Pepper catalase: a broad analysis of its modulation during fruit ripening and by nitric oxide.

Catalase is a major antioxidant enzyme located in plant peroxisomes that catalyzes the decomposition of H2O2. Based on our previous transcriptomic (RNA-Seq) and proteomic (iTRAQ) data at different stages of pepper (Capsicum annuum L.) fruit ripening and after exposure to nitric oxide (NO) enriched atmosphere, a broad analysis has allowed us to characterize the functioning of this enzyme. Three genes were identified, and their expression was differentially modulated during ripening and by NO gas treatment. A dissimilar behavior was observed in the protein expression of the encoded protein catalases (CaCat1-CaCat3). Total catalase activity was down-regulated by 50% in ripe (red) fruits concerning immature green fruits. This was corroborated by non-denaturing polyacrylamide gel electrophoresis, where only a single catalase isozyme was identified. In vitro analyses of the recombinant CaCat3 protein exposed to peroxynitrite (ONOO-) confirmed, by immunoblot assay, that catalase underwent a nitration process. Mass spectrometric analysis identified that Tyr348 and Tyr360 were nitrated by ONOO-, occurring near the active center of catalase. The data indicate the complex regulation at gene and protein levels of catalase during the ripening of pepper fruits, with activity significantly down-regulated in ripe fruits. Nitration seems to play a key role in this down-regulation, favoring an increase in H2O2 content during ripening. This pattern can be reversed by the exogenous NO application. While plant catalases are generally reported to be tetrameric, the analysis of the protein structure supports that pepper catalase has a favored quaternary homodimer nature. Taken together, data show that pepper catalase is down-regulated during fruit ripening, becoming a target of tyrosine nitration, which provokes its inhibition.

Ascorbate peroxidase in fruits and modulation of its activity by reactive species.

Ascorbate peroxidase (APX) is one of the enzymes of the ascorbate-glutathione cycle and is the key enzyme that breaks down H2O2 with the aid of ascorbate as an electron source. APX is present in all photosynthetic eukaryotes from algae to higher plants and, at the cellular level, it is localized in all subcellular compartments where H2O2 is generated, including the apoplast, cytosol, plastids, mitochondria, and peroxisomes, either in soluble form or attached to the organelle membranes. APX activity can be modulated by various post-translational modifications including tyrosine nitration, S-nitrosation, persulfidation, and S-sulfenylation. This allows the connection of H2O2 metabolism with other relevant signaling molecules such as NO and H2S, thus building a complex coordination system. In both climacteric and non-climacteric fruits, APX plays a key role during the ripening process and during post-harvest, since it participates in the regulation of both H2O2 and ascorbate levels affecting fruit quality. Currently, the exogenous application of molecules such as NO, H2S, H2O2, and, more recently, melatonin is seen as a new alternative to maintain and extend the shelf life and quality of fruits because they can modulate APX activity as well as other antioxidant systems. Therefore, these molecules are being considered as new biotechnological tools to improve crop quality in the horticultural industry.

Characterization of leucine aminopeptidase (LAP) activity in sweet pepper fruits during ripening and its inhibition by nitration and reducing events.

