Assessing the physiological S nitrosoproteome reveals nitric oxide mediated regulatory networks in rice

Nitric oxide metabolism-based protein post-translational modifications, such as reversible S- nitrosylation, have been at the pinnacle of plant redox research owing to their significant correlation with seed dormancy, interaction with other signaling molecules, plant development and metabolism, biotic and abiotic plant stress responses, immune defense responses against plant pathogens, and senescence. The rapid interconversion of reactive nitrogen species, the abrogation of nitric oxide homeostasis by exogenous supplementation of NO donors and scavengers, the lack of spatio-temporal specificity of NO signaling, and the limited bioavailability or assay sensitivity for detection often limit the effectiveness of identifying and characterizing S-nitrosothiols in plants. Hitherto unknown, we report the first experimental evidence of the total in vivo S-nitrosoproteome in Oryza sativa L. subsp. indica, comprising 134 PSNOs, enriched with 169 putative sites susceptible to S-nitrosylation, without any exogenous supplementation of NO donors. In the present study, mercuric salt- driven facile decomposition of S-nitrosoproteins in the presence of nitrone spin trap 5,5- dimethyl-1-pyrroline N-oxide, resulting in the synthesis of DMPO-nitrone adducts with PSNO-derived protein thiyl radicals in O. sativa, has been demonstrated as an efficient and novel strategy for characterizing the PSNOs using mass spectrometry analysis. The evidence of physiological levels of PSNOs was further re-examined in a bi-directional qualitative and quantitative approach involving the 2,3-diaminonaphthalene assay in tandem with fluorescence-based visualization and fluorometric quantification. In silico analyses, involving both functional enrichment and pathway prediction analyses, have furthermore revealed unique protein-protein interaction networks and signaling pathways among the S- nitrosoproteome candidates and their predictable physiological roles in O. sativa indica, awaiting further in vitro validation for their functional correlation in response to S- nitrosylation. In conclusion, the present study provides novel evidence of nitric oxide signaling in rice cultivars under physiological conditions, bringing new insights into the potential in vivo transnitrosylation of regulatory or active-site cysteine thiols.