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¹ Center for Gene Research, Nagoya University, Furo, Chikusa, Nagoya 464-8602, Japan
² Bio-oriented Technology Research Advancement Institution (BRAIN), Nagoya University, Furo, Chikusa, Nagoya 464-8602, Japan
³ Division of Biological Science, Graduate School of Science, Nagoya University, Furo, Chikusa, Nagoya 464-8602, Japan
⁴ Aichi Science and Technology Foundation, 2-4-7 Marunouchi, Naka, Nagoya 460-0002, Japan

	Abstract

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Previously, we screened 50 000 seedlings of Arabidopsis thaliana carrying a P_GI::LUC⁺ bioluminescence reporter gene mutagenized with ethylmethanesulfonate for mutants with phenotypes of extensively altered circadian rhythms, and identified three loci, PHYTOCLOCK 1 (PCL1), PCL2 and PCL3, whose mutations cause arrhythmia. Here we succeeded to clone the PCL1 gene and show that the PCL1 gene encodes a novel DNA binding protein belonging to the GARP protein family and is essential for a functional clock oscillator in A. thaliana. The PCL1 gene satisfies the requirements for the clock oscillator gene: (i) pcl1 null mutations caused arrhythmia in multiple circadian outputs, including expression of potential clock genes TOC1, CCA1 and LHY, and flowering lacked a photoperiodic response; (ii) PCL1 expression showed circadian rhythm in both constant light and constant dark; (iii) over-expression of the PCL1 gene gradually caused arrhythmicity in all the multiple circadian outputs examined; and (iv) the PCL1 gene controlled its own expression via negative feedback. Therefore, the PCL1 gene is the clock oscillator gene essential to the generation of clock oscillation in the higher plant.

	Introduction

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Circadian rhythms are endogenous daily fluctuations in physiological and biological activities that are observed in organisms from cyanobacteria to humans (Bünning 1973; Dunlap et al. 2004). In higher plants, the clock regulates many processes, including stomatal aperture, leaf movement, hypocotyl elongation, photosynthetic activity and photoperiodic flowering induction (Lumsden & Millar 1998; McClung 2001). Circadian oscillations are generated by both positive and negative feedback control of the clock genes (Ishiura et al. 1998; Dunlap 1999; Young & Kay 2001; Salomé & McClung 2004).

In Arabidopsis, potential circadian clock genes, CCA1 (Wang & Tobin 1998), LHY (Schaffer et al. 1998) and TOC1 (Makino et al. 2000; Strayer et al. 2000) and clock-associated genes, GI (Fowler et al. 1999; Park et al. 1999) and ELF4 (Doyle et al. 2002), have been cloned, and a clock feedback model has been proposed (Young & Kay 2001; Hayama & Coupland 2003; Yanovsky & Kay 2003; Salomé & McClung 2004). However, these genes are not essential to the generation of clock oscillation because none of the null mutations of these genes ever cause arrhythmia. Previous explanation is that because several analogous clock genes have duplicated functions, any null mutation in one of these genes could not cause arrhythmia.

Previously, we screened 50 000 M₂ seedlings of Arabidopsis carrying a P_GI::LUC⁺ bioluminescence reporter gene mutagenized with ethylmethanesulfonate and isolated five arrhythmic mutants belonging to three complementation groups: PHYTOCLOCK 1 (PCL1), PCL2 and PCL3. All mutants had a recessive pcl mutation and did not show any rhythmicity in the circadian rhythms examined, suggesting that the PCL genes encode clock components (Onai et al. 2004).

Here we succeeded to clone a clock oscillator gene, PCL1, in Arabidopsis and characterize it. The PCL1 gene is the first clock gene reported that is essential to the generation of the circadian clock oscillation in the higher plant because the gene satisfied the requirements for the clock oscillator gene. Furthermore, the PCL1 gene regulates its own expression via negative feedback, and this feedback loop is an essential process for the generation of clock oscillation. We also found PCL1 homologs in rice, tobacco, tomato, potato and pine.

