拟南芥SUMO E3连接酶SIZ1调控的植物盐胁迫反应

Post-translational modification is one of the major mechanisms that regualting the biological function or activity of a protein in eucaryotes. SUMO (small ubiquitin-like modifier) is a kind of small peptides that can conjugate to protein substrates. Unlike ubiquitination causes protein degradation, SUMOylation may affect the protein localization, protein stability and activity, protein interaction with other proteins or molecules. Therefore SUMOylation regulates diverse cellular functions, such as innate immunity, DNA repair, cell division, subnuclear targeting. E3 ligase contributes to the substrate specificity and plays key role in protein SUMOylation. Arabidopsis genome has four genes encoding SUMO E3 ligase, only one has been determined in its function and named as AtSIZ1. SIZ1 conducting its ligase function has been confirmed in protein modification assay. It facilitates SUMOylation of transcription factors such as ICE1 and ABI5 in vivo, which plays key roles in signal transduction pathways by controling the down-stream target gene expression. Moreover, disruption of SIZ1 function has been reported to affect plant growth development, pathogen defense, ABA reaction, flowering time control, phosphate deficiency response and abiotic stress response,etc., suggesting that SIZ1 plays important roles in diverse cellular events. In the current project, we are planing to investigate the protein SUMOylation change in response to salt stress. By using the proteomic approaches, identify salt stress responsible proteins that are directily or indirectly regulated by the function of E3 ligase AtSIZ1. To identify the substrates of AtSIZ1, with the aim at elucidating the regulatory mechanisms of protein SUMOylation in plant development and salt stress tolerance.

翻译后修饰是真核细胞中蛋白质发挥生物学功能的重要调节机制之一。SUMO化修饰是SUMO分子与靶蛋白的共价结合。与泛素化修饰主要介导蛋白质的降解功能不同，SUMO化修饰参与了蛋白质在细胞内的定位、稳定性与活性的调节，底物蛋白同其他分子的相互作用，因而更广泛地参与了细胞内代谢过程和信号转导途径。其中E3连接酶是SUMO化修饰反应中的一个关键酶，负责识别修饰底物。拟南芥的研究表明E3连接酶SIZ1通过对逆境胁迫信号转导途径中关键转录因子的SUMO化修饰，启动或关闭下游基因，实现对胁迫信号响应的调控。本项目拟应用蛋白质组学、生物化学和分子生物学等技术手段，研究蛋白SUMO化修饰在植物盐胁迫反应中的变化，在蛋白质组水平上分离受SUMO化修饰调控的盐胁迫响应蛋白，分离E3连接酶SIZ1的作用靶蛋白，揭示植物体内SUMO化修饰的机制以及在植物生长发育、适应逆境中的作用机理。

本项目研究了拟南芥SIZ1在植物抗盐反应中的作用，重点研究了RACK1蛋白SUMO化修饰模式以及RACK1B的SUMO化修饰介导逆境信号的作用机制。通过研究发现E3连接酶SIZ1是盐胁迫响应的正调控因子，盐胁迫条件下，拟南芥蛋白SUMO化水平升高，siz1突变体中蛋白SUMO化对盐胁迫的响应显著降低。项目细致研究了脚手架蛋白RACK1的SUMO化修饰模式。发现了RACK1A和RACK1B但不包括另一个同源蛋白RACK1C可以在体外被SUMO1/2分子修饰，并且这一修饰并不依赖E3连接酶SIZ1。RACK1B蛋白序列中存在4个SUMO化修饰赖氨酸位点，其中K276是主要的修饰位点。在体内实验中发现，ABA处理促进了RACK1B蛋白的SUMO化修饰，并且SUMO化修饰通过竞争相同的泛素化修饰赖氨酸位点，提高了RACK1B的稳定性。SUMO修饰的RACK1B增加了对转录因子RAP2.6的结合，并且改变了转录因子对下游不同顺式作用元件的新合力。另外，RACK1B对钾内流离子通道活性具有抑制作用。在盐胁迫应答上，RACK1B通过抑制AKT1活性，抑制根细胞中钾内流，因此在植物抗盐中具有副调控作用。在应对干旱胁迫时，RACK1B抑制保卫细胞中KAT1活性，从而降低保卫细胞膨压，促进气孔关闭，因此在植物抗旱中具有正调控作用。