Plant fertility is fundamental to plant survival and requires the coordinated interaction of developmental pathways and signaling molecules. Nitric oxide (NO) is a small, gaseous signaling molecule that plays crucial roles in plant fertility as well as other developmental processes and stress responses. NO influences biological processes through S-nitrosation, the posttranslational modification of protein cysteines to S-nitrosocysteine (R-SNO). NO homeostasis is controlled by S-nitrosoglutathione reductase (GSNOR), which reduces S-nitrosoglutathione (GSNO), the major form of NO in cells. GSNOR mutants (hot5-2/gsnor1) have defects in female gametophyte development along with elevated levels of reactive nitrogen species and R-SNOs. To better understand the fertility defects in hot5-2, we investigated the in vivo nitrosoproteome of Arabidopsis (Arabidopsis thaliana) floral tissues coupled with quantitative proteomics of pistils. To identify protein-SNOs, we used an organomercury-based method that involves direct reaction with S-nitrosocysteine, enabling specific identification of S-nitrosocysteine–containing peptides and S-nitrosated proteins. We identified 1102 endogenously S-nitrosated proteins in floral tissues, of which 1049 were unique to hot5-2. Among the identified proteins, 728 were novel S-nitrosation targets. Notably, specific UDP-glycosyltransferases and argonaute proteins are S-nitrosated in floral tissues and differentially regulated in pistils. We also discovered S-nitrosation of subunits of the 26S proteasome together with increased abundance of proteasomal components and enhanced trypsin-like proteasomal activity in hot5-2 pistils. Our data establish a method for nitrosoprotein detection in plants, expand knowledge of the plant S-nitrosoproteome, and suggest that nitro-oxidative modification and NO homeostasis are critical to protein quality control in reproductive tissues.

中文翻译：

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一氧化氮稳态被破坏通过包括蛋白质质量控制在内的多个过程影响生育能力


植物肥力是植物生存的基础，需要发育途径和信号分子的协调相互作用。一氧化氮 （NO） 是一种小的气态信号分子，在植物肥力以及其他发育过程和胁迫反应中起着至关重要的作用。NO 通过 S-亚硝化影响生物过程，S-亚硝化是蛋白质半胱氨酸向 S-亚硝基半胱氨酸 （R-SNO） 的翻译后修饰。NO 稳态受 S-亚硝基谷胱甘肽还原酶 （GSNOR） 控制，GSNOR 可降低 S-亚硝基谷胱甘肽 （GSNO），这是细胞中 NO 的主要形式。GSNOR 突变体 （hot5-2/gsnor1） 在雌配子体发育方面存在缺陷，同时活性氮和 R-SNO 水平升高。为了更好地了解 hot5-2 的生育缺陷，我们研究了拟南芥 （Arabidopsis thaliana） 花组织的体内亚硝基蛋白质组与雌蕊的定量蛋白质组学。为了鉴定蛋白质 SNO，我们使用了一种基于有机汞的方法，该方法涉及与 S-亚硝基半胱氨酸的直接反应，从而能够特异性鉴定含 S-亚硝基半胱氨酸的肽和 S-亚硝酸盐蛋白质。我们在花组织中鉴定了 1102 个内源性 S-亚硝化蛋白，其中 1049 个是 hot5-2 独有的。在鉴定的蛋白质中，728 种是新的 S-亚硝化靶标。值得注意的是，特异性 UDP-糖基转移酶和精氨酸蛋白在花组织中被 S-亚硝化，并在雌蕊中受到差异调节。我们还发现了 26S 蛋白酶体亚基的 S-亚硝化，以及蛋白酶体成分丰度的增加和热 5-2 雌蕊中胰蛋白酶样蛋白酶体活性的增强。 我们的数据建立了一种植物亚硝基蛋白检测方法，扩展了植物 S-亚硝基蛋白质组的知识，并表明硝基氧化修饰和 NO 稳态对生殖组织中的蛋白质质量控制至关重要。