Reversible S-nitrosylation of bZIP67 by peroxiredoxin IIE activity and nitro-fatty acids regulates the plant lipid profile.

Nitric oxide (NO) is a gasotransmitter required in a broad range of mechanisms controlling plant development and stress conditions. However, little is known about the specific role of this signaling molecule during lipid storage in the seeds. Here, we show that NO is accumulated in developing embryos and regulates the fatty acid profile through the stabilization of the basic/leucine zipper transcription factor bZIP67. NO and nitro-linolenic acid target and accumulate bZIP67 to induce the downstream expression of FAD3 desaturase, which is misregulated in a non-nitrosylable version of the protein. Moreover, the post-translational modification of bZIP67 is reversible by the trans-denitrosylation activity of peroxiredoxin IIE and defines a feedback mechanism for bZIP67 redox regulation. These findings provide a molecular framework to control the seed fatty acid profile caused by NO, and evidence of the in vivo functionality of nitro-fatty acids during plant developmental signaling.

Functions of nitric oxide-mediated post-translational modifications under abiotic stress.

Environmental conditions greatly impact plant growth and development. In the current context of both global climate change and land degradation, abiotic stresses usually lead to growth restriction limiting crop production. Plants have evolved to sense and respond to maximize adaptation and survival; therefore, understanding the mechanisms involved in the different converging signaling networks becomes critical for improving plant tolerance. In the last few years, several studies have shown the plant responses against drought and salinity, high and low temperatures, mechanical wounding, heavy metals, hypoxia, UV radiation, or ozone stresses. These threats lead the plant to coordinate a crosstalk among different pathways, highlighting the role of phytohormones and reactive oxygen and nitrogen species (RONS). In particular, plants sense these reactive species through post-translational modification (PTM) of macromolecules such as nucleic acids, proteins, and fatty acids, hence triggering antioxidant responses with molecular implications in the plant welfare. Here, this review compiles the state of the art about how plant systems sense and transduce this crosstalk through PTMs of biological molecules, highlighting the S-nitrosylation of protein targets. These molecular mechanisms finally impact at a physiological level facing the abiotic stressful traits that could lead to establishing molecular patterns underlying stress responses and adaptation strategies.

Role of electrophilic nitrated fatty acids during development and response to abiotic stress processes in plants.

Nitro-fatty acids are generated from the interaction of unsaturated fatty acids and nitric oxide (NO)-derived molecules. The endogenous occurrence and modulation throughout plant development of nitro-linolenic acid (NO2-Ln) and nitro-oleic acid (NO2-OA) suggest a key role for these molecules in initial development stages. In addition, NO2-Ln content increases significantly in stress situations and induces the expression of genes mainly related to abiotic stress, such as genes encoding members of the heat shock response family and antioxidant enzymes. The promoter regions of NO2-Ln-induced genes are also involved mainly in stress responses. These findings confirm that NO2-Ln is involved in plant defense processes against abiotic stress conditions via 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 protein function. NO2-Ln is a NO donor that may be involved in NO signaling events and is able to generate S-nitrosoglutathione, the major reservoir of NO in cells and a key player in NO-mediated abiotic stress responses. This review describes the current state of the art regarding the essential role of nitro-fatty acids as signaling mediators in development and abiotic stress processes.

Endogenous Biosynthesis of S-Nitrosoglutathione From Nitro-Fatty Acids in Plants.

Nitro-fatty acids (NO2-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 NO2-FAs trigger an antioxidant and a defence response against stressful situations. Among the properties of NO2-FAs highlight the ability to release NO therefore modulating specific protein targets through post-translational modifications (NO-PTMs). Thus, based on the capacity of NO2-FAs to act as physiological NO donors and using high-accuracy mass-spectrometric approaches, herein, we show that endogenous nitro-linolenic acid (NO2-Ln) can modulate S-nitrosoglutathione (GSNO) biosynthesis in Arabidopsis. The incubation of NO2-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 15N-labeled NO2-Ln throughout the roots, and 15N-labeled GSNO (GS15NO) was detected in the leaves. With the aim to go in depth in the relation of NO2-FA and GSNO in plants, Arabidopsis alkenal reductase mutants (aer mutants) which modulate NO2-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 NO2-FAs, increasing knowledge about S-nitrosothiols and GSNO-signaling pathways in plants.