	Results

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Effects of pcl1 mutations on circadian rhythms and photoperiodic flowering induction

Although several potential clock genes have been identified in Arabidopsis, none of their null mutations cause arrhythmia (Salomé & McClung 2004). In contrast, the pcl1-1 and pcl1-2 mutants abolish the circadian bioluminescence rhythms of plants carrying a P_GI::LUC⁺ reporter gene (a fusion gene connecting an Arabidopsis GI promoter to a modified firefly luciferase gene) in both constant light (LL) and constant dark (DD) (Figs 1A and 6E; Onai et al. 2004). They also show arrhythmia in leaf movement, which is distinct from the circadian output of GI expression rhythms (Onai et al. 2004). Moreover, pcl1 mutations affect photoperiodic flowering induction: wild-type strains flowered earlier in long day than in short day, but this photoperiodic response is abolished in the mutants (Fig. 2). Since both the pcl1-1 and pcl1-2 mutants expressed the same phenotype for circadian rhythms and photoperiodic flowering, we elected to use mainly the pcl1-1 mutant for our experiments.

View larger version (40K): [in this window] [in a new window]   Figure 1 Arrhythmia of circadian rhythms in pcl1 mutants. (A) Bioluminescence of wild-type (blue), pcl1-1 (red), and pcl1-2 (brown) plants carrying a PGI::LUC+ reporter gene in LL. After exposure to three cycles of LD (12 h light/12 h dark), plants were transferred to LL (time 0). (B) Bioluminescence of wild-type (blue) and pcl1-1 (red) plants carrying the PGI::LUC+ reporter gene in LD. Hatched white bars, light periods; solid bars, dark periods. (C) Bioluminescence of wild-type (blue) and pcl1-1 (red) plants carrying the PGI::LUC+ reporter gene in HL (22 °C for 12 h/17 °C for 12 h) cycles in LL. Hatched yellow bars, 22 °C; solid blue bars, 17 °C. (A–C) Data points and error bars represent the mean and SD for 96 seedlings. (D–I) Northern blot and quantification of GI(D), CAB2(E), TOC1(F), ELF4(G), CCA1(H) and LHY(I) mRNAs in wild-type (WT, blue) and pcl1-1 (red) plants grown in LL. Data represent one of two (D–I) or three (A–C) independent experiments that yielded essentially the same results. (B–I) pcl1-2 mutants yielded essentially the same data.

View larger version (17K): [in this window] [in a new window]   Figure 2  Flowering time of pcl1 mutants. We examined the flowering time of wild-type Col-0 and G-38 plants and the homozygous F4 plants of pcl1 mutants (pcl1-1 and pcl1-2) under long-day (16L8D) and short-day (10L14D) conditions. Data are mean ± SEM (n = 16–53). The data represent one of two independent experiments that yielded similar results.

The bioluminescence of the pcl1-1 mutant carrying the P_GI::LUC⁺ reporter gene was arrhythmic and constitutively high in LL (Fig. 1A), which is consistent with GI mRNA levels (Fig. 1D). Using Northern blot analysis to examine expression of other genes in the pcl1-1 mutant, we found that the photosynthetic gene CAB2 (Millar et al. 1995), the potential clock genes TOC1, CCA1 and LHY, and the clock-associated gene ELF4 were expressed arrhythmically, although mRNA levels were transiently changed on the 1st day (Fig. 1E–I). The levels of CAB2, TOC1 and ELF4 mRNA were constitutively high (Fig. 1E–G) whereas those of CCA1 and LHY were constitutively low (Fig. 1H,I). These results suggest that PCL1 is essential for the generation of circadian rhythms and that the gene controls the expression of the potential clock and clock-associated genes.

