Nitric oxide (NO), a new addition to plant hormones, affects numerous processes in planta. It is produced as a part of stress response, but its signaling is poorly understood. S-nitrosylation, a PTM, is currently the most investigated modification of NO. Recent studies indicate significant modulation of metabolome by S-nitrosylation, as the identified targets span major metabolic pathways and regulatory proteins. Identification of S-nitrosylation targets is necessary to understand NO signaling. By combining biotin switch technique and MS, 20 S-nitrosylated proteins including four novel ones were identified from Brassica juncea. Further, to know if the abiotic stress-induced NO evolution contributes to S-nitrosothiols (SNO), the cellular NO reservoirs, SNO content was measured by Saville method. Low temperature (LT)-stress resulted in highest (1.4-fold) SNO formation followed by drought, high temperature and salinity. LT induced differentially nitrosylated proteins were identified as photosynthetic, plant defense related, glycolytic and signaling associated. Interestingly, both the subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) showed an increase as well as a decrease in nitrosylation by LT. Inactivation of Rubisco carboxylase by LT is well documented but the mechanism is not known. Here, we show that LT-induced S-nitrosylation is responsible for significant (∼40%) inactivation of Rubisco. This in turn could explain cold stress-induced photosynthetic inhibition.