Coordination of Circadian Clock in Chilling Stress Response of Rice (Oryza sativa L.). Coordination of Circadian Clock in Chilling Stress Response of Rice (Oryza sativa L.)

Chilling stress is a major abiotic stress that affects rice growth and development. Rice seedlings are quite sensitive to chilling stress and this harms global rice production. Comprehensive studies of the molecular mechanisms for response to low temperature are of fundamental importance to chilling tolerance improvement. The number of identified cold regulated genes (CORs) in rice is still very small. Circadian clock is an endogenous timer that enables plants to cope with forever changing surroundings including light–dark cycles imposed by the rotation of the planet. Previous studies have demonstrated that the circadian clock regulates stress tolerances in plants show circadian clock regulation of plant stress tolerances. However, little is known about coordination of the circadian clock in rice chilling tolerance. In this study, we investigated rice responses to chilling stress under conditions with natural light-dark cycles. We demonstrated that chilling stress occurring at nighttime significantly decreased chlorophyll content and photosynthesis efficiency in comparison with that occurring at daytime. Transcriptome analysis characterized novel CORs in indica rice, and suggested that circadian clock obviously interferes with cold effects on key genes in chlorophyll (Chl) biosynthesis pathway and photosynthesis-antenna proteins. Expression profiling revealed that chilling stress during different Zeitberger times (ZTs) at nighttime repressed the expression of those genes involved Chl biosynthesis and photosynthesis, whereas stress during ZTs at daytime increases their expression dramatically. Moreover, marker genes OsDREBs for chilling tolerance were regulated differentially by the chilling stress occurring at different ZTs. The phase and amplitude of oscillation curves of core clock component genes such as OsLHY and OsPRR1 are regulated by chilling stress, suggesting the role of chilling stress as an input signal to the rice circadian clock. Our work revealed impacts of circadian clock on chilling responses in rice, and proved that the effects on the fitness costs are varying with the time in a day when the chilling stress occurs. Overall design: For RNA sampling, 10-day-seedlings were treated with chilling temperature (13°C) for 4 h at different Zeitgeber Time (ZT) (ZT0-ZT36) before harvesting for RNA extraction on the 11th day. For transcriptome analyses, CT12 group seedlings experienced 4 hrs treatment of chilling temperature from ZT8-ZT12, while NT12 group seedlings were kept at 28°C before harvesting at ZT12 as controls. CT24 group seedlings were chilling-treated from ZT20 to ZT24 before being harvested, while NT24 group seedlings were harvested at the same time at normal growth temperature.

冷胁迫（chilling stress）是影响水稻生长发育的主要非生物胁迫之一。水稻幼苗对冷胁迫极为敏感，该胁迫会对全球水稻生产造成严重损害。深入解析水稻低温响应的分子机制，对于改良水稻耐冷性具有核心理论与应用价值。目前已在水稻中鉴定出的冷调控基因（cold regulated genes, CORs）数量仍然极少。

生物钟（circadian clock）是一类内生计时系统，可帮助植物适应不断变化的外界环境，其中包括由地球自转形成的光暗循环。已有研究表明，生物钟可调控植物的胁迫耐受性，即植物的胁迫响应过程受生物钟的直接调控。然而，目前对于生物钟如何协同调控水稻冷胁迫耐受性的相关研究仍较为匮乏。

本研究在自然光暗循环条件下，探究了水稻对冷胁迫的响应机制。研究发现，相较于日间施加的冷胁迫，夜间施加的冷胁迫会显著降低水稻的叶绿素含量与光合效率。转录组分析不仅在籼稻中鉴定出了新的冷调控基因，还揭示生物钟显著干扰了低温对叶绿素（Chl）生物合成通路及光合天线蛋白关键基因的调控作用。

表达谱分析结果显示，夜间不同授时因子时间（Zeitberger times, ZTs）下施加的冷胁迫会抑制叶绿素生物合成及光合相关基因的表达，而日间不同ZTs下施加的冷胁迫则会显著上调这些基因的表达水平。此外，耐冷性标记基因OsDREBs的表达会因施加冷胁迫的ZTs不同而呈现差异化调控。核心生物钟组分基因（如OsLHY与OsPRR1）的振荡曲线相位与振幅，会受到冷胁迫的调控，这表明冷胁迫可作为输入信号作用于水稻生物钟系统。

本研究揭示了生物钟对水稻冷胁迫响应的调控作用，并证实冷胁迫对水稻适应成本的影响会因胁迫施加的日间时段不同而存在差异。

实验设计如下：
用于RNA取样的样品制备：将生长10天的水稻幼苗置于不同授时因子时间（Zeitgeber Time, ZT，ZT0-ZT36）下进行4小时的13℃低温处理，于第11天收获幼苗并提取RNA。
转录组分析分组设置如下：CT12组幼苗于ZT8-ZT12时段接受4小时13℃低温处理；NT12组作为对照，于ZT12时段在28℃正常生长条件下收获。CT24组幼苗于ZT20-ZT24时段接受低温处理后收获；NT24组则于相同时段在正常生长温度下收获并作为对照。