拟南芥EPP2介导光信号与生长素信号的机理研究

Identification of new regulatory factors in the light signaling pathway is of important for elaborating the molecular mechanisms underlying plant growth and development. We obtained a photomorphogenesis-enhanced mutant epp2 using forward genetics approach. By map-based cloning, we cloned the EPP2 gene which encode a putative transcriptional cofactor. Overexpression of EPP2 could rescue the short.hypocotyl phenotypes of the epp2 mutant. Co-IP/MS experiment showed that EPP2 interact with an E3 ubiqutin ligase U1 with unknown function and U1 might mediate protein degradation of EPP2. EPP2 interact with PIF4, a key factor involved in light signaling. Whole Transcriptome Sequencing results showed that EPP2 and PIF4 could co-regulate the expression of a lot of genes, including the auxin-responsive gene family. By using the approaches of molecular biology, biochemistry, genetics and cell biology, we propose to investigate the biochemical function of EPP2.We will explor the mode of action and the molecular mechanism of between EPP2 and PIF4. The purpose of this study is to clarify the function of EPP2 and PIF4 in the light signaling and auxin pathway. We believe that this study will identify a new factor of the light signaling pathway and provide new insight into the mechanism that different signaling networks interact.

寻找光信号途径的调控因子对阐述光调控植物生长发育的分子机理有重要意义。我们通过正向遗传学手段筛选获得了一个光形态建成增强的突变体epp2（enhanced hotomorphogenic2）。epp2突变体在红光、远红光及蓝光下下胚轴均比野生型短。经图位克隆，EPP2可能编码一个转录辅助因子。Co-IP/MS结果表明EPP2与一个未知功能的E3泛素连接酶U1互作，U1可能介导EPP2的降解。另外，EPP2与光信号关键因子PIF4也互作。转录组结果表明，EPP2与PIF4共同调控很多基因表达，包括生长素通路相关基因。另外，PIF4发挥功能有可能部分依赖于EPP2。本项目拟采用分子生物学、生物化学、遗传学和细胞生物学等研究手段，探究EPP2的生化功能，阐释EPP2与PIF4互作机理，以期阐明EPP2与PIF4在调控光形态建成与生长素信号转导中的作用，为不同信号网络的互作提供新的见解。

寻找光信号途径的调控因子对阐述光调控植物生长发育的分子机理有重要意义。我们通过正向遗传学手段筛选获得了一个光形态建成增强的突变体epp2（enhanced hotomorphogenic2）。经图位克隆，EPP2编码一个转录辅助因子SEUSS（SEU）。我们将epp2突变体命名为seu-6突变体。seu-6突变体在红光、远红光及蓝光下下胚轴均比野生型短，是温度形态建成的正向调控因子。seu-6在环境高温（28°C）条件下，下胚轴比野生型短，是温度形态建成的正向调控因子。SEU与光温信号的关键转录因子PIF4互作。SEU可以结合到众多参与生长素合成及信号转导基因的启动子上并激活它们的表达，但SEU结合下游基因不依赖于PIF4，而PIF4激活相关基因的表达依赖于SEU。同时，SEU的突变导致生长素含量显著下降，表明SEU调控了生长素合成而影响光形态建成。我们研究发现了光信号与温度信号整合的新因子SEU，揭示了SEU通过与PIF4互作形成转录调控复合物，对生长素合成与信号相关的靶基因进行调节，从而改变植物体生长素的含量和信号过程，实现对生长发育的调控，使植物更好地适应光温环境。我们研究还发现，SEU含有4个能够被SUMO化的赖氨酸残基位点，体内SUMO化实验证明SEU确实是SUMO底物。酵母双杂交以及免疫共沉淀实验发现，SEU的SUMO化位点突变导致SEU与PIF4的互作增强，说明PIF4极有可能与非SUMO化形式的SEU互作。结果表明，光信号由光受体phyB 接收，通过SIZ1 介导SEU的SUMO化来影响SEU蛋白水平以及与PIF4的相互作用进而调控拟南芥光形态建成。本文研究通过SEU在转录水平和翻译后修饰水平调节拟南芥光温形态建成反应的新机制，为研究植物如何通过调节精细的生物学过程使其在不同光照条件下更好得生长发育、适应不断变化的环境提供了非常重要的理论依据。