KEY MESSAGE: Pepper fruits contain two leucine aminopeptidase (LAP) genes which are differentially modulated during ripening and by nitric oxide. The LAP activity increases during ripening but is negatively modulated by nitration. Leucine aminopeptidase (LAP) is an essential metalloenzyme that cleaves N-terminal leucine residues from proteins but also metabolizes dipeptides and tripeptides. LAPs play a fundamental role in cell protein turnover and participate in physiological processes such as defense mechanisms against biotic and abiotic stresses, but little is known about their involvement in fruit physiology. This study aims to identify and characterize genes encoding LAP and evaluate their role during the ripening of pepper (Capsicum annuum L.) fruits and under a nitric oxide (NO)-enriched environment. Using a data-mining approach of the pepper plant genome and fruit transcriptome (RNA-seq), two LAP genes, designated CaLAP1 and CaLAP2, were identified. The time course expression analysis of these genes during different fruit ripening stages showed that whereas CaLAP1 decreased, CaLAP2 was upregulated. However, under an exogenous NO treatment of fruits, both genes were downregulated. On the contrary, it was shown that during fruit ripening LAP activity increased by 81%. An in vitro assay of the LAP activity in the presence of different modulating compounds including peroxynitrite (ONOO-), NO donors (S-nitrosoglutathione and nitrosocyteine), reducing agents such as reduced glutathione (GSH), L-cysteine (L-Cys), and cyanide triggered a differential response. Thus, peroxynitrite and reducing compounds provoked around 50% inhibition of the LAP activity in green immature fruits, whereas cyanide upregulated it 1.5 folds. To our knowledge, this is the first characterization of LAP in pepper fruits as well as of its regulation by diverse modulating compounds. Based on the capacity of LAP to metabolize dipeptides and tripeptides, it could be hypothesized that the LAP might be involved in the GSH recycling during the ripening process.

Soybean (Glycine max L.) Lipoxygenase 1 (LOX 1) Is Modulated by Nitric Oxide and Hydrogen Sulfide: An In Vitro Approach.

Hydrogen sulfide (H2S) and nitric oxide (NO) are two relevant signal molecules that can affect protein function throughout post-translational modifications (PTMs) such as persulfidation, S-nitrosation, metal-nitrosylation, and nitration. Lipoxygenases (LOXs) are a group of non-heme iron enzymes involved in a wide range of plant physiological functions including seed germination, plant growth and development, and fruit ripening and senescence. Likewise, LOXs are also involved in the mechanisms of response to diverse environmental stresses. Using purified soybean (Glycine max L.) lipoxygenase type 1 (LOX 1) and nitrosocysteine (CysNO) and sodium hydrosulfide (NaHS) as NO and H2S donors, respectively, the present study reveals that both compounds negatively affect LOX activity, suggesting that S-nitrosation and persulfidation are involved. Mass spectrometric analysis of nitrated soybean LOX 1 using a peroxynitrite (ONOO-) donor enabled us to identify that, among the thirty-five tyrosine residues present in this enzyme, only Y214 was exclusively nitrated by ONOO-. The nitration of Y214 seems to affect its interaction with W500, a residue involved in the substrate binding site. The analysis of the structure 3PZW demonstrates the existence of several tunnels that directly communicate the surface of the protein with different internal cysteines, thus making feasible their potential persulfidation, especially C429 and C127. On the other hand, the CysNO molecule, which is hydrophilic and bulkier than H2S, can somehow be accommodated throughout the tunnel until it reaches C127, thus facilitating its nitrosation. Overall, a large number of potential persulfidation targets and the ease by which H2S can reach them through the diffuse tunneling network could be behind their efficient inhibition.

Thiol-based Oxidative Posttranslational Modifications (OxiPTMs) of Plant Proteins.

The thiol group of cysteine (Cys) residues, often present in the active center of the protein, is of particular importance to protein function, which is significantly determined by the redox state of a protein's environment. Our knowledge of different thiol-based oxidative posttranslational modifications (oxiPTMs), which compete for specific protein thiol groups, has increased over the last 10 years. The principal oxiPTMs include S-sulfenylation, S-glutathionylation, S-nitrosation, persulfidation, S-cyanylation and S-acylation. The role of each oxiPTM depends on the redox cellular state, which in turn depends on cellular homeostasis under either optimal or stressful conditions. Under such conditions, the metabolism of molecules such as glutathione, NADPH (reduced nicotinamide adenine dinucleotide phosphate), nitric oxide, hydrogen sulfide and hydrogen peroxide can be altered, exacerbated and, consequently, outside the cell's control. This review provides a broad overview of these oxiPTMs under physiological and unfavorable conditions, which can regulate the function of target proteins.

