拟南芥蓝光受体CRY与AP2类转录因子TOE互作调控光周期开花的分子机理研究

Plants undergo a major physiological change as they transit from vegetative growth to reproductive development. Flowering time is regulated by complex and fine regulatory molecular networks that monitor the ever changing environment and ensure reproductive development at an appropriate season. Light is a critical environmental cue, which regulates photoperiodic flowering in plants. Cryptochromes interact directly with COP1 and CIB1 to mediate blue light signaling and regulate floral initiation. To aim to decipher a new CRY1-mediated blue light signaling transduction pathway, we initiated yeast two-hybrid screening and identified a candidate of CRY1-interacting proteins, TOE1, a member of AP2 family transcription factors that interacts with CO and inhibit its activation of FT to regulate flowering. Through BiFC, in vitro, and semi-in vivo pull-down assays, we confirmed that CRY1 and CRY2 interact physically with TOE1 and TOE2 in a blue-light dependent manner, respectively. Based on these findings, we are planning to generate cry1 cry2 toe1 toe2 quadruple mutant and cry1 cry2 toe1 toe2 smz snz hextuple mutant to verify the genetic interaction between CRY and TOE. EMSA、ChIP-qPCR and Dual-LUC assays will be performed to test whether CRY inhibit the DNA-binding activity of TOE or transcriptional activity. Further pull-down and co-immunoprecipitation assays will be performed to test whether CRY influences flowering by interfering with the interaction of TOE with CO. Through these studies, we will hopefully demonstrate the biological function of the CRY-TOE-CO pathway in the regulation of photoperiodic flowering, and further our understanding of the molecular mechanism by which light signal finely regulates floral initiation via multiple regulatory levels.

开花是高等植物从营养生长阶段向生殖生长阶段转变的发育过程，复杂而精细的调控途径可以保证其在合适时间开花以及成功繁殖。光信号是调控光周期开花时间的重要环境信号。目前的研究表明，光信号可以通过蓝光受体隐花素CRY分别与COP1和CIB1互作来调控开花时间。为了解析CRY信号转导的新机制，我们通过酵母双杂交筛选得到了拟南芥CRY1的互作蛋白TOE1，该蛋白抑制开花。通过pull-down、BiFC和半体内pull-down等方法证明CRY1和CRY2与TOE1和TOE2互作。本项目计划通过多突变体构建探索CRY和TOE之间的遗传关系；通过蛋白-DNA结合手段探索CRY是否会影响TOE的DNA结合能力和转录活性；通过蛋白互作手段探索CRY是否会影响TOE与CO相互作用。本项目将可望揭示CRY通过抑制TOE促进光周期开花的新机制，从而进一步认识光信号通过多个层次精细调控植物开花时间的本质。

隐花素是一种在很多物种中发现的光裂解酶样的蓝光受体。拟南芥CRYs（cryptochromes, CRY1 and CRY2）介导了很多植物的光反应过程，其中包括光周期开花的过程。CRYs与COP1-SPA1复合物互作，促进CO蛋白稳定性，从而促进FT的转录和开花。AP2类转录因子TOE1通过抑制CO的转录活性并且直接结合FT基因序列来负调控FT的表达。在项目的资助下，我们阐明了CRY1和CRY2可以直接与TOE1和TOE2（TOEs）发生依赖于蓝光的相互作用。遗传实验表明TOEs的突变可以部分地回复cry1 cry2的晚花表型。CRY2-TOEs的互作促进TOEs-CO复合物的解离，导致CO转录活性抑制的解除，从而增强FT的转录。另外我们发现CRY2可以抑制TOE1结合FT的Block E区域。我们揭示了CRY2-TOEs互作调控光周期开花的分子机制，加深了我们对于植物调控光周期开花调控精密性的认识。