Radiation Interception, Chlorophyll Fluorescence and Senescence of Flag leaves in Winter Wheat under Supplemental Irrigation

Water shortage threatens agricultural sustainability in China, effective water-saving technologies urgently need to be developed. In this study, five treatments were conducted: rainfed (W0), a local supplemental irrigation (SI) practice (W1), and three treatments in which soil water content was tested prior to SI, specifically at 0–20 (W2), 0–40 (W3) and 0–60 cm (W4) soil layers. Soil water consumption in W3 had no differ with W2 but was higher than W1 and W4. Crop evapotranspiration in W1, W3 and W4 treatments were higher than that in W2. W3 treatment had higher leaf area index than W1 and W4 at later grain filling stages. The mean photosynthetically active radiation capture ratio in W3, especially at 20, 40 and 60 cm plant heights, were significantly higher than those in W1, W2 and W4. The chlorophyll content index, actual photosynthetic activities, catalase and superoxide dismutase activities of flag leaves from W3 were the highest after the middle grain filling stages. W3 treatment obtained the highest grain yield (9169 kg ha−1) and water use efficiency (20.8 kg ha−1 mm−1) in the two seasons. These benefits likely accrued through created a suitable soil moisture environment in W3 treatment.

Winter wheat (Triticum aestivum L.) is one of the major crops in the Huang-Huai-Hai Plain of China. More than 60% of wheat produced in China is grown here 1 . The climate is a warm-temperate continental monsoon type with an annual average temperature range of 3.1 °C in the north to 16.8 °C in the south 2 . In this region, the total water consumption required by winter wheat is approximately 400-500 mm in a growing season, but only 150-180 mm of precipitation falls during this period 3 . Additional irrigation is thus required. Farmers using traditional practices in this region irrigate wheat crops with up to 310 mm of water, leading to the low water use efficiency (WUE) 4 . Therefore, effective water-saving technologies urgently need to be developed to maintain a high production of winter wheat.
In recent years, supplemental irrigation (SI) has been studied as a highly efficient practice with great potential for increasing agricultural production and improving livelihoods, which was widely used to improve crop yield and WUE 5 . Abourached et al. suggested that SI applied at heading and/or after heading could reduce water shortage stress during the grain-filling period and increase grain yield and WUE 6 . To obtain higher grain yield and WUE than the traditional irrigation practices, Wang et al. recommended two applications, at jointing and booting, with 75 mm each time, whereas Li et al. recommended two applications, at jointing and heading, with 60 mm each time, which was considered as one of the water-saving irrigation regimes in the Huang-Huai-Hai Plain of China [7][8][9] . However, most of the studied focused on fixed amounts of irrigation and, there are some limitations, as did not consider the effect of soil water conditions before irrigation (particularly those at different soil depths) on the irrigation amount, water consumption and winter wheat production. Therefore, irrigation practices based on the consideration of precipitation, soil water supply, and the physiological requirement of wheat grown in this region to increase water-saving in wheat production need to be developed.

Results
Irrigation, soil water consumption and crop evapotranspiration. The irrigation amount increased with increasing the measure depth from 20 cm (W2) to 60 cm (W4) (Table 1), the mean irrigation in W4 was 128.6 mm, which was higher by 33.4 mm and 73.0 mm than W3 and W2, respectively. The soil water content (SWC) in 0-200 cm soil layers at maturity in both seasons were presented at Fig. 1, the SWC in 60-140 cm soil layers of W3 was significantly lower than that in W1, W2 and W4, indicating that W3 had the highest soil water absorption in 60-140 cm soil layers among SI treatments.
The soil water consumption (ΔW) from the W0 treatment was higher than that of the other treatments in 2012/2013 and 2013/2014 (Fig. 2). For the SI treatments, in 2012/2013, the highest ΔW was obtained from the W3 treatment, followed by the W2, the lowest ΔW was obtained in the W1 and W4 treatments. In 2013/2014, there was no significant difference in ΔW between W2 and W3 treatments, but the values were significantly higher than those from W1 and W4. The crop evapotranspiration (ET c ) from the W1, W3 and W4 treatments did not differ in 2012/2013, but the ET c in the W3 treatment was lower than that of the W1 and W4 treatments in 2013/2014. The W2 treatment had the lowest ET c among the irrigation treatments. W0 had the lowest ET c values in both growing seasons.
