Physiological and molecular characterization of the cytochromes b561 protein family in Arabidopsis thaliana: the role of the redox homeostasis in the transitions between developmental stages

Ascorbate is a key redox metabolite in plants, primarily known for its antioxidant properties in response to biotic and abiotic stresses, but also involved in numerous physiological growth and development processes. To perform these functions, ascorbate must be kept in its reduced form, and plants have evolved enzymatic systems to regenerate reduced ascorbate from its oxidized forms (monodehydroascorbate and dehydroascorbate reductases), which can only be found in specific cellular subcompartments. Among the enzymes using ascorbate as a substrate are cytochromes b561 (CYB561s), a family of highly conserved eukaryotic proteins that catalyze transmembrane electron transfer, reducing ascorbate on one side of the membrane and oxidizing monodehydroascorbate or ferrichelates on the opposite side. However, the biological function of these proteins in plants remains unclear. The Arabidopsis thaliana genome contains four CYB561-encoding genes, CYB561-A, CYB561-B, CYB561-C and CYB561-D. In this thesis, using an electrophysiological approach, we demonstrate that the tonoplast-located CYB561-A acts as a transmembrane monodehydroascorbate reductase, transporting electrons from cytosolic ascorbate to vacuolar monodehydroascorbate, although in vitro it can catalyze the same reaction in the opposite direction. Furthermore, the subcellular localization of CYB561-A and CYB561-B was examined by confocal microscopy and their physiological role was investigated by molecular phenotyping of T-DNA mutant plants at different developmental stages. Under control growth conditions, cyb561-a and cyb561-b mutants showed phenotypic differences compared to wild-type plants, including higher ascorbate peroxidase activity, lower ROS content, delayed flowering, and impaired primary root development; preliminary high-light stress experiments revealed an increased anthocyanin content in cyb561-a mutants. Our data suggest that CYB561s could act as transmembrane monodehydroascorbate reductases, contributing to the redox homeostasis of the ascorbate pool in compartments lacking soluble enzymes. The lack of CYB561-A and CYB561-B correlates with pleiotropic phenotypes indicating alterations in ascorbate redox homeostasis across different compartments and possibly ROS-mediated signaling.