Functional AOMT1 genes coupled with AOMT1 promoter alterations: shaping methoxylated anthocyanin accumulation in grape cultivars

Anthocyanin methoxylation, a key enzymatic modification catalyzed by anthocyanin O-methyltransferases (AOMTs), significantly influences color stability and intensity in grape berries. However, the molecular mechanisms underlying varietal differences in methoxylation patterns between colored and colorless grape cultivars remain unclear. In this study, we characterized the function and promoter activity of VvAOMT1 across multiple grape varieties. Phylogenetic and structural analyses revealed that AOMT1 was highly conserved among fruit species. Expression profiling demonstrated that AOMT1 transcript levels correlated with anthocyanin accumulation and skin color. Functional assays via transient and stable transformation in tobacco showed that AOMT1 enzymes from both colored and colorless cultivars effectively catalyzed anthocyanin methoxylation when co-expressed with the regulator MYBA1, significantly increasing methoxylated anthocyanin proportions. In vitro enzymatic assays further confirmed the methoxylation activity of AOMT1 toward anthocyanin substrates. Notably, promoter activity assays and electrophoretic mobility shift assays (EMSA) revealed that natural variations in cis-regulatory elements, especially MYB binding sites (MBS), lead to differential promoter activities and MYBA1 transactivation among cultivars, ultimately affecting AOMT1 expression and methoxylated anthocyanin accumulation. These findings elucidate the regulatory mechanism behind anthocyanin methoxylation in grapes and provide insights for molecular breeding aimed at improving fruit color traits.