拟南芥转录因子MYB30的泛素化修饰和SUMO化修饰共同调节ABA信号转导的机理研究

The phytohormone abscisic acid (ABA) plays an essential role in plant development, including seed development, dormancy, germination, vegetative growth and during the response of the plant to biotic and abiotic stresses. We found that the Arabidopsis transcription factor MYB30 negatively regulates the ABA response, myb30 mutants are hypersensitive to ABA during germination and seedling growth. We also found the Arabidopsis SUMO E3 ligase SIZ1 mediates the sumoylation of MYB30 and the sumoylation of MYB30 plays an important role in ABA signaling. In wild-type seedlings, the protein stability of MYB30 is not affected by ABA, but ABA induces the degradation of MYB30 in siz1 mutant, suggesting that the sumoylation alters protein stability of MYB30. A further study found that the degradation of MYB30 is controlled by the ubiquitination. Post-translational control of protein degradation by the ubiquitin proteasome system (UPS) is a highly regulated process essential for the proper growth and development, several E3 ubiquitin ligases have been found to play a variety of roles in ABA signaling. We hope we can identify the ubiquitination sites in MYB30 and find out how ubiquitination functions in ABA response. In mammals, the sumoylation can promote the ubiquitination and ultimately led to protein degradation via the proteasome pathway, but the relationship between these two modifications in plant is still unclear. The study of MYB30 is a good chance to make the relationship clear, because MYB30 can be modified by these two modifications and both of them mediate the degradation of MYB30. We hope that we can figure out how sumoylation and ubiquitination regulate the degradation of MYB30 together, and how these modifications regulate the MYB30 downstream genes expression together to mediate the ABA response, so we can understand the relationship between these two modifications in plant through the study of ubiquitination and sumoylation of MYB30.

植物激素ABA在植物生长发育和外界信号响应过程中具有重要的调控作用。而泛素介导的蛋白质降解和SUMO化修饰，在生物体维持自身正常生长发育和适应环境过程中同样起着重要作用。在动物细胞中，已发现SUMO化修饰可以诱导蛋白的泛素化修饰，但植物中这两种修饰的关系并不清楚。之前的研究发现，拟南芥转录因子MYB30负调控ABA信号转导，而SUMO化修饰可以通过抑制MYB30蛋白的降解参与调节ABA信号途径。进一步研究发现，MYB30蛋白的降解是由泛素化修饰途径介导的。我们推测MYB30蛋白的SUMO化修饰可能是通过抑制MYB30蛋白的泛素化修饰起作用的。本项目希望通过鉴定MYB30蛋白的泛素化修饰位点，通过突变SUMO化修饰与泛素化修饰的关键位点了解两种修饰间如何相互调节，让我们对它们如何共同调节MYB30蛋白的降解进而参与ABA信号转导，以及植物中SUMO化修饰与泛素化修饰的关系有更加深入的了解。

植物激素ABA在植物生长发育和外界信号响应过程中具有重要的调控作用。在种子萌发及萌发后生长过程中，拟南芥转录因子MYB30负调控ABA信号的转导，SUMO化修饰可以增强ABA处理条件下MYB30蛋白的稳定性。本项目前期的研究发现，SUMO化修饰对MYB30蛋白的保护作用是通过抑制26S蛋白酶体介导的泛素化降解实现的。本项目希望通过对MYB30蛋白泛素化降解过程的研究，一方面希望明确MYB30蛋白的泛素化修饰在ABA信号途径中的作用；另一方面，通过确定两种修饰如何共同参与调节MYB30蛋白的稳定性，进而参与ABA信号途径，使我们对植物中SUMO化修饰与泛素化修饰两者之间的关系有进一步的了解。.通过本项目的研究，我们首先明确了ABA处理条件下，MYB30的降解是由泛素E3连接酶RHA2b介导的。共表达MYB30和RHA2b蛋白，会导致MYB30蛋白稳定性和转录活性下降。在野生型和rha2b-1突变体中过表达MYB30蛋白，会导致转基因植株具有相似的ABA不敏感性，而rha2b-1 myb30-2双突变体的ABA表型与myb30-2突变体的相同，这些都表明在ABA信号传导过程中，MYB30位于RHA2b的下游。.与此同时，项目还鉴定了MYB30的泛素化位点K283以及K165，其中K283位点的突变可以严重的抑制MYB30蛋白的泛素化降解。研究发现这两个泛素化位点存在功能的冗余，在myb30突变体中表达两种突变体蛋白都不能互补其ABA表型，而且转基因植株中MYB30调控的下游基因的表达也不能被完全恢复。SUMO-MYB30在各种条件下的降解被严重的抑制，同时由于K283位点也是MYB30的主要SUMO化位点，所以我们推测SUMO化修饰通过占据K283位点来阻止MYB30的降解。ABA处理会增强MYB30蛋白和RHA2b的相互作用，而与SUMO E3连接酶SIZ1的互作没有增强。这些结果表明ABA处理条件下，MYB30蛋白可能通过RHA2b介导降解，而SUMO化修饰则起到维持MYB30稳定性的作用，两者共同调控了MYB30在ABA信号途径中的功能。本项目通过对ABA信号途径中MYB30蛋白稳定性的研究，揭示了植物泛素化和SUMO化修饰共同调节蛋白稳定性的一种可能机制。