JXB Advance Access originally published online on July 1, 2003
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Journal of Experimental Botany, Vol. 54, No. 389, pp. 1879-1887,
August 1, 2003
© 2003 Oxford University Press
Molecular cloning and expression analysis of a CONSTANS homologue, PnCOL1, from Pharbitis nil
Received 18 February 2003; Accepted 16 May 2003
1 Department of Life Science, Sogang University, Seoul 121-742, Korea
2 School of Biological Sciences, Seoul National University, Seoul 151-742, Korea
3 Plant Metabolism Research Center, Kyung Hee University, Suwon 449-701, Korea
* To whom correspondence should be addressed. Fax: +822 704 3601. E-mail: sungkim{at}sogang.ac.kr
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Abstract |
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The Arabidopsis CONSTANS (CO) gene is a key regulator of the long day (LD)-dependent flowering pathway and two CO homologous genes COL1 and COL2 are involved in the regulation of the circadian rhythm. In order to understand the role of CO and COL in short-day plants, a CO homologue, PnCOL1, was isolated and characterized from Japanese morning glory (Pharbitis nil). The deduced PnCOL1 protein of 386 amino acid residues contained two putative zinc finger motifs at the N-terminal region and a conserved CCT domain at the C-terminal region. The deduced amino acid sequence of PnCOL1 was 34% identical to that of PnCO, but 32%, 34%, and 34% identical to those of CO, COL1, and COL2, respectively. Expression of PnCOL1 was barely detected in the cotyledons of plants grown under continuous light (CL), but highly expressed in the cotyledons of plants grown under SD. Expression of PnCOL1 showed a pattern of circadian rhythm as well as daily oscillation. The overexpression of PnCOL1 by a 35S promoter did not overcome the late-flowering phenotype of Arabidopsis co mutants. The results provided in this study suggest that PnCOL1 may have a role in the circadian rhythm in Pharbitis nil.
Key words: cDNA, floral primordia, Pharbitis nil, PnCOL1, short-day plant.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Flowering has long been a subject of plant biology due to its biological importance and agronomic impact. The transition to flowering is defined as the conversion of shoot meristems from vegetative to reproductive development and flower initiation is a key step of the reproductive processes. Plants have evolved multiple pathways to regulate flowering time, which is influenced by both environmental factors such as day length and temperature, and developmental factors associated with the age of the plant (David, 1997; Martinez-Zapater et al., 1994; Blazquez, 2000).
The genetic control of flowering has been extensively studied in Arabidopsis, a quantitative long-day plant (LDP) that flowers faster under long days (LD) than short days (SD). Molecular genetic analyses using Arabidopsis revealed many genes regulating flowering time upon LD induction. Among them, CONSTANS (CO), that encodes a zinc finger protein, has been recognized as a genetic component of the LD-dependent flowering pathway (Putterill et al., 1995). The co mutant flowers later than the wild type under LD, but shows similar flowering time to the wild type under SD. Consistently, the CO gene shows higher expression under LD than SD during the day and overexpression of CO causes early flowering even under SD (Putterill et al., 1995; Suarez-Lopez et al., 2001). Therefore, the CO gene has been assigned to the photoperiod-dependent pathway (Putterill et al., 1995). It was reported that CO activates meristem identity genes such as LEAFY and APETALA 1 (Simon et al., 1996). However, FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), not LEAFY, were found to be the immediate target of CO proteins (Samach et al., 2000). In addition, screening of mutations that suppress the early flowering effect of 35S::CO plants revealed that FT and SOC1 are downstream genetic factors of CO (Onouchi et al., 2000). These results suggest that CO has a pivotal role for regulating the LD-dependent pathway.
