritical antisolvent process applied this technique to encapsulate bioactive extracts with high encapsulation efficiencies. The main advantages of supercritical fluids are related to their physical properties such as viscosity, density, solvating power, diffusion and mass transfer. The solubilisation of the core and shell materials is therefore faster as microcapsule formation is facilitated, i.e. they are formed by using lower temperatures and in the absence of water. 39 2.2.7. Ultrasound based process. Ultrasound based processes, such as sonication and ultrasound, are also reliable techniques for food applications, mostly being used with the double functions of extracting the bioactives and forming the microcapsules. 131,132 However, Kalogeropoulos et al. 133 used sonication to aggregate the inclusion complex of propolis extract and β-cyclodextrins to form microcapsules. 2.2.8. Others. Despite all those described above, there are other methods not so common for food applications. An example is the fluidized bed, a microencapsulation technique for powder compounds. It needs the preparation of a suspension with the coating material ( polysaccharides, proteins, emulsifiers and fats) and subsequent spraying, offering a more effective controlled release of the core material than with other existing technologies. 30,37,39 Li et al. 134 used this technology achieving good integrity and stability of the core compound after the drying process. Molecular inclusion is another process that is not so commonly used, generally referred to as a supramolecular method in the sense that the bonding between the encapsulated compound and the shell material occurs in a cavity-bearing substrate by hydrogen bonds, van der Waals forces or entropy-driven hydrophobic effects. Cyclodextrins and hydrophobic vitamins are the most commonly used shell materials in molecular inclusion methods. 39 Spinning-disk and centrifugal co-extrusion are new atomisation methods, possibly used in modified spray encapsulation methods; the difference lies in the formation of the capsule, involving the creation of a film with much smaller dimensions than those obtained in common atomisers. 39 Akhtar et al. 135 showed the reduction of the particle size using a spinning-disk reactor to encapsulate flavonoids by means of a double emulsion technique, achieving better stabilization of the prepared emulsions by this technique. Other microencapsulation methods that are not commonly used in the food sector are cocrystallization, 136 Encapsulation materials When designing an experiment protocol for the development of encapsulated products (Fig. 4), the shell material choice is one of the most important steps, firstly because it has to be non-toxic to the organism, its preparation should consider environmental issues and use clean solvents (water soluble materials are therefore preferable) and, finally, because it plays a crucial role in the bioactive release behaviour. Conditions such as pH, temperature, salts and ion concentration also have to be taken into account and defined in accordance with the ultimate objective of the developed microcapsules. In this work the materials were divided into four categories (Table 2), according to Kuang et al. 30 which discriminate them as water and non-water soluble materials, and as polymer and nonpolymer materials. Within each category it was also possible to sub-divide them into carbohydrate and its derivatives, protein and its derivatives, synthetic polymers and other types of materials. The coating material and its physical structure strongly influence the product development; nevertheless there are some constraints since law does not allow the application of some materials in food. They must be considered "generally recognized as safe" (GRAS), biodegradable and efficient as the protective barrier between the nucleus and the surrounding medium. Both EU through the EFSA and the US through FDA have many strict rules about material usage for food applications. 37,147 The most commonly used materials are carbohydrate polymers (starch and cellulose and their derivatives), plant exudates and extracts (gum, galactomannans, pectins and soybean polysaccharide), marine extracts (carrageenan and alginate), microbial and animal derived polysaccharides (xanthan, gellan, dextran and chitosan), and also proteins, lipids and others ( paraffin and some inorganic materials). 148 This is in accordance with our survey, where it can be observed that water soluble materials, both polymer (e.g. alginate and chitosan) and non-polymer (e.g. cyclodextrins) types, are the most commonly used, followed by non-water soluble polymers (e.g. starch and casein) and, finally, non-water soluble nonpolymers (e.g. sucrose and lecithin). Concerning the EU, no access is provided to a list of authorized materials for food product development by EFSA. There is a lack of information, as the existing list is under construction. They include only food additives and nutrient sources, listing only those who are not considered food additives (e.g. starch), but without any reference to whether they are authorized or not. 149 Regarding the US, the FDA has a list of approved food ingredients that allows the companies and academia to design microencapsulation protocols more suitable to serve food industry purposes. Despite the above listed compounds, identified as the most commonly used, not all have been approved by the FDA (or they were not considered for review or the assessment is pending). From Table 2, and following the guidelines of FDA, it can be observed that the approved materials are stearic acid, sucrose, amylopectin, maize starch, calcium caseinate, casein, FHCO (fully hydrogenized canola oil), PGPR, β-cyclodextrin, ethanol, lactose, PEG ( polyethylene glycol), alginate, chitosan, whey protein, cellulose, xanthan, ethyl cellulose, soy protein, inulin, pectin and lysozyme. The materials with pending requests for assessment are lecithin, caffeine, arabic gum, milk proteins and poloxamers. For the remaining materials no information is available. It is also necessary to understand that some investigations are conducted to find new encapsulation materials, meaning that although they are not currently present in the FDA list, they could be added in the future. Many of them are of natural origin such as starch from Araucaria angustifolia (Bertol.) Kuntze seeds, 100,101 mucilage extract from Opuntia ficus Indica 72 and gelatinized sweet potato starch, 150 and therefore further studies are needed to establish the safety of these materials. Bioactive extracts The main reason to consider a bioactive extract is related to synergistic effects occurring among their components that often result in increased bioactive characteristics. The information regarding microencapsulated bioactive extracts obtained from different plant materials and other natural matrices after extraction with various solvents is summarized in Table 3. Crude extracts represent a significant part of the microencapsulation studies, followed by polyphenols (as also anthocyanins), essential oils, vitamins, proteins and fat extracts. The majority of the microencapsulation studies for food purposes have focused on the technique development itself which includes the definition of the best suitable materials and the achievement of microcapsules with the adequate morphology, encapsulation efficiency, stability and release behaviour. The studies calling up the development of final applications, i.e. the test of the microencapsulated materials with real food matrices, are much scarcer. Chiou & Langrish 47 used the crude extract (water) of Hibiscus sabdariffa L. for encapsulation with the fibres extracted from the same fruit as the wall material, aiming at developing a novel nutraceutical product using a by-product usually not consumed. A similar study conducted by Berg et al. 70 in which pectin (natural polysaccharide) was used as the encapsulation wall material to protect anthocyanins extracted from Vaccinium genus fruits showed that the addition of gelling substances provided a higher encapsulation efficiency. The optimization of encapsulation methodologies is constantly evolving, as is the case of supercritical fluid-based processes, which were used to encapsulate green tea extract from Camellia sinensis L. leaves with polycaprolactone (PCL), by high pressure antisolvent coprecipitation demonstrating high retention of catechins in the co-precipitates, and also to encapsulate ethanolic extracts from Rosmarinus officinalis L. leaves with poloxamer polymers, with similar results. 87,130 With a different goal, but intending to improve encapsulation and delivery of bioactive extracts, Averina & Allémann 111 developed pH sensitive micro-and nanoparticle containing natural sources of polyunsaturated fatty acids, namely oils extracted from Thymallus baikalensis Dybowski muscle and Pinus sibirica Du Tour seeds, and commercial fish oil, by using the emulsification-diffusion and nanoprecipitation techniques with promising results. Barras et al. 124 developed lipid nanoparticles loaded with polyphenol extracts to enhance their solubility and stability. Many of the studies with phenolic compounds are performed with the main objective to optimize the encapsulation process, 80,118,125,131 using different types of extracts (e.g. alcoholic, aqueous, hydroalcoholic etc.). In fact, there is no specific standard protocols for the extraction of each class of phenolic compounds, depending on the nature of the sample and the objective of the work (structure elucidation and quantification). 151 In terms of proteins, 138,152 vitamins, 88 phytosterols 153 and essential oils, 57,59,60 the majority of the studies was also conducted with the aim of developing new encapsulating methodologies and materials, and optimizing the process. After optimization of the encapsulation process, it is necessary to establish whether the extracts maintain, reduce or increase their bioactive characteristics. Therefore, several bioactivity assays can be conducted to evaluate the antioxidant and antimicrobial activities, and quantify total phenolic compounds. To assess the antioxidant activity, the DPPH (2,2diphenyl-1-picrylhydrazyl) scavenging activity is the most commonly used assay not only to characterize a given sample, but also to evaluate the bioactivity maintenance. The studies performed by López-Córdoba et al. 136 and Chan et al. 154 with crude extracts of Ilex paraguariensis A. St. Hil. aerial parts and Piper sarmentosum Roxb., respectively, showed that encapsulation did not affect, positively or negatively, the antioxidant activity of the extracts. On the other hand, in the studies conducted by Igual et al. 49 and Parthasarathi et al. 43 with Solanum quitoense L. pulp and Garcinia cowa Roxb. fruit, respectively, the encapsulation proved to be very effective, since an increase in the antioxidant activity of the extracts was observed, which can be explained by protection of the bioactives from degradation. Anthocyanin extracts obtained from Garcinia indica Choisy fruit pulp, 68 Euterpe oleracea Mart. fruit pulp 71 and Daucus carota L. roots 67 were encapsulated with maltodextrins, which proved to be efficient in protecting these extracts whose stability and antioxidant activity increased after microencapsulation. With another goal Deladino et al. 90 used the DPPH assay to assess the diffusion and kinetic behaviour of the produced microencapsulated system. The oxygen radical absorbance capacity (ORAC), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid assay (ABTS) and trolox equivalent antioxidant capacity (TEAC) assays have also been used to evaluate the antioxidant activity of microencapsulated extracts. 50,62,76,82,105,115,117 As previously mentioned, the quantification of phenolic compounds is also a very common methodology to assess the effectiveness of the encapsulation process. 42,44,46,48,64,78,79,85,92,98,141,155 Some studies also describe the use of carotenoids to infer the efficacy of the microencapsulation process. 94,141 Antibacterial and antifungal properties are among the most studied and important bioactivities not only due to the increasing resistance of the microorganisms to commercially available synthetic antibiotics, but also because natural matrices present great potential for the discovery of new drugs. There are several studies focusing on the microencapsulation of natural extracts presenting antibacterial and antifungal activities. Sansone et al. 52 Studies considering the improvement of bone quality in rats 121 and in vitro cytotoxicity 107 were performed with micro- encapsulated C. sinensis tea. The antioxidant α-glucosidase inhibitory activity of microencapsulated aqueous extracts from Punica granatum L. peel and the anti-inflammatory effect of commercial polyphenols and oil extracts were also studied. 77,128 As can be observed in Fig. 4, in vitro release studies are among the most important steps to consider when developing and validating a microencapsulated product. A successful microencapsulated system not only has to protect the bioactive compound ensuring its bioavailability, but also needs to guarantee the intended release behaviour (temporal and target oriented). In vitro release studies can be performed by simulating the gastrointestinal environment using pH buffers mimicking the conditions of digestion, 106,156 or using in vitro gastrointestinal models comprising enzymes and pH buffers. 110,133,143,150 Tavano et al. 156 showed, by in vitro release studies, that curcumin and quercetin when microencapsulated in niosomes improved the solubility after gastrointestinal digestion. Frank et al. 110 and Park et al. 150 reported that after in vitro gastrointestinal digestion, microencapsulated anthocyanin extracts of V. myrtillus and a commercial oil extract, respectively, presented good resistance to pH change during digestion, being released only under intestinal conditions. This corroborates the efficacy of microencapsulation in designing adequate delivery systems for water and non-water soluble compounds to be incorporated in innovative food products. Bioactive compounds The importance of studying individual bioactive compounds lies in their powerful bioactivities, with different applications, such as in pharmaceutical and food industries. In this context their isolation from the original matrix is an interesting topic of study and provides an added value to the developed pro-ducts. A set of microencapsulated individual bioactive compounds used for food application purposes is described in Table 4. The number of articles concerning the encapsulation of individual compounds is markedly lower than that for bioactive extracts. However, phenolic compounds are once again the individual molecules most commonly used in microencapsulation experiments. Most of these studies are focused on the development and optimization of microencapsulation techniques, 74,82,132,140,144,145,157 including new encapsulation materials. An example is the work performed by Medina-Torres et al., 72 in which commercial gallic acid was encapsulated using mucilage extracted from O. ficus Indica. Robert et al. 73 also encapsulated gallic acid using acetylated starch and inulin, obtaining higher encapsulation efficiency with the first material. On the other hand, for quercetin and vanillin phenolic compounds, inulin gave the best results. 81 Despite the beneficial health effects of phenolic compounds, their stability and bioavailability are severely compromised during food processing, storage and digestion, as mentioned in the previous sections. So, microencapsulation of individual phenolic compounds could provide a way to maintain or increase their antioxidant activity, 114,139 stability 75,97 and bioavailability. 96,127 The antimicrobial activity was also tested in microcapsules containing chlorogenic acid isolated from Nicotiana tabacum L. leaves, indicating that its activity was not affected by microencapsulation, being an alternative in the development of food products with antimicrobial properties. 158 Polyunsaturated fatty acids are also the target of microencapsulation studies. Their known beneficial health effects make them very appealing to enrich food matrices. However, their lipophilic nature and rancidity tendency are obstacles in the development of efficient delivery systems. Naik et al. 102 developed an encapsulation technique for the delivery of α-linoleic acid isolated from the seeds of Lepidium sativum Linn. using freeze drying to achieve a stable and bioavailable compound. On the other hand, Shaw et al. 66 and Rasti et al. 129 developed different lipophilic delivery systems for commercial ω-3-fatty acids. Shaw et al. 66 used the spray-drying technique with lecithin and chitosan as the wall material, to prevent lipid oxidation and to study the reconstruction of the enriched microcapsules in aqueous medium, showing that this multilayer system is very promising. Rasti et al. 129 used liposome based delivery systems to microencapsulate ω-3-fatty acids, using soybean phospholipids as the wall material. The authors demonstrated that the formation of liposomes in aqueous medium, combined with the antioxidant protection of the phospholipids, increased the stability and prevented fatty acid peroxidation. Other compounds, also very unstable and there-fore benefiting from microencapsulation, are essential oils or their constituents. In addition to the lipophilic character they are also very volatile, needing the protection by microencapsulation. Lipid carriers involve the formulation of a lipidic solution containing solid lipids, surfactants and drying carriers (e.g. polysaccharides) and have provided high encapsulation efficiencies for eugenol and eugenyl acetate isolated from Syzygium aromaticum L. buds. 61 Microencapsulation by co-crystallization of cardamom oleoresin also protected their major components, 1,8-cineole and α-terpinyl acetate; nevertheless, some degradation occurred during packaging and storage. 137 Carotenoids are a family of compounds largely used for food coloration in place of synthetic dyes, presenting additionally antioxidant and antiangiogenic effects. Nevertheless, their tendency to oxidation and isomerization is high. Qv et al. 104 159 studied the stability of lutein and curcumin, respectively, after microencapsulation by complex coacervation with Ca-alginate/k-carrageenan, and Ca-alginate/lysozyme, respectively. Both achieved good encapsulation efficiencies and demonstrated the efficacy of the used method. Spada et al. 100,101 microencapsulated commercial β-carotene in starch obtained from Araucaria angustifolia (Bertol.) Kuntze seeds, and concluded that a modified gelation form of this starch led to higher carotenoid encapsulation efficiency ensuring protection against adverse conditions. Aissa et al. 54 tested microcapsules enriched with β-carotene for its genotoxic and antiangiogenic effects, using arabic gum as the wall material. The authors observed preservation of the genotoxic effects, but a decrease in antiangiogenic activity, maybe due to the loss of bioavailability during microencapsulation. Organic acids, 83 Vitamin B 2 (riboflavin) and vitamin B 9 (folic acid) have also been microencapsulated for food purposes. Due to their known beneficial health effects, coupled with a high tendency to degradation and loss of bioavailability, in vitro release tests were used to evaluate new delivery systems. Chen & Subirade 113 tested the release of riboflavin using simulated gastric, intestinal and pancreatic fluids, concluding that riboflavin microcapsules made of alginate/whey protein are semi-destroyed by the intestinal fluid and completely released with the pancreatic fluid. To estimate the product shelf life, Wichchukit et al. 89 studied the release of riboflavin incorporated into a food product, a model beverage. Prasertmanakit et al. 146 studied the in vitro release of folic acid from ethyl cellulose microcapsules, a material that had good encapsulation efficiency. The addition of a water soluble polymer, sucrose, caused swelling of the polymer matrix, which allowed better controlled release of folic acid. An improvement in delivery system development is the encapsulation of a mixture of bioactive compounds within the same microcapsule, thereby obtaining several beneficial effects. Augustin et al. 112 developed an oil-in-water emulsion to stabilize commercial fish oil, resveratrol and tributyrin using caseinate, glucose and starch, to study their behaviour in the gastrointestinal tract, obtaining increased bioavailability for all the compounds. Pan et al. 109 studied the oxidative stability of curcumin (carotenoid) and retinol (essential oil) in oil-in-water emulsions, with very satisfactory results. Incorporation in food matrices Some examples of applicability studies with microencapsulated bioactive extracts or individual compounds are described in Table 5. After an exhaustive search in the literature, it was confirmed that the vast majority of the studies do not include the validation of the developed microencapsulated bioactives through their incorporation into food matrices. Only twelve studies were found where this final step, so important for the food industry, was included. In general, milk and dairy products such as cheese and yoghurt, and ice creams are the pre-ferable food matrices under study. The sector of cereals, bread and pasta, is also significant in applicability studies. Tea, soup and meat are also food matrices that have been tested for incorporation of bioactive microcapsules. Phenolic extracts of Punica granatum L. peels were studied by Çam et al. 77 and were added to ice cream to enhance antioxidant and α-glucosidase inhibitory activities. Martins et al. 92 and Robert et al. 85 also incorporated phenolic extracts in yogurt using Rubus ulmifolius Schott flowers and Punica granatum L. fruits, respectively. Martins et al. 92 obtained a higher antioxidant activity in yogurt with microencapsulated extracts, compared with the use of extracts in the free form and with the control (yogurts without extracts); on the other hand, Robert et al. 85 also reported a higher content of phenolic compounds and anthocyanins in yogurt with microencapsulated extracts. The incorporation technique developed by Barbosa-Pereira et al. 122 to add phenolic extracts in active packaging to extend the shelf-life of meat products gave promising results retarding lipid oxidation and microbial growth. In terms of individual phenolic compounds, a water soluble isoflavone was microencapsulated in a polyglycerol monostearate emulsion and further incorporated in milk to study its stability during storage and after in vitro digestion. It was demonstrated that the microencapsulated isoflavone did not affect milk taste and that its absorption in the intestine increased. 116 Citric acid and its derivative, (−)-hydroxycitric acid, were also used in incorporation studies; in particular, the derivative extracted from the fruits of Garcinia cowa Roxb. was incorporated into bread 83,99 and pasta; 84 in both cases, bread and pasta enriched with microencapsulated bioactives showed good sensory and quality attributes, which proves the viability of using such strategies in food product development. Citric acid was also incorporated in chewing gum at a micronized scale, using a technique based on casein and inulin to form bioactive microcapsules, to develop chewing gums with health promoting properties. 142 Soups, among the most highly consumed food products worldwide, also served as the matrix for the incorporation study developed by Rubilar et al. 65 Microcapsules containing fatty acids (linseed oil) were added to an instant soup in powder form in order to develop a new functional product; moreover, since the linseed oil was microencapsulated in a polymeric matrix consisting of arabic gum and maltodextrin, a higher controlled release of the lipophilic core was successfully achieved. Sardar et al. 137 also encapsulated a lipophilic compound, cardamom oleoresin. Since the stability of this compound for spray-drying was very poor, a sucrose wall matrix was used with a co-crystallization method giving rise to small flavouring sugar cubes for tea beverages. The produced cubes were stable to storage when packed in a three-layer metalized laminate. Cheese, although appreciated by many consumers, is rich in fat and, therefore, there have been efforts for the addition of vegetable oils to this matrix. However, oils degrade very quickly, benefiting from the addition of antioxidants such as vitamins A and E and coenzymes. In this context, the work of Stratulat et al. 160 was intended to inhibit lipid peroxidation (rancidity), by formulat-ing emulsions, stabilized with calcium caseinate, containing vitamins A and E, and Coenzyme Q10. The results showed that the vegetables oils did not affect the cheese stability, due to the presence of antioxidants. Conclusion Nowadays, food not only serves to satisfy the primal urge of hunger but is also intended to overcome dietary flaws and/or impart health benefits. Bioactives are sources of functional molecules with recognized health effects in populations that otherwise would not be able to benefit from them. Nevertheless, they show organoleptic constraints and instability to food processes, storage and ingestion, which has led to research in the field of bioactive protection and controlled release. Among the proposed technologies, microencapsulation emerged as a viable route to valorise natural bioactives in functional foods, thus extending their benefits to a wider population. According to the present review, there are several examples available of microencapsulation of bioactives using a wide range of processes and encapsulating materials. Among the various possibilities, the spray-based processes, e.g. spraydrying, are the most commonly used techniques. The advantages are its easy implementation, namely at the industrial level, and the fact of being inexpensive. Nevertheless, green techniques, such as supercritical and ultrasound based processes, are nowadays attracting much attention. Water soluble materials, both polymer and non-polymer ones, are the most commonly used encapsulation materials. They include carbohydrate polymers (starch and cellulose and their derivatives), plant exudates and extracts (gum, galactomannans and pectins), marine extracts (carrageenan and alginate), and microbial and animal derived polysaccharides (xanthan, gellan, dextran and chitosan). In most of the cases, the industrial applicability in the field of food production is prevented by current regulations. Crude and phenolic extracts, together with individual phenolic compounds, are the most studied bioactives for food purposes. Nevertheless, studies dealing with final food applications are scarce, demanding investment from academia, industry and regulatory agencies. Finally, the consumers also have a crucial role in the acceptance of new products in the market.

v3-fos

2019-04-13T13:12:26.694Z

2015-03-01T00:00:00.000Z

110147032

s2

Study on the Improvement of Natto-production Process To improve the Natto flavor, in this experiment, different ratios between black soybean and soybean as raw materials were used to explore the suitable fermentation conditions for Natto producing. The results of orthogonal test show that the best condition is: the ratio of black soybean to soybean is 1:4, boiling for 30 min, Bacillus natto inoculation amount 10%, fermentation temperature 38C and fermentation time 13 h. INTRODUCTION Natto is a kind of functional food with rich nutrition, which is made from the fermentation of boiled soybeans by inoculated Natto bacillus. Natto is rich in nutrients such as various organic acids and oligosaccharides, which can be easily absorbed and utilized by the human and it contains a large number of physiologically active substances, such as Nattokinase, vitamin K, vitamin E, superoxide dismutase, soy isoflavones, saponin, tocopherol and B vitamins (wherein its vitamin B2 content is more than 6 times higher than that of the steamed or boiled soybeans) (Ma, 2007); therefore, after being processed into Natto, the nutrient value of soybeans is multiplied. In the course of Natto's fermentation, the Nattokinase is generated, which is a kind of protein kinase of hay bacillus. Sumi Hiroyuki, a Japanese cardiovascular specialist-doctor, found that Nattokinase has the strong function of dissolving thrombus in the study and experiment of dissolving thrombus medicines, which can reduce blood viscosity, strengthen blood circulation and increase the blood vessel elasticity (Wei et al., 2007). So for a long time, Natto has been popular among Japanese consumers. It has been reported as edible and medicinal food and even the unique Nattokinase was made into capsules for thrombosis patients. China has rich soybean resources, non-genetically modified small soybean used for making Natto has higher yield in Northeast China, so we should actively explore the development of soybean products and promote Natto, a kind of richly nutritious health care product. It is not accepted by part of Chinese because of the ammonia smell of Natto, so it is necessary to improve its taste in the case of keeping the nutrients invariable and make it more suitable for Chinese. It was found that Natto produced by fermentation from soybean has more Nattokinases and ammoniacal smell is heavier, but the Natto produced from black bean as the raw material has less Nattokinases and ammoniacal smell is light with good taste. If black bean and yellow bean could be mixed in certain ratio and then ferment to explore an optimal fermentation condition to produce and to improve the taste, a Natto product suitable for Chinese could be developed. MATERIALS AND METHODS Experiment materials: Black beans (purchased from the market), soybeans (a new soybean variety X73 provided by the Agricultural Science Institute of Zigong) and purebred bacillus Natto. The manufacturing craft of Natto: Weigh Accurately five fractions fifty gram soybeans, clean two to three times, clean and decorticate after soaking twelve hours, all of these are put respectively into five stainless steel boxes which are sterilized, steam and boil under the high-pressure 121C, inoculate seed solution when the natural cooling reaches to the indoor temperature, cover the sterile gauze after mixing equably, train respectively in the constant temperature incubator, take out and put them into the 4° freezer to be ripe 24 h. Culture of bacillus Natto seed solution: Make BPY culture medium, use inoculating loop to carry bacillus Natto inoculation to nutrient solution under aseptic conditions and set rotating speed 150 r/min, temperature 37C and culture for 16 h through shaking method. Single-factor experiment: Respectively select ferment time, culture temperature, Natto strain's inoculation amount, stewing time, black bean: yellow bean and beans' peel removed or not, six single factors' influence on Natto's quality to carry on research, each factor selects five gradient variance. Orthogonal experiments: Experiment with three factors and three levels were selected according to the experiment results of the univariate analysis (Table 1). Sensory assessment indicators: Sensory assessment adopts three indicators: drawing situation, color and taste and smell. Seven persons are to carry out the sensory assessment on a scale where the full mark is 100 and the lowest mark is 0. Then the average mark is taken. The ammonia taste generated by Natto during fermentation has an impact on the popularity of this healthy food in our country, so we weight higher on smell (Table 2). RESULTS AND DISCUSSION The effect of fermentation time on the quality of Natto: Figure 1 shows that the quality of Natto changes evidently with the increase of fermentation time. After it reaches the best incubation time, the quality of Natto declines little by little. From the morphological characteristics, we can see that the satins of Natto and its mucus decrease gradually and the colour changes from golden yellow to dark yellow, while the ammonia odor gradually becomes thick and bitter taste appears. Comprehensively assessing the result, the optimal fermentation time of 13 h is chosen. The effect of fermentation temperature on Natto's quality: Figure 2 shows that the sensory score of Natto rises gradually and then declines with the fermentation Fig. 1: Effect of fermentation on Natto quality by sensory evaluation Fig. 2: Effect of fermentation temperature on the quality of Natto temperature increasing, with the peak value of 82.5 points at 39C. At this moment, the grain of Natto are in full sizes, with crisp and soft texture and moist, but it has long drawing, large amount of mucus, a strong stickiness and relatively weak ammonia odor. When the temperature goes gradually up to 43C, the color of Natto is dark with bad texture and strong ammoniacal odor. The consolidated sensory assessment result is that Natto's optimal fermentation temperature ranges from 37 to 39C. The effects of inoculation amount of bacillus Natto on Natto's quality: In Fig. 3, the results showed that the influence of inoculation quantity on the quality of Natto is significant. With too little inoculation amount, the sticky substance on Natto's surface is quite less and drawing is easily strain, the Natto products have some beany flavor. When increasing the inoculation amount, the surface of Natto is dry, drawing decrease, its color goes dark yellow with strong ammonia odor and bitter taste grows. According to the assessment result of the sensory assessment indicator, the optimal inoculation amount should be 9%. Effect of cooking time on quality of Natto: As seen in Fig. 4, the cooking time has certain influence on the quality of the Natto. As the cooking time increases, the Natto tastes tenderer and the color is golden yellow. When the cooking time reaches 30 min, the taste is the best. Continue increasing the cooking time, the texture of Natto goes worse and ammonia odor grows, which affects Natto's sensory assessment. The findings of overall sensory assessment are that, the best cooking time is 30 min. The effects of peeled and not peeled of raw beans on the Natto quality: It can be seen from Table 3, black soybean without peeling can produce Natto products with no ammonia odor and nice tender, but nearly no draw bench grow, worse stickiness and low scores in sensory assessment. Natto products fermented from peeled black soybean contain a small amount of drawing with crisp and soft texture and improved quality. Soybean without peeling produce Natto that has soft texture, moisture content, much mucus, strong in viscous, but the Natto product has ammonia smell, which affects the quality; Natto fermented from peeled soybeans tastes crisp and soft, with much mucus, strong viscidity and large amounts of drawings and slight odor of ammonia. It is shown in the overall sensory assessment findings that choosing peeling process craft after soaking black soybean and soybean can help Natto ferment and improve Natto's quality. The effect of the ratio of black soybean to soybean on the quality of Natto: As it can be seen from Fig. 5, when the black soya beans account for 20% (namely, the ratio of black soya beans to soybeans is 1:4), the sensory evaluation is the highest. At this moment, the Natto tastes soft, is moist, much mucus, strong viscosity and high content of drawing, namely, Nattokinase, so the quality of Natto is the best. With the increasing scale of black soybean, the drawbench of Natto gradually decrease and less mucus, but well texture. The findings of consolidated sensory assessment are that the best scale of black soybean and soybean is 1:4. Orthogonal test: According to observations of the single factor experiment, it can be seen that more drawings were produced when the seed peels of soybeans were removed. In particular, without removing seed peels, the Natto made from black soya beans produced nearly no drawing. And the quality of the Natto produced under the condition of 30-min cooking and 13-h cultivation was significantly better than that of other groups. Therefore, in determining the optimal production process of compound Natto, an orthogonal experiment with three levels or three factors, which are fermentation temperature, inoculation amount and the proportion of black beans and soybeans, was conducted and its combinations is shown in Table 4. Nine experiments were divided into 3 groups as showed in Table 3 and 2. Considering limited lab equipment, the 3 groups would be done at 3 times with the same interval. Take the first group as an example: 3 sets of 50 g black soybean and soybean with the ratio of 1:3, 1:4 and 1:5 were weighed out accurately. All beans in each set were cleaned, soaked for 12 h and peeled, then placed in three stainless plates respectively, steamed for 30 min under high pressure and 121C, cooled down to the room temperature naturally, inoculated with the seed solutions of 4.5 mL (9%), 5.0 mL (10%) and 5.5 mL (11%), respectively in sterile environment. After mixing, the plates were covered with disinfected gauzes and then placed in a thermostatic incubator of 37C for incubation. After 13 h, the plates were taken out and put into a refrigerator of 4C for 24 h for after-ripening. The results are shown in Table 5. It can be concluded from Table 5 that the optimal craft for the composite beans to ferment Natto is A2B2C2, which is fermentation temperature of 38 degree Celsius, inoculation amount of 10%, the ratio of black soya bean to soybean of 1:4. This configuration was evaluated by 7 persons and the selected Natto products had features of weak ammoniacal smell, long drawings and rich mucus. They were of the best quality and scored the highest mark of 65.69 points as shown in the orthogonal experiment table, higher than any other combination. From Table 5, it can also be concluded that the primary factor that affects the fermentation of composite Natto is the fermentation temperature, the secondary factor is the ratio of black bean to yellow bean and then follows the inoculum concentration. Analyze the relationship between the factors of experiments and the levels from this, the range of the overall mark of composite Natto fermentation indicates that the optimal condition of Natto production obtained from this orthogonal experiment is the fermentation temperature of 38°C, the inoculum concentration of 10% and the ratio of black bean to yellow bean of 1:4. Protein content, bacillus Natto and nattokinase activity in produced Natto: From Table 6, it can be seen that different fermentation conditions have significant effects on both the content of bacillus Natto and the activity of Nattokinase. The content of bacillus Natto in Natto products is positively correlated with the activity of Nattokinase. When the Natto bacteria content is higher in Natto products, Nattokinase activity is also higher, such as the third group; when the Natto bacteria content is lower in Natto products, Nattokinase activity is also lower, such as the eighth group. CONCLUSION On the basis of the single factor assay, orthogonal test and sensory evaluation, the following conclusions were drawn. The best production process of excellent Natto is: the ratio of peeled black soya bean and peeled soybean is 1:4; the steaming and boiling time is 30 min; the inoculation amount is 10%; the fermentation temperature is 38C; and the fermentation time is 13 h. It can be seen that the protein content of fermented Natto is increased by 4%, compared with that of steamed and boiled beans. Different processing condition of fermentation has significant effect on the content of Natto bacteria. The order of magnitude of content of Natto bacteria in each gram of Natto prodcts measured in the experiment reaches 109 cfu/g, suggesting the Natto bacteria strain has higher activity. The bacillus Natto content in Natto products through different fermentation technologies positively correlates with Nattokinase activity. When bacillus Natto content is relatively higher, the Nattokinase activity is more active; When the Bacillus subtilis content is relatively low, Nattokinase activity is also relatively low. Natto prepared in the research is golden brown, whole-grained with profuse mucus, plentiful and long drawings, soft taste and acceptable ammonia smell. At the same time, it is found that this kind of Natto contains high content of Bacillus subtilis Natto, with high Nattokinase activity and high content of protein, which is a health food in line with the modern concept of healthy eating.

v3-fos

2018-04-03T02:40:33.585Z

2015-12-01T00:00:00.000Z

13590332

s2

Composition and antifungal activity of Zhumeria majdae essential oil Background and Purpose: Essential oils extracted from different plants are extensively used in perfume, beverage, and food industries and are reported to exhibit antimicrobial activities against a variety of fungi. Zhumeria majdae belonging to the Lamiaceae family is a rare and endemic medicinal plant species in Iran, with a strong and pleasant odor. The leaves of this plant have been used for many years as an antiseptic carminative agent for the treatment of stomachache (especially in infants) and dysmenorrhea. Materials and Methods: Gas chromatography/mass spectrometry (GC/MS) analysis was performed to determine the main constituents of the essential oil extracted from the aerial parts of Z. majdae. Also, the minimum inhibitory concentrations (MICs) were determined, using serial dilution method. Results: Based on the GC/MS analysis, 31 compounds representing 95.36% of the essential oil, extracted from the aerial parts of the plant, were identified, among which linalool (63.40%) and camphor (27.48%) were recognized as the major constituents. The total phenolic content was 42.74 GAE (mg)/DW (g). The hydro-distilled essential oil from the aerial part of the plant displayed potential antifungal activities against all the tested pathogenic fungal species (i.e., Candida albicans, Trichophyton mentagrophytes, Aspergillus flavus, Trichophyton rubrum, Microsporum canis, Microsporum gypseum, and Epidermophyton floccosum). Based on the inhibition zone (29 mm) and MIC value (0.015 μl/ml), all the tested strains were sensitive to Z. majdae essential oil. Conclusion: The present results support the traditional and possible use of Z. majdae essential oil in food, pharmaceutical, and cosmetic industries. Introduction espite modern advances in slaughter hygiene and food production techniques, food safety has become an increasingly important public health concern. Nearly 30% of the population in industrialized countries is estimated to suffer from a food-borne disease each year. In 2000, at least two million people died from diarrhoeal diseases, worldwide [1,2]. In recent years, use of natural substances has gained considerable attention in medical commu-nities, and further research on plant resources has been encouraged to answer questions regarding the safety of synthetic compounds. Essential oils, which are odorous and volatile products of plant secondary metabolism, are extensively applied in traditional medicine, food flavoring and preservation, and fragrance industries [3]. Essential oils, also called volatile or ethereal oils [4], are composed of lipophilic and highly volatile secondary plant metabolites, reaching a mass below a molecular weight of 300 g/mol. Essential oils can be separated from other plant components or membrane tissues. The Inter-national Organization for Standardization (ISO 9235, 1997) has defined essential oil as "a product obtained from vegetable raw material, either by distillation with water or steam, or from the epicarp of Citrus fruits by a mechanical process, or by dry distillation" [5]. So far, nearly 3000 types of essential oils have been recognized, 300 of which are of commercial importance, particularly for pharmaceutical, agronomic, food, sanitary, cosmetic, and perfume industries [6]. Plants D belonging to the Lamiaceae family have been more frequently used as flavoring or medicinal agents, considering the significant amount of the extracted essential oils, compared to other medicinal plants [7]. Zhumeria majdae, which is locally known as "Mehrkhosh" in Hormozgan Province, grows in southeastern Iran. This plant with a strong and pleasant odor belongs to the Lamiaceae family [8,9]. The use of this plant for stomachache and dysmenorrhea has been reported in traditional medicine [10] and its anti-nociceptive and antiinflammatory activities have been recognized by researchers [8]. Z. majdae is used for the treatment of various disorders including diarrhea, cold, acid reflux, and headache and is applied as a carminative for wound healing [11,12]. The present study aimed to describe the chemical composition, total phenolic content, and antifungal activity of essential oils extracted from the aerial parts of Z. majdae. Plant materials The aerial parts of Z. majdae were harvested in the flowering stage from the natural habitat of this plant under artificial plant growth conditions. The harvested plants were dried at room temperature (25°C) for two weeks. Then, the air-dried plants from each habitat were ground and powdered with a mixer for essential oil extraction and other experiments. Preparation of the extracts The plant powder was extracted via hydrodistillation for 3 h, using a Clevenger apparatus. For the infusion, one liter of hot water was added to 100 g of the plant material, boiled for 15 min, and filtered through a cloth. Preparation of the test samples The essential oil (100 μl) was diluted with sterile distilled water to prepare a 5 ml stock solution, which was further diluted to prepare the test samples. Gas chromatography/mass spectrometry (GC/ MS) and GC analyses The analysis of essential oils was carried out, using GC and GC/MS methods. The GC apparatus was Agilent Technology (Model 6890USA, HP) with an HP-5MS capillary column (60 m×0.25 mm, film thickness of 0.25 μm). The oven temperature was initially set at 40°C for 1 min and then raised up to 230°C for 10 min (at a rate of 3°C per min). Helium was used as the carrier gas at a flow rate of 1.0 ml/min. The detector and injector temperatures were set at 250°C and 230°C, respectively. The GC/MS analysis was conducted on the GC system (Model 6890, HP), coupled with a 5973 Network Mass Selective Detector and a capillary column (HP-5MS capillary column). Total phenolic content The total phenolic content of Z. majdae essential oil was specified, using the Folin-Ciocalteu reagent and the method proposed by Spanos and Wrolstad (1990), which was later revised by Lister and Wilson (2001) [13,14]. Afterwards, 2.5 ml dilution (1:10) of Folin-Ciocalteu reagent and 2 ml of Na2CO3 (7.5%, w/v) were added to 50 μl of each sample (three replicates) and incubated at 45°C for 15 min. The absorbance of all the samples was measured at 765 nm, using a SpectraMax-Plus 384 ultraviolet-visible spectrophotometer. The values were expressed as gallic acid equivalent (GAE) in milligram per gram of dry weight (DW). Fungal strains For the bioassays, seven species of different fungi were used: Candida albicans (ATCC Serial dilution method Antifungal activities of essential oils, prepared in a diluent containing dimethyl sulfoxide (DMSO), were determined, using the serial dilution method. For adequate growth, all the strains were grown on Sabouraud's dextrose broth (SDB; Sigma-Aldrich, USA). The aqueous essential oil was mixed with the liquefied agar medium at 45-50°C, poured in a microtube, and left to solidify. Moreover, a microtube series was prepared through increasing the concentration of the essential oil. By using the applicator, different strains were inoculated on each plate. Following overnight incubation, the minimum inhibitory concentration (MIC) endpoint was calculated by placing the microtube against a dark background and determining the lowest concentration of derivatives impeding visible growth; the MIC of essential oil was recorded in μg/ml. The test was repeated in case two or more colonies persisted beyond the determined endpoint or if growth was observed at a higher concentration (not a lower concentration). The punch well/cup plate diffusion method In this technique, the melted agar, inoculated with a variety of microorganisms, was poured in Petri dishes of the agar medium. After the agar settled, cups were placed in the agar Petri dishes. Based on the results obtained by serial dilution method, Z. majdae essential oil solutions were prepared at the following concentrations: 0.007, 0.015, 0.031, 0.062, 0.125, 0.25, 0.5, 1, 2, and 4 μg/ml, respectively. Statistical analysis All experimental measurements were carried out in triplicate and the values were expressed as the average of three analyses (± standard deviation). The correlation between variables was assessed, using SPSS version 19 (Chicago, IL, USA). Essential oil composition The GC/MS analysis of essential oils led to the identification of 17 different organic compounds, representing 99.13% of the total content of oils, extracted from the aerial parts of Z. majdae. The identified chemical compounds are listed in Table 1, according to their elution order on the capillary column. The essential oils mainly contained a complex mixture of oxygenated mono-and sesquiterpene hydrocarbons. Total phenolic content and antioxidant activity The total phenolic content of Z. majdae essential oil was measured by Folin-Ciocalteu reagent and expressed as GAE (standard curve equation: y=0.04812x+0.0452, R 2 =0.9901; data not shown). The total phenolic content of compounds in the extracts was 42.74 GAE (mg)/DW (g). Antifungal activity The antifungal activities of Z. majdae essential oil are presented in tables 2 and 3. The antifungal (−) Represents no growth inhibition/resistance; (+) represents growth inhibition/susceptibility activity was assessed by the measurement of inhibition zone, using a film disk containing the antifungal agent [15]. The results showed that the control films did not inhibit the growth of pathogenic fungal strains (n=7). Z. majdae essential oil showed antifungal effects against all the studied fungal strains. In general, the essential oil showed significant inhibitory effects (7.84-29.05 mm). Based on the findings, the essential oil was most effective against C. albicans (inhibition zone diameter= 29.05 mm and MIC= 0.031 μl/ml), while A. flavus (inhibition zone diameter= 7.84 mm and MIC= 0.25 μl/ml) was the most resistant species. It should be noted that all the samples showed very strong antifungal activities. Essential oil composition The present results showed that linalool and camphor are the main components of Z. majdae essential oil. Linalool (a terpene alcohol chemical) and camphor (a terpenoid) are naturally occurring compounds, which can be found in Multiple commercial applications are attributed to these compounds, the majority of which are based on the induced pleasant odor [16][17][18]. In this study, the composition of Z. majdae essential oil was similar to that described by other researchers. According to a study by Rustaiyan (1992), Z. majdae essential oil mainly consists of monoterpenes (about 97 %). The ratio of linalool to camphor in this plant was nearly 1:1 in 1988, whereas in 1990, this ratio was reported to be approximately 2:1 [19]. Some sesquiterpenes (about 1%) occur only in traces of Z. majdae [19]. Based on a study by Javidnia et al. (2006), the yield of Z. majdae essential oil was 0.04% (w/w) and linalool and camphor, as the main compounds of the essential oil from the aerial parts of the plant, accounted for 2.1% and 0.8% of the stem oil, respectively [20]. Total phenolic content and antioxidant activity Antioxidants are able to reduce or prevent lipid oxidation in food products [23]. In fact, synthetic antioxidants have been extensively applied to impede lipid oxidation in foods [24]. However, use of such synthetic antioxidants is not preferred due to toxicological concerns. Based on previous findings, at a concentration of 50 mg/kg/day (500 times higher than the mentioned value), butylated hydroxyanisole and butylated hydroxytoluene seem to be free of any apparent adverse effects [25]. Based on the aforementioned background, there has been an increasing interest in identifying plant extracts to minimize or inhibit lipid oxidation in lipid-based food products [26]. Most of these natural antioxidants originate from fruits, vegetables, spices, grains, and herbs [27]; consequently, detection and application of more effective antioxidants are essential [28]. Antioxidant supplements may be used to help reduce oxidative damage in the human body [29]. Plants belonging to the Lamiaceae family have been used more frequently as antioxidant agents, compared to other plants, given the significant amount of the extracted essential oils [9]. According to a study by Moein and Moein (2010), the highest amount of phenolic compounds (1.98±0.01 mg/g) was detected in ethyl acetate extracts of Z. majdae [12], while in a study by , the total phenolic content was the highest in the methanolic extracts (50.1±2.3 μg/mg) [9]. The variability in the total phenolic content in the conducted studies could be attributed to the changing solubility of phenolic compounds; also, the variation in solubility may be related to the solvent polarity [30,31]. In some previous studies, methanol and ethanol were considered as more valuable solvents for phenolic extraction from plant materials, compared to less polar solvents such as acetone and hexane [31][32][33]. The present study suggested that Z. majdae has a high phenolic content and exhibits significant antioxidant activities. These findings suggest that the major part of antioxidant activities in Z. majdae results from the phenolic compounds. Antifungal activity An antifungal agent selectively eliminates fungal pathogens from the host while inducing minimal toxicity [34]. According to recent studies, the essential oils of various plants belonging to the Lamiaceae family have a broad range of biological activities, notably antifungal potency, which is generally correlated with the chemical composition of the essential oil [35]. Overall, terpenes and flavonoids (natural phenols) constitute the active antifungal compounds of essential oils. It seems that the mechanism of antifungal or antibacterial action may be related to that of other compounds [36]. In the present study, Z. Majdae essential oil showed antifungal effects against all the tested fungi. Based on some earlier studies on the antibacterial properties of essential oils extracted from several species (from various genera), diverse degrees of growth inhibition were reported against some Staphylococcus and Bacillus species due to varying chemical compositions of the essential oils [37][38][39]. In a study by Burt (2004), Z. majdae essential oil, given its antifungal properties, could be potentially used in aromatherapy, pharmaceutical sciences, and pathogenic systems for the prevention of microbial growth. Therefore, Z. majdae could become an alternative to synthetic fungicides for application in agro-industries. Moreover, this plant could be used to screen and develop selective and natural fungicides for the treatment of many microorganisms, causing severe destruction of crops, vegetables, and ornamental plants. Conclusion The present results support the traditional and possible use of Z. majdae essential oil in food, pharmaceutical, and cosmetic industries. the research and edited the final manuscript. H. F. helped analyze the data. Conflicts of interest No potential conflicts of interest were reported in this study. All authors are responsible for the content and writing of the paper. Financial disclosure No financial interests related to the material of this manuscript were declared.

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2015-05-19T00:00:00.000Z

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Will selenium increase lentil (Lens culinaris Medik) yield and seed quality? Lentil (Lens culinaris Medik), a nutritious traditional pulse crop, has been experiencing a declining area of production in South East Asia, due to lower yields, and marginal soils. The objective of this study was to determine whether selenium (Se) fertilization can increase lentil yield, productivity, and seed quality (both seed Se concentration and speciation). Selenium was provided to five lentil accessions as selenate or selenite by foliar or soil application at rates of 0, 10, 20, or 30 kg Se/ha and the resulting lentil biomass, grain yield, seed Se concentration, and Se speciation was determined. Seed Se concentration was measured using inductively coupled plasma optical emission spectrometry (ICP-OES) after acid digestion. Seed Se speciation was measured using ICP-mass spectrometry with a high performance liquid chromatography (ICP-MS-LC) system. Foliar application of Se significantly increased lentil biomass (5586 vs. 7361 kg/ha), grain yield (1732 vs. 2468 kg /ha), and seed Se concentrations (0.8 vs. 2.4 μg/g) compared to soil application. In general, both application methods and both forms of Se increased concentrations of organic Se forms (selenocysteine and selenomethionine) in lentil seeds. Not surprisingly, the high yielding CDC Redberry had the highest levels of biomass and grain yield of all varieties evaluated. Eston, ILL505, and CDC Robin had the greatest responses to Se fertilization with respect to both grain yield, seed Se concentration and speciation; thus, use of these varieties in areas with low-Se soils might require Se fertilization to reach yield potentials. Introduction Selenium (Se) is an essential element for humans, animals, and certain algae to make selenoproteins. However, no physiological Se requirement has been shown for higher plants (Pilon-Smits and Quinn, 2010). Nevertheless, some studies have demonstrated benefits of Se application with respect to the productivity of certain vascular plants (Hu et al., 2003;Hartikainen, 2005;Smrkolj et al., 2006;Turakainen, 2007;Lyons et al., 2009). For example, Se application increased Se concentration in potato tubers, tea leaves, and field pea seeds (Hu et al., 2003;Smrkolj et al., 2006;Turakainen, 2007). The metabolic basis of such effects is unclear. Plants take up Se from the soil primarily as selenate or selenite, which they translocate and assimilate into organic forms (Lauchli, 1993;Ellis and Salt, 2003;Pilon-Smits and Quinn, 2010). In the chloroplast, adenosine-5-phosphoselenate is formed by the activation of ATP sulfurylase. Selenate is reduced to selenide to form selenocysteine (SeCys), which can then be converted into selenomethionine (SeMet), and methylated metabolites including Se-methylselenocysteine (methylSeCys), and dimethylselenide (Terry et al., 2000;Pilon-Smits and Quinn, 2010). Selenomethionine and SeCys are the major organic forms of Se found in legumes (Wu et al., 1997). Plants grown on seleniferous soils can be broadly categorized as Sehyperaccumulators, which incorporate high concentrations of Se (100s of ppm), or Se-non-accumulators, which accumulate relatively low concentrations of Se (<50 ppm). Lentil (Lens culinaris Medik) is a non-accumulator grain legume, with concentrations reaching 0.6-1.0 ppm Se when grown in the high-Se soils of western Canada and the midwestern USA (Brown and Shrift, 1982;Thavarajah et al., 2008). Notably, Se in naturally grown lentils is present as 90% of organic and 10% of inorganic Se (Thavarajah et al., 2007(Thavarajah et al., , 2008. Detailed analyses of lentil seeds indicate most organic Se is present as the seleno-amino acid SeMet, with small concentrations of SeCys and other seleno-oligopeptides such as gammaglutamylselenocysteine (Thavarajah et al., 2008). International lentil samples vary significantly with respect to organic and inorganic Se forms, suggesting differences in the bioavailability of Se to human consumers (Thavarajah et al., 2011). Interestingly, a recent field study indicates that Se application increased grain yield, antioxidant activity, and seed Se concentration (Ekanayake et al., 2015). However, this study was conducted in high Se mid-western soils with high yielding lentil cultivars grown in the USA and Canada. Thus, it appears that Se-fertilization can improve Se nutrition, making lentils a good source of Se for Sedeficient populations. Globally, 30-100 M people are Se-deficient, mainly due to low concentrations of Se in commonly eaten foods (Combs, 2001;Ellis and Salt, 2003). The nutritional benefits of Se were first reported in Schwarz and Foltz (1957), since then, Se necessity in enzymes, cofactors, antioxidants, and protective pathways have been discovered (Combs, 2015). Selenium has multiple biological activities in mammals, depending on the level of Se intake. Low dietary Se intakes determine the expression of selenoenzymes, and higher intakes have been shown to have anti-tumorigenic potential; and very high Se intakes can produce adverse effects including selenosis and type 2 diabetes (Combs, 2015). The recommended tolerable upper Se intake level for adults is 400-450 µg/day (Food and Nutrition Board, 2000). Enriching lentils with Se may thus be an effective and sustainable means of increasing Se intakes (Thavarajah et al., 2008(Thavarajah et al., , 2011. Vascular non-accumulator species respond metabolically to low dosage Se fertilization. Germ et al. (2005) demonstrated a significant (1.6-fold) yield increase in pumpkin (Cucurbita pepo L.) grown in the field after foliar Se application. In lettuce (Lactuca sativa L.), Se application was reported to increase shoot yield and increase energy reserves, produce structural changes in cell walls, increase tissue antioxidant capacity, confer protection of chloroplast enzymes, and increase senescence (Pennanen et al., 2002). Similarly, foliar application of Se has been found to increase grain yield, plant height, number of pods per plant, and harvest index of canola (Brassica napus L.; Lyons et al., 2009;Zahedi et al., 2009). Rahman et al. (2014) demonstrated that foliar application of a single dosage of Se fertilizer increased lentil seed Se concentration from 201 to 2772 µg kg −1 ; however, application of Se fertilizer did not effect on lentil grain yield (Rahman et al., 2014). In contrast, Ekanayake et al. (2015) showed application of Se at seeding and flowering increased lentil grain yield, seed Se concentration, and antioxidant levels. Pulse crops, mainly lentil, are staples in developing countries and part of the daily diets of many vegetarians. About, two-thirds of the world's lentils are produced by smallholders in resource-poor countries where soil is deficient in Se (Thavarajah et al., 2011). Soil application of the trace element Se can increase lentil grain yield by a significant amount (Ekanayake et al., 2015) challenging the current thinking that Se is not essential for plants. Therefore, detailed control environmental studies are required to confirm the response of lentil accessions selected for international lentil breeding program with response to low dosage of Se influence on plant growth, grain yield, seed Se concentration, and seed Se speciation. Therefore, the objectives of this study were to determine the effect of Se fertilization (form, Se application method, and rate) on agronomic (biomass production and grain yield) and compositional (seed Se concentration and Se speciation) characteristics of selected lentil genotypes under controlled conditions. Materials Standards, chemicals, and high-purity solvents used for seed digestion and analysis were purchased from VWR International, Sigma Aldrich Co. (St. Louis, MO, USA), and Alfa Assar-A Johnson Matthey Company (Ward Hill, MA, USA) and used without further purification. Greenhouse Experiment Lentil genotypes included three cultivars widely grown in the USA and Canada (Eston, CDC Redberry, and CDC Robin) and two lentil accessions (ILL 7537 and ILL 505). Seeds were obtained from the USDA-ARS Grain Legume Genetics and Physiology Research Unit, Washington State University, WA, USA, and were multiplied using single plants at the former Pulse Quality and Nutrition Laboratory, North Dakota State University (NDSU), Fargo, ND, USA. These lentil genotypes were chosen based on their current agricultural use in the USA and Canada, as well for the potential for follow-up genetic studies. More than 100 surface-sterilized seeds from each genotype were placed on sterile petri dishes with absorbent paper saturated with deionized water. Pre-germination was conducted in a dark wooden drawer at 22 • C for 2 days until radical emergence. Three pre-germinated seeds from each genotype were sown in 6 plastic pots filled with ∼150 g of a peat-perlite-vermiculite mixture (Sunshine Grow Mix Number 1, Sun Gro Horticulture Canada Inc., Toronto, ON, Canada) containing 10-15 µg Se/kg (equivalent to 51-76 g of Se/ha). The soil in each pot was saturated with deionized water and allowed to drain overnight before the weight was recorded. At seeding, pots were at 70% field capacity. Greenhouse conditions were as follows: day/night temperatures of 22/16 • C, photosynthetically active radiation levels of 300 µmol/m 2 /s using a 16 h photoperiod, and 50-60% relative humidity. A total of 240 pots were seeded: three replicates of the five genotypes at four deferent Se fertilizer rates [0 (control), 10 (low), 20 (moderate), and 30 (high) kg of Se/ha], using two application methods [at seeding as a soil treatment and at flowering (10th node stage) as a foliar treatment] and two chemical forms (potassium selenate and potassium selenite). Blocks of 120 pots were separated for soil and foliar Se treatment, respectively. Soil Se Treatment Aqueous solutions of potassium selenate or potassium selenite were made to provide the above-mentioned application rates. At seeding, 1 mL of Se solution was added to each pot followed by 100 mL distilled, deionized water. Control and foliar treatment groups received no Se at seeding. All pots were watered to ∼70% of free draining moisture content every day and 250 mL of nutrient solution without Se were added to all pots every 2 weeks, as per standard procedures for lentils at the NDSU Pulse Quality and Nutrition program. Nutrient concentrations of the all-purpose 20-20-20 fertilizer solution (Plant Products Co. Ltd., Brampton, ON, Canada) were 20% total N, 20% total P, 20% soluble K, 0.02% B, 0.05% chelated Cu, 0.1% chelated Fe, 0.05% Mo, 0.05% Zn, and 1% EDTA. Foliar Se Application Plants were allowed to grow for 43 days, by which time all had begun flowering. Each Se rate (0, 10, 20, 30 kg of Se/ha) was carefully prepared in a 1 L, hand-held sprayer and each plant received one application of ∼80 mL applied within 5 s to the foliage. Control plants were treated with deionized water containing non-detectable (<0.01 ppm) concentrations of Se. During the foliar treatment, plants of the soil treatment series were covered and removed to a distant part of the greenhouse to avoid cross-treatment contamination. Sample Preparation At physiological maturity, all plants from both treatment series were hand-harvested and air-dried (40 • C); biomass was recorded as dry weight (DW). Plants were then hand-threshed and the total seed weight per pot recorded. Seeds were stored at −40 • C until analysis. Se Analysis Total Se concentration in lentil seeds was determined using inductively coupled plasma optical emission spectrophotometry (ICP-OES) after nitric acid-hydrogen peroxide digestion (Thavarajah et al., 2008). Finely ground seed samples (500 mg) were digested in nitric acid (70% HNO 3 ) at 90 • C for 1 h. Samples were then further digested with hydrogen peroxide (30%) before being diluted to 10 mL with nanopure water. Se concentrations were measured using ICP-OES (ICP-6500 Duo, Thermo Fisher Scientific, Pittsburg, PA, USA). Total Se measurements using this method were validated using National Institute of Standards and Technology (NIST) standard reference material 1573a (apple leaves; [Se] = 0.054 ± 0.003 mg kg −1 ). A homogenized laboratory reference material (CDC Redberry: Se = 400 ± 100 mg kg −1 ) was also used periodically for quality control. A calibration curve for Se concentration was produced using serial dilutions from 1 to 40 mg L −1 . The limit of detection for this method was 10 ppt. Se Speciation Seeds from control and low Se (10 kg Se/ha) treatments for both application methods and both Se forms were selected for analysis of organic Se species. Composite triplicate samples were ground to a fine powder, 250 mg of which was then mixed with 4 mL of nanopure water. Samples were digested with 10 mg of protease XIV (Streptomyces griseus) at 38 • C for 90 min, after which they were centrifuged for 5 min (5000 g) and filtered through a 0.5 µm polytetrafluoroethylene (PTFE) membrane (Thavarajah et al., 2008). Selenium species were determined by high performance liquid chromatography (HPLC)/vapor generation/ICP-MS as previously described (Kuehnelt et al., 2006). A sample size of 30 µL was injected. The mobile phase flowed at 1 mL/min and was comprized of a phosphate buffer adjusted to pH 3.0 (5 mM NH 4 H 2 PO 4 adjusted with H 3 PO 4 ) with 1% NaBH 4 and 5% HCl. Speciation of different Se forms was determined by ICP-MS (Elan DRCII, ICP-MS, Perkin Elmer Waltham, MA, USA) with a PE Series 200 HPLC fitted with micro pumps (Perkin Elmer, Waltham, MA, USA), a Waters Spherisorb 5 µm ODS2 4.6 × 250 mm column (Waters Corporation, Milford, MA, USA), and a Varian VGA-77 gas liquid separator (Agilent Technologies, Santa Clara, CA, USA; Kuehnelt et al., 2006). Standards (selenate, selenite, SeCys, SeMet, and Se-methylSeCys) were prepared at 40 ng Se mL −1 . Statistical Analysis The experiment used a completely randomized design with five lentil cultivars, three replicates per cultivar, four Se rates, two Se forms, and two Se application methods (n = 240). Data from replicates were combined and data error variances tested for homogeneity. For combined analysis, a mixed model analysis of variance was performed using the PROC GLM procedure of SAS version 9.3 (SAS, 2010), with genotypes, Se forms, Se rates, and application method as the class variables and replicates as a random factor. A separate analysis of variance was performed for each class variable to examine the effect of lentil genotype on biomass, seed yield, and total seed Se concentration. Means were separated by Fisher's protected least significant difference (LSD) at p < 0.05. For Se speciation, data presented as a mean with SE (n = 8). Results Combined analysis of variance showed that Se fertilization method, rate, and lentil genotype significantly affected lentil biomass, grain yield, and seed Se concentration at P < 0.05. Se form significantly affected lentil grain yield at P < 0.1 and seed Se concentration at P < 0.05. Interaction terms including lentil genotype, application method, rate, and forms were significant in some cases (Table 1). Selenium application significantly increased lentil biomass and grain yield as well as the Se concentration of the edible portion of the plant, i.e., the seed. The magnitude of the effect varied with the method and rate of Se application as well as lentil genotype ( Table 2). Se fertilizer form did not affect lentil biomass or grain yield, but lentil seed Se concentration was significantly greater after selenate fertilization (2.2 µg/g) than after selenite fertilization (1.1 µg/g; Table 2). Foliar application of Se was the most effective method of Se fertilization with respect to increasing lentil biomass, grain yield, and seed Se concentration ( Table 2). Both low (10 kg/ha) and high (30 kg/ha) rates of Se fertilization increased lentil biomass and grain yield compared to the control (Table 2). Consequently, low rate of Se fertilization was adequate in lentils to achieve optimum biomass (6538 kg/ha) and grain yield (2122 kg/ha). Se addition at the lowest rate (10 kg/ha) increased the seed Se concentration from 0.2 (control value) to 1.3 µg/g. As expected, CDC Redberry (a high yielding red lentil genotype) showed significantly higher biomass (10,522 kg/ha) and grain yield (2,815 kg/ha) than all other lentil genotypes and had moderate concentrations of seed Se (1.5 µg/g; Table 2). Overall, foliar application of Se at low rate (10 kg/ha) significantly increase lentil biomass and grain yield with adequate amounts of seed Se levels. Concentrations of different Se forms present in lentils were affected by the form of Se added, rate (control and low), and application method. In general, both application methods and both forms of Se increased concentrations of organic Se (SeCys, SeMet, and methyl-SeCys). Selenium fertilization significantly increased SeMet concentrations in most lentil genotypes, with the highest concentration noted in ILL7537 (1484 µg/kg) and the lowest in CDC Robin (13 µg/kg; Table 3). Soil selenate fertilization increased SeMet levels in Eston, CDC Redberry, CDC Robin, and ILL 7537 compared to ILL505 (Table 3). Foliar Se fertilization was more effective with respect to assimilation of SeMet in ILL7537. Response to added Se fertilizer varies with lentil genotypes. Low rate of soil selenate fertilization significantly increased biomass growth in all lentil genotypes except ILL 7537 compared to controls ( Figure 1A). However, low rate of soil selenate significantly increased lentil grain yield in Eston and CDC Robin (Figure 1C). High rate of soil selenate fertilization increased yields in all genotypes except CDC Redberry grain yield. Overall, low rate of selenate fertilization was adequate to increase lentil grain yield for small seed size lentil cultivars, Eston and CDC Robin. Biomass yields of Eston, ILL505, and CDC Redberry responded positively to soil selenite fertilization at moderate and high rates (Figure 1B), however, soil selenite fertilization did not effect on grain yields of lentil genotypes except Eston (Figure 1D). Low and high rates of foliar selenate application increased biomass yield for Eston, ILL 7537, and CDC Robin compared to controls (Figure 2A). Low rate of foliar selenate significantly increased lentil grain yield in Eston, ILL7537, and CDC Robin ( Figure 2C). In contrast, foliar selenite treatment did not influence biomass or grain yield for most genotypes except ILL 7537 for both yields and CDC Robin only at biomass (Figures 2B,D). Overall, ILL 7537 was the least responsive and Eston was the most responsive to Se fertilization. Lentil seed Se concentration vary with Se fertilizer form, application method, and rate. In addition, most lentil genotypes preferred selenate as a fertilizer form compared to selenite. Low and moderate rates of soil selenate significantly increased seed Se concentrations in Eston and ILL 7537 compared to other three lentil genotypes (Figure 3A). Eston and ILL7537 showed the most response to added Se in most cases ( Figure 3A); CDC Redberry and CDC Robin increased seed Se concentrations with added Se, and ILL505 showed moderate responses to all Se rates. Foliar application of selenate increased the seed Se concentration in all lentil genotypes compared to selenite foliar treatment ( Figure 3B). ILL 7537 showed the most significant response to added foliar Se at the low rate; ILL505 showed the least significant responses to all Se rates ( Figure 3B). Discussion Selenium is an essential element for mammals but has not been considered an essential element for higher plants, although benefits from Se application have been documented (Hartikainen and Xue, 1999;Turakainen et al., 2004;Lyons et al., 2009). A recent study indicated that application of selenite and selenate as a single dosage increased field grown lentil grain yield by 10 and 4%, respectively, compared to the control (Ekanayake et al., 2015). In order to investigate the impact of soil Se levels, their study should be repeated in soils with low and high levels of Se. These findings are consistent with previous research that shows crop plants respond to Se fertilization, e.g., increased tuber yield in potato (Solanum tuberosum L.), growth in lettuce (Lactuca sativa L.; Hartikainen and Xue, 1999;Turakainen et al., 2004), seed yield in mustard (Brassica rapa L.; Lyons et al., 2009), and lentils (Ekanayake et al., 2015). The application of Se, particularly as selenate, increased the nutritional value of lentil seed, as evidenced by the general increase in seed concentration of total Se and SeMet. This approach has been used in Finland to increase the Se content of foods and thereby the nutritional Se status of the general population (Wang et al., 1995). Approximately, 86-95% of Se in naturally grown lentil seeds is present in organic forms, which we modeled as SeMet with a smaller amount (5-14%) as selenate (Thavarajah et al., 2007(Thavarajah et al., , 2008. Both forms are likely to be readily bioavailable to humans. The present findings indicate that Se fertilization can increase the amounts of Se metabolites in the seed, with responses varying among lentil cultivars. Eston and ILL7537 increased seed SeMet concentration about 4-10 folds with soil application of selenate fertilizer at a low rate. ILL 505 was the least responsive lentil cultivar to Se fertilizer for increase seed SeMet concentration. Our results clearly indicated that Eston and ILL 7537 would be the most suitable cultivars for further Se enhancement studies. The Se concentrations of lentils produced from different parts of the world vary with soil Se level (Thavarajah et al., 2008(Thavarajah et al., , 2011. Lentils produced in Nepal (147-254 µg kg −1 ), Turkey (30-67 µg kg −1 ), Morocco (6-65 µg kg −1 ), and Australia (110-174 µg kg −1 ) have lower Se levels than those produced in Canada FIGURE 1 | Genotypic variation of lentil biomass yield (A,B) and grain yield (C,D) with response to soil application of selenate and selenite fertilizer at different rates. Each genotype (bars) for each Se rate followed by different letters are significantly different at P < 0.05. (425-673 µg kg −1 ; Combs, 2001;Thavarajah et al., 2008Thavarajah et al., , 2011. Rahman et al. (2013) conducted a farmers' lentil field survey in Bangladesh in 2010-2011 to determine the correlation between seed Se and soil Se concentration (Rahman et al., 2013). Their study indicated mean soil and lentil seed Se concentrations of 163 and 312 µg kg −1 , respectively. Our study data, comparing added Se fertilizer rates and seed Se concentrations, suggest that the application of Se fertilizer substantially increased lentil seed Se levels (Figures 3A,B). Furthermore, our data clearly showed that adding Se boost lentil grain yield. Therefore, we suspect that Se fertilization can be most effective in contributing to seed Se and grain yield in low-Se soils, i.e., <5 ppm Se. Soil Se is uneven in distribution and bioavailability to plants (Combs, 2001). This suggests that soil Se concentration might play an important role in determining the final grain yield and seed Se concentration. Lentil is one of the oldest domesticated pulse crops, originating in the Mediterranean region, Asia, and Europe in the Bronze Age. Lentil was introduced to the USA in 1916 (Baun et al., 2008). Of the five genotypes considered here, Eston, CDC Robin, and CDC Redberry are commercial genotypes with a broad range of genetic diversity for Se uptake in high Se soils (Thavarajah et al., 2008). The two selected breeding lines (ILL505 and ILL 7537) are currently used in ICARDA's lentil breeding program for Se biofortification. Eston and CDC Robin are closely related, as CDC Robin was created from a cross between CDC Matador and Eston. Eston originated from line ILL176 found in Turkey. ILL505 originated in Germany and ILL7537 in Syria. All of these parental lines were well adapted to low Se soils. CDC Redberry is a popular high yielding genotype from the Crop Development Centre, Canada, the parental information for which is not currently available. Our findings show that Eston, CDC Robin, and ILL7537 are likely candidates for Se biofortification in low Se soils. The physiological basis of the positive response of crop plants to Se is puzzling in view of the fact that genomic analyses of higher plants have not revealed sequences for selenoproteins (Lobanov et al., 2009). However, this view has changed after the first de novo assembly and annotation of a complete mitochondrial genome in American cranberry (Vaccinium macrocarpon Ait.; Fajardo et al., 2014). Fajardo et al. (2014) revealed the presence of two copies of tRNA-Sec and a SeCys insertion sequence element which were FIGURE 2 | Genotypic variation of lentil biomass yield (A,B) and grain yield (C,D) with response to foliar application of selenate and selenite fertilizer at different rates. Each genotype (bars) for each Se rate followed by different letters are significantly different at P < 0.05. FIGURE 3 | Seed Se concentration in lentil genotypes grown under greenhouse conditions with soil application (A) and foliar application (B) of different rates and forms of Se. Each genotype (bars) for each Se rate and form followed by different letters are significantly different at P < 0.05. lost in higher plants during evolution. Therefore, the positive response to Se fertilization must involve Se metabolites. Others have noted that Se application increases antioxidant activities in plant tissues (Hartikainen, 2005;Pilon-Smits et al., 2009;Pilon-Smits and Quinn, 2010); this may enhance plant tissue growth and, subsequently, grain yield due to the redox scavenging of electrophiles, and radicals produced during photosynthesis (Cartes et al., 2005;Lyons et al., 2009). Selenium fertilization of lentils offers the potential dual benefits of increasing both yield and nutritional valueoutcomes with particular relevance to smallholder farms in low and middle-income countries. The identification of Se as a critical trace mineral and its provision through low dose fertilization may remove a barrier to the ability of smallholder farms in developing countries to achieve increased lentil yields. Such effects offer prospects for reversing the decline in lentil acreages in areas of greatest need. The resulting increase in lentil yield will benefit farmers and may contribute to reductions in malnutrition in general and micronutrient malnutrition in particular. Ultimately, progress in this direction may positively affect food security in developing countries, many of which consume 70% of their annual lentil production.

