Figure 5.

ARF2 Directly Binds to the HAK5 Promoter.

(A) Diagram of the HAK5 promoter region. Gray lines represent the positions of AuxREs in the HAK5 promoter. Black lines indicate three fragments (P1 to P3) containing AuxREs in the HAK5 promoter that were used in the ChIP-qPCR assay.

(B) ChIP-qPCR analyses of ARF2 DNA binding activity to the HAK5 promoter. Chromatin was isolated from 7-d-old seedlings grown on K⁺-sufficient medium. Chromatin was immunoprecipitated with ARF2 antibody (anti-ARF2). Then, the three fragments (P1 to P3) were tested using qPCR. The ratios of immunoprecipitated DNA over the input DNA were calculated and presented as percentage of input (% IP). Chromatin without ARF2 antibody was used as a negative control. Data are means ± se (n = 3).

(C) EMSA to analyze the DNA binding activity of ARF2 N-terminal to P1 and P3 fragments in the HAK5 promoter. The purified CKS-ARF2-N-His proteins were incubated with P1 or P3 probes labeled with biotin. An excess of unlabeled probes was added to compete with biotin-labeled probes. ARF2-N can bind to P1 and P3 fragments, but not interact with the fragments with mutated AuxREs. The AuxREs were labeled in red. CKS-His protein was used as a negative control. The positions of hysteresis bands and free labeled probes are indicated with arrows.