Photoperiod-dependent flowering requires an endogenous timekeeper, the circadian clock, that measures the duration of the day or night. The circadian clock regulates many aspects of the rhythmic processes in plants such as the daily rhythm of leaf movement and the cyclic expression of several genes, such as CAB2 and CCR2 (Somers, 1999). Several Arabidopsis mutations, for example, TIMING OF CAB 1 (TOC1), LONG HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) affect both circadian rhythm and flowering time (Somers et al., 2000; Strayer et al., 2000; Schaffer et al., 1998; Wang and Tobin, 1998; Hicks et al., 1996). In addition, the expression of CO is regulated by the circadian rhythm and CO was recently proposed to mediate between the circadian clock and the control of flowering (Suarez-Lopez et al., 2001). It has also been suggested that the interaction of CO and light signal through phytochrome A and cryptochrome 2 is the molecular basis of seasonal time measurement in Arabidopsis (Yanovsky and Kay, 2002).
In addition to CO, CO homologous genes, CONSTANS-LIKE 1 and 2 (COL1, COL2) were isolated from Arabidopsis (Putterill et al., 1997; Ledger et al., 1996). Similar to CO, COL1 and COL2 have two zinc finger motifs at the N-terminus and have a C-terminal basic region containing a putative nuclear localization signal called the CCT domain (the domain conserved in CO, COL, TOC1). By contrast with CO, the overexpression of COL1 and COL2 did not change the flowering time in Arabidopsis (Ledger et al., 2001). However, the expressions of COL1 and COL2 were under the control of the circadian clock. In addition, the overexpression of COL1 and COL2 changed the circadian rhythmicity of leaf movement and the expression of the CAB gene (Ledger et al., 2001).
Genes affecting flowering time have been isolated mainly in Arabidopsis, an LDP. Therefore, it is an interesting question if the functional roles of the flowering time genes and the relevant circadian clock components are conserved in SDP. Recently, a CO orthologue has been isolated and characterized from a qualitative SDP, P. nil (Liu et al., 2001). Expression of PnCO, a CO orthologue of P. nil showed the circadian rhythm and also complemented the Arabidopsis co mutant (Liu et al., 2001). Therefore, it has been suggested that the CO orthologue in SDP has a similar role to CO in Arabidopsis (Samach and Gover, 2001). P. nil is an ideal plant for the study of the photoperiodic induction of flowering, because young, light-grown seedlings can be induced to flower qualitatively by the exposure to a single dark period of 16 h, but fails to flower under LD (Vince-Prue and Gressel, 1985; Ono et al., 1998). The isolation and characterization of the CO homologue, PnCOL1, from P. nil is reported here. PnCOL1 showed strong homology with CO and PnCO, having two conservesd zinc finger domains at the N-terminus and a CCT domain at the C-terminus. Although PnCOL1 could not complement the co mutant, the expression of PnCOL1 was regulated by the circadian clock.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Plant samples
Seeds of P. nil Choisy cv. Violet (purchased from Marutane Co., Kyoto, Japan) were soaked in concentrated sulphuric acid (H2SO4) for 30 min, then rinsed in running tap water for 20 h and sown for germination. Conditions in the plant growth chamber were set at 26±1 °C with continuous fluorescent light (80 µmol m–2 s–1). When the cotyledons had opened maximally (6 d after the H2SO4 treatment), the seedlings were subjected to SD (8/16 h light/dark) for 3 d. Afterwards, the seedlings were grown under continuous light for 8 d at 26 °C. The emerging young floral buds (<2 mm long) that were produced in the fourth or fifth node were then harvested.
Construction and screening of cDNA librarys
Total RNAs were isolated using the methods developed by Davis et al. (1986) and Wang and Vodkin (1994). To isolate poly (A)+ RNA, oligo d(T)-cellulose spin columns (Pharmacia) were used. Approximately 5 µg of poly (A)+ RNA was used as the template and oligo d(T) as the primer for cDNA synthesis. The cDNA was ligated into the Uni-ZAP XR vector (Stratagene), then in vitro packaged using Gigapack II Gold packaging extracts (Stratagene). Escherichia coli strain XL-1 Blue MRF' [(F' Tn10, proAB, lacI qZ 
M15) (mcrA)183, (mcrCB-hsdSMR-mrr) recA1, endA1, gyrA96 (NaIr), thi-1, hsdR17 (rk–mk+), supE44, relA1, lac] served as a host for molecular clonings. The ExAssist helper phage (M13) was used for in vivo excision of the pBluescript plasmid vector from the 
ZAP II phage (Stratagene).