v3-fos

2019-03-31T13:42:57.236Z

2015-01-01T00:00:00.000Z

87914762

s2

Prediction of Standard Lactation Curves for Primiparous Holstein Cows by Using Corrected Regression Models Prediction of the expected milk yield is important for the management of the primiparous cows (PPC) with a few or no data on their own milk productivity. We developed a system of regression equations for predicting milk yields in standard lactation. The models include the systematic effects of the calving season, the five-year rolling herd average of milk yield of PPC, the breeding values of the parents for milk production, and daily milk recordings. A total of 21,901 lactations of Holstein PPC were collected during the regular monthly milk recordings of cows in the Republic of Slovenia. By including daily milk recordings in the model, the coefficients of determination of regression models for the prediction of milk yield increase: without known recordings (M0) R2=0.80; with one recording (M1) R2=0.82; with two first consecutive recordings (M2) R2=0.86; and with three recordings (M3) R2=0.89. Deviations of milk yield up to 500 kg in a standard lactation (<1.6 kg/day) were as follows: with the model M0, they occurred in 53.4% of PPC; with M1, they occurred in 56.3% of PPC; with M2, they occurred in 64.5% of PPC; and with M3, they occurred in 70.9% of PPC. We concluded that the developed system of regression models is an appropriate method for milk yield prediction of PPC. Introduction To achieve good economic efficiency of breeding, as well as to ensure good health and well being of dairy cows at the farm setting real milk production targets in accordance with the genetic potential of the herd and the level of its management is undoubtedly essential. Within the management framework, nutrition of dairy cows is a key factor that directly influences milk production, health status, fertility, and quality of milk of dairy cows (Friggens et al., 2007, Sova et al., 2013. Without the expected milk yield predictions it is difficult to plan a rationale and physiologically balanced ration in accordance with the needs of the animals. Primiparous cows (PPC) in the initial stage of lactation present a special challenge, because breeders have very limited data on their milk productivity, which makes such milk yield predictions rather unreliable. To classify PPC into milk production groups based on their predicted milk yield, we need not only data on their current milk production, but also data on the expected milk yield in a standard lactation and data on the lactation curve trajectory in all stages of lactation. Various methods have been proposed for predicting milk yield in lactation with a limited number of milk recordings (Macciotta et al., 2002). Jones (1997) suggested the Empirical Bayes prediction method, which weights the test-day milk production from the lactation in progress with lactation curves from a historical database of completed lactations of cows with the same characteristics. An Autoregressive Integrated Moving Average (ARMA) model was determined by Macciotta et al. (2002), and it requires many repetitions of adjustments by time series for the estimation of parameters. The test-day model (TDM) that is generally used for estimating breeding values is another method (Mayeres et al., 2004). The model includes a fixed herd-year-season (HYS) influence, which cannot be used directly in the algorithms for predicting milk yields, as it does not predict trends of shifting milk yields. Mayeres et al. (2004) addressed this by decomposing the HYS influence into three effects related to the herd, which determine the trend of shifting milk yields according to month, year, and a random herd effect on a test-day. When estimating daily milk yield on the basis of the expected standard lactation curve (SLC) trajectory, the random effect of daily milk fluctuations due to management errors and animal health problems will be lower than when estimating milk yield with the TDM model (Nordlund, 2006). This is important for managing nutrition of a dairy herd, since that allows us to monitor the planned lactation curve trajectory, which reflects the phenotypic milk producing ability of an animal. In addition to the above-mentioned methods for estimating the lactation curve trajectory and the expected milk yield, several other models exist (Grzesiak et al., 2003), which are based on a minimum number of milk recordings. When using the SLC, we encountered the issue of unreliability of milk yield predictions based on first milk recordings (Jeretina et al., 2013). When there is insufficient milk production data available, milk yield predictions for PPC can be doubtful (Macciotta et al., 2005;Silvestre et al., 2006). Consequently, it is reasonable to use breeding value data of the parents and the average milk yield of the herd, as well as the first milk recordings, which are collected during regular milk recordings (Mayeres et al., 2004). We hypothesized that the milk yield of PPC in the herd depends on the level of herd management, while the breeding value of the parents reflects their genetic potential transferred to the PPC. Moreover, the first daily milk recordings provide additional information, which can be used to improve the reliability of our predictions. The aim of the present study was to develop regression equations for predicting milk yield in a standard lactation of PPC, which can be used together with the SLC to estimate the standard lactation curve trajectory on a daily level. The method is simple and allows realtime corrections to the herd management. Data The survey was limited to the data of primiparous Holstein dairy cows, which calved between 1 January 2008 and 31 December 2012. The data were collected during the regular monthly milk recordings, which are per-formed by the Slovenian Agency for the Control of Milk Production of Cows. The calculation of lactations was carried out using the test interval method (ICAR, 2012), based on which breeding values for milk production are calculated (Potočnik et al., 2001). We processed data on lactations longer than 304 days with a normal shape of the lactation curve, as determined by the parameters of the model for estimating a standard lactation curve trajectory (Wood, 1967). The interval between the calving and the first milk recording usually had to be between 5 and 37 days; in exceptional cases (sickness, injury, animal under treatment, disaster -the reason must be reported) it could be up to 80 days. All the consecutive milk recordings were performed between day 22 and day 37; in exceptional cases (because of holidays or veterinary limitations), they could be performed up to day 75. In addition, the PPC had to have both parents known (i.e., with available breeding value data for milk production over 305 days), as well as more than nine herd mates with completed standard lactations in the 5 years before the calving of the primiparous cow. On the basis of these conditions, we processed data of 21,901 PPC, for which 271,988 milk recordings were performed (Table 1). Regression models Based on the data of standard lactations (MY305), we analysed four regression models. All of them included the influence of the season (S) and the age at first calving (FCA, categorized in eight classes: ≤23 months, 24, 25, ≥30 months), as well as the rolling herd average of PPC in a standard lactation (RPHA), breeding value of the father (BVF), and breeding value of the mother (BVM). We presupposed that the residuals were normally distributed. Model M0 did not include data on the milk yield of the PPC. The other models used milk yields from the first milk recording (M1), the first and the second milk recordings (M2), and the first three consecutive milk recordings in the initial stage of lactation (M3). The model equations can be written in scalar form as: The estimates of the fixed effects and the regression coefficients of the models allowed us to estimate milk yield in a standard lactation for an individual model (MY305M). Model specification and the choice of fixed effects in the model were based on checking their significance by using the lm function in R software (R Development Core Team, 2006), and the variables that were significant at P<0.001 were included in the model. Variance components were estimated separately for S, FCA, RPHA, BVF, BVM, and TD using the ANOVA function in R software. At the same time, we estimated total variance for comparisons of the models. Checking the reliability of predictions of the models We checked the reliability of the predictions of the models by calculating the correlations between MY305M and MY305, residual variance (RV), and percentage-squared bias (PSB) (Ali and Schaeffer, 1987), and by k-fold crossvalidation (Hastie et al., 2002). In k-fold crossvalidation, part of the available data is used to fit the model, and a different part to test it. Overall, typically 10-fold cross-validation is sufficient for testing the reliability of model predictions (Kohavi, 1995). The PPC were randomly classified into 10 subsets of approximately equal size. In 10 repetitions, the model parameters were estimated on a learning set of 9 subsets and the predictions were compared with the observed values in the remaining subset. For all the repetitions, the correlations between MY305M and MY305 were calculated. The average correlation coefficient across the 10 repetitions and the standard deviation of the correlation were determined. A total of 2491 animals were included in calibration set with exception of set ten, where 2486 animals were involved. All animals were classified in calibration sets randomly (using Oracle random function). In ten repetitions, the regression parameters were estimated for all four models on nine calibration sets and at the same time were estimated regression parameters on set 10. Regardless of whether they have been animals randomly split between calibration sets was herd distribution within calibration set randomly and balanced as well. Animals that were included in present study derive from 2009 farms. All analyses were carried out in R software (R Development Core Team, 2006). Additional correction of the models Expected milk yields of PPC were estimated using models M0, M1, M2, and M3. Because of high milk yield variation of PPC within herds, we expected a biased prediction of milk yield. To improve the prediction of milk yields, we adjusted the predictions using the regression equation: where: DMY305M, additional correction to MY305M; a, intercept of the regression model; β1, regression coefficient for milk yield in standard lactation predicted by the model (MY305M). Results and discussion The prediction of milk yield in a standard lactation is very unreliable in the initial stage of lactation because of very few daily milk recordings and their distribution. We approached the issue by using four regression models that include the average milk yield in a standard lactation of PPC in a herd, as well as breeding values of the fathers and the mothers for milk production in a standard lactation. In these models, the age of cows at their first calving, as well as milk yields upon the first, second, and third milk recording were included. For predictions of milk yield that take into consideration first milk recordings, the PPC that calve in autumn showed higher milk yields than PPC calving in the other seasons. For cows calving during the summer months, lower milk yields were mostly the result of heat stress, as confirmed by several authors (Jenko, 2012;Hammami et al., 2013;Smith et al., 2013). This can be alleviated by using suitable breeding technologies. Before setting up the models, we tested the influence of the number of days between calving and an individual milk recording. This influence was not significant and so was not included in the model. As the consequence of the highest unexplained percentage of residual value, the lowest coefficient of determination (R 2 ) was estimated with model M0 (R 2 =0.45) ( Table 2). The calculated correlation between MY305M from model M0 and MY305 was 0.66 (Table 2), which was greater than in a comparable study (Mayeres et al., 2004), where the value was r=0.575. Including the data of own production in the first, second and third milk recordings increased R 2 even more (M1, R 2 =0.67; M2, R 2 =0.78; M3, R 2 =0.85) ( Table 2). In these cases, we also obtained better correlations r=0.82, 0.89, and 0.92 (for models M1, M2, and M3, respectively) ( Table 2) compared with Mayeres et al. (2004). The influence of the calving season was statistically significant in all of the models (P<0.001) ( Table 3). The largest percentage of phenotypic variance of the models can be explained by the influence of RPHA (35.4%) ( Table 3). The average milk yield of PPC in a herd depends on the level of management, which comprises a variety of influences that cannot be broken down, but influence the milk yield in a standard lactation. Caccamo et al. (2008) showed high variance between average herd milk yield curves around the peak, which indicates differences in management between herds. We assume that these influences include the nutrition of the animals, the size of the animals, individual or group approach to management, health and general well being of the animals, as well as breeding intensity of heifers. Several authors have established that the age of cows at first calving influences milk yield in a standard lactation (Ettema and Santos, 2004;Mohd Nor et al., 2013), as well as the number of somatic cells in the initial stage of lactation (Archer et al., 2013). However, in our study, the influence of the somatic cells was not statistically significant and was therefore not included in further analyses. Only a low percentage (0.4%) of the variability of the model was explained by the influence Jeretina et al. (Table 3). We included it as a fixed influence in the regression models and we came to similar conclusions as in previous studies (Nilforooshan and Edriss, 2004;Mohd Nor et al., 2013). In model M0, the milk yield of PPC that calved at age 23 months was 143 kg lower than that of cows that calved at age 24 months ( Table 2). Cows that calved at age 25 months had milk yield 48 kg higher than for cows that calved at age 24 months (Table 2). We observed the same trend in the other models. Effects The increase in milk yield with higher calving age is related to the increase in frame size of PPC and the development of their mammary glands (Mohd Nor et al., 2013). Milk yield most significantly increased with PPC that calved between 23 and 25 months of age. With cows that calved at a later age, the increase in milk yield became less pronounced. In all the models, the influence of breeding values of the father and the mother accounted for 3.3% and 4.9% of the total variance, respectively (Table 3). This is due to low heritability of first test day records, which depicts the evolution of heritability of milk over days in milk (DIM), with an average of 0.197 (SE=0.015 to 0.022) (Bastin et al., 2011). Consequently, these two influences explain 8.2% of the total variance. The influence of breeding values of the father and the mother were most pronounced in model M0. However, after including the first milk recordings in the models, their influence became less pronounced. In model M0, the regression factor BVM was bigger than the regression factor BVF (β3=0.63 vs β2=0.54) ( Table 2), but with additional recordings BVF weighted more than BVM (Table 3). The milk yield upon first recording explained 21.4% of the total variance (Table 3). With each additional kilogram of milk upon first recording, we can expect an increase of milk yield in MY305M by 128.6 kg from the model using one known milk recording (Table 2). When cows have two known milk recordings, the expected milk yield increase is 65.8 kg per kilogram of the first milk recording and 119.7 kg per kilogram of the second milk recording (Table 2). Consequently, with two known milk recordings, we can explain 33.7% of the total variance (Table 3). With three known milk recordings, the expected milk yield in MY305M increases by 49.8 kg with each additional kilogram of milk upon first recording; by 68 kg with each additional kilogram of milk upon the second recording; and by 101.1 kg with each additional kilogram of milk upon the third recording ( Table 2). As a result, the total percentage of explained variance is 39.6% (Table 3). The unexplained residual variance decreased with the increase of the number of independent variables in the models (M0=55.3%, M1=33.9%, M2=21.6%, and M3=14.6%) (Table 3). After the analysis of variance between the models, we established that the mean values of the models showed statistically significant differences (P<0.001). The percentage of squared bias was reduced with the inclusion of milk recordings, which demonstrates that, by increasing the number of milk recordings, the model becomes more reliable. Using multiple cross-validations in 10 repetitions of 2491 PPC on each subset except the last where it was 2486 PPC, the average correlations and their standard deviations were calculated. The values for individual models were as follows: M0, r=0.66, SD=0.010; M1, r=0.82, SD=0.008; M2, r=0.89, SD=0.004; and M3, r=0.92, SD=0.004 (Table 2). The high correla-tions and low standard deviations indicate that the models for predicting of milk yield in a standard lactation are relatively stable. The distribution of differences between MY305 and MY305M calculated from each model were analysed and are presented in Table 4 and Figure 1. The prediction is biased because herd averages include PPC with low milk yield and PPC with high milk yield, which is confirmed by the high variability within the herd (coefficient of variation ranged from 5.5 to 29.2%). To improve the prediction of milk yields in a standard lactation, as well as to reduce the bias, the models were adjusted in several different ways. By re-parameterizing the models, the average herd milk yields of PPC were classified into classes. Milk yield in a standard lactation was expressed by the quotient between milk yield of a primiparous cow and average milk yield of PPC in the herd. Contrary to our expectations, the reliability of prediction of the models was reduced, as well as the percentage of the explained variance from the total variance. Using additional regression analysis and correction of the already predicted milk yield significantly improved the prediction of milk yield in a standard lactation. Regressions for the residuals were estimated and the new corrected values in relation to the milk yield were calculated (Table 4). Prediction of standard lactation curves The results from the additional analysis were compared with the results from the original four models, and an improvement in the reliability of the estimated standard lactation with models M0 and M1 was established. The effect of additional correction with models M2 and M3 was slightly smaller because of the smaller bias of the models. Nevertheless, it was sufficient to be considered. PSB was significantly reduced with models M0 and M1 (PSB from 2.57 to 0.96 at M0 and PSB from 1.39 to 0.87 at M1), but slightly less with models M2 and M3 (PSB from 0.85 to 0.65 at M2 and PSB from 0.57 to 0.48 at M3) ( Table 4). Values of R 2 for models M0, M1, M2, and M3 after the additional correction were 0.80, 0.82, 0.86, and 0.89 (Table 4). The additional correction of the regression model was checked with 10-fold cross-validation (Table 4). The results showed sufficient accuracy of prediction, which confirms the suitability of the additional correction method. Figure 2 shows the percentages of PPC with deviations of milk yield up to 500 kg in a standard deviation. Model M0 without first milk recordings comprises only 53.4% of all PPC; model M1 with one milk recording comprises 56.3% of PPC; model M2 with two milk recordings comprises 64.5% of PPC, and model M3 with three milk recordings comprises almost 70.9% of PPC. A deviation of milk yield up to 500 kg in a standard lactation is equivalent to average 1.6 kg milk per day, which is acceptable variation for management. Because the milk yield prediction based on models that include the first milk recording is more reliable, it would be reasonable to include data on daily milk yields of PPC in the prediction as soon as possible. This is especially recommended for herds with daily milk recordings, e.g. with robotic milking or in milking parlours with automated milk yield recording. Conclusions Regression models can reliably and simply Jeretina et al. predict the expected milk yield of PPC using calculated breeding values of the parents, calving period, age at calving, and the five-year rolling average herd milk yield of PPC. The rolling herd average of standard lactation of PPC is the most important factor for predicting milk yield in standard lactation of cows without their own test day data. Adding the first and all the consecutive milk recordings in the model can further improve the prediction of milk yield and enable checking of herd management success in real time. In order to facilitate computations, it is reasonable to adopt an approach which applies breeding values of parents -whose data are pre-corrected for additive genetic and environmental effects -from population-wide breeding value estimation. However, owing to the great variability in milk yields of cows within a herd, the prediction of milk yield without an additional correction is biased.

v3-fos

2016-05-04T20:20:58.661Z

2015-03-04T00:00:00.000Z

11763002

s2

MiR-486 Regulates Lactation and Targets the PTEN Gene in Cow Mammary Glands Mammary gland development is controlled by several genes. Although miRNAs have been implicated in mammary gland function, the mechanism by which miR-486 regulates mammary gland development and lactation remains unclear. We investigated miR-486 expression in cow mammary gland using qRT-PCR and ISH and show that miR-486 expression was higher during the high-quality lactation period. We found that miR-486 targets phosphoinositide signaling in the cow mammary gland by directly downregulating PTEN gene expression and by altering the expression of downstream genes that are important for the function of the mammary gland, such as AKT, mTOR. We analyzed the effect of β-casein, lactose and triglyceride secretion in bovine mammary gland epithelial cells (BMECs) transfected by an inhibitor and by mimics of miR-486. Our results identify miR-486 as a downstream regulator of PTEN that is required for the development of the cow mammary gland. Introduction Bovine mammary glands arise from the ectoderm during embryonic development and continue to develop postnatally through puberty, pregnancy, lactation, and subsequent involution. Most developmental and functional differentiation in the mammary gland occurs after the birth of offspring [1]. During lactation, the mammary gland secretes milk, which provides nearly all the nutrient requirements of the newborn offspring during the transition from pregnancy to lactation [2]. The gland develops primarily postnatally, and its development is mainly controlled by steroids, peptide hormones, and cell matrix interactions during different stages. Several pathways have been shown to modulate the progression of mammary gland development. Additionally, more than 100 genes have been shown to modulate various aspects of mammary physiology, from the formation of the fetus to remodeling of the gland during involution [2][3]. MicroRNAs (miRNAs) have also been shown to regulate cell processes, and many miRNAs are involved in mammary gland development and tumorigenesis [4]. Due to the unique developmental features found during distinct stages of lactation, the mammary gland represents an important model for use in studies to elucidate signaling related to cell cycle progression, survival, proliferation, differentiation, and cell death. Despite the relatively recent recognition of miRNAs as key regulators of cellular function, little research has groups by developmental stage: the lactation with high quality milk stage (milk yield 30.6±0.78 kg/d, milk protein >3.0%, and milk fat >3.5%; n = 3), the lactation with poor quality milk stage (milk yield 30.6±0.78 kg/d, milk protein <3.0%, and milk fat <3.5%; n = 3), and the pregnancy stage (n = 3). All of the cows were fed with standard foodstuffs consisting of 30% roughage and 70% concentrate. The cows were slaughtered by exsanguinations, and their mammary tissue was aseptically excised 5 cm from the base of the healthy breast and 3 cm from the midline that divides the core of the secretory gland tissue. After removing the connective tissue, the remaining tissue was cut into small blocks with a thickness of 1 cm. The mammary tissue samples were immediately frozen in liquid nitrogen and stored at-80°C for later analysis. All animal experiments were approved by the Institutional Animal Care and Use Ethics Committee of Northeast Agricultural University and conducted in accordance with the Guidelines for Experimental Animals from the Ministry of Science and Technology (Beijing, China). The indicated cells were transfected with 50 nmol miR-486 mimics, inhibitors, or the corresponding negative control constructs (miR-NC, Anti-NC) (GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions, respectively. Twenty-four hours after transfection, the cells were treated with cell differentiation medium. Small RNA sequencing We extracted total RNA from mammary gland tissues at 3 different developmental stages using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The RNA samples were sent to the Beijing Genomics Institute (Shenzhen, Guangdong, China), where a small RNA library was constructed and sequenced using a Genome Analyzer (Illumina, San Diego, USA). All of the data were stored in the Short Archive section of the NCBI GEO under accession number GSE57991. The sequencing data were first filtered into mRNA using the Rfam 10.1 and Genebank databases and then mapped to miRBase 18. The mapped data were used to identify significant differences in miRNA expression [18]. Luciferase activity assay HEK-293 cells were cultured in 24-well plates, and each plate was transfected with 80 ng of pGL3/PTEN vector or pGL3/PTEN/mut vector containing firefly luciferase and 4 ng of the pRL-TK vector (Promega Madison, WI, USA) containing 4 pmol miR-486 mimics or negative control. The cells were transfected using Lipofectamine 2000 (Invitrogen). After 48 h of transfection, relative luciferase activity was calculated by normalizing the firefly luminescence to the renilla luminescence using the Dual-Luciferase Reporter Assay (Promega Madison, WI, USA) according to the manufacturer's instructions. The experiments were performed in triplicate. Quantitative polymerase chain reaction (qPCR) assays After the transfection of the cells with the miR-486 mimics/inhibitors, total RNA was purified using Trizol reagent (Invitrogen) following the manufacturer's instructions. cDNA (complementary DNA) was synthesized using the PrimerScript RT-PCR kit (TaKaRa) according to the manufacturer's instructions. The expression of miR-486 and the 5S internal control gene were quantified using real-time PCR quantification and the Hairpin-it miRNAs qPCR Quantitation Kit (GenePharma, Shanghai, China) according to the manufacturer's protocol. Specific primers for miR-486 and 5S were designed by GenePharma. The expression of these genes was analyzed using a 7300 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The expression of PTEN, AKT, mTOR, and β-actin was quantified using Real-Time-PCR quantification and SYBR Premix Ex Taq II (TaKaRa) according to the manufacturer's instructions. β-actin was used as a loading control. The primers that were used for qRT-PCR are listed in Table 1. All qPCR reactions were performed in triplicate. In Situ hybridization In situ hybridization (ISH) experiments with 5-μm paraffin sections of bovine mammary gland tissues were performed using a DIG-labeled locked nucleic acid probe against miR-486 (5-CTCGGGGCAGCTCAGTACAGGA-3), used scramble (Exiqon, USA) as the negative control probe. Tissues were dewaxed, postfixed in 4% paraformaldehyde (PFA) for 1 h, and digested in proteinase K solution (5 μg/ml) at 37°C for 5 min. The slides were pre-hybridized at 37°C for 1 h in 50% formamide. The LNA probe was diluted with ISH buffer (1.5 pM), and the probe was denatured at 95°C for 8 min. Hybridization was performed overnight at 55°C, followed by post-hybridization washes in 5×SSC and 0.2×SSC at 60°C. After exposure for 10 min to 3% H 2 O 2 , the slides were incubated with a mouse monoclonal antibody against digoxin (Abcam Technology, Cambridge, MA, USA) at 37°C for 30 min and then incubated with an HRPconjugated goat-anti-murine secondary antibody (Abcam). After antibody treatment, the sections were incubated with 0.04% DAB + 0.03% H 2 O 2 to develop the color for 30 min, after which the sections were counterstained slightly with Mayer's hematoxylin. An Olympus CX31 microscope was used. Western blot analysis After the overexpression and inhibition of miR-486, the cell lysates were separated on a 10% SDS-PAGE gel (30 μg protein per sample), and the proteins were then transferred onto nitrocellulose membranes (Bio-Rad, Shanghai, China). Nonspecific binding sites were blocked using a 5% skim milk solution for 1 h at 37°C, and the membranes were then incubated with a rabbit polyclonal antibody against PTEN (Santa Cruz Biotechnology Inc., Dallas, TX, USA), AKT, phospho-AKT (Ser473), MTOR (Cell Signaling Technology, Beverly, MA, USA), or phospho-MTOR (S2448) or a murine polyclonal antibody against β-actin (Santa Cruz Biotechnology Inc.) overnight at 4°C; this was followed by incubation with goat-anti-rabbit or goatanti-murine secondary antibodies conjugated to HRP (Zhongshan-Bio, Beijing, China), and the proteins were visualized using Super ECL Plus (ApplyGEN, Beijing, China). β-actin was used as a loading control. Cell cycle analysis Cell cycle analysis was performed using flow cytometry (FCM) as follows: harvested cells were washed with cold PBS and fixed with 70% ethanol overnight at 4°C, and washed with cold PBS. The cells were then incubated with PI (50 μg/ml) and mixed with 2 μl/ml TritonX-100 for 20 min at room temperature in the dark. The cells were then suspended in 500 μl PBS. Samples were analyzed using flow cytometry on a Cytomics TMFC500 flow cytometer, and the data were analyzed using Mod Fit LT 3.2 software (Verity Software House, USA). The FCM analysis was carried out in triplicate. Cell Proliferation Cell proliferation was determined using the colorimetric water-soluble tetrazolium salt (WST-8) assay using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Japan) according to the manufacturer's instructions. After 24 h of transfection with miR-486 mimics/inhibitors, BMECs were seeded onto a 96-well plate (4×10 4 cells per 100 μl per well), and 10 μl of CCK-8 solution was added to each well. Cells were incubated for 2 h, and cell proliferation was documented. Absorbance was measured at 450 nm using a microplate reader with a reference wavelength of 650 nm. Averages of 6 replicates were analyzed, and statistical analysis was performed using the t-test. Cell proliferation assay BMECs were seeded in glass-bottomed cell culture dishes. After 48 h of transfection, cell proliferation was quantified based on the incorporation of 5-ethynyl-2-deoxyuridine (EdU) into Statistical analysis The results were analyzed using SPSS 17.0 statistics software (Chicago, IL, USA). All results were expressed as the means ± standard deviation (SD) of separate experiments (n3). P values less than 0.05 were considered significant. Expression of miR-486 in bovine mammary gland tissues with different milk qualities To examine whether miR-486 expression depends on the stage of mammary development specifically during the high-milk-quality lactation period (H) (Fig. 1A-1D), the low-milk-quality lactation period (L) (Fig. 1E-1H), and the pregnant period (P) (Fig. 1I-1L), we used in situ hybridization to evaluate the expression of miR-486 in bovine glandular tissue and adipose tissue (Fig. 1A). In situ hybridization using a DIG-labeled locked nucleic acid probe against miR-486 revealed that miR-486 was primarily expressed in bovine mammary glandular epithelium tissue ( Fig. 1A: H-a, L-a, P-a) and was not expressed in bovine mammary adipose tissue ( Fig. 1A: H-c, L-c, P-c). Additionally, to determine if miR-486 expression was different between the high-milk quality and low-milk quality lactation periods we used a small RNA sequencing approach developed by our laboratory. And we found that the glandular expression of miR-486 was higher in high milk quality cows than in low milk quality cows (P<0.01) and pregnant cows (P<0.01). These results are shown in Fig. 1B. Furthermore, qRT-PCR analysis showed that the expression of miR-486 was higher in the H group than in the L (P<0.01) and P groups (P<0.01) (Fig. 1C). MiR-486 targets the PTEN gene MiR-486 is conserved in mammals and is the sole miRNA with no known family members [14]. Using the prediction software TargetScan6.2, we found that miR-486 target sites are present at nt 725-732 and nt 3,183-3,190 of the PTEN 3 0 UTR; these two sites are conserved in mammals. The 3 0 UTR of wild-type PTEN was linked to pGL3 Luciferase Reporter Vectors (Promega) and examined in HEK-293 cells that had been transfected with miR-486 mimics or a negative control. A significant decrease in reporter activity was observed when using the miR-486 mimics compared to the negative control. Transfection with miR-486 mimics had little effect on the luciferase activity of the mutant 3 0 UTR-reporter of PTEN compared with the negative control. Moreover, the luciferase signal of the pGL3-control vector with miR-486 was similar to that obtained with the negative control as shown in Fig. 2. MiR-486 inversely correlates with PTEN in BMECs We found that the expression of miR-486 increased after treatment with miR-486 mimics (Fig. 3 A, P<0.0001) and decreased after treatment with miR-486 inhibitors (Fig. 3 B, P<0.0001). To investigate whether miR-486 affects the regulation of the PTEN in mammary epithelial cells, we measured the relative expression of PTEN using qRT-PCR and western blotting. We found that the level of miR-486 changed concomitantly with the mRNA level of PTEN and that miR-486 inhibited the expression of PTEN (Fig. 3 C, Fig. 3 E, P<0.0001). Transfecting BMECs with a miR-486 inhibitor increased the expression of PTEN (Fig. 3 D, P<0.01; Fig. 3F, P<0.0001). We assessed the expression of PTEN after treatment with miR-486 mimics/inhibitors in BMECs using immunohistochemistry. We observed the expression of PTEN both in the cell nuclei and cytoplasm of mammary gland tissues (Fig. 3 G). A high expression of miR-486 repressed the expression of PTEN in mammary epithelium cell nuclei. However, a reduced expression of miR-486 stimulated PTEN expression in mammary epithelial cell nuclei. Thus, the data above suggest that miR-486 negatively affects PTEN. MiR-486 increases the expression of AKT and MTOR in bovine mammary epithelial cells MiR-486 significantly repressed PTEN expression in cell culture and induced AKT and MTOR expression in BMECs. We observed that, compared to a negative control, the high-level expression of miR-486 stimulated the expression of AKT and MTOR (Fig. 4 A, P<0.0001). Moreover, the knockdown miR-486 repressed the expression of AKT and MTOR (Fig. 4 B, P<0.0001). The relative protein expression of AKT, phospho-AKT (p-AKT), MTOR, and phospho-MTOR (p-MTOR) increased as the expression of miR-486 was increased (Fig. 4 C, P<0.0001). However, decreasing the levels of miR-486 reduced the relative protein expression of AKT, p-AKT, MTOR, and p-MTOR (Fig. 4 D; P<0.0001, P<0.0001, P<0.01, respectively). To assess whether miR-486 affects the proliferation of bovine mammary epithelial cells, these cells were transfected with miR-486 mimics/inhibitor and analyzed using CCK-8 assays after 48 h. We measured the proliferation rate of the miR-486 mimics group and the miR-486 inhibitor group; miR-486 mimics increased the ability of the cells to proliferate, and the miR-486 inhibitor suppressed the ability of the cells to proliferate (Fig. 5 A). Additionally, we used the EdU incorporation assay to determine the role of miR-486 in the proliferation of BMECs. The percentage of Edu-positive cells was significantly higher in the miR-486 mimic-treated cells than in the negative control group (P<0.05). Additionally, the percentage of Edu-positive cells was lower in the miR-486 inhibitor group than in the negative control group (Anti-NC) (Fig. 5 B, P<0.05). Next, BMECs transfected with miR-486 mimics, inhibitor, or negative control were examined for changes in the cell cycle using flow cytometry analysis (Fig. 6). Transfection with miR-486 mimics resulted in an increased population of cells in the S phase and a decreased population of cells in G0/G1 compared to transfection with the negative control ( Fig. 6 A, P<0.05). Furthermore, miR-486 inhibitors caused the opposite effect compared to the negative control (Anti-NC), (Fig. 6 B, P<0.05). The data above indicate that miR-486 is a negative modulator of the G1 to S transition. β-casein levels, lactose secretion, and triglyceride levels are upregulated by miR-486 Mammary epithelial cells synthesize and secrete proteins, lactose, and lipids. Thus, we examined the supernatant for changes in the levels these key components. The overexpression of miR-486 increased the concentrations of triglycerides, β-casein and lactose (Fig. 7 A, 7 C, and 7 E, P<0.0001; P<0.0001; P<0.001); conversely, inhibition of miR-486 suppressed the concentrations of triglycerides, β-casein and lactose (Fig. 7 B, 7 D, and 7 F, P<0.0001). Discussion Currently, many miRNAs have been identified using algorithmic and experimental methods in animals, plants, and viruses. Many miRNAs have been found to play roles in cell differentiation, proliferation, apoptosis, development, tumorigenesis, and host-pathogen interactions [19][20][21][22][23][24]. Although experiments have been performed to identify the roles of an increasing number of miRNAs, few studies have been conducted to examine the role of miRNAs in the bovine mammary gland. Here, we present a study on the role played by miR-486 in the bovine mammary gland. This study shows that the tumor suppressor PTEN is negatively regulated by miR-486 via two specific sites (nt 725-732 and nt 3,183-3,190) within the 3`UTR. MiR-486 has been sparsely studied to date, and its relevance to the mammary gland has only begun to be elucidated. Using a small RNA sequencing approach, we observed that the levels of miR-486 were higher in lactation gland tissue during the high-quality milk stage than in the low-quality BMECs after treatment with miR-486 inhibitors or negative control (Anti-NC). E: Western blotting analysis of PTEN protein expression in BMECs after treatment with miR-486 mimics or negative control (miR-NC). F: Western blotting analysis of PTEN protein expression in BMECs after treatment with miR-486 inhibitors or negative control (Anti-NC). All of the above experiments were performed in triplicate. G: Confocal microscopy analysis of PTEN expression in BMECs after treatment with miR-486 mimics (30 μM), negative control (miR-NC) ( milk and pregnancy stages and that the expression of miR-486 was almost unchanged from the low-quality lactation stage to the pregnancy stage. We further validated this result using qRT-PCR and small RNA sequencing. In situ hybridization showed that miR-486 was found primarily in mammary gland tissue and not in adipose tissue. Therefore, we concluded that the post-transfection effects of miR-486 mimics or a miR-486 inhibitor affected mammary epithelial cells more than fat cells. The results were similar for almost all stages of bovine mammary gland development. Therefore, we believe that miR-486 is a key biomarker in bovine mammary gland epithelial cells. Heterozygous PTEN mice were crossed with MMTV-wnt1 transgenic mice, resulting in the formation of breast tumors earlier in life than in the parental strains [25]. Some studies have examined the role of PTEN in normal mammary glands, but few studies have examined the secretion of these glands, which might prove that the tumor suppressor protein PTEN controls mammary gland development [17]. PTEN is commonly inactivated in human cancers and acts as a key regulator of the PIP3/AKT/mTOR pathway. One of study showed PTEN functions as an inhibitor during mammary gland development and lactation in dairy cows [26]. Indeed, our study confirmed that levels of AKT, phosphorylated AKT, mTOR and phosphorylated mTOR change in response to PTEN and that PTEN levels appeared to depend on miR-486 levels. The overexpression of miR-486 inhibited the expression of PTEN both at the mRNA and protein levels and thus increased the levels of AKT, phosphorylated AKT, mTOR and phosphorylated mTOR. PTEN is a well-known tumor suppressor in numerous cancers and normal tissues and regulates the AKT and mTOR signaling pathways. Our study provided information about the levels of nuclear PTEN. Bovine mammary epithelial cells overexpressing miR-486 had decreased PTEN expression in their nuclei; however, BMECs lacking miR-486 had increased PTEN levels. In general, despite its key role at the plasma membrane, PTEN is present in the nucleus of diverse cell types [27][28] (including tissue cells and cell lines [29][30][31]) and participates in many cellular processes that are relevant to tumorigenesis [32]. The regulation of PTEN nuclear MiR-486 Regulates Lactation and Targets PTEN Gene in Cow Mammary Gland localization is complex because PTEN plays many roles within the nucleus [17]. MiR-486 inhibited the expression of PTEN, and as same time cytoplasmic PTEN cellular staining towards nuclear staining, for PTEN/AKT/MTOR pathway plays prominent roles in development and lactation of mammary gland [26]. Here we suppose that miR-486 regulates expression of cytoplasmic PTEN, PTEN shift from cellular towards nuclear in mammary epithelial cells increase AKT/ MTOR pathway, and regulate mammary epithelial cells secretion of β-casein, triglyceride, and lactose, and plays a critical role in lactation related signaling pathways. During the lactation period, the quality of milk that is secreted depends on milk fat (mostly triglyceride), milk protein (mostly β-casein) [33] and lactose. The results of this study show that triglyceride, β-casein and lactose levels were regulated by the transfection of miR-486 mimics or miR-486 inhibitors, indicating that miR-486 stimulated the lactation of milk fat, milk protein and lactose in the cow mammary gland. The number and activity of mammary gland secreting cells in cows have been reported to affect the quality of milk [34]. We found that overexpressing miR-486 increased the amount of mammary epithelial cell proliferation as determined by CCK-8 assays. However, the inhibition of miR-486 showed the opposite effect. Edu incorporation experiments illustrated that miR-486 stimulated DNA proliferation by increasing the percentage of Edu-positive cells. Furthermore, Analysis of secretion in mammary supernatants. A: Secretion of triglyceride following overexpressive miR-486, compared with negative control (miR-NC); B: Secretion of triglyceride following inhibiting miR-486, compared with negative control (Anti-NC); C: Secretion of β-casein following overexpressive miR-486, compared with negative control (miR-NC); D: Secretion of β-casein following inhibiting miR-486, compared with negative control (Anti-NC); E: Secretion of lactose following overexpressive miR-486, compared with negative control (miR-NC); F: Secretion of lactose following inhibiting miR-486, compared with negative control (Anti-NC). Data were averages of at least three independent runs; Values are means±SD, **P<0.01, ***P<0.0001. our studies showed that miR-486 regulated the cell cycle of BMECs. MiR-486 mimics increased the population of BMECs in the G0/G1 phase and decreased the population of BMECs in the S stage. Therefore, we propose that miR-486 is a novel regulator that stimulates the proliferation of BMECs. The role of miR-486 in the cell cycle is an ongoing area of study. Conclusion MiR-486 plays a key role in the mammary gland and directly downregulates the PTEN gene and affects downstream genes, such as AKT, mTOR, and β-casein, which play important roles in the mammary gland. Our data suggest that the repression of PTEN by miR-486 is associated with lactation and in this way increases AKT and mTOR. Additionally, miR-486 stimulates cell proliferation and increases some crucial secretory elements, such as β-casein, lactose, and lipids. Taken together, our findings indicate that miR-486 functions primarily in bovine mammary gland epithelium tissue and cells and promotes milk synthesis and secretion.

v3-fos

2018-04-03T00:50:23.049Z

2015-06-23T00:00:00.000Z

18613542

s2

The Glycine max cv. Enrei Genome for Improvement of Japanese Soybean Cultivars We elucidated the genome sequence of Glycine max cv. Enrei to provide a reference for characterization of Japanese domestic soybean cultivars. The whole genome sequence obtained using a next-generation sequencer was used for reference mapping into the current genome assembly of G. max cv. Williams 82 obtained by the Soybean Genome Sequencing Consortium in the USA. After sequencing and assembling the whole genome shotgun reads, we obtained a data set with about 928 Mbs total bases and 60,838 gene models. Phylogenetic analysis provided glimpses into the ancestral relationships of both cultivars and their divergence from the complex that include the wild relatives of soybean. The gene models were analyzed in relation to traits associated with anthocyanin and flavonoid biosynthesis and an overall profile of the proteome. The sequence data are made available in DAIZUbase in order to provide a comprehensive informatics resource for comparative genomics of a wide range of soybean cultivars in Japan and a reference tool for improvement of soybean cultivars worldwide. Introduction Soybean (Glycine max) is one of the world's most important leguminous crops being a major source of edible proteins and vegetable oils. In terms of global production, soybean ranks fourth, following the major cereal crops such as rice, wheat, and corn. It is also a major source of nutritionally and physiologically active substances such as saponins, isoflavones, phytosterols, and tocopherols. Consumption of soybeans as food is largely concentrated in Asia. Soybean has been a part of the Japanese diet and eaten from ancient times as a valuable source of traditional fermented products such as miso, soy sauce, and natto and nonfermented products such as edamame (boiled soybean), kinako (toasted soybean flour), tofu, and soymilk. As in other major crops, the main targets of soybean breeding in Japan are high yield, high quality (absence of seed coat cracking, seed size, hilum color, uniformity of seed size, and food processing adaptability) to compete with imported soybean, and resistance to biotic/abiotic stress for stable production. Additionally, the chemical component of seeds including high protein content, modification of storage proteins, absence of lipoxygenases and saponin, high isoflavone content, and high sucrose have been given much consideration in many soybean breeding programs [1]. The domesticated soybean has its origin from Glycine soja, a wild soybean species found mainly in northern China, Japan, Korea, and the eastern part of Russia [2]. Archaeological studies indicate that the word for soybean first appeared in China about 3,700 years ago in bone inscriptions dating back to the Yin and Shang dynasties and carbonized soybean 2 International Journal of Genomics seeds found about 2,600 years ago [2]. Estimation of archaeological records indicates a widespread early association of small seeded soybean to be as old as 9,000-8,600 calibrated years before the present (cal BP) in northern China and 7,000 cal BP in Japan [3]. Direct radiocarbon dates on charred soybean seeds indicate selection resulted in large seed sizes in Japan by 5,000 cal BP (Middle Jomon) and in Korea by 3,000 cal BP (Early Mumun) [3]. Extensive genome analysis also indicates that the G. soja/G. max complex diverged from the most recent common ancestor at 0.27 Mya [4] or 0.8 Mya [5]. In a more recent study, the genetic variation and population structure among 1,603 soybean accessions indicated a clear genetic differentiation among Japanese soybean landraces, exotic and cultivated soybeans, and wild soybeans [6]. From the genomics point of view, soybean has been used as a model plant for comparative studies of legumes in terms of root nodulation, oilseed production, and secondary metabolism. It is also a valuable material for genome research because of the availability of many genomic and germplasm resources. In 2010 a great deal of effort in the USA culminated with the sequencing of the paleopolyploid soybean genome based on a soybean cultivar Williams 82 [7]. This cultivar was derived from backcrossing a Phytophthora root rot resistance locus from the donor parent Kingwa which was selected in 1921 from the cultivar Peking introduced from Beijing, China, in 1906 [8]. In Japan, however, domestic cultivars have been developed to suit a variety of conditions and applications of specific importance to Japanese growers. Although the G. max cv. Williams 82 reference soybean genome sequence could be useful in understanding the diversity among many cultivars, it is necessary to have genomic resources that could be directly applied to Japanese soybean cultivation. The Japanese soybean cultivar Enrei was derived from cultivars Norin number 2 and Higashiyama number 6 (also known as cv. Shiromeyutaka) and was developed in 1971 at Kikyogahara Branch of the Nagano Agricultural Experiment Station (presently known as Nagano Vegetable and Ornamental Crops Experiment Station) [9]. In this paper, we described the analysis of the genome sequence of the Japanese soybean cultivar Enrei focusing on the phylogenetic analysis and major traits for soybean breeding including anthocyanin and flavonoid biosynthesis and proteome profile. Genome Sequencing. The plant material was provided by the Genebank of the National Institute of Agrobiological Sciences (NIAS). High-quality nuclear DNA with reduced organellar DNA was extracted from young leaves using a protocol designed for BAC DNA extraction with some modifications [10]. All sequencing reads were obtained using the Illumina HiSeq2000 at Operon Biotechnologies, Inc. (Eurofins Genomics). Standard short-read libraries and mate-paired libraries with 8 kbp insertion were built using the TruSeq SBS v5 for sequencing runs at 2 × 100 bp or 200 bp total. After sequencing, HiSeq Control Software v.1.4.8 and CASAVA 1.8.1 (Illumina) were utilized for base calling. Single-ended libraries and 3 kbp pair-ended libraries constructed with the GS FLX Titanium General Library Preparation Kit and Rapid Library Preparation Kit (Roche) were sequenced on Roche 454 FLX Titanium at the NIAS, and base calling was performed using the 454 FLX Titanium base caller. Assembly and Reference Mapping. We constructed a de novo genome assembly (G. max Enrei1) and reference genome assembly (G. max Enrei2) to facilitate comprehensive analysis of the genome. The G. max Enrei1 assembly was constructed from the Roche 454 FLX Titanium single-ended reads and pair-ended reads with 3 kbp insert, the Illumina HiSeq2000 pair-ended reads with 300 bp insert and matepair reads with 8 kbp insert, and the ABI 3730xl BAC-end reads using the Roche Newbler 2.7. The G. max Enrei2 assembly was derived from Roche 454 FLX Titanium single-ended reads and Illumina HiSeq2000 pair-ended reads were used for reference mapping with the BWA 0.7.5a (Li H. Aligning sequence reads, clone sequences, and assembly contigs with BWA-MEM, 2013; http://bio-bwa .sourceforge.net/), SAMtools 0.1.19 [11], and NIG script (NGS Surfer's wiki, http://cell-innovation.nig.ac.jp/wiki/tiki-index .php?page=samtools#mpileup ). The G. max cv. Williams 82, also referred to as Gmax275 genome assembly, was used for reference mapping. The pseudomolecules and scaffolds in the G. max Enrei2 were searched for marker sequences by BLASTn (NCBI BLAST, ftp://ftp.ncbi.nih.gov/blast/). Then marker sequences were mapped in the regions with clear sequences, gap regions (indicated as N's in the sequence), BAC-end sequences (BES) hit position, marker hit position, and scaffold derived from de novo assembly hit position. Subsequently, the cutting points were identified to reconstruct the pseudomolecules and scaffolds. Gene Models. The soybean parameter files were built from chromosome 16 region of hard masked Gmax275 genome [30,000,000-37,887,014 bps] using Augustus program [12]. The transposable elements in the scaffolds and pseudomolecules of the G. max Enrei2 genome assembly were masked using RepeatMasker (Smit AFA, Hubley R., and Green P. RepeatMasker Open-3.0, 1996-2010, http://www .repeatmasker.org/) and the gene models were built using Augustus. These gene models were used as queries in BLASTn search using the soyTE as a database [13]. The filtered gene models with bit score of 100 and above were selected. Additionally, we used available RNAseq data (PRJDB3582) assembled by Trinity version 2014-07-17 [14]. A total of 172,753 gene models were extracted. For each gene, the longest ORF was identified using EMBOSS getorf [15]. Anthocyanin and Flavonoid Biosynthesis. All gene models in Gmax275 and G. max Enrei2 associated with anthocyanin and flavonoid biosynthesis were extracted and clustered using OrthoMCL [19]. Then these gene models were associated by BLASTn. 2.6. Proteome Analysis. The proteome analysis of Enrei cultivar was performed using seeds. The cotyledons from ten seeds were grounded in liquid nitrogen and purified by phase separation using standard procedures [24]. The purified proteins were digested with trypsin. For mass spectrometry analysis, the eluted peptides were analyzed on a nanospray LTQ XL Orbitrap mass spectrometer and the MS spectra were used for protein identification. Identification of proteins was performed using the Mascot search engine version 2.4.1 (Matrix Science, London, UK) and Proteome Discoverer software version 1.4.0.288 (Thermo Fisher Scientific) against 54,175 soybean peptide sequences [7]. Mascot results were filtered with Mascot Percolator software to improve the accuracy and sensitivity of the peptide identification [25]. The protein abundance was analyzed using emPAI value as described in Shinoda et al. [26]. Furthermore, the protein gene models derived from Gmax275 and G. max Enrei2 genome assemblies were associated using the clustered data obtained from OrthoMCL [19]. Additionally, these gene models were associated by BLASTn. Table S6). In total, 11 gene models had no ORF hit sequences, 20,542 gene models had more than 50% coverage, 5,950 gene models had more than 90% coverage, and 2,269 gene models had 100% coverage. As mean coding sequence length was 1,168.1 bps, mean number of exons per gene 5.0, and mean exon length 231.5 bps of 56,044 Gmax275 gene models without variant, Enrei number of exons per gene was shorter and CDS was longer. The difference in the gene models between Gmax275 and G. max Enrei2 may be attributed to SNPs between the two cultivars as well as several parameters used in building the gene models. Phylogenetic Analysis. We applied OrthoMCL [19] to the clustered and aligned gene models of A. thaliana [16], A. lyrata [17], G. max cv. Williams 82 [7], G. max cv. Enrei, M. truncatula [18], and O. sativa (annotation data on Os-Nipponbare-Reference-IRGSP-1.0.). A set of filtered single copy genes was selected to calculate the phylogenetic relationships and divergence time among these species (Supplementary Table S1). Based on the phylogenetic divergence of [28]. A whole genome duplication (WGD) which occurred around 58 Mya had been a major factor in shaping the M. truncatula genome [18]. The complex of G. max and its wild relative, G. soja, diverged from the most recent common ancestor around 0.27 Mya [4] or 0.8 Mya [5]. Assuming that G. max diverged from G. soja at around 0.8 Mya, the divergence of the branch for both Williams 82 and Enrei at around 0.34 Mya was much later than previously estimated. As Li et al. [5] pointed out, divergent selection may have contributed to the differentiation of G. soja and G. max before domestication of G. max. The divergent selection as adaptation to different environments must have contributed to the differentiation of both cv. Williams 82 and cv. Enrei from the most recent common ancestor. 3.6. Protein. Using gene models associated with seed proteome data, a total of 164 protein gene models corresponding to storage proteins, lipid synthesis/degradation enzymes, sorting/folding-related proteins, late embryogenesis abundant (LEA) protein, glycolysis pathway enzymes, protease/ protease inhibitors, and others were identified (Supplementary Table S8). The protein content of dry seeds was 35-42% [34,35] of the dry weight, and 70% of protein consists of 7S and 11S globulins [34,36], which are part of the cupin superfamily (http://www.ebi.ac.uk/interpro/entry/IPR006045), corresponding to beta-conglycinin and glycinin, respectively [37]. To identify the proteins associated with grain filling of soybean seeds, we conducted a proteome analysis of the cotyledon. A total of 160 protein gene models in Gmax189 correspond to Enrei protein gene models ranging from 7.87 mol% to 0.03 mol% (Supplementary Table S8). Most of these proteins are storage proteins and cupin including beta-conglycinin and glycinin representing about 42% of total mol% and about 55% of total weight (sum of mass * mol) ( Table 2). Genes controlling the content of seed storage proteins were also highly represented [38,39]. Genes associated with lipid metabolism such as lipoxygenase 1, peroxygenase 2, and oleosin family protein genes [40]; gene associated with sorting/folding-related protein such as HSP20-like chaperone, PDI-like, SNF7 family, and vacuolar sorting receptor proteins; and LEA protein genes which may be important in protecting other proteins from aggregations were highly represented in the Enrei genome. In addition, some genes involved in glycolysis pathway, enzymes, and proteinase/protease inhibitors, which may play an important role in germination stage, were also found. This proteome profile may provide the basis for understanding cultivar diversity and adaptation to cultivation condition. Enrei Genome Database. All sequencing data can be accessed in DAIZUbase (http://daizu.dna.affrc.go.jp/enrei/), an informatics resource for soybean genomics focusing on the Japanese soybean cultivar Enrei. The database is provided with a GBrowse [41] with interactive pages for displaying the Enrei genome sequence as well all aligned Enrei BAC clones and accompanying annotations. DAIZUbase also includes a unified map, which indicates the relationship between the linkage map and the physical map of the Enrei cultivar. Conclusion The genome sequence of the Japanese cultivar Enrei will provide valuable information for improvement of soybean cultivars adapted to domestic cultivation. The genome sequence will complement emerging strategies for effective soybean breeding through analysis of the genome structure of Japanese (domestic) soybean, development of DNA markers serving as landmarks of agronomically important traits, development of research resources for the identification of important genes in soybean, and isolation of genes controlling important traits such as disease and pest resistance, productivity, and regional adaptability. Detailed knowledge of the genes controlling specific traits will allow for more efficient soybean improvement enabling researchers to develop plant types adaptable to various environmental conditions.

v3-fos

2019-03-20T13:04:06.735Z

2015-10-28T00:00:00.000Z

54878092

s2

Ecology of Basal Stem Rot Disease of Oil Palm (Elaeis guineensis Jacq.) in Cameroon Basal stem rot (BSR) disease caused by species of Ganoderma is of immense importance in oil palm production. Although much is known on the occurrence of this devastating disease, fundamental studies on the ecology in oil palm in plantations are rather limited. This study sought to determine the incidence, severity, distribution and spread pattern of BSR disease in oil palm plantations and relate disease parameters to climatic and edaphic factors. Surveys were carried out for two years on two–hectare plots in each of five oil palm estates of the Cameroon Development Corporation. Data for disease incidence and severity in each estate were recorded. Disease spread patterns were generated from Arc GIS version 9.3 using GIS coordinates of diseased plants. A correlation between disease parameters and soil physicochemical properties and multivariate analyses were done. Typical BSR disease symptoms were observed including unopened spear leaves, skirt–like appearance of leaves, basidiocarp formation, bole creation and death of the palm. The disease incidence ranged from 5.4% in 16-year old palms at Bota to 39.0% in palms of the same age in Mungo were about 50% of infected plants had extreme severe symptoms. Although principal component analysis showed that six soil properties account for variation in BSR disease incidence and severity, only fine sand content was positively correlated (P≤0.05) with disease incidence and severity, while C/N ratio was negatively correlated. This study has established the occurrence and spread of basal stem rot disease in five oil palm plantations in South Western Cameroon. Introduction Oil palm, Elaeis guineensis Jacq is an important oil crop in Cameroon where over 230,000 tons of crude palm oil is produced in about 190,000ha [1]. In the past, production was based on the Dura type palm whose production was low, but with the development of plantation agriculture, the more productive Tenera hybrid is widely cultivated. This hybrid produces up to eight times the amount of oil produced by other vegetable oil seeds like soybean and sunflower [2]. The inflorescence is also tapped for the much cherished palm wine. Generally, oil palm suffers from relatively few important diseases in each of the different environments where it has been planted commercially. In Southeast Asia, basal stem rot (BSR) disease caused by species of Ganoderma is the most important disease of oil palm [1]. Although in several African countries, vascular wilt caused by Fusarium oxysporum f.s. elaeidis was thought to be the only disease causing serious problems in some plantations, BSR disease has become one of the major diseases of oil palm, especially in Cameroon [4]. The disease was first recorded in Malaysia where it was initially considered a disease of older palms as it occurred in palms of over 25 years [5]. The BSR disease has also been recorded in Malaysia, Indonesia, Nigeria, Ghana, Zaire, Angola, Tanzania, North Mozambique, Papua New Guinea and Cameroon [5,6]. The causal agent of basal stem rot disease in Malaysia was first identified as G. lucidum (W. Curt.) Karst [5]. At least seven species of Ganoderma have been associated with BSR of oil palm in Malaysia, Indonesia, Papua New Guinea and Cameroon including G. boninense Pat., G. miniatocinctum Steyaert, G. chalceum (Cooke) Steyaert, G. tornatum (Pers.) Bers., G. zonatum Murill, G. xylonoides Steyaert, G. ryvardense Tonjock and Mih and G. lobenense [7,5,6,8,9]. BSR disease has been found to infect oil palms as young as 1 to 2 years of age, and is serious on palms aged 4 -5 years of age, particularly in replanted areas [10]. In new oil palm planted from jungle or old rubber plantations, BSR incidence of 25% has been recorded after 25 years while in that planted from old coconut plantations, an incidence of 60% occurred after 16 years [11], whereas oil palm to oil palm under planting has resulted in 33% infection after 15 years. The highest disease incidence is in coastal areas [5,12]. In Malaysian coastal areas, a 50% loss of yield was recorded from 80% disease incidence on 13 year old plantings [13]. A survey has also reported typical levels of disease incidence of 30% on 13 -year old palms in both inland and peat soils [14]. In North Sumatra (Indonesia), by the time of replanting (25 years) 40 -50% of palms are lost in some fields with the majority of standing palms showing disease symptoms. Where oil palm stumps were left in the ground at replanting then more serious palm losses due to Ganoderma have been observed in some fields with up to 25% incidence that occurred within 7 years [15]. The natural infection with Ganoderma in oil palm occurs as a result of contact between healthy roots and diseased tissues left buried in the soil [16]. Subsequent spread occurs by root to root contact once a few palms are infected [17]. In young palms, the external symptoms of basal stem rot normally comprise a one sided yellowing, or mottling of the lower fronds, followed by necrosis [10]. The newly unfolded leaves are shorter than normal and chlorotic, and additionally the tips maybe necrotic. As the disease progresses within the plant, the diseased palm may take on an overall pale appearance, with retarded growth and the spear leaves remain unopened [10]. Affected leaves die, necrosis sets in, beginning with the oldest leaves and extending progressively upwards through the crown. Dead dessicated fronds droop at the point of attachment to the trunk or fracture at some point along the rachis and hang down to form a skirt of dead leaves. Although there have been sporadic reports of basal stem rot in Cameroon, there is no comprehensive study on the ecology of the disease that could guide its management. The objectives of this work was therefore to determine the incidence, severity, distribution and spread pattern of basal stem rot disease in south western Cameroon and see how the disease relates to climatic and edaphic factors. Establishment of Sampling Plots The ecology of basal stem rot was studied in five oil palm estates belonging to the Cameroon Development Corporation (CDC) in south western Cameroon (Fig. 1). The means of various environmental parameters are shown on Table 1. In each estate, four 2ha plots were mapped out, each of the same age as shown on Table 2. Location of plot was done through a stratified random sampling technique. Disease Scoring Each plant in the sampling plots was observed for symptoms of BSR during the wet (June -August) and dry (October -December) seasons of 2010 and 2011 respectively, and scored for BSR severity on a scale of 0 -4 as shown on (Table 3) according to the method of Abdullah et al. [18]. Adapted from Abdullah et al. [18] The disease incidence was calculated as follows; . 100% (1) Where I= Incidence To assess the severity of the BSR disease, the disease severity index (DSI) was calculated using the method of Abdullah et al. [18] thus: (2) Where: A -Disease class (0, 1, 2, 3 or 4) B -Number of plants showing that disease class per estate. The Geographical Positioning System (GPS) point of each symptomatic plant was recorded. The data were processed using arc GIS version 9.3, to generate the disease spread pattern map. Soil Sampling and Analysis For each of the estates surveyed, five core soil samples were collected at a depth of 0 -10 cm, bulked, mixed thoroughly, air dried and sieved through a 2mm sieve. Results and Discussion Both asymptomatic and symptomatic plants of various levels of severity were observed in the field. Symptoms observed in the field are shown on Fig. 2. These ranged from presence of multiple unopened spear leaves at the centre, to skirt-like appearance of the leaves. Generally, the symptomatic palms had a pale appearance. Other symptoms observed were, production of fruiting bodies, bole creation on the base of the trunk, and finally death of the palms. The symptoms were typical of those described for the disease [19]. These are different from symptoms of vascular wilt of oil palm in that there is dryness of the lower leaves, the breaking of the rachis at about one third the length from the trunk, the hanging of the dry leaves along the trunk for the typical or acute form, the narrowing of the trunk at the top taking a "pencil -point" appearance, and the cracking of the trunk resulting from deterioration of the vessels for the chronic form [20,4]. During the first year of observation, the incidence ranged from 4.6% in Beneo estate to 38.0% in 16 year old palms at the Mungo estate ( Table 4). The incidence was generally high in young palms (≤16years) when compared to old palms (>30years) which recorded an incidence range of 14.2 -20.7%. The second year of observation showed a general trend of increasing incidence from 6.8% in Bota to as high as 40.0% in Mungo. Considering the two years of observation, the average disease incidence was highest at Mungo in palms that are at about their peak production age. Except for Mungo, the incidence of basal stem rot was generally low when compared to reports from other parts of the world on palms of comparable age. For example, values of 85% have been reported in 25 year old palms in Malaysia [21] and 30% in 13 years old palms in Malaysia [14]. The basal stem rot severity index was highest in Mungo where the mean for the period of observation was 80%, rated as very severe (Fig. 3). This is supported by the fact that the proportion of symptomatic plants with a score of five was highest there (Fig. 4). The high severity indices are an indication of the threat of this disease to production. Other observations on small holder fields in the Mungo area have similarly been shown to be highly infected, resulting in death of palms [1]. Basal stem rot disease spread showed a cluster pattern of spatial dispersion with only Bota showing a sparse pattern (Fig. 5). The disease had a dense cluster pattern in Mungo, Mondoni, Beneo, and Idenau estates. The spread pattern of BSR disease in this study was typical of soil-borne diseases which occurred in patches [22]. They typically appear in clusters or patches. The clustered pattern of infected plants was recorded at a number of sites and this was analogous to the pattern of basal stem rot reported by Rao et al. [14]. The mode of survival of Ganoderma as hyphae in oil palm residues would favour a clustered pattern of infected plants since dispersion of the residues of each plant would tend to overlap with that of other plants and a continuum of infested residue may result as the incidence of infected plants increases. Thus, under environmental conditions which favour a high incidence of infected plants, the pattern of infected plants would become regular. Soil physicochemical properties varied across the estates ( Table 6). Of these properties, fine sand had a significant positive correlation with disease incidence and severity at 0.01 probability level, while C/N ratio had a significant negative correlation at 0.05% probability level. There was no significant correlation between any of the climatic factors and disease incidence. This does not preclude the indirect effect of climate through the vigour of the plant and soil. This observation is expected because the disease is soil borne and systemic. Results of the principal component analysis (PCA) showed that the first four principal components explained 100% of the total variation ( Table 7). The PC 1 is strongly associated with fine silt, pH in KCl, organic carbon and total organic matter. PC 2 is strongly associated with pH in H 2 O, base saturation and Sodium. PC 3 is strongly associated with total nitrogen, clay content, base saturation and fine sand, and PC 4 is strongly associated with coarse silt, moisture content, cation exchange capacity and water holding capacity ( Table 8). The climatic data for Mungo was comparable to those of other sites. However, the edaphic factors were unique. The soils have a very high sand content, low clay content and consequently, low water holding capacity. The soil factors may partly contribute to this high incidence, given that there was a strong positive correlation between fine sand and incidence. Also, it may be due to the low nutrient status since there was a significant negative correlation between C/N ratio and disease incidence and severity. The low percentage of clay content of soil in Mungo estate might have been more conducive to the development of the basal stem rot disease than those with high clay contents. Soils low in clay content have been shown to be favourable to some soil borne diseases as is the case for damping-off of tomato seedlings caused by Sclerotium rolfsii in the Nigerian Savanna [23]. The incidence of basal stem rot would be much higher in future than the values obtained in the present survey because detection was based only on symptomatology. Plants at the early stages of infection generally do not manifest any symptoms. More elaborate techniques may be required to detect such plants [24]. Fig. 2. Field symptoms of BSR disease of oil palm in plantations of the CDC. A) Asymptomatic plant, B) Unopened spear leaves at the centre, C) Skirt-like appearance, D) Basidiocarp formation, E) Bole creation, F) Death of palm. Tengoua and Bakoume [4] recorded a 40% incidence level of basal stem rot in Mussaka palm plantation of the CDC planted between 1967 and 1969 but they actually projected a 60% incidence level of basal stem rot disease. However, these were very old palms and their result was not comparable to the present result in terms of percentage disease incidence but it shows that this disease had been present in oil palm plantations of the CDC. The high disease severity indices observed in the palms was expected. Plants with severity scores 3 and 4 constituted over 50% of the plants, thus resulting in the high disease indices observed. There was no management strategy put in place in the oil palm estates surveyed. The effects of the disease could easily be mitigated in Cameroon with proper management efforts. A preliminary survey in plantations of SOCAPALM where a rigorous eradication scheme was supplemented with trenching to curb spread revealed an extremely low incidence of less than 1%. However, in Malaysia a basal stem rot incidence of 80% and more were recorded in areas with attempted management strategies [14]. Fine sand was primarily the edaphic factor that influenced basal stem rot disease, having a significant positive correlation. High fine sand content (45.86%) found in Mungo estate allows for easy growth of roots thus enhancing contact and subsequent spread of basal stem rot. A very strong significant negative correlation was observed between incidence of basal stem rot and C/N ratio. Soils with low C/N ratios tend to release nutrients fast [25]. These nutrients easily leach away in the low water holding capacity soils thus starving plants and predisposing them to attack. Unsuitable soil conditions for plant development generally arise from lack of organic matter content in the soil [26,27]. Higher content of easily decomposable organic matters might be associated with higher microbial activity and ultimately lead to the decline of Ganoderma species population. Thus higher population of Ganoderma may aggravate the disease situation in locations where soil reaction is more acidic and organic carbon content is less as in the case of Mungo estate. The lower disease incidence in other estates may be attributed to high organic carbon contents and weak acidity. Similar results were obtained by Sharma et al. [28] for ginger rhizome rot. Conclusion This study documents the first comprehensive study on the incidence, severity and associated factors of Ganoderma disease of oil palm in South Western Cameroon, after the initial report by Tengoua and Bakoume [4]. The incidence and severity of the disease shows that it is of increasing importance in oil palm production such that its management needs to be considered in the oil palm production plan, given that the disease is very devastating in Malaysia and other oil palm producing countries of the Indian subcontinent. The pattern of disease occurrence and distribution was attributed to variations in edaphic factors which are important in pathogen survival and host predisposition. Although there was no correlation between incidence and severity of BSR with climatic factors, of Oil Palm (Elaeis guineensis Jacq.) in Cameroon principal component analyses showed the indirect influence of climate on the disease. Although no resistant clones are known for the disease, seed producers in Cameroon should screen for resistance in different progeny crosses. Also, there is need to monitor the disease on a continuous basis.

v3-fos

2016-03-22T00:56:01.885Z

2015-05-27T00:00:00.000Z

129695132

s2

Improving Water Use in Fodder Production Water deficit in semi-arid regions limits the future of the livestock sector. Also, its high price represents a percentage of the total cost of forage production. Non-conventional water resources applied by subsurface drip irrigation (SDI), in which the safe use lies in the management and not on the level of water treatment, would enhance the ruminant production sustainability. To obtain the optimal benefit, the transformation of water per kilogram of dry matter produced must have a high grade of effectiveness. Under this premise, a maralfalfa crop (Penissetum sp, hybridum) has been established with an SDI system and reclaimed water. Forage yield is analyzed with respect to a 40% irrigation reduction. This study shows that, with the use of these good irrigation management practices, it is possible to harvest an annual production of 90 to 72 t·ha in the warmer regions of the Canary Islands. This implies water consumption between 13,200 and 8100 m·ha. A water consumption of 21,000 m·ha per year for the same production, at a ratio of 230 L·t, can be estimated for the rest of the Canary Islands coastal regions. The use of the water management described in this paper can be profitable in the Canary Islands for fodder production. Introduction "White" water or the efficient use thereof is defined as biomass production per square meter of water consumed, including both "green" water (effective rainfall) and "blue" (surface and groundwater) in the case of irrigated areas. The production of livestock feed consumes large amounts of white water, so a new concept appears: "livestock water productivity" [1]. Water scarcity has limited the livestock development in semiarid regions, as high price of water has an elevated percentage of the total cost of forage production. On the other hand, this sector is unable to compete with other sectors for the use of blue water. In this sense, water management is essential, especially if using non-conventional water resources (such as reclaimed water) as origin of irrigation water in fodder production. Moreover, using these resources, much abandoned land could be recovered by reducing the environmental impact of erosion. In current Spanish legislation [2], reclaimed water quality requirements for fodder irrigation (quality 2.2) are lower than the ones required for other uses. However, this legislation does not take into account the water management used in the field, for example: some irrigation types are not considered, and dosage and agro-environmental conditions are neglected. The soil could act as advanced water treatment system, allowing lower water quality in fodder irrigation, thereby avoiding the health problems. In fact, an FAO study concluded that rather than focusing only on the quality of wastewater, it would be better to ass and manage the risks of using reclaimed water to achieve the same goal of health guarantee [3]. This study determined that subsurface drip irrigation (SDI) allows the use of effluent with a lower level of treatment to reaching the same health security. In this sense, Palacios [4] concluded that SDI optimizes water by preventing water loss and providing greater health guarantee. Jensen [5] linked the crop yields with water use. This author cited several factors that affect the water efficiency, such as climatic factors, water efficient application and the crop physiology. Má rquez [6] demonstrated that maralfalfa grass (Pennisetum sp) is an alternative to increase forage availability for livestock due to its high dry matter productivity and nutritive values, provided once the minimum requirements for this species are satisfied. However, few studies [7] mentioned the water needs of this fodder, which often conduces to misuse of this resource. The aim of this study was to optimize the reclaimed urban water use in the maralfalfa crop in the Canary Islands agro-climatic conditions. Experimental Plot During 2014, the Maralfalfa grass (Pennisetum sp) experiment was conducted in a field of 272 m 2 situated in Granja Agrí cola Experimental del Cabildo de Gran Canaria. An automatic weather station, used to calculate reference Penman-Monteith Evapotranspiration (ETo), was already set in the field. During the study, three maralfalfa harvests were obtained: first productive period, from February to May (harvest on 22 May), the second: May to July (17 July) and the third: July to September (17 September). Rainfall was low: 3.7, 3.8 and 7.4 mm respectively, compared with the average of 83.37 during the period from February to September (191.6 of annual value average for the 14 last years, which is the period with available information). Mean of minimum temperatures (Tm) was 17 °C and mean of maximum temperatures (TM) was 25 °C . Mean temperature (Tmed) is presented in Figure 1. During the previous 14 years, the average values of the minimum and maximum temperatures were 11 °C and 31 °C, respectively. The field is classified as Anthrosol [8] or Torriarent [9]. An experimental plot of 272 m 2 was divided in two blocks with two treatments in each (T1 and T2). Each treatment consists of five lines of approximately 21 m long, coinciding with the irrigation lines. These lines are spaced at 0.75 m. Therefore, both T1 (100% of dose) and T2 (60% of dose), are irrigated in each block. During the third harvest, a fertilization compatible with organic farming management was applied (Lignoser, n° CE/19287), at a rate of 1.1 g/m 2 N. Fertilizer also provides fulvic acid, K and trace elements. Soil Analysis Sample soils were taken from the first 0.2 m, coinciding with the transplant and harvest days. Table 1 shows the results in each block. Organic carbon (OC, %) and nitrogen (N, %) were determined by dry combustion with a LECO CNS 2000 analyzer. Soluble salts were estimated by the electrical conductivity EC1:5 (soil:water ratio; dS/m). Available nitrate was determined by extraction of soil, also at 1:5 ratio, with 0.01 M calcium chloride, and analyzed by ionic chromatography. Available soil P (mg/kg) was determined by sodium bicarbonate extraction, according to Olsen method [10]. Exchangeable cations (K, Ca, Mg, and Na, meq 100 g −1 ; B, Fe, Cu, Mn and Zn, mg· kg −1 ) were extracted with buffered 1 M ammonium acetate at pH 7, and were analyzed by ICP. Table 1. Determination of organic matter (OM, expressed in %), total nitrogen (N tot, expressed in %), electrical conductivity 1:5 (EC, dS/m), nitrate (expressed in mg· kg −1 , available phosphorus (P, expressed in mg· kg −1 ), extracted cations (expressed as me 100 g −1 ): potassium (K), calcium (Ca), magnesium (Mg), sodium (Na), and the rest of analyzed nutrients (expressed in mg•kg −1 ): boron (B), copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn), for T1 and T2, expressed by mean and standard deviation. Forage Characterization Fresh matter production was weighed in field in each harvest for the different lines. Composite samples of the different treatments were taken to determine the percentage of dry matter (DM), macro-and microelements ( Table 2). Dry matter (DM) was determined by drying in an oven at 60 °C to constant weight. Plant samples were subjected to microwave digestion with nitric acid. The following elements were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES): P, K, Ca, Mg, Na (expressed in %), B, Cu, Fe, Mn and Zn (expressed in mg/kg). Nitrogen was determined by dry combustion in an LECO CNS 2000. Irrigation System Ground water was applied by subsurface drip irrigation (SDI) system with integral drippers: pressure-compensating at range 1.5-4.0 bar, anti-Siphon and high anti-drain mechanism (Techline mod) spaced 0.5 m with delivery rates at 2.3 L· h −1 , being an abnormal low flow for drip irrigation. The lateral lines were spaced to 0.75 m. Irrigation was provided daily two times a day during 20 min and 12 min (T1 and T2 respectively) on each irrigation event. The weather station information was used for irrigation in this experiment. Each treatment has a flow meter, read weekly. Table 3 shows water consumption (L· m −2 ) for each harvest. Statistical Analysis Analyses of variance (ANOVA) were carried out using statistical Packaged SPSS (version 22) by the Generalized Linear Model. The model includes soil parameters, leaf nutrients and yield (expressed as kg· DM· m −2 ) from each harvest, the irrigation doses (100% and 60%) and their interactions. Mean separation was tested using the least significant difference (LSD), considering p = 0.05 Figure 1 shows the climatic data during the experimental periods. As observed, there are a progressive increase in the Tmed and a relatively low radiation values at the time of study (higher radiation in June than in July and August). Results and Discussion The soil nutrients, present in Table 1, show the high content of OM while Ntot content is stable (it does not present significant differences), which is consistent with the low relation C/N. Although not significant, there is a salinization trend on the surface horizon, compatible with water and irrigation system management. Although it does not show significant differences, nitrate content decreased slightly over time, but it remained at acceptable levels (between 321 and 114 mg· kg −1 for September) to ensure an adequate forage growth. However, N deficiency symptoms in plant were observed, coinciding with low values of leaf N content (Figure 2, T1), so fertilizer was applied. These N contents correspond to values of crude protein (CP) between 5.3% and 7.7%, lower values than would be expected for harvesting at 60 days (8.8%, [11]). This apparent contradiction between adequate nitrate values in soil and low N leaf contents can be explained because the soil sampling was done from top soil to 0.2 m and, due to the localization of the irrigation system (buried), the root system is not able to absorb the nitrate ascended by capillary rise. Trace elements are increased for the third cut, which seems to be influenced by the fertilizer used. Table 2. Determination of dry matter (DM, expressed in %), nitrogen (N), phosphorus (P) potassium (K), calcium (Ca), magnesium (Mg) and sodium (Na), expressed in% and other elements (expressed in mg/kg): boron (B), copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn) for forage harvested at different dates and for each of the treatments, T1 and T2, expressed by mean and standard deviation. Table 3. Period of study (days); thermal integral (TI); radiation (rad, MJ· m −2 ), water consumption, Wcons, (L· m −2 ); accumulated (Ac) and mean evapotranspiration (ETo, L· m −2 ); water consumption/ETo (%); production (kg· m −2 DM) and water consumption per kg of dry matter (Water consumption coefficient, L· kg −1 DM) produced for the different harvest periods and treatments, for T1 and T2 expressed by mean and standard deviation. Nutrient contents in leaves ( Table 2) remain relatively stable, although decreasing slightly over time. As observed in Figure 2, reduction dose treatment (T2) shows higher N contents in leaves, although these differences were not significant. Adequate P contents were measured (between 0.2% and 0.4%). No relationship was found between increased contents in soil, after fertilizer application, and leaf contents. A great increase in DM harvested in the third period (September 2014) was obtained (Figure 3a), yielding significantly more than the other two ones. This production could be the result from the higher temperatures in this period (see T med in Figure 1). To explain this result, the thermal integral (TI) for the experimental periods was calculated (Table 3), as a sum of daily temperatures above 10° C (zero vegetation for this forage, [12]). The TI for the first period was very high, because it corresponded to the establishment phase (100 days). Although periods 2 and 3 were similar in terms of days, the lower TI for the second period vs. the third (75% versus 100%), showed a significantly lower yield than obtained by the third period. Table 3 shows the water consumption (L· m −2 ) and yield (kg· DM· m −2 ) for the different periods. Water consumption divided by ETo, shows that the ratio of consumption average were 0.9, 1 and 0.9 for the three periods in T1, whereas for T2 were 0.7, 0.6 and 0.5 respectively. Therefore, water consumption coefficients are slightly higher for T1 and slightly lower for T2 to those obtained by Murillo Solano [7], since these authors quote to ET 3 L· m −2 per day, and Kc from 1 to 0.7. In fact, as shown in Figure 3b, water consumption divided by dry matter produced (water consumption coefficient) demonstrated significant differences between treatment and dates since the second harvest (once the plant was established). Higher values were obtained in both dates for controlling dose (T1), (516 and 182 L· kg −1 · DM) versus T2 (355 and 134 L· kg −1 DM). The significant differences between yield obtained in T1 and T2 in September (Figure 3a) were high, demonstrating that water, although used very efficiently in both treatments (Figure 3b), was the limiting factor for this period. However, in the establishment period (May), no differences were found. Depending on the water and fodder price, the farmer could or could not use a reduction in the irrigation dose. Water consumption coefficients obtained for the third cut, once the plant is established and with very supportive temperatures for this crop, are exceptionally favorable (Figure 4 left). This figure shows that in September, the best yields were obtained, although the water applied was limited. This was possible because the water consumption coefficient is extremely favorable (182 and 134 L· kg −1 DM, respectively for T1 and T2 treatments, Table 3). This fact contributes to the high water use efficiency (WUE), which was calculated using the ratio of dry mass per area (kg· ha) and the amount of water consumed (mm), whose values are shown in Figure 4 right), values above those mentioned for a C3 grass, [13]. This high WUE can be explained by the subsurface drip irrigation system (practically all the water supplied is absorbed by the plant), water management (low dose twice daily) and plant physiology (C4 metabolism). Under these conditions, maralfalfa production is very efficient in water use and can be estimated that, for a production of 72 to 90 t of hay· ha −1 respectively (with or without a reduction in irrigation) the amount of water would be 13,200 m 3 · ha −1 or 9600 m 3 · ha −1 . Conclusions Fodder production is possible in warm areas of the Canary Islands with a good water management, providing the appropriate technology, despite the high price of water. In this sense the farmers need to use a high efficiency irrigation system, a crop that take advantage of the favorable conditions of temperature and radiation and no-conventional water resources whose safety use is filed in the water management and not in the treatment level. When subsurface drip irrigation systems are used, the soil profile explored by roots is modified. This factor must be taken into account when sampling the soil and providing nutrients, especially when the fodder demands high quantities of these elements. For the warmer regions of the Canaries, whose average monthly temperatures are at or above 22 °C, our studies allow us to estimate an accumulated production (annual) of 90 or 72 t· ha −1 · hay (control or dose reduction of 40%), with water applied between 13,200 and 8100 m 3 · ha −1 . For other coastal regions, where this species can be cultivated, but in which the use of water is not as effective (as occurred in July), the amount of water needed to obtain the same yield can be estimated in about 21,000 m 3 · ha −1 .

