Starch Granules In Endosperm Research Articles

Effects of thermophilic and acidophilic microbial consortia on maize wet-milling steeping

To understand the ecology of species and promote biotechnology through beneficial strain selection for improving starch yield in maize wet-milling steeping, bacterial diversity and community structure during the counter-current steeping process in a commercial steeping system were characterized and investigated. The microbial diversity in the steeping liquor, which consisted of 16 phyla, 131 families, and 290 genera, was more abundant compared to those present on the surface of unsteeped maize. As the counter-current steeping progressed, exposing newer maize to the older steepwater, Lactobacillus dominated, replacing Rahnella, Pseudomonas, Pantoea, and Serratia. The thermophilic and acidophilic microbial consortia were enriched through adaptive evolution engineering and employed to improve starch yield. Several steeping strategies were evaluated, including water alone, SO2 alone, mono-culture of B. coagulans, microbial consortia, and a combination of consortium and SO2. Combining the microbial consortium with SO2 significantly increased the starch yield to, about 66.4 ± 0.5%, a 22% and 46% increase over SO2 alone and the consortium alone, respectively. Scanning electron microscope (SEM) of steeped maize structure indicated that the combination of consortium and SO2 disrupted the protein matrix and widened gaps between starch granules in maize endosperm. This released proteins into the steepwater and left starch granules in the aleurone layer. The steeping strategy of using thermophilic and acidophilic microbial consortium as additives shows potential application as an environmentally friendly alternative to conventional maize steeping procedures.

Relationship between the nanometer-scale structures of amylopectin molecules and temperature dependence of internal structures of starch granules in endosperm of starch branching enzyme 2b (be2b) allelic mutant lines from japonica rice

We analyzed the relationship between the nanometer-scale structure of amylopectin and physicochemical properties such as thermal properties of starch granules and higher-order internal structure of starch granules evaluated by using synchrotron radiation X-ray scattering and scanning electron microscopy. In this study, a line of allelic mutants of rice lacking in starch branching enzyme IIb (BEIIb) activity in their endosperm were used as starch sources. The crystal lattice changed from A-type to C- and B-type. The nanoscale cluster size, and higher-order structures of the starch granules varied among mutant lines. The thermal gelatinization properties of the rice-derived starch samples, as well as the dependence of this process on their crystal structure, were evaluated using differential scanning calorimetry and synchrotron X-ray scattering. After gelatinization, several nm-scaled density fluctuations owing to the formation of gel balls were observed. These fluctuations of all starch samples correlated with the cluster size decreased with the temperature.

Initiation of B-type starch granules in wheat endosperm requires the plastidial α-glucan phosphorylase PHS1.

The plastidial α-glucan phosphorylase (PHS1) can elongate and degrade maltooligosaccharides (MOSs), but its exact physiological role in plants is poorly understood. Here, we discover a specialized role of PHS1 in establishing the unique bimodal characteristic of starch granules in wheat (Triticum spp.) endosperm. Wheat endosperm contains large A-type granules that initiate at early grain development and small B-type granules that initiate in later grain development. We demonstrate that PHS1 interacts with B-GRANULE CONTENT1 (BGC1), a carbohydrate-binding protein essential for normal B-type granule initiation. Mutants of tetraploid durum wheat (Triticum turgidum) deficient in all homoeologs of PHS1 had normal A-type granules but fewer and larger B-type granules. Grain size and starch content were not affected by the mutations. Further, by assessing granule numbers during grain development in the phs1 mutant and using a double mutant defective in both PHS1 and BGC1, we demonstrate that PHS1 is exclusively involved in B-type granule initiation. The total starch content and number of starch granules per chloroplast in leaves were not affected by loss of PHS1, suggesting that its role in granule initiation in wheat is limited to the endosperm. We therefore propose that the initiation of A- and B-type granules occurs via distinct biochemical mechanisms, where PHS1 plays an exclusive role in B-type granule initiation.

