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The class III peroxidases (PODs) catalyze the oxidation of several substrates coupled to the reduction of H2O2 to water, and play important roles in diverse plant processes. The POD family members have been well-studied in several plant species, but little information is available on sweet pepper fruit physiology. Based on the existing pepper genome, a total of 75 CaPOD genes have been identified, but only 10 genes were found in the fruit transcriptome (RNA-Seq). The time-course expression analysis of these genes showed that two were upregulated during fruit ripening, seven were downregulated, and one gene was unaffected. Furthermore, nitric oxide (NO) treatment triggered the upregulation of two CaPOD genes whereas the others were unaffected. Non-denaturing PAGE and in-gel activity staining allowed identifying four CaPOD isozymes (CaPOD I-CaPOD IV) which were differentially modulated during ripening and by NO. In vitro analyses of green fruit samples with peroxynitrite, NO donors, and reducing agents triggered about 100% inhibition of CaPOD IV. These data support the modulation of POD at gene and activity levels, which is in agreement with the nitro-oxidative metabolism of pepper fruit during ripening, and suggest that POD IV is a target for nitration and reducing events that lead to its inhibition.

Nitric oxide (NO) and hydrogen sulfide (H2S) are two key molecules in plant cells that participate, directly or indirectly, as regulators of protein functions through derived post-translational modifications, mainly tyrosine nitration, S-nitrosation, and persulfidation. These post-translational modifications allow the participation of both NO and H2S signal molecules in a wide range of cellular processes either physiological or under stressful circumstances. NADPH participates in cellular redox status and it is a key cofactor necessary for cell growth and development. It is involved in significant biochemical routes such as fatty acid, carotenoid and proline biosynthesis, and the shikimate pathway, as well as in cellular detoxification processes including the ascorbate–glutathione cycle, the NADPH-dependent thioredoxin reductase (NTR), or the superoxide-generating NADPH oxidase. Plant cells have diverse mechanisms to generate NADPH by a group of NADP-dependent oxidoreductases including ferredoxin-NADP reductase (FNR), NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH), NADP-dependent malic enzyme (NADP-ME), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and both enzymes of the oxidative pentose phosphate pathway, designated as glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). These enzymes consist of different isozymes located in diverse subcellular compartments (chloroplasts, cytosol, mitochondria, and peroxisomes) which contribute to the NAPDH cellular pool. We provide a comprehensive overview of how post-translational modifications promoted by NO (tyrosine nitration and S-nitrosation), H2S (persulfidation), and glutathione (glutathionylation), affect the cellular redox status through regulation of the NADP-dependent dehydrogenases.

Our research is supported by a European Regional Development Fund cofinanced grants from the Ministry of Science and Innovation (PID2019-103924GB-I00) and Junta de Andalucia (P18-FR-1359), Spain., Nitric oxide (NO) is a free radical which modulates protein function and gene expression throughout all stages of plant development. Fruit ripening involves a complex scenario where drastic phenotypical and metabolic changes take place. Pepper fruits are one of the most consumed horticultural products worldwide which, at ripening, undergo crucial phenotypical and biochemical events, with NO and antioxidants being implicated. Based on previous transcriptomic (RNA-Seq), proteomics (iTRAQ), and enzymatic data, this study aimed to identify the ascorbate peroxidase (APX) gene and protein profiles in sweet peppers and to evaluate their potential modulation by NO during fruit ripening. The data show the existence of six CaAPX genes (CaAPX1–CaAPX6) that encode corresponding APX isozymes distributed in cytosol, plastids, mitochondria, and peroxisomes. The time course expression analysis of these genes showed heterogeneous expression patterns throughout the different ripening stages, and also as a consequence of treatment with NO gas. Additionally, six APX isozymes activities (APX I–APX VI) were identified by non-denaturing PAGE, and they were also differentially modulated during maturation and NO treatment. In vitro analyses of fruit samples in the presence of NO donors, peroxynitrite, and glutathione, showed that CaAPX activity was inhibited, thus suggesting that different posttranslational modifications (PTMs), including Snitrosation, Tyr-nitration, and glutathionylation, respectively, may occur in APX isozymes. In silico analysis of the protein tertiary structure showed that residues Cys32 and Tyr235 were conserved in the six CaAPXs, and are thus likely potential targets for S-nitrosation and nitration, respectively. These data highlight the complex mechanisms of the regulation of APX isozymes during the ripening process of sweet pepper fruits and how NO can exert fine control. This information could be useful for postharvest technology; NO regulates H2O2 levels through the different APX isozymes and, consequently, could modulate the shelf life and nutritional quality of pepper fruits., European Regional Development Fund from the Ministry of Science and Innovation PID2019-103924GB-I00 Junta de Andalucia, European Commission P18-FR-1359

Potassium (K) is an essential macro-element for plant growth and development given its implication in major processes such as photosynthesis, osmoregulation, protein synthesis, and enzyme function. Using 30-day-old Cakile maritima plants as halophyte model grown under K deprivation for 15 days, it was analyzed at the biochemical level to determine the metabolism of reactive oxygen species (ROS), key photorespiratory enzymes, and the main NADPH-generating systems. K starvation-induced oxidative stress was noticed by high malondialdehyde (MDA) content associated with an increase of superoxide radical (O) in leaves from K-deficient plants. K shortage led to an overall increase in the activity of hydroxypyruvate reductase (HPR) and glycolate oxidase (GOX), as well as of antioxidant enzymes catalase (CAT), those of the ascorbate-glutathione cycle, peroxidase (POX), and superoxide dismutase (SOD), and the main enzymes involved in the NADPH generation in both leaves and roots. Especially remarkable was the induction of up to seven CuZn-SOD isozymes in leaves due to K deficiency. As a whole, data show that the K starvation has associated oxidative stress that boosts a biochemical response leading to a general increase of the antioxidant and NADPH-generating systems that allow the survival of the halophyte Cakile maritima., F.J.C. and J.M.P. research is supported by ERDF-cofinanced grant from the Ministry of Science and Innovation (BIO2012-33904), Ministry of Economy and Competitiveness (PID2019- 103924GB-I00), the Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020) (P18-FR1359), and Junta de Andalucía (group BIO192).