v3-fos

2018-05-21T21:28:06.993Z

2015-12-01T00:00:00.000Z

21705472

s2

Effects of catechins on litter size, reproductive performance and antioxidative status in gestating sows This study was conducted to investigate the effects of catechins on reproductive performance, antioxidative capacity and immune function of gestating sows. A total of 60 cross-bred (Landrace × Large White) multiparious sows were blocked by body weight, parity and backfact and randomly allocated to 1 of 5 treatments: 0, 100, 200, 300, or 400 mg/kg catechins. Dietary treatments were imposed from mating to d 40 of gestation of sows. At farrowing, litter total born, born alive, dead, and normal-(healthy piglets, ≥0.85 kg) and low-birth weight piglets (<0.85 kg) were recorded. Within 3.00 ± 0.50 days after farrowing litter size was standardized to 8.00 ± 1.50 piglets within treatment. The piglets were weighed at birth (d 1) and weaning (d 28). Sows serum samples were obtained from blood samples collected on d 40 of gestation for analyses of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), hydrogen peroxide (H2O2), nitric oxide synthetase (NOS) and nitrogen monoxide (NO). Our results showed that supplementation of catechins at levels of 200 or 300 mg/kg led to improvements in litter born alive (P < 0.01) and piglet born healthy (P < 0.01) and a decrease in stillborn (P < 0.05) at farrowing when compared with the control. In comparison with the control, catechins at any supplemental levels all enhanced the serum SOD (P < 0.05) and CAT (P < 0.01) activities of sows at farrowing but no obvious differences in the serum GSH-Px and NOS activities were observed in this trial (P > 0.05). Sows received 200 mg catechin per kg diets showed a reduction (P < 0.05) of the serum MDA level at farrowing compared with all other treatments. Sows received all the levels of catechin showed a reduction (P < 0.05) of serum H2O2 level compared with sows received the control diet on both d 40 of gestation and farrowing. Our results demonstrated that the catechins may be a potential antioxidant to increase the reproductive performance and antioxidative capacity of sows when it was added into diets during the early gestation. Introduction Rapid fetal development during the gestation led to a catabolic status of pregnant women or dams which is known to contribute to the production of excessive free radicals including superoxide and hydrogen peroxide and the induction of systemic oxidative stress (Herrera and Ortega-Senovilla, 2010;Kim et al., 2013). Increased oxidative stress was reported to be an important factor causing decreased availability of antioxidants during late gestation, which could impaired placenta and fetal growth (Prater et al., 2008) and trigger a disrupted antioxidant system that was involved in a variety of pregnancy complications such as preterm labor, fetal growth restriction, preeclampsia and miscarriage Sugino et al., 2007). This elevated oxidative stress during gestation and lactation was likely to influence not only the litter performance, but also the well-being and health status of sows including impaired milk production, reproductive performance, and longevity (Agarwal et al., 2003;Jabbour et al., 2009;Zhao et al., 2011Zhao et al., , 2013. Therefore, much attention has been paid to how to reduce maternal oxidative stress levels and inflammatory responses of highly prolific sows in late gestation by feed antioxidant additives. In numerous previous studies, antioxidants such as vitamin E, vitamin C, carotenoids, and selenium (Lykkesfeldt and Svendsen, 2007), fish oil and olive oil (Shen et al., 2015) and soy isoflavones (Hu et al., 2015) were added into the diet during gestation period in order to compensate for the substantial loss of these feed antioxidant additives. Excessive reactive oxygen and radical were actually produced from placental and maternal metabolism during the early pregnancy of sows. Although oxidative stress in late gestation was more serious than that in early gestation with the course of pregnancy (Berchieri-Ronchi et al., 2011;Casanueva and Viteri et al., 2003;Myatt and Cui, 2004), indicating that early pregnancy may be a key phase for prevention of oxidative damage. Catechins are members of the flavonoid family and belong to plant polyphenolic constituents (Uzun et al., 2010), which are not only existing in a high concentration within tea, but also present in many foods, such as apples, grapes, vine and their processed beverages (Tichopad et al., 2005;Suzuki et al., 2007). Previous studies showed that catechins has a certain degree of hydrophobicity and can capture the OH À1 , which protect the DNA from the oxidant damage (Yoshioka et al., 1996); And catechins also alleviated the damage by up-regulating the expression of genes of some antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) so that increase those enzyme's production and activity (Mkimura et al., 2002). Previous studies showed that catechins prevented metal ions from participating in peroxidase reactions by binding them and had the potential to scavenge reactive oxygen and nitrogen species, thus reducing their damage to lipid membranes, proteins, and nucleic acids in cell-free systems (Wiseman et al., 1997). These findings indicated that catechins prevented metal ions from participating in peroxidase reactions by binding them and had the potential to scavenge reactive oxygen and nitrogen species (Wiseman et al., 1997). It was reported that catechins administration significantly decreased malondialdehyde (MDA) level and noticeably increased activities of CAT, GSH-Px and SOD, suggesting that catechins provided effective protection from oxidative damages through their antioxidant properties (Tarek et al., 2012). Therefore, it is believed that catechins have a beneficial role on physiological functions and biotransformation of physiological processes involved in the detoxification activities and preventing oxidative damage as a result of their ability to scavenge reactive oxygen species such as hydroxyl radical and superoxide anion (Galati et al., 2002) and metal chelating (Pedrielli and Skibsted, 2002), thereby providing some protection from toxic metabolic actions of oxidative stress (Suhel et al., 2006). Considering the antioxidant properties of catechins and oxidative stress for highly prolific sows in early pregnancy, the aim of this study was to determine the effect of catechins supplementation in diets fed to sows in early pregnancy on reproductive performance and antioxidative status of gestating sows. Materials and methods This experiment was conducted at the Zhenghong Swine Research Farm in Miluo District, Hunan Province, China. This study was performed in accordance with Chinese Animal Welfare Act guidelines and approved by the Animal Care and Use Committee of the Institute of Subtropical Agriculture, the Chinese Academy of Sciences . Animals and experimental design A total sixty of multiparous lactating sows (Landrace  Large White) were used in this experiment. The sows were evenly allocated by BW, back fat, expected farrowing date and parity into 5 dietary treatments, with 12 replicates. Litter size was standardized to 8.00 ± 1.50 piglets within 3.00 ± 0.50 days after farrowing. The sows were housed individually in crate stalls from mating. Five days before the expected date of farrowing/parturition, all the sows were moved into the individual farrowing crates with a heated piglet nest on d 109 of pregnancy. Rooms were ventilated mechanically. The sows receive a gestational diet ( Table 1) with levels of nutrients and minerals based on NRC (1998) recommendations from mating to d 40 of gestation, which is only different in the dose of catechins: 0 (Group I), 100 (Group II), 200 (Group III), 300 (Group IV) or 400 (Group V) mg catechins per kg diet. The sows were given 1.8, 2.3, and 2.5 kg/d during the pregnancy period, respectively, and thin sows were provided an extra amount of feed (0.3 kg/d). The day of farrowing sows did not receive any feed and the daily amount of feed was increased by 0.75 kg/d until ad libitum at d 4 of lactation, and thereafter the sows were fed twice daily, in the morning and evening, and had free access to water from nipple drinkers. The lactation length was 28 days. The litter total born, born alive, stillborn, and low-birth weight piglets (birth weight 0.8 kg) were recorded. Pigs born healthy number was obtained by the difference between the litter total born and stillborn and mummies. The piglets were weighed at farrowing. No creep feed was provided. Natural catechins (99% purity) were obtained from National Research Center of Engineering Technology For Utilization of Functional Ingredients From Botanicals (Hunan, China). Sample collection and chemical analysis On farrowing day, colostrum samples were drawn manually from every active teat of a sow after injection of 15 IU oxytocin. Blood samples (10 mL) were taken from ear veins into vacuum blood collection tubes on farrowing and d 40 of gestation. The blood samples were allowed to clot at room temperature for 30 min, and then centrifuged at 3,000  g for 15 min at room temperature with the resulting serum stored at À20 C until analysis . The activities of SOD, GSH-Px, catalase, nitric oxide synthetase (NOS) and MDA were determined by assay kits according to the manufacturer's instructions (Nanjing Jiancheng Technology LTD., China). The contents of serum hydrogen peroxide (H 2 O 2 ), nitric oxide (NO) were also determined by assay kits in accordance with the protocols provided by the manufacture (Nanjing Jiancheng Technology LTD., China). Statistical analysis All data were analyzed by ANOVA using the GLM procedures of SPSS11.0. The statistical model consisted of the effect of diet. Pen was used as the experimental unit for the piglet performance data, whereas individual sow was used as the experimental unit for reproductive performance and blood analysis. Multiple comparisons were carried out by the Tukey test. Differences were considered significant when P < 0.05. The results are reported as the means and standard errors (means ± SE). Litter performance Sows received 200 and 300 mg catechin per kg diets had improved (P < 0.01) pigs born alive and pig born health compared with sows fed the control diet. Sows received 200, 300, and 400 mg catechin per kg diets had reduced (P < 0.05) stillborn compared with sows fed the control diet (Table 2). Antioxidative status indicators in sow serum The serum GSH-Px activity did not differ among all treatments on d 40 of gestation and at farrowing (P > 0.05). But all levels of catechin improved (P < 0.05) activities of sow serum SOD and CAT at farrowing. In addition, no significant changes (P > 0.05) in sow serum NOS activity was observed on d 40 of gestation and at farrowing. At farrowing, serum MDA level decreased with the increase of catechins and the lowest MDA content was observed in group III (200 mg/kg catechins) (P < 0.01). And the serum MDA level in groups IV and V didn't show any significant difference compared with the group I even the content of diet catechins increased from 300 to 400 mg/kg. However, no effects (P > 0.05) of catechins on serum MDA concentration were noticed on d 40 of gestation. Serum H 2 O 2 content decreased (P < 0.05) with the increase of catechins on d 40 of gestation and at farrowing compared with the control. Similar to NOS activity in serum, there was no difference (P > 0.05) in serum level of NO among treatments (Table 3). Discussions Oxidative stress leads to low reproductive performance through the pregnancy complications, preterm labor, fetal growth restriction, miscarriage or abnormal fetal development (Dennery, 2004;Agarwal et al., 2003), which adversely affects the production of highly prolific sows (Berchieri-Ronchi et al., 2011). Previous results showed that oxidative stress led to both lipid and protein oxidation, impaired normal endothelial cell functions (Serdar et al., 2003), and altered placenta and fetal development as well (Hansen et al., 2001;Prater et al., 2008). Litter size at birth is determined by the implantation of fertilized eggs and fetal development in uterus during the early gestation, which directly affects the reproductive performance of breeding sows and production of their offsprings (Herrera and Ortega-Senovilla, 2010;Berchieri-Ronchi et al., 2011). Therefore, it is very important to decrease oxidative damage caused by excessive free radicals including reactive oxygen species (ROS) and nitrogen species (RONS) on fetal implantation and development in wombs of sows during the early gestation. One of the primary aims of this study was to investigate whether oxidative stress and its damage on the litter performance could be reduced by providing the catechins during the early gestation of sows. Our results demonstrated that catechins showed a beneficial effect on the sow reproductive performance, including significant improvements in Values are means ± SE, n ¼ 12. Means within a row without common lower-case letters (P < 0.05) and upper-case letters (P < 0.01) differ. Values are means ± SE, n ¼ 12. Means within a row without common lower-case letters (P < 0.05) and upper-case letters (P < 0.01) differ. the litter size (total and alive), piglet born healthy and average piglet birth weight, and a substantial decrease in stillborn. Additionally, sows receiving 200 or 300 mg catechins per kg diets showed higher litter performance compared with sows from other treatments, indicating that there may be a doseeeffect relationship in a certain range between catechins and sow performances. Moreover, free radical-induced birth defects and other situations such as abortions, stillborn and mummies (Lagod et al., 2001;Loeken, 2004) could be avoided or decreased when the catechins is added into the gestation diets of sows during the early gestation. Animals' antioxidant defense systems can cope with ROS including hydroxyl radicals, superoxide radicals, hydrogen peroxide and the oxidative metabolites such as MDA (Gielgiel et al., 2012). However, during the whole gestation, there is an increased energy demand and oxygen requirement with heavy metabolic burden, which favors a state of oxidative stress resulted from the overproduction of reactive oxygen species (Agarwal et al., 2003;Reyes et al., 2006). The excessive ROS and NOS and disrupted antioxidant system caused by the deficiency of antioxidant such as SOD, GSH-Px, CAT, NOS were reported to be involved in a variety of reproductive problems Sugino et al., 2007;Berchieri-Ronchi et al., 2011;Hana et al., 2015). This indicates that it may be necessary to provide or supplement some antioxidants such as vitamin E and vitamin A, fish oil, linseed oil and catechins from plant extracts in the diet during the gestational period in order to compensate for the substantial loss of these nutrients. The effect of catechins on the above mentioned oxidative enzymes and oxidative metabolites including MDA, H 2 O 2 and NO were assessed in this trial. The results showed that, activities of SOD and CAT in serum of sows at farrowing were increased by 12 to 77% relative to the control, despite that only a numerical increase in activities of serum GSH-Px and NOS caused by dietary supplementation of catechins was observed in the present study. This result demonstrated that the anti-oxidant ability of sows at farrowing were mainly modulated by SOD and CAT, which consist of sows' defense system with other anti-oxidant enzymes in order to cope with the oxidative damage caused by excessive radicals. Corresponding to the change of serum enzyme activities of sows was the variation of oxidative metabolites such as MDA, H 2 O 2 and NO in serum of sows. Compared with the improvement on the serum SOD and CAT activities by catechins, both serum MDA and H 2 O 2 levels were significantly decreased on d 40 of gestation and at farrowing. This indicated that catechins had a good effect on the anti-oxidant ability of sows by increasing the serum enzyme activities and decreasing the levels of oxidative metabolites including MDA, H 2 O 2 and NO in serum of sows. The similar tendency of serum NO level and NOS activity as affected by catechins suggested a fact that the catechins had little effects on anti-oxidative capacity modulated through NOS and NO of sows under our experimental conditions. Conclusions Catechin had a positive effect on the reproductive performance, antioxidant and health status of sows when added into the diet during the early gestation. The optimal supplementation level is between 200 and 300 mg catechin per kg diet based on the relationship between catechin and reproductive performance and antioxidative indicators of gestation sows. Conflicts of interest None of the authors have any conflict of interest to declare.

v3-fos

2019-03-31T13:42:35.771Z

2015-12-10T00:00:00.000Z

87574052

s2

Egg Production and Physical Quality in Cortunix Cortunix Japonica Fed Diet Containing Piperine as Phytogenic Feed Additive The objective of this study was to determine the effect of piperine as a phytogenic feed additive on quail performances and egg quality. The experiment used a completely randomized design with five treatments and four replications and used ten quails with one week of age in each replication. The piperine was added to the diets at concentrations of 0 (T0), 15 (T1), 30 (T2), 45 (T3), and 60 mg/kg body weight (T4) for 8 consecutive weeks. The results showed that addition of 60 mg/kg body weight (T4) of piperine significantly (P<0.05) reduced feed consumption, egg production, egg mass, income over feed cost (IOFC), and increased water consumption as compared to the other treatments. The addition of 15-60 mg piperine/kg body weight significantly (P<0.05) reduced eggshell weight and increased egg yolk color score. The conclusion of this experiment was that the addition of piperine at 15-45 mg/kg body weight could be used as phytogenic feed additive to improve performance, IOFC, haugh unit, and yolk color. INTRODUCTION Quail is one of the poultry that should be developed and enhanced its production potential because this species has a very short live cycle and efficient to produce animal protein needed in the form of eggs and meat for the community. According to BPS (2013), population of quails in Indonesia from 2009 to 2013 increased from 7,667,586 to 12,596,043 heads. There are some advantages in business of quail such as the species starts laying egg at the age of six weeks, does not require large capital, easy on maintenance and need a smaller land area to rear the quails. However, to produce the production and good quality of eggs, many farmers use synthetic antibiotics in the feed causing antibiotics residues in the eggs and meat. According to Saeid & Al-Nasry (2010), food safety issues occurring in society, some of those are originally from animals include contamination of microbial pathogens and antibiotic residues in meat and eggs as a side effect of antibiotics in the feed that serves as Antibiotic Growth Promoters (AGPS). The measure taken to anticipate that issue was the use of phytogenic feed additive. Phytogenic feed additive is an additional material for feed that was derived from plants drug (herb) and spices as the replacement of antibiotic growth promoters (Lee et al., 2013;Saki et al., 2014), which is able to improve performance, feed conversion ratio, digestibility, and weight gain in poultry (Perić et al., 2009;Saki et al., 2014). One of the potential phytogenic feed additives is black pepper (Piper nigrum). One of the active components contained in black pepper (P. nigrum) is piperine. Piperine is an alkaloid substance which can increase the absorption of selenium, vitamin B complex, beta-carotene, and curcumin as well as other nutrients (Khalaf et al., 2008) that eventually improve performance, productivity, and carcass quality and increase the body's immune status (Cardoso et al., 2012;Ravindran & Kallupurackal, 2012). Piperine is also an active ingredient which works to increase thermogenesis in the body (Bhutani et al., 2009;Ahmad et al., 2012), increases digestion and energy metabolism in the body and increases the bioavailability of certain drugs in the organism (Ahmad et al., 2012) so as to improve immunity. Cardoso et al. (2012) suggested that supplementation of piperine into the feed at the dosage of 60 mg/kg BW could increase performance and health status of broilers. The aim of this study was to determine the effectiveness of piperine as a phytogenic feed additive on production and egg's physical quality in Coturnix coternix japonica. MATERIALS AND METHODS The black pepper (P. nigrum) was obtained from the farmers at Sukadana, Lampung Province. The black pepper was dried in the oven with a temperature of 60 o C for 24 h and then grounded to be a powder. The content of phytochemical of the powder was 5.18% piperine (Balitro, 2014). According to Madhavi et al. (2009), the content of piperine in the black pepper is 5%-9% of the total nutrient. The feeding trial in this experiment used 200 laying quails with 6 weeks of age and the average weight of 101.8 g/bird. Piperine doses calculation was based on a standard requirement of quail per day i.e., feed consumption of 25 g. Piperine content in black pepper is 5.18% so that each treatment dose was obtained as follows 15 mg/kg body weight: 1.18 g piperine/kg feed, 30 mg/kg body weight: 2.36 g piperine/kg feed, 45 mg/ kg body weight: 3.54 g piperine/kg feed, and 60 mg/kg body weight: 4.72 g piperine/kg feed. The experiment used a completely randomized design with five treatments and four replications with ten quails of each replication. The experimental quails were placed in 60 x 60 x 40 cm pen. The treatment diets were T0: Control diet (without piperine), T1: T0 + piperin 15 mg/kg body weight, T2: T0 + piperin 30 mg/kg body weight, T3: T0 + piperin 45 mg/kg body weight, T4: T0 + piperin 60 mg/kg body weight. The diet were formulated according to the nutrients requirement recommendation of Leeson & Summers (2005). The diet contained 18% crude protein and 2950 kcal/kg. Composition and nutrients content of the control diet are presented in Table 1. Quails were reared for 10 wk, 1 wk for environmental adaptation, 1 week for diet treatment adaptation, and 8 wk for diet treatment. Feed and water were provided ad libitum. Feed consumption and water consumption were measured every week, while egg production and egg weight were recorded every day during the treatment. Physical quality of egg was observed at the first, second, third, fourth, and eighth week of the treatment. Three eggs were taken from each replication for physical quality analysis. Parameters observed in this study were quail performance: feed consumption, water consumption, quail day egg production, egg mass, feed conversion, income over feed cost (IOFC), and egg physical quality. All data were analyzed by using one way analysis of variance (ANOVA) according to General Linear Models (GLM) procedure for completely randomized design using SAS 9.3 (SAS, 2010). If there was a significant different, the data was further analyzed by using Duncan's multiple range test (Mattjik & Sumertajaya, 2006 Quail Performances Supplementation of piperine with the dose of 60 mg/kg BW (T4) significantly (P<0.05) decreased feed consumption and increased water consumption compared to the other treatments (Table 2). Water consumption of control (T0) quail was lower (P<0.05) as compared to the other treatments. The decrease in feed consumption of T4 was due to a strong and spicy flavor that could reduce the palatability of the feed. Windisch et al. (2008) stated that the content of the active substances contained in black pepper caused the feed was very pungent and spicy flavor which may limit its use for animal feed. According to Amad et al. (2011), the addition of phytogenic feed additive derived from spices could affect the flavor, such as piperine (spicy flavor) from phytogenic compounds so that feed consumption would decline. The decreased feed consumption will have an impact on increasing water consumption due to the high content of piperine in the feed. The high content of piperine in the feed could trigger the process of thermogenesis in the body so that it will have an impact on increasing water consumption. The increased water consumption will increase the release of heat loss from the body. According to Boschmann et al. (2007), consumption of large amounts of water increase heat loss so that the release of the heat generated by the process of thermogenesis would be able to balance the body temperature. Adding piperine as much as 15-45 mg/kg BW did not affect the egg production (quail day production). Adding piperine with the dosage of 30 mg/kg BW increased egg production significantly (P<0.05) compared to T4 but not significantly different from T0, T1, and T3. Egg production in quails supplemented with piperine with a dosage of 60 mg/kg BW (T4) was significantly (P<0.05) lower than the other treatments. The decreased egg production was due to the low feed consumption which affected the consumption of nutrients. According to Leeson & Summers (2005), egg production was affected by the strain, age, feed consumption, water consumption, the consumption of mineral, and protein content of feed, while Widjastuti & Kartasudjana (2006) explained that the low feed and energy consumption during the production phase of quail would decrease egg production. The average of egg mass in this study was 409.23-531.96 g/quail. Supplementation of piperine at the level of 15% up to 45% did not affect egg mass. Addition of piperine at the dose of 60 mg/kg BW (T4) decreased the egg mass significantly (P<0.05) compared to the other treatments. The decreased egg mass was due to the decrease in percentage of egg production that finally decreased egg mass. In contrast to results of the study conducted by Al-Harthi et al. (2009), the addition of spices such as black pepper, cumin, and cardomon could increase the egg mass and egg weight. The egg mass would be positively correlated with the decrease in egg production, while egg mass was calculated as the mean of egg weight produced by mean of laying rate during the production days so that would be positively correlated (Sh et al., 2013). Furthermore Vercese et al. (2012) stated that the mass of egg was influenced by egg weight, egg production, and heat stress. The treatments did not affect the feed conversion. The average feed conversion in this study was 2.10-2.41 (Table 2). Feed conversion in quails supplemented piperine with a dose of 60 mg/kg BW (T4) was significantly (P<0.05) lower than the other treatments. This lower feed conversion was due to the lower feed consumption with the higher weight of the eggs produced. Feed conversion values closely associated with the ability to change the feed into meat and eggs. The lower the figure the more efficient is the conversion of feed, since the amount of feed used to produce meat and egg is lower. According to Leeson & Summers (2005), the factors affecting feed conversion was egg production, nutrient content of feed, egg weight, and temperature. Supplementation of piperine at the doses of 15 up to 45 mg/kg BW did not affect IOFC. Addition of piperine at a dose of 30 mg/kg BW (T2) increased IOFC significantly (P<0.05) compared to T4 but did not different significantly as compared to T0, T1, and T3. IOFC in quails supplemented with piperine at a dose of 60 mg/kg BW was significantly (P<0.05) lower than other treatments. Declining value of IOFC in the addition of piperine at a dose of 60 mg/kg BW was caused by the decrease of egg production and egg mass that impaired the income (Safingi et al., 2013 Table 2. The average of feed consumption, water consumption, egg production, and feed conversion of laying quail during 8 weeks treatment influenced by the increased of feed intake and the decreased of egg production and egg mass. Physical Quality of Egg The supplementation of laying quails with piperine as a phytogenic feed additive did not affect egg weight. The average of egg weight was 8.97 g/egg. This results is in accordance with the standard weight of quail egg recommended by Tserveni-Goussi & Fortomaris (2011) and it ranges from 6 to 16 g/egg. The factors affecting egg weight is protein consumption (Tuleun & Adenkola, 2013), hormones (Latifa, 2007), and age (Tserveni-Goussi & Fortomaris, 2011). Furthermore Leeson & Summers (2005) states that protein or amino acid is a nutrient that plays an important role in controlling the egg weight. The average of albumens weight was 5.29 g (55.99%), which showed that supplementation of piperine as a phytogenic feed additive did not affect albumen weight. Albumen weight is generally influenced by egg weight, age, genetic (Zita et al., 2009), and hormones (Latifa, 2007). The average of albumens weight in this research were lower than reported by Tserveni-Goussi & Fortomaris (2011) that albumen weight was 7.80 g (56.83%). The percentage of normal quail egg albumen ranged between 60%-63% of the egg weight (Li-Chan & Kim, 2007) or 52%-62% of the egg weight (Nys & Guyot, 2011). The supplementation of piperine as a phytogenic feed additive did not affect the yolk weight. The average of yolk weight was 3.07 g (32.09%) ( Table 3). The averages of yolk weights in this research were lower than reported by Kumari et al. (2008) that yolk weight was 4.74 g (34.61%). According to Reyes-Herrera & Donoghue (2012), the yolk percentage is around 30%-32% of egg weight. As proposed by Darmawan et al. (2013), egg size was more related to the size of the yolk compared with albumen, despite the fact that albumen was still important to determine the size of an egg. According to Subekti et al. (2006), the decreased of yolk cholesterol levels could decrease yolk weight. Percentage of yolk ranged between 28% and 29% of the egg weight (Li-Chan & Kim, 2007), meanwhile according to Nys & Guyot (2011) the range was between 30% and 33%. According to Zita et al. (2013), yolk weight was influenced by genotype and age as well as the cholesterol. Supplementation of piperine in this study significantly (P<0.05) affected eggshell weight. The result showed that eggshell weight in T0 (control) was significantly (P<0.05) higher than the other treatments. The average of eggshell thickness in T0 (control) was significantly (P <0.05) thicker as compared to those supplemented with piperine at the dose of 60 mg/kg BW (T4). However, there was no significant difference in eggshell thickness among treatments of T1, T2, and T3. This result was due to the addition of piperine into the feed could inhibit calcium transport into the mitochondria; the mitochondria will release calcium and stimulate the ATP-ase activity (SPI) (Duke et al., 2012). Furthermore Yoon et al. (2015) stated that piperine would inhibit the enzyme activity of Ca 2-ATP-ase in the transport of the calcium ions across the cell membrane that had an impact on decreasing calcium absorption. Inhibition of the calcium absorption resulted in the declining of egg quality such as egg weight and eggshell strength. According to Kebreab et al. (2009), the content of calcium and phosphorus in the diet can affect the weight and thickness of the eggshell. Eggshell quality and egg shape could be influenced by age and the content of mineral in the feed, such as calcium, magnesium, and phosphorus as inorganic constituent (Hincke et al., 2011;Darmawan et al., 2013). The mineral nutrients that contribute to the eggshell thickness and strength were calcium, magnesium, carbonates, phosphorus, vitamin D3, and other organic nutrients including protein (Leeson & Summers, 2005;Karoui et al., 2009). Haugh unit value is a measure of the quality of the eggs inside the shell. Haugh unit value obtained from the relationship between height of egg white (albumen) and egg weight. Supplementation of piperine at the dose of 30 mg/kg BW significantly (P<0.05) increased Haugh unit compared to T0 (control) and T4, but it was not significantly different as compared to T1 and T2. The average of haugh unit was 91.81-94.45 (Table 3). The addition of piperine could improve the bioavailability of the nutrients, such as amino acid, glucose, Table 3. Egg quality of laying quail at 9-13 weeks of age and beta-carotene (Atal & Bedi, 2010;Ahmad et al., 2012). According to Nugraha et al. (2013), the increased absorption of amino acids could sustain ovomucin and lecithin that finally enhanced the quality of eggs, and amino acids were used to raise the viscosity of egg albumen that eventually increased Haugh unit. Furthermore, Honkatukia et al. (2013) explained that ovomucin was able to control the quality of the protein albumen and assisted the process of egg albumen viscosity. The content of ovomucin in the egg albumen affected the value of haugh units; the higher egg albumen resulted in the higher haugh unit values (Nugraha et al., 2013). In this research, results showed that the egg produced in this experiment were included in grade AA. According to United States Department of Agriculture (USDA, 2011), egg haugh unit values above 72 are categorized as egg with grade AA, 60-72 as a grade A, 31-60 as a grade B, and less than 31 as a grade C. The average of yolk score in this study was 7.53-8.98 (Table 3). Supplementation of piperine at a dose of 60 mg/kg BW significantly (P<0.05) increased yolk score compared to the other treatments. Piperine supplementation at the doses of 15 and 30 mg/kg BW significantly (P<0.05) increased yolk score as compared to T0 (control). The addition of piperine into the feed was able to increase the absorption of beta-carotene contained in the feed. According to Ahmad et al. (2012), piperine was able to increase the absorption of beta-carotene (a carotenoid) and other nutrients within the body. Carotenoid (beta-carotene) supported the high yolk color score since it had the same function with xanthophylls (Hermana et al., 2014). Furthermore, Hammershoj et al. (2010) stated that egg yolk color was influenced by the consumption of zeaxanthin, lutein, alpha-carotene, beta-carotene, and carotenoids. CONCLUSION The addition of piperine at 15-45 mg/kg body weight could be used as phytogenic feed additive to improve performance, income over feed cost (IOFC), haugh unit and a yolk color.

v3-fos

2017-08-03T01:18:30.406Z

2015-05-19T00:00:00.000Z

18090502

s2

The genetic architecture of growth and fillet traits in farmed Atlantic salmon (Salmo salar) Background Performance and quality traits such as harvest weight, fillet weight and flesh color are of economic importance to the Atlantic salmon aquaculture industry. The genetic factors underlying these traits are of scientific and commercial interest. However, such traits are typically polygenic in nature, with the number and size of QTL likely to vary between studies and populations. The aim of this study was to investigate the genetic basis of several growth and fillet traits measured at harvest in a large farmed salmon population by using SNP markers. Due to the marked heterochiasmy in salmonids, an efficient two-stage mapping approach was applied whereby QTL were detected using a sire-based linkage analysis, a sparse SNP marker map and exploiting low rates of recombination, while a subsequent dam-based analysis focused on the significant chromosomes with a denser map to confirm QTL and estimate their position. Results The harvest traits all showed significant heritability, ranging from 0.05 for fillet yield up to 0.53 for the weight traits. In the sire-based analysis, 1695 offspring with trait records and their 20 sires were successfully genotyped for the SNPs on the sparse map. Chromosomes 13, 18, 19 and 20 were shown to harbor genome-wide significant QTL affecting several growth-related traits. The QTL on chr. 13, 18 and 20 were detected in the dam-based analysis using 512 offspring from 10 dams and explained approximately 6–7 % of the within-family variation in these traits. Conclusions We have detected several QTL affecting economically important complex traits in a commercial salmon population. Overall, the results suggest that the traits are relatively polygenic and that QTL tend to be pleiotropic (affecting the weight of several components of the harvested fish). Comparison of QTL regions across studies suggests that harvest trait QTL tend to be relatively population-specific. Therefore, the application of marker or genomic selection for improvement in these traits is likely to be most effective when the discovery population is closely related to the selection candidates (e.g. within-family genomic selection). Electronic supplementary material The online version of this article (doi:10.1186/s12863-015-0215-y) contains supplementary material, which is available to authorized users. Background Traditional selective breeding has rapidly improved economically important traits in aquaculture species, such as growth and disease resistance in aquaculture species [1]. Atlantic salmon have been more extensively studied than most other aquaculture species due to its high economic value and the significant scientific interest in salmonid species [2]. However, the genetic factors affecting some complex traits of economic importance, such as size, morphology and composition, are not yet well known. The limitations to detecting and defining these genetic factors may include a previous lack of genomic resources, the polygenic nature of the traits in question, and the relatively recent whole genome duplication (e.g. [3,4]) in the salmonid lineage. Understanding the genetic basis of phenotypic variation is a fundamental goal of biological research. Quantitative genetic analysis has been widely used to apportion variation in the traits of interest into genetic and environmental factors [12]. A further goal is to ascertain the genetic architecture of these traits, and quantitative trait loci (QTL) mapping is useful for this purpose. This approach has been widely applied in most farmed animal and plant species to improve genetic breeding programs [13][14][15][16]. To date, QTL mapping relating to the growth performance of farmed salmonid species have been undertaken in Atlantic salmon [17][18][19][20][21], Coho salmon [22,23], Arctic char [24], Chinook salmon [25] and Rainbow trout [26,27]. The loci associated with these apparently polygenic growth traits tend to vary between studies, which may reflect population differences or gene by environment interaction. Traits of economic interest in aquaculture species include those pertaining to the efficient production of high quality fillets. As such, overall growth rate is important, alongside the relative proportion of particular components of the fish (fillet, guts, and head, etc.). Ultimately, fillet weight is a key economic trait, and variation in this characteristic significantly depends on the proliferation and composition of white and red muscle. Muscle cell development and proliferation are part of a complex regulatory process and intricately linked with the development of the skeleton. These processes are typically controlled by networks involving many genes and biological pathways [28]. As such, a polygenic architecture of variation in this trait may be expected. Previous studies have shown that the less desirable parts of Atlantic salmon (e.g. head weight and vertebral weight) have a significant positive correlation with desirable traits such as harvest and fillet yields [29]. By detecting and selecting haplotypes at specific QTL, it may be possible to improve the proportion of fillet within the fish for any given growth rate (albeit caution should also be applied to ensure overall wellbeing and robustness of the fish). The objective of this study was to detect and characterize QTL affecting growth and fillet characteristics in farmed salmon, using SNP markers genotyped in several large families reared under commercial aquaculture conditions. Due to the lower recombination rate observed throughout much of the genome in male salmon, compared to female salmon [30], the efficiency of QTL detection is increased by using a two stage analysis. In this strategy, QTL are first detected in a sire analysis using few markers per chromosome, and the chromosomes harbouring significant QTL are then genotyped for additional markers and analysed using dam mapping parents. Here, we use this approach with the overall target of improving understanding of the genetic regulation of growth and fillet characteristics in Atlantic salmon, and providing candidate regions for potential application in marker-based selection to capture within-family variation in these traits. Animals and phenotype measurement A commercial salmon population comprising 198 fullsibling families derived from 136 sires and 198 dams (Landcatch Natural Selection, Ormsary, UK) was utilized in this experiment. Details of this population have been previously published [31][32][33]. Briefly, approximately 5000 fish were harvested at~3 years of age and measured for overall and component weight traits: harvest weight (kg), gutted weight (kg), deheaded weight (kg), fillet weight (kg), gutted yield (%), fillet yield (%), head weight (kg), gut weight (kg), body waste weight (kg) and total waste weight (kg), fat percentage [% as estimated using a Torry Fatmeter (Distell Ltd, Aberdeen, Scotland)]; and fillet color [assessed visually using the Roche SalmoFan scale (Hoffmann-La Roche, U.K.), ranging from 20 (Yellow) to 34 (Red)]. Details of trait measurements at harvest are given in Powell et al. [29]. A fin clip sample of each fish was retained for DNA extraction. All animals were reared and harvested in accordance with all relevant national and EU legislation concerning health and welfare. Landcatch are accredited participants in the RSPCA Freedom Foods standard, the Scottish Salmon Producers Organization Code of Good Practice, and the EU Code-EFABAR Code of Good Practice for Farm Animal Breeding and Reproduction Organizations. The traits of fat percentage and gut weight were log 10 transformed to approximate a normal distribution. Two generation pedigree records were available for all fish and the sex of the offspring was not observable at harvest and processing. Heritability estimates for some of the traits have been estimated previously in the larger population from which the QTL families were sampled [31,32]. For gut, head, waste and total waste weight, the polygenic heritability was estimated in this larger population using a simple animal model, Y ij = μ + A i + e ij , where Y ij is the trait value measured in the individual i, μ is the overall mean value of the trait, A i is the additive genetic effect of the individual based on the pedigree information and e ij is the residual error. The heritability for each of the traits was estimated using the above model, and the procedure was described in Tsai et al. [32]. SNP marker selection and genotyping To account for the large differences in recombination rate between male and female salmon, a two-stage QTL detection and mapping strategy was employed [30,34]. Stage 1 used sire mapping parents (low recombination), with few markers per chromosome to detect chromosomes containing putative QTL. Stage 2 used dam mapping parents, with a denser marker coverage, to confirm QTL on significant chromosomes and estimate QTL position. For stage 1, the 20 sires in the population with the most progeny were chosen for analysis (total n = 1695). The sparse panel of SNP markers described in Gonen et al. [35], largely taken from Moen et al. [36], were provided to LGC Genomics (Herts, U.K.) for the design of Kompetitive Allele Specific PCR (KASP) assays (see details at http:// www.lgcgroup.com/products/kasp-genotyping-chemistry/kasp-technical-resources/#.VVUKo_1waM8) for genotyping. From these, a total of 51 informative SNPs, with one to three SNPs per chromosome, were genotyped in all 1695 offspring (Additional file 1: Table S1). Stage 2 aimed to confirm the QTL detected in stage 1 and to estimate their position on the chromosome. Therefore, stage 2 focused on three putative QTL-containing chromosomes (chr. 13, 18, and 20) detected in stage 1. Thirty additional segregating SNP markers (Additional file 1: Table S1) [9] were chosen to be positioned at regular (~10 cM) intervals across the candidate chromosome according to published linkage maps. As such, it was anticipated that this marker density would be sufficient to estimate approximate position of QTL on chromosomes. These SNPs were selected for genotyping in the ten dams with the largest number of offspring. A total of ten, eight and eight informative SNPs from chr. 13, 18 and 20 respectively were genotyped in the 512 offspring of these dams (which were a subset of the offspring genotyped offspring in stage 1). Linkage and QTL mapping Sex-specific genetic maps were constructed using Crimap version 2.4 [37]. The 'prepare' option was used to create the input files (markers had previously been assigned to linkage groups based on a LOD score of >4.0), followed by the 'build' option to estimate marker order, and 'fixed' option to estimate the map distance between the markers. Where relevant, the 'flipsn' option was used to test different order permutations and determine the most likely marker order. For both sire and dam based QTL detection, a two stage linear regression-based linkage analysis was performed using the GridQTL software [38]. The conditional probability of inheriting a particular haplotype from the sire or dam was inferred from the marker genotypes in all offspring, at 1 cM intervals. Subsequently, the trait value was regressed on the probability that a particular haplotype allele was inherited from the sire (stage 1) or the dam (stage 2). At each genomic location, the model containing a single QTL is compared to a model with no QTL resulting in an F Ratio statistic. The chromosome-wide significance thresholds for each trait were computed by permutation using 10,000 iterations per chromosome. With 29 chromosomes, the expected number of false positive was 1.45 at the 5 % significance threshold, and 0.29 at the 1 % significance threshold per genome scan respectively. The genome-wide thresholds were determined by applying the Bonferroni correction [39] to 29 independent chromosomes. In addition, in the stage 2 dam-based analysis, the confidence intervals for the QTL were estimated using bootstrapping with 10,000 permutations. In order to estimate the size of the effect of the significant QTL on the traits, the withinfamily variation explained by the QTL (PVE) was calculated using the following equation: h 2 QTL = 4[1-(MSE full / MSE reduced )] for sire-based analysis. For the dam-based analysis, because the dams were nested within sires (fullsibling families), the estimated equation was revised to h 2 QTL = 2[1-(MSE full / MSE reduced )], where the MSE full is the mean square error of the performed model including the QTL and MSE reduced is the model including the family mean only. For traits related to the component weights of the fish, the QTL analyses were repeated including harvest weight as a covariate in the analysis. This was done to assess and distinguish QTL associated with an overall growth effect on the fish, versus QTL associated with proportional growth of specific components (e.g. fillet and waste, etc.) Results Trait records of 1695 offspring derived from 20 sire families were obtained from a larger dataset of~5000 salmon measured at harvest (~3 years old). The heritability of the weight traits was significant and consistent with previous estimated (h 2 = 0.52 to 0.53). For the traits not previously analysed in this population (i.e. gut, head, waste and total waste weight) the heritabilities ranged from 0.15 to 0.32. Summary statistics from the QTLmapping offspring and population-wide estimates of heritability for these traits are given in Table 1. The weight traits showed a high phenotypic and genetic correlation ( Table 2) and fitting overall harvest weight as a covariate in the animal model reduced the estimated h 2 for the component traits to 0.02 -0.05 (although these were still significantly different from zero). In total, 51 SNP markers dispersed over all 29 chromosomes were successfully genotyped in the parents and offspring. In the sire-based QTL mapping analysis, a total of 13 chromosomes showed suggestive evidence for a QTL (chromosome-wide p < 0.05), while four chromosomes showed a significant effect on growth-related traits at the genome-wide leve1 (chr. 13,18,19, and 20; Table 3, Figure 1). The QTL typically affected several of the weight measurements and, given the high phenotypic correlations between these traits (r~0.97-1.00), it is plausible that these results reflect single pleiotropic QTL on each chromosome, rather than distinct linked QTL. Interestingly, when harvest weight was fitted as a covariate (as a proxy for an overall measure of growth), the QTL affecting the component traits on chr. 18, 19 and 20 were no longer significant, suggesting these QTL affect overall growth of the fish. In contrast, on chr. 13, most of the QTL effects for the component traits remained after fitting the covariate, suggesting putative proportional differences in the growth of components of the fish. In addition, four new QTLs (chr. 12, 22, 23, and 25) reached chromosome-wide significance in the sire-based analysis with the inclusion of harvest weight as a covariate in the analysis ( Table 3). The proportion of within-family phenotypic variance explained (PVE) varied between 8 and 10 % for the genome-wide significant QTL in the sire-based analysis, suggesting QTL of moderate but not large effect in this population. Three of the genome-wide significant QTL in the sirebased analysis (chr. 13, 18, and 20) were tested in a dam-based analysis using 512 offspring from ten dams, and a denser SNP marker map of the significant chromosomes (Additional file 1: Table S1). The genome-wide significant QTL affecting gutted, deheaded and total waste weight on chr. 20 was confirmed in the dambased analysis and mapped to a best estimated position of 21, 19 and 14 cM respectively, although the 95 % confidence intervals encompassed the entire linkage map for this chromosome ( Table 4). The evidence for QTL on chr. 13 and 18 was not as strong in the dam-based analysis, with only gut weight (chr. 13) and gutted weight (chr. 18) showing chromosome-wide significance (in the analysis with harvest weight included as a covariate). For the chr. 20 QTL, there were three sires and three dams segregating for a QTL affecting at least one weight trait, and the average size of the allelic substitution effect for deheaded weight of the salmon in segregating parents was consistent across all segregating parents, with an average effect of 620 grams (Table 5). Discussion In this study, the genetic basis and architecture of growth-related traits was investigated in a large commercial population of Atlantic salmon using a two-stage QTL mapping approach. All traits measured showed significant evidence for heritability and significant weightrelated QTLs were observed on chr. 13, 18, 19 and 20 in the sire-based analysis. These QTL typically affected several of the weight measurements taken at harvest, which reflects the high positive correlation between these traits and suggests that their effect is related to overall size of the fish. However, the QTL on chr. 13 may have effects on the weight of components of the fish independent of an overall growth effect, as indicated by an analysis including harvest weight as a covariate. A QTL affecting several of the growth-related traits on chr. 20 was confirmed in the dam-based analysis. This chromosome has previously been shown to harbor QTL affecting body weight of Atlantic salmon at younger age (10 months; [16]). However, a comparison of the QTL detected in the current study with those observed in previous studies (Table 6) shows that, even amongst populations of salmon measured at similar age, QTL tend to be rather population-specific. This may reflect differing underlying quantitative trait nucleotide affecting growth of the populations, genotype by environment interaction, or simply that a proportion of QTL identified in most studies are likely to be 'false positives'. The weight traits measured at harvest had high positive genetic and phenotypic correlations (r~0.97-1.00 in phenotypic and~0.99-1.00 in genetic correlation), and this is generally reflected in the QTL results, because individual QTL tended to affect the weight of several components of the fish. This is a phenomenon observed in several other studies (e.g. [18]) and suggests that improvement of the growth of all components of the fish in breeding programs can be made by simply measuring overall harvest weight. This will improve harvest weight, the most important trait, but is likely to also improve potentially undesirable traits such as gut weight. Achieving different rates of gain in individual components of the fish using QTL or conventional family-based selection is likely to be challenging and may require more detailed or accurate measures of these component traits. However, the existence of QTL affecting fillet weight seemingly independent of overall harvest weight (e.g. chr. 11) suggests that there are potentially some genes affecting component traits partially independently of harvest weight that could be targets for further study. Atlantic salmon are closely related to rainbow trout and previous studies in trout have reported several QTLs affecting body mass [25,[40][41]. There was some overlap between these QTL and the genome-wide significant QTL identified in the current study, in particular for body mass QTL mapped to trout chromosomes 1q and 16q/12p [26], chr. 9p and 21p [42] and chr. 16q [40], which correspond to chr. 13 and 18 in salmon. In addition, corresponding QTL regions showing chromosome-wide significance with body weight were also discovered between Chinook salmon (chr. 25) [25] and Atlantic salmon (chr. 28) (this study). These results raise the possibility that some of the QTL affecting complex growth traits may be conserved across salmonid species. However, clearly some overlap between studies will occur by chance and the likelihood of the underlying QTL being common in both species will become more apparent with further studies and a finer mapping resolution. The confidence intervals associated with the QTL in the current study were large which precluded the meaningful identification of potential underlying candidate genes. However, known candidate genes explaining a small percentage of variation in growth in this population (myostatin [31] and IGF1 [32]) do not coincide with the QTL identified here. The size of the QTL effects in the current study was typically around 5-9 % and 6-7 % of the within-family phenotypic variance in the sire and dam-based analysis respectively. While this may be an overestimate due to the Beavis effect [41], it is certainly plausible that markers linked to these QTL may be of use in selective breeding programs. However, the confidence intervals were large and this indicates that while the two-stage mapping approach employed appears to be effective at detecting QTL, the fine mapping to smaller chromosome regions in the dam analysis may benefit from additional markers. The results of this and other studies support the hypothesis that complex traits such as weight are polygenic, which may reflect the involvement of diverse regulation pathways related to energy balance, muscle cell proliferation and skeletal growth. The fact Fig. 1 The distribution of PVE according to chromosome in the sire-based analysis for the representative weight trait of gutted weight. Gray represents the chromosome showing genome-wide significance (p < 0.05) in sire-based analysis. Chromosome 20 also showed chr-wide significance in dam-based analysis (p < 0.05) that the proportion of variation explained by the QTL is smaller than in previous studies (e.g. [19]) is probably due to the large sample size of the current study (i.e. 1700 offspring for the sire-based analysis), and hence potentially more reliable estimates of QTL effect size [41]. Further, the two-step approach provided some degree of within-study validation for the detected QTL on chr. 18. The traits of most commercial interest in salmon production, such as fillet weight were affected by the QTL on chr. 13,18,19, and 20 (genome-wide significance) in the sire-based analysis. Notably, except chr. 19 in sire-based analysis -for which further study may be merited -all of these QTL regions showed a significant effect on gutted weight and deheaded weight. No QTL affecting fat content were detected in our study. Interestingly, components of fat content of salmon, such as n-3 long chain polyunsaturated acid, are highly heritable [43]. Therefore, perhaps more consideration could be given to the investigation of the genetic architecture of the specific components of the fat content of the fillet, as opposed to a more crude overall measure of fat levels. Naturally, this refinement of phenotype would incur a greater cost. In addition, only three QTL (chr. 3, 7 and 26) were shown to affect fillet colour at the chromosome-wide significance level. Chromosomes 3 and 26 have previously been suggested to harbor QTL associated with fillet colour traits [18]. The heritability of this trait is relatively low in this study (h 2 The sign + oris arbitrary when compared across families but indicates the direction of the allelic effect within families (e.g. an allele decreasing harvest weight in sire J9L2M0088 also decreased fillet, gutted, deheaded, head and total waste weight) [44], although recently published studies have given higher heritabilities [45] and fillet color has been suggested to show a significant association with a single locus SCAR marker [46]. It has also been suggested that fillet colour is positively correlated with overall body weight in farmed Coho salmon (r~0.4 ± 0.5) [46] and Atlantic salmon (r~0.49) [47]. This may be related to the inclusion of dietary additives such as astaxanthin, canthaxanthin and carotenoid, which are included in feed to enhanced fillet pigmentation [48]. As such, protein/muscle gains may be accompanied by an associated increase in colour additives. However, we did not observe a correlation between harvest weight and fillet colour in our study. In part, this may be due to a lack of fillet colour variation observed in the population (coefficient of variation~0.025). Of the putative colour QTL in the current study, only chr. 17 showed some evidence for an effect on growth-related traits, while chr. 3 and 26 were associated with fillet colour independent of the other traits measured. Given the economic importance of this trait, further study of these putative QTL and other aspects of the genetic regulation of colour are merited. Marker-assisted selection has been applied in the salmon aquaculture industry for several traits, the foremost example being resistance to the Infectious Pancreatic Necrosis virus [34,49,50]. However, the genetic architecture Table 6 Comparison of harvest weight QTL chromosomes in Atlantic salmon from this and previous studies Gutierrez et al. [17] Baranski et al. [18] Houston et al. [19] This study of resistance to this disease was unusually monogenic, with a single QTL explaining most of the genetic variation. For more typical complex traits such as growth or fillet component traits, the optimal use of markers in selective breeding programs has yet to be established. Clearly, the advantages of using markers in selection for aquaculture are maximal where the traits are difficult or impossible to measure on the selection candidates themselves, and some of the harvest traits fall into this category. However, due to the lack of large-effect QTL and the putative populationspecificity of those QTL, it is unlikely that QTL-targeted, across population marker-assisted selection will be a highly effective tool for breeding. With the recent development of high density SNP arrays (e.g. [7]), genomic selection may be a more effective (albeit expensive) means of capturing variation at QTL of small effect, but is likely to be the most effective when the training and selection population are closely related. Within family genomic selection using lower marker density may be a more costeffective method of capturing the within-family genetic variation associated with QTLs that are relatively population-specific [51]. The large full-sibling family sizes and routine sib-testing in salmon breeding schemes makes such approaches feasible and powerful.

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2015-11-19T00:00:00.000Z

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Fish oil prevents excessive accumulation of subcutaneous fat caused by an adverse effect of pioglitazone treatment and positively changes adipocytes in KK mice Pioglitazone, a thiazolidinedione (TZD), is widely used as an insulin sensitizer in the treatment of type 2 diabetes. However, body weight gain is frequently observed in TZD-treated patients. Fish oil improves lipid metabolism dysfunction and obesity. In this study, we demonstrated suppression of body weight gain in response to pioglitazone administration by combination therapy of pioglitazone and fish oil in type 2 diabetic KK mice. Male KK mice were fed experimental diets for 8 weeks. In safflower oil (SO), safflower oil/low-dose pioglitazone (S/PL), and safflower oil/high-dose pioglitazone (S/PH) diets, 20% of calories were provided by safflower oil containing 0%, 0.006%, or 0.012% (wt/wt) pioglitazone, respectively. In fish oil (FO), fish oil/low-dose pioglitazone (F/PL), and fish oil/high-dose pioglitazone (F/PH) diets, 20% of calories were provided by a mixture of fish oil and safflower oil. Increased body weight and subcutaneous fat mass were observed in the S/PL and S/PH groups; however, diets containing fish oil were found to ameliorate these changes. Hepatic mRNA levels of lipogenic enzymes were significantly decreased in fish oil-fed groups. These findings demonstrate that the combination of pioglitazone and fish oil decreases subcutaneous fat accumulation, ameliorating pioglitazone-induced body weight gain, through fish oil-mediated inhibition of hepatic de novo lipogenesis. Introduction Lifestyle-related diseases, particularly type 2 diabetes, are increasingly becoming a major global health issue associated with excessive caloric intake and decreasing physical activity [1]. Type 2 diabetes epidemic has grown rapidly worldwide and is known to affect an estimated 387 million people (Diabetes Atlas 2014 update, International Diabetes Federation, IDF, 2014). The major pathogenic mechanism underlying type 2 diabetes is thought to be insulin resistance (IR) associated with abdominal obesity [2]. Excessive amounts of unconsumed calories result in triglyceride accumulation in adipocytes, particularly in visceral fat tissue, leading to cellular hypertrophy and stimulation of inflammatory cytokine production [3][4][5]. In addition, large amounts of free fatty acids (FFAs) derived from hypertrophic adipocytes are transferred to insulin responsive organs, such as the liver, skeletal muscle, and pancreas, as "ectopic fat" leading to decreased insulin sensitivity [6,7]. Adiponectin, an adipose tissue-derived secreted protein with insulin-sensitizing activity, has been shown to be decrease in abdominal obesity, with a negative correlation found between plasma adiponectin levels and body mass index [8]. Through the interactions of these complex mechanisms, obesity-induced accumulation of visceral fat is thought to contribute to IR through "lipotoxicity" [2]. Thiazolidinediones (TZDs), potent and selective ligands for peroxisome proliferator-activated receptor gamma (PPAR␥), are widely used clinically in the treatment of type 2 diabetes as insulin-sensitizing agents [9]. TZDs promote the differentiation of preadipocytes into mature adipocytes and apoptosis of hypertrophic adipocyte, a process termed "adipose tissue remodeling." Further, TZDs increase plasma adiponectin levels and reduce the production of adipocytokines, such as tumor necrosis factor-␣ (TNF-␣) and monocyte chemoattractant protein-1 (MCP-1), thereby ameliorating adipocytokine-mediated IR [10][11][12][13]. Thus, the antihyperglycemic effects of TZDs are thought to be mediated independently of increased insulin production, suggesting the greatest utility of TZDs lies in the treatment of type 2 diabetic patients without pancreatic beta cell failure [14,15]. Many studies have demonstrated TZDs have multifaceted utility including the improvement of lipid profiles [16,17], amelioration of nonalcoholic steatohepatitis [18,19], systemic anti-inflammatory effects [20], and the prevention of arteriosclerosis [21,22]. And, TZD-induced "adipose tissue remodeling" is thought to lead increased absorption and storage of excess lipids by small adipocytes newly derived in response to the effects of TZDs [13,23]. However, body weight gain is considered a major adverse effect of TZD therapy following results from animal studies and several clinical trials in patients with type 2 diabetes [24,25]. Moreover, subcutaneous fat accumulation is frequently observed in patients administered TZDs [26]. Fish oil contains eicosapentaenoic acid (EPA, 20-5) and docosahexaenoic acid (DHA, , which have been shown to have beneficial effects in hyperlipidemia, fatty liver, atherosclerosis, and cardiovascular disease in both animal models and clinical trials [27][28][29][30]. Hepatic de novo lipogenesis is mainly controlled by sterol regulatory element-binding proteins (SREBPs), transcription factors that regulate the expression of genes involved in lipogenesis. SREBP-1c, one of the SREBPs isoforms, plays a particularly critical role in fatty acid synthesis through the regulation of several target genes including fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase 1 (SCD-1) [31]. Fish oil consumption has been shown to decrease mRNA expression and mature protein levels of SREBP-1c [32,33]. Additionally, fatty acid oxidation has been shown to be stimulated by fish oil consumption through stimulation of peroxisome proliferator-activated receptor alpha (PPAR␣), a nuclear receptor involved in the regulation of numerous target genes, such as acyl-CoA oxidase (AOX) and uncou-pling protein 2 (UCP-2) [32,34]. In our previous study of female KK mice, we demonstrated a diet with 25% of total calories from fish oil had numerous beneficial effects including suppression of body weight gain associated with whole-body adiposity, improvements in lipid metabolism dysfunction, the prevention of hyperinsulinemia due to increased fatty acid oxidation, and decreased lipid synthesis [35]. Therefore, we hypothesized that the combination of pioglitazone and fish oil prevents pioglitazone-induced body weight gain attributed to the accumulation of subcutaneous fat and exerts synergistic beneficial effects on glucose and lipid metabolism. In this study, we evaluated the combined effects of pioglitazone and fish oil in mice of type 2 diabetes to test the above hypothesis and inform the development of novel TZD treatment approaches. Animals and diets All animal studies were approved by the Guidelines of Institutional Animal Care and Use Committee at the Josai University Life Science Center performed in accordance with the "Standards Relating to the Care and Management of Experimental Animals" (Notice No. 6 of the Office of the Prime Minister of Japan dated March 27, 1980). Male KK mice at 6 weeks of age were obtained from Tokyo Laboratory Animals Science Co. (Tokyo, Japan). All mice were individually housed and allowed free access to water and feed under the conditions of a 12-h light-dark cycle, a temperature of 22 ± 2 • C, and humidity of 55 ± 10% at Josai University Life Science Center. Mice were fed a standard commercial rodent diet for 1 week to stabilize metabolic conditions and divided into 6 groups (n = 5 in each group). All groups were fed experimental diets with contents composed of 60% carbohydrates, 20% fats, and 20% protein for 8 weeks. Dietary fats used safflower oil (Benibana Foods, Tokyo, Japan) and/or fish oil (NOF, Tokyo, Japan). Safflower oil contained 78 wt% oleic acid; fish oil contained 40.4 wt% polyunsaturated fatty acids, especially 6.6 wt% EPA and 24.7 wt% DHA. SO diet contained 20% of calories from safflower oil, and FO diet contained 10% of calories from safflower oil and 10% from fish oil. SO and FO diets were supplemented with 0.006% wt/wt (low-dose pioglitazone [PL]) or 0.012% wt/wt (high-dose pioglitazone [PH]) pioglitazone and designated S/PL, S/PH, F/PL, or F/PH accordingly. Dietary details are shown in Table 1. The feed were changed once in every 2-3 days, and the residual amounts were recorded and showed as total food intake during experimental periods. All diets were stored at −30 • C until each meal is supplied freshly. Computed tomography Radiographic estimations of abdominal composition were performed by computed tomography (CT) for experimental animals using the mouse mode of the CT scanner (La Theta LCT100; ALOKA, Tokyo, Japan). At the end of the experiment, mice fasted for 3 h were anesthetized with intraperitoneal injections of pentobarbital sodium (Dainippon Sumitomo Pharma, Osaka, Japan) before scanning. Abdominal compositions of visceral and subcutaneous fats were estimated by fat slice images at 2 mm intervals between the second lumbar vertebra (L2) and L4 using La Theta software (version 2.10). Collection of blood and tissue samples After CT scanning, blood samples were obtained from tail veins of anesthetized mice. Glucose levels were measured using a blood glucose monitoring system (One Touch Ultra; Johnson & Johnson, Inc.). Mice were then immediately weighed prior to dissection. Blood samples were obtained from the inferior vena cava and treated with EDTA-2Na. Tissue samples, including liver, white adipose tissue (WAT) around the epididymis, and brown adipose tissue (BAT), were immediately removed, weighed, frozen in liquid nitrogen, and then stored at −80 • C until further use. Representative images of harvested whole livers from each group were taken using a digital camera to assess macroscopic appearance. For histopathological examination or morphometric analysis, samples of liver and WAT were collected from 4 to 5 mice per group, fixed in 10% neutral buffered formalin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and stained with hematoxylin and eosin (H&E, Kotobiken Medical Laboratoties, Inc., Tokyo, Japan). Blood samples were centrifuged at 900 × g for 10 min to collect plasma, which was stored at −80 • C until further use. Oral glucose tolerance tests Oral glucose tolerance tests (OGTTs) were performed in 3 h fasted mice at 14 weeks of age. Glucose solutions were prepared by diluting glucose in ultrapure water and orally administrated at a dose of 1 g glucose/kg body weight. Blood samples were obtained before (0 min) and 30, 60, and 120 min after glucose administration, and glucose levels were immediately measured using a blood glucose monitoring system (One Touch Ultra; Johnson & Johnson, Inc.). Insulin tolerance tests Insulin tolerance tests (ITTs) were performed in 3 h fasted mice at 14 weeks of age by intraperitoneal injection of insulin (Humulin R; Lilly USA, Indianapolis, USA) diluted in saline at a dose of 0.75 units/kg body weight. Blood glucose levels were determined using a similar protocol to OGTT. Blood glucose levels in response to insulin were expressed as percentages of initial values prior to insulin administration. Measurement of hepatic lipid content and plasma biochemical parameters Hepatic lipid extraction was performed using approximately 100 mg of liver tissue per mouse according to a previously described method [36]. Hepatic triglyceride and total cholesterol levels, and plasma triglyceride, total cholesterol, high-density lipoprotein cholesterol (HDL-C), and FFA levels, were quantified by an enzymatic colorimetric method using commercial kits (Wako E-Test kits; Wako Pure Chemical Industries Ltd., Osaka, Japan). Plasma insulin and leptin levels were determined by enzymelinked immunosorbent assay (ELISA) using Insulin ELISA kits and Leptin/mouse ELISA kits (Morinaga Institute of Biological Science, Tokyo, Japan), respectively. Plasma adiponectin levels were measured using Mouse/rat adiponectin ELISA kits (Otsuka Pharmaceutical, Tokyo, Japan). Homeostasis model assessment of insulin resistance (HOMA-IR) indices and plasma non-HDL-C levels were calculated using the following formula: HOMA-IR, fasting blood glucose (mg/dl) × fasting plasma insulin (U/ml)/405; plasma non-HDL-C, plasma total cholesterol (mg/dl) − plasma HDL-C (mg/dl). Liver and WAT histopathology and adipocyte percentage area quantification H&E-stained liver and epididymal WAT sections were examined under a microscope at 100-fold magnification. For morphometric analysis of epididymal WAT, adipocytes were manually traced and quantitated with an image analysis system (ImageJ, Wayne Rasband, NIH). Four different fields per group were randomly captured and white adipocyte areas were measured from more than 900 cells per group. RNA isolation and measurement of mRNA levels by real-time polymerase chain reaction TRIzol Reagent (Invitrogen Co.) was used to extract total RNA from liver and WAT samples from each mouse according to the manufacturer's protocol. Quantitative real-time polymerase chain reaction (RT-PCR) was performed using 1 g of total RNA per sample on an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA) using QuantiTect SYBR Green RT-PCR kits (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Primer sequences used in the present study are shown in Table 2. Thermal cycling conditions were set as follows: 1 cycle of reverse transcription at 50 • C for 30 min, initial activation at 95 • C for 15 min, then 40 cycles of denaturation at 94 • C for 15 s, annealing at 55 • C for 30 s and extension at 72 • C for 1 min. Statistical analysis Values are presented as mean ± SD. Comparisons between experimental groups were performed using one-way ANOVA followed by the Tukey-Kramer test (Statview 5.0; SAS Institute Inc., USA). Means with different letters are different at P < 0.05. Combination of pioglitazone and fish oil suppresses weight gain associated with subcutaneous fat accumulation in response to pioglitazone in male KK mice Greater body weight gain was observed in the pioglitazonetreated S/PL and S/PH groups compared with the non-treated SO group; however, F/PL and F/PH groups had approximately 3 g less body weight gain compared with the S/PL and S/PH groups, respectively (Table 3). Epididymal WAT weights in the S/PL, FO, F/PL, and F/PH groups were significantly lower than the SO group (Table 3). There was a trend toward lower epididymal WAT weights in S/PH compared with the SO group. In pioglitazone-treated groups, BAT weights were almost twice as great compared with nontreated mice. No significant difference in BAT weights was observed between the SO and FO groups (Table 3). CT scanning analysis revealed visceral fat mass weights were unchanged in response to either pioglitazone or fish oil (Fig. 1A, B). However, subcutaneous fat mass in the S/PL and S/PH groups was increased by 40% and 57% compared with the SO group, respectively. The accumulation of subcutaneous fat in the F/PL and F/PH groups was comparable with the SO group, indicating amelioration of the effect of pioglitazone by fish oil (Fig. 1A, C). Combination of pioglitazone and fish oil improves glucose metabolisms in male KK mice To investigate the effects of the combination of pioglitazone and fish oil on glucose metabolism, we performed OGTT and ITT on obese and diabetic male KK mice. In OGTT, no significant differences in blood glucose levels at 30, 60, or 120 min were observed between groups. No significant differences in blood glucose area under the curve (AUC) values were observed between groups ( Fig. 2A). For ITT, blood glucose levels at 30 min after insulin injections were reduced to approximately 80% and 60% of initial values in the F/PL and F/PH groups, respectively, with differences maintained until 120 min. In the F/PH group, blood glucose levels were significantly lower at 120 min after insulin injection than in the S/PH group (Fig. 2B). Table 2 Primers for real-time polymerase chain reaction. Genes Forward Insig-1 TCACAGTGACTGAGCTTCAGCA TCATCTTCATCACACCCAGGAC FAS TCACCACTGTGGGCTCTGCAGAGAAGCGAG TGTCATTGGCCTCCTCAAAAAGGGCGTCCA SCD-1 CCGGAGACCCCTTAGATCGA TAGCCTGTAAAAGATTTCTGCAAACC ACC TGACAGACTGATCGCAGAGAAAG TGGAGAGCCCCACACACA GPAT TCATCCAGTATGGCATTCTCACA GCAAGGCCAGGACTGACATC AOX TCAACAGCCCAACTGTGACTTCCATTA TCAGGTAGCCATTATCCATCTCTTCA Plasma insulin levels were significantly decreased in all pioglitazone-treated groups compared with the SO group. In the FO group, a trend toward decreased plasma insulin levels was observed compared with the SO group (Table 4). Similarly, HOMA-IR indices were significantly decreased in the S/PL, S/PH, F/PL, and F/PH groups, with a trend toward a decrease in the FO group, compared with the SO group (Table 4). No significant differences in plasma adiponectin levels were observed between the SO and FO groups. However, plasma adiponectin levels were higher in pioglitazone-treated groups compared with non-treated groups, with significant differences observed in some groups. In particular, the F/PH group had a 2.2-fold significant increase in plasma adiponectin levels compared with the S/PH group (Table 4). Hepatic expression levels of genes related to gluconeogenesis are shown in Fig. 6. Phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels were significantly decreased in the S/PL and S/PH groups compared with the SO group, whereas no changes were observed in fish oil-fed groups. No significant differences in glucose-6-phosphatase (G6pase) mRNA levels were observed between all groups. Combination of pioglitazone and fish oil improves plasma lipid profiles in male KK mice To examine the effects of the combination of pioglitazone and fish oil on plasma lipid profiles, plasma lipid levels were measured, with the results shown in Table 4. No significant differences in triglyceride levels were observed among the SO, S/PL, S/PH, and FO groups. Triglyceride levels were significantly lower in the F/PL and F/PH groups compared with the SO group. No significant differences in total plasma cholesterol levels were observed between pioglitazone-treated and untreated safflower oil-fed groups. Total plasma cholesterol levels were significantly decreased in fish oil-fed groups treated with pioglitazone (F/PL and F/PH) compared with the FO group. HDL-cholesterol levels were significantly decreased in fish oil-fed groups compared with safflower oil-fed groups, regardless of pioglitazone treatment (Table 4). Plasma FFA levels were significantly decreased in all groups, except the S/PL group, compared with the SO group. Plasma FFA levels in the F/PH group were significantly lower than in the S/PH group. Pioglitazone treatment induces accumulation of liver triglyceride, but fish oil feeding suppresses that in male KK mice To examine the effects of the combination of pioglitazone and fish oil on hepatic lipid accumulation, we performed histological analysis on liver specimens and measured hepatic lipid contents. Liver tissues had the palest appearance, characteristic of severe steatosis, in the S/PL group (Fig. 3A). A greater severity steatosis was observed in liver sections from the S/PL and S/PH groups than the SO group, with minimal steatosis observed in the FO group (Fig. 3B). Similarly, liver triglyceride levels were significantly increased in pioglitazone-treated S/PL (2.2-fold) and S/PH (1.6-fold) groups compared with those in the SO group. Liver triglyceride levels in the FO group were significantly decreased compared with those in the SO group. Increased liver triglyceride levels in response to pioglitazone were significantly reduced to the level of the SO group by the combination of pioglitazone and fish oil (Fig. 3C). Although liver total cholesterol levels were significantly increased in the S/PL group than the SO group, liver total cholesterol levels in fish oil-fed groups (FO, F/PL, and F/PH) were significantly lower than in the SO group (Fig. 3D). Combination of pioglitazone and fish oil ameliorates adipocyte hypertrophy and increases the number of small adipocytes in male KK mice To clarify the interaction between the amelioration of insulin resistance in response to pioglitazone and fish oil treatment and changes in visceral fat parameters, we performed histological analysis on WAT sections (Fig. 4A). Mean adipocyte areas were significantly lower in the S/PH, F/PL, and F/PH groups compared with groups not treated with pioglitazone. No difference in mean adipocyte areas was observed between the FO group and SO group. Mean adipocyte areas were significantly lower in the F/PH group compared with the S/PH group (Fig. 4B). Evaluation of adipocyte size distributions demonstrated lower peak sizes in the F/PL and F/PH groups (2500-3600 m 2 ) compared with other groups. The percentages of small adipocytes (400-3600 m 2 ) and larger adipocytes (>6400 m 2 ) were increased and decreased, respectively, in the F/PL and F/PH groups compared with other groups (Fig. 4C). Fish oil feeding decreases hepatic mRNA levels of genes involved in lipogenesis with or without pioglitazone treatment To elucidate the mechanisms underlying the amelioration of weight gain and subcutaneous fat accumulation in response to pioglitazone by the combination of pioglitazone and fish oil, we measured hepatic mRNA expression of genes involved in de novo lipogenesis (Fig. 5). SREBP-1c mRNA levels were significantly decreased in all pioglitazone-treated groups compared with the SO group, with no difference observed between the SO and FO groups. Insulin-induced gene 1 (Insig-1) mRNA levels were significantly decreased in fish oil-fed groups. Insig-1 mRNA levels in the F/PL and F/PH groups were approximately 60% lower than in the S/PL and S/PH groups, respectively. Expression levels of SREBP-1c target genes, such as FAS, SCD-1, and ACC, were significantly decreased in fish oil-fed groups, regardless of pioglitazone treatment (Fig. 5). In addition, mRNA levels of glycerol-3-phosphate acyltransferase (GPAT), a lipogenic rate-controlling enzyme, were increased in the S/PL and S/PH groups compared with the SO group; however, increased expression of GPAT was markedly suppressed by the combination of pioglitazone with fish oil (Fig. 5). Pioglitazone, but not fish oil, increases hepatic mRNA levels of genes involved in ˇ-oxidation To elucidate the association between ␤-oxidation and suppression of fat accumulation by the combination of pioglitazone and fish oil, we measured hepatic mRNA expression of genes involved in fatty acid ␤-oxidation and metabolism. AOX mRNA levels were significantly increased in the S/PL, S/PH, and F/PH groups compared with the SO group. No difference in AOX mRNA levels was observed between the SO and FO groups (Fig. 6). Although pioglitazone and fish oil did not significantly affect UCP-2 mRNA expression, there was a trend toward increased levels of UCP-2 mRNA levels in the S/PL and S/PH groups compared with the SO group (Fig. 6). Carnitine palmitoyltransferase 1 (CPT-1) and medium-chain acyl-CoA dehydrogenase (MCAD) mRNA levels were unaffected by pioglitazone or fish oil (Fig. 6). Pioglitazone and fish oil decreases cytokine mRNA expression in WAT To elucidate the relationship between histological changes and chronic inflammation in response to the combination of pioglitazone and fish oil, cytokine mRNA expression levels were measured in epididymal fat. No significant differences in adiponectin mRNA levels were observed between groups; however, there was a trend toward increased adiponectin expression in the S/PH and F/PH groups compared with the SO group. TNF-␣ mRNA levels were lower in pioglitazone-treated groups compared with the SO group, with significant differences observed in some groups. No significant difference in TNF-␣ mRNA levels was observed in response to fish oil. Interleukin 6 (IL-6) mRNA levels were significantly decreased in the S/PL, FO, F/PL, and F/PH groups and tended to attenuate in the S/PH group compared with the SO group. MCP-1 mRNA levels were significantly lower in pioglitazone and/or fish oil treated groups compared with the SO group (Fig. 7). Discussion Pioglitazone is widely used as an insulin sensitizer in the treatment of type 2 diabetes. However, body weight gain accompanied by an increase in subcutaneous fat is frequently observed in patients treated with pioglitazone and is considered a major adverse effect of pioglitazone. To address this substantial clinical issue, we examined the utility of fish oil consumption in ameliorating pioglitazone-induced body weight gain and further elucidated the beneficial effects of the combination of pioglitazone and fish oil on glucose and lipid metabolism. In the present study, we found that the combination of pioglitazone and fish oil ameliorated body weight gain and subcutaneous fat accumulation in response to pioglitazone treatment. In addition, pioglitazone and/or fish oil-treated mice had significantly lower epididymal fat weight. However, no significant differences in visceral fat mass were observed between the treatment groups. Majority of FFAs released from the triglycerides of chylomicron and VLDL particles are re-esterified to triacylglycerol in adipose tissue for storage [37]. Thus, fish oil consumption is thought to decrease triacylglycerol supply to adipose tissue by reducing VLDL produc-tion, thereby inhibiting the accumulation of lipids in adipose tissue [33,35]. Our results demonstrate fish oil consumption potently decreases mRNA expression of lipogenic enzymes, including FAS, SCD-1, ACC, GPAT, and SREBP-1c. The majority of lipogenic gene expression levels were unchanged or increased in S/PL and S/PH groups despite decreased expression of SREBP-1c mRNA. These results are coincident with previous study used KKAy mice by Oribe et al. [38]. On the other hands, it has reported that pioglitazone inhibits nuclear translocation of SREBP-1c and represses lipogenic gene expression in lean C57BL6/J mice [39]. These data showed that lipogenic gene expressions in response to pioglitazone vary considerably depending on obesity or non-obesity. Expression levels of fatty acid ␤-oxidation related genes, including AOX, UCP-2, CPT-1, and MCAD, were unaffected by fish oil consumption. These results suggest diets with 10% of total calories provided by fish oil had no effect on ␤-oxidation in the present study. Our previous studies demonstrated diets with 2% of total calories provided by fish oil significantly increased AOX and UCP-2 mRNA levels in C57BL/6 mice [40]; however, diets with 2-25% of total calories provided by fish oil were not found to stimulate ␤oxidation in obesity-induced KK mice fed a high fat diet [41]. These reports suggested fish oil-induced effects on stimulating fatty acid ␤-oxidation may be attenuated by obesity in female KK mice. In the present study used male KK mice, also, diets with 10% of total calories provided by fish oil were found to potently inhibit lipogenesis without activation of fatty acid ␤-oxidation, resulting in decreased accumulation of subcutaneous fat and body weight gain in response to pioglitazone treatment. TZDs have been reported to ameliorate IR by increasing plasma adiponectin levels in obese and diabetes murine models and clinical trials [12,[42][43][44]. Our data demonstrated plasma adiponectin levels were increased in pioglitazone-treated groups with significantly decreased HOMA-IR indices compared with corresponding pioglitazone-untreated groups. On the other hand, fish oil treatment alone was found to have no marked effect on these parameters. Fish oil supplementation reportedly increases circulating adiponectin levels in non-obese rodents [45,46]. However, our previous study demonstrated diets with 2.5%, 12.5%, and 25% of total calories provided by fish oil had no beneficial effects on plasma adiponectin and insulin levels in KK mice [41]. These findings suggest the antidiabetic effects of fish oil related to circulating adiponectin levels may be attenuated by obesity. In the present study, adiponectin levels in the F/PH group were 2.2-fold higher than in the S/PH group despite few notable effects on other parameters observed. Increased plasma adiponectin levels may contribute to the amelioration of IR in male KK mice; however, adiponectin levels above a certain level may not necessarily correspond to an insulin sensitizing effect. The KK mouse model has significantly greater severity of hyperinsulinemia and IR compared with wild-type mice, with maximum dysfunction observed at 16-20 months of age [47]. In the present study, treatment of male KK mice with pioglitazone led to significantly decreased plasma insulin levels and improved IR compared with pioglitazone-untreated groups. However, these changes may be insufficient to greatly decrease blood glucose levels in OGTT. Pro-inflammatory cytokines produced by adipose tissue macrophages (ATMs) in adipose tissue are closely associated with obesity and IR [48]. ATMs can be classified into two phenotypes: the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype. M1 ATMs are seen to accumulate in obese adipose tissue and secrete pro-inflammatory cytokines including TNF-␣, IL-6, and MCP-1. M1 ATMs have been shown to promote adipose tissue lipolysis via activation of mitogen-activated protein kinases resulting in increased FFA production, particularly saturated fatty acids. In addition, the FFAs have been shown to act as a ligand for toll-like receptor-4 (TLR-4) expressed by macrophages and cause further exacerbation of chronic inflammation in adipose tissue [49]. Dietary n-3 polyunsaturated fatty acids, such as EPA and DHA, inhibited TLR-induced inflammatory signaling pathways [50]. And, pioglitazone treatment increased the ratio of M2-to-M1 ATMs in epididymal fat of diet-induced obese mice, and changes in the number of ATMs were closely correlated with IR [51]. In this study, fish oil consumption or/and pioglitazone treatment decreased expression of pro-inflammatory cytokines, such as TNF-␣, IL-6, and MCP-1, in epididymal fat. Our results suggest that fish oil and/or pioglitazone ameliorated inflammation in adipose tissue and improved IR. Hepatic lipid accumulation is closely related to IR and compensatory hyperinsulinemia. TZDs have been reported to potently stimulate the differentiation of adipocytes and induce a redistribution of FFAs from extra-adipose tissues, including the liver and pancreas, to newly derived adipocytes, thereby ameliorating whole-body IR and fatty liver [6,18]. In contrast, several studies have demonstrated the anti-steatotic effect of TZDs in both animals and humans [19,39,52]. Bedoucha et al. reported markedly increased hepatic levels of PPAR␥ mRNA and protein in diabetic KKAy and ob/ob mice compared with lean C57BL6/J mice [53]. Further, prominent microvesicular steatosis due to lipid accumulation was observed in KKAy mice treated with TZDs, suggesting obese mice are more susceptible to TZD-induced steatosis than lean mice. In the present study, pioglitazone monotherapy induced dramatic hepatic triglyceride accumulation in male KK mice, particularly in the S/PL group, in addition to stimulating expression of genes related to fatty acid synthesis. And, pioglitazone-induced hepatic lipogenesis was potently inhibited by fish oil consumption, contributing to the efficacy of the combination of TZD and fish oil in the treatment of type 2 diabetes. Conclusions In conclusion, we demonstrated that the combination of pioglitazone and fish oil may prevent the adverse effects of pioglitazone treatment on promoting excessive body weight gain and subcutaneous fat accumulation. Fish oil consumption was found to potently inhibit hepatic lipogenesis in KK mice. The results of this study suggest that the combination of pioglitazone and fish oil has particular efficacy in diabetic patients with fatty liver and adipose tissue inflammation. Thus, we provide novel data regarding the utility of nutritional therapy in patients with type 2 diabetes being treated with pioglitazone. Conflict of interest The authors have nothing to declare. Transparency document The http://dx.doi.org/10.1016/j.toxrep.2015.11.003 associated with this article can be found in the online version.