PCL1 encodes a novel GARP protein

To further analyze PCL1 function in the Arabidopsis clock, we performed map-based cloning (Fig. 3) and identified gene At3g46640 as PCL1. Both pcl1-1 and pcl1-2 had a nonsense mutation (Figs 3 and 4A). The predicted PCL1 protein contained a motif (Hosoda et al. 2002) that is widely observed in plant transcription factors belonging to the GARP family, including Arabidopsis type-B response regulators such as ARR1 (Sakai et al. 1998) and ARR10 (Imamura et al. 1999) (Fig. 4A,B). The GARP motif contains a nuclear localization signal; and ARR1 and ARR10 localize in the nucleus and bind to DNA in vitro through the GARP motif (Sakai et al. 1998; Hosoda et al. 2002). In ARR10, this motif has a Myb-related helix-loop-helix structure (Hosoda et al. 2002). Because both pcl1-1 and pcl1-2 cause truncation of the PCL1 protein proximal to the GARP motif, arrhythmia in both mutants should exhibit a loss-of-function phenotype. PCL1 localized in the nucleus in onion epidermal cells, as demonstrated by transient expression of GFP (green-fluorescent protein)-PCL1 and PCL1-GFP fusion proteins (Fig. 4C–H). PCL1 is therefore likely to localize in the nucleus of Arabidopsis cells and function as a transcription factor. Neither the N-terminus nor C-terminus of PCL1 has a homology to known proteins. However, we identified a PCL1-LIKE (PCLL) gene in the Arabidopsis genome, a putative PCL1 homolog genes (OsPCL1) in the rice genome, and expression sequence tags (ESTs) in tobacco (NbPCL1), tomato (LePCL1), potato (StPCL1), and pine (PtPCL1) (Fig. 5).

View larger version (20K): [in this window] [in a new window]   Figure 3  Map-based cloning of PCL1. Percent of recombination between physical markers and pcl1-1 mutation and number of recombinants per chromosome tested are shown under each physical marker. Contigs of BAC clones that are available in TAIR database are also shown. PCL1 mapped to the bottom of chromosome 3 between SSLP marker NIT1.2 and CAPS marker ALS. Fine physical mapping delimited PCL1 to a 150 kb interval (blue arrow) between SNPs F18L15-1 and CAPS marker TOPP5. F18L15-1 contains SNP no. CER468139 to CER468143, released from the Monsanto Arabidopsis Polymorphism Collection. We sequenced the 150 kb region in both pcl1 mutants and found that each had a single nucleotide substitution resulting in a nonsense codon (an opal codon in pcl1-1 and an ochre codon in pcl1-2) on a predicted PCL1 gene (At3g46640 in TAIR database). PCL1 had no intron, as shown at the bottom of the figure. The 5'- and 3'-untranslated regions are shown as open boxes, the coding sequence as a shaded box. Positions of the pcl1 mutations in full-length PCL1 cDNA are also shown.

View larger version (58K): [in this window] [in a new window]   Figure 4  Structure and nuclear localization of PCL1 protein. (A) Predicted protein structure of PCL1 showing positions of pcl1 mutations and GARP motif (gray box) (Hosoda et al. 2002). (B) Comparison of the GARP motifs of PCL1, Arabidopsis ARR1 (Sakai et al. 1998) (accession no. NM_112561 [GenBank] ), and ArabidopsisARR10 (Imamura et al. 1999; Hosoda et al. 2002) (NM_119343 [GenBank] ). Identical residues are boxed. Numbers in parentheses indicate position of the GARP motif. (C–H) Nuclear localization of GFP-PCL1 and PCL1-GFP fusion proteins. Plasmid DNA carrying a PCaMV35S::::sGFP(S65T)::Tnos (C,D), PCaMV35S::PCL1::::sGFP(S65T)::Tnos (E,F), or PCaMV35S::::sGFP(S65T)::PCL1:: Tnos (G,H) gene cassette was transferred into onion epidermal cells. (C,E,G) Images of GFP fluorescence. (D,F,H) Images of Normarsky.

View larger version (61K): [in this window] [in a new window]   Figure 5  Amino acid sequences of PCL1 and its Arabidopsis homologs (PCLL, PCL1-LIKE), Oryza sativa (OsPCL1), Nicotiana benthamina (NbPCL1), Lycopersicon esculentum (LePCL1), Solanum tuberosum (StPCL1) and Pinus taeda (PtPCL1). The sequences of LePCL1 and PtPCL1 are partial. We aligned the sequences using the CLUSTAL W program (Thompson et al. 1994). Asterisks (*) and dots (.) under the residues indicate identical and similar residues, respectively. Red letters in the PCL1 sequence indicate the position of the mutant stop codons. The accession numbers for the PCL1, PCLL, OsPCL1, NbPCL1, LePCL1, StPCL1 and PtPCL1 are AB206576, AB206577, AB206578, AB219069, AB219070, AB219071 and AB219072, respectively.