Short-Term Low Temperature Induces Nitro-Oxidative Stress that Deregulates the NADP-Malic Enzyme Function by Tyrosine Nitration in Arabidopsis thaliana.

Low temperature (LT) negatively affects plant growth and development via the alteration of the metabolism of reactive oxygen and nitrogen species (ROS and RNS). Among RNS, tyrosine nitration, the addition of an NO2 group to a tyrosine residue, can modulate reduced nicotinamide-dinucleotide phosphate (NADPH)-generating systems and, therefore, can alter the levels of NADPH, a key cofactor in cellular redox homeostasis. NADPH also acts as an indispensable electron donor within a wide range of enzymatic reactions, biosynthetic pathways, and detoxification processes, which could affect plant viability. To extend our knowledge about the regulation of this key cofactor by this nitric oxide (NO)-related post-translational modification, we analyzed the effect of tyrosine nitration on another NADPH-generating enzyme, the NADP-malic enzyme (NADP-ME), under LT stress. In Arabidopsis thaliana seedlings exposed to short-term LT (4 °C for 48 h), a 50% growth reduction accompanied by an increase in the content of superoxide, nitric oxide, and peroxynitrite, in addition to diminished cytosolic NADP-ME activity, were found. In vitro assays confirmed that peroxynitrite inhibits cytosolic NADP-ME2 activity due to tyrosine nitration. The mass spectrometric analysis of nitrated NADP-ME2 enabled us to determine that Tyr-73 was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. The in silico analysis of the Arabidopsis NADP-ME2 protein sequence suggests that Tyr73 nitration could disrupt the interactions between the specific amino acids responsible for protein structure stability. In conclusion, the present data show that short-term LT stress affects the metabolism of ROS and RNS, which appears to negatively modulate the activity of cytosolic NADP-ME through the tyrosine nitration process.

The function of S-nitrosothiols during abiotic stress in plants.

Nitric oxide (NO) is an active redox molecule involved in the control of a wide range of functions integral to plant biology. For instance, NO is implicated in seed germination, floral development, senescence, stomatal closure, and plant responses to stress. NO usually mediates signaling events via interactions with different biomolecules, for example the modulation of protein functioning through post-translational modifications (NO-PTMs). S-nitrosation is a reversible redox NO-PTM that consists of the addition of NO to a specific thiol group of a cysteine residue, leading to formation of S-nitrosothiols (SNOs). SNOs are more stable than NO and therefore they can extend and spread the in vivo NO signaling. The development of robust and reliable detection methods has allowed the identification of hundreds of S-nitrosated proteins involved in a wide range of physiological and stress-related processes in plants. For example, SNOs have a physiological function in plant development, hormone metabolism, nutrient uptake, and photosynthesis, among many other processes. The role of S-nitrosation as a regulator of plant responses to salinity and drought stress through the modulation of specific protein targets has also been well established. However, there are many S-nitrosated proteins that have been identified under different abiotic stresses for which the specific roles have not yet been identified. In this review, we examine current knowledge of the specific role of SNOs in the signaling events that lead to plant responses to abiotic stress, with a particular focus on examples where their functions have been well characterized at the molecular level.

Post-Translational Modification of Proteins Mediated by Nitro-Fatty Acids in Plants: Nitroalkylation.

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.

Thioredoxin-mediated redox signalling in plant immunity.

