S-Nitrosylation Analysis in Brassica juncea Apoplast Highlights the Importance of Nitric Oxide in Cold-Stress Signaling

Abstract

Reactive nitrogen species (RNS) including nitric oxide (NO) are important components of stress signaling. However, RNS-mediated signaling in the apoplast remains largely unknown. NO production measured in the shoot apoplast of Brassica juncea seedlings showed nonenzymatic nitrite reduction to NO. Thiol pool quantification showed cold-induced increase in the protein (including S-nitrosothiols) as well as non protein thiols. Proteins from the apoplast were resolved as 109 spots on the 2-D gel, while S-nitrosoglutathione-treated (a NO donor), neutravidin-agarose affinity chromatography-purified S-nitrosylated proteins were resolved as 52 spots. Functional categorization after MALDI-TOF/TOF identification showed 41 and 38% targets to be metabolic/cell-wall-modifying and stress-related, respectively, suggesting the potential role(s) of S-nitrosylation in regulating these responses. Additionally, identification of cold-stress-modulated putative S-nitrosylated proteins by nLC-MS/MS showed that only 38.4% targets with increased S-nitrosylation were secreted by classical pathway, while the majority (61.6%) of these were secreted by unknown/nonclassical pathways. Cold-stress-increased dehydroascorbate reductase and glutathione S-transferase activity via S-nitrosylation and promoted ROS detoxification by ascorbate regeneration and hydrogen peroxide detoxification. Taken together, cold-mediated NO production, thiol pool enrichment, and identification of the 48 putative S-nitrosylated proteins, including 25 novel targets, provided the preview of RNS-mediated cold-stress signaling in the apoplast.

Identification of nuclear target proteins for S-nitrosylation in pathogen-treated Arabidopsis thaliana cell cultures

“…The number of identified S-nitrosylated candidate proteins is higher in the nucleus than in other organelles. For example in mitochondria 11 proteins were identified as targets for S-nitrosylation [13], 6 candidate proteins were identified in peroxisome [20], and 48 candidates in apoplast [49]. However, as mentioned above a computational prediction of target proteins for S-nitrosylation in Arabidopsis thaliana showed that most candidates were appointed to the "nucleus" (5924 proteins) [52].…”

“…In plants, the biotin switch assay has becoming a credible method for identification of candidate proteins for S-nitrosylation [8]. Proteomic analysis of S-nitrosylation has been studied in different plant species as well as in isolated organelles like mitochondria [13,21], peroxisome [20], and apoplast [49]. The nucleus is an important sub-cellular compartment, which consists of almost all information needed for the regulation of gene expression.…”

Assessing Nitric Oxide (NO) in Higher Plants: An Outline

“…In plants, the number of identified proteins susceptible to undergo this NO-mediated modification under physiological and stress conditions, as well as how this NO-PTM could affect the function of the target proteins, is continually increasing [28][29][30][31][32][33][34][35]. Taking into consideration that this process is reversible, S-nitrosation should be assessed as a relevant mechanism of NO signal transduction in the cells.…”

Asada-Halliwell pathway maintains redox status in Dioscorea alata tuber which helps in germination

“…Furthermore, these NO-derived molecules mediate post-translational modifications (PTMs) like S-nitrosylation which is the binding of an NO group to thiol in a protein cysteine residue [19]. Proteomic studies have identified S-nitrosylated proteins belonging to diverse functional categories such as redox, stress, metabolism and signalling [20][21][22]. The identified targets also included components of the ASC-GSH cycle [23].…”