in CKB4-MYC-ox and CCA1-YFP-ox/CKB4-MYC-ox plants. This inhibitory function is not exclusive of the plant circadian system as a phosphorylation-dependent inactivation of transcriptional function was previously reported in other circadian systems [62]. To be effective as a key mechanism of clock progression, the interplay between CK2 and CCA1 should be in turn precisely regulated. Indeed, we found that different temperatures affect CK2 regulatory function. The higher amplitude of TOC1:LUC expression in CKB4-MYC-ox plants was mostly observed at 22uC and 27uC while the activating function of CK2 on TOC1 expression was not so clearly observed at 12uC. The lower amplitude of TOC1:LUC expression in CKB4-MYC-ox plants at 12uC is difficult to explain, but denotes that a different mechanism is engaged at low temperatures and emphasizes the complexity of the temperature response within the Arabidopsis circadian network. This notion is reinforced by the results showing that CK2 also phosphorylates LHY [26]. Thus, it is possible that the previously described role of LHY-GI in clock temperature compensation [40] might be also regulated by CK2. Notably, CCA1 binding activity appeared to be also modulated by temperature in CCA1-ox plants. Despite the lower binding at 12uC, CCA1 is still able to repress TOC1 expression at 12uC as evidenced by our luminescence assays. Furthermore, CK2 activity might also have a residual function as it was able to interfere with CCA1 binding. Therefore, there is a parallelism between the temperature regulation of CCA1 binding and CK2 activity. We proposed that the dynamic regulation of these activities by temperature is important for clock function, as altering the functional expression by over-expression, mutation or pharma- cological inhibition deregulates the temperature-dependent modu- lation of gene expression. Unlike the temperature dependency of most biological and biochemical activities, the circadian clock sustains period length over a range of constantly maintained temperatures [66]. When we examined clock outputs, we found that the temperature compensation profiles were altered in CKB4-MYC-ox plants compared to those observed in WT. A simple explanation would be that CK2 activity might be compromised only when CKB4 abundance is increased above physiological limits. However, decreased CK2 activity by pharmacological inhibitors also led to altered temperature compensation. Additionally, CKB4 protein abundance did not vary between 12uC and 27uC. Together, these findings indicate that CK2 contributes to proper temperature responses in Arabidopsis: increased CK2 activity led to under- compensation whereas decreased activity resulted in a slightly over-compensated clock. This is noteworthy because previous studies in Neurospora have also implicated CK2 in temperature compensation [30]. Therefore, CK2 integrates a molecular clock component with a regulatory function that is conserved among different circadian systems. However, the specific mechanisms of CK2 function differ among organisms. In Neurospora, the stability of FRQ is controlled, at least in part, by a CK2-dependent phosphorylating mechanism, which facilitates the FRQ protein degradation preferentially at high temperatures [30]. In Arabi- dopsis, CK2 does not affect CCA1 protein accumulation but rather its transcriptional activity. Our results suggest that a precise temperature regulation of CK2 and CCA1 activity shapes the temperature compensation profile. Altering this delicate balance by over-expression, mutation or pharmacological inhibition affects the period compensation. In addition to the CK2-CCA1 counterbalancing loop, other loops and/or different mechanism should also account for precise regulation of temperature compensation. In fact, prior studies have proposed that LHY and GI contribute to temperature compensation in Arabidopsis [40]. Our findings focusing on CCA1 binding and CK2 activities Clock Temperature Compensation in Arabidopsis add exciting insights into the mechanisms of temperature compensation in Arabidopsis. It would be also interesting to perform similar studies with the other three CK2 regulatory subunits in order to highlight functional similarities or divergences in the control of temperature compensation. Our findings are also in line with the notion that evolution might enhance fitness under different climate conditions without directly affecting the expres- sion of central oscillator components but rather modulating their activity. Circadian clock adaptation to different environments might thus rely on key regulatory factors that modulate the oscillator activity. Further studies using different Arabidopsis ecotypes can provide new insights into how this regulation might have shaped plant adaptation to different climate areas around the globe. The parallelism in temperature dependency of CCA1 and CK2 activities and the inhibitory effect of CK2 on CCA1 function is in agreement with a previously described model for temperature compensation [33]. The model proposes that the temperature independence of circadian period by the clock occurs through the balance between two biochemical activities, each of which has a similar temperature dependency [33]. Our studies would be also in consonance with the notion that temperature compensation is not only determined by central clock components but also by other elements that function in trans to regulate the core proteins [40]. A recent computer modeling study has proposed that a switch-like mechanism might regulate period sensitivity through the control of two parameters that are a function of processes such as phosphorylation, ubiquitination or complex formation [67]. It would be interesting to identify a role of these regulatory processes as key modules shaping the temperature compensation profiles. Specifically for phosphorylation, studies in Neurospora [30] and mammals [68] indicate that in contrast to CK2, the activities of other kinases and clock-related phosphorylation events are temperature-insensitive. Our study showing that the CK2 activity antagonizes CCA1 function in a temperature-dependent manner highlights only one of the many aspects contributing to temperature compensation. Further analysis focusing on addition- al components and mechanisms would aid in our understanding of the intricate interacting networks responsible for temperature compensation in Arabidopsis and in other organisms. Materials and Methods Plant material, luminescence assays, flowering time, and hypocotyl length analysis Arabidopsis thaliana seedlings were stratified at 4uC in the dark for 3 days on Murashige and Skoog agar medium supplemented with 3% sucrose and then transferred to light:dark conditions (LD, 12 h light:12 h hours dark) with 60 mmol m22s21 of cool white fluorescent light at 22uC. The list with the different plants and constructs used in this study is shown in Table S1 and S2. For CCA1-ox/CKB4-ox studies, lines 9 and 14 were used (see Figure S1 and Figure S2). For cca1/lhy/CKB4-ox studies, lines 4 and 5 were used (see Figure 3D and Figure S1). For Luminescence analyses were performed as previously described [28]. In experiments with CK2 inhibitors, one-week old plants were transferred to 96-well plates containing MS medium supplemented with the specified concentration of DRB or DMAT. Luminescence was recorded 24 hours after the seedlings were transferred to the plates. For Dexamethasone induction, 1 mM was added to each well at Zeitgeber Time 2 (ZT 2). For flowering time analysis, seeds were stratified in the dark at 4uC for 3 days on soil. Seedlings were grown under Short-Day (ShD, 8 h light:16 h dark) or Long-Day (16 h light:8 h dark) conditions with 60 mmol m22s21 of cool white fluorescent light at 22uC. Flowering time was scored by PLoS Genetics | www.plosgenetics.org 10 November 2010 | Volume 6 | Issue 11 | e1001201