We examined PCL1 expression under constant conditions by Northern blot analysis and by bioluminescence monitoring of plants carrying a P_PCL1::LUC⁺ reporter gene. In LL, the level of PCL1 mRNA in wild-type plants oscillated rhythmically in a circadian manner with peaks at subjective dusk (Fig. 6A). In pcl1-1 mutants, in contrast, the levels were arrhythmic and constitutively high. Wild-type plants carrying the P_PCL1::LUC⁺ reporter gene showed robust circadian bioluminescence rhythms with a saw-toothed waveform; the increase was rapid and the decrease was gradual (Fig. 6B). The period length of the rhythm was 23.5 ± 0.3 h (mean ± SD, n = 48), and the phase was circadian time (CT) 11.5 ± 0.3. In DD, the level of bioluminescence of the wild-type plants decreased to about 30% of the LL level within 24 h of the light to dark transition. After 24 h, it showed robust circadian bioluminescence rhythms with a longer period length (25.9 ± 0.7 h, n = 48) than it had in LL (Fig. 6C).

View larger version (28K): [in this window] [in a new window]   Figure 6  Circadian expression of PCL1 and its disruption by over-expression of PCL1. Data points and error bars represent means and SDs. (A) Northern blot and quantification of PCL1 mRNA in wild-type (WT, blue) and pcl1-1 (red) plants grown in LL. (B,C) Bioluminescence of wild-type plants carrying a PPCL1::LUC+ reporter gene in LL (B) or DD (C). (D) Bioluminescence of wild-type (blue) and PCL1-ox (green) plants carrying the PGI::LUC+ reporter gene in LL. (E) Bioluminescence of wild-type (blue), pcl1-1 (red) and PCL1-ox (green) plants carrying the PGI::LUC+ reporter gene in DD. After exposure to three cycles of LD or DL (12 h dark/12 h light), plants were transferred to LL or DD (time 0), respectively. n = 48. (B,C) Other transgenic strains carrying the PPCL1::LUC+ reporter gene yielded similar results, varying somewhat in bioluminescence levels with strains. (D,E) Other PCL1-ox plants yielded similar results. (A,D) The pcl1-2 mutant yielded essentially the same data. (F) Leaf movement in wild-type (blue) and PCL1-ox (green) plants in LL. The vertical positions of cotyledon tips at the initial time point in each strain are plotted as zero. n = 12. (G) Northern blot analysis and quantification of PCL1 mRNA from endogenous PCL1 in wild-type (WT, blue), pcl1-1 (red) and PCL1-ox (green) plants grown in LL. Data represent one of two (A,F,G) or three (B–E) independent experiments that yielded essentially the same results.

Rhythmic expression of the clock oscillator gene generates circadian oscillation, and disruption of the rhythmic expression abolishes multiple circadian outputs (Ishiura et al. 1998; Dunlap 1999). We constructed PCL1-over-expressing (PCL1-ox) plants carrying a P_GI::LUC⁺ reporter gene (Fig. 7A) and examined their bioluminescence rhythms (Fig. 6D,E). In both LL and DD, the rhythms are gradually lost within four cycles and bioluminescence levels were then expressed constitutively at less than half the wild-type level. PCL1 over-expression also gradually caused arrhythmicity in the rhythms of leaf movement (Fig. 6F) and of the mRNA levels of GI, TOC1, ELF4, CCA1 and LHY (Fig. 7B–F) within four cycles.

View larger version (77K): [in this window] [in a new window]   Figure 7  Northern blot analysis of potential clock and clock-associated genes in PCL1-ox plants. Northern blot and quantification of PCL1(A), GI(B), TOC1(C), ELF4(D), CCA1(E) and LHY(F) mRNAs in wild-type (WT, blue), pcl1-1 (red) and PCL1-ox (green) plants grown in LL. Data represent one of two independent experiments that yielded essentially the same results.

Most clock oscillator genes (for example Per and Tim in animals, frq in Neurospora and kaiC in cyanobacteria) are expressed rhythmically in a circadian manner under constant conditions and control their own expression level by negative feedback (Ishiura et al. 1998; Dunlap 1999; Young & Kay 2001; Salomé & McClung 2004). In the present study, over-expression of PCL1 repressed endogenous PCL1 expression and disrupted its circadian expression rhythm (Fig. 6G). These results suggest that PCL1 forms an autoregulatory negative feedback loop and that oscillation of expression is essential for the Arabidopsis clock.