Cloning of PnCOL1 by polymerase chain reaction
For cloning of PnCOL1, a nested-PCR amplification was performed using sense primer (T3; 5'-AAT TAA CCC TCA CTA AAG GG-3') and two antisense degenerated primers (CoA1: 5'-C(A/G)T A(C/T)C (G/T)(A/T)A T(C/T)G TCT TCT C-3', CoA2: 5'-(C/T)TT (C/T)TC TCT (A/G)TA (C/T)CT (G/C)A-3'). Recombinant phage DNA from the cDNA library of the floral primordia was used as PCR templates. The PCR condition was 1 min at 94 °C, 1 min at 54 ° C, and 1 min at 72 ° C for 30 cycles. The amplified fragments were analysed by DNA sequencing.
Southern and northern blot analysis
Genomic DNA was extracted from young leaves by cethyltrimethyl ammonium bromide methods (Rogers and Bendich, 1988). Ten micrograms of DNA, digested with restriction enzymes (20 unit µg–1 DNA) for 6 h at 37 °C, were separated on a 0.8% agarose gel, and transferred to a Hybond-N membrane (Amersham), using a vacuum transfer system (Hoefer). For northern blot analysis, 10 µg of total RNA was resolved on a 1.3% formalehyde agarose gel and blotted onto a nylon membrane (Sambrook et al., 1989). DNA and RNA blot analyses were performed using the radiolabelled PnCOL1 probe. DNA fragments for hybridization were purified by electro-elution and radioactively labelled using [
-32P] dCTP (3000 Ci mmol–1) by the random priming method (Feinberg and Vogelstein, 1983). The hybridization and washing were performed according to Sambrook et al. (1989). Hybridization signals were either detected with a BAS-1500 image analyzer (Fuji) or exposed on Agfa RP1 film.
In situ hybridization
Young floral buds (0.2 cm) of P. nil were fixed in a solution containing 5% glacial acetic acid, 47.5% ethanol, and 3.7% formaldehyde. The fixed tissues were dehydrated with ethanol and embedded in paraffin (Leica). The paraffin-embedded tissues were sliced into 8 µm sections with a rotary microtome (Leica). The sections were attached to glass slides that were coated with VECTABONDTM Reagent (VECTOR). The in situ hybridizations were carried out essentially as described by Cox and Goldberg (1988). The dioxygenin-labelled sense or antisense RNA probes were prepared from the linearized pBluscript carrying a PnCOL1 cDNA insert using either T3 or T7 RNA polymerase. The sections were hybridized with the probes at 42 °C for 16 h in a hybridization solution (50% formamide, 300 mM NaCl, 10 mM TRIS-HCl pH 7.5, 10 mM EDTA pH 7.5, 1x Denhardt’s, 10% dextran sulphate, and 30 mM DTT) and washed in a solution containing 2x SSC for 15 min at 37 °C and 1x SSC for 15 min at RT. The hybridizing signals were detected colorimetrically using an anti-DIG Fab fragment that was conjugated to alkaline phosphatase (Boehringer Mannheim). Photographs were taken under bright-field microscope (Nikon eclipse E600).