Accumulation characteristics and structural properties of starch in different spatial positions of wheat endosperm under nitrogen application

Starch accumulation in wheat endosperm has spatial differences. In this study, the accumulation, granule size distribution, physicochemical properties of starch in different spatial positions in wheat endosperm and response to nitrogen treatment were analysed. Results indicated that the starch content in the inner endosperm was higher than that in the outer endosperm. The total starch and amylose contents in the inner and outer endosperms decreased and increased after nitrogen treatment, respectively. The starch granules that accumulated in the inner endosperm were larger than those in the outer endosperm 10 days after anthesis. The amount of C-type starch granules in the inner endosperm was less than that in the outer endosperm, and nitrogen treatment increased the C-type starch granules in the inner endosperm. After nitrogen treatment, the short chains decreased, whereas the middle and long chains increased in the inner and outer endosperms. Nitrogen treatment also increased the crystal area in the inner and outer endosperms, especially the single helix in the inner endosperm. These conclusions provide new insights into the mechanism of starch accumulation in wheat endosperm and its response to the external environment.

The relationship between the fine structure of amylopectin and the type of crystalline allomorph of starch granules in rice endosperm

AbstractBackground and ObjectivesIt is known that any wild‐type varieties of cereals so far examined have A‐type starch crystals in their endosperm whereas their starch branching enzyme 2b (be2b) mutants that are usually referred to as amylose‐extender (ae) mutants produce B‐type starch crystals. The present study aimed to examine the structural features of amylopectin which are responsible for the A‐type or B‐type crystalline allomorphs of starch granules by using starches in wild‐type japonica and indica rice varieties and their be2b (ae) mutants.FindingsThe average length of chains of A‐type amylopectin was markedly shorter than that of B‐type amylopectin. It was also thought that A‐type amylopectin had two types of branches, namely the first branches were formed mainly by BEI in the basal region of the cluster and the second branches were synthesized specifically by BEIIb inside the cluster. These differences caused the average length of double helices formed by cluster constituent chains to be shorter in wild‐types than that in their be2b mutants, and these differences changed the crystalline allomorph of starch granules to B‐type in the mutants from A‐type in their wild‐types, as examined by wide angle X‐ray diffraction and sum frequency generation spectroscopy.ConclusionsCombined with these observations and calculations of chain‐length of hypothetical A chains in four types of amylopectin molecules, it was concluded that both the average length of external segments of cluster chains and the formation of the second branches inside the cluster greatly affect the crystal types of starch granules.Significance and NoveltyThe novel findings in the present study can provide new insights into the structural features of amylopectin which determine the A‐type or B‐type crystalline allomorph of starch granules of cereals.

Phenotypic differences in the appearance of soft rice and its endosperm structural basis.

In view of the significant differences among genotypes in the appearance of soft rice, it is necessary to conduct research on the differences in the appearance quality of soft rice and their mechanisms. It can provide a theoretical basis for the selection and breeding of superior appearance varieties at a later stage. In order to clarify the differences in appearance phenotypes between different soft rice genotypes and structural basis of endosperm structures behind the differences, four soft rice varieties were selected in this study, including two varieties with good-appearance and two varieties with cloudy appearance. The differences in appearance phenotypes and endosperm structure in mature grains of soft rice with different appearance phenotypes were scientifically analyzed. The development process of their endosperm differences at the filling stage was investigated. The results show that the difference in the rice appearance of soft rice varieties mainly lay in the chalk-free seed transparency and chalkiness. These differences were caused by two completely different types of endosperm structure. Fewer and smaller starch grain cavities were responsible for higher chalk-free transparency of soft rice grains, denser starch granules arrangement caused lower chalkiness of soft rice grains. Ten days after flowering, the starch granules in the back and heart of good-appearance soft rice were already significantly fuller and more closely packed than those of cloudy soft rice. At the same time, the number and area of starch granule holes were significantly smaller than those of cloudy soft rice. This difference gradually increased until maturity. Therefore, based on appearance evaluation, soft rice with good-appearance should have higher transparency and lower chalkiness. The endosperm starch granules should be full and tightly arranged. The number of starch grain cavities and the area should be smaller. These differences develop in the early stages of grouting and gradually increase.

Characteristics of the composite film of arabinoxylan and starch granules in simulated wheat endosperm

We found that cell wall components of wheat grains differed significantly across different grain-filling stages; specifically, we observed significant differences in water content and water migration rate (p < 0.05). A composite film of arabinoxylan and starch granules was prepared to simulate wheat endosperm structure. Scanning electron microscopy (SEM), X-ray diffractometer (XRD), and thermogravimetric analysis (TGA) showed that the crystallinity and structural stability of the film increased with increasing starch content. Water diffusion experiments of the films revealed that the water diffusion rate gradually decreased with increasing starch content. Therefore, the water mobility of the starch endosperm was lower than that of the aleurone layer. These findings provide a basis for further studies in the context of wheat grain water regulation.

