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

Abstract

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.

Keywords: Fruit ripening; Hydrogen sulfide; NADP-isocitrate dehydrogenase; Nitration; Nitric oxide; Pepper; Persulfidation; S-nitrosation; S-nitrosocyteine; S-nitrosylation; S-sulfhydration.