Nitrate fatty acids (NO2-FAs) are considered reactive lipid species derived from the non-enzymatic oxidation of polyunsaturated fatty acids by nitric oxide (NO) and related species. Nitrate fatty acids are powerful biological electrophiles which can react with biological nucleophiles such as glutathione and certain protein–amino acid residues. The adduction of NO2-FAs to protein targets generates a reversible post-translational modification called nitroalkylation. In different animal and human systems, NO2-FAs, such as nitro-oleic acid (NO2-OA) and conjugated nitro-linoleic acid (NO2-cLA), have cytoprotective and anti-inflammatory influences in a broad spectrum of pathologies by modulating various intracellular pathways. However, little knowledge on these molecules in the plant kingdom exists. The presence of NO2-OA and NO2-cLA in olives and extra-virgin olive oil and nitro-linolenic acid (NO2-Ln) in Arabidopsis thaliana has recently been detected. Specifically, NO2-Ln acts as a signaling molecule during seed and plant progression and beneath abiotic stress events. It can also release NO and modulate the expression of genes associated with antioxidant responses. Nevertheless, the repercussions of nitroalkylation on plant proteins are still poorly known. In this review, we demonstrate the existence of endogenous nitroalkylation and its effect on the in vitro activity of the antioxidant protein ascorbate peroxidase.

Nitro-fatty acids (NO 2-FAs) are novel molecules resulting from the interaction of unsaturated fatty acids and nitric oxide (NO) or NO-related molecules. In plants, it has recently been described that NO 2-FAs trigger an antioxidant and a defence response against stressful situations. Among the properties of NO 2-FAs highlight the ability to release NO therefore modulating specific protein targets through post-translational modifications (NO-PTMs). Thus, based on the capacity of NO 2-FAs to act as physiological NO donors and using high-accuracy mass-spectrometric approaches, herein, we show that endogenous nitro-linolenic acid (NO 2-Ln) can modulate Snitrosoglutathione (GSNO) biosynthesis in Arabidopsis. The incubation of NO 2-Ln with GSH was analyzed by LC-MS/MS and the in vitro synthesis of GSNO was noted. The in vivo confirmation of this behavior was carried out by incubating Arabidopsis plants with 15 N-labeled NO 2-Ln throughout the roots, and 15 N-labeled GSNO (GS 15 NO) was detected in the leaves. With the aim to go in depth in the relation of NO 2-FA and GSNO in plants, Arabidopsis alkenal reductase mutants (aer mutants) which modulate NO 2-FAs levels were used. Our results constitute the first evidence of the modulation of a key NO biological reservoir in plants (GSNO) by these novel NO 2-FAs, increasing knowledge about S-nitrosothiols and GSNO-signaling pathways in plants.

Heat stress is one of the abiotic stresses that leads to oxidative stress. To protect themselves, yeast cells activate the antioxidant response, in which cytosolic peroxiredoxin Tsa1 plays an important role in hydrogen peroxide removal. Concomitantly, the activation of the heat shock response (HSR) is also triggered. Nitro-fatty acids are signaling molecules generated by the interaction of reactive nitrogen species with unsaturated fatty acids. These molecules have been detected in animals and plants. They exert their signaling function mainly through a post-translational modification called nitroalkylation. In addition, these molecules are closely related to the induction of the HSR. In this work, the endogenous presence of nitro-oleic acid (NO2-OA) in Saccharomyces cerevisiae is identified for the first time by LC-MS/MS. Both hydrogen peroxide levels and Tsa1 activity increased after heat stress with no change in protein content. The nitroalkylation of recombinant Tsa1 with NO2-OA was also observed. It is important to point out that cysteine 47 (peroxidatic) and cysteine 171 (resolving) are the main residues responsible for protein activity. Moreover, the in vivo nitroalkylation of Tsa1 peroxidatic cysteine disappeared during heat stress as the hydrogen peroxide generated in this situation caused the rupture of the NO2-OA binding to the protein and, thus, restored Tsa1 activity. Finally, the amino acid targets susceptible to nitroalkylation and the modulatory effect of this PTM on the enzymatic activity of Tsa1 are also shown in vitro and in vivo. This mechanism of response was faster than that involving the induction of genes and the synthesis of new proteins and could be considered as a key element in the fine-tuning regulation of defence mechanisms against oxidative stress in yeast.

Nitro-fatty acids (NO2-FAs), generated from the interaction of unsaturated fatty acids and nitric oxide (NO)-derived molecules, have been recently proposed to act as mediators of cell signaling in plant systems. Following in this line, the endogenous occurrence and modulation throughout plant development of nitro-linolenic acid (NO2-Ln) and nitro-oleic acid (NO2-OA) suggest a key role of these molecules in initial development stages. In addition, NO2-Ln content significantly rises in different stress situations like wounding, salinity, cadmium or low temperature. Interestingly, NO2-Ln induces the expression of the genes mainly related to abiotic stress such as different members of the heat shock response (HSR) family genes, and antioxidant enzymes like ascorbate peroxidase (APX) and methionine sulfoxide reductase B (MSRB). The promoter regions of NO2-Ln-induced genes are also involved mainly in stress responses. All these findings confirm that NO2-Ln is involved in plant defense processes against abiotic stress conditions via the induction of the chaperone network and antioxidant systems. NO2-Ln signaling capacity lies mainly in its electrophilic nature and allows it to mediate a reversible post-translational modification called nitroalkylation, which is capable of modulating the protein function. NO2-Ln is a NO donor that may be involved in NO-signaling events and is able to generate S-nitrosoglutathione (GSNO), the major reservoir of NO in cells and a key player in NO-mediated abiotic stress responses. Given this background, this review describes the current state of the art regarding the essential role of NO2-FAs as signaling mediators in development and abiotic stress processes.

The non-enzymatic interaction of polyunsaturated fatty acids with nitric oxide (NO) and derived species results in the formation of nitrated fatty acids (NO2-FAs). These signaling molecules can release NO, reversibly esterify with complex lipids, and modulate protein function through the post-translational modification called nitroalkylation. To date, NO2-FAs act as signaling molecules during plant development in plant systems and are involved in defense responses against abiotic stress conditions. In this work, the previously unknown storage biomolecules of NO2-FAs in Arabidopsis thaliana were identified. In addition, the distribution of NO2-FAs in storage biomolecules during plant development was determined, with phytosterol esters (SE) and TAGs being reservoir biomolecules in seeds, which were replaced by phospholipids and proteins in the vegetative, generative, and senescence stages. The detected esterified NO2-FAs were nitro-linolenic acid (NO2-Ln), nitro-oleic acid (NO2-OA), and nitro-linoleic acid (NO2-LA). The last two were detected for the first time in Arabidopsis. The levels of the three NO2-FAs that were esterified in both lipid and protein storage biomolecules showed a decreasing pattern throughout Arabidopsis development. Esterification of NO2-FAs in phospholipids and proteins highlights their involvement in both biomembrane dynamics and signaling processes, respectively, during Arabidopsis plant development.