counting the number of days and number of leaves at the time of a 1 cm-high flower bolt. For hypocotyl length assays, seeds were stratified in the dark at 4uC for 4 days on MS medium supplemented with 3% Sucrose. Germination was induced by exposing the seeds to white light (60 mmol m22 s21) for 6 h followed by 18 h under darkness. Seeds were placed under ShD or LgD conditions (60 mmol m22 s21) or under continuous white light (LL) with the specified light intensities. Hypocotyl length was measured after seven days by using the Image J software (http:// rsb.info.nih.gov/ij/). Western-blot and co-immunoprecipitation analysis Western-blot assays were essentially performed as previously described [69]. Briefly, nine day-old seedlings were ground in liquid nitrogen and proteins were extracted in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40 0.1% SDS, 0.5% Sodium Deoxycholate, 0.5% Polyvinylpolypyrrolidone (PVPP), 50 mm MG132, 10 mM NaF, 1 mM PMSF, 5 mg/ml Leupeptin, 1 mg/ml Aprotinin, 5 mg/ml Antipain, 1 mg/ml Pepstatin, 5 mg/ml Chymostatin). Protein concentration was calculated using the Bradford method (Bio-Rad) and 20-60 mg of total protein was loaded per lane. Proteins were transferred to nitrocellulose membranes and stained with Red Ponceau following standard protocols. Anti-MYC (clone 9E10; Sigma) and Anti-GFP (A11122; Invitrogen) antibodies were used to detect CKB4-MYC and CCA1-YFP proteins, respectively. Protein accumulation was quantified using the LAS-4000 imaging system (Fujifilm-GE Healthcare). In experiments with CK2 inhibitors, 48 hours before sampling, plants were transferred to medium supplemented with 150 mM of DRB, 150 mM DMAT or 1 mM Dex. For co- immunoprecipitation assays, nine day-old seedlings were ground in liquid nitrogen and proteins extracted in RIPA buffer. Extracts were incubated for 4 h at 4uC with Protein G–Sepharose beads (Amersham Biosciences) conjugated with Anti-GFP antibody. Immunocomplexes were washed 5 times with RIPA buffer followed by additional washing with PBS (Phosphate buffer saline). Immunoprecipitated proteins were eluted by adding Laemmli buffer followed by 4 min incubation at 95uC. Anti- MYC and Anti-GFP antibodies were used to detect CKB4-MYC and CCA1-YFP respectively. The anti-phosphoserine antibody (Anti-PSer, 4A3, Calbiochem) was used to detect phosphorylated isoforms of CCA1. The antibody recognizes serine-phosphorylated residues (in a positively charged amino acid context directly neighbouring the phosphoserine). For interaction of proteins expressed at endogenous levels, we used a polyclonal anti-CK2B antibody to the human CK2B regulatory subunit CK2B (CSNK2B; Abnova). Detection of CCA1 was performed with the Anti-MYC antibody. Two-dimensional gels and immunoblotting For two-dimensional gels experiments, nine day-old seedlings were ground in liquid nitrogen and proteins extracted in lysis buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 18 mM Tris-HCl pH 8.0, 50 mm MG132, 10 mM NaF, 1 mM PMSF, 5 mg/ml Leupeptin, 1 mg/ml Aprotinin, 5 mg/ml Antipain, 1 mg/ml Pepstatin, 5 mg/ml Chymostatin). Protein concentration was determined using the Bradford method (Bio-Rad) and 40 mg of total protein was loaded onto immobilized pH gradient (IPG) strips (7 cm, pH 3–10, Amersham Biosciences) for the first dimension separation. Strips were rehydrated for 6 h at room temperature and the isoelectric point focusing was performed at 30 V for 6.5 h, 500 V for 1 h, 1000 V for 1 h and 5000 V