Fig. 7: MYB4 participates in a negative feedback mechanism to balance the accumulation of anthocyanins and PAs.

a Transient expression of MYBA, bHLH3, and MYB4 in tobacco leaves. SK, empty vector. b Dual-luciferase reporter assays demonstrated that MYB4 can inhibit the activity of the ANS promoter. The data represent averages of four experiments. c The positive transgenic lines were determined by semiquantitative RT-PCR. AtActin served as an internal standard. d DMACA-stained (bottom row) and unstained (top row) seeds of non-transgenic wild-type Arabidopsis and transgenic Arabidopsis plants overexpressing MYB4. WT: wild-type, OE-MYB4: T2 seed of MYB4-transgenic plants. Scale bars are 1 mm. e Dual-luciferase reporter assays demonstrated that MYB4 can inhibit the activity of the LAR promoter. The data represent averages of four experiments. f Effect of MYB activators together with different bHLH cofactors on the activation of the MYB4 promoter. SK, empty vector. The data represent averages of four experiments. g Validation of the activation effect of MYB/bHLH binary complexes on the MYB4 promoter using a transgenic tobacco transient expression system. Transgenic tobacco plants expressing GUS driven by the MYB4 promoter (transgenic line 1). GUS was expressed in transgenic tobacco leaves transiently transformed with candidate transcription factors and Renilla luciferase at a ratio of 10:1. The bar is 1 mm. h Measurement of GUS activity in transgenic tobacco leaves transiently transformed with candidate TFs and Renilla luciferase. i Measurement of Renilla luciferase activity in transgenic tobacco leaves transiently transformed with candidate TFs and Renilla luciferase. j Activation of the MYB4 promoter by anthocyanin and PA activation complexes. A signal from Renilla luciferase was used as an internal control to normalize GUS activity. The data represent averages of three experiments. Asterisks (*) indicate the statistical significance of the difference between the experimental and the control groups. (Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001). Significant differences between treatments were determined using one-way ANOVA (P < 0.05)