repressive function should become also most apparent at the higher range of temperatures. Indeed, our analysis with the double CCA1-YFP-ox/CKB4-MYC-ox plants revealed very similar waveforms of TOC1:LUC expression in WT and CCA1-YFP-ox/ CKB4-MYC-ox plants at both 27uC and 22uC (Figure S8) suggesting that over-expression of CKB4 efficiently reverted the repressive function of CCA1 at these temperatures. However, at 12uC, the amplitude of TOC1:LUC expression in CCA1-YFP-ox/ CKB4-MYC-ox plants was lower than in WT plants (Figure S8) suggesting that CK2 activity interfering with CCA1 repressive function is less effective or that additional mechanisms are engaged to regulate TOC1 expression at low temperatures. We also observed an earlier declining phase, particularly evident at 27uC. The mechanism behind this phenotype might rely on the direct/ indirect regulation of TOC1 declining phase by CK2/CCA1 and/ or other clock components. In view of our findings and based on previous studies assigning a major role for CK2 in temperature compensation within the Neurospora circadian system [30], we next examined the possible connection between temperature compensation and CK2 activity in Arabidopsis. As the direct effects of temperature on TOC1 expression might mask real clock period compensation, we monitored overall clock output rates by using the morning- expressed reporter CAB2:LUC and the evening-expressed reporter CCR2:LUC (COLD-CIRCADIAN RHYTHM–RNA BINDING2). We determined the free-running period (FRP) in plants entrained at 22uC and then transferred to constant light at 12uC, 17uC, 22uC or 27uC. As expected, our results showed that circadian clock function in WT plants was temperature compensated with similar period length at the different temperatures (Figure 5A, 5B, 5D, 5E, and Figure S11). In CKB4-MYC-ox plants, the FRP was shorter than that of WT plants at all temperatures examined. However, the period shortening for CAB2:LUC (Figure 5A) and CCR2:LUC (Figure 5D) became more severe at the higher end of the temperature range. The temperature dependency of period shortening was also evident when the inverse of the period (as an indication of the oscillator rate) was plotted against temperature (Figure 5B and 5E). The linear regression analysis showed that the Clock Temperature Compensation in Arabidopsis slope was significantly deviated from zero (p-values,0.0001) in CKB4-MYC-ox plants but not in WT (Figure 5B and 5E). These results were in agreement with our studies using CK2 inhibitors in which the increased FRP could be correlated with increasing amounts of the inhibitor (Figure 5C and 5F). Noticeably, period lengthening was more severe at high temperatures (Figure 5C and 5F and Figure S11). Therefore, manipulation of CK2 activity by over-expression of CKB4 or by pharmacological inhibition modulates the temperature compensation profiles. Increased CK2 activity leads to under-compensated clock function (i.e. the clock runs faster at high temperatures) whereas decreased CK2 activity results in a slightly over-compensation (i.e. the clock runs slower at high temperatures). The temperature dependent function of CK2 is manifested by the different phenotypes of clock outputs at various temperatures such that period compensation is not properly achieved when CK2 activity is mis-regulated. Temperature modulates both CK2 and CCA1 regulatory activities In a last part of our study, we were interested in determining the mechanism underlying the temperature-dependent CK2 function in the Arabidopsis circadian clock. Our results indicated that CK2 interferes with the binding of CCA1 to the promoters of its target genes. Therefore, we next explored whether the temperature- dependent phenotypes of CK2 could be mechanistically linked with altered CCA1 activity. We examined by ChIP assays the CCA1 binding activity in CCA1-YFP-ox and CCA1-YFP-ox/ CKB4-MYC-ox plants entrained at 22uC and then transferred to 12uC or 27uC. Q-PCR analysis of CCA1-YFP-ox plants revealed significantly increased amplification at 27uC compared to 12uC (Figure 6A and Figure S12) suggesting that CCA1 binding is regulated by temperature. Previous studies have shown that the temperature dependence of the heat capacity is a thermodynamic property of the majority of sequence-specific DNA-protein interactions [63]. Compared to CCA1-YFP-ox, a much reduced amplification was observed when CCA1-YFP-ox/CKB4-MYC-ox plants were examined (Figure 6A), with reduced differences in CCA1 binding between 12uC and 27uC (Figure S12). These Figure 5. A link between CK2 and temperature compensation in Arabidopsis. Free-running periods of CAB2:LUC (A) and CCR2:LUC (D) in WT and CKB4-MYC-ox plants entrained under LD cycles at 22uC and subsequently transferred to LL conditions at 12uC, 17uC, 22uC or 27uC. Data are represented as means 6 SEM of the period estimated of approximately 12 plants (from two independent experiments). The inverse of the period (oscillator rate) from CAB2:LUC (B) and CCR2:LUC (E) was used to extrapolate the linear regression of the temperature response. Free-running periods for CAB2:LUC (C) and CCR2:LUC (F) of WT plants entrained under LD cycles at 22uC and subsequently transferred to LL conditions at 17uC, 22uC and 27uC. Plants were treated with 0, 75, 150, 200 mM DRB. Data are the means 6 SEM of the period estimated of approximately 12 plants. doi:10.1371/journal.pgen.1001201.g005 PLoS Genetics | www.plosgenetics.org 7 November 2010 | Volume 6 | Issue 11 | e1001201