REGULATION OF ANTHOCYANIN BIOSYNTHESIS IN TOMATO FRUITS

Major interest of this research was to study Aft mutation, primarily responsible for the anthocyanin accumulation in tomato fruit peel. Candidates for the Aft locus are SlANT1, SlAN2 and SlANT1-like genes, all encoding MYB transcription factors regulating transcription of the late anthocyanin biosynthetic genes. To investigate the possible different role of these three genes, diverse experimental approaches were used. amiRNA- and CRISPR-Cas9-mediated gene silencing of SlAN2, SlANT1 and SlANT1-like genes; gene expression analysis in amiRNA-SlAN2, SlANT1 and SlANT1-like transgenic plants; gene expression analysis in tomato plants ectopically expressing SlANT1-like gene; dual luciferase reporter assay on tomato leaf protoplasts with the transient expression of SlAN2, SlANT1 and SlANT1-like, independently or in combination with other factors in the biosynthetic pathway. An additional objective of the research was to study, in more detail, how the expression of these genes can affect the quality of SunBlack purple tomatoes, with a particular attention on pigmentation patterns and shelf life. Wild type amiRNA-SlAN2 transgenic lines showed a low anthocyanin phenotype. In SB amiRNA-SlAN2 plants, likely due to a partial silencing of SlAN2, the phenotype was patchy, with an irregular presence of low anthocyanin areas both in leaves and fruits. In leaves of WT plants ectopically expressing SlANT1-like, anthocyanin synthesis appeared inhibited, while fruit mesocarp of the same plants showed a slight anthocyanin accumulation. In vegetative tissues of amiRNA-SlAN2 transgenic plants, silencing of SlAN2 was efficient and led to reduced expression of SlDFR target gene and reduced anthocyanin accumulation. In fruit peel of amiRNA-SlAN2 SB plants, silencing of the gene was not enough to switch off the anthocyanin biosynthesis or a different regulation, including another MYB master gene, has to be hypothesized. Results obtained in WT plants ectopically expressing SlANT1-like gene indicate a complex role of this transcription factor, with both activating and repressing functions in different organs. Additionally, postharvest results indicated that storage of SunBlack fruits collected at the breaker stage up to two-three weeks under moderate light and cool temperature can be advantageous since fruits can continue to accumulate anthocyanins in their peel without negatively affecting other qualitative parameters involved in taste and flavour.