JXB Advance Access originally published online on August 13, 2004
Journal of Experimental Botany 2004 55(405):2015-2027; doi:10.1093/jxb/erh226
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RESEARCH PAPER |
Identification of ASK and clock-associated proteins as molecular partners of LKP2 (LOV kelch protein 2) in Arabidopsis
1Gene Research Center, Kagawa University, 2393 Ikenobe, Miki-cho Kita-gun, Kagawa 761-0795, Japan
2Department of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho Kita-gun, Kagawa 761-0795, Japan
3Laboratory of Plant Molecular Biology, RIKEN (Institute of Physical and Chemical Research), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
To whom correspondence should be addressed. Fax: +81 87 891 3405. E-mail: tkiyosue{at}ag.kagawa-u.ac.jp
Received 25 November 2003; Accepted 8 June 2004
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Abstract |
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The ADO/FKF/LKP/ZTL family of proteins of Arabidopsis thaliana Heynh. have a LOV domain, an F-box motif, and a kelch repeat region. LKP2 is a member of this family and functions either within or very close to the circadian oscillator in Arabidopsis. Promoter–GUS fusion studies revealed that the LKP2 gene was highly active in rosette leaves. In CaMV 35S:LKP2-GFP plants, GFP-associated fluorescence was detected in nuclei, suggesting that LKP2 is a nuclear protein. Yeast two-hybrid analysis demonstrated that LKP2 interacted with some Arabidopsis Skp1-like proteins (ASK), as do other ADO/FKF/LKP/ZTL family proteins, suggesting that LKP2 can form an SCF (Skp1-Cullin-F-box protein) complex that functions as a ubiquitin E3 ligase. LKP2 interacted not only with itself but also with other members of the family, LKP1 and FKF1. The two-hybrid analysis also demonstrated that LKP2 interacted with TOC1, a clock component, but not with CCA1 or LHY, negative regulators of TOC1 gene expression. The LOV domain of LKP2 was shown to be necessary and sufficient for the interaction with TOC1. An interaction between LKP2 and APRR5, a paralogue of TOC1, was also observed, but LKP2 did not interact with APRR3, APRR7, or APRR9, other paralogues of TOC1.
Key words: Circadian clock, F-box, kelch repeat, LOV domain, yeast two-hybrid analysis
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Introduction |
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Circadian rhythms, cycles of about 24 h within living organisms, are conserved from cyanobacteria to higher plants and are thought to have developed through evolution as an adaptation to a 24 h day. The circadian rhythms are driven or co-ordinated by biological clocks, also called circadian oscillators. The expressions of a large number of genes are under circadian regulation in higher plants. Genes include those for photosynthesis, photoreceptors, photoprotection, and cold-protection; and those for carbon, nitrogen, and sulphur pathways, and starch-mobilizing enzymes (Harmer et al., 2000
). Recently, the circadian oscillator has been shown to be very important not only for daily life but also for the recognition of seasonal changes, such as in the control of plant flowering time. A flowering-time gene called CONSTANS (CO) is a key regulator in the photoperiodic pathway in Arabidopsis. The expression of CO is under the control of a circadian clock. CO expression occurs in the evening. CO levels in the presence of light seem to be the key to flowering (Schultz and Kay, 2003; Yanovsky and Kay, 2003).
The only well-characterized circadian oscillator in higher plants is a feedback loop system between TOC1 and two Myb-like DNA-binding proteins, CCA1 and LHY. TOC1 positively affects CCA1 and LHY gene expression, but CCA1 and LHY negatively regulate the expression of TOC1 (Alabadi et al., 2001). TOC1 belongs to a small protein family of pseudo-response regulators of a two-component system in Arabidopsis; it resembles response regulators of other two-component systems but lacks the invariant phospho-accepting aspartate site (Makino et al., 2000; Strayer et al., 2000). Among the members of this protein family, the accumulation of mRNAs starts sequentially after dawn at intervals of 2–3 h in the order of APRR9, APRR7, APRR5, APRR3, and APRR1 or TOC1. The expression of the APRR9 gene is induced by red light, and it is down-regulated in plants that overexpress APRR1/TOC1 (Makino et al., 2001, 2002). Members of the APRR family may modulate the circadian rhythm to fit the local environment (Michael et al., 2003).
