Clock Temperature Compensation in Arabidopsis Figure 2. Molecular interaction of CCA1 and CKB4. (A) Western-blot analysis of protein extracts immunoprecipitated with anti-GFP antibody (a-GFP) and subsequent detection of CKB4-MYC (a-MYC), CCA1-YFP (a-GFP) or phosphorylated CCA1 isoforms (CCA1-P)(a-PSer). Lower panels show the immunoblot analysis of two-dimensional gels detecting CCA1-YFP with a-GFP. In all cases, plants were grown under LD conditions and samples were collected at ZT3. All the experiments were performed at least twice with similar results to those shown here. (B) Confocal microscopy analysis of seedlings over-expressing CCA1 fused to the N-terminal fragment of YFP and CKB4 fused to the C-terminal fragment (top panel). Images of CCA1 (middle panel) and CKB4 (lower panel) fused to full-length YFP are also shown. Scale bar 0.2 mm. doi:10.1371/journal.pgen.1001201.g002 to the localization observed in plants expressing the proteins fused to the full-length YFP. In both cases, the localization was mostly nuclear, in speckles and homogeneous distribution throughout the nucleoplasm for CKB4 (Figure 2B, CKB4-YFP) and mostly in nuclear speckles for CCA1 (Figure 2B, CCA1-YFP). Similar fluorescent signals were observed in CCA1-ox plants over- expressing CKB4 fused to full-length YFP or in CKB4-MYC-ox plants over-expressing CCA1 fused to full-length YFP (Figure S4). In contrast, no evident fluorescence could be detected when BiFC experiments were performed with plants over-expressing both TOC1 (TOC1-nYFP-ox) and CKB4-cYFP-ox (Figure S4). CK2 activity antagonizes CCA1 regulatory function without affecting CCA1 protein accumulation As phosphorylation targets many clock proteins for degradation [55], we compared by Western-blot analysis, CCA1 protein abundance in CCA1-YFP-ox and CCA1-YFP-ox/CKB4-MYC- ox plants over a diurnal cycle. Our results showed a very similar pattern of CCA1 accumulation in both genotypes at every time point examined (Figure 3A–3C). Furthermore, analysis of CKB4 and CCA1 protein abundance in different CCA1-YFP-ox lines transformed with the CKB4-MYC-ox construct showed a lack of correlation between increasing concentrations of CKB4 and decreasing amounts of CCA1 (Figure 3D). These results strongly suggest that over-expression of CKB4 does not modulate CCA1 protein accumulation. Our previous studies have shown that CKB4-MYC-ox plants exhibit increased CK2 activity that correlates with the circadian phenotypes observed in CKB4- MYC-ox plants [28]. Next we explored whether decreasing CK2 activity could alter the CCA1 protein accumulation or CCA1 repressive function. As a long-term and constant depletion of CK2 activity is lethal for plants [56], the CCA1 protein abundance was analyzed in the presence or in the absence of specific CK2 inhibitors such as DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) or DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimid- azole) [57]. In a complementary approach, CCA1 protein abundance was examined in plants expressing a dominant negative CK2 alpha subunit (CKA3-) under the control of the Dexameth- asone (Dex) inducible promoter [56]. Our studies showed that treatment with the inhibitors (Figure 3E) or the induction of CKA3- by Dex (Figure 3F) did not significantly alter CCA1 protein accumulation as compared with non-treated plants. In clear contrast, TOC1:LUC expression was markedly affected by the decreased pattern of CK2 activity. Indeed, treatment with DMAT or with DRB progressively reduced the amplitude and lengthened the period of TOC1:LUC expression in a dose-dependent manner (Figure 3G and Figure S5). Similar effects were observed after treatment of CKA3- plants with Dex (Figure 3H) while TOC1:LUC expression was not affected when WT plants were treated with Dex (not shown). This is noteworthy, as decreasing CK2 activity leads to the opposite phenotypes of those observed in CKB4-MYC-ox plants. Therefore, CK2 activity is important in controlling the circadian waveform of TOC1:LUC expression. As CKB4-MYC-ox reduces the severity of CCA1 repressive function on TOC1 expression, we reasoned that decreasing CK2 activity should have the opposite effects. To explore this hypothesis, we examined TOC1:LUC expression in CCA1-YFP-ox/CKA3- plants before and after treatment with Dex. Our studies showed a very reduced amplitude of TOC1:LUC expression in CCA1-YFP-ox/CKA3- plants treated with Dex (Figure 3H). Therefore, and consistently with our hypothesis, the repressive function of CCA1 appears to be enhanced by decreasing CK2 activity. Together, these results show that CK2 and CCA1 have opposing functions in the regulation of TOC1 expression and suggest a possible role for CK2 antagonizing CCA1 regulatory activity. CK2 antagonizes CCA1 binding to the promoters of its target genes As CK2 activity reduces the severity of CCA1-YFP-ox phenotypes but this effect is not due to increased degradation of CCA1, we reasoned that CK2 might modulate CCA1 transcrip- tional regulatory function. To explore this possibility, we examined PLoS Genetics | www.plosgenetics.org 4 November 2010 | Volume 6 | Issue 11 | e1001201