Abstract
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 floral tissues coupled with quantitative proteomics of pistils. To identify protein-SNOs we employed, for the first time in plants, 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 UGT-glycosyltransferases and argonaute proteins are S-nitrosated in floral tissues and differentially regulated in pistils. We also discovered S-nitrosation of proteins 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 new 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.