v3-fos

2016-05-16T03:46:31.260Z

2015-06-23T00:00:00.000Z

594682

s2

Genome-wide transcriptional profiling of wheat infected with Fusarium graminearum Fusarium head blight (FHB) is a destructive disease in wheat caused by Fusarium graminearum (F.g). It infects during the flowering stage favored by warm and highly humid climates. In order to understand possible wheat defense mechanism, gene expression analysis in response to F.g was undertaken in three genotypes of wheat, Japanese landrace cultivar Nobeokabouzu (highly resistant), Chinese cv. Sumai 3 (resistant) and Australian cv. Gamenya (susceptible). For microarray analysis, 3 and 7 days after inoculation (dai) samples were used in Agilent wheat custom array 4x38k. At 3 dai, the highest number of genes was up-regulated in Nobeokabouzu followed by Sumai 3 and minimum expression in Gamenya. Whereas at 7 dai, Sumai 3 expressed more genes compared to others. Further narrowing down by excluding commonly expressed genes in three genotypes and grouping according to the gene function has identified differentially high expression of genes involved in detoxification process such as multidrug resistant protein, multidrug resistance-associated protein, UDP-glycosyltransferase and ABC transporters in Nobeokabouzu at 3 dai. However in Sumai 3 many defense-related genes such as peroxidase, proteases and genes involved in plant cell wall defense at 7 dai were identified. These findings showed the difference of molecular defense mechanism among the cultivars in response to the pathogen. The complete data was accessed in NCBI GEO database with accession number GSE59721. Specifications Subject area Biology More specific subject area Plant-pathogen interaction Organism Triticum aestivum L. (common wheat) and Fusarium graminearum (fungus) Tissue Wheat-fungus inoculated florets Time points 3 and 7 days after inoculation ( Materials The experiment was carried out by selecting three wheat genotypes that differ with regard to their disease response against Fusarium graminearum (F. g) (Nobeokabouzu, Sumai 3 and Gamenya were selected as highly resistant, resistant and susceptible cultivars respectively). The plant materials were grown in glass house condition. At early anthesis time, florets of each spike were inoculated with F. g strain 'H-3' by pipetting 10 μl of the fungal suspension (1 × 10 5 macroconidia ml −1 ). Mock samples were prepared by inoculating 10 μl of distilled water. In order to develop conducing environment for disease development, the inoculated spikes were covered with a plastic bag for 72 h. Temperature and moisture content in the glass house were maintained at 25°C and 50% respectively. At 3 and 7 days after inoculation (dai), six spikes per genotype/ treatment/time point were sampled for RNA extraction. Three biological replications were done for each sample. Microarray experiment Total RNA was extracted by using Nucleo Spin RNA plant kit (Macherey-Nagel, Germany) then converted to cRNA and labelled Contents lists available at ScienceDirect Genomics Data j o u r n a l h o m e p a g e : h t t p : / / w w w . j o u r n a l s . e l s e v i e r . c o m / g e n o m i c s -d a t a / using Low Input Quick Amp Labeling kit (Agilent Technologies) and fluorophore cy3-CTP. Agilent wheat custom array 4x38k (G2514F) was used to measure the gene expression changes among three different genotypes with and without (mock) FHB infection at 3 and 7 dai. In total, 12 samples were hybridized and biologically replicated three times (12 samples × 3 replication = 36 samples). Gene intensities were extracted from the scanned images, and the data were analyzed using Gene spring 12.6 software (Agilent Technologies). Data analyses Genome wide gene expression analyses of three genotypes were carried out in a systematic manner. After normalization and statistical analysis, the data were grouped by Venn diagram to categorize the up-regulated genes. The groups were made into three categories, a) common F. g responsive genes in wheat genotypes, b) genotypicspecific F. g responsive genes for susceptible, resistant and highly resistant wheat genotypes in specific time point (Table 1, Supplementary Table 1) and c) FHB resistance-related genes was picked out by selective comparison of resistant and highly resistant genotypes. Further the expressed genes were functionally assigned to 11 different classes based on previous patho-transcriptomic studies [1][2][3][4]: (1) JA-and ET-related genes; (2) cysteine-rich antimicrobial peptides (AMPs) including serine-protease inhibitors; (3) jasmonate-regulated proteins (JRP); (4) GDSL-lipases; (5) proteolysis including serine proteases; (6) peroxidases (POD); (7) genes related to cell wall defense, such as polygalacturonase inhibiting proteins, xylanase inhibitors and glucan endo-1,3-beta-glucosidase precursors; (8) secondary metabolism and detoxification involved genes; (9) miscellaneous defense-related genes, for example disease resistance-responsive family protein, NBS-LRR disease resistance protein; (10) transcription and signaling related genes and (11) hormone (auxin, gibberellins, abscisic acid and salicylic acid) metabolism related genes. In order to pinpoint difference in molecular mechanism among genotypes, genes were categorized into three functional groups. They were (I) systemic defense-related genes, this includes genes which are known to play important role in plant immunity by eliciting systemic resistance such as, JA & ET related genes, JRP, GDSL-lipase and miscellaneous defense-related genes; (II) local defense-related genes, composed of genes which interact directly with the pathogen avoiding fungal spread such as AMPs, POD, proteolysis and genes related to cell wall defense, and (III) detoxification involved genes, in this group includes secondary metabolism and detoxification process involved genes (Fig. 1). Based on gene expression analysis the disease reaction model of wheat against F. g was developed (Fig. 2). Specific genes and their possible molecular mechanism related to disease was explained in Ayumi et al. [5].

v3-fos

2018-04-03T02:37:38.666Z

2015-08-04T00:00:00.000Z

19801482

s2

Large-Scale Transcriptome Analysis of Two Sugarcane Genotypes Contrasting for Lignin Content Sugarcane is an important crop worldwide for sugar and first generation ethanol production. Recently, the residue of sugarcane mills, named bagasse, has been considered a promising lignocellulosic biomass to produce the second-generation ethanol. Lignin is a major factor limiting the use of bagasse and other plant lignocellulosic materials to produce second-generation ethanol. Lignin biosynthesis pathway is a complex network and changes in the expression of genes of this pathway have in general led to diverse and undesirable impacts on plant structure and physiology. Despite its economic importance, sugarcane genome was still not sequenced. In this study a high-throughput transcriptome evaluation of two sugarcane genotypes contrasting for lignin content was carried out. We generated a set of 85,151 transcripts of sugarcane using RNA-seq and de novo assembling. More than 2,000 transcripts showed differential expression between the genotypes, including several genes involved in the lignin biosynthetic pathway. This information can give valuable knowledge on the lignin biosynthesis and its interactions with other metabolic pathways in the complex sugarcane genome. Introduction Sugarcane (Saccharum spp.) is a member of the Poaceae family with the unique property to accumulate up to 50-60% of dry weight of the mature stem as sucrose [1]. Such ability makes sugarcane economically an important for the production of sugar and bioethanol. At sugarcane mills, the juice is extracted from stalks and used for both sugar crystallization and fermentation to produce the so-called first generation ethanol [2]. So far sugarcane is among the most efficient biomass producers [3], so the industry could also benefit from the production of ethanol from the lignocellulosic feedstock represented by the bagasse, which is promptly available after sucrose extraction and is currently used for heat generation and electricity production [4]. Lignocellulose is the plant biomass composed of cellulose, hemicellulose and lignin polymers and is a renewable resource for the production of biofuels and bio-based materials [5]. However, the highly complex nature and compact structure of lignocellulose, in which cellulose microfibrils are embedded in a matrix of hemicellulose polysaccharides covalently cross-linked with the heterogenic lignin polymer, constitutes a physical barrier that limits the release of fermentable sugars, a fact known as biomass recalcitrance [6,7]. The distinctive non-linear structure of lignin, built with chemically diverse and poorly reactive linkages and a variety of monomer units, makes this phenolic polymer the major plant cell wall component responsible for biomass recalcitrance [8][9][10]. Consequently, biomass-to-biofuel conversion requires costly and harsh pre-treatments to remove lignin and allow the access to polysaccharides for saccharification [11]. Genetic engineering of bioenergy crops has emerged as an alternative to optimize this process, with research efforts aiming to produce plants that either accumulate less lignin or produce lignins that are more amenable to chemical degradation [12,13]. While drastic reductions in lignin deposition normally impacts plant growth and development and consequently results in yield penalties [14], even large shifts in lignin composition are generally well tolerated without negative effects on plant development and morphology [15]. Despite the recent advances in our understanding of lignin biosynthesis, many aspects remain to be explored in terms of gene expression [16], the regulation of the pathway [17,18] and lignin polymerization, in which peroxidases and laccases play an important role [19,20]. For example, despite the fact that most researchers thought that the lignin biosynthetic pathway had been fully elucidated over a decade ago, a new biosynthetic enzyme called caffeoyl shikimate esterase (CSE) was recently discovered in Arabidopsis [21]. In addition, recently the flavonoid tricin was identified as an important component of the cell wall and suggested to be a true monomer, acting as a nucleation site for lignification in monocots [22]. The information on lignin metabolism in sugarcane is still very scarce and only a few genes have been identified from large scale transcription profiling [23,24]. Only very recently, the first comprehensive study on lignin biosynthesis in sugarcane was published, reporting histological, biochemical, and transcriptional data derived from two sugarcane genotypes with contrasting lignin contents [25]. Despite of sugarcane's economic importance, its highly complex polyploid genome has not been sequenced so far [26]. Because possibly more than five thousand sugarcane genes remain undiscovered [27] there is a need for new sequencing efforts of sugarcane transcriptomes derived from several tissues and treatments, in order to increase the basic set of sequence information and further assist biotechnological strategies. Emerging genomic technologies, such as next generation sequencing, will allow more efficient trancriptomic studies and will help in laying a platform for sugarcane genomics [28]. Here, we used RNA-seq analysis to compare the transcript profiles of developing internodes of two sugarcane genotypes contrasting for lignin content. We present an overview of the transcriptional profile of the fifth internode of sugarcane stem, and a set of more than 2,000 transcripts that shows differential expression between the contrasting genotypes. Plant material, RNA extraction, library construction and Illumina sequencing The sugarcane genotypes IACSP04-065 and IACSP04-627, contrasting for lignin content, were used in this study. The two genotypes belong to a F1 segregating progeny produced by a breeding program developed at the Sugarcane Center of Agronomic Institute of Campinas (IAC). The genotypes were evaluated for agronomic traits such as sucrose, fiber and lignin content during a three years period. Lignin content was indirectly obtained from the determination of acid and neutral detergent fibers [29]. Lignin averaged on a dry weight basis 4.32% in genotype IACSP04-065 and 8,12% in genotype IACSP04-627. Mature stalks of non-flowering plants cultivated in the field were used to obtain the setts for both cultivars. Setts were allowed to grow in the greenhouse for 9 months without light or temperature control, in containers (1 m 3 ) filled with a mixture of sand and a commercial organic soil (1:1, v/v). The 5 th internode of each genotype was harvested and immediately separated in central (pith) and peripheral (rind) regions, and then frozen. Total RNA was extracted from these tissues using a CTAB protocol [30]. Prior to the preparation of the RNA library, the integrity and quantity of isolated RNA were assessed on Bioanalyser (Agilent Technologies). To avoid underrepresentation of transcripts that show low abundance in the pith, total RNA were mixed (1:1) and used in cDNA synthesis. Single-end Illumina mRNA libraries were generated from total RNA following the manufacturer's instructions (Illumina Inc, San Diego, CA). The libraries were sequenced on Illumina HiSeq2000 at Fasteris SA (Fasteris Inc, Geneva, Switzerland). Raw reads (100 bp length) were retrieved in a FASTQ format. Data processing, de novo assembly and expression analysis The raw reads were cleaned by removing low-quality sequences (Phred quality score < Q20) and sequences with less than 70 bp using the FASTX-Toolkit (FASTQ/A short-reads pre-processing tools, Cold Spring Harbor Laboratory). The output from the pre-processing step, a total of 22,900,400 high-quality reads, was de novo assembled using the Velvet/Oases suite [31,32] to generate sugarcane transcripts. Assembly results were evaluated using different hash lengths (k = 21, 23, 27, 29 and 47), and the optimal assembly of reads had hash length equal to 29. Evaluated parameters included the total number of obtained transcripts, distribution of transcripts lengths, N50 value, and mainly the results of phylogenetic analysis of previously selected genes from lignin biosynthesis pathway, as described below. GENE-counter software [33] was used to quantify gene expression, in which reads from each library were mapped against the performed assembly. Finally, the differential expression analysis were conducted with the Bioconductor edgeR package [34], with a p-value 1e -4 . The raw reads were deposited in the National Center for Biotechnology Information (NCBI) under accession number SRP056824. Orthology relationship, MapMan annotation, clustering and enrichment analysis The orthology relationship analysis was performed as described by Bottcher et al. [25]. Briefly, a blastx search against proteins previously shown to be involved in lignin biosynthesis was performed (e-value cutoff e -5 ) and the selected transcripts were subsequently used in a new blastx alignment including sequences from representative species of the Viridiplantae (Arabidopsis thaliana, Populus trichocarpa, Oryza sativa, Sorghum bicolor, Selaginella moellendorffii and Physcomitrella patens), whose output was further used as the data set to phylogenetic analyses [25]. The coding-sequences of the selected transcripts were deduced from the amino acids alignment with the blastx best-match hit and then aligned with the 40 first blastx hits by MAFFT [35]. The phylogenetic relationship of the aligned protein sequences was then inferred by maximum-likelihood using PhyML [36]. This process allowed identifying the putative orthologous sugarcane lignin gene for each selected transcript. Functional annotation of the transcripts was done with MapMan categories [37] using blastx (e-value 10 −5 ) against Arabidopsis thaliana proteins. The clustering analysis of transcriptional expression profile was conducted in R package (http://www.r-project.org) using the K-means clustering algorithms, with best results obtained with 100 clusters. In a second step, the clusters were tested for MapMan terms enrichment with Fisher's exact test (p-value < 0.05), using the PageMan application [38]. The gene test sets consisted of all transcripts in each cluster, and reference set is the whole MapMan annotation of sugarcane transcriptome described above. The Self-organizing maps (SOM) were obtained using the Flora software, http://www-microarrays.u-strasbg.fr [39]. Results and Discussion In a previous study, we performed a characterization of lignin deposition during sugarcane stem development, using histological, biochemical, and transcriptional data derived from two sugarcane genotypes with contrasting lignin contents [25]. Putative lignin genes were identified and their spatiotemporal expression patterns were evaluated using quantitative RT-PCR in an attempt to identify the gene family members that are likely involved in constitutive lignification in sugarcane. Here, we compared the whole transcriptome of developing internodes of two contrasting sugarcane genotypes from the same segregating progeny using next-generation sequence. Lignin content of genotype IACSP04-065 was 4.32%, whereas IACSP04-627 showed 8.12% lignin. The aim was to evaluate large-scale differences in gene expression in a plant tissue undergoing active lignification between the genotypes contrasting to lignin deposition. De novo assembly and quantification of the sugarcane transcriptome Normalized cDNA libraries from a developing internode of both sugarcane genotypes were constructed and sequenced using Illumina HSeq2000 platform, generating around 28 million single-end 100 bp long reads. The pre-processing step removed 18% of the raw data, in which low-quality sequences with ambiguous bases and reads with less than 70 bp were discarded, resulting in a set of 22,900,400 high-quality reads (10,555,189 from IACSP04-627 library, and 12,345,211 from IACSP04-065). The high-quality reads were de novo assembled and resulted in a set of 85,151 transcripts of sugarcane. This data set can be accessed in FASTA format in the S1 File and it includes all transcripts isoforms of 51,068 assembled unigenes. These unigenes have a mean length of 987 bp and a N50 equal to 1,385 bp. In total, there were 8,315 (16%) unigenes with size between 500 bp and 1,000 bp, and 7,679 (15%) unigenes between 1 kb and 3 kb. The assembled transcripts were functional annotated with MapMan categories, using blastx against Arabidopsis thaliana proteins. Subsequently, the expression levels of all transcripts in IACSP04-627 and IACSP04-065 genotypes were inferred by mapping reads from each library against the performed assembly (S2 File). Identification and expression quantification of genes associated with phenylpropanoid biosynthesis The RNA-seq data were used to quantify and study sugarcane genome-wide changes in gene expression related with the phenylpropanoid biosynthesis, in particular the lignin biosynthetic pathway. Applying the previously described orthology workflow [25] it was possible to identify 21 sugarcane transcripts that encode for enzymes involved in lignin biosynthesis (Fig 1) 1.195). The total number of identified phenylpropanoid-monolignol transcripts represents the members of lignin gene families expressed in sugarcane stems, but most likely does not represent the total number of lignin genes present in the highly polyploidy sugarcane genome. Accordingly, the total number of lignin genes present in closely related species is significantly higher. Xu and colleagues [40] performed a comparative genome analysis of lignin biosynthesis gene families across the plant kingdom, including representative species from Bryophytes, Lycophytes, Dicot Angiosperms and Monocot Angiosperms. While Arabidopsis genome harbors 63 lignin genes, a total of 141, 149 and 157 genes were found for sorghum, poplar and rice, respectively. Noteworthy, the results of Xu et al. [40] differ significantly from the data previously published by Raes et al. [41] and Shi et al. [42], in which 34 and 95 genes were annotated as monolignol genes in Arabidopsis and poplar, respectively. This observation highlights the fact that different approaches applied to identify homolog genes in a given genome leads to significantly different outputs. A novel enzymatic step involving caffeoyl shikimate esterase (CSE) was recently identified as central to the lignin biosynthetic pathway in Arabidopsis thaliana [21]. CSE hydrolyses caffeoyl-shikimate into caffeic acid, which in turn is used by 4CL to produce caffeoyl-CoA, bypassing the so-called second reaction performed by HCT. Orthologues of bona fide CSE have been found in a wide range of plant species, especially dicots such as poplar, eucalyptus, tomato and Medicago truncatula [21]. However, although orthologues for this gene were also found in bryophytes (Physcomitrella patens), lycophytes (Selaginella moellendorfii) and gymnosperms (Picea abies) [43], only a few grasses with available genomic data seem to harbor bona fide CSE genes in their genome. Within the Poaceae family, CSE genes were not found in species belonging to the Pooideae subfamily, which includes Brachypodium, wheat, rye, barley and oat, while rice (Oryzodeae) and switchgrass (Panicoideae) harbor 3 and 1 bona fide CSE genes, respectively [21,44]. The Panicoideae subfamily is one of the most economically important grass groups, since it includes important grain and bioenergy crops such as switchgrass (Panicum virgatum) and foxtail millet (Setaria italic) as members of the tribe Paniceae and maize (Zea mays), sorghum (Sorghum bicolor) and sugarcane (Saccharum spp.) as members of the tribe Andropogoneae [45]. Switchgrass is the only member of the Panicoideae subfamily whose genome harbors a bona fide CSE gene, while members of the Andropogoneae tribe seem to lack orthologues of this gene. Accordingly, transcripts coding for CSE were not found in the transcriptomic datasets of the sugarcane genotypes used in our study, suggesting that sugarcane also does not possess a bona fide CSE gene and that this enzymatic step is not important for lignin biosynthesis in this plant species. Alternatively, a phylogenetically distant CSE-like gene might be responsible for this enzymatic activity in plants lacking a bona fide CSE gene. For instance, a similar scenario has been characterized for the biosynthesis of chlorogenic acids (CGAs) in switchgrass [46]. In solanaceous plants, the enzyme hydroxycinnamoyl-CoA: quinate hydroxycinnamoyltransferase (HQT) is responsible for CGA biosynthesis, but there are no close HQT orthologues in switchgrass. Escamilla-Treviño and colleagues [46] demonstrated that a switchgrass HCT-like enzyme exhibits HQT activity, preferring quinic acid instead of shikimate as acyl acceptor, and is most likely responsible for CGA biosynthesis. Therefore, before any further conclusions, the role for CSE in lignification of plants other than Arabidopsis awaits experimental confirmation. The analysis of the transcriptome data showed that, with the exception of ShC4H3, all the genes from the general phenylpropanoid pathway (i.e. from PAL to 4CL) show relatively low expression levels in both sugarcane genotypes (Fig 1), which is expected to result in low availability of monolignols. PAL is known to be differentially expressed through time and does not act at capacity when the demand for lignin is relatively low, for example in young internodes [47]. Although lignification is an active process in the fifth internode of sugarcane stem, this developing internode is still considered a relatively young tissue and, therefore, shows only limited lignin deposition. In accordance with this observation, the expression levels of many of these key genes significantly increased with stem maturity, which correlates with the increase of lignin content during sugarcane stem development [25]. The transcript levels of all sugarcane CCoAOMT genes were conspicuously high, especially in the high-lignin genotype IACSP04-627 (Fig 1). This is in contrast to what was found in Arabidopsis, in which only one family member AtCCoAOMT1 was highly expressed in the inflorescence stem [41]. In Populus trichocarpa, from six identified CCoAOMT genes, three showed significant expression levels in developing xylem, but PtrCCoAOMT1 was clearly the most abundant, showing more than two-fold higher expression levels when compared to PtrCCoAOMT2 and four-fold when compared to PtrCCoAOMT3 [42]. Since CCoAOMT was shown, together with COMT, to be involved in methylation steps necessary for the biosynthesis of not only monolignols but also soluble phenolics like flavonoids and sinapoyl malate in Arabidopsis, we could hypothesize that the high expression levels of sugarcane CCoAOMT genes might also be related to the production of other phenolic compounds in sugarcane. Indeed, sugarcane stem produces an array of different phenolic compounds, as revealed by the analysis of the phenolic profiles during sugarcane stem development [25]. Among the thirtyfive identified compounds, all annotated flavonoids were tricin and tricin O-glycosides. Interestingly, the biosynthesis of tricin occurs through the sequential methylation of its precursor tricetin, in two consecutive reactions catalyzed by the same O-metyltransferase that shows significant similarity to COMTs and, to a lower extent to CCoAOMTs [48]. Among the ShCAD genes, the high expression of ShCAD2 and ShCAD8-1 contrasted with the very low transcript levels of ShCAD6 and ShCAD7 (Fig 1). It is important to mention that ShCAD2 is the putative orthologue of SbCAD2 from Sorghum bicolor, which was recently identified as the Brown midrib6 (Bmr6) gene [49]. The bmr6 mutant is affected in the phenylpropanoid metabolism, which results in reduced lignin content and altered lignin composition in sorghum. A nonsense mutation in SbCAD2 truncates the reading frame prior to the catalytic domains of the protein, resulting in a nonfunctional enzyme and is, consequently, responsible for the bmr6 phenotype [50]. In addition, phylogenetic analysis showed that SbCAD2 belongs to an evolutionarily conserved group of CAD proteins involved in lignin biosynthesis [50]. The high expression levels of ShCAD2 and its close phylogenetic relationship to the ligninrelated SbCAD2 from sorghum support a role for this gene in the constitutive lignification in sugarcane. In accordance with previous results, S branch-specific genes showed the same discrepancy in transcript levels, with very low expression levels of ShF5H1 and much higher for both ShCOMT genes (Fig 1). Lower levels of ShF5H1 expression correlates with lower S/G ratio found in younger internodes of sugarcane. This observation is in line with the fact that syringyl units can be decreased or increased simply by down-regulation or up-regulation of F5H, respectively [51]. Several lines of evidence suggest that F5H expression might be differentially regulated than other lignin biosynthetic genes [52]. First, AC elements (i.e. cis-elements that determine xylem-specific expression) are present in all G-branch lignin genes in Arabidopsis, but they cannot be found in the promoter region of S-branch-specific F5H and COMT [41]. Second, F5H is the only lignin biosynthetic gene directly regulated by the secondary cell wall master switch SND1 in Medicago truncatula, while all the other genes are directly regulated by SND1 targets MYB46 and its functionally redundant pair MYB58 [17]. Third, secondary wall master switches SND1/NST1 are exclusively expressed in fibers, a xylem cell type enriched in Slignin, while the transcripts of master switches VND6/VND7 are only found in G-rich vessels [53][54][55]. In contrast to the low expression of ShF5H1, the high transcript levels of ShCOMT genes already in younger internodes might be related to the above-mentioned production of soluble phenolics such as tricin, tricin O-glycosides and other flavonoids. In order to evaluate whether the difference in lignin content between the genotypes is related to differential expression of lignin-specific genes, the transcript levels for each gene was compared between the contrasting sugarcane genotypes. Surprisingly, only three genes showed differential expression: ShPAL3 and ShC4H1 were higher in the low-lignin genotype IACSP04-065 and ShCOMT1 showed higher expression in the high-lignin genotype IACSP04-627 ( Fig 1). When comparing two genotypes contrasting for lignin content, one would expect that most of the lignin biosynthetic genes would show increased transcript levels in the high lignin genotype. However, many studies have shown that a simple correlation between lignin content and differential expression of lignin genes is not always straightforward. ShPAL3, ShC4H1 and ShCOMT1 had their expression analyzed in different tissues of two sugarcane genotypes also differing for lignin content [25] and in general the expression of the first gene was higher in the genotype containing less lignin, while the other two genes had similar expression in both genotypes. Recently we demonstrated that in sugarcane culm, the differential lignin deposition between genotypes, tissue types and at different developmental stages is under transcriptional regulation [56]. The comparison of eleven guinea grass genotypes differing in cell wall digestibility and lignin content revealed that the expression of only two lignin genes, C4H and CCoAOMT, was increased in the high-lignin genotypes [57]. Similarly, a microarray-based approach showed that only CCoAOMT was up-regulated in the high-lignin genotype of Eucalyptus grandis when compared to low lignin genotypes [58]. Even more surprising, when RNA-seq was used to compare two alfalfa genotypes with divergent cell wall composition in stems, some lignin genes (e.g. CAD and COMT) were up-regulated while others (e.g. PAL) were down-regulated in the high lignin content genotype [59]. Noteworthy, while the formers encode enzymes involved in downstream steps of the pathway, the later is the entry point enzyme that directs the carbon flux into the phenylpropanoid pathway and therefore, would be more likely to control the overall lignin content in the plant. Recent studies have demonstrated that lignin deposition is not only regulated at the transcriptional level but also at the post-transcriptional and post-translational levels and through the control of enzyme catalytic activities. In the stem differentiating xylem of Populus trichocarpa, the microRNA ptr-miR397a was shown to be a negative regulator of laccase genes involved in lignin polymerization. Overexpression of ptr-miR397 resulted in the down-regulation of 17 laccase genes without any effect on the transcript levels of monolignol biosynthetic genes, which led to a reduction in lignin content of up to 22% [60]. In Arabidopsis, Kelch repeat F-box (KFB) proteins physically interact with PAL isoenzymes and mediate their ubiquitination and subsequent proteolytic degradation via the 26S proteasome pathway. The expression of KFB genes varies among plant tissues and responds to developmental and environmental cues, which contributes to the dynamic control of PAL activity. By affecting the stability of the PAL enzymes, KFBs post-translationally regulate the levels of phenylpropanoids and lignin [61]. Finally, many studies have shown that phenylpropanoid intermediates (e.g. hydroxycinnamates and shikimate esters) are able to inhibit different steps of the pathway, working as regulators of the carbon flux towards the different branches of the phenylpropanoid-monolignol metabolic grid [62][63][64]. As lignification represents a non-recoverable investment of carbon and energy [65], it is understandable that the regulatory mechanism controlling lignin deposition is so complex, involving a multi-leveled feed-forward loop transcriptional network that modulates the expression of biosynthetic genes [66] and post-transcriptional and post-translational processes that fine-tune the regulatory system. Despite of the orthology relationship previously showed, the use of high-throughput sequencing technology can also be useful to estimate the diversity of transcripts isoforms and gene families. As sugarcane is a polyploid species, polymorphisms can be generated from a different number of allelic copies present in each genotype [27]. Therefore, sugarcane cultivars are highly heterozygous, with several different alleles at each locus, and this high level of genetic complexity creates challenges during conventional and molecular breeding [67]. Some studies showed that a mutation in a single lignin biosynthesis gene can affect the expression of several other genes of the exact same biosynthetic pathway [16,68,69]. In this type of study with sugarcane it is possible to detect many transcripts that show high homology to functional genes. The MapMan analysis of sugarcane transcripts related with the phenylpropanoid pathway (Fig 2) shows transcripts of genes that encode the same enzyme and had different expression pattern between the two genotypes (higher or lower expression levels). However some gene families show a clear pattern. For instance, all transcripts of genes encoding ShPAL genes showed lower expression levels (or no differential expression) in the high lignin content genotype IACSP04-627 (Fig 1). ShHCT genes also showed strong divergent expression patterns between both genotypes, with some transcripts more expressed in the high-lignin genotype (in red) and others showed higher expression in the low-lignin genotype (in green). expression ratio values were false color-coded using a scale of -1 to 1 and indicate the genotype that showed highest expression for each transcript. The intensity of green and red indicates the degree of down-and up-regulation of the corresponding lignin gene. doi:10.1371/journal.pone.0134909.g002 Whole sugarcane transcriptome differential expression and functional enrichment analysis Besides the expression profile of phenylpropanoid biosynthetic genes, the transcriptome obtained from both contrasting genotypes was also analyzed for the whole differential expression. This analysis resulted in 2,162 differentially expressed transcripts (S3 File) that were clustered by self-organizing maps (Fig 3). This clustering analysis shows four different expression patterns for these 2,162 differentially expressed transcripts. The Cluster 1 (C1, with 425 sugarcane transcripts) represents the profile of transcripts with higher expression levels in the IACSP04-065 sugarcane genotype. Cluster 2 (C2, n = 604) has the profile of transcripts that had a minimal abundance in IACSP04-627. Cluster 3 (C3, n = 629) and Cluster 4 (C4, n = 504) shows the opposite of C1 and C2, where transcripts are more abundant in the IACSP04-627 genotype. A general overview of the whole sugarcane transcriptome of both genotypes allowed the establishment of 100 clusters that share genes with similar expression pattern (Fig 4), in which several clusters have a significant enrichment for certain MapMan classes (according to Page-Man) [38]. S4 File shows the K-means cluster associated with each transcript, while S5 File presents the MapMan term enriched in each cluster. The MapMan system was used to annotate the whole sugarcane transcriptome dataset (S6 File). This analysis results in clusters (Fig 5) enriched for flavonoids metabolism, glutathione-S-transferases (GSTs), abscisic acid and brassinosteroids metabolism, cell wall proteins, trehalose metabolism and response to biotic stress. Table 1. Transcripts involved with flavonoids metabolism (Fig 5A) showed a very similar expression pattern between both genotypes, with a higher expression in IACSP04-627 (high-lignin) than IACSP04-065 (low-lignin). Thus, although we have not observed a general and significant differential expression of lignin genes, the expression pattern of flavonoid-related genes and the higher lignin content in genotype IACSP04-627 suggest that both phenolic routes might be transcriptionally coordinated. Table 1 shows the transcripts that are present in the cluster enriched for flavonoid metabolism, as derived from the K-means analysis, in which all the transcripts are more expressed in the IACSP04-627 genotype. The IACSP04-627 genotype showed higher levels of transcripts related with GSTs (Fig 5B), while genotype IACSP04-065 showed higher expression of genes involved with biotic stress (Fig 5G). GSTs catalyze the detoxification of xenobiotics by their conjugation with the reduced form of glutathione (γ-glutamil-cistenil-glicine; GSH) and an electrophilic substrate, such as reactive oxygen species (ROS) [70,71]. GSTs are also thought to play major roles in oxidative stress metabolism induced by abiotic and biotic stresses [72]. The transcripts of genes involved with trehalose metabolism (Fig 5F) showed higher levels in genotype IACSP04-065. Trehalose metabolism is apparently associated with the ability to resist to abiotic stresses, in particular desiccation, although there are indications that it may also play a role in biotic stresses induced by microorganisms [73]. In this regard, the higher expression of transcripts of trehalose metabolism (Fig 5F) in the genotype IACSP04-065 is in agreement with the higher expression of biotic stress-related genes in the same genotype ( Fig 5G). Further experiments would confirm the capacity of this genotype to better cope with different stress conditions. Conclusions Most of the current knowledge about lignin metabolism was obtained with studies in dicotyledonous plants like Arabidopsis and poplar, but the mechanisms underlying several aspects of lignin biology such as biosynthesis, polymerization and regulation are not necessarily conserved among all vascular plants [74]. For instance, despite of its economical importance for bioethanol production, genetic information on lignin metabolism in sugarcane is limited. Only recently, the first lignin-specific gene was characterized in sugarcane using reverse genetics, by down-regulating the expression of a COMT [75]. In addition, evidence for the involvement of a specific laccase in the lignification process in sugarcane was obtained by the complementation of lac17 mutant in Arabidopsis with a sugarcane laccase gene, SofLAC [19,76]. Despite these recent advances, there is still much to be explored and determined in terms of gene expression and pathway regulation of lignin biosynthesis in sugarcane. In this regard, the data reported here provide a comprehensive resource for lignin studies in sugarcane. In addition, several clusters of co-expressed genes potentially involved in flavonoids and carbohydrate metabolism, stress response, hormone metabolism were identified between contrasting lignin content genotypes ( Fig 5 and Table 1). In general, most of the lignin genes were less expressed on the low lignin content genotype IACSP04-065, with exception for differentially expressed PAL3 and C4H1 genes. Finally, the expression profile obtained (Figs 1 and 2) for both contrasting lignin content varieties should indicate targets for further biotechnological approaches.

v3-fos

2018-12-05T08:57:52.244Z

2015-06-12T00:00:00.000Z

54861342

s2

Comparative Studies on the Physicochemical and Sensory Properties of Watermelon (Citrullus lanatus) and Melon (Citrullus vulgaris) Seed Flours Used in “EGUSI” Soup Preparation A comparative study on the physicochemical and sensory properties of watermelon (Citrullus lanatus) and melon (Citrullus vulgaris) seed flours in food preparation were investigated. A composite flour containing equal parts of watermelon seed flour and melon seed flour were prepared. Egusi soups were prepared from the melon seed flour; watermelon seed flour and a combination of the two flours in equal proportions. Sensory properties of the three soups were evaluated. The results of the investigation showed that the equal proportions of watermelon/melon seed flours had higher crude protein of 27.73% and crude fat of 47.85% than the water melon seed and melon seed flours. There was no significant difference (P>0.05) in water absorption, foam capacity, viscosity and least gelation properties of the melon seed flour compared to the 50:50 flour sample. The sensory properties showed no significant difference (P>0.05) in appearance, taste, thickness and overall acceptability of egusi soup from melon seed flour and 50:50 flour sample. Therefore watermelon seed flour can be used to replace 50% melon seed flour in the preparation of egusi soup Introduction Melon (Citrullus vulgaris) is a member of the cucurbitaceous family and is the biological ancestor of water melon (Citrullus lanatus). It is a large plant family which includes many economic species such as melon, various gourds, pumpkins and cucumbers (Oyeleke et al., 2012). Melon (Citrullus vulgaris) is grown widely in the tropics and its seeds popularly known as "egusi" in Nigeria is consumed in various forms such as egusi soup, melon ball snacks and ogiri fermented melon seed (King & Onuora, 1983;Achinewhu, 1987). Melon seeds have nutritive and caloric values which make them necessary in diets. Ojieh et al. (2008) reported that melon seeds (egusi) contain 3.7% ash, 45.7% ether extract, 23% crude protein, 12% crude fibre and 10% total carbohydrate. Similar proximate composition had been reported by Kiin-Kabari and Akusu (2014) on watermelon seed flour using different processing methods. The oils from melon seed and watermelon had been characterized by other researchers Oresanya et al. (2000), Ebuehi and Avwobobe (2006) and they observed that melon seed oil contained more of unsaturated fatty acids than watermelon seed oils. The consumption of watermelon fruit in Nigeria had increased tremendously in recent years due to the increased awareness on the health benefits. The watermelon juice contains important carotenoids such as β-carotene, carotene and Lycopene which are important in neutralizing free radicals in the body (Oseni & Okoye, 2013;Penuel et al., 2013). In Nigeria, the utilization of watermelon fruits is limited to the direct consumption of the fresh fruits. The fruits contain seeds which are un-utilized fruit by-products. The seeds are discarded and not eaten but are consumed in other parts of the world either roasted and served as snacks or milled into flour for incorporation into wheat flour and baked into bread (El-Adway & Taha, 2011). Watermelon seeds are high in proteins and fats and can find applications as a protein source in various food formulations and preparation (El-Adway & Taha, 2011). Shadrach and Oyebiodun (1999) reported that the ultimate success of utilizing plant proteins as ingredients largely depends upon the beneficial qualities they impact to foods which also depend largely on their nutritional and functional properties. Melon seeds (egusi) is used traditionally as the basis for a number of soup preparation especially the popular "egusi" soup in Nigeria, were the melon seed act as a thickener in the soup. Water absorption, viscosity and the least gelation concentration are all important functional properties in egusilike soup preparation (Kiin-Kabari & Akusu, 2014). One of the major sensory attributes of the egusi soup is the thickening properties of the soup, whether it is prepared from melon seeds flour or water melon seed flour. However, melon (egusi) seeds are becoming very expensive in Nigeria whereas water melon seeds are discarded after the consumption of the water melon fresh fruits. Improvement in the utilization of both melon seeds and water melon seeds can be achieved if we understand their proximate and functional behaviour of the seed flour in food preparation. The functional properties are important in determining the organoleptic properties of "egusi" and "egusi-like" soup preparation. Combining melon seeds and watermelon seeds in "egusi-like" soup preparation may be of interest in reducing the costs of "egusi" soup preparation. This study was aimed at evaluating the potential food uses of water melon seeds by comparing its proximate, functional and sensory properties with that of melon (egusi) seed in soup preparation as well as evaluating the functional impact on the organoleptic/ acceptability of "egusi-like" soup prepared from a 50:50 melon: Water melon seed flour blends. Materials Watermelon fruits (Citrullus lanatus) were purchased from a local market in Port Harcourt and transported to the Department of Food, Science and Technology laboratory for processing. All chemicals used were of the analytical grades, products of BDH chemical Ltd pool, England. Preparation of Watermelon Seed Flour Watermelon seeds were removed from the pod, washed, pre-boiled for 5 min and sun-dried at 34 °C for 3 days. The sun-dried seeds were dehaulled and milled into flour as shown in Figure 1a. Preparation of Melon Seed Flour Five (5) kg of melon seed was shelled, sorted, cleaned and oven dried (50 °C, 24 h) in a hot -air fan circulating oven (model QUB,305010G, Gallenkamp, U.K), ground to pass through a 0.25 mm British standard sieve (Model B5410, Endecoths Lt, London, UK), as shown in Figure 1b. Both watermelon seed flour and melon seed flour were divided separately into fifteen lots for each; stored in air tight containers in a refrigerator. A 50:50 ratio was used based on the preliminary study on various blends of melon/watermelon seed flour that was most acceptable. Sensory Evaluation A panel of 20 people who are used to egusi soup were used for the sensory evaluation of the soup prepared with watermelon seed flour, melon seed flour and melon seed flour. A 9-point hedonic scale with 9 = lie extremely, 5 = neither like nor dislike and 1 = dislike extremely was used for the evaluation of the prepared soups for colour, appearance, thickness, taste, aroma and overall acceptability. The panelists were served the "egusi" like soup and the egusi soup in the food and nutrition laboratory of the food science and technology department at room temperature (28 ± 2 °C). Three soups were prepared: watermelon seed flour soup, melon seed flour soup (egusi soup) as control and a mixture of 50% water melon seed flour and 50% melon seed flour as shown in Table 1. Statistical Analysis The data obtained were subjected to analysis of variance (ANOVA) using Statistical Package for Social Science (SPSS) version 20.0 software 2011. All analysis was done in triplicate and means separated using Duncan Multiple Range Test. The selected functional properties of watermelon seed flour, melon seed flour and the 50:50 blended sample are shown in Table 3. There was no significant difference (P>0.05) in water absorption capacity, foam capacity, viscosity and least gelation concentration between melon seed flour and the 50:50 blended sample; however, watermelon seed flour gave lower values for these functional properties. Ige et al. (1984) reported that proteins are linked to some functional properties such as foaming, water absorption, viscosity and gelation. Matti (1970) reported that the desirability of carrying out functional test is to predict how the plant proteins will affect the food system in which they are incorporated. The sensory properties of "egusi" soups prepared with watermelon seed flour, melon seed flour and the 50:50 blended sample is shown in Table 4. The result revealed that there was no significant difference (P>0.05) between melon seed flour soup and the 50:50 blended sample soup in terms of appearance, taste, flavour, thickness and overall acceptability. The viscosity of the melon seed flour is higher which may affect the thickness and functional properties such as foaming capacity, water absorption capacity and least gelation concentrations. This is similar to the observation made by Akusu and Kiin-Kabari (2013) on the relationship between viscosity and other functional properties of watermelon seed flour. Conclusion Equal proportions of melon/watermelon seed flours compared favourably well in viscosity, water absorption, foam capacity and least gelation concentration when compared to melon seed flour alone. Similar patterns were observed in sensory properties of appearance, taste and overall acceptability of egusi soups. Thus the cost of egusi preparation can be reduced by substituting upto 50% of melon seed flour with watermelon seed flour which before now is un-utilized.

v3-fos

2019-01-03T04:35:14.944Z

2015-06-13T00:00:00.000Z

95409532

s2

Pretreatment of rapid detection of veterinary drug residue in eggs Eggs in the standard method for rapid detection of veterinary drug residue has not been formed, therefore, it is necessary to explore a rapid, highly effective, the total amount and qualitative method of rapid detection of antibiotic residues in eggs. Many different kinds of veterinary drugs on the market, this study selected: tetracycline, beta lactam, sulfa, fluoroquinolone for veterinary drug residue detection pretreatment research, finally to strip test results prior to determine the best INTRODUCTION Veterinary drug residue is refers to the accumulation of livestock animal medication after or stays in the body or prototype drugs in animal products such as eggs or their metabolites, including related to the veterinary drug residue [1]. These drugs can enter the human food chain through transfer, enrichment of the animal body, eventually leading to human in the process of eating the accumulation of veterinary drugs. Control of veterinary drug residues in eggs plays an important role in human life and health. Veterinary drug residue detection is an important means of eggs food safety, the rapid detection of standard has not been formed and its preparation method, therefore the experiment was carried out to explore, use is made of the existing market the rapid detection of veterinary drug residue detection milk strip for eggs of veterinary drug residues detection, the need for a series of eggs before treatment, including removal of the fat, to protein and so on. In theory, many different kinds of veterinary drug residue in eggs, this study selected: tetracycline, beta lactam, sulfa, fluoroquinolone for veterinary drug residue detection research. Control of veterinary drug residues in eggs plays an important role in human life and health. Veterinary drug residue detection is an important means of eggs food safety, the existing detection methods at home and abroad for capillary electrophoresis, fluorescence method, uv spectrophotometry, chemiluminescence method, atomic absorption spectrophotometry, microbiological method, high performance liquid chromatography (HPLC) method and liquid mass usage, etc, but has not been a rapid detection of veterinary drug residue in eggs standard method, therefore, it is necessary to explore a rapid, highly effective, the total amount and qualitative method of rapid detection of antibiotic residues in eggs. There are many kinds of veterinary drugs on the market, this study selected: tetracycline, beta lactam, sulfa, fluoroquinolone for veterinary drug residue detection research II. MATERIALS AND METHODS Eggs, northern suburb of Beijing changping farmers market to buy; The rapid detection of strip, Beijing often state biological technology co., LTD.; Organic reagents, analytical pure, west long chemical co., LTD. III. EXPERIMENT A. Tetracycline class antibiotic residues detection of egg sample pretreatment Chloramphenicol, oxytetracycline, tetracycline for common three kinds of tetracycline antibiotics. Tetracycline class of antibiotics, tetracyclines, TCs) is yellow crystalline powder, taste bitter, solubility in the alcohol (such as methanol and ethanol), such as ethyl acetate, acetone, acetonitrile dissolved in organic solvent is smaller. This kind of antibiotic produced by Streptomyces, on the chemical structure are hydrogenated pian and four benzene derivatives (pictured) basic structure, near the visible area near (350 nm) with strong ultraviolet absorption. Composition is complicated in biological samples, interference of tetracycline class of antibiotics on material is numerous, high concentration of salt in the sample and the existence of large molecular weight proteins will seriously affect the rapid detection of tetracycline class of antibiotics, so the pretreatment of the sample become one of the most important step in the sample analysis, adopt the method of reasonable and effective will tetracycline antibiotics extracted from the matrix of complex biological samples and to purify is particularly important. By tetracycline compounds in weak acid solution is stable, so often need to extract the target under the condition of weak acid composition, molecular weight protein precipitation and biological sample cuhk, generally USES the extraction solvents including citric acid salt, phosphate, trichloroacetic acid and perchloric acid, etc [2]. B. Beta lactam class residue detection of egg sample pretreatment Aminoglycoside antibiotics will act as a veterinary drug used to treat meat and dairy animals, so we need an analysis of the residual effective analysis method in these goods. This class of antibiotics for residue analysis put forward a huge challenge. Unlike most other antibiotics, these compounds could not using acetonitrile, or other organic solvent extracted from the organization or dairy products. In this study, with trichloroacetic acid (TCA) aqueous buffer will aminoglycoside antibiotics extracted from meat or dairy products. Join the TCA can make protein precipitation and inhibit protein with the analyte. Before the LC/MS analysis, the use of effective solid phase extraction (SPE) purification operation to remove residual TCA, to minimize the total extract interference. Through the use of the Oasis HLB (efficient, water can be invasive inverting absorber), in the milk and meat can obtain good solid phase extraction yield and purification effect.Ampicillin, the free acid containing 3 molecular crystal water. White crystalline powder. Taste slightly bitter. Slightly soluble in water, insoluble in ethanol, dissolve in the dilute acid dilute alkali solution. Soluble in water, soluble in ethanol. [3]. Ampicillin and ampicillin. The free acid containing 3 molecular crystal water (for internal use); White crystalline powder. Taste slightly bitter. Slightly soluble in water, insoluble in ethanol, dissolve in the dilute acid dilute alkali solution. PKa of 2.5 and 7.3. 0. 25% aqueous solution of pH 3. 5 ~ 5. 5. The structural formula below. Salt, the sodium for injection is white or kind of white powder or crystal. Odourless or slight odor, taste bitter. Have led to wet. Soluble in water, slightly soluble in ethanol. 10% aqueous solution of pH for 8 ~ 10. Xudong will whole egg, egg white and yolk homogenate respectively, accurately say 5 g and grout, put in 50 mL with centrifugal pipe plug, add 5 mL of acetonitrile solution set spiral mixer blender 1 min, then add 15 mL of acetonitrile solution, the swirling disaster 2 min, oscillation and mixed with 8000 x g centrifuge for 10 min, the temperature is set to 10 °C. Will clear liquid transferred to another 50 mL plug in the centrifuge tube, to add 20 mL with ultrapure water saturated me curse the methylene chloride solution blending 2 min (drug dissolved in the upper water, acetonitrile, soluble in methylene chloride solution Yan-hong song said samples from 2.0 g homogenate of egg yolk and egg white, accurate to 0.1 g, put in 50 ml with centrifugal pipe plug, add 10 ml of acetonitrile, intense blend for 5 minutes on the vortex mixer, oscillator with low speed oscillation omin, 3 to 5400 r/min, the centrifugal 10 minutes, supernatant fluid in a centrifuge tube, and add 8 ml of acetonitrile to slag, repeat extraction time, clear liquid on merger, 35 °C water bath nitrogen blow dry. In 2 ml (mobile phase of methanol/l % ice acetic acid solution: 23/77, V/V) dissolve yolk residue, add 3 ml n-hexane, oscillation let stand for 5 minutes, 10 minutes at 3000 r/min, the centrifugal, remove n-hexane layer through a straw, add the 3 ml n-hexane repeat to fat, 0.22 mu m organic membrane, for the liquid chromatographic; Egg white without removal of the fat, with LML mobile phase (1% methanol/ice acetic acid solution: 28/72, V/V) dissolved residue, after 0.22 mu m membrane filter, the liquid chromatograph. According to take in the homogenizer is homogeneous whole egg sample 2 g, put in 50 ml with centrifugal pipe plug, add 5, 10, 15, 20 ml of acetonitrile, intense mixing 5 min on the vortex mixer, oscillator with low speed oscillation, 10, 15, 20, 25 min to 5400 r/min, the centrifugal 10 min, supernatant fluid in a centrifuge tube, and add 8 ml of acetonitrile to slag, repeat extraction time, clear liquid on merger, 35 °C water bath nitrogen blow dry. Add 3 ml n-hexane, oscillation let stand for 5 minutes, 10 minutes at 3000 r/min, the centrifugal, remove nhexane layer through a straw, add the 3 ml n-hexane repeat to fat, after 0.22μm organic membrane on rapid test. Sulfa drugs, its molecular weight between 170 ~ 300, soluble in water, soluble in ethanol and acetone, almost insoluble in chloroform and ether. Except for alkaline sulfaguanidine, sulfa drugs because of containing the primary amine and sulfonamide based in acid and alkali sex, soluble in acid and alkali solution. Acid is weak and easy to absorb carbon dioxide from the air and carbonate precipitation. Because of its structure with a benzene ring, all have the ultraviolet absorption [4]. Add egg samples samples from 2 g to 5, 10, 15, 20 ml ethyl acetate oscillating up and down 5, 10, 15, 20 min, at room temperature to 5000 r/min centrifugal 5 min, take the lower liquid, after 0.22 mu m organic membrane on rapid immunochromatographic test. According to the article late rapid test results, it is concluded that under the condition of the following the fastest the most accurate test result. Add egg samples from 2 g to 5 ml 5 min, ethyl acetate and oscillations at room temperature to 5000 r/min centrifugal 5 min, take the lower liquid, after 0.22 μm organic membrane on rapid test. C. Fluoroquinolone residues detection of egg sample pretreatment Fluoroquinolone drugs (FQs) are white or light yellow crystal powder, usually free acid soluble in dilute alkali and glacial acetic acid, dilute acid solution, the water in the pH6 ~ 8 solubility, minimum in methanol, chloroform, ether and most difficult soluble or insoluble in the solvent. The structure of benzene and heterocyclic or heterocyclic skeleton and carbonyl and carboxyl, heteroatom such as chromophore or help base group of conjugate system, in the ultraviolet region characteristic and strong absorption, the ultraviolet spectrum contains several absorption peak: 240 ~ 300 nm. 330 ~ 350 nm. FQs itself also have fluorescent properties, the Ex = 280 ~ 330 nm, Em =410~430 nm. FQs acid-base amphoteric compounds, ultraviolet absorption spectrum in different pH medium will be slightly different [5]. In fresh egg, after the break with low speed homogenizer homogeneous egg samples, egg white and yellow when fully mixing, as the blank sample. After taking homogenate of blank sample, add the appropriate concentration of standard solution as the blank to add sample. In 2.0 g egg samples to 15 ml polystyrene in centrifuge tube, add 5, 10, 15, 20 ml of acetonitrile, fully use oscillator oscillation 5 min. Learned that 2 ml supernatant fluid to 10 ml of clean glass tube, 50 to 60 °C dry nitrogen flow. Add 2, 5, 8, 10 ml n-hexane, using vortex finder vortex 1 min, centrifugal 5 min at room temperature, remove the upper organic phase, take 50μl for analysis. According to the article late rapid immunochromatographic test results, it is concluded that under the condition of the following the fastest the most accurate test result. In 2.0 g egg samples to 15 ml polystyrene in centrifuge tube, add 5 ml of acetonitrile, fully use oscillator oscillation 5 min. Learned that 2 ml supernatant fluid to 10 ml of clean glass tube, 50 to 60 °C dry nitrogen flow. Add 5 ml n-hexane, using vortex finder vortex 1 min. D. Sulfonamides residues detection of egg sample pretreatment Sulfa drugs, its molecular weight between 170 ~ 300, its chemical structure is as follows. Slightly soluble in water, soluble in ethanol and acetone, almost insoluble in chloroform and ether. Except for alkaline sulfaguanidine, sulfa drugs because of containing the primary amine and sulfonamide based in acid and alkali sex, soluble in acid and alkali solution. Most pKa sulfa drugs within 5 ~ 8, isoelectric point for 3 ~ 5, a few pKa is 8.5 ~ 10.5. Acid is weak and easy to absorb carbon dioxide from the air and carbonate precipitation precipitation. Because of its structure with a benzene ring, so all have the ultraviolet absorption. Zhong Ziqing said in 5 g egg samples, accurate to 0.01 g, 10 copies of put in 50 ml centrifuge tube. Each addition concentration set A, B, C three parallel, each sample set A blank control. Add quantity respectively for 10, 100, 200 ng/g. Add the sample after 30 min. Each centrifugal pipe add 20 g anhydrous sodium sulfate and 20 ml of acetonitrile, homogeneous 2 min, to 3000 r/min, the centrifugal 3 min. Supernatant fluid in 100 ml bottle heart, residue add 20 ml of acetonitrile, repeat the above for 1 times. Merge extract, to the heart in a bottle and add 10 ml isopropyl alcohol, using rotary evaporator in 50 °C water bath to dry, accurate flow with 1 ml and 1 ml n-hexane soluble residues. Transferred to the 5 ml centrifuge tube, the vortex 1 min, 3000 r/min, the centrifugal 3 min, abandon to upper n-hexane, add 1 ml n-hexane, repeat the above steps. Take the lower solution, after 0.2 mu m membrane filter, using liquid chromatography -tandem mass spectrometry determination. Add egg samples samples from 2 g to 4 ml ethyl acetate oscillation for 3 minutes, up and down at 5000 r/min, the centrifugal 5 minutes, take 300 mu l upper liquid 80 °C water bath top up or down in nitrogen blow dry, with 150 mu l 0.01 mol/l pH7.4 slightly dissolve the residue of phosphate buffer. A. Ampicillin, tetracycline class antibiotics residue sample detection Beta lactam and tetracycline class rapid test strip applied the principle of the competitive inhibition of immune chromatography samples in the beta lactam classes and tetracycline drugs in the process of flow and colloidal gold marked specificity monoclonal antibody, suppresses the antibodies and lines of NC membrane (B, T line) on the beta lactam classes and tetracycline drugsa combination of BSA coupling, leading to lines of depth changes. When the samples have no beta lactam classes and tetracycline drugs or beta lactam and tetracycline drugs concentrations below the detection limit, B, T line color; When the samples in the beta lactam classes and tetracycline drugs concentration equal to or higher than the detection limit, B, T line color; And no matter whether the sample contains beta lactam type and tetracycline drugs, color quality control line C line, to test effectively. Sample preparation extraction buffer (Na2EDTA NH4OOCH3 10 mM / 0.4 mM / 1% NaCl / 2% TCA) : 0.77 g ammonium acetate (NH4OOCH3) in 1 l of the volumetric flask. Add about 900 mL of reagent water, dissolve. Use 1 N 1 N HCl or NaOH to adjust pH value to 4.0. Add 0.15 g ethylenediamine tetraacetic acid disodium (Na2EDTA. 2 h2o), 5 g of sodium chloride (NaCl) and 20 g trifluoroacetic acid (TCA). Mix, make the solid solution, add reagent water to scale. Preliminary extraction: 2 g even cattle tissues or 10 mL milk in 50 mL centrifuge tube. Add 20 mL extraction buffer, vortex 10 seconds, and then fully shake the 1 minute. Sample of the centrifugal 5 minutes under 4000 RPM, clear fluid collection. According to the need to use dilute HCl or NaOH will clear liquid pH adjustment to 6.5 + / -0.5. B. Fluoroquinolone, sulfonamides residue sample detection Sulfa and fluoroquinolone combined rapid test strip applied the principle of the competitive inhibition of immune chromatography samples of sulfa drugs and fluoroquinolone drugs in the process of flow combined with colloidal gold marked specificity monoclonal antibody, suppresses the antibodies and lines of NC membrane (T) on the sulfa and fluoroquinolone -a combination of BSA coupling, resulting in lines of color depth changes. When there is no sample of sulfa drugs and fluoroquinolone drugs or sulfa drugs and fluoroquinolone drugs concentrations below the detection limit, T1, T2 line color; When sulfa drug concentration in the sample is equal to or higher than the detection limit, a T1 line color, T2 line color; When the sample of fluoroquinolone drugs concentration equal to or higher than the detection limit, T2 line color, not a T1 line color; And no matter whether samples containing sulfa drugs and fluoroquinolone drugs, quality control line (C) all can color, to show detection effectively. Marry remove all reagent from cold storage environment, balance at room temperature (25 °C) 20-30 min rapidly. The kits and standard sample of the corresponding microporous serial number, each sample and standard substance do two parallel holes. Add to the corresponding standard/sample 50 mu l microporous, soon to join enzyme mark two 50 mu l/hole, oscillation blending, 25 °C avoid light reaction after 60 min in the environment with washing liquid is 250 mu l/hole, full of washing 4-5 times. Join the substrate liquid A liquid 50 mu l/hole, then add the substrate liquid B liquid 50 mu l/hole, oscillation and mixed 25 °C avoid light environment reaction after 30 min. Add terminated liquid 50 mu l/hole, blending, enzyme standard instrument set in each hole OD value at 450 nm. To standard percentage absorbance (samples/zero absorbance value of standard absorbance value) as the ordinate, standard concentration (PPB), the abscissa denotes the semilog, draw standard curve. The percentage of sample absorbance generation into the standard curve, read the concentration of the sample corresponds to the standard curve, multiplied by the corresponding dilution ratio . SPE purification: this study used the Oasis HLB 96 -well plates (30 mg). If necessary, can use a 1 cc / 30 mg extraction column. Successively used 1.5 mL of methanol, 1.5 mL water, balance board hole or extraction column. The flow velocity is set to 1 mL/min or less. Join the preliminary extraction to get supernatant after pH adjustment, for tissue samples, add 1 mL solution, samples for dairy products, to join a 1.5 mL solution. 1 mL water to rinse. With 0.5 mL 10:5:8 5 formate/isopropanol/water elution. To join (including 1.5 L HFBA, using UPLC/MS/MS analysis [6]. C. Sulfonamides residue sample detection Li Kui by using immune chromatography rapid determination of sulfonamides residues in eggs. From 4 °C strip out of the fridge, return to room temperature. Blank respectively 120 mu l sulfa drug standard samples and waiting for inspection, in turn, add to the MPP hole, insert the strip sample end microporous plate hole, make holes in the liquid by capillary action up swimming, reaction after 10 minutes, judge the results. Sulfa drugs standard strip blank lines appear red stripe; If waiting for the sample strip lines appear similar to the blank lines of standard red stripe, sulfa drugs, not detected in the samples that sentence to negative; Visible light on the blank lines of standard color light red stripe, description of sulfanilamide in oxygen pyrimidine (SMM), sulfanilamide for oxygen pyrimidine (SMD), sulfanilamide dimethyl oxygen pyrimidine (SDM) concentration in the 20 ~ 100 mu g/kg range, or SDZ concentration within 40 ~ 160 mu g/kg, jailed for weakly positive; If the inspected samples without color strip line, SMM, SMD, concentration of SDM in the sample that is more than 100 mu V. PROSPECT Laying hens production in our country in the world, but more than 95% of the eggs are produced by farmers and small businesses, as a result of the limitation of capital, technology, management and other aspects of vicious competition and market prices, the price of eggs was far below the normal production cost. Feed producers as the pursuit of profit, and extensive use of cheap inferior raw material; In order to reduce disease risk, abuse of antibiotics; For high yield, and extensive use of synthetic hormones. Individual farmers to buy cheap feed, and the imperfection of the supervision strength, have an opportunity to make illegal activities. Resulting in a decline in egg quality and nutrient unbalance, pathogenic bacteria, antibiotics, hormones and pesticide residues exceeds bid badly. The use of veterinary drugs not science, not specification, led to the occurrence of drug residues. To prevent avian disease, in the case of uncertain etiology abuse of antibiotics, optional increasing dosage, dosage change, do not obey to take medicine. All in all, First is looking at home and abroad research status, can include physical and chemical properties of the target component, harm, types of existing research methods, and the pros and cons of each method, the current methods of GB and components limited threshold. Research status in a certain extent, have the latest research results. Research focus, and the light spot and so on also can have certain embodiment. And in the veterinary drug detection, drugs, veterinary drugs can be divided into categories of methods summary (similar components of structure, a lot of time there may be a lot of consistency test method).

v3-fos

2016-10-31T15:45:48.767Z

2015-02-01T00:00:00.000Z

4887002

s2

Mitigation of Prion Infectivity and Conversion Capacity by a Simulated Natural Process—Repeated Cycles of Drying and Wetting Prions enter the environment from infected hosts, bind to a wide range of soil and soil minerals, and remain highly infectious. Environmental sources of prions almost certainly contribute to the transmission of chronic wasting disease in cervids and scrapie in sheep and goats. While much is known about the introduction of prions into the environment and their interaction with soil, relatively little is known about prion degradation and inactivation by natural environmental processes. In this study, we examined the effect of repeated cycles of drying and wetting on prion fitness and determined that 10 cycles of repeated drying and wetting could reduce PrPSc abundance, PMCA amplification efficiency and extend the incubation period of disease. Importantly, prions bound to soil were more susceptible to inactivation by repeated cycles of drying and wetting compared to unbound prions, a result which may be due to conformational changes in soil-bound PrPSc or consolidation of the bonding between PrPSc and soil. This novel finding demonstrates that naturally-occurring environmental process can degrade prions. Introduction Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of fatal neurodegenerative diseases which impact a number of species including cattle (bovine spongiform encephalopathy, BSE), sheep and goats (scrapie), deer, elk and moose (chronic wasting disease, CWD), and humans (Creutzfeldt-Jakob disease, CJD, and others) [1]. The infectious agent of prion diseases, PrP Sc , is a misfolded isoform of a non-infectious cellular prion protein, PrP C . CWD and scrapie prions can remain infectious over long time periods [2][3][4][5] in the environment. The increasing incidence and geographic range of CWD in cervids and its unknown host range makes this disease of particular concern in North America. Prions enter the environment from infected hosts. Prions are shed into the environment via antler velvet [6], blood, saliva [7], urine [8][9][10], feces [11,12], and birthing matter [13]. Prions also enter the environment through decomposition of infected animal carcasses [5]. Prions can be present in these excreta during the presymptomatic phase of disease, therefore, infected animals can shed prions into the environment over wide areas of the host's home range. The amount of infectivity that is introduced into the environment is difficult to assess since prion titer is operationally defined with the route of infection, the age of the animal, the number of doses, and the PrP genotype of the host all making significant contributions in establishment of infection [14][15][16][17]. After release from an infected host, PrP Sc binds to a wide range of soil and soil minerals [18][19][20]. Clay and clay soils have higher affinity for prions and adsorb PrP Sc at a faster rate compared to sand or sandy soils [18,19]. Binding of PrP Sc to soil in a competitive matrix such as brain homogenate is slow and reduced compared to non-competitive environments (i.e. purified PrP Sc ) [18,19]. Once bound to soil, prions remain highly infectious although soil-induced changes in in vitro PrP Sc conversion efficiency and infectivity in animals have been measured [21,22]. Further work is needed to fully elucidate the effect of PrP Sc binding to surfaces on prion infectivity and transmission. The contribution of soil bound prions to the natural transmission of prion disease is incompletely understood. Modeling studies conducted in CWD-endemic areas indicate that locations with a preponderance of organic soils correspond with an increased incidence of CWD [23][24][25]. Since the soil type that is bound to PrP Sc has a relatively small influence on prion infectivity, other factors may play a role in prion transmission. Prions are primarily retained in surface soils [26] and the close contact of ruminant animals with soils renders soil-bound prions a likely source for prion disease transmission through ingestion or inhalation [27][28][29][30]. Therefore, PrP Sc binding to soil may increase the bioavailability of prions for transmission. Inactivation of soilbound prions will be required to control and prevent the spread of prion diseases in the environment. Prion degradation under environmentally-relevant conditions is poorly understood. To date, the majority of studies have investigated degradation and inactivation of prions that are not bound to soil. Microorganisms and isolated enzymes, sometimes associated with harsh digestion conditions (high temperature and extreme pH), effectively reduce PrP Sc abundance [31][32][33][34]. Exposure of prions to intact lichens at room temperature and neutral pH can reduce the abundance of PrP Sc [35]. The loss of PrP Sc immunoreactivity does not always correspond with a measurable reduction of prion infectivity [33,36] therefore, studies that rely solely on changes in PrP Sc abundance must be interpreted with caution. Prionzyme, a commercially-available enzyme, degraded soil-adsorbed prions under environmentallyrelevant conditions and is the first evidence to suggest that mitigation of soil-bound prions is possible [37,38]. Prions retained in surface soils are exposed to ambient environmental processes that have the potential to inactivate prions. Naturally-occurring cycles of drying and wetting alter soil aggregate stability and can influence interactions between soil particulate organic matter and dissolved organic compounds [39][40][41]. They can also change microbial activity and population dynamics [39,40,42]. Additionally, dehydration can unfold the native protein structure [43]. It is not known if these processes alter the biologic properties of soil-bound prions. To address this important question, we investigated the effects of repeated cycles of drying and wetting on the fitness of prions bound to various soil types. Results Repeated cycles of drying and wetting reduced the abundance of total protein in hamster brain homogenate The abundance of total protein in the brain homogenate (BH) of hamster infected with HY TME before or after binding to silty clay loam (SCL) was shown in Fig. 1. The amount of total protein of unadsorbed BH was significantly reduced (p<0.05) by 13% (Fig. 1). After binding to SCL, the amount of total protein remained unchanged ( Fig. 1) suggesting protection of proteins from degradation of repeated drying and wetting by adsorption to soil surface. Repeated cycles of wetting and drying did not result in changes in pH or conductivity. Repeated cycles of drying and wetting alter the resistance of HY PrP Sc to digestion with proteinase K Soil-or soil mineral-adsorbed HY PrP Sc (HY) was prepared as described in Table 1 and subjected to 0 (control), 1, and 10 repeated cycles of drying and wetting. Significant differences (p>0.05) were not observed in normalized PrP Sc immunoreactivity between samples after 1 drying and wetting cycle (Dry 1) compared to the negative control (no drying and wetting treatment, Dry 0) (Fig. 2). PrP Sc abundance was significantly decreased (p<0.05) between the negative control and 10 repeated cycles of drying and wetting of unbound HY, silty clay loam (SCL)-, bentonite-, and silicon dioxide (SiO 2 )-adsorbed HY (Fig. 2). The average reductions are 51%, 53%, 72%, and 73%, respectively (Fig. 2B). The PrP Sc abundance of sandy loam soil (SLS)-adsorbed and sand-adsorbed HY PrP Sc were not significantly (p>0.05) changed following 10 repeated cycles of drying and wetting (Fig. 2). Repeated cycles of drying and wetting reduced the PMCA conversion efficiency of soil bound HY TME After 1 round of PMCA, PrP Sc amplification in all samples subjected to 1 cycle of drying and wetting was not significantly (p>0.05) different compared to unbound HY (Fig. 3). The amplification of unbound HY, SCL-, SiO 2-, bentonite-, and sand-adsorbed HY subjected to 10 drying/wetting cycles was significantly (p<0.05) reduced by 48%, 95%, 100% (negative value for bentonite-HY was corrected to 0%), 74%, and 95%, respectively (Fig. 3). However, the PMCA conversion efficiency of SiO 2 HA-adsorbed HY was not changed (p>0.05) after 10 cycles of drying and wetting (Fig. 3). Sandy loam soil inhibits HY PrP Sc PMCA conversion independent of HY adsorption resulting in low PrP Sc abundance (Fig. 3). Influence of repeated cycles of drying and wetting on proteinase K resistance and amplification efficiency of DY TME Samples were prepared as described in Table 1. A significant (p>0.05) difference in PrP Sc immunoreactivity was not observed for unbound DY or SCL-adsorbed DY after 1 drying and wetting cycle compared to the control ( Fig. 4A and 4C). A significant (p<0.05) reduction in PrP Sc abundance of 48% was observed for unbound DY treated with 10 cycles of drying and wetting while the PrP Sc abundance of SCL-bound DY was not significantly (p>0.05) changed ( Fig. 4A and 4C). After 3 rounds of PMCA, DY and SCL-bound DY amplified to similar (p>0.05) levels after 1 cycle of drying and wetting compared to controls ( Fig. 4B and 4D). When exposed to 10 drying/wetting cycles, a significant reduction (p<0.05) in amplification was only observed for SCL-bound DY by 68% and not for unbound DY (Fig. 4B and 4D). Reduced total protein abundance in HY TME brain homogenate. UV scanned polyacrylamide gel stained with SYPRO Ruby (A) and quantification of total proteins (B). Samples were not digested with proteinase K. Star indicates significant difference (p<0.05; n = 3) between treated and untreated sample. Influence of repeated cycles of drying and wetting on proteinase K resistance and amplification efficiency of CWD SCL-adsorbed CWD was prepared as described in Table 1. Compared to the untreated samples (Dry 0), the PrP Sc abundance did not significantly (p>0.05) differ between SCL-bound and unbound CWD with up to 10 cycles of drying and wetting ( Fig. 5A and 5C). The PMCA conversion efficiency of unbound CWD after 3 rounds of PMCA was not significantly (p>0.05) different through 10 repeated cycles of drying and wetting compared to controls ( Fig. 5B and 5D). In contrast, a significant (p<0.05) reduction of 83% in PMCA conversion efficiency of SCL-bound CWD after 10 cycles of drying and wetting treatment was observed ( Fig. 5B and 5D). Sorption of HY to soil reduces the number of wet dry cycles that result in a decrease in PMCA conversion activity Unbound HY or SCL-adsorbed HY were subjected to 3, 5 or 7 repeated rounds of drying and wetting. Samples were then subjected to 1 round of PMCA and the abundance of amplified PrP Sc was quantified. Unbound HY had similar (p>0.05) conversion efficiency at 1, 3, 5 and 7 repeated cycles of drying and wetting. However, 10 cycles of repeated drying and wetting resulted in a significant (p<0.05) reduction in HY PrP Sc amplification compared to the sample treated with 1 cycle of drying and wetting ( Fig. 6A and 6C). In contrast, after 3 repeated rounds of drying and wetting of SCL-bound HY amplification was significantly (p<0.05) inhibited ( Fig. 6B and 6D). These results demonstrate that, under the conditions tested, binding to SCL enhances the reduction in conversion efficiency induced by repeated cycles of drying and wetting. PMCA conversion efficiency is decreased by sorption to soil Unabsorbed and SCL-adsorbed HY was subject to 0 or 10 serial rounds of wetting and drying. The samples were adjusted to equalize the abundance of PrP Sc between the samples. Ten-fold serial dilutions, ranging from 10 -2 to 10 -8 , of these samples were subject to one round of PMCA. PMCA reactions seeded with untreated HY-SCL resulted in detectable PrP Sc though the 10 -4 dilution, while PMCA reaction seeded with an equal amount of HY-SLC PrP Sc that was treated with 10 serial rounds of wetting and drying resulted in detectable PrP Sc though the 10 -2 dilution (Fig. 7). These results indicate that 10 serial rounds of wetting and drying reduce the specific activity of SCL absorbed HY PrP Sc by two logs. Repeated cycles of drying and wetting extend the incubation period of prion infection Selected drying and wetting treated samples and untreated controls were intracerebrally inoculated into Syrian hamsters. Incubation periods for hamsters inoculated with HY, SCL-, SiO 2-, and SLS-bound HY subjected to 0, 1, or 10 drying/wetting cycles are summarized in Table 2 and the survival results are presented in Fig. 8. Consistent with PK-resistance and PMCA results ( Fig. 2 and 3), the incubation period of hamsters inoculated with SCL-HY subjected to 1 cycle of drying and wetting did not significantly (p>0.05) differ compared to hamsters inoculated with untreated SCL-HY ( Fig. 8 and Table 2). The incubation period of HY-and SCL-HYinoculated hamsters subjected to 10 cycles of drying/wetting was significantly (p<0.05) extended 13 days compared to that of hamsters inoculated with the untreated control ( Fig. 8 and Table 2). This extension of the incubation period is consistent with a 2 log reduction in prion titer [21]. The incubation period of hamsters inoculated with SLS-HY and SiO 2-HY treated with 10 cycles did not significantly (p>0.05) differ compared to hamsters inoculated with untreated control ( Fig. 8 and Table 2). Discussion Prions have been detected in the environment [2-5, 44, 45] and they can survive for years in soils [2][3][4][5]. Prion shedding and persistence in the environment is well documented, however, little is known about environmental degradation of prions. In this study we investigated if prion infectivity is mitigated by natural environmental conditions. Under natural conditions of repeated wetting and drying we found evidence of PrP Sc degradation, decreased PrP Sc conversion activity, and an increase in prion incubation period suggesting reduced prion infectivity. This effect was dependent on the soil type that was bound to PrP Sc and the source of prions, underscoring the complexity of the interaction of prions with soil. Reduced PrP Sc abundance after repeated cycles of drying and wetting Repeated cycles of drying and wetting degraded not only PrP Sc but also other proteins in the brain homogenate whereas binding to soil prior to repeated cycles of wetting and drying eliminated this effect. (Fig. 1). While the exact mechanism of prion protein degradation due to repeated cycles of drying and wetting are not known, several possibilities exist. We hypothesize that exposure to repeated cycles of drying and wetting results in protein conformational changes that render PrP Sc more susceptible to degradation. Loss of water and changes in ion concentrations and pH in solution may occur during dehydration which can affect the secondary structure of proteins [43,[46][47][48], however, changes in ionic strength or pH were not observed between cycles in this study. Although poorly understood, changes in soil properties such as surface charge or cation exchange capacity occur after repeated drying and wetting cycles [49,50] that can result in desorption and/or reorganization of adsorbed compounds including proteins. Alternatively, consolidation of the bonding between soil and the adsorbed PrP Sc after drying may make PrP Sc desorption more difficult resulting in reduced PrP Sc detection and prion infectivity. The implication of this possibility is that PrP Sc desorption is required for prion and quantification (C and D) of PK digested and PMCA amplification (3 rounds) of DY PrP Sc alone or adsorbed to SCL before (Dry 0) and after 1 (Dry 1) and 10 (Dry 10) serial rounds of drying and wetting. Negative PMCA samples were diluted from corresponding PMCA seeding with a dilution factor of 80. Migration of 29 and 19 kDa molecular weight marker is indicated on the right of the Western blot. Star indicates significant difference (p<0.05; n = 3) between treated and untreated sample. conversion and western blot detection. This explanation is consistent with previous findings that PrP Sc attached to stainless steel surfaces (dried onto surface or as incubation solution) becomes more resistant to decontamination when exposed to extended drying compared with no drying or wet storage condition [51,52]. We speculate more compact stacking of PrP Sc aggregates on the surface may occur during drying as water molecules evaporate, minimizing PrP Sc exposure to the surroundings. Repeated drying and wetting renders soil-bound prion less infectious The reduced PMCA conversion coefficient in combination with the extended incubation period in hamster bioassay compared to untreated prions ( Fig. 3 and 8, Table 2) are consistent with a 2 log reduction in prion infectivity of soil-bound prions [21] after 10 cycles of drying/ wetting. While both PMCA and bioassay data are in agreement with a 2 log reduction in titer following 10 cycles of drying/wetting, since titer was not directly calculated, this value should be interpreted with caution. It is unknown if additional cycles of wetting will further reduce infectivity or if complete prion inactivation is possible. The observed reduction in infectivity is not entirely due to loss of PrP Sc . When standardized for the amount of starting PrP Sc , the PMCA conversion coefficient of HY bound to SCL that has not been treated to repeated cycles of wetting and drying is two orders of magnitude greater compared to HY-SCL that has been repeatedly wetted and dried for 10 cycles (Fig. 6). Since the reduction in the specific activity of PrP Sc is enhanced when HY is bound to SCL, we hypothesize at the PrP Sc -SCL interface, the wetting and drying process is altering a property of PrP Sc that renders it less infectious. Effect of repeated drying and wetting on the properties of soil-bound prions is soil type and strain dependent Changes in PrP Sc abundance and PMCA conversion efficiency after repeated cycles of drying and wetting are observed for a subset of unbound and soil-bound PrP Sc . HY and DY PrP Sc are more susceptible to PK digestion following repeated cycles of drying and wetting compared to CWD (Figs. 2, 3, 4, and 5). Binding of DY PrP Sc to SCL protects DY PrP Sc by enhancing PK resistance but results in a reduction in DY and CWD PrP Sc conversion activity (Figs. 4 and 5). Adsorption of the same PrP Sc to different soils resulted in a greater or lesser effect of repeated drying/wetting on PrP Sc properties (Figs. 2 and 3). Factors that may contribute to the variation include soil surface properties and soil-prion bonding. Since the percentage of sand and clay in a soil resulted in different soil stability changes in response to drying/wetting cycles we hypothesize that clay-clay and clay-sand interactions can indirectly affect the soil-prion interface [41]. Overall, this dynamic can affect which prion strains persist and the relative titer in any given environment. Since the relative ratios of prion strains can affect strain emergence, the Environmental factors affecting the efficiency of repeated drying and wetting Since prions are likely to be immobile in surface soils [26], soil-bound prions are readily accessible to ambient weather conditions which can include changes in surface soil moisture. In some CWD-endemic areas such as Phantom Valley (Latitude: 40.4°N; Longitude: 105.9°W) close to Rocky Mountain National Park, the number of moisture change in surface soil (soil depth is 0.5 m) can be up to 28 per month [53]. Based on our findings, drying/wetting cycles which repeatedly change soil moisture may be a natural degradation pathway for soil-bound prions. Additionally, wetting and drying can change microbial activity in soils [39,40] which may affect prion-soil interactions. Ambient temperature likely does not contribute to natural prion degradation since most heat induced decontamination (incomplete inactivation) occurs at temperatures well over 100°C [54][55][56][57]. Enzymes secreted from soil microorganisms, such as serine proteases, may be auxiliary for soil-bound prion degradation and inactivation, however, they were only found to be effective at high temperature, high pH or both [58,59]. Overall, this study provides the first evidence that natural processes can reduce prion infectivity. Since the total environmental prion load is a balance between addition of prions to the environment and clearance of prions from the environment, efforts to limit prion input into the environment may positively affect this balance and have meaningful results in reducing environmental transmission of prion diseases. Additionally, the soil composition and hydrology of an area may shape the overall transmission dynamics and alter strain prevalence of prions. Prion sources and tissue preparation Prion-infected brain tissues were collected from hamsters infected with either the hyper (HY) or drowsy (DY) strain of transmissible mink encephalopathy (TME) or from a naturally infected elk with CWD agent as described previously [20]. Prion adsorption to soils and soil minerals Soils and soil minerals used in this study included sterile Rindasilty clay loam (SCL) soil (a Ver-ticEpiaqualf); sterile Dickinson sandy loam soil (SLS, a TypicHapludoll); sodium bentonite clay (CETCO, Arlington Heights, IL); silicon dioxide powder (Sigma Aldrich, St. Louis, MO); humic acid (HA)-coated silica gel particles (SiO 2 HA); and gamma-irradiated fine white sand (Fisher Scientific, Pittsburgh, PA). Physicochemical properties of these soils and soil minerals have been described previously [19,38]. To obtain soil bound prions, 10% brain homogenate (BH) was added to soil and for each soil or soil mineral, the incubation time and prion to soil ratios, were selected based on previous studies [19,20] (Table 1). Each BH-soil combination was prepared in triplicate. The BH-soil mixture was rotated at 24 rpm (Mini Labroller, Edison, NJ) at room temperature. Samples were removed after incubation and centrifuged at 100× g for 5min. The supernatant was removed and the pellets were washed a minimum of three times with 1× DPBS. The soil pellets were resuspended in 1× DPBS at concentrations described in Table 1 and were stored at -80°C until use. HY TME, DY TME, and elk CWD BH were used as unbound controls. Drying and wetting treatment Each sample was placed in an uncapped 200 µL PCR tube (Thermo Scientific) and incubated at 40°C. Samples were dried and weighed periodically until there was less than a 0.5% change in weight resulting in a minimum of 7 hr drying time. To perform consistently, around 12 hours' drying is selected for one cycle. Dried samples were rehydrated with 10 µl of ultrafiltered deionized water and mixed thoroughly. The drying followed by rewetting constituted one drying/ wetting cycle. Conductivity and pH were measured with an Oakton 700 bench top meter using a 3-point calibration curve. After the desired number of treatment cycles, samples were stored at -80°C until use. Animal bioassay Intracerebral (i.c.) inoculations of Syrian hamsters (Harlan Sprague-Dawley, Indianapolis, IN) were conducted as described previously [30,60]. Silty clay loam soil HY TME (untreated, and 1-and 10-drying/wetting-cycle treated) and silicon dioxide powder adsorbed HY TME (untreated, and 10-drying/wetting-cycle treated) were selected as inocula. The incubation period was determined as the length of time in days between inoculation and the onset of clinical signs of HY TME. PMCA Protein misfolding cyclic amplification (PMCA) was performed as described previously [61]. Sonication was performed with a Misonix (Farmingdale, NY) 4000 sonicator with amplitude set to level 75, generating an average output of 160 W during sonication treatment. Samples were diluted with 10% (w/v) uninfected hamster or elk brain homogenate at 1:100 for HY TME and CWD, and 1:20 for DY TME for the first round. After one round, homogenate from round 1 was diluted at 1:20 for HY TME, 1:10 for elk CWD, and 1:1 for DY TME for the subsequent rounds. Each round was performed at 37°C for either 24 hr for HY TME and DY TME consisted of 144 cycles of 5 s sonication followed by 9min 55s of incubation or 48 hr for elk CWD consisted of 288 cycles with the same sonication/incubation time as HY TME and DY TME. Before each PMCA round, an aliquot was placed at -80°C as an unsonicated control. Samples containing only 10% (w/v) uninfected brain homogenate were included with each PMCA round as negative controls. Western blot analysis Western blot analysis was performed as described previously [62]. Briefly, samples were incubated and digested with 22.5 µg/ml (HY TME/DY TME) or 45 µg/ml (elk CWD) proteinase K (PK) (Roche Diagnostics Corporation, Indianapolis, IN) at 37°C for 30 min (HY TME/DY TME) or 1 hr (CWD) with constant agitation. The PK digestion was terminated by boiling in 1x SDS-PAGE sample buffer (final concentration). The samples were size fractionated with 12.5% SDS-PAGE and transferred to a polyvinylidenedifluoride membrane (NuPage; Invitrogen, Carlsbad, CA). The membrane was blocked with 5% w/v nonfat dry milk in 1× TTBS (Bio-Rad Laboratories, Hercules, CA) for 30 min. Hamster samples were immunoblotted with MAb 3F4 (Chemicon, Temecula, CA; 1:10,000). Elk/Tg(CerPrP) 1536 samples were immunoblotted with 8H4 (1:10,000). The blots were developed with Supersignal West Femto maximum sensitivity substrate, according to the manufacturer's instructions (Pierce, Rockford, IL), imaged on a 4000R imaging station (Kodak, Rochester, NY), and analyzed using Kodak (New Haven, CT) molecular imaging software, V.5.0.1.27. Densities of sample replicate (n3) intensities were standardized to brain homogenate controls on the same gel to control for inter-gel variance. PMCA amplification is determined as the absolute difference of intensity density between unamplified and sonicator-amplified samples. For each type of soil, unbound prions and untreated samples (Dry 0) were used as controls to determine the effect of drying/wetting (Dry 1 and Dry 10) on PrP Sc seeding efficiency. Intensities of the same control were averaged out through the entire study. Statistical analysis (Student's t test with Welch's correction, two-tailed P value) was performed using Prism 6.0 (GraphPad Software, Inc., San Diego, CA) by separately comparing samples with each treatment to the untreated control or samples with the least treatment. SYPRO Ruby gel staining Separated proteins in the polyacrylamide gel were stained with SUPRO Ruby following the protocol provided by the manufacturer. Briefly, the gel was placed in a clean plastic dish and incubated in a fixative solution containing 10% methanol and 7% acetic acid at room temperature with gentle agitation for two 15 minutes. Then the gel was incubated in undiluted SYPRO Ruby staining solution overnight without being exposed to light. Stained gel was then transferred to a clean plastic dish and washed with 10% methanol and 7% acetic solution for two times followed by one time rinse with MQ H 2 O. The gel was imaged in an electrophoresis gel imaging imager cabinet (Bio-rad Universal Hood ii) using UV epi-illumination and analyzed by a 1-D analysis software Quantity One version 4.6.7. The lightness density of samples of interest were compared and significance was analyzed with build-in t test (two-tailed p value with unequal variance) in Microsoft Excel 2013. Ethics statement All procedures involving animals were approved by Creighton University Institutional Animal Care and Use Committee (protocol number 0 872 and 0881) and comply with the Guide for the Care and Use of Laboratory Animals.

