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This study presents the first direct evidence of nitric oxide ( • NO) production in ripening pepper fruits ( Capsicum annuum L.) and identifies the specific enzyme responsible for its generation. By clarifying the enzymatic origin of • NO, this research enhances our understanding of the redox signaling mechanisms involved in the development of non-climacteric pepper fruits. Using electron paramagnetic resonance (EPR) spectroscopy combined with a spin-trapping technique, • NO production was directly detected at various stages of ripening. Enzymatic assays confirmed that the nitrite- and NADH (reduced nicotinamide adenine dinucleotide)-dependent nitrate reductase (NR) activity is responsible for the • NO generation, while RNA sequencing (RNA-Seq) identified a single nitrate reductase ( CaNR ) gene as the likely source of • NO. A recombinant CaNR protein was expressed and demonstrated, through EPR, to produce • NO from nitrite. Quantitatively, the nitrite-dependent NR activity in pepper fruit extracts varied from 1.40 to 0.39 nmol • NO · min -1 · mg -1 of protein throughout the ripening process. This activity was entirely inhibited by peroxynitrite (ONOO - ) and reduced by 78% in the presence of hydrogen sulfide (H 2 S), indicating redox regulation. In summary, these findings reveal CaNR as a physiological source of • NO in non-climacteric pepper fruits and uncover previously unknown regulatory roles of ONOO - and H 2 S in modulating • NO biosynthesis through redox-based post-translational mechanisms. This work defines a novel redox regulatory network that links reactive nitrogen and sulfur species to fruit ripening.
Corpas et al. (Tue,) studied this question.