H2S-Generating Cytosolic L-Cysteine Desulfhydrase and Mitochondrial D-Cysteine Desulfhydrase from Sweet Pepper (Capsicum annuum L.) Are Regulated During Fruit Ripening and by Nitric Oxide

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.

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.

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