Clock Temperature Compensation in Arabidopsis Figure 4. CK2 antagonizes CCA1 binding to the promoters of its target genes. ChIP analysis of CCA1 binding to the promoters of the morning-expressed genes PRR7 and PRR9 (A, C and E) or the evening-expressed genes TOC1 and LUX (B, D and F). Analysis was performed in CCA1- YFP-ox and CCA1-YFP-ox/CKB4-MYC-ox plants (A and B) or in CCA1-YFP-ox plants collected after treatment for 48 h with 150 mM DRB (+DRB) (C and D). As a control, samples were similarly processed in the absence of DRB (-DRB). (E and F) ChIP analysis of CCA1-YFP-ox/CKA3- plants collected after induction with 1 mM Dex for 48 h (+Dex). As a control, samples were similarly processed but in the absence of Dex (-Dex). Input DNA was used as a control. Plants were synchronized under LD cycles and samples were collected at ZT3. In all cases, the experiments were performed at least three times with similar results to those shown here. doi:10.1371/journal.pgen.1001201.g004 circadian period (Figure S7). However, the effects of DRB were considerably reduced in cca1-11 mutant plants as compared to cca1-11 untreated plants (Figure S7). A similar trend was observed for TOC1:LUC expression in cca1-1/lhy-11 mutant plants [61], in which the up-regulation of TOC1 by the absence of the CCA1 and LHY repressors was not importantly affected by DRB treatment (Figure S7). The alteration of TOC1:LUC expression in cca1-1/lhy- 11 mutant plants was more severe than the circadian phenotypes previously described [61]. This might be due to different growth conditions, different intensities of light different regimes of entrainment and/or the different reporters. Together, our results indicate that the circadian function of CK2 is mostly mediated by CCA1/LHY, which reinforces the link between these components. Our results also indicate that CK2 activity antagonizes CCA1 regulation of circadian gene expression by interfering with the binding of CCA1 to the promoters of the oscillator genes. A phosphorylation-dependent inactivation of transcriptional activity was previously reported to be important in other circadian systems [62]. Proper regulation of CK2 activity is important for temperature compensation in Arabidopsis Our results show that CK2 activity regulates TOC1 expression. As TOC1 is also modulated by temperature [40], we next explored whether CK2 regulatory functions were affected by temperature. To that end, we compared the waveforms of TOC1:LUC expression in WT, CKB4-MYC-ox and CCA1-YFP-ox/CKB4- MYC-ox plants synchronized for 7 days under LD cycles at 22uC and then transferred to 12uC, 22uC or 27uC. Our results showed that over-expression of CKB4 resulted in higher amplitude and slight advanced phase of TOC1 promoter activity at 27uC (Figure S8), a phenotype slightly more severe but following the same trend than that observed at 22uC (Figure S8). Analysis of TOC1:LUC expression at 12uC revealed that CKB4-MYC-ox plants exhibited a lower amplitude and a slightly advanced phase compared to WT (Figure S8). The differential changes in gene expression by temperature could be fully appreciated in Figure S9 showing point-by-point comparisons of TOC1:LUC expression in WT and CKB4-ox at 27uC and 12uC. These results indicate that CK2 function regulating TOC1 expression is differentially modulated by temperature. This notion was in agreement with our pharmaco- logical studies using the CK2 inhibitor DRB. Treatment with DRB at 27uC resulted in a delayed phase and lower amplitude of TOC1:LUC expression whereas at 12uC, only subtle phenotypes of TOC1:LUC phase or amplitude were observed (Figure S10). Thus, decreasing CK2 activity has stronger effects at high temperatures. If CK2 regulation of TOC1 expression is differentially modulated by temperature, then the CK2 activity counteracting CCA1 PLoS Genetics | www.plosgenetics.org 6 November 2010 | Volume 6 | Issue 11 | e1001201