RNA-seq analysis of NIL-GAF1 and NIL-gaf1 grains of 15 DAF at filling stage
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ABSTRACT: Favorable grain filling ability is crucial for seed development and plant yield1, with less fertilizer applying is the most urgent goals to meet the growing demands of green and safe food. However, the balance mechanism between grain filling and nutrient elements is still unclear so far. Here, we describe a gene GAF1, specially expressed in endosperm aleurone layer, encoding a phosphate transporter, positively controls rice grain filling and seed development and also contributes to phosphate balance was mapped in our study. To study the regulation pattern of GAF1, we performed the RNA-seq analysis of NIL-GAF1 and NIL-gaf1 at middle grain filling stage in seeds. The data shows that many pathways related to starch and sugar metabolism were enriched, and many starch synthesis related genes and phosphate response genes were up-regulated or down-regulated. These data further support that seed specific expressed GAF1 plays a key role in regulating both phosphate homeostasis and seed development. More importantly, overexpression of GAF1 can significantly improve grain filling and thus yield enhanced plant production in field.
Project description:Grain development is a key life cycle stage of many plants. The development of seeds is the basis of agriculture and the primary source of calories consumed by humans. Here, we employ laser micro dissection (LMD) combined with shotgun proteomics to generate a cell-type proteome atlas of developing wheat endosperm at the early and late grain filling stages. We identified 1803 proteins from four different cell layers (aleurone (AL), sub-aleurone (SA), starchy endosperm (SE), and endosperm transfer cells (ETCs)) of developing endosperm at 15 Days after anthesis (DAA) and 26 DAA. Sixty-seven differentially expressed proteins in the aleurone, 31 in the sub-aleurone, 27 in the starchy endosperm, and 50 in the endosperm transfer cells were detected between these two-time points. The results revealed highly distinguishable proteome dynamics in the different cell layers of endosperm over the time course. We observed high general metabolic activity of the grain with regard to carbohydrate metabolism, defence against oxidative stress, and signalling in the different cell layers during the grain filling process. Cell-specific identification of SUT and GLUT transporters suggest a grain filling model via nucellar projections and endosperm transfer cells (ETCs) initiating starch biosynthesis in the starchy endosperm (SE). The identification and regulation dynamics of proteins in the different cell layers demonstrate a functional switch of the proteome from the early to the late grain filling stage. Based on these data, we proposed a model for sugar loading and starch biosynthesis in wheat developing endosperm, including an abundance switch of cell-type-specific key proteins.
Project description:Grain filling and proper grain development are essential biological processes in the plant’s life cycle, which majorly contributes to the final seed yield and quality in all cereal crop. However, very scarcely this knowledge is available in the literature regarding how the different wheat grain components contribute to the overall development of the seed. We performed a proteomics and metabolomics analysis in four different developing components of the wheat grain (seed coat, embryo, endosperm and cavity fluid) to characterize molecular processes during early and late grain development. In-gel shotgun proteomics analysis in 12, 15, 20 and 25 days after anthesis (DAA) lead us to identify and quantify 15,484 proteins out of which 410 differentially expressed proteins (DEPs) were identified in the seed coat, 815 in embryo, 372 in endosperm and 492 in cavity fluid. The abundance of selected protein candidates revealed spatially and temporally resolved protein functions associated with development and grain filling. Multiple proteins such as pyruvate phosphate dikinase (PPDK) and 14 -3- 3 undergo a major change in abundance during wheat grain development. Proteins binned into the functional category of cell growth /division were highly expressed during early stages (12 and 15 DAA) whereas those of starch biosynthesis in the middle or late stages. At the metabolome level all tissues and especially the cavity fluid showed highly distinct metabolite profiles. The tissue specific data are integrated with biochemical networks to explore a comprehensive map of molecular processes during grain filling and developmental processes.
Project description:We study the expression level change of candidate genes associated with seed filling in maize, through RNA-seq for maize seeds in different filling stages. The results show that the change of the expression of these candidate genes would lead to the change of grain filling trajectory.
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:Tissue-specific transcriptional profiling of the abscission layer (AL) at the base of young flower in rice using laser micro-dissection: NIL(qSH1) vs. Nipponbare. We used two rice varieties, NIL(qSH1) and Nipponbare. NIL(qSH1) is a nearly isogenic line containing the seed shattering gene qSH1. Seed shattering is easy in NIL(qSH1), but it is not in Nipponbare. So, we used some stages of young flower in NIL(qSH1) and some in Nipponbare. Four regions: 1. abscission layer region of NIL(qSH1), 2. upper abscission region of NIL(qSH1), 3. lower abscission layer region of NIL(qSH1), and 4. abscission layer region of Nipponbare. Sample experiments: NIL(qSH1) AL vs. Nipponbare AL, NIL(qSH1) AL vs. NIL(qSH1) upper region of AL, and NIL(qSH1) AL vs. NIL(qSH1) lower region of AL.
Project description:RNAi mediated suppression of MADS29 severely affects seed set; the surviving seeds are smaller in size with reduced grain filling, abnormal starch grains and aberrant embryo development. To identify the affected pathways due to suppression of this transcription factor in the transgenic seeds, transcriptome analysis using microarray was carried out.
Project description:Analysis of gene expression level. The hypothesis tested in the present study was that OsNF-YB1 is specifically expressed in aleurone layer of developing endosperm and suppressed expression of OsNF-YB1 results in a reduced grain-filling rate and small grains. Of the down-regulated genes, the enrichment of transmembrane transport and ATP biosynthetic process is consistent with the decreased grain-filling rate.
Project description:OsNF-YB1 is a transcription factor that plays important roles during rice grain filling. OsNF-YB1 is specifically expressed in aleurone layer of developing endosperm and OsNF-YB1 RNAi lines showed retardation in grain filling and produced small grains with chalky endosperm as well as the altered starch quality. To reveal the transcriptional regulatory framework of OsNF-YB1, we determined OsNF-YB1 DNA binding targets using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq). ChIP-Seq analysis detected 933 binding peaks distributed 743 neighbor genes. OsNF-YB1 directly regulates genes involved in nutrient transport including sugar and amino acids, and interestingly, different from the reported binding site of NF-Y complex, the GCC-box (a binding motif of ERF transcription factors) was enriched in the binding peaks of OsNF-YB1.