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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.

Fruits are unique to flowering plants and confer a selective advantage as they facilitate seed maturation and dispersal. In fleshy fruits, development and ripening are associated with numerous structural, biochemical, and physiological changes, including modifications in the general appearance, texture, flavor, and aroma, which ultimately convert the immature fruit into a considerably more attractive and palatable structure for seed dispersal by animals. Treatment with exogenous nitric oxide (NO) delays fruit ripening, prevents chilling damage, promotes disease resistance, and enhances the nutritional value. The ripening process is influenced by NO, which operates antagonistically to ethylene, but it also interacts with other regulatory molecules such as abscisic acid, auxin, jasmonic acid, salicylic acid, melatonin, and hydrogen sulfide. NO content progressively declines during fruit ripening, with concomitant increases in protein nitration and nitrosation, two post-translational modifications that are promoted by reactive nitrogen species. Dissecting the intimate interactions of NO with other ripening-associated factors, including reactive oxygen species, antioxidants, and the aforementioned phytohormones, remains a challenging subject of research. In this context, integrative 'omics' and gene-editing approaches may provide additional knowledge of the impact of NO in the regulatory processes involved in controlling physiology and quality traits in both climacteric and non-climacteric fruits., Financing by ERDF funds through the Ministry of Economy and Competitiveness (AGL2015-65104-P) and by the Junta de Andalucía (group BIO192), Spain, is acknowledged. The work of LF is supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant nos #2016/01128-99 and #2018/16389-8).

Ripening is a complex physiological process that involves changes in reactive nitrogen and oxygen species that govern the shelf-life and quality of fruits. Nitric oxide (NO)-dependent changes in the sweet pepper fruit transcriptome were determined by treating fruits at the initial breaking point stage with NO gas. Fruits were also harvested at the immature (green) and ripe (red) stages. Fruit ripening in the absence of NO resulted in changes in the abundance of 8805 transcripts whose function could be identified. Among these, functional clusters associated with reactive oxygen/nitrogen species and lipid metabolism were significantly modified. NO treatment resulted in the differential expression of 498 genes framed within these functional categories. Biochemical analysis revealed that NO treatment resulted in changes in fatty acid profiling, glutathione and proline contents, and the extent of lipid peroxidation, as well as increases in the activity of ascorbate peroxidase and lipoxygenase. These data provide supporting evidence for the crucial role of NO in the ripening of pepper fruit., The work of FJC and JMP is supported by a European Regional Development Fund-cofinanced grant from the Ministry of Economy and Competitiveness (AGL2015-65104-P) and Junta de Andalucia (group BIO192), Spain. SGG acknowledges a ‘Formacion de Personal Investigador’ contract from the Ministry of Economy and Competitiveness. The provision of pepper fruits by Syngenta Seeds Ltd (El Ejido, Almeria, Spain) is acknowledged. GC-MS analyses were done at the Instrumental Technical Services of the Estacion Experimental del Zaidin (CSIC) and special thanks are given to Dr Rafael Nunez-Gomez. The valuable technical assistance of Mrs Maria J. Campos and Mr Carmelo Ruiz-Torres is deeply acknowledged. The authors also gratefully acknowledge the computer resources and technical support provided by the Plataforma Andaluza de Bioinformatica of the University of Malaga.

Nitric oxide (NO) has been implicated as part of the ripening regulatory network in fleshy fruits. However, very little is known about the simultaneous action of NO on the network of regulatory events and metabolic reactions behind ripening-related changes in fruit color, taste, aroma and nutritional value. Here, we performed an in-depth characterization of the concomitant changes in tomato (Solanum lycopersicum) fruit transcriptome and metabolome associated with the delayed-ripening phenotype caused by NO supplementation at the pre-climacteric stage. Approximately one-third of the fruit transcriptome was altered in response to NO, including a multilevel down-regulation of ripening regulatory genes, which in turn restricted the production and tissue sensitivity to ethylene. NO also repressed hydrogen peroxide-scavenging enzymes, intensifying nitro-oxidative stress and S-nitrosation and nitration events throughout ripening. Carotenoid, tocopherol, flavonoid and ascorbate biosynthesis were differentially affected by NO, resulting in overaccumulation of ascorbate (25%) and flavonoids (60%), and impaired lycopene production. In contrast, the biosynthesis of compounds related to tomato taste (sugars, organic acids, amino acids) and aroma (volatiles) was slightly affected by NO. Our findings indicate that NO triggers extensive transcriptional and metabolic rewiring at the early ripening stage, modifying tomato antioxidant composition with minimal impact on fruit taste and aroma., This work was supported by the São Paulo Research Foundation (FAPESP) (grants 2018/16389-8, 2016/04924-0, 2017/17935-3 and 2016/01128-9), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grants 422287/2018-0, 305012/2018-5, 303332/2019-0 and 300986/2018-1), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001. The research work of FJC and JMP is supported by a European Regional Development Fund cofinanced grant from the Ministry of Economy and Competitiveness (AGL2015-65104-P and PID2019-103924GB-I00), Spain.

