Figure 7.

MYB4 Binds to Its Own Promoter in EMSAs and Represses Its Own Transcription.

Recombinant MYB4 protein was expressed in bacteria as an MBP fusion and purified by affinity chromatography.

(A) EMSAs were performed with radiolabeled or unlabeled MYB4 promoter fragments and recombinant MYB4 protein; specific combinations are shown above the autoradiograph. Unlabeled fragments were added in 100-fold excess as indicated.

(B) MYB4 binding cis-elements (W) in the MYB4 promoter and their mutant forms (M).

(C) EMSA with MYB4 and the cis-elements identified in (B). Competitors were added in 100-fold excess as indicated. Bands in the boxes show the specific binding.

(D) MYB4 inhibits its own transcription in the wild type but not in sad2. Relative GUS activities were measured after transfection of MYB4:GUS with or without 35SP:MYB4 in wild-type and sad2 protoplasts. The values represent averages of three transfections, and error bars indicate sd.

(E) Overexpression of MYB4 repressed endogenous MYB4, CHS, and C4H expression in the wild type but not in the sad2 mutant. Total RNA was extracted from 12-d-old seedlings of the wild type, the sad2 mutant, and overexpression MYB4 transgenic plants in both the C24 and sad2 background. The resulting cDNAs were used for real-time PCR analysis. Error bars indicate sd (n = 3).