Outside the box: the role of B-BOX proteins (BBX) in tomato fruit ripening

Tocopherols, lipid-soluble antioxidants compounds exclusively synthesized in the chloroplast of photosynthetic organisms, are essential for the maintenance of the integrity of thylakoids membranes and, consequently, of photosynthesis. The tocopherol capability of inhibiting the propagation of lipid peroxidation and scavenge oxygen singlet is not only important for the plant physiology, but is also beneficial for human health. Thus, understanding the tocopherol metabolism is interesting for plant breeding with economic and nutritional implications. For tocopherol synthesis, a chromanol ring, produced by shikimic acid pathway, is condensed to phytyl diphosphate (PDP). The latter is originated by de novo synthesis via the methyl erythritol 4-phosphate (MEP) pathway or provided by the recycling of chlorophyll-derived phytol after a two-step phosphorylation sequentially catalysed by PHYTOL KINASE (VTE5) and PHYTYL-PHOSPATE KINASE (VTE6), respectively. During the ripening of tomato fruit, Solanum lycopersicum, chloroplasts turns into chromoplast, what is marked by an intense MEP activity and chlorophyll degradation, turning the tomato an interesting model for the study of tocopherol accumulation. The PDP availability has been identified as a limiting factor for tocopherol synthesis in tomato fruits. While in the green stages of fruit development PDP can be obtained by de novo synthesis via MEP and by the phytol recycling, ripe fruits rely only in the latter pathway, as during ripening the MEP is diverted towards carotenoid accumulation by the downregulation of the GERANYLGERANYL DIPHOSPHATE REDUTASE (SlGGDR). Aiming the improvement of tocopherol accumulation in tomato fruits, two approaches were taken to increase PDP availability: the enhancement of the de novo synthesis and the phytol recycling. For the first, either 1-DEOXY-D-XYLULOSE-5-PHOSPHATE SYNTHASE (DXS), the enzyme involved in the first step of the MEP pathway, or GGDR were targeted; while for the latter, VTE5 or VTE6 were targeted. In all four cases, the ortholog from Arabidopsis thaliana were overexpressed under the control of a fruit specific promoter. The results from the phenotypic chararcterization of the obtained transgenic lines, including the content of chlorophylls, carotenoids and tocopherols, will be presented. The data will bring tools for the development of breeding strategies aiming to enhance the content of an important nutraceutical in tomato edible fruit.