Scanning Electron Microscopic Observation of Endosperm Chalkiness Traits of OsAAP1 Mutant in Rice Gains

In order to investigate the effect of OsAAP1 gene mutation on chalkiness traits of rice endosperm and the microstructure of starch granules in endosperm, we used rice OsAAP1 mutants as experimental materials, with the help of ordinary optical microscopes and scanning electron microscopes, the morphological structure and arrangement of chalkiness traits and starch granules in the endosperm of Zhonghua 11 rice were observed in rice OsAAP1 mutants and controls. The results showed that the chalkiness rate, chalkiness area and chalkiness degree in rice endosperm of the OsAAP1 mutants were significantly increased, and the morphological structure, arrangement and development of chalkiness traits and starch granules were closely related. Compared with the control Zhonghua 11, there were also significant differences in the shape, structure and arrangement of starch granules between chalky rice and non-chalky rice. However, there was no significant difference between the starch granules in the transparent part of chalky rice and non-chalky rice, and the starch granules in the areas with low chalkiness degree developed well. Thus, the OsAAP1 mutation of rice could significantly increase the chalkiness traits of rice endosperm, and have an important impact on the morphology, structure and development of starch granules in its endosperm.

Fine Mapping of qWCR4, a Rice Chalkiness QTL Affecting Yield and Quality

Rice (Oryza sativa L.) chalkiness greatly reduces the rice quality and the commercial value. In this study, qWCR4, a previously reported quantitative trait locus (QTL) of white-core rate (WCR), was confirmed by a BC5F2 segregation population and further fine mapped to a 35.26 kb region. In the qWCR4 region, LOC_Os04g50060 and LOC_Os04g50070 showed significant differences in expression level in endosperm between two NILs, whereas four other genes had no expression. Starch granules in the central endosperm of chalky grains from NIL(J23B) with higher WCR exhibited a typically round and loosely packed morphology. NIL(J23B) with higher WCR accompanied a higher seed filling speed. Moreover, qWCR4J23B (qWCR4 allele in J23B) increased WCR, grain numbers per plant, seed setting rate, grain width, and thousand-grain weight, contributing to a superior yield per plant. All in all, our research results not only lay a foundation for map-based cloning of qWCR4 but also provide new genetic resources for rice yield and quality breeding.

Starch synthases SSIIa and GBSSI control starch structure but do not determine starch granule morphology in the absence of SSIIIa and SSIVb.

High levels of two major starch synthases, SSIIa and GBSSI, in ss3a ss4b double mutant rice alter the starch structure but fail to recover the polygonal starch granule morphology. The endosperm starch granule is polygonal in wild-type rice but spherical in double mutant japonica rice lacking genes encoding two of the five major Starch synthase (SS) isozymesexpressed in endosperm, SSIIIa and SSIVb. Japonica rice naturally has low levels of SSIIa and Granule-bound SSI (GBSSI). Therefore, introduction of active SSIIa allele and/or high-expressing GBSSI allele from indica rice into the japonica rice mutant lacking SS isozymes can help elucidate the compensatory roles of SS isozymes in starch biosynthesis. In this study, we crossed the ss3a ss4a double mutant japonica rice with the indica rice to generate three new rice lines with high and/or low SSIIa and GBSSI levels, and examined their starch structure, physicochemical properties, and levels of other starch biosynthetic enzymes. Lines with high SSIIa levels showed more SSI and SSIIa bound to starch granule, reduced levels of short amylopectin chains (7 ≤ DP ≤ 12), increased levels of amylopectin chains with DP > 13, and consequently higher gelatinization temperature. Lines with high GBSSI levels showed an increase in amylose content. The ADP-glucose content of the crude extract was high in lines with low or high SSIIa and low GBSSI levels, but was low in lines with high GBSSI. Addition of high SSIIa and GBSSI altered the starch structure and physicochemical properties but did not affect the starch granule morphology, confirming that SSIIIa and SSIVb are key enzymes affecting starch granule morphology in rice. The relationship among SS isozymes and its effect on the amount of substrate (ADP-glucose) is discussed.

