拟南芥脱落酸和干旱信号转导中O-连接糖基转移酶SPINDLY与CPN20蛋白的交互作用

SPINDLY (SPY) and SECRET AGENT (SEC) are two putative O-linked N-acetylglucosamine transferases (OGTs) in Arabidopsis. It has been previously reported that the mutation of both SPY and SEC causes embryo lethality, indicating that they play a crucial role in embro development. While spy mutant plants have defects in response to gibberllin and cytokinin, SPY is also involved in light, meristem activity, root growth, circadian rhythms, and response to drought stress. In addition, it remains unclear whether abscisic acid (ABA) is involved in SPY regulation of drought response. If SPY is involved in ABA signaling, it is also elusive what role SPY may play and how SPY is related to the established major ABA signaling pathways. This project would employ two strategies to address above questions. We investigate how SPY is involved in ABA and drought signaling to uncover the relationship between SPY and the key proteins of major ABA signaling pathways. Another strategy is to screen for SPY-interacting proteins by using yeast-two hybrid assay. In the previous attempt, we have successfully screened a SPY-interacting protein, CPN20, a co-chaperone protein that locates in stroma of chloroplasts. We will make use of multiple approaches including pull-down, co-immunoprecipitation, BiFC and split-LUC assays to confirm the interaction between SPY (SEC) and CPN20. We also make genetic and phenotypical analyses to address the genetic and functional relationship between SPY (SEC) and CPN20. We explore the cellular localization of SPY (SEC) and CPN20 and its relationship with ABA. We detect the possible O-GlcNAc modification of CPN20 and its dependence on SPY (SEC) in mammalian and bacteria cells, and eventually in plants. We aim to elucidate the molecular basis for the involvement of SPY in ABA and drought signaling, and to reveal the biological function of SPY-CPN20 interaction and its biochemical and cytological basis.

SPY和SEC是拟南芥中两个可能的O-连接糖基化转移酶，有报道称SPY蛋白参与干旱响应，但其分子机理及脱落酸（ABA）是否参与尚不清楚。本课题通过遗传学方法探讨SPY蛋白如何参与ABA和干旱信号转导通路，揭示SPY与ABA信号通路关键蛋白之间的关系。前期工作筛选到SPY互作蛋白—辅分子伴侣蛋白CPN20，拟用多种方法验证这种互作的真实性；通过SPY（SEC）与CPN20相互作用的表型分析和遗传分析，分析它们的遗传和功能关系；研究SPY（SEC）与CPN20的定位及其与ABA的关系；用哺乳动物（和大肠杆菌）细胞体系检测CPN20的O-连接糖基化及其对SPY（SEC）的依赖性；进而检测植物体内CPN20的O-连接糖基化及其对SPY和SEC的可能依赖性。目的在于揭示SPY蛋白参与ABA和干旱信号转导途径的分子基础，弄清楚SPY与CPN20相互作用的生物学功能及其生化和细胞学基础。

项目背景：O-连接糖基化在动物细胞发育中起重要调节作用，其在植物中研究较少。已经发现拟南芥中的SECRET AGENT（SEC）和SPINDLY（SPY）分别具有O-GlcNac糖基化和O-fucosylation糖基化活性。但对它们在植物发育和逆境响应中的作用及其机制的了解甚少。..主要研究内容、重要结果、关键数据：通过酵母双杂交和BiFC（双分子荧光互补技术）证明了SPY和SEC均与CPN20发生相互作用，而且ABA促进SPY与CPN20的相互作用；通过化学生物学方法和质谱技术证明CPN20可在哺乳动物细胞和植物中发生O-GlcNAc糖基化； 体内实验证明SEC O-GlcNAc糖基化 CPN20，且鉴定了CPN20中的O-GlcNAc糖基化位点；在细菌和植物中、以及体外系统中证明CPN20可被SPY O-岩藻糖化；证明SPY通过对CPN20的O-岩藻糖化调节其在叶绿体中的定位；证明O-岩藻糖化位点对CPN20在叶绿体中定位的作用；证明spy突变不影响CPN20的蛋白稳定性，佐证了SPY对CPN20的O-岩藻糖化调节其往叶绿体的运输；突变体表型分析证明SPY在ABA调节种子萌发和幼苗发育中起正的调节因子的作用；遗传分析发现cpn20突变部分抑制spy-3对ABA的不敏感表型，证明CPN20部分介导SPY的作用；遗传分析发现SPY位于ABA受体ABAR的上游；遗传分析发现SPY与ABI5、ABI3和ABI4之间存在复合的关系；遗传分析发现SPY不位于PYR/PYLs/RCARs介导的ABA信号通路中。..以上研究结果，已经发表一篇研究论文，正在整理一篇研究论文。..科学意义：本项目研究SPY对脱落酸（ABA）信号转导通路的负调节因子CPN20的O-fucosylation及其在脱落酸和干旱信号转导中的作用机制，对理解植物ABA和干旱信号转导机制具有重要意义。