Figure 4.

WRKY23 acts downstream of ARF7 and ARF19 and directly activates transcription of PLT3 and PLT7. A) Relative WRKY23 transcript levels in response to CIM in arf7 arf19 and lbd16-2 air1-2 seedlings. Seven-day-old seedlings were incubated in liquid CIM for the indicated times, and the relative expression level at 0 h was set to 1; data are shown as means ± Sd (n = 3 biological replicates). B) Callus-forming and shoot-regenerating phenotypes of WT, arf7 arf19, and arf7 arf19 35S:WRKY23 explants and derived calli (n = 20 explants). Scale bar, 10 mm. C) Callus-forming and shoot-regenerating phenotypes of WT, 35S:WRKY23, plt3 plt7, and plt3 plt7 35S:WRKY23 explants (n = 20 explants) and derived calli. Scale bar, 10 mm. Three independent transgenic lines of arf7 arf19 35S:WRKY23 and plt3 plt7 35S:WRKY23 named #1, #2, and #3 were examined in B) and C), respectively. D) Induction of PLT3 and PLT7 transcript levels by WRKY23. Five-d-old transgenic ProXVE:WRKY23 seedlings were incubated in liquid B5 medium containing 10 μm 17-β-estradiol or DMSO (Mock) for 3 h, and relative expression levels are shown as means ± Sd (n = 3 biological replicates). E) ChIP-qPCR assay showing the WRKY23 binding activity to the PLT3 and PLT7 promoters. ChIP-qPCR was performed with 10-d-old WT and wrky23-3 ProWRKY23:gWRKY23-GFP seedlings incubated in liquid CIM for 48 h with an anti-GFP antibody, and the enrichment of PLT3 or PLT7 promoter fragments F-1 to F-9 are indicated. A fragment of the UBQ10 promoter was used as a negative control. Data are shown as means ± Sd (n = 3 biological replicates). F) Transcriptional activation of PLT3 and PLT7 by WRKY23. The activity of the ProPLT3:LUC or ProPLT7:LUC reporter was determined in Arabidopsis protoplasts cotransfected with an empty vector (EV), or 35S:Ω:WRKY23-GFP (WRKY23) construct, together with each LUC reporter. Data are shown as means of the original LUC/REN ratios × 100 ± Sd (n = 3 biological replicates). *P < 0.05, **P < 0.01, and ***P < 0.001 by t-test.