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RNA-Seq Analysis of Laser-Capture Microdissected Cells of the Developing Central Starchy Endosperm of Maize

ABSTRACT: Endosperm is a product of double fertilization, and provides nutrients and signals to the embryo during seed development in flowering plants. Early stages of endosperm development are critical for the development of its storage capacity through synthesis and accumulation of starch and storage proteins. Here we report on the isolation and sequencing of mRNAs from the central portion of the starchy endosperm of Zea mays (maize) B73 at 6 days after pollination. We detected high correlation among the four biological replicates of RNAs isolated using laser-capture microdissection of the cell type. Because the assayed stage of development precedes the synthesis and accumulation of the major storage proteins and starch in the endosperm, our dataset likely include mRNAs for genes that are involved in control and establishment of these developmental programs.

ORGANISM(S): Zea mays

PROVIDER: GSE58504 | GEO | 2014/06/17

SECONDARY ACCESSION(S): PRJNA252770

REPOSITORIES: GEO

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Project description:Barley (Hordeum vulgare) is one of the major food sources for humans and forage source for animal livestock. Barley endosperm is structured into three distinct cell layers: the starchy endosperm, which acts essentially as storage tissue for starch, the subaleurone, which is characterized by a high accumulation of endoplasmic reticulum (ER)-derived seed storage proteins (SSP) and finally the aleurone beside the seed coat with a prominent role during seed germination. Prolamins account for more than 50% of the total protein amount in mature seeds. Together with other seed storage proteins (SSPs) they are important for both grain quality and flour quality. Prolamins are synthesized on the rough ER, translocated into the ER lumen and accumulate in distinct, ER-derived protein bodies (PBs) that are most abundant in the SE. PB formation is regulated by the protein disulfide isomerase (PDI) that is involved in the disulfide transfer pathway. Here, we used laser microdisection (LMD) to characterize spatio-temporal molecular and morphological differences of the ER during barley endosperm development. We revealed by nanoLC-MS/MS proteomic analyses performed on whole seeds and collected tissues at different seed development stages that the protein level of the protein disulfide isomerase HvPDIL1-1 is spatio-temporally regulated in developing barley endosperm. Our microscopic studies showed that HvPDIL1-1 preferentially accumulates in SE, especially at 12 days after pollination (dap). HvPDIL1-1 re-localized from PBs to the protein matrix at the periphery of starch granules along grain filling process. Detailed analysis of SE proteome dynamics identified clusters of proteins with similar expression pattern as HvPDIL1-1, which were analysed in a protein-protein network. It revealed a strong functional interconnection between transcription and translation, protein folding and amino acid synthesis with sucrose and starch metabolism. Our data indicate a role of HvPDIL1-1 in the coordination of protein synthesis and prolamins deposition during grain filling processes in developing barley endosperm. These results are discussed in relation to the putative role of HvPDIL1-1 for cereal food end-product quality and recombinant protein production in cereal seeds.

Project description:Heat stress occurrence during endosperm development and seed filling forms chalky portion in the limited zone of starchy endosperm of rice grains. In this study, isolation of aleurone, dorsal, central and lateral tissues of developing endosperm by laser-microdissection (LM) coupled with gene expression analysis of 44K microarray was performed to identify key regulatory genes involved in the formation of milky-white (MW) and white-back (WB) chalky grains during heat stress. Gene regulatory network analysis classified the genes changed under heat stress into five modules. the modules of genes changed in developing starchy endosperm corresponding to MW and WB portion under heat stress suggested different regulatory genes involved in each type of grain chalk. The most distinct expression pattern was observed in a M1 and M2 modules where most of the small heat shock proteins and cellular organisation genes being upregulated under heat stress in dorsal aleurone cells and dorsal starchy endosperm zones The histological observation supported the significant increase in cell number and size of dorsal aleurone cells in WB grains. With regard to the central zone of starchy endosperm, preferential downregulation of high molecular weight heat shock proteins (HMW HSPs) including a prominent member encoding for endoplasmic reticulum (ER) chaperones by heat stress were observed, while expression of starch-biosynthesis genes remained unaffected. Characterization of transgenic plants supressing endosperm lumenal binding protein (BiP1), an ER chaperone preferentially downregulated at MW portion under heat stress, showed an evidence of forming the chalky grains without disturbing the expression of starch-biosynthesis genes. The present LM-based comprehensive expression analyses provides novel inferences that HMW HSPs play important role in controlling the redox, nitrogen and amino aicd metabolism in endosperm leading to the formation of MW and WB chalky grains.Keywords ; developing endosperm, gene expression, grain chalkiness, heat stress, laser-microdissection, Oryza sativa.

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RNA-Seq Analysis of Laser-Capture Microdissected Cells of the Developing Central Starchy Endosperm of Maize

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