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Protein persulfidation is a post-translational modification (PTM) mediated by hydrogen sulfide (H2S), which affects the thiol group of cysteine residues from target proteins and can have a positive, negative or zero impact on protein function. Due to advances in proteomic techniques, the number of potential protein targets identified in higher plants, which are affected by this PTM, has increased considerably. However, its precise impact on biological function needs to be evaluated at the experimental level in purified proteins in order to identify the specific cysteine(s) residue(s) affected. It also needs to be evaluated at the cellular redox level given the potential interactions among different oxidative post-translational modifications (oxiPTMs), such as S-nitrosation, glutathionylation, sulfenylation, S-cyanylation and S-acylation, which also affect thiol groups. This review aims to provide an updated and comprehensive overview of the important physiological role exerted by persulfidation in higher plants, which acts as a cellular mechanism of protein protection against irreversible oxidation. [ABSTRACT FROM AUTHOR]

At the cellular level, the generation of reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ), due to different abiotic or biotic stress, causes oxidative stress that induces an imbalance in the metabolism. Among the different H 2 O 2 -scavenging enzymatic antioxidants, ascorbate peroxidase (APX) is a heme-peroxidase that plays an important role in the ascorbate-glutathione pathway using ascorbate to reduce H 2 O 2 to water. Using non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with a spectrophotometric assay for APX activity, the protocol allows identifying diverse APX isozymes present in different organs and plant species., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 (H 2 S) are recognized signal molecules that can exert regulatory functions in diverse plant processes. This study aims at analyzing the interrelationship between NO and H 2 S during fruit ripening. Results: Our data indicate that the H 2 S-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 H 2 S-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 H 2 S 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.

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 (H 2 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 H 2 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 H 2 S, we observed that, while NADP-ME activity was inhibited by up to 29-32% using 2 and 5 mM Na 2 S as H 2 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 H 2 S and NO partially modulate the NADPH-generating system., (© 2019 Scandinavian Plant Physiology Society.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)