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Title:Global transcriptional, post-transcriptional, and translational regulation during soybean seed and seedling development
Author(s):Shamimuzzaman, Md
Director of Research:Vodkin, Lila O.
Doctoral Committee Chair(s):Vodkin, Lila O.
Doctoral Committee Member(s):Huber, Steven; Briskin, Donald P.; Zielinski, Raymond E.
Department / Program:Crop Sciences
Discipline:Crop Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Ribosome profiling
Chromatin Immunoprecipitation Sequencing (ChIP-Seq)
Degradome sequencing
Transcription factors
Gene regulation
Soybean seed development
Abstract:The regulation of gene expression is maintained in a highly organized fashion to ensure specific genes are expressed at the appropriate levels during soybean seed and seedling developmental stages. To define the developmental shifts in gene expression and begin to understand the gene regulatory networks, high-throughput RNA sequencing (RNA-Seq) was carried out using cotyledons from seven developmental stages of soybean seedlings. A total of 154 genes demonstrated higher level of expression exclusively in the early seedling stages. About 25% of those genes with known annotations were involved in carbohydrate metabolism. A significant number (approximately 50%) of the highly expressed genes whose expression peaked in the mid-developmental stages encoded ribosomal family proteins. The analysis also identified 219 gene models with high expression at late developmental stages. The majority of these genes are involved in photosynthesis. The findings revealed approximately 460 transcription factors with notable expression in at least one stage of the developing soybean seedling. Relatively over-represented transcription factor genes encode AP2, zinc finger, NAC, WRKY and MYB families. Specific members of NAC and YABBY transcription factors showed a bell shaped expression pattern during soybean seedling development. The highest level of their expression was found in seedling developmental stage 4 when cotyledons undergo a physiological transition from non-photosynthetic storage tissue to a metabolically active photosynthetic tissue. In order to identify genome-wide binding sites of specific members of the NAC and YABBY transcription factors and co-regulated genes, Chromatin Immunoprecipitation Sequencing (ChIP-Seq) was performed using seedling cotyledons at stage 4 and 5. The ChIP-Seq data identified 72 genes potentially regulated by the NAC and 96 genes by the YABBY transcription factors examined. The RNA-Seq data revealed highly differentially expressed candidate genes regulated by the NAC transcription factor include lipoxygense, pectin methyl esterase inhibitor, DEAD/DEAH box helicase and homeobox associated proteins. YABBY-regulated genes include AP2 transcription factor, fatty acid desaturase and WRKY transcription factor. Additionally, DNA binding motifs were identified for the NAC and YABBY transcription factors. Post-transcriptional regulation is another layer of gene regulation. In order to dissect miRNA guided gene regulation in soybean developing seeds, a transcriptome-wide experimental method called degradome sequencing was carried out to directly detect cleaved miRNA targets. In this study, degradome libraries were constructed from immature soybean cotyledons representing three stages of development and from seed coats of two stages. Sequencing and analysis of 10 to 40 million reads from each library resulted in identification of 183 different targets for 53 known soybean miRNAs. Among these, some were found only in the cotyledons representing cleavage by 25 miRNAs and others were found only in the seed coats reflecting cleavage by 12 miRNAs. A large number of targets for 16 miRNAs families were identified in both tissues irrespective of the stage. Interestingly, more miRNA targets were found in the desiccating cotyledons of late seed maturation than in immature seed. Four different auxin response factor genes were validated as targets for gma-miR160 via RNA ligase mediated 5’ rapid amplification of cDNA ends (RLM-5’RACE). Gene Ontology (GO) analysis indicated the involvement of miRNA target genes in various cellular processes during seed development. To understand the translational regulation, ribosome profiling was performed in combination with RNA sequencing using soybean cotyledons and seed coats from different seed developmental stages. A total of 148 genes demonstrated higher translational efficiencies (TEs) during early stage. A number of genes annotated as cysteine and serine proteases showed higher translational efficiencies in cotyledons during early seed development. A specific group of zinc finger transcription factors demonstrated higher translational efficiencies in cotyledons during mid and late stages of seed development. Interestingly, no ribosome footprints were identified for 22 genes in all three developmental stages, although they have at least 10 RPKMs of mRNA in at least one developmental stage. Genes encoding seed storage proteins such as lectin, glycinin, beta-conglycinin showed similar expression patterns in cotyledons. The highest level of transcripts were found at the mid stage whereas the highest ribosome footprint level was found at the late developmental stage even though there is not much difference in translational efficiencies across development. The experimental findings suggest that the seed storage protein coding genes are primarily regulated at the transcriptional level. So, the multiple levels of gene regulation is very important in setting or maintaining the developmental program leading to high quality soybean seeds that are one of the dominant sources of protein and oil in world markets.
Issue Date:2015-03-10
Rights Information:Copyright 2015 Md Shamimuzzaman
Date Available in IDEALS:2015-07-22
Date Deposited:May 2015

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