Evolutionary Analysis and Bifunctionality of VcCCoAOMT5: A Key <i>O</i> -Methyltransferase Involved in Both Lignin and Anthocyanin Biosynthesis in Highbush Blueberry

Caffeoyl-CoA <i>O</i>-methyltransferase (CCoAOMT) is a core enzyme in plant specialized metabolism. This study investigated the <i>CCoAOMT</i> gene family in highbush blueberry (<i>Vaccinium corymbosum</i>, HB), revealing that whole-genome duplications have driven its evolutionary history. Among the family members, <i>VcCCoAOMT5</i> was identified as a central regulator of fruit quality. <i>In vitro</i> enzymatic assays demonstrated that VcCCoAOMT5 is a bifunctional enzyme with broad substrate selectivity, capable of methylating precursors for both anthocyanin (e.g., delphinidin-3-<i>O</i>-glucoside) and lignin (e.g., caffeic acid) pathways. Functionally, transient overexpression of <i>VcCCoAOMT5</i> in blueberry fruit significantly enhanced methylated anthocyanin levels and increased fruit firmness, with more immature seeds observed. Conversely, its silencing impaired these traits. The gene's role in lignification was further verified through heterologous expression in <i>Nicotiana tabacum</i>. These findings establish <i>VcCCoAOMT5</i> as a critical link between the anthocyanin and lignin biosynthetic pathways, making it an important contributor to blueberry fruit quality and a promising target for molecular breeding.