Leaf area index. The LAI of plants from the W0 treatment was the lowest at May 2, May 16, and June 1 in 2013 (Fig. 3). For the SI treatments, LAI from the W3 treatment were higher than those from W1 and W4, and the differences were significant at June 1; the lowest LAI was obtained in the W2 treatment. Compared with the  Fig. 4. On May 2 and May 16, the PAR capture ratios at a height of 0 cm did not differ among the W1, W3 and W4 treatments, but these were higher than that from W2; the highest PAR capture ratios at heights of 20, 40 and 60 cm were obtained in W3, followed by W1 and W4, and then W2. On June 1, the PAR capture ratios at heights of 0, 20, 40 and 60 cm from W3 were the highest, followed by the W1 and W4, and the W2 treatment had the lowest values among the SI treatment. Compared to the SI treatments, the lowest PAR capture ratio were obtained in W0 at heights of 0, 20, 40 and 60 cm on the three measure days.
Chlorophyll content and chlorophyll fluorescence. In 2012/2013, the CCI and F v /F m of flag leaves from 0 to 21 days after anthesis (DAA) from W1, W3 and W4 treatment had no differ, but the values from 28 to 35 DAA from W3 were significantly higher than those from W1 and W4 (Fig. 5); there were no differ in the ΦPSII of flag leaves between W1, W3 and W4 treatments from 0 to 7 DAA, however, W3 had higher ΦPSII than those from W1 and W4 from 14 to 35 DAA. In 2013/2014, the CCI of flag leaves from 0 to 21 DAA from W1, W3 and W4 treatment had no differ, but the values from 28 to 35 DAA from W3 were significantly higher than those from W1 and W4; there were no differ in the F v /F m and ΦPSII of flag leaves between W1, W3 and W4 treatments from 0 to 7 DAA, however, W3 had higher F v /F m and ΦPSII than those from W1 and W4 from 14 to 35 DAA. The CCI, F v /F m and ΦPSII of flag leaves from the W0 treatment were lowest during the grain filling stage in both growing seasons.
Leaf senescence characteristics. There were no significant differences among treatments in malondialdehyde (MDA) concentration of flag leaves at the beginning of grain filling in 2012/2013 growing season (Fig. 6). However, the highest MDA concentrations in flag leaves were obtained from the W0 treatment from 7 to 28 DAA. The MDA concentrations in flag leaves from W2 were significantly higher than those from W1, W3 and W4 from 21 to 28 DAA, but the values between W1, W3 or W4 did not differ. By contrast, the lowest catalase activities, SOD activities and soluble protein concentrations in flag leaves were obtained from the W0 treatment. The catalase, SOD activities and soluble protein concentrations in flag leaves from W2 were significantly lower than those from W1, W3 and W4 during the grain filling stage. The CAT, SOD activities and soluble protein concentrations in flag leaves between the W1 and W3 or W4 treatments from 0 to 14 DAA were not differ, but from 21 to 28 DAA, those parameters from the W3 were significantly higher than those from W1 and W4 treatments.    Table 3. The grain yield, WUE and HI from W3 were higher than those from W1 and W4, and the lowest values were from W2 among SI treatment in both growing seasons. The mean grain yield and WUE in the W3 treatment in both growing seasons were higher by 3.4% and 2.8% than those from W1 and higher by 4.4% and 6.0% than those from W4, respectively. Compared to the SI treatments, W0 had the lowest grain yield, WUE and HI in both growing seasons.

