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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.

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.

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 (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., JCBM wishes to thank the Ministry of Economy and Competitiveness (Spain) for postdoctoral research funding within the Juan de la Cierva-Incorporación programme. This study was supported by the ERDF grants co-financed by the Ministry of Economy and Competitiveness (projects BIO2015-66390-P and AGL2015-65104-P) and the Junta de Andalucía (groups BIO286 and BIO192) in Spain.

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.

Abiotic stress is one of the principal factors that affect plant growth and productivity and is characterized by the overproduction of reactive oxygen species (ROS) and reactive nitrogen specie (RNS). These molecules trigger the defense mechanisms sustaining the redox homeostasis of the cells. Nonetheless, when the stress is persistent for a long time, the elevated contents of ROS and RNS could cause impairment of cellular macromolecules such as proteins, nucleic acids, and membrane lipids that finally results in a nitro-oxidative stress which produces cell death. In this sense, it is necessary to understand how plants survive with the change of the environment stress that could be essential to improve plant growth and crop yield. Increasing evidences suggest the relevance of nitric oxide (NO) and other RNS as signaling molecules in response to different environmental insults. In this regard, RNS can mediate signaling processes by different posttranslational modifications (PTMs) including protein S-nitrosylation, protein tyrosine nitration, and nitroalkylation. Many candidate proteins have been identified as potential cellular targets of S-nitrosylation and nitration under physiological processes and stress conditions. However, there is no information regarding protein nitroalkylation in plant systems. Until now, there is few data about the effect of these NO-dependent PTMs on protein function; therefore the identified candidates need to be characterized in more detail to better understand the function of these modifications in higher plants. The present chapter describes the current knowledge of the NO-PTMs effects on protein function and its outcome on plant response to adverse environmental stress conditions.

Plants usually face adverse environmental conditions that can compromise their viability and therefore have a high influence on crop productivity and a great economic impact. It is predicted that food shortage will become a real concern in the forthcoming decades. For this reason, understanding how plants survive under these environmental changes will be of an invaluable help for breeding programs to develop crop with an improved tolerance to abiotic stresses. The adverse environmental changes are characterized by a fast burst of signaling molecules, which induces a complex response mechanism in plants such as reactive oxygen- and nitrogen-derived species (ROS) (RNS), highlighting hydrogen peroxide (H2O2) and nitric oxide (NO), respectively. NO is a fundamental molecule involved in the regulation of key processes during the whole life cycle of plants, including plant response to stress. Most studies available have used pharmacological approaches showing that exogenous NO positively regulates plant development under abiotic stresses via control of ROS and antioxidant systems. However, endogenous NO source(s) is(are) still under debate in plants, and therefore efforts should be focused on the identification of the specific NO source under different abiotic stresses. Moreover, NO mainly transmits its bioactivity by posttranslational modifications of proteins, chief among them being S-nitrosylation and tyrosine nitration. A high number of proteins have been identified as NO targets during plant response to stress, but a deeper characterization of these modifications at molecular level such as the impact on plant structure and/or function are still required. In this chapter, we will summarize the most recent information on NO function during plant response to several abiotic stresses.

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 NADPbinding 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 (150 mM 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., Spanish Government, ERDF - Ministry of Economy and Competitiveness BIO2012-33904, Junta de Andalucía BIO286 BIO192

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.

Posttranslational modifications (PTMs) of proteins can be considered an additional step in protein metabolism that greatly expands the proteome of living organisms. In plant systems, PTMs affect protein structure and activity which are associated with concomitant changes in signaling and gene expression that make cellular systems more dynamic.

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., Technical and human support provided by CICT of Universidad de Jaén (UJA, MINECO, Junta de Andalucía, and FEDER) is gratefully acknowledged.

Hydrogen peroxide (H2O2) and nitric oxide (NO) are biological messengers that control a plethora of physiological functions integral to plant biology such as seed germination, growth, development, flowering, or plant response to stress. Furthermore, the interplay between the signaling pathways governed by these redox molecules has emerged as crucial during plant response to different stress situations. In recent years, to gain in the knowledge of the mode of action of these signaling molecules at molecular levels, different NO donors and H2O2 have been used in medium- and large-scale transcriptomic analyses including microarray, cDNA-amplification fragment length polymorphism (AFLP), and high-throughput sequencing (RNA-seq technology). Following this strategy, a high transcriptional reprogramming induced by both NO and H2O2 has been proposed. In this regard, thousands of NO- and H2O2-cell targets have been identified in different plant species and organs and predicted to be related to a wide diversity of biological processes. However, some authors have identified by comparing different transcriptomic analysis that there is a low overlap in the transcriptomic data available under different treatment conditions as well as different organ analyzed. In this sense, more transcriptomic data comparisons will help in the identification of the NO- and H2O2-specific targets and even the common genes involved in both H2O2- and NO-dependent signaling events.

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 (NO

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.

