SlMYB76, an SlANS-Repressing R2R3-MYB Transcription Factor, Regulates Anthocyanin Accumulation in ‘Black Pearl’ Tomato (Solanum lycopersicum)

Background/Objectives: Anthocyanins, water-soluble flavonoid pigments with critical roles in plant stress resistance, are not naturally accumulated in cultivated tomato (Solanum lycopersicum) due to an incomplete flavonoid metabolism pathway. In contrast, ‘Black Pearl’ tomato exhibits distinct peel color transitions (from indigo rose to deep purple–red) during ripening, making it an ideal model for investigating the regulatory mechanisms of anthocyanin synthesis. A comprehensive strategy was employed to elucidate these mechanisms, involving transcriptomic (Illumina HiSeq), metabolomic (UPLC-MS/MS), and functional analyses of the ‘Black Pearl’ tomato peel across four developmental stages: mature green (S1), coloring (S2), purple immature (S3), and fully ripened (S4). Results: Transcriptome profiling identified 597 core differentially expressed genes (DEGs) associated with anthocyanin accumulation. Temporal analysis indicated that structural genes and activators peaked at S3, whereas repressive MYBs, including SlMYB76 which peaked at S2, exhibited staged expression. In parallel, metabolomic analysis identified 36 metabolites, with cyanidin and pelargonidin derivatives being characterized as the principal pigments. Functionally, SlMYB76 was confirmed to be a negative regulator, as its transient overexpression reduced anthocyanin content and downregulated SlANS. Mechanistically, direct binding and repression of the SlANS promoter by SlMYB76 were confirmed through yeast one-hybrid and dual-luciferase assays. Furthermore, its physical interaction with the bHLH factor SlJAF13 in the nucleus was demonstrated by Y2H, BiFC, LCI, and Co-IP, supporting the formation of a repressive complex that co-regulates SlANS. Conclusions: A novel SlMYB76-SlJAF13-SlANS regulatory module controlling anthocyanin accumulation in the peel of ‘Black Pearl’ tomato was identified. This discovery enhances the current understanding of the tomato flavonoid regulatory network and provides strategic targets for the genetic improvement of fruit color and anthocyanin content through molecular breeding.