Three bilberry bHLHs of subgroup IIIf play divergent roles in fruit anthocyanin and flavonoid biosynthesis

Bilberry (Vaccinium myrtillus L.) fruit are one of the best natural sources of anthocyanins. Anthocyanin and flavonoid biosynthesis are transcriptionally regulated by the conserved MBW complex, including R2R3 MYB, basic helix-loop-helix (bHLH) and WD40 proteins. In this study, homology-search in the bilberry genome identified three putative anthocyanin biosynthesis-regulating bHLH genes, all containing a MYB binding domain, indicating their possible interaction with R2R3 MYBs. Phylogenetic analysis assigned the VmbHLHs to three different clusters within the IIIf subgroup of bHLHs. The expression of VmbHLH2 elevated at berry ripening, while VmbHLH1 and VmbHLH3 showed decreasing expression towards berry maturation. When transiently overexpressed together with MdMYB10 in Nicotiana benthamiana leaves, VmbHLH2 and VmbHLH3 were able to complement MdbHLH3 and upregulate a full set of anthocyanin biosynthetic genes leading to anthocyanin accumulation. In contrast, VmbHLH1 induced a reduced set of flavonoid pathway genes. Our results provide the first functional characterization of flavonoid-regulating bHLHs that contribute to the MBW complex in the economically important genus Vaccinium. We propose that the TT8-type VmbHLH2 is involved in anthocyanin biosynthesis in ripening bilberry fruit, while the MYC1-type VmbHLH1 and GL3/EGL3-type VmbHLH3 are associated with anthocyanin and PA biosynthesis, mainly in tissues other than berries.