Regulation of anthocyanin-related biosynthetic genes by bHLH transcription factors in Rubus idaeus and Fragaria vesca

Red coloration on berry fruits is caused due the presence of anthocyanin pigments mainly produced during the late stages of the fruit maturation process and is an important phenotypic feature, making plant breeders around the world consider these plant metabolites as an important trait to follow up and understand. The anthocyanin biosynthesis is regulated through the interaction of the well known MYB-bHLH-WD40 complex (1), which has already been characterized in several crops from diverse plant families, including some Rosaceae species and even on some commercial varieties of Fragaria (2). Using genome wide phylogenetic analysis, we identified three putative bHLH candidates from F vesca and two from R. idaeus based on sequence homology with reported bHLH genes from Malus, Arabidopsis and Fragaria. To date it is not clear which of the encoded bHLH proteins are regulators of anthocyanin biosynthesis, and how this regulation is occurring in strawberries and raspberries. Here we show the results of expression studies of these bHLH genes as well as of putative anthocyanin biosynthetic genes at several fruit developmental stages. In addition, we show the results of promoter activation studies determined by co-transformation of the promoter regions of biosynthetic genes fused to the luciferase gene and bHLH genes under 35S promoter expression in Nicotiana benthamiana; and we establish that some of the bHLH candidate proteins are essential components for activation of key genes inolved in the anthocyanin pathway.