Arabidopsis transformation with 35S::PnCOL1
The 35S::PnCOL1 construct was made as follows. PnCOL1 cDNA was amplified by using two primers, 5'-CGC GGA TCC ATG GTT ATT GC-3', 5'-CGC GGA TCC CTA CCA TAT CAA GG-3', and digested with BamHI and inserted into the BamHI site of the pCGN18 binary vector (Jack et al., 1994). Arabidopsis co mutants were vacuum infiltrated with ASE strain (Lee et al., 2000) of Agrobacterium containing the 35S::PnCOL1 construct as described by Bechtold et al. (1993). The transformants were selected on the kanamycin (50 µg ml–1) MS plate and transferred to soil. The flowering time was compared with co mutants.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Cloning of PnCOL1
To obtain the CO homologue from P. nil, nested PCR was performed. Using the recombinant phage DNA from the floral bud library as the template and T3 and degenerated oligonucleotides as primers, a PCR product of 450 bp was obtained. Sequence analysis of the PCR product showed that the clone has homology of 52% with Brassica napus CO. RNA blot analysis was performed using the PCR clone as a probe. Since the clone strongly hybridized with SD-treated cotyledon mRNA (data not shown), the clone was used as a probe for screening a full-length cDNA from the SD-treated cotyledon library. About 1.5x105 plaques were screened and five positives were detected, indicating that the clone was present at 0.003% of the total library. After secondary screening, 11 positives were picked up and excised in vivo as described in the Materials and methods. Among 11 positives, six were selected and analysed by DNA sequencing. Four clones contained sequences identical to the PCR clone. One of them was named as PnCOL1 (Pharbitis nil CONSTANS LIKE 1) based on the result of sequence analysis.
Sequence analysis of PnCOL1
DNA sequence analysis revealed that PnCOL1 has a full length putative open reading frame (Fig. 1). The clone was 1265 bp long and contained 105 bp 3'-untranslated region, and a poly(A) tail. The deduced PnCOL1 protein had 386 amino acid residues with a molecular mass of 41.6 kDa which is similar to those of PnCO or Arabidopsis CO (Putterill et al., 1995; Liu et al., 2001). The PnCOL1 protein was 34% and 32% identical to PnCO and CO, respectively, at the overall amino acid level. The homology between PnCOL1 and PnCO increases to 59% and 89% at the regions of the N-terminal zinc motif and the C-terminal nuclear localizing domain, respectively. PnCOL1 contains two putative zinc finger motifs, which are separated by 19 amino acid residues, in the amino terminal region. Each of these motifs contains a C-X2-C-X16-C-X2-C arrangement, which is similar to the zinc finger domains of GATA-1 transcription factors (C-X2-C-X17-C-X2-C) and nuclear hormone receptors (C-X2-C-X13-C-X2-C) (Putterill et al., 1995; Ramain et al., 1993; Schmiedeskamp and Klevit, 1994). These amino acids may be involved in DNA binding or protein–protein interaction and appear to be critical to the CO function as five out of seven co mutants carry mutations in the zinc finger region (Coupland, 1997). As shown in Fig. 2, both N-terminal zinc-finger domains and C-terminal NLS region of CO members are most highly conserved indicating the importance of the regions for CO function.
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Genomic organization of the PnCOL1
The Arabidopsis genome has 16 genes that belong to the CO family (Robson et al., 2001). Also at least five ESTs from rice showed significant homology to the CO (Coupland, 1997). These results indicate that the CO-related genes are members of a large gene family in the plant kingdom (Coupland, 1997; Jeong et al., 1999).
To determine if the genome of P. nil has a gene family of PnCOL1, genomic DNA was analysed by Southern blot hybridization (Fig. 3). Ten µg of DNA samples from cotyledons were digested with EcoRI, HindIII, and BamHI. The Southern blot was hybridized with the radiolabelled 1.1 kb PnCOL1 cDNA fragment. Because PnCOL1 cDNA has no HindIII and BamHI restriction sites, the result of the Southern analysis indicates that the P. nil genome may contain another close member of PnCOL1. It is suggested that this close member is not PnCO which was recently isolated as a CO orthologue (Liu et al., 2001) because the homology between PnCO and PnCOL1 is very low. Considered together, the CO genes are also composed of a gene family in the P. nil genome.
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PnCOL1 transcript analysis
The PnCOL1 mRNA level was examined in different tissues and organs. The expression of PnCOL1 was high in seedlings grown under SD, but the mRNA was less abundant in seedlings grown under continuous light (CL) (Fig. 4A). The cotyledons from SD-treated plants also had higher expression than that from plants grown under CL (Fig. 4B). The size from the hybridizing signal examined was about 1.4 kb. The abundance of the PnCOL1 transcript was different from that of CO. Whereas the CO transcript is very rare, the PnCOL1 transcript was relatively abundant and was detectable by northern blot analysis using total RNA. The Hd1 gene, a homologue of CO in rice also showed very low expression and the transcript was only detectable by RT-PCR (Yano et al., 2000). The significance of such a difference in abundance is currently not known, but may indicate the difference in the sensitivity to CO and PnCOL1 of each plant species.
