software (Version 1.5.0.39, Roche). Melting peak analysis using the LightCycler 480 Basic software module (Roche) and gel electrophoresis confirmed that primer-dimers or other non-specific products were not present. Crossing point (Cp) calculation was used for quantification using the Absolute Quantification analysis by the 2nd Derivative Maximun method (LightCycler 480 Basic software module, Roche). ChIP values for each set of primers were normalized to Input values. Primers were designed using the PrimerExpress 2.0 software (Applied Biosystems) with lengths of 18-25 nucleotides, PCR amplicon lengths of 80 to 180 bp, 40-60% G:C content and melting point of 58-62uC. The list of primers used for promoter (prom) amplification and for Q-PCR analysis is shown in Table S3. In experiments with CK2 inhibitors or with plants expressing the CK2 inducible mutant, 48 hours before sampling, plants were transferred to medium supplemented with 150 mM of DRB, 150 mM DMAT or 1 mM Dex. Double-ChIP assays [70] were performed following the ChIP procedure for chromatin extraction and immunoprecipitation followed by one wash with low salt buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS and 2 mM EDTA) and two washes with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Immunocomplexes were eluted using 10 mM DTT and incubat- ing at 37uC for 30 min. Chromatin was diluted in 40 volumes of Double-ChIP buffer (15 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton-X-100, 1 mM EDTA, 1 mM PMSF, 5 mg/ml Leupeptin, 1 mg/ml Aprotinin, 5 mg/ml Antipain, 1 mg/ml Pepstatin, 5 mg/ml Chymostatin and 50 mm MG132) and incubated overnight at 4uC with Protein G–Sepharose beads (Amersham Biosciences) conjugated with Anti-PSer or Anti-GFP antibody. Immunocomplexes were washed, eluted, purified and amplified as described for the ChIP protocol. Statistical analysis Statistical analyses were performed using the GraphPad Prism software (GraphPad Software, Inc). For hypocotyl length and flowering time experiments, two-tailed t-tests with 99% of confidence were performed. For multiple comparisons, two-way ANOVA followed by Bonferroni post-tests were performed; photoperiod and genotype were considered as variables. For ChIP quantifications, two-way ANOVA tests followed by Bonferroni post-tests were used. Gene and genotype or temperature and genotype were considered as variables. GraphPad Prism software was also used to extrapolate the linear regression of the temperature response. Profiles for bioluminescence experiments, the estimated period length was determined by Fast Fourier Transform/Nonlinear Least Squares method (FFT/NLLS) [71] using a window of 96 hr of data excluding the first 24 hr to avoid any transient effects after transferring to constant conditions. Supporting Information Figure S1 CKB4 and CCA1 expression in different CCA1-ox/ CKB4-ox and cca1/lhy/CKB4-ox lines. (A) RT-PCR analysis of CKB4, CCA1 and ACTIN2 (ACT2) expression in WT and double CCA1 and CKB4 over-expressing plants. Lines 14 and 9 were used for subsequent studies. (B) RT-PCR analysis of CKB4 and ACTIN2 (ACT2) expression in WT and in cca1-1/lhyRNAi/CKB4- ox plants. Lines 5 and 4 were used for subsequent studies. Seedlings were entrained under LD cycles and samples were collected at Zeitgeber Time 2 (ZT2). (C) Immunodetection of CKB4 protein accumulation in cca1-1/lhyRNAi/CKB4-ox plants. CKB4 protein was detected using the a-MYC antibody. Seedlings were entrained under LD cycles and samples were collected at ZT2. Clock Temperature Compensation in Arabidopsis Found at: doi:10.1371/journal.pgen.1001201.s001 (0.21 MB PDF) Figure S2 Analysis of CCA1 and CKB4 genetic interaction. Flowering time of WT, cca1-1/lhyRNAi, cca1-1/lhyRNAi/CKB4- MYC-ox and CKB4-MYC-ox plants grown under (A, B) Long- Day (LgD, 16 h light:8 h dark) or (C, D) Short-Day (ShD, 8 h light:16 h dark) conditions. Flowering time was measured as the number of leaves at flowering or the number of days to flowering (1-cm-high bolt). Data are shown as means 6 SEM of three independent experiments. Similar results were obtained when flowering time was examined in cca1-1/lhy-11 and cca1-1/lhy-11/ CKB4-MYC-ox plants. Found at: doi:10.1371/journal.pgen.1001201.s002 (0.12 MB PDF) Figure S3 Analysis of TOC1:LUC diurnal expression in different genetic backgrounds. TOC1:LUC luminescence in seedlings maintained under LD (12 h light:12 h dark) cycles. Plots represent means 6 SEM of at least 12 individual seedlings. The white and solid boxes correspond to the light and dark periods, respectively. The experiment was performed three times with similar results to those shown here. Found at: doi:10.1371/journal.pgen.1001201.s003 (0.09 MB PDF) Figure S4 Analysis of CCA1 and CKB4 molecular interaction. (A) Western-blot analysis of WT, CCA1-ox and CCA1- prom:CCA1-MYC/cca1-1 plants using an antibody to the human CK2B subunit (a-CKB). The antibody efficiently recognizes the CK2 regulatory subunits of Arabidopsis. (B) Western-blot analysis of Co-IP experiments with plants expressing CCA1 under its own promoter (CCA1prom:CCA1-MYC/cca1-1). Protein extracts were immunoprecipitated with the a-CKB antibody followed by detection with the a-MYC antibody. Plants were grown under LD conditions and samples were collected at ZT1.5. Nuclear localization analysis by confocal microscopy at ZT11 of plants expressing (C) CCA1-ox/CKB4-YFP-ox, (D) CCA1-YFP-ox/ CKB4-MYC-ox, (E) TOC1 fused to the N-terminal fragment of YFP (TOC1-nYFP-ox) and CKB4 fused to the C-terminal fragment of YFP (CKB4-cYFP-ox) or (F) TOC1 fused to full- length YFP (TOC1-YFP-ox). Found at: doi:10.1371/journal.pgen.1001201.s004 (0.40 MB PDF) Figure S5 Effects of the CK2 inhibitors DRB and DMAT on TOC1:LUC expression. (A) Free-running periods estimated from TOC1:LUC luminescence signals in WT plants under LL conditions treated with increasing concentrations of DRB. Period was estimated from individual seedlings plotted against their relative amplitude errors. (B) TOC1:LUC luminescence in WT plants treated with increasing concentrations of DMAT. Lumi- nescence was recorded under LD (12 h light:12 h dark) cycles. Data are represented as means 6 SEM of luminescence signals from at least 12 independent plants. Found at: doi:10.1371/journal.pgen.1001201.s005 (0.11 MB PDF) Figure S6 Effects of CK2 activity on the in vivo CCA1 binding to the promoters of the oscillator genes. (A) ChIP analysis of CCA1 binding to the promoters of the morning-expressed genes, PRR7 and PRR9 and the evening-expressed genes TOC1 and LUX. Analysis was performed with CCA1-YFP-ox plants entrained under LD cycles and samples were collected at ZT3 and ZT15. The binding regions of TOC1, LUX and PRR9 promoters contain the Evening Element (EE) motif. No conserved motifs were identified in the CCA1 binding region of PRR7 promoters. No amplification was obtained with the promoter of a clock-unrelated gene (At5g55840) or when samples were similarly processed in the absence of antibody. The experiments were performed three times with similar results to those shown here. Q-PCR analysis of CCA1 binding in (B) CCA1-YFP-ox and CCA1-YFP-ox/CKB4-MYC- PLoS Genetics | www.plosgenetics.org 12 November 2010 | Volume 6 | Issue 11 | e1001201