Protein persulfidation is a post-translational modification (PTM) mediated by hydrogen sulfide (H S), which affects the thiol group of cysteine residues from target proteins and can have a positive, negative or zero impact on protein function. Due to advances in proteomic techniques, the number of potential protein targets identified in higher plants, which are affected by this PTM, has increased considerably. However, its precise impact on biological function needs to be evaluated at the experimental level in purified proteins in order to identify the specific cysteine(s) residue(s) affected. It also needs to be evaluated at the cellular redox level given the potential interactions among different oxidative post-translational modifications (oxiPTMs), such as S-nitrosation, glutathionylation, sulfenylation, S-cyanylation and S-acylation, which also affect thiol groups. This review aims to provide an updated and comprehensive overview of the important physiological role exerted by persulfidation in higher plants, which acts as a cellular mechanism of protein protection against irreversible oxidation., F.J.C. and J.M.P. research is supported by a European Regional Development Fund cofinanced grant from the Spanish Ministry of Science, Innovation and Universities (PID2019-103924GBI00), the Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020) (P18-FR-1359) and Junta de Andalucía (group BIO192), Spain.

The peroxisome is a single-membrane subcellular compartment present in almost all eukaryotic cells from simple protists and fungi to complex organisms such as higher plants and animals. Historically, the name of the peroxisome came from a subcellular structure that contained high levels of hydrogen peroxide (H2O2) and the antioxidant enzyme catalase, which indicated that this organelle had basically an oxidative metabolism. During the last 20 years, it has been shown that plant peroxisomes also contain nitric oxide (NO), a radical molecule than leads to a family of derived molecules designated as reactive nitrogen species (RNS). These reactive species can mediate post-translational modifications (PTMs) of proteins, such as S-nitrosation and tyrosine nitration, thus affecting their function. This review aims to provide a comprehensive overview of how NO could affect peroxisomal metabolism and its internal protein-protein interactions (PPIs). Remarkably, many of the identified NO-target proteins in plant peroxisomes are involved in the metabolism of reactive oxygen species (ROS), either in its generation or its scavenging. Therefore, it is proposed that NO is a molecule with signaling properties with the capacity to modulate the peroxisomal protein-protein network and consequently the peroxisomal functions, especially under adverse environmental conditions, Our research is supported by a European Regional Development Fund cofinanced grant from the Ministry of Science and Innovation (PID2019-103924GB-I00), the Plan Andaluz de I + D + i (PAIDI 2020) (P18-FR-1359) and Junta de Andalucía (group BIO192), Spain. SG-G acknowledges a “Formación de Personal Investigador” contract (BES-2016-078368) from the Ministry of Economy and Competitiveness, Sp

Fruit ripening is a complex physiological process involving many external modifications affecting shape, color, flavor, and metabolite composition, among others, which are consequence of deeper changes at biochemical, molecular, and cellular level. Hydrogen sulfide (H2S) is a gasotransmitter molecule that is endogenously produced in plant cells by enzymatic processes during sulfur metabolism. At present, H2S is recognized as a new signaling molecule because it has the capacity to affect protein function through a posttranslational modification (PTM) designated persulfidation. The present chapter has the goal to provide an updated comprehensive overview of the H2S metabolism and its implication in the ripening of climacteric and non-climacteric fruits. Moreover, the beneficial effects exerted by the exogenous application of H2S during the ripening period and postharvest storage are also overviewed.

Nitric oxide (NO) is a free radical which exerts a myriad of functions in the physiology of higher plants either under physiological and environmental stress conditions. NO, and derived molecules designated as reactive nitrogen species (RNS), can mediate posttranslational modifications (PTMs) of proteins which can affect their functionality. Among these NO/RNS-derived PTMs, it can be highlighted S-nitrosation, metal nitrosylation and nitration. This last one involves the addition of a nitro group (-NO) to some specific amino acids such as tyrosine or tryptophan. An increase in the content of protein nitration has been recognized as a suitable marker of nitro-oxidative stress which is frequently associated with oxidative stress under diverse environmental stress conditions. This mini-review aims to provide a comprehensive overview of protein nitration and its significance in higher plants., SG-G acknowledges a “Formación de Personal Investigador” contract (BES-2016-078368) from the Ministry of Economy and Competitiveness, Spain. Our research is supported by a European Regional Development Fund-cofinanced grant from the Ministry of Economy and Competitiveness (PID2019-103924GB-I00), the Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020) (P18-FR-1359) and Junta de Andalucía (group BIO192), Spain.

Tryptophan (Trp) is an aromatic amino acid which is synthesized through the shikimate/chorismate pathway. Behind that this amino acid is part of proteins; the relevance of Trp resides as a precursor of secondary metabolism which includes relevant molecules such as auxin (indole-3-acetic acid, IAA), serotonin and melatonin which have a wide range of functions in higher plants including physiological processes such as seed germination, root growth and development, senescence, flowering or fruit ripening as well as in the mechanism of response against biotic and abiotic stresses. The main goal of this chapter is to provide a comprehensive overview of these pleiotropic signalling molecules and its implication in physiological processes as well as stress environmental conditions in higher plants.

© 2020 by the authors., Capsicum is the genus where a number of species and varieties have pungent features due to the exclusive content of capsaicinoids such as capsaicin and dihydrocapsaicin. In this work, the main enzymatic and non-enzymatic systems in pepper fruits from four varieties with different pungent capacity have been investigated at two ripening stages. Thus, a sweet pepper variety (Melchor) from California-type fruits and three autochthonous Spanish varieties which have different pungency levels were used, including Piquillo, Padrón and Alegría riojana. The capsaicinoids contents were determined in the pericarp and placenta from fruits, showing that these phenyl-propanoids were mainly localized in placenta. The activity profiles of catalase, total and isoenzymatic superoxide dismutase (SOD), the enzymes of the ascorbate–glutathione cycle (AGC) and four NADP-dehydrogenases indicate that some interaction with capsaicinoid metabolism seems to occur. Among the results obtained on enzymatic antioxidants, the role of Fe-SOD and the glutathione reductase from the AGC is highlighted. Additionally, it was found that ascorbate and glutathione contents were higher in those pepper fruits which displayed the greater contents of capsaicinoids. Taken together, all these data indicate that antioxidants may contribute to preserve capsaicinoids metabolism to maintain their functionality in a framework where NADPH is perhaps playing an essential role., This work was supported by the ERDF-cofinanced grants AGL2015-65104-P from MINECO, PID2019-103924GB-I00 from MICIT, and P18-FR-1359 from the Plan Andaluz de Investigación, Desarrollo e Innovación, Spain.