v3-fos

2019-05-28T13:15:37.386Z

2015-09-18T00:00:00.000Z

166377042

s2

Toward resilient food systems through increased agricultural diversity and local sourcing in the Carolinas Biological and agricultural diversity are connected to food security through strengthened resilience to both anthropogenic and natural perturbations. Increased resilience to stress via increased biodiversity has been described in a number of natural systems. Diversity in food production can be considered on the following three levels: (a) genetic diversity as reflected in the range of cultivars which can be selected for production; (b) species diversity, captured through production of a wide range of crops on each farm; and (c) broad ecosystem diversity, described by the diversity of production between farms and within the broader food system. A network of locally based food producers and entrepreneurs provides opportunity for high diversity at each network stage, with increased adaptive capacity and the ability for rapid response to disturbance. We argue that production techniques that use carefully planned diverse plantings, such as biointensive cultivation, increase resilience by increased water use efficiency, yield and nutrient retention while reducing pressure from pests and pathogens. We present a model for a diverse, distributed food system in the North Carolina Piedmont and analyze an existing distributed network by a food hub in South Carolina. Through these models, we argue that a shift in the food network has the potential to increase local food security by having food more reliably available where it is needed and by contributing to local resilience through community economic development. The shift in food production and distribution systems serves multiple goals: When crop loss occurs, other crops still contribute to overall harvest, reducing net loss. Diverse on-farm production can support a more distributed network of food aggregators, processors, and markets than the current approach of large-scale consolidation. Finally, a distributed food supply network supported with diverse agricultural products can increase resilience by providing access to diversified markets for producers and improved food access to consumers with more food choices, while expanding the need for skilled jobs supporting the regionally based food industry. Introduction The term Bresilience^has been used in many social, economic, and ecological contexts with varied meanings and interpretations. In its broadest form, the term resilience refers to the ability to recover from or to adjust to change (Gunderson 2000). Uncertainties in an era of rapid change in agriculture have led to uncertainty in the capacity to meet growing food needs. Concerns about chronic malnutrition, environmental degradation, livelihood security, food safety and hygiene, and equitable access raise important questions about how and where food is grown and eaten. In this paper, resilience is defined as the capacity for a system to absorb disturbance and to reorganize, while retaining system function and self-organizing processes (Gunderson 2000;Folke et al. 2004;Folke et al. 2010). Resilience can be measured by the magnitude of disturbance that can be absorbed before the system is redefined into a different state (Gunderson 2000). In the case of agriculture and food security, resilience can be considered from the perspective of food availability with a return to a predictable and adequate supply following a disturbance. In this case, disturbances may occur in production (as with the loss of a crop following a plant disease or pest outbreak, severe weather, or climate change), transportation, processing, distribution, and access/supply. Locally based and controlled food production systems with high diversity can provide opportunities for adaptive capacity through the ability to rapidly respond to disturbance and to changing conditions in production and market conditions (Hendrickson and Heffernen 2002). The prevailing perception is that food production is predictable with a constancy of relationships. As anyone who has been involved with food production, processing, and distribution has experienced, uncertainty in food production and post-harvest handling is common-and risks are high. Adaptive capacity and adaptive management acknowledge that the system being managed will always change, so humans can respond by adjusting the system quickly (Gunderson 2000). A growing body of evidence highlights the importance of biodiversity for ecosystem functionality (Peterson et al. 1998;Fischer et al. 2006;Kerkhoff and Enquist 2007). Although food and agricultural systems are highly managed, they are still guided by ecological principles through provision of essential natural resources, ecological function, and ecosystem services. Just as with natural systems, biodiversity within food systems can be considered across a range of spatial scales from single farms to larger landscapes. Food system diversity also includes social dimensions such as food distribution and access. Sustaining ecosystem function in agricultural activities is essential for sustaining farm livelihoods and the food supply (Tomimatsu et al. 2013). Functional redundancy within an ecosystem may increase resilience to environmental fluctuations, facilitating successful reorganization of ecological systems. High response diversity may ensure ecosystem functionality, providing a range of ways to respond to environmental change and uncertainty (Folke et al. 2004). Biodiversity in agricultural systems can improve crop protection and soil fertility through these expanded functionalities (Altieri 1999). Below, we discuss two examples of risks from biodiversity loss, the Southern corn leaf blight epidemic of 1970 and porcine epidemic diarrhea, which demonstrate the negative possible outcomes of genetic uniformity in food systems. Building on this, the insurance hypothesis for ecosystem resilience through conservation of functional diversity and responsive scale may be transferable to food systems. In the case of food systems, diversity in farm scale and number, diversity of crops, type of market opportunities, and higher numbers of farms equate to high numbers of species that respond differently to external pressures in ecosystems where diversity provides Binsurance^if one component of the system declines or is lost. To overcome such risks, species with similar traits may be functionally redundant; if a crop is lost because of high disturbance sensitivity (drought or disease, for example), other crops will still provide agricultural product (Mori et al. 2013). Some techniques such as biointensive agriculture which emphasize high biological diversity are most easily adaptable to small acreage. A concern can be about yield loss under such production techniques. However, intensively managed biointensive farms can produce yields which are comparable and often greater than conventional methods, while enhancing ecosystem structure and functions such as biogeochemical cycles and pest population controls. The contemporary food system is built on a complex network of related activities, ranging from on-farm production to harvest and sale to distribution, processing, and marketing, ending with consumer access, purchase, consumption and resource, and waste recovery. Many related factors including environmental, social, and economic disruptions have the potential to contribute both chronic and acute disturbance to all points in this complex system. It is important to consider resilience at all points in the food system, minimizing vulnerability in each individual sector. Therefore, it is crucial to link the shift in food production systems with a shift in the food distribution mechanisms. Indeed, greater crop diversity offers opportunities for more diverse markets, and a locally embedded network of markets for food sale and access may provide greater resilience by insuring multiple points of entry for sale and access if the system is disrupted. As the two models below, North Carolina Central Piedmont Network and the South Carolina Food Hub demonstrate, decentralized models that link producers to consumers provide opportunities for farmers that utilize high-yield, low input techniques such as biointensive and other agroecological techniques a convenient and reasonable access to markets. Since the techniques can be developed both in urban and rural areas on smaller acreage farms, they can reduce the upfront capitalization costs for a start-up farm and also provide access to food related entrepreneurial opportunities and to food for low-income, ethnically and racially diverse consumers. This paper explores the link between diversity and resilience in our food networks-from production to distribution. We first illustrate risks from loss of biological diversity in food production systems and discuss how biointensive techniques can help overcome such risks. We then discuss two models of food value chains in the Carolinas and how a shift in food distribution mechanisms built on principles of biological diversity can help build food security and community resilience. We also discuss the challenges of establishing such models on the ground and thus offer guidelines for practitioners on how to establish decentralized/local/ networked food production, distribution, and access systems. conditions of extreme genetic uniformity for production efficiency. The outcome was the 1970 epidemic of Southern Corn Leaf Blight, causing a crop loss of over $1 billion (priced at 1970 dollars) (American Phytopathological Society 2015). Corn leaf blight is caused by a fungus, an ascomycete, Cochliobolus heterostrophus (also known as Bipolaris maydis, or its name at the time of the epidemic, Helminthosporium maydis). The fungus was common across the southern corn production areas, with annual losses of around 2.3 % under normal production. Cultural practices to reduce leaf wetness and maintain general resistance throughout the corn population were the most affordable and consistently effective means of control. Stubble left following harvest was plowed under to enhance decay, with corn production moved to other fields the following year (Levings III 1990;Schumann 1991). Early in the twentieth century, corn breeders found that greatest yields were produced by progeny from the crossing of two genetically different inbred parental lines (Buckner 2014). Production of seed from defined crosses, however, required carefully controlled pollination and removal of the corn tassels. The detasseling process is very labor intensive and must be done by hand before pollen shedding. A genetic factor was discovered that caused plants to become male sterile, the Texas male-sterile (TMS) cytoplasm (Levings III 1990). This meant that plants used for corn production no longer had to be de-tasseled prior to pollination-a huge economic benefit to the corn industry. By 1970, nearly 85 % of the corn produced in the USA contained TMS cytoplasm. Genetically, two changes occurred simultaneously in the host (the corn) and the pathogen populations. The corn crop now had the new TMS gene throughout the population, and a genetic change occurred in the population of the fungal pathogen. A new race, named Race T, evolved which had genes to produce a toxin that only affected the TMS plants. The fungus could now complete its life cycle more quickly and was able to infect not only the leaves and husks but also the developing ears. For disease to occur, three conditions must be met-a susceptible host, a virulent pathogen contacting the host, and an appropriate environment. The genetic uniformity of the corn with the TMS genes introduced the susceptible host. The genetic change in the pathogen, evolved in response to changes in the host genetics, greatly increased the virulence. The weather during the summer of 1970 was warm and wet, ideal conditions for disease to develop. The infection started in early summer in the Southeastern United States. By early fall, corn across the entire east coast and westward past the Mississippi River had become infected, with 80-100 % crop loss in many areas. The result was near complete loss of the corn crop that year, with a huge economic impact. Fortunately, producers returned to still available non-TMS lines of corn, again requiring manual detasseling. With the return to production of corn varieties lacking the TMS genes, epidemics of this disease have not returned. However, as current corn production becomes less diverse, similar risks may again become important. Porcine epidemic diarrhea Most pork producers raise hogs for specific markets, resulting in limited genetic variability. Breeds and hybrid lines are often selected for uniformity and product predictability, such as size, feed conversion, time to market, and/or meeting the requirements of the integrator or marketing company (Martinez and Zering 2004). The disease porcine epidemic diarrhea has had widespread and damaging impacts throughout the pork industry. The disease was first confirmed in the USA in April 2013. It is caused by a highly transmissible coronavirus genetically related to strains found in China (Stevenson et al. 2013). This disease can be contracted by pigs of all ages; 100 % mortality often occurs with suckling pigs. The incubation period is only 12-24 h. Symptoms include vomiting and severe diarrhea, frequently followed by dehydration and death. There are no effective vaccines or pharmaceutical therapies for the disease at this time. The disease is highly contagious and environmentally stable. A tiny amount of material taken from intestines of an infected animal can be highly diluted (diluted 10 -8 ) and still remain infective. Air-borne transmission is under investigation. Trucks and trailers used for dead haul, transport of animals to processing plants, feed deliveries, trash removal and other activities on infected farms have likely been associated with disease spread (National Pork Board 2014). The toll on the swine industry has been high. Of the 63 million hogs in the USA, about 7 million have been lost to this disease (US Department of Agriculture 2014). Some individual swine farms have lost as many as 30,000 pigs. In response to the rapid disease spread, quarantine measures were put into place in fall 2013. Farm visits were not allowed, and movement of people and materials between farms and processors was restricted and monitored. Biosecurity measures, isolating one farm from another and preventing movement of infectious material between farms, have been the only effective method for control (National Pork Board 2014). Separation of farms across North Carolina and across the country is comparable to high landscape level diversity with species separation in natural systems, similar to the space between farms and sanitation providing a physical barrier to pathogen movement. Most hogs are grown by independent farmers, who are producing on contract for sale of the animals to a larger integrator (animal processor). For example, one such integrator has contracts with over 2000 independent farmers for swine production across the USA. The integrators are requiring quarantine between farms as part of the contractual agreement. Many of these independent farmers are small to mid-sized concentrated animal feeding operations (US Environmental Protection Agency 2015). When compared to production on only a few very large swine farms (>100,000 animals), this distributed national network of independent, smaller farms provides opportunities for implementation of the needed biosecurity measures and physical separation. Farms can be widely spaced across the landscape, reducing potential for aerial transmission, and strict sanitation measures have been effectively implemented with farm isolation. In addition, risk is distributed across the 2000 farmer network, rather than being concentrated in only a few centralized production farms. This suggests that resilience in the hog industry may be dependent on small farms producing animals independently of each other, rather than depending on large, centralized Concentrated Animal Feeding Operations. It also argues that genetic diversity between growers and by each independent grower should be increased to add potential disease resistance and resilience within the industry. Biological and Agricultural diversity in farm production In agricultural systems focused on local market channels, resilience can be enhanced by a diversification of on-farm products as well as by distribution of more smaller farms across the landscape, as suggested by the above two examples (Thrupp 2000). On farms, production risk is distributed across the products being grown for harvest and sale. Each product represents an ecologically functional group, with varied ranges of tolerance for conditions within the production environment. For example, some crop plants may have a wider or shifted range of tolerance for temperatures or available moisture. Based on observations from natural systems, biological and agricultural diversity can be considered from several perspectives: & Diversity of markets based on diverse crops and production Some crop production techniques mimic and enhance ecological processes and function within agricultural settings. Biointensive production is an example of one approach which is focused on application of principles of agroecology (Grow Biointensive 2014). Biointensive production emphasizes high crop spatial and temporal diversity via crop selection and multi-cropping practices. Soil is highly managed for fertility and biological activity through an initial deep tillage and frequent carbon and nitrogen additions through green manures and compost. These techniques are often most suitable for small-scale farming and are able to provide product to the local food supply network for sale and distribution. There is also efficient land use and minimal reliance on mechanized equipment. These options are severely limited in conventional agriculture which is dependent on high acreage, uniform mechanized production (Jeavons 2006). A number of studies in natural systems have shown a correlation between high biodiversity and pressures from pests and pathogens (Janzen 1970;Connell 1971;Wright 2002;Peterman et al. 2008;Terborgh 2012;Bagchi et al. 2014). In some ecosystems, there is a feedback between pest and pathogen populations and biological diversity, with pests/ pathogen pressures reduced as individuals of each species become more widely separated with increasing biological diversity, reducing the potential for disease and insect spread. Production methods which can enhance agricultural diversity such as organic production have been compared to conventional production in a number of studies. Two large-scale meta-analysis studies have suggested that high acreage crop yield with organic production is generally lower when conventional methods are used. However, the yield differences between the two methods varied widely and were highly contextual, differing between crop groups, regions, site characteristics, and cropping techniques (di Ponti et al. 2012;Seufert et al. 2012). In addition, organic production can be done in large acreage monoculture, minimizing biodiversity (Mission 2014). In contrast, some techniques that are most easily adaptable to small acreage, intensively managed farms can produce yields which are comparable and often greater than conventional methods, while enhancing ecosystem structure and functions such as biogeochemical cycles and pest population controls. Biointensive techniques with high density diverse plantings have shown a 25-400 % increase in production with improved water use efficiency, as for tomato, basil, and brussel sprouts (Jeavons 2001;Jeavons 2006;Bomford 2009;Grow Biointensive 2014). In another study evaluating yield of nine onion cultivars, 45 kg/10 m 2 were produced with conventional practice compared to 160 kg/10 m 2 with biointensive techniques (Moore 2010). In addition, energy efficiency was improved from an energy efficiency ratio of 0.9:1 with conventional tillage compared to 51:1 with biointensive production-meaning 51.5 cal of food were produced for 1 cal used for production. With biointensive production, most of the energy input was from direct human input (renewable) rather than from fossil fuels (Moore 2010). High biological diversity is a characteristic of biointensive production, with at least 13 different types of plants grown together, including food for local consumption, food for sale, and compost crops. One farm in California, Woodleaf Farm, grows over 200 different crops on 3 ha of land with 7 ha in woodland and meadow, using similar cultivation techniques. This farm notes anecdotal evidence that populations of beneficial insects have increased while pests and pathogens are minimized (Woodleaf 2014). Enhanced productivity, water use efficiency, creation of unique microclimates, and pest/pathogen management are also attributed to high crop diversity (Woodleaf 2014). Soil management in biointensive systems is directed toward enhancing ecosystem processes (e.g., nitrogen fixation, mineral immobilization) which are associated with soil fertility (Perrings et al. 2006;D'Haene 2012). Common practices include double-digging soils (loosening two layers of soil) to a depth of 0.7 m with annual amendments of cured compost and green manures. Communities of bacteria acting synergistically can suppress plant pathogens, promoting plant growth and crop production (Kinkel et al. 2011;Mendes 2011;Hadar and Papadopoulou 2012;Gaiero 2013) and regulating carbon flux (Fitter et al. 2005). Biologically active soil amended with organic materials will also provide ecosystem services by retaining water in soil, regulating biogeochemical processes, increasing cation exchange capacity to enhance nutrient retention and plant availability and reducing material loss from the production beds (Cooperband 2002). Environmental benefits with biointensive and similar types of agricultural production include (Jeavons 2001 From 2007 to 2012, average farm size in the USA increased from 418 to 434 acres, while the number of farms declined by about 100,000. Significantly, the number of small farms, those most suitable for biointensive and similar types of production (1-9 acres) declined nationally from 232,849 to 223,634. The number of large farms (≥2000 acres) increased from 80,393 to 82,207 during the same period of time (US Department of Agriculture 2014). These trends suggest that agricultural production in the USA has the potential for decreased resilience, as farming becomes more consolidated into large acreage operations growing a small number of crops on each farm. Biointensive and similar practices have the potential to greatly enhance the adaptive capacity of the food production network, increasing crop/product diversity, while shortening the supply chain from production to consumption. They also have the potential to increase productivity per unit area, while protecting and enhancing ecosystem health. Resilience in economic and social systems through local food networks A proposed model for the North Carolina Central Piedmont Through more diversified agricultural production, as described above, greater resilience can also be attained within locally based food systems and associated economies. A distributed and regional network of small-scale aggregators, processors, distributors, and markets has the capacity to make venues for sale of farm products more accessible to small farmers, especially as they diversify their crops. Greater crop diversity offers opportunities for more diverse markets, especially in specialty and ethnic foods. A distributed network can foster regional job growth within the food system and enhance food access to underserved communities. A distributed network of producers, buyers, processors, and consumers that share adjacency also have more opportunity for sharing information and knowledge such as technical advice, information about buyers and markets, new regulations, market opportunities, and new innovative practices. The distributed network can also support current models of centralized markets by consolidating products from small growers into larger quantities for sale/purchase. Currently, large aggregators leave little room for small producers who cannot meet minimum quantities required for sale. They also usually require a substantial supply of single, uniform crops, creating a disincentive for on-farm crop diversification. In contrast, small producers have found much of their success by selling directly to consumers through farmers markets and Community Supported Agricultural (CSA) models (Low and Vogel 2011). Among US farmers, in 2012, nearly 8 % of farms sold primarily through direct-to-consumer (DTC) markets, including farmer markets and CSAs while another 30 % used DTC marketing in addition to other venues. The number of American farms with DTC sales increased by 17 % and sales increased by 32 % between 2002 and 2007; however, between 2007 and 2012, the number of farms with DTC sales increased 5.5 %, with no change in total DTC sales. That DTC sales did not increase may be a reflection that consumer demand has been met (Low and Vogel 2011). Effectiveness of these direct market outlets for small farmers can be place-based and the cost of marketing can be high. Additionally, most farmers benefit by having a combination of sale approaches and types of crops to market. As in other industries, market diversification in the produce business is a means to minimize risks-a diversity of market connections supports a financially sustainable farm enterprise (Izumi et al. 2010;Vogt and Kaiser 2008; US Department of Agriculture 2010). Additionally, the food system benefits by having a diversity of local and global channels for the distribution, storage, and value-added processing of food through an increased base of skilled labor. In the event of a disruption (natural disaster, economic collapse, contamination of a supply chain), there are multiple alternative channels that can supply a population's food needs, including supplies closer to communities needing food. Disaster notwithstanding, the resilience of the local economy may be increased because of the geographically-linked, skilled jobs that are created: farming, logistics, marketing, storage and distribution, value-added processing, equipment maintenance, input supplier, and many other jobs that cannot be outsourced to non-local entities. In the current national food system, where food is grown and harvested several states away and aggregated by large, centralized corporations, these jobs and skills are not available locally. An increase in non-transferable, geographically dependent, skilled jobs increases the resilience of the local economy immediately. Opportunities/current challenges in the North Carolina Piedmont According to the the North Carolina Commission on Workforce Development, State of the North Carolina Workforce report (2007), rural areas of North Carolina continue to lose employment opportunities, and Bmiddle jobs^that supplied a family-sustaining wage for workers with little formal education are disappearing rapidly. The median family income in North Carolina is $46,450, substantially below the national median of $53,004, with 17 % of the population living in poverty. Median incomes in rural counties are often 10 % or more below the state median (U.S. Census Bureau 2014). Central North Carolina is also challenged by many communities having limited access to food. North Carolina is ranked 10th in the nation for degree of food hardships, and Greensboro, North Carolina in the North Carolina Piedmont, has been identified as the most food-limited metropolitan area in the nation (Food Research and Action Center 2015). For many small farmers in the Piedmont region of North Carolina, farm income is a supplemental, yet crucial, part of their household economy. In 2012, about half of the people identified as farmers (50,218 individuals) in North Carolina by the US Department of Agriculture did not farm full time. Of those, 19,563 worked more than 200 days off farm (US Department of Agriculture 2014). For many of these families, small farm operations provide much needed household income to supplement off-farm, low-wage job earnings (US Department of Agriculture 2014). Small farming operations also present economic opportunities for partially-or fullyretired growers, as well as opportunities for beginning producers interested in scaling up to full-time farm businesses. When farmers utilize high-yield, low input techniques such as biointensive and other agroecological methods, farming businesses can be developed in both urban and rural areas, as smaller acreage farms (typically 0.5-5 acres) are proving their viability. Reduced land and equipment requirements also reduce the upfront capitalization costs for a start-up farm. One of the barriers that small farmers experience when developing a farm enterprise is identifying convenient and reasonable access to markets for the sale of their products. Generally, individual small growers do not have the physical infrastructure (e.g., cold storage), product volume or market access to sell to institutions or buyers that can pay a fair wholesale price and provide a consistent market for their products. There is room for growth and diversification in the local food sector. According to the US Department of Agriculture, in 2004 North Carolinians spend $35 billion per year on food (US Department of Agriculture 2004, Center for Environmental Farming Systems 2013, 2014a, b). Although spending on local foods is difficult to track, CEFS is encouraging North Carolina consumers to spend at least 10 % of their food dollars on locally produced food. The North Carolina 10 % campaign is in the third year of its promotion to bring local foods spending closer to this benchmark. At 10 %, $3.5 billion would be generated for the local economy, increasing opportunities for entrepreneurial jobs, skilled employment, and healthy foods. At the same time, there is a renewed interest in local foods to foster healthier North Carolinians and protect and preserve the rural landscape (Curtis et al. 2010). There is a tremendous untapped opportunity for selling locally grown produce to indirect markets such as stores, restaurants, institutions, and distributors, particularly for small growers that cannot market their produce directly to consumers. Developing a marketing network geared toward these small and micro-farmers simultaneously builds the resilience of local economies through job creation, the resilience of the food system through a diversified, decentralized network of small suppliers, and resilience in production through cultivation of diversified crops. Many of the jobs that have been created since the end of the recession have been low-wage service jobs. Rural areas of North Carolina continue to lose jobs, while metropolitan areas slowly begin adding new jobs (Forter et al. 2013). This slow economic recovery is a reflection of a lack of resilience in the local economy-largely in response to changes in the manufacturing foundation of the region. Fostering locally embedded sustainable, high-yield/low-input agriculture and markets for product sale has the potential to replace the former manufacturing economic foundation. Potential for distribution, sale and access to locally produced foods in the North Carolina Piedmont region To assist local governments in fostering sustainable neighborhoods through food, a model was designed to describe a decentralized, produce aggregation network serving smalland micro-producers within the North Carolina Piedmont (Piedmont Together 2013; Walker 2014). Questions of economic and food system resilience were addressed by asking: What opportunities exist to build the supply and demand for local produce while engaging and involving rural and urban, low-resource, diverse communities in ways that generate individual and community wealth and security? More specifically, what sectors are not currently engaged in local food system efforts that hold potential for growing their businesses while contributing to a more robust local food system? (Walker 2014). The economy of the Piedmont region of North Carolina during the latter half of the 20th century-dependent largely on low-skilled manufacturing jobs in textiles, furniture, and tobacco in urban areas and tobacco production in rural areasis a prime example of low resilience, as attested by the lingering effects of the recent economic downturn. Similarly, while the prevalence and growth of farmers markets across the Piedmont is certainly a welcomed addition, a food system dependent on a small number of relatively high-priced, geographically distant markets with limited hours lacks the resilience possible in a food system that includes markets characterized by a high diversity of product, price, location, and methods of sale. The goal of the model designed to address the participation gaps in the local food system described above was to enhance resilient economic development through creative and sustainable diversification of employment opportunities and market types within the local foodshed through opportunities for aggregation, storage, and sale of local food products, exploiting the growing diversity of locally grown agricultural products. Figure 1 shows the model developed. Dashed boxes, focusing on micro-, small-, and medium-scale aggregators, are assets that could provide opportunities for sale and distribution of local foods from small-and micro-scale producers that currently lack many opportunities for indirect sales of their products. These small-scale aggregators would fill a niche that links a diverse set of producers (part-and full-time, rural and urban) and diverse agricultural products to retail outlets that cater to populations that currently lack access to local food, such as small rural grocers, urban convenience stores, locally owned mid-priced restaurants, and city-and county-managed institutions. Diversifying crops also provides opportunities for consumer access to more diverse produce and other foods, such as culturally important or desired foods. This model demonstrates one method by which resilience is increased through supplementing and integrating with the existing, larger-scale food system, not creating a parallel, stand-alone Blocal^system. Resilience, defined earlier as the capacity for a system to absorb disturbance and to reorganize, while retaining system function, structure, and self-organizing processes, can best be achieved within the food system by more food produced, distributed, and consumed within local geographies, while keeping the larger-even national-structures that fill gaps which cannot be filled locally or where those structures inform and support local efforts. Another important concept model is illustrated in Fig. 2, where the inner ring of the diagram shows a direct-toconsumer relationship within a typical local food system. While this smaller model is more highly favored by local food system activists who place a priority on Bknowing one's farmer,^it can exclude opportunities for generating community wealth, resilience, and new jobs through the multiplier effect: increasing the number of times a dollar cycles through a community increases the economic impact of that dollar on the local economy (Morgan 2006). A study by the Iowa State University concluded that buying local food has a multiplier effect of 1.4-2.6 throughout the wider local economy, depending on the rural or urban context and commodities and scale of the community economy (Swenson 2007). Additionally, current research suggests that adding one skilled job in the tradable sector generates 2.5 jobs in local goods and services, with high potential for the non-tradable sector (Moretti 2010). The larger circle in Fig. 2 illustrates the inclusion of these Bvalue-added^industries, including aggregators, institutions and wide variety of retail outlets. The larger circle also illustrates multiple points of entry for community members into a food production/aggregation/processing/distribution network. Programs such as farm incubators can provide training and technical assistance in sustainable production techniques (e.g., biointensive, agroecology), access to on-farm resources and infrastructure, initial entry into post-harvest processing/marketing, and land access opportunities to facilitate the start of new businesses. Additionally, underutilized facilities such as convenience stores, restaurants, grocery stores, and local institutions can provide the capacity for storage, post-harvest processing, and/or distribution that can serve as points of entry for people interested in starting non-farm food system enterprises. Retail outlets such as restaurants, small grocers, institutions, and innovative outlets, for example mobile or pop-up markets, can widen the distribution of the local food network increasing (Walker 2014) access for community members who would not normally be a part of a regional foodshed based only around direct farm-toconsumer marketing. Currently, there are a number of projects, programs, and initiatives geared toward assisting mid-scale farmers who wish to enter mainstream markets. For example, started in 1997, the North Carolina Farm to School Program now facilitates sale of locally grown food to 87 of the 100 counties in North Carolina. During the 2013-2014 school year, $1,350, 263 was spent purchasing fruits and vegetables grown in North Carolina, which were then served in schools (North Carolina Department of Agriculture and Consumer Services 2014). Both approaches are necessary to build a resilient local food system that can absorb disturbances such as natural disasters and disruptions to the national economy and transportation network. The models illustrated in Figs. 1 and 2 highlight the opportunities for new, small-scale, locally-based markets that can be the portal for sale of products by locallyconnected producers, thus increasing the resilience of the food system through dynamic, adaptive, and responsive enterprises. Challenges To achieve this increase in locally sourced food through decentralized, networked distribution channels, three main issues must be addressed. 1) Many of the resources for the promotion of local foods development over the past decade have gone to supporting mid-and larger-scale farms within North Carolina. Large foundations and universities have focused on helping farmers who were affected by the tobacco buyout program transition to other viable crops, as well as the renewed public health focus on obesity, diabetes, and stroke prevention. This led to much public support for getting fresh, local foods into a variety of markets and food access programs. Working with larger farmers and distributors has made a difference in the prevalence of local foods available to consumers. However, smallscale farmers are often not able to sell to these markets. For example, during the 2012-2013 school year, the North Carolina Farm to School Program purchased strawberries from only 7 farms and sweet potatoes from 11 farms. For comparison, there are 4155 North Carolina farms of 1-9 acres identified in the US Department of Agriculture Agricultural Census, who are also potential suppliers for this program (North Carolina Department of Agriculture and Consumer Services 2014; US Department of Agriculture 2014). In order to engender greater resilience of the local food system, more attention needs to be paid to the contribution potential for smalland micro-scale farms. 2) Nationally, the local food movement has often been hegemonic in its lack of inclusion of minority participantsbe they underrepresented racial, ethnic, class, or genders (Hinrichs 2002;Jarosz 2008). Greater attention paid to the barriers for participation by individuals, businesses, and communities that are not currently part of the local food movement will help in creating greater equity of food access, economic opportunity, and environmental health (Holley 2012). It will also expand the range of products available for production, processing, and purchase, increasing both social and economic resilience within the local network. In the same way that a diversity of scale and market type will strengthen the ecosystem of the local food system in the Piedmont region, greater involvement from a diversity of stakeholders throughout the system will engender more dynamism and resilience (Page 2007;Holley 2012). Expanding and diversifying the community of growers and non-farm entrepreneurs will also expand markets and market access to parts of the community now underserved by a centralized food marketing network. 3) For many small farmers, resources and equipment that would facilitate sale to a network are not available. For example, the cost of an 8 × 10′ cold storage unit is typically $4000-$10,000. For many farmers who have substantial capital, off-farm income, or access to credit, this is attainable. However, need for capital is a barrier for most low-resource farmers. Guidelines for a decentralized storage and distribution network The local food models outlined above for central North Carolina's small and mid-sized farmers and aggregators can be described in a set of five Bdesign guidelines^that increase economic, social, and agricultural resilience. These guidelines are intended as a beginning resource for people interested in starting or adding to some aspect of the decentralized aggregation and cold storage local food network. They are suggestions that can be utilized by decision makers, designers, and planners that may be working on other community-wide issues such as transportation, housing, and economic development. Having design guidelines that summarize a model of diversification and resilience based on food system localization allows for the inclusion of ideas generated through academic research into practical, applied community practice. Guideline #1: Promote networks and nodes Intent: Develop a regional food system that utilizes complexity (a system of networks and nodes) as a mechanism for achieving greater economic and food system resiliency. A. Look for opportunities to situate aggregation and storage facilities throughout the region and close to production farms, while encouraging strong interconnectedness throughout. B. Cluster small aggregation and storage facilities near one another, forming Bnodes^of the local food supply chain. Fifty miles has been defined as a maximum distance between aggregation facilities in rural North Carolina (Bruno and Hossfeld 2009). Facilities may be closer in the more urban Piedmont. Guideline #2: Engender equity and inclusivity Intent: Build on the strengths of the Piedmont Triad region by integrating cross-barrier collaborations into the aggregation or cold storage business initiative. A. Understand the political geography of your community. While doing business in a new area may present challenges, new business opportunities, collaborators, and markets also await. B. Build new lines of community rapport across racial, economic, class, and language boundaries. C. Work to promote access to financing and entrepreneurial assistance for diverse communities that are specific to current trends in food system development. Guideline #3: Plan for appropriate transportation options Intent: Ensure that the cold storage chain of all produce is maintained from farm to fork and that handling is appropriately documented. Each segment of the supply chain should work in tandem with other segments. A. Ensure that there is access to a refrigerated truck or trailer to maintain the cold chain. B. Know the acceptable upper and lower temperature and humidity limits of the produce being aggregated. C. Manage traceability of products. Keep all appropriate records regarding the place of origin, place of sale, and storage notes for all produce that moves through the aggregation facility. Guideline #4: Design an effective management plan Intent: Design a management structure and plan that addresses the goals and limitations of your aggregation or cold storage business, while adhering to all regulatory requirements. A. Choose a management structure that fits with your business. A simple cold storage unit leased to several farmers who are marketing their own produce to aggregators will still require facility management, rent collection, and marketing to new farmers. B. Ensure that your facility is compliant with all regulatory requirements, including appropriate recordkeeping. Track and document temperature and humidity through the duration of the entire supply chain. C. Develop procedures that allow access to storage space by clients when needed and ensure that everyone who enters keeps appropriate logs for food safety requirements. Guideline #5: Build appropriately-sized cold storage facilities Intent: Invest in infrastructure wisely. Cold storage units can be re-configured, and their temperature and humidity changed with relative ease if planned for up front. However, there is an economy of scale, even when working exclusively with small-scale farmers and buyers. Develop a business plan that helps to determine what and when your enterprise should build. A. Estimate how much cold storage space you need based on the amount of produce your suppliers will need to store. B. Approximate energy costs ahead of time and investigate alternative energy as a way to lower the utility bill. C. Know what the optimal temperature and humidity settings are for each produce type you will store and aggregate. D. Determine what storage system you will use within the cold storage units based on the farmers and buyers you are working with. Some smaller growers who are storing their own produce to sell at a local farmers' market may prefer industrial shelves where they can store produce boxes, while larger growers may prefer palettes and use palette jacks to move their product around. Also, there is a need to consider the storage of certified organic and GAP-certified produce, which need to remain segregated from non-certified foods in order to maintain their certification. Food production and access through a South Carolina food hub The distributed network of aggregation and distribution centers proposed for the Piedmont of North Carolina provides an integrated, regional model, reducing transport needs for farmers and expanding distribution, sale, and access. A South Carolina food hub has been successful in making local foods more available, providing an aggregation/distribution/service network to benefit farmers and consumers. GrowFood Carolina is presented below as a case study of a food hub that is helping the Charleston food system move toward a fledgling level of resilience. GrowFood Carolina provides a model for one of the nodes in Fig. 2-an aggregation/distribution hub that connects small-scale growers to consumers in the same region. It also has the potential to serve as a central hub networked to smaller aggregation and value-added processing centers throughout the region, like the spokes of a wheel. Charleston, South Carolina is the center of one of the Southeast's real estate development booms, aided by an influx of retirees moving to the area due to its climate and the availability of still-relatively affordable land. Charleston County has grown from 350,000 in 2010 to almost 373,000 in 2013, a 6.5 % increase (US Census 2014). A corollary to the above growth and international recognition for Charleston's reputation is the recent development of a vibrant, local food culture. Green Grocer Farms on Wadmalaw Island, located outside of Charleston is representative of this growing local food culture. In the 1990s, the farm grew a variety of row crops under regional organic certification, which were sold in Savannah and Atlanta. Now, 15 years later, pastured eggs and pastured raw milk are produced, and the farm is unable to keep up with local demand for their products. The growth in demand for local foods is also seen at the Charleston Farmers Market. Ten years ago there were approximately five farmers; now there are over twenty-six farms selling a variety of local and regional products, including meat, eggs, milk and cheese, vegetables, flowers, shrimp, rice and fruit. One other area of growth is in the local restaurant industry, where almost every local restaurant advertises that they support local farmers and purchase local food, with one nationally recognized restaurant, Husk, advertising a strictly regional menu. GrowFood Carolina-a regional food hub GrowFood Carolina is a working example of how a food hub contributes to resilience in the Lowcountry of South Carolina. The US Department of Agriculture defines a food hub as Ba centrally located facility with a business management structure facilitating the aggregation, storage, processing, distribution, and/or marketing of locally/regionally produced food products^(US Department of Agriculture 2010). Food hubs have operational services which can range from aggregation to distribution and sale, including branding, promotion, packaging, and product storage. They can also assist with producer services, ranging from transportation, training, business guidance, value-added product development, and providing liability insurance, to an often key service, linking buyers with producers. Food hubs can also help with the promotion of community/social missions, such as distribution of products to food deserts, raising awareness about benefits of supporting local agricultural businesses, providing opportunities to community youth, offering composting and other forms of regionally-appropriate farming/agricultural workshops. GrowFood Carolina is a subsidiary of South Carolina's leading environmental advocacy group, Coastal League (CCL), started in 1989 with the mission to Bprotect the natural environment of the South Carolina coastal plain and to enhance the quality of life in our communities by working with individuals, businesses, and government to ensure balanced solutions( Coastal Conservation League 2014). To realize this mission, in 2009, CCL started two Bin-house^policy and advocacy groups to help protect the coastal corridor of South Carolina. One group focuses on climate change and energy, while the other group focuses on sustainable agriculture. The CCL Sustainable Agriculture committee helps CCL's overall strategy of land conservation: if farming is not economically viable, then small and mid-sized farmers will be squeezed out of the market with subsequent changes in land use. Thus, CCL actively supports local, sustainable agriculture. To support their mission, GrowFood was created to provide infrastructural help to regional small-and mid-sized farmers. In 2010, a 6500-ft 2 warehouse in downtown Charleston was donated to CCL. After renovating it and hiring a general manager, GrowFood officially opened for business in 2011. The warehouse contains 1400ft 2 of refrigerated space. Although not climate controlled, the building meets Leadership in Energy and Environmental Design (LEED) standards. GrowFood also owns a 15-ft refrigerated truck purchased with funds from a US Department of Agriculture grant and has an urban garden demonstration plot on site where they host various workshops and school groups. The warehouse is located directly off interstate I-26 leading inland to Columbia, SC, and is close to Hwy 17, a North/South artery. This location is strategic, allowing GrowFood to meet its mission as a food hub, consolidating production and distribution within a 120-mile radius. This radius allows GrowFood to work with farmers growing produce from Georgia to North Carolina, and CCL has plans to open a second hub in Greenville, South Carolina that will service the upstate, mountain region of South Carolina. Farms within the 120-mile radius contact the GrowFood General Manager, who visits the farm for Bfull transparency. restaurants and retail stores, and GrowFood either delivers the produce, or retail customers come to purchase it from the warehouse. After a sale, 80 % of the purchase price returns to the grower, and 20 % remains with GrowFood to help cover operating costs. GrowFood does all sales, marketing, and distribution via a two-person marketing team, and they work with over 300 local businesses, distributing 5 days per week. This requires five fulltime staff and two part-time drivers. In 2011, they had five growers and 15 customers, and as of 2013, they have 35 growers with 85 regular restaurant and 10 retail customers. Currently, demand far outstrips supply, demonstrating demand for additional local foods and food hub services in the South Carolina foodshed. With 2135 farms from 1 to 9 acres in size in South Carolina, clearly, there is additional production and distribution capacity (US Department of Agriculure 2014). The goal of the CCL staff is to expand both GrowFood as well as the local farming community, helping them to diversify, increase production and transition to organic production. Therefore, besides retail distribution, GrowFood is an advocate for small-scale, sustainable farming. South Carolina currently has 15 certified organic growers, compared to over 700 in North Carolina. To expand the number of SC organic growers, CCL does outreach and education about the benefits of sustainable agriculture, focusing especially on the next generation of farmers. The average age of farmers in South Carolina is 63. There are currently not enough young farmers to supply current demand, let alone create new farms that can help meet growing regional food needs. But this also presents an entrepreneurial opportunity for increasing the resilience of the regional food system through creation of new, small, sustainable farms if land, training, and resources for start-up business are made available. Dirt Works Incubator Farm on Johns Island outside of Charleston is working to support development of new farmers starting their new farm businesses in the area. GrowFood as a food hub is helping the community in three important areas. & BThe environment.^Use of sustainable production techniques that improves the soil with each cropping cycle and use of the food near the production farms, reducing transportation impacts & BDiversify local agriculture.^Smaller farms have the adaptive capacity for increased diversity of crops in production both on-farm and between farms, also increasing nutritional and preference opportunities for consumers. & BRural economic development.^As the manager of GrowFood stated, BCash flow is the most important thing for a grower.^This cash flow allows farmers to be economically solvent, allowing them to continue farming. Median income in South Carolina is $44,623, well below the national average, with 18 % living in poverty. Despite the ready support of local restaurants, it is recognized the client base needs to expand, so further goals are to sell to local schools, hospitals, and retail supermarkets, and to advocate for a year-round farmers' market. Lastly, their larger goal is to have 80 % of Charleston's food come from within the 120-mile radius of the food hub by 2030. If successful, Charleston and the Lowcountry will demonstrate a powerful move toward food resilience as a disturbance response. Thus, CCL's goal is to develop an aggregated food hub network in the state, as is proposed for the North Carolina Piedmont in the previous section, while working on education and training, advocating for policy shifts at the state level, and creating food network market resilience. Food hubs can potentially add redundancy and resilience at regional scales and across industries (production, distribution, consumption) and thus act as catalysts fostering reorganization of regional food networks. As anyone who has farmed knows, if a farm cannot make money, then there will be no farm. Food hubs can become key nodes in food networks, and because they interact with farmers and retailers on a daily basis, they can serve as a platform to quickly disseminate information, creating synergies between and among the diverse members of a food system. Rapid information transfer throughout the food network strengthens adaptive capacity for growers, processors, and distributors, allowing rapid response to changes in market and/or environmental conditions. However, a note of caution: food hubs may not be appropriate in all places, and local production may not fill all food needs. Even GrowFood is years away, at best, from helping create a critical mass of local farms that can provide even a small percentage of the Lowcountry's daily food needs. The food hub model in South Carolina and the proposed model for North Carolina may also be appropriate for other regions, extending the capacity of the regional food networks with strengthened resilience. Conclusion It is an open question as to whether our global food system contains the capacity to respond to a variety of disturbances, which may threaten the integrity of system function and structure and the ability to provide needed food to all people. By definition, if such structure and function were to collapse and be unable to reorganize, such a food system lacks resilience, with potentially severe human consequences. However, adaptive capacity and adaptive management acknowledge that a system being managed will always change, so humans can respond by adjusting the system in response to disturbance and changing conditions (Gunderson 2000(Gunderson , 2001(Gunderson , 2002. In the case of agriculture and food security, resilience can be considered from the perspective of food availability with a return to a predictable, dependable, safe supply following disturbance. This issue of the Journal of Environmental Studies and Science grapples with diverse perspectives of this challenge (Marten and Atalan-Helicke 2015). While appearing to make agricultural production more consistent and predictable, genetic uniformity also has the potential to increase risk from pests, pathogens, and other disturbances, as highlighted earlier in this paper. In contrast, high biological diversity has the capacity to decrease risk with genetic diversity within a single crop and through distribution of risk between a variety of products being grown for harvest and sale. These insights should help us think more clearly about what are long-term goals for a truly resilient food system. Small-scale, locally-managed farm operations using agroecological techniques such as biointensive agriculture have the adaptive capacity for increasing biological and ecological diversity both on and between farms, increasing resilience through the ability to rapidly respond to changing environmental and market conditions. Highly managed, low acreage production systems such as the biointensive technique have shown greater yields per unit area than conventional production systems reliant on larger acreage. These highly managed systems also result in more efficient water use, reduced pest/pathogen pressures, and protection of adjacent environments. Interspersing small farms with large farms and returning some lands to a non-farmed condition to provide localized ecosystem services has the potential to engender social, economic, and environmental resilience through regionally-enhanced, responsive, and more highly-integrated food networks. Development of small, diverse farming operations can help communities respond to chronic economic stress by providing supplemental income for some growers and opportunities to build larger, full-time farm businesses for others. High-yield, low acreage techniques can also make farming accessible to a diverse community of people who had not been involved with agriculture, such as urban residents, minorities, and women. However, one barrier to small farmers is often inconvenient access to markets for sale of their products. To have a resilient food system, you have to make it economically viable to farm. Therefore, it is crucial to link the shift in food production systems with the shift in food distribution networks. There is a growing emphasis for the need for new, locally based markets that can be the portal for sale of products by small-scale, locally connected producers. Indeed, several regional food hubs and networks have emerged in the USA that aim to close the gap between producers and consumers. Regional food network and hub models can increase opportunities for direct consumer purchase, or they can serve as aggregation centers for food to be passed into a centralized distribution network, connecting with large volume sellers such as grocery stores, processing facilities, and wholesale distributors. A food system that is predicated upon supporting and cultivating farmers of all scales, who utilize various sustainable production methods for a diversity of crops, and have a variety of market outlets will inevitably be stronger, more resilient, and more adaptable to change than a system that only responds to one scale or type of farmer or one type of market. Using biological and agricultural diversity to expand locally based, sustainable farming systems, foster new farmers and food entrepreneurs, and build distributed aggregation, processing and marketing networks that focus on triple bottom line benefits-environmental, social, and economic-have the potential to strengthen our food security and our communities, providing resilience to both acute and long-term stress.

v3-fos

2019-04-11T13:15:12.031Z

2015-06-22T00:00:00.000Z

106716502

s2

Development of Ozone Technology Rice Storage Systems (OTRISS) for Quality Improvement of Rice Production This research has been carried out by using ozone to address the rapidly declining quality of rice in storage. In the first year, research has focused on the rice storage with ozone technology for small capacity (e.g., household) and the medium capacity (e.g., dormitories, hospitals). Ozone was produced by an ozone generator with Dielectric Barrier Discharge Plasma (DBDP). Ozone technology rice storage system (OTRISS) is using ozone charateristic which is a strong oxidizer. Ozone have a short endurance of existence and then decompose, as a result produce oxygen and radicals of oxygen. These characteristics could kill microorganisms and pests, reduce air humidity and enrich oxygen. All components used in SPBTO assembled using raw materials available in the big cities in Indonesia. Provider of high voltage (High Voltage Power Supply, 40-70 kV, 23 KH, AC) is one of components that have been assembled and tested. Ozone generator is assembled with 7 reactors of Dielectric Barrier Discharge Plasma (DBDP). Rice container that have been prepared for OTRISS have adjusted so can be integrated with generator, power supply and blower to blow air. OTRISS with a capacity of 75 kg and 100 kg have been made and tested. The ability of ozone to eliminate bacteria and fungi have been tested and resulted in a decrease of microorganisms at 3 log CFU/g. Testing in food chemistry showed that ozone treatment of rice had not changed the chemical content that still meet the standard of chemical content and nutritional applicable to ISO standard milled rice. The results of this study are very likely to be used as an alternative to rice storage systems in warehouse. Test and scale-up is being carried out in a mini warehouse whose condition is mimicked to rice in National Rice Storage of Indonesia (Bulog) to ensure quality. Next adaptations would be installed in the rice storage system in the Bulog. Introduction Technology of rice storage in Indonesia has not been able to keep the quality of rice in a long time. Often, distribution of rice in large quantities had to be canceled by the damage of rice. due to the quality of the rice that was not fit for consumption. It is very worrying the government in this case Bulog can not replace the rice in a large amount in a short time. On the other side the needs of the community may not be delayed. In remote regions that are not producing rice this problem may be more fatal, such as famine. The rapid of decay of rice occurs due to high water levels and breeding of microorganisms contained in the rice storage container. Rice that has been reduced quality is also easily become breeding places of which often called mite pests of rice. So far, in rice storehouses owned Bulog, fumigation and spraying insecticides have been done as efforts for maintaining the quality of rice. Fumigation is a method of insect pest control with specific gases generated by the fumigant. With this method, pest insects that were in or at surface of a sack of rice can be eradicated. To improve the effectiveness of the insect pests eradication on rice and sacks, fumigation collaborated with insecticide spraying method. Insecticides was sprayed on surface of the sack, sack piles sidelines, warehouse floors, and the surrounding environment that serves to eradicate insects that exist in the area. So the insect pest population can be reduced and also can prevent the insects return to the rice barn. Technology of rice storage has not been able to keep the quality of rice. The new technology is needed to resolve the issue. The alternative is ozone technology. Based on research, this technology proved can maintain dan even increase the quality of cereals (especially wheat). Utilization of appropriate ozone generating technology and proper dosage of ozone is believed to be used for rice storage technology. Ozone can also be generated by using a plasma reactor for a particular purpose. Ozone consists of three oxygen atoms and has chemical formula O3. Ozone is an unstable molecule with a very short life span (20-30 minutes) before return to be oxygen, therefore ozone will always seek 'target' to able release one oxygen atom by means of oxidation, so it can be turned into oxygen molecules stable (O2). The precise dose of ozone is determined by configuration of the ozone reactor and the generator. [1]. Ozone as an oxidant has many potential applications in food industry due to the advantages of ozone technology compared to traditional food preservation techniques. Ozone has been known to be extensive utilization for microbial inactivation. Ozone has been reported to eliminate micro-organisms including bacteria, fungi, viruses, protozoa, fungi and bacteria spores. Another advantage of the use of ozone for food is that ozone compounds rapidly decompose and produce oxygen so it does not leave residues in food [2]. Ozone treatments for wheat and corn with ozone concentration of 50 ppm for (3)(4)(5) days treatment can kill insect pests reach 70% -100% [2]. The factors that determine the ability of ozone to kill insects, eliminate fungus and decontamination of toxins, depends on: ozone concentration, exposure time, pH and moisture content of the grains (e.g., wheat, and corn) [3] [4] [5]. In recent years, Diponegoro University has developed research of applications of plasma technology including ozone produced by the plasma technology [6] [7]. Utilization of ozone to maintain the shelf life of rice [8]. In 1995, ozone expressed by Generalize Recognized as Safe (GRAS) or is generally recognized as safe by FDA (U.S. Food and Drug Administration) for the treatment of drinking water bottles. These applications are then developed as "GRAS" for processing food by some experts in last few years [9]. Disinfectant properties of ozone have been tested by several researchers. For example Tiwari et al. [2], which examined the effectiveness of ozone against grain pests such as Tribolium castaneum and Ephestia elutella and the coliform bacteria, Micrococcus, Bacillus spores, fungi and the like. In his journal Tiwari et al. [2] write that the respiratory system be the entrance of ozone to the inside the body of the insect. Ozone causes oxidative tissue damage (even at low concentrations), causing DNA damage, changes in pulmonary system, bronchial sensitivity, membrane oxidation or mutation in vivo. Increased respiratory system with an increase in temperature can result excessive gas exchange due to the increase in metabolism and respiration rate. [10]. In a different study, Ran et al. [11] examined the effectiveness of ozone against Criptosporidium (intestinal protozoa, one of the causes of diarrhea) and protozoan parasite Giardia. In addition, Ran also investigated the mechanism of cell destruction Cryptosporidium by ozone using scanning electron microscopy (SEM) at treatment of time 0, 60, 120, and 480 seconds. As a result, the cell Cryptosporidium suffered the greatest damage in 480 seconds. Research on microbial inactivation of bacteria and molds (fungi) using ozone has been done by several researchers. For example, Zorlugenc et al. [12] who observed the effectiveness of ozone gas and liquid against the bacteria E. coli and coliform bacteria as well as fungi. The tests proved, disinfectant properties (against E. coli and coliform bacteria) ozone gas is lower than the liquid ozone. Similarly, when testing against molds (fungi), which perfectly reduced the mold in 15 minutes using liquid ozone while in ozone gas still leaving approximately 1 log CFU/g. Types of mold that identified having degraded populations include Aspergillus flavus, Aspergillus niger, Aspergillus parasiticus [12]. Test effectiveness of ozone against E. coli was also conducted by Patil et al. [13], where they use ozone to sterilize orange juice from bacterial contamination. The bacteria can be symbiotic mutualism and/or parasitism. In many ways, the bacteria associated with causing disease (parasites and pathogens), accelerating the decay and the like. Overcome the things that adverse caused by bacteria, it is used various types of disinfecting agents. Disinfectant agent that is quite popular in use today is chlorine. Chlorine can kill bacteria with a low concentration, in quick time, and little risk. Chlorine kills bacteria in a way, destabilize cell membranes and inhibit biochemical reactions. Besides chlorine, ozone and ultraviolet is also used. Disinfecting ability of ozone has been studied by several experts in recent years. Ozone, in some cases can kill the bacteria in which chlorine is quite difficult to use. Effectiveness of the disinfectant agent is different, depending on the type of microbes, concentration, and the like [14]. The ability of ozone to kill bacteria, fungi, and insects used as basis for utilizing ozone in rice storage system. In addition, due to ozone within 40 minutes will decompose and possible to bind water molecules, the water content in rice will also be reduced. This paper discusses the use of ozone for rice storage. The discussion is about ozone generation systems, the ability to hold the growth of microorganisms and the quality of rice that has undergone ozonized. In this paper also discussed adaptation of ozonized system into the storage system in Bulog warehouse. Method Ozone generators are made in the laboratory can be adapted for rice storage system which is based on techniques Dielectric Barrier Discharge Plasma (DBDP) and raised with an AC voltage source; 40-70 kV, frequency of 23 kHz. Active electrode is made of spiral wire copper and passive electrodes made from tube-shaped copper plate. Between the two electrodes is given dielectric made of pyrex glass with varying diameter (2 cm, 3 cm and 5 cm). Air pump (Amara AA-250) was used to deliver the air into the reactor, and ozone from recombination process, into treatment media. Probe (Ser. No. 20048737) was used to divide the power supply voltage to 1000 times smaller before being connected to an oscilloscope. Oscilloscope (Instek, GOS-620 20Mhz) was used to monitor the voltage, and operating frequency of the power supply. Ozone detector (Model OS-4, serial # 1208) was used to test the generated ozone levels for treatment (figure 1). Ozone generator was integrated of DBDP reactor with a spiral configuration of copper and copper-dielectric cylindrical pyrex pipes (diameter 3 cm and length 15 cm for research on rice with small and medium capacity). Adjustments to the requirements of adaptation to SPBTO containers, using one generator unit with 7 reactor DBDP. Rice samples were placed in a cylindrical media. Ozone is supplied using an air pipe through the bottom of rice samples. Variation of ozone exposure time was 30, 60, 90, 120, 150 and 180 minutes. Each treatment was performed 4 times, so to be obtained at least 20 data from each object, where the objects of this study are bacteria and mold or fungi which contaminate rice. Testing of samples for chemical analysis (proximate), biological analysis (bacteria and mold/fungi), physical analysis (amilografi, hardness, viscosity and whiteness) and SEM analysis (SEM morphology and SEM EDS). Characterization of Current-Voltage and Ozone concentration The influence of voltage to electrical current average is presented in Figure 2 for the integrated generator which consists of 7 pieces reactor Spiral Cylinder (SC) with 3 cm of diameter pyrex tube. Total electric current has two components: capacitive electric current from the capacitor barrier and capacitive electrical current from the capacitor gas. Therefore, the electric current that actually being measured is the average capacitive electrical current. Influence of electric voltage to ozone concentrations are presented in figure 3 for spiral cylinder reactor of pyrex tube with a diameter of 3 cm. Inactivation of Bacteria and Fungi In Figure 4, we find that the effect of ozone on the amount of bacteria that contaminate rice showed a negative correlation. Bacterial growth is inversely related to ozone exposure time. The average number of bacteria decreased 0.41 log CFU / g every 30 minutes during treatment lasts beyond. This condition is caused by the amount of contaminating bacteria do not survive to breeding process. Resulting slower in population growth of bacteria. The curve above shows, the optimum point decline in bacteria occurs on treatment for 180 minutes. The number of bacteria decreased to 2.48 log CFU / g from the initial state (5.39 log CFU/g). Bacterial growth at 180 minutes, only reached 2.91 log CFU/g indicate that the highest number of bacteria which died before the proliferation (in vitro incubator) done. Remaining bacteria after treatment for 180 minutes is 54.02% of the total number of bacteria before treatment, or can be said to be 45.98% of the bacteria died before breeding. The most significant decrease in the yield curve occurs in treatment between 0 to 30 minutes, in which the number of bacteria decreased 1.44 log CFU/g compared to the transition time of 30-60 minutes. The influence of ozone to the number of fungi is presented in Figure 5. Research of ozone treatment for inactivation of fungi indicate decline from an average of 3.44 log CFU/g to an average of 1.88 log CFU/g. The curve above shows the optimal reduction in the number of fungi occurred at 180 minutes. Where the number of fungi population decreased 1.56 log CFU/g compared with no treatment. The number of fungi surviving 180 minutes after treatment was 1.88 log CFU/g (approximately 54.56% than without treatment). The most significant decrease in the curve occurs at the transition 0 to 30 minutes, in which the number of fungi fell 0.52 log CFU/g. The curve is a significant decrease in the number of fungi in the first 30 minutes also occur in bacteria. This decrease may be due to some fungi can not survive environmental changes abruptly. Content of Carbohydrate, Amylose and Amylopectin Chemical content in rice are being subjected to ozone should be studied seriously Application of ozone at sufficient doses for effective decontamination of rice can affect various quality parameters. Utilization of ozone are not all beneficial and in some cases can lead to oxidative degradation of the chemicals present in rice. Surface oxidation, discoloration or change unwanted odors can be occur from excessive use of ozone [5]. The main part of rice is carbohydrate, then the rice used to meet nutritional needs as a source of carbohydrate. Carbohydrate content in rice, corn, and potatoes are 78.9; 72.4; 19.1% respectively [16]. In different exposure time tends to decrease carbohydrate content, but in relatively small amounts. Carbohydrate in rice consists of amylose and amylopectin. The content both of the compounds is commonly used as a standard of rice quality ( figure 6, figure 7). The results shows that there is no change in significant chemical content in rice for conditions in this study. Table 1 shows chemical composition Nutritional value per 100 grams rice. There are 4 classes of rice [17], the first class is rice with high amylose content about 25-33%, the second class is rice with intermediate amylose content about 20-25%, the third class is rice with low amylose content about 9-20%; the fourth class is rice with very low amylose content about < 9%. The analysis result of amylose and amylopectin content in rice known that generally, amylose content increased with increasing of exposure time by ozone. The treated rice by ozone content 16.26 % of amylose. As previously described, based on the amylose content, the quality of rice is classified into 4 classes [17], the first class is rice with high amylose content about 25-33%, the second class is rice with intermediate amylose content about 20-25%, the third class is rice with low amylose content about 9-20%; the fourth class is rice with very low amylose content about < 9%. The samples that used in this research are rice with low amylose and high amylopectin. Therefore, when it cooked into cooked rice, it has high adhesion level. Content of amylose and amylopectin in rice are one one of the factors that determine the quality of rice. Glutinous rice has a low amylose content of less than 2%, it caused the glutinous rice more sticky than rice. Content of Fat and Protein From the data of test result known that the fat content tended to decrease after treat by ozone irradiation. Changes in fat content in rice were being treatment by ozone are still in a very small amount such that there is no significant change after rice being treat by ozone ( Figure 8). Fat content is generally below the analytical results of the fat content of milled rice [16]. This is not being problem because the rice used as a source of carbohydrate. Raw white rice contains about 6.7% protein, while if it is cooked, it contain about 2%. Rice contains glutelin protein group that is orizenin and in the protein contained 24.1% of glutamate amino acid and slightly lysine. Physical properties of protein of cereal grains is very important, although the protein content is not so high. The size of protein content depends on: species, type of soil, fertilizers, and climate. In general, changes in protein content is very small due to the irradiation is done at room temperature about 29 °C, less than 50 o C. While the character of protein is easy to denatured at temperatures about 50 °C and having surface denaturation in range of pH of 4.5 to 6.0. In the Figure 9 shown that there is an Content of Ash and Water The analysis result of ash content on treated rice by ozone is shown in figure 10. It shows that there is no significant change of ash content, and still in the range of milled rice standard. From the data analysis it appears that the water content before and after irradiation are not much different (figure 11). Rice with minimum water contain will be more resistant to microbial attack due to the properties of microbial that tend to live in high humid condition. In addition, rice with minimum water contain is also will be more resistant to insect attack. The level of water contain reduced by 12.4%, and according to the experience on Bulog, it can be stored for more than 3 years. Time of Treatment (minutes) Water content (initial) Water content (final) Figure 10. The test result of ash content on rice with variation of time of treatment by ozone, compared to control, for the same rice condition Figure 11. The test result of water content on rice with variation of time of treatment by ozone, compared to control, for the same rice condition Conclusion In this study has successfully developed ozone technology rice storage system (SPBTO). Ozone was generated with application of plasma technology by Dielectric Barrier Discharge Plasma (DBDP) reactor. Development of the product begins with the electro mechanical testing of DBDP plasma reactor and ozone generator. The results indicate that the DBDP reactor can produce ozone properly and can be adjusted to the desired concentration to maintain the quality of rice. The test results on treated rice by ozone indicate that the bacteria in rice greatly reduced over a periode time, and the most effective decrease is in 180 minutes. Utilization of ozone have also been able to reduce the growth of fungi, it also obtained that the optimal reduction in the number of fungi occurred at 180 minutes. This time will be used for each repetition period of ozonation on rice. There are no insects on treated rice by ozone although it has been stored for 12 months. The analysis of fat, water, and protein, ash, and carbohydrate that content in rice before and after treatment by ozone results that there is no significant change.

