NADPH Oxidase (Rboh) Activity is Up Regulated during Sweet Pepper (Capsicum annuum L.) Fruit Ripening

Abstract

In plants, NADPH oxidase (NOX) is also known as a respiratory burst oxidase homolog (Rboh). This highly important enzyme, one of the main enzymatic sources of superoxide radicals (O2•−), is involved in the metabolism of reactive oxygen and nitrogen species (ROS and RNS), which is active in the non-climacteric pepper (Capsicum annuum L.) fruit. We used sweet pepper fruits at two ripening stages (green and red) to biochemically analyze the O2•−-generating Rboh activity and the number of isozymes during this physiological process. Malondialdehyde (MDA) content, an oxidative stress marker, was also assayed as an index of lipid peroxidation. In red fruits, MDA was observed to increase 2-fold accompanied by a 5.3-fold increase in total Rboh activity. Using in-gel assays of Rboh activity, we identified a total of seven CaRboh isozymes (I–VII) which were differentially modulated during ripening. CaRboh-III and CaRboh-I were the most prominent isozymes in green and red fruits, respectively. An in vitro assay showed that CaRboh activity is inhibited in the presence of nitric oxide (NO) donors, peroxynitrite (ONOO−) and glutathione (GSH), suggesting that CaRboh can undergo S-nitrosation, Tyr-nitration, and glutathionylation, respectively. In summary, this study provides a basic biochemical characterization of CaRboh activity in pepper fruits and indicates that this O2•−-generating Rboh is involved in nitro-oxidative stress associated with sweet pepper fruit ripening.

Inhibition of NADP‐malic enzyme activity by H2S and NO in sweet pepper (Capsicum annuum L.) fruits

“…Given that pepper fruit ripening is subject to nitro‐oxidative stress (Corpas et al , Chu‐Puga et al ), to gain further insight into the regulation of NADP‐ME activity in pepper fruit, the effect of increasing concentrations of NO and peroxynitrite donors (Begara‐Morales et al , , Chaki et al , de Freitas‐Silva et al , Mun et al ) was also evaluated. Fig.…”

“…In previous studies, we found that the nitric oxide (NO) metabolism is significantly affected during sweet pepper fruit ripening (Chaki et al , Corpas et al ) and, more recently, that NADP‐ICDH is inhibited by both H 2 S and NO, suggesting that NADPH generation is compromised during pepper ripening (Muñoz‐Vargas et al ). We have also shown that NADPH oxidase (Rboh) activity increases during the ripening process (Chu‐Puga et al ), thus highlighting the important role played by NADPH in the fruit physiology. This study aims to analyze the other plant NADPH‐generating systems (G6PDH, 6PGDH and NADP‐ME) during ripening and to evaluate how their activity is affected by H 2 S.…”

“…In addition, NADPH, which is critical for the conversion of ribonucleotide (RNA) to deoxy‐ribonucleotide (DNA), is an electron donor for certain enzymatic components of ROS and RNS metabolism (Ying 2008, Corpas and Barroso ). For example, it enables the glutathione reductase and the monodehydroascorbate reductase enzymes to regenerate reduced glutathione (GSH) and ascorbate, respectively (Blanch et al ) and facilitates NADPH‐dependent superoxide radical generation by NADPH oxidase (NOX), also known as respiratory burst oxidase homolog (Rboh; Chu‐Puga et al ). In addition to the NO metabolism in fruit (Corpas and Palma ), hydrogen sulfide (H 2 S) is also important in physiological terms due to its signaling properties (Corpas et al ).…”

“…On the other hand, we have previously reported several examples of NADPH requirements during pepper fruit ripening. For example, NADPH oxidase (Rboh) activity involved in superoxide generation increased 5.3‐fold, which closely correlates with an increase in lipid peroxidation, a marker of oxidative stress associated with fruit ripening (Chu‐Puga et al ). On the other hand, proline (Pro) content, whose biosynthesis from glutamate by the Δ 1 ‐pyrroline 5‐carboxylate synthase (P5CS) enzyme specifically requires NADPH, has also been reported to increase during ripening (Signorelli and Monza , González‐Gordo et al ).…”

“…Sweet pepper fruit ripening is a complex physiological process in which ROS and RNS metabolism is dynamically involved. Previously, we observed an increase in lipid peroxidation, protein nitration and in the activity of NADPH oxidase (NOX), also known as a respiratory burst oxidase homolog (Rboh), which is one of the enzymes responsible for superoxide radicals (O 2 .− ) (Chu‐Puga et al ). These findings suggest that ripening is physiologically associated with nitro‐oxidative stress.…”