Figure 3.

WDL4 is an EIN3 target gene. A, EMSA assay for EIN3 binding to the WDL4 promoter. Each biotin-labeled DNA fragment was incubated with recombinant GST-EIN3. Free GST was used as a control. Competition for the labeled promoter fragments was performed by adding an excess (30×) of unlabeled WT or mutated probes. Two putative EBSs (P1 and P2) in the WDL4 promoter were tested. White arrowhead indicates free probe and black arrowhead indicates bound complex. B, ChIP assay for EIN3 binding to the WDL4 promoter. Chromatin was immunoprecipitated from etiolated seedlings expressing EIN3-3 × FLAG under the β-estradiol-inducible promoter using an anti-FLAG antibody. Seedlings treated with mock buffer (half-strength MS medium with the same volume of DMSO used for β-estradiol treatment) were used as a control. The amount of indicated DNA in the immune complex was determined by qPCR. Values represent mean ± sd from three individual biological repeats. C, Transient expression of WDL4pro:GUS with or without EIN3 in N. benthamiana leaves. LUC was expressed from the Super promoter as an internal control. GUS and LUC activity was quantified, and the GUS/LUC ratio used to estimate the binding activity of EIN3 to the WDL4 promoter. At least three independent experiments were performed with similar results. D and E, Representative images and quantification of the hook curvature in the ein3 eil1 wdl4-1 triple mutant compared to that in WT and the ein3 eil1 double mutant. Two independent ein3 eil1 wdl4-1 triple mutant lines were used for analysis. Values represent mean  ± sd (n > 43 independent seedlings; ^**P < 0.01). Scale bar = 0.5 mm.