REVEILLE2 is a cold-induced component of the circadian system that regulates how Arabidopsis thaliana responds to night-time chilling (constant light)

Dataset: RNA-seq diel and temperature time series experiment of constant light (LL)All plant material was the Columbia (Col-0) ecotype. Plants were harvested at 4 h intervals over a period of 56 h for a cool constant light RNA-seq experiment, saving an additional time-point during the first subjective dark phase (t =18h; anticipated peak of RVE2 in constant light). A single biological replicate consisted of 9-13 plants in hydroponic containers, aerial tissue harvested and pooled per condition/time point. Three biological replicates were generated for each time-point in separate experiments. The same growth cabinet was used for all repeats to eliminate the potential effects of minor changes in light intensities and light quality on gene expression.Project SummarySplicing mediated reversals of the relative abundances of transcript isoforms of the same gene - 'isoform switching' - is a striking feature of plants' rapid response to cooling. We find the flip-flop dynamism of the REVEILLE2 isoform switch particularly striking; it is detectable on the minute-timescale and unusually is an 'off:on' switch with the shift to translatable transcript message evident during the transition to cool nights.The physiological relevance of this circadian clock cold-switch was explored using molecular genetic approaches centred around a newly characterised T-DNA insertion line, rve2-2, that is unable to cold-switch.We demonstrate altered cooling-associated rhythmic behaviour in plants bearing a clock promoter reporter (CCA1::LUC) in the rve2-2 background. RNA-seq experiments with rve2-2 identify potential RVE2 target genes and temporal profiling show they comprise a cascade of gene expression initiated shortly after peak RVE2 expression mid-nocturnal phase extending to the imminent dawn. Surprisingly we find a subset of chloroplast-encoded candidates with modified expression in rve2-2 and consequently we find altered photosynthetic capacity a rve2-2 characteristic.The RVE2 cold switch extends our understanding of plants' rapid response to cooling. We propose that RVE2 integrates nocturnal temperature with priming plants' photosynthetic capacity in anticipation of the break of the day.

数据集：恒光照（LL）条件下的RNA测序昼夜节律与温度时间序列实验

所有植物材料均为哥伦比亚（Col-0）生态型。本恒冷光RNA-seq实验以4小时间隔在56小时周期内采集植物样本，并在首个主观暗期（t=18h；恒光条件下RVE2的预期表达峰值时刻）额外增设一个采样时间点。单个生物学重复由水培容器中培养的9至13株植物组成，采集各条件/时间点的地上组织并混合为一份样本。每个时间点均通过独立重复实验设置3个生物学重复。所有重复实验均使用同一生长箱，以消除光照强度与光质的细微变化对基因表达的潜在影响。

项目摘要：可变剪接介导的同一基因转录本异构体相对丰度反转——即‘异构体转换（isoform switching）’——是植物快速响应低温的显著特征之一。我们尤为关注REVEILLE2（RVE2）异构体转换的翻转动态：该转换可在分钟级时间尺度上被检测到，且其为罕见的‘关-开’型转换，在向低温夜晚过渡的过程中，可翻译转录本的表达量显著上调。

我们以新鉴定的T-DNA插入突变体rve2-2（该突变体无法发生低温转换）为核心，通过分子遗传学方法探究了该生物钟低温转换的生理学相关性。我们证实，在rve2-2遗传背景中携带生物钟启动子报告基因（CCA1::LUC）的植物，其与低温应答相关的节律行为发生了改变。

对rve2-2进行RNA-seq实验，鉴定出潜在的RVE2靶基因；时间维度表达谱分析显示，这些靶基因构成了一个基因表达级联反应，其启动时间略晚于夜间中期RVE2的表达峰值，并持续至临近黎明。令人意外的是，我们发现部分叶绿体编码的候选基因在rve2-2中的表达发生改变，因此rve2-2植株的光合能力也出现异常表型。

RVE2的低温转换机制加深了我们对植物快速低温响应的理解。我们提出，RVE2可整合夜间温度信号，并在黎明来临前预激活植物的光合能力。