In genome analysis of Arabidopsis, in an attempt to identify new blue-light receptors, two genes were found that encode unique proteins with a LOV domain, an F-box, and a kelch repeat region each (Kiyosue and Wada, 2000; Schultz et al., 2001). The LOV domain is a blue-light-sensing motif found in PHOT1 and PHOT2, which are involved in phototropism, chloroplast movement, and stomatal opening (Briggs et al., 2001; Briggs and Christie, 2002). The F-box is a motif found in F-box proteins, which are adapters that bring specific substrates to core ubiquitin protein ligase subunits for ubiquitination and subsequent degradation (Craig and Tyers, 1999; Xiao and Jang, 2000). In yeasts and mammals, F-box proteins form SCF complexes together with Cul1, Rbx1, and Skp1. These three proteins form a core ubiquitin ligase (E3); F-box proteins function in the recognition of the targets for ubiquitination, and their F-box motif regions are involved in the binding to Skp1. The ubiquitinated target proteins are subsequently degraded by the 26S proteasome in an ATP-dependent manner. The kelch repeats are speculated to be organized as a one-domain superbarrel (ß-propeller) structure that is similar to the structure formed by repeats of WD40, a motif found at the C terminus of both yeast and mammalian F-box proteins (Patton et al., 1998; Winston et al., 1999). These repeats are postulated to be involved in protein–protein interactions (Andrade et al., 2001). These proteins were named LKP1 and LKP2 for LOV kelch protein 1 and 2 (Kiyosue and Wada, 2000; Schultz et al., 2001). The LKP1 gene is identical to ZTL, a mutation of which resulted in a slowed rhythm of clock-regulated gene expression and leaf movement (Somers et al., 2000). LKP1/ZTL is also identical to ADO1, the T-DNA knock-out of which resulted in arrhythmia of cotyledon movement in red light (Jarillo et al., 2001). ADO1/LKP1/ZTL was shown to interact with the C-terminus region of PHYB and CRY1 (Jarillo et al., 2001). The function of LKP2 was analysed by using 35S:LKP2 plants (Schultz et al., 2001). Those plants exhibited arrhythmic phenotypes for circadian clock outputs in both constant light and darkness, long hypocotyls, and a late-flowering phenotype in a long-day condition but not in a short-day condition, which suggests that LKP2 functions either within or very close to the circadian oscillator in Arabidopsis. LKP2 is identical to ADO2 and FKL2 (Nelson et al., 2000; Somers et al., 2000; Jarillo et al., 2001). The other protein in this family is FKF1 or ADO3. The ADO3/FKF1 gene was identified as a mutated gene in an fkf1 mutant that flowers late in a long-day condition, but can be rescued by vernalization or gibberellin treatment (Nelson et al., 2000).
In this report, the promoter activity of LKP2, the intracellular localization of LKP2, and the molecular interactions of LKP2 with Arabidopsis Skp1-like proteins (ASK) and with circadian clock components and possible accessories are shown. The importance of the interaction with respect to the function of LKP2 is also discussed.
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Plant materials and growth conditions
Seeds of Arabidopsis thaliana (Columbia ecotype) were sown axenically on germination medium (GM) (Valvekens et al., 1988
) containing 0.8% agar and incubated at 4 °C in the dark for 3 d to break dormancy, and then grown under long-day conditions of 16 h of light (c. 100 µmol m–2 s–1) and 8 h of dark at 22 °C.
Analysis of DNA sequences
Plasmid DNA templates for sequencing were prepared in automatic plasmid isolation systems (models PI-200 and PI-50
, Kurabo, Osaka, Japan). DNA sequences were determined by the BigDye Terminator Cycle Sequencing method on a DNA sequencer (ABI PRISM 3100 Genetic Analyzer; Applied Biosystems, Foster City, CA, USA). The GENETYX (Software Development, Tokyo, Japan) and Sequencher (Gene Codes Corporation, Ann Arbor, MI, USA) software systems were used for the analysis of DNA sequences.