Background Protein tyrosine nitration is a post-translational modification (PTM) mediated by nitric oxide-derived molecules. Peroxisomes are oxidative organelles in which the presence of nitric oxide (NO) has been reported. Methods We studied peroxisomal nitroproteome of pea leaves by high-performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) and proteomic approaches. Results Proteomic analysis of peroxisomes from pea leaves detected a total of four nitro-tyrosine immunopositive proteins by using an antibody against nitrotyrosine. One of these proteins was found to be the NADH-dependent hydroxypyruvate reductase (HPR). The in vitro nitration of peroxisomal samples caused a 65% inhibition of HPR activity. Analysis of recombinant peroxisomal NADH-dependent HPR1 activity from Arabidopsis in the presence of H2O2, NO, GSH and peroxynitrite showed that the ONOO− molecule caused the highest inhibition of activity (51% at 5 mM SIN-1), with 5 mM H2O2 having no inhibitory effect. Mass spectrometric analysis of the nitrated recombinant HPR1 enabled us to determine that, among the eleven tyrosine present in this enzyme, only Tyr-97, Tyr-108 and Tyr-198 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Site-directed mutagenesis confirmed Tyr198 as the primary site of nitration responsible for the inhibition on the enzymatic activity by peroxynitrite. Conclusion These findings suggest that peroxisomal HPR is a target of peroxynitrite which provokes a loss of function. General significance This is the first report demonstrating the peroxisomal NADH-dependent HPR activity involved in the photorespiration pathway is regulated by tyrosine nitration, indicating that peroxisomal NO metabolism may contribute to the regulation of physiological processes under no-stress conditions.

Protein tyrosine nitration is a post-translational modification mediated by reactive nitrogen species (RNS) that is associated with nitro-oxidative damage. No information about this process is available in relation to higher plants during development and senescence. Using pea plants at different developmental stages (ranging from 8 to 71 days), tyrosine nitration in the main organs (roots, stems, leaves, flowers, and fruits) was analysed using immunological and proteomic approaches. In the roots of 71-day-old senescent plants, nitroproteome analysis enabled the identification a total of 16 nitrotyrosine-immunopositive proteins. Among the proteins identified, NADP-isocitrate dehydrogenase (ICDH), an enzyme involved in the carbon and nitrogen metabolism, redox regulation, and responses to oxidative stress, was selected to evaluate the effect of nitration. NADP-ICDH activity fell by 75% during senescence. Analysis showed that peroxynitrite inhibits recombinant cytosolic NADP-ICDH activity through a process of nitration. Of the 12 tyrosines present in this enzyme, mass spectrometric analysis of nitrated recombinant cytosolic NADP-ICDH enabled this study to identify the Tyr392 as exclusively nitrated by peroxynitrite. The data as a whole reveal that protein tyrosine nitration is a nitric oxide-derived PTM prevalent throughout root development and intensifies during senescence.

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., Graphical abstract Nitro-fatty acid signaling in plants. Pathway 1 indicates that NO2-Ln is able to induce a defense mechanism through the induction of the chaperone network and the increase in the expression of some antioxidant enzymes such as APX. This latter may be involved in the alleviation of the oxidative stress generated by the overproduction of ROS such as H2O2. Furthermore, NO2-Ln is a NO donor being therefore implicated in the wide range of actions in which this molecule is involved (pathway 2). The electrophilic ability of this nitro-fatty acid could contribute to the signaling actions involving NO2-FAs (pathway 3) and, under nitro-oxidative stress conditions, the oxidation of Michael adducts may occur with subsequent NO2-FA release (pathway 4) and the observed antioxidant properties of these molecules. NO: nitric oxide; HSP: heat shock protein; APX: ascorbate peroxidase.fx1, Highlights • Nitro-Linolenic acid is an endogenous molecule whose levels are modulated throughout Arabidopsis development. • Nitro-Linolenic acid induces the molecular chaperone network in Arabidopsis. • Nitro-fatty acids are able to act as a signaling mediators in the plant-defense mechanism in abiotic and oxidative stress situations, setting up a defense response against cell damage. • Nitro-fatty acids have been demonstrated to be in vitro and in vivo NO donors. • The nitro-fatty acid detection in other plant species highlight a ubiquitous distribution of nitro-fatty acids in plant kingdom and the potential signaling actions of these molecules in plant systems.

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., This study was supported by an ERDF grant co-financed by the Ministry of Economy and Competitiveness (project BIO2015-66390-P) and Junta de Andalucía (groups BIO286 and BIO192). Research in FJC laboratory is supported by an ERDF grant co-financed by the Ministry of Economy and Competitiveness (AGL2015-65104-P).

Stress situations are characterized by a rise in reactive oxygen (ROS) and nitrogen (RNS) species levels. Nitro-fatty acids (NO

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.

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., JB-M would like to thank the Ministry of Science and Innovation for funding the Ph.D. fellowship (F.P.U.). This study was supported by an ERDF grant co-financed by the Ministry of Economy and Competitiveness (project BIO2012-33904), Junta de Andalucía (P10-AGR-6038 and groups BIO286 and BIO192) and RECUPERA2020 in Spain.

