Figure 2: EAR motif mediates BZR1 regulation of gene expression.

(a,b) The deletion of EAR motif abolishes BZR1 regulation of gene expression. Seedlings were grown on the medium containing 2 μM PPZ in the dark for 5 days before harvesting for RNA extraction. Gene expression levels are normalized to that of PP2A and are shown relative to the expression levels in wild type (Col-0). (c,d) The EAR motif-deleted bzr1-1D neither represses CPD expression (c) nor activates SAUR15 expression (d). Four-week-old leaves of transgenic plants expressing bzr1-1D-GR or bzr1-1DΔEAR-GR were treated with mock or 40 μM of dexamethasone (DEX) for 4 h. Ratios of expression levels in DEX-treated to mock-treated are shown. Each bar represents independent transgenic plant. (e,f) The EAR motif-deleted bzr1-1D cannot activate PRE5 promoter activity (e) and cannot repress CPD promoter activity (f) in a transient gene expression assay. The 2 kb of PRE5 promoter or 1 kb of CPD promoter fused to firefly luciferase reporter gene was co-transfected with 35S::bzr1-1D or 35S::bzr1-1DΔEAR into Arabidopsis mesophyll protoplast. The firefly luciferase activities were normalized by Renilla luciferase as an internal control and are shown relative to that of protoplasts transfected with 35S::GFP (GFP). **P<0.01 and *P<0.05 by Student’s t-test. (g) The deletion of EAR motif does not affect BZR1 DNA binding. Transgenic plants overexpressing BZR1 and BZR1ΔEAR were treated with 100 nM BL and cross-linked for the ChIP assays. In the ChIP assays, the enrichment of DNA was calculated as the ratio between transgenic plants and wild-type control (Col-0), normalized to that of the PP2A coding region as an internal control. Error bars in (a,b,e,f,g) indicate the s.d. (n=3).