E3泛素连接酶基因SAEL调控拟南芥叶片衰老机制的研究

As two important posttranslational modification mechanisms, protein ubiquitination and phosphorylation play fundamental roles and cross talk with each other in the regulation of plant growth and development as well as many other signalling networks. The involvement of reversible phosphorylation in the signal transduction and progression-control of leaf senescence has been well-known, however, the role of protein ubiquitination has rarely been studied and whether there are synergistic cross-talks in this field between the two types of protein posttranslational modification remains elusive. We have previously reported that a leucine-rich repeat receptor-like kinase AtSARK and a PP2C-type protein phosphatase SSPP could interact with each other and were involved in the positive and negative regulation of leaf senescence respectively. Recently we identified an E3 ubiquitin ligase encoding gene SAEL as a positive regulator of leaf senescence. In addition, preliminary study has shown that SAEL could interact with SSPP and reversed the delayed senescence caused by the SSPP overexpression. In the current project of great scientific and innovative significance, we aim to functionally characterize the SAEL gene using multiple biochemical and molecular approaches, comprehensively analyze the relationship between SAEL, AtSARK and SSPP, and explore layer by layer the interplay between protein ubiquitination modifications and reversible protein phosphorylation during the regulation of Arabidopsis leaf senescence.

泛素化和可逆磷酸化作为两种重要的蛋白质翻译后修饰机制，在植物生长发育及多种信号网络调控中发挥重要作用，且两者在多种生命活动的调控中存在相互作用与协同控制。可逆磷酸化反应参与叶片衰老信号传递与衰老进程控制已广为人知，而关键因子的泛素化修饰在叶片衰老调控中的研究则相对较少；两者在叶片衰老调控中是否存在互作与协同，目前还未见报道。我们前期发现类受体蛋白激酶AtSARK和蛋白磷酸酶SSPP能直接互作并分别正向和负向调控拟南芥的叶片衰老，近期又分离鉴定了一种E3泛素连接酶基因SAEL，发现SAEL能够诱导叶片早衰，且与 SSPP发生直接互作并逆转SSPP诱导的叶片晚衰。在此基础上将综合利用多种技术手段，深入研究SAEL基因的功能，全面解析SAEL与AtSARK和SSPP在拟南芥叶片衰老调控中的相互关系，逐层探索蛋白质泛素化修饰与可逆磷酸化反应在叶片衰老调控中的协同作用机制，具有重要科学意义和创新性。

泛素化和可逆磷酸化作为两种重要的蛋白质翻译后修饰机制，在植物生长发育及多种信号网络调控中发挥重要作用，且两者在多种生命活动的调控中存在相互作用与协同控制。可逆磷酸化反应参与叶片衰老信号传递与衰老进程控制已广为人知，而关键因子的泛素化修饰在叶片衰老调控中的研究则相对较少；两者在叶片衰老调控中是否存在互作与协同，目前还未见报道。本项目在前期发现拟南芥类受体蛋白激酶AtSARK和蛋白磷酸酶SSPP能够直接互作并分别正向和负向调控拟南芥叶片衰老的基础上，进一步分离鉴定了一种E3泛素连接酶基因SAEL，发现SAEL与SSPP和AtSARK与SSPP之间两两互作。SAEL能够单泛素化SSPP并抑制其延缓衰老的功能，而SAEL对叶片衰老的促进作用则依赖于AtSARK的激酶活性，三者之间的复杂关系充分体现了关键因子的泛素化与可逆磷酸化这两种重要修饰机制在拟南芥叶片衰老调控中的协同作用。进一步通过文库筛选分离鉴定参与SARK、SSPP和SAEL协同作用的关键组分，发现SAUR49能够与SSPP互作并逆转其延缓叶片衰老的功能，而转录调节因子HBI1可以分别结合SAEL和SAUR49等的启动子，通过促进SAEL和SAURs的表达而负调控SSPP的功能。利用对多种材料的转录组分析，初步构建SAEL、AtSARK 和SSPP协同调控拟南芥叶片衰老的信号网络，开展对三者共同调控关键基因的筛选，发现拟南芥ACS家族成员AtACS7在SARK和SAEL诱导的早衰中都发挥重要功能，是“衰老乙烯”合成的主要贡献者，并进一步发现AtACS7具有ACS和C-S裂解酶双酶活性，两种酶活的转换可能对其在叶片发育中的功能起重要调节作用。上述研究成果的取得，为深入理解泛素化和可逆磷酸化修饰对叶片衰老信号传递与衰老进程控制的精细、协同调控，拓展和完善人们对叶片发育机制的全面认知，奠定了重要基础。