转录因子SRS5参与调控植物光形态学建成机理研究

Higher plants exhibit two distinct morphogenesis in dark and light, and the knowledge of the underlying mechanisms is important for us to understand how plants cope with environment and choose the appropriate morphogenesis. By screening Arabidopsis mutant library, a mutant SRSox, in which SRS5 is over-expressed, exhibits photomorphogenesis in dark. Our further study demonstrated that blue light induces SRS5 expression. Under blue light, SRSox has shorter hypocotyl compared with wild-type, while the knock-out mutant (srs5) shows higher hypocotyl than wild-type. In addition, the phenotype of short hypocotyls in SRSox is partially rescued in two double mutants SRSox hy5 and SRSox sth3. Furthermore, while the expression of HY5 and STH3 is induced in SRSox, they are repressed in srs5, suggesting that HY5 and STH3 function in SRS5-mediated photomorphogenesis. Also, ChIP-PCR experiments indicated that SRS5 may bind to STH3 promoter and yeast two-hybrid showed possible interaction of COP1 and SRS5. In this project, we plan to carry our further experiments to demonstrate that SRS5 plays an important role in blue light-mediated photomorphogeneses. The project will explore whether SRS5 directly regulates the expression of HY5 and STH3 and identify its binding motifs, indicate that HY5 and STH3 are essential for SRS5-mediated photomorphogenesis, investigate a role of COP1-mediated ubiquitin degradation pathway for SRS5 stabilization, and isolate other factors involved in SRS5-meidated photomorphogeneses. Taken together, our findings will show how SRS5 modulates blue light-mediated photomorphogeneses and enhance our understanding of the mechanisms underlying light-regulated plant morphogenesis.

高等植物在黑暗和光照条件下会展现两种截然不同的形态，其调控机理研究对了解植物生长发育调控有重要意义。我们前期实验筛选获得蓝光诱导基因SRS5过表达突变体SRSox。SRSox在黑暗条件下表现出光形态建成表型,而在蓝光下srs5下胚轴比野生型长。双突变分析表明，SRSox的短下胚轴在SRSox hy5和SRSox sth3中被部分回复；而且SRS5能调控HY5和STH3的表达。此外，初步ChIP-PCR表明SRS5能结合到STH3的启动子区；酵母双杂交显示COP1能与SRS5互作。我们计划进一步验证SRS5在光形态学建成中的作用，检验HY5和STH3是否为SRS5的直接下游并深入了解它们间的协同关系，探究SRS5是否受COP1介导的泛素化降解途径调控,通过回复突变体的深入分析获得其它因子。研究结果将明确证明转录因子SRS5参与调控光形态学建成，并明晰其在蓝光介导的光形态建成中如何发挥作用。

高等植物在黑暗与光照下会展现出两种截然不同的形态建成。已有研究显示，ELONGATED HYPOCOTYL 5 (HY5), B-BOX PROTEIN 21 (BBX21)和BBX22为光形态建成促进基因, 在多种光受体下游发挥重要作用。然而，在黑暗条件下，这些基因的过量表达却不能表现出光形态建成的表现型。.我们通过筛选突变体库获得新的拟南芥突变体SRS5ox。该突变体由雌激素诱导SHI-RELATED SEQUENCE5 （SRS5）基因过量表达所致，在黑暗条件下表现为光形态建成。SRS5属于SHI基因家族转录因子，在光形态学建成中的作用未见报道。我们发现，SRS5基因的表达在蓝光诱导下显著增强。蓝光下，SRS5ox和Pro35S: SRS5-GFP的下胚轴比野生型更短，而SRS5插入失活突变体srs5-2下胚轴则更长，这些结果表明SRS5是光形态建成的正调控因子。为了探索SRS5与光形态建成促进基因的关系，将hy5突变体与SRS5ox杂交获得 SRS5ox hy5。分析发现此双突变使得SRS5ox短下胚轴表型得到部分回复。SRS5ox中HY5表达上升，而srs5-2中HY5表达下降。ChIP和EMSA实验证明SRS5能直接结合到HY5启动子上。这些结果表明SRS5调控拟南芥光形态学建成应是部分通过促进HY5来进行。此外，我们还发现SRS5能够也能直接结合到其它光形态建成促进基因包括 BBX21和BBX22 的启动子上，并促进其表达。 因此，SRS5是光信号途径上游重要的正调控因子，同时调控HY5, BBX21和BBX22等来促进光形态建成。当野生型材料从光下转入黑暗后，SRS5蛋白积累减少；但是， CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1)突变后，这种减少被抑制从而积累较多SRS5蛋白。反之，过表达COP1能使得SRS5蛋白量减少。COP1同SRS5直接相互作用，并且COP1能直接泛素化SRS5使其降解。这些结果表明，在黑暗条件下，SRS5能够被 COP1蛋白介导的泛素化系统降解。.本项目研究明确证明转录因子SRS5位于光信号上游，通过直接调控光形态发生促进基因的表达参与植物光形态学建成调控，并且此蛋白本身也受COP1蛋白介导的泛素化系统调控。研究结果对我们深入了解植物复杂的光信号调控网络和光形态建成机制有重要意义。