Figure 4. The circadian clock gates thermomorphogenesis.

(a) Effects of warm temperature at different ZTs on the expression of PIF4 and YUC8. Wild-type seedlings were grown in 12 h light/12 h dark cycles (12L:12D) at 20 °C for 4 days. On the 5th day, the seedlings were treated with warm temperature (29 °C) for 4 h at different ZT (ZT0–ZT36) before harvesting for RNA extraction. At different ZTs, the growth temperature was increased to 29 °C or kept at 20 °C for 4 h. Gene expression levels were normalized to PP2A and presented as values relative to that of wild type at ZT0. Error bars indicate s.d. (n=3). *P<0.05 (Student's t-test). (b) Effects of warm temperature at different ZTs on the expression of PIF4 and YUC8. Wild-type seedlings were entrained in 12L:12D cycles at 20 °C for 4 days and then transferred under the continuous light. At different ZTs, the growth temperature was increased to 29 °C or kept at 20 °C for 4 h. Gene expression levels were normalized to PP2A and presented as values relative to that of wild type at ZT0. Error bars indicate s.d. (n=3). *P<0.05 (Student's t-test). (c,d) Warm-temperature effects on PIF4 protein levels. PIF4p::PIF4-Myc seedlings (c) or 35S::PIF4-Myc seedlings (d) were grown in the same condition as (b). Immunoblotting was probed using an anti-Myc antibody and stained with Ponceau S. The experiments were repeated with similar results. The molecular weight (kDa) is indicated on the right side of the gel. (e) ChIP-quantitative PCR assays of PIF4-Myc binding to YUC8, IAA19 and IAA29 promoters. The PIF4p::PIF4-Myc seedlings were grown in the same condition as (d). The enrichment of DNA was normalized to that of the PP2A coding region as an internal control. Error bars indicate s.d. (n=3). *P<0.05 (Student's t-test).