Figure 4.

Interaction with MPK4 Is Required for MYB75 Function in Anthocyanin Accumulation.

(A) Schematic representation of the MYB75 truncations and their interaction with CA-MPK4 in yeast two-hybrid assays. The numbers indicate the positions of the amino acids in the constructs. “+” and “−” denote positive and negative results for interaction, respectively.

(B) Yeast two-hybrid assay showing that mutation R84K of MYB75, but not W85A, abolishes its interaction with CA-MPK4.

(C) Quantitative real-time PCR analysis showing comparable expression levels of the transgenes MYB75^WT and MYB75^R84K in 35S:MYB75^WT/myb75-c and 35S:MYB75^R84K/myb75-c transgenic seedlings. The forward and reverse primers for MYB75 and EGFP, respectively, were designed to detect transgene transcripts of MYB75^WT and MYB75^R84K fused with EGFP, but not endogenous MYB75 transcripts. Results were normalized to ACTIN8, and expression levels of the genes in 35S:MYB75^WT/myb75-c transgenic seedlings were set at one unit. Error bars indicate sd of three replicates (as described in Figure 2B).

(D) Anthocyanin contents of 12-d-old Arabidopsis seedlings of myb75-c, 35S:MYB75^WT/myb75-c, and 35S:MYB75^R84K/myb75-c grown on plates under low light and moderate high light (high light). FW, fresh weight. Error bars represent sd of three replicates (as described in Figure 2B).

(E) Quantitative real-time PCR analysis of DFR, LDOX, and UF3GT transcript levels in 12-d-old seedlings grown on plates exposed to low light and moderate high light (high light) for 9 h, respectively. Results were normalized to ACTIN8, and expression levels of the genes in myb75-c under low light were set at one unit. Error bars indicate sd of three replicates (as described in Figure 2B).