v3-fos

2022-11-26T14:43:08.163Z

2015-11-07T00:00:00.000Z

253894082

s2

Long-term effect of lime application on the chemical composition of soil organic carbon in acid soils varying in texture and liming history There is ample evidence to suggest that liming can regulate soil organic carbon (SOC) pools either directly through influencing the solubility of SOC or indirectly by altering total organic C input as crop residue and SOC loss via change in microbial activity. The aim of this study was to determine the long-term impact of lime application on the quantity and quality of SOC in acid soils. Soils were collected at depths of 0–10, 10–20, 20–30, 30–40, and 40–50 cm from four long-term lime trials with various lime rates (0–25 t ha−1), lime histories (5–35 years), and soil textures (clay content 5–36 %). Surface application of lime was effective in ameliorating both topsoil and subsoil acidities at sites with low clay content. Liming decreased dissolved organic C (DOC) at 0–30 cm but increased its aromaticity. Total SOC at 0–10 cm decreased or remained unchanged following long-term liming, depending on the rates of lime application and crop management. Changes in the contents of particulate organic C (POC) and humic organic C (HOC) predicted by mid-infrared spectroscopy (MIR) and partial least squares regression (PLSR) showed a similar trend to total SOC at all sites. Lime application had no significant effect on SOC below 10-cm layers and on the MIR-predicted resistant organic C (ROC) fraction. Solid-state 13C nuclear magnetic resonance (NMR) spectra indicated that the alkyl C content and alkyl/O-alkyl C ratio were lower in the limed than unlimed plots. Liming possibly had a marked effect on regulating the decomposition and preservation of certain C compounds. The apparent accumulation of alkyl C in the unlimed soil could indicate the potential ability of acid soils to store SOC. Introduction Soil organic C (SOC) is formed through repeated recycling of new and existing plant and animal residues by soil microorganisms. The final product is a complex mixture of different types of organic C differing in decomposability, chemical recalcitrance, and interactions with soil matrix. Some components of SOC (e.g., fragments of plant residue) are more susceptible to decomposition, and some (e.g., charcoal) are less susceptible than the weighted average behavior noted for total SOC. The dynamics of total SOC may thus not be a sensitive indicator of short-or long-term impacts of agricultural management practices on SOC stability and storage (Krull et al. 2003). Various chemical or physical fractionation schemes have been proposed to separate SOC into pools differing in solubility, particle size and density, or biological stability and to provide insight into the mechanisms accounting for changes in SOC due to factors such as land -use, tillage, or fertilization (Six et al. 2001;Skjemstad et al. 2004;Yagi et al. 2003). In addition, solid-state 13 C nuclear magnetic resonance (NMR) spectroscopy has become a popular and important tool for direct chemical characterization of C in whole soil or in each C fraction (Baldock et al. 2013b;Courtier-Murias et al. 2014;Simpson et al. 2012). Recently, the potential of agricultural soils to sequester C has received significant attention, considering its potential contribution to mitigate emissions of greenhouse gases and to improve soil sustainability. The application of lime, a common agricultural practice for ameliorating soil acidity, has also been shown to affect SOC preservation and decomposition. For example, liming can enhance soil C loss by increasing C solubility, microbial activity, and thus the rates of C decomposition (Bezdicek et al. 2003;Fuentes et al. 2006). In contrast, other studies have found that liming enhanced SOC stabilization through redistribution of C from labile to more humified soil pools and/or the complexation of SOC with Ca 2+ thereby enhancing its resistance to decomposition (Manna et al. 2007; Morris et al. 2007). Also, lime-induced increases in root and shoot growth and thus organic residue inputs to soil could contribute to observed increases in SOC storage (Briedis et al. 2012;Bronick and Lal 2005). The final change in total SOC content following liming depends on the balance between SOC gains and losses. However, it is still not clear under what conditions a particular mechanism dominates. Liming could also influence the chemical composition of SOC in addition to altering the overall soil C balance. Soil pH is known to impact the selective preservation of some macromolecules (e.g., lipids) during SOC decomposition (Bull et al. 2000;Guggenberger et al. 1995;van Bergen et al. 1998). Moreover, liming has been reported to reduce the C:N ratio of soil organic matter (Fornara et al. 2011;Tonon et al. 2010), indicating an alteration to the chemical composition of soil organic matter. It is not clear how lime affects different pools of SOC that are lost or accumulated. For example, liming can increase or decrease the allocation of SOC to the humus C fraction irrespective of changes in SOC content (Fornara et al. 2011;Yagi et al. 2003). To date, studies examining the long-term impact of liming have focused on assessing the change in total SOC storage or physically separated C fractions. Little information is available on lime-induced change in the chemical composition of SOC, e.g., amount and proportional distribution of each type of SOC. Knowledge of the chemical structure of SOC is essential as it could reflect SOC stability and provide an indication of long-term C sequestration mechanisms. This paper aimed to quantify the long-term effects of liming on SOC content, the allocation of SOC to component fractions, and the chemical composition of SOC using diffuse reflectance mid-infrared (MIR) spectroscopy and solid-state 13 C nuclear magnetic resonance (NMR) spectroscopy. Soils with various liming histories (5-35 years) and soil textures (clay content 5-36 %) were sampled from four long-term field trials. We hypothesized that (1) liming would decrease soil C storage by increasing C loss through enhanced microbial decomposition, (2) SOC in limed soils would be dominated by C in a more decomposed state than unlimed soils, and (3) alkyl C would be preferentially accumulated in the limed soils due to enhanced SOC decomposition and its high chemical recalcitrance. Materials and methods Liming trial sites and soil sampling details Four long-term liming trials were used in the study. Two sites were located on the La Trobe University farm (37°42′ S, 145°0 2′ E), Victoria, Australia. The soil was similar between the two sites (100 m apart) and was classified as a Sodosol (Isbell 2002) with 36 % clay content. The first trial was established in 1979 and consisted of a completely randomized design with 1×2-m plots replicated three times and was mainly sown with legumes including lentils and medic pasture. The second site was established in 2008 and consisted of a randomized complete block design with three replicates, and the site had been under unmanaged pasture before the liming trial. Three lime treatments were selected from each site, 0, 12.5, and 25 t ha −1 . For both sites, the organic matter inputs had been minimal. The third trial (Wongan Hills site) was established in 1984 on a paddock, 15 km east of Wongan Hills, Western Australia (30°54′ S, 116°51′ E). The soil was classified as an acidic Tenosol (Isbell 2002) characterized by low clay content (6 %), low organic matter (7 g kg −1 ), with gravel (∼5 %) below 10 cm. The trial consisted of 12 plots of 1.8×22 m (Tang et al. 2003). Soils were sampled in May 2013 from the following four lime treatments: no lime (no lime), lime applied at 1.5 t ha −1 in 1999 (lime99), lime applied at 6.2 t ha −1 in 1984 (lime84), and lime applied at 6.2 t ha −1 in 1984 and 1.5 t ha −1 in 1999 (lime84+99), with three replicates. The annual crop rotation on the trial and surrounding paddock were wheat and barley. Crop residues (straw) were returned to the field after each harvest. The fourth trial (Kellerberrin site) was located in a paddock 17 km north of Kellerberrin, Western Australia (31°29′ S, 117°47′ E). The soil was classified as an acidic Tenosol (Isbell 2002) and had clay content of 6.8 % at 0-10 cm, 10.4 % at 10-30 cm, and 8.4 % below 30 cm. The trial consisted of a completely randomized design with 15×100m plots replicated three times. Soil samples were taken in May 2013 from three liming treatments; lime applied in 1991 at 0, 2.5, and 5 t ha -1 with a further 1 t ha -1 applied to all treatments in 2000. The trial was cropped to wheat, barley, lupins, or canola annually, with crop residue returned to the field after harvest. At the Wongan Hills and Kellerberrin sites, soils were sampled from each plot by taking five cores (4 cm diameter) at depth intervals of 0-10, 10-20, 20-30, 30-40, and 40-50 cm. At the La Trobe University sites, only the top 0-10 cm was sampled. All soils were air-dried and passed through a 2-mm sieve. Basic soil physiochemical analyses Soil pH was measured in 0.01 M CaCl 2 (1:5 soil solution ratio, 1 h end-over-end shaking, centrifuging at 700g for 10 min). Soil pH buffer capacity (pHBC) was determined using the method described by Wang et al. (2015). Soil texture was measured using a laser particle size analyzer (Malvern Mastersizer 2000, Worcester, UK). Dissolved organic C (DOC) was determined for all soils collected from Kellerberrin and Wongan Hills sites. Briefly, 20 g of airdried soil and 20 mL of 0.5 M K 2 SO 4 were shaken endover-end for 10 min, centrifuged at 2800g for 10 min, and filtered through a 0.45-μm nylon membrane. The concentration of DOC in the extracts was determined colorimetrically after wet digestion with K 2 Cr 2 O 7 (49 g L −1 ) and concentrated H 2 SO 4 (1:2) (Heanes 1984). The aromaticity of DOC was quantified by measuring the specific UV absorbance at 280 nm (Weishaar et al. 2003). SOC was determined by dry combustion using a CHNS Analyzer (PerkinElmer EA2400, Shelton, USA). Prior to SOC analysis, soils were ball-milled (MM400, Retsch GmbH, Haan, Germany) and tested for the presence of CaCO 3 using 4 M HCl. Only one soil collected from the plot limed at 25 t ha -1 lime of the second trial showed visible effervescence and was pretreated with H 2 SO 3 to remove inorganic C (Schmidt et al. 2012). SOC characterization using MIR and NMR analyses MIR spectroscopic analyses were carried out using the ballmilled soils on a Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific Inc., MA, USA) equipped with a PIKE AutoDiff-automated diffuse reflectance accessory (Pike Technologies, WI, USA). Samples (approximately 100 mg) were packed into stainless steel cups and loaded into the 60 position PIKE AutoDiff sample wheel. MIR spectra were acquired over 8000-400 cm −1 with a resolution of 8 cm −1 using a KBr beam splitter and a DTGS detector. The background signal intensity was quantified by collecting 240 scans on a silicon carbide disk prior to analyzing each set of 60 soil samples and used to correct the signal obtained for the soil samples. A total of 60 scans were acquired and averaged to produce MIR reflectance spectra for each individual sample, and the OMNIC software (version 8.0) was used to convert the acquired reflectance spectra into absorbance spectra (log transform of the inverse of reflectance). All chemometric analyses (spectral transformation and predictions using partial least squares regression, PLSR, prediction algorithms) were completed using Unscrambler 10.3 (CAMO software, Olso, Norway). All acquired MIR spectra were truncated to a spectral range of 6000-1030 cm −1 , and a baseline offset transformation was applied. The square root of the gravimetric contents of particulate organic C (POC) (SOC associated with 50-2000-μm particles excluding poly-aromatic C), humic organic C (HOC) (SOC associated with particles ≤50 μm excluding poly-aromatic C), and resistant organic C (ROC) (poly-aromatic C associated with particles ≤2 mm) were predicted from the transformed MIR spectra using the MIR/ PLSR algorithms developed by Baldock et al. (2013a) using measured POC, HOC, and ROC contents derived using an automated size fractionation protocol (Baldock et al. 2013b). Soils for solid-state 13 C NMR analysis were prepared by forming single composite samples for each treatment at each site by combining a mass of soil normalized to measured bulk densities from each field replicate for the 0-10-cm depth layer. To remove the sand particles, all composite soils were dispersed (20 g soil in 80 mL of a 50 g L − 1 sodium hexametaphosphate solution shaken for 16 h on a flatbed orbital shaker) and passed through a 50-μm sieve. The coarse organic materials retained on the 50-μm sieve were separated from sand by backward pushing the organic material on the sieve with a stream of water into a separate container. The collected coarse organic materials were combined with the collected <50 μm suspension and then freeze-dried (Baldock et al. 2013b). The final lyophilized and ground samples were treated with 2 % hydrofluoric acid (HF) to further concentrate SOC and remove paramagnetic materials (Skjemstad et al. 1994). Solid-state 13 C NMR analyses were completed on a Bruker 200 Avance spectrometer equipped with a 4.7-T wide-bore superconducting magnet operating at a resonance frequency of 50.33 MHz. Weighed samples (150-600 mg) were packed into 7-mm diameter zirconia rotors with Kel-F end caps and spun at 5 kHz. Chemical shift values were calibrated to the methyl resonance of hexamethylbenzene at 17.36 ppm, and a 50-Hz Lorentzian line broadening was applied to all spectra. Three separate 13 C NMR experiments were performed. An inversion recovery pulse sequence using eight inversion recovery times varying from 0.001 to 3.0 s and a recycle delay (d 1 ) of 5 s was applied to each sample. T 1 H values were calculated for each sample from the inversion recovery data and indicated that a recycle delay of 1 s was adequate (>5 times calculated T 1 H values) to avoid saturation in the subsequent cross-polarisation (CP) 13 C NMR analyses. The CP analyses used a 3.2 μs, 195 w, 90°pulse, and a contact time of 1 ms. Between 10,000 and 20,000 scans were collected for each CP analysis. The number of scans was increased as the amount of C contained in the rotor declined across the various samples. A variable spinlock experiment using an array of spinlock times (1, 2, 6, 10, 15, and 20 ms), a contact time of 1 ms, and a recycle delay of 1 s was performed to calculate sample specific T 1ρ H values and allow the CP NMR observability of organic C in the samples to be quantified as described by Baldock and Smernik (2002). The observability of C was close to 100 % (99-108 %) for samples collected from Wongan Hills site and ranged from 75 to 85 % for other three sites. All spectral processing including the calculations of T 1 H and T 1ρ H and integration of spectral regions were completed using the Bruker TopSpin 3.2 software. After phasing and baseline corrections were applied, the absolute NMR signal intensities acquired for each sample were divided by the number of scans collected and corrected for empty rotor background signals. The resultant spectra were integrated using the chemical shift limits and calculations defined by Baldock et al. (2013b) and used to quantify the allocations of soil C to alkyl, N-alkyl/methoxyl, O-alkyl, di-O-alkyl, aryl, O-aryl, carbonyl/amide, and ketone forms. In this integration process, any signal intensity present in spinning sidebands was mathematically added to the parent resonances from which they were derived. Statistical analyses The effects of liming on soil pH, DOC, SOC, C:N ratio, MIRpredicted C fractions, and soil pHBC at each depth were analyzed using a one-way analysis of variance (ANOVA). Significant (P=0.05) differences between means were identified using the least significant difference (LSD) test. For NMR analysis, duplicates were performed for one treatment at each site to assess the experimental error. Results The magnitude of the increase in soil pH reflected both liming rates and soil pHBC. Applying lime at 12.5 and 25 t ha −1 increased the soil pH by 1.0 and 1.6 pH units, respectively, at the university sites (data not shown). At the Kellerberrin site, liming at 5 t ha −1 increased the soil pH by nearly 1 pH unit to a depth of 30 cm, when compared with the unlimed control ( Fig. 1). At the Wongan Hills site, marked increases in soil pH (1.5-1.8 pH units) were detected even below 30 cm, when lime was applied at 6.2 or 6.2+1.5 t ha −1 . Liming at lower rates increased soil pH by less than 0.5 pH units at both sites ( Fig. 1). Liming significantly decreased DOC concentration at 0-30 cm (Fig. 2a, c). However, when compared with the unlimed control, DOC concentration increased at 40-50 cm at the Wongan Hills site following liming at 6.2 or 6.2+1.5 t ha −1 . Similarly, liming increased the aromaticity of DOC, except at the depth below 30 cm at the Wongan Hills site, where higher specific UV absorbance of DOC was observed in the plots limed at 6.2 or 6.2+1.5 t ha −1 than the control (Fig. 2b, d). In general, SOC content at 0-10 cm decreased by increasing lime rates at all sites (Tables 1 and 2); however, SOC remained unchanged when lime was applied at 6.2 + 1.5 t ha −1 at the Wongan Hills site. Changes in C/N ratio, MIR-predicted POC, and HOC fractions showed a similar trend to total SOC at all sites (Tables 1, 2, and 3). For example, liming decreased soil C/N ratio in most cases but not when lime was applied at 6.2+1.5 t ha −1 at the Wongan Hills site (Tables 1 and 2). At the university farm, MIR-predicted POC and HOC decreased consistently with SOC in response to liming. Lime application had no significant effect on SOC below 10-cm soil layers and on the MIR-predicted ROC fraction at all depths (data not shown for below 10 cm). The pHBC of soils was also decreased by liming, except where residual lime was detected in the university farm limed at 25 t ha −1 in 2008. As revealed by the NMR spectra, O-alkyl C (24.6-27.7 %) and alkyl C (22-25.4 %) were the dominated C types in the topsoils at all sites (Figs. 3 and 4). Change in each C type was affected by the change in total organic carbon (TOC), with the content of each type of C lower in the limed than unlimed soils, except at the Wongan Hills site where lime applied at 6.2+1.5 t ha −1 did not affect the C content. Only alkyl C showed an apparent difference in relative intensity distribution in the spectra of the limed and unlimed soils (Fig. 4). The relative alkyl C content and alkyl/O-alkyl C ratio were lower in the limed plots, compared with the unlimed plots established at the university farm in 1979 (Figs. 4 and 5). There was also an apparent trend of decreased proportion of alkyl C and alkyl/O-alkyl ratio with liming at the other three sites. The experiment error (≤0.5 %), as revealed by the duplicate measurements, was lower than the magnitude of change in the relative content of alkyl C (1.7-2 %). Effect of surface liming The apparent pH increase due to liming to a depth of 30 cm at Kellerberrin and 50 cm at Wongan Hills sites suggested that surface application of lime was effective in ameliorating subsoil acidity in soils with low clay content in the long term. High liming rates, low soil pHBC, and the presence of gravel in the subsurface layers could account for the significant downward movement of lime at the Wongan Hills site. Little movement of surface-applied lime was frequently detected on soils with relatively higher soil pHBC as indicated by their higher clay (24-32 %) and organic C content (>20 mg kg −1 ) (Godsey et al. 2007;Conyers et al. 2003). Given the soil pHBC of 6.3 mmol c kg −1 pH −1 at the Wongan Hills site, applying lime at 6.2 and 6.2+ 1.5 t ha −1 equated to twice the amount of lime required to increase the soil pH to a depth of 50 cm by 1 and 1.4 pH units, respectively. Blevins et al. (1978) also observed a vertical movement of lime to a depth of 30 cm when lime was applied at a rate of three times the lime requirement. Thus, liming at relatively high rates on sandy soils with low pHBC allows vertical movement and amelioration of subsoil acidity. Moreover, when compared with soil pH data obtained in 2003 (Tang et al. 2003), re-acidification of whole soil profile (0.1-0.9 pH units) had occurred during last 10 years of continuous cropping, indicating that re-liming every 5-10 years would be essential to prevent pH decrease due to re-acidification. DOC The DOC concentration of limed soils decreased proportionally to the amount of added lime. Lower amounts of DOC in the limed soil could be due to enhanced decomposition or leaching associated with higher soil pH. It is well recognized that soil pH can modify the quantity of DOC through affecting its production, decomposition, solubility, and adsorption by soil minerals. Liming could increase the production and and Kellerberrin (c, d), Western Australia. Lime treatments at Wongan Hills include no lime (no lime), liming at 1.5 t ha −1 in 1999 (lime99), liming at 6.2 t ha −1 in 1984 (lime84), and liming at 6.2 t ha −1 in 1984 and 1.5 t ha −1 in 1999 (lime84+99). Lime treatments at Kellerberrin include no lime (0), liming at 2.5 t ha −1 (2.5), and liming at 5 t ha −1 (5). Horizontal bars indicate ± the standard error of three replicates decomposition rates of DOC by stimulating microbial activity (Andersson et al. 1994(Andersson et al. , 2000. Erich and Trusty (1997) found that DOC in the limed plots was more susceptible to microbial attack as Al-organic complexes might have been replaced by Ca-organic complexes. Furthermore, enhanced solubility and leaching of DOC due to deprotonation or desorption have also been observed after liming (Andersson et al. 1994(Andersson et al. , 2000. Therefore, liming could increase or decrease DOC concentration in soil, depending on which processes dominate. In this study, lower DOC contents of limed soils suggest that DOC loss via enhanced microbial mineralization or leaching was greater than DOC production via decomposition or processes that increased DOC availability such as deprotonation or desorption. However, lime-induced increases in DOC have been frequently reported in some short-term field and laboratory studies (Andersson and Nilsson 2001;Erich and Trusty 1997;Garbuio et al. 2011). Possibly, DOC did increase immediately following liming, but this increase was temporary and could not be sustained against enhanced microbial degradation or leaching in these lightly textured soils over long periods. Leaching of DOC down the soil profiles at the Wongan Hills and Kellerberrin sites is likely given their coarse texture. Many studies detected rapid adsorption of DOC by soil minerals (Kaiser and Guggenberger 2000;Kawahigashi et al. 2006), which could lead to low rates of DOC transport to deeper layers. However, vertical movement of DOC had been detected in sandy soils with limited sorption sites (Ahmad et al. 2013;Li and Shuman 1997). The higher DOC, together with the relatively higher clay content, at 20-30 cm at the Kellerberrin site could indicate that DOC was leached down from the surface layer and adsorbed by clay minerals at subsurface layers. At Wongan Hills site, increased DOC at a depth of 40-50 cm due to liming at 6.2 or 6.2+1.5 t ha −1 could provide evidence of DOC migration to deep layers. In addition to lower DOC content, DOC within the limed soil was more aromatic and more chemically recalcitrant than in the unlimed soil. The DOC could be derived from both recent litter and humus, but most studies suggested that DOC consisted mainly of highly altered humified material (Karltun et al. 2005;Sanderman et al. 2008). The aromatic signal in DOC was found to be dominated by lignin-derived compounds which were highly chemically recalcitrant (Nakanishi et al. 2012;Sanderman et al. 2008). Other studies also revealed a higher proportion of labile hydrophilic material (e.g., carbohydrates) in DOC of the unlimed soils, compared with a higher proportion of aromatic hydrophobic material in the limed soils (Andersson and Nilsson 2001;Andersson et al. 2000). The higher aromaticity of DOC in the limed than unlimed soils could be attributed to two possible reasons: (1) DOC was more decomposed in the limed soil due to enhanced microbial activity at higher soil pH (Andersson et al. 2000) and (2) aromatic C compounds containing less polar hydrophobic acids became soluble as pH increased (Guggenberger et al. 1994). The lower aromaticity of DOC at a depth of 30-50 cm at the Wongan Hills site in the limed than unlimed soils possibly reflected the labile or mobile nature of DOC leached. It has been well documented that only structurally simple molecules, such as carbohydrates held by minerals through weaker bonds, could be more easily leached down to the soil profile than aromatic compounds bound via ligand exchange (Corvasce et al. 2006;Kawahigashi et al. 2006). The susceptibility of this labile Table 1 Soil organic C (SOC) (mg g −1 ) and C/N ratio at depths of 0-10, 10-20, 20-30, 30-40, and 40-50 (lime84), and liming at 6.2 t ha −1 in 1984 and 1.5 t ha −1 in 1999 (lime84+99) n.s. not significant at P=0.05 Table 2 Soil organic C (SOC) (mg g −1 ) and C/N ratio at depths of 0-10, 10-20, 20-30, 30-40, and 40-50 C to microbial mineralization would be relatively low due to reduced microbial biomass and activity at deeper soil layers. Overall, the quality of DOC could be greatly affected by liming, and DOC in the limed soil showed higher stability against either microbial degradation or leaching than in the unlimed soil. Total SOC and MIR-predicted SOC fractions There was a substantial loss of SOC from limed soils, except when lime was applied at 6.2+1.5 t ha -1 at the Wongan Hills site. Liming has been shown to decrease (Chan and Heenan 1999;Marschner and Wilczynski 1991), increase (Briedis et al. 2012;Fornara et al. 2011), or not impact SOC content (Garbuio et al. 2011;Kemmitt et al. 2006). Liming-induced decreases in SOC are mainly attributed to enhanced C mineralization following increased C solubility, microbial activity, or both (Ahmad et al. 2013;Bezdicek et al. 2003;Fuentes et al. 2006;Kemmitt et al. 2006). However, greater C inputs as crop residues following liming would be expected to increase SOC (Bronick and Lal 2005;Briedis et al. 2012). The rate and direction of changes in SOC following liming depend on the balance between SOC gains and losses. In our study, lower SOC content in the limed than unlimed plots would suggest that additional C inputs, if they occurred, were too low to counterbalance the C losses from enhanced decomposition. On the other hand, the TOC content of Wongan Hills site receiving the highest lime rates (6.2+1.5 t ha −1 ) was not different from that of the unlimed plots, indicating that lime-induced increase in the C input matched the enhanced decomposition of SOC. Tang et al. (2003) showed that, 18 years after liming at the Wongan Hills site, shoot biomass at maturity increased by 60 % due to liming at 6.2+1.5 t ha −1 , compared with 48 and 46 % following liming at the 1.5 and 6.2 t ha −1 , respectively. This suggests that less than 50 % increase in shoot biomass, or possibly in C input as plant residues, could not counteract the C losses from limed soils. The relative high shoot biomass production following liming at 6.2 + 1.2 t ha −1 could be related to the great alleviation of soil acidity at both topsoil and subsoil layers (Fig. 1). The rates of liming were supposed to have a profound effect on regulating the balance and size of total SOC in the limed plots through affecting their total C input. Lime treatments at Wongan Hills include no lime (no lime), liming at 1.5 t ha −1 in 1999 (lime99), liming at 6.2 t ha −1 in 1984 (lime84), and liming at 6.2 t ha −1 in 1984 and 1.5 t ha −1 in 1999 (lime84+99). Lime treatments at Kellerberrin include no lime (0), liming at 2.5, and liming at 5 t ha −1 . Lime treatments at the university farm include no lime (0), liming at 12.5 and 25 t ha −1 in 1979, and liming at 12.5 and 25 t ha −1 in 2008 n.s. not significant at P=0.05 It has been proposed that liming can promote SOC storage via increased aggregate stability and the formation of Ca 2+ complexes with SOC and clay (Baldock et al. 1994;Bronick and Lal 2005). These processes could reduce the decomposition of both soil organic matter and added plant residues (Chan and Heenan 1999); Muneer and Oades 1989). However, the loss of C following liming at all sites except at Wongan Hills where lime was applied at 1.5+6.2 t ha −1 does not support this. Also, the low clay content (5-7 %) at Kellerberrin and Wongan Hills would render the amount of SOC that could potentially be protected through the formation of Ca 2+ bridges between clay and SOC very low. Baldock et al. (1994) and Briedis et al. (2012) found that increases in aggregate stability and SOC storage become more significant when lime was incorporated with straw, compared with lime alone. Therefore, the direct or indirect stabilizing effects of Ca 2+ ions may contribute little to long-term C storage in plots where C inputs and/or clay contents were low. Decrease in TOC following liming occurred mainly in the POC and HOC pools. The ROC, mainly in the form of char, was the most chemically protected and biologically recalcitrant pool (Skjemstad et al. 1996) and was not affected by liming. Similar to TOC, the levels of POC and HOC pools should be governed by the following two main processes: C migration from plant residue to POC and then onto HOC with continued decomposition of POC. Following liming, more C from the POC pool could have been processed and incorporated into HOC as a consequence of fast C turnover (Baldock et al. 1994;Marschner and Wilczynski 1991;Tonon et al. 2010). This could explain the decreased C:N ratio and decreased proportion of POC fraction by liming in the present study. Previous findings on changes in HOC after liming are contradictory, with both decreases (Marschner and Wilczynski 1991;Yagi et al. 2003) and increases reported (Chan and Heenan 1999); Fornara et al. 2011). The net decrease in HOC in this study indicates that the loss of HOC outweighed the newly incorporated C from POC. In contrast, little C loss from HOC when limed at 6.2+1.5 t ha −1 at the Wongan Hills site might be attributed to a greater C flux from POC to HOC associated with high C input or enhanced abiotic C sequestration through Ca-humus complex. Although HOC is considered to be relatively chemically recalcitrant, our results suggest that liming could accelerate its decomposition and decrease its contribution to long-term C storage. Lime-induced solubilization or desorption of humic substances from mineral surface were possibly responsible for the decreased stability of HOC pool (Garbuio et al. 2011). Nevertheless, liming at high rates may result in an increase in total C input as plant residue or enhanced humus stabilization which may offset the C losses from the HOC pool due to enhanced microbial decomposition. The humus fraction enriched with carboxyl functional groups was expected to be closely related to change in soil pHBC. Nevertheless, while soil pHBC decreased consistently with liming, HOC was not affected by the lime applied at 6.2+1.5 t ha −1 at the Wongan Hills site. We speculate that liming, at high rates, will favor the formation of new complexes between functional groups of the humus and Ca 2+ , in addition to the replacement of the Al with Ca in the organic complexes. Subsequently, the availability of functional groups for soil pH buffering might be lowered. After liming, soil pHBC probably depended more on the availability rather than the quantity of functional groups indicated by HOC content or carbonyl groups. This can also partly account for the low correlation between soil pHBC and carbonyl groups. Soil organic C composition A lower proportion of alkyl C or alkyl/O-alkyl C ratio was observed in the limed than unlimed plots. This suggests enhanced decomposition of alkyl C in the limed soils or the selective accumulation of alky C in the unlimed acid soil. However, without the baseline chemical data prior to liming, the exact process cannot be identified. A greater chemical recalcitrance of alkyl C suggests that it should be selectively preserved during SOC decomposition (Baldock et al. 1992). Thus, if the enhanced SOC decomposition following liming acted as the main mechanism, a greater decrease in the O-alkyl than alkyl C would be expected. A consistent increase in alkyl/O-alkyl ratio with increasing the degree of decomposition of fresh organic materials has been frequently observed (Baldock et al. 1992(Baldock et al. , 1997. This indicates that the alkyl/Oalkyl C ratio may not be a good indicator of the extent of C decomposition between the limed and unlimed soils. On the other hand, alkyl C which is sourced from lipids, cutin, and suberin polyesters (Winkler et al. 2005) can be selectively preserved in acidic soils. For example, numerous studies reported that lipids or lipid-derived fatty acids (van Bergen et al. 1998;Nierop et al. , 2005 or cutin-and suberin-derived moieties (Nierop 1998;Nierop and Buurman 1999) accumulated during long-term SOC formation under acidic conditions. The selective preservation of cutin and suberin was attributed to the low cutinase or suberinase activity at low soil pH . Also, lime-induced changes in microbial community composition, particularly a shift from fungi to bacteria (Bååth and Anderson 2003), could also be responsible for the different C compounds accumulated in soils differing in pH. Several studies have shown that, in the long-term, lignin can be effectively degraded by fungi in acid soils, whereas lipids and suberin-derived moieties accumulated and remained relatively unaltered (Stevenson 1994;. To conclude, the relative proportion of alkyl C was possibly controlled by the following two contrasting processes: selective preservation of alkyl C due to the accumulation of lipid or polyester in the unlimed soil and selective preservation of alkyl C during the decomposition of plant residues in both limed and unlimed soils. Litter decomposition in the limed soil at the two cropping sites was expected to contribute more to the increase in alkyl C than at the trials at the university farm with little C input. This would have narrowed the difference in the proportion of alkyl C between limed and unlimed soils where the accumulation of lipid or polyester dominated but the decomposition of litter was limited by low pH. The duration of the liming treatments at the university farm limed in 2008 might not have been long enough for greater changes in SOC composition to be detected. Rosenberg et al. (2003) also found that liming decreased the proportion of alkyl C and the alkyl/O-alkyl C ratio in a mature Norway spruce forest. They attributed the decrease of alkyl-C in the limed soil to the greater root biomass production, which was lower in alkyl C than aboveground litter. This was less likely to occur at our university site with low C input. Other studies by Lorenz et al. (2001) and Tonon et al. (2010) revealed that the 13 C NMR spectra of limed and unlimed soils were very similar and concluded that pH did not significantly affect the chemical and structural composition of SOC. Among all studies, soil microbial communities were expected to differ among soils with different environmental physico-chemical properties (Delmont et al. 2014), which could possibly account for the observed discrepancies. Our study suggests that differences in the quantity and quality of C inputs associated with liming rates or land management practices might also have a great impact on the final SOC composition. Conclusion Total SOC at 0-10 cm decreased or remained unchanged following long-term liming, depending on the rates of lime application. Decreases in SOC due to liming occurred mainly in the labile and moderately labile C pools and only in the surface soil layer. To maximize C input as crop residues and to prevent the loss of C from limed soil, lime should be applied periodically at relatively high rates or more frequently at lower rates. Liming practices should also be accompanied by retention of crop residues, considering enhanced mineralization associated with high soil pH. On the other hand, liming possibly had a marked effect on regulating the decomposition and preservation of certain C compounds. The apparent accumulation of alkyl C in the unlimed soil could indicate the potential ability of acid soils to store SOC. Less changes in the chemical composition of SOC of the two cropping sites in Western Australia was supposed to be partly masked by the continuous replenishment of C via residue return, although further research is required to confirm this speculation. DP120104100). We thank Dr. Nick Uren for the establishment of 1979 lime plots at the first site.

v3-fos

2018-04-03T04:13:02.659Z

2015-05-28T00:00:00.000Z

18550962

s2

Oral pharmacokinetics of acetaminophen to evaluate gastric emptying profiles of Shiba goats The pharmacokinetics of acetaminophen was investigated following oral dosing to Shiba goats in order to evaluate the properties of gastric emptying. Acetaminophen was intravenously and orally administered at 30 mg/kg body weight to goats using a crossover design with a 3-week washout period. The stability of acetaminophen in rumen juice was also assessed. Acetaminophen concentrations were measured by HPLC. Since acetaminophen was stable in rumen juice for 24 hr, the extremely low bioavailability (16%) was attributed to its hepatic extensive first-pass effect. The mean absorption time and absorption half-life were unexpectedly short (4.93 and 3.35 hr, respectively), indicating its marked absorption from the forestomach, which may have been due to its smaller molecular weight. Therefore, acetaminophen was considered to be unsuitable for evaluating gastric emptying in Shiba goats. Although the entire gastrointestinal tract is capable of drug absorption, the main site of absorption of orally administered drugs is the proximal part of the gut. Several factors have been shown to influence the absorption of drugs from the gastrointestinal tract, with the gastric emptying rate being identified as important [5,6,16,17]. The rate of gastric emptying determines the time taken to reach the absorption site, and thus, significantly affects the rate and extent of drug absorption. Delays in the gastric emptying time was previously reported to significantly decrease the rate of absorption of acetaminophen (AAP) and aspirin, whereas stimulating the gastric emptying accelerated the absorption of these drugs [11,12]. Gastric emptying in ruminants necessitates drug transit from the rumen through the reticulum, omasum and abomasum, leading to the long residence of orally administered drugs in the forestomach. Therefore, the rate of drug absorption in ruminants may be the slowest of all animals due to the time required for drug particles to pass through the fourchambered stomach [1]. This finding explains why drugs with a very short half-life by the intravenous route, such as salicylic acid (1 hr), may nevertheless give sustained plasma concentrations in ruminants when administered by the oral route [5,17]. Although the oral route is considered to be inappropriate for ruminants, we previously demonstrated the effectiveness of this route for diclofenac (DF) and sulphamonomethoxine (SMM) in Shiba goats [4]; the mean absorption time (MAT) of DF (6.05 hr) was less than half that of SMM (15.1 hr). These findings suggested that the short MAT of DF was due to its marked absorption from the forestomach while the long MAT of SMM was due to a long gastric emptying time; however, gastric emptying time needs to be estimated in order to confirm this. AAP is mainly absorbed from the small intestine of humans and most animal species, and not from the stomach [2,14,15,18]. The AAP absorption test, which involves measuring plasma AAP concentrations in short time intervals following its oral administration, is considered a reliable method to evaluate gastric emptying rates in the stomachs of humans [2] as well as ponies and horses [3,9]. Therefore, the present study was undertaken to examine the pharmacokinetics of AAP after oral dosing in order to evaluate the properties of gastric emptying in Shiba goats. AAP was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). AAP was dissolved in 70% propylene glycol at a high temperature (approximately 70°C) for its intravenous administration. AAP was dissolved in 90% ethanol, mixed with three hay cubes and then allowed to dry before its oral administration. These solutions were prepared at a concentration of 200 mg/ml. All other reagents and chemicals used in this study were of HPLC or analytical grade. The present study was performed using five clinically healthy male Shiba goats, weighing 21-44 kg and aged 2-3 years. All goats were maintained in accordance with the recommendations of the 'Guide for the Care and Use of Laboratory Animals' approved by the Ethics Committee of the Faculty of Agriculture, Tokyo University of Agriculture and Technology (approval number 76/25). These goats were housed in pens at an ambient temperature and with good ventilation. Animals were fed hay cubes (#1A Cubes, Eckenberg Farms Inc., Mattawa, WA, U.S.A.) at 0.8 kg/head twice a day, and water was available ad libitum. The oral pharmacokinetics of AAP, its stability in rumen juice and the octanol-buffer (pH 6.5) partition coefficient were investigated in the present study. In the pharmacokinetics study, AAP was administered into the left jugular vein or orally at a dose of 30 mg/kg body weight to five male goats using a crossover design with at least a 3-week washout period. Blood samples (3 ml) were collected from the right jugular vein immediately prior to and 0.5, 1, 2, 3, 4, 6, 9 and 12 hr following an intravenous injection of AAP, and 0.5, 1, 2, 4, 6, 9, 12 and 16 hr after its oral administration. Plasma samples were separated by the centrifugation of blood at 1,600 g for 10 min and stored at −20°C until later analyses. The stability of AAP in the rumen juice was determined as described previously [4]. Briefly, 40 ml of rumen fluid was collected from two goats using a catheter, pooled and processed for incubation immediately after its collection. Two hundred microliters of the AAP solution (1 mg/ml) was added to 1.8 ml of the rumen juice to give a final concentration of 100 µg per ml of the incubation mixture. Five samples were prepared from this mixture and incubated in a thermostatic shaking water bath at 39°C for 24 hr under anaerobic conditions. The incubated mixture was then centrifuged at 20,000 g for 10 min, and the supernatant was collected. The octanol-buffer partition coefficient of AAP was determined by the shake flask method as recommended by the Organization for Economic Cooperation and Development [13]. Before partitioning, the two solvents were mutually saturated at 25°C for 24 hr. Solutions of AAP (10 µg/ml) were prepared in octanol-saturated phosphate buffer (50 mM, pH 6.5). These solutions were then equilibrated at 25°C with an equivalent, double and half volume of buffer-saturated octanol. Two separating funnels were used in all three runs. After equilibration, the buffer phase was collected and centrifuged at 1,600 g for 10 min. The concentration of AAP in the buffer phase was then determined. The concentration of AAP in the octanol phase was obtained by mass balance. The apparent and intrinsic octanol/buffer partition coefficients were then determined from these data. AAP concentrations in plasma, rumen juice and buffer samples were determined by HPLC with UV detection, as described previously [8] with some modifications. Briefly, 200 µl of perchloric acid (0.15 M) was added to 200 µl of the plasma or rumen juice samples and stirred. The mixtures were centrifuged at 20,000 g for 10 min. The supernatants were obtained and filtered using a 0.45-µm HPLC filter (Chromatodisc ® , 4P, Kurabo Biomedical Industries, Ltd., Osaka, Japan). Fifty microliters of the filtrate was injected into the HPLC column. The HPLC system (Shimadzu Corporation, Kyoto, Japan) consisted of a pump (LC-10AD), UV detector (SPD-6A), integrator (Chromatopac C-R7A plus) and loop injector (model 7125). The mobile phase was a mixture of 0.1 M acetate buffer (pH 4) and acetonitrile (90:10, v/v). Triethylamine 150 µl/l mobile was added. Analytical separation was accomplished using a reversed-phase ODS column (TSKgel ODS-120T ® , 4.6 µm × 250 mm, TOSOH Co., Tokyo, Japan). The flow rate was 1 ml/min. The wavelength of the detector was 248 nm. Sample preparation and analysis were conducted at room temperature. AAP was found to be accurately resolved as a single sharp peak with a retention time of 5-6 min. The recovery of AAP from plasma samples was 100.1 ± 2.65% at 1 µg/ml (mean ± SD, n=5), while that from rumen juice samples was 97.0 ± 2.03% at 25 µg/ml (mean ± SD, n=5). The inter-day CV values ranged from 2.24 to 3.20% for plasma samples and from 1.44 to 3.05% for rumen juice samples (n=5, 3 times). The plasma concentration-time curves of AAP after the intravenous injection fit well with the two compartment model. Therefore, the curves obtained after the intravenous injection (Cp iv (t)) and oral administration (Cp po (t)) were described by Eq. 1 and 2, respectively. (Eq. 1) (Eq. 2) Equations 1 and 2 were simultaneously fit to the plasma concentration-time curves of AAP after it was intravenously and orally administered to the same goats, respectively, in order to calculate pharmacokinetic parameters by the nonlinear least-squares method using the curve fitting program, MULTI [19]. Several pharmacokinetic parameters were calculated by a non-compartmental analysis. The area under the concentration versus time curve (AUC) was calculated using the trapezoidal method (from time zero to the last sampling time) and integration (from the last sampling time to infinity). Total body clearance (CL tot ), bioavailability (F), mean residence time (MRT), MAT and the distribution volume at a steady state (V dss ) were calculated by conventional methods. The plasma concentrations of AAP rapidly increased and peaked 0.90 ± 0.22 hr after being orally administered, and this was followed by its slow elimination. On the other hand, plasma concentrations were eliminated rapidly after the intravenous injection with short half-lives (1.14 ± 0.46 hr), as presented in Fig. 1. The calculated average values with SD of MAT and absorption half-life (t 1/2ka ) of AAP were 4.93 ± 0.87 and 3.35 ± 0.50 hr, respectively (Table 1). These values are similar to those of DF (6.75 ± 2.74 and 4.13 ± 1.94 hr, respectively) in a previous study using Shiba goats [4]. These results suggested that AAP was absorbed more from the forestomach, similar to DF. The partition coefficient of AAP was markedly lower than that of DF at the pH of rumen fluid (pH 6.5), as shown in Table 2. This result indicated that factors other than lipophilicity predominantly influenced the absorption of AAP from the forestomach, for example, molecular size, as has already been suggested by Morishita et al. [10]. They compared the gastrointestinal absorption of several sulfonamides in rats and found that sulfanilamide had a fast absorption rate that was unexplainable from its smaller partition coefficient than other sulfonamides. They concluded that the fast absorption of sulfanilamide may have been due to its small molecular weight (172.21). Because the molecular weight of AAP (151.2) is similar to that of sulfanilamide and is markedly smaller than that of DF (318.1), as listed in Table 2, the faster absorption of AAP may have been due to its smaller molecular weight, similar to sulfanilamide. Fast oral absorption of AAP has been found in dairy cows by Grünberg et al. [7]. In their experiment, peak concentrations of AAP were observed less than 2 hr after oral administration. This fact may suggest that AAP is markedly absorbed from forestomach also in daily cows like in Shiba goats. The bioavailability of AAP was less than 20%. The recoveries of AAP from rumen juice samples at 100 µg/ml (n=5) after a 12-and 24-hr incubation at 39°C were 90.5 ± 1.5 and 88.7 ± 0.8% (mean ± SD), respectively. Since AAP is stable in rumen juice, its low bioavailability after its oral administration may have been due to its extensive first-pass effect in the liver. This may also be attributed to the large metabolic capacity of Shiba goats [1,17]. In conclusion, the results of the present study suggested that AAP was markedly absorbed from the forestomach of Shiba goats, which may have been due to its small molecular weight. Therefore, AAP was considered unsuitable for evaluating gastric emptying in Shiba goats. 0.210 ± 0.032 0.194 ± 0.073 [4] pk a : Dissociation constant. Referred from reference [9] (AAP) and [16] (DF). f u %: The ratio of the unionized fraction (calculated at pH 6.5). P: Apparent partition coefficient between octanol and phosphate buffer at pH 6.5. P*: Intrinsic partition coefficient between octanol and phosphate buffer calculated from apparent partition coefficient and pk a in the table. MAT: Mean absorption time. k a : Absorption rate constant. a) Measured by the same method used for AAP in the present study.

v3-fos

2020-05-21T09:08:27.989Z

2015-05-15T00:00:00.000Z

223824572

s2

1101 Development of an ecological strategy for the control of downy mildew (Pseu-doperonospora cubensis) in cucumber cultivation (Cucumis sativus L.) : Downy mildew is a severe disease of cucumber worldwide. The oomycete Pseudoperonospora cubensis cause it and once it is established in a region, the infection spreads rapidly, causing significant loss of yield and fruit quality. The objective of the research was to develop an ecological strategy for the control of downy mildew in cucumber. The treatments were arranged in a completely randomized experimental design with an alternation of chemical and biological fungicides. The treatments were: T1: systemic fungicide (Ridomil Gold, 2.5 g/l) alternating with a contact fungicide (Bravo 2.5 ml/l), T2: CustomBio 5 (Bacillus-based fungicide, 3ml/l), T3: control (water), T4: Trichoderma sp. (3 ml/l), T5: systemic fungicide (Ridomil Gold 2.5g/l) alternating with CustomBio 5 (3 ml/l), and T6: systemic fungicide (Ridomil Gold 2.5g/l) alternating with Trichoderma sp. (3 ml/l). The following variables were evaluated: stem thickness, plant height, number and weight of fruits, yield, the area under the relative progress curve (AUDPCr), and economic analysis of each treatment. The results showed that the best treatments were T1 and T6, with an AUDPCr of 11.89% and 12.10%, respectively. Treatments T6 and T1 showed the best yield, as well. The profitability analysis showed that all the alternatives were profitable with a Benefit/Cost>1 ratio. However, the treatments T6 and T1 were the most useful. We recommend this control strategy to reduce the use of chemical fungicides and, at the same time, obtain an efficient control of the disease, which guarantees a significant yield of high-quality fruit. Introduction Cucumber (Cucumis sativus L.) is the fourth most important vegetable in the world 1 . It was originated in India, where two botanical cultivars arose in the Himalayas, the domesticated (Cucumis sativus var. sativus), and wild cucumber (Cucumis sativus var. hardwickii Royle, Alef) 2 . Cucumber is a short-cycle crop (3-4 months) that could be grown in the open field, or under greenhouse conditions 3 . Although the cucumber can be reproduced by vegetative propagation, it has substantial genetic diversity because it also spreads sexually 4 . Current cultivars show a remarkable variety of agricultural features, including the color, shape, rib, diameter, spines, and the brightness of the fruit and the overall resistance to stress. Some cucumber cultivars are well adapted to specific environments because of genetic selection 5 . In Ecuador, this vegetable is cultivated in the warm valleys of the Andean mountains and the dry tropics of the coastline corresponding to 87.20% and 12.80% of total land devoted to this vegetable, respectively 6 . The Ecuadorian production reaches an annual average of 3,045 tons, from an area of 364 hectares with an average yearly growth of 1.03% 6 . Various diseases affect cucumber. Among the most critical conditions is downy mildew caused by the oomycete Pseudoperonospora cubensis, which is one of the most severe threats to cucumber production worldwide and in Ecuador. It is spread in temperate and semiarid regions, and the pathogen infects plants of all ages, although the disease is more severe at early stages. Usually, the disease infects foliage, causing a reduction in early photosynthetic activity, which affects plant development, delaying growth, and reducing yield. The inflicted chlorotic lesions on the leaf surface could progress until they become necrotic, engaging the entire leave, which may die within days after the infection 7 . The disease cause premature defoliation and then the sunburn of the fruit due to overexposure to direct sunlight. In the United States, host resistance in past decades successfully controlled the downy mildew; however, the pathogen has overcome this resistance and now has become a very severe threat to cucumber growers, mainly because it is also evolving fungicide resistance 8 . In Ecuador, the disease severely affects cucumber production damaging more than 60% of the output 7,9 . The application of chemical pesticides is the most commonly used control method, either through using contact and systemic fungicides or the combination of both 10 . However, the new trends are directed towards the integrated pest management (IPM) 11 , which includes the application of alternative products such as resistance inductors (i.e., Trichoderma atroviride) 12,13 or beneficial microorganisms such as Bacillus subtilis, Pseudomonas fluorescens, Derxia gummosa and Trichoderma harzianum 12,14 . These microorganisms show the potential to interact with local microflora, and usually, their products are biodegradable in situ to non-toxic compounds by environmental organisms. The search for new and varied products of natural origin for disease management programs represents an essential alternative in sustainable agriculture 15 . Therefore, the objective of this work is to develop an ecological strategy for the control of downy mildew (Ps. cubensis) in cucumber farming. There is a need to develop strategies for the control of Ps. cubensis in an ecological and environmentally friendly way, thereby safeguarding people's health, and contributing to reducing production costs due to the lower application of pesticides. Here, we present preliminary data on the use of a combination of chemical fungicides and microorganisms suitable for control of Ps. cubensis in cucumber production under greenhouse conditions 16 . Study site This investigation was carried out from June to November 2017 in the 'Puerto La Boca' community in Manabi province, Ecuador (1°18'20''S, 80°45'42" W, 53 m altitude). Its climate has of 24.8°C and 298 mm of average temperature and annual rainfall, respectively, corresponding to dry tropical forest ecosystems. Treatments, growth conditions and research management Sexual seeds of the hybrid Humocaro were pre-germinated in blotting paper moistened with distilled water. The seeds were placed in a closed environment with white light, RH > 80% and a temperature of 20°C±2. For the transplanting of the seedlings, plastic trays with 128 alveoli (15 x 20 10 cm/ alveolus) were used. The seedbed was prepared with topsoil (60%), Biocompost (30%), and Earthworm Humus (10%). The pre-germinated seeds were transplanted and then sprayed to field capacity. Nine days after the seedlings were put on plastic trays, they were transplanted to soil beds (36 x 1.20 x 0.60 m), containing topsoil supplemented with Biocompost in a 3:1 ratio. The transplant was performed to holes 0.15 m deep, at a distance of 1.2 m and 0.20 m between rows and plants, respectively, totaling 12 rows. Each experimental unit had an area of 26 m 2 with 55 plants/units. The total area of the experiment was hosted in a 500 m 2 greenhouse with a photoperiod of 12h light:12h dark, an average temperature of 22°C, and RH>60%. The chemical fungicides and biological organisms that we tested in various treatments are described in Table 1. The organic fungicide CustomBio 5 contains five species of the genus Bacillus (B. subtilis. B. laterosporus. B. licheniformus. B. megaterium y B. pumilus at 8x10 8 CFU/ml) and is produced by Ecuadorian company Naturalite S.A., Guayaquil, Ecuador. The treatments followed a completely randomized design (DCA) 17 , and the experimental units of each treatment consisted of 330 plants, totaling about 1980 plants. Twenty-five random plants were chosen from each experimental group in each treatment for evaluation. Response Variables The area under the relative disease progress curve (AU-DPCr) was calculated using five affected leaves of the upper middle third of the plant and a -100% damage scale 18 . Stem diameter (SD, in mm) was measured with a Vernier caliper at 30 cm from the ground when the crop had 50% flowering. Fruit weight (FW, in g) was measured by weighting fruits in each harvest. The number of fruits (NF) was measured by counting all fruits of each harvest. The number of not-set fruits (NNSF) was assessed, by counting the number of dead fruits in each treatment. Plant height (PH, in cm) was measured when the plant had 50% of the flowering. Profit/cost analysis was estimated using the methodology suggested by CIMMYT 19 that analyzes all the benefits and costs of the different treatments. Statistical analysis We performed an analysis of variance to test hypotheses about the fixed effects of treatments and the comparisons of means to determine the response of the best treatment using SAS University software, Cary, NC. For this, we contrasted ways using one degree of freedom and α=0.01. Likewise, the analysis of variance allowed estimating the variance components for random effects. The comparison of means was performed using the multiple Tukey tests at α = 0.05, and the association between the variables studied were analyzed using Pearson's correlation test 17 . Results Before the analysis of variance, analysis of normality and homogeneity of variances of the variables and the treatments were made. This analysis showed that the variables AUDPCr, NF, FW, and NNSF were not normally distributed, so the data was transformed to square root to normalize it. For AUDPCr, severity data were arcsine transformed 17 . The analysis of variance of the following variables, AUDPCr, SD, NF, and FW, showed that the coefficients of variation (CV) are within the ranges allowed for this type of research (CV of Plants were fertilized with Biocompost Organic Fertilizer (Organicgreen S.A., Ecuador) (50 g/hole), and EcoFungi (Eco-Microbials, Florida, USA) a mix of mycorrhizae (20 g/20 l of water) and Starlite bioregulator (Organicgreen S.A., Ecuador) (50 ml/20 l of water) applied to the ground at the first day of transplanting and along the crop cycle totaling six fertilizations. Nine harvests were made at intervals of four to five days for each one. This began when the cucumber fruits presented the right color and size. The second, third, fourth, fifth, sixth, and seventh applications of the treatments were performed at 27, 34, 41, 48, 55, 62, and 68 days after the transplant, respectively. In total, seven crops were made throughout the crop cycle. 7.61 to 35.43%) ( Table 2). Highly significant differences were observed at the probability of p <0.01 for all the variables evaluated. This result indicates that at least one of the treatments was different from the others ( Table 2). The analysis of variance of the variables NNSF and PH showed that the CV, are within the ranges allowed for this type of investigation (CV from 8.08 to 29.44 %) ( Table 2). Highly significant differences were observed at the probability of p <0.01 only for PH. This indicated that at least one of the treatments is different in PH. In contrast, there were no significant differences in NNSF ( Table 2). The analysis of means performed by the Tukey test at p <0.05 probability for the variables AUDPCr, SD, NF, FW, NNSF, and PH, Table 1. Treatments analyzed in this work. different from the T2 and T5 treatments. The T3 treatment was not statistically different from the T2, T5, T6, and T1 treatments for this variable. Regarding the FW, the T4 treatment was significantly different from the T3, T6, and T1 treatments at p <0.05 probability. The treatments T2, T5, and T4 were not significantly different from each other, nor treatments T3, T2, T5, T6, and T1. Concerning PH, it was found ( Table 4) that treatments T3, T2, T5, T4, and T1 were not significantly different from each other, but were significantly different from the T6 treatment. There were no significant differences for NNSF for any of the treatments. The Pearson's correlation analysis (Table 4) showed a high and significant negative correlation for AUDPCr concerning to NF (-0.94) and FW (-0.94). The profitability analysis (Table 5) showed that, in general, all showed significant differences in all cases except for NNSF, where all treatments are statistically equal ( Table 3). The best disease reduction was achieved with treatments T1 and T6 relative to T3 (control), observing AUDPCr of 11.89%, 12.10%, and 29.84 %, respectively. It is worthy to note that other secondary diseases caused by different fungi were also controlled. Regarding the SD, results indicate that the T4 treatment showed a higher diameter (36.80 mm) compared to the T1 treatment (32.01 mm), being significantly different at a p<0.05 probability. The T3 and T6 treatments were statistically the same instead;, treatments T4, T2, T5, and T6 were not statistically different from each other. About NF, the T4 treatment was significantly different from the T3, T6, and T1 treatments at p <0.05 probability, but not substantially Table 2. Analysis of variance for AUDPCr, SD, NF, FW, NNSF and PH in cucumber plants, Puerto La Boca, Ecuador, 2017. Table 3. Analysis of means for AUDPCr, SD, NF, FW, NNSF and PH, Puerto la Boca, Ecuador, 2017. Table 4. Correlation analysis. Development of an ecological strategy for the control of downy mildew (Pseudoperonospora cubensis) in cucumber cultivation (Cucumis sativus L.) the alternatives were profitable with a benefit to cost ratio (B/C> 1). However, the treatments T6 (Systemic fungicide + Trichoderma sp) and T1 (Systemic fungicide + contact fungicide) were distinguished, as the most profitable with USD 1606 and USD 1600 respectively. Discussion The downy mildew of cucumber is traditionally controlled with chemical fungicides; however, chemical control is not always feasible because of the high costs associated with the application especially for poor farmers of South American countries and the high environmental liabilities that the use of chemical fungicides means. In this work, we developed an ecological control strategy for Ps. cubensis based on the alternation of systemic and contact fungicides and the replacement of the contact fungicide by a microorganisms-based one. This strategy allowed satisfactorily controlling the pathogen and reaching a high production of fruits. Likewise, it helped to reduce production costs due to the lower application of pesticides. Although we present preliminary results, we undoubtedly think this is a promising strategy to control fungal pathogens in cucumber that can be adopted in IPM programs because of their affability with the environment and lost cost 20,21 . We also determined that all control alternatives evaluated here were profitable with a benefit/cost ratio greater than 1 (B/C> 1). Still, particularly the treatments alternating systemic fungicide with Trichoderma, as well as systemic with contact fungicide, were the most profitable. This suggests that applying an ecological strategy with microorganisms is useful and beneficial. The contact fungicides that we used (Chlorothalonil), acted in the first hours after the application. Still, once oomycete penetrates the plant, systemic fungicides (Metalaxyl) should be used to control fungal infection because these are mobilized in the internal tissues and organs of the plant after their application 22,23 . In our trials, the alternate form of Bacillus-based fungicide (CustomBio 5) with chemical counterparts allowed reducing the use of pesticides at least 50% of the applications. The mode of action may rely on the production of antibiotic molecules because it is well known that the Bacillus species contained in CustomBio 5 are efficient producers of antibiotic molecules 24,25 and antifungal volatiles 26 . Likewise, Trichoderma sp. worked properly when it was applied alone or in alternate applications with the systemic and contact fun-gicide 27 . Adnan et al. 27 mention that the species of the genus Trichoderma are the most commonly used antagonists for the control of plant diseases caused by fungi, due to their rapid growth in a large number of substrates, ample abiotic stress tolerance, quick ability to colonize, easy establishment after inoculation and sufficient capacity to compete for space and nutrients with pathogens. Besides, Trichoderma can stimulate plant growth due to its ability to produce plant growth regulators, vitamins, and recycle nutrients such as P (phytate) and Zn in the soil 28 . In our experiments, the systemic fungicide did not harm Trichoderma since the applications were alternate, letting at least one week pass between applications. Additionally, some Trichoderma strains show resistance or tolerance to fungicides 29,30 , although this particular property was not tested in the Trichoderma strains used in this work. Conclusions Ps. cubensis is a significant fungal disease for cucumber farming in Ecuador and worldwide, causing the disease called downy mildew. Traditionally, the application of fungicides has helped to hinder the spreading of the disease; however, chemical control is not always feasible because of the high costs associated with the implementation of fungicides and the implied environmental damage. In this work, we developed an ecological control strategy for Ps. cubensis based on the alternation of systemic and contact fungicides and the replacement of the contact fungicide by a microorganisms-based one. This strategy allowed satisfactorily controlling the pathogen and reaching a high production of fruits. Likewise, it helped to reduce production costs due to the lower application of pesticides. Undoubtedly, this strategy constitutes an excellent alternative to control pathogens in cucumber, and it can be adopted in IPM programs because of their affability with the environment and lost cost

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2018-04-03T02:17:15.455Z

2015-05-19T00:00:00.000Z

15524662

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Land use change influences soil C, N, and P stoichiometry under ‘Grain-to-Green Program’ in China Changes in land use might affect the combined C, N and P stoichiometry in soil. The Grain-to-Green Program (GTGP), which converts low-yield croplands or abandoned lands into forest, shrub, and/or grassland, was the largest land reforestation project in China. This study collected the reported C, N and P contents of soil in GTGP zones to achieve the factors driving the changes in the C:N, C:P, and N:P values. The results showed that the annual average precipitation exerted significant effects on the C:P value, and on the N:P value became significant 20 years after the change in land use. The annual average temperature was the main factor affecting the C:N value during the first 10 years, while the annual average precipitation strongly affected this value afterwards. In addition, “Redfield-like” interactions between C, N, and P in the soil may exist. A linear regression revealed significant positive correlations between the C:N, C:P, and N:P values and the restoration age, temperature, and precipitation after a change in land use. Therefore large-scale changes in land use under the ‘GTGP’ program might significantly affect the C:N, C:P and N:P ratios in soil. . Distribution of soil C:N value (a), C:P value (b) and N:P value (c) value under 'Grain-to-Green Program related zones' . The map plotted by Arcgis9.3 using inverse distance weighting (IDW) method. fertilization 18 . Additionally, Tian et al. reported that the P supply in soil depends on the total P content and the weathering stage of the parent material, which is characterized by spatial heterogeneities 19 . In addition, this work proposed a 'Redfield ratio' in soil 19 . Cleveland and Liptzin 12 reported that the C, N, and P stoichiometry in soil remains relatively stable at 186:13:1 on the global scale. Lal 20 suggested that the humus C:N:P:S ratio is 10,000:833:200:143. Tian et al. 19 found a well-constrained C:N molar ratio (14.4), as well as relatively consistent C:P (136) and N:P (9.3) ratios, with a general C:N:P ratio of 134:9:1. Cristina et al. 21 also demonstrated that the interactions among the season, vegetation type and structure, and soil properties affect microbial nutrient immobilization under a Mediterranean-type climate to influence the biogeochemical cycles for C, N and P in Mediterranean forest ecosystems. To date, studies on the soil C, N, and P stoichiometry at different scales are lacking, and information about their influences on the global or regional scale are scarce, particularly in China. In China, widespread ecological degradation has constrained sustainable socioeconomic development in recent decades, particularly before the end of the 20th century 22 . After the 1950s, the Chinese government has made great efforts to control soil erosion and restore ecosystems 23 . More than 9.27 million ha of cropland and abandoned land have been afforested in China through the "Grain to Green Program" (GTGP), which has required more than 28.8 billion USD and involved 0.12 billion farmers; the GTGP has implemented large-scale ecological rehabilitation since 1999 22 . Currently, this is the first and most ambitious "payment-for-ecosystem-services" program in China 22,24 . Although the initial goal of the GTGP was to control soil erosion, the program strongly affects the C, N, and P cycling in soil 25,26 . However, few studies have reported the soil C, N, and P stoichiometries under GTGP. Therefore, this study aims to accomplish the following: a) illustrate the distribution of the soil C:N, C:P, N:P values under the GTGP; b) establish the changes in the soil C:N, C:P, N:P values after the change in land use; and c) study the factors driving the changes in the C:N, C:P, N:P ratios. Results Changes in the soil C:N, C:P, and N:P values due to the 'Grain-to-Green Program'. The distribution of the soil C:N, C:P, and N:P values from 0-20 cm measured after the change in land use under the 'Grain-to-Green Program' Program in China is displayed in Fig. 1. The highest soil C:N and C:P values were obtained in Southern China (Guangdong and Guangxi province) (Fig 1a,b), while the highest soil N:P values were obtained in Northern China (Jiling and Heilongjiang province) (Fig 1c). The frequency distribution of the soil C:N, C:P, and N:P values (Fig. 2) revealed that most of the soil C:N, C:P, and N:P values were ranged from 8 to 16, 16 to 32, 0 to 2, respectively. Changes in the soil C:N, C:P, and N:P ratios in response to the changes in land use. For forest The effects of the change in land use from cropland or abandon land to forest on the soil C:N, C:P, and N:P values changes are shown in Fig. 3a-c. The changes of soil C:N ratio increased during the first 10 years and after 20 years (Fig. 3a) but decreased from 10 to 20 years. In contrast, the changes of soil C:P ration decreased over the first 10 years before increasing (Fig. 3b). The changes of soil N:P ratio constantly increased after the change in land use; the highest changes of ratios were achieved during the first 10 years. Meanwhile, the changes of soil C:N, C:P, and N:P ratios were differently following annual average temperature ( Fig. 3d-f). The changes of soil C:N ratio was highest in > 16 °C, whereas the changes of C:P, and N:P ratio were highest in 9-16 °C. However, the changes of soil C:N, and C:P ratio were highest in > 1350 mm following annual average precipitation, while the changes in the soil N:P ratio was found in < 584 mm ( Fig. 3g-i). For shrubland generated by a change in the land used for cropland or abandoned land, the changes of soil C:N ratio decreased slightly during the first 10 years and decreased sharply during years 10 to 20 before increasing after 20 years (Fig. 4a). The changes in the soil C:P ratio increased continuously after the changes in land use; the highest changes of ratios were obtained from years 10 to 20 (Fig. 4b). Additionally, the changes of soil N:P ratios increased during the first 10 years before decreasing continuously (Fig. 4c). The changes in the soil C:N, C:P, and N:P ratios were different with annual average temperature ( Fig. 4d-f). the changes in the soil C:P, and N:P ratio were highest in 9-16 °C, but the changes in C:N ratio was highest in > 16 °C. Moreover, the changes in the soil C:N, C:P, and N:P ratios were all highest in > 1350 mm following average precipitation ( Fig. 4g-i). For grassland generated from cropland or abandoned land, the changes in the soil C:N ratio decreased slightly during the first 10 years before decreasing sharply during 10 to 20 years and increasing after 20 years, similarly to shrubland (Fig. 5a). Meanwhile, the changes in the soil C:P ratio increased during the first 10 years and after 20 years but decreased during 10 to 20 years (Fig. 5b). However, the changes in the soil N:P ratio increased during all stages after the changes in land use, and the highest value appeared during years 10 to 20 (Fig. 5c). Meanwhile, The changes of soil C:N, C:P, and N:P ratios were highest in > 16 °C following annual average temperature ( Fig. 5d-f), however, there was no obvious regularity following average precipitation ( Fig. 5g-i). Factors affecting the soil C:N, C:P, and N:P values. Stepwise regressions revealed that the annual average temperature was the main factor affecting the soil C:N value during the first 10 years, and the annual average precipitation had significant effects on the soil C:N value after 10 years (Table. 1). The annual average precipitation also significantly affected the soil C:P value continuously after the change in land use. The restoration age was the major factor affecting the soil N:P value during the first 20 years, whereas the annual average precipitation strongly affected the soil N:P value 20 years after the change in land use. ANOVA shown that significant positive correlations between the soil C:N, C:P, and N:P values and the restoration age, temperature, and precipitation after the change in land use (p < 0.05), but their interactions were significant in few of case (Table. 2). Discussion The soil C:N, C:P, and N:P ratios are good indicators of the status of soil nutrients during development 19 . the high C:N ratios (> 25 on a mass basis) indicate that organic matter is accumulating faster than it is decomposing. Our results showed that most of the soil C:N value ranged from 8 to 16 (Fig. 2), indicating that the organic matter is thoroughly broken down. Similar results were reported by Bui and Henderson 27 . Our synthesis also revealed that most of the soil C:P value ranged from 16 to 32 (Fig. 2), implying a net mineralization of nutrients. Paul 16 also reported that C:P <200 implies a net mineralization, C:P >300 implies a net immobilization, and a C:P between 200 and 300 reveals little change in the soluble P concentrations. Cleveland and Liptzin 12 estimated that the global soil C:P and N:P ratios for surface soil (0-10 cm) are 186 and 13.1, respectively. Tian et al. 19 reported that the C:P and N:P values were 136 and 9.3 at the same depth in China. Our analysis revealed lower values. These differences might occur because the soil samples used by Cleveland and Liptzin 12 and Tian et al. 19 have a humified litter layer, generating higher C:N, C:P, and N:P values. Additionally, the correlation between the total soil C, N and P was obtained based on more than 592 soil samples (Table. 3). The results revealed that the C:N ratio was highly constrained based on the relatively high correlation coefficient (0.71) for the C and N concentrations. Relatively constrained C:P and N:P ratios were observed with correlation coefficients of 0.28 and 0.48, respectively, implying a relatively constrained C:N:P ratio, similarly to that reported by Cleveland and Liptzin 12 and Tian et al. 19 . Therefore, we agree with Cleveland and Liptzin 12 that "Redfield-like" interactions may exist among C, N, and P in soil. The C, N, and P stoichiometry in soil varies based on the type of land use, and these variations are highly complex 8 . Our results indicated that the effects on the soil C:N, C:P, and N:P values in the forest, shrub and grassland varied (Figs. 3,4,5). For example, the conversion of cropland or abandoned land to forest increased the C:N value over the first 10 years (Fig. 3a), these values decreased during the same period in land that was converted into shrub and grassland (Figs. 4a,5a). These differences most likely occur for two reasons. Plants change the C, N, and P ratios by absorbing/releasing these elements from/ to soil 28,29 . However, soils with different vegetation undergo different litter decomposition processes and rates, meaning that the release of nitrogen and phosphorus to soil differs 6 . Similar results were reported by Elisabeth and Brent 27 . However, our results contrasted sharply with those of Cleveland and Liptzin 12 , who reported that the soil nutrient ratios did not vary significantly between forests and grasslands. We speculate that vegetation covers, plant communities, and geomorphology all affect the nutrient stoichiometry in soil. Li et al. 7 reported that the different types of land use exhibited different soil C:N:P ratios due to differences in elevation, vegetation type and land management practices. Aponte et al. 30 presented a Spanish dataset indicating that the average soil C:N:P ratio in forests is slightly greater than that in woodlands; this ratio varied based on the type of land use. Additionally, compared to other countries, the soil C:N, C:P, and N:P values in China also vary between forest, shrub and grassland (Table. 4). For example, the soil C:N, C:P, and N:P values in China were lower than in some countries (i.e., USA, Germany) and were higher than those found in other countries (i.e., UK). These variations likely arose from the different climatic zones, soil orders, soil depth and weathering stages, which affect the soil C:N, C:P, and N:P values. Similar results were reported by Tian et al. 19 and Zhang et al. 31 . Table 3. Correlations among soil organic C (g/kg), total N (g/kg) and total P (g/kg) under 'Grain-to-Green Program' . The restoration age, temperature and precipitation are important factors that must be considered when estimating the soil C, N, and P stoichiometry after changing the land use (Table. 1 Table. 2). A stepwise regression revealed that the annual average temperature was the primary factor affecting the soil C:N value over the first 10 years. However, the restoration age became the major factor affecting the soil N:P value during first 20 years (Table. 1). In addition, the annual average precipitation also significantly affected the soil C:P value during all of the stages after the land use change (Table. 1). Therefore, the C, N, and P ecological stoichiometry is highly complex in soils. The climate may affect the soil C, N, and P stoichiometry that accumulates through biotic processes, relying on the productivity of the vegetation and the decomposition of organic matter. Several studies have illustrated that the climate imposes important controls on the biota and its interaction with the soil nutrients 15,[30][31][32] . Moreover, the restoration of the soil C content after afforestation varies with the climate 26,33-36 . The annual average temperature and precipitation also affected the soil organic carbon during some stages after the change in land use 27 . In addition, Zhang et al. 30 reported that the soil C:N value was primarily affected by the total phosphorus, the soil C:P value was primarily affected by the total nitrogen, and the soil N:P value was primarily affected by the soil organic carbon. Therefore, the C, N, and P stoichiometry in soils is also affected by the soil elements, which are largely influenced by the annual average temperature and precipitation. Methods All of the available publications concerning the changes in the C, N, and P contents in soil from forest, shrub, and grassland that was converted from cropland and/or abandoned land from 1999-2013 under the GTGP in China were collected. The following criteria were used to select publications for analysis: • data existed for the all land use types (grasslands and shrublands as well as forest) sites; • The soil C, N, which were measured by the K 2 Cr 2 O 7 -H 2 SO 4 oxidation method and the Kjeldahl digestion procedure method, the phosphorus method was used complexation with ammonium molybdate and antimony potassium tartrate followed by quantification using a spectrophotometer. The contents of at least two of the elements (C, N, and P) were provided or could be calculated; • paired sites were used within their chronosequence (sampled many replicate plots and paired plots over a landscape, those plots with the same age, edaphic conditions and land use were pooled); • similar conditions (i.e., soil types, elevation); • the restoration age (year), temperature (°C), and precipitation (mm) were clearly given; • additionally, studies that lack replication or provide unclear information were excluded; • The reported sites were distributed across the GTGP zones shown in Fig. 6. • The final dataset was composed of 92 publications that included 592 observations (Appendix 1). In our study, the C:N, C:P, and N:P ratios are calculated on a molar basis. The raw data were either obtained from tables or extracted by digitizing graphs with the GetData Graph Digitizer (version 2.24, Russian Federation) as reported by Deng et al. 37 . The following information was compiled from each publication: location (longitude and latitude), annual temperature and precipitation, types of land use (forest, shrub, or grassland), and restoration age after the change in land use. The soil layer was set to 0-20 cm because most works only documented the changes in the C, N, and P contents within this layer, and significant differences were only observed in the topsoil 23 . Moreover, Tian et al. 19 reported that the soil C:N, C:P, and N:P ratios in organic-rich topsoil might be a good indicator of the soil nutrient status during soil development. Studies utilizing different soil depths (for example, 5 publications utilized 27 observations at 0 -15 cm; and 7 publications utilized 49 observations at 0 -10 cm and 10 -20 cm) were adjusted to encompass 0~20 cm. In this study, we assumed that no differences were observed in the C, N, and P contents from 15 to 2 cm. Specifically, the changes in the C, N, and P contents in the 0~15 cm layer equaled those in the 0~20 cm layer. Averaged values from 0 -10 cm and 10 -20 cm were used to represent the 0 -20 cm layer. The restoration age of afforestation was divided into three groups: < 10, 10 -20, and > 20 years. The previous type of land use was cropland or abandoned land in all cases. If a sample only reports the SOM, the SOC value is calculated using the following formula 38 : where SOC is the soil organic carbon and SOM is the soil organic matterIf the bulk density (BD) of the soil is not reported, it is calculated 28 as follows: where Δ R CN,CP,NP is the change in the C:N, C:P, and N:P ratios, y 0 is a constant, k represents the slope in Equation 3, and Δ Age is the restoration age. The same method was used by Deng et al. 37 . A stepwise regression analysis was used to analyze the relationship between the C:N, C:P, and N:P ratios and the annual average temperature (T, °C), the annual average precipitation (P, mm) and the restoration age (A, year) in each restoration age group. Pearson's correlation coefficients were used to study the relationship between the soil C:N, C:P, and N:P values and the restoration age, temperature, and precipitation measured after the change in land use. Statistical analyses were performed using SPSS, ver. 17.5 (SPSS Inc., Chicago, IL, USA). The Figures were plotted using Origin 7.5 and Arcgis9.3.

