Pan‐genome analysis reveals the evolutionary dynamics and functional divergence of the <i>O‐</i> methyltransferase gene family in tomato

O-Methyltransferases (OMTs) play crucial roles in plant defense, environmental adaptation, and quality formation by catalyzing the biosynthesis of diverse methylated metabolites. Although OMT (COMT and CCoAOMT) genes have been functionally characterized in various plant species, the evolutionary trajectory of the entire OMT gene family and the functional divergence of the CCoAOMT subfamily remain to be systematically elucidated. In this study, we performed pan-genome analysis of the OMT gene family in 61 tomato (Solanum spp.) accessions and conducted phylogenetic analysis across 20 plant species (from algae to angiosperms), identifying 2,882 OMT genes. Phylogenetic reconstruction revealed that all extant plant CCoAOMT genes evolved from a single ancestral lineage (Clade I) originating before the divergence of red and green algae. In tomato, 2,199 OMT genes were classified into 42 orthogroups: nine core, five soft-core, 22 dispensable, and six private orthogroups, with 52.4% classified as dispensable genes. OMT genes in the Solanum genus have predominantly undergone purifying selection. Among all COMT orthogroups, a single tandem duplicate cluster stands out as exclusively conserved. Members of this cluster have evolved a distinct catalytic role, as evidenced by the finding that SlCOMT2c exclusively catalyzes the formation of kaempferide via the 4'-O-methylation of kaempferol. Ion mobility spectrometry showed that SlAOMT, a member of the CCoAOMT-like subfamily, catalyzes the methylation of luteolin to produce two isomeric products identified as diosmetin and chrysoeriol while losing the canonical catalytic function of the CCoAOMT subfamily. In addition, we identified a potential gene regulatory network associated with methylated flavonoid biosynthesis. This study establishes an integrative framework for elucidating OMT evolution and provides analytical tools for identifying genes involved in isomeric methylated flavonoid biosynthesis, paving the way for studying adaptive evolution and specialized metabolic pathways in plants.