Interactions of melatonin, reactive oxygen species, and nitric oxide during fruit ripening: an update and prospective view.

Fruit ripening is a physiological process that involves a complex network of signaling molecules that act as switches to activate or deactivate certain metabolic pathways at different levels, not only by regulating gene and protein expression but also through post-translational modifications of the involved proteins. Ethylene is the distinctive molecule that regulates the ripening of fruits, which can be classified as climacteric or non-climacteric according to whether or not, respectively, they are dependent on this phytohormone. However, in recent years it has been found that other molecules with signaling potential also exert regulatory roles, not only individually but also as a result of interactions among them. These observations imply the existence of mutual and hierarchical regulations that sometimes make it difficult to identify the initial triggering event. Among these 'new' molecules, hydrogen peroxide, nitric oxide, and melatonin have been highlighted as prominent. This review provides a comprehensive outline of the relevance of these molecules in the fruit ripening process and the complex network of the known interactions among them.

H2S in Horticultural Plants: Endogenous Detection by an Electrochemical Sensor, Emission by a Gas Detector, and Its Correlation with L-Cysteine Desulfhydrase (LCD) Activity.

H2S has acquired great attention in plant research because it has signaling functions under physiological and stress conditions. However, the direct detection of endogenous H2S and its potential emission is still a challenge in higher plants. In order to achieve a comparative analysis of the content of H2S among different plants with agronomical and nutritional interest including pepper fruits, broccoli, ginger, and different members of the genus Allium such as garlic, leek, Welsh and purple onion, the endogenous H2S and its emission was determined using an ion-selective microelectrode and a specific gas detector, respectively. The data show that endogenous H2S content range from pmol to µmol H2S · g-1 fresh weight whereas the H2S emission of fresh-cut vegetables was only detected in the different species of the genus Allium with a maximum of 9 ppm in garlic cloves. Additionally, the activity and isozymes of the L-cysteine desulfhydrase (LCD) were analyzed, which is one of the main enzymatic sources of H2S, where the different species of the genus Allium showed the highest activities. Using non-denaturing gel electrophoresis, the data indicated the presence of up to nine different LCD isozymes from one in ginger to four in onion, leek, and broccoli. In summary, the data indicate a correlation between higher LCD activity with the endogenous H2S content and its emission in the analyzed horticultural species. Furthermore, the high content of endogenous H2S in the Allium species supports the recognized benefits for human health, which are associated with its consumption.

Lipoxygenase (LOX) in Sweet and Hot Pepper (Capsicum annuum L.) Fruits during Ripening and under an Enriched Nitric Oxide (NO) Gas Atmosphere.

Lipoxygenases (LOXs) catalyze the insertion of molecular oxygen into polyunsaturated fatty acids (PUFA) such as linoleic and linolenic acids, being the first step in the biosynthesis of a large group of biologically active fatty acid (FA)-derived metabolites collectively named oxylipins. LOXs are involved in multiple functions such as the biosynthesis of jasmonic acid (JA) and volatile molecules related to the aroma and flavor production of plant tissues, among others. Using sweet pepper (Capsicum annuum L.) plants as a model, LOX activity was assayed by non-denaturing polyacrylamide gel electrophoresis (PAGE) and specific in-gel activity staining. Thus, we identified a total of seven LOX isozymes (I to VII) distributed among the main plant organs (roots, stems, leaves, and fruits). Furthermore, we studied the FA profile and the LOX isozyme pattern in pepper fruits including a sweet variety (Melchor) and three autochthonous Spanish varieties that have different pungency levels (Piquillo, Padrón, and Alegría riojana). It was observed that the number of LOX isozymes increased as the capsaicin content increased in the fruits. On the other hand, a total of eight CaLOX genes were identified in sweet pepper fruits, and their expression was differentially regulated during ripening and by the treatment with nitric oxide (NO) gas. Finally, a deeper analysis of the LOX IV isoenzyme activity in the presence of nitrosocysteine (CysNO, a NO donor) suggests a regulatory mechanism via S-nitrosation. In summary, our data indicate that the different LOX isozymes are differentially regulated by the capsaicin content, fruit ripening, and NO.

