(A) Illustration of the YUC5, YUC8, YUC9 and YUC10 promoter regions showing the presence of G-box DNA motifs. The arrows indicate positions of primers used for ChIP-PCR experiment. Shown are 2-kb upstream sequences of the YUC genes. The translational start site (ATG) is shown at position +1. (B) Gel photographs showing the amplified products from the ChIP assay. The ChIP assays were performed using 6-d-old seedlings expressing the PIF4-HA fusion protein untreated or treated with 29°C for 6 h. Antibody to the HA tag was used to immunoprecipitate PIF4-HA and associated DNA fragments. DNA was amplified by using primers specific to the region containing the G-box element or control regions in ACT2 promoter as indicated. Shown are representative data from one biological replicate; this experiment was conducted for three biological replicates, yielding similar results. (C) EMSA assay showing that PIF4 binds the G-box motifs present in the YUC8 promoter in vitro. The YUC8 promoter fragments containing the G-box motifs were incubated with in vitro TNT-expressed PIF4 protein as indicated. Competition for PIF4 binding was performed with 10×, 20× and 50× cold YUC8 probes containing G-box (G-wt, CACGTG) or mutated G-box (G-mut, CACGGG), respectively. FP, free probe. TnT indicates in vitro-expressed luciferase proteins used as a control.