Examinando por Autor "Chaki, Mounira"
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Ítem Computational Prediction of Candidate Proteins for S-Nitrosylation in Arabidopsis thaliana(PUBLIC LIBRARY SCIENCE, 2014-10-21) Chaki, Mounira; Kovacs, Izabella; Spannagl, Manuel; Lindermayr, ChristianNitric oxide (NO) is an important signaling molecule that regulates many physiological processes in plants. One of the most important regulatory mechanisms of NO is S-nitrosylation—the covalent attachment of NO to cysteine residues. Although the involvement of cysteine S-nitrosylation in the regulation of protein functions is well established, its substrate specificity remains unknown. Identification of candidates for S-nitrosylation and their target cysteine residues is fundamental for studying the molecular mechanisms and regulatory roles of S-nitrosylation in plants. Several experimental methods that are based on the biotin switch have been developed to identify target proteins for S-nitrosylation. However, these methods have their limits. Thus, computational methods are attracting considerable attention for the identification of modification sites in proteins. Using GPS-SNO version 1.0, a recently developed S-nitrosylation site-prediction program, a set of 16,610 candidate proteins for S-nitrosylation containing 31,900 S-nitrosylation sites was isolated from the entire Arabidopsis proteome using the medium threshold. In the compartments ‘‘chloroplast,’’ ‘‘CUL4-RING ubiquitin ligase complex,’’ and ‘‘membrane’’ more than 70% of the proteins were identified as candidates for S-nitrosylation. The high number of identified candidates in the proteome reflects the importance of redox signaling in these compartments. An analysis of the functional distribution of the predicted candidates showed that proteins involved in signaling processes exhibited the highest prediction rate. In a set of 46 proteins, where 53 putative S-nitrosylation sites were already experimentally determined, the GPS-SNO program predicted 60 S-nitrosylation sites, but only 11 overlap with the results of the experimental approach. In general, a computer-assisted method for the prediction of targets for S-nitrosylation is a very good tool; however, further development, such as including the three dimensional structure of proteins in such analyses, would improve the identification of S-nitrosylation sites.Ítem Differential molecular response of monodehydroascorbate reductase and glutathione reductase by nitration and S-nitrosylation(Oxford Academic, 2015-06-25) Begara-Morales, Juan Carlos; Sánchez-Calvo, Beatriz; Chaki, Mounira; Mata-Pérez, Capilla; Valderrama, Raquel; Padilla-Serrano, María Nieves; López-Jaramillo, Javier; Luque-Vázquez, Francisco; Corpas, Francisco Javier; Barroso-Albarracín, Juan BautistaThe ascorbate–glutathione cycle is a metabolic pathway that detoxifies hydrogen peroxide and involves enzymatic and non-enzymatic antioxidants. Proteomic studies have shown that some enzymes in this cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) are potential targets for post-translational modifications (PMTs) mediated by nitric oxide-derived molecules. Using purified recombinant pea peroxisomal MDAR and cytosolic and chloroplastic GR enzymes produced in Escherichia coli, the effects of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO) which are known to mediate protein nitration and S-nitrosylation processes, respectively, were analysed. Although ONOO– and GSNO inhibit peroxisomal MDAR activity, chloroplastic and cytosolic GR were not affected by these molecules. Mass spectrometric analysis of the nitrated MDAR revealed that Tyr213, Try292, and Tyr345 were exclusively nitrated to 3-nitrotyrosine by ONOO–. The location of these residues in the structure of pea peroxisomal MDAR reveals that Tyr345 is found at 3.3 Å of His313 which is involved in the NADP-binding site. Site-directed mutagenesis confirmed Tyr345 as the primary site of nitration responsible for the inhibition of MDAR activity by ONOO–. These results provide new insights into the molecular regulation of MDAR which is deactivated by nitration and S-nitrosylation. However, GR was not affected by ONOO– or GSNO, suggesting the existence of a mechanism to conserve redox status by maintaining the level of reduced GSH. Under a nitro-oxidative stress induced by salinity (150mM NaCl), MDAR expression (mRNA, protein, and enzyme activity levels) was increased, probably to compensate the inhibitory effects of S-nitrosylation and nitration on the enzyme. The present data show the modulation of the antioxidative response of key enzymes in the ascorbate–glutathione cycle by nitric oxide (NO)-PTMs, thus indicating the close involvement of NO and reactive oxygen species metabolism in antioxidant defence against nitro-oxidative stress situations in plants.