Dynamic transcript abundance during cellularisation of the developing barley endosperm: Temporal regulation of cell wall synthesis
ABSTRACT: Grain development in the Poaceae defines important end-use properties such as yield, quality and nutritive value. Microarray analyses have been performed on barley grain endosperm extracts from three to eight days after pollination (DAP), when cellularization of the syncytium occurs through the growth of cell walls around individual nuclei. Profiling of transcripts differentially expressed over time reveal 56 specific modules of genes that cluster into 15 groups. Expression patterns have been superimposed upon microscopy data, which identify the timing of key stages in grain development. Thus, cellularization is complete at six DAP, aleurone-related genes can be detected at seven to eight DAP, and starch synthase and hordein genes increase dramatically at seven and eight DAP, respectively. Genes known to be involved in cell wall metabolism are found predominantly in a single module, but analysis using a gene ontology approach splits these genes into four modules, which remain in a single cluster. Transcript levels of the cell wall-related genes peak at seven DAP and the developmental patterns of genes involved in arabinoxylan and (1,3;1,4)-β-glucan synthesis are defined. The transcript data are publicly available (www.etc.) and can be used to interrogate co-expression and differential expression patterns for other groups of genes. In addition, the examination of transcription factor genes that are co-expressed in modules of genes involved in specific processes, such as aleurone differentiation, can be used to identify candidate genes for the control of those particular processes during barley grain development.
Project description:We used microarrays to detail the global programme of gene expression underlying cellularization and identified distinct classes of regulated genes during this process. Rice endosperm at 2 days after pollination(DAP) was selected for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain the endosperm expression profile at 2 DAP. To that end, we hand-selected endosperm according to morphological criteria at 2 DAP before cellularization.
Project description:Plant seeds prepare for germination already during seed maturation. We performed a detailed transcriptome analysis of barley grain maturation, desiccation and germination in two tissue fractions (endosperm/aleurone = e/a and embryo = em) using the Affymetrix barley1 chip. Experiment Overall Design: Barley developing and germinating seeds were harvested at different time points after flowering (developing) and imbibition (germinating). To further disseect the influence of different tissues, seeds were dissecte and tissues were analyzed individually.
Project description:Purple-grain wheat are caused by anthocyanin accumulation in the seed coat. The anthocyanin biosynthesis and accumulation were affected by light in purple-grain wheat. The spikes of purple-grain wheat Luozhen No.1 were bagged with four-layer Kraft paper bags after pollination. To identify genes involved in the anthocyanin biosynthesis, we sequenced two pericarp cDNA libraries, D20 (20 DAP) of shading treatment, and L20 (20 DAP) of untreated control using an Illumina HiSeqTM 2000.
Project description:Purple-grain wheat are caused by anthocyanin accumulation in the seed coat. But little is known about molecular mechanism of anthocyanin biosynthesis. The anthocyanin biosynthesis and accumulation were affected by light in purple-grain wheat. The spikes of purple-grain wheat Luozhen No.1 were bagged with four-layer Kraft paper bags after pollination. To identify genes involved in the anthocyanin biosynthesis, we sequenced four pericarp cDNA libraries, D15 (15 DAP), D20 (20 DAP) of shading treatment, and L15 (15 DAP), L20 (20 DAP) of untreated control using an Illumina HiSeqTM 2000. After quality control, raw reads are filtered into clean reads which will be aligned to the reference sequences. The alignment data is utilized to calculate distribution of reads on reference genes and mapping ratio, and proceed with downstream analysis including gene and isoform expression, deep analysis based on gene expression (PCA/correlation/screening differentially expressed genes and so on),exon expression, gene structure refinement, alternative splicing, novel transcript prediction and annotation, SNP detection, Indel detection. Further, we also perform deep analysis based on different expression genes, including Gene Ontology (GO) enrichment analysis, Pathway enrichment analysis, cluster analysis, and finding transcriptor factor.
Project description:Eight tissues of cultivar Morex (three biological replications each) earmarking stages of the barley life cycle from germinating grain to maturing caryopsis were selected for deep RNA sequencing (RNA-seq)
Project description:The objective of the current study is to unravel the gene regulatory networks controlled by the nkd genes during maize endosperm development. We compared wild type (B73) vs. nkd mutant (introgressed into B73 background) transcriptomes in aleurone vs. starchy endosperm cell types captured by laser capture microdissection technology. We performed RNA seq analysis of mid-mature (15DAP) endosperm in two cell types [aleurone (A) and starchy endosperm (S)] of wild type B73 (B) and nkd mutant (N) kernels with three independent biological replicates.
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.
Project description:To summarize the impact of high temperature on rice grain filling, we performed the rice 44k oligo microarray analysis. Total RNA was extracted from developing caryopses ripened under 33˚C/28˚C (high temperature) and 25˚C/20˚C (Control), and subjected to 44k oligo-DNA microarray with 3 biological replicates.
Project description:Nuclear proteins of developing wheat grains collected during the cellularization, effective grain-filling and maturation phases of development were analysed.. Nuclear proteins were extracted and separated by two-dimensional gel electrophoresis. Image analysis revealed 371 and 299 reproducible spots in gels with first dimension separation along pH 4-7 and pH 6-11 isoelectric gradients, respectively. The relative abundance of 464 (67%) protein spots changed during grain development. Abundance profiles of these proteins clustered in six groups associated with the major phases and phase transitions of grain development. Using nano liquid chromatography-tandem mass spectrometry to analyse 387 variant and non-variant protein spots, 114 different proteins were identified that were classified into 16 functional classes. We noted that some proteins involved in the regulation of transcription, like HMG1/2-like protein and histone deacetylase HDAC2, were most abundant before the phase transition from cellularization to grain-filling, suggesting that major transcriptional changes occur during this key developmental phase. The maturation period was characterized by high relative abundance of proteins involved in ribosome biogenesis.
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. Overall design: Aleurone layer mRNA profiles of 8 DAF ZH11 and OsNF-YB1 RNAi plants were generated by deep sequencing, using Illumina Hiseq 2500 (Oryza sativa).