Transgenic plants
A 1.6 kb 5' non-coding region with a 20 bp overlap with the LKP2 coding sequence from the ATG initiation codon was PCR-amplified by Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA, USA) from genomic DNA of Arabidopsis. The primers used were 5'-GAGAGCTAGCTCTCCGGCAAAGTCTCGACC-3' and 5'-TCTCCCCGGGCACTCCATTTGATTTTGCAT-3', which introduced an NheI site and an SmaI site, respectively, at each end. The PCR fragment was subcloned into pCR-Blunt II-TOPO (Invitrogen, Carlsbad, CA, USA), sequenced entirely to verify the sequence, and then ligated into a promoterless ß-glucuronidase (GUS) expression vector, pBI101 (Clontech, Palo Alto, CA, USA). The resultant construct was sequenced to verify in-frame fusion of the first six amino acids of the LKP2 to GUS protein region.
The S65T modification version of GFP (smRS-GFP) (Davis and Vierstra, 1996) was used to make a GFP–LKP2 construct. The GFP coding region was PCR-amplified from psmRS-GFP from the Arabidopsis Biological Resource Center (Columbus, OH, USA) with the primers 5'-GAGATCTAGACAATGAGTAAAGGAGAAGAA-3' and 5'-TCTCAGGCCTTTGTATAGTTCATCCATGCC-3', which introduced an XbaI site and an StuI site, respectively, at each end. The LKP2 coding region was also PCR-amplified from the cDNA with the primers 5'-GAGAAGGCCTTATGCAAAATCAAATGGAGT-3' and 5'-TCTCGGATCCGATCAAGTACTTGCAGTGGT-3', which introduced an StuI site and a BamHI site, respectively, at each end. Both PCR fragments were subcloned, sequenced entirely to verify the sequence, ligated at the StuI site, and sequenced again. Then the GFP–LKP2 fragment was introduced into pBE2113, an expression vector that contains the cauliflower mosaic virus (CaMV) 35S promoter (Mitsuhara et al., 1996).
Agrobacterium-mediated transformation of Arabidopsis plants was performed by a simplified in planta infiltration method (Clough and Bent, 1998). Transgenic lines were selected on GM agar that contained 50 µg ml–1 kanamycin.
GUS staining, GFP, and DAPI observation
Histochemical localization of GUS activity in the transgenic plants was performed as described previously (Nakashima et al., 1977). Optical images of GFP activity and 4',6-diamidino-2-phenylindole (DAPI) staining were obtained by using an Olympus BX51 microscope (Olympus, Tokyo, Japan) with a fluorescent unit and suitable filter units. GFP fluorescence was also visualized by using a Leica TCS-E confocal laser scanning microscope with an air-cooled argon ion laser system (Leica Microsystems GmbH, Mannheim, Germany).
RNA gel blot analysis
Total RNA was isolated from whole plants as described earlier (Kiyosue et al., 1992), fractionated in a 1% agarose gel containing formaldehyde, and blotted onto a nylon filter. A DIG-labelled RNA probe prepared from the coding region of LKP2 cDNA or GFP was used for the hybridizations according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Chemiluminescence signals were detected by a Light Capture system (model AE-6962; Atto, Tokyo, Japan).
Yeast two-hybrid clones and assays