Activation of plant immune responses is associated with rapid production of vast amounts of reactive oxygen and nitrogen species (ROS/RNS) that dramatically alter cellular redox homeostasis. Even though excessive ROS/RNS accumulation can cause widespread cellular damage and thus constitute a major risk, plant cells have evolved to utilise these molecules as important signalling cues. Particularly their ability to modify redox-sensitive cysteine residues has emerged as a key mechanism to control the activity, conformation, protein-protein interaction and localisation of a growing number of immune signalling proteins. Regulated reversal of cysteine oxidation is dependent on activities of the conserved superfamily of Thioredoxin (TRX) enzymes that function as cysteine reductases. The plant immune system recruits specific TRX enzymes that have the potential to functionally regulate numerous immune signalling proteins. Although our knowledge of different TRX immune targets is now expanding, little remains known about how these enzymes select their substrates, what range of oxidized residues they target, and if they function selectively in different redox-mediated immune signalling pathways. In this review we discuss these questions by examining evidence showing TRX enzymes exhibit novel activities that play important roles in diverse aspects of plant immune signalling.

Nitro-Fatty Acid Detection in Plants by High-Pressure Liquid Chromatography Coupled to Triple Quadrupole Mass Spectrometry.

In the last few years, the role of nitric oxide (NO) and NO-related molecules has attracted attention in the field of plant systems. In this sense, the ability of NO to mediate several posttranslational modifications (NO-PTM) in different biomolecules, such as protein tyrosine nitration or S-nitrosylation, has shown the involvement of these reactive nitrogen species in a wide range of functions in plant physiology such as the antioxidant response or the involvement in processes such as germination, growth, development, or senescence. However, growing interest has focused on the interaction of these NO-derived molecules with unsaturated fatty acids, yielding nitro-fatty acids (NO2-FAs). It has recently been shown that these molecules are involved in key signaling pathways in animal systems through the implementation of antioxidant and anti-inflammatory responses. Nevertheless, this interaction has been poorly studied in plant systems. Very recently, the endogenous presence of NO2-FAs in the model plant Arabidopsis thaliana has been demonstrated as well as the significant involvement of nitro-linolenic acid (NO2-Ln) in the defence response against several abiotic and oxidative stress conditions. In this respect, the detection of NO2-FAs in plant systems can be a useful tool to determine the importance of these molecules in the regulation of different biochemical pathways. Using high-pressure liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS), the methods described in this chapter enable the determination of the NO2-FA content in a pM range as well as the characterization of these nitrated derivatives of unsaturated fatty acids in plant tissues.

Biological properties of nitro-fatty acids in plants.

Nitro-fatty acids (NO2-FAs) are formed from the reaction between nitrogen dioxide (NO2) and mono and polyunsaturated fatty acids. Knowledge concerning NO2-FAs has significantly increased within a few years ago and the beneficial actions of these species uncovered in animal systems have led to consider them as molecules with therapeutic potential. Based on their nature and structure, NO2-FAs have the ability to release nitric oxide (NO) in aqueous environments and the capacity to mediate post-translational modifications (PTM) by nitroalkylation. Recently, based on the potential of these NO-derived molecules in the animal field, the endogenous occurrence of nitrated-derivatives of linolenic acid (NO2-Ln) was assessed in plant species. Moreover and through RNA-seq technology, it was shown that NO2-Ln can induce a large set of heat-shock proteins (HSPs) and different antioxidant systems suggesting this molecule may launch antioxidant and defence responses in plants. Furthermore, the capacity of this nitro-fatty acid to release NO has also been demonstrated. In view of this background, here we offer an overview on the biological properties described for NO2-FAs in plants and the potential of these molecules to be considered new key intermediaries of NO metabolism in the plant field.

Nitric oxide buffering and conditional nitric oxide release in stress response.

Nitric oxide (NO) has emerged as an essential biological messenger in plant biology that usually transmits its bioactivity by post-translational modifications such as S-nitrosylation, the reversible addition of an NO group to a protein cysteine residue leading to S-nitrosothiols (SNOs). In recent years, SNOs have risen as key signalling molecules mainly involved in plant response to stress. Chief among SNOs is S-nitrosoglutathione (GSNO), generated by S-nitrosylation of the key antioxidant glutathione (GSH). GSNO is considered the major NO reservoir and a phloem mobile signal that confers to NO the capacity to be a long-distance signalling molecule. GSNO is able to regulate protein function and gene expression, resulting in a key role for GSNO in fundamental processes in plants, such as development and response to a wide range of environmental stresses. In addition, GSNO is also able to regulate the total SNO pool and, consequently, it could be considered the storage of NO in cells that may control NO signalling under basal and stress-related responses. Thus, GSNO function could be crucial during plant response to environmental stresses. Besides the importance of GSNO in plant biology, its mode of action has not been widely discussed in the literature. In this review, we will first discuss the GSNO turnover in cells and secondly the role of GSNO as a mediator of physiological and stress-related processes in plants, highlighting those aspects for which there is still some controversy.

