Figure 4: Cis-element binding ability and transcriptional activation assays of McMYB12a and McMYB12b with anthocyanin and PAs biosynthetic genes.

(A) Interaction of McMYB12 proteins with the promoters of flavonoids biosynthetic genes as revealed by yeast one-hybrid assays. The panel shows yeast cells containing distinct effector and reporter constructs grown on an SD/-Trp/-Ura medium plate. Interaction of McMYB12a or McMYB12b, fused to the GAL4 activation domain (pJG4-5-McMYB12a or b), with LacZ driven by the flavonoid biosynthetic gene promoters (pLacZi-promoter of flavonoid biosynthetic gene) is shown in left top panel. Yeast transformed with pJG4-5-McMYB12s/pLacZi, pJG4-5/pLacZi-promoter of flavonoid biosynthetic genes and pJG4-5/pLacZi were used as controls. (B) Gel-shift analysis of McMYB12a binding to the promoters of six flavonoid biosynthetic genes. Purified proteins (10 μg) were incubated with 25 picomoles biotin-labeled probe or mutant probe (mP). For the competition test, non-labeled probes at varying concentrations (from 10- to 1,000-fold excess) or labeled mutant probes (1,000-fold) were added to the above experiment. (C) Gel-shift analysis of McMYB12b binding to the promoters of five flavonoid biosynthetic genes.