Columbia versions of full-length cDNAs for LKP1, LKP2, FKF1, ASK, TOC1, CCA1, LHY, APRR3, APRR5, APRR7, and APRR9 were obtained by cDNA library screening (Kiyosue and Wada, 2000; Schultz et al., 2001), by RT-PCR with ReverTra Dash (Toyobo, Osaka, Japan), or from the RIKEN Bio Resource Center (Tsukuba, Japan). The clones were PCR-amplified to generate suitable restriction enzyme sites, subcloned into pCR4-TOPO (Invitrogen), and sequenced entirely to verify the sequences. The primers used for PCR were the following:
5'-AGGATCCGTATGGAGTGGGACAGTGGTTCC-3' and 5'-AGGATCCTTACGTGAGATAGCTCGCTAGTG-3' for LKP1; 5'-AGGATCCGTATGCAAATCAAATGGAGTGGG-3' and 5'-TCTCGGATCCGATCAAGTACTTGCAGTGGT-3' for LKP2; 5'-AGGATCCGTATGGCGAGAGAACATGCGATC-3' and 5'-AGGATCCCTTTACAGATCCGAGTCTTGCCG-3' for FKF1; 5'-AGGATCCGTATGTCTGCGAAGAAGATTGTG-3' and 5'-AGGATCCTCATTCAAAAGCCCATTGGTTCT-3' for ASK1, 5'-AGGATCCGTATGTCGACGGTGAGAAAAATC-3' and 5'-AGGATCCTCATTCAAACGCCCACTGATTCT-3'for ASK2; 5'-AGGATCCGTATGGCAGAAACGAAGAAGATG-3' and 5'-AGGATCCTCACTCGAACGCCCACCTGTTCT-3' for ASK3; 5'-AGGATCCGTATGGCAGAAACGAAGAAGATG-3' and 5'-AGGATCCTCACTCGAACGCCCACTTGTTCT-3' for ASK4; 5'-AGGATCCGTATGTCGACGAAGATCATGTGG-3' and 5'-AGGATCCTCATTGAAAAGCCCATTGATTCT-3' for ASK5; 5'-AGGATCCGTATGTCGACAAAAAAGATCATG-3' and 5'-AGGATCCTCATTCAAAAGCCCATTTATTGT-3' for ASK7; 5'-AGGATCCGTATGTCGACGAAAAAGATCATG-3' and 5'-AGGATCCTCATTCAAAAGCCCATTTATTCT-3' for ASK8; 5'-AGGATCCGTATGTCGACGAAGAAGATCATA-3' and 5'-AGGATCCTCATTCAAAAGCCCATTTATTCT-3' for ASK9; 5'-AGGATCCGTATGTCGACGAAGAAGATCATA-3' and 5'-AGGATCCTCATTCAAAACCCCATTGATTCT-3' for ASK10, 5'-AGGATCCGTATGTCTTCGAAGATGATCGTG-3' and 5'-AGGATCCTCATTCAAAAGCCCATTGATTCT-3' for ASK11; 5'-AGGATCCGTATGTCTTCGAAGATGATCGTG-3' and 5'-AGGATCCTCATTCAAAAGCCCATTGATTCT-3' for ASK12; 5'-AGGATCCGTATGTCGAAGATGGTTATGTTG-3' and 5'-AGGATCCTCATTCAAAAGCCCATTGATTCT-3' for ASK13; 5'-AGGATCCGTATGTCTTCCAACAAGATTGTT-3' and 5'-AGGATCCCTATTCAAAAGCCCATGCGTTTT-3' for ASK14; 5'-AGGATCCGTATGTCTTCTAACAAGATTGTG-3' and 5'-AGGATCCCTAGGGCTTTGGATCTTCGTGTT-3' for ASK15; 5'-AGGATCCGTATGTCTTCGAAGAAGATTGTG-3' and 5'-AGGATCCTTAATTGAAAGCCCATTCGTTCT-3' for ASK17; 5'-AGGATCCGTATGGCTTCTTCTTCCGAAGAG-3' and 5'-AGGATCCTTACTCATTAAAAGTCCAAGCAT-3' for ASK18; 5'-AGGATCCGTATGTCTTCGAAAAAGATTGTG-3' and 5'-AGGATCCCTAGGGTTTTGGAACTTGTTGTT-3' for ASK19; 5'-AAGATCTGTATGTCAGAAGGTGATTTGGCC-3' and 5'-TAGATCTCAGCCTTGTGATCTGTGAAACAG-3' for ASK20A; 5'-AAGATCTGTATGTCAGAAGGTGATTTGGCC-3' and 5'-TAGATCTTGGAGATTGACCTGTATGCCGTC-3' for ASK20B; 5'-AGAGCTCGTATGGATTTGAACGGTGAGTGT-3' and 5'-AGAGCTCTCAAGTTCCCAAAGCATCATCCT-3' for TOC1; 5'-AGGATCCGTATGGAGACAAATTCGTCTGGA-3' and 5'-ACGGATCCCTCATGTGGAAGCTTGAGTTTC-3' for CCA1; 5'-AGGATCCGTATGGATACTAATACATCTGGA-3' and 5'-ACGGATCCTCATGTAGAAGCTTCTCCTTCC-3' for LHY; 5'-AGGATCCGTATGTGTTTTAATAACATTGAA-3' and 5'-AGGATCCTCAATTGTCTTCACTTCCTGATT-3' for APRR3; 5'-AGGATCCGTATGTGGCAAACGTGGCCACGT-3' and 5'-TGGATCCTATGGAGCTTGTGTGGATTGGAC-3' for APRR5; 5'-ACCCGGGTATGAATGCTAATGAGGAGGGGG-3' and 5'-ACCCGGGTTAGCTATCCTCAATGTTTTTTA-3' for APRR7; and 5'-AGGATCCGTATGGGGGAGATTGTGGTTTTA-3' and 5'-TGGATCCTCATGATTTTGTAGACGCGTCTG-3' for APRR9.
