TOC1-PIF4 interaction mediates the circadian gating of thermoresponsive growth in Arabidopsis

Abstract

Arabidopsis adapts to elevated temperature by promoting stem elongation and hyponastic growth through a temperature-responsive transcription factor PIF4. Here we show that the evening-expressed clock component TOC1 interacts with and inactivates PIF4, thereby suppressing thermoresponsive growth in the evening. We find that the expression of PIF4 target genes show circadian rhythms of thermosensitivity, with minimum responsiveness in the evening when TOC1 level is high. Loss of function of TOC1 and its close homologue PRR5 restores thermosensitivity in the evening, whereas TOC1 overexpression causes thermo insensitivity, demonstrating that TOC1 mediates the evening-specific inhibition of thermoresponses. We further show that PIF4 is required for thermoadaptation mediated by moderately elevated temperature. Our results demonstrate that the interaction between TOC1 and PIF4 mediates the circadian gating of thermoresponsive growth, which may serve to increase fitness by matching thermoresponsiveness with the day-night cycles of fluctuating temperature and light conditions.

Figure 1. TOC1 directly interacts with PIF4.

(a,b) Box diagram of various fragments of PIF4 and TOC1 used in c,d. (c,d) Yeast two-hybrid assays. Yeast clones were grown on the synthetic dropout medium (+HIS) or synthetic dropout medium without histidine plus 1 mM 3-amino-1,2,4-Triazol (3-AT) (−HIS). The experiments were replicated with additional yeast clones (Supplementary Fig. 1). (e) Co-IP assays. Protein extracts from protoplasts expressing TOC1-Myc or TOC1-Myc and PIF4-GFP were immunoprecipitated with anti-GFP antibody and analysed by immunoblottings with anti-GFP or anti-Myc antibody. The molecular weight (kDa) is indicated on the right side of the gel. (f) Overlap between target genes of PIF4 and TOC1 identified in the previous ChIP-Seq studies is statistically significant (Fisher's exact test P<2 × 10⁻¹⁶). (g) Distribution of distances between the binding sites of PIF4 and TOC1 in their common target genes identified in f. (h) The most enriched motif in the TOC1-binding sites of the PIF4 and TOC1 common target genes. (i) Frequency of G-box motif per 1 kb in the PIF4-specific binding sites (PIF4 only) and TOC1-binding sites of the PIF4 and TOC1 common target genes (TOC1 & PIF4) or TOC1-specific target genes (TOC1 only).

Figure 2. TOC1 represses PIF4 ability to activate target gene transcription.

(a) Transient gene expression assays. IAA19p::Luc was co-transfected with PIF4-GFP, TOC1-GFP and 35S::renilla luciferase into Arabidopsis mesophyll protoplasts. Luciferase activity levels were normalized to Renilla luciferase activity. Error bars indicate s.d. (n=3). *P<0.05 (Student's t-test), numbers indicate fold induction by PIF4. (b) Western blotting with anti-Myc antibodies showing PIF4-Myc protein levels in PIF4-OX (PIF4-Myc) and PIF4-OX;TOC1-OX samples. Equal loading of samples is shown by Ponceau S staining. The molecular weight (kDa) is indicated on the right side of the gel. (c) ChIP-quantitative PCR assays of PIF4 binding to YUC8, IAA19 and IAA29 promoters. Five-day-old 35S::PIF4-MYC or 35S::PIF4-MYC;TOC1-OX seedlings were treated with 29 °C for 4 h and then used for ChIP assays using an anti-Myc antibody. The enrichment of DNA was normalized to that of the PP2A coding region. Error bars indicate s.d. (n=3); NS, not significant (Student's t-test P≥0.05). (d,e) Transient gene expression assays. The UAS-GUS reporter construct was co-transfected with GBD-PIF4 with or without TOC1, and 35S::Luc as an internal control. The GUS reporter activities were normalized to the luciferase activity and then to the empty vector control. GAL4 BS, GAL4-binding site; 35S MP, 35S minimal promoter; GBD, GAL4 DNA-binding domain. Error bars indicate s.d. (n=3). *P<0.05 (Student's t-test), numbers indicate fold induction (GBD-PIF4/GBD).

Figure 3. TOC1 suppresses thermomorphogenesis and warm-temperature activation of PIF4 target genes.

