NO诱导桃果实抗病中亚硝基谷胱甘肽还原酶及其介导的蛋白亚硝基化修饰的作用

Postharvest diseases in peach fruit cause great loss. Induced disease resistance is a kind of effective and promising method in controlling these diseases. More and more research in mode plants indicated that S-nitrosoglutathione reductase (GSNOR) was part of defense responses of plant by regulating the level of S-nitrosothiols (SNOs). Our previous study showed that nitric oxide (NO) could induce disease resistance of peach fruit, and several S-nitrosylated proteins were detected by surface plasmon resonance (SPR) and mass spectrometry (MS). Based on that evidence, this project is intended to (1) investigate the dynamic change of GSNOR and its relationship with endogenous NO and SNOs by the techniques of liquid chromatography- electrospray/MS (LC-ES/MS) and confocal laser scanning microscopy; (2) determine the key agents and gens in signal pathway regulated by GSNOR by molecular biology methods; (3) analyze the S-nitrosylated proteins mediated by GSNOR, target sites and peptide sequences of S-nitrosulated modification in proteins during the pathway of induced disease resistance by proteomics and SPR/MS techniques; (4) study the effects of S-nitrosylation or denitrosylation reactions on the structures of key transcriptional factors and their bonding activities of DNA by analyzing the interactions between DNA and protein or protein and protein. The mechanisms of the role of GSNOR and protein S-nitrosylation modification mediated by it in disease resistance induced by nitric oxide in peach fruit will be deeply elucidated by the research. The successful conduction of this project will be important for providing theoretical foundation for the application of new elicitors of induced disease resistance in postharvest fruits.

采后病害导致桃严重损失。模式植物中亚硝基谷胱甘肽还原酶（GSNOR）可通过调控亚硝基硫醇的转移参与多种防御反应，GSNOR是否在诱导桃果实抗病中有重要作用，国内外鲜见报道。我们前期研究证实，NO可诱导桃果实提高抗病性，并检测到蛋白亚硝基化修饰现象。由此推测GSNOR及其介导的蛋白亚硝基化修饰可能在诱导桃抗病中起重要作用，因此，本项目拟利用激光共聚焦扫描显微镜和LC-ES/MS技术研究桃果实抗病中GSNOR的动态变化及与亚硝基硫醇、NO的关系；研究受GSNOR调控的诱导抗病途径关键因子的变化；通过蛋白组学、SPR-MS技术研究诱导抗病中GSNOR介导的亚硝基化靶标蛋白、作用位点和肽段序列；通过蛋白质互作研究抗病途径亚硝基化修饰对关键转录因子结构和DNA结合活性的影响，以期揭示GSNOR及其介导的蛋白亚硝基化修饰在NO诱导抗病中的作用机理，为探寻诱导采后果实抗病性的新方法提供理论基础和新思路。

桃果实采后腐烂严重，其中由链核盘菌（Monilinia fructicola）引起的褐腐病是采后的主要病害之一。诱导抗病性为果实病害安全防治提供了一种新思路。前期研究证实，一氧化氮（nitric oxide, NO）可诱导采后桃果实提高抗病性，NO作为抗病反应的第二信使，在模式植物中可以通过亚硝基硫醇（S-nitrosothiols, SNOs）的转移和蛋白亚硝基化修饰作用完成整个信号传导过程。亚硝基谷胱甘肽还原酶（S-nitrosoglutathione reductase, GSNOR）参与NO信号传导，通过催化调节SNOs的水平和蛋白亚硝基化作用参与植物防御反应。但采后桃果实内GSNOR的水平变化与NO诱导抗病性的关系尚未见研究报道。.本研究发现，NO诱导果实抗病过程可抑制GSNOR的活性，但是对其基因表达的影响是瞬时的，与对照相比，只在处理后6h之前有显著性差异。但是通过亚硝基化蛋白组学结合质谱分析发现，NO处理后6h和12h均可引起GSNOR半胱氨酸85号位（Cys-85）发生巯基亚硝基化反应，说明NO在诱导桃果实抗病过程中主要通过蛋白亚硝基化作用调控GSNOR活性。对NO处理后4 h的差异蛋白进行GO/Domain/KEGG Pathway富集分析，发现在差异蛋白中，上调蛋白显著富集在“叶绿体”、“质体”等细胞组成中，显著富集在“酪氨酸代谢”等代谢途径；下调蛋白显著富集在“金属离子跨膜转运蛋白活性”和“锌离子结合”等分子功能中，显著富集在 “谷胱甘肽代谢”等代谢途径。用N6022抑制GSNOR活性可以提高桃果实抗褐腐病的能力，同时抑制了GSNOR基因的表达和酶活性。NO和N6022均可以提高果实GSNO、内源NO和亚硝基硫醇（SNOs）的含量，果实病程相关蛋白（PR1）、PR1非表达子（NPR1）以及水杨酸结合蛋白（TGA1）的表达量显著上升，说明NO和N6022处理均提高了果实抗病性。用VIGS方法对GSNOR基因进行沉默后发现，果实抗病性降低，PR1表达量下降，蛋白亚硝基化组学结果发现，沉默果实中上调差异蛋白和差异亚硝基化位点数增多，说明桃果实抗病能力需要一定水平的GSNOR。. 本项目揭示了NO诱导桃果实抗病过程中GSNOR的作用，不仅为阐明果实诱导抗病性分子机理提供了理论依据，也为减少果实采后损失提供了实践基础。