Clock Temperature Compensation in Arabidopsis Figure 1. Genetic interaction of CCA1 and CKB4. Flowering time of plants grown under LgD (16 h light:8 h dark) (A) or ShD (8 h light:16 h dark) (B). The number of leaves at flowering and the number of days to flowering are presented. Data is shown as means 6 SEM of three independent experiments. (C) Hypocotyl lengths of seedlings grown under LgD or ShD. Data is shown as means 6 SEM of 15–20 seedlings. (D) Hypocotyl lengths of seedlings under different intensities of white light (LL). Data is shown as means 6 SEM of 15–20 seedlings. All the experiments were performed at least twice with similar results to those shown here. doi:10.1371/journal.pgen.1001201.g001 As CCA1 regulates the expression of the evening-expressed clock gene TOC1 [51,52], we next explored whether over- expression of CKB4 altered the repressive function of CCA1. Luminescence from WT, CCA1-YFP-ox, CKB4-MYC-ox and CCA1-YFP-ox/CKB4-MYC-ox plants expressing the TOC1 promoter fused to the luciferase (TOC1:LUC) was examined under 12 h light/12 h dark (LD) conditions. As expected, TOC1:LUC expression in CCA1-YFP-ox plants exhibited a very reduced amplitude compared to WT plants (Figure S3). In contrast, higher amplitude and slightly advanced phase of TOC1 promoter activity was observed in CKB4-MYC-ox plants (Figure S3). Noticeably, the double over-expressing CKB4 and CCA1 plants displayed circadian waveforms very similar to those observed in WT plants (Figure S3). Consistent with the studies of hypocotyl length and flowering time, these results suggest that the TOC1:LUC repression by CCA1-YFP-ox is reduced by over-expression of CKB4. These results are also in agreement with the notion that CKB4 interferes with CCA1 function within the circadian clock. Molecular interaction of CCA1 and CKB4 in the nucleus Previous studies have shown the interaction of CCA1 with CKB1, CKB2 and CKB3, three members of the CK2 regulatory subunit family with high sequence homology to CKB4 [26]. To examine the possible physical association of CCA1 with CKB4, we performed in vivo co-immunoprecipitation assays using the CCA1- YFP-ox/CKB4-MYC-ox plants. Immunoprecipitation of CCA1 with the anti-GFP antibody (a-GFP) and subsequent detection with the anti-MYC antibody (a-MYC) revealed a band with a relative molecular mass of about 40 kDa, coincident with the expected size of CKB4-MYC (Figure 2A, a-MYC) indicating that CCA1 and CKB4 can be found in the same protein complex in Arabidopsis plants. The absence of signal in single CCA1-YFP-ox extracts (Figure 2A, a-MYC) and single CKB4-MYC-ox (not shown) revealed the specificity of the interaction. Detection using the GFP antibody showed a protein band with a molecular mass of 110 kDa that coincided with the predicted size of the CCA1:YFP fusion protein and confirmed the immunoprecipitation of CCA1 (Figure 2A, a-GFP). The interaction was also examined in plants expressing CCA1 under its own promoter (CCA1pro:CCA1- MYC/cca1-1) [53]. The results confirmed that CCA1 and CK2 regulatory subunits are present in the same protein complex (Figure S4) as revealed after immunoprecipitation with an antibody to the human regulatory subunit CK2B (a-CKB) followed by detection with the a-MYC antibody. The a-CKB antibody efficiently recognizes the Arabidopsis CK2 regulatory subunits (Figure S4). In agreement with previous studies showing that CK2 phosphorylates CCA1 [26], our immunoprecipitation assays with anti-GFP and subsequent detection with an antibody that specifically recognizes phosphorylated serine residues (a-PSer) revealed that the pattern of CCA1 phosphorylation was increased in CCA1-YFP-ox/CKB4-MYC-ox plants as compared with single CCA1-YFP-ox plants (Figure 2A, a-PSer). The data was reinforced by two-dimensional protein gel analysis followed by immunoblotting with the GFP antibody (Figure 2A, lower panels). The results showed that over-expression of CKB4 enriched CCA1 spots with a lower isoelectric point, which most likely correspond to CCA1 phosphorylated isoforms. Together, these results are consistent with previous published studies and suggest that CK2 phosphorylates CCA1 most likely by direct interaction with the CK2 regulatory subunits. We next examined the in vivo subcellular localization of CCA1 and CKB4 interaction by exploiting Bimolecular Fluorescent Complementation (BiFC) assays [54]. We analyzed plants over- expressing both CKB4 fused to the C-terminal fragment of the Yellow Fluorescent Protein (YFP) and CCA1 fused to the YFP N- terminal fragment. Confocal microscopy analysis revealed fluo- rescent signals accumulating in the nucleus (Figure 2B, CCA1- nYFP-ox, CKB4-cYFP-ox). As fluorescence would be only visualized if the two fragments of YFP are in such a close proximity that the fluorophore is reconstituted, our results reflect the in vivo interaction of CCA1 and CKB4 in the nucleus. The subcellular localization of CCA1 and CKB4 interaction is similar PLoS Genetics | www.plosgenetics.org 3 November 2010 | Volume 6 | Issue 11 | e1001201