Pepper catalase: a broad analysis of its modulation during fruit ripening and by nitric oxide.

Catalase is a major antioxidant enzyme located in plant peroxisomes that catalyzes the decomposition of H2O2. Based on our previous transcriptomic (RNA-Seq) and proteomic (iTRAQ) data at different stages of pepper fruit ripening and after exposure to NO enriched atmosphere, a broad analysis has allowed to characterize the functioning of this enzyme. Three genes were identified, and their expression was differentially modulated during ripening and by NO gas treatment. A dissimilar behavior was observed in the protein expression of the encoded protein catalases (CaCat1-CaCat3). Total catalase activity was downregulated by 50% in ripe fruits concerning immature green fruits. This was corroborated by non-denaturing polyacrylamide gel electrophoresis, where only a single catalase isozyme was identified. In vitro analyses of the recombinant CaCat3 protein exposed to peroxynitrite confirmed, by immunoblot assay, that catalase underwent a nitration process. Mass spectrometric analysis identified that Tyr348 and Tyr360 were nitrated by peroxynitrite, occurring near the catalase active center. The data indicate the complex regulation at gene and protein levels of catalase during the ripening of fruits, with activity significantly downregulated in ripe fruits. Nitration plays a key role in this downregulation, favoring an increase in H2O2 during ripening. This pattern can be reversed by the exogenous NO application. While plant catalases are generally reported to be tetrameric, the analysis of the protein structure supports that pepper catalase has a favored quaternary homodimer nature. Taken together, data show that pepper catalase is downregulated during fruit ripening, becoming a target of tyrosine nitration, which provokes its inhibition.

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