Plant peroxisomes are organelles enclosed by a single membrane whose biochemical composition has the capacity to adapt depending on the plant tissue, developmental stage, as well as internal and external cellular stimuli. Apart from the peroxisomal metabolism of reactive oxygen species (ROS), discovered several decades ago, new molecules with signaling potential, including nitric oxide (NO) and hydrogen sulfide (HS), have been detected in these organelles in recent years. These molecules generate a family of derived molecules, called reactive nitrogen species (RNS) and reactive sulfur species (RSS), whose peroxisomal metabolism is autoregulated through posttranslational modifications (PTMs) such as S-nitrosation, nitration and persulfidation. The peroxisomal metabolism of these reactive species, which can be weaponized against pathogens, is susceptible to modification in response to external stimuli. This review aims to provide up-to-date information on crosstalk between these reactive species families and peroxisomes, as well as on their cellular environment in light of the well-recognized signaling properties of HO, NO and HS., This work was supported by the European Regional Development Fund co-financed grant from the Ministry of Economy and Competitiveness (AGL2015-65104-P and PID2019-103924GB-I00), the Plan Andaluz de Investigación, Desarrollo e Innovación (P18-FR-1359), and the Junta de Andalucía (group BIO192), Spain.

SGG acknowledges a ‘Formación de Personal Investigador’ contract from the Ministry of Economy and Competitiveness, Spain., Catalase is a powerful antioxidant metalloenzyme located in peroxisomes which also plays a central role in signaling processes under physiological and adverse situations. Whereas animals contain a single catalase gene, in plants this enzyme is encoded by a multigene family providing multiple isoenzymes whose number varies depending on the species, and their expression is regulated according to their tissue/organ distribution and the environmental conditions. This enzyme can be modulated by reactive oxygen and nitrogen species (ROS/RNS) as well as by hydrogen sulfide (H2S). Catalase is the major protein undergoing Tyr-nitration [post-translational modification (PTM) promoted by RNS] during fruit ripening, but the enzyme from diverse sources is also susceptible to undergo other activity-modifying PTMs. Data on S-nitrosation and persulfidation of catalase from different plant origins are given and compared here with results from obese children where S-nitrosation of catalase occurs. The cysteine residues prone to be S-nitrosated in catalase from plants and from bovine liver have been identified. These evidences assign to peroxisomes a crucial statement in the signaling crossroads among relevant molecules (NO and H2S), since catalase is allocated in these organelles. This review depicts a scenario where the regulation of catalase through PTMs, especially S-nitrosation and persulfidation, is highlighted., European Union (EU), Spanish Government AGL2015-65104-P PID2019103924GB-I00, Plan Andaluz de Investigacion, Desarrollo e Innovacion P18-FR-1359, Junta de Andalucia BIO 192, Health Strategy Action (Spain's National Plan for Science and Technology Research, Development and Innovation) PI18-01316

The concept of antioxidants refers to a substance with the capacity to either directly scavenge or indirectly prevent the formation of pro-oxidant molecules, basically associated to the so called reactive oxygen species (ROS) [...], This research received no external funding

Glyphosate is a broad-spectrum systemic herbicide used worldwide. In susceptible plants, glyphosate affects the shikimate pathway and reduces aromatic amino acid synthesis. Using Arabidopsis seedlings grown in the presence of 20 μM glyphosate, we analyzed H2O2, ascorbate, glutathione (GSH) and protein oxidation content as well as antioxidant catalase, superoxide dismutase (SOD) and ascorbate-glutathione cycle enzyme activity. We also examined the principal NADPH-generating system components, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH). Glyphosate caused a drastic reduction in growth parameters and an increase in protein oxidation. The herbicide also resulted in an overall increase in GSH content, antioxidant enzyme activity (catalase and all enzymatic components of the ascorbate-glutathione cycle) in addition to the two oxidative phase enzymes, G6PDH and 6PGDH, in the pentose phosphate pathway involved in NADPH generation. In this study, we provide new evidence on the participation of G6PDH and 6PGDH in the response to oxidative stress induced by glyphosate in Arabidopsis, in which peroxisomal enzymes, such as catalase and glycolate oxidase, are positively affected. We suggest that the NADPH provided by the oxidative phase of the pentose phosphate pathway (OxPPP) should serve to maintain glutathione reductase (GR) activity, thus preserving and regenerating the intracellular GSH pool under glyphosate-induced stress. It is particularly remarkable that the 6PGDH activity was unaffected by pro-oxidant and nitrating molecules such as H202, nitric oxide or peroxynitrite.

© The Author(s)., During the ripening of sweet pepper (Capsicum annuum L.) fruits, in a genetically controlled scenario, enormous metabolic changes occur that affect the physiology of most cell compartments. Peroxisomal catalase gene expression decreases after pepper fruit ripening, while the enzyme is also susceptible to undergo post-translational modifications (nitration, S-nitrosation, and oxidation) promoted by reactive oxygen and nitrogen species (ROS/RNS). Unlike most plant catalases, the pepper fruit enzyme acts as a homodimer, with an atypical native molecular mass of 125 to 135 kDa and an isoelectric point of 7.4, which is higher than that of most plant catalases. These data suggest that ROS/RNS could be essential to modulate the role of catalase in maintaining basic cellular peroxisomal functions during pepper fruit ripening when nitro-oxidative stress occurs. Using catalase from bovine liver as a model and biotin-switch labeling, in-gel trypsin digestion, and nanoliquid chromatography coupled with mass spectrometry, it was found that Cys377 from the bovine enzyme could potentially undergo S-nitrosation. To our knowledge, this is the first report of a cysteine residue from catalase that can be post-translationally modified by S-nitrosation, which makes it especially important to find the target points where the enzyme can be modulated under either physiological or adverse conditions., This work was supported by the ERDF-cofinanced grant AGL2015-65104-P from MINECO, Spain.

