Proteomics Analysis Of Flag Leaves In Response To Heat Stress And Genome Wide Analysis Of The Important Heat Responsive Gene,HSP90,During Grain Filling Stage In Wheat

High temperature is frequent during grain filling stage of wheat,and causes severe grain yield and quality penalty.Thus,unveiling the molecular mechanism of wheat thermotolerance becomes an urgent task.In this study,the wheat variety,“Chinese Spring” is subjected to 37 °C for 3 days at 15 days after flowering,and then an iTRAQ-based proteomics analysis of flag leaves is performed to explore the global proteome changes,combined with physiological studies.In proteomics analysis,many chaperones are accumulated especially heat shock proteins(HSPs),thus,we perform a comprehensive analysis of the 90-kDa HSPs(HSP90s)and their corresponding transcript isoforms to explore the how HSP90 s in response to heat,to provide useful information for further functional characterization.The results are as following.1)258 heat responsive proteins are characterized in the proteomics analysis of wheat flag leaves under heat stress.Functional annotation and enrichment analysis show that the heat responsive proteins are mainly participated in processes related to protein synthesis and turnover,stress response,redox regulation,and photosynthesis.In heat stress,protein translation is not only inhibited at initiation but also at elongation stage,however,molecular chaperones particularly HSPs are significantly accumulated,thus the wheat leaf is preferentially protected existing proteins by chaperones.Heat stress also causes oxidative stress and results in severe membrane damage,interestingly,an unusual redox regulation system in which the glutathione S-tranferases and thioredoxins are the major players is activated in wheat flag leaves under heat stress.2)In photosynthesis process,besides the already known heat suppressed Calvin cycle,the proteomics results also reveals that biosynthesis of protochlorophyll,precursor of chlorophyll,is impeded in heat stress.Consistently,contents of chlorophyll a and chlorophyll b are both reduced under in heat-stressed leaves,suggesting that their common precursor biosynthesis is inhibited.Further qRT-PCR analysis of the chlorophyll biosynthesis and photosynthesis related genes are performed,when 10-days old wheat seedling are subjected to temperature rigimes of 24 °C,37 °C,40 °C,these genes are quickly inhibited,and the inhibition is severer under either longer stress duration or higher temperature condition.However,the rapid downregulation of genes involved in chlorophyll precursor synthesis does not result in substantial chlorophyll loss until 4-5 days later.These results demonstrate an accumulated effect of heat stress.3)The wheat genome contains 18 HSP90 s which are only distributed on homologous group 2,5 and 7.The 18 genes are conserved and present as triplet in a subfamily.Transcriptome analysis of flag leaves and grains during filling stage finds that expression patterns of genes in a triplet are similar within a subfamily,but distinct between subfamilies.In normal condition,the HSP90 ABs are constitutively ubiquitous expressed,while the HSP90 AAs and HSP90C2 s are lowly expressed.In heat stress,the HSP90 AAs are dramatically induced,in terms of transcript abundance and fold changes.All HSP90 s are heat responsive in at least one condition.Surprisingly,in grain,the HSP90 ABs in homologous group 5 and the HSP90C2 s are not in response to heat,and none gene is responsive to heat in 10 minutes.4)The HSP90 s produce totally 126 transcripts,in which 36 are annotated in the genome annotation files,and 90 are novel in this study.Each genes encodes 2-13 transcripts,in which 1-2 major transcript isoforms defined by the the isoform expression percentage represent the major gene expression.Whereas under heat stress,in each gene the transcript with the largest fold change is always not the major isoform.Twelve out of the 18 HSP90 genes are regulated not only at transcription level but also at alternative splicing(AS)level,though the HSP90C2 s are not regulated at AS level.In grains,7 genes are regulated at AS level within 10 min of heat stress,and among the genes those are not regulated at transcription level,one is regulated at AS level.In flag leaves,only the HSP90 AAs and HSP90 ABs are regulated at AS level,in addition to the transcription regulation.These results demonstrate that the HSP90 s undergo extensive alternative splicing events,diversifying the transcriptome reprogramming of HSP90 in heat stress.And some of these genes are both regulated at transcriptional and alternative splicing level,improving the understanding of regulation of these important heat responsive genes.