Heterologous expression of gentian MYB1R transcription factors suppresses anthocyanin pigmentation in tobacco flowers

Single-repeat MYB transcription factors, GtMYB1R1 and GtMYB1R9 , were isolated from gentian. Overexpression of these genes reduced anthocyanin accumulation in tobacco flowers, demonstrating their applicability to modification of flower color. RNA interference (RNAi) has recently been used to successfully modify flower color intensity in several plant species. In most floricultural plants, this technique requires prior isolation of target flavonoid biosynthetic genes from the same or closely related species. To overcome this limitation, we developed a simple and efficient method for reducing floral anthocyanin accumulation based on genetic engineering using novel transcription factor genes isolated from Japanese gentians. We identified two single-repeat MYB genes-GtMYB1R and GtMYB1R9-predominantly expressed in gentian petals. Transgenic tobacco plants expressing these genes were produced, and their flowers were analyzed for flavonoid components and expression of flavonoid biosynthetic genes. Transgenic tobacco plants expressing GtMYB1R1 or GtMYB1R9 exhibited significant reductions in floral anthocyanin accumulation, resulting in white-flowered phenotypes. Expression levels of chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) genes were preferentially suppressed in these transgenic tobacco flowers. A yeast two-hybrid assay demonstrated that both GtMYB1R1 and GtMYB1R9 proteins interacted with the GtbHLH1 protein, previously identified as an anthocyanin biosynthesis regulator in gentian flowers. In addition, a transient expression assay indicated that activation of the gentian GtDFR promoter by the GtMYB3-GtbHLH1 complex was partly canceled by addition of GtMYB1R1 or GtMYB1R9. These results suggest that GtMYB1R1 and GtMYB1R9 act as antagonistic transcription factors of anthocyanin biosynthesis in gentian flowers. These genes should consequently be useful for manipulating anthocyanin accumulation via genetic engineering in flowers of other floricultural plant species.