NADPH is an essential cofactor in many physiological processes. Fruit ripening is caused by multiple biochemical pathways in which, reactive oxygen and nitrogen species (ROS/RNS) metabolism is involved. Previous studies have demonstrated the differential modulation of nitric oxide (NO) and hydrogen sulfide (HS) content during sweet pepper (Capsicum annuum L.) fruit ripening, both of which regulate NADP-isocitrate dehydrogenase activity. To gain a deeper understanding of the potential functions of other NADPH-generating components, we analyzed glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), which are involved in the oxidative phase of the pentose phosphate pathway (OxPPP) and NADP-malic enzyme (NADP-ME). During fruit ripening, G6PDH activity diminished by 38%, while 6PGDH and NADP-ME activity increased 1.5- and 2.6-fold, respectively. To better understand the potential regulation of these NADP-dehydrogenases by HS, we obtained a 50–75% ammonium-sulfate-enriched protein fraction containing these proteins. With the aid of in vitro assays, in the presence of HS, we observed that, while NADP-ME activity was inhibited by up to 29–32% using 2 and 5 mM NaS as HS donor, G6PDH and 6PGDH activities were unaffected. On the other hand, NO donors, S-nitrosocyteine (CysNO) and DETA NONOate also inhibited NADP-ME activity by 35%. These findings suggest that both NADP-ME and 6PGDH play an important role in maintaining the supply of NADPH during pepper fruit ripening and that HS and NO partially modulate the NADPH-generating system., This work was supported by the ERDF-cofinanced grant from the Ministry of Economy and Competitiveness (AGL2015-65104-P) and Junta de Andalucia (group BIO192), Spain. The technical assistance of Carmelo Ruiz-Torres and Maria J. Campos is deeply acknowledged. The provision of pepper fruits by Syngenta Seeds Ltd. (El Ejido, Almeria, Spain) is acknowledged.

Plant peroxisomes are subcellular compartments involved in many biochemical pathways during the life cycle of a plant but also in the mechanism of response against adverse environmental conditions. These organelles have an active nitro-oxidative metabolism under physiological conditions but this could be exacerbated under stress situations. Furthermore, peroxisomes have the capacity to proliferate and also undergo biochemical adaptations depending on the surrounding cellular status. An important characteristic of peroxisomes is that they have a dynamic metabolism of reactive nitrogen and oxygen species (RNS and ROS) which generates two key molecules, nitric oxide (NO) and hydrogen peroxide (HO). These molecules can exert signaling functions by means of post-translational modifications that affect the functionality of target molecules like proteins, peptides or fatty acids. This review provides an overview of the endogenous metabolism of ROS and RNS in peroxisomes with special emphasis on polyamine and uric acid metabolism as well as the possibility that these organelles could be a source of signal molecules involved in the functional interconnection with other subcellular compartments., Research in our laboratory is supported by an ERDFcofinanced grant from the Ministry of Economy and Competitiveness (AGL2015-65104-P) and Junta de Andalucía (group BIO-192), Spain.

Nitric oxide (NO) is a gaseous free radical endogenously generated in plant cells. Peroxisomes are cell organelles characterized by an active metabolism of reactive oxygen species (ROS) and are also one of the main cellular sites of NO production in higher plants. In this mini-review, an updated and comprehensive overview is presented of the evidence available demonstrating that plant peroxisomes have the capacity to generate NO, and how this molecule and its derived products, peroxynitrite (ONOO−) and S-nitrosoglutathione (GSNO), can modulate the ROS metabolism of peroxisomes, mainly throughout protein posttranslational modifications (PTMs), including S-nitrosation and tyrosine nitration. Several peroxisomal antioxidant enzymes, such as catalase (CAT), copper-zinc superoxide dismutase (CuZnSOD), and monodehydroascorbate reductase (MDAR), have been demonstrated to be targets of NO-mediated PTMs. Accordingly, plant peroxisomes can be considered as a good example of the interconnection existing between ROS and reactive nitrogen species (RNS), where NO exerts a regulatory function of ROS metabolism acting upstream of H2O2, Research in our laboratory is supported by an ERDF-co-financed grant from the Ministry of Economy and Competitiveness (AGL2015-65104-P) and Junta de Andalucía (group BIO-192), Spain, We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Hydrogen peroxide (H2O2) and nitric oxide (NO) are two key molecules representative of two families of related compounds designated as reactive oxygen and nitrogen species (ROS and RNS, respectively). Our present knowledge about where, when, and how these molecules are produced in a specific plant tissue either under physiological or stress conditions and how they interact support the relevant crosstalk between these molecules which in many cases are autoregulated throughout posttranslational modifications. Thus, either S-nitrosation or nitration of different enzymes of the ROS metabolism including superoxide-generating NADPH oxidase (NOX) or antioxidant enzymes such as catalase and superoxide dismutase (SOD) and components of the ascorbate-glutathione cycle may take place under diverse situations. However, H2O2 and NO may react among them giving rise to a more powerful toxic species, the hydroxyl radical (·OH), which may react with most biomolecules (nucleic acids, proteins, and lipids), leading to irreversible damages within cells. This chapter will provide a comprehensive and easy overview about H2O2 and NO production, on how these molecules are generated within different cell compartments, and about their metabolic interaction. A proposed model on how such interaction between H2O2 and NO may influence the organelles’ signaling network under normal physiological and stress conditions and/or developmental metabolic shifts is discussed.

