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.

Overwintering plants secrete antifreeze proteins (AFPs) to provide freezing tolerance. These proteins bind to and inhibit the growth of ice crystals that are formed in the apoplast during subzero temperatures. Antifreeze activity has been detected in more than 60 plants and AFPs have been purified from 15 of these, including gymnosperms, dicots and monocots. Biochemical characterization of plant antifreeze activity, as determined by the high ice recrystallization inhibition (IRI) activities and low thermal hysteresis (TH) of AFPs, showed that their main function is inhibition of ice crystal growth rather than the lowering of freezing temperatures. However, recent studies showed that antifreeze activity with higher TH also exists in plants. Calcium and hormones like ethylene and jasmonic acid have been shown to regulate plant antifreeze activity. Recent studies have shown that plant AFPs bind to both prism planes and basal planes of ice crystals by means of two flat ice binding sites. Plant AFPs have been postulated to evolve from the OsLRR-PSR gene nearly 36 million years ago. In this review, we present the current scenario of plant AFP research in order to understand the possible potential of plant AFPs in generation of freezing-tolerant crops.

Glyoxalases I and I1 have been studied and characterized in animals and in micro-organisms [ 11, but the work on glyoxalases in plants is still restricted to a few reports. Resides having a role in growth and development [ 1, 21, the enzymes help in the protection against ketoaldehyde toxicity [3, 41, as methylglyoxal, which is cytotoxic at high concentrations [S] is used as the substrate. Glyoxalases also affect microtubule assembly in cell-free systems [6] and are involved in vesicle mobilization [7, 81 and in various diseases [9-131 as well. Establishment of the presence of the glyoxalase system in plantsThe activity of glyoxalase I has been measured in a gymnosperm and a role for it in needle development has been suggested [ 141. Glyoxalase I and I1 levels were measured in Douglas Fir needles and calli. It was observed that a vigorously dividing system, like the callus, had more glyoxalase I and less methylglyoxal than a more quiescent system, like the needles, which had a very high methylglyoxal level and undetectable levels of glyoxalase I. Hesides glyoxalase I and 11, the level of methylglyoxal synthetase and reductase were measured as well in the same system. Methylglyoxal synthetase was detected in mature needles, while methyl reductase was present in young, dividing needles and in calli. It was suggested that it is the level of methylglyoxal and glyoxalase that controls proper needle development [ 141. W e have investigated the function and regulation of glyoxalase I in higher plants over the last few years. In 1983 we showed the presence of the enzyme in germinating pea seedlings [ 151. Later the enzyme activity was also detected in wheat, Datura, Brassica, Nicotiana and Amaranthus[ 16-181. The effect of various exogenous factors like temperature, pH, light, hormones, growth inhibitors, cell-division inhibitors and cell-division stimulators, that is, polyamines was studied in a number of plant systems. T o identify a source from which to purify the enzyme, a number of species of Brassica were screened for enzyme levels. B. juncea, B. oleracea, B. napus and B. carinata showed comparable activities while the activity was lower in B. nigra and B. campestris (Table 1). Interestingly it has been observed that the latter two mustard species are quite difficult to grow in in vitro cultures and it is possible that high levels of the enzyme make the other species easy to work with in vitro. The enzyme was purified to homogeneity from 7-dayold B. juncea [ 191. The enzyme migrated as a single band of 27 kDa on a 10% SDSIPAGE gel. W e have I993

Plants produce secondary metabolites in response to various external signals. Coordinated transcriptional control of biosynthetic genes emerges as a major mechanism dictating the accumulation of secondary metabolites in plant cells. However, information about stress regulation of secondary metabolites and the molecular mechanisms regulating these specialized pathways are poorly understood. Here, we show that terpenoid indole alkaloid (TIA) biosynthetic pathway is differentially regulated in response to different abiotic stresses in Catharanthus roseus, a model medicinal plant producing important anticancer and antihypertensive drugs. Semiquantitative RT-PCR analysis of TIA and related primary pathway genes in response to dehydration, low temperature, salinity, UV-light and wounding revealed their negative regulation in response to low temperature. HPLC analysis further supports the notion that TIA biosynthetic pathway is negatively controlled by low temperature stress. Furthermore, we report the cloning of a C-repeat binding transcription factor from C. roseus (CrCbf), belonging to AP2 class of transcription factor and possessed the NLS and CBF signature sequence characteristic of CBFs. CrCbf was found to be similar to Brassica Cbfs, whereas it was distant to monocot Cbfs. Southern analysis of CrCbf revealed the presence of more than one copy of CrCbf gene or other Cbf homologues in C. roseus genome. The transcription of CrCbf was found to be constitutive in response to low temperature but it showed differential distribution. The need for identifying novel transcription factors in understanding secondary metabolite biosynthesis is discussed.