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When the tissue specificity of PnCOL1 was checked, PnCOL1 mRNA was highly present in SD-treated cotyledons, which is the organ responsible for the photoperiodic induction of flowering and the production of transmissible flower-promoting substances in P. nil (Kopcewicz and Tretyn, 1998). The PnCOL1 mRNA was also detected in floral buds, but leaves, stems and roots showed barely detectable amount of expression (Fig. 4B). Since the expression of PnCOL1 was detected in the floral bud, which is a complex organ having both flower and leaf primordia, the organ was subject to in situ hybridization. Strong expression was detected in both leaf primordia and floral meristem (Fig. 5A). During vegetative development, the PnCOL1 transcript was detected in the shoot apical meristem, but was rarely detected in the rib meristem (Fig. 5C). The results of CO expression also showed that the transcript was detected in young floral buds as well as in the shoot apical meristems (Simon et al., 1996). The similarity of expression pattern between CO and PnCOL1 suggests that the function of PnCOL1 may be associated with the induction of flowering.
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Daily oscillation of PnCOL1 expression
As shown in Fig. 4, the amount of the PnCOL1 transcript was lower in seedlings or cotyledons under CL than those under SD. In order to see if the difference in the amount of the PnCOL1 transcript is due to the effect of the light regime, the daily oscillation of PnCOL1 mRNA was examined under different photoperiods. Seedlings grown under continuous light were subject to treatments of either LD (16/8 h light/dark) or SD (8/16 h light/dark). After 3 d of the treatment, the cotyledons of 12 plants were harvested every 4 h under the same photoperiodic condition. As shown in Fig. 6, the daily rhythm of PnCOL1 expression was different under LD and SD. Under inductive SD, PnCOL1 was highly expressed during the day, but the expression disappeared during the dark period (Fig. 6A). In contrast to this, under LD, PnCOL1 expression began to be detected 4 h after dawn, thus the onset of expression was delayed 4 h compared to SD (Fig. 6B). In addition, the expression of PnCOL1 was not detected 12 h after dawn, hence the period showing abundant expression of PnCOL1 was relatively shorter under LD compared to SD conditions (Fig. 6B). Such a result was somewhat different from the daily rhythm of CO expression in Arabidopsis (Suarez-Lopez et al., 2001; Yanovsky and Kay, 2002). The daily rhythm of CO expression is well correlated with external coincidence model (Davis, 2002). That is, CO expression exceeded certain threshold levels during the day under inductive LD, but never exceeded the threshold level during the day under SD, although the peak expression levels of CO under LD and SD were similar (Suarez-Lopez et al., 2001; Yanovsky and Kay, 2002). However, PnCOL1 expression could not explain such an external coincidence model because both LD and SD showed high expression during the day. This result may suggest that PnCOL1 is not directly involved in the regulation of flowering time. Similar to PnCOL1, COL1 and COL2 genes also showed a daily rhythm in the expression and high expression just after dawn (Coupland, 1997). Whether the high level of transcripts present at dawn is important for the flowering in both SDP and LDP would be an interesting subject to study.
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Control of PnCOL1 expression by the circadian clock
Recent studies of the genes involved in the regulation of flowering time in Arabidopsis revealed a close relationship between the circadian clock and photoperiodic flowering. The genes LHY, CCA1, GI, ELF3, TOC1, ZTL, and FKF1 are involved in the control of both circadian rhythm and flowering time (Somers et al., 2000; Strayer et al., 2000; Schaffer et al., 1998; Wang and Tobin, 1998; Hicks et al., 1996; Fowler et al., 1999; Park et al., 1999; Nelson et al., 2000). In addition, two genes involved in the photoperiod-dependent pathway, CO and GI, show circadian rhythmicity in the mRNA expression (Suarez-Lopez et al., 2001; Fowler et al., 1999; Park et al., 1999).
