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Analysis of the protein distribution within the wheat endosperm

ABSTRACT: Starchy endosperm proteins determine wheat quality and exhibit, besides a quantitative protein gradient, a qualitative protein gradient from the outer to inner starchy endosperm. The goal was to investigate the relative differences in protein composition between the aleurone, sub-aleurone and inner endosperm. Using laser microdissection followed by nanoLC-MS/MS, an innovative method combining a high spatial specificity and analytical selectivity in sample-limited situations, 780 proteins were detected and classified by function. Relatively more gluten proteins were detected in the sub-aleurone compared to inner endosperm. Gluten composition-wise, the sub-aleurone is relatively more enriched in ω-gliadins, but impoverished in LMW-GS and γ-gliadins. While a basic set of albumins and globulins is detected across the entire endosperm, some proteins, like puroindoline-B, display an increasing (or decreasing) gradient. Histological origin and relative positioning of the endosperm cells are hypothesized to drive the protein gradient. Knowledge on this gradient provides major opportunities for the wheat manufacturing industry.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Triticum Aestivum (wheat)

TISSUE(S): Endosperm

SUBMITTER: Sabrina Geisslitz

LAB HEAD: Katharina Anne Scherf

PROVIDER: PXD038743 | Pride | 2023-12-15

REPOSITORIES: Pride

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Project description:In species with exalbuminous seeds such as crucifer oilseeds and legumes, the endosperm is eventually consumed and its space occupied by the embryo during seed development. However, the main constituent of the early developing seed is the liquid endosperm, and most of the carbon resources for the ensuing stages of seed development arrive at the embryo through the endosperm. In contrast to the extensive study of species with persistent endosperm, little is known about the global gene expression pattern in the endosperm of exalbuminous seed species. We took a multiparallel approach that combines ESTs, protein profiling and microarray analyses to look into the gene expression landscape in the endosperm of the oilseed crop Brassica napus. An EST collection of over 30,000 entries allowed us to detect close to 10,000 unisequences expressed in the endosperm. A protein profile analysis of more than 800 proteins corroborated several signature pathways uncovered by abundant ESTs. Using microarray analyses, we identified genes that are differentially or highly expressed across all developmental stages. These complementary analyses provided insight on several prominent metabolic pathways in the endosperm. We also discovered that LEC1 was highly expressed in the endosperm and that the regulatory cascade downstream of LEC1 operates in the endosperm. The endosperm EST collection and the microarray dataset provide a basic genomic resource for dissecting metabolic and developmental events important for oilseed improvement. Our findings on the featured metabolic processes and the LEC1 regulatory cascade offer new angles for investigation on the integration of endosperm gene expression with embryo development and storage product deposition in seed development. Keywords: seed development We defined the developmental phase of the endosperm based on the stages of the embedded embryos. Under our growth conditions, the globular-shape embryo stage is reached approximately six days after flowering (DAF), the heart-shape embryo stage is reached after eight days, and by day 14 the seed enters the cotyledon stage. The collected tissues allowed the construction of two cDNA libraries: one derived from endosperm of heart-shape embryo developing stage seeds and the other from endosperm of a mix of globular-shape embryo and cotyledon stage developing seeds. Heart-shaped-embryo endosperm is also ready for protein profiling analysis. For microarray analysis, endosperms were harvested from young seeds at the globular-shape embryo, heart-shape embryo and cotyledon stages, respectively. Two biological duplicates were set up, including four technological repeats in each duplicate. A dye-swap hybridization experiment was performed for each pair of target RNA comparisons. We performed four independent aRNA labelings for each tissue pair. For example, we used globular-shape embryo stage endosperm RNA coupled with Cy5 dye vs. heart-shape embryo stage endosperm RNA coupled with Cy3 dye, and the dye-swap experiment was with globular-shape embryo stage endosperm RNA with Cy3 dye vs. the heart-shape embryo stage endosperm RNA with Cy5 dye. Each tissue pair experiment was done with four repeats, using a total of 24 microarrays for the whole experiment.

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Analysis of the protein distribution within the wheat endosperm

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