v3-fos

2016-05-15T08:32:35.797Z

2015-09-10T00:00:00.000Z

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Prevalence and distribution of soil-borne zoonotic pathogens in Lahore district of Pakistan A multidisciplinary, collaborative project was conducted to determine the prevalence and distribution of soil-borne zoonotic pathogens in Lahore district of Pakistan and ascertain its Public Health Significance. Using a grid-based sampling strategy, soil samples (n = 145) were collected from villages (n = 29, 5 samples/village) and examined for Bacillus anthracis, Burkholderia mallei/pseudomallei, Coxiella burnetii, Francisella tularensis, and Yersinia pestis using real time PCR assays. Chemical analysis of soil samples was also performed on these samples. The relationship between soil composition and absence or presence of the pathogen, and seven risk factors was evaluated. DNA of B. anthracis (CapB), B. mallei/pseudomallei (chromosomal gene), C. burnetii (IS1111, transposase gene), and F. tularensis (lipoprotein/outer membrane protein) was detected in 9.6, 1.4, 4.8, and 13.1% of soil samples, respectively. None of the samples were positive for protective antigen plasmid (PA) of B. anthracis and Y. pestis (plasminogen activating factor, pPla gene). The prevalence of B. anthracis (CapB) was found to be associated with organic matter, magnesium (Mg), copper (Cu), chromium (Cr), manganese (Mn), cobalt (Co), cadmium (Cd), sodium (Na), ferrous (Fe), calcium (Ca), and potassium (K). Phosphorous (P) was found to be associated with prevalence of F. tularensis while it were Mg, Co, Na, Fe, Ca, and K for C. burnetii. The odds of detecting DNA of F. tularensis were 2.7, 4.1, and 2.7 higher when soil sample sites were >1 km from animal markets, >500 m from vehicular traffic roads and animal density of < 1000 animals, respectively. While the odds of detecting DNA of C. burnetii was 32, 11.8, and 5.9 higher when soil sample sites were >500 m from vehicular traffic roads, presence of ground cover and animal density of < 1000 animals, respectively. In conclusion, the distribution pattern of the soil-borne pathogens in and around the areas of Lahore district puts both human and animal populations at a high risk of exposure. Further studies are needed to explore the genetic nature and molecular diversity of prevailing pathogens together with their seroconversion in animals and humans. The incidence of B. anthracis, F. tularensis, B. mallei/ pseudomallei and Y. pestis have been reported worldwide (Ayyadurai et al., 2008;Oyston, 2008;Butler, 2009). Prevalence of B. anthracis and B. mallei in humans and animals has been previously reported in Punjab province of Pakistan based on clinical findings and not supported by laboratory based confirmation. Further, little is known about the epidemiology of F. tularensis and Y. pestis in human and animal populations in Pakistan. Determination of the true prevalence of these pathogens is further complicated due to lack of a reporting system in the province of Punjab. The objectives of this study were to; (1) determine the prevalence of soil-borne zoonotic pathogens including B. anthracis, B. mallei/pseudomallei, F. tularensis, Y. pestis and C. burnetii in soil samples collected FIGURE 1 | Geospatial distribution of Burkholderia mallei/pseudomallei, Bacillus anthracis, Francisella tularensis, and Coxiella burnetii DNA in soil samples collected in Lahore district, Pakistan. from villages in Lahore district, (2) identify soil components that favor or deter the presence of pathogens, and (3) identify risk factors associated with the presence or absence of the pathogen in the soil. It is anticipated that the findings of the study will provide information to undertake rigorous epidemiologic investigations focused on prevalence and distribution of soil-borne zoonotic pathogens in humans and domestic animals in Pakistan. Study Area The Lahore district in Punjab Province of Pakistan was selected for conducting the study. Lahore (31 • 15 ′ -31 • 45 ′ N and 74 • 01 ′ -74 • 39 ′ E) is bound in the north and west by the Sheikhupura district, Kasur district in the south and India in the east. The river Ravi flows on the north-western side of Lahore city (Figure 1). The study area is 217 m above sea level and covers a total land area of 1772 km 2 . With a population exceeding 10 million, administratively the district is comprised of 10 towns, 271 union councils and 301 villages. Villages represent the lowest administrative unit, while a collection of villages represent a union council. Sample Collection Lahore district has 301 villages. Ten percent of these villages (n = 29) were randomly selected using Open Epi version 2.3.1 (http://www.openepi.com/Menu/OE_Menu.htm) software using a 5% confidence limit and 80% level of significance (Figure 1). From each village, five sites were identified and sampled by removing 3-5 inches of the top soil. Samples from four sites were locations that had both animals and humans dwelling in close proximity while the fifth site was located in an area where there was no evidence of apparent recent human and animal activity. Samples were collected using personal protective equipment (PPE). From each sample site, three separate samples were collected approximately within one square meter of the sample site. The three samples were pooled, mixed thoroughly and divided into three aliquots for soil chemistry (∼500 g), real time (RT) PCR assays for pathogens (∼200 g) and sample archives (∼500 g). Using a sample collection survey questionnaire, necessary information pertaining to risk factor analysis for a given sample collection site including GIS location, presence or absence of domestic animals, animal density, distance from animal market, main road and water source such as river, canal, streams, and drains (canal or channel system which carries sewage and rain water), presence or absence of vegetation and human dwelling was obtained. DNA Extraction from Soil Samples DNA from soil samples was extracted using PowerSoil R DNA Isolation Kit (MoBio, USA) as per the manufacturer's instructions. The quality (A 260/280 and A 260/230 ) of the DNA was assessed by spectrophotometry (NanoDrop, USA) while the quantity (ng/µL) was determined by Qubit fluorometer (Invitrogen, USA) using the DNA BR Assay kit (Invitrogen, USA) as per the manufacturer's instruction. RT PCR assay was optimized and validated using the positive controls (dsDNA PCR products) and proficiency testing samples provided by Drs. Francesconi and Patel Naval Medical Research Unit, Frederick, Maryland. RT PCR assays for detection of B. anthracis, B. mallei/pseudomallei, F. tularensis and Y. pestis were performed in 25 µL of reaction volume comprising of final concentration of 1X PCR buffer, 5 mM of MgCl 2 , 0.25 mg/mL of bovine serum albumin, 0.25 mM of dNTPs, 0.6 µM of each forward and reverse primer, 0.025 µM of probe and 0.5 U of Taq polymerase along with DNA extracted from soil (10-30 ng). The cycling condition was as follows; one cycle of 95 • C for 5 min followed by 45 cycles each of denaturation at 94 • C for (FP, forward primer; RP, reverse primer) and probes used in the study were as described by Christensen et al. (2006). b this study. c Primers and probes used in the study were as described by Tozer et al. (2014). 5 s and annealing at 60 • C for 20 s; and then one cycle of cooling at 40 • C for 1 min. For detection of C. burnetii, the 25 µL of reaction volume contained 12.5 µL of qPCR mix (Thermo Scientific, USA), 10 pM of each primer and probe and 10-30 ng of template DNA. The cycling condition was as follows: 15 min incubation at 95 • C followed by 50 cycles of 95 • C for 15 s and 60 • C for 1 min. Diethylpyrocarbonate (DEPC)-H 2 O was used as a negative control in all the assays. DNA from the same extract underwent two independent RT PCR reaction for the studied pathogens. Soil samples that exhibited a positive RT PCR result were assayed a third time beginning from genome extraction and the PCR products (presence and size in bp) were assessed by agarose gel electrophoresis (3.0%) with the appropriate controls and a 50 bp ladder (Fermentas, Germany). Data Analysis A total of 145 samples from 29 villages in Lahore district were examined for the presence of five pathogens. Results of the RT PCR analysis, soil chemistry and seven risk factors for each sample was compiled in a single Microsoft Excel spreadsheet. The presence or absence of a pathogen in relation to soil chemistry and seven potential risk factors was determined through student t distribution (T-test) and odd ratio (OR), respectively. Soil chemistry values (average count) were compared using the Tukey-Kramer (equal variance) or Dunnett's T3 (unequal variance) procedures. These two procedures were used due to unequal sample sizes observed in the two categories of a given value. The Tukey-Kramer procedure performs all pair-wise comparisons, testing whether the means are significantly different. The Dunnett's T3 procedure performs all comparisons with a control category. P < 0.05 was considered significant. ORs were used to evaluate risk factors associated with pathogen. Observations to the seven risk factors were grouped by their categorical response (e.g., yes, no) to estimate if an observation had an influence on the presence or absence of the pathogen. For a potential risk factor, OR > 1 was considered to be associated with the outcome (presence or absence of pathogen) while it was considered vice versa for OR < 1. Confidence interval (CI, 95%) was used to estimate precision of the OR where a large CI value was considered a low level of precision and small CI was considered with higher precision. Statistical analyses related to soil chemistry were performed with SPSS (version17.0; SPSS Inc., Chicago, IL) and STATA (version 12.0, College Station, Texas, USA) while ORs were calculated through two by two frequency table available with OpenEpi version 2.3.1. Prevalence of DNA of Bacterial Pathogens in Soil Samples Collected in Lahore District A total of 29 of 301 villages in Lahore district were examined for soil-borne pathogens including B. anthracis, B. mallei/pseudomallei, C. burnetii, F. tularensis and Y. pestis. None of the soil samples were positive for DNA of Y. pestis. Soil samples from 25 of 29 (86.2%) villages and 40 of 145 (27.6%) sample sites were positive for DNA of atleast one of the four pathogens. DNA of B. anthracis (CapB), F. tularensis and C. burnetii was detected in 11 (37.9%), 14 (48.3%) and 4 (13.8%) of 29 villages, respectively. None of the samples were positive for the DNA encoding for protective antigen (pOX1). Burkholderia mallei/pseudomallei was detected in two of 29 (6.9%) villages and two of 145 (1. 4%) soil samples. Both these villages (Medhipur and Nasir Gurj) shared the same major roadway and were adjacent to a water canal ( Table 2). Soil Chemistry The pH of soil samples collected from different sites ranged from near neutral to alkaline (6.50-9.85). A total of 20 soil analytes were examined including moisture (0.50-42.24%) , soluble salts Table 3). The presence of B. anthracis (CapB) DNA was significantly associated with elevated levels of organic matter, chromium, cobalt and cadmium while it was significantly associated with low concentrations of magnesium, copper, manganese, sodium, ferrous, calcium and potassium. F. tularensis DNA was associated with low concentrations of phosphorous. Except cobalt, the presence of DNA for C. burnetii was associated with low concentration of magnesium, sodium, ferrous, calcium and potassium. None of the soil chemistry variables was found to be associated with presence of B. mallei/pseudomallei ( Table 4). Determination of Risk Factors Associated with Soil-borne Pathogens Risk factors including presence of domestic animals, distance between the sampling site and animal market, main road and water source, presence of ground cover, animal density and the number of households in the village were evaluated to determine if any of these factors could be associated with the presence or absence of the pathogen at the sampling site (Table 5). It was observed that B. anthracis (CapB) and F. tularensis were identified in four villages located along the Lahore-Multan road (Figure 1) Discussion Presumptive diagnosis of a disease condition in humans or animals is largely based on clinical manifestations of the disease for a given geographic area and its population. The process of disease diagnosis is greatly enhanced when there is prior history of a similar disease condition, and this is greatly augmented with accurate clinical, laboratory and epidemiologic data. Therefore, timely identification of the etiologic agent using a validated and approved diagnostic test is critical to developing and implementing the most responsive disease prevention and control practices for a given geographic location and population. Although in recent years, considerable efforts have been made to improve disease reporting, monitoring and surveillance in Pakistan; much needs to be done with respect to addressing emerging and re-emerging diseases. The Directorate of Animal Disease Reporting and Surveillance (ADRS) and Punjab Health Department in Punjab province of Pakistan periodically collect data on various disease conditions; however, the data collected is largely based upon clinical symptoms and/or with little laboratory diagnostic support. Further reporting for B. anthracis and B. mallei is exclusively done by ADRS. In our study, real time PCR was the test of choice for the following reasons: (a) culture-based methods for highly dangerous pathogens such as B. anthracis, B. mallei/pseudomaillei, C. burnetii, F. tularensis, and Y. pestis requires a highly contained laboratory and trained personnel as these bio-threat pathogens could pose catastrophic human and animal threat in the event there is a lapse in chain of custody and biosafety practices, (b) the RT PCR assays used in the study are validated and are of very high sensitivity and specificity as compared to conventional PCR, and (c) the RT PCR assays allows simultaneous examination of several samples with high throughout and rapid turnaround time. The RT PCR assay and protocol used in our study is highly sensitive (97%) with detection limit as low as <100 genome copies in a given sample and can be used for a variety of matrices such as tissue, blood, soil and other environmental samples (Christensen et al., 2006). The RT-PCR assay for C. burnetii targeted a multicopy gene (IS1111 gene) that has much greater sensitivity than single copy gene targets (Tozer et al., 2014). The use of manure, plowing during plantation season, or use of waste water and water from small canals used for irrigation of crops/fields could be attributed to prevalence of the soil-borne pathogens. Based on the observations of our study, it is difficult to derive a causal relationship between soil characteristics and the presence/absence of studied pathogens. However, the findings do provide some insights into the distribution of these pathogens in soils of the Lahore region. For instance, pH, organic matter, water activity (a w , available water within microenvironment), availability of oxygen, CO 2 /CO 3 and the presence of certain cations particularly calcium are considered important for the ecology, endemicity and virulence of B. anthracis in a given geographical area (Dragon and Rennie, 1995;Shen et al., 2002;Hugh-Jones and Blackburn, 2009;Koehler, 2009;Hammerstrom et al., 2011). While determining the historical distribution and molecular diversity of B. anthracis in Kazakhastan, Aikembayev et al. (2010) attributed higher frequency of disease outbreaks in southern and northern portions of the country to alkaline soil rich in organic matter than to central regions which are dominated by desert and where soil does not support the survival of spores. Although we found lower concentration of cations particularly calcium (1.61 mg/Kg or 1610 mEq/gram) at places where DNA to B. anthracis was detected than places where it was not detected, it was adequate to support sporulation and its survival in soil. For example, in an effort to determine a possible link between soil calcium and ecology of B. anthracis, Smith (personal communication 2003, http://www.oie.int/doc/ ged/D7115.PDF, page 12) concluded that areas with calcium (>150 mEq/gram) and pH (>7) had an incidence of disease occurrence seven time more than the places lacking these parameters. Anthrax has been considered endemic in northern Punjab districts such as Chakwal and Jhelum in particular, where sporadic cases do occur during the rainy season. Epp et al. (2010) concluded that within high-risk regions, flooding in spring followed by hot and dry conditions, wet pastures, short grass length and high animal density could result in the persistence and subsequent occurrence of outbreak. Based on our study it can be inferred that soil with alkaline pH and increased organic contents could be suitable for persistence of B. anthracis. Virulent strains of B. anthracis contain plasmid pXO1 and pXO2 that encode toxins and capsule, respectively (Fouet and Mock, 1996). In our study, DNA of B. anthracis that encodes for capsular gene (pXO2, capsular antigen CapB) was identified, while the plasmid for protective antigen (pXO1) was not detected. This could explain the presence of non-virulent type of B. anthracis in soil samples from Lahore district and could perhaps explain the lack of any documented evidence of anthrax cases in humans and animals. The river Ravi which originates from the Himachal Pradesh, India serves as the northwest border of Lahore district (Figure 1). Through recorded history, this fertile river basin has nurtured and supported civilizations and, even in modern times, is fundamental to agriculture, livestock and human habitation. In our study, soil samples from 14 and 19 sample sites were positive for DNA of B. anthracis and F. tularensis, of which 6 and 7 sampling sites were adjacent to a road way or a canal. Further, both the villages where DNA of B. mallei/pseudomallei was identified were exclusively located on "Lahore-Multan road." This time traveled highway serves as major interstate road that joins the river Ravi at several places of its course. The said road is key to transport people, animals, agricultural and industrial products to other regions of Pakistan. Certain places and villages around the road serve as animal holding areas, auction markets, butcher shops and rest/shelter area for animals and their herders. Many of the positive sample sites were close to private and government-owned slaughter houses as well as animal markets along/around the road. Annually, several thousand animals pass/sheltered for a day or two along the road as well as villages around it. These animals are either sold alive in a nearby animal market or slaughtered for meat. It was also observed that these locations lacked designated areas for waste disposal and animal refuse for slaughter houses in particular. The waste is dispersed into adjacent fields and canals and is being used to irrigate the agriculture land in villages around this interstate road. It is therefore not surprising that we identified pathogens even away from places with more frequent human-animal activity. It was also noted that suburban housing developments and well established industries bordered this road. Based on the findings of the study, it can be inferred that the soil in this area is subject to considerable perturbations through animal, human and industrial activities. The high frequency of detection of all major pathogens in this region of Lahore district is of particular concern to public health and puts human and animal populations at a higher risk of exposure to these pathogens. None of the soil samples showed the presence of DNA of Y. pestis, a vector-borne pathogen transmitted by rat fleas to humans. The long-term persistence of Y. pestis in soil and environmental factor contributing its survival is still yet to be fully understood. Perry and Fetherston (1997) reported that Y. pestis perishes quickly outside its host or vector, temperature exceeding 40 • C and exposure to desiccation. Ayyadurai et al. (2008) showed that Y. pestis can be isolated from hydrated soils. In our study, with the exception of May-July, the temperature in the Lahore district is typically below 40 • C and the soil remains hydrated through all seasons of the year. Historically, incidence of Y. pestis has been reported in coastal borders of sub-continent (India and adjoining areas). However, there has been no reported incidence of plague in the study area, which is consistent with the absence of Y. pestis soil DNA reported here. F. tularensis was detected in soil samples from 14 of the 29 villages. As observed with Anthrax, there are no reported incidences or records of disease outbreaks or cases suggestive of tularemia in Lahore district. The incidence of tularemia has been reported globally with varying relative virulence (Low/moderate/high) caused by the Francisella species or subspecies involved in the particular geography (Oyston, 2008). A number of cases of clinical infection and subsequent isolation as well as identification of F. tularensis has been reported from many countries of the Northern Hemisphere. Further, the strain isolated in North America has been found highly virulent as compared to the strains isolated from Europe or Asia (Sjöstedt, 2007;Oyston, 2008). Though it needs further molecular characterization at subspecies level in future, it is for the first time that DNA to F. tularensis has been detected in the environment from this part of the world and thus expanding its known range of occurrence worldwide. It has also been reported that F. tularensis may persist in the environment in a given geographical area without concomitant disease outbreaks (Sjöstedt, 2007). Several factors favor the persistence of F. tularensis in endemic areas and subsequent infection. These factors include climatic conditions, heat stress, limitation of potassium, cysteine/sulfur, CO 2 and iron that affects virulence and its survival in the soil (Olsufiev, 1966;Bernard et al., 1994;Deng et al., 2006;Sjöstedt, 2007;Lindgren et al., 2009;Alkhuder et al., 2010). Furthermore, Francisella spp. has been found to have affinity to low temperature, moisture, organic matter and hay/straw (Dennis et al., 2001). The role of potential reservoirs cannot be ignored where transmission of virulent strains (F. tularensis subspecies tularensis) is associated with rabbit, ticks and sheep, whereas, transmission of less virulent strain (F. tularensis subspecies holarctica) is associated with ponds, streams, lakes, river, and water associated species (Ulu-Kilic and Doganay, 2014). Interestingly, of the total soil sample examined, we found DNA of F. tularensis more at places <100 m of water canals/drains (n = 15/19, Table 5). Burkhoderia mallei and B. pseudomallei are shown to represent a single genomic species by DNA-DNA hybridization, however each exhibit distinct biochemical properties, epidemiology and manifest different clinical symptoms in humans and animals (Rogul et al., 1970;Coenye and Vandamme, 2003). BLAST analysis was performed for the B. mallei/pseudomallei primers and probe used in our study and they were found to be equally applicable to the detection of chromosomal gene of both species. Two soil samples collected along the "Lahore-Multan" road were positive for this pathogen. We anticipated that the likelihood of isolating or identifying this organism was much higher in this region of Lahore compared to other sites as this area has many horses and mule stables and is well traveled for goods/material transport by them. B. mallei has been isolated from Pakistan (Hornstra et al., 2009), and asymptomatic horses and mules could transmit this organism without being detected (Hornstra et al., 2009;Khan et al., 2013b). Burkhoderia mallei/pseudomallei has been shown to survive in the soils and artificial environments involved in animal husbandry such as water troughs (Coenye and Vandamme, 2003;Hornstra et al., 2009) that were observed to be abundant along the roads and in communal stables within our sampling region. To date, there have been no reported cases/outbreaks of Qfever in humans and animals in Lahore district. Even in the absence of a reported outbreak, detection of C. burnetii in soil (Kersh et al., 2010) or dust and aerosols is not unusual (Schulz et al., 2005;Astobiza et al., 2011). The clinical signs of Qfever mimic many other diseases with undifferentiated clinical symptoms including fever, nasal and chest congestion, myalgia, neuralgia, nasal and ocular discharges which, in the absence of a confirmed laboratory diagnosis, makes it very difficult to identify the disease (Tozer et al., 2014;Vanderburg et al., 2014). In conclusion, the findings of our study demonstrate the presence of DNA of B. anthracis, B. mallei/pseudomallei, C. burnetii, and F. tularensis in soil samples collected from Lahore district of Punjab Province. Although we observed an association between the concentration of certain soil analytes and the presence of soil-borne pathogens, a more comprehensive study with a larger sample size will be required to fully examine this observation. Based on the collective experience of the investigators involved in this study, a unified human and animal active and passive surveillance program is key to understand the prevalence and distribution of the pathogens in humans and animals in Pakistan. The surveillance program must be complemented with a contemporary national and region laboratory network system to detect and identify zoonotic diseases of public health importance.

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2019-04-12T13:54:34.591Z

2015-01-01T00:00:00.000Z

109461272

s2

Degradation of Acid Cyanide Poison in Rubber Seed (Hevea brasiliensis) after Treatment with Rice Husk Ash Rubber seed ( Hevea brasiliensis) contains protein (17.41 %) and non-essential amino acid cysteine (0.78 %) and acid cyanide poison (186.00 mg/kg). The purpose of this research was to determine the effect of rice husk ash on degradation of acid cyanide in rubber seed. This research used Completely Randomized Design (CRD) using treatment of rice husk ash concentration with 5 levels of treatments (45; 60; 75; 90; 105 %) and 4 replications. The result showed nonsignificant differences (p>0.05) in degradation of acid cyanide level during aging and significant differences (p<0.05) during soaking. Rubber seed treated with 90% of rice husk ash during aging period contained cyanide 47.25 ppm and rubber seed treated with 60% of rice husk ash during soaking period contained cyanide 40.37 ppm. Keywords— rubber seed, HCN, rice husk ash, aging, soaking. A wide variety of different methods to reduce HCN content was soaking, boiling, drying [8] and adsorbent [9]. The improving processing methods or a combination of them (peeling, slicing, soaking, boiling, drying etc.) could be reducing cyanide content of cassava [10]. The naturally occurring materials which are used as adsorbents such as the minerals, zeolites, clays and synthetic materials which include Al 2 O 3 , SiO 2 [11], biomass materials and agricultural by-products such as walnut waste, maize cobs, peanut shell, cassava waste, wheat bran, maize husk, coconut shell and bagasse [12]. Rice husk ash could be used as adsorbent to remove cyanide, good scavenger and lowcost [9]. Absorbent material can adsorb HCN poison on Gadung corm using ash of wood 15 % can reduce HCN by 63.78% [13]. Aging in 30% rice husk ash solution for 24 hour could reduce HCN Lindur fruit to 3.435 ppm [14]. Soaking of Cassava chips in water for about 24 h reduced 90% HCN [10]. The effect of rice husk ash on degradation of acid cyanide as a function of contact time, pH, adsorbate concentration and temperature [9]. A. Material The fresh rubber seeds used in this study were collected from rubber plantation in Sarolangun, Jambi, Indonesia. The samples were used for this experiment where from the same plantation. The seeds were stored in freezer (-10°C), until further required. Preparation of Adsorbent, the rice husk used was obtained from rice Mill in Sarolangun, Jambi, Indonesia. The rice husk was incineration from 2-3 hours until dark color and sieved with 60 mesh sieve. Then the husk ash was thoroughly stored in plastic bags and sealed. B. Method The research used Completely Randomized Design (CRD) consisted of five level of treatment of rice husk ash concentrations (45; 60; 75; 90; 105 %) in three processing technique and four replications. HCN Analyses were carried out using Spectrophotometry method [15]. C. Processing Techniques 1) Aging: A set of rubber seed (250 g) was boiled in distilled water (100°C) for 15 min. After boiling, the water was drained off, the boiled seeds were peeled and then aging with rice husk ash on different concentration for 24 hours and then wash in water. 2) Soaking: A set of rubber seed (250 g) was boiled in distilled water (100°C) for 15 min. After boiling, the water was drained off, the boiled seeds were peeled and then aging with rice husk ash on different concentration for 24 hours and then wash in water. Rubber seed soaked in 500 ml of water (1:2 w/v) for 24 hours for each level of treatment. 3) Boiling: A set of rubber seed (100 g) was boiled in distilled water (100°C) for 15 min. After boiling, the water was drained off, the boiled seeds were peeled and then aging with rice husk ash on different concentration for 24 hours and then wash in water. Rubber seed soaked in 500 ml of water for 24 hours for each level of treatment, then boiled (100±5°C) for 1.5 hour for each level of treatment. A. Aging The concentration of rice husk ash were not significantly differences (p>0.05) in degradation of HCN poison level in rubber seed, but the average value of HCN poison were decreasing by increasing of rice husk ash concentration (Table 1). Rice husk ash is adsorptive properties, contains strong base such as CaO (0.5-1.4%) and K 2 O (2.46-3.68%) [16]. It reacted with HCN and O 2 around it yielding CaCN and KCN salt which easily soluble in water. Carbon could extract cyanide from the samples then transfer thought the porous carbon and absorb in to the boundary carbon so it could reduce the cyanide concentration [17]. Means with the same superscript in the same column were not significantly different (p>0.05). B. Soaking Degradation of HCN poison level in soaking rubber seed were significantly differences (p<0.05). The average values of HCN poison were decreasing by increasing of rice husk ash concentration ( Table 2). The decreasing HCN on soaking in the water processing technique lowest than aging alone ( Table 2 and Table 1) Means with the same superscript in the same column were not significantly different (p>0.05). Rubber seed treated with 60%-105% of rice husk ash during soaking-period contained cyanide 40.37-30.54 ppm, it is under standard from BPOM as 53.76 ppm [7]. These values are lowest than the rubber seed treated with soaking in running water for 24 hours values of 52.60 ppm [18]. The soaking in water caused diffusion and osmosis process, which diluted the HCN in rubber seed and produced foam and opaque solution. Rubber seed during soaking process degraded HCN level because CaO + HCN reacted to CaCN + H 2 O. CaCN will be dissolved in water to decrease HCN poison level during soaking process. Aging rubber seed with rice husk ash followed by soaking in the water is better than aging alone in removing cyanide. C. Boiling Degradation of HCN poison level in boiling rubber seed were very significantly differences (p<0.01). The average values of HCN poison were decreasing by increasing of rice husk ash concentration ( Table 3). The decreasing HCN on boiling processing technique lowest than soaking and aging alone ( Table 2 and Table 1). Means with the same superscript in the same column were not significantly different (p>0.05). Rubber seed treated with 45-105% of rice husk ash followed soaking in the water and boiling contained cyanide 33.32-11.26 ppm, it is lowest than cyanide standard from BPOM as 53.76 ppm [7]. These values are lowest than the rubber seed treated with boiling for 10-20 minutes values of 71.78-54.15 ppm [18]. Ninety precent of free cyanide is removed within 15 minutes of boiling fresh cassava chips [19]. The adsorption of cyanide increases with increasing temperature [9]. HCN rubber seed boiled decreasing is influenced by aging rice husk ash in the previous stage in which the rice husk ash can inhibit the oxidation of toxic and carcinogenic nature neutralizes acid in the material [20]. Rice husk ash as well as an adsorbent for a porous material that can act as hydrolyzed crude fiber [9]. Processing with soaking in hot water it is one form of physical treatment that can be used to eliminate the HCN content because will reduce the activity of the enzyme so that the liberation of HCN linamarinase also be reduced. HCN can be easily lost by way of warming due to HCN is readily soluble in water and volatile [21]. D. Comparison HCN in Some the Processing Techniques Rubber seed not treated with rubber husk ash contained cyanide the highest than treated with rice husk ash in all processing technique (Fig. 1), contained cyanide 150.16 ppm (aging), 124.34 ppm (soaking), 101.28 ppm (boiling). It is also higher than standard from BPOM as 53.76 ppm [7]. Rubber seed treated with 90% of rice husk ash during aging contained cyanide 47.25 ppm. Rubber seed treated with 60% of rice husk ash followed soaking in the water contained cyanide 40.37 ppm. Rubber seed treated with 45% of rice husk ash followed soaking in the water and boiling contained cyanide 33.32 ppm. The addition of rice husk ash greatly affects the reduction of cyanide poison rubber seeds in the all-processing techniques. IV. CONCLUSIONS Rice husk ash decreased acid cyanide level in aging, soaking and boiling rubber seed. The concentrations of rice husk ash were significantly in degradation of HCN poison level in rubber seed soaking and boiling. Aging rubber seed with rice husk ash followed by soaking in the water and boiling is better than aging alone in removing cyanide. Rubber seed treated with 90% of rice husk ash during aging contained cyanide 47.25 ppm. Rubber seed treated with 60% of rice husk ash followed soaking in the water contained cyanide 40.37 ppm. Rubber seed treated with 45% of rice husk ash followed soaking in the water and boiling contained cyanide 33.32 ppm.

v3-fos

2018-04-03T01:49:28.506Z

2015-12-26T00:00:00.000Z

207277542

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Mercury bio-extraction by fungus Coprinus comatus: a possible bioindicator and mycoremediator of polluted soils? The Shaggy Ink Cap (Coprinus comatus), which is a common in wild in northern hemisphere was examined in field for potential to be used as possible bio-extractor of Hg from polluted grounds but also as possible bioindicator of urban soils (roadside, barren lands, lawns) pollution with Hg. The contents of Hg in caps and stipes of C. comatus from the grounds examined in this study correlated positively with the levels of soil contamination. Analysis of sets of data available worldwide on Hg in C. comatus and soils beneath-fruiting bodies showed on a positive correlation between degree of soil and mushroom contamination. Hence, C. comatus could be considered as a sensitive species and with bioindication and bioremediation potency for soils polluted with Hg in further studies. Young-fruiting bodies of C. comatus are edible and considered excellent if consumed soon after pick-up. Eating them when foraged from the urban places can provide to a consumer Hg at relatively high dose, while unresolved question is absorption rate of Hg compounds contained in ingested mushroom meal. Introduction Environmental pollution with heavy metals such as Cd, Hg, Pb, and their accumulation in soils due to the industrial activities, urbanization, and traffic is an ongoing process, and various efforts are undertaken to effectively reduce and eliminate the sources as well as to restore degraded grounds (McGrath and Zhao 2003;Xu et al. 2015). One of the biological techniques of soil restoring discussed in scientific literature is mycoremediation using macrofungi (Gadd et al. 2012). Macrofungi are well known for their ability to efficiently absorb various metallic elements and metalloids from the substrata and to sequester them in their fruiting bodies (Byrne and Tušek-Žnidarič 1990). Hence, fruiting bodies of edible and inedible mushrooms can be relatively rich in inorganic constituents, and data published on metals and minerals composition and content of macrofungi is much more when compared to that on their potential to bio-extract elements (Falandysz and Borovička 2013;Falandysz et al. 2001b;Tel et al. 2014). Nevertheless, practical solutions are lacking and fishing for most suitable species continues. Recently, it has been shown in a pot study that Coprinus comatus in presence of chelating agents such as ethylenediaminetetraacetic acid (EDTA) or nitrilotriacetate (NTA) very efficiently take-ups of Cd, Cu, and Pb from soil (Cen et al. 2012), and similar study with added chelators has been conducted for Tricholoma lobayense Heim (Wang et al. 2012). A potential for bio-extraction of Pb from low-polluted soils has been shown for Oudemansiella radicata (Zhang et al. 2012). Mercury is a particular example of environmental and food toxicant because of high toxicity and biomagnification of methylmercury (MeHg) in food chains. Mercury, because is highly volatile could be emitted due to any high temperature process (e.g., combustion of biomass or waste, production of cement, metal ore refining). At the local or regional scale, soil can become a hot spot polluted with Hg because of fumes from the nonferrous metals producing facilities, use of the Hg compounds as the catalyst in an organic chemicals manufacturing, or an improper storage and disposal of the Hg containing materials, products, and wastes (Hu and Cheng 2012). Hence, both identifications of current and forgotten places polluted with this element as well as development of remediation techniques are the actual needs. As mentioned, an exceptional feature of mushrooms when compared to vascular plants is efficient accumulation by them of mercury (Cibulka et al. 1996), and some examples are available on bioconcentration of Hg by several species both the ectomycorrhizal and saprophytic mushrooms (Falandysz 2002;Falandysz et al. 2001aFalandysz et al. , 2002aFalandysz et al. , b, 2003aFalandysz et al. , b, c and 2014bKrasińska and Falandysz 2015a, b;Melgar et al. 2009;Nasr and Arp 2011;Rieder et al. 2011). The rates of transfer (bioconcentration) of Hg sequestered in fruiting bodies by mushrooms depend on species and decrease with increasing soil/substratum Hg content because of limited natural capacity for accumulation (Bargagli and Baldi 1984;Falandysz et al. 2012;Falandysz and Drewnowska 2015a), while some species will tolerate highly polluted grounds because of cinnabar mining and can accumulate Hg at remarkably great concentration (Árvay et al. 2014). Also, mushrooms with deeper mycelia when emerged from red and yellow lateritic soils enriched with Hg in the mineral layer because of occurrence of the Circum-Pacific Mercuriferous Belt could accumulate Hg in fruiting bodies at highly elevated concentration (Falandysz et al. 2015a, b;Kojta et al. 2015;Wiejak et al. 2014). Nevertheless, this is difficult to find in nature a such good example positive relationship, at local scale but exceptions probably happen if there is diversity of Hg concentration in soil substrate to a given species of mushroom. For example, when studding mercury (Hg)-total Hg for some mushrooms and places, a good correlation has been found between soil (substrata) Hg and mushroom Hg for Macrolepiota procera (Falandysz and Chwir 1997). The same was for methylmercury (MeHg) and a set of four samples (three individuals of Boletus (Xerocomus) badius -current name Imleria badia and one of Leccinum scabrum) form abandoned Hg mine (Fischer et al. 1995) but this was not exactly the same for inorganic Hg. A layer(s) in soil where mycelia lives and layer(s) in soil where Hg is contained and available matter. Deposition of airborne mercury from a long-range transport in pristine region of the Himalayan size Mountain Gongga (Minya Konka) in the eastern Tibetan plateau is the only explanation for elevated Hg content in the Gymnopus erythropus and Marasmius dryophilus, which both have shallow mycelia (Falandysz et al. 2014a). In a case of mushrooms that have deeper mycelia a diversification of Hg contents, as observed for several species of Leccinum and Boletus mushrooms from the Yunnan Province in China, could rather reflect the local/regional differences of Hg in the mineral layer of soil due to the geochemical anomalies but not because of fallout from the anthropogenic emissions from the beginning of the era of industrialization of the world (Falandysz et al. 2015a, b). Coprinus comatus (O.F. Müll.) Pers. that is commonly named as Shaggy Ink Cap, Shaggy Mane, Shaggy Parasol, or Lawyer's Wing and is popular saprophytic mushroom in moderate climate, and its young-fruiting bodies are edible (Lassoe et al. 1996). This mushroom can be found emerging from the ground on the city lawns, abandoned grounds, parks, along the roads, and waste areas, and hence is rather rare example of edible wild mushroom that survives unhostile urban condition and can be picked-up both in the cities and in villager areas. This species is picked up by some consumers even if emerged at grounds in populated city. The C. comatus is also cultivated in China as food. Aim of this study was to examine, if the Shaggy Ink Cap, which is a common in wild in northern hemisphere, has any potential to be used as possible bio-extractor of Hg from polluted grounds but also as possible bioindicator of urban soils (roadside, barren lands, lawns) pollution with Hg. Also, estimated was the possible intake rate of total mercury by consumers of C. comatus at the region investigated. Data available on Hg accumulated in C. comatus from Europe and Asia were summarized. Materials and methods The specimens of fruiting bodies of C. comatus and topsoil (0-10-cm layer) samples beneath to them were collected at several sites from an area of the town of Kartuzy (14866 people) in Kaszuby region of the Pomerania land in the northern part of Poland in 2011 ( Fig. 1; Table 1). Two sampling places assigned respectively with number 1, and two other with number 2 (Fig. 1) were considered as of the similar character. Hence, mushrooms and soil samples collected at two places with number 1 were integrated to make composite samples, and the same was in the case of material collected at the places assigned with number 2. The region of Kartuzy is surrounded by farmland, woodland and lakes, while apart from tourism in summer an industrial activity is little and restricted to small wood processing facilities and processing of crops. A Byoung^(white) of edible quality fresh-fruiting bodies, after clean up with a plastic knife from any visible plant vegetation and soil substrate debris and bottom part of the stipe was cut off, was separated into two parts-cap and stipe. Next, they were in situ sliced and dried (initially in ambient temperature for 0.5-2 h) and further were placed into plastic basket of an electrically heated commercial dryers (dehydrator for mushrooms, fruits, vegetables and herbs; model: MSG-01; MPM Product, Milanówek, Poland) and dried at 65°C to constant mass. Dried mushrooms parts were respectively pooled and pulverized in a porcelain mortar and kept in brand new sealed polyethylene bags under dry conditions. The soils (0-10-cm layer) and litter samples free of any visible organisms, small stones, sticks, and leaves were air dried at room temperature for several weeks under clean condition. Next, the soil samples were sieved through a pore size of 2-mm plastic sieve and sealed in brand new polyethylene bags and kept under dry and clean condition. Mushrooms and soil samples were pooled, respectively (Table 1). Mercury was determined using a direct sample matrices thermal decomposition and cold-vapor atomic absorption spectroscopy (CV-AAS; Mercury analyzer type MA-2000, Nippon Instruments Corporation, Takatsuki, Japan) Nnorom et al. 2013). The accuracy of the method was evaluated and further controlled by examination of the fungal-certified reference materials (CRM) and reagent blanks (both with every set of 5 fungal or soil samples). The CRM fungal material used were driedfruiting bodies of Cow Bolete (Suillus bovinus; code: CS-M- Fig. 1 Localization of the sampling places of C. comatus and soils in the town of Kartuzy (54°20'06" N and 18°12'05" E; 1-5) and elsewhere: Kczewo (54°23'00" N and 18°20'00" E; 6), Pępowo (54°38'66" N and 18°40'23" E; 7) and Leźno (54°21'0" N and 18°26'0" E; 8) in Pomerania land in northern part of Poland (Google maps; color figure available in online version) Samples and total number of fruiting bodies per place (in parentheses) a numeration of the sampling place (see Figs 1 and 2) b number of pooled 1) and the Basma 5 tobacco leaves code: INCT-OBTL-5, both produced by the Institute of Nuclear Technology and Chemistry (ICHTJ), Warsaw, Poland). The content of Hg in CS-M-1 declared by the producer is 0.174 ± 0.018-mg kg −1 dry matter (dm), while our measurements obtained in separate trials showed 0.168 ± 0.009 mg kg −1 dm (n = 5). Declared content of Hg in INCT-OBTL-5 is 0.021 ± 0.001-mg kg −1 dm, and our result showed 0.020 ± 0.006-mg kg −1 dm (n = 5). For mushrooms and the soil substrates, the limit of detection was 0.005-mg kg −1 dm, and the quantification limit was 0.0015-mg kg −1 dm. One blank sample and one certified reference material sample were examined with each set of 3-10 samples studied. Intake rate of total mercury was estimated based on median the values of mercury concentrations noted in fruiting bodies, possible intake rates of mushroom, and a provisionally tolerable intake limits of element to adult human. The computer software Statistica, version 10.0 (Statsoft Polska, Kraków, Poland), was used for statistical analysis of data and for graphical presentation of the results of twodimensional multiple scatter plot relationships between the variables. Results and discussion The Shaggy Ink Cap seems to be a sensitive boindicator of urban soils pollution with Hg that is efficiently sequestered by this species in fruiting bodies-both caps and stipes ( Table 2). The median values of BCF from the stand where topsoil showed Hg at 0.13-mg kg −1 dm (no 3, Fig. 1; Table 1) reached up to 73 for caps and 30 for stipes. Also, Hg content of fruiting bodies from that stand was high and median value was 9.2mg kg −1 dm in caps and 5.2-mg kg −1 dm in stipes, An elevated content of Hg in topsoil and hence also in flesh of mushrooms at the stand no 3 could be explained by character of the ground usage there, where a scrap-heap and recycling company is active from a year. At two other places in Kartuzy (nos. 2 and 4, Fig. 1; Table 1), the median values of Hg in caps were between 2.5 and 3.0-mg kg −1 dm (Table 2), which are values within a range of the concentrations determined in C. comatus from several urban places in Europe (Table 3). At the places west (no 1) and north (no 5) of the center of the town (Fig. 1)fruiting bodies of C. comatus were less contaminated, while much less were the mushrooms and soil collected in outskirts of two villages east of the town (nos. 6 and 7). At the Leźno Table 2). The geochemical background value of Hg (total Hg) suggested for soils of Poland is 0.05-mg kg −1 dm (PIG 2013), while the forest soils did contain in a top 0-10-cm layer much less of Hg than 0.05-mg kg −1 dm Drewnowska and Falandysz 2015). In view of those figures, the soil sampled in the town of Kartuzy as well as other places (Fig. 1) can be considered as more or less contaminated with Hg from the anthropogenic emissions. Finland (n = 2) # 5.6 (1.5-9.8) Laaksovirta and Lodenius 1979 Finland, rural (n = 3) 2.7 (2.5-2.9) Kuusi et al. 1981 Finland, urban (n = 37) 4.7 (0.68-17) Kuusi et al. 1981 Finland, urban (n = 55) 3.8 (1.4-10) Lodenius et al. 1981 Finland, lead processing area (n = 2) 2.1 (0.6-3.5) Liukkonen-Lilja et al. 1983 Finland (n = 1) 6.7 Kojo and Lodenius 1989 Germany (n = 6) 1. The contents of Hg in caps and stipes of C. comatus in this study correlated positively with the levels of soil contamination (Fig. 2). Earlier studies showed that C. comatus has a potential to accumulate mercury in fruiting bodies, e.g., for specimens from the cinnabar (HgS) mining place in Germany with Hg in soil at 83-mg kg −1 dm, the content of Hg in two fruiting bodies was at 144-mg kg −1 dm, while for cinnabar mining place in Italy with Hg in soil at 210-mg kg −1 dm, the content of Hg in a single fruiting body was much less, i.e., at 23-mg kg −1 dm. Also high was Hg content in C. comatus emerged at the urbanized places in Finland, which showed up to 17-mg kg −1 dm (Table 3). In one study, the C. comatus, when compared to inorganic Hg has much better potential for bioconcentration of MeHg with a value of BCF reaching 198 (Fischer et al. 1995). Analysis of all sets of data available on Hg in C. comatus and soils (Tables 2 and 3) beneath fruiting bodies showed on a positive correlation between degree of soil and mushroom contamination. Hence, C. comatus can be considered as a sensitive species and with bioindication potency for soils polluted with Hg. As mentioned in the introductory section, young fruiting bodies of C. comatus are edible and considered excellent if consumed soon after pick-up, while kept in ambient temperature for longer period undergo auto digestion. Given data showed in Tables 2 and 3, the specimens of C. comatus emerged in urban soils can be considerably or highly contaminated with mercury. Eating them can provide to a consumer Hg at relatively high dose, while unresolved question is absorption rate of Hg compounds contained in ingested mushroom meal. Mercury is largely retained in flesh of culinary processed (blanched) mushrooms (Falandysz and Drewnowska 2015b), and this phenomenon seems to be more or less a species-specific feature, and if based on a dry matter content, no loss or even a pseudo-enrichment of Hg in a blanched mushrooms could be observed when compared to substrate product (unpublished, JF). Retention of Hg in blanched mushrooms implies on its occurrence in large portion in form of species hardly soluble in water (HgS, HgSe), and this may suggest on limited absorption of Hg contained in a mushroom meal from the alimentary tract.

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Recessive Resistance Derived from Tomato cv. Tyking-Limits Drastically the Spread of Tomato Yellow Leaf Curl Virus The tomato yellow leaf curl disease (TYLCD) causes severe damage to tomato (Solanum lycopersicum L.) crops throughout tropical and subtropical regions of the world. TYLCD is associated with a complex of single-stranded circular DNA plant viruses of the genus Begomovirus (family Geminiviridae) transmitted by the whitefy Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae). The tomato inbred line TX 468-RG is a source of monogenic recessive resistance to begomoviruses derived from the hybrid cv. Tyking F1. A detailed analysis of this germplasm source against tomato yellow leaf curl virus-Israel (TYLCV-IL), a widespread TYLCD-associated virus, showed a significant restriction to systemic virus accumulation even under continuous virus supply. The resistance was effective in limiting the onset of TYLCV-IL in tomato, as significantly lower primary spread of the virus occurred in resistant plants. Also, even if a limited number of resistant plants could result infected, they were less efficient virus sources for secondary spread owing to the impaired TYLCV-IL accumulation. Therefore, the incorporation of this resistance into breeding programs might help TYLCD management by drastically limiting TYLCV-IL spread. Introduction The tomato yellow leaf curl disease (TYLCD) is one of the major yield-limiting factors of tomato (Solanum lycopersicum L.) crops in tropical and subtropical regions [1,2]. This devastating disease is caused by a complex of single-stranded circular DNA plant viruses of the genus Begomovirus (family Geminiviridae) that are transmitted in a persistent circulative manner [3] by whitefly (Hemiptera: Aleyrodidae) members of the Bemisia tabaci Gennadius cryptic species. Among them, the monopartite tomato yellow leaf curl virus (TYLCV) is the most widespread and economically important worldwide [4]. Tomato plants affected by TYLCD exhibit characteristic symptoms of stunting, yellowing, upward curling of leaves, and suffer premature dropping of flowers and reduction of marketable fruits that can result in 100% yield loss when infections occur during early growth stages [1,5]. The control of TYLCD is often based on intensive chemical treatments to limit vector population and/or by using physical barriers, both with limited success. In addition, chemical control result in deleterious environmental effects and can determine selection of insecticide-resistant B. tabaci populations [6,7]. Therefore, in this scenario, the use of resistant tomato cultivars is the most environmentally sustainable and economically viable approach to reduce TYLCD damage. Susceptibility to TYLCV requires virus replication in the plant cell nucleus via a double-stranded DNA intermediate, movement to adjacent cells through plasmodesmata, long-distance movement through the phloem, and further acquisition by vectors for transmission from plant to plant to reinitiate the infection cycle. A block at any of these steps, either by active defense responses or by incompatible interactions of viral and host factors, may lead to virus resistance [8]. A number of virus resistance factors have been derived from wild tomato relative species that can help restricting TYLCD-associated virus infection and limit disease damage [9]. The partially dominant Ty-1 resistance gene derived from the S. chilense accession LA1969 [10] is so far the most widely used commercially. However, the performance of Ty-1 or other plant host resistance genes varies depending upon the TYLCD-causing virus [11,12]. Also, breakdown of Ty-1 resistance can occur under high inoculum pressure [13] and it often shows lower effectiveness in heterozygous plants used commercially [14]. Furthermore, emergence of recombinant TYLCD-viruses with novel pathogenic characteristics [15,16] might pose a threat to available resistance genes. Recently, we reported an alternative tomato source of begomovirus resistance named TX 468-RG, which was derived via selfing from the commercial F 1 hybrid "Tyking" (released by Royal Sluis, The Netherlands). This inbred line displayed high levels of resistance to bipartite and monopartite begomoviruses associated to TYLCD based on single recessive gene control [17,18]. A previous work allowed us to clarify the genetic control of the TX 468-RG resistance and to demonstrate its effectiveness to a range of TYLCD-associated monopartite begomoviruses [17]. However, the resistance mechanism remains unsolved. In the present study, a detailed set of analyses was conducted to understand the restriction to accumulation of an isolate of the Israel strain of TYLCV (TYLCV-IL) in TX 468-RG and to evaluate to what extent this resistance can help to limit virus spread under field conditions. We conclude that although no effect in inoculated leaves was observed, systemic infection of TYLCV-IL was impaired in TX 468-RG plants, resulting in reduced virus accumulation. As a consequence, TX 468-RG resistance was effective to limit primary and secondary spread of the virus. Therefore, this resistance is highly recommended for breeding purposes to control damage caused by TYLCV-IL in tomato. Tomato Plants, Virus Isolate, and Whitefly Population A tomato F 8 inbred line (named as "TX 468-RG") was derived via repeated selfing and selection steps from the commercial F 1 hybrid "Tyking" (released in the 1990s by Royal Sluis, The Netherlands) and used in all assays. This germplasm displayed high levels of resistance to bipartite begomoviruses [18] as well as to a range of TYLCD-associated monopartite begomoviruses [17]. The open-pollinated tomato cv. Moneymaker (MM) (IHSM-UMA-CSIC seed bank) was used as susceptible control in the experiments. The infectious clone of the isolate [ES:Alm:Pep:99] of TYLCV-IL (TYLCV-IL [ES:Alm:Pep:99], from now on, TYLCV-IL) (GenBank accession number AJ489258), has been described elsewhere [19]. Healthy B. tabaci adult individuals were obtained from a colony of the Mediterranean (MED) species (formerly known as Q biotype) originated from field individuals collected in Malaga, Spain. Whiteflies were reared on melon (Cucumis melo L. cv. ANC42, IHSM-UMA-CSIC seed bank) plants within wooden cages covered with insect-proof nets, in an insect-proof glasshouse with temperature control (22-27 • C day and 17-20 • C night) and supplemental light when needed. Virus Inoculation Agrobacterium tumefaciens-mediated stem puncture inoculation (agroinjection) with TYLCV-IL was conducted on plants at the three-leaf growth stage as described previously [20]. Also, for TYLCV-IL local accumulation studies, A. tumefaciens-mediated leaf tissue infiltration (agroinfiltration) studies were conducted following Tomás et al. [21]. Either for agroinjection or for agroinfiltration, bacterial suspensions at OD 600 = 1.0 were used. For B. tabaci-mediated inoculation, viruliferous whiteflies were obtained by providing insect adults with a 48 h acquisition access period (AAP) on systemically infected young leaves of MM plants agroinjected with TYLCV-IL three weeks earlier. For graft inoculation, healthy scions of TX 468-RG and MM were grafted onto MM and TX 468-RG plants previously infected with TYLCV-IL by agroinjection. Scions consisted of a stem piece containing a leaf with its associated lateral shoot meristem obtained from healthy test plants. Grafts of healthy MM or TX 468-RG scions were made on both MM and TX 468-RG infected rootstocks by splice-grafting. The grafted plants were then kept in a shaded and humid environment within a growth chamber for seven days and then moved to the insect-proof glasshouse (see below) until evaluation . Evaluations of TYLCD symptom and presence/accumulation of TYLCV-IL in young developing leaves of the scions was done at 28 days post-grafting by visual observation and by tissue blot and dot blot hybridization analyses of individual plants, respectively. Plant inoculations were performed in a growth chamber (25 • C day and 20 • C night, 70% relative humidity, with a 16 h photoperiod at 250 µmol· s −1 · m −2 photosynthetically active radiation), and the inoculated plants were then kept until analyzed in an insect-proof glasshouse with temperature control (approximately 16 h day length, at 22 to 27 • C during the day and 17 to 20 • C at night) and supplemental light when needed. Primary and Secondary TYLCV-IL Spread Experiments Primary and secondary spread experiments were conducted at "La Mayora" Experimental Station (Malaga, southern coastal Spain) essentially as described by Rodríguez-López et al. [22]. Briefly, primary spread of TYLCV-IL, i.e., virus spread to healthy plants from external source of viruliferous vectors [23], was simulated in medium-scale experiments conducted within insect-proof net, walk-in structures Virus Detection and Determination of TYLCV-IL Accumulation Levels in Infected Tomato Plants Virus presence was analyzed in young leaves of inoculated plants by tissue-blot hybridization of freshly cross-sectioned leaf petioles or by Southern blot hybridization of total nucleic acid (TNA), according to Tomás et al. [21] using a probe specific to TYLCV-IL. To monitor virus accumulation in young leaf tissues of TX 468-RG and MM plants inoculated with TYLC-IL, dot blot hybridization analyses were conducted. For this, TNA extracts were prepared from the second leaf from the apex, as described by Celix et al. [25], except that TNA were dissolved in 50 µL of sterile H 2 O in the final step. TNA extracts were quantified, the concentrations standardized at 200 ng/µL, and used for dot blot hybridization. For estimation of TYLCV-IL accumulation levels, different groups of inoculated plants were sampled in a destructive manner at 7, 14, 21, and 28 days post inoculation (dpi) (about 14 plants per evaluation date). Two microliter per sample of each dilution of a dilution series 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, and 1:512 of TNA were applied to positively charged nylon membranes (Roche Diagnostics GmbH, Mannheim, Germany). Two replicated membranes were prepared for DIG-labeled DNA-probe hybridization, one for hybridization with a probe to TYLCV [26] and the other with a probe to a gene fragment coding for 18S ribosomal RNA (18S rRNA) [27], as loading control. Healthy MM plants were used as negative controls. Assessment of TYCLV-IL DNA content was done by densitometric measure of hybridization signals obtained in autoradiographs. Plant ribosomal RNA signals were used as an internal standard to equilibrate TNA loading among samples. Densitometry measurements were expressed in pixels measured using Quantity One Software v 4.6.7 (VersaDoc MP 4000 Imaging System; BioRad), giving an arbitrary value of 1000 to that of one of the MM infected plants per date analyzed, and referring the other values to this one. In every case, the densitometry values used for estimation of TYLCV-IL accumulation levels were those from a dilution that fell within the linear range of the relationship between dilution and densitometry measure. Distribution of values was represented by Box-and-Whisker plots [24]. Data Analysis Statistical effects of genotypes and/or treatments in the different experiments were analyzed with the IBM SPSS Statistics v. 22 software by applying Generalized Linear Models (GzLM), in which all possible pair-wise comparisons were performed using the sequential Bonferroni method for error correction. For the case of comparisons of the TYLCV-IL accumulation levels estimated, the GzLM used Logarithm as the link function and Normal as the underlying distribution. For TYLCV incidence in the primary and secondary spread experiments, data set was expressed as the number of infected and non-infected plants at each time point and were analyzed by GzLM using Logit as the link function and Binomial as the underlying distribution. Also, a general disease incidence pressure was estimated for combinations of genotypes and treatments in those experiments by calculating the Area Under the Disease Progression Curve (AUDPC) using the formula AUDPC = Σ((Yi + Y(i+1))(T(i+1) -Ti))/2, where Yi = proportion of infected plants at date i, and Ti = time (in days) at date i. For comparing means of AUDPC values, the GzLM used Identity as the link function and Normal as the underlying distribution. Finally, for comparison of the number of whiteflies visiting the two tomato genotypes in the preference experiment, whiteflies per plant data were analyzed by GzLM using Logarithm as the link function and Negative Binomial as the underlying distribution. TYLCV-IL Accumulation is Highly Restricted in TX 468-RG Systemic TYLCV-IL infection was observed in all inoculated MM plants, whereas lower incidence levels were observed in TX 468-RG plants, especially at initial evaluation dates (see number of plants infected vs. number of plants inoculated at the bottom of boxes, Figure 1A). Also, no symptoms were observed in any TX 468-RG-infected plant, whereas typical TYLCD symptoms were observed in all infected MM plant from 21 dpi. Interestingly, viral DNA accumulation in infected plants revealed a strong restriction in TX 468-RG. Although viral DNA could be detected in young tissues of a number of these plants, confirming that they are not immune (sensu [28]) to TYLCV-IL, significantly lower accumulation levels were observed ( Figure 1A) when compared to plants of the susceptible MM control (see e.g., dot blot hybridization for two representative plants from the two genotypes at 28 dpi in Figure 1B). Thus, at 28 dpi, about eight times lower viral DNA accumulation was detected in young tissues of systemically infected TX 468-RG than in the equivalent ones of MM plants. By contrast, no major differences in accumulation levels of viral forms derived from the input DNA was observed at local agroinfiltrated tissues of TX 468-RG and MM plants (Figure 2), suggesting that constraints at initial steps of viral infection does not seem to be the basis of the resistance mechanism. Therefore, differences in virus accumulation might occur during the systemic infection process. To investigate whether virus resistance in TX 468-RG occurred during the process of systemic virus translocation, grafting of TX 468-RG and MM healthy scions onto TYLC-IL-infected rootstock were analyzed. Although tissue blot hybridization is not a quantitative assay, the results shown in Figure 3 strongly suggest that resistance in TX 468-RG impaired TYLC-IL accumulation during systemic translocation and was operative even under a continuous virus supply. Thus, only traces of virus were observed in all TX 468-RG scions either grafted onto MM or TX 468-RG rootstocks infected with TYLCV-IL, supporting that the level of virus accumulation in this genotype was not related to the amount of inoculum supplied. Also, no TYLCD symptoms were ever observed in any of the TX 468-RG grafted scions. In contrast, severe symptoms and higher virus accumulation was observed in MM scions grafted either onto MM or onto TX 468-RG rootstocks infected with TYLCV-IL, even if in the latter case low virus titers occurred in the rootstock (see Figure 1). Therefore, the begomovirus-resistance present in TX 468-RG was able to impair the TYLCV-IL systemic translocation within the plant even under high viral loads. TX 468-RG Results in a Reduced Primary Spread of TYLCV-IL The results of the study conducted to estimate the effect of plant genotype on primary virus spread showed that significantly lower incidence levels were observed in TX 468-RG in relation to MM, either in choice or in no-choice conditions in all the three independent experiments conducted ( Figure 4). Over 80% incidence was achieved at 28 dpi in MM, whereas about half of this value was observed for TX 468-RG. The resistant line also displayed a significant delay in the onset of the infection with a significantly reduced number of infected plants at 10 dpi. All this resulted in significantly lower AUDPC values for TX 468-RG when compared to MM (5.2 ± 0.3 vs. 18.3 ± 0.6 for no-choice and 2.7 ± 1.4 vs. 18.4 ± 0.9 for free-choice) and, therefore, lower disease pressure in TX 468-RG under our experimental conditions. Thus, the use of TX 468-RG-derived resistance would result in a significantly reduced primary spread of TYLCV-IL and lower virus pressure under field conditions. TX 468-RG Limits Secondary Spread of TYLCV-IL from Infected Source Plants to either TX 468-RG or MM Because the previous results indicated that TYLCV-IL could infect TX 468-RG plants during virus spread, secondary spread of TYLCV-IL to both TX 468-RG and MM healthy test plants was assessed. As summarized in Figure 5, the results of the three independent experiments conducted clearly showed that significantly lower secondary spread occurred when TX 468-RG was present, as source and/or test plant, with no infections observed when this genotype was used as source and test plant. Therefore, theTYLCV-IL resistance of TX 468-RG is strongly effective in reducing the secondary spread of this virus. As a result, a significantly lower infection pressure occurred in test plants (AUDPC 16.0 ± 2.9 for secondary spread from MM to MM vs. 5.5 ± 1.3, 0.8 ± 0.8, and 0, for secondary spread from TX 468-RG to MM, MM to TX 468-RG, and TX 468-RG to TX 468-RG, respectively). Therefore, lower TYLCD infection pressure under field conditions is expected when using this resistance. B. tabaci Exhibited no Preference for either MM or TX 468-RG No significant difference in whitefly preference for either MM or TX 468-RG tomato genotypes was observed based on the data obtained from the no-choice preference experiment (MM, 9.5 ± 1.4 vs. TX 468-RG, 8.3 ± 1.3 whiteflies per plant; p = 0.553) ( Figure 6). Therefore, results from spread experiments were not influenced by differences in vector behavior depending on the tomato genotype. Discussion The use of tomato cultivars with genetic resistance has been the most effective strategy to minimize losses caused by viral diseases, including pathosystems involving Begomovirus species [9]. The current study advances our understanding about the benefits of using virus resistant genotypes to limit TYLCV spread in the field. A compatible virus interaction with a plant involves effective multiplication and movement of the virus from the site of infection and throughout the plant as well as effective transmission to other plants to guarantee maintenance in nature [29]. However, during the virus/host plant co-evolution process, distinct host plant mechanisms to restrict virus infection can be selected and sources of resistance can become predominant in natural plant populations under continuous disease pressure due to their selective advantages [28]. Here, we demonstrated that the previously reported monogenic recessive resistance against TYLCV-IL present in TX 468-RG [17] strongly impairs systemic virus infection, resulting in a significantly reduced primary and secondary spread of the virus. Therefore, this resistance is proposed as a good and effective alternative for breeding purposes to reduce TYLCD damage in commercial tomatoes. selective advantages [28]. Here, we demonstrated that the previously reported monogenic recessive resistance against TYLCV-IL present in TX 468-RG [17] strongly impairs systemic virus infection, resulting in a significantly reduced primary and secondary spread of the virus. Therefore, this resistance is proposed as a good and effective alternative for breeding purposes to reduce TYLCD damage in commercial tomatoes. Figure S1. Previous observations for the usefulness of TX 468-RG resistance to control TYLCV-IL [17] were confirmed by the detailed studies conducted here. This is important because TYLCV-IL is one of the most widespread TYLCD-associated virus worldwide [4] causing severe economic losses to tomato production where present. TX 468-RG plants, however, are not immune and can be infected with this virus. Nevertheless, we show here that a severe restriction to systemic virus accumulation occurred in infected plants harboring this recessive resistance. Leaf agroinfiltration experiments demonstrated that local accumulation of TYLCV-IL was not impaired in TX 468-RG plants, suggesting no effect on the initial steps of infection and that the restriction occurred during the systemic infection process [30]. Based on our studies, however, we cannot rule out whether the latter restriction is due to impairment of viral movement and/or to the triggering of plant defenses, such as gene silencing [31]; further research will be needed to address this issue. Similar restriction to systemic infection was shown to be conferred to another monopartite begomovirus, Tomato leaf curl virus by the recessive resistance tgr-1 gene present in the line FLA-653 [32], derived from a cross between the resistant genotypes "Tyking" and S. chilense LA2779. In this case, however, Bian et al. [32] observed that in addition to a limitation of long-distance translocation a restriction also occurred at local level, which was associated with impaired cell-to-cell movement of the virus. Restriction to systemic virus accumulation has also been reported for the cultivar "Tyking" when confronted by other begomoviruses [33]. Interestingly, we showed that similarly to that reported for the breeding tomato line TY172 resistant to TYLCV [34], the plant defense mechanism operating in TX 468-RG against TYLCV-IL is not overcome by continuous supply of high loads of virus. Therefore, contrary to that observed for the Ty-1 gene widely used commercially [13], effectiveness of the recessive resistance studied here is expected even under high disease pressure, in accordance with preliminary field studies [17]. Previous observations for the usefulness of TX 468-RG resistance to control TYLCV-IL [17] were confirmed by the detailed studies conducted here. This is important because TYLCV-IL is one of the most widespread TYLCD-associated virus worldwide [4] causing severe economic losses to tomato production where present. TX 468-RG plants, however, are not immune and can be infected with this virus. Nevertheless, we show here that a severe restriction to systemic virus accumulation occurred in infected plants harboring this recessive resistance. Leaf agroinfiltration experiments demonstrated that local accumulation of TYLCV-IL was not impaired in TX 468-RG plants, suggesting no effect on the initial steps of infection and that the restriction occurred during the systemic infection process [30]. Based on our studies, however, we cannot rule out whether the latter restriction is due to impairment of viral movement and/or to the triggering of plant defenses, such as gene silencing [31]; further research will be needed to address this issue. Similar restriction to systemic infection was shown to be conferred to another monopartite begomovirus, Tomato leaf curl virus by the recessive resistance tgr-1 gene present in the line FLA-653 [32], derived from a cross between the resistant genotypes "Tyking" and S. chilense LA2779. In this case, however, Bian et al. [32] observed that in addition to a limitation of long-distance translocation a restriction also occurred at local level, which was associated with impaired cell-to-cell movement of the virus. Restriction to systemic virus accumulation has also been reported for the cultivar "Tyking" when confronted by other begomoviruses [33]. Interestingly, we showed that similarly to that reported for the breeding tomato line TY172 resistant to TYLCV [34], the plant defense mechanism operating in TX 468-RG against TYLCV-IL is not overcome by continuous supply of high loads of virus. Therefore, contrary to that observed for the Ty-1 gene widely used commercially [13], effectiveness of the recessive resistance studied here is expected even under high disease pressure, in accordance with preliminary field studies [17]. Spread of TYLCD by whiteflies in the field generally occurs in two phases. In a first instance, onset of the disease in a crop occurs from external sources of inoculum, i.e., primary spread, which usually leads to a random distribution pattern of primary infections foci [23]. Then, secondary spread from these primary sources of infection can occur within the crop, whose intensity will strongly depend on the magnitude of the insect vector population present in the crop. Here, we demonstrate that the resistance present in TX 468-RG was effective to severely limit primary spread of TYLCV-IL, thus resulting into a first barrier to reduce TYLCD field epidemics. As no significant difference on the B. tabaci preference was observed between TX 468-RG and MM, spread restriction was mainly based on the resistance to TYLCV-IL present in the former genotype. We observed, however, that a small number of TX 468-RG plants could get infected, even though no virus damage occurs in these plants due to the resistance factor [17]. These infected TX-468-RG plants constitute potential sources for secondary virus spread within the crop or even for primary spread to nearby susceptible crops. In fact, there are reports supporting the threat of some tomato genotypes resistant but not immune to TYLCV as efficient sources for secondary virus spread [35,36]. However, we demonstrate here that the level of resistance expressed by TX 468-RG was effective to drastically restrict secondary spread of the virus. This finding suggests that TX 468-RG plants restricted virus accumulation to levels that resulted in impaired insect transmission [37], and is compatible with the low transmission rates observed by Lapidot et al. [35] from tomato plants highly resistant to TYLCV. This is an important aspect for TYLCV management, as virus transmission from infected resistant plants can determine the field success of the resistant genotype to control TYLCD epidemics [35,36]. In conclusion, the resistance present in TX 468-RG was effective to reduce primary and secondary spread of TYLCV-IL. Therefore, this resistance can help in effective management of TYLCD under field conditions. Even though some TYLCD-associated virus-resistant cultivars are available commercially, owed to the resistance breakdown occasionally observed under high disease pressure [13], control measures traditionally have emphasized reducing vector populations through chemical control [38]. Concerns exist, however, about intensive use of insecticides due to the environmental damage caused and to development of pesticide resistance in insect population [6,7]. Therefore, based on the results shown here, the TYLCV-IL resistance present in TX 468-RG offers a good alternative for TYLCD-resistance breeding programs to incorporate this character into new hybrids and cultivars. Moreover, since TX 468-RG has proven to also be resistant to bipartite begomoviruses [18] with a similar reduction in viral accumulation, the same epidemiological impact on their spread is expected. Therefore, this source of resistance can be an important component of broad management of begomoviruses in tomato. wrote the manuscript. R.F.-M. performed statistical analyses of data. E.M. conceived and designed field experiments, supervised the studies and wrote the manuscript and R.O., supervised the studies and wrote the manuscript. All authors read and approved the final manuscript.

v3-fos

2016-05-12T22:15:10.714Z

2015-01-17T00:00:00.000Z

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s2

Helicoverpa zea (Lepidoptera: Noctuidae) and Spodoptera frugiperda (Lepidoptera: Noctuidae) Responses to Sorghum bicolor (Poales: Poaceae) Tissues From Lowered Lignin Lines The presence of lignin within biomass impedes the production of liquid fuels. Plants with altered lignin content and composition are more amenable to lignocellulosic conversion to ethanol and other biofuels but may be more susceptible to insect damage where lignin is an important resistance factor. However, reduced lignin lines of switchgrasses still retained insect resistance in prior studies. Therefore, we hypothesized that sorghum lines with lowered lignin content will also retain insect resistance. Sorghum excised leaves and stalk pith Sorghum bicolor (L.) Moench (Poales: Poaceae) from near isogenic brown midrib (bmr) 6 and 12 mutants lines, which have lowered lignin content and increased lignocellulosic ethanol conversion efficiency, were examined for insect resistance relative to wild-type (normal BTx623). Greenhouse and growth chamber grown plant tissues were fed to first-instar larvae of corn earworms, Helicoverpa zea (Boddie) and fall armyworms Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), two sorghum major pests. Younger bmr leaves had significantly greater feeding damage in some assays than wild-type leaves, but older bmr6 leaves generally had significantly less damage than wild-type leaves. Caterpillars feeding on the bmr6 leaves often weighed significantly less than those feeding on wild-type leaves, especially in the S. frugiperda assays. Larvae fed the pith from bmr stalks had significantly higher mortality compared with those larvae fed on wild-type pith, which suggested that bmr pith was more toxic. Thus, reducing lignin content or changing subunit composition of bioenergy grasses does not necessarily increase their susceptibility to insects and may result in increased resistance, which would contribute to sustainable production. There is global interest in developing fuels for the transportation sector from renewable resources as a means to reduce dependency on petroleum and other finite fuel sources. One of the impediments to converting biomass to biofuels is the presence of the cell wall polymer lignin, which interferes with the release of sugars from the corresponding cell wall polysaccharides, cellulose, and hemicelluloses during enzymatic saccharification (Dien et al. 2009). Therefore, reducing lignin levels through traditional breeding or genetic engineering is a target for bioenergy feedstock improvement but reducing lignin in cell walls may impair pest resistance where lignin is a major component of resistance. There are mutants of many plant species that have altered lignin content and subunit composition. The brown midrib phenotype has long been associated with reduced lignin content of maize, Zea mays L. (referred to as bm), and sorghum, Sorghum bicolor (L.) Moench (Poales: Poaceae) (referred to as bmr) (Sattler et al. 2010). Although bm plants have reduced lignin and improved forage digestibility, maize bm plants can have increased stalk breakage caused by insect damage or pathogens when compared with wild-type lines (Barriére and Agillier 1993). However, brown midrib mutants of other grass species are acceptable agronomically (Pedersen et al. 2005). The sorghum bmr6 phenotype is due to a nonsense mutation that causes premature truncation of the cinnamyl alcohol dehydrogenase (CAD) open reading frame. The bmr6 plants have reduced levels of lignin relative to the wild-type and incorporate phenolic aldehydes into the lignin polymer in addition to phenolic alcohols as in wild-type but a lignin relative ratio of two major subunits (syringyl and guaiacyl lignin; S/G ratio) similar to wildtype (Palmer et al. 2008, Saballos et al. 2009). The sorghum bmr12 phenotype is due to a nonsense mutation that causes premature truncation of caffeic O-methyl transferase (COMT). The bmr12 plants have reduced lignin levels, and syringyl subunits within lignin are greatly reduced relative to other subunits compared with wild-type plants (Bout andVermerris 2003, Palmer et al. 2008). These two bmr mutants have increased ethanol conversion efficiency compared with wild-type (Dien et al. 2009). However, the effects of these two mutations on pest resistance have not been examined. On the basis of prior work with switchgrass (Dowd et al. 2013), we hypothesized that the bmr6 and bmr12 sorghum lines will retain insect resistance compared with the near isogenic wild-type line. Here, we report on studies with corn earworms, Helicoverpa zea (Boddie) and fall armyworms, Spodoptera frugiperda) (J.E. Smith) (both Lepidoptera: Noctuidae) that indicate bmr leaves and pith were generally as resistant or more resistant to feeding by these two insects compared with the wild-type near isogenic line. These two species of insects can be serious pests of sorghum and other grasses (Metcalf and Metcalf 1993), and thus impaired insect resistance would impede use of bmr6 or bmr12 to improve lignocellulosic biofuels production. Materials and Methods Insects. The H. zea and S. frugiperda were obtained from colonies reared on pinto bean-based diet at 27 6 1 C, 50 6 10% relative humidity (RH), and a photoperiod of 14:10 (L:D) h, as described previously (Dowd 1988). First instars without any prior feeding experience were randomly selected and used in all assays. Plant Phenolic Bioassays. The plant phenolics adipic acid (99%), p-coumaric acid (98.0%), ferulic acid (99%), sinapic acid (98%), syringic acid (95%), and vanillic acid (97%) were obtained from Sigma/Aldrich (St. Louis, MO) (www.sigmaaldrich.com). They were incorporated into warm, still liquid pinto bean diets by blending with a vortex mixer as described previously (Dowd 1988). Phenolic acids were incorporated into diet at concentrations reported previously from near isogenic wild-type, bmr6, and bmr12 RTx430 stalks (Palmer et al. 2008) as follows for compounds listed above in order, in mg/g wet weight: "wild-type" was 67.8, 30.0, 8.8, 0.0, 5.6, and 17.7; "bmr6" was 36.1, 84.0 42.8, 13.8, 21.0, and 9.3; and "bmr12" was 51.5, 30.1, 21.1, 4.8, 3.2, and 20.7. Leaf disk diets were prepared by substituting wild-type leaf tissue (12 leaf stage plants; fourth leaf from the top) for pinto beans, wheat germ, and brewer's yeast and a proportional amount of water from the pinto bean diet (Dowd 1987) to provide similar nutritional composition as leaves for examining the effects of added phenolics under nutrient stress conditions. Diet was dispensed between two metal plates spaced 1 mm apart, cut into 4-mm disks when firm and freeze dried (Dowd et al. 2011). Because the wild-type stalks already contained some phenolics at higher levels than the bmr lines, only those phenolics that were at a higher concentration in the bmr lines were added to the disks, and only those phenolics that had levels at least 10% higher in the bmr compared with wild-type plants were used. For the "bmr6"-simulated leaf disks, ferulic, vanillic, sinapic, and syringic acids were added at 54.0, 34.0, 13.8, and 16.0 mg/g, respectively. For the "bmr12"-simulated disks, only vanillic acid was added at 12.3 mg/g wet weight. For the "wild-type"-simulated disks, no additional phenolic acids were added (only solvent control). Freeze-dried disks were used as described previously (Dowd et al. 2011). The phenolics acids were added in acetone to each dry disk, the acetone was evaporated in a chemical fume hood for 30 min, and the diet disks were rehydrated with sterile distilled water. Each treatment of the pinto bean diet was cut into pieces sufficient for ad libitum feeding, and each piece was placed in a separate well of a 24-well tissue culture plate (Dowd 1988). An individual larva was added to each well. For leaf diet disk assays, rehydrated sorghum leaf disk materials were placed on a Teflon disk inside a Petri dish containing 3% water agar. Ten first-instar larvae were added to each dish. The surviving larvae were weighed after 5 d (pinto bean diet pieces) or 3 d (sorghum leaf disks). Plants. Wild-type, bmr6, and bmr12 near isogenic plants in the background BTx623 were used in the present experiments. The near isogenic lines containing the bmr6 and bmr12 alleles were previously developed by crossing source of the bmr6 or bmr12 allele with BTx623 and four generations of backcrossing to BTx623 (Pedersen et al. 2006). When plants were grown in the Peoria greenhouse or a growth room (Peoria only), pots containing a previously reported soil and fertilizer mix, which contained bark mix (Dowd et al. 2007), were used and grown under previously reported conditions (Dowd et al. 2007). Growth conditions in the Peoria, Illinois greenhouse (which has supplemental lighting, heating, and cooling) and growth room were 24 6 2 C day and 18 6 2 C night, with a photoperiod of 14:10 (L:D) h at 50 6 10% RH. The soil mix for plants grown in the Lincoln, Nebraska greenhouse, consisted of soil:peat moss:vermiculite:perlite:sand (4:1:1:1:1), and plants were fertilized approximately every 7 d at 5 mL per pot with 11:15:11 (N:P:K; Ferti-lome Gardener's Special, VPG, Bonham, TX). Temperatures were maintained at 29.5 6 0.5 C during the day and 26.5 6 0.5 C during the night with a photoperiod of 16:8 (L:D) h and 50 6 10% RH. Leaves from plants grown in the greenhouse in Lincoln, NE, were shipped overnight on flaked ice and used the day of arrival. From 10 to 20 plants of each genotype were used in each experiment. Plant Tissue Bioassays. All bioassays were performed in Peoria. Leaf section bioassays were performed using Petri dishes with tight fitting lids as described previously (Dowd et al. 2007). Immature leaves (2nd from top, 12 cm of the leaf including the tip) of five leaf plants and more mature leaves (4th from top, 20 cm of the leaf including the tip) of 10-12 leaf stage plants were used in the bioassays. After 2 d, additional 4 cm long leaf pieces were removed from the initial harvest point of the 10-12 leaf plants to investigate wounding induced factors. Approximately 2 cm 2 leaf pieces were used in the assays, which were removed 0-6 cm from the leaf base, except for the additional piece of 4-cm long piece removed 2 d later from previous harvest site. There was sufficient leaf material for ad libitum feeding for the duration of each experiment. Leaf pieces were placed in Petri dishes with tight fitting lids, containing moistened filter paper, and 10 newly hatched firstinstar caterpillars without prior feeding experience were added to each dish. For pith assays, stalks sections were harvested from growth room grown plants after other assays were completed. The plants were postflowering, at soft dough stage of grain filling, and the stalk were green and turgid. Approximately 1 cm in diameter internodes between the first and second leaf from the top were harvested, which had color differences in the pith amongst the respective lines. Approximately 2-cm-long pith sections were placed in the Petri dishes as described for leaf piece bioassays with 10 first-instar caterpillars. Feeding damage on leaves was evaluated by determining the total number of 0.25 mm 2 or 1 mm 2 hole equivalents after 2 d as described previously (Dowd et al. 2007(Dowd et al. , 2011; caterpillars often molted to the second instar during that period. Because little mortality occurred in leaf assays, survivors were weighed using a Mettler AE104 analytical balance (Mettler Instrument Corp, Hightstown, NJ), which is accurate to 0.01 mg (Dowd et al. 2007(Dowd et al. , 2011. For the pith assays, insect feeding damage was not determined due to the difficulty of quantifying varying lengths and depths of feeding damage within the pith sections. Only mortality was determined after a 3-day feeding period due to highly variable weights resulting from cannibalism of dead larvae. Statistical Analysis. Statistically significant differences in overall insect mortality for each main effect plant variety treatment were determined by Chi square analysis, and differences in feeding rates and weights of survivors were determined by analysis of variance. SAS Proc Freq was used for Chi square analysis, and SAS Proc GLM was used for the other analyses. Windows Version 8.0 of the SAS software (SAS Institute 1999) was used. Results Consistent with our hypothesis, both first-instar H. zea-and S. frugiperda-fed sorghum leaves at different plants stage generally did not show statistically significant differences (P < 0.05) in feeding damage, mortality (very limited, so data not shown), or survivor weights indicative of reduced resistance for bmr compared with wild-type lines after 2 d of feeding on leaves (Table 1, Supp Table S1 [online only]). However, there was one exception; H. zea-fed leaves from five-leaf stage bmr12 plants; leaves from this plant stage had significantly (P < 0.01) greater feeding damage (mm 2 ) relative to wild-type leaves in both experiments (F ¼ 21.08, P < 0.001, df ¼ 1, 19 and F ¼ 14.06, Table 1). In contrast, there were several cases where the leaves of mutant lines were significantly more resistant to one or both of the insect species, based on significantly lower amounts of leaf damage or lower weights of survivors (Table 1). The leaves from both bmr6 5 leaf and 10-12 leaf plants had significantly less (P < 0.05 or P < 0.01) feeding damage from S. frugiperda compared with wildtype leaves in nearly all experiments. The amounts of S. frugiperda feeding damage on bmr12 leaves were not significantly different from wild-type leaves. Similar results were obtained whether plants were grown in the greenhouse or plant growth room, although some variation of some results was noted between the first and second experiments for some components. Damaging the leaves mechanically did not change relative resistance trends for the S. frugiperda that were observed for bmr versus wild-type undamaged leaves. Results with the H. zea were more variable but significantly (P < 0.05 or 0.01) less leaf material was removed from bmr6 leaves than wild-type leaves in several cases, which indicated bmr6 leaves were more resistant. For one experiment, in contrast to observations from first assay, when another piece was removed from the same leaf a few days later, H. zea feeding damage on bmr12 leaf pieces was significantly (P < 0.01) (F ¼ 10.14, P ¼ 0.004, df ¼ 1,19) less compared with wild-type leaves, whereas no effect of S. frugiperda feeding damage was observed. After 2 d of feeding, weights of S. frugiperda larvae that fed on bmr6 leaves were generally significantly (P < 0.05 or 0.01) lower than larvae that fed on wild-type leaves. Weights of S. frugiperda that fed on bmr12 leaves were generally similar to those that fed on wild-type leaves, although in a few cases, the weights of larvae fed on the bmr12 leaves were lower than larvae fed on the wild-type leaves. Some of the same trends were observed with H. zea, although weights of larvae that fed on bmr6 leaves were less often significantly lower than larvae fed on wild-type leaves ( Table 1). The H. zea larvae tended to consume significantly more of the leaf tissue from the 5 leaf bmr plants compared with the wild-type leaf tissue but did not weigh significantly more. The stalk pith was selected because its color was similar to the reddish-brown leaf veins of bmr mutants, and more uniformly lignified tissue may provide better insight into the effects of bmr mutants on insect susceptibility. H. zea and S. frugiperda larvae had significantly higher mortality (P < 0.05 and P < 0.01) when fed the pith from both bmr lines when compared with wild-type pith for both experiments (Table 2, Supp Table S2 [online only]). The difference was most dramatic for H. zea larvae where very little mortality occurred when they fed on wild-type pith in both experiment, but nearly 50% mortality of larvae occurred when they were fed bmr6 pith in the second experiment (Table 2). Discussion Consistent with our hypothesis, no general increased susceptibility of either bmr6 or bmr12 leaves to the insect species tested in this study were observed, although in some cases younger bmr leaves from fiveleaf stage plants had more feeding damage than wild-type leaves. Interestingly, there were several instances where bmr leaves and pith were more resistant to these larvae compared with the wild-type tissues based on higher mortality (pith) or lower amounts of feeding (leaves). The enhanced resistance was especially evident for S. frugiperdalarvae-fed bmr6 tissue. Variations between experiments may have been due to environmental factors (temperatures and light intensity in the different growth locations) or subtle differences plant developmental stage. Genotype by environment effects can influence agronomic traits in brown midrib lines (Cassler et al. 2003, Palmer et al. 2008, which can also influence insect resistance (Dowd and Johnson 2009). The relative trend for resistance was generally consistent whether the insects fed on undamaged or previously damaged leaves, but we cannot rule out that the relationship may change over time due to resistance factors induced specifically by insect feeding, which can vary depending on the insect species involved (Rodriguez-Saona et al. 2010). However, based on reports in maize (Shen et al. 2000), the time frame we used for the assays should have been sufficient to observe induced responses. In some cases, feeding damage to leaves was similar between wildtype and bmr lines, but postfeeding weights were significantly less for larvae-fed bmr leaves compared with larvae-fed wild-type leaves. In other cases, feeding damage on bmr leaves was significantly greater than on wild-type leaves, but weights of survivors were not significantly different. Both of these situations suggest that the bmr lines were less nutritious than the wild-type, and compensatory feeding was Twelve-leaf plant, mature leaf-recut (growth room) experiment 1 Wild-type 56 6 3a 0.23 6 0.01a 71 6 4a 0.42 6 0.02a bmr6 45 6 3b* 0.21 6 0.01ab 58 6 3b* 0.36 6 0.01b* bmr12 41 6 3b* 0.20 6 0.01b 61 6 4ab 0.38 6 0.02ab Twelve-leaf plant, mature leaf-recut (growth room) experiment 2 Wild-type 29 6 2a 0.21 6 0.01a 38 6 2a 0.48 6 0.02a bmr6 18 6 1b 0.16 6 0.01b* 29 6 2b 0.33 6 0.01b* bmr12 29 6 1a 0.24 6 0.01a 42 6 2a 0.55 6 0.03a At least 10 leaves of each line were used. Mean 6 standard error values reported are in mm 2 (feeding) and mg (weights) after 2 d of feeding. Values in columns for the same experiment followed by different letters are significantly different at P < 0.05 by analysis of variance. Values of bmr (low lignin) lines in columns for the same experiment followed by a "*" are significantly different from wild-type (normal lignin) values at P < 0.01. 37.3b * 34.0b* Pith from at least eight plants of each line was used. Values followed by different letters for the same experiment are significantly different at P < 0.05 by Chi square analysis. Values of bmr (low lignin) lines in columns for the same experiment followed by a "*" are significantly different from wild-type (normal lignin) values at P < 0.01. occurring in some cases, but it did not result in greater larval weights. A compensatory feeding response has also been reported when S frugiperda were fed diet with increased levels of non-nutritional cellulose (Wheeler and Slansky 1991). In some cases, resistance was due to antibiosis (toxic compounds), which is indicated when reduced feeding results in lower larval weights. This phenomenon was observed in several cases when the larvae fed on bmr6 compared with wild-type leaves. Lignin can be an important insect resistance factor in plants (Swain 1979), although the complex composition of lignin and interconnected metabolic network involved the synthesis of its precursors makes it difficult to predict how altering lignin concentration or composition of plants will affect insect resistance. Reduced ferulate crosslinking in fescue (Festuca sp.) resulted in increased damage by fall armyworms (Buanafina and Fescemyer 2012), which illustrates the importance of ester and ether-linked ferulic acid that are separate from lignin polymers in grass cell walls. However, low lignin lines of switchgrass (Panicum virgatum L.) (where lignin levels were reduced from 7 to 4%) that had higher rates of saccharification and fermentation retained resistance to S. frugiperda compared with high lignin lines Johnson 2009, Dowd et al. 2013). Age of plants and tissues can also influence resistance levels (Smith et al. 1994), which we also observed in evaluating feeding on leaves from 5-leafed compared with 10-or 12-leafed plants. Similarly, a significant positive association was observed between degree of insect resistance and lignin levels in younger switchgrass plants but not older plants (Dowd et al. 2013). This study indicated sorghum lines with lowered lignin can result in greater resistant to insects than lines unimpaired in their ability to synthesize lignin. Unexpectedly, the pith of both bmr lines was highly resistant relative to wild-type pith, based on higher rates of mortality observed for larvae-fed bmr pith. Previous studies have indicated impairing different steps in monolignol biosynthesis results in reddish brown to tan stem and stalk pigmentation, including the CAD and COMT mutants examined in this study (Mackay et al. 1997, Tsai et al. 1998, Sibout et al. 2005, Zhang et al. 2006). There are differences in pith and midrib (leaf vein) coloration between bmr6 and bmr12 (Porter et al. 1978, Saballos et al. 2009), probably because bmr6 and bmr12 block different steps of the monolignol biosynthetic pathway. However, the increased mortality of both H. zea and S. frugiperda on both bmr mutant pith types suggests a common chemical or biochemical resistance factor(s), yet to be identified, which is not related to differences in pith color. The greater resistance observed in bmr pith and bmr leaves at times suggest the similar phenolic compounds or pathway intermediates may accumulate in both tissues due to altered phenylpropanoid metabolisms caused by these mutations. Alternatively, the changes in phenylpropanoid metabolism could induce the expression of unrelated resistance genes not involved in this metabolic pathway. Additionally, the loss of these biosynthetic enzymes in the bmr6 and bmr12 lines may have a previously unrecognized role, which is not unprecedented. Transgenic overexpression of a peroxidase altered expression of other, unrelated defensive genes in tomato, Solanum lycopersicum L. (Suzuki et al. 2012). In addition, the lignin biosynthetic enzyme Cinnamoyl-CoA reductase (CCR) is also involved in defense signaling in rice, Oryza sativa L. (Kawasaki et al. 2006). The same unknown factors could be present in both bmr lines, and these same factors could be responsible for the increased morality, reduced leaf damage, and lower larval weights in the pith and leaf feeding assays, respectively. Considering that monolignol pathway is active in both leaves and stalks, and bmr6 and bmr12 both affect this pathway, the accumulation of phenolic compounds resulting from alteration to this pathways may partly play a role in resistance. The studies with diets involving added phenolic acids that simulated levels observed in bmr stalks (Table 3) suggest that changes in phenolic composition found in bmr mutants may increasing toxicity or interfering with nutrient absorption. No significant differences in insect weights were noted for experiments with nutritionally complete pinto bean based artificial diet was used, but weights of both H. zea and S. frugiperda were lower when they fed on the simulated "bmr" compared with "wild-type" diet made from sorghum leaf material. This information suggests that diet nutrient composition influences the level of resistance conferred by potential resistance molecules. No effect was observed with the nutritionally rich pinto bean diet, but a significant effect was detected with the nutrient poor sorghum leaf diet, which suggests the phenolic acids are interfering with nutrient absorption. S. frugiperda caterpillars that fed upon the simulated "bmr6" and "bmr12" leaf diet disks weighed less than those fed the "wild-type" leaf diet disks. However, the "bmr12" diet disks only had additional vanillic acid, whereas the "bmr6" leaf diet also had ferulic and syringic acid added. This result suggests that increased levels of vanillic acid may be associated with increased insect resistance in the bmr lines. Stem and pith resistance to the stalk borer, Sesamia nonagrioides (Lefebvre) in several lines of maize was correlated with p-coumaric content but not with several other phenolics, including ferulic, sinapic, syringic, and vanillic acids (Santiago et al. 2005). Syringic and vanillic acid, but not ferulic or p-coumaric acid concentration were correlated with pigeon pea, Cajanus cajan (L.) Millsp., resistance to the pod borer, Helicoverpa armigera (Hübner) (Verulkar and Singh 2000). Why increased vanillic acid and apparently not ferulic or syringic (i.e., expected similar mode of action) affect S. frugiperda caterpillars remains to be determined. This study indicates that sorghum lines bmr6 and bmr12, which are easier to enzymatically saccharify, generally do not reduced levels of insect resistance. The increased resistance of bmr6 leaves and pith to insects observed suggests the bmr6 mutant may actually have sufficient enhanced insect resistance, such that it would require fewer insecticide applications in field production. This information suggests that sorghum bmr traits, which enhance conversion of lignocellulosic biomass to ethanol, or other biofuels are viable objections for sustainable bioenergy feedstock production. Further evaluations under field conditions are needed to better assess the potential of the bmr sorghum lines to be sustainably produced, for which insect resistance is an important component. The toxicity of the pith from the bmr lines to caterpillars noted in this study suggests it may promote resistance to stalk boring insects, and field studies are in progress to evaluate this potential. Chemical and molecular analyses of the potential resistance mechanisms within bmr pith are in progress. This study represents the first effort to evaluate insect resistance in sorghum lines with potential bioenergy uses. This study also provides incentive to continue investigating insect resistance of these lines under field conditions. Overall, the results of this study and other studies examining effects plants with altered lignin content and composition on insect resistance indicate that changes to this Artificial pinto bean-based diet Solvent control 0.0a 6.3 6 0.3a 0.0a 4.6 6 0.2a "Wild-type" 0.0a 6.7 6 0.4a 0.0a 4.4 6 0.4a "bmr6" 0.0a 6.8 6 0.2a 0.0a 3.4 6 0.3b "bmr12" 0.0a 6.1 6 0.4a 0.0a 3.5 6 0.4b Sorghum leaf disk diet "Wild-type" 0.0a 0.21 6 0.03a 0.0a 0.20 6 0.01a "bmr6" 0.0a 0.20 6 0.02a 0.0a 0.13 6 0.01b* "bmr12" 0.0a 0.15 6 0.02a 0.0a 0.13 6 0.01b* Values are after 3 d for leaf disk diets and 5 d for artificial diet. Weights are means 6 standard errors in mg. Values followed by different letters for like studies are significantly different at P < 0.05 by Chi square analysis (mortality) or analysis of variance (weights). Values of "bmr" diets in columns for the same experiment followed by a "*" are significantly different from "wild-type" diet values at P < 0.01. See Materials and Methods for phenolic additions that simulate the phenolic acid compositions found in wild-type, bmr6, and bmr12 stalks (Palmer et al. 2008). pathway to augment biofuel production should be evaluated on a case by case basis for both the plant production and the insect resistance.