Discussion
Irrigation increases crop evapotranspiration, while soil water consumption has a negative relationship with irrigation 22 . In this study, the irrigation amount was determined by the water content of different soil layers, which was used in our previous studies 21, 23 . The mean irrigation amount from the W3 treatment was 95.2 mm, significantly higher than that of the W2 treatment but lower than that of the W1 and W4 treatments, the crop evapotranspiration from W3 was higher than that of W2 but lower that of W1 and W4, however, the mean soil water consumption from the W3 treatment was higher than that of the W1 and W4 treatments by 27. Soil water use by plant is associating with root growth and development. Studies found that reasonable irrigation regimes with lower water stress could facilitate root growth, especially in deep soil layers, which is conductive to water absorption from soil 7, 24 . Xue et al. showed that the water uptake rate in 0-100 cm soil layers was significantly lower in rainfed than in irrigation treatments because of low root density 26 . Li et al. reported that irrigation of 120 mm only at jointing results in the highest root length density, leading to the highest soil water consumption in 0-160 cm soil layers 9 . In this study, the irrigation amount in W3 ranges from 89.6 mm to 100.8 mm, which was significantly higher than that in W2 but lower than that in W4 (Table 1). However, the soil water use in W3 was the highest (Fig. 2), particularly in the 60-140 cm soil layers (Fig. 1), which are likely because that the irrigation determined by measuring the moisture in 0-40 cm soil layer facilitate root growth in the 60-140 cm soil layer, improve water use from soil 27 .
Suitable irrigation amount could improve grain yield and water use efficiency 28,29 . Karam et al. reported that irrigation with 50% of the full SI (based on the SWC in 0-90 cm soil layers) achieved 340 kg ha −1 greater yield than that of the 100% SI treatment 28 . While, Boutraa et al. concluded that plants grown under 80% FC in the 0-120 cm soil layer have the highest grain yield and WUE 29 . Here, our results showed that the highest grain yield, WUE and HI were obtained in the W3 treatment, with mean values of 9169 kg ha −1 , 20.8 kg ha −1 mm −1 and 44.5%, respectively, in both growing seasons, which indicate that the appropriate irrigation amount obtained by measuring the 0-40 cm soil layer moisture created a suitable soil environment (approximately 65% FC and 70% FC after SI at jointing and anthesis, respectively), which increased the use of soil water and improved the grain yield and WUE.
Leaf area index and PAR are the main factors determining crop growth in wheat, which is significantly affected by irrigation 9, 30 . Ram et al. reported that LAI increased with increased irrigation in wheat, but the difference was not significant as more than 225 mm of irrigation 11 , we obtained the same results in this study, and we also found that the LAI dropped at later stages of winter wheat as the total irrigation amount over 95.2 mm. The LAI was one of the most important factors on affecting the amount of incoming PAR absorbed by the canopy, and the greater the crop LAI, the greater is its PAR interception 31 . In this study, the LAI from the W3 treatment was higher than that of the W1, W2 and W4 treatments, the PAR capture ratio was also higher than those of the W1 and W4 treatments, especially at later grain filling stages, the results indicating that suitable soil water conditions could improve the LAI and PAR at these stages. We also found that the PAR capture ratio at heights from 20 cm to 60 cm in the W3 treatment was higher than that of the other treatments during the grain filling stage (Fig. 3), which indicates that the PAR capture ratio in the upper canopy of winter wheat had a large contribution to the final grain yield.  Table 2. Photosynthetically active radiation (PAR) capture ratio, penetration ratio, and reflection ratio in winter wheat canopy of each treatment (%) The data were the average values on May 1, May 16, and June 1 in 2013. W0: rainfed, W1: a local supplemental irrigation practice at jointing and anthesis with 60 mm each time, W2, W3 and W4 are supplemental irrigation determined by measuring 0-20 cm, 0-40 cm, and 0-60 cm soil layer moisture, respectively, and brought the soil moisture to 65% FC at jointing and 70% FC at anthesis. Within a column values followed by different letters differ significantly at the 0.05 level by the LSD test.
