Unknown,Transcriptomics,Genomics,Proteomics

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Expression data from microdissected endosperm transfer cells (ETCs) of barley seeds during different developmental stages

ABSTRACT: Endosperm transfer cells (ETCs) of barley grains/seeds are located at the maternal-filial boundary in seeds and fascilitate nutrient transfer from the mother plant into the filial endosperm tissue. Due to the difficult accessibility of ETCs inside the seed, signalling and metabolic pathways involved in differentiation processes of ETCs are poorly understood. To analyse ETC differentiation, we applied laser microdissection pressure catapulting (LMPC) coupled to transcriptome analysis at various developmental stages from cellularization to full functionality and ongoing modifications. ETC-specific transcriptome data identified candidate genes and associated pathways involved in distinct differentiation processes. Barley grains were harvested at 5, 7, 10 and 12 days after flowering/pollination (DAF) and immediately frozen in liquid nitrogen. In a cryostat (-20M-BM-0C), middle parts were cut by a razor blade, glued onto sample plates by Tissue-TekM-BM-. O.C.TM-bM-^DM-" and 20 M-BM-5m sections were mounted on PEN membrane slides (PALM, Bernried, Germany). Cryosections were stored for 7 days at -20M-BM-0C until complete dryness. LMPC-based isolation of ETC tissue from cryosections has been done using the PALMM-BM-. MicroBeam laser system. For each biological replicate, 50-70 sections from three independent grains were collected. Three biological replicates were generated from each time point (total number of samples = 12).

ORGANISM(S): Hordeum vulgare

SUBMITTER: Johannes Thiel

PROVIDER: E-GEOD-35639 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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.

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Expression data from microdissected endosperm transfer cells (ETCs) of barley seeds during different developmental stages

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