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.

In this work the manganese superoxide dismutase (Mn-SOD) bound to peroxisomal membranes of watermelon cotyledons (Citrullus lanatus Schrad.) was purified to homogeneity and some of its molecular properties were determined. The stepwise purification procedure consisted of ammonium sulphate fractionation, batch anion-exchange chromatography, and anion-exchange and gel-filtration column chromatography using a fast protein liquid chromatography system. Peroxisomal membrane Mn-SOD (perMn-SOD; EC 1.15.1.1) was purified 5600-fold with a yield of 2.6 mug of enzyme g(-1) of cotyledons, and had a specific activity of 480 U mg(-1) of protein. The native molecular mass determined for perMn-SOD was 108 000 Da, and it was composed of four equal subunits of 27 kDa, which indicates that perMn-SOD is a homotetramer. Ultraviolet and visible absorption spectra of the enzyme showed a shoulder at 275 nm and two absorption maxima at 448 nm and 555 nm, respectively. By isoelectric focusing, a pI of 5.75 was determined for perMn-SOD. In immunoblot assays, purified perMn-SOD was recognized by a polyclonal antibody against Mn-SOD from pea leaves, and the peroxisomal enzyme rapidly dissociated in the presence of dithiothreitol and SDS. The potential binding of the Mn-SOD isozyme to the peroxisomal membrane was confirmed by immunoelectron microscopy analysis. The properties of perMn-SOD and the mitMn-SOD are compared and the possible function in peroxisomal membranes of the peripheral protein Mn-SOD is discussed.

Leaf senescence and associated changes in redox components were monitored in commercial pea (Pisum sativum L. cv. Phoenix) plants grown under different nitrogen regimes for 12 weeks until both nodules and leaves had fully senesced. One group of plants was inoculated with Rhizobium leguminosarum and grown with nutrient solution without nitrogen. A second group was not inoculated and these were grown on complete nutrient solution containing nitrogen. Leaf senescence was evident at 11 weeks in both sets of plants as determined by decreases in leaf chlorophyll and protein. However, a marked decrease in photosynthesis was observed in nodulated plants at 9 weeks. Losses in the leaf ascorbate pool preceded leaf senescence, but leaf glutathione decreased only during the senescence phase. Large decreases in dehydroascorbate reductase and catalase activities were observed after 9 weeks, but the activities of other antioxidant enzymes remained high even at 11 weeks. The extent of lipid peroxidation, the number of protein carbonyl groups and the level of H(2)O(2) in the leaves of both nitrate-fed and nodulated plants were highest at the later stages of senescence. At 12 weeks, the leaves of nodulated plants had more protein carbonyl groups and greater lipid peroxidation than the nitrate-fed controls. These results demonstrate that the leaves of nodulated plants undergo an earlier inhibition of photosynthesis and suffer enhanced oxidation during the senescence phase than those from nitrate-fed plants.

