Plant peroxisomes have the capacity to generate different reactive oxygen and nitrogen species (ROS and RNS), such as H 2 O 2 , superoxide radical (O 2 Á À ), nitric oxide and peroxynitrite (ONOO -). These organelles have an active nitrooxidative metabolism which can be exacerbated by adverse stress conditions. Hydrogen sulfide (H 2 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 H 2 S. H 2 S was also detected in chloroplasts under glyphosate-induced oxidative stress conditions. Peroxisomal enzyme activities, including catalase, photorespiratory H 2 O 2 -generating glycolate oxidase (GOX) and hydroxypyruvate reductase (HPR), were assayed in vitro with a H 2 S donor. In line with the persulfidation of this enzyme, catalase activity declined significantly in the presence of the H 2 S donor. To corroborate the inhibitory effect of H 2 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 H 2 S in plant peroxisomes which appears to regulate catalase activity and, consequently, the peroxisomal H 2 O 2 metabolism. . In plant systems, several enzymes are capable of generating H 2 S, which is part of the cysteine (Cys) metabolism. These enzymes, including L-and D-cysteine desulfhydrase (L-DES/D-DES), sulfite reductase (SiR), cyano alanine synthase (CAS) and cysteine synthase (CS) (Li et al. 2013; Calderwood and Kopriva 2014; Hancock and Whiteman 2014), are present in different subcellular compartments, such as cytosol, chloroplasts

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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 (H2S) 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 H2S, we obtained a 50–75% ammonium‐sulfate‐enriched protein fraction containing these proteins. With the aid of in vitro assays, in the presence of H2S, we observed that, while NADP‐ME activity was inhibited by up to 29–32% using 2 and 5 mM Na2S as H2S 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 H2S and NO partially modulate the NADPH‐generating system.

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 regulating the gene and protein expression but also through post-translational modifications (PTMs) of the involved proteins. Ethylene is the distinctive molecule that regulates the ripening of fruits, thus allowing their classification as climacteric and non-climacteric, according to their dependence or not of this phytohormone, respectively. However, in recent years it has been found that other molecules with signaling potential also exert regulatory roles not only by individually, but also due to the interactions among them. This implies mutual and hierarchical regulations that sometimes make it difficult to identify the initial triggering event. Among these “new” molecules, hydrogen peroxide (H2O2), nitric oxide (NO), and melatonin are highlighted. 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.