Dual-Luciferase Assays

Dual-luciferase transactivation activity of TFs on target promoters was performed according to Cao et al. (2019). The full-length coding sequences of MrMYB candidates were individually cloned into pGreenII0029 62_SK vectors. Primers are listed in Supplementary Table 2. Promoters of MrDFR1^–1557, MrFLS1^–1705, MrLAR1^–1534, and MrANR1^–1512 were isolated from ‘BQ’ genomic DNA and cloned individually into pGreen II0800_LUC vectors. Primers are listed in Supplementary Table 3. All constructs were electroporated into Agrobacterium tumefaciens GV3101. The bacteria were prepared in infiltration buffer (10 mM MES, 10 mM MgCl₂, 150 mM acetosyringone, pH 5.6) when the optical density at 600 nm reached approximately 0.75. The culture mixtures of bacteria containing TFs (1 ml) and promoters (100 μl) were infiltrated into leaves of 4-week-old Nicotiana benthamiana plants. The luminescence from Firefly luciferase (LUC) and Renilla luciferase (REN) was detected by Dual-Luciferase Reporter Assay System (Promega, Madison, WI, United States) on the third day after infiltration, and six biological replicates were used. The ratios of LUC and REN were expressed as activation or repression of the promoters by the TFs.