Ítem Dual regulation of cytosolic ascorbate peroxidase (APX) by tyrosine nitration and S-nitrosylation(OXFORD UNIV PRESS, 2014-02) Begara-Morales, Juan Carlos; Sanchez-Calvo, Beatriz; Chaki, Mounira; Valderrama, Raquel; Mata-Pérez, Capilla; López-Jaramillo, Jaime; Padilla-Serrano, María Nieves; Carreras, Alfonso; Corpas, Francisco Javier; Barroso-Albarracín, Juan BautistaPost-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 (150 mM 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.Ítem Endogenous Biosynthesis of S-Nitrosoglutathione From Nitro-Fatty Acids in Plants(FRONTIERS MEDIA SA, 2020-06) Mata-Pérez, Capilla; Padilla-Serrano, María Nieves; Sánchez-Calvo, Beatriz; Begara-Morales, Juan Carlos; Valderrama, Raquel; Chaki, Mounira; Aranda-Caño, Lorena; Moreno-González, David; Molina-Díaz, Antonio; Barroso-Albarracín, Juan BautistaNitro-fatty acids (NO2-FAs) are novel molecules resulting from the interaction of unsaturated fatty acids and nitric oxide (NO) or NO-related molecules. In plants, it has recently been described that NO2-FAs trigger an antioxidant and a defence response against stressful situations. Among the properties of NO2-FAs highlight the ability to release NO therefore modulating specific protein targets through post translational modifications (NO-PTMs). Thus, based on the capacity of NO2-FAs to act as physiological NO donors and using high-accuracy mass-spectrometric approaches, herein, we show that endogenous nitro-linolenic acid (NO2-Ln) can modulate S nitrosoglutathione (GSNO) biosynthesis in Arabidopsis. The incubation of NO2-Ln with GSH was analyzed by LC-MS/MS and the in vitro synthesis of GSNO was noted. The in vivo confirmation of this behavior was carried out by incubating Arabidopsis plants with 15N-labeled NO2-Ln throughout the roots, and 15N-labeled GSNO (GS15NO) was detected in the leaves. With the aim to go in depth in the relation of NO2-FA and GSNO in plants, Arabidopsis alkenal reductase mutants (aer mutants) which modulate NO2-FAs levels were used. Our results constitute the first evidence of the modulation of a key NO biological reservoir in plants (GSNO) by these novel NO2-FAs, increasing knowledge about S-nitrosothiols and GSNO signaling pathways in plants.Ítem Función de las especies de Nitrógeno reactivo en la modulación de respuesta de plántulas de girasol (Helianthus annuus L.) frente a procesos de estrés biótico y abiótico(Jaén : Universidad de Jaén, 2012) Chaki, Mounira; Barroso-Albarracín, Juan Bautista; Carreras, Alfonso; Universidad de Jaén. Departamento de Biología ExperimentalEl óxido nítrico (NO) es la especies de nitrógeno reactivo más relevante, tiene un papel importamte en la regulación de funciones de planta. Así, se mostró su participación en los mecanismos de respuesta de planta frente al estrés biótico y abiótico. Sin embargo, poco es conocido sobre la participación de otras moléculas derivadas de NO como S-nitrosotioles, S-nitrosoglutation, peroxinitrito y 3-nitrotirosina. Por lo tanto, este trabajo se ha enfocado en el estudio del metabolismo de NO y moléculas derivadas de NO en hipocótilos de plantas de girasol (Helianthus annuus L.) sometidas a estrés biótico y abiótico. Para el estrés biótico, se ha estudiado la inoculación por el patógeno Plasmopara halstedii usando dos líneas de girasol HA89 susceptible donde el patógeno desarrolla su virulencia (interacción compatible) y la línea X55 donde el patógeno no puede desarrollarse (interacción incompatible). Por otro lado, se ha estudiado el estrés abiótico usando las mismas plantas que fueron sometidas a diferentes condiciones de estrés incluyendo alta y baja temperatura, daño mecánico, alta intensidad luminosa, luz continua y oscuridad.