Nitric oxide and hydrogen sulfide modulate the NADPH-generating enzymatic system in higher plants.

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.

Nitric Oxide (NO) Scaffolds the Peroxisomal Protein-Protein Interaction Network in Higher Plants.

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.

Identification of Compounds with Potential Therapeutic Uses from Sweet Pepper (Capsicum annuum L.) Fruits and Their Modulation by Nitric Oxide (NO).

Plant species are precursors of a wide variety of secondary metabolites that, besides being useful for themselves, can also be used by humans for their consumption and economic benefit. Pepper (Capsicum annuum L.) fruit is not only a common food and spice source, it also stands out for containing high amounts of antioxidants (such as vitamins C and A), polyphenols and capsaicinoids. Particular attention has been paid to capsaicin, whose anti-inflammatory, antiproliferative and analgesic activities have been reported in the literature. Due to the potential interest in pepper metabolites for human use, in this project, we carried out an investigation to identify new bioactive compounds of this crop. To achieve this, we applied a metabolomic approach, using an HPLC (high-performance liquid chromatography) separative technique coupled to metabolite identification by high resolution mass spectrometry (HRMS). After chromatographic analysis and data processing against metabolic databases, 12 differential bioactive compounds were identified in sweet pepper fruits, including quercetin and its derivatives, L-tryptophan, phytosphingosin, FAD, gingerglycolipid A, tetrahydropentoxylin, blumenol C glucoside, colnelenic acid and capsoside A. The abundance of these metabolites varied depending on the ripening stage of the fruits, either immature green or ripe red. We also studied the variation of these 12 metabolites upon treatment with exogenous nitric oxide (NO), a free radical gas involved in a good number of physiological processes in higher plants such as germination, growth, flowering, senescence, and fruit ripening, among others. Overall, it was found that the content of the analyzed metabolites depended on the ripening stage and on the presence of NO. The metabolic pattern followed by quercetin and its derivatives, as a consequence of the ripening stage and NO treatment, was also corroborated by transcriptomic analysis of genes involved in the synthesis of these compounds. This opens new research perspectives on the pepper fruit's bioactive compounds with nutraceutical potentiality, where biotechnological strategies can be applied for optimizing the level of these beneficial compounds.

Inhibition of NADP-malic enzyme activity by H2 S and NO in sweet pepper (Capsicum annuum L.) fruits.

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 (H2 S) 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 H2 S, we obtained a 50-75% ammonium-sulfate-enriched protein fraction containing these proteins. With the aid of in vitro assays, in the presence of H2 S, we observed that, while NADP-ME activity was inhibited by up to 29-32% using 2 and 5 mM Na2 S as H2 S 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 H2 S and NO partially modulate the NADPH-generating system.

Nitric oxide and hydrogen sulfide in plants: which comes first?

Nitric oxide (NO) is a signal molecule regarded as being involved in myriad functions in plants under physiological, pathogenic, and adverse environmental conditions. Hydrogen sulfide (H2S) has also recently been recognized as a new gasotransmitter with a diverse range of functions similar to those of NO. Depending on their respective concentrations, both these molecules act synergistically or antagonistically as signals or damage promoters in plants. Nevertheless, available evidence shows that the complex biological connections between NO and H2S involve multiple pathways and depend on the plant organ and species, as well as on experimental conditions. Cysteine-based redox switches are prone to reversible modification; proteomic and biochemical analyses have demonstrated that certain target proteins undergo post-translational modifications such as S-nitrosation, caused by NO, and persulfidation, caused by H2S, both of which affect functionality. This review provides a comprehensive update on NO and H2S in physiological processes (seed germination, root development, stomatal movement, leaf senescence, and fruit ripening) and under adverse environmental conditions. Existing data suggest that H2S acts upstream or downstream of the NO signaling cascade, depending on processes such as stomatal closure or in response to abiotic stress, respectively.

