Tocopherol metabolic engineering in tomato fruits

• Grant number: 18/19020-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2019
• End date: July 31, 2019
• Field of knowledge: Biological Sciences - Botany
• Principal Investigator: Bruno Silvestre Lira
• Grantee: Paula Cristina Amaral Silva
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Tocopherols, lipid-soluble compounds exclusively synthesized by photosynthetic organisms, are essential for the maintenance of the integrity of lipid membranes of the thylakoids and, consequently, of photosynthesis, this is due to their capability of inhibiting the propagation of lipid peroxidation and of protecting the photosystem II from oxidative damage. Moreover, from a nutritional point of view, they are important in human diet due to their antioxidant potential, what was found to be beneficial for the maintenance of the integrity of the genetic material, and in prophylaxis and treatment of several diseases. Thus, the understanding of tocopherol metabolism is interesting in a plant breeding perspective with economic and nutritional implications. For tocopherol synthesis, a chromanol ring, product of the shikimic acid pathway, is condensed a side chain derived from phytyl diphosphate (PDP), which is originated by de novo synthesis via the methyl erythritol 4-phosphate (MEP) pathway or provided by the recycling of chlorophyll-derived phytol . During the ripening of tomato fruit, Solanum lycopersicum, the chloroplasts are turned into chromoplast, what is also marked by an intense MEP activity and chlorophyll degradation, turning the tomato an interesting model for the study of tocopherol accumulation. The metabolism of this compound in tomato has been studied by our research group. First, studies identified all genes involved in tocopherol biosynthesis and elucidated critical points for its accumulation. This is the case of PDP availability, during fruit green stages occurs de novo synthesis of this compound, feeding the biosynthesis of chlorophyll, which in turn provides the side chain for biosynthesis of tocopherols. However, throughout ripening, the MEP products are channeled towards carotenoid synthesis, together with a drastic reduction in the transcript levels of the GERANYLGERANYL DIPHOSPHATE REDUTASE (SlGGDR), the enzyme responsible for the formation of PDP from geranyl geranyl diphosphate, and chlorophyll synthesis ceases. In this scenario, the intense chlorophyll degradation allows the increase in the content of tocopherols in the ripe fruits by providing PDP. This shows that the recycling of the phytol derived from chlorophyll is essential for tocopherols synthesis, what occurs by two phosphorylation reactions catalyzed by PHYTOL KINASE (SlVTE5) and PHYTYL-PHOSPHATE KINASE (SlVTE6). This project aims to promote the accumulation of tocopherols in tomato fruits by increasing the availability of PDP. To achieve this, transgenic tomato lines will be generated by overexpressing, under control of a fruit-specific promoter, genes encoding two enzymes that act on MEP, SlGGDR and 1-DEOXI-D-XYLULOSE-5-PHOSPHATE SINTASE (SlDXS). Together with plants overexpressing SlVTE5 and SlVTE6, green and mature fruits of these lines will be phenotyped in relation to their levels of chlorophylls, carotenoids and tocopherols, as well by analyzing the transcriptional profile of the genes related to these compounds.