Plant peroxisomes have the capacity to generate different reactive oxygen and nitrogen species (ROS and RNS), such as HO, superoxide radical (O), nitric oxide and peroxynitrite (ONOO). These organelles have an active nitro-oxidative metabolism which can be exacerbated by adverse stress conditions. Hydrogen sulfide (HS) is a new signaling gasotransmitter which can mediate the posttranslational modification (PTM) persulfidation. We used Arabidopsis thaliana transgenic seedlings expressing cyan fluorescent protein (CFP) fused to a canonical peroxisome targeting signal 1 (PTS1) to visualize peroxisomes in living cells, as well as a specific fluorescent probe which showed that peroxisomes contain HS. HS was also detected in chloroplasts under glyphosate-induced oxidative stress conditions. Peroxisomal enzyme activities, including catalase, photorespiratory HO-generating glycolate oxidase (GOX) and hydroxypyruvate reductase (HPR), were assayed in vitro with a HS donor. In line with the persulfidation of this enzyme, catalase activity declined significantly in the presence of the HS donor. To corroborate the inhibitory effect of HS on catalase activity, we also assayed pure catalase from bovine liver and pepper fruit-enriched samples, in which catalase activity was inhibited. Taken together, these data provide evidence of the presence of HS in plant peroxisomes which appears to regulate catalase activity and, consequently, the peroxisomal HO metabolism., Mr. Carmelo Ruiz-Torres is deeply acknowledged to for his technical help. The “Centro de Instrumentación Científico-Tecnica” (CICT) of University of Jaen is also recognized for the technical and human support (UJA,MINECO, Junta de Andalucía, FEDER). The Project AGL2015-65104-P (ERD F grants co-financed by the Spanish Ministry of Economy and Competitiveness) supports the research in the laboratory of FJC, SGG,MAMV and JMP, and the grant BIO2015-66390-P forJBBs laboratory.

Nitric oxide (NO) is a free radical and a component of the N-cycle. Nevertheless, NO is likewise endogenously produced inside plant cells where it participates in a myriad of physiological functions, as well as in the mechanism of response against abiotic and biotic stresses. At biochemical level, NO has a family of derived molecules designated as reactive nitrogen species (RNS) which finally can interact with different bio-macromolecules including proteins, lipids, and nucleic acids affecting their functions. The present review has the goal to provide a comprehensive and quick overview of the relevance of NO in higher plants, especially for those researchers who are not familiar in this research area in higher plants., Research in our laboratory is supported by the ERDF-co-financed grant AGL2015-65104-P from the Ministry of Economy and Competitiveness, Spain.

Despite of their economical and nutritional interest, the biology of fruits is still little studied in comparison with reports of other plant organs such as leaves and roots. Accordingly, research at subcellular and molecular levels is necessary not only to understand the physiology of fruits, but also to improve crop qualities. Efforts addressed to gain knowledge of the peroxisome proteome and how it interacts with the overall metabolism of fruits will provide tools to be used in breeding strategies of agricultural species with added value. In this work, special attention will be paid to peroxisomal proteins involved in the metabolism of reactive oxygen species (ROS) due to the relevant role of these compounds at fruit ripening. The proteome of peroxisomes purified from sweet pepper (Capsicum annuum L.) fruit is reported, where an iron-superoxide dismutase (Fe-SOD) was localized in these organelles, besides other antioxidant enzymes such as catalase and a Mn-SOD, as well as enzymes involved in the metabolism of carbohydrates, malate, lipids and fatty acids, amino acids, the glyoxylate cycle and in the potential organelles’ movements.

Superoxide dismutases (SODs) are a family of metalloenzymes that catalyze the dismutation or disproportionation of superoxide radicals (\( {{\text{O}}_{2}}^{ \cdot - } \)) into molecular oxygen (O2) and hydrogen peroxide (H2O2). In plants, essentially, there are three groups of SODs depending on the prosthetic metals in their active sites, either: copper and zinc (Cu,Zn-SODs); manganese (Mn-SODs); or iron (Fe-SODs). Different plant SODs have been isolated and characterized, and many cDNAs and genes for SODs have been identified and characterized. SODs have an important function in plant physiology as a result of the double role of reactive oxygen species (ROS), as signals in important transduction pathways and as inducers of cellular damage when overproduced at high concentrations. In metabolic reactions, superoxide radicals are modulated by SODs but in their enzymatic reaction the key metabolite and signaling molecule H2O2 is produced, an important transduction signal in response to abiotic and biotic stresses and in diverse physiological processes. In general, abiotic stresses in plants induce the generation of ROS that can produce cellular oxidative damage when overproduced in high amounts. After abiotic stress treatment, those cultivars more resistant/tolerant usually show an enhanced activity of antioxidative enzymes, including SODs. Different reports are described on the response of SODs to abiotic stress produced in plants by heavy metals, salinity and drought, xenobiotics, low and high temperature, high light intensity, ozone and atmospheric contaminants, and mechanical stress. The genetic manipulation of plants with altered SOD activity to produce more oxidative stress-tolerant phenotypes that could be used to improve the stress tolerance of economically important plants are briefly examined. Finally, the effect of nitric oxide-mediated post-translational modifications of SODs on their enzymatic activity is discussed.

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.

Background and aims The development of seedlings involves many morphological, physiological and biochemical processes, which are controlled by many factors. Some reactive oxygen and nitrogen species (ROS and RNS, respectively) are implicated as signal molecules in physiological and phytopathological processes. Pepper (Capsicum annuum) is a very important crop and the goal of this work was to provide a framework of the behaviour of the key elements in the metabolism of ROS and RNS in the main organs of pepper during its development. Methods The main seedling organs (roots, hypocotyls and green cotyledons) of pepper seedlings were analysed 7, 10 and 14 d after germination. Activity and gene expression of the main enzymatic antioxidants (catalase, ascorbate-glutathione cycle enzymes), NADP-generating dehydrogenases and S-nitrosoglutathione reductase were determined. Cellular distribution of nitric oxide ((·)NO), superoxide radical (O2 (·-)) and peroxynitrite (ONOO(-)) was investigated using confocal laser scanning microscopy. Key results The metabolism of ROS and RNS during pepper seedling development was highly regulated and showed significant plasticity, which was co-ordinated among the main seedling organs, resulting in correct development. Catalase showed higher activity in the aerial parts of the seedling (hypocotyls and green cotyledons) whereas roots of 7-d-old seedlings contained higher activity of the enzymatic components of the ascorbate glutathione cycle, NADP-isocitrate dehydrogenase and NADP-malic enzyme. Conclusions There is differential regulation of the metabolism of ROS, nitric oxide and NADP dehydrogenases in the different plant organs during seedling development in pepper in the absence of stress. The metabolism of ROS and RNS seems to contribute significantly to plant development since their components are involved directly or indirectly in many metabolic pathways. Thus, specific molecules such as H2O2 and NO have implications for signalling, and their temporal and spatial regulation contributes to the success of seedling establishment.