(a,b) Hypocotyl length of seedlings grown under the continuous white light at 20 °C for 4 days followed by the indicated time of warm temperature (29 °C) treatment (a) or 12 h-warm-temperature treatment (b) and then grown at 20 °C till hypocotyl measurement on the seventh day. Error bars in a–e indicate s.d. (n=10 plants). *P<0.05 (Student's t-test). Numbers in b indicate ratios of hypocotyl lengths (29 °C/20 °C). (c–e) Hypocotyl lengths of wild-type (WT), TOC1-OX, PRR5-OX and ztl-105 seedlings grown under the continuous white light at 20 °C for 7 days or 20 °C for 4 days followed by 29 °C for 3 days. *P<0.05 (Student's t-test); NS, not significant (P≥0.05); numbers in e indicate ratio of hypocotyl lengths (29 °C/20 °C). (f,g) qRT–PCR analysis of YUC8 (f) and PIF4 (g) expression in WT and TOC1-OX seedlings grown at 20 °C for 5 days then incubated at 20 °C or 29 °C for 4 h. Gene expression levels were normalized to PP2A and presented as values relative to that of wild type at 20 °C. Error bars in f–i indicate s.d. (n=3). *P<0.05 (Student's t-test); NS, not significant (P≥0.05); numbers in g indicate ratio of PIF4 expression levels (29 °C/20 °C). (h,i) qRT–PCR analysis of the expression levels of PIF4 and its target genes YUC8 and IAA29 in WT and PRR5-OX seedlings grown at 20 °C for 5 days then incubated at 20 °C or 29 °C for 4 h. *P<0.05 (Student's t-test); NS, not significant (P≥0.05); numbers in i indicate ratio of PIF4 expression levels (29 °C/20 °C). (j) TOC1 ChIP assays showing TOC1 binds to the promoters of PIF4 target genes. Five-day-old TOC1p::TOC1-YFP seedlings were used for ChIP assay. Enrichment of DNA was calculated as the ratio between TOC1p::TOC1-YFP and WT control, normalized to that of the PP2A coding region as an internal reference. Error bars indicate s.d. (n=3). *P<0.05 (Student's t-test). (k) Hypocotyl lengths of TOC1-OX;elf3-1 seedlings grown under continuous white light at 20 °C for 7 days or 20 °C for 4 days followed by 29 °C for 3 days. Error bars indicate s.d. (n=10 plants). *P<0.05 (Student's t-test); numbers indicate ratio of hypocotyl lengths (29 °C/20 °C). (l) qRT–PCR analysis of YUC8 expression in seedlings grown at 20 °C for 5 days then incubated at 20 °C or 29 °C for 4 h. *P<0.05 (Student's t-test); NS, not significant (P≥0.05); numbers indicate ratio of YUC8 expression (29 °C/20 °C).

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).

Figure 5. TOC1 and PRR5 mediate the circadian gating of thermomorphogenesis.

(a,b) Effects of warm temperature at different ZTs on the expression of PIF4 (a) and YUC8 (b). Seedlings were entrained in 12 h light and 12 h dark (12L:12D) cycles at 20 °C for 4 days and then transferred to continuous light. At different ZTs, the growth temperature was increased to 29 °C or kept at 20 °C for 4 h. Seedlings were then harvested at ZT4, ZT16 and ZT20 for RNA extraction. Gene expression levels quantified by qRT–PCR were normalized to PP2A and to that of wild type at ZT0 and 20 °C. Grey dots in b indicate the ratio of YUC8 expression levels between 29 °C and 20 °C samples. Error bars indicate s.d. (n=3). *P<0.05 (Student's t-test).

Figure 6. PIF4 is required for thermo-adaptation.

(a,b) PIF4 is required for thermo-adaptation. Seedlings of WT, pif4 and PIF4-OX genotypes were grown at 20 °C or 29 °C under the continuous light and then were subjected to a 45 °C heat shock for different time periods. After recovery under the constant light at 20 °C for 5 days, survival rate was measured for over 40 seedlings in each sample. The experiments were repeated for three times. Error bars indicate s.d. (n=3). *P<0.05 (Student's t-test). (c) The survival rate of WT, TOC-OX and toc1;prr5 seedlings grown at 20 °C or 29 °C, after 45 °C heat-shock treatment for the indicate time periods. Error bars indicate s.d. (n=3). *P<0.05 (Student's t-test). (d) A model of the circadian gating of thermomorphogenesis through the TOC1–PIF4 interaction. During the day, warm temperature activates PIF4, which in turn activates auxin biosynthesis genes including YUC8 and promotes hypocotyl growth. However, in the evening and at early night, TOC1 accumulates at high levels and directly inhibits PIF4, suppressing thermomorphogenesis.