Since PnCOL1 expression showed the daily oscillation pattern, it was further examined whether the abundance of PnCOL1 is also regulated by the circadian clock. Seedlings grown under continuous light were transferred to continuous darkness. Right after the transfer to darkness, cotyledons were harvested every 4 h until 72 h of the dark treatment. Expression of PnCOL1 increased at 8 h and reached a peak at 12 h (Fig. 7). These data were different from PnCO, which was expressed at high levels between 14 h and 20 h of darkness (Liu et al., 2001). The circadian expression pattern of PnCOL1 was maintained in this 3 d experiment, although the level of the expression gets lower on the third day. The peak expression was also maintained for an exact 24 h interval. As a circadian clock-regulated positive control, PnCcr cDNA which is a homologue of grapefruit ccr (Abied and Holland, 1994) was also hybridized to the same northern blot. Accumulation of PnCcr mRNA increased slowly reaching a peak at 20 h.
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Overexpression of PnCOL1 could not rescue late-flowering phenotype in the co mutation
PnCOL1 showed similar molecular characteristics with CO such that it showed higher expression under inductive photoperiod than non-inductive photoperiod, the PnCOL1 mRNA abundance showed a daily rhythm and PnCOL1 expression was localized to the floral bud. Therefore, it was tested if the overexpression of PnCOL1 could reverse the late-flowering phenotype of the co mutant of Arabidopsis. 35S::PnCOL1 was introduced into the co-1 mutants and 44 transgenic plants were obtained. None of the transgenic plants showed earlier flowering than co-1 (Fig. 8A). By northern blot analysis using PnCOL1 cDNA as a probe, overexpression of PnCOL1 in some of the transgenic plants was confirmed (Fig. 8B). This result shows that PnCOL1 cannot complement co mutation.
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By contrast with this study’s results, PnCO could reverse the late-flowering phenotype of the co mutation when overexpressed (Liu et al., 2001). This suggests that the function of PnCOL1 is slightly different with CO and PnCO may be more functionally relevant to CO. The predicted PnCOL1 protein showed 32% and 34% amino acid homology to CO and PnCO proteins, respectively. However, the predicted protein of PnCO showed 47% homology to CO protein indicating that PnCO is more similar to the Arabidopsis CO family. Like PnCOL1, the expression of COL1 and COL2 showed a circadian rhythm (Ledger et al., 2001). However, the overexpression of COL1 and COL2 in transgenic Arabidopsis had little effect on flowering time, suggesting that COL1 and COL2 do not have a function in regulating flowering time. Although the expression of PnCOL1 showed a circadian rhythm, the molecular characteristics of PnCOL1 were different with COL1 and COL2. That is, PnCOL1 showed specificity in both temporal and spatial expression, as PnCOL1 was only detected in SD-induced cotyledon and floral buds, but COL1 and COL2 were expressed throughout development in most aerial plant organs. These results suggest that PnCOL1 may have a different function from COL1 or COL2. Further molecular characterization of the PnCOL1 gene may reveal the divergence of flowering regulatory mechanism between SDP and LDP.
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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A CO homologue, PnCOL1 was isolated from a SDP, P. nil. The sequence of PnCOL1 is highly conserved in zinc finger domains and a CCT domain, which are very important for the biological function of CO. SD, an inductive photoperiod of P. nil induced a higher expression of PnCOL1 than continuous light. The abundance of PnCOL1 transcript showed circadian rhythm as well as daily oscillation, which is similar to CO family members in Arabidopsis. The characterization of CO homologous genes in both SDP and LDP would help to understand the role of the genes in the flowering process linked to the circadian regulation of those genes.
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Acknowledgement |
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This work was supported by Korea Research Foundation Grant (KRF-2001-013-D00069) and a grant (1999G0102; 98-0401-06-01-3) from the Korea Science and Engineering Foundation to S-RK. S-JK has been supported by the Biogreen 21 Program, Rural Development Administration.
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