Author Summary Most organisms are able to rhythmically coordinate their physiology and metabolism in consonance with the day- night cycle. The cellular mechanism responsible for generating the biological rhythms is known as circadian clock. In contrast to many other biochemical reactions, the pace of the clock does not change with temperature (i.e. the clock does not run faster at high temperatures). This is a property known as clock temperature compensation, and despite its importance for circadian function, we know few details about the mechanisms responsible for the temperature-dependent regulation of circadian period length. Plants, as sessile organisms, are particularly sensible to maintaining the clock running at the proper pace, regardless the external temperature. In our study, we have identified that the activity of two key plant clock components (CK2 and CCA1) is regulated by temperature. Notably, these temperature-dependent activities are an- tagonistic, and they counterbalance each other at increasing temperatures so that the clock does not run faster at these high temperatures. When we alter the expression and activity of CK2 and CCA1, we observe that the clock is not longer able to maintain the proper pace, thus demonstrating that the balance between these two activities is essential for clock temperature compensation in plants. Despite all these advances, little is known about the actual mechanism of CK2 function within the Arabidopsis circadian clock. In contrast, a recent study on the Neurospora crassa circadian system has importantly advanced our knowledge of CK2 role controlling a defining property of circadian function [30]. Indeed, to be effective as a timing mechanism, the circadian system must be relatively independent of temperature changes in order to avoid running faster at higher temperatures and slower at lower temperatures [31]. This property was proposed to rely on a compensating mechanism, which would allow the clock to buffer its period length against changes in temperature [32]. The recent study by Mehra et al., assigns a role for CK2 in the mechanism underlying temperature compensation in Neurospora. The mechanism seems to involve CK2-mediated phosphorylation at specific sites of the period- controlling clock protein FREQUENCY (FRQ). The CK2- mediated phosphorylation of FRQ targets the protein for degradation preferentially at high temperatures, thereby contribut- ing to temperature compensation in Neurospora [30]. Experimental data and computer modeling studies have suggested different mechanisms contributing to temperature compensation. One of the mechanisms include the counterbalance of opposing biochemical functions that have similar temperature coefficients [33]. Consistently, the contrasting effects of inter- and intra-molecular interactions of PER were proposed as a basic mechanism underlying temperature compensation in Drosophila [34]. The key role of Drosophila PER was further reinforced by natural variation studies showing that per polymorphisms might help to fine-tune the circadian clock to different thermal environments [35]. A more recent study has also shown that the interaction between the Drosophila circadian photoreceptor CRYPTO- CHROME (CRY) and the protein complex composed of PER and TIM are also critical for temperature compensation in Drosophila [36]. In other model organisms, the mechanisms by which the circadian clock compensates its period length over a range of temperatures are less well known. In the cyanobacterium Synechococcus elongatus, the ATPase activity of the essential clock Clock Temperature Compensation in Arabidopsis component KaiC was found to be temperature compensated. The authors suggested that temperature compensation in Synechococ- cus could be driven by this biochemical reaction [37]. In plants, the temperature compensation of leaf movement rhythms was uncov- ered by a quantitative genetic approach [38]. The identified genes included the flowering time regulator FLOWERING LOCUS C [39] and the flowering and clock-related gene GIGANTEA (GI) [40]. GI was proposed to play a role extending the temperature range over which rhythms can be maintained [40]. By using mutant plants and analyzing the expression of the central clock genes CCA1 [41], and LHY [42] and TIMING OF CAB EXPRESSION 1 (TOC1 or PRR1) [43,44], the authors also concluded that a balance between GI and the core component LHY was important at high temperatures, while CCA1 would preferentially function at low temperatures [40]. Although quantitative genetic studies have been a successful approach for the identification of possible elements contributing to temperature compensation, the mechanisms responsible for these clock responses remain to be discovered. Here, we provide evidence for a role of CK2 modulating temperature responses in Arabidopsis. This function is achieved by antagonizing the clock component CCA1. CK2 does not alter protein accumulation or subcellular localization, as CK2 does in other circadian systems, but affects the transcriptional activity of CCA1. Over-expression, mutation or pharmacological inhibition reveal that both CK2 regulatory function and CCA1 binding are more effective at high temperatures, which provide a mechanism by which plants buffer clock period length against temperature changes. Results Genetic interaction of CCA1 and CKB4 in the control of flowering time, hypocotyl elongation, and circadian gene expression The circadian clock is responsible for the integration of temporal and photic information that regulates hypocotyl elongation [45,46] and flowering time in Arabidopsis [47–49]. Previous studies have shown similar circadian phenotypes of cca1 mutant and CKB4 over-expressing (CKB4-MYC-ox) plants [29]. We therefore explored the inverse correlation between phenotypes and expression by conducting a genetic study in which CCA1 over-expressing (CCA1-YFP-ox) and cca1-1/lhyRNAi mutant plants [50] were transformed with the CKB4-MYC-ox construct [29]. We used homozygous, single insertion lines (Figure S1) to examine hypocotyl elongation and flowering time as an indication of clock function on these developmental outputs. Analysis of CCA1-YFP-ox/CKB4-MYC-ox plants revealed that over-expres- sion of CKB4 reduced the delayed flowering phenotype of CCA1- YFP-ox plants such that flowering in CCA1-YFP-ox/CKB4- MYC-ox plants occurred at almost the same time than in Wild- Type (WT) plants under both Long-Days (LgD,16 h light:8 h dark) (Figure 1A) or Short-Days (ShD, 8 h light:16 h dark) (Figure 1B). In contrast, the early flowering phenotype of cca1-1/ lhyRNAi mutant plants was not significantly affected by over- expression of CKB4 (Figure S2), which indicates a non-additive interaction and suggests that the effect of CKB4-MYC-ox might require the functional expression of CCA1 and LHY. When hypocotyl elongation was examined under light:dark (LD) cycles, we found that over-expression of CKB4 significantly reduced the long hypocotyl phenotype of CCA1-YFP-ox plants under both LgD (p-value,0.0001) or ShD (p-value,0.0001)(Figure 1C). A similar trend was observed at different fluence rates of constant white light (Figure 1D). Together, these results indicate that the severity of the flowering and hypocotyl phenotypes of CCA1-YFP- ox plants is considerably reduced by over-expression of CKB4. PLoS Genetics | www.plosgenetics.org 2 November 2010 | Volume 6 | Issue 11 | e1001201