Diel and circadian gating of Arabidopsis thermotolerance and transcriptional response to heat stress.. Diel and circadian gating of Arabidopsis thermotolerance and transcriptional response to heat stress.

Purpose: To understand time of day variation in Arabidopsis heat shock responses. We have observed significant differences in terms of plant survivability in response to a heat shock across various times of day. Because of this, we believe that transcriptional responses may be a significant underlying factor which contributes to the observed physiology and surivability after heat stress. Methods: Whole plant tissue mRNA profiles in Col-0 Arabidopsis were generated by deep sequencing using Illumina HiSeq 2500. Tissue samples of four replicates were collected at two times of day (Dawn and Dusk, ZT0/ZT12) and were either control or heat stressed (30oC) for 1 hour. An additional 4 samples were collected in constant light conditions (single replicate) to quantify circadian effects. Sequence reads from each sample were mapped to the TAIR10 reference genome with Tophat2 and counts were obtained using Htseq-count. Differential expression analysis was done using DeSeq2 to calculate differential expression levels of each gene in the dataset. Results: We identified 5512 differentially expressed transcripts with our DESeq2 based data analysis pipeline that involves characterizing gene expression profiles based on basal expression level, time of differential expression, and direction of differential expression. An additional interaction term analysis revealed 572 genes which showe time of day dependent responses to heat stress, meaning the response to heat stress differs depending on the time of day that the heat stress is perceived by the plant. Downstream analysis using gene ontology analysis tools (Pantherdb.org) and Cytoscape revealed strong differences in terms of response happening in the AM and PM gene expression networks. The significance of this work is that it is the first study to show that there is a significant difference in terms of transcriptional responses to heat stress in Arabidopsis where the differences were quantified by levels of mRNA expression. Overall design: Whole tissue mRNA profiles were measured by deep sequencing with four replicates in Col-0 ecotype Arabidopsis. Samples collected were control samples at two times of day (dawn and dusk) and heat stressed samples at two times of day (dawn and dusk). Additionally, 4 single replicate samples were collected in contant light conditions (two times of day/control and treatment) to quantify circadian effects. Reads were collected using Illumina Hiseq 2500 and analyzed with dowstream computational analysis.

研究目的：解析拟南芥（Arabidopsis）热激响应的日间时间变异规律。我们观察到，在一天的不同时段施加热激后，植株的存活率存在显著差异。据此我们推测，转录响应可能是导致热胁迫后植株生理状态与存活率出现差异的关键内在因素。
实验方法：以Col-0生态型拟南芥的全植株组织为材料，采用Illumina HiSeq 2500测序平台进行深度测序，获取mRNA表达谱。实验设置两个采样时段（黎明与黄昏，对应ZT0/ZT12），每组设置4次生物学重复，样本分为对照组与热胁迫组（30℃处理1小时）。此外，在持续光照条件下采集了4份单重复样本，用于量化昼夜节律效应。使用Tophat2将测序读段比对至TAIR10参考基因组，通过Htseq-count获得基因计数数据。采用DeSeq2进行差异表达分析，以计算数据集内各基因的差异表达水平。
实验结果：基于DeSeq2的数据分析流程，我们共鉴定出5512个差异表达转录本，该流程基于基因基础表达水平、差异表达时段以及差异表达方向对基因表达谱进行表征。额外的互作项分析显示，共有572个基因呈现出日间时间依赖性的热胁迫响应，即植物感知热胁迫的时段不同，其热胁迫响应模式也存在差异。通过基因本体论（gene ontology）分析工具（Pantherdb.org）与Cytoscape进行下游分析，发现上午与下午的基因表达网络在响应模式上存在显著差异。本研究的重要意义在于，它首次通过mRNA表达水平量化了拟南芥热胁迫转录响应的显著差异。
整体实验设计：以Col-0生态型拟南芥的全组织mRNA表达谱为研究对象，通过深度测序完成检测。其中，两个采样时段（黎明与黄昏）的对照组与热胁迫组样本各设置4次生物学重复。此外，在持续光照条件下采集4份单重复样本（覆盖两个时段的对照与处理组），用于量化昼夜节律效应。测序数据通过Illumina HiSeq 2500平台获取，并经后续生物信息学分析完成处理。