Ítem High temperature triggers the metabolism of S-nitrosothiols in sunflower mediating a process of nitrosative stress which provokes the inhibition of ferredoxin-NADP reductase by tyrosine nitration(WILEY, 2011-06) Chaki, Mounira; Valderrama, Raquel; Fernández-Ocaña, Ana; Carreras, Alfonso; Gómez-Rodríguez, María Victoria; López-Jaramillo, Jaime; Begara-Morales, Juan Carlos; Sánchez-Calvo, Beatriz; Luque-Vázquez, Francisco; Leterrier, Marina; Corpas, Francisco Javier; Barroso-Albarracín, Juan BautistaHigh temperature (HT) is considered a major abiotic stress that negatively affects both vegetative and reproductive growth. Whereas the metabolism of reactive oxygen species (ROS) is well established under HT, less is known about the metabolism of reactive nitrogen species (RNS). In sunflower (Helianthus annuus L.) seedlings exposed to HT, NO content as well as S-nitrosoglutathione reductase (GSNOR) activity and expression were down-regulated with the simultaneous accumulation of total S-nitrosothiols (SNOs) including S-nitrosoglutathione (GSNO). However, the content of tyrosine nitration (NO2-Tyr) studied by highperformance liquid chromatography with tandem mass spectrometry (LC–MS/MS) and by confocal laser scanning microscope was induced. Nitroproteome analysis under HT showed that this stress induced the protein expression of 13 tyrosine-nitrated proteins. Among the induced proteins, ferredoxin–NADP reductase (FNR) was selected to evaluate the effect of nitration on its activity after heat stress and in vitro conditions using 3-morpholinosydnonimine (SIN-1) (peroxynitrite donor) as the nitrating agent, the FNR activity being inhibited. Taken together, these results suggest that HT augments SNOs, which appear to mediate protein tyrosine nitration, inhibiting FNR, which is involved in the photosynthesis process.Ítem Mechanical wounding induces a nitrosative stress by down-regulation of GSNO reductase and an increase in S-nitrosothiols in sunflower (Helianthus annuus) seedlings(OXFORD UNIVERSITY PRESS, 2011-03) Chaki, Mounira; Valderrama, Raquel; Fernández-Ocaña, Ana; Carreras, Alfonso; Gómez-Rodríguez, María Victoria; Pedrajas, José Rafael; Begara-Morales, Juan Carlos; Sánchez-Calvo, Beatriz; Luque-Vázquez, Francisco; Leterrier, Marina; Corpas, Francisco Javier; Barroso-Albarracín, Juan BautistaNitric oxide (NO) and related molecules such as peroxynitrite, S-nitrosoglutathione (GSNO), and nitrotyrosine, among others, are involved in physiological processes as well in the mechanisms of response to stress conditions. In sunflower seedlings exposed to five different adverse environmental conditions (low temperature, mechanical wounding, high light intensity, continuous light, and continuous darkness), key components of the metabolism of reactive nitrogen species (RNS) and reactive oxygen species (ROS), including the enzyme activities L-argininedependent nitric oxide synthase (NOS), S-nitrosogluthathione reductase (GSNOR), nitrate reductase (NR), catalase, and superoxide dismutase, the content of lipid hydroperoxide, hydrogen peroxide, S-nitrosothiols (SNOs), the cellular level of NO, GSNO, and GSNOR, and protein tyrosine nitration [nitrotyrosine (NO2-Tyr)] were analysed. Among the stress conditions studied, mechanical wounding was the only one that caused a down-regulation of NOS and GSNOR activities, which in turn provoked an accumulation of SNOs. The analyses of the cellular content of NO, GSNO, GSNOR, and NO2-Tyr by confocal laser scanning microscopy confirmed these biochemical data. Therefore, it is proposed that mechanical wounding triggers the accumulation of SNOs, specifically GSNO, due to a downregulation of GSNOR activity, while NO2-Tyr increases. Consequently a process of nitrosative stress is induced in sunflower seedlings and SNOs constitute a new wound signal in plants.Ítem Metabolism of reactive nitrogen species in pea plants under abiotic stress conditions(OXFORD UNIV PRESS, 2008-09-18) Corpas, Francisco Javier; Chaki, Mounira; Fernández-Ocaña, Ana; Valderrama, Raquel; Palma, José Manuel; Carreras, Alfonso; Begara-Morales, Juan Carlos; Airaki, Morad; del-Río, Luis Alfonso; Barroso-Albarracín, Juan BautistaNitric oxide (NO) is a key signaling molecule in different physiological processes of animals and plants. However, little is known about the metabolism of endogenous NO and other reactive nitrogen species (RNS) in plants under abiotic stress conditions. Using pea plants exposed to six different abiotic stress conditions (high light intensity, low and high temperature, continuous light, continuous dark and mechanical wounding), several key components of the metabolism of RNS including the content of NO, S-nitrosothiols (RSNOs) and nitrite plus nitrate, the enzyme activities of L-arginine dependent nitric oxide synthase (NOS) and S-nitrosogluthathione reductase (GSNOR), and the profile of protein tyrosine nitration (NO2-Tyr) were analyzed in leaves. Low temperature was the stress that produced the highest increase of NOS and GSNOR activities, and this was accompanied by an increase in the content of total _NO and S-nitrosothiols, and an intensification of the immunoreactivity with an antibody against NO2-Tyr. Mechanical wounding, high temperature and light also had a clear activating effect on the different indicators of RNS metabolism in pea plants. However, the total content of nitrite and nitrate in leaves was not affected by any of these stresses. Considering that protein tyrosine nitration is a potential marker of nitrosative stress, the results obtained suggest that low and high temperature, continuous light and high light intensity are abiotic stress conditions that can induce nitrosative stress in pea plants.Ítem Metabolism of reactive oxygen species and reactive nitrogen species in pepper (Capsicum annuum L.) plants under low temperature stress(WILEY, 2012-02) Airaki, Morad; Leterrier, Marina; Mateos, Rosa María; Valderrama, Raquel; Chaki, Mounira; Barroso-Albarracín, Juan Bautista; del-Río, Luis Alfonso; Palma, José Manuel; Corpas, Francisco JavierLow temperature is an environmental stress that affects crop production and quality and regulates the expression of many genes, and the level of a number of proteins and metabolites. Using leaves from pepper (Capsicum annum L.) plants exposed to low temperature (8°C) for different time periods (1 to 3d), several key components of the metabolism of reactive nitrogen and oxygen species (RNS and ROS, respectively) were analysed. After 24h of exposure at 8°C, pepper plants exhibited visible symptoms characterized by flaccidity of stems and leaves. This was accompanied by significant changes in the metabolism of RNS and ROS with an increase of both protein tyrosine nitration (NO2-Tyr) and lipid peroxidation, indicating that low temperature induces nitrosative and oxidative stress. During the second and third days at low temperature, pepper plants underwent cold acclimation by adjusting their antioxidant metabolism and reverting the observed nitrosative and oxidative stress. In this process, the levels of the soluble non-enzymatic antioxidants ascorbate and glutathione, and the activity of the main NADPH-generating dehydrogenases were significantly induced. This suggests that ascorbate, glutathione and the NADPH-generating dehydrogenases have a role in the process of cold acclimation through their effect on the redox state of the cell.Ítem Nitrated Fatty-Acids Distribution in Storage Biomolecules during Arabidopsis thaliana Development(MDPI, 2022-09) Valderrama, Raquel; Chaki, Mounira; Begara-Morales, Juan Carlos; Melguizo, Manuel; Barroso-Albarracín, Juan Bautista; Aranda-Caño, LorenaThe non-enzymatic interaction of polyunsaturated fatty acids with nitric oxide (NO) and de rived species results in the formation of nitrated fatty acids (NO2-FAs). These signaling molecules can release NO, reversibly esterify with complex lipids, and modulate protein function through the post translational modification called nitroalkylation. To date, NO2-FAs act as signaling molecules during plant development in plant systems and are involved in defense responses against abiotic stress conditions. In this work, the previously unknown storage biomolecules of NO2-FAs in Arabidopsis thaliana were identified. In addition, the distribution of NO2-FAs in storage biomolecules during plant development was determined, with phytosterol esters (SE) and TAGs being reservoir biomolecules in seeds, which were replaced by phospholipids and proteins in the vegetative, generative, and senescence stages. The detected esterified NO2-FAs were nitro-linolenic acid (NO2-Ln), nitro-oleic acid (NO2-OA), and nitro-linoleic acid (NO2-LA). The last two were detected for the first time in Arabidopsis. The levels of the three NO2-FAs that were esterified in both lipid and protein storage biomolecules showed a decreasing pattern throughout Arabidopsis development. Esterification of NO2-FAs in phospholipids and proteins highlights their involvement in both biomembrane dynamics and signaling processes, respectively, during Arabidopsis plant development.