Figure 9.

Phosphorylation of ARF2 Relieves the Repression of HAK5 Transcription under Low-K⁺ Conditions.

(A) ChIP-qPCR analyses of ARF2 DNA binding activity to P1 and P3 fragments in the HAK5 promoter with or without low-K⁺ treatment. Wild-type seedlings were germinated and grown on K⁺-sufficient medium for 5 d and then transferred to K⁺-sufficient medium or low-K⁺ medium for 6 h. Data are means ± se (n = 3).

(B) ARF2 can be phosphorylated after low-K⁺ treatment. arf2-7/ProARF2:ARF2-Flag (COM1) transgenic plants were germinated and grown on K⁺-sufficient medium for 5 d and then transferred to K⁺-sufficient medium or low-K⁺ medium for 2, 4, and 6 h. Total proteins of the plants at different time points were extracted. ARF2 proteins were immunoprecipitated using anti-Flag mAb agarose. Phosphorylated ARF2 proteins were detected by immunoblotting (IB) using biotinylated Phos-tag. CIAP can dephosphorylate ARF2, which was used as a control.

(C) GUS activity measurement in tobacco leaves after transient expression of ProHAK5:GUS and Pro35S:ARF2 with point mutations (S689A and S689D). ARF2^S689D can relieve the repression of HAK5 transcription. Data are means ± se (n = 6). Student’s t test (**P < 0.01) was used to analyze statistical significance, and “#” represents control.

(D) Ser-689 on ARF2 is phosphorylated after low-K⁺ treatment. Wild-type ARF2 peptide (ARF2^501-700) and mutated peptide (ARF2^{501-700 S689A}) were expressed in E. coli and purified, respectively. Wild-type plants were germinated and grown on K⁺-sufficient medium for 5 d and then transferred to K⁺-sufficient medium or low-K⁺ medium for 2, 4, and 6 h. Total proteins were extracted from wild-type plants at the indicated time points. The purified ARF2 peptides were incubated with the total proteins for 40 min. Then the phosphorylation status of ARF2 peptides was tested using Phos-tag SDS-PAGE. ARF2 peptides were detected by immunoblot analysis using His antibody. CIAP can dephosphorylate ARF2 peptides, which were used as a control.

(E) EMSA to analyze the DNA binding activity of ARF2^S689A (SA) and ARF2^S689D (SD) to P1 and P3 fragments. The purified CKS-ARF2-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. The CKS-His protein was used as a negative control. The positions of hysteresis bands and free labeled probes are indicated with arrows.