Figure 3.

Red light inhibits the binding of PIF3 and PIF1 to their target promoters in plants possessing full-length phyB. (A) The binding of PIF3 to PIL1 and RGA promoters in Dc and Rc samples of the PIF3-OX determined by ChIP. The rDNA fragment was used as a non-binding control. The co-IPd DNA was expressed as relative values of PIF3-OX, Dc (SD, n=2, biological replicates). Dc and Rc indicate dark-grown and red light-grown PIF3-OX respectively. The upper panel indicates PIF3 protein levels after the ChIP (After IP). (B) MG132 treatment increases PIF3 protein levels both in Dc- and Rc-grown samples, but the binding of PIF3 to its target promoters increases only in Dc-grown PIF3-OX. DMSO is dimethyl sulfoxide used to dissolve MG132. The co-IPd DNA was expressed as relative values of PIF3-OX, Dc+DMSO (SD, n=2, biological replicates). (C) MG132 treatment increases PIF1 protein level and the subsequent binding of it to its target promoter in Dc-grown, but not in Rc-grown PIF1-OX. The co-IPd DNA was expressed as relative values of PIF1-OX, Dc+DMSO (SD, n=2, biological replicates). (D) A far-red pulse followed by a short dark incubation increases PIF3 and PIF1 protein levels mildly but the binding of them to their target promoter increases strongly in PIF3-OX and PIF1-OX. Red light-grown seedlings were irradiated with far-red light pulse (5′FR, 3 μmol m⁻² s⁻¹), transferred to dark for 10 (10′Dark) or 30 minutes (30′Dark), and sampled for ChIP analysis. The co-IPd DNA was expressed as relative values of either PIF3-OX, Rc, or PIF1-OX, Rc (SD, n=2, biological replicates).