STARCH SYNTHASE 4 is required for normal starch granule initiation in amyloplasts of wheat endosperm.

Starch granule initiation is poorly understood at the molecular level. The glucosyltransferase, STARCH SYNTHASE 4 (SS4), plays a central role in granule initiation in Arabidopsis leaves, but its function in cereal endosperms is unknown. We investigated the role of SS4 in wheat, which has a distinct spatiotemporal pattern of granule initiation during grain development. We generated TILLING mutants in tetraploid wheat (Triticum turgidum) that are defective in both SS4 homoeologs. The morphology of endosperm starch was examined in developing and mature grains. SS4 deficiency led to severe alterations in endosperm starch granule morphology. During early grain development, while the wild-type initiated single 'A-type' granules per amyloplast, most amyloplasts in the mutant formed compound granules due to multiple initiations. This phenotype was similar to mutants deficient in B-GRANULE CONTENT 1 (BGC1). SS4 deficiency also reduced starch content in leaves and pollen grains. We propose that SS4 and BGC1 are required for the proper control of granule initiation during early grain development that leads to a single A-type granule per amyloplast. The absence of either protein results in a variable number of initiations per amyloplast and compound granule formation.

Перикарпы боялған күріш генотиптеріне амилоза мөлшері бойынша скрининг

Rice is one of the oldest crops. Starch and protein are among the main components of grains. In addition, it contains vitamins, minerals and fats. The criteria for high quality rice vary from country to country. For example, in the Philippines, Bangladesh and India, a preference is given to rice with a high amylose content. In Japan, Egypt and Korea, a preference is given to low-amylose varieties. All varieties are used in China, Myanmar, Nepal and Malaysia. Starch is a major contributor to the culinary and nutritional quality of rice. In cereal grains, starch consists of C6 sugar, which is polymerized into endosperm starch granules. There are two types of polymerization: straight-chain amylose and amylopectin with branched-chain sugar molecules that are linked by glycosidic bonds. The light-optical properties of the caryopsis depend on the type of starch polymerization. In caryopses that contain 20-30% amylose, the endosperm is transparent, and with waxy starches, it is cloudy. According to the composition of starch, grains are divided into glutinous and non-glutinous. However, there are many intermediate forms. One of the important indicators and a good indicator for assessing the quality of grain is the content of amylose. The culinary advantages of rice cereals (coefficient of water absorption and weld, taste and consistency of porridge) depend on the content of amylose. In Kazakhstan, targeted studies have not been carried out to determine the quantitative content of amylose in rice genotypes with a colored pericarp. Therefore, the authors undertook biochemical screening for amylose content in rice genotypes with a stained pericarp and their parental forms. The article presents results of biochemical analysis of generations F6-F7 hybrids with stained pericarp for amylose content. As a result of the study, it was revealed that among the studied rice samples there are no high amylose genotypes, and the amylose content ranges from 0 to 24.5%. In terms of amylose content, the studied rice genotypes were classified into 4 groups.

Effects of BEIIb-Deficiency on the Cluster Structure of Amylopectin and the Internal Structure of Starch Granules in Endosperm and Culm of Japonica-Type Rice.

It is known that one of starch branching enzyme (BE) isoforms, BEIIb, plays a specific role not only in the synthesis of distinct amylopectin cluster structure, but also in the formation of the internal structure of starch granules in rice endosperm because in its absence the starch crystalline polymorph changes to the B-type from the typical A-type found in the wild-type (WT) cereal endosperm starch granules. In the present study, to examine the contribution of BEIIb to the amylopectin cluster structure, the chain-length distributions of amylopectin and its phosphorylase-limit dextrins (Φ-LD) from endosperm and culm of a null be2b mutant called amylose-extender (ae) mutant line, EM10, were compared with those of its WT cultivar, Kinmaze, of japonica rice. The results strongly suggest that BEIIb specifically formed new short chains whose branch points were localized in the basal part of the crystalline lamellae and presumably in the intermediate between the crystalline and amorphous lamellae of amylopectin clusters in the WT endosperm, whereas in its absence branch points which were mainly formed by BEI were only located in the amorphous lamellae of amylopectin. These differences in the cluster structure of amylopectin between Kinmaze and EM10 endosperm were considered to be responsible for the differences in the A-type and B-type crystalline structures of starch granules between Kinmaze and EM10, respectively. The changes in internal structure of starch granules caused by BEIIb were analyzed by wide angle X-ray diffraction, small-angle X-ray scattering, solid state 13C NMR, and optical sum frequency generation spectroscopy. It was noted that the size the amylopectin cluster in ae endosperm (approximately 8.24 nm) was significantly smaller than that in WT endosperm (approximately 8.81 nm). Based on the present results, we proposed a model for the cluster structure of amylopectin in WT and ae mutant of rice endosperm. We also hypothesized the role of BEIIa in amylopectin biosynthesis in culm where BEIIb was not expressed and instead BEIIa was the major BE component in WT of rice.