v3-fos

2017-10-14T04:14:24.527Z

2015-08-26T00:00:00.000Z

41649002

s2

Isoflavone and Mineral Content in Conventional and Transgenic Soybean Cultivars The objective of this study was to evaluate the differences in composition among six brands of conventional soybean and six genetically modified cultivars (GM). We focused on the isoflavones profile and mineral content questioning the substantial equivalence between conventional and GM organisms. The statement of compliance label for conventional grains was verified for the presence of genetic modified genes by real time polymerase chain reaction (PCR). We did not detect the presence of the 35S promoter in commercial samples, indicating the absence of transgene insertion. For mineral analysis, we used the method of inductively coupled plasma-optical emission spectrometry (ICP-OES). Isoflavones quantification was performed by high performance liquid chromatography (HPLC). The results showed no statistical difference between the conventional and transgenic soybean groups concerning isoflavone content and mineral composition. The concentration of potassium, the main mineral component of soy, was the highest in conventional soybeans compared to that in GM soy, while GM samples presented the highest concentraCorresponding author. The presence of the transgene was initially verified by the detection of the 35S promoter and, when present, detection of the Roundup Ready (RR) transgene, according to ISO 21570:2005 [12]. The quantitation limit (LOD) was 0.1%. For mineral analysis, the methodology used was that recommended by the Instituto Adolfo Lutz [13]. The minerals quantified were the following: K (potassium), Ca (calcium), P (phosphorus), Mg (magnesium), Na (sodium), Cu (Copper), Fe (iron ), Mn (manganese), Zn (zinc), Cd (cadmium), Pb (lead), Ni (nickel), Ba (barium), and Cr (chromium). A standard curve was obtained by using standard solutions of these minerals at a concentration of 1000 mg/L (Merck) prepared in aqueous solution acidified at 10% (v/v). The soybeans were ground, weighed, and placed in an oven with air renewal and circulation (Marconi, mod. MA035) at 70˚C, and after 12 h they were calcinated in an oven (Vulcan, mod. 3 -1750) using a heating ramp up to 530˚C. After cooling the samples, 1.0 mL of concentrated HNO 3 was added. This mixture was heated on a hot plate at 100˚C to dryness and placed again in an oven at 375˚C for 1 h. HCl (10 mL) was added to the ash obtained and gauged in a 100-mL volumetric flask with water purified with a Milli-Q system. The samples were analyzed directly by inductively coupled plasma-optical emission spectrometry (ICP-OES; Perkin Elmer, mod. Optima 2000 DV-sampler, mod. As90plus) in Axial configuration, at 1400 kW radiofrequency power, and 0.60 L•min −1 gas flow. For analysis of isoflavones, the samples were crushed in a micromill and degreased with n-hexane (HPLC grade). The extraction of isoflavones was performed according to the method proposed by Carrão-Panizzi [8] and colleagues for the extraction of isoflavones in soybeans. The separation and quantification of isoflavones were performed according to the changes in the method proposed by Berhow [14], by using a liquid chromatograph (Waters model 2690) equipped with W 600 model pump, and an automatic sample injector (model W 717 plus). An octadecyl-silica (ODS)-type reverse phase C18 column (YMC-Pack ODS AM column; 250 mm length × 0.4 mm internal diameter, particle size 5 µm) was used for this purpose. For the separation of isoflavones we adopted a binary linear gradient system, using the following as the mobile phases: 1) methanol containing 0.025% trifluoroacetic acid (TFA) (solvent A) and 2) ultrapure deionized distilled water containing 0.025% TFA (solvent B). The initial condition of the gradient was 20% solvent A, which reached 100% after 40 min, then reduced to 20% after 41 min, and remained in this condition up to 60 min. Therefore, the total time of analysis for each sample was 60 min. The mobile phase flow rate was 1.0 mL•min −1 and the temperature throughout the analysis was maintained at 25˚C. The detection of isoflavones was performed using a photo diode array detector (Waters model W 996) adjusted to the wavelength of 260 nm. To identify the peaks corresponding to each of the twelve different forms of isoflavones, the following standards were used: daidzin, daidzein, genistin, genistein, glycitin, glycitein, and also standards of their acetyl and malonyl conjugates, (Sigma and Fuji) solubilized in methanol (HPLC grade) at the following concentrations: 0.00625 mg/mL, 0.0125 mg/mL, 0.0250 mg/mL, 0.0500 mg/mL, and 0.1000 mg/mL. To quantify the 12 types of isoflavones by external standardization (peak area), standards were used as reference. The identification of the peaks corresponding to each of the twelve forms of isoflavones in the samples was performed by comparison with the spectra and retention times of the standards. Statistical analysis was performed using analysis if variance (ANOVA) and Tukey's test for comparison of the means, as well as the Statistica 7.0 program (STATSOFT, 2004), with a significance level of 5%. Results and Discussion Article 40 of Law No. 11.105/2005 defines that "food and food ingredients for human consumption or animal feed containing or produced from GMOs or derivatives should contain information on their labels accordingly" [4]. Decree 4680/2003 regulates the mandatory labeling of foods and food ingredients for human consumption or animal feed containing or produced from GMOs that contain GMOs above the limit of 1% of the product. These products should bear on their labels prominently the words: "(name of product) transgenic", "contains (name of ingredient or ingredients) transgenic (s)", or "product produced from (name of product) transgenic" as well as the symbol for GM, which consists in a yellow triangle with the letter "T" inside, defined by Ordinance No. 2658, December 22, 2003 [4] [5]. Initially, we observed that the label statements in the commercial soybean samples did not comply with the law. However, the results of the genetic analysis did not show the presence of the 35S promoter. Therefore, there was no need to investigate RR gene alterations, as described in the methodology. Thus, since the tested samples effectively contained no detectable amounts of GMOs, the brands sampled in local suppliers were in accordance with the law. It is interesting to note that although 76% of soybeans grown in Brazil are from transgenic cultivars, the conventional product continues to be prominent in local trades. In a study by Branquinho and colleagues [15], it was shown that 28.3% of the samples tested contained transgenic soybean [(n = 68) in 240 foods derived from soybeans analyzed between 2004 and 2007]. Quantitative analysis revealed GMO content between 0.05% and 1% in 43 (63.2%) samples, and more than 1% in 25 (36.8%) samples. There was no indication on the label of the presence of GM material and; therefore, the labels were not in accordance with the law. These results differ from ours probably because the authors analyzed products derived from soy (type 1 products), and not soybeans, which were the object of our study. Over the last decade, other studies have assessed the presence of GMOs in food available in the consumer market. Cardarelli et al. [16] analyzed 89 food products that contained soy and/or corn ingredients in samples originating from different cities in Brazil. The presence of soybean RR was found in 16 samples such as soy extract, pastes, sauces, dehydrated soups, and uncooked soybean feed. Of the total number of samples analyzed by Cardarelli and colleagues [16], 15 were raw soybeans and only one was positive for the presence of the 35S promoter. The results and methodology of this study are similar to those of the present study. Subsequently, the positive sample found by Cardarelli et al. [16] was evaluated for the presence of the RR transgene, which was then confirmed. Brod and colleagues [17] have produced a series of articles evaluating the presence of GM soy in different food products marketed in Florianópolis, Brazil. In 2007, they analyzed 37 samples of soy products including six samples of flour, six samples of infant formula, and 25 samples of soymilk powder. The results were positive for the presence of RR soybean in four samples of defatted soy flour and 15 samples of soymilk powder [18]. In 2007, Brod and Arisi [18] analyzed 32 meat additives containing soy proteins. Twenty-five were positive for lecithin, confirming the presence of soy in the amplified DNA, and 15 of these were positive for RR, confirming the presence of GM soy. In 2008, Brod and Arisi [19] analyzed 62 samples of soy isolates, of which 37 were of textured soy protein (TSP) and 25 were of soybean extract powder. Forty samples were positive for RR but only two contained more than 1% RR soybean. It is interesting to note that these studies were conducted primarily with soy products, in which the presence of GM soy lecithin and RR was detected but not the 35S promoter. These results suggest that the use of GM soybeans is directed to processed foods. Regarding the glycoside daidzin (SC 4.61 ± 1.88 and ST 6.76 ± 3.74 mg•100 g −1 of defatted sample) and its corresponding aglycone form daidzein (SC 55.40 ± 12.64 and ST 67.14 ± 31.95 mg•100 g −1 of defatted sample), there was no statistically significant difference (p > 0.05) when comparing the ST (GM soy) and SC (commercial soybean) sample groups, which is in accordance with the results of Zhou and colleagues [11], who also found no significant difference between samples of transgenic soybeans and conventional soybeans for these forms of isoflavones. There were detected all forms of isoflavones in this study; therefore, the three forms of isoflavones and aglycone glycitein were not quantified. Because the study focused on unprocessed soybeans, the finding of acetyl conjugates was expected, as they are only present in processed soy products. The work of Zhou and colleagues [11] demonstrated, by a mixed model, regional differences in isoflavone content; all levels of isoflavones were higher in southern Brazil when compared with the levels found in the samples from northern Brazil. This model attributed the variation in the composition of the isoflavones mostly to a combination of parameters such as region, year, and phenotype, which were statistically different (p < 0.05) for all the isoflavones evaluated. These data confirm that multiple factors are associated with the variation in the content of isoflavones, including environmental factors such as local culture and especially the temperature during grain filling; genetic factors inherent to the soybean cultivars also influence the accumulation of isoflavones in the grains. This has been consistently observed in recent studies [7]- [10], establishing that the formation and accumulation of isoflavones is affected by many biotic and abiotic factors. The absence of statistically significant difference (p > 0.05) between the samples of GM and conventional soy samples found in this study for these two isoflavones (daidzin and daidzein) shows the absence of any effect attributed to the transgene. In contrast, when we compared the levels of other isoflavones in samples of transgenic cultivars and conventional commercial soy we observed a significant difference (p < 0.05), despite the large intragroup variability observed ( , the levels observed were statistically higher for the transgenic cultivars. The mean levels of genistein observed were 2.95 ± 1.24 mg•100 g −1 in transgenic cultivars, and 4.83 ± 2.41 mg•100 g −1 for conventional commercial cultivars, which indicated that these, on average, had higher levels of this bioactive isoflavone. However, the wide range of contents of isoflavones in soybeans and transgenic cultivars, especially in conventional commercial cultivars, as verified by the intragroup standard deviation, may have interfered with this significance. The variation in the concentration of isoflavones, especially in the conventional commercial cultivars, can be explained by the potential heterogeneity both with regard to genotype and the place and time of planting, growing conditions, and even storage conditions. , who found that, even though lower levels of these compounds were observed in GM varieties, the total isoflavone content was higher. It is important to note that, in our study, this difference was statistically significant. In another study by Barbosa et al. [20], the authors found 381 ± 3 mg 100 g −1 of total isoflavones in defatted soybean flour obtained from conventional commercial soybeans, which is similar to our results for conventional commercial soybean, because isoflavones were assessed on degreased samples. Duke et al. [21] performed a study using a different approach to evaluate the response of transgenic cultivars DP 5806 RR and Asgrow 3701 RR subjected to different environmental conditions but similar herbicide applications. The authors observed a wide variation in the levels of isoflavones between the two cultivars, but not within the same cultivar with different herbicide applications, which suggests that this wide range of variation among cultivars is independent of the presence of the transgene. When grouping isoflavones by their radical groups, the average percentages observed for the different cultivars (19.82 and 38.61 mg•100 g −1 of β-glycosides, 78.68 and 58.11 mg•100 g −1 of malonylglycosides, 1.49 and 3.28 mg•100 g −1 of aglycones and no acetylglycosides, for transgenic soybean cultivars and conventional commercial cultivars, respectively) were in agreement with the findings of Carrão-Panizzi [9] for raw grains when the values were converted into percentage of total isoflavones (19.15, 79.26, and 1.55 mg•100 g −1 ). However, Barbosa et al. [20] found different proportions of total isoflavones: 42.8 and 39.1 mg•100 g −1 of β-glycosides, 52.5 and 50.6 mg•100 g −1 of malonylglycosides, 4.0 and 7.2 mg•100 g −1 of aglycones, and 1.0 and 2.9 mg•100 g −1 of acetylglycosides in soybeans and defatted flour, respectively. However, these authors did not have information about the presence of transgenes in the tested samples. Bavia et al. [22] using the same methodology, found different values for different cultivars and variation in the proportion in raw grains: β-glycosides from 37.9% to 47.8%, malonylglycosides from 40.8% to 48.1%, and aglycones from 11.4% to 16.7%; total isoflavones from 89.63% to 200.24 mg•110 g −1 of dry matter. Regarding the aglycones, the values found in this study were similar to those obtained by Benedetti [23] in defatted soybeans, which represented 1.8% of total isoflavone content, and to those found by Wang & Murphy [24], in which the aglycone levels were between 1% and 3% of total isoflavones. The average total aglycone was 9.6 mg•100 g −1 . This result was similar to that found by Silva et al. [25], which was 9.19 mg•100 g −1 , and higher than that reported by Carrão-Panizzi et al. [8], which was 4.0 mg•100g −1 . These variations are explained by the cultivar studied and the environmental cultivation conditions. The results of the analysis of micro-and macrominerals are summarized in Table 2 and Table 3, respectively. When comparing the results of the analysis of macro-and micro-minerals, with the samples divided into conventional and transgenic cultivars, it was possible to observe significant differences between groups for the following minerals: potassium (p < 0.003), chromium (p < 0.007), and iron (p < 0.013). The minerals Pb, Ni, and Cd were not detected or were classified as trace elements. In study by Zobiole et al. [26] found lower values for macro-and micro-nutrients analyzed in soybeans that had undergone genetic modification compared to their near-isogenic cultivars, regardless of the application of glyphosate; however, these studies evaluated the plant (shoot and root) and not the seeds. In study by Shinonaga et al. [27], the uptake and translocation of trace elements (Co, Se, Rb, Sr, Ru, Rh, and Cs) in maturing soybean plants cultivated on soil were studied over 360 h under diurnal conditions after the administration of a multitracer. In study by Vieira et al. [28] evaluated six soybean cultivars and observed that K was the most abundant mineral in all cultivars, with a maximum of 1824.02 mg•100 g −1 in the Iguaçu cultivar, and a minimum of 1567.05 mg•100 g −1 for the EMBRAPA-4 cultivar. The contents of P, Ca, and Mg ranged from 454.71 to 503.84 mg•100 g −1 , 170.19 to 313.93 mg•100 g −1 and 214.36 to 259.97 mg•100 g −1 , respectively. The concentrations of Fe, Mn, and Na varied between 13.39 and 19.12 mg•100 g −1 , 1.75 and 2.79 mg•100 g −1 , and 11.73 and 12.08 mg•100 g −1 , respectively. In this study, the high variation in mineral contents between cultivars may be related to genetic factors intrinsic to each cultivar as well as growing conditions, climate, soil, and fertilizers. We also observed that for these minerals, the values obtained in this study were lower than those found by Vieira et al. [28] in their samples. Conclusions The six brands of conventional soybean marketed in the municipality of Belo Horizonte were labeled for the presence of genetically modified organisms, in accordance with current legislation. Although differences among groups were observed for some minerals, the same trend was observed within the group, which could be explained by genetic differences among cultivars as well as by environmental conditions during cultivation. The highest levels of potassium and the main mineral present in soy were found in conventional cultivars when compared with transgenic cultivars, which in turn had higher content of iron. The variation in total isoflavone contents of soybeans from local suppliers confirms the need for labels to bear information regarding these levels. In addition, information is needed regarding unknown parameters such as variety, cultivation region, and maturation of the commercially available grains.

v3-fos

2016-05-12T22:15:10.714Z

2015-06-12T00:00:00.000Z

3109482

s2

Regulation of flavonol content and composition in (Syrah×Pinot Noir) mature grapes: integration of transcriptional profiling and metabolic quantitative trait locus analyses Highlight Novel candidate genes for the fine regulation of flavonol content in ripe berries are identified through integration of transcriptional profiling and metabolic QTL analyses of a segregating grapevine progeny. Introduction Flavonols in grapes, as in almost all higher plants, are an important class of bioactive compounds. In grapes they are most abundant in the flowers and skins of the berries, although they have also been reported in leaves and stems (Hmamouchi et al., 1996). They are produced by the flavonoid biosynthetic pathway, which also gives rise to anthocyanins and condensed tannins. Among flavonoids, they represent one of the most important classes in terms of concentration, especially in white grapes (Flamini et al., 2013). From an agronomic perspective, flavonols are important phytochemicals for viticulture and enology. They stabilize the colour of red wines, forming molecular complexes with anthocyanins, a phenomenon known as co-pigmentation (Boulton, 2001). In addition, they confer antioxidant properties to the wine (Yang et al., 2009), which translates into health benefits for the consumer (Nassiri- Asl and Hosseinzadeh, 2009). From a taxonomic perspective, flavonols are useful markers since their profiles vary considerably among grapevine cultivars, ultimately contributing to wine typicity, and, as such, they have been used for the authentication and the varietal differentiation of grapes and wine (Mattivi et al., 2006;Castillo-Munoz et al., 2007. Flavonols possess a 3-hydroxyflavone backbone and differ in the number and type of substitutions on the B ring. They usually occur as 3-O-glycoside derivatives with the sugar attached to the 3′ position of the flavonoid skeleton. Although the presence of up to six different aglycons is expected in grape berry skin (Fujita et al., 2006), the lack of expression of the flavonoid 3′,5′-hydroxylase genes restricts them to the mono-and di-substituted derivatives kaempferol (Kaemp), quercetin (Que), and isorhamnetin (Isor) in white grapes. In contrast, red grapes usually also accumulate the tri-substituted flavonols myricetin (Myr), laricitrin (Lar), and syringetin (Syr) (Castillo-Munoz et al., 2007. Flavonols accumulate exclusively in the outer epidermis of the grape berry pericarp and in some layers of the seed coat (Fontes et al., 2011). In particular, they are stored in the inner, thick-walled layers of hypodermis, and the thickness of the berry skin influences their concentration (McDonald et al., 1998). The localization of flavonols is related to their role as protectants against UV, extreme temperatures, as well as free radicals (Winkel-Shirley, 2002). As a consequence, synthesis of flavonols is a light-dependent process regulated by lightdependent modulation of the main genes involved in their biosynthesis (Czemmel et al., 2009). It was recently demonstrated that UV-B light has a greater effect on mono-and di-substituted flavonol accumulation through the up-regulation of flavonoid 3′-hydroxylase genes (Martinez-Luscher et al., 2014). During development, flavonol biosynthesis begins in the flower buttons, decreases between flowering and berry set, and then reaches a peak at 3-4 weeks post-véraison (Downey et al., 2003). In general, the total amount of flavonols varies extensively among Vitis vinifera varieties, ranging from 1 mg kg -1 to 80 mg kg -1 of fresh berry weight, with red cultivars usually being richer than white ones (Mattivi et al., 2006;Castillo-Munoz et al., 2007, and reaching >100 mg kg -1 in some wild Vitis species (Liang et al., 2012). The level and the distribution of the different classes of flavonols can be strongly affected by agronomic and environmental conditions which impact sunlight exposure and water status. However, there is evidence that flavonol profiles, as in general phenolic profiles, are also highly influenced by genetic factors playing a role at the cultivar level (for reviews, see Flamini et al., 2013;Teixeira et al., 2013). The general flavonol biosynthetic pathway has been genetically and biochemically elucidated in many plant species, and recently it was also described in grapevine: their synthesis is catalysed by the action of flavonol synthase (VvFLS), an enzyme which uses di-hydroflavonols as substrates and whose encoding gene is temporally and specifically regulated by a R2R3-MYB transcription factor, named VvMYBF1 (Downey et al., 2003;Czemmel et al., 2009). Also determining steps in flavonol accumulation are reactions driven by chalcone isomerase (VvCHI) and by flavonoid 3′-hydroxylase (VvF3′H) and flavonoid 3′,5′-hydroxylase (VvF3′5′H), which mediate the addition of hydroxyl groups to the B-ring of flavanones, flavones, and dihydroflavonols (Jeong et al., 2006). In particular, VvF3′5′H competes with VvF3′H and VvFLS for dihydroflavonol substrates and, therefore, changes in any of these enzymatic activities by external factors, such as an increase of F3′H and FLS activities by UV-B, may affect the flavonol profile in favour of mono-and di-substituted derivatives (Martinez-Luscher et al., 2014). Subsequently, glycosylation of flavonols driven by the glucosyltranferases 5 (VvGT5) and 6 (VvGT6) enhances their water solubility, allowing the accumulation of high concentrations of these compounds (Ono et al., 2010). Despite this knowledge, understanding of the fine control of flavonol biosynthesis requires further investigation, especially at the stage of technological maturity of the grapes, the most relevant for industry. In this study, advantage was taken of a segregating population derived from a cross between two V. vinifera cultivars with significantly divergent flavonol content in the skin at the mature berry stage ('Syrah'×'Pinot Noir'). The progeny were characterized in four seasons by analysing the flavonol content and composition. Overall, 22 traits were considered for analysis using two parallel approaches: (i) individuals of the population exhibiting the extremes of flavonol content were analysed at the transcriptional level using microarrays; and (ii) the metabolic data were used in combination with a genetic map (Costantini et al., 2015) for quantitative trait locus (QTL) analysis. This enabled the identification and characterization of seven mQTLs exclusively associated with flavonol biosynthesis. The study revealed a large effect QTL controlling all the traits under analysis which consistently co-localized with the major locus for anthocyanin berry content on linkage group 2 (LG 2; Costantini et al., 2015). The identification of a common genetic control was completely novel. By integrating the results of the two approaches, a list of candidate genes for the regulation of flavonol content and composition in mature grapes was produced based on co-localization of significantly modulated transcripts with metabolic QTLs (mQTLs) and on available knowledge on flavonol metabolism. The identification of new genetic determinants of flavonol varietal composition at technological maturity represents a valuable result for the wine growing industry. Plant material and sampling The content of flavonols in the berry skin was evaluated in 170 F 1 individuals derived from the cross 'Syrah'×'Pinot Noir' (clone ENTAV 115) and in the parental lines. All plants were grown at the Giaroni experimental field of FEM (Edmund Mach Foundation, San Michele all'Adige, Italy, 46°18′N, 11°13′E). Plants were grafted on the rootstock Kober 5BB and trained according to the Guyot system. Two clusters of the same plant were sampled for each genotype at technological maturity (18 °Brix) in four different seasons (2007-2008-2009-2011) and stored at -80 °C until use. Anthocyanin content was analysed on the same samples in a parallel study (Costantini et al., 2015). After ranking the median value obtained considering the concentration of all the compounds in all the seasons, eight individuals of the cross were divided into two groups significantly divergent for their content (HFPs,high flavonol producers,codes 16,56,63,223;and LFPs,low flavonol producers,codes 64,256,260,Pinot Noir) and subjected to gene expression profiling. The selection of the individuals was statistically supported by analysis of variance (ANOVA) followed by Tukey HSD test (P-value <0.05) using Statistica 9 software (StatSoft, Tulsa, OK, USA). For microarray analysis, a representative sample of 50 berry skins was collected from each of the eight individuals (during the 2007 season) at three berry developmental stages: hard green berry (33E-L, PV), véraison (35E-L, 50% coloured berries, VER), and maturity (38E-L, 18 °Brix, MAT). For real-time reverse transcription-PCR (RT-PCR) analysis, the same samples used for microarray analysis were used for technical validation, while a representative sample of 50 berry skins was collected from three biological replicates of the eight individuals (during the 2011 season) at the same three developmental stages for the assessment of the biological variability in the gene expression. HPLC-DAD-MS analysis of flavonols For each genotype of the population, 20 berries representative of the two collected clusters were considered for flavonol analysis. Flavonol extraction, acid hydrolysis of flavonol glycosides, and high-performance liquid chromatography-diode array coupled to mass spectrometer detector (HPLC-DAD-MS) analysis were performed as previously described (Mattivi et al., 2006). Each flavonol was identified by comparison of retention time, UV spectra at 370 nm, and MS spectra in positive mode with those of the corresponding standards. The content was quantified in mg kg -1 of fresh berry weight by means of the external standard method, specific for each compound. Values under the limit of quantification (LOQ) were imposed equal to zero in the final quantification: Myr LOQ=0.66 mg l -1 , Que LOQ=0.46 mg l -1 , Lar LOQ=0.55 mg l -1 , Kaemp LOQ=0.51 mg l -1 , Isor LOQ=0.63 mg l -1 , Syr LOQ=0.54 mg l -1 . Statistical analyses of biochemical data were performed with the software SPSS 11.0 (IBM SPSS Statistics). The normality of trait distribution was checked by a Kolmogorov-Smirnov test. If necessary, data were log-transformed with a ln(1+x) function. Correlations between traits within years and between years within traits were determined using the non-parametric Spearman correlation coefficient. QTL analysis For QTL identification, a genetic linkage map based on the segregation of 690 simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers in 170 F 1 individuals (Costantini et al., 2015) was used. QTL analysis was performed using MapQTL 6.0 (Van Ooijen, 2009) with simple interval mapping and multiple QTL mapping functions, adopting the same procedure as in Costantini et al. (2015). As a general trend, QTLs for the different flavonols were identified by analysing the whole progeny, while QTLs for flavonol ratios were identified by considering the coloured progeny only. In contrast, in the case of Kaemp, Que, and Isor, the analysis was run both in the whole and in the white progeny, to test if the co-existence of anthocyanins together with flavonols in the mature berry skin of coloured individuals could affect the regulation of flavonol biosynthesis. Suffixes (a and b) were adopted to indicate different regions on the same LG where QTLs were found for flavonol (this work) and anthocyanin (Costantini et al., 2015) content. The 12× grape reference genome (http://genomes.cribi.unipd. it/) was used to extract version 1 of the gene predictions underlying QTLs. A Fisher's exact test was applied to evaluate the over-representation of specific functional categories in the QTL regions as in Costantini et al. (2015). The combination of all the regions controlling a given trait was tested. Adjusted P-values obtained applying the Benjamini-Hochberg method (Benjamini and Hochberg, 1995) for multiple testing correction were considered significant when ≤0.05. RNA isolation, microarray hybridization, and data analysis RNA isolation, microarray hybridization, and data analysis were carried out as described in detail at the GEO database under the accession GSE42909 (http://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE42909 accessed 19 December 2014). Briefly, a total of 24 hybridizations were performed (eight F 1 individuals×three developmental stages) on the custom Affymetrix GrapeGen GeneChip ® , representing ~18, 711 unique genes. Information about the GeneChip ® is available in ArrayExpress (Pontin et al., 2010) and GEO (Ali et al., 2011) under the accession numbers E-MEXP-2541 and GSE24561, respectively. The Robust Multi-array Analysis (RMA) algorithm was used for background correction, normalization, and expression level summarization (Irizarry et al., 2003). Probe sets with significant differential expression (DEPs) between LFPs and HFPs were selected in two steps and under the assumption that the four individuals of the LFP and HFP groups can be treated as biological replicates (pseudoreplicates). Step 1: within each group (LFPs and HFPs), the data were analysed through pairwise comparisons of developmental stages (PV versus VER, PV versus MAT, and VER versus MAT). Differential expression analysis was performed with the Bayes t-statistics from LIMMA (Linear Models for Microarray Data), and P-values were corrected for multiple testing using the Benjamini-Hochberg method (Benjamini and Hochberg, 1995). Three lists of DEPs for each group were thus obtained [with a false discovery rate (FDR) <5% and a cut-off of 2-fold change (FC) between each pair of stages]. Step 2: the lists of DEPs were then compared between LFPs and HFPs to provide a final list of probe sets either exclusively modulated in LFPs or in HFPs, or modulated in both groups but with low correlation [−0.5>(logFC LFPs -logFC HFPs ) pairwise comparison >0.5]. The two-step analysis was preferred to the direct comparison between LFPs and HFPs at each developmental stage, to reduce the number of false DEPs caused by a nonperfect phasing of the samples of the two groups. Probe set annotation was updated according to the 12Xv1 prediction (http://genomes.cribi.unipd.it/ accessed 19 December 2014), and functional categorization was inferred from Lijavetzky et al. (2012). In particular, each probe set was assigned to one of 516 categories of a custom-made catalogue based on plant-related terms from the GO vocabulary and MIPS FunCat (Ashburner et al., 2000;Ruepp et al., 2004). Therefore, hypergeometric tests (P<0.05) were applied to evaluate the enrichment of a functional category of the third level among the DEPs, in comparison with the distribution of the 23, 096 GrapeGen probe sets as calculated from Lijavetzky et al. (2012). Quantitative RT-PCR analysis Total RNA from all the samples of the eight individuals collected in both the 2007 (also used in microarray experiments) and 2011 seasons was treated with DNase I (Ambion, Life Technologies), and the first-strand cDNA was synthesized from 1.5 μg of treated total RNA using Vilo™ reverse transcriptase (Life Technologies Ltd, Paisley, UK). Reactions were carried out with Platinum SYBR Green qPCR SuperMix-UDG (Life Technologies Ltd) and specific primers (details are given in Supplementary Table S5 available at JXB online) using a ViiA7 real-time PCR machine (Applied Biosystems, Foster City, CA, USA). Plates were set up according to the sample maximization strategy proposed in Hellemans et al. (2007). Each sample was examined in three technical replicates, and dissociation curves were analysed to verify the specificity of each amplification reaction. The conditions of PCRs and the protocol of analysis were the same as reported in Giacomelli et al. (2012). Non-baseline-corrected data were imported in LinReg software to calculate reaction efficiencies (Ruijter et al., 2009). Six housekeeping genes (EF1α, GAPDH, ACTIN, UBIQUITIN, SAND, and α-TUBULIN; Supplementary Table S5) were tested for their stability in each experiment using the GeNorm software (Vandesompele et al., 2002). Normalized relative quantities (NRQs) were then calculated by dividing the RQ by a normalization factor, based on the expression of the two most stable reference genes (GAPDH and ACTIN) (Reid et al., 2006). Candidate gene selection Candidate genes were selected among the 12Xv1 predictions included in the confidence interval of reliable QTLs in the presence of at least one of the following functional pieces of evidence: (i) involvement in trait regulation based on the literature; (ii) differential expression between LFPs and HFPs (Supplementary Table S4 at JXB online); (iii) expression profile consistent with the accumulation of flavonols during berry development; in particular it was considered that Kaemp and Que show the highest peak of accumulation at flowering, and Isor and tri-hydroxylated flavonols at maturity (Sternad Lemut et al., 2013); expression in berry skin higher than in berry flesh (data from (Fasoli et al., 2012;Lijavetzky et al., 2012;Gouthu et al., 2014); (iv) co-expression with genes involved in the regulation of flavonol biosynthesis or in flavonoid metabolism [data from MarcoPaolo (provisional link, http://vitis.colombos.fmach.it/ accessed 19 December 2014), a grapevine compendium built based on COLOMBOS technology (Meysman et al., 2014), and from the VTC database (Wong et al., 2013); co-expression analyses by COLOMBOS were essentially based on contrast relevance and gene similarity scores, as described in depth in Supplementary Text S1, from Engelen et al. (2011); in both databases, the microarray and RNA-seq experiments related to berry and berry development were considered as data sets; and (v) assignment to functional categories significantly over-represented in QTL regions (Supplementary Table S6). Results and Discussion The population obtained by crossing the two red-skinned varieties, Syrah and Pinot Noir, comprises individuals with white (25%) and coloured (75%) berries in agreement with a 1:3 segregation of a major locus and with the bi-modal distribution of white-to-coloured grapes for anthocyanin content as reported by Costantini et al. (2015). The total flavonol content in the four seasons was on average three times higher in 'Syrah' than in 'Pinot Noir', ranging from 34 mg kg -1 to 57 mg kg -1 for 'Syrah' and from 5 mg kg -1 to 23 mg kg -1 for 'Pinot Noir'. In particular, Que and Myr were the most abundant flavonols in all the seasons, with Que being on average three and four times higher than Myr in 'Pinot Noir' and 'Syrah', respectively (Fig. 1A). The profiles among years were relatively stable in both cultivars, with few exceptions: in 2009, Kaemp, Isor, and Lar were completely undetectable in 'Pinot Noir', possibly compensated by an increase in Myr, with the latter being very low in 'Syrah' in 2011 (Fig. 1A). Moreover, looking at the ratios between tri-hydroxylated and di-hydroxylated flavonols (triOH/diOH), between 3′-methylated and 3′-hydroxylated flavonols (3′Meth/3′OH), and between 3′,5′-methylated and 3′,5′-hydroxylated flavonols (5′Meth/5′OH), it appears that they were on average higher in 'Syrah' than in 'Pinot Noir'. Considering the progeny, all six flavonol aglycons could be detected in almost all the red-skinned individuals, while the tri-hydroxylated flavonols (Myr, Syr, and Lar) were under the detection limit in the white-skinned progeny. The total flavonol content varied a lot in the progeny, ranging from 1 mg kg -1 to 76 mg kg -1 in the white-skinned offspring, and from 5 mg kg -1 to 142 mg kg -1 in the red-skinned individuals (Supplementary Table S1 at JXB online). These intervals are consistent with the reported range of variability of V. vinifera varieties: from 1 mg kg -1 to 132 mg kg -1 for white cultivars, and from 4 mg kg -1 to 176 mg kg -1 for red cultivars (Mattivi et al., 2006;Liang et al., 2011). The di-hydroxylated flavonol Que represented on average >70% of total flavonols in the white progeny, while Que and Myr together represented on average 80% of total flavonols in the coloured progeny, as previously observed (Mattivi et al., 2006;Castillo-Munoz et al., 2007. The Kaemp concentration was much higher in the white (max value=21 mg kg -1 of fresh berry in 2007) than in the coloured progeny (max value=8 mg kg -1 of fresh berry in 2011). In contrast, Isor was much higher in the coloured (max value=15 mg kg -1 of fresh berry in 2007) than in the white progeny (max value=1 mg kg -1 of fresh berry in 2011). In all years, the content of the six flavonols in the berries of the whole progeny showed a continuous variation, although the distribution was specific for each single compound and year (Fig. 1B). In addition, the progeny showed a transgressive segregation for all compounds in all vintages; that is, the two tails of the flavonol distributions were populated by individuals displaying a phenotype out of the range delimited by the parents. A significant number of offspring presented a content of each single flavonol much higher than 'Syrah', while 'Pinot Noir' displayed an average value of Kaemp, Lar, and Syr close to the minimum observed in the whole coloured progeny in all seasons (Fig. 1B). The concentration of the different compounds was in general stable across years (correlation r>0.80, P<0.01, when considering the whole progeny), supporting a substantial genetic control of their biosynthesis. However, Kaemp and Que appeared more influenced than the others by the season (r=0.5 and r=0.4, respectively; Fig. 1B). This tendency might be linked in general to the effect of the variability of the environmental conditions among seasons and also to the developmental regulation of the different classes of flavonols. Harvest is the time with the highest accumulation of trihydroxylated flavonols, matching well with the specific expression of flavonoid 3′,5′-hydroxylase genes (Jeong et al., 2006); flowering, on the other hand, is the time when the 'early peaking' flavonols Kaemp and Que, as named by Sternad Lemut et al. (2013), have a peak of abundance (Fujita et al., 2006). Metabolite analysis has also clearly indicated that some flavonoid compounds are highly associated at the biosynthetic level. Indeed, high Spearman rank-order correlations (significance of 0.01) were found both within flavonols and between flavonols and anthocyanins (Supplementary Table S2 at JXB online), supporting a common enzymatic and/or transcriptional regulatory system, as previously suggested (Mattivi et al., 2006;Martinez-Luscher et al., 2014). This was so in the case of tri-substituted flavonols (r>0.7 between Lar and Syr, and Myr and Lar, and r>0.6 between Myr and Syr), di-substituted flavonols (r>0.7, between Que and Isor), and mono-and di-substituted flavonols (0.6<r<0.8). Furthermore, the analysis of the correlation between triOH/ diOH, 3′Meth/3′OH, and 5′Meth/5′OH flavonols and anthocyanins suggests that some specific traits may be controlled by the same reaction(s). This could be the case of a common hydroxylase for the synthesis of Myr and delphinidin (r>0.6), or the same methyltransferase for the correlated 3′Meth/3′OH and 5′Meth/5′OH ratios (0.5<r<0.6). Conversely, the synthesis of Kaemp and diOH flavonols showed little or no correlation with that of anthocyanins and of triOH flavonols, as discussed above. QTL analysis In this study, several QTLs for the content of each single flavonol, for the sum of and for the ratio between the concentrations of specific classes of flavonols have been identified. When analysing the whole progeny, a major QTL for all the traits under analysis and for their sum (Tot_Flav) was identified on LG 2. In the case of Kaemp and Que, this QTL explained from 21% to 25% of phenotypic variance averaged over 3 and 2 years, respectively, and from 42% to 67% of variance averaged over 4 years in the case of Isor and triOH flavonols, respectively. The same QTL also regulates the ratio between specific classes of flavonols (explained variance from 15% to 35%). However, the position of the region on LG 2 associated with Kaemp and Que content was not stable over the different seasons (Supplementary Table S3 at JXB online). It is noteworthy that the QTL on LG 2 coincides with the major locus controlling anthocyanin berry content (Costantini et al., 2015) and co-localizes with a cluster of four MYBA genes, known to be essential for anthocyanin production via specific activation of the UFGT promoter (Kobayashi et al., 2002). Although there was some indirect evidence that these two classes of flavonoids are at least partially connected (Mattivi et al., 2006;Martinez-Luscher et al., 2014) and there is a clear strict interdependency of all the classes belonging to flavonoid metabolism, this finding of a common genetic control was completely novel. Further support for this regulatory link comes from the observation that the 'late peaking' flavonols, namely Isor and the triOH compounds, which show an accumulation profile mimicking those of the anthocyanins, are those where a greater extent of the phenotypic variation is explained by the QTL on LG 2. To investigate further the genetic control of this chromosomal region on flavonol content, advantage was taken of the data on the white-skinned individuals where MYBA genes are not expressed, anthocyanins are absent, and Kaemp, Que, and Isor are the only flavonols detected. For these compounds the analysis was run considering both the data of the whole progeny and the data of the white-skinned individuals (Kaemp_white progeny, Que_white progeny, and Isor_white progeny), independently. Interestingly, different regions (diverse from the QTL on LG 2) were found associated with the synthesis of Que, Isor, and their ratio, depending on the data set, while no QTL was found for Kaemp synthesis considering only the white-skinned individuals. A major QTL for Que_white progeny mapped to LG 10 (explaining 39% of the total variance in 2 years), while two different QTLs for Que in the whole progeny were detected on LG 2 and on LG 17 (explaining 25% and 11% of the total variance in 2 years). In the same way, a QTL for Isor_white progeny mapped to LG 11 (explaining 39% of the total variance, 3 years), while three different QTLs for Isor_whole progeny mapped to LG 2 (explaining 67% of the total variance) and to LGs 5 and 15 (explaining in both cases 5% of total variance). Finally, four different QTLs for 3′OMT_white progeny were identified on LGs 4a, 11b, 14, and 18b (explaining 33, 43, 22, and 19% of total variance, respectively). The regions associated with Isor/Que_whole progeny were completely different and mapped on LGs 1a, 15, 16, and 17 (explaining 25, 10, 15, and 13% of the total variance, respectively). These results suggest that the genetic regulation of the synthesis of non-triOH flavonols may be different in whiteskinned individuals compared with red-skinned ones, where the influence of the LG 2/MYBA region is predominant. Of the flavonol-specific QTLs, only a few regulated a specific trait: QTLs on LGs 4a and 14 controlling the 3′Meth/3′OH ratio (only on the white progeny), and a QTL on LG 18a controlling the Kaemp/Que+Isor ratio. All other QTL displayed a pleiotropic effect, as suggested by their influence on many traits visually highlighted in Fig. 2. Interestingly, the region on LG 7a associated with Kaemp (explaining 14% of total variance) co-localizes with the locus of VvMYBF1, whose expression shows a peak at early berry development (Czemmel et al., 2009), in agreement with the profile of the 'early peaking' flavonols such as Kaemp (Sternad Lemut et al., 2013). All the regions specifically related to flavonol content represent a valuable resource to search for new candidate genes potentially involved in the fine regulation of flavonols in the mature berry. Except for the major QTL on LG 2, the other regions shared with anthocyanins were related to more general activities. For example, the QTL on LG 1a primarily regulates the methylation level (23-35% of explained variance), the QTL on LG 6 the hydroxylation level (28% of explained variance), and the QTL on LG 10 the 5′-methylation level (10% of explained variance). Gene expression analysis Of the coloured offspring, four HFPs and four LFPs were selected, and compared at the transcriptomic level by means of a custom Affymetrix GeneChip using an approach based on bulked segregant gene expression analysis. Probe sets with significant differential expression (DEPs) between LFPs and HFPs were selected in a two-step analysis and under the assumption that individuals of the LFP and HFP groups can be treated as four biological replicates (pseudoreplicates). Since the expression levels reflect the average of each pool and expression outliers have reduced effect, genes showing differential expression between the two groups can be associated with the trait under investigation. This type of approach has been successfully used in previous works (Guo et al., 2006;Fernandez-Del-Carmen et al., 2007;Chen et al., 2010Chen et al., , 2011Kloosterman et al., 2010;Pons et al., 2014). Comparative analysis of the transcriptome at three developmental stages (PV versus VER, VER versus MAT, and PV versus MAT) within each bulk revealed the differential expression of 4834 and 3930 probe sets in HFPs and in LFPs, respectively. Of these, 1579 and 675 were exclusively modulated in HFPs and LFPs, while 3109 of 3255 were modulated in both, but with a difference in FC >50% (Supplementary Table S4 at JXB online). Differentially expressed probe sets were assigned to 84 functional categories at the third level of definition taken from the annotation of the GeneChip probe sets (Ashburner et al., 2000;Ruepp et al., 2004). Although the group of probe sets that could not be associated with any biological process ('unknown', 'unclear', and 'unclassified') or that could not find any significant hit ('no hit') was the largest in the lists of both the HFP and the LFP DEPs, 10 categories differently represented when compared with the GeneChip categories were identified (Fig. 3). In the HFP data set, the categories of the cellular process 'Oil body organization and biogenesis', of the primary metabolism 'Amino acid metabolism', 'Carbohydrate metabolism', 'Lipid metabolism', of the response to stress 'Abiotic stress response', and of transport 'a-Type channels' and 'Lipid transport' were significantly over-represented, while the 'no hit' category was somewhat surprisingly under-represented (Fig. 3). Noteworthy is the case of the 24 probe sets of the 'Abiotic stress response' category corresponding to genes mainly encoding dehydration-and light stress-responsive proteins, known to affect the flavonol profiles in the grape berry skin (Flamini et al., 2013;Teixeira et al., 2013): three abscisic acid (ABA)-responsive dehydration-responsive proteins 22 (Yamaguchi-Shinozaki and Shinozaki, 1993;Hanana et al., 2008) and one ELIP1 (early light-inducible protein) were identified whose activity in Arabidopsis was related to the presence of photoprotective flavonoids (Heddad et al., 2006). On the other hand, the LFP data set was significantly and exclusively enriched in two categories, 'Cell wall organization and biogenesis' and 'ABA signalling'. The large fraction of differentially expressed probe sets, corresponding to genes with unknown function potentially relevant for the fine control of the flavonol pathway, highlights that knowledge of the process is still scarce and is a valuable source for further investigation. The differential expression between HFPs and LFPs was used as the main criterion to support the choice of candidates within QTLs for the genes represented on the GeneChip. Candidate gene identification The number of genes within each QTL confidence interval varied from a minimum of 101 (LG 18b) to a maximum of 740 (LG 18a) (Fig. 2). The criteria adopted for candidate gene selection are detailed in the Materials and methods. However, functional annotation deriving from previous knowledge present in the literature was particularly considered, in the case of transcription regulators and structural enzymes related to phenylalanine, phenylpropanoid, flavonoid, and flavonol metabolism, as well as transport and signalling/response to stimulus. The selected candidate genes are listed in Table 1 and Supplementary Table S7 at JXB online, together with functional evidence from this study or previous studies. Costantini et al. (2015) controlling anthocyanin content are also presented. In both cases, only the traits regulated by at least two regions are visualized as coloured connections between chromosomes. External numbers and axes, for each blue arc, indicate the chromosomes and the physical length in megabases (Mbp), respectively. The concentric orange coloured circles represent three different levels of the explained variance for each trait (10, 50, and 90% outwards), while the red and violet squares correspond to flavonols and anthocyanins, respectively. A single colour code is associated with each trait as in the key. Gene names indicate the identified candidate genes to which particular attention was paid in the text. Table 1. Genes with a potential role in the regulation of flavonol content and composition Only QTL regions exclusively associated with flavonol content are shown, while those associated with both flavonol and anthocyanin content are listed in Supplementary Table S7 at JXB online.  Glutathione S-transferase 26 GSTF12: VIT_04s0079g00690* Induced at maturity both in HFPs and LFPs (slightly higher fold change in HFPs); higher expression in skin than in flesh (Lijavetzky et al., 2012); expressed in the skin from véraison (Fasoli et al., 2012); co-expressed with genes of the "quercetin O-glucoside metabolic process".  Cyclic nucleotide-regulated ion channel (CNGC14): VIT_04s0069g00790* Induced at maturity both in HFPs and LFPs; higher expression in skin than in flesh (Lijavetzky et al., 2012); co-expressed with genes of the "flavonoid metabolic process"; involved in the response to biotic stimulus.  LG Transparent testa 8 TT8: VIT_07s0104g00090 BrTT8 is involved in controlling the late biosynthetic genes of the flavonoid pathway (Li et al., 2012); higher expression in skin than in flesh (Lijavetzky et al., 2012).  Among the selected genes, special attention was paid to those encoding specific enzymes of the flavonol metabolism pathway as well as to fine regulators of the trait, which represent novel candidates identified in this study (Table 1). Glycosylation is an important step in flavonol biosynthesis, enhancing their water stability and accumulation in plant cells. This process is regio-specific and is driven by specific glycosyltransferases (UGTs) for each flavonoid class (Ono et al., 2010). Two previously characterized flavonol UGTs (VIT_11s0052g01630=VvGT5 and VIT_11s0052g01600=VvGT6) are located within a QTL on LG 11 (11b) that was found to be specific for flavonol biosynthesis. The conversion of Myr into Lar and Syr by 3′ and 5′ methylation is driven by the O-methyltransferases (OMTs) that convert delphinidin into petunidin and malvidin (LG 1; Hugueney et al., 2009;Fournier-Level et al., 2011;Costantini et al., 2015). However two new putative caffeoyl-CoA OMTs for the methylation of Myr to Syr (VIT_11s0016g02600 and VIT_11s0016g02610; VvCCOAMT genes in the present work) were identified in this study on LG 11a. These two genes are very similar to AT4G26220, which encodes an enzyme preferentially methylating the para position of flavanones and dihydroflavonols (Wils et al., 2013). Considering hydroxylation, it was confirmed that two of the five flavonoid 3′,5′-hydroxylases identified as candidates within the QTL on LG 6 are involved in the hydroxylation of both anthocyanins and flavonols. This is in agreement with the significant correlation between the triOH/diOH ratio in the two classes observed in the 'Syrah'×'Pinot Noir' progeny (r=0.55; Supplementary Table S2 at JXB online) and in the grapevine germplasm (r=0.45; Mattivi et al., 2006)). Three diphenol oxidases (VIT_18s0164g00090, VIT_18s0164g00110, and VIT_18s0164g00170; VvLAC14 in the present work) and the putative homologue of AtPrx12 (VIT_18s0072g00160; VvPrx12 in the present work) (QTL on LG 18b, common to anthocyanins) were considered as potentially involved in the oxidative turnover of flavonols among flavonoids (Pourcel et al., 2005;Oren-Shamir, 2009;Zipor et al., 2014). Genes playing a role in flavonoid transport in many plants including grapevine (Zhao and Dixon, 2010) were also listed among the best candidates for flavonol sequestration in the vacuole and the cell wall: priority was GSTs have been associated to flavonoids' transport .  ER6 protein universal stress protein (USP) family: VIT_18s0001g07360* Involved in ethylene signalling; induced in green versus ripe berries in HFPs.  Ethylene-inducible CTR1: VIT_18s0001g07700* Involved in ethylene signalling. Induced in green versus ripe berries in HFPs and more expressed in the skin than in the flesh (Lijavetzky et al., 2012).  LIM domain protein WLIM: VIT_18s0001g09040* Induced in green versus ripe berries in in HFPs and LFPs; co-expressed with VvMYBF1.  The symbols * and # indicate genes found to be differentially expressed or not in this study. given to glutathione S-transferases on LGs 1a, 4a, and 15 and glutamate receptor proteins (LG 4), whose relationship with flavonoids is due to their neuroprotective effect under stress (Nakayama et al., 2011). At the same time, genes encoding regulatory factors or proteins whose expression is known to be modulated by external or endogenous factors are identified. Flavonol accumulation has been shown to be highly influenced by UV-B light and auxin and ethylene signalling, due to the important role played by flavonols as UV protectants during berry ripening (reviewed by Flamini et al., 2013;Teixeira et al., 2013) and as modulators of auxin transport (Lewis et al., 2011). It is worth noting that most of them are located within QTLs specifically involved in flavonol variation. This is true in the case of VIT_07s0005g01210 (VvMYBF1), VIT_14s0066g01090 (MYB24), VIT_17s0000g06930 (VvUNE10 in the present work), VIT_18s0122g00450 (VvCAD6 in the present work), and VIT_18s0001g03470 (VvFLS4) as these transcripts were found to be up-regulated by UV-radiation in the skin of ripe berries of the cultivar Tempranillo (Carbonell-Bejerano et al., 2014). VvFLS4 and VvFLS5 are the only two isoforms of flavonol synthase that accumulate in the skin of berries at the ripening stage (Fujita et al., 2006). As potential candidate genes, attention was also focused on the bZIP family members falling within the identified QTL regions, since they are known to be part of light-mediated regulation of some flavonoid biosynthetic genes (Hartmann et al., 2005;Czemmel et al., 2009). VIT_05s0020g01090 (VvbZIP15 in Liu et al., 2014) is the putative homologue of AtHY5, which regulates PFG1/MYB112, a flavonol-specific activator of flavonoid biosynthesis in response to light (Stracke et al., 2010). One bZIP gene probably involved in flavonol metabolism is VIT_07s0005g01450 (VvbZIP22 in Liu et al., 2014), in proximity to VvMYBF1 within a QTL on LG 7 and whose expression during berry development and upon UV-light treatment correlates well with flavonol accumulation (G. Malacarne et al., unpublished). A very similar bZIP in terms of sequence and of expression profile in HFPs and LFPs is VIT_05s0077g01140 (VvbZIP14 in Liu et al., 2014) which was found in a QTL on LG 5. Both sequences are highly similar to that of AtbZIP53, coding for a bZIP related to amino acid metabolism during plant starvation (Dietrich et al., 2011). Three genes coding for a proton-dependent oligopeptide transport family protein (VIT_17s0000g05550), for the putative homologue of AtMYB66 (VIT_17s0000g08480), and for a chlorophyll a-b binding protein 4 (VIT_17s0000g06350) were localized within the QTL on LG 17 which appeared to be associated with different metabolic traits. These genes were coexpressed with VvMYBF1 during berry development (Fasoli et al., 2012;Lijavetzky et al., 2012;Dal Santo et al., 2013) and they are differentially expressed in HFPs versus LFPs. Finally, genes such as VIT_01s0150g00300 and VIT_01s 0026g02620, VIT_07s0141g00290 and VIT_07s0141g00270, VIT_11s0016g03640 and VIT_12s0059g00190, although located within different QTL intervals, all appear to be involved in auxin-mediated flavonoid regulation of plant growth and development (Lewis et al., 2011), or during response to UV-B radiation (Berli et al., 2010). There is indeed evidence of flavonols influencing auxin transport and auxin-dependent physiological processes (Buer and Muday, 2004), as well as auxin and ethylene tuning flavonol accumulation (Lewis et al., 2011). Moreover, factors that induce flavonol biosynthesis such as the light levels also affect auxin transport (Buer et al., 2006). . Only the functional categories significantly enriched in HFP or LFP data sets (black and grey, respectively) compared with the GrapeGen Chip (white) according to a hypergeometric test (P<0.05) are shown. Conclusions In this work, an integrative approach based on transcriptional profiling and metabolic QTL analyses was adopted to examine the regulation of berry flavonol content and composition in wine cultivars at the ripe berry stage. The seven QTL regions associated exclusively with flavonol biosynthesis represent a valuable resource for breeding and selection of new high quality cultivars. Supplementary data Supplementary data are available at JXB online. Supplementarty Text. Figure S1. (A) Comparison of microarray and real-time RT-PCR analyses. (B) Consistency of expression tested among replicates for VvMYBF1 and VvFLS4 genes by real-time RT-PCR. Table S1. Flavonol variation in the white and coloured individuals of the 'Syrah'x'Pinot Noir' progeny. Table S2. Correlations between metabolites in the coloured progeny. Table S3. QTLs for flavonol content and composition identified in the 'Syrah'x'Pinot Noir' progeny. Table S4. GrapeGen Chip probe sets differentially expressed during development in high-and low-flavonol producers. Table S5. Primers used in quantitative RT-PCR analysis for microarray validation. Table S6. Test for over-representation of specific functional categories in QTL regions. Table S7. Genes with a potential role in the regulation of both flavonol and anthocyanin content and composition. Text S1. Validation of microarray results by real-time RT-PCR analysis.

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Grape Seed Proanthocyanidin Extract Improved some of Biochemical Parameters and Antioxidant Disturbances of Red Blood Cells in Diabetic Rats. Grape seed proanthocyanidin extract (GSPE) has a broad spectrum of biologic properties against oxidative stress. This study aimed to investigate the effects of GSPE on biochemical factors and antioxidant enzymes of erythrocyte in diabetic rats. Diabetes was induced through single injection of streptozotocin (50 mg.Kg-1, i.p). Forty Male Sprague-Dawley rats were divided into four Groups: Group 1, healthy control group; Group 2, healthy group treated with GSPE (200 mg.Kg-1); Group 3, diabetic control group; Group 4, diabetic group treated with GSPE (200 mg.Kg-1) for 4 weeks. At the end, the experimental animals were sacrificed and blood samples were collected and plasma parameters and erythrocytes antioxidant status were evaluated. The results show, treatment with GSPE significantly reduced (P<0.001) urine volume, proteinuria and biochemical factors such as blood urea nitrogen, creatinine, triglyceride, total cholesterol, low density lipoprotein and very low density lipoprotein as well as malondialdehyde. Also GSPE treatment significantly (P<0.005) increased high density lipoprotein, total protein and albumin. Moreover GSPE significantly increased antioxidant enzymes activity such as: superoxide dismutase, glutathione peroxidase and catalase. These results suggest that GSPE can ameliorate biochemical abnormalities and antioxidant system status in streptozotocin- induced diabetic rats probably by its potent antioxidant features. Introduction Diabetes mellitus (DM) is an important metabolic disorder. Due to the increasing over the world incidence of diabetes mellitus creates a financial burden for global public health (1). It is distinguished with hyperglycemia and longterm complications which involves the nerves, kidneys, eyes and blood vessels. Although the mechanisms causing diabetic complications are still unknown, but more attention has been focused to the function of oxidative stress. It has been suggested that oxidative stress may be involved in the development of various diabetic complications (2, 3). Moreover, diabetes is associated with many features such as, increased lipid peroxidation (4), change of the glutathione redox situation, and decreased activity of antioxidant enzymes. These alterations show that oxidative stress induced by hyperglycemia (5). Several defense systems participate against oxidative damages caused by diabetes. One of these system are antioxidants destroy free radicals (6). Nowadays to minimize oxidative damage, there is an increasing interest to use Glucose Monitoring system, Infopia, Korea). Rats with fasting blood glucose levels above 250 mg/dl were used as the diabetic animals. The treatment was started on the fourth day after the STZ injection and this was considered as the first day of treatment. The treatment was continued daily for 4 weeks. The rats were divided into four Groups of comprising 10 animals in each group as follows: Group 1, healthy control group; Group 2, healthy group treated with GSPE (200 mg.Kg -1 ); Group 3, diabetic control group; Group 4, diabetic group treated with GSPE (200 mg.Kg -1 ). Rats were orally administered with GSPE (Hangzhou Joymore Technology Co., Ltd China) dissolved in normal saline. At the end of 4 th week six rats selected randomly from every investigated group then rats were kept individually in metabolic cages to collect 24 hour urine for measurements of urine output and excreted protein. Assessment of excreted protein was done by using commercially available kit on spectrophotometer (Pars Azmon, Iran). Then rats were scarified under ether anesthesia and their blood was collected and processed Preparation of erythrocyte lysate Whole blood was obtained by cardiac puncture and collected into heparinized tubes. Erythrocytes were separated from plasma by centrifugation at 3500 rpm for 10 min. To get packed erythrocytes, the remaining erythrocytes were washed tree or four times with an isotonic solution of NaCl (0.9%) until a colorless supernatant was observed. To obtain erythrocyte hemolysate, 500 µL packed erythrocyte was crushed by addition of four volumes of cold redistilled water. Then was centrifuged twice to remove all of the cell membranes: first for 10 minutes in the tube centrifuge at 3500 rpm at 4 °C, then in an Eppendorf centrifuge at 7800 rpm for 5 minutes at 4 °C. Clear supernatant was obtained as hemolysate. plasma was used for the determination of biochemical parameters and malondialdehyde (MDA) and hemolysate used for study of antioxidant status. Biochemical parameters Biochemical factors such as fasting blood sugar (FBS), total protein (TP), albumin (Alb), blood urea nitrogen (BUN), creatinine (Cr), and natural antioxidants. The researchers showed that many of damaging effects of oxidative stress are reduced with intake of dietary antioxidant such as vitamins and other nonfood antioxidants such as flavonoids and polyphenols (7). Proanthocyanidin is a compound extracted from grape seed and its basic structure unit is the catechin. Proanthocyanidins are containing monomer, dimer and trimer catechin, all of which are water-soluble molecules and contain a number of phenolic hydroxyls (8). Polyphenolic compounds having a very important function of antioxidant, they can clean off the free radicals, and reduce the membrane lipid peroxidation, so they can reduce the occurrence of free radical-related diseases and delay aging (9, 10). Current researches have revealed that grape seed proanthocyanidin extract can clear off free radicals, protect the over-oxidative damage caused by free radicals, (11, 12) and prevent a range of diseases caused by free radicals, such as myocardial infarction, atherosclerosis, druginduced liver and kidney injury; moreover, it has functions of anti-thrombotic, anti-tumor, anti-mutagenic, anti-radiation, and anti-fatigue (13-15). Therefore, we investigated the effects of grape seed proanthocyanidin extract (GSPE) on some biochemical parameters and antioxidant enzymes in blood of diabetic rats. Animals and experimental design This study was reviewed and approved by the Ethics Committee of Ahvaz University of Medical Sciences. Forty male Sprage-Dawley rats (150-170 g) were prepared from animal house central of Ahvaz Jundishapur University of Medical Sciences (AJUMS). All animals were housed in cages with 12/12 h light/dark cycle at 21 ± 2 • C. The animals were fasted overnight and diabetes was induced by a single intraperitoneal injection STZ (50 mg/Kg body weight) (Sigma-U.S.A.) a freshly dissolved in citrate buffer (o.1 M pH 4.5), while control rats were injected with vehicle buffer only. Blood samples were obtained from the tail vein of the animals at 72 hours after STZ injection and fasting blood glucose levels were determined with a glucose strip test in a glucometer (Easy Gluco Blood lipid profile study such as, triglycerides (TG), cholesterol (Chol), high density lipoprotein (HDL), low density lipoprotein (LDL) and very low density lipoprotein (VLDL) were determined by auto analyzer (Vita lab Selectra E, Netherland) in Golestan Hospital, Ahvaz using commercially available kits (Pars Azmon, Iran). Estimation of MDA in plasma MDA was assayed according to the method of Satho (16). 0.5 mL of plasma was mixed with 1.5 mL trichloroacetic acid (TCA) (10%) and was centrifuged (4000× g for 10 minutes) then 1.5 mL of supernatant was blended with 2 mL thiobarbituric acid (TBA) (0.67%) and was kept for 30 minutes at 100 °C. After cooling, 2 mL n-butanol was added to the solution and then centrifuged at 4000× g for 15 min, finally pink supernatant absorbance was read at wavelengths 535 nm. Values were expressed as nmol/mL. As 99% of the TBARS is malondialdehyde (MDA), TBARS concentrations of the samples were calculated from a standard curve using 1,1,3,3-tetramethoxypropane. Determination of antioxidant enzymes activity in erythrocyte lysate Superoxide dismutase (SOD) and Glutathione peroxidase (GPX) activities were determined using the diagnostic kits RANSOD and RANSEL produced by RANDOX (Randox Labs, Crumlin, UK) and were expressed in unit per gram of haeomglobin (Hb). Catalase (CAT) activity was measured by the method of Aebi (17). The final reaction volume of 1 mL contained 50 mM potassium phosphate (pH 7.0), 19 mM H 2 O 2 , and a 20-50 µL sample. The reaction was initiated by the addition of H 2 O 2 , and absorbance changes were measured at 240 nm (20 °C) for 30 s. Catalase activity was estimated by using the molar extinction coefficient of 43.6M -1 cm -1 for H 2 O 2 . The level of CAT was expressed in terms of µmoles H 2 O 2 consumed/min per gram of haeomglobin. Statistical analysis Data are expressed as the mean ± SD. Statistical significance of differences was assessed with one-way analysis of variance (ANOVA) by SPSS for Windows version 18 (IBM, USA) followed by Tukey´s -test. P<0.05 was assumed as statistically significant. Changes in urine volume (UV), urinary protein 24-hour (UP24h) and biochemical parameters As shown in the Table 1, urine volume (UV) and UP24h were higher in group 3 when compared with the group 1 (P<0.001) but in group 4, GSPE administration significantly (P<0.001) reduced urine volume and urinary protein excretion as compared to Group 3. There was no significant difference between the two groups 1 and 2 (P>0.05). Assessment of biochemical factors at the end of the 4-week period showed that the GSPE in group 4, significantly (P<0.001) decreased the elevated levels of FBS, BUN, Cr, TG, LDL, VLDL, Chol and increased the reduced levels TP, Alb and HDL levels in the group 3(P<0.005), However, no significant difference was observed between 1 and 2 (P>0.05) ( Table 1). Table 2, demonstrates the differences in plasma MDA and erythrocyte SOD, GPx and CAT activities. A significant decrease (p<0.001) of plasma MDA and significant increase in erythrocyte GPx, CAT and SOD were observed in group 4 after administration GSPE as compared with group 3. Also group 2 showed no significant difference with group 1 (P>0.05). Discussion STZ by increasing of free radicals formation and/or impaired antioxidant defense leads to generation of oxidative which can cause intensive injury in various tissues and may lead to various diseases such as diabetes (18). Antioxidants are substances which postpone or arrest the oxidation of cellular substrates that can be oxidized. Function of the different antioxidants is to eliminate superoxide, and/ or activating of detoxifying/defensive proteins (3). This study was designed to investigate the effect of GSPE Each value is expressed as mean ± standard deviation (n = 6), differences at p < 0.05 were considered significant. a -different than the healthy control group; b -different than the diabetic control group. Each value is expressed as mean ± standard deviation (n = 6), differences at p < 0.05 were considered significant. a -different than the healthy control group; b -different than the diabetic control group on biochemical factors and antioxidant changes in STZ-induced diabetic rats. We observed that diabetic control rats developed severe polyuria as a result of osmotic diuresis. However, the diabetic rats that were treated with GSPE in group 4 showed significant decrease in urinary volume, probably as a result of the normalization of plasma glucose level or synergistic effect with insulin as shown in previous study (19,20). Our study also showed, an increase UP24h in diabetic control rat. Proteinuria, a hallmark feature of early glomerular damage in patients with diabetes, is associated with renal hemodynamic and histologic changes (21) also increased synthesis of reactive oxygen species, or loss of nephrin in podocytes as shown by different investigators (22). GSPE-treated diabetic rats showed an impressive decrease in the amount of proteinuria like the previous study (23). Our findings indicate that FBS had a meaningful decrease in diabetic rats treated with GSPE as compared with diabetic control rats. It is likely that GSPE decreased glucose levels in diabetic rats by increasing circulating insulin levels (20). This findings supported by previous study (24). The results in Table 1 showed, reduction in plasma total protein and albumin level in diabetic control rats. The decrease in protein and albumin may be due to microproteinuria and albuminuria, and/or may be due to increased protein catabolism (25). Also we observed significant increase in the level of plasma BUN and Cr in the diabetic control rats when compared respective with healthy control group rats. Increased blood urea nitrogen and serum creatinine in diabetic rats indicates progressive renal damage (26) also increased urea nitrogen production in diabetes may be accounted for by Groups enhanced catabolism of both liver and plasma proteins (27). In current study, serum levels of TG, LDL, VLDL and TC were significantly elevated in diabetic control rats when compared with healthy control group rats. Alterations in plasma lipoprotein metabolism are common in diabetes, which tend to exaggerate any preexisting tendencies towards elevated lipid levels (28). In addition, diabetes is associated with increased dyslipidemia (29). Elevation of serum lipids indicate either the defective removal or overproduction (or both) of one or more lipoproteins (30). Administration of GSPE significantly improved all of these changes of biochemical factors caused by diabetes. Moreover, the serum level of HDL-C, which was significantly decreased in diabetic rats, was also improved by GSPE. These effects of GSPE are in agreement with the previous studies (20, 31, 32). Our results showed an increased MDA level in the plasma of diabetic control rats and a significantly decreased erythrocyte enzymatic antioxidant activities such as GPx, SOD and CAT. Hyperglycemia, reduces production and activities of some of antioxidant enzymes such as: SOD and GPx, likely by glycation (33). It is well known that people with diabetes have a lower antioxidant defense, enzymatic (SOD, catalase and GPx) and non-enzymatic (vitamin C, E or A, free radical scavengers). Also, increase of MDA information is probably due to the additional production of divers radical species. These radicals have been suggested to stimulate destruction of lipids and carbohydrates leading to hyperglycemia and related glucose autooxidation (34). All of these found changes in the antioxidant system and MDA were restored by the administration of GSPE. Conclusion We suggest that oral administration of GSPE provides a new and effective approach to attenuating metabolic disorders and defect of antioxidant system defense that induced by STZ. These effects are probably due to powerful antioxidant property of GSPE. Therefore, in view of these properties GSPE may be a good candidate to prevent the progression of diabetes complications.

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