Cold modulated nuclear S-nitrosoproteome analysis indicates redox modulation of novel Brassicaceae specific, myrosinase and napin in Brassica juncea

Stress leads to production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Among the RNS, nitric oxide (NO) is an important signaling molecule that mediates its effect majorly via a reversible post-translational modification (PTM) S-nitrosylation. However, information on the NO mediated cold stress signaling is still missing for nucleus. Therefore, in the present study an attempt was made to analyze NO signaling, particularly, Snitrosylation in the nuclei of Brassica juncea seedlings. Cold stress (4 degrees C) induced nitrate reductase (NR) mediated NO production along with the accumulation of available protein thiols (5 fold, at 96 h, 4 degrees C) and low molecular weight thiols (1.2 fold at 12h, 4 degrees C). S-nitrosoglutathione (GSNO, a NO donor) and cold (endogenous, S-nitrosylation without the addition of NO donor) treated, neutravidin-agarose affinity chromatography purified S-nitrosylated proteins were resolved as 55- and 32-spots respectively on the 2-D gels. Functional categorization after MALDI-TOF/TOF identification showed 67% targets to be stress/defense/redox related and 33% to be metabolic, suggesting the potential role(s) of S-nitrosylation in these responses. Cold stress increased S-nitrosylation of Cu/Zn SOD, L-ascorbate oxidase, fructose bisphosphate aldolase (FBA2) and malate dehydrogenase 1 (MDH1), therefore, indicating NO mediated regulation of redox and metabolism related pathways. Identification of targets of glucosinolate hydrolysis pathway (myrosinase and myrosinase-binding protein 2-like) indicated the role of S-nitrosylation in regulating mustard oil bomb. Presence of myrosinase was confirmed by activity measurements and western blotting. Increased S-nitrosylation of myrosinase in cold stress in the nucleus is reported for the first time. Here, we proposed S-nitrosylation mediated nuclear trafficking of non-nuclear targets such as beta-D-xylosidase, daikon cysteine protease, and beta-glucosidase to the nucleus. Similarly, identification of FBA2 and MDH1 in the nucleus, indicated the role of NO in modulating their moonlighting function via S-nitrosylation in stress. Present study provided the first repertoire of cold modulated S-nitrosylated proteins in the nucleus.