Nitric oxide in the physiology and quality of fleshy fruits.

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.

Nitric oxide-dependent regulation of sweet pepper fruit ripening.

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.

Hydrogen sulfide: A novel component in Arabidopsis peroxisomes which triggers catalase inhibition.

Plant peroxisomes have the capacity to generate different reactive oxygen and nitrogen species (ROS and RNS), such as H2 O2 , superoxide radical (O2 · - ), nitric oxide and peroxynitrite (ONOO- ). These organelles have an active nitro-oxidative metabolism which can be exacerbated by adverse stress conditions. Hydrogen sulfide (H2 S) 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 H2 S. H2 S was also detected in chloroplasts under glyphosate-induced oxidative stress conditions. Peroxisomal enzyme activities, including catalase, photorespiratory H2 O2 -generating glycolate oxidase (GOX) and hydroxypyruvate reductase (HPR), were assayed in vitro with a H2 S donor. In line with the persulfidation of this enzyme, catalase activity declined significantly in the presence of the H2 S donor. To corroborate the inhibitory effect of H2 S 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 H2 S in plant peroxisomes which appears to regulate catalase activity and, consequently, the peroxisomal H2 O2 metabolism.

Nitro-oxidative metabolism during fruit ripening.

Pepper (Capsicum annuum L.) and tomato (Solanum lycopersicum L.), which belong to the Solanaceae family, are among the most cultivated and consumed fleshy fruits worldwide and constitute excellent sources of many essential nutrients, such as vitamins A, C, and E, calcium, and carotenoids. While fruit ripening is a highly regulated and complex process, tomato and pepper have been classified as climacteric and non-climacteric fruits, respectively. These fruits differ greatly in shape, color composition, flavor, and several other features which undergo drastic changes during the ripening process. Such ripening-related metabolic and developmental changes require extensive alterations in many cellular and biochemical processes, which ultimately leads to fully ripe fruits with nutritional and organoleptic features that are attractive to both natural dispersers and human consumers. Recent data show that reactive oxygen and nitrogen species (ROS/RNS) are involved in fruit ripening, during which molecules, such as hydrogen peroxide (H2O2), NADPH, nitric oxide (NO), peroxynitrite (ONOO-), and S-nitrosothiols (SNOs), interact to regulate protein functions through post-translational modifications. In light of these recent discoveries, this review provides an update on the nitro-oxidative metabolism during the ripening of two of the most economically important fruits, discusses the signaling roles played by ROS/RNS in controlling this complex physiological process, and highlights the potential biotechnological applications of these substances to promote further improvements in fruit ripening regulation and nutritional quality. In addition, we suggest that the term 'nitro-oxidative eustress' with regard to fruit ripening would be more appropriate than nitro-oxidative stress, which ultimately favors the consolidation of the plant species.

Endogenous hydrogen sulfide (H2S) is up-regulated during sweet pepper (Capsicum annuum L.) fruit ripening. In vitro analysis shows that NADP-dependent isocitrate dehydrogenase (ICDH) activity is inhibited by H2S and NO.

