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. 2020 Mar 9;32(5):1464–1478. doi: 10.1105/tpc.19.00981

Figure 3.

Figure 3.

PIF5 Directly Binds to the CCA1 Promoter and Represses Its Expression.

(A) Yeast one-hybrid assay showing that PIF3 and PIF5 directly bind to the CCA1 promoter. PIF3 and PIF5, but not PIF1 and PIF4, activated the AbAr reporter gene driven by the wild-type CCA1 promoter but not the AbAr reporter gene driven by the CCA1 promoter with a mutated G-box element. Empty vector expressing the activation domain (AD) alone was used as the negative control.

(B) EMSA showing that GST-PIF3 and GST-PIF5 bHLH (DNA binding domain) specifically bind to the biotin-labeled CCA1p-G-box (right) probe. The arrowheads indicate GST-PIF3 and GST-PIF5 bHLH proteins.

(C) Transient expression assay showing that PIF5 represses CCA1 expression in N. benthamiana leaf cells (**, P < 0.05, Student’s t test). Values are means ± sd (n = 3 technical replicates). Three independent biological replicates showed similar results.