	Discussion

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The mechanism proposed for the generation of circadian oscillations in all organisms is negative and positive feedback control of clock genes (Ishiura et al. 1998; Dunlap 1999; Young & Kay 2001; Salomé & McClung 2004) although, in cyanobacteria, a transcription and translation feedback loop is not the only way to make circadian oscillation (Nakajima et al. 2005). In Arabidopsis, potential circadian clock genes CCA1, LHY and TOC1 and clock-associated genes GI and ELF4 may be involved in clock feedback (Yanovsky & Kay 2003; Salomé & McClung 2004). CCA1 and LHY expression is out of phase with TOC1, GI and ELF4 expression: CCA1 and LHY expression peak in the morning whereas TOC1, GI and ELF4 expression peak in the evening. TOC1, GI and ELF4 maintain high CCA1 and LHY mRNA levels in the morning whereas CCA1 and LHY repress TOC1 transcription in the evening (Yanovsky & Kay 2003; Salomé & McClung 2004). Mutations in these genes, however, including null mutations, do not cause complete arrhythmia (Wang & Tobin 1998; Makino et al. 2000; Strayer et al. 2000; Mizoguchi et al. 2002). Moreover, a double null mutation of CCA1 and LHY, cca1-1/lhy-12, also does not cause arrhythmia although rhythms damped out within three cycles (Mizoguchi et al. 2002). Here we demonstrated that PCL1 satisfied the requirements for the clock oscillator gene in plants, establishing it as an essential clock oscillator gene. We proposed a model of the Arabidopsis circadian clock (Fig. 8). In the morning, PCL1 up-regulates CCA1 and LHY while it down-regulates GI, TOC1, ELF4 and PCL1 itself. CCA1 and LHY mRNA levels were much lower in the pcl1-1 mutant than in the wild-type (Fig. 1H,I), but their levels were not substantially increased in PCL1-ox plants (Fig. 7E,F). Therefore, PCL1 activation of CCA1 and LHY may require not only PCL1, but also another yet unidentified factor(s) (‘X’ in Fig. 8). Expressions of PCL1, GI, TOC1 and ELF4 were correlated in LL (Figs 1D,F,G and 6A), and their products localized in the nucleus (Fig. 4C–H; Makino et al. 2000; Strayer et al. 2000; Huq et al. 2000; Khanna et al. 2003). Of those proteins, only PCL1 has an obvious DNA binding motif (Fig. 4A,B; Fowler et al. 1999; Park et al. 1999; Makino et al. 2000; Strayer et al. 2000; Doyle et al. 2002). It is possible therefore that PCL1 directly interacts, or closely associates, with GI, TOC1, and/or ELF4 to function as a transcription activator for CCA1 and LHY, given that their genes were required for the maintenance of high morning CCA1 and LHY mRNA levels. PCL1 must also be required for the morning decrease of GI, TOC1, ELF4 and PCL1 mRNA levels because they were constitutively high in the pcl1-1 mutant (Figs 1D,F,G and 6A). TOC1 and ELF4 regulation by PCL1, however, may be distinct from GI and PCL1 regulation by PCL1 because PCL1 over-expression did not affect TOC1 and ELF4 mRNA levels, whereas it repressed GI and PCL1 mRNA levels (Fig. 6D,E,G and Fig. 7B,D). Therefore, repression of TOC1 and ELF4 by PCL1 may require not only PCL1 but also another yet unidentified factor(s) (‘Y’ and ‘Z’ in Fig. 8). In contrast, expression of GI and PCL1 itself may be repressed by a pathway(s) other than the CCA1/LHY-regulating pathway described above.

View larger version (26K): [in this window] [in a new window]   Figure 8  Model of the Arabidopsis circadian clock. PCL1 up-regulates CCA1 and LHY in the morning and down-regulates GI, TOC1, ELF4 and itself in the morning. Expression of PCL1 is regulated by its own expression via negative feedback, and the feedback regulation may be essential for the generation of circadian oscillation although the feedback mechanism is unknown.