Both Saeidi et al. and Mu et al. reported that more than 70% of winter wheat yield is produced by photosynthesis in spikes and leaves after heading 32,33 . Photosynthesis is directly affected by chlorophyll content and chlorophyll fluorescence, which decreased significantly in water deficit conditions and resulted in a reduction in photosynthetic capacity 34,35 . In this study, the CCI, F v /F m , and ΦPSII of flag leaves from the W3 treatment were higher than those from W1, W2 and W4 treatments after 21 DAA (Fig. 5). This result indicated that SI applied at jointing and anthesis by measuring the soil moisture of the 0-40 cm layers created a suitable soil environment, which improved the photosynthetic capacity at the middle and later grain filling stages.
Loss of leaf viability during senescence closely links the duration of photosynthetically active leaf area and grain yield in wheat 36 . Leaf photosynthesis declines during grain filling, when leaves start to senesce and the photosynthetic apparatus disassembles rapidly within chloroplasts 17 . This senescence-associated decline in photosynthetic capacity of leaves can be exacerbated by water stress or waterlogging 19,37 . Here, our results also found that the MDA concentrations in W0 flag leaves were higher than those in flag leaves from SI treatments. On the contrary, the lowest SOD and catalase activities were observed in the W0 treatment. Saeedipour and Moradi also observed that irrigation with 50% FC after anthesis enhanced the senescence by accelerating loss of leaf chlorophyll and soluble proteins 38 . In this study, there were no significant differences in MDA in flag leaves between the W1 and W3 or W4 treatments, but the SOD and catalase activities and soluble protein concentrations in flag leaves from the W3 treatment were significantly higher than those from the W1 and W4 treatments after 14 DAA (Fig. 4). These findings are likely the causes of the high chlorophyll content and fluorescence in flag leaves from the W3 treatment at the middle and later stages of grain filling, which are beneficial to wheat production 18,36 .

Conclusions
Supplemental irrigation amount (SI) at jointing and anthesis, determined by measuring the soil moisture of the 0-40 cm layer (W3), enhanced the soil water consumption, increased the leaf area index at the later grain filling stages, and improved the photosynthetically active radiation capture ratio, especially at plant heights from 20 to 60 cm. The highest chlorophyll content index, maximum quantum yield of the PSII, and actual photochemical efficiency at the middle and later stages of grain filling were obtained in W3, which were likely because of the high superoxide dismutase and catalase activities and soluble protein concentrations of flag leaves during the stages, and finally increased the grain yield and water use efficiency. It is hypothesized that these benefits accrued through created a suitable soil moisture environment in W3 treatment.  clay, 37.3% silt, and 33.1% sand as determined by the classification of soil in the USDA taxonomy 40 . The altitude of this area is 33.2 m.

Materials and Methods
The organic matter, total nitrogen, available phosphorus and available potassium in the topsoil (0-20 cm) of the experimental plots were 15.9 g kg −1 , 1.2 g kg −1 , 30.9 mg kg −1 , and 114.5 mg kg −1 , respectively, according to potassium dichromate colorimetric method, the Kjeldahl method, the sodium bicarbonate method and the ammonium acetate method, respectively. The soil dry bulk density, is defined as the ratio of dry soil weight to bulk soil volume was measured by cutting ring method according to MOA 41 , the equation is  Table 4, the SWC at maturity in different treatments are shown in Fig. 1. The groundwater depth is 25 m. Experimental design. Five treatments were designed: a rainfed (W0) treatment with no irrigation, a local SI practice treatment (W1, 60 mm of irrigation each at jointing and anthesis), and three treatments in which soil layers at specified depth were measured for SWC prior to SI: 0-20 cm (W2), 0-40 cm (W3), and 0-60 cm (W4). SI brought the mean SWC in each measured soil layers to 65% FC at jointing (Z31, first node detectable) and 70% of FC at anthesis (Z61, beginning of anthesis) 43 .     Table 4. Soil bulk density and soil water in the top 0-200 cm of the soil (in 20-cm increments) of the experimental field.
Three soil samples were collected using a soil corer (length 20.0 cm and diameter 5.0 cm) down to 20, 40, and 60 cm depth at the middle part in each plot of W2, W3, and W4 treatments, respectively. The SWC (gravimetric water content) of each treatment (average SWC of 0-20 cm in W2, 0-40 cm in W3, and 0-60 cm in W4 treatment, respectively) was determined using the oven-drying method 44 .
