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 ( approximately 40%) inactivation of Rubisco. This in turn could explain cold stress-induced photosynthetic inhibition.

Nitric oxide (NO), a water-and lipid-soluble gaseous free radical, has emerged as a key signaling molecule in plants. Pharmacological investigations using NO donors and inhibitors have implicated NO in diverse processes, from seed germination to cell death [1][2][3]. However, information about the NO-mediated signal transduction pathway(s) or the components involved is limited. An important biological role of NO may involve post-translational modification of proteins by: (i) S-nitrosylation of thiol groups, (ii) nitration of tyrosine and tryptophan (biological nitration), (iii) oxidation of thiols and tyrosine, and (iv) binding to metal centers [4]. S-nitrosylation of cysteine residues in the target protein is a principle and reversible modification by NO mediating its cyclic guanosine monophosphate (cGMP)-independent effects [5].NO nitrosylates transition metals, whereas NO-derived species such as NO 2 , N 2 O 3 and transition metal-NO adducts nitrosylate cysteine residues in proteins. Low-molecular-weight nitrosothiols such as S-nitrosoglutathione (GSNO) nitrosylate target proteins via transnitrosation, which involves direct transfer of a NO group [6]. S-nitrosylation further promotes disulfide bond formation in the neighboring Nitric oxide (NO) is a signaling molecule that affects a myriad of processes in plants. However, the mechanistic details are limited. NO post-translationally modifies proteins by S-nitrosylation of cysteines. The soluble S-nitrosoproteome of a medicinal, crassulacean acid metabolism (CAM) plant, Kalanchoe pinnata, was purified using the biotin switch technique. Nineteen targets were identified by MALDI-TOF mass spectrometry, including proteins associated with carbon, nitrogen and sulfur metabolism, the cytoskeleton, stress and photosynthesis. Some were similar to those previously identified in Arabidopsis thaliana, but kinesin-like protein, glycolate oxidase, putative UDP glucose 4-epimerase and putative DNA topoisomerase II had not been identified as targets previously for any organism. In vitro and in vivo nitrosylation of ribulose-1,5-bisphosphate carboxylase ⁄ oxygenase (Rubisco), one of the targets, was confirmed by immunoblotting. Rubisco plays a central role in photosynthesis, and the effect of S-nitrosylation on its enzymatic activity was determined using NaH 14 CO 3 . The NO-releasing compound S-nitrosoglutathione inhibited its activity in a dose-dependent manner suggesting Rubisco inactivation by nitrosylation for the first time.Abbreviations

Plants growing in temperate regions are able to survive freezing temperatures from -5 degrees to -30 degrees C, depending on the species, through a process known as cold acclimation. In the last decade much work has been done on the molecular mechanisms of low temperature (LT) signal transduction and cold acclimation. Mutant studies and microarray analyses have revealed C-Repeat binding factor (CBF) -dependent and -independent signaling pathways in plants. Experimental evidence suggests the existence of 'potential LT sensors' but as yet there is no direct proof. A number of signal transducers such as various kinases/phosphatases have been demonstrated but the signal transduction pathways have not been elucidated. An understanding of the molecular basis of the signaling process, however, is of potential practical application. Designing new strategies to improve cold tolerance in crop varieties could increase the plant productivity and also expand the area under cultivation.

Plants' distribution and productivity are adversely affected by low temperature (LT) stress. LT induced proteins were analyzed by 2-DE-nano-LC–MS/MS in shoot secretome of Hippophae rhamnoides (seabuckthorn), a Himalayan wonder shrub. Seedlings were subjected to direct freezing stress (−5 °C), cold acclimation (CA), and subzero acclimation (SZA), and extracellular proteins (ECPs) were isolated using vacuum infiltration. Approximately 245 spots were reproducibly detected in 2-DE gels of LT treated secretome, out of which 61 were LT responsive. Functional categorization of 34 upregulated proteins showed 47% signaling, redox regulated, and defense associated proteins. LT induced secretome contained thaumatin like protein and Chitinase as putative antifreeze proteins (AFPs). Phase contrast microscopy with a nanoliter osmometer showed hexagonal ice crystals with 0.13 °C thermal hysteresis (TH), and splat assay showed 1.5-fold ice recrystallization inhibition (IRI), confirming antifreeze activity in LT induced secretome. A 41 kDa polygalacturonase inhibitor protein (PGIP), purified by ice adsorption chromatography (IAC), showed hexagonal ice crystals, a TH of 0.19 °C, and 9-fold IRI activity. Deglycosylated PGIP retained its AFP activity, suggesting that glycosylation is not required for AFP activity. This is the first report of LT modulated secretome analysis and purification of AFPs from seabuckthorn. Overall, these findings provide an insight in probable LT induced signaling in the secretome.

Although in the last few years good number of S-nitrosylated proteins are identified but information on endogenous targets is still limiting. Therefore, an attempt is made to decipher NO signaling in cold treated Brassica juncea seedlings. Treatment of seedlings with substrate, cofactor and inhibitor of Nitric-oxide synthase and nitrate reductase (NR), indicated NR mediated NO biosynthesis in cold. Analysis of the in vivo thiols showed depletion of low molecular weight thiols and enhancement of available protein thiols, suggesting redox changes. To have a detailed view, S-nitrosylation analysis was done using biotin switch technique (BST) and avidin-affinity chromatography. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is S-nitrosylated and therefore, is identified as target repeatedly due to its abundance. It also competes out low abundant proteins which are important NO signaling components. Therefore, RuBisCO was removed (over 80%) using immunoaffinity purification. Purified S-nitrosylated RuBisCO depleted proteins were resolved on 2-D gel as 110 spots, including 13 new, which were absent in the crude S-nitrosoproteome. These were identified by nLC-MS/MS as thioredoxin, fructose biphosphate aldolase class I, myrosinase, salt responsive proteins, peptidyl-prolyl cis-trans isomerase and malate dehydrogenase. Cold showed differential S-nitrosylation of 15 spots, enhanced superoxide dismutase activity (via S-nitrosylation) and promoted the detoxification of superoxide radicals. Increased S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase sedoheptulose-biphosphatase, and fructose biphosphate aldolase, indicated regulation of Calvin cycle by S-nitrosylation. The results showed that RuBisCO depletion improved proteome coverage and provided clues for NO signaling in cold.