	Experimental procedures

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Plant materials, growth conditions and measurement of circadian rhythms

We used bioluminescence reporter strain G-38 carrying a P_GI::LUC⁺ reporter gene (ecotype Col-0) (Onai et al. 2004) as the wild-type strain unless otherwise indicated. The pcl1-1 and pcl1-2 mutants were originally isolated as arrhythmic mutants 23-15D9 and 32-5E2, respectively, and have the G-38 genetic background (Onai et al. 2004). We used homozygous F₄ plants of the mutants. They were grown on solid MS medium at 22.0 ± 0.3 °C as previously described (Onai et al. 2004). We measured bioluminescence rhythms and leaf movement rhythms as previously described (Okamoto et al. 2005a,b,c; Onai et al. 2004). Light intensity was 70 µmol m^–2 s^–1 from white fluorescence lamps.

Measurement of flowering time

Seeds were placed in Milli-Q water (Millipore K.K., Tokyo, Japan) at 4 °C for 2 days and grown on soil at 22.0 ± 0.3 °C under long-day (16 h light/8 h dark) or short-day (10 h light/14 h dark) conditions. Light intensity in the light period was 100 µmol m^–2 s^–1 from white fluorescence lamps. We determined the flowering time by counting the number of leaves (rosette and cauline leaves) when the inflorescence was 1.5-cm high (Ohto et al. 2001).

Map-based cloning of PCL1

We crossed F₃ homozygotes of the pcl1-1 mutant (ecotype Col-0) to Ler and used F₂ homozygotes from the cross for genetic mapping. We selected the F₂ homozygotes by measuring the bioluminescence in LL of plants carrying a P_GI::LUC⁺ reporter gene and confirmed their phenotypes in F₃ plants. We used publicly available cleaved amplified polymorphic sequences (CAPS) and simple sequence length polymorphisms (SSLP) as markers between Col-0 and Ler in TAIR database (http://www.arabidopsis.org/) and single nucleotide polymorphisms (SNPs) between the two strains released from the Monsanto Arabidopsis Polymorphism Collection (Jander et al. 2002). We examined SNPs by PCR amplification following direct sequencing of the genomic DNA. Fine mapping delimited PCL1 to a 150 kb interval between SNPs F18L15-1 and CAPS marker TOPP5. We sequenced the 150 kb region containing PCL1 in the genomes of both the pcl1-1 and pcl1-2 mutants and identified the two mutations by comparing their sequences to the genomic sequences of wild-type Col-0 and G-38 strains. We identified the nucleotide sequences of full-length PCL1 cDNAs from TAIR and RIKEN (RARGE; http://rarge.gsc.riken.go.jp/) EST databases and determined PCL1 gene structure by comparison with the genomic sequence. Details are described in the legend for Fig. 3.

Cellular localization of PCL1

We constructed P_CaMV35S::::PCL1::sGFP(S65T)::T_nos and P_CaMV35S::::sGFP(S65T)::PCL1::T_nos fusion genes in the plasmid pACYC177 (New England Biolabs, Beverly, MA, USA) and transferred them into onion epidermal cells by particle bombardment. After 24-h incubation at 28.0 ± 0.5 °C in DD, we observed the localization of each GFP fusion protein with confocal laser scanning microscope (FluoView 500; Olympus, Tokyo, Japan). P_CaMV35S::::sGFP(S65T)::T_nos cassette was derived from plasmid pTH2 (Chiu et al. 1996; Niwa et al. 1999).

Construction of a P_PCL1::LUC⁺ reporter strain and a P_CaMV35S::PCL1 strain

The P_PCL1::LUC⁺::T_nos reporter cassette consisted of an upstream region of PCL1 (P_PCL1; nucleotides (nt) 1–1020 under accession no. AB206576), the coding region of a modified firefly luciferase gene (LUC⁺ Promega Japan, Tokyo), and the transcriptional terminator sequence of the Agrobacterium nopaline synthase gene (T_nos). The P_PCL1::LUC⁺::T_nos reporter cassette was inserted into the SmaI site of binary vector pBIB-Hyg (Becker 1990) in the same direction as an HPT (hygromycin B phosphotransferase) gene cassette, giving pBIB/PCL1::LUC⁺. The P_CaMV35S::PCL1 cassette consisted of a cauliflower mosaic virus 35S promoter (P_CaMV35S; nt 4951–5815 in AF485783) derived from pBI121 (Jefferson et al. 1987) and the coding region of PCL1 (nt 997–2001 in AB206576). The P_CaMV35S::PCL1 cassette was inserted just upstream of T_nos in binary vector pBIB-Hyg (HindIII-SmaI region) in the same direction as T_nos, giving pBIB/35S::PCL1. We transferred the T-DNA regions of pBIB/PCL1::LUC⁺ and pBIB/35S::PCL1 into the genome of Col-0 and G-38 strains, respectively, by the Agrobacterium tumefaciens-mediated floral dip method (Clough & Bent 1998). We selected strains containing the T-DNA in a single locus as a homozygote by standard techniques (Weigel & Glazebrook 2002) and used homozygous T₃ plants as PCL1-bioluminescence reporter plants or PCL1-over-expressing (PCL1-ox) plants.

