Studies On The Mechanism By Which MdTTG1, MdMYB9and MdMYB11Regulate Anthocyanin Biosythesis In Apple

Fruit color is one of the most important quality traits. It determines the diet and commercial values of apple fruit to a great extent. Apple fruit coloration results from the accumulation of anthocyanins which synthesized through flavonoid secondary metabolic pathway. The biosynthesis of anthocyanins is greatly influenced by various stimuli, however, the molecular mechanism through which those stimuli regulate anthocyanins biosynthesis is largely unknown.In these study, three genes, i.e. MdTTGl, MdMYB9and MdMYBll, were isolated from apple fruit(Malus×Domestica Borkh cv. Starkrimson). To characterize their functions, they were genetically transformed into Arabidopsis and apple callus, respectively. Main results are shown as follows:1. MdTTGl promotes anthocyanins accumulation in transgenic ArabidopsisTo characterize the function of MdTTGl in planta, we constracted35S::MdTTGl construct, and then transformed it into Arabidopsis. The results showed that ectopic expression of MdTTGl in Arabidopsis promoted anthocyanins accumulation, especially with continuous light treatment. The structural genes associated with anthocyanins biosynthesis were remarkably up-regulation compared to WT. These results indicated that MdTTGl involved in anthocyanins accumulation in planta.2. MdTTGl is a member of the WBM complexY2H and BiFC assay showed that MdTTGl interacts with MdbHLH3and MdbHLH33, respectively, but not with MdMYB1protein. In apple, MdMYB1, MdbHLH3and MdbHLH33were proved to be involved in the regulation of anthocyanins biosythesis. The transcription level of MdTTG1was associated with fruit pigment, and transgenic Arabidopsis overexpressing MdTTGl produced more anthocyanins than the WT control. Therefore, it may come to a conclusion that MdTTGl works together with MdMYB1, MdbHLH3and MdbHLH33to form WBM complex and is involved in the regulation of anthocyanins biosynthesis.3. MdMYB9and MdMYB11were involved in the regulation of anthocyanins and proanthocyanidins biosynthesisIn this study, two new MYB transcription factor genes named MdMYB9and MdMYB11were isolated from apple fruit. Transgenic callus overexpressing MdMYB9and MdMYB11, respectively, generated more transcripts of structural genes associated with flavonoids biosynthesis, and therefore produced more anthocyanins and proanthocyanins than control. ChIP-PCR assay demonstrated that MdMYB9and MdMYB11bind to the promoters of the structural genes, respectively, which leads to anthocyanins and proanthocyanins accumulation in transgenic callus.4. JA regulates the accumulation of anthocyanins and proanthocyanins by modulating the expression MdMYB9and MdMYB11The expression levels of MdMYB9and MdMYB11were induced by MeJA. Both MdMYB9and MdMYB11transgenic calluses accumulated more anthocyanins and proanthocyanins under MeJA treatment, compared with untreatment. In addition, MeJA enhanced the binding of MdMYB9and MdMYB11to the promoters of anthocyanins and proanthocyanins structural genes. Therefore, MdMYB9and MdMYB11are involved in the JA-induced anthocyanins and proanthocyanins accumulation in apple.