Nitro-fatty acids in plant signaling: New key mediators of nitric oxide metabolism.

Recent studies in animal systems have shown that NO can interact with fatty acids to generate nitro-fatty acids (NO2-FAs). They are the product of the reaction between reactive nitrogen species and unsaturated fatty acids, and are considered novel mediators of cell signaling based mainly on a proven anti-inflammatory response. Although these signaling mediators have been described widely in animal systems, NO2-FAs have scarcely been studied in plants. Preliminary data have revealed the endogenous presence of free and protein-adducted NO2-FAs in extra-virgin olive oil (EVOO), which appear to be contributing to the cardiovascular benefits associated with the Mediterranean diet. Importantly, new findings have displayed the endogenous occurrence of nitro-linolenic acid (NO2-Ln) in the model plant Arabidopsis thaliana and the modulation of NO2-Ln levels throughout this plant's development. Furthermore, a transcriptomic analysis by RNA-seq technology established a clear signaling role for this molecule, demonstrating that NO2-Ln was involved in plant-defense response against different abiotic-stress conditions, mainly by inducing the chaperone network and supporting a conserved mechanism of action in both animal and plant defense processes. Thus, NO2-Ln levels significantly rose under several abiotic-stress conditions, highlighting the strong signaling role of these molecules in the plant-protection mechanism. Finally, the potential of NO2-Ln as a NO donor has recently been described both in vitro and in vivo. Jointly, this ability gives NO2-Ln the potential to act as a signaling molecule by the direct release of NO, due to its capacity to induce different changes mediated by NO or NO-related molecules such as nitration and S-nitrosylation, or by the electrophilic capacity of these molecules through a nitroalkylation mechanism. Here, we describe the current state of the art regarding the advances performed in the field of NO2-FAs in plants and their implication in plant physiology.

In vitro nitro-fatty acid release from Cys-NO2-fatty acid adducts under nitro-oxidative conditions.

Stress situations are characterized by a rise in reactive oxygen (ROS) and nitrogen (RNS) species levels. Nitro-fatty acids (NO2-FAs), or nitroalkenes, are produced by the interaction of RNS and unsaturated fatty acids, stored in cells, mostly as part of protein-adducted NO2-FAs, and are esterified in complex lipids. These molecules, which have been shown to play a pivotal role as anti-inflammatory and pro-survival players, have been widely characterized in animal systems. Recently, it has been reported that NO2-FAs play an important role in plant defense against several stress conditions. Furthermore, a significant increase in NO2-FA levels has been observed under various inflammatory and stressful conditions in both animal and plant systems. In this study, we describe the in vitro release of NO2-FAs from protein-adducts under nitro-oxidative stress conditions. The findings of this study highlight the ability of hydrogen peroxide and peroxynitrite, as representative ROS and RNS molecules induced under stress conditions, to oxidize cysteine-adducted NO2-FAs, which is followed by the release of free nitroalkenes. This release may be partly responsible for the increase in NO2-FA content observed under different stressful conditions in both animal and plant systems as well as the activation of antioxidant and anti-inflammatory properties attributed to these molecules.

Protein Tyrosine Nitration during Development and Abiotic Stress Response in Plants.

In recent years, the study of nitric oxide (NO) in plant systems has attracted the attention of many researchers. A growing number of investigations have shown the significance of NO as a signal molecule or as a molecule involved in the response against (a)biotic processes. NO can be responsible of the post-translational modifications (NO-PTM) of target proteins by mechanisms such as the nitration of tyrosine residues. The study of protein tyrosine nitration during development and under biotic and adverse environmental conditions has increased in the last decade; nevertheless, there is also an endogenous nitration which seems to have regulatory functions. Moreover, the advance in proteome techniques has enabled the identification of new nitrated proteins, showing the high variability among plant organs, development stage and species. Finally, it may be important to discern between a widespread protein nitration because of greater RNS content, and the specific nitration of key targets which could affect cell-signaling processes. In view of the above point, we present a mini-review that offers an update about the endogenous protein tyrosine nitration, during plant development and under several abiotic stress conditions.

