蛋白泛素化修饰调控植物HsfA2热激转录因子表达及耐热性形成的作用机制研究

Little is known about the molecular mechanisms of protein ubiquitination by which heat stress signaling is regulated although the expression of polyubiquitin genes was found to be induced by heat stress in initial studies on effects of ubiquitination on abiotic stress responses. We found that mutations of EHT1, encoding an E3 ubiquitin ligase, lead to enhanced heat tolerance as exhibited in the eht1 mutant plants. Importantly, the expression of heat stress transcription factor HsfA2 was maintained at much higher levels during heat stress in eht1, indicating that EHT1 is likely to function as a negative regulator during heat stress responses. In this study, we will identify the downstream substrate protein(s) of EHT1 and reveal the molecular mechanisms how EHT1 regulates the heat-responsive expression of HsfA2 through ubiquitinating its substrate protein(s). Our findings will bring insights into better understanding the contribution of protein ubiquitination to formation of thermotolerance in plants.

虽然泛素化修饰参与植物逆境响应的研究最初发端于人们发现多聚泛素化基因的表达受高温胁迫调控，但是泛素化修饰调控热胁迫信号转导的研究却十分有限。我们前期研究发现一个E3泛素连接酶基因EHT1突变后，导致突变体呈现耐热性显著增强的表型，且eht1突变体HsfA2表达在高温胁迫条件下维持在相对较高的水平，表明E3泛素连接酶EHT1为一个植物热激信号转导和耐热性形成的负调节因子。本项目围绕以E3泛素连接酶EHT1为节点的泛素化修饰与植物热激信号转导的遗传关系进行深入和全面的探索和阐述。项目的研究目标是通过鉴定E3泛素连接酶EHT1泛素化修饰下游关键的靶标蛋白，揭示EHT1调控其靶蛋白活性的生化特性，建立较为明确的EHT1调控下游靶蛋白泛素化修饰水平，进而调节植物热激转录因子表达及耐热性形成的分子调控路径。

我们从一个E3泛素连接酶EHT1入手，证实它为一个植物热激信号转导和耐热性形成的负调节因子。我们主要研究内容是围绕以E3连接酶EHT1为节点的泛素化修饰与植物热激信号转导的遗传关系进行了深入和全面的探索和阐述。综合运用分子遗传和生化等实验手段分别鉴定ETP1(EHT1 TARGET PROTEIN 1)和ETP2 (EHT1 TARGET PROTEIN 2)为E3泛素连接酶EHT1的修饰底物蛋白, 二者具有较高的同源性，同为乙烯合成途径的关键酶。我们的研究结果支持如下的结论：EHT1 通过泛素化修饰增强ETP1的蛋白稳定性。在对照条件下WT中ETP1-GFP蛋白量的积累是EHT1通过K63类型泛素化修饰ETP1-GFP来实现的；而在eht1-1突变体中，由于EHT1功能的缺失，导致对照和热处理条件下ETP1-GFP的K63类型泛素化修饰被抑制，进而导致ETP1-GFP稳定性显著下降。研究结果揭示了E3连接酶EHT1通过泛素化修饰下游关键的靶标蛋白ETP1和ETP2的分子机制，明确EHT1调控其靶蛋白活性的生化特性，建立了较为明确的EHT1调控下游靶蛋白泛素化修饰水平，进而调节植物耐热性形成的分子调控路径。