Clock Temperature Compensation in Arabidopsis Figure 3. CK2 regulates TOC1:LUC expression without altering CCA1 protein accumulation. Immunodetection of CCA1 protein with a-GFP in CCA1-YFP-ox (A) or CCA1-YFP-ox/CKB4-MYC-ox plants (B). Seedlings were entrained under LD cycles and samples were collected at the indicated ZT. Quantification of CCA1-YFP protein accumulation is shown in (C). Means 6 SD of two independent experiments are represented relative to the maximum value and normalized to the RUBISCO protein. (D) Western-blot analysis of CKB4-MYC (a-MYC) or CCA1-YFP (a-GFP) in CCA1-YFP-ox, CKB4- MYC-ox and different CCA1-YFP-ox/CKB4-MYC-ox lines. Western-blot analysis of CCA1-YFP protein accumulation in CCA1-YFP-ox plants in the presence (+) or absence of 150 mM DRB (E) or in CCA1-YFP-ox/CKA3- plants with (+) or without 1 mM Dex (F). Samples were collected at ZT3 and ZT15. (G) TOC1:LUC luminescence in WT plants treated with DRB (0, 50, 75, 150 mM) or (H) in CKA3- and CCA1-YFP-ox/CKA3- plants in the absence or in the presence of 1 mM Dex added at CT2 (arrow). Seedlings were entrained under LD cycles and transferred to LL prior luminescence recording. Data are represented as means 6 SEM of luminescence signals from at least 12 independent plants. All the experiments were performed at least twice with similar results to those shown here. doi:10.1371/journal.pgen.1001201.g003 by chromatin immunoprecipitation (ChIP) assays, the in vivo binding of CCA1 to the promoters of its target genes. CCA1 was proposed to be part of a morning loop regulating the expression of both morning- and evening-expressed genes [58]. However, direct in vivo binding to oscillator genes was only demonstrated for TOC1 [52]. Our ChIP assays with CCA1-YFP-ox plants confirmed the binding of CCA1 to the TOC1 promoter (Figure S6) and also revealed the physical association of CCA1 to the promoters of the oscillator genes PRR7, PRR9 and LUX (LUX ARRHYTHMO) (Figure S6). We next examined the effects of over-expressing CKB4 by comparing the binding of CCA1 in single CCA1-YFP- ox and double CCA1-YFP and CKB4-MYC over-expressing plants. Our results showed that over-expression of CKB4 considerably decreased the binding of CCA1 to the promoters of the morning-expressed genes PRR7 and PRR9 (Figure 4A) as well as the evening-expressed genes TOC1 and LUX (Figure 4B). Conversely, decreasing CK2 activity by treatment with DRB (Figure 4C and 4D) or by inducing the dominant negative CK2 mutant (Figure 4E and 4F) had the opposite effect, with an evident increment of CCA1 binding to these promoters. Q-PCR analysis revealed the significance of the binding changes (Figure S6) with p- values,0.001 in all cases. Furthermore, DRB treatment of CCA1- YFP-ox/CKB4-MYC-ox plants significantly decreased the effects of CKB4 over-expression as compared with non-treated plants (Figure S6), suggesting that phosphorylation is indeed important for CKB4 regulation of CCA1 activity. The link between CCA1 phosphorylation and function was also reinforced in studies in which WT and cca1-11 mutant plants [59] were treated with DRB. Our results showed that treatment of WT plants expressing the promoter of the clock-controlled gene CAB2 (CHLOROPHYLL A/ B BINDING PROTEIN 2, or LIGHT HARVESTING COMPLEX B1, LHCB1*1) fused to luciferase [60] considerably lengthened PLoS Genetics | www.plosgenetics.org 5 November 2010 | Volume 6 | Issue 11 | e1001201