The ectopic expression of apple MYB1 and bHLH3 differentially activates anthocyanin biosynthesis in tobacco

The apple (Malus domestica) MdMYB1 gene encodes an R2R3-MYB transcription factor (TF) that regulates anthocyanin biosynthesis. The transient and stable expression of the apple basic helix-loop-helix 3 gene, MdbHLH3, produces anthocyanin pigmentation in apple fruit and calli. In order to understand the MdMYB1- and MdbHLH3-related regulatory mechanisms of anthocyanin biosynthesis, we established and characterized the expression of those two genes in stably transformed tobacco (Nicotiana tabacum NC89) plants, including the phenotypic differences in anthocyanin-containing flowers, ovaries, anthers and anthocyanin-free leaves. A real-time PCR analysis showed that the expression levels of the MdMYB1 and MdbHLH3 transgenes were up-regulated in all tobacco organs. However, the bHLH and MYB TFs, NtAN1 and NtAN2, were not detected in tobacco leaves, respectively, indicating that neither can individually activate anthocyanin biosynthesis. However, MdMYB1 induced pigment accumulation in ovaries because NtAN1 is active in that organ. Thus, the endogenous bHLH TF is necessary for anthocyanin biosynthesis in tobacco. Furthermore, MdMYB1 binding to the promoters of the anthocyanin biosynthesis genes MdDFR and MdUFGT activated their expression in apple. However, because NtAN2 is not activated in leaves or ovaries, the pigmentation patterns were not significantly altered in these organs in MdbHLH3-overexpressing tobacco. Thus, the data indicated that MdMYB1 regulated anthocyanin biosynthesis directly by binding the MdDFR and MdUFGT promoters, and interacted with the tobacco bHLH TF, triggering anthocyanin accumulation.