Disrupted Nitric Oxide Homeostasis Impacts Fertility Through Multiple Processes Including Protein Quality Control
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.