In plant cells, the analysis of protein S-nitrosothiols (SNOs) under physiological and adverse stress conditions is essential to understand the mechanisms of Nitric oxide (NO)-based signaling. We adapted a previously reported protocol for detecting protein SNOs in animal systems ( King et al., 2005 ) for plant samples. Briefly, proteins from plant samples are separated via non-reducing SDS-PAGE, then the NO bound by S-nitrosylated proteins is released using UV light and, finally, the NO is detected using the fluorescent probe DAF-FM (Rodriguez-Ruiz et al., 2017). Thus, the approach presented here provides a relatively quick and economical procedure that can be used to compare protein SNOs content in plant samples and provide insight in NO-based signaling in plants.

6-Phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) catalyzes the third and irreversible reaction of the pentose phosphate pathway (PPP). It carries out the oxidative decarboxylation of the 6-phosphogluconate to yield ribulose-5-phosphate, carbon dioxide and NADPH. In higher plants, 6PGDH has several subcellular localizations including cytosol, chloroplast, mitochondria and peroxisomes ( Corpas et al., 1998 ; Krepinsky et al., 2001 ; Mateos et al., 2009 ; Fernandez-Fernandez and Corpas, 2016; Holscher et al., 2016 ). Using Arabidopsis thaliana as plant model and sweet pepper (Capsicum annuum L.) fruits as a plant with agronomical interest, this protocol illustrates how to prepare the plant extracts for the separation of the potential 6PGDH isoforms by electrophoresis on 6% polyacrylamide non-denaturing gels. Thus, this method allows detecting three 6PGDH isoforms in Arabidopsis seedlings and two 6PGDH isoforms in sweet pepper fruits.

Mechanical wounding in plants, which are capable of generating defense responses possibly associated with nitro-oxidative stress, can be caused by (a)biotic factors such as rain, wind, herbivores and insects. Sea rocket (Cakile maritima L.), a halophyte plant belonging to the mustard family Brassicaceae, is commonly found on sandy coasts throughout Europe. Using 7-day-old Cakile maritima L. seedlings, mechanical wounding was induced in hypocotyls by pinching with a striped-tip forceps; after 3 h, several biochemical parameters were analyzed in both the damaged and unwounded organs (green cotyledons and roots). We thus determined NO production, HO content, lipid oxidation as well as protein nitration patterns; we also identified several antioxidant enzymes including catalase, superoxide dismutase (SOD) isozymes, peroxidases, ascorbate-glutathione cycle enzymes and NADP-dehydrogenases. All these parameters were differentially modulated in the damaged (hypocotyls) and unwounded organs, which clearly indicated an induction of CuZnSOD V in the three organs, an increase in protein nitration in green cotyledons and an induction of NADP-isocitrate dehydrogenase activity in roots. On the whole, our results indicate that the wounding of hypocotyls, which showed an active ROS metabolism and oxidative stress, causes long-distance signals that also trigger responses in unwounded tissues with a more active RNS metabolism. These data therefore confirm the existence of local and long-distance responses which counteract negative effects and provide appropriate responses, enabling the wounded seedlings to survive., MRR acknowledges an FPI contract (BES-2012-055904) from the Ministry of Economy and Competitiveness, Spain. Work in the FJ Corpas laboratory is supported by the ERDF-cofinanced grant from the Ministry of Economy and Competitiveness (AGL2015-65104-P) and Junta de Andalucía (groupBIO192), Spain.

Pepper fruit is one of the highest vitamin C sources of plant origin for our diet. In plants, ascorbic acid is mainly synthesized through the L-galactose pathway, being the L-galactono-1,4-lactone dehydrogenase (GalLDH) the last step. Using pepper fruits, the full GalLDH gene was cloned and the protein molecular characterization accomplished. GalLDH protein sequence (586 residues) showed a 37 amino acids signal peptide at the N-terminus, characteristic of mitochondria. The hydrophobic analysis of the mature protein displayed one transmembrane helix comprising 20 amino acids at the N-terminus. By using a polyclonal antibody raised against a GalLDH internal sequence and immunoblotting analysis, a 56 kDa polypeptide cross-reacted with pepper fruit samples. Using leaves, flowers, stems and fruits, the expression of GalLDH by qRT-PCR and the enzyme activity were analyzed, and results indicate that GalLDH is a key player in the physiology of pepper plants, being possibly involved in the processes which undertake the transport of ascorbate among different organs. We also report that an NO (nitric oxide)-enriched atmosphere enhanced ascorbate content in pepper fruits about 40% parallel to increased GalLDH gene expression and enzyme activity. This is the first report on the stimulating effect of NO treatment on the vitamin C concentration in plants. Accordingly, the modulation by NO of GalLDH was addressed. In vitro enzymatic assays of GalLDH were performed in the presence of SIN-1 (peroxynitrite donor) and S-nitrosoglutahione (NO donor). Combined results of in vivo NO treatment and in vitro assays showed that NO provoked the regulation of GalLDH at transcriptional and post-transcriptional levels, but not post-translational modifications through nitration or S-nitrosylation events promoted by reactive nitrogen species (RNS) took place. These results suggest that this modulation point of the ascorbate biosynthesis could be potentially used for biotechnological purposes to increase the vitamin C levels in pepper fruits., Graphical abstract Application of exogenous nitric oxide (NO) gas to pepper fruits provokes enhancements of the ascobate levels and the gene expression and enzyme activity of the galactono-1,4-lactone dehydrogenase (GalLDH). This last enzyme of the of the biosynthetic pathway, which converts galactono-1,4-lactone (GalL) into ascorbate, is characterized by a mitochondrial targeting signal and a transmembrane domain both located at the N-teminus, and is likely to be regulated at transcriptional and post-transcriptional levels by reactive nitrogen species (RNS).fx1, Highlights • Treatment with nitric oxide (NO) enhances ascorbate levels in pepper fruits. • NO induces the galactono-1,4-lactone dehydrogenase (GalLDH), the last enzyme in the ascorbate biosynthetic pathway. • GalLDH seems to be modulated by NO at transcriptional and post-translational levels • This enzyme is bound to the mitochondrial membrane through a 20 amino acid transmembrane helix located at the N-terminus