Like nitric oxide (NO), hydrogen sulfide (H2S) has been recognized as a new gasotransmitter which plays an important role as a signaling molecule in many physiological processes in higher plants. Although fruit ripening is a complex process associated with the metabolism of reactive oxygen species (ROS) and nitrogen oxygen species (RNS), little is known about the potential involvement of endogenous H2S. Using sweet pepper (Capsicum annuum L.) as a model non-climacteric fruit during the green and red ripening stages, we studied endogenous H2S content and cytosolic l-cysteine desulfhydrase (L-DES) activity which increased by 14% and 28%, respectively, in red pepper fruits. NADPH is a redox compound and key cofactor required for cell growth, proliferation and detoxification. We studied the NADPH-regenerating enzyme, NADP-isocitrate dehydrogenase (NADP-ICDH), whose activity decreased by 34% during ripening. To gain a better understanding of its potential regulation by H2S, we obtained a 50-75% ammonium sulfate-enriched protein fraction containing the NADP-ICDH protein; with the aid of in vitro assays in the presence of H2S, we observed that 2 and 10 mM NaHS used as H2S donors resulted in a decrease of up to 36% and 45%, respectively, in NADP-ICDH activity, which was unaffected by reduced glutathione (GSH). On the other hand, peroxynitrite (ONOO-), S-nitrosocyteine (CysNO) and DETA-NONOate, with the last two acting as NO donors, also inhibited NADP-ICDH activity. In silico analysis of the tertiary structure of sweet pepper NADP-ICDH activity (UniProtKB ID A0A2G2Y555) suggests that residues Cys133 and Tyr450 are the most likely potential targets for S-nitrosation and nitration, respectively. Taken together, the data reveal that the increase in the H2S production capacity of red fruits is due to higher L-DES activity during non-climacteric pepper fruit ripening. In vitro assays appear to show that H2S inhibits NADP-ICDH activity, thus suggesting that this enzyme may be regulated by persulfidation, as well as by S-nitrosation and nitration. NO and H2S may therefore regulate NADPH production and consequently cellular redox status during pepper fruit ripening.

Comparative Analysis of Catalase Activity in Plants: Spectrophotometry and Native PAGE Approaches.

Catalase, a pivotal enzyme in plant antioxidative defense mechanisms, plays a crucial role in detoxifying hydrogen peroxide, a reactive oxygen species (ROS). In this chapter, a comparative analysis of catalase activity was conducted using two distinct methodologies: spectrophotometry and non-denaturing polyacrylamide gel electrophoresis (PAGE). The spectrophotometric approach allowed the quantification of catalase activity by measuring the breakdown rate of hydrogen peroxide, while native PAGE enabled the separation and visualization of catalase isozymes, based on their native molecular weight and charge characteristics, and specific staining assay. Both methods provide valuable insights into catalase activity, offering complementary information on the enzyme's functional diversity and distribution within different plant tissues. This study integrates different techniques, previously described, to comprehensively elucidate the role of catalase in plant metabolism. Furthermore, it provides the possibility of obtaining a holistic understanding of antioxidant defense mechanisms by considering both total activity and isoenzyme distribution of catalase enzyme.

Persulfidome of Sweet Pepper Fruits during Ripening: The Case Study of Leucine Aminopeptidase That Is Positively Modulated by H2S.

Protein persulfidation is a thiol-based oxidative posttranslational modification (oxiPTM) that involves the modification of susceptible cysteine thiol groups present in peptides and proteins through hydrogen sulfide (H2S), thus affecting their function. Using sweet pepper (Capsicum annuum L.) fruits as a model material at different stages of ripening (immature green and ripe red), endogenous persulfidated proteins (persulfidome) were labeled using the dimedone switch method and identified using liquid chromatography and mass spectrometry analysis (LC-MS/MS). A total of 891 persulfidated proteins were found in pepper fruits, either immature green or ripe red. Among these, 370 proteins were exclusively present in green pepper, 237 proteins were exclusively present in red pepper, and 284 proteins were shared between both stages of ripening. A comparative analysis of the pepper persulfidome with that described in Arabidopsis leaves allowed the identification of 25% of common proteins. Among these proteins, glutathione reductase (GR) and leucine aminopeptidase (LAP) were selected to evaluate the effect of persulfidation using an in vitro approach. GR activity was unaffected, whereas LAP activity increased by 3-fold after persulfidation. Furthermore, this effect was reverted through treatment with dithiothreitol (DTT). To our knowledge, this is the first persulfidome described in fruits, which opens new avenues to study H2S metabolism. Additionally, the results obtained lead us to hypothesize that LAP could be involved in glutathione (GSH) recycling in pepper fruits.