拟南芥AtGAAPs 对非生物胁迫诱导的程序性细胞死亡与植株整体抗性的调控机制

Understanding how the program cell death (PCD) and plant survival with adaption are regulated in response to abiotic stress signals in plant is still in its infancy. Bcl-2 family members integrate pro- and anti-apoptotic signals in healthy and stressed cells and therefore constitute one of the main signaling nodes in the life or death decision. While core regulators controlling and executing program cell death (PCD) in plants remain largely unknown. However conserverd interactors of Bcl-2 such as Beclin-1 and Bax inhibitor-1 (BI-1) related proteins exist in plants. BI-1 and like proteins are one class of the most intensively characterized cell death suppressors conserved between plants and mammals. In mammals, BI-1 like proteins play roles in cell protective through regulating unfolded protein response (UPR) signaling and autophagy in response to different ER-stress. In addition, they perform functions in regulation PCD via interaction with pro-apoptosis factors. GAAPs (Golgi anti-apoptotic protein) are a novel, evolutionarily conserved sub-group of BI-1 related proteins. Human and viral GAAPs have been shown to inhibit apoptosis induced by a variety of pro-apoptotic stimuli, while the function of GAAPs in plants has not been reported. We found that AtGAAP1/AtGAAP3 protected cells from death induced by ER stress or NaCl stress. While AtGAAP4 mutation enhanced plant survival upon ER stress. In addation, AtGAAP1/AtGAAP3 negatively regulated the expression of UPR genes against mild ER stress. To further elucidate the molecualr mechanism of PCD and plant adaption mediated by GAAPs under abiotic stress, this project aims to characterize Arabidopsis AtGAAPs concentrating on their putative roles as regulator of PCD during ER stress. Genetics, biochemical, molecular and cellular studies will be conducted to address how AtGAAPs affect PCD and UPR signaling pathways in addition to autophagy and vesicle changes. The effects on second message factors such as reactive oxigen and Ca2+ are also analyzed. Additionally, the split-ubiquitin yeast-two hybrid assay will be adopted to isolate AtGAAPs interactors. And their interactions will be verified by fluorescent co-localization and co-immunoprecipitation respectively. Then genetics, biochemical, molecular and cellular studies will be conducted to address how AtGAAPs execute its role in the regulation of UPR and PCD through interacting with different partners. These studies will provide the fundamental information about the molecular mechanisms uderlying PCD and plant adaption strategy during stress in plants.

由胁迫诱导的程序性细胞死亡（PCD）是植物抗性的重要手段。目前植物PCD的调控机制还不清楚，但植物中存在保守的BI-1类因子，动物中BI-1与决定细胞生死的核心元件互作抑制PCD，也通过未折叠蛋白响应(UPR)通路调节细胞抗性与死亡的转变。我们前期研究发现，拟南芥BI-1类因子AtGAAPs对细胞死亡、植株抗性和UPR的调控关系密切，为阐明其在PCD与植株抗性方面的作用机制，本项目拟从细胞和生理水平分析内质网胁迫下AtGAAPs对PCD和植株抗性的调控关系；进而分析确定AtGAAPs如何调节UPR来决定PCD的分子机制；同时通过酵母双杂交手段获得AtGAAPs互作因子，并采用遗传学等手段，结合芯片分析，阐明AtGAAPs与互作因子的相互作用对UPR、PCD和植株抗性的调节。本研究对阐明逆境下植物生存适应策略具有重要科学意义，也有利于人们有更多手段培育或提高经济植物在逆境下的综合性能和产量。

合成加工蛋白质的重要细胞器内质网（Endoplasmic reticulum，ER）对各种胁迫非常敏感，胁迫下可导致ER中堆积大量异常折叠或未折叠蛋白，即内质网胁迫（ER stress），细胞会通过激活未折叠蛋白信号通路（Unfolded Protein Response，UPR）重建内质网稳态，胁迫过强超过细胞修复能力时就会诱导细胞死亡发生。但人们对植物中UPR在细胞稳态维持和细胞死亡之间的调控机制认识还非常有限。GAAPs（Golgi anti-apoptotic protein）是保守的抗凋亡因子BI-1亚家族，拟南芥中有5个GAAPs同源蛋白。本项目围绕拟南芥GAAPs在抗ER胁迫中的功能展开，通过突变体、多级突变体和过量表达转化子的构建，在细胞水平上和整体水平上分析了GAAPs在细胞死亡诱导发生与器官、整体水平的存亡关系，结果发现，GAAP1-GAAP4在抵抗ER胁迫方面功能冗余，既抵抗ER胁迫诱导的细胞死亡，也负调控UPR的细胞保护性反应；结合胁迫恢复实验分析说明GAAPs推迟弱的ER胁迫条件下UPR的启动，促进胁迫修复后UPR的关闭，从而有利于能量的再分配；细胞原位组化分析、RNAseq分析结合分子生物学研究表明，GAAPs既抑制胁迫下细胞活性氧的产生积累，同时也削弱水杨酸通路的上调从而抑制细胞死亡的发生；GAAPs通过与受体IRE1蛋白质互作负调控UPR，遗传学研究表明GAAPs在抗胁迫中的功能发挥既有依赖于IRE1也有不依赖于IRE1的途径；通过酵母双杂技术、生化分析结合双分子荧光互补分析筛选出GAAPs的互作因子MPPR3和PAS2；GAAPs与MAPR3双突变体对ER胁迫敏感性增强，自噬减弱，RNAseq分析结合分子生物学分析表明GAAPs与MAPR3互作参与胁迫下IRE1-RIDD通路和细胞自噬的调控；PAS2突变体和PAS2与GAAPs双突变体在整体水平和细胞水平都对于ER胁迫和灰霉菌侵染高度敏感；PAS2及其与GAAPs互作在ER胁迫下抑制ROS的过度积累，正调控ER胁迫诱导的细胞自噬。本项目的研究结果不仅阐明了动植物种保守因子GAAPs的功能及其在ER胁迫下细胞稳态与细胞死亡的调控机制，为进一步阐明不同胁迫条件下细胞稳态、细胞自噬和细胞死亡之间命运转换的分子机制奠定了很好的基础，同时本项目获得了一些与GAAP互作的重要抗性调控因子，对这

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