Mitochondrial protein expression during sweet pepper (Capsicum annuum L.) fruit ripening: iTRAQ-based proteomic analysis and role of cytochrome c oxidase

The physiological process of fruit ripening is associated with the late developmental stages of plants in which mitochondrial organelles play an important role in the final success of this whole process. Thus, an isobaric tag for relative and absolute quantification (iTRAQ)-based analysis was used to quantify the mitochondrial proteome in pepper fruits in this study. Analysis of both green and red pepper fruits identified a total of 2284 proteins, of which 692 were found to be significantly more abundant in unripe green fruits as compared to red fruits, while 497 showed lower levels as the ripening process proceeded. Of the total number of proteins identified, 2253 (98,6%) were found to share orthologs with Arabidopsis thaliana. Proteomic analysis identified 163 proteins which were categorized as cell components, the major part assigned to cellular, intracellular space and other subcellular locations such as cytosol, plastids and, to a lesser extent, to mitochondria. Of the 224 mitochondrial proteins detected in pepper fruits, 78 and 48 were more abundant in green and red fruits, respectively. The majority of these proteins which displayed differential abundance in both fruit types were involved in the mitochondrial electron transport chain (mETC) and the tricarboxylic acid (TCA) cycle. The abundance levels of the proteins from both pathways were higher in green fruits, except for cytochrome c (CYC2), whose abundance was significantly higher in red fruits. We also investigated cytochrome c oxidase (COX) activity during pepper fruit ripening, as well as in the presence of molecules such as nitric oxide (NO) and hydrogen peroxide (H₂O₂), which promote thiol-based oxidative post-translational modifications (oxiPTMs). Thus, with the aid of in vitro assays, cytochrome c oxidase (COX) activity was found to be potentially inhibited by the PTMs nitration, S-nitrosation and carbonylation. According to protein abundance data, the final segment of the mETC appears to be a crucial locus with regard to fruit ripening, but also because in this location the biosynthesis of ascorbate, an antioxidant which plays a major role in the metabolism of pepper fruits, occurs.