Peroxisomes are subcellular organelles with an essentially oxidative type of metabolism. Like chloroplasts and mitochondria, plant peroxisomes also produce superoxide radicals (O .- 2 ) and there are, at least, two sites of superoxide generation: one in the organelle matrix, the generating system being xanthine oxidase, and another site in the peroxisomal membranes dependent on NAD(P)H. In peroxisomal membranes, three integral polypeptides (PMPs) with molecular masses of 18, 29 and 32 kDa have been shown to generate O .- 2 radicals. Besides catalase, several antioxidative systems have been demonstrated in plant peroxisomes, including different superoxide dismutases, the ascorbate-glutathione cycle, and three NADP-dependent dehydrogenases. A CuZn-SOD and two Mn-SODs have been purified and characterized from different types of peroxisomes. The four enzymes of the ascorbate-glutathione cycle (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase) as well as the antioxidants glutathione and ascorbate have been found in plant peroxisomes. The recycling of NADPH from NADP + can be carried out in peroxisomes by three dehydrogenases: glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and isocitrate dehydrogenase. In the last decade, different experimental evidence has suggested the existence of cellular functions for peroxisomes related to reactive oxygen species (ROS), but the recent demonstration of the presence of nitric oxide synthase (NOS) in plant peroxisomes implies that these organelles could also have a function in plant cells as a source of signal molecules like nitric oxide (NO), superoxide radicals, hydrogen peroxide, and possibly S-nitrosoglutathione (GSNO).

Tyrosine nitration is recognized as an important post-translational protein modification in animal cells that can be used as an indicator of a nitrosative process. However, in plant systems, there is scant information on proteins that undergo this process. In sunflower hypocotyls, the content of tyrosine nitration (NO(2)-Tyr) and the identification of nitrated proteins were studied by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and proteomic approaches, respectively. In addition, the cell localization of nitrotyrosine proteins and peroxynitrite were analysed by confocal laser-scanning microscopy (CLSM) using antibodies against 3-nitrotyrosine and 3'-(p-aminophenyl) fluorescein (APF) as the fluorescent probe, in that order. The concentration of Tyr and NO(2)-Tyr in hypocotyls was 0.56 micromol mg(-1) protein and 0.19 pmol mg(-1) protein, respectively. By proteomic analysis, a total of 21 nitrotyrosine-immunopositive proteins were identified. These targets include proteins involved in photosynthesis, and in antioxidant, ATP, carbohydrate, and nitrogen metabolism. Among the proteins identified, S-adenosyl homocysteine hydrolase (SAHH) was selected as a model to evaluate the effect of nitration on SAHH activity using SIN-1 (a peroxynitrite donor) as the nitrating agent. When the hypocotyl extracts were exposed to 0.5 mM, 1 mM, and 5 mM SIN-1, the SAHH activity was inhibited by some 49%, 89%, and 94%, respectively. In silico analysis of the barley SAHH sequence, characterized Tyr448 as the most likely potential target for nitration. In summary, the present data are the first in plants concerning the content of nitrotyrosine and the identification of candidates of protein nitration. Taken together, the results suggest that Tyr nitration occurs in plant tissues under physiological conditions that could constitute an important process of protein regulation in such a way that, when it is overproduced in adverse circumstances, it can be used as a marker of nitrosative stress.

Peroxisomes are subcellular organelles with an essentially oxidative type of metabolism. Like chloroplasts and mitochondria, plant peroxisomes also produce superoxide radicals (O2*(-)) and there are, at least, two sites of superoxide generation: one in the organelle matrix, the generating system being xanthine oxidase, and another site in the peroxisomal membranes dependent on NAD(P)H. In peroxisomal membranes, three integral polypeptides (PMPs) with molecular masses of 18, 29 and 32 kDa have been shown to generate radicals O2*(-). Besides catalase, several antioxidative systems have been demonstrated in plant peroxisomes, including different superoxide dismutases, the ascorbate-glutathione cycle, and three NADP-dependent dehydrogenases. A CuZn-SOD and two Mn-SODs have been purified and characterized from different types of peroxisomes. The four enzymes of the ascorbate-glutathione cycle (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase) as well as the antioxidants glutathione and ascorbate have been found in plant peroxisomes. The recycling of NADPH from NADP(+) can be carried out in peroxisomes by three dehydrogenases: glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and isocitrate dehydrogenase. In the last decade, different experimental evidence has suggested the existence of cellular functions for peroxisomes related to reactive oxygen species (ROS), but the recent demonstration of the presence of nitric oxide synthase (NOS) in plant peroxisomes implies that these organelles could also have a function in plant cells as a source of signal molecules like nitric oxide (NO*), superoxide radicals, hydrogen peroxide, and possibly S-nitrosoglutathione (GSNO).