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.

Nitrated Fatty-Acids Distribution in Storage Biomolecules during Arabidopsis thaliana Development.

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.

Nitro-Oleic Acid-Mediated Nitroalkylation Modulates the Antioxidant Function of Cytosolic Peroxiredoxin Tsa1 during Heat Stress in Saccharomyces cerevisiae.

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.

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.

Oxidative Stress in Plants.

Environmental stresses negatively affect plant growth, development and crop productivity [...].

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.

Identification of Tyrosine and Nitrotyrosine with a Mixed-Mode Solid-Phase Extraction Cleanup Followed by Liquid Chromatography-Electrospray Time-of-Flight Mass Spectrometry in Plants.

In higher plants, there is a growing interest in the study of protein tyrosine nitration (NO2Tyr) as well as the identification of in vivo nitrated proteins. Different methods have been developed for identifying nitrotyrosine in biological samples. However, these analyses are difficult because tyrosine nitration is a very low-abundance posttranslational protein modification (PTM) and the lack of efficient enrichment methods for detection. The identification and quantification of NO2Tyr in proteins has represented a challenge for researchers.In this chapter a new method for determining NO2Tyr and tyrosine (Tyr) in Arabidopsis thaliana cell-suspension culture extracts is proposed. The quantification was performed using a simple, sensitive, and specific sample preparation assay based on mixed-mode solid-phase extraction (SPE) which was developed for the quantification of trace NO2Tyr in Arabidopsis extracts by liquid chromatography-electrospray time-of-flight mass spectrometry (LC-TOFMS).

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.

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.

Quantification and Localization of S-Nitrosothiols (SNOs) in Higher Plants.

S-nitrosothiols (SNOs) are a family of molecules produced by the reaction of nitric oxide (NO) with -SH thiol groups present in the cysteine residues of proteins and peptides caused by a posttranslational modification (PTM) known as S-nitrosylation (strictly speaking S-nitrosation) that can affect the cellular function of proteins. These molecules are a relatively more stable form of NO and consequently can act as a major intracellular NO reservoir and, in some cases, as a long-distance NO signal. Additionally, SNOs can be transferred between small peptides and protein thiol groups through S-transnitrosylation mechanisms. Thus, detection and cellular localization of SNOs in plant cells can be useful tools to determine how these molecules are modulated under physiological and adverse conditions and to determine their importance as a mechanism for regulating different biochemical pathways. Using a highly sensitive chemiluminescence ozone technique and a specific fluorescence probe (Alexa Fluor 488 Hg-link phenylmercury), the methods described in this chapter enable us to determine SNOs in an nM range as well as their cellular distribution in the tissues of different plant species.

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.

Peroxisomal NADP-isocitrate dehydrogenase is required for Arabidopsis stomatal movement.

Peroxisomes are subcellular organelles characterized by a simple morphological structure but have a complex biochemical machinery involved in signaling processes through molecules such as hydrogen peroxide (H2O2) and nitric oxide (NO). Nicotinamide adenine dinucleotide phosphate (NADPH) is an essential component in cell redox homeostasis, and its regeneration is critical for reductive biosynthesis and detoxification pathways. Plants have several NADPH-generating dehydrogenases, with NADP-isocitrate dehydrogenase (NADP-ICDH) being one of these enzymes. Arabidopsis contains three genes that encode for cytosolic, mitochondrial/chloroplastic, and peroxisomal NADP-ICDH isozymes although the specific function of each of these remains largely unknown. Using two T-DNA insertion lines of the peroxisomal NADP-ICDH designated as picdh-1 and picdh-2, the data show that the peroxisomal NADP-ICDH is involved in stomatal movements, suggesting that peroxisomes are a new element in the signaling network of guard cells.

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

Nitric oxide (NO) is a significant signalling molecule involved in the regulation of many different physiological processes in plants. One of the most imperative regulatory modes of action of NO is protein S-nitrosylation--the covalent attachment of an NO group to the sulfur atom of cysteine residues. In this study, we focus on S-nitrosylation of Arabidopsis nuclear proteins after pathogen infection. After treatment of Arabidopsis suspension cell cultures with pathogens, nuclear proteins were extracted and treated with the S-nitrosylating agent S-nitrosoglutathione (GSNO). A biotin switch assay was performed and biotin-labelled proteins were purified by neutravidin affinity chromatography and identified by mass spectrometry. A total of 135 proteins were identified, whereas nuclear localization has been described for 122 proteins of them. 117 of these proteins contain at least one cysteine residue. Most of the S-nitrosylated candidates were involved in protein and RNA metabolism, stress response, and cell organization and division. Interestingly, two plant-specific histone deacetylases were identified suggesting that nitric oxide regulated epigenetic processes in plants. In sum, this work provides a new collection of targets for protein S-nitrosylation in Arabidopsis and gives insight into the regulatory function of NO in the nucleus during plant defense response. Moreover, our data extend the knowledge on the regulatory function of NO in events located in the nucleus.

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.

Ripening of pepper (Capsicum annuum) fruit is characterized by an enhancement of protein tyrosine nitration.

BACKGROUND AND AIMS: Pepper (Capsicum annuum, Solanaceae) fruits are consumed worldwide and are of great economic importance. In most species ripening is characterized by important visual and metabolic changes, the latter including emission of volatile organic compounds associated with respiration, destruction of chlorophylls, synthesis of new pigments (red/yellow carotenoids plus xanthophylls and anthocyanins), formation of pectins and protein synthesis. The involvement of nitric oxide (NO) in fruit ripening has been established, but more work is needed to detail the metabolic networks involving NO and other reactive nitrogen species (RNS) in the process. It has been reported that RNS can mediate post-translational modifications of proteins, which can modulate physiological processes through mechanisms of cellular signalling. This study therefore examined the potential role of NO in nitration of tyrosine during the ripening of California sweet pepper. METHODS: The NO content of green and red pepper fruit was determined spectrofluorometrically. Fruits at the breaking point between green and red coloration were incubated in the presence of NO for 1 h and then left to ripen for 3 d. Profiles of nitrated proteins were determined using an antibody against nitro-tyrosine (NO2-Tyr), and profiles of nitrosothiols were determined by confocal laser scanning microscopy. Nitrated proteins were identified by 2-D electrophoresis and MALDI-TOF/TOF analysis. KEY RESULTS: Treatment with NO delayed the ripening of fruit. An enhancement of nitrosothiols and nitroproteins was observed in fruit during ripening, and this was reversed by the addition of exogenous NO gas. Six nitrated proteins were identified and were characterized as being involved in redox, protein, carbohydrate and oxidative metabolism, and in glutamate biosynthesis. Catalase was the most abundant nitrated protein found in both green and red fruit. CONCLUSIONS: The RNS profile reported here indicates that ripening of pepper fruit is characterized by an enhancement of S-nitrosothiols and protein tyrosine nitration. The nitrated proteins identified have important functions in photosynthesis, generation of NADPH, proteolysis, amino acid biosynthesis and oxidative metabolism. The decrease of catalase in red fruit implies a lower capacity to scavenge H2O2, which would promote lipid peroxidation, as has already been reported in ripe pepper fruit.