Study on the Chalkiness Character of Giant Embryo Rice and Observation on Its Microstructure in Grains

Grain chalkiness greatly affects the grain appearance and milling, eating, cooking, and nutritional qualities, thus it is one of the most important traits of grain qualities. Based on the study of chalkiness characters in endosperm of different giant embryo rice, the relationship between the differences of chalkiness characters in endosperm of different giant embryo rice and the morphological structure, arrangement and development of starch granules in endosperm was clarified, which will lay a solid foundation for further revealing the formation mechanism of chalkiness characters in giant embryo rice and cultivating excellent new varieties of giant embryo rice. In this study, the chalkiness of endosperm (including chalkiness rate, chalkiness degree and chalkiness area) of 16 different rice varieties were investigated and analyzed. The results showed that the chalkiness of endosperm was closely related to the morphological structure and arrangement of starch granules in endosperm cells of different kinds of giant embryo rice, and there were significant differences between the chalkiness of different parts of the same giant embryo rice and the morphological structure and arrangement of starch granules in endosperm cells. Therefore, the results of this study will provide important information for the improvement of quality characters and the breeding of new rice varieties.

Optimization of isolation and transfection conditions of maize endosperm protoplasts

BackgroundEndosperm-trait related genes are associated with grain yield or quality in maize. There are vast numbers of these genes whose functions and regulations are still unknown. The biolistic system, which is often used for transient gene expression, is expensive and involves complex protocol. Besides, it cannot be used for simultaneous analysis of multiple genes. Moreover, the biolistic system has little physiological relevance when compared to cell-specific based system. Plant protoplasts are efficient cell-based systems which allow quick and simultaneous transient analysis of multiple genes. Typically, PEG-calcium mediated transfection of protoplast is simple and cost-effective. Notably, starch granules in cereal endosperm may diminish protoplast yield and integrity, if the isolation and transfection conditions are not accurately measured. Prior to this study, no PEG-calcium mediated endosperm protoplast system has been reported for cereal crop, perhaps, because endosperm cells accumulate starch grains.ResultsHere, we showed the uniqueness of maize endosperm-protoplast system (EPS) in conducting endosperm cell-based experiments. By using response surface designs, we established optimized conditions for the isolation and PEG-calcium mediated transfection of maize endosperm protoplasts. The optimized conditions of 1% cellulase, 0.75% macerozyme and 0.4 M mannitol enzymolysis solution for 6 h showed that more than 80% protoplasts remained viable after re-suspension in 1 ml MMG. The EPS was used to express GFP protein, analyze the subcellular location of ZmBT1, characterize the interaction of O2 and PBF1 by bimolecular fluorescent complementation (BiFC), and simultaneously analyze the regulation of ZmBt1 expression by ZmMYB14.ConclusionsThe described optimized conditions proved efficient for reasonable yield of viable protoplasts from maize endosperm, and utility of the protoplast in rapid analysis of endosperm-trait related genes. The development of the optimized protoplast isolation and transfection conditions, allow the exploitation of the functional advantages of protoplast system over biolistic system in conducting endosperm-based studies (particularly, in transient analysis of genes and gene regulation networks, associated with the accumulation of endosperm storage products). Such analyses will be invaluable in characterizing endosperm-trait related genes whose functions have not been identified. Thus, the EPS will benefit the research of cereal grain yield and quality improvement.

The defective effect of starch branching enzyme IIb from weak to strong induces the formation of biphasic starch granules in amylose-extender maize endosperm.

