Shekariesfahlan Azam, Lindermayr Christian
Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran.
Helmholtz Zentrum München-German Research GmbH, Center for Environment Health, Institute of Biochemical Plant Pathology, Ingolstädter Landstraße 1, 85764, Munich-Neuherberg, Germany.
Methods Mol Biol. 2018;1747:205-221. doi: 10.1007/978-1-4939-7695-9_16.
Nitric oxide (NO) is an important signaling molecule involved in various plant physiological processes. The main effect of NO arises from its reaction with proteins. S-Nitrosation is the most studied NO-mediated protein posttranslational modification in plants. Via S-nitrosation, NO derivatives react with thiol groups (SHs) of protein cysteine residues and produce nitrosothiol groups (SNOs). From the time of discovering the biological function of NO in plants, an interesting case of study has been the detection of the endogenous S-nitrosated proteins in different plants, tissues, organelles, and various conditions. Maps of S-nitrosated proteins provide hints for deeper studies on the function of this modification in specific proteins, biochemical pathways, and physiological processes. Many functions of NO have been found to be related to plant defense; on the other hand the involvement of nuclear proteins in regulation of plant defense reactions is well studied. Here, an approach is described in which the Arabidopsis cell cultures first are treated with P. syringae, afterward their bioactive nuclear proteins are extracted, then the nuclear proteins are subjected to biotin switch assay in which S-nitrosated proteins are specifically converted to S-biotinylated proteins. The biotin switch technique (BST) which was introduced by Jaffrey et al. (Nat Cell Biol 3:193-197, 2001) solves the instability issue of SNOs. Additionally, it provides detection and purification of biotinylated proteins by anti-biotin antibody and affinity chromatography, respectively.
一氧化氮（NO）是参与多种植物生理过程的重要信号分子。NO的主要作用源于其与蛋白质的反应。S-亚硝基化是植物中研究最多的NO介导的蛋白质翻译后修饰。通过S-亚硝基化，NO衍生物与蛋白质半胱氨酸残基的巯基（SHs）反应，产生亚硝基硫醇基团（SNOs）。从发现NO在植物中的生物学功能之时起，一个有趣的研究案例就是检测不同植物、组织、细胞器以及各种条件下的内源性S-亚硝基化蛋白质。S-亚硝基化蛋白质图谱为深入研究这种修饰在特定蛋白质、生化途径和生理过程中的功能提供了线索。已发现NO的许多功能与植物防御有关；另一方面，核蛋白在植物防御反应调控中的作用也得到了充分研究。在此，描述了一种方法，即先用丁香假单胞菌处理拟南芥细胞培养物，然后提取其生物活性核蛋白，接着对核蛋白进行生物素转换分析，其中S-亚硝基化蛋白质被特异性转化为S-生物素化蛋白质。Jaffrey等人（《自然细胞生物学》3:193 - 197，2001）引入的生物素转换技术（BST）解决了SNOs的不稳定性问题。此外，它分别通过抗生物素抗体和亲和色谱提供了生物素化蛋白质的检测和纯化方法。
