Evolutionary Analysis and Bifunctionality of VcCCoAOMT5: A Key O‑Methyltransferase Involved in Both Lignin and Anthocyanin Biosynthesis in Highbush Blueberry

Caffeoyl-CoA O-methyltransferase (CCoAOMT) is
a core enzyme in plant specialized metabolism. This study investigated
the CCoAOMT gene family in highbush blueberry (Vaccinium corymbosum, HB), revealing that whole-genome
duplications have driven its evolutionary history. Among the family
members, VcCCoAOMT5 was identified as a central regulator
of fruit quality. In vitro enzymatic assays demonstrated
that VcCCoAOMT5 is a bifunctional enzyme with broad substrate selectivity,
capable of methylating precursors for both anthocyanin (e.g., delphinidin-3-O-glucoside) and lignin (e.g., caffeic acid) pathways. Functionally,
transient overexpression of VcCCoAOMT5 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 Nicotiana tabacum. These findings establish VcCCoAOMT5 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.