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Fig. 4 | BMC Biology

Fig. 4

From: ERF49 mediates brassinosteroid regulation of heat stress tolerance in Arabidopsis thaliana

Fig. 4

Expression of ERF49, a direct target gene of BZR1, is repressed by BZR1 and BR. a Yeast one-hybrid assays for binding of BZR1 to the ERF49 promoter. Diagram depicting the segmentation of ERF49 promoter. Black and red asterisks indicate the positions of putative E-box and BRRE motifs, respectively. The region from −1964 to −1 bp was named ERF49pro and the region from −1102 to −420 bp was named ERF49pro-2 (upper panel). The activity of β-galactosidase when BZR1 and ERF49pro-2 were transformed into the yeast cells together (lower panel). b ChIP-qPCR assays for direct binding of BZR1 to the ERF49 promoter. Relative enrichment of PP2A coding region was normalized as an internal control. DWF4 and CPD were identified as target genes of BZR1. ERF49-F1 indicates the sequence analyzed by ChIP-qPCR. c Dual-luciferase reporter assay of ERF49 in Arabidopsis protoplasts. Schematic diagram of effector (35S::BZR1) and reporter (ERF49pro::LUC) constructs used in the protoplast transcription system (upper panel). Expression activity assay of LUC reporter genes in Arabidopsis protoplasts transformed with the effector and reporter plasmids (lower panel). The plasmid pUC18-3HA was empty vector (EV). d Transcriptional activity assays of the interaction between BZR1 protein and ERF49 promoter fragments. Tobacco transient expression of the LUC reporter gene (under the control of the ERF49 promoter fragments with native or mutant BRRE motif) by the BZR1. e Quantification of the dual-luciferase assays of LUC expression in d. The expression of REN (Renilla luciferase) was used as the internal control. The LUC/REN ratio indicates the relative activity of the promoter. Asterisks represent statistical significance (**, P < 0.01; one-way ANOVA followed by post hoc Tukey’s multiple comparison test). f Yeast one-hybrid assay of the interaction between BZR1 protein and ERF49 promoter fragments. Systematic yeast one-hybrid assay showing the binding of BZR1 to the ERF49 promoter fragments with native or mutant BRRE motif. g The relative expression level of ERF49 in Col-0, bzr1-1D, and det2. h Relative expression level of ERF49 in Col-0 after 100 nM 2, 4-eBL for 0, 1, 2, 4, 8, and 12 h. i GUS activities of the ERF49pro::gERF49-GUS transgenic Arabidopsis plants after soaking in a solution of 100 nM 2, 4-eBL for 0 and 4 h. CK was a solution containing 80% ethanol. Scale bars, 2 mm. Asterisks represent statistical significance (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA followed by post hoc Tukey’s multiple comparison test). UBC30 was used as a reference gene. Numerical data is provided in Additional file 6. The experiments were repeated three times, error bars indicate standard deviation

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