These PCR primers were also used for the RT-PCR.
Each domain construct of LKP2 was also constructed by PCR, subcloned into pCR4-TOPO, and sequenced entirely to verify the sequences. The primers used for PCR were the following:
5'-AGGATCCGTATGCAAATCAAATGGAGTGGG-3' and 5'-TGGATCCTTAGGGCCCAGGCCTTCTAGGAATTTCTTTTGC-3' for the LOV domain (L) of LKP2; 5'-AGGATCCGTATATCTCGCTCATTTACTTCT-3' and 5'-TGGATCCTTACCTTTTTGCACCGGGAACAC-3' for the F-box region (F) of LKP2; 5'-AGGATCCGTATTGGTTGGGTGCGACTGGCC-3' and 5'- TCTCGGATCCGATCAAGTACTTGCAGTGGT-3' for the kelch repeat region (K) of LKP2; 5'-AGGATCCGTATGCAAATCAAATGGAGTGGG-3' and 5'-TGGATCCTTACCTTTTTGCACCGGGAACAC-3' for LF of LKP2; and 5'-AGGATCCGTATATCTCGCTCATTTACTTCT-3' and 5'-TCTCGGATCCGATCAAGTACTTGCAGTGGT-3' for FK of LKP2.
For the LK (L+K) construct of LKP2, two PCR fragments were generated by using primer sets 5'-AGGATCCGTATGCAAATCAAATGGAGTGGG-3' and 5'-AGATCTAGGCCTTCTAGGAATTTCTTTTGC-3' for L, and 5'-AAGATCTATTGGTTGGGTGCGACTGGCCCG-3' and 5'-TCTCGGATCCGATCAAGTACTTGCAGTGGT-3' for K; subcloned into pCR4-TOPO; and sequenced entirely for verification. Then the L fragment was cut out by BamHI and BglII double digestion, and ligated into the BglII site of the pCR4-subcloned K fragment. The junction region was sequenced to confirm the in-frame ligation. The subcloned fragments were cut out with appropriate restriction enzymes and introduced into the multi-cloning sites of either pGADT7, which contains the GAL4 activation domain, or pGBKT7, which contains the GAL4 DNA-binding domain (Clontech). The ligated junction regions were sequenced to verify the in-frame ligation. Appropriate pairs of constructs were introduced into AH109 yeast cells. Transformants were grown on synthetic complete (SD) medium that lacked leucine and tryptophan, and were assayed on SD medium that lacked adenine (Ade), histidine (His), leucine (Leu), and tryptophan (Trp), but was supplemented with or without 3-amino-1H-1,2,4-triazole (3-AT). Interaction tests were performed as described in the Matchmaker GAL4 Two-Hybrid System 3 user's manual and the Yeast Protocols Handbook from Clontech by using at least three independent transformant colonies. In this system, the reporter genes ADE2, HIS3, and MEL1/lacZ are under the control of three completely heterologous, GAL4 upstream-activating sequences and TATA box elements. The HIS3 nutritional marker can be used for the identification of weak positive interactions, while the ADE2 marker is very stringent and reduces the incidence of false positives (James et al., 1996). Expression of the lacZ reporter further verifies the two-hybrid interactions and reduces the false positives. In the spot assay, overnight liquid cultures (OD600=1.5) in SD medium without Leu and Trp were spotted (5 µl) onto SD agar medium without Ade, His, Leu, and Trp, with or without 3-AT. Yeast colonies were cultured at 30 °C under red, far-red, blue, green, or white illumination (photon flux density 40 µmol m–2 s–1) (LED system: Sanyo, Osaka, Japan), or in the dark. For the ß-galactosidase activity assay, the yeast cells were pelleted by centrifugation from each overnight culture, and permeabilized by three cycles of freeze–thawing. The crude extracts were incubated with O-nitrophenyl-ß-