Northern blot analysis

Plants were grown on solid MS medium containing 1.5% sucrose and 0.3% gelrite for 11 days at 22.0 ± 0.3 °C in LL, exposed to three cycles of LD (12 h light/12 h dark) to synchronize the circadian clock, and returned to LL. Light intensity in the light period was 50 µmol m^–2 s^–1 from white fluorescence lamps. At 3- or 4-h intervals, we harvested ten plants of each strain in LL and immediately froze the whole plants in liquid nitrogen. We isolated total RNA from the frozen plants using RNeasy Midi kit (QIAGEN K.K., Tokyo, Japan) according to the manufacturer's instructions. The total RNA (5 µg) was separated on 1.2% formaldehyde gels, blotted on to BIODYNE Plus membranes (Pall, New York, NY, USA), hybridized with ³²P-labeled gene-specific probes with Perfect-Hyb (Toyobo, Osaka, Japan) according to the manufacturer's instructions. Signals from hybridized bands were detected and quantified with a Bio-Image Analyzer (BAS2000; Fuji Photo Film, Tokyo, Japan). We normalized signals against the densities of Coomassie Brilliant Blue-stained 18S rRNA. Probes for TOC1, CCA1 and LHY were labeled with [-³²P] dCTP as described by Makino et al. (2002). Genomic DNA fragments of GI (nt 8 064 660–8 066 052 in NC_003070 [GenBank] ), CAB2 (nt 10 474 729–10 475 025 in NC_003070 [GenBank] ), ELF4 (nt 16 741 294–16 742 019 in NC_003071 [GenBank] ), and PCL1 (nt 1021–1992 in AB206576 [GenBank] ) were labeled with [-³²P] dCTP by the random primer method. To detect only PCL1 mRNA derived from endogenous PCL1 but not from the P_CaMV35S::PCL1 transgene, we synthesized an [-³²P] UTP-labeled RNA probe from the 3'-untranslated region of endogenous PCL1 (nt 1992-2237 in AB206576 [GenBank] ) by in vitro transcription using a MAXIscript kit (Ambion KK, Tokyo, Japan).

	Note added in proof

Top Abstract Introduction Results Discussion Experimental procedures Note added in proof References

 
Recently, PCL1 was cloned independently by Dr. Steve A. Kay and his colleagues as LUX (Hazen et al. (2005) Plant Physiol. 138, 990–997).

	Acknowledgements

 
We thank Yasuo Niwa (University of Shizuoka) for his kind gift of vectors containing the P_CaMV35S::::sGFP(S65T)::T_nos gene cassette, Hironaka Tsukagoshi and Kenzo Nakamura (Nagoya University) for their help with microscopic observation of GFP-PCL1 fusion proteins, Harumi Nishimoto and Chisato Morioka (Nagoya University) for technical assistance, Kazuhisa Okamoto and Nobunori Kami-ike (Nagoya University) for improvement of the rhythm measuring and analyzing systems, and Miriam Bloom (SciWrite Biomedical Writing & Editing Services) for professional editing. We are grateful for use of TAIR and RIKEN databases and the Monsanto Arabidopsis Polymorphism Collection. This study was supported by grants to M.I. from MEXT, JSPS, BRAIN, the Japan Space Forum, and the Aichi Science and Technology Foundation. The Division of Biological Science, Graduate School of Science, Nagoya University is supported by a 21st-century COE grant from MEXT.

	Footnotes

 
Communicated by: Shunsuke Ishii

^* Correspondence: E-mail: ishiura{at}gene.nagoya-u.ac.jp

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