Redox regulation of 6-phosphogluconate dehydrogenase (6PGDH) isozymes and gene regulation by nitric oxide (NO) and melatonin during pepper fruit ripening.

6-Phosphogluconate dehydrogenase (6PGDH) functions in the oxidative phase of the pentose phosphate pathway (oxiPPP), together with glucose-6-phosphate dehydrogenase (G6PDH). These two enzymes play central roles in cellular NADPH generation. In pepper fruit, two genes were identified in the transcriptome, designated Ca6PGDH1 and Ca6PGDH2, which appear to encode a peroxisomal and a plastidial putative isozyme, respectively. During ripening, Ca6PGDH1 exhibited only minor changes in its expression, whereas Ca6PGDH2 was markedly downregulated. The expression patterns of both genes diverged in response to exposure to a nitric oxide (NO)-enriched atmosphere and melatonin, while both NO and melatonin treatments partially mitigated the downregulation of Ca6PGDH2 but not that of Ca6PGDH1. At the enzymatic level, the total 6PGDH activity exhibited a modest decline during ripening. Isoenzymatic analysis using non-denaturing polyacrylamide gel electrophoresis revealed three distinct isozymes, designated 6PGDH I to III, in order of increasing electrophoretic mobility. In vitro assays examining the effects of various reducing agents, including glutathione (GSH) and l-cysteine (L-Cys), as well as signaling molecules such as NO, peroxynitrite (ONOO-), hydrogen sulfide (H2S), and cyanide, showed that the NO donors and H2S exerted inhibitory effects on some isozymes, with the strongest inhibition observed for Ca6PGDH III whereas reducing compounds (GSH and Cys) protected against these inhibitory effects. Together, these results highlight Ca6PGDH as a redox-sensitive enzyme that may contribute to metabolic and signaling homeostasis during pepper fruit ripening.