ox plants; in (C) CCA1-YFP-ox with 150 mM of the CK2 inhibitor DRB; in (D) CKA3-/CCA1-YFP-ox plants induced with 1 mM Dexamethasone (Dex) or in (E) CCA1-YFP-ox/CKB4-MYC-ox plants treated with 150 mM of the CK2 inhibitor DRB. Seedlings were grown under LD cycles and collected at ZT3. Data are presented as means 6 SEM relative to the input and to the maximum value of at least three independent experiments (*** p-value, 0.001). Found at: doi:10.1371/journal.pgen.1001201.s006 (0.20 MB PDF) Figure S7 Effects of temperature and CK2 kinase function on circadian gene expression. Bioluminescence of CAB2:LUC expres- sion in WT (A) and cca1-11 mutant (B) plants in the presence or in the absence of 100 mM DRB. Luminescence was measured in plants entrained under LD conditions at 22uC and transferred to LL conditions. Plots are means 6 SEM of 8-12 individual seedlings. (C) Analysis of circadian period length of CAB2::LUC expression in WT and cca1-11 mutant plants in the presence or in the absence of DRB. Estimated period length was determined as described in Supplemental experimental procedures. (D) TO- C1:LUC luminescence in WT and cca1-1/lhy-11 double mutant plants in the presence or in the absence of 100 mM DRB. Luminescence was measured in plants entrained under LD conditions at 22uC and transferred to LL conditions. Plots are means 6 SEM of 8-12 individual seedlings. The experiments were repeated at least twice with similar results to those shown here. Found at: doi:10.1371/journal.pgen.1001201.s007 (0.14 MB PDF) Figure S8 Analysis of TOC1:LUC diurnal expression under different temperatures. Luminescence analysis of TOC1:LUC expression in plants entrained under LD cycles at 22uC and subsequently transferred to LD cycles at 27uC (A, D), 22uC (B, E) or 12uC (C, F). Data is shown as means 6 SEM of at least 12 individual seedlings. The experiments were performed at least twice with similar results to those shown here. Found at: doi:10.1371/journal.pgen.1001201.s008 (0.13 MB PDF) Figure S9 Analysis of TOC1:LUC diurnal expression in CKB4- ox plants under different temperatures. Point-by-point lumines- cence analysis of TOC1:LUC expression in plants entrained under LD cycles at 22uC and subsequently transferred to LD cycles at 27uC (A) or 12uC (B). Data is shown as means 6 SEM of at least 12 individual seedlings. The experiments were performed at least twice with similar results to those shown here. Found at: doi:10.1371/journal.pgen.1001201.s009 (0.13 MB PDF) Figure S10 Effects of DRB treatment on TOC1:LUC diurnal expression at different temperatures. TOC1:LUC luminescence in WT plants in the presence or in the absence of 100 mM DRB at 27uC or 12uC. Luminescence was measured in plants entrained under LD conditions at 22uC and transferred to LD cycles at 27uC (A) or 12uC (B). Plots are means 6 SEM of 12 individual seedlings. The experiments were repeated at least twice with similar results to those shown here. The white and solid boxes correspond to the light and dark periods, respectively. Found at: doi:10.1371/journal.pgen.1001201.s010 (0.11 MB PDF) Figure S11 Effects of temperature and CK2 kinase function on the free-running period of the clock-output CAB2:LUC. Free- running periods estimated from CAB2:LUC luminescence signals in WT, CKB4-MYC-ox and WT plants treated with 100 mM DRB at 12uC (A), 22uC (B) and 27uC (C). Period was estimated from individual seedlings plotted against their relative amplitude errors. Data come from two independent experiments with approximately 6-12 plants per phenotype or treatment. Found at: doi:10.1371/journal.pgen.1001201.s011 (0.12 MB PDF) Clock