Biphasic starch granules in maize ae mutant underwent the weak to strong SBEIIb-defective effect during endosperm development, leading to no birefringence in their exterior due to extended long branch-chains of amylopectin. Biphasic starch granules are usually detected regionally in cereal endosperm lacking starch branching enzyme (SBE). However, their molecular structure, formation mechanism, and regional distribution are unclear. In this research, biphasic starch granules were observed in the inner region of crown endosperm of maize ae mutant, and had poorly oriented structure with comb-like profiles in their exterior. The inner endosperm (IE) rich in biphasic starch granules and outer endosperm (OE) without biphasic starch granules were investigated. The starch had lower amylose content and higher proportion of long branch-chains of amylopectin in IE than in OE, and the exterior of biphasic starch granules had less amylose and more long branch-chains of amylopectin than the interior. Compared with OE, the expression pattern of starch synthesis related enzymes changed significantly in IE. The granule-bound starch synthase I activity within biphasic starch granules decreased slightly. The IE experienced more severe hypoxic stress than OE, and the up-regulated anaerobic respiration pathway indicated an increase in carbon consumption. The starch in IE underwent the SBEIIb-defective effect from weak to strong due to the lack of sufficient carbon inflow, leading to the formation of biphasic starch granules and their regional distribution in endosperm. The results provided information for understanding the biphasic starch granules.

Overexpression of HvCslF6 in barley grain alters carbohydrate partitioning plus transfer tissue and endosperm development.

In cereal grain, sucrose is converted into storage carbohydrates: mainly starch, fructan, and mixed-linkage (1,3;1,4)-β-glucan (MLG). Previously, endosperm-specific overexpression of the HvCslF6 gene in hull-less barley was shown to result in high MLG and low starch content in mature grains. Morphological changes included inwardly elongated aleurone cells, irregular cell shapes of peripheral endosperm, and smaller starch granules of starchy endosperm. Here we explored the physiological basis for these defects by investigating how changes in carbohydrate composition of developing grain impact mature grain morphology. Augmented MLG coincided with increased levels of soluble carbohydrates in the cavity and endosperm at the storage phase. Transcript levels of genes relating to cell wall, starch, sucrose, and fructan metabolism were perturbed in all tissues. The cell walls of endosperm transfer cells (ETCs) in transgenic grain were thinner and showed reduced mannan labelling relative to the wild type. At the early storage phase, ruptures of the non-uniformly developed ETCs and disorganization of adjacent endosperm cells were observed. Soluble sugars accumulated in the developing grain cavity, suggesting a disturbance of carbohydrate flow from the cavity towards the endosperm, resulting in a shrunken mature grain phenotype. Our findings demonstrate the importance of regulating carbohydrate partitioning in maintenance of grain cellularization and filling processes.

Contributions of Three Starch Branching Enzyme Isozymes to the Fine Structure of Amylopectin in Rice Endosperm.

Three starch branching enzyme (BE) isozymes, BEI, BEIIa, and BEIIb, are involved in starch biosynthesis in rice endosperm. Past in vivo and in vitro studies have suggested that each BE isozyme plays a distinct role in forming the fine structure of amylopectin. To elucidate more details of their roles, we prepared DNA constructs in which all the possible combinations of the expressions of these three isozymes were suppressed in developing rice endosperm. Analysis of the chain-length distributions of amylopectin produced under these various conditions confirmed the contributions of the individual BE isozymes to the fine structure of amylopectin in rice endosperm. Among these isozymes, the impact of loss of BEIIb activity on amylopectin fine structure was most remarkable and indicated that it plays a specific role in the synthesis of short chains with a 6–13 degree of polymerization (DP). The contribution of BEI to the amylopectin synthesis was unclear when only BEI activity was reduced. It was clear, however, when both BEI and BEIIb activities were substantially inhibited. The DP11-22 intermediate chains were markedly reduced in the ΔBEI/BEIIb line compared with the ΔBEIIb line, indicating that BEI plays a distinct role in the synthesis of these intermediate chains. Although no substantial change in amylopectin chain profile was detected in the ΔBEIIa line, the role of BEIIa could be deciphered by analyzing amylopectin fine structure from the ΔBEI/BEIIa/BEIIb line in comparison to that from ΔBEI/BEIIb line. This strongly suggests that BEIIa compensates for the role of BEI, rather than that of BEIIb, by forming intermediate chains of DP11-22. In addition, the new possibility that BEIIa is involved in the formation of starch granules in rice endosperm was suggested because the onset temperature for gelatinization of starch granules in the ΔBEIIa/BEIIb line was significantly higher than that in the ΔBEIIb line. In summary, the present study highlights the distinct roles of BEI, BEIIa, and BEIIb in the synthesis of amylopectin in developing rice endosperm.