The amount of SI matched the crop irrigation requirement (CIR), which was calculated from the relative soil water content in the corresponding soil layers. The CIR was calculated using the following equation as described by Jalilian et al. 45 .
where CIR (mm) is the amount of SI; γ bd (g cm −3 ) is the soil bulk density; D h (cm) is the depth of the soil layer; SWC t (%) is the target soil water content after SI; SWC n (%) is the soil water content before irrigation. SWC t was calculated use equation (4): where FC (%) is field water-holding capacity; SWC tr (%) is the target relative soil water content (it was 65% at jointing and 70% at anthesis in this study). Uniform flood irrigation method was used, and a flow meter (accuracy: 0.001 m 3 , type: N15, linyi-mingquan Inc., China) was used to measure the amount of water applied. The relative SWC and SWC before and after irrigation, and the CIR for different treatments are shown in Table 1.
The experiment followed a randomised scheme, and all treatments were replicated three times. Each experimental plot was 4 × 4 m in size, and a 2.0-m-wide unirrigated zone was maintained between adjacent plots to minimize the effects of adjacent treatments.
Crop management. All plots were supplied with 240 kg N ha −1 , 150 kg P 2 O 5 ha −1 and 150 kg K 2 O ha −1 . All P and K fertiliser and half the N fertiliser were applied pre-sowing, and the remaining N fertiliser was topdressed at the jointing stage. The high-yielding wheat (Triticum aestivum L.) cultivar Jimai22 was used in the experiments. Wheat seeds were sown at a density of 180 plants m −2 on October 10, 2012, and October 9, 2013. Wheat seedling shoots ceased growth at the beginning of December and started to grow again at the end of February. During this period, the average daily temperature was below 0 °C. Wheat plants were harvested on June 12, 2013, and June 6, 2014.
Crop water use. Crop evapotranspiration (ET c ) was calculated using the soil water balance equation 46 for the growing season as: c where ET c (mm) is the total crop evapotranspiration during a growing season; P (mm) is the precipitation; CIR (mm) is the amount of SI; ΔW (mm) is the soil water consumption, which was defined as the difference of soil water storage between sowing and harvesting, the equations are: Where ΔW (mm) is the soil water consumption; S i (mm) is soil water storage (S s and S h are the soil water storage in 0-200 cm soil layers at sowing and harvesting, respectively); γ bd (g cm −3 ) is the soil bulk density; D h (cm) is the depth of the soil layer (D h = 200 cm in this study); SWC i (%) is the SWC on a weight-basis at sowing and harvesting, respectively. No account was taken of capillary rise, runoff and drainage. When the groundwater table is lower than 2.5 m below the soil surface, as it is at the experimental site, the capillary rising of groundwater is negligible 47 ; runoff can be ignored because of the terrier around the border in the North China Plain, including this experimental site 46 ; the drainage is very little in this study, we assumed that the irrigation and precipitation was absorbed by winter wheat completely, therefore, the drainage was ignored here 21,23 .
The water use efficiency of winter wheat was calculated using the method described by Wang et al. 48 , the equation is: where WUE (kg ha −1 mm −1 ) is the water use efficiency for grain yield; Y (kg ha −1 ) is the grain yield; ET c (mm) is the total crop evapotranspiration (water consumption) over the winter wheat growing season. The harvest index (HI) is the grain yield over total above-ground biomass at maturity.