Temperature Compensation in Arabidopsis Figure S12 Effects of temperature and CK2 kinase function on CCA1 binding to the promoters of the oscillator genes. Q-PCR analysis of CCA1 binding to TOC1 (A), LUX (B), PRR7 (C) or PRR9 (D) promoters. CCA1-YFP-ox and CCA1-YFP-ox/CKB4- MYC-ox plants were grown under LD cycles at 22uC and transferred to continuous light (LL) at 12uC or 27uC. Samples were collected after 35 h under LL conditions. Data are presented as means 6 SD relative to the input and to the maximum value of two independent experiments. (E) Q-PCR analysis of CCA1 binding to TOC1, LUX, PRR7, PRR9 and a clock unrelated gene (At5g55840) in plants expressing CCA1 under its own promoter (CCA1pro:CCA1-HA-YFP). Plants were grown under LD cycles at 22uC and transferred to continuous light (LL) at 12uC or 27uC. Samples were collected after 49.5 h under LL conditions. (F) Western-blot analysis of CCA1 protein accumulation in CCA1- YFP-ox plants at 12uC and 27uC. Similar protein transference in each lane was verified by staining with Red Ponceau. The experiments were performed twice with similar results to those shown here (** p-value,0.01; *** p-value,0.001). Found at: doi:10.1371/journal.pgen.1001201.s012 (0.17 MB PDF) Figure S13 A role for CCA1 regulating circadian gene expression at high temperatures. TOC1:LUC luminescence in WT and CCA1-YFP-ox plants at 27uC (A) or 12uC (B). TOC1:LUC luminescence in WT and CCA1-YFP-ox plants at 27uC (C) or 12uC (D) in the presence or in the absence of 100 mM DRB. (E) CAB2:LUC luminescence in WT and cca1-11 mutant plants at 27uC and in the presence or in the absence of 100 mM DRB (F). Luminescence was measured in plants entrained under LD conditions at 22uC and transferred to LL conditions at the indicated temperatures. Plots are means 6 SEM of 12 individual seedlings. The experiments were repeated at least twice with similar results to those shown here. Found at: doi:10.1371/journal.pgen.1001201.s013 (0.15 MB PDF) Figure S14 Effects of temperature and CK2 kinase function on CCA1 binding to the promoters of the oscillator genes. Q-PCR analysis of CCA1 binding to TOC1, LUX, PRR7 and PRR9 promoters in plants over-expressing CCA1 in the absence (-) or in the presence (+) of 150 mM of the CK2 inhibitor DRB. Plants were grown under LD cycles at 22uC and transferred to continuous light (LL) at 12uC (A) or 27uC (B). Data are presented as means 6 SD relative to the input and to the maximum value. Samples were collected after 50 h under LL conditions. Found at: doi:10.1371/journal.pgen.1001201.s014 (0.11 MB PDF) Figure S15 Double ChIP analysis. (A) Schematic representation depicting a summary of the Double-ChIP assay described in Figure 7. A double round of immunoprecipitation (IP) was performed. In the first round, the a-GFP antibody was used to detect total CCA1-YFP protein bound to chromatin. In a second round, the a-PSer antibody was used to specifically detect the phosphorylated isoforms of CCA1 (P). PCR amplification would be obtained only if the phosphorylated CCA1 isoforms are preferentially bound to chromatin. (B) Double-ChIP assays with the combination of a-H3/anti-PSer or a-H3/anti-H3 antibodies Data are presented as means 6 SD relative to the maximum value of two independent experiments. Found at: doi:10.1371/journal.pgen.1001201.s015 (0.13 MB PDF) Table S1 Plant Material used in this study. Found at: doi:10.1371/journal.pgen.1001201.s016 (0.07 DOC) Table S2 Constructs used in this study. Found at: doi:10.1371/journal.pgen.1001201.s017 (0.05 DOC) MB MB PLoS Genetics | www.plosgenetics.org 13 November 2010 | Volume 6 | Issue 11 | e1001201