Post-translational modifications (PTMs) mediated by nitric oxide (NO)-derived molecules have become a new area of research, as they can modulate the function of target proteins. Proteomic data have shown that ascorbate peroxidase (APX) is one of the potential targets of PTMs mediated by NO-derived molecules. Using recombinant pea cytosolic APX, the impact of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO), which are known to mediate protein nitration and S-nitrosylation processes, respectively, was analysed. While peroxynitrite inhibits APX activity, GSNO enhances its enzymatic activity. Mass spectrometric analysis of the nitrated APX enabled the determination that Tyr5 and Tyr235 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Residue Cys32 was identified by the biotin switch method as S-nitrosylated. The location of these residues on the structure of pea APX reveals that Tyr235 is found at the bottom of the pocket where the haem group is enclosed, whereas Cys32 is at the ascorbate binding site. Pea plants grown under saline (150mM NaCl) stress showed an enhancement of both APX activity and S-nitrosylated APX, as well as an increase of H2O2, NO, and S-nitrosothiol (SNO) content that can justify the induction of the APX activity. The results provide new insight into the molecular mechanism of the regulation of APX which can be both inactivated by irreversible nitration and activated by reversible S-nitrosylation.
ORCID IDs: 0000-0002-6609-7242 (C.M.-P); 0000-0002-2210-9547 (B.S.-C.); 0000-0001-5284-8837 (J.J.-R.); 0000-0002-1814-9212 (F.J.C.); 0000-0002-9477-9195 (J.J.B.).Nitro-fatty acids (NO 2 -FAs) are the product of the reaction between reactive nitrogen species derived of nitric oxide (NO) and unsaturated fatty acids. In animal systems, NO 2 -FAs are considered novel signaling mediators of cell function based on a proven antiinflammatory response. Nevertheless, the interaction of NO with fatty acids in plant systems has scarcely been studied. Here, we examine the endogenous occurrence of nitro-linolenic acid (NO 2 -Ln) in Arabidopsis and the modulation of NO 2 -Ln levels throughout this plant's development by mass spectrometry. The observed levels of this NO 2 -FA at picomolar concentrations suggested its role as a signaling effector of cell function. In fact, a transcriptomic analysis by RNA-seq technology established a clear signaling role for this molecule, demonstrating that NO 2 -Ln was involved in plant defense response against different abiotic-stress conditions, mainly by inducing heat shock proteins and supporting a conserved mechanism of action in both animal and plant defense processes. Bioinformatics analysis revealed that NO 2 -Ln was also involved in the response to oxidative stress conditions, mainly depicted by H 2 O 2 , reactive oxygen species, and oxygen-containing compound responses, with a high induction of ascorbate peroxidase expression. Closely related to these results, NO 2 -Ln levels significantly rose under several abiotic-stress conditions such as wounding or exposure to salinity, cadmium, and low temperature, thus validating the outcomes found by RNA-seq technology. Jointly, to our knowledge, these are the first results showing the endogenous presence of NO 2 -Ln in Arabidopsis (Arabidopsis thaliana) and supporting the strong signaling role of these molecules in the defense mechanism against different abiotic-stress situations.
The effect of ripening stage and water regimen on oleate desaturase gene expression levels in the fruit of different olive ( Olea europaea L.) varieties was investigated to elucidate the contribution of each to the linoleic acid content in virgin olive oil. To this end, fatty acid analysis and quantitative real time PCR were performed using distinct olive tissues and different developmental stages from the Picual and Arbequina cultivars. The results showed that the olive FAD2-1, FAD2-2, and FAD6 genes were spatial and temporally regulated. In addition, the data indicated that FAD2-2 seems to be the main gene responsible for the linoleic acid content in the olive fruit mesocarp tissue. This conclusion was also confirmed when the study was extended to Hojiblanca, Picudo, and Manzanilla varieties. With regard to the water regimen, unlike the Picual cultivar, a small increase of linoleic acid was observed in the Arbequina variety cultivated with irrigation, which correlated well with the increase detected for the FAD2-2 gene expression level. All of these data strongly suggest that FAD2-2 is the main gene that determines the linoleic acid content in the virgin olive oil.
HighlightNitration and S-nitrosylation, two post-translational modifications (PTMs) mediated by nitric oxide, differentially regulate MDAR and GR activities which are key components of the ascorbate–glutathione cycle.
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