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:Transcription profiling by high throughput sequencing of wheat starchy endosperm, aleurone layer and transfer cells at three different developmental stages
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: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:Microbiota from rats fed with wheat aleurone and plant omega fatty acids In this study we investigated how an AX-rich WA and ALA from linseed oil (LO) modulate the gut microbiota of rats. Wistar rats were fed a standard diet and received either an iso-energetic control oil (PO), control oil + aleurone (A+PO), linseed oil (LO) or linseed oil + aleurone (A+LO) during 12 weeks. Feacal samples were recovered after the 12 week treatments. DNA extractions were performed using using the Qiagen's DNA Stool Kit (Qiagen, West Sussex, UK). 10ng of DNA template were amplified by PCR (16S gene) and purified using Qiagen's Qiaquick PCR purification kit (Qiagen, West Sussex, UK). 1ug of purified PCR product were labelled with either Cy3 or Cy5 using Genomic DNA ULS Labelling kit (Agilent Technologies, Palo Alto, CA). 250ng of labelled DNA were hybridized on the microarray for 24h at 65°C. Washings were performed as recommended by the manufacturer. Microarray scanning was performed on a Surescan Microarray scanner (Agilent Technologies, Palo Alto, CA). Data were extracted using the Feature extraction software (Agilent Technologies, Palo Alto, CA). The retained intensity value for each probe was the ratio between the spot’s median intensity signals and the median of background signals.
Project description:Microbiota from rats fed with wheat aleurone and plant omega fatty acids In this study we investigated how an AX-rich WA and ALA from linseed oil (LO) modulate the gut microbiota of rats. Wistar rats were fed a standard diet and received either an iso-energetic control oil (PO), control oil + aleurone (A+PO), linseed oil (LO) or linseed oil + aleurone (A+LO) during 12 weeks. Feacal samples were recovered after the 12 week treatments. DNA extractions were performed using using the Qiagen's DNA Stool Kit (Qiagen, West Sussex, UK). 10ng of DNA template were amplified by PCR (16S gene) and purified using Qiagen's Qiaquick PCR purification kit (Qiagen, West Sussex, UK). 1ug of purified PCR product were labelled with either Cy3 or Cy5 using Genomic DNA ULS Labelling kit (Agilent Technologies, Palo Alto, CA). 250ng of labelled DNA were hybridized on the microarray for 24h at 65M-BM-0C. Washings were performed as recommended by the manufacturer. Microarray scanning was performed on a Surescan Microarray scanner (Agilent Technologies, Palo Alto, CA). Data were extracted using the Feature extraction software (Agilent Technologies, Palo Alto, CA). The retained intensity value for each probe was the ratio between the spotM-bM-^@M-^Ys median intensity signals and the median of background signals. A 13 chip study was realized to analyze the feacal microbiota of rats treated with either an iso-energetic control oil (PO), control oil + aleurone (A+PO), linseed oil (LO) or linseed oil + aleurone (A+LO) during 12 weeks. Each microarray corresponding to hybridization with 250ng of labelled 16S rRNA gene amplicons from 2 rat DNA faecal samples. Microbiota structure and diversity were assessed using the HuGChip (Tottey et al., 2013). Each probe (4441) was synthetized in three replicates. On the same array, 2 different samples were hybridized. One labelled with the Cy3 dye and one with the Cy5 dye. The results were processed as single channel (13 raw data files available on Series records for 25 samples).
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.