Project description:PHS1 is a plastidial α-glucan phosphorylase that can elongate and degrade maltooligosaccharides (MOS), but its exact physiological role in plants is poorly understood. Here, we discover a specialised role of PHS1 in establishing the unique bimodal characteristic of starch granules in the wheat 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 BGC1 – a carbohydrate-binding protein essential for normal B-type granule initiation. Mutants of tetraploid durum wheat deficient in all homeologs of PHS1 had normal A-type granules, but fewer and larger B-type granules. Further, using a double mutant defective in both PHS1 and BGC1, we show that PHS1 is exclusively involved in B-type granule initiation. Grain size and starch content were not affected by the mutations. In leaves, the total starch content and number of starch granules per chloroplast 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 occur by distinct biochemical mechanisms, where PHS1 plays an exclusive role in B-type granule initiation.
Project description:Transcriptome of starchy endosperm of hexaploid wheat var. Cadenza at 5 stages during grain-fill. This provides a reference set of all genes which are expressed in this single cell type during development which is of huge importance for human nutrition and for industrial uses of wheat grain. Here we focus on genes in glycosyl transferase and glycosyl hydrolase families which are responsible for the non-starch polysaccharide composition of wheat flour.
Project description:Plant-based adhesives, such as the ones made from wheat, have been prominently used for books and paper-based objects and are also used as conservation adhesives. Starch paste originates from starch granules, whereas flour paste encompasses the entire wheat endosperm proteome, offering strong adhesive properties due to gluten proteins. From the conservation perspective, understanding the precise nature of the adhesive is vital, as the longevity, resilience, and reaction to environmental changes can differ substantially between starch and flour-based pastes. We devised a proteomics method to discern the protein content of these pastes. Protocols involved extracting soluble proteins using 0.5 M NaCl and 30 mM Tris-HCl solutions, then targeting insoluble proteins, such as gliadins and glutenins, with a buffer containing 7M urea, 2M thiourea, 4% CHAPS, 40 mM Tris, and 75 mM DTT. Flour paste's proteome is diverse (1942 proteins across 759 groups), contrasting with starch paste's predominant starch-associated protein makeup (218 proteins in 58 groups). Transformation into pastes reduces proteomes' complexity. Testing on historical bookbindings confirmed the use of flour-based glue, rich in gluten and serpins. High levels of deamidation were detected, particularly for glutamine residues, which can impact the solubility and stability of the glue over time.
Project description:Background: Waterlogging was one of the most serious abiotic stresses in wheat-growing regions of China. There were great differences in waterlogging tolerance among different wheat varieties, and the mechanism of waterlogging tolerance of wheat seeds during germination was unclear. Methods: In order to reveal the adaptability of wheat to waterlogging stress during germination, we analyzed the germination rate and anatomical structure of three wheat seeds, ‘Zhoumai 22’, ‘Bainong 207’ and ‘Bainong 607’. At the same time, Illumina sequencing technology was used to determine the transcriptome of these three wheat varieties during germination. Results: The results showed that there was no significant difference between the germination rate of ‘Bainong 207’ after 3 days of waterlogging treatment and that of the control seeds. However, under waterlogging stress, the degree of emulsification and degradation of endosperm cells was higher than that of the control treatment, and starch granules in endosperm were significantly reduced. Transcriptome data were obtained from seed samples (a total of 18 samples) of three wheat varieties under waterlogging and control treatment. A total of 2,775 differentially expressed genes (DEGs) were identified by comprehensive analysis. In addition, by analyzing the correlation between the expression levels of DEGs and seed germination rates in three wheat varieties under waterlogging stress, it was found that the relative expression levels of 563 and 398 genes were positively and negatively correlated with the germination rate of wheat seeds, respectively. The GO and KEGG analysis found that the difference in waterlogging tolerance of the three wheat varieties was related to the abundance of key genes involved in the glycolysis pathway, the starch and sucrose metabolism pathway, and the lactose metabolism pathway. The ethanol dehydrogenase (ADH) gene in the endosperm of ‘Bainong 607’ was immediately induced after a short period of waterlogging, and the energy provided by glycolysis pathway enabled the seeds of ‘Bainong 607’ to germinate as early as possible, while the expression level of AP2/ERF transcription factor was up-regulated to further enhance its waterlogging tolerance. Conclusions: In general, this study provided a deeper understanding of the mechanisms by which different wheat varieties respond to waterlogging stress during germination.
