Project description:Transcription profiling by high throughput sequencing of wheat starchy endosperm, aleurone layer and transfer cells at three different developmental stages
Project description:This reports the transcript profiling of the aleurone and starchy endosperm layers of wheat seed over 3 time points critical in the development of the aleurone layer. Wheat is a critical food source globally. The aleurone layer develops from the starchy endosperm and is a concentrated source of vitamins and minerals, essential for the germination of the plant embryo. However the molecular mechanisms behind the development of this layer remain poorly understood. Here we present the first direct systematic comparison of the transcriptomes of the aleurone and starchy endosperm tissues of the wheat seed (Triticum aestivum) at time points critical to the development of the aleurone layer of 6, 9 and 14 days post anthesis. Gene expression patterns reflect the changing role of these tissues in seed development. Illumina sequencing gave 25 to 55 million sequence reads per tissue, of the trimmed reads, 70 – 81% mapped to reference expressed sequence transcripts. To quantify transcript abundance, RNA-Seq normalisation was performed to generate RPKM values, these were used in comparative analyses between the tissues at each time point using Kals Z-test. Sequences with significantly different RPKM values were categorised on the basis of tissue and time point expression and functionally annotated using standardised gene ontology vocabularies, revealing two very distinct tissues. In conclusion we show the relationships between and the fundamental biological reprogramming of the two major biologically and economically significant tissues of the wheat seed over time. Understanding these changes in gene expression profiles is essential to mining the potential these tissues hold for human nutrition and contributing to foundational and systems biology of this important crop. Examines the transcript profile of aleurone and starchy endoperm tissues at 6,9 and 14DPA. A minimum of 5 individual seeds from 3 separate spikes from three individual plants were pooled for each tissue preparation.
Project description:This reports the transcript profiling of the aleurone and starchy endosperm layers of wheat seed over 3 time points critical in the development of the aleurone layer. Wheat is a critical food source globally. The aleurone layer develops from the starchy endosperm and is a concentrated source of vitamins and minerals, essential for the germination of the plant embryo. However the molecular mechanisms behind the development of this layer remain poorly understood. Here we present the first direct systematic comparison of the transcriptomes of the aleurone and starchy endosperm tissues of the wheat seed (Triticum aestivum) at time points critical to the development of the aleurone layer of 6, 9 and 14 days post anthesis. Gene expression patterns reflect the changing role of these tissues in seed development. Illumina sequencing gave 25 to 55 million sequence reads per tissue, of the trimmed reads, 70 – 81% mapped to reference expressed sequence transcripts. To quantify transcript abundance, RNA-Seq normalisation was performed to generate RPKM values, these were used in comparative analyses between the tissues at each time point using Kals Z-test. Sequences with significantly different RPKM values were categorised on the basis of tissue and time point expression and functionally annotated using standardised gene ontology vocabularies, revealing two very distinct tissues. In conclusion we show the relationships between and the fundamental biological reprogramming of the two major biologically and economically significant tissues of the wheat seed over time. Understanding these changes in gene expression profiles is essential to mining the potential these tissues hold for human nutrition and contributing to foundational and systems biology of this important crop.
Project description:Aleurone layer differentiation completes by 9 DAF, and most of the nutrient filling occurs between 6-21 DAF, (Wu et al., 2016a, 2016b), so we decided to use 10 DAF time point for collection of pericarp, aleurone, embryo and endosperm tissues for RNA-Seq analysis. Since aleurone is mostly single layer, and it’s almost impossible to separately collect pure pericarp and aleurone layers manually, so firstly we developed a method to collect different tissue types of developing grain through LCM and RNA extraction and Sequencing
Project description: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.
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.