Ítem Nitro-Oleic Acid-Mediated Nitroalkylation Modulates the Antioxidant Function of Cytosolic Peroxiredoxin Tsa1 during Heat Stress in Saccharomyces cerevisiae(MDPI, 2022-05-14) Aranda-Caño, Lorena; Valderrama, Raquel; Pedrajas, José Rafael; Begara-Morales, Juan Carlos; Chaki, Mounira; Padilla-Serrano, María Nieves; Melguizo, Manuel; López-Jaramillo, Francisco Javier; Barroso-Albarracín, Juan BautistaHeat stress is one of the abiotic stresses that leads to oxidative stress. To protect themselves, yeast cells activate the antioxidant response, in which cytosolic peroxiredoxin Tsa1 plays an important role in hydrogen peroxide removal. Concomitantly, the activation of the heat shock response (HSR) is also triggered. Nitro-fatty acids are signaling molecules generated by the interaction of reactive nitrogen species with unsaturated fatty acids. These molecules have been detected in animals and plants. They exert their signaling function mainly through a post-translational modification called nitroalkylation. In addition, these molecules are closely related to the induction of the HSR. In this work, the endogenous presence of nitro-oleic acid (NO2-OA) in Saccharomyces cerevisiae is identified for the first time by LC-MS/MS. Both hydrogen peroxide levels and Tsa1 activity increased after heat stress with no change in protein content. The nitroalkylation of recombinant Tsa1 with NO2-OA was also observed. It is important to point out that cysteine 47 (peroxidatic) and cysteine 171 (resolving) are the main residues responsible for protein activity. Moreover, the in vivo nitroalkylation of Tsa1 peroxidatic cysteine disappeared during heat stress as the hydrogen peroxide generated in this situation caused the rupture of the NO2-OA binding to the protein and, thus, restored Tsa1 activity. Finally, the amino acid targets susceptible to nitroalkylation and the modulatory effect of this PTM on the enzymatic activity of Tsa1 are also shown in vitro and in vivo. This mechanism of response was faster than that involving the induction of genes and the synthesis of new proteins and could be considered as a key element in the fine-tuning regulation of defence mechanisms against oxidative stress in yeast.Ítem Post-Translational Modification of Proteins Mediated by Nitro-Fatty Acids in Plants: Nitroalkylation(MDPI, 2019-03-29) Aranda-Caño, Lorena; Sánchez-Calvo, Beatriz; Begara-Morales, Juan Carlos; Chaki, Mounira; Mata-Pérez, Capilla; Padilla-Serrano, María Nieves; Valderrama, Raquel; Barroso-Albarracín, Juan BautistaNitrate fatty acids (NO2-FAs) are considered reactive lipid species derived from the non-enzymatic oxidation of polyunsaturated fatty acids by nitric oxide (NO) and related species. Nitrate fatty acids are powerful biological electrophiles which can react with biological nucleophiles such as glutathione and certain protein–amino acid residues. The adduction of NO2-FAs to protein targets generates a reversible post-translational modification called nitroalkylation. In di erent animal and human systems, NO2-FAs, such as nitro-oleic acid (NO2-OA) and conjugated nitro-linoleic acid (NO2-cLA), have cytoprotective and anti-inflammatory influences in a broad spectrum of pathologies by modulating various intracellular pathways. However, little knowledge on these molecules in the plant kingdom exists. The presence of NO2-OA and NO2-cLA in olives and extra-virgin olive oil and nitro-linolenic acid (NO2-Ln) in Arabidopsis thaliana has recently been detected. Specifically, NO2-Ln acts as a signaling molecule during seed and plant progression and beneath abiotic stress events. It can also release NO and modulate the expression of genes associated with antioxidant responses. Nevertheless, the repercussions of nitroalkylation on plant proteins are still poorly known. In this review, we demonstrate the existence of endogenous nitroalkylation and its effect on the in vitro activity of the antioxidant protein ascorbate peroxidase.Ítem Protein targets of tyrosine nitration in sunflower (Helianthus annuus L.) hypocotyls(OXFORD UNIV PRESS, 2009-08-28) Chaki, Mounira; Valderrama, Raquel; Fernández-Ocaña, Ana; Carreras, Alfonso; López-Jaramillo, Jaime; Luque-Vázquez, Francisco; Palma, José Manuel; Pedrajas, José Rafael; Begara-Morales, Juan Carlos; Sánchez-Calvo, Beatriz; Gómez-Rodríguez, María Victoria; Corpas, Francisco Javier; Barroso-Albarracín, Juan BautistaTyrosine 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 (NO₂-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 NO₂- Tyr in hypocotyls was 0.56 mmol 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.