拟南芥WHIRLY3和WRKY57互作调控JA诱导叶片衰老的分子机理

Leaf senescence is regulated by diverse developmental and environmental factors which are bridged by members of Arabidopsis WRKY family. Our recent study revealed that Arabidopsis WRKY57 transcription factor functions as a node of convergence for JA- and auxin-mediated signaling in JA-induced leaf senescence. To further explore the signaling pathway of JA-induced leaf senescence, the Arabidopsis WRKY57 protein was used as a prey to screen the cDNA library. As a result, an interacting protein, Arabidopsis WHY3, was obtained from Y2H and further confirmed by CoIP and BiFC assays. Expressive pattern of WHY3 showed that the relative expression of WHY3 was repressed by exogenetic JA treatment as well as was high in senescent leaves. Further phenotype analysis showed that loss of WHY3 function accelerates leaf senescence in response to JA, indicating a negative role of WHY3 in regulating JA-induced leaf senescence. Based on these findings, we proposed that WHY3 may modulate JA signaling and leaf senescence through physically interacting with WRKY57. To test this possibility, we will focus on the relationships between WHY3 and WRKY57 in JA-induced leaf senescence. Y2H will be used to verify the sequence of amino acids involved in the interaction of WHY3 and WRKY57. GUS stain will be used to clarify the expressive pattern of WHY3 under exogenous JA treatment. Furthermore, why3wrky57 and WHY3OE lines will be constructed to investigate the functions of WHY3 in regulating JA-induced leaf senescence. To determine the biological consequence of WHY3-WRKT57 interaction, we will analyze whether WHY3 interferes with the DNA-binding activity, transcriptional function, and/or stability of WRKY57. Moreover, ChIP-Seq analysis will be used to gain the direct target genes of WHY3. Through the above experiments, we attempt to uncover possible regulatory effects of WHY3 on WRKY57, and finally explore the molecular mechanisms underlying the involvement of WHY3-WRKY57 complex in regulating JA-induced leaf senescence.

我们最新研究发现拟南芥WRKY57参与介导JA与Auxin之间的动态平衡，进而调控叶片衰老过程。为深入揭示WRKY57调控叶片衰老的分子机理，我们进一步用WRKY57作为诱饵进行酵母双杂交筛选并获得其相互作用蛋白WHY3。表达分析表明，WHY3基因主要在衰老叶片中表达，并受外源JA抑制。表型分析表明，WHY3负调控JA诱导的叶片衰老过程。本项目我们将详细研究WRKY57与WHY3互作参与调控JA诱导叶片衰老的分子机理。利用Y2H技术确定介导WHY3与WRKY57互作的氨基酸序列；利用GUS染色验证WHY3响应外源JA的表达情况；构建why3 wrky57及WHY3高表达植物，确定WHY3调控JA诱导叶片衰老的功能；分析WHY3对WRKY57的调控功能；利用ChIP-Seq技术获得WHY3调控的直接靶基因。通过上述研究，以期揭示WRKY57-WHY3转录复合物调控JA诱导叶片衰老的分子机理。

WRKY57是茉莉酸和生长素信号交互调控的关键调控因子，参与了JA诱导的叶片衰老过程。筛选和鉴定WRKY57的相互作用蛋白，并阐明它们与WRKY57之间的调控关系，对于深入理解WRKY57调控的叶片衰老过程具有非常重要的意义。在前期研究中，我们发现单链DNA结合蛋白Whirly3 (WHY3)能与转录因子WRKY57相互作用形成蛋白复合体。WHY3主要在衰老叶片中表达，并负调控茉莉酸诱导的叶片衰老过程。为了深入研究WRKY57-WHY3蛋白复合体调控茉莉酸诱导的植物叶片衰老的分子机理，该项目采用一系列遗传学、分子生物学及基因功能分析的方法和手段，系统解析了WHY3与WRKY57转录因子相互作用形成蛋白复合体，共同调控茉莉酸诱导的叶片衰老的生物学功能。. 在前期研究基础上，本项目进一步构建了WHY3的高表达转基因植物材料，并详细分析了它们在茉莉酸诱导的叶片衰老方面的表型，明确了WHY3负调控茉莉酸诱导的叶片衰老的生物学功能；构建了why3wrky57的双突变体并分析其表型，确定了它们之间的遗传关系；验证了WHY3和WRKY57之间的相互作用关系，结果表明WHY3与WRKY57在植物细胞核内相互作用形成蛋白复合物；重点研究了WHY3与WRKY57之间可能存在的调控关系，结果表明WHY3能促进WRKY57对下游靶基因的转录抑制功能。综合上述研究结果表明，WHY3通过跟WRKY57相互作用形成蛋白复合体，从而促进了WRKY57对下游靶基因SAG12和SEN4的抑制作用，进而调控了茉莉酸诱导的叶片衰老过程。. 本项目的研究不仅有助于人们全面理解茉莉酸诱导的植物叶片衰老过程的网络调控模型，为叶片衰老调控的研究和作物产量增加提供理论基础。，还有助于人们深入了解Whirly家族成员的生物学功能。