Nitro-linolenic acid is a nitric oxide donor.

Nitro-fatty acids (NO2-FAs), which are the result of the interaction between reactive nitrogen species (RNS) and non-saturated fatty acids, constitute a new research area in plant systems, and their study has significantly increased. Very recently, the endogenous presence of nitro-linolenic acid (NO2-Ln) has been reported in the model plant Arabidopsis thaliana. In this regard, the signaling role of this molecule has been shown to be key in setting up a defense mechanism by inducing the chaperone network in plants. Here, we report on the ability of NO2-Ln to release nitric oxide (NO) in an aqueous medium with several approaches, such as by a spectrofluorometric probe with DAF-2, the oxyhemoglobin oxidation method, ozone chemiluminescence, and also by confocal laser scanning microscopy in Arabidopsis cell cultures. Jointly, this ability gives NO2-Ln the potential to act as a signaling molecule by the direct release of NO, due to its capacity to induce different changes mediated by NO or NO-related molecules such as nitration and S-nitrosylation or by the electrophilic capacity of these molecules through a nitroalkylation mechanism.

Antioxidant Systems are Regulated by Nitric Oxide-Mediated Post-translational Modifications (NO-PTMs).

Nitric oxide (NO) is a biological messenger that orchestrates a plethora of plant functions, mainly through post-translational modifications (PTMs) such as S-nitrosylation or tyrosine nitration. In plants, hundreds of proteins have been identified as potential targets of these NO-PTMs under physiological and stress conditions indicating the relevance of NO in plant-signaling mechanisms. Among these NO protein targets, there are different antioxidant enzymes involved in the control of reactive oxygen species (ROS), such as H2O2, which is also a signal molecule. This highlights the close relationship between ROS/NO signaling pathways. The major plant antioxidant enzymes, including catalase, superoxide dismutases (SODs) peroxiredoxins (Prx) and all the enzymatic components of the ascorbate-glutathione (Asa-GSH) cycle, have been shown to be modulated to different degrees by NO-PTMs. This mini-review will update the recent knowledge concerning the interaction of NO with these antioxidant enzymes, with a special focus on the components of the Asa-GSH cycle and their physiological relevance.

Nitric oxide release from nitro-fatty acids in Arabidopsis roots.

In recent years, research on the involvement of nitric oxide (NO) in plant systems has remarkably grown. However, most of the interest in this molecule has been focused on its ability to mediate different post-translational modifications (NO-PTM) in biomolecules, mainly nitration and S-nitrosylation of proteins, and its involvement in physiological and stress situations. Nevertheless, very recently the nitration of other molecules such as fatty acids has commanded increasingly greater attention. In the last February issue of Plant Physiology, we again reported on the endogenous occurrence of nitro-fatty acids (NO2-FAs), specifically nitro-linolenic acid (NO2-Ln), in the model plant Arabidopsis thaliana. The analysis of the presence of this nitro-fatty acid showed that levels of NO2-Ln decreased throughout the plant development with the higher levels detected in seeds and young seedlings of this plant. Furthermore, through a transcriptomic analysis by RNA-seq technology applying NO2-Ln to A. thaliana cell-suspension cultures, we found high induction in the transcriptional expression of several heat-shock proteins (HSPs) and the enzymes ascorbate peroxidase (APX) and methionine sulfoxide reductase (MSR). Based on these findings, the involvement of NO2-Ln in the NO metabolism was analyzed showing a significant NO formation in roots from 7-day-old Arabidopsis thaliana seedlings and standing out that NO generated from NO2-Ln could have an important role at the beginning of plant development. Therefore, these findings highlight the importance of these novel NO-derived molecules in plant systems playing a pivotal role in development and in the antioxidant defense response against different abiotic stress conditions.