Ítem Protein tyrosine nitration in pea roots during development and senescence(OXFORD UNIV PRESS, 2013-01-28) Begara-Morales, Juan Carlos; Chaki, Mounira; Sánchez-Calvo, Beatriz; Mata-Pérez, Capilla; Leterrier, Marina; Palma, José Manuel; Barroso-Albarracín, Juan Bautista; Corpas, Francisco JavierProtein 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.Ítem Ripening of pepper (Capsicum annuum) fruit is characterized by an enhancement of protein tyrosine nitration(OXFORD UNIV PRESS, 2015-03-26) Chaki, Mounira; Álvarez-de-Morales, Paz; Ruíz-Torres, Carmelo; Begara-Morales, Juan Carlos; Barroso-Albarracín, Juan Bautista; Corpas, Francisco Javier; Palma, José ManuelPepper (Capsicum annuum, Solanaceae) fruits are consumed worldwide and are of great economic importance. In most species ripening is characterized by important visual and metabolic changes, the latter including emission of volatile organic compounds associated with respiration, destruction of chlorophylls, synthesis of new pigments (red/yellow carotenoids plus xanthophylls and anthocyanins), formation of pectins and protein synthesis. The involvement of nitric oxide (NO) in fruit ripening has been established, but more work is needed to detail the metabolic networks involving NO and other reactive nitrogen species (RNS) in the process. It has been reported that RNS can mediate post-translational modifications of proteins, which can modulate physiological processes through mechanisms of cellular signalling. This study therefore examined the potential role of NO in nitration of tyrosine during the ripening of California sweet pepper. The NO content of green and red pepper fruit was determined spectrofluorometrically. Fruits at the breaking point between green and red coloration were incubated in the presence of NO for 1 h and then left to ripen for 3 d. Profiles of nitrated proteins were determined using an antibody against nitro-tyrosine (NO2-Tyr), and profiles of nitrosothiols were determined by confocal laser scanning microscopy. Nitrated proteins were identified by 2-D electrophoresis and MALDI-TOF/TOF analysis. Treatment with NO delayed the ripening of fruit. An enhancement of nitrosothiols and nitroproteins was observed in fruit during ripening, and this was reversed by the addition of exogenous NO gas. Six nitrated proteins were identified and were characterized as being involved in redox, protein, carbohydrate and oxidative metabolism, and in glutamate biosynthesis. Catalase was the most abundant nitrated protein found in both green and red fruit. The RNS profile reported here indicates that ripening of pepper fruit is characterized by an enhancement of S-nitrosothiols and protein tyrosine nitration. The nitrated proteins identified have important functions in photosynthesis, generation of NADPH, proteolysis, amino acid biosynthesis and oxidative metabolism. The decrease of catalase in red fruit implies a lower capacity to scavenge H2O2, which would promote lipid peroxidation, as has already been reported in ripe pepper fruit.Ítem Role of electrophilic nitrated fatty acids during development and response to abiotic stress processes in plants(Oxford University Press, 2020-11-07) Begara-Morales, Juan Carlos; Mata-Pérez, Capilla; Padilla-Serrano, María Nieves; Chaki, Mounira; Valderrama, Raquel; Aranda-Caño, Lorena; Barroso-Albarracín, Juan BautistaNitro-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.Ítem Short-Term Low Temperature Induces Nitro-Oxidative Stress that Deregulates the NADP-Malic Enzyme Function by Tyrosine Nitration in Arabidopsis thaliana(MDPI, 2019-10-01) Begara-Morales, Juan Carlos; Sánchez-Calvo, Beatriz; Gómez-Rodríguez, María Victoria; Chaki, Mounira; Valderrama, Raquel; Mata-Pérez, Capilla; López-Jaramillo, Francisco Javier; Corpas, Francisco Javier; Barroso-Albarracín, Juan BautistaLow temperature (LT) negatively a ects plant growth and development via the alteration of the metabolism of reactive oxygen and nitrogen species (ROS and RNS).AmongRNS, 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 a ect 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 e ect 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 a ects the metabolism of ROS and RNS, which appears to negatively modulate the activity of cytosolic NADP-ME through the tyrosine nitration process.Ítem The function of S-nitrosothiols during abiotic stress in plants(Oxford University Press, 2019-04-27) Begara-Morales, Juan Carlos; Chaki, Mounira; Valderrama, Raquel; Mata-Pérez, Capilla; Padilla-Serrano, María Nieves; Barroso-Albarracín, Juan BautistaNitric 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.