Clock Temperature Compensation in Arabidopsis Figure 6. Effects of temperature and CK2 kinase function on CCA1 binding activity. (A) ChIP analysis of CCA1 binding to the promoters of TOC1, LUX, PRR7 and PRR9 in plants entrained under LD cycles at 22uC and transferred to 12uC or 27uC. Samples were collected after 35 h under LL. Input DNA was used as a control. Immunodetection of (B) CCA1-YFP and (C) CKB4-MYC protein accumulation in CCA1-YFP-ox/CKB4-MYC-ox plants grown under the same conditions described in (A). Similar protein transference in each lane was verified by staining with Red Ponceau. (D) Western- blot analysis of CCA1-YFP-ox/CKB4-MYC-ox protein extracts immunoprecipitated with anti-GFP antibody (a-GFP) with subsequent detection of phosphorylated CCA1 isoforms (CCA1-P)(a-PSer) at 12uC or 27uC. Luminescence of TOC1:LUC expression in WT and cca1-1/lhy-11 plants grown under LD cycles at 22uC and subsequently transferred to 27uC (E) or 12uC (F). Plots are means 6 SEM of 12 individual seedlings. The white and solid boxes correspond to the light and dark periods, respectively. The experiments were performed at least twice with similar results to those shown here. doi:10.1371/journal.pgen.1001201.g006 results indicate a temperature-dependent regulation of both CCA1 binding activity and the CK2 antagonistic function. The differences in binding cannot be attributable to changes in CCA1 and CKB4 mRNA (not shown) or protein accumulation (Figure 6B, 6C, and Figure S12) did not vary at different temperatures. In contrast, the CCA1 phosphorylated isoforms were considerably increased at 27uC, as revealed by the immunoprecipitation assays with anti-GFP antibody followed by detection with anti-P-Ser antibody (Figure 6D). To verify that the observed effects were not due to artefactual protein over- expression, we performed ChIP assays with a CCA1prom:CCA1- HA-YFP line, which expresses CCA1 under its own promoter [53]. This line displays a circadian period (25.660.6) slightly longer than WT plants (24.360.1) [53]. Using this line, we confirmed the CCA1 binding to its target loci and verified a regulatory role of temperature on this binding (Figure S12). Our conclusions were also in agreement with studies of TOC1:LUC-expressing double mutant cca1-1/lhy-11 plants [61]. The results showed that the luminescence signals damped high at 27uC (Figure 6E) indicating that TOC1 repression was clearly alleviated by the absence of CCA1 and LHY. However, this function was not so evident at 12uC, with cca1-1/lhy-11 plants displaying intermediate lumines- cence signals (Figure 6F). These results also suggest that at 12uC and in the absence of CCA1 and LHY, additional factors contribute to TOC1 repression. The previously postulated major role for CCA1 at low temperatures [40] might be reflecting the decreased regulatory function of CK2 at the lower range of temperatures. In consonance with this hypothesis, our studies showed that circadian gene expression was clearly affected in CCA1-ox and in cca1-11 mutant plants at high temperatures (Figure S13) and the DRB effects were more severe at 27uC than at 12uC (Figure S13). In agreement with these results, we observed more evident effects of DRB on CCA1 binding at high than at low temperatures (Figure S14). Altogether, our results suggest that the temperature-dependent balance between CCA1 binding activity and CK2 opposing function contributes to proper temperature compensation in Arabidopsis. CCA1 dephosphorylated isoforms are preferentially bound to the promoters of the oscillator genes Our results indicate that CK2 interferes with CCA1 function and suggest an inverse correlation between CK2 activity and CCA1 binding. We next performed Double ChIP assays at temperatures of maximal CCA1 binding (22uC and 27uC) in an attempt to estimate the phosphorylated CCA1 fraction that is associated with the promoters. We performed a double round of PLoS Genetics | www.plosgenetics.org 8 November 2010 | Volume 6 | Issue 11 | e1001201