Peroxiredoxins (Prxs) constitute a group of thiol-specific antioxidant enzymes which are present in bacteria, yeasts, and in plant and animal cells. Although Prxs are mainly localized in the cytosol, they are also present in mitochondria, chloroplasts, and nuclei, but there is no evidence of the existence of Prxs in plant peroxisomes. Using soluble fractions (matrices) of peroxisomes purified from leaves of pea (Pisum sativum L.) plants, the immunological analysis with affinity-purified IgG against yeast Prx1 revealed the presence of an immunoreactive band of about 50 kDa. The apparent molecular mass of the peroxisomal Prx was not sensitive to oxidizing and reducing conditions what could be a mechanism of protection against the oxidative environment existing in peroxisomes. Postembedment, EM immunocytochemical analysis with affinity-purified IgG against yeast Prx1 antibodies, confirmed that this protein was present in the peroxisomal matrix, mitochondria, and chloroplasts. In pea plants grown under oxidative stress conditions, the protein level of peroxisomal Prx was differentially modulated, being slightly induced by growth of plants with 50 µM CdCl2, but being significantly reduced by treatment with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The presence in the matrix of peroxisomes of a protein immunorelated to Prx of about 50 kDa, which is in the range of molecular mass of the dimeric form of other Prxs, opens new questions on the molecular properties of Prxs, but also on their function in the metabolism of reactive oxygen and nitrogen species (ROS/RNS) in these plant cell organelles, where they could be involved in the regulation of hydrogen peroxide and/or peroxynitrite.

Although peroxisomes are very simple organelles, research on different species has provided us with an understanding of their importance in terms of cell viability. In addition to the significant role played by plant peroxisomes in the metabolism of reactive oxygen species (ROS), data gathered over the last two decades show that these organelles are an endogenous source of nitric oxide (NO) and related molecules called reactive nitrogen species (RNS). Molecules such as NO and H2O2 act as retrograde signals among the different cellular compartments, thus facilitating integral cellular adaptation to physiological and environmental changes. However, under nitro-oxidative conditions, part of this network can be overloaded, possibly leading to cellular damage and even cell death. This review aims to update our knowledge of the ROS/RNS metabolism, whose important role in plant peroxisomes is still underestimated. However, this pioneering approach, in which key elements such as β-oxidation, superoxide dismutase (SOD) and NO have been mainly described in relation to plant peroxisomes, could also be used to explore peroxisomes from other organisms., Graphical abstract Guard cells are specialized plant cells located in photosynthetic organs, mainly in leaves, that are involved in the gas exchange. The confocal laser scanning microscope pictures show in vivo peroxisomal localization of nitric oxide (NO) and peroxynitrite (ONOO-) in guard cells of transgenic Arabidopsis thaliana. Green fluorescence is attributable of either NO or ONOO- and the green fluorescence punctate corresponds to peroxisomesfx1, Highlights • Plant peroxisomes possess unusual metabolic plasticity. • Plant peroxisomes enclose a very active ROS and RNS metabolism. • NO and H2O2 could act as retrograde signals among the different cellular compartments.

Arsenic (As) contamination is a major environmental problem which affects most living organisms from plants to animals. This metalloid poses a health risk for humans through its accumulation in crops and water. Using garlic (Allium sativum L.) plants as model crop exposed to 200 μM arsenate, a comparative study among their main organs (roots and shoots) was made. The analysis of arsenic, glutathione (GSH), phytochelatins (PCs) and lipid peroxidation contents with the activities of antioxidant enzymes (catalase, superoxide dismutase, ascorbate-glutathione cycle), and the main components of the NADPH-generating system, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH) was carried out. Data showed a correlation among arsenic accumulation in the different organs, PCs content and the antioxidative response, with a general decline of the NADPH-generating systems in roots. Overall, our results demonstrate that there are clear connections between arsenic uptake, increase of their As-chelating capacity in roots and a decline of antioxidative enzyme activities (catalase and the ascorbate peroxidase) whose alteration provoked As-induced oxidative stress. Thus, the data suggest that roots act as barrier of arsenic mediated by a prominent sulfur metabolism which is characterized by the biosynthesis of high amount of PCs., This work was supported by ERDF-cofinanced grants from the Ministry of Economy and Competitiveness (Recupera 2020-20134R056) and Junta de Andalucía (group BIO 192), Spain. MRR acknowledges a fellowship from the Ministry of Economy and Competitiveness, Spain. LC-ES/MS, and mineral analyses were carried out at the Instrumental Technical Services of the Estación Experimental del Zaidín (CSIC).

Pepper (Capsicum annuum, L.) fruit ripening is characterized by important phenotypic and metabolic changes, with emission of volatile compounds, cell wall softening, chlorophyll degradation, and new pigments and proteins’ synthesis. Recent data have shown that reactive oxygen and nitrogen species (ROS/RNS) are involved in fruit ripening by regulating both gene expression and protein functions through post-translational modifications. In this work, using immature (green) and ripe (red) pepper fruits, RNA-sequencing was performed through an Illumina platform. Raw reads were pre-processed and assembled resulting in a representative transcriptome of 63,353 tentative transcripts (TTs), of which 29,716 had a unique orthologue. Differential expression analysis between green and red pepper fruits showed that 4,325 and 3,269 transcripts were down-and up-regulated respectively at ripening. Moreover, isobaric tags for relative and absolute quantification (iTRAQ®) was used as a proteomic approach, identifying 2,574 proteins, of which 1,189 showed differential expression. In this study, we have combined transcriptomic and proteomic data focusing on oxidative metabolism and antioxidative systems to gain insight into their role on fruit ripening.