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.

Abiotic stress, one of the main factors affecting crop yield, is characterized by a rapid burst of redox molecules, especially belonging to reactive oxygen (ROS) and nitrogen (RNS) species. These molecules can act as molecular cues that trigger the defense mechanisms leading to maintaining the cellular redox balance. However, when the stress persists over time, a high concentration of ROS and RNS can overwhelm the capacity of protection of the antioxidant systems, thereby perturbing cellular redox homeostasis. This situation can induce a nitro-oxidative stress that ultimately causes cell damage and compromises plant survival. Therefore, understanding how plants cope with the changing environment can be essential for improving crops. In this regard, cysteine residues appear to be crucial to perceive the environmental signals and to orchestrate plant responses, which are usually mediated by redox posttranslational modifications (PTMs) such as S-nitrosylation and S-glutathionylation. Increasing evidence suggests that these redox PTMs could be key players in maintaining the cellular redox homeostasis by regulating the antioxidant systems. However, although hundreds of proteins, including some main antioxidants, have been reported to be targets of S-nitrosylation and/or S-glutathionylation under physiological and/or abiotic stress, there is still little information on the specific impact of these changes on the protein function and their physiological relevance. In this book chapter, we will explore recent knowledge concerning the involvement of these modifications in response to abiotic stress, with special attention to characterizing these modified proteins at the molecular level.

Nitro-fatty acids (NO-FAs) are the product of the reaction between reactive nitrogen species derived of nitric oxide (NO) and unsaturated fatty acids. In animal systems, NO-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-Ln) in Arabidopsis and the modulation of NO-Ln levels throughout this plant’s development by mass spectrometry. The observed levels of this NO-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-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-Ln was also involved in the response to oxidative stress conditions, mainly depicted by HO, reactive oxygen species, and oxygen-containing compound responses, with a high induction of ascorbate peroxidase expression. Closely related to these results, NO-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-Ln in Arabidopsis (Arabidopsis thaliana) and supporting the strong signaling role of these molecules in the defense mechanism against different abiotic-stress situations., C.M.-P. thanks the University of Jaén for funding the Ph.D. fellowship. LC-MS/MS analyses were carried out at the Technical Services Department of the University of Granada, Spain. ACSCs were kindly provided by Dr. Juan Bautista Arellano from the Institute of Natural Resources and Agrobiology (IRNASA-CSIC, Salamanca, Spain).

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.

Environmental stresses cause a rapid burst of second messengers belonging to reactive oxygen (ROS) and nitrogen (RNS) species, mainly hydrogen peroxide (H2O2) and nitric oxide (NO), respectively. H2O2 can act as a signal molecule or become toxic at high levels. Plants have developed different antioxidant tools, such as the ascorbate–glutathione (Asa–GSH) cycle, a key antioxidant system involved in the finely tuned regulation of H2O2 in cells, in order to control H2O2 overproduction. In recent years, a growing body of evidence points to the existence of a link between NO and physiological and stress responses in plants. NO activity is mainly conveyed through posttranslational modifications (PTMs) such as S-nitrosylation and/or tyrosine nitration. Over the last 10 years, the number of S-nitrosylated and nitrated proteins subjected to physiological and a stress condition has been observed to increase significantly in higher plants, suggesting that NO-PTMs are involved in plant physiology. Emerging evidence shows that ROS and NO interact during plant responses to (a)biotic stress, and proteins linked to ROS metabolism have been reported to be regulated by NO-related PTMs. Furthermore, using proteomic analytical techniques, enzymes involved in the Asa–GSH cycle have been identified as NO targets. However, little information exists on the specific impact of NO-PTMs on the structure and activity of these antioxidant enzymes. In this chapter, we will discuss recent findings concerning the regulation of the Asa–GSH cycle antioxidant capacity by NO-PTMs, particularly in relation to the role played by the NO target residues identified under stress conditions.

JBM acknowledges a PhD fellowship (F.P.U.) from the Ministry of Science and Innovation. This work was supported by an ERDF-co-financed grant from the Ministry of Science and Innovation (BIO2009-12003-C02-01, BIO2009-12003-C02-02, and BIO2012-33904) and Junta de Andalucia (group BIO286 and BIO192), Spain. LC/MS/MS analyses were carried out at the Laboratorio de Proteomica LP-CSIC/UAB, a member of the ProteoRed network. Technical and human support provided by CICT of Universidad de Jaen (UJA, MINECO, Junta de Andalucia, FEDER) is gratefully acknowledged. We acknowledge Mr Carmelo Ruiz-Torres for his excellent technical support., 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., Spanish Government, ERDF from the Ministry of Science and Innovation BIO2009-12003-C02-01 BIO2009-12003-C02-02 BIO2012-33904, Junta de Andalucia BIO286 BIO192, CICT of Universidad de Jaen (UJA, MINECO, Junta de Andalucia, FEDER)