Figure 9.

Direct Activation of Monolignol Biosynthetic Genes by MYB58.

Arabidopsis leaf protoplasts were transfected with MYB58 fused with the regulatory region of HER under the control of the CaMV 35S promoter. After transfection, the protoplasts were treated with estradiol, cycloheximide (CHX), or cycloheximide plus estradiol. The expression of the monolignol biosynthetic genes was analyzed by real-time quantitative RT-PCR. The quantitative differences of the expression of the tested genes between the control and treated samples are statistically significant (P < 0.001). The expression level of each gene or GUS activity in the mock-treated (control) or cycloheximide-treated protoplasts (CHX) is set to 1. Error bars represent se of three biological replicates.

(A) Diagrams of the MYB58-HER construct used for direct target analysis and the 4CL1 promoter-driven GUS reporter gene construct.

(B) GUS activity in leaf protoplasts cotransfected with 35S:MYB58-HER and 4CL1P:GUS. Addition of the protein synthesis inhibitor cycloheximide at 2 μM completely abolished the estradiol-induced GUS activity, indicating that new protein synthesis was completely inhibited by the treatment.

(C) Induction of the monolignol biosynthetic genes by estradiol treatment of protoplasts expressing MYB58-HER. Addition of estradiol releases MYB58-HER from the cytoplasm and its subsequent entry into the nucleus leads to activation of its downstream target genes.

(D) Induction of the monolignol biosynthetic genes by estradiol activation of MYB58 occurred in the presence of cycloheximide.