Pomegranate (Punica granatum L.) is a common edible fruit. Its juice can be used as a source of antioxidative compounds, primarily polyphenols and vitamin C, in addition to other vitamins and minerals. Nevertheless, little is still known about how the enzymatic machinery, mainly that related to oxidative metabolism, is influenced by the genotype and the environmental and climate conditions where pomegranate plants grow. In this work, seeds and juices from two pomegranate varieties (Valenciana and Mollar) grown in two different Spanish locations were assayed. Both varieties showed clear differences in their respective polypeptide profiles. The analysis of the isoenzymatic superoxide dismutase (SOD) activity pattern displayed oneMn-SODand five CuZn-SODs (I-V) whose abundances depended on the variety. Furthermore, by immunoblot assays, at least one additional Fe-SOD with a subunit size of about 23 kDa was also detected in both varieties. Besides this, the presence of the H2O2-scavenging peroxisomal catalase in seeds and juice indicates that an active metabolism of reactive oxygen species (ROS) takes place in this fruit, but the two pomegranate varieties showed opposite activity profiles. The activities of the main NADPH-regenerating enzymes, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphlogluconate dehydrogenase (6PGDH), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and NADP-dependent malic enzyme (NADP-ME), were studied in the same plant materials, and they behaved differently depending on the genotype. Finally, our data demonstrate the presence of two specific enzymes of the peroxisomal glyoxylate cycle, malate synthase (MS) and isocitrate lyase (ICL). These enzymes participate in oilseeds by channeling the lipid catabolism to the carbohydrate synthesis for further use in seed germination and early seedling growth. The results obtained in this work indicate that a similar mechanism to that reported in oilseeds may also operate in pomegranate., This research was achieved by an ERDF-cofinanced grant from the Ministry of Economy and Competitiveness (AGL2015-65104-P) and the Junta de Andalucía (group BIO192), Spain.

In plants, NADPH oxidase (NOX) is also known as a respiratory burst oxidase homolog (Rboh). This highly important enzyme, one of the main enzymatic sources of superoxide radicals (O2&bull, &minus, ), is involved in the metabolism of reactive oxygen and nitrogen species (ROS and RNS), which is active in the non-climacteric pepper (Capsicum annuum L.) fruit. We used sweet pepper fruits at two ripening stages (green and red) to biochemically analyze the O2&bull, generating Rboh activity and the number of isozymes during this physiological process. Malondialdehyde (MDA) content, an oxidative stress marker, was also assayed as an index of lipid peroxidation. In red fruits, MDA was observed to increase 2-fold accompanied by a 5.3-fold increase in total Rboh activity. Using in-gel assays of Rboh activity, we identified a total of seven CaRboh isozymes (I&ndash, VII) which were differentially modulated during ripening. CaRboh-III and CaRboh-I were the most prominent isozymes in green and red fruits, respectively. An in vitro assay showed that CaRboh activity is inhibited in the presence of nitric oxide (NO) donors, peroxynitrite (ONOO&minus, ) and glutathione (GSH), suggesting that CaRboh can undergo S-nitrosation, Tyr-nitration, and glutathionylation, respectively. In summary, this study provides a basic biochemical characterization of CaRboh activity in pepper fruits and indicates that this O2&bull, generating Rboh is involved in nitro-oxidative stress associated with sweet pepper fruit ripening.

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.

Pepper (Capsicum annuum L.) is an annual plant species of great agronomic importance whose fruits undergo major metabolic changes through development and ripening. These changes include emission of volatile organic compounds associated with respiration, destruction of chlorophylls and synthesis of new pigments (red/yellow carotenoids plus xanthophylls and anthocyans) responsible for color shift, protein degradation/synthesis and changes in total soluble reducing equivalents. Previous data have shown that, during the ripening of pepper fruit, an enhancement of protein tyrosine nitration takes place. On the other hand, it is well known that S-nitrosoglutathione reductase (GSNOR) activity can modulate the transnitrosylation equilibrium between GSNO and S-nitrosylated proteins and, consequently, regulate cellular NO homeostasis. In this study, GSNOR activity, protein content and gene expression were analyzed in green and red pepper fruits. The content of S-nitrosylated proteins on diaminofluorescein (DAF) gels was also studied. The data show that, while GSNOR activity and protein expression diminished during fruit ripening, S-nitrosylated protein content increased. Some of the protein candidates for S-nitrosylation identified, such as cytochorme c oxidase and peroxiredoxin II E, have previously been described as targets of this posttranslational modification in other plant species. These findings corroborate the important role played by GSNOR activity in the NO metabolism during the process of pepper fruit ripening.

NADP-dependent isocitrate dehydrogenase (NADP-ICDH) catalyses the production of NADPH, which is an essential component in the cellular homeostasis. In Arabidopsis, the kinetic parameters (Km and Vmax) of cytosolic NADP-ICDH were different in leaves and roots. In vitro applied H2O2 did not affect the NADP-ICDH activity in either organ, however, the reduced glutathione inhibited the activity in leaves but not in roots. On the other hand, S-nitrosoglutathione (a NO donor) and peroxynitrite depressed NADP-ICDH activity in leaves and roots.

Environmental contamination by arsenic constitutes a problem in many countries, and its accumulation in food crops may pose health complications for humans. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved at various levels in the mechanism of responding to environmental stress in higher plants. Using Arabidopsis seedlings exposed to different arsenate concentrations, physiological and biochemical parameters were analyzed to determine the status of ROS and RNS metabolisms. Arsenate provoked a significant reduction in growth parameters and an increase in lipid oxidation. These changes were accompanied by an alteration in antioxidative enzymes and the nitric oxide (NO) metabolism, with a significant increase in NO content, S-nitrosoglutathione reductase (GSNOR) activity and protein tyrosine nitration as well as a concomitant reduction in glutathione and S-nitrosoglutathione (GSNO) content. Our results indicate that 500 μM arsenate (AsV) causes nitro-oxidative stress in Arabidopsis, being the glutathione reductase and the GSNOR activities clearly affected.

Glutathione (GSH) is one of the major, soluble, low molecular weight antioxidants, as well as the major non-protein thiol in plant cells. However, the relevance of this molecule could be even greater considering that it can react with nitric oxide (NO) to generate S-nitrosoglutathione (GSNO) which is considered to function as a mobile reservoir of NO bioactivity in plants. Although this NO-derived molecule has an increased physiological and phytopathological relevance in plants cells, its identification and quantification in plant tissues have not be reported so far. Using liquid chromatography-electrospray/mass spectrometry (LC-ES/MS), a method was set up to detect and quantify simultaneously GSNO as well reduced and oxidized glutathione (GSH and GSSG, respectively) in different pepper plant organs including roots, stems and leaves, and in Arabidopsis leaves. The analysis of NO and GSNO reductase (GSNOR) activity in these pepper organs showed that the content of GSNO was directly related to the content of NO in each organ and oppositely related to the GSNOR activity. This approach opens up new analytical possibilities to understand the relevance of GSNO in plant cells under physiological and stress conditions.