Leaf area index and radiation interception. Green area of leaves from 30 plants in each plot was measured on May 2, May 16, and June 1 in 2013 using a leaf area meter (Winfolia Analysis System, Regent Instruments Inc., Canada), and the green leaves were scanned through the leaf area meter, the total leaf area were recorded, LAI is the leaf area for the 1-m 2 . The photosynthetically active radiation interception was measured on typical sunny days (May 2, May 16, and June 1 in 2013) at heights of 0, 20, 40, 60, and 80 cm above the ground using the AccuPAR Ceptometer, model LP-80 (Decagon Devices, Inc. USA). The data were acquired using a 0.87-m linear sensor placed at the middle of wheat inter-rows parallel to wheat rows and at the vertical direction of wheat rows, respectively 49 . The PAR capture ratio was calculated as the ratio of the difference between incident and transmitted radiation to incident radiation. The PAR penetration ratio was calculated as the ratio of transmitted radiation to incident radiation, and the PAR reflection ratio was calculated as the ratio of PAR reflection measured at 50 cm above the canopy to incident radiation. These values were obtained from instantaneous measurements taken from 11:00 to 13:00 on sunny days 50 . Chlorophyll content and chlorophyll fluorescence. Flag leaf chlorophyll content index was measured by the CCM-200 Chlorophyll Content Meter (Opti-Science, Inc. USA.) on 10 flag leaves from each experimental plot. The fluorescence parameters (CFP) of flag leaves on which CCI were measured were determined using a pulse-modulated fluorimeter (FMS-2, Hansatech, UK). The minimum and maximum fluorescence (F o and F m ) were determined after a full-dark adaptation for 30 min. Steady state fluorescence (F s ) was determined under actinic light. A saturating light pulse was applied to obtain F m after each actinic light episode. The F v /F m and ΦPSII were calculated according to Mu et al. 34 . and Zivcak et al. 12 . Measurements were made between 9:30 and 11:00 on days with full sunlight at seven-day intervals from anthesis to 35 DAA.
Biochemical assays on flag leaves. Flag leaves from each experimental plot were sampled at seven-day intervals from anthesis to 28 DAA. At each sampling date, 20 flag leaves from each plot were detached, immediately submerged in liquid nitrogen, and then stored at −80 °C until biochemical assays were performed. The whole flag leaf (including the leaves and veins) was used to measure catalase and SOD activities, MDA and soluble protein concentrations in the leaf.
Catalase and SOD activities were extracted from flag leaves by grinding 2 g of leaf tissue in 5 ml extraction buffer (0.1 M phosphate, pH 7.5, containing 1.5 mM EDTA and 1 mM ascorbic acid) at 0 °C. The mixture was then centrifuged at 13000 × g for 20 min, and enzyme assays were performed on the supernatant 51 .
Catalase activity was assayed by measuring the initial rate of H 2 O 2 disappearance 52 . A 3-ml reaction mixture contained 0.1 M sodium phosphate buffer (pH 7.0), 2 mM H 2 O 2 and 0.1 ml of crude extract. The breakdown of H 2 O 2 was followed by measuring the absorbance change at 240 nm, and enzyme activity was calculated using the extinction coefficient for H 2 O 2 (40 mM cm −1 at 240 nm) according to Wang et al. 23 .
Superoxide dismutase activity was assayed by measuring inhibition of the photoreduction of nitro blue tetrazolium (NBT) following the method of Giannopolitis and Ries 53 . A 3-ml reaction mixture contained 50 μM NBT, 13 mM methionine, 75 μM NBT chloride, 0.1 mM EDTA, 50 mM phosphate buffer (pH 7.8), 50 mM sodium carbonate, and 0.1 ml crude extract. Test tubes containing this reaction mixture were placed under a light bank (15 fluorescent lamps) delivering 78 μmol m −2 s −1 for 15 min. Absorbance was determined at 560 nm using a spectrophotometer (Hitachi U-1100, Tokyo, Japan). One unit of SOD activity was defined as the amount of enzyme that inhibited NBT photoreduction by 50%.
Malondialdehyde concentrations of flag leaves were assayed according to Quan et al. 54 . MDA concentration was expressed as nmol g −1 fresh weight (FW).
Soluble protein concentrations of flag leaves were measured according to the Coomassie brilliant blue G250 method described by Read and Northcote 55 . Protein concentration was expressed as mg g −1 FW. Statistical analysis. Statistical analysis employed standard analysis of variance (ANOVA) using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA.). The least significant difference (LSD) method was used to determine whether treatment means differed. The probability level for determination of significance was 0.05.