Project description: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 tounderstand 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 dap, 12 dap and ≥ 20 dap)) as well as from aleurone, subaleurone and starchy endosperm at two (12 dap 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 indicats 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 high 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.
Project description:The starch, acting as the major energy-producing component of the daily diet, is the main carbohydrate in mammal nutrition. However, the nutritional value of starch can vary widely depending upon its source and site of digestion. The distinct physiological responses were previously observed both in human and other mammals, but still little is known about the underlying mechanisms regarding the metabolic shifts due to the intake of various dietary starches. Here, we assessed the overall metabolic changes in weaned pigs induced by different dietary starch sources at the transcriptome level. Sixteen weaned pigs (DurocÃLandraceÃYorkshire) were selected and randomly allotted to diets containing either wheat (WH) or cassava (CA) starch as the energy source (n=8). We measured serum metabolites and hormones and generated transcriptional profiles of liver. 648 genes in liver were differentially expressed in response to dietary starch sources. Pathway analysis indicated that dietary starch sources altered both carbohydrate and lipid metabolism in liver. In contrast, CA may be more healthful as dietary energy source than WH by down-regulating lipogenesis and steroidogenesis in liver. Sixteen weaned pigs (DurocÃLandraceÃYorkshire) with an average initial body weight of 7.37±0.25 kg were selected and randomly allotted to two dietary treatments (either wheat or cassava starch as the energy source) for 21 d. At the end of the trial, the liver tissue were collected for transcriptome analysis using Agilent porcine microarrays.
Project description:We report the transcriptome profile of the developing maize endosperm of nacRNAi. The nacRNAi is the transgenic maize which knocks down the expression of both ZmNAC128 and ZmNAC130 in the developing endosperm.ZmNAC128 and ZmNAC130 are the two maize endosperm-specific NAC-type transcription factors. This study finds that nacRNAi has a broad effect on the accumulation of starch and protein by regulating their main synthetic genes.
Project description:OsbZIP58 is a regulator of starch synthesis in rice endosperm. T-DNA insertion null mutants of this gene showed a white belly phenotype indicating an altered starch composition and content. We further investigated how reduction of OsbZIP58 gene expression caused these changes by analyzing the transcriptomes in the immature endosperm at 9 DAF of the wild-type and osbzip58-1 mutants by microarray analysis.
Project description:Genetic imprinting is an epigenetic phenomenon that describes unequal expression of paternal and maternal alleles of a gene in sexually reproducing organisms including mammals and flowering plants. The function of imprinted genes was rarely reported. We report genome-wide analysis of gene expression, DNA methylation, and small RNAs in the rice endosperm and functional tests of five imprinted genes in seed development using CRISPR/Cas9 editing technology. We identified 162 maternally expressed genes(MEGs) and 95 paternally expressed genes (PEGs) in the rice endosperm, which were associated with miniature inverted-repeat transposable elements, imprinted differentially methylated loci, and some 21-22-siRNAs and lncRNAs. Remarkably, one-third of MEGs and nearly half of PEGs were associated with grain-yield quantitative trait loci and enriched in the endosperm-expressed genes. Disrupting two MEGs increased the amount of small starch granules and reduced grain size, weight, and embryo size, while mutating three PEGs reduced starch content and seed fertility. Our data support both MEGs and PEGs in rice are required for starch and nutrient accumulation, mediating offspring fitness and optimal seed size. This imprinting strategy provides potential means for improving grain yield of rice and other cereal crops.
Project description:OsbZIP58 is a regulator of starch synthesis in rice endosperm. T-DNA insertion null mutants of this gene showed a white belly phenotype indicating an altered starch composition and content. We further investigated how reduction of OsbZIP58 gene expression caused these changes by analyzing the transcriptomes in the immature endosperm at 9 DAF of the wild-type and osbzip58-1 mutants by microarray analysis. 9 DAF endosperm of Dongjin (wild type) and osbzip58-1 were used to compare the gene expresion, and three independent biological replicates for each material.