for 7 h. Strips were subsequently equilibrated for 15 min with equilibra- tion buffer I (50 mM Tris-HCl pH 8.8, 6 M Urea, 30% Glycerol, Clock Temperature Compensation in Arabidopsis 2% SDS, 10 mg/ml DTT) followed by a 15 min wash with equilibration buffer II (50 mM Tris-HCl pH 8.8, 6 M Urea, 30% Glycerol, 2% SDS, 25 mg/ml Iodoacetamide). For the second dimension, strips were loaded onto SDS-PAGE 8% polyacryl- amide gels followed by blotting to nitrocellulose membranes. Anti- GFP antibody was used to detect CCA1-YFP protein. Bimolecular Fluorescence Complementation (BiFC) and confocal microscopy Plants over-expressing both the CCA1 protein fused to the N- terminal fragment of the Yellow Fluorescent Protein (YFP) (nucleotides: 1–462) and the CKB4 protein fused to the C-terminal part of the YFP protein (nucleotides: 463–741) were grown on MS- agar medium supplemented with 3% Sucrose under LD cycles. Fluorescence signals of hypocotyl cells were imaged using an Olympus Fluoview FV1000 confocal microscope using a 515 nm argon excitation laser. CKB4-YFP-ox, CCA1-YFP-ox, TOC1-YFP- ox, CCA1-ox/CKB4-YFP-ox, CCA1-YFP-ox/CKB4-MYC-ox, TOC1-nYFP-ox/CKB4-cYFP-ox were similarly imaged. ChIP and double-ChIP assays ChIP assays were performed essentially as previously described [52]. Briefly, fourteen day-old seedlings were fixed in fixation buffer (0.4 M Sucrose, 10 mM Tris-HCl pH 8.0, 1 mM EDTA,1 mM PMSF, 1% Formaldehyde, 0.05% Triton X-100) for 10 min, followed by addition of Glycine 0.125 M and vacuum incubation during 10 min. Seedlings were subsequently ground in liquid nitrogen and extracted in extraction buffer I (0.4 M Sucrose, 10 mM Tris-HCl pH 8.0, 5 mM b-mercaptoethanol, 1 mM PMSF, 5 mg/ml Leupeptin, 1 mg/ml Aprotinin, 5 mg/ml Anti- pain, 1 mg/ml Pepstatin, 5 mg/ml Chymostatin and 50 mm MG132). Nuclei were then purified by centrifugation and washed with extraction buffer II (0.25 M Sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1% Triton X-100, 5 mM b-mercapto- ethanol, 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). Nuclei were resuspended in nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 5 mg/ml Leupeptin, 1 mg/ml Aprotinin, 5 mg/ml Antipain, 1 mg/ml Pepstatin, 5 mg/ml Chymostatin and 50 mm MG132). Chromatin was sonicated to approximately 500–1000 bp fragments with a sonicator (Branch). After centrifugation, soluble chromatin was diluted in ChIP dilution 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-GFP antibody. Immunocomplexes were washed with low salt buffer (20 mM Tris- HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA), high salt buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA), LiCl wash buffer (10 mM Tris-HCl pH 8.0, 0.25 M LiCl, 1% NP-40, 1% Sodium Deoxycholate, 1 mM EDTA) and 2x TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Immunocomplexes were eluted with 1% SDS and 0.1 M NaHCO3 followed by overnight reverse cross-link at 65uC and proteinase K treatment for 1 h at 45uC. Immunoprecipitated DNA was isolated using the QIAquick kit (Qiagen) following the manufacturer instructions. ChIP samples were amplified by PCR, stained with SYBR Green (Molecular Probes) and resolved by electrophoresis on 2% agarose gel. Images were captured with the LAS-4000 imaging system (Fujifilm-GE Healthcare). ChIPs were quantified by Q-PCR analysis using a 96- well Lightcycler 480 system (Roche) with the Lightcycler 480 PLoS Genetics | www.plosgenetics.org 11 November 2010 | Volume 6 | Issue 11 | e1001201