The R2R3-MYB transcription factor VcMYB4a inhibits lignin biosynthesis in blueberry (Vaccinium corymbosum)

Lignin biosynthesis occurs via the phenylpropanoid pathway and is regulated by transcription factors (TFs) including R2R3-MYB family members. In this study, we functionally characterized the R2R3-MYB TF VcMYB4a from blueberry (Vaccinium corymbosum) in lignin biosynthetic pathway. Phylogenetic analysis indicated that VcMYB4a clusters in a subclade with other TFs that act as transcriptional repressors of lignin and phenolic acid biosynthesis. Furthermore, lignin accumulation appeared to be negatively correlated with VcMYB4a expression during fruit development. Heterologous expression of VcMYB4a repressed lignin accumulation in Arabidopsis. Overexpression of VcMYB4a decreased lignin content in blueberry calli, whereas inhibition of VcMYB4a expression increased lignin accumulation in blueberry leaves. Finally, the transcriptome sequencing showed that overexpressing VcMYB4a in blueberry calli downregulated the expression of Vc4CL (Vc4CL5 and Vc4CL7), VcCOMT (VcCOMT1 and VcCOMT2), and VcCAD (VcCAD1 and VcCAD9) genes involved in lignin biosynthetic pathway. The heterologously expressing VcMYB4a in Arabidopsis downregulated the expression of genes, including AtC4H, At4CL (At4CL1 and At4CL5), AtCAD (AtCAD5 and AtCAD9), and AtCOMT1. The promoter sequences of these genes all contain MYB binding sites, and VcCAD9 and AtCAD9 genes have the most MYB binding sites. At the same time, VcCAD9 is more closely related to AtCAD9 than other CAD homologs from blueberry and Arabidopsis according to phylogenetic analysis. These findings suggested that VcMYB4a functions as a repressor of lignin biosynthesis by downregulating expression of 4CL, COMT, and CAD family members, especially CAD9 homologs. Our studies provide prospects for breeding new blueberry varieties with high lignin contents.