EPR-detected NO biosynthesis via nitrate reductase in non-climacteric pepper fruit: downregulation by ripening,...

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 (H₂S), 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 H₂S 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.