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Aims: Pepper fruit is a horticultural product worldwide consumed that has great nutritional and economic relevance. Besides the phenotypical changes that undergo pepper fruit during ripening, there are many associated modifications at transcriptomic, proteomic, biochemical, and metabolic levels. Nitric oxide (NO) and hydrogen sulfide (H2S) are recognized signal molecules that can exert regulatory functions in diverse plant processes. This study aims at analyzing the interrelationship between NO and H2S during fruit ripening. Results: Our data indicate that the H2S-generating cytosolic L-cysteine desulfhydrase (LCD) and the mitochondrial D-cysteine desulfhydrase (DCD) activities are downregulated during ripening but this effect was reverted after NO treatment of fruits. Innovation and Conclusion: Using as a model the non-climacteric pepper fruits at different ripening stages and under an NO-enriched atmosphere, the activity of the H2S-generating LCD and DCD was analyzed. LCD and DCD activities were downregulated during ripening, but this effect was reverted after NO treatment of fruits. The analysis of LCD activity by non-denaturing polyacrylamide gel electrophoresis (PAGE) allowed identifying three isozymes designated CaLCD I to CaLCD III, which were differentially modulated by NO and strictly dependent on pyridoxal 5′-phosphate (PLP). In vitro analyses of green fruit samples in the presence of different compounds including NO donors, peroxynitrite (ONOO−), and reducing agents such as reduced glutathione (GSH) and L-cysteine (L-Cys) triggered an almost 100% inhibition of CaLCD II and CaLCD III. This redox adaptation process of both enzymes could be cataloged as a hormesis phenomenon. The protein tyrosine (Tyr) nitration (an NO-promoted post-translational modification) of the recombinant LCD was corroborated by immunoblot and by mass spectrometry (MS) analyses. Among the 11 Tyr residues present in this enzyme, MS of the recombinant LCD enabled us to identify that Tyr82 and Tyr254 were nitrated by ONOO−, this occurring near the active center on the enzyme, where His237 and Lys260 together with the cofactor PLP are involved. These data support the relationship between NO and H2S during pepper fruit ripening, since LCD and DCD are regulated by NO during this physiological event, and this could also be extrapolated to other plant species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 S-nitrosation, 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. [ABSTRACT FROM AUTHOR]

Abstract Like nitric oxide (NO), hydrogen sulfide (H 2 S) 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 H 2 S. Using sweet pepper (Capsicum annuum L.) as a model non-climacteric fruit during the green and red ripening stages, we studied endogenous H 2 S 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 H 2 S, 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 H 2 S, we observed that 2 and 10 mM NaHS used as H 2 S 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 H 2 S 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 H 2 S inhibits NADP-ICDH activity, thus suggesting that this enzyme may be regulated by persulfidation, as well as by S -nitrosation and nitration. NO and H 2 S may therefore regulate NADPH production and consequently cellular redox status during pepper fruit ripening. Graphical abstract Outline of endogenous H 2 S and NO during pepper (Capsicum annuum L.) fruit ripening. Image 1 Highlights • Hydrogen sulfide is up-regulated during sweet pepper fruit ripening. • The NADP-isocitrate dehydrogenase (ICDH) activity is down regulated during ripening. • The NADP-ICDH is regulated by persulfidation, as well as by S-nitrosation and nitration. [ABSTRACT FROM AUTHOR]