Nitro-Fatty Acids in Plant Signaling: Nitro-Linolenic Acid Induces the Molecular Chaperone Network in Arabidopsis.

Nitro-fatty acids (NO2-FAs) are the product of the reaction between reactive nitrogen species derived of nitric oxide (NO) and unsaturated fatty acids. In animal systems, NO2-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 (NO2-Ln) in Arabidopsis and the modulation of NO2-Ln levels throughout this plant's development by mass spectrometry. The observed levels of this NO2-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 NO2-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 NO2-Ln was also involved in the response to oxidative stress conditions, mainly depicted by H2O2, reactive oxygen species, and oxygen-containing compound responses, with a high induction of ascorbate peroxidase expression. Closely related to these results, NO2-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 NO2-Ln in Arabidopsis (Arabidopsis thaliana) and supporting the strong signaling role of these molecules in the defense mechanism against different abiotic-stress situations.

Transcriptomic Analyses on the Role of Nitric Oxide in Plant Disease Resistance.

Nitric oxide (NO) is a gaseous molecule having key roles in many physiological processes such as germination, growth, development and senescence. It has been also shown the important role of NO as a signaling molecule in the response to a wide variety of stress situations, including both biotic and abiotic stress conditions. In the last few years, a growing number of studies have focused on NO-cell targets by several approaches such as transcriptomic and proteomic analyses. This review is centered on offering an update about the principal medium- and large-scale transcriptomic analyses performed with several NO donors including microarray, cDNA-amplification fragment length polymorphism (AFLP) and high throughput sequencing (RNA-seq technology) approaches mainly focused on the role of this reactive nitrogen species in relation to plant disease resistance. Different putative NO-responsive genes have been identified in different plant tissues and plant species by application of several NO donors suggesting the implication of NO-responsive genes with plant adaptive responses to biotic stress processes. Finally, it is also provided an overview about common transcription factor-binding sites of NO-responsive genes and the need to further analyze the different NO-targets by other omics studies.

Differential molecular response of monodehydroascorbate reductase and glutathione reductase by nitration and S-nitrosylation.

The ascorbate-glutathione cycle is a metabolic pathway that detoxifies hydrogen peroxide and involves enzymatic and non-enzymatic antioxidants. Proteomic studies have shown that some enzymes in this cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) are potential targets for post-translational modifications (PMTs) mediated by nitric oxide-derived molecules. Using purified recombinant pea peroxisomal MDAR and cytosolic and chloroplastic GR enzymes produced in Escherichia coli, the effects of peroxynitrite (ONOO(-)) and S-nitrosoglutathione (GSNO) which are known to mediate protein nitration and S-nitrosylation processes, respectively, were analysed. Although ONOO(-) and GSNO inhibit peroxisomal MDAR activity, chloroplastic and cytosolic GR were not affected by these molecules. Mass spectrometric analysis of the nitrated MDAR revealed that Tyr213, Try292, and Tyr345 were exclusively nitrated to 3-nitrotyrosine by ONOO(-). The location of these residues in the structure of pea peroxisomal MDAR reveals that Tyr345 is found at 3.3 Å of His313 which is involved in the NADP-binding site. Site-directed mutagenesis confirmed Tyr345 as the primary site of nitration responsible for the inhibition of MDAR activity by ONOO(-). These results provide new insights into the molecular regulation of MDAR which is deactivated by nitration and S-nitrosylation. However, GR was not affected by ONOO(-) or GSNO, suggesting the existence of a mechanism to conserve redox status by maintaining the level of reduced GSH. Under a nitro-oxidative stress induced by salinity (150mM NaCl), MDAR expression (mRNA, protein, and enzyme activity levels) was increased, probably to compensate the inhibitory effects of S-nitrosylation and nitration on the enzyme. The present data show the modulation of the antioxidative response of key enzymes in the ascorbate-glutathione cycle by nitric oxide (NO)-PTMs, thus indicating the close involvement of NO and reactive oxygen species metabolism in antioxidant defence against nitro-oxidative stress situations in plants.