Superoxide Radical Metabolism in Sweet Pepper (Capsicum annuumL.) Fruits Is Regulated by Ripening and by a NO-Enriched Environment

Superoxide radical (O-2(center dot-)) is involved in numerous physiological and stress processes in higher plants. Fruit ripening encompasses degradative and biosynthetic pathways including reactive oxygen and nitrogen species. With the use of sweet pepper (Capsicum annuumL.) fruits at different ripening stages and under a nitric oxide (NO)-enriched environment, the metabolism of O(2)(center dot-)was evaluated at biochemical and molecular levels considering the O(2)(center dot-)generation by a NADPH oxidase system and its dismutation by superoxide dismutase (SOD). At the biochemical level, seven O-2(center dot-)-generating NADPH-dependent oxidase isozymes [also called respiratory burst oxidase homologs (RBOHs) I-VII], with different electrophoretic mobility and abundance, were detected considering all ripening stages from green to red fruits and NO environment. Globally, this system was gradually increased from green to red stage with a maximum of approximately 2.4-fold increase in red fruit compared with green fruit. Significantly, breaking-point (BP) fruits with and without NO treatment both showed intermediate values between those observed in green and red peppers, although the value in NO-treated fruits was lower than in BP untreated fruits. The O-2(center dot-)-generating NADPH oxidase isozymes I and VI were the most affected. On the other hand, four SOD isozymes were identified by non-denaturing electrophoresis: one Mn-SOD, one Fe-SOD, and two CuZn-SODs. However, none of these SOD isozymes showed any significant change during the ripening from green to red fruits or under NO treatment. In contrast, at the molecular level, both RNA-sequencing and real-time quantitative PCR analyses revealed different patterns with downregulation of four genesRBOH A,C,D, andEduring pepper fruit ripening. On the contrary, it was found out the upregulation of aMn-SODgene in the ripening transition from immature green to red ripe stages, whereas aFe-SODgene was downregulated. In summary, the data reveal a contradictory behavior between activity and gene expression of the enzymes involved in the metabolism of O(2)(center dot-)during the ripening of pepper fruit. However, it could be concluded that the prevalence and regulation of the O(2)(center dot-)generation system (NADPH oxidase-like) seem to be essential for an appropriate control of the pepper fruit ripening, which, additionally, is modulated in the presence of a NO-enriched environment.