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Science 336 (6077): 75-79

Copyright © 2012 by the American Association for the Advancement of Science

Mapping the Core of the Arabidopsis Circadian Clock Defines the Network Structure of the Oscillator

W. Huang1, P. Pérez-García1, A. Pokhilko2, A. J. Millar2,3, I. Antoshechkin4, J. L. Riechmann1,4,5, and P. Mas1,*

1 Center for Research in Agricultural Genomics, Barcelona 08193, Spain.
2 School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JH, UK.
3 Centre for Systems Biology at Edinburgh, C. H. Edinburgh EH9 3JD, UK.
4 California Institute of Technology, Division of Biology, Pasadena, CA 91125, USA.
5 Institució Catalana de Recerca i Estudis Avançats, Barcelona 08010, Spain.


Figure 1
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Fig. 1. Analysis of TOC1 ChIP-Seq circadian targets. Global trend of expression phases within TOC1 circadian targets (A and B) and for a random list of circadian genes (C). Phase enrichments were graphically portrayed within a range of ChIP-Seq P values at different Zeitgeber times (radial axis: 0-dawn; 12-dusk). The white and gray areas represent day and night, respectively. Peak traces of LHY (D), PRR7 (E), PRR9 (F), and GI (G) from TOC1 ChIP-Seq data. The exon-intron–untranslated region structure of the different gene models above and below each panel indicates forward and reverse orientation, respectively. Position weight matrix representation of consensus motifs present within the TOC1 target oscillator sequences. The Gbox-expanded (H) and EE-like–expanded (I) motifs were identified using the SCOPE motif finder. Both strands were used to compute the significance value. ChIP-Seq, peak visualization, circadian phase analysis, and motif discovery were performed as described in the supporting online material (SOM).

 

Figure 2
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Fig. 2. TOC1 binds to the promoters of the oscillator genes and inhibits their expression. ChIP-QPCR assays of TMG plants that were sampled every 4 hours over a 24-hour LD cycle are shown. Primers encompassing target sequences obtained in the ChIP-Seq analysis were used to amplify CCA1 and LHY (A), PRR7 and PRR9 (B), GI and PRR5 (C), or LUX/PCL1 and ELF4 (D). Binding was also examined under LL conditions following entrainment in TMG (E and F) and in TOC1-ox plants (G and H). Values are represented as means ± SEM. Expression profiling of PRR9 (I), PRR7 (J), GI (K), and ELF4 (L) in WT, TMG, and TOC1-ox plants. Gene expression was analyzed in plants grown under LD cycles. mRNA abundance was normalized to IPP2 expression. Values represent means ± SEM. White shading, day; gray shading, night.

 

Figure 3
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Fig. 3. Effects of TOC1 transient induction on oscillator gene expression. Analysis of CCA1 (A) and GI (B) in WT and TOC1-GR plants mock-treated or treated with 5 μM dexamethasone (+Dex). Seedlings were treated at ZT10, and samples were analyzed at the indicated ZT. (C) Analysis of CCA1::LUC luminescence in TOC1-GR seedlings under 8-hours light/16-hours dark cycles. Luminescence was recorded 24 hours after the seedlings were transferred to a medium containing 5 μM of Dex. Data are presented as means ± SEM of luminescence signals from six to seven independent plants. (D to F) Comparisons of CCA1 expression in TOC1-GR plants mock-treated or treated with 5 μM of Dex at ZT10 and analyzed at ZT21 and ZT23 in the absence (-CHX) or presence (+CHX) of 50 μM of cycloheximide. The statistical relevance of the differences is presented (*P < 0.05; **P < 0.01; ***P < 0.001). (G to I) Time-course analysis of TOC1, CCA1, and PRR9 expression over the diurnal cycle in TMG plants in the presence or absence of the hormone ABA. Seedlings were sprayed with 100 μM (±) ABA at ZT5. The y axes of the CCA1 and PRR9 graphs were divided into segments so that repression at trough can clearly be observed. White shading, day; gray shading, night.

 

Figure 4
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Fig. 4. Gene expression analysis in TOC1 RNAi plants. Analysis of LHY (A) and PRR7 (B) expression under 12-hours light:12-hours dark conditions. Gene expression analysis of PRR9 (C) and GI (D) during the night in TOC1 RNAi under 12-hours light/12-hours dark conditions. Values are represented as means ± SEM. Analysis was performed as described in the SOM. Schematic diagrams depicting the waveform of CCA1/LHY expression and the different waves of PRRs repression. Schemes depict the regulations in the wild type (black line) (E), toc1 mutant (blue line), (F) and TOC1-ox (red line) (G). The small lines ending in perpendicular dashes represent repression. White boxes, day; black boxes, night.

 


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