Grafting is a critical agricultural practice in citrus growing. The effectiveness of grafting not only depends on the technique but also on the stock–scion combinations. In this study, we investigated the grafting compatibility of five interstock combinations based on physiological and biochemical traits. The results revealed that the grafting compatibility in the early stages of grafting mediated by interstocks was related to the genetic relationship. The leaf chlorophyll content  and soluble sugar could be employed as preselected indicators to assess compatibility in the late stage of grafting. Our findings lay the foundation for the further research on rootstock–scion interaction mechanism.The interstock, a stock between the rootstock and scion, has a significant regulatory effect on the stock and scion, and its function is highly dependent on graft compatibility. To assess the graft compatibility of the interstock and scion, ‘Yuanxiaochun’ was top grafted onto ‘Ponkan’, ‘Shiranuhi’, ‘Harumi’, ‘Tarocco’, and ‘Kumquat’. The results showed significant differences in the survival ratio and preservation ratio among different combinations. Grafting compatibility in the early stages of grafting was associated with the genetic relationship. The biomass accumulation revealed that the interstock could influence both the rootstock and the scion. The physiological and biochemical traits analysis suggested that SPAD reading and soluble sugar could be employed as preselected indices to evaluate graft compatibility in the late stage of grafting. These results indicated that the evaluation of graft compatibility was a dynamic process. The findings provided a new approach for studying the stock and scion interaction mechanisms mediated by interstock, and directly provided a theoretical and practical basis for the high-grafting of ‘Yuanxiaochun’ citrus. Grafting compatibility between the interstock and scion of citrus has become a limiting factor for high-grafting. Therefore, it is particularly important to select suitable stock and scion combinations.Grafting, an asexual propagation technique used in agriculture for more than 2500 years, can effectively improve the propagation, production, and multiple stress tolerance of plants ,2,3. CitThe compatibility mechanisms of stock and scion grafting have become one of the research hotspots. Previous studies have found that stock–scion combinations exhibit an incompatibility phenomenon in grafting, because the combinations have a certain degree of exclusivity , and the2O2 and O2. The POD with CAT breaks down H2O2 into water. The accumulation of malonaldehyde (MDA) is a sign that the plant is suffering from free radical poisoning , ‘Harumi’ [Citrus reticulata × (Citrus reticulata × Citrus sinensis)], ‘Tarocco’ (Citrus sinensis L. Osbeck) and ‘Kumquat’ (Fortunella margarita Lour. Swingle), were widely cultivated or about to be replaced as interstocks. For example, the cultivation area of ‘Harumi’ has exceeded 130,000 hectares. Sichuan Province is the most suitable ecological zone for late-maturing hybrid citrus in China [Citrus unshiu Marcov × Citrus sinensis Osbeck) × (Citrus reticulata × Citrus paradisi)], a new hybrid citrus variety selected and bred in recent years, has shown good performance in Sichuan Province. To achieve the efficient performance of ‘Yuanxiaochun’ citrus, ‘Yuanxiaochun’ citrus was grafted onto five interstocks, with the aim of understanding the effects of different interstocks on the compatibility of scion grafting. After grafting, we determined the survival and preservation ratio, nutritional development, antioxidant enzyme activities and osmoregulatory substance content in the leaves for each combination. The graft compatibility of the stock and scion combinations was also thoroughly assessed using a mix of observational and anatomical approaches. This study provided an important reference for the interstock graft compatibility and guided high-grafting of ‘Yuanxiaochun’.In the present study, we selected ‘tability . Five ciin China . ‘YuanxiTrifoliate orange’. The grafting height was fixed at 5 cm, the stem diameter was 0.6–0.8 cm, and each cultivar was grafted with 150 plants. Plant spacing was 30 × 30 cm after grafting for colonization and unified management. These plants were employed as an interstock material after survival.In March 2018, ‘Ponkan’, ‘Shiranuhi’, ‘Harumi’, ‘Tarocco’ and ‘Kumquat’ scions were grafted onto ‘On 15 March 2019, 100 grafted seedlings of ‘Ponkan’, ‘Shiranuhi’, ‘Harumi’, ‘Tarocco’, and ‘Kumquat’ were chosen for unified planting, and the separation distance between varieties was 0.5 m. In the same varieties, the plant spacing and row spacing was 30 × 30 cm, and the diameter of each plant’s graft union was between 0.6 and 0.8 cm. The interstock materials were grafted with ‘Yuanxiaochun’ 30 days after plantation, with a grafting height (interstock length) of 10 cm. At 60 days after grafting (DAG), 60 plants of each interstock variety that survived grafting and grew almost identically were chosen as the test materials, with 20 plants per plot, for three replicates. At 60–180 DAG, the survival and preservation ratios of grafting, determination of vegetative growth, enzyme activities and osmotic regulatory substances in leaves, and graft compatibility were evaluated. All seedlings were handled according to standard procedures in Zhuangjia village, Hongyuan Township, and Jianyang City, Sichuan Province, China.Referring to previous research and the genetic relationship of materials, a genetic relationship map a was creThe rootstock, interstock, scion, and lower graft union (graft union of the rootstock and interstock)/upper graft union (graft union of the interstock and scion) diameters were measured with vernier calipers at 240 DAG. In addition, the morphological characteristics of appearance and leaf quality of ‘Yuan Xiaochun’ were counted according to Najla Safaa et al. . The chlMature functional leaves were collected from each treatment every 30 days from 60 DAG to 240 DAG. The leaf veins were removed and stored in an ultra-low temperature refrigerator at −80 °C until use.SOD and POD activities were evaluated according to Liao et al. , and CATAt 240 DAG, the ‘Translocated’ incompatibility was categorized according to Moreno et al. , whereasp < 0.05 level of significance.The data were analyzed using Duncan’s multiple range test in SPSS software (ver. 22.0) at the In terms of genetic relationship, ‘Yuanxiaochun’ was the most closely related to ‘Harumi’ and ‘Shiranuhi’, while ‘Kumquat’ was the most distant a. IntersThe preservation and survival ratios showed different trends. The highest preservation ratio was recorded in the grafting combination with ‘Shiranuhi’ as the interstock, while the lowest was observed in the combination with ‘Kumquat’ as the interstock. The ‘Kumquat’ treatment exhibited the most significant difference in preservation and survival ratio. When the data were combined with a genetic relationship diagram of variations, it was discovered that the closer the genetic relationship with ‘Yunaixiaochun’, the higher the survival and preservation ratios. These findings suggested a relationship between graft compatibility and the genetic relationship in the studied cultivars.Rootstock, interstock, scion diameters, and the lower and upper graft union diameters all responded significantly to the interaction between stock and scion at 240 DAG . SignifiThe exterior morphological traits and leaf quality of ‘Yuanxiaochun’ were affected by different interstocks . The valThe relationships between SPAD reading and trunk diameter were investigated in this study. They were significantly positively correlated with interstock diameter and upper graft union diameter . In addiAntioxidant enzyme activities demonstrated a tendency of ‘decrease-increase-decrease-finally climb’ at 60–240 DAG a–c. SOD The variations in the MDA content of various treatments were shown in Only ‘Yuanxiaochun’ with ‘Harumi’ as interstock displayed ‘Translocated’ incompatibility signs . The ‘YuAs an ancient method of asexual propagation, grafting is widely used for plant propagation, increasing the yield or resistance of horticultural crops . GraftinGrafting is a complex process, in which the genetic relationship is the most important influencing factor . PreviouPrunus dulcis [Stock–scion interactions have been shown to affect scion biomass accumulation in apples and s dulcis ,45, whics dulcis ,46.Leaf characteristics affect the photosynthetic capacity of plants and further affect the yield . The dryThe physiological and biochemical indicators could reflect the graft compatibility ,53. The Soluble sugar plays an important role in the evaluation of grafting compatibility. Previous studies have found that sugar promotes graft union development in the heterograft of cucumber onto pumpkin . HoweverIn addition, the soluble proteins are important for the early growth stages of linkage between rootstock combinations, and facilitate the formation of initial adhesions between rootstocks ,57. It aAmong these indicators, antioxidant enzymes showed different dynamic trends with grafting time, which was not favorable for the evaluation of grafting compatibility. In contrast, soluble sugar showed better agreement with grafting compatibility, and we suggest that soluble sugar in the scion leaves might be one of the preselected indicators of grafting compatibility.The evaluation of grafting compatibility is a long and complex process. This study revealed that grafting compatibility was associated with genetic relationship, SPAD reading, and soluble sugar content of leaves. The closer the genetic relationship, the higher the survival ratio of grafting in the early stages of grafting. In the late stages of grafting, the grafted combinations with lower SPAD reading and higher soluble sugar content of leaves displayed worse compatibility and growth characteristics. Our experimental results should be further understood by setting up more controlled experiments, increasing the variety sample and continuing to extend the evaluation time. Overall, ‘Tarocco’ and ‘Kumquat’ were the most suitable as interstocks for ‘Yuanxiaochun’.


We here examined the linearity assumption to see if it can be used to predict properties at conditions that are not close to those under which they were measured. For this we used both common domesticated rices (DRs) and Australian wild rices (AWRs), the latter having significantly different structural parameters and properties compared to DRs. The results showed that (1) the properties were controlled by more than just the amylopectin or amylose chain-length distributions or amylose content, other structural features also being important, (2) the linear model can predict the enthalpy ΔHg of both AWRs and DRs from the structural parameters to some extent but is often not accurate; it can predict the ΔHg of indica rices with acceptable accuracy from the chain length distribution and the amount of longer amylose chains (degree of polymerization > 500), and (3) the linear model can predict the stickiness of both AWRs and DRs to acceptable accuracy in terms of the amount of longer amylose chains. Thus, the commonly used linearity assumption for structure-property correlations needs to be regarded circumspectly if also used for quantitative prediction.Many properties of starch-containing foods are significantly statistically correlated with various structural parameters. The significance of a correlation is judged by the  This involves the assumption that the relation between the selected properties and structural parameters are linear. While mathematically this has to hold over sufficiently small changes in structure and properties , this assumption is likely to become invalid with large changes. It would be useful if one could use the coefficients resulting from these correlations predictively, e.g., to see what fraction of amylose (Am) long chains would produce a desired reduction in the rate of digestion of a starch-based food to glucose, as a guide to plant breeding and variety selection. A badly inaccurate prediction here could prove a costly error for a plant breeder.To understand and improve the processing and quality  of starch-containing foods, it is important to have statistically valid and physically meaningful correlations between starch structure and functional properties. These correlations are often obtained using the Here we examine the predictive power of this common hypothesis for an important food system: the relation between structural parameters and functional properties (such as gelatinization behavior) for rice samples with diverse ranges of structures and properties.Oryza sativa L.) is a widely consumed staple food and can be divided into two commonly domesticated rice species: Oryza sativa L. (from Asia), Oryza glaberrima Steud (from Africa), and 24 wild species  The composition of three AWRs and 70 DRs were studied.(2) The CLDs of rice starch (obtained after enzymatic debranching of the whole starch) were characterized with size-exclusion chromatography (SEC) and the results were fitted to biosynthesis-based models. These reduce the structural data to a small number of biosynthesis-related parameters suited to the types of analysis pursued here.in-vitro digestibility of cooked rice flours, which were subsequently fitted to kinetic models, and the texture of cooked rices. These treatments provide a small number of parameters describing these properties, which are suitable for the present aims.(3) We measured the thermal properties of starch, the (4) Linear regression was performed between properties and their related structural parameters.(5)All parameters from the linear fits were then used to predict properties not included in the original data set, to test how well the linear regression treatment can be extrapolated beyond the conditions under which the linear coefficients were obtained.indica  and japonica . For performing the linear regressions, here rice samples were grouped into three categories  for each property. These are: for thermal properties, in Group A, sample AWR S01–S03, DR S04–S13, and S14–S23, in Group B, sample AWR S01–S03 and DR S04–S13, and in Group C, sample AWR S01–S03 and DR S14–S23; for in vitro digestion properties, in Group A, sample AWR S01–S03, DR S04, S06, S13, S28–S29, S31, S40–S43, and S14, S17, S19, S21, S23, S37–S39, S44–S45, in Group B, sample AWR S01–S03 and DR S04, S06, S13, S28–S29, S31, S40–S43, in Group C, sample AWR S01–S03 and DR S14, S17, S19, S21, S23, S37–S39, S44–S45; for textural properties, in Group A, sample AWR S01–S03, DR S13, S31, S40–S43, S46–S47, S52–S53, and S37–S38, S44, S54–S60, in Group B, sample AWR S01–S03 and DR S13, S31, S40–S43, S46–S47, S52–S53, in Group C, sample AWR S01–S03 and DR S37–S38, S44, S54–S60. After the linear regressions were performed, different samples were used in Groups A, B, and C (described below) to test the predictability of the linear regressions at conditions that were not close to those used to find the linear correlations. The order of DR varieties was determined by the order of their properties. AWR S01, Oryza meridionalis (Taxa B) (T.B.) was collected at Global Positioning System (GPS) latitude S 15°29'48.6” and GPS longitude E: 144°18'47.6”, in Queensland, Australia, on 11th May 2018. AWR S02, Oryza officinalis (O.O.) and AWR S03, Oryza australiensis (O.A.) were both provided by Australian GeneBank without detailed information. The AWR was dehulled manually, followed by polishing with a rice polisher . These three AWR varieties are the only ones currently available. Collecting sufficient amounts of samples for characterization is not straightforward, as they grow in the wild interspersed with many other plants, requiring laborious manual collection in a geographically remote location. In addition, the important need to request permission from traditional owners, following the legal protocols ensuring that their rights are protected, is time-consuming.Rice types used in this study consisted of AWRs (S01–S03) and DRs (S04–S73) . Varietiin-vitro digestibility was measured as part of the present study. All sample details are shown in Some DR data  were obtained from the literature from sources which used the same methodology, while the thermal properties, and indica rice variety: S13, S31, S40–S43, S46–47, S52–53, S61–62; japonica rice variety: S37–S38, S44, S54–S60, S68–S72. From Cheng Li and co-workers : indica rice varieties S63–S67. From Li et al. : japonica rice varieties S39, S45, S73. From Zhu et al. (indica rice varieties S48–S51)  . The colStreptomyces griseus type XIV) , α-amylase from human saliva , pepsin from porcine gastric mucosa , and pancreatin from porcine pancreas  were purchased from Sigma-Aldrich Chemical Co. . Amyloglucosidase (from Aspergillus niger)  and isoamylase (from Pseudomonas sp.) (200 Units/mL) were purchased from Megazyme International Ltd. . Pullulan SEC standards with known peak molecular weights ranging from 180 to 1.22 × 106 were obtained from Polymer Standards Service (PSS) GmbH . Other chemical reagents were analytical grade and used as received.Protease  assay kit . The cru−1) before being filtered with a 75-μm sieve. The flour was immersed in 0.45% sodium metabisulfite solution (the volume ratio of rice flour to solution being 1:3) at 4°C for 0.5 h. Proteins were removed by protease treatment [2.5 Units mL−1 of protease in tricine buffer ] at 37°C overnight. After centrifuging at 4,000 × g for 10 min, the supernatant was discarded. The treated flour was washed with deionized water six times, then twice precipitated in ethanol. Finally, the extracted starch was freeze-dried  for 48 h.The extraction of starch from the rice grains followed a previously described method . BrieflyRh). Briefly, native starch was dissolved in dimethyl sulfoxide (DMSO) with 0.5% (w/w) LiBr (DMSO/LiBr) at 80°C before centrifugation, the supernatant then mixed with absolute ethanol, and the resulting precipitate was debranched with isoamylase prior to freeze-drying. The freeze-dried sample was dissolved in DMSO/LiBr and the resulting supernatant was then injected into the SEC. The CLDs were obtained using a LC20AD SEC system  equipped with three columns in sequence : GRAM pre-column, GRAM 100 and GRAM 1000 columns, and a RID-10A refractive index detector . DMSO/LiBr solution was used as the mobile phase with a flow rate of 0.6 mL/min.SEC was used to measure the CLDs of debranched starches as described in detail previously . SEC sepi, the contribution to the CLD from this set can be calculated from the values of two parameters: βAm, i and hAm, i  for Am and βAp, i and hAp, i  for Ap. The βi are the ratios of the activity of SBE to that of SS in set i and the hi are the relative activities of the SS in the enzyme set.The Ap and Am starch CLDs were each fitted to biosynthesis-based models using publicly available codes. This enables a reduction of the CLD data into a small number of biologically-relevant parameters, suitable for finding statistically valid correlations , 19. BriTo), peak temperature (Tp), conclusion temperature (Tc) and crystal melting enthalpy (ΔHg) were measured.The gelatinization properties of rice starches were measured by differential scanning calorimetry . Starch  and water (at a weight ratio of 1:2) were weighed in a DSC aluminum crucible and sealed. After equilibrating at 4°C for 24 h and then at room temperature for 1 h, the crucibles were held at 20°C for 1 min in the DSC oven, and then heated from 20 to 100°C at a rate of 10°C/min, followed by cooling at 20°C/min from 100 to 20°C. An empty crucible was used as a reference. The onset temperature  was added to each tube and incubated for 20 s, followed by incubation with porcine pepsin (1 mg/mL) in HCl solution  for 30 min. The digesta were then neutralized with 1 mL NaOH (0.02 M) and mixed with 5 mL sodium acetate buffer  containing 200 mM CaCl2, 0.49 mM MgCl2 and 0.02% w/v NaN3. Pancreatin (2 mg/mL) and 28 U/mL amyloglucosidase in the same sodium acetate buffer solution (1 mL) were added to the digesta. After times ranging from 0 to 300 min, a 0.1 mL aliquot of solution was quickly transferred to a microcentrifuge tube containing 0.9 mL absolute ethanol, to stop the reaction. The glucose content of each sample was determined by the GOPOD method with a Megazyme D-Glucose  assay kit. The whole digestion progress was carried out at 37°C with constant shaking at 100 rpm. The in vitro digestion results were first fitted to a first order equation:An o et al. . Brieflyo et al. , ~70 mg]C(t) is the fraction of total starch digested at time t, C∞ is the fraction of starch digested at very long reaction time and k is the digestion rate coefficient of the starch. The digestion curves were further treated with the logarithm-of-slope (LoS) method, which identifies the region(s) following first-order loss kinetics , equipped with a P36R cylindrical probe, following a previously described method . BrieflyThe textural data of cooked DRs, obtained under the same conditions as the cooked AWRs, were those provided by the same sources as those providing the samples, detailed above.indica rices), Group C (three AWRs and 10 japonica rices)} (mentioned above) were used to build linear correlations between properties parameters and their related structural parameters by a backward multiple linear regression  ], Group B (three AWRs and 10 erature) , 23–34. aij are constants whose values depend on the values of the sj and Pi. Non-linear behavior would be when one has to include terms in sisj, p < 0.05) on the subset of our samples given above, another 16 DRs (8 indica and 8 japonica), were treated in the same way as the samples used to build the linear regressions and used in Groups A, B, and C, respectively. For thermal properties, sample DR S24–S31, and S32–S39 were used in Group A, samples DR S24–S31 were used in Group B, and samples DR S32–S39 were used in Group C. For in vitro digestion properties, samples DR S05, S10, S46–S51, and S18, S20, S22, S32–S36 were used in Group A, samples DR S05, S10, S46–S51 were used in Group B, and samples DR S18, S20, S22, S32–S36 were used in Group C. For textural properties, samples DR S10, S61–S67, and S39, S45, S68–S73 were used in Group A, samples DR S10, S61–S67 were used in Group B, and samples DR S39, S45, S68–S73 were used in Group C. These last were used to test the predictability of the linear regressions over significant displacements from the conditions used to find the linear correlations.Here the It is noted that this paper concerns structure-property relations; how these structures were formed  is irrelevant to the present aims, although of interest in other contexts.R2) and root mean square errors (RMSE) were used as indicator of significance for the regression models.Statistical analysis was conducted using SPSS software . Analysis of Variance (ANOVA) was used to determine significant differences in starch molecular fine structural parameters, moisture content, AC and property parameters. A backward regression approach was employed in the multiple linear regression, and coefficient of determination , which are the weight (not molecular weight) of chains as a function of their degree of polymerization (DP) X. Normalization of such distributions is arbitrary; here, the data were normalized to the highest Ap branch peak. Generally, chains shorter than DP 100 are taken as Ap branches and those longer than DP 100 as Am branches . This lower amount for X > 5,000 in AWR starches could be due to the lower enzyme activity of an isoform of granule-bound starch synthase ((1) In the SEC results used to build structure-thermal properties relations , generalsynthase .Am, 2 than those of domesticated ones. The hAm, 1 and hAm, 2 values were generally in the order indica rice starches>AWR starches>japonica rice starches, which might be because of the different activity of an isoform of SS in different starches ((2) The SEC results  showed tstarches .Am, 1 and higher βAm, 2 compared to domesticated ones.(3) The SEC results used to build structure-textural properties relations  showed tThese noticeable differences in structural properties are all desirable from the point of view of providing a wide “space” of structural parameters to test the linearity assumption, which is the aim of the present paper.To and Tp) than those of DR starches, showing that AWR starches had more ordered crystallinity than DR starches. The AWR starches had higher ΔHg than those of DR starches, but the ACs of AWR starches were in the range of those of DR.The thermal data of different starches are shown in in-vitro digestion curves of typical rice flours are shown in kL is the digestion rate coefficient of starch of LoS and CL∞ is the percentage of starch digested at long times. Generally, the values of kL of AWR flours were the slowest among all samples. The AC had significant negative correlations with kL (indica rice varieties > AWRs > japonica rice varieties. This indicated that AC was the dominant but not sole factor determining kL. No obvious differences can be seen in CL∞ between AWR flours and DR flours.The  with kL  and the kN is/are the digestion rate coefficient(s) of starch of NLLS over the first-order region(s) and Cres is the fraction of residual starch (starch remaining after an extended digestion period). Generally, the kN values were in the order of AWRs < indica rice varieties < japonica rice varieties. There were no significant differences in Cres between AWR flours and DR flours. In the parallel digestion model, kp is the digestion rate coefficient of starch and Cp∞ is the fraction of starch digested at very long reaction time. Generally, the kp of AWR flours were slightly less than the DR counterparts. No clear differences were found in Cp∞ between AWR flours and DR flours.In the NLLS model, the value(s) of Generally, the AWR flours had slower digestion rate coefficients, but no noticeable differences in digestion degree, compared to DRs.japonica rices. The results are in agreement with the finding that hardness was not only affected by AC . It has been reported that ΔHg were significantly correlated with βAp, 1, βAp, 2, βAp, 3, hAp, 1, βAm, 2 and hAm, 2 in rice starches  by a backward regression approach. We applied a linear regression model for Group A-ΔHg to yield ΔHg (J g−1) = −10+10βAm, 2 −0.04hAm, 2   . It was To, and Tp had significant correlations with βAp, 1, βAp, 2, hAp, 1, hAp, 2, hAm, 1 and hAm, 2, while Tc and ΔHg were significantly correlated with βAp, 1, βAp, 2, βAp, 3, hAp, 1, βAm, 2 and hAm, 2 , while the linear model cannot predict Tc to acceptable accuracy. In addition, the linear model can acceptably predict ΔHg of Group B in terms of the CLDs of long-to-extra long Am chains (X > 500) , but cannot predict that of Group C to acceptable accuracy. This might be because, as shown in Hg and Group B-ΔHg had similar structural parameters, but different from those of Group C – ΔHg. The R2 of linear regression of the Tc results for Group B was higher than that for Group A, but the number of variables in establishing linear regression of Group B was less than that of Group A. This suggests that although using similar varieties can improve the accuracy of linear regression analysis of structure-property relations, this may overlook important structural parameters, by restricting the “space” of structures. Compared to the linear regression model for gelatinization properties which confined the Am structural parameters to AC alone   . Nearly Ap, 2, βAp, 3, hAp, 2, hAp, 3, hAm, 1, hAm, 2 and protein content, while stickiness was significantly correlated with βAp, 2, βAp, 3, hAp, 2, hAp, 3, βAm, 1, hAm, 1, hAm, 2 and protein content (X > 500)  accounted for 73.9% of the total variation of stickiness). The linear regression value of R2 was always much lower than 1, but some linear regression models cannot be applied to additional samples, suggesting that the properties were controlled by more than just the CLDs, such as other structural features and food components also being important. The linear model cannot predict either Group B stickiness or Group C hardness to acceptable accuracy.For texture properties, hardness had significant correlations with β content , 14, 25.In conclusion, it is apparent that while the assumption of structure-property linearity is useful for determining statistical correlations, it is only occasionally useful for quantitative prediction of these properties. The linearity assumption is often only applicable for changes close to the conditions under which the linear coefficients are determined.The original contributions presented in the study are included in the article/YZ: conceptualization, methodology, investigation, formal analysis, writing—original draft, and writing—review and editing. RH: conceptualization, supervision, and writing—review and editing. RG: conceptualization, supervision, formal analysis, and writing—review and editing. All authors contributed to the article and approved the submitted version.The authors acknowledge the financial support of a University of Queensland Research Training Scholarship, China Scholarship Council Scholarship and National Natural Science Foundation of China grant C1304013151101138 – a project funded by the Priority Academic Program of Jiangsu Higher Education Institutions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ros 119, among 105 callus-sourced clones, recorded a higher tuberization potential than the parent cv Lady Rosetta in a field experiment. Although this somaclone was similar to the parent in the number of tubers produced, it exhibited tubers with 42 and 61% higher fresh and dry weights, respectively. Additionally, this clone recorded 10 and 75% increases in starch content based on the dry weight and average content per plant, respectively. The enhanced starch accumulation was associated with the upregulation of six starch-synthesis-related genes, namely, the AGPase, GBSS I, SBE I, SBE II, SS II and SS III genes. AGPase affords the glycosyl moieties required for the synthesis of amylose and amylopectin. GBSS is required for amylose elongation, while SBE I, SBE II, SS II and SS III are responsible for amylopectin.Starch content is one of the major quality criteria targeted by potato breeding programs. Traditional potato breeding is a laborious duty due to the tetraploid nature and immense heterozygosity of potato genomes. In addition, screening for functional genetic variations in wild relatives is slow and strenuous. Moreover, genetic diversity, which is the raw material for breeding programs, is limited due to vegetative propagation used in the potato industry. Somaclonal variation provides a time-efficient tool to breeders for obtaining genetic variability, which is essential for breeding programs, at a reasonable cost and independent of sophisticated technology. The present investigation aimed to create potato somaclones with an improved potential for starch accumulation. Based on the weight and starch content of tubers, the somaclonal variant  Solanum tuberosum) is the most crucial non-graminaceous food crop cultivated in 17.34 million hectares yielding 370 million tons . Tubers are an important source for carbohydrates, proteins and other essential nutrients including minerals , vitamins , and dietary fibers . The. The8]. −DDCt method [The relative transcript abundance of tublin was used as an endogenous control, to which the transcription levels of the AGPase, GBSS I, SBE I, SBE II, SS II and SS III genes were normalized using the 2t method . The expp = 0.05 using SPSS v. 14.The results of all clones are presented as the mean of five replicates ± standard deviation (SD). One-way ANOVA with the LSD post hoc statistical test was utilized to compare the different clones on the basis of tuber yield per plant manifested in number of tubers, the fresh and dry weights of the tubers, the starch content on dry-weight and per-plant bases and the expressions of six starch-related genes at Somaclonal variation is an efficient breeding tool. It can provide breeders with new genotypes with favorable traits, which is essential for breeding programs targeting yield improvements. Somaclonal variation is a time-efficient alternative to conventional breeding, and it is able to provide potato clones with an outstanding potential for starch accumulation at a reasonable cost. It is an acceptable approach that is able to manipulate starch synthesis genes without issues arising regarding genetic transformation. However, the emerging clone requires genetic characterization to participate effectively in breeding programs targeting qualitative and quantitative yield improvements, which is the aim of our future work.


Clinical outcomes after anterior cruciate ligament reconstruction (ACLR) have improved remarkably. However, residual rotational instability of the knee joint remains a major concern. The anterolateral ligament (ALL) has recently gained interest as a secondary stabilizer of knee joint rotatory instability, and this has led to the attempt of ALL reconstruction (ALLR) in combination with ACLR to restore rotational stability in patients with anterior cruciate ligament (ACL) injury. Although several techniques for ALLR have recently been introduced, the ideal graft type and surgical technique for combined ACLR and ALLR are yet to be established. This technical note therefore aimed at introducing a novel surgical procedure involving the use of a single Achilles tendon allograft as a relatively simple and minimally invasive procedure for combined ALL and ACL reconstruction. Clinical outcomes after anterior cruciate ligament reconstruction (ACLR) have remarkably improved due to the accumulating knowledge of the anatomy and biomechanics of the anterior cruciate ligament (ACL) and advances in surgical techniques ,2. HowevThe anterolateral ligament (ALL) is an extra-articular ligamentous structure located on the anterolateral aspect of the knee joint. Since the re-discovery of the ALL by Claes et al. , there hSeveral surgical techniques have recently been suggested for combined ALL and ACL reconstruction. Helito et al. recommended the use of a tripled semitendinosus auto- or allograft in conjunction with a gracilis auto- or allograft for a combined ACL and ALL reconstruction . SonneryThe purpose of this technical note is to introduce a novel surgical procedure using a single Achilles tendon allograft as a relatively simple and minimally invasive procedure for combined ALL and ACL reconstruction.The patient is anesthetized  and positioned supine in the standard arthroscopy position with a lateral post proximal to the knee. The knee joint is positioned slightly past the distal break point of the operating table to place the limb in a 90° flexion position. As such, the knee can freely undergo its full range of motion. A thigh tourniquet is applied and maintained inflated throughout the procedure. Physical examination, including the Lachman and pivot shift tests, are performed to evaluate anteroposterior and rotatory instability.After setting up the patient, routine arthroscopic examination is performed through the standard anterolateral and anteromedial portals. After complete intra-articular examination, any concomitant pathology of the menisci or articular cartilage is managed prior to ACLR. Subsequently, a femoral tunnel is created for both the ACLR and ALLR using the ACL RetroConstruction System and FlipCutter drill  through the anatomic outside-in retrograde-reaming technique. The tip of the femoral aiming guide is placed through the anterolateral portal at the ACL femoral footprint under direct arthroscopic visualization through the anteromedial portal. After palpation of the LFE, a 15 mm incision is made just proximal and posterior to the LFE. The guide’s angulation is adjusted to allow the tip of drill sleeve placement in the incision at the femoral footprint of the ALL  . FluorosA small transverse skin incision is made on the tibial footprint of the ALL, which is located midway between Gerdy’s tubercle and the tip of the fibular head (approximately 20 mm posterior to the center of Gerdy’s tubercle), approximately 10 mm below the lateral joint line [After creating the bony tunnels and sockets for ACLR and ALLR, the footprint distances and tunnel lengths are measured to determine the appropriate length of the tendon graft. The Achilles tendon allograft is trimmed into a Y-shaped graft consisting of two portions: an ACL portion (9–10 mm in diameter) and an ALL portion with two bundles (5 mm in diameter each). The end of the ACL portion is sutured using the whip-stitch method, with an Ethibond suture . The ALL portion is divided into two bundles (with each end sutured in the same manner) for later fixation into the two ALL tibial sockets. At the bifurcation site from the ACL portion to the ALL portion, an additional whip-stitched suture is made, approximately 30 mm in length, in the direction of the ACL portion, to prevent possible tendon splitting .The prepared Achilles tendon allograft is attached to the loop outside the tibial tunnel and then pulled through the tibial and femoral tunnels passing over the joint. The two bundles of the ALL graft are pulled and positioned outside the femoral tunnel outlet. The graft is then sequentially fixed for ACL and ALL reconstructions. First, the bifurcated transitional portion of the graft is fixed at the femoral tunnel using a bioabsorbable interference screw . For ACLR, after graft pre-tensioning, the distal portion of the ACL graft is fixed at the tibial tunnel  using a bioabsorbable interference screw  followed by a post-tie fixation. Subsequently, for the ALLR, the two bundles of the ALL graft from the femoral tunnel outlet are directed deep to the iliotibial band (ITB) percutaneously and delivered through a distal skin incision to the tibial sockets . The kneAs a potential solution for residual rotatory instability after isolated ACLR, combined ACL and ALL reconstruction has recently gained interest among orthopedic knee specialists. Different surgical techniques for combining ACLR and ALLR have already been reported to have favorable clinical outcomes ,19,20. TThe proposed technique offers several advantages. First, by trimming a single Achilles tendon allograft into two parts consisting of the ALL and ACL portions, there is no need to use additional graft tissue. As the Achilles tendon allograft is sufficient in width and length, it is possible to produce a single graft sufficient to realize the combined ACL and ALL reconstruction by performing graft preparation as described above. This may also be beneficial to the patient as it may help reduce the cost of the surgery. Second, by performing the combined ALLR and ACLR through a common single femoral tunnel, the potential risk of overlap between the two femoral tunnels (in case tunnels for the ALLR and ACLR procedures are made separately) can be avoided. Furthermore, sharing a common femoral tunnel for the fixation of the ALL and ACL graft could contribute to a decrease in the operative time, thus preventing possible intra- and post-operative complications. Third, through a minimally invasive approach, this technique can minimize complications such as ITB injuries. Fourth, as a technique using only a single allograft tendon, donor site morbidity such as weakness of the hamstring muscle and anterior knee pain, caused by the harvesting of the autografts, can be avoided. Additionally, this technique has the advantage of being easy to apply in cases of revisional reconstructions, where the use of autografts is difficult. Finally, although some studies reported ALL reconstruction techniques using a single bundle graft ,21, the The potential risks of this technique include irritation of the iliotibial band (by the protruding interference screws), anterolateral tibial plateau fracture, and tibial tunnel collision during the creation of the bony tunnels. To avoid these possible complications, it is crucial to insert the screws such that their ends do not protrude outside the cortex when fixing the interference screw in the lateral femoral tunnel. It is also recommended to maintain the two tibial sockets at a distance of approximately 10 mm from each other (so that the two sockets do not overlap), as well as to consider the direction of the sockets so as to prevent tibial tunnel collision or tibial plateau fracture. Lastly, this novel procedure uses an Achilles tendon allograft. Graft selection is one of the important issues in ACLRs. Although ACLRs using autografts have consistently yielded good clinical outcomes, the incidence of post-operative donor site morbidity and the desire to avoid autologous tissue sacrifice have prompted a consideration of allografts ,27,28. AAlthough several techniques for ALLR have recently been introduced, the ideal graft type and surgical technique for combined ACL and ALL reconstruction are yet to be established. To the best of our knowledge, this is the first surgical technique that combines ACLR and ALLR using a single Achilles tendon allograft. This relatively facile and minimally invasive procedure will be beneficial in restoring the function of the native ACL and ALL in patients who present with both anteroposterior and anterolateral rotatory knee instabilities. Further biomechanical and long-term clinical studies are necessary to validate this novel technique for combined ACL and ALL reconstruction by using a single Achilles tendon allograft.


Many concerns are being expressed about the biodegradability, biocompatibility, and long-term viability of polymer-based substances. This prompted the quest for an alternative source of material that could be utilized for various purposes. Starch is widely used as a thickener, emulsifier, and binder in many food and non-food sectors, but research focuses on increasing its application beyond these areas. Due to its biodegradability, low cost, renewability, and abundance, starch is considered a “green path” raw material for generating porous substances such as aerogels, biofoams, and bioplastics, which have sparked an academic interest. Existing research has focused on strategies for developing biomaterials from organic polymers , but there has been little research on its polysaccharide counterpart (starch). This review paper highlighted the structure of starch, the context of amylose and amylopectin, and the extraction and modification of starch with their processes and limitations. Moreover, this paper describes nanofillers, intelligent pH-sensitive films, biofoams, aerogels of various types, bioplastics, and their precursors, including drying and manufacturing. The perspectives reveal the great potential of starch-based biomaterials in food, pharmaceuticals, biomedicine, and non-food applications. One of the standard methods suggested as a lasting solution to the adverse setback of non-biodegradable materials (NBMs) is the use of plant-based materials (PBMs) from compostable sources, especially those of plant origin ,2. PBMs Starch has been used in areas where low density, high surface area, and porosity are of the utmost importance ,14. To tIn this paper, we comprehensively present the valorization of starch to biobased materials. The method we adopted was to divide our findings into two sections: the first was to present information about starch, including its history, structure, extraction, and modification, and the second was to emphasize the unique applications of starch. A more in-depth look into the application of starch-based sources in nanofillers, smart/intelligent packaging films, aerogels, and biofoams, and their manufacturing techniques, was offered. The literatures considered in this review were indexed, with a preference for those published within the past five years. However, a few older studies that are important for this study were also incorporated. Finally, research gaps were identified and highlighted, and the outlook in the forthcoming years was also presented.Amylose makes up 5–35% of most natural starches and significantly impacts starch characteristics . AmyloseAmylopectin, a component of starch (70–80%) , is one Retrogradation, gelatinization, gel rheology , solubilL-cysteine) has solved this negative effect. L-cysteine is an amino acid that is both safe and natural. It is commonly used in food processing as a food additive and acts as a reducing agent [Proteins, carbohydrates, and lipids, which are potential ingredients for the manufacture of enzymes, gelatin, collagen, and other bioactive compounds, are known to be present in discarded and underutilized by-products . This sing agent . The wetng agent  with excng agent , the prong agent , which cng agent ,67. Althng agent .2) and ethanol were used in a study to extract starch from pulses (peas) [Having conducted a critical overview of these distinct wet milling starch extraction types, novel extraction strategies that match the given parameters while also mitigating associated drawbacks are required. Supercritical carbon dioxide (ScCOs (peas) . The orgs (peas) . Their s2 drying as a means of modification of starch.Starch modification refers to efforts to increase starch synthesis while also modifying the content and structure of the starch, as well as inducing essential qualities to satisfy specific end goals . AccordiChanges in non-digestible carbohydrates could result from physical and chemical starches, affecting their technical qualities and nutritional value. Zieba and colleagues studied an extrusion method for starch modification. The scholars observed a deeper color, more excellent resistance to amylolytic enzyme action, and lower solubility and phase transition heat. They proposed their method as a panacea for overcoming barriers of resistant starch. The physical, thermal, paste attributes, and resistant starch content (RS) of rice and potato starches were investigated after extrusion at 100 °C and 15 rpm with 5% and 10% oleic acid (OA) in the experiment of Cabrera-Ramirez and his co-workers . FindingChemical modification is the process of changing the physicochemical properties of starch by introducing new chemical or functional groups into the molecule without changing its structure or size. Various chemical modification methods include oxidation, etherification, esterification, and cationization by introducing some cationic molecules, cross-linking, and grafting among the plethora of chemical modification routes . RegardlIn a closed-loop system for starch, genetic modification, seed creation, growth, and delivery to the processing mill are all perfectly controlled. Genome editing  and genePolysaccharides continue to be most researched, naturally available, and abundant precursors for developing biopolymers . They arThe use of starch for various applications cannot be overemphasized. Starch is the second most abundant polysaccharide on the planet  and remaIn the previous sections of this review, we have shown how the extraction and modification of starch could be performed. However, the use of these low-value products into biobased products with economic value is of particular interest. Although starch is a prominent and sustainable source for producing biodegradable food packaging materials ,146, staOrganic nanofillers have been widely used in starch, and the establishment of a hydrogen bonding network has resulted in high interfacial adhesion between the matrix and the nanofiller. Typically, nanochitin, nanochitosan, and nanocellulose have been used as fillers in various applications. Majorly sourced from skeletal components of insects and broadly crustaceans, chitin and chitosan are naturally abundant green materials, and their application with starch-based sources have been documented in several articles ,153. TheE. coli. Compared to chitin, chitosan on the other hand is also considered a green material obtained from the deacetylation of chitin and has a higher solubility when dissolved in solvents such as lactic acid, acetic acid, water, acetone, and methanol. Considering the preference for mechanical and chemical stabilities and surface area, nanochitosan presents more advantages, and, because of this, they are adopted as a packaging material in food applications. It is worth noting that the preparation method of nanochitosan is completely different from its counterparts (nanochitin). As reported by Mary and co-workers, the crosslinking of tripolyphosphate and protonated chitosan yielded chitosan nanoparticles, which were employed as reinforcement in potato starch TPS films. The tensile strength of thermoplastic films increased (2.84–10.80 MPa) with the increase of chitosan nanoparticles (0–6%) [2), suggesting that SNT and ZrO2 may be more successful in enhancing the ductility of the polyimide nanocomposites for applications where the more brittle polyimide/MMT nanocomposites films are unusable. In addition, in oriented nanocomposite fibers, Ivankova and colleagues reported that vapor-grown carbon nanofibers (VGCFs) and single-wall carbon nanotubes (SWCNTs) boosted tensile strength and modulus by 275 MPa and 5 GPa, respectively, but decreased deformation at break. The researchers discovered that SWCNTs are more effective reinforcers than VGCFs [Outstandingly, the characteristics of starch-based bionanocomposite films are significantly influenced by the shape and size of the chitin nanoparticles. The inclusion of nanowhiskers  improved the mechanical, thermal, barrier, and antifungal properties of the resulting films. High pressure homogenization was used to extract the additional chitin nanofibers, while acid hydrolysis was used to extract the chitin nanocrystals. Using different weight of starch, and different weights  of nanowhiskers,  reporteds (0–6%) . Similars (0–6%)  maintains (0–6%)  and carbs (0–6%) , amongsts (0–6%) . Besidesan VGCFs . More scan VGCFs ,166 and an VGCFs . Figure Consumers can benefit from better preservation of food materials due to smart packaging using starch-based materials, thus serving as an excellent response to mitigate the preservation of foods using chemicals. Starch, particularly corn starch, is considered one of the most promising biopolymer materials since it is abundant, inexpensive, biodegradable, tasty, and has good film-forming potential ,169. TheL. ruthenicumanthocyanins into cassava starch enhanced the water vapor permeability and antioxidant potential and reduced the ultraviolet light barrier of the starch film [To mention a few, the addition of anthocyanin-rich bayberry extract to cassava starch produced an excellent colorimetric film with exceptional antioxidant properties and tensile strength , whereasrch film . In a morch film .2), toughness (2.54 MJ/m3), Young’s Modulus (73.96 MPa), and water vapor permeability (0.00026 gm/day. kPa.m2) were all lowest in the film with the highest concentration of TGE. However, when compared to other films, it had the highest elongation at break (85.14%), moisture content (0.27%), and water solubility (37.92%). The films containing TGE extract change color when the pH is changed, according to pH sensitivity analysis. As the pH increased from 4 to 9, the color of the films changed from pink to slightly green [In order to critically assess the mechanical properties of pH-sensitive films, Mei and colleagues evidenced that the flexibility of sago starch increased with the addition of anthocyanin obtained from torch ginger extract (TGE) . In theily green . Propertly green . Howeverly green .Aerogel is a solid, ultra-lightweight substance with high porosity and other exceptional features . It consAerogels are distinguished by their drying process and precursors. The drying techniques necessitate the use of specific parameters and precursors in the production of aerogels. Broadly, organic and inorganic aerogels are the two types of supercritically-dried aerogels. However, recent research combined organic and inorganic aerogel precursors, resulting in hybrid aerogels resulting in different aerogel nomenclature such as ceramic, carbon, cellulose, and clay. Given these differences, it is necessary to distinguish existing aerogels and their possibilities in industrial application.2) process in the sol-gel processes of silicon and vanadium oxide (V2O5). Tetramethoxysilane (TMOS) and tetraethyl orthosilicate (TEOS) are typical precursors in silica aerogels. TMOS is dissolved in methanol, then hydrolyzed and condensed in a precise amount of water in this method. Recent studies have reported the use of silica and metal oxide aerogels. Silica is an exciting material for high-performance insulation applications due to its aesthetics, non-flammability, non-reactivity, and low thermal conductivity [2O5—fibrous and Al2O3—leaf-like). At comparable densities, metal oxides such as ZrO2 have a higher Young’s modulus (10.7 MPa), whereas most common SiO2 materials have a modulus of 2 to 3 MPa.These sorts of aerogels were the forerunners of today’s aerogels. They were first described by S.S. Kistler, who used the supercritical drying , high porosity, and low thermal conductivity (0.020.05 W/m K) [2 (m K). In the freeze-drying process, the compressive strength and fracture resistance of CNF/starch/clay aerogels were enhanced at reduced moisture (2%) without compromising the structure of the aerogel. The density (0.05 g/cm3) and thermal conductivity (41.5 mW/mK) were drastically lowered [Aerogel of this form was created using low-cost, non-toxic components using an environmentally favorable freeze-drying technology, making them more desirable for sustainability and environmental issues. Clay aerogels are distinguished by their low density (0.1 g/cm5 W/m K) , but the5 W/m K) . Clay ae lowered . However2 drying process. Nonetheless, freeze-drying is also applicable to polymer aerogels. Uniquely, supercritical drying presents higher porosity, while the freeze-dried technique, on the other hand, is frequently used because of its environmentally benign qualities and inexpensive cost. Extant studies on the supercritical drying of aerogels use biobased materials such as alginate, chitin, chitosan, carrageenan, agar, and cellulose gels as part of their component [2-dried aerogels from these biobased sources was determined to be macroporosity (pore width >50 nm), mesoporosity (2–50 nm), and microporosity (less than 2 nm) during the examination of the synthesis and characterization of tannin/lignin-based aerogels [The demand for polymer aerogels arose from industries’ unquenchable quest for materials with extremely low density, outstanding thermal characteristics, and a large specific surface area (aerospace). National Aeronautics and Space Administration (NASA), for example, has a high demand for materials such as polyimides due to their insulating qualities . Polymeromponent . Neverthaerogels . Class Iaerogels .Starch could be obtained from common agricultural materials such as rice, wheat, corn, potato, cassava, etc. ,198. ThoTrisodium citrate prepared by the addition of citric acid to an alkali solution has a better reaction when incorporated into the gelatinization process of aerogels because of their eco-friendliness and non-toxicity. Using a freeze-drying method, Camani and co-workers prepared starch-based aerogels using corn starch and trisodium citrate as the crosslinking agent at different levels of concentration. The authors evidenced a microporous structure (>50 nm) but observed that a higher concentration of crosslinking agents led to a larger pore diameter, which invariably affects the porous structure .The amylose content of starches (potato and peas) was varied for starch-based aerogels . The autAs discussed, aerogels could be organic, inorganic, or hybrid. The transition between these classes of aerogels is triggered by possible limitations associated with the inorganic aerogel, which S.S. Kistler first synthesized. The development of organic aerogels also has drawbacks related to their weak mechanical framework. Thus, to address these overall limitations, hybrid aerogels became a panacea. Though it could be assumed that the advent of hybrid aerogels has met the setbacks of organic and inorganic aerogels, the mechanical properties of polymeric aerogels must still be methodically tailored. Some scholars have undertaken efforts to manipulate the density and pore volume of polymeric aerogels, primarily by altering the precursor polymer concentration . TherefoThe manufacturing of aerogels via the sol-gel method has quickly become a fascinating new subject of material science research. Practically all aerogels are made using the sol-gel route ,202. ThiIn applying the sol-gel process first used for the production of silica aerogels, the excellent hydrophobicity and flexibility of aerogels  are critical for long-term usage. We discovered that the sol-gel technique is highly prominent and the only method reported so far in forming a three-dimensional (3D) porous network structure in an aerogel after reviewing published articles on the subject. However, sol-gel techniques have several drawbacks, including high material costs, extended processing times, and poor mechanical properties. Nonetheless, due to its flexibility, which allows for the adjustment of the pore structure, it is one of the greatest approaches for generating extremely porous solids.Removing the underlying liquid in aerogels without disturbing the solid structural framework is vital in aerogel synthesis. The purpose of drying solid materials is to remove moisture from their pores, which would otherwise cause the silica framework to cave in and succumb, causing the gel structure to collapse completely . Among t2/g [2SiO3 as precursors in the freeze-drying process, an ultralight aerogel with a pore volume capable of absorbing about 1.49 mmol/g of CO2 was produced [Freeze-drying or lyophilization releases solid, generally frozen, water from the pores of a wet precursor . Freeze-2/g ; while uproduced .2) solvents have been used in most scenarios [2 drying, thermodynamic parameters such as pressure and temperature can alter the gel’s liquid phase condition. As a result, ScCO2 drying has been discovered to be the ideal drying procedure for replacing the liquid with gas in the pores of the produced gels solely because CO2 is non-reactive and has a critical temperature that is very close to room temperature. In addition, CO2 benefits from its mild supercritical conditions, particularly the 304.13 K supercritical temperature. Sahin’s co-workers reported the ScCO2 procedure and stressed the importance of the solvent requirement and its benefits [2. Solvent exchange can cause gel shrinkage due to solvent-gel interactions. The type of solvent used to reduce shrinkage is very important. The critical parameters of an organic solvent are temperature (K), pressure (MPa), density (kg/m3), molar volume (cm3), compressibility factor, and permittivity [The most important step in the production of aerogels is supercritical drying, which preserves the three-dimensional pore structure, resulting in unique material properties such as high porosity, low density, and large surface area . This prcenarios . In supebenefits . As a reittivity .2 drying have a strong affinity for moisture over time, causing a capillary effect and eventually resulting in textural degradation. As a result, some researchers who have modified the supercritical drying process arguably emphasized that ScCO2 is inefficient, hazardous, costly, and unscalable. Nevertheless, ScCO2 drying has thus been the mainstream drying process of gel substances mainly utilized in functional areas such as medicines, culinary science, and the textile industry.Aerogels manufactured by ScCO2/g, a total pore volume of 1.2 cm3/g, an average pore diameter of 5.5 nm, a penetration ratio of 70%, and a thermal conductivity of 0.02 W/mK [3/g, with a pore size of 18.5 nm, a high hydrophobicity of 144.2 ± 1.1°, and low thermal conductivity of 0.031 ± 0.001 W/m.K. [Although APD is a less energy-intensive alternative, it frequently relies on refilling the initial gel preparation solvent . Cheng d.02 W/mK . In a si1 W/m.K. .Although water is largely not used in this drying process, except as an initial solvent for the precursor, scholars researched the eco-friendliness of water-based hydrophobicity silica aerogels using the APD route . Specifi2/g, respectively, compared to ScCO2 drying with a porosity and specific surface area of 95% and 353 m2/g, respectively. The density of FD was as low as 0.003 g/cm3, which is much lower than ScCO2 (0.08 g/cm3) [2 drying had specific surface areas between 60 to 100 m2/g [2/g [In the drying of cellulose aerogels, FD is widely employed, while ambient pressure drying is rarely used. The porosity and specific surface area of freeze-dried material from nanocellulose sources could rise as high as 99.97% and 700.1 m8 g/cm3) . All dev100 m2/g ,222 but 2/g [2/g . Technic2/g [2/g . AlthougNon-biodegradable synthetic polymer-based foams, such as polyethylene (PE), polystyrene (PS), polyurethanes (PU), and polypropylene (PP), are known to cause more environmental issues than their biodegradable counterparts ,223, makBiopolymers  derived from renewable resources such as plants and animals can assist in addressing both the challenges of diminishing oil reserves and the environmental issues associated with the rising use of petroleum-based plastics . In econTPS, also known as plasticized starch, is a substance generated by altering the structure of starch granules. It is recognized to have various advantages, including renewability and flexibility in the manufacturing process. The global thermoplastic starch market reached 179.58-kilometric tons in 2019. Between 2020 and 2025, the market is expected to reach a value of 255.82-kilometric tons, with a CAGR of 7.01% . Despitew/w). Biodegradable thermoplastic starches such as polybutylene succinate adipate (PBSA), polyhydroxybutyrate (PHB), and polycaprolactone (PCL) provide outstanding flexibility, a low melting point, and good compatibility [According to many reported studies, sorbitol, glycols, maltodextrin, amino acid, citric acid, formamide, and urea are the most commonly used plasticizers for TPS . Howevertibility . Succincw/w ratio) in a flask at 130 °C for 2 h with agitation. Second, starch particles (56 wt%) were added to the PEG and MA mixture, and the reaction was kept at 150 °C for an additional 2 h. The compatibilizer improved interfacial adhesion, which they discovered was due to interactions between the compatibilizer’s free end carboxylic acid groups and the active groups of the TPS and PLA phases [Polymer blending is the incorporation of hydrophilic starch with hydrophobic polymers . Starch-filled biodegradable polymer blends to replace inert plastic materials have attracted considerable interest due to the compatibility and degradation drawbacks of filler starch . CompatiA phases . Since tA phases . For insA phases . SolutioThis section and subsequent sub-sections highlight the processing methods for the manufacture of biofoams. Mechanical and thermal processes are the two main categories of these processes. However, because the drying processes of aerogels and biofoams are nearly identical, the drying process of aerogels is partially helpful in this regard. As a result, we are more concerned with the mechanical aspects of biofoam production. The preferred methods of producing biofoams include extrusion, injection, and compression molding (baking) . Foaming2) and nitrogen (N2). However, the type of gases produced are dependent on the utilized blowing agent [Extrusion is the process of compressing, conveying, and expelling a material through a specially designed outlet. Extrusion creates high pressure near the die output because of the expansion caused by injected gas or in situ generated gas. The pressure of the material drops significantly as it comes into contact with ambient air, causing the substance to foam. The gas bubbles that form in the starch paste are predominantly carbon dioxide  is another preferred method for producing biofoams. Because of its distinct advantages, this technology is very widely used. An injection molding machine usually has numerous components that allow for distinct unit activities such as injection, molding, ejection, core pulling, and cooling. The screw chamber comprises a helical screw and a heating element, while the substrate (polymeric ingredients) is fed through a specifically built hopper. The starch-based polymer melts when heat is applied, and the screw homogenizes the ingredients, allowing for increased flowability. IM maximizes production rates without compromising strength, brittleness, shrinkage, and aesthetic qualities . Further2 and nitrogen as a byproduct. Foaming agents from organic and inorganic compounds could be used for chemical foaming. However, for glass materials, chemical foaming agents and their advantages are CaSO4 , carbonates, sulfates, and carbon black [2HPO4 (reduces pore nonuniformity of foams) is used as a stabilizer [Foamed products are manufactured using physical, chemical, and mechanical techniques . The difon black , while Nabilizer .Meanwhile, attaining better foaming efficiency is influenced by enthalpy reactions that produce the gas in the foaming process, which may be exothermic, endothermic, or a combination (exo-endothermic). Exothermic foaming agents have the most favorable foaming efficiency and are thus widely used in industrial processes. From an economic and efficiency viewpoint, chemical foaming agents offer superior advantages over physical foaming agents. Chemical foaming agents also provide consistent monitoring and nucleating effects, which can help alleviate moisture problems and improve mechanical characteristics. However, chemical agents react negatively by producing dangerous fumes when stored at higher temperatures.Aerogels and biofoams have unique appearances. These two are nanocellulose; however, they exhibit differing porosities. Biofoam is any multi-phase porous material with a porosity of more than 50%, in which gas  is distributed in a liquid, solid, or hydrogel. Pore size (or bubble diameter) is usually more than 50 nm. An aerogel is a mesoporous material with high porosity (>90%) with pore sizes ranging from 2 nm to 50 nm . BiofoamAerogels and biofoams are also used in restoring, maintaining, or repairing the biological function of wounded tissues or complete organs . This idAerogels and biofoams also have wound healing applications because they absorb large amounts of water while dry and donate water when hydrated . Wound hSince biodegradability, biocompatibility, and sustainability are essential in conserving materials and ensuring their practical use, an in-depth survey of underutilized agricultural materials is essential to access their economic values.Most literature did not characterize  the raw starch used as a precursor for manufacturing biomaterials. Likewise, modified starch-based aerogel with unique mechanical properties and hydrophobicity could be a potential delivery system and food packaging option yet to be fully explored.Most scale-up processes have associated limitations because of the processes and parameters involved. Therefore, critical evaluation unit operations such as drying and solvent exchange are crucial.The modeling of a continuous process will improve the understanding of the process, since all available data in this research area are empirical-based.Though there is considerable success in measuring the morphological and structural characteristics of biomaterials, most tests have not been standardized, and thus comparability of the values becomes difficult. In addition to available data on mechanical properties, there is a scanty survey of literature on the acoustic properties of biomaterials, even though biomaterials (aerogels and biofoams) are reported as a sound absorption material.Aerogels and biofoams, developed from both renewable and non-renewable sources, are integral components of biomaterials. They have been deployed as a vital tool for diverse industrial uses such as food, medicine, and pharmaceuticals, making them potentially relevant and gaining widespread attention. Nonetheless, these materials are currently more popular in academia because a more significant percentage of literature surveys revealed that these biomaterials had been manufactured on a laboratory scale. As a result, non-biodegradable polymers  are dominant in many areas. Thankfully, the precursors  for the manufacturing of aerogels and biofoams are commercially abundant. Our standpoint suggests that using organic precursors to develop biomaterials will mitigate the ineffective utilization of raw materials and fulfill the gaps within the circular economy concept geared towards the reuse and remanufacture of underutilized agricultural materials. Furthermore, the competitiveness of most biomaterials requires the scalability of all processes involved; thus, careful handling of the materials is crucial to its eventual marketability. After carefully reviewing the published articles, we, therefore, summarized our identified research gaps below:Having surveyed the literature loop, we (the authors) are unarguably convinced that inorganic aerogels, and a few organic aerogels with resorcinol formaldehyde as a precursor, dominate the polymer sector because of their suitability for some engineering and non-engineering applications. However, because of their poor degradability and possibility of releasing harmful toxic substances, attention has now been shifted to complete natural biobased polymers such as starch. Notably, polysaccharides , lignin, proteins, and other biopolymers have been employed as natural precursors for biobased polymers. The findings of numerous academics revealed that they exhibited being microporous and mesoporous, having low densities, and having high surface areas, among other features. However, if laboratory cost comes with many routine processes, it is evident that the industrial price of these biomaterials will have associated exorbitant fees. Therefore, the pursuit of suitable raw materials with improved sustainability and lower pricing must continue, especially when biopolymers are desired for various applications. Similarly, the weak mechanical properties of natural precursors are one of the concerns of using them for biomaterial processes. Nevertheless, incorporating fillers provides a better framework, which is a panacea for their weak mechanical strength. On a molecular level, synergetic effects between distinct precursors and additions must be understood and predicted. In most cases, this is the only approach to creating novel hybrid materials rationally. Biomaterials have been used in pharmaceuticals  and food (packaging). The expectation of these industries will be met if there is a reduction in the processes of these biomaterials. Therefore, the fundamental knowledge of the thermodynamics and kinetics of the solvent exchange and drying processes and their translation into multiscale models are essential to achieving this goal. We expect rising concerns of biomaterials (aerogels and biofoams) in the coming years. One of those concerns that might trigger scholars’ interest is how to reduce the processing time of all the stages involved in the manufacture of biomaterials. The retrogradation step in monolithic-manufactured aerogels cannot be a semi-continuous process, which usually necessitates a lengthy autoclaving time. The ability to directly produce aerogels in the form of particles or thin fibers, on the other hand, has a unique advantage: the solvent extraction time for small particles is faster than for monoliths, allowing for significant cost savings. Lastly, there is a considerable demand for biodegradable materials in numerous scientific fields. Unfortunately, while the synthetic porous materials have gained commercial acceptance, not minding their waste contribution, natural biobased materials are still seldomly transitioning from laboratory to industrial scale. Therefore, the development of scale-up techniques and novel process changes could mitigate this drawback. Furthermore, the research on the techno-economic analysis and life cycle assessment of aerogels and biofoams is minimal. As a result, we anticipate increased interest in these identified areas in the forthcoming years.


Starch–gluten interactions are affected by biopolymer type and processing. However, the differentiation mechanisms for gluten–starch interactions during heating have not been illuminated. The effects of glutens from two different wheat flours  and a medium-strong gluten ) on starch’s (S) structural and physicochemical properties during heating and their molecular interactions were investigated in this study. The results showed that gluten hindered the gelatinization and swelling of starch during heating when temperature was below 75 °C, due to competitive hydration and physical barriers of glutens, especially in Y22. Thus, over-heating caused the long-range molecular order and amylopectin branches of starch to be better preserved in the Y22-starch mixture (Y22-S) than in the Y16-starch mixture (Y16-S). Meanwhile, the starch’s degradation pattern during heating in turn influenced the polymerization of both glutens. During heating, residual amylopectin branching points restricted the aggregation and cross-linking of gluten proteins due to steric hindrance. More intense interaction between Y16 and starch during heating mitigated the steric hindrance in starch–gluten networks, which was due to more residual short-range ordered starch and hydrogen bonds involved in the formation of starch–gluten networks in Y16-S during heating. Starch and gluten are the most important macromolecules in wheat flour, which greatly affect the functional and processing characteristics of wheat flour . Wheat fThe properties of starch and gluten usually change with temperature. When processed under different heating temperature, protein configuration and molecular weight changed, which are driven by the release and rearrangement of chemical forces  . GelatinThe interactions between macromolecules under the action of heat are complex. For example, un-gelatinized starch granules may affect the gluten and interact with gluten, differing with granule size . GelatinHowever, the presence of lipids in real cereal-based food systems may affect the physicochemical behaviors of starch and gluten during processing through the potential formation of starch–lipid or starch–lipid–protein complexes, causing great difficulties in investigating the starch–gluten interaction mechanism. Use of purified starch and gluten as a model material has led to a deep and comprehensive understanding of the interaction mechanisms of starch granules and the gluten network during thermal food processing. In this study, gluten from two wheat varieties with the same high-molecular weight (HMW) glutenin profile but of different strength was used to probe the transformation and interaction between the transient and continuous states of starch and gluten during a gradual heating process. This study may help us better understand the structure–function relationship of gluten–starch during food processing, thereby relating the thermal processing of starch and gluten to further provide guidance for the manufacturing of wheat flour products.The pasting parameters of native starch and pre-heated mixtures are shown in Thermal properties of native starch and pre-heated mixtures are summarized in As shown in The XRD patterns and crystallinity of starch during heating are illustrated in −1 is indicative of the degree of order in the outer regions of starch while the ratio of 1022/995 cm−1 denotes proportions of amorphous versus ordered structures. As shown in −1 and significantly lower ratio of 1022/995 cm−1 than all Y22-S samples. In these samples, the 1047/1022 cm−1 and 1022/995 cm−1 ratios progressively decreased and increased as soon as samples were heated. In contrast, the ratios were only significantly different between Y16-S and native starch after samples had been heated to 50 °C (1047/1022 cm−1) or 75 °C (1022/995 cm−1). Moreover, while the 1047/1022 cm−1 ratio decreased and the 1022/995 cm−1 ratio also increased in Y16-S over heating, the ratios were, at all temperatures, higher and lower, respectively, than in Y22-S samples.FT-IR and FT-Raman spectroscopy were performed to provide information on the short-range order of starch. The absorbance ratio of 1047/1022 cm−1 was calculated to characterize the short-range ordered structures in starch, and higher FWHM values at 480 cm−1 represent a lower proportion of short-range ordered structures. As shown in −1 decreased when the temperature increased from 25 to 50 °C, and then increased with the further increasing in temperature in both mixtures. The FWHM at 480 cm−1 increased from 16.31 to 17.74 in the Y22-S mixture and from 16.14 to 17.54 in the Y16-S mixture with the increase of temperature from 25 to 100 °C, respectively. Additionally, the IR ratio of 1022/995 cm−1 significantly increased compared with native starch in both mixtures with the increase in temperature, while there was no significant difference in the IR ratio of 1022/995 cm−1 for the Y16-S mixture between 75 and 100 °C.Although some vibrations may give rise to signals in both the Raman and FTIR regions of the electromagnetic spectrum, FTIR spectroscopy is best for asymmetrical vibrations of polar groups, whereas Raman spectroscopy is best at detecting symmetrical internal vibrations of nonpolar groups. The full width at half maximum (FWHM) of the Raman band at 480 cmThe morphology of starch during heating was observed by CLSM . For theNon-reducing and reducing SDS-PAGE were performed to analyze the protein polymerization during heating. As shown in non-reducing SDS-PAGE A, the baThe changes in chemical interactions in two mixtures during heating are shown in A molecular docking program was used to probe the in-silico protein–starch interaction. Previous research hypothesized that glutenin comprises the network and embeds the starch granules which are covered by gliadin ,16. This−1 and lower values for the 1022/995 cm−1 ratio as well as FWHM at 480 cm−1 than Y22-S, suggesting that more of the short-range ordered structure of starch remained in Y16-S over heating than in Y22-S. Previous works revealed that less change in the structural order of starch is mainly attributed to the close link between starch and other ingredients, i.e., lipid or protein [Since proteins can interact with starch molecules, the mixture or addition of protein can affect the structural changes of starches at the molecular level . No gela protein . Our datWith the increase in temperature, chemical interactions changed significantly among gluten proteins, resulting in new surface properties and enlarged protein aggregates . TherefoTriticum aestivum L.) cultivars, a weak-gluten wheat Yangmai 22 and a medium-gluten wheat Yangmai 16, were used in this study. Yangmai16 and Yangmai22 are two widely cultivated varieties in the middle and lower reaches of the Yangtze River. They have the same high-molecular-weight glutenins (HMW-GSs) but with different gluten strength. Wheat grains were milled using flour miller  with a 100 μm mesh sieve. The gluten indices from medium and weak gluten wheat flour were 88.60 and 63.00, respectively. The glutenin to gliadin ratio was 1.09:1 for weak-gluten wheat flour and 1.52:1 for medium-gluten wheat flour. The ratio of high-molecular-weight glutenin subunit to low-molecular-weight glutenin subunit (HMW/LMW-GS) was 0.29:1 for weak-gluten wheat and 0.38:1 for medium-gluten wheat flour. The glutens from two different wheat variety flours were isolated and extracted following the method described by Wang et al. [Commercial wheat starch  was purchased from Yuanye Biological Co., Ltd., Shanghai, China. The contents of moisture, ash, lipid, protein, amylose, and A- and B-type starch granule were 13.11%, 0.19%, 0.45%, 0.76%, 23.4%, 70.32%, and 29.68%, respectively. Two winter wheat  of starch and gluten samples were accurately weighed in an aluminum case to prepare the starch suspension via adding with 25 mL of distilled water to prepare starch and gluten suspensions, respectively. The starch suspension was added to 0.50 g (dry basis) of gluten followed by 30 s of homogenization to prepare the starch–gluten mixture, then, the starch suspension, the starch–gluten mixture, and gluten suspension were sealed in aluminum case and allowed to stand for 12 h before heating with Rapid Viscosity Analyzer (RVA) program  at 25, 50, 75, and 100 °C with a shear speed of 160 rpm/s for 10 min. The suspensions treated with different temperatures were sampled and stored in liquid nitrogen. After lyophilizing, the samples were ground into powder using a high throughput tissue grinder TL 2020  and passed through an 80-mesh sieve for further analysis. The control of native starch was designated as S. The treatments of Y16 and S mixture at 25, 50, 75, and 100 °C were designated as Y16-S-25 °C/50 °C/75 °C/100 °C, and Y22 and S mixture at 25, 50, 75, and 100 °C were designated as Y22-S-25 °C/50 °C/75 °C/100 °C, respectively. The moisture contents of lyophilized Y16-S and Y22-S mixtures were 10.52% and 10.88%, respectively.The pasting property of the prepared sample was analyzed with Rapid Viscosity Analyzer 130 , following the method described by Chang et al. . The thew/v) and heated at 100 °C for 45 min with stirring and then immediately cooled. After centrifugation , the supernatant was discarded and dried at 105 °C to a constant weight. Swelling power (g/g) was calculated as the ratio of the wet weight of precipitated colloid to its dry weight, and solubility (%) was calculated as the ratio of dry weight of water-soluble starch in supernatant to the initial dry weight of starch.Starch swelling power and solubility were determined according the method described by Leach . The preThe XRD patterns of the prepared sample were determined by an X-ray diffractometer , which was operated at 40 KV and 40 mA with Cu Kα radiation (λ = 0.154 nm). The sample was operated with diffraction angle (2θ) ranged at 5°–40° at a speed of 0.02° at room temperature. The relative crystallinity (%) was calculated by software MDI Jade 5.0.−1 were used to further calculate the IR ratio of 1047/1022 cm−1 and 1022/995 cm−1. The ratio of absorbance 1022/995 cm−1 was used to measure the ratio of amorphous to ordered carbohydrate structure in starch, and the ratio of absorbance 1047/1022 cm−1 was used to quantify the ordered degree of starch external region of starchThe short-range ordered structure of the starch sample was determined with Thermo Nicolet Nexus FTIR , following the method previously described by Yang et al. . The det−1 was obtained using the WIRE 2.0 software, and higher FWHM values at 480 cm−1 represent a lower proportion of short-range ordered structures.The short-range ordered structure was also determined by FT-Raman spectra , as reported by Brandt et al. . The fulThe morphological distribution of starch in different mixture samples before and after hydrothermal treatment was observed with CLSM . The starch suspension or paste was dyed with fluorescein isothiocyanate (FITC) (20 μL at 2 mg/mL) on a glass slide; meanwhile, it was washed three times with deionized water to remove the unbonded fluorescent dye, then covered with a microscopic glass. The images were recorded using CLSM with an emission range of 500–525 nm.w/v), 10% glycerol (v/v) and 0.01% bromophenol blue (w/v)) for 3 h at room temperature, and the reducing SDS-PAGE was performed using the extraction buffer contained 5% (v/v) β-mercaptoethanol (β-ME). After extraction and centrifugation , the supernatant was heated for 5 min in boiling water and loaded after cooling. The supernatant (matched in protein content) was loaded for SDS-PAGE analysis. Grey scale measurements were carried out with Image J software to denote the electrophoresis bands.SDS-PAGE was conducted in a vertical electrophoresis cell using 10% separating gel and 6% stacking gel. A 50 mg sample was mixed with 1.5 mL of extraction buffer . The chemical interactions were analyzed based on the protein content in different solutions, where ionic bonds were presented as the difference in the protein content between S1 and S2 solutions; similarly, the difference in the protein content between S2 and S3, S3 and S4, and S4 and S5 were used to estimate the contributions of hydrogen bond, hydrophobic interaction, and disulfide bond, respectively.Chemical interactions between the protein molecules of the prepared sample were determined according to the method described by Gómez-Guillén et al. , with miThe Autodock vina program  was used to study the binding information between two gluten proteins and starch. The protein amino acid sequences for HMW-GSs of 1Bx7 (GenBank: SCW25213.1) from Yangmai 16 and 1By9 (Accession: CAA43361.1) from Yangmai 22 were obtained from the protein data bank, and tertiary structures of HMW-GSs were predicted using I-TASSER online server, based on the multiple-threading alignments by LOMETS and iterative TASSER simulations . An amylp < 0.05 was considered significant.All data were expressed as mean ± standard deviation (SD) of three replicates. Data were analyzed using one-way analysis of variance (ANOVA), and Duncan’s multiple range test was used to compare the means with a significance using an SPSS package . The probability value of The present study has shown that the disruptive effects of starch and aggregation behaviors of protein caused by heat processing could be accompanied by interactions with each other during heating. The multi-scale structural and physicochemical properties of starch during heating were significantly affected by different gluten proteins. More degradation of amylopectin and disruption of long-range molecular order was detected in the Y16-S mixture during heating, which was due to less competitive hydration and more water mobility in the Y16-S mixture compared with the Y22-S mixture, as the starch gelatinization was influenced by water availability. Similarly, the polymerization behaviors of Y22 and Y16 in corresponding mixtures were significantly and differentially affected by starch due to steric hindrance, especially at higher temperature. Fewer amylopectin branching points and a higher degree of short-range order in Y16-S allowed for more gluten–starch interactions in this system than in Y22-S mixtures.


Arabidopsis thaliana) mutants lacking enzymes of the phosphoglucose isomerase-phosphoglucose mutase-ADP-glucose pyrophosphorylase starch synthesis pathway in leaf mesophyll chloroplasts or sugar transporters at the plastid membrane, such as glucose-6-phosphate/phosphate translocators, which are active in heterotrophic tissues. We demonstrate that GCs have metabolic features of both photoautotrophic and heterotrophic cells. GCs make starch using different carbon precursors depending on the time of day, which can originate both from GC photosynthesis and/or sugars imported from the leaf mesophyll. Furthermore, we unravel the major enzymes involved in GC starch synthesis and demonstrate that they act in a temporal manner according to the fluctuations of stomatal aperture, which is unique for GCs. Our work substantially enhances our knowledge on GC starch metabolism and uncovers targets for manipulating GC starch dynamics to improve stomatal behavior, directly affecting plant productivity.The pathway of starch synthesis in guard cells (GCs), despite the crucial role starch plays in stomatal movements, is not well understood. Here, we characterized starch dynamics in GCs of Arabidopsis ( Guard cells synthesize starch using carbon precursors originating in the plastid and/or imported from the cytosol depending on the time of the day. Starch is the most abundant form in which plants store carbohydrates. It is composed of homopolymers of glucose (Glc), which form dense, insoluble, semi-crystalline granules within plastids. The way starch is synthesized and metabolized may vary upon the tissue in which it is found. In autotrophic tissues, such as the leaf mesophyll, starch is gradually formed during the day using a portion of the carbon fixed through photosynthesis; at night, starch is degraded to support non-photosynthetic leaf metabolism and the export of sucrose (Suc) . In heteIn the leaf mesophyll, starch is the end-product of a biosynthetic pathway that takes place exclusively within the chloroplast and is directly linked to the Calvin–Benson–Bassham (CBB) cycle by means of the phosphoglucose isomerase (PGI) enzyme. PGI generates Glc-6-phosphate (G6P) from the primary photosynthetic product fructose-6-phosphate (F6P). Phosphoglucose mutase (PGM) further converts G6P into Glc-1-phosphate (G1P), which is ultimately used for the ATP-consuming generation of the activated glucosyl donor ADPGlc by the ADPGlc pyrophosphorylase AGPase; . Each enZea mays) endosperm  (Vitis vinifera) (Solanum tuberosum) tubers (Oryza sativa), wheat (Triticum aestivum), and potato, G1P is directly added to elongating glucan chains via the α-glucan phosphorylase (PHS1) . This transmembrane protein was initially detected in the plastidial envelope membranes of maize   and grapinifera) . Besides) tubers . Alternae (PHS1) . Lastly,e (PHS1) .2 uptake for photosynthesis, while limiting water loss through transpiration. These highly specialized cells possess several characteristics of heterotrophic tissues, such as high respiratory rates   that surry rates , fewer cry rates , low levry rates , callingry rates , it was ry rates . Unlike ry rates .In GCs, starch shows a distinct temporal pattern of accumulation and degradation that differs in several aspects from that of MCs. In Arabidopsis, GC starch is abundant during the night and is rapidly mobilized within 1-h of illumination, after which starch progressively accumulates until the middle of the night . Starch PGI, PGM, and the AGPase, as well as genes linked to uptake of sugars to the chloroplast, including the GPTs. Although GCs are limited in photosynthetic activities , suggesting that PGI is not required for GC starch synthesis. However, starch granules were only visualized at the end of the day (EoD)  compared with WT  . Here, w with WT . Upon litant GCs  to reachpgi mutant had reduced expression of PGI, it was not a complete knockout   . We deteve to WT , pointinan in WT .pgi mutants prompted us to examine whether GPTs were involved in GC starch metabolism. The GPT1 gene was approximately four-fold upregulated in WT GC-enriched epidermal peels relative to leaves (GPT1 in GC protoplasts compared with MC protoplasts (GPT2 was expressed to similar levels as the GC marker genes + inward rectifier channel 1K (KAT1) and Myb transcription factor 60 (MYB60), therefore displaying a more pronounced preferential GC expression than GPT1  and GPT2  and quantified stomatal starch amounts during the 24 h light/dark cycle  in combination with the reduced starch contents throughout the day indicate that both PGI and GPT1 are required for proper GC starch synthesis. Residual accumulation of starch in  the EoN .gpt1pgi or gpt1gpt2 double mutants, respectively, we generated plants lacking all three enzymes. Due to the leaky pgi mutation, as described above, we transcriptionally downregulated PGI in gpt1gpt2 mutant background, using artificial microRNA-based silencing  gene from potato , are devoid of leaf starch  and G1PT2 (SALK_123601) and analyzed stomatal starch dynamics throughout the 12 h light period  accumulated starch similarly to WT  and apl4 (SALK_108632) throughout the 12 h light period. As expected, GC starch dynamics were indistinguishable between WT and the apl1 mutant , which is structurally similar to that of maize. BT1 localizes to the plastidial membrane and was described to mediate AMP, ADP, and ATP transport into the chloroplast. However, in vitro experiments showed that BT1 does not accept ADPGlc as a substrate  or pea reported otherwise  represents the major carbon source for GC starch biosynthesis  the PGM-catalyzed conversion of G6P within the chloroplast stroma and (2) G1P imported from the cytosol via G1PTs.pgm mutants suffer from severely reduced growth and disturbed carbohydrate metabolism . Interestingly, in The unexpected accumulation of starch in GCs of amiRNA-of light  can be eof light . More reof light . Using tof light . HoweverG1PT genes were highly expressed in GCs  failed to accumulate starch in GCs specifically between 6 and 12 h of light  plants in the accession Columbia (Col-0 = WT) background. Transfer DNA (T-DNA) insertion lines SALK_026943 (BT1), GABIKAT_454H06 (gpt2), GABI_099E03 (g1pt1) and SALK_123601 (g1pt2) and GABI_257A06 (phs1) were obtained from Nottingham Arabidopsis Stock Centre (NASC). SALK_108632 (apl4-3) line was provided by Samuel Zeeman . Alison Smith  provided FLAG_458A07 (apl3-1) in the accession Wassilewskija (Was) background. Ethyl methanesulfonate (EMS) mutants apl1 , which were characterized to have unaltered GPT1 transcript amounts  found substantial reductions of GPT1 transcripts in different flower organs along with reduced starch contents , LED panels (ClimeCab 1400) and halogen lamps (Klimaschrank).Plants were cultivated in soil in controlled-climate chambers  under a 12-h/12-h light/dark photoperiod, with a temperature of 21°C/19°C day/night, a relative humidity of 45%/55% day/night and an irradiance of 150 µmol mPGI and PGM, sequences of pre-miRNAs were designed using the Web MicroRNA Designer tool . The primer set listed in KST1 (PGM) and pSF21 (amiR-PGI) constructs were transformed into Arabidopsis WT (pSF10) and gpt1gpt2 (pSF21) backgrounds followed by selection of independent lines (For GC-specific gene silencing of  in KST1 . The resnt lines .http://rsbweb.nih.gov/ij/). To avoid overlapping signals with starch granules from MCs, images of GC starch granules were acquired only from mesophyll-free parts of the epidermal peels. Four biological replicates were analyzed per genotype and time point for each experiment.GC starch was quantified at the indicated time points. Epidermal peels were manually obtained from leaf number 5 or 6. GC starch granules were fixed and stained as previously described . SubsequTo extract leaf RNA, three entire rosettes per genotype and time point  were harvested at the indicated time points and frozen in liquid nitrogen.To extract RNA from GC-enriched epidermal peels, the middle veins of 12 rosettes per genotype and time point  were excised at the indicated time points and the remaining leaf material was blended in 100 mL ice-cold water using a kitchen blender . Blended sample was filtered through a 200-µm nylon mesh (Sefar), and the remaining epidermal peels were dried, collected in a tube, and immediately frozen in liquid nitrogen.Subsequently, the epidermal peels were ground to a fine powder using a tissue grinder . For each experiment, two or three biological replicates per genotype and time point were harvested. Two independent experiments were performed for each extraction .Actin2 gene . Error calculations were done according to Applied Biosystems guidelines . Primers and PCR efficiencies for RT-qPCR are listed in Total RNA was extracted from ≥30 mg of ground tissue using the SV Total RNA Isolation Kit (Promega) following the manufacturer’s instructions. RNA quality and quantity were analyzed with a NanoDrop ND-1000 spectrophotometer . A total of 1 µg of RNA was used for cDNA first-strand synthesis using the M-MLV Reverse Transcriptase RNase H Minus Point Mutant and oligo(dT)15 primer (Promega). Transcript levels were examined by RT-qPCR using the SYBR Green Master Mix  and the 7500 Fast Real-Time PCR System (Applied Biosystems). RT-qPCR was performed in triplicates. Transcript levels were calculated according to the comparative CT method  and wereP-value < 0.05). All data are indicated as means ± SEM.Statistical differences between genotypes and time points were determined by ANOVA with post hoc Tukey’s Honest Significant Difference test (All data supporting the findings of this study are available within the paper and within its At4g17090 (BAM3), At3g18780 (ACT2), At5g46240 (KAT1), At1g08810 (MYB60), At5g54800 (GPT1), At1t61800 (GPT2), At4g24620 (PGI), At5t51820 (PGM), At3t29320 (PHS1), At4g39210 (APL1), At4g39210 (APL3), At2g21590 (APL4), At4g32400 (BT1), At1g34020 (G1PT1), and At4g09810 (G1PT2).Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: The following materials are available in the online version of this article. Genotyping of pgi and gpt1pgi mutants. Gene expression of GPT2 in gpt1pgi mutants. Growth retardation of pgi mutants. GC gene expression of PHS1 and starch contents in phs1 mutants. Leaf starch contents in apl mutants. GC gene expression of BRITTLE1 and starch contents in brittle1 mutants. Starch synthesis rates of WT, pgi and gpt mutant GCs. Starch synthesis rates of WT and gpt1gpt2 GCs. Starch synthesis rates of WT and PGI silencing lines GCs. Starch synthesis rates of WT and PGM silencing lines GCs. Starch synthesis rates of WT and g1pt mutant GCs. Starch synthesis rates of WT, phs1 and apl mutant GCs. Oligonucleotides used in this study. Plasmids generated in this study.kiac087_Supplementary_DataClick here for additional data file.