Using RT-qPCR, Proteomics, and Microscopy to Unravel the Spatio-Temporal Expression and Subcellular Localization of Hordoindolines Across Development in Barley Endosperm.

Hordeum vulgare (barley) hordoindolines (HINs), HINa, HINb1, and HINb2, are orthologous proteins of wheat puroindolines (PINs) that are small, basic, cysteine-rich seed-specific proteins and responsible for grain hardness. Grain hardness is, next to its protein content, a major quality trait. In barley, HINb is most highly expressed in the mid-stage developed endosperm and is associated with both major endosperm texture and grain hardness. However, data required to understand the spatio-temporal dynamics of HIN transcripts and HIN protein regulation during grain filling processes are missing. Using reverse transcription quantitative PCR (RT-qPCR) and proteomics, we analyzed HIN transcript and HIN protein abundance from whole seeds (WSs) at four [6 days after pollination (dap), 10, 12, and ≥20 dap] as well as from aleurone, subaleurone, and starchy endosperm at two (12 and ≥20 dap) developmental stages. At the WS level, results from RT-qPCR, proteomics, and western blot showed a continuous increase of HIN transcript and HIN protein abundance across these four developmental stages. Miroscopic studies revealed HIN localization mainly at the vacuolar membrane in the aleurone, at protein bodies (PBs) in subaleurone and at the periphery of starch granules in the starchy endosperm. Laser microdissetion (LMD) proteomic analyses identified HINb2 as the most prominent HIN protein in starchy endosperm at ≥20 dap. Additionally, our quantification data revealed a poor correlation between transcript and protein levels of HINs in subaleurone during development. Here, we correlated data achieved by RT-qPCR, proteomics, and microscopy that reveal different expression and localization pattern of HINs in each layer during barley endosperm development. This indicates a contribution of each tissue to the regulation of HINs during grain filling. The effect of the high protein abundance of HINs in the starchy endosperm and their localization at the periphery of starch granules at late development stages at the cereal-based end-product quality is discussed. Understanding the spatio-temporal regulated HINs is essential to improve barley quality traits for high end-product quality, as hard texture of the barley grain is regulated by the ratio between HINb/HINa.

Waterlogging and simulated acid rain after anthesis deteriorate starch quality in wheat grain

The synthesis and accumulation of starch is greatly affected by environmental stress. Wheat grown in the downstream area of the Yangtze River is easily subjected to stress of waterlogging and acid rain. In order to probe the effect of waterlogging and acid rain on yield and starch characteristic, we used winter wheat cultivars of Yangmai 16 (more resistant) and Wennong 17 (relatively sensitive) to sole stress of acid rain or waterlogging and to their combinations during grain filling. The responses of grain yield, the physiochemical properties and morphology of starch granules in endosperm to the stresses were investigated. Compared with CT (control), grain yield in Wennong 17 were significantly decreased by both pH 4.0 acid rain and pH 2.5 acid rain, while in Yangmai 16 only by pH 2.5 acid rain. Waterlogging combined with acid rain significantly reduced grain weight and grain yield in both wheat cultivars. Acid rain, waterlogging and their combination depressed activities of ADP glucose pyrophosphorylase and soluble starch synthase (SSS) in grains of both cultivars. Acid rain and waterlogging damaged endosperm cell structure and caused abnormal starch granules. Starch granules at maturity became fragile and failed to keep in shape and some granules were even totally submerged in the protein matrix and other tissue under acid rain and waterlogging. Content of amylopectin and amylopectin/amylose ratio was also decreased, while content of amylose was increased under acid rain and waterlogging. As a result, swelling power and most viscosity parameters decreased, while the pasting temperature increased in both cultivars due to stresses. In sum, acid rain and waterlogging and their combination damaged cell structure and depressed synthesis of amylopectin, and led to the formation of abnormal fragile starch granules, and finally reduced grain weight and then yield, and deteriorated starch quality.