| Heat, drought and their combination dramatically reduce wheat yield and quality, but the molecular mechanisms underlying wheat tolerance to extreme environments are largely unknown. To explore the transcriptional response to the individual and combined stress, we performed high-throughput transcriptome sequencing of wheat seedling subjected to drought stress (DS), heat stress (HS) and their combination (HD) after1h and6h, and presented a global gene expression reprograms in response to these three environmental stresses. Additionally, to investigate the association of heat tolerance between wheat seedling stage and adult stage, seedling leaf and flag leaf of wheat cultivar TAM107were subjected to heat stress for1h and transcriptome analysis was performed by using Affymetrix microarray platform. Specifically, main results are as follows:1. DEG (Differential Expression Genes) analysis showd that although most of (-64-83%) the differentially expressed genes were commonly responsive to DS, HS and HD,1738(16%of up-regulated genes) and2,482(23%of down-regulated genes) genes exhibited specific response to HD. Further GO (Gene Ontology) enrichment analysis confirmed our observation that, abiotic and biotic stress related pathways such as "response to water deprivation","heat acclimation","response to wounding" and "defense response to bacterium" were significantly enriched in common up-regulated genes of DS, HS and HD, and plant development and primary metabolism related pathways such as "leaf morphogenesis","photosynthesis","regulation of cell size" were enriched in common down-regulated genes.In contrast, two distinct functional categories of HD specifically up-regulated genes exhibited significantly higher enrichments compared to the individual stress, namely RNA processing and epigenetic regulation (e.g."methylation dependent chromatin silencing","maintenance of DNA methylation ","chromatin assembly or disassembly ","histone modification").2. Totally,4,375transcription factor genes (TFs) were identified from wheat genome by using domain searching method, distributing among51gene families. Among which,1,328were differentially expressed under DS, HS and HD, and seven largest TF families accounted for approximately half of stress responsive TF genes, including FAR1, NAC, bZIP, bHLH, AP2/ERF, WRKY, Myb-related and Myb. The differentially expressed TFs can be classified into20clusters based on their expression patterns under DS, HS and HD, which included common or specific responsive TF clusters and TFs responsive at different time point after stress treatments. Furthermore, by using cis-element and co-expression analysis,1,503and564target genes of HSFs and DREBs were predicted respectively and transcriptional regulation networks of the two types of TFs were analyzed. Function annotation and enrichment analysis of target genes showed that, some stress response related pathways (e.g. cold, salt, drought and wound response, ABA responsive pathway) were regulated by both types of TFs, and also, some pathways were specifically regulated by HSFs or DREBs. Additionally, some important TFs (e.g. RAP2.4^STZ and HB-7) involved in plant heat and drought response were included in the target genes of HSFs and DREBs.3. We developed a new algrathm for quantitative analysis of homeologous gene expression in wheat based on high through-put RNA-Seq and genome information released by IWGSC. And performed an expression partitioning analysis of wheat homeologous genes under DS, HS and HD on a whole-genome scale. The confidence of our new algorithm was validated by both simulation test and quantitative RT-PCR. The results of homeologous genes analysis showed that (1) about42and55%homologous genes in wheat seedling leaf displayed significant expression partitioning under normal and stress condition respectively;(2) the proportion of partitioned expressed homologous genes increased when stress applied more severely, e.g. stressed> unstressed, combined stress> single stress,6h>1h;(3) A, B and D homeologs of27genes were identified showing different expression patterns under different type of stresses (DS, HS or HD);(4) A, B and D homeologs of5,17and21genes were identified showing different expression patterns at different time (1h or6h) after DS, HS and HD, respectively;(5) clustering analysis showed that the overall gene expression patterns of A-and D-subgenome shared more similarity, and B-subgenome was distinct from them.4. We performed comparative transcriptome analysis of wheat seedling leaf and flag leaf subjected to heat stress, and observed that most (-87.5%) heat responsive genes showed consistent expression trends in the two growth stages. Correspondingly, Gene Ontology enrichment analysis of heat responsive genes in seedling leaf and flag leaf represented similar functional categories between each other, enriched mainly in "response to heat","response to tempreture stimulus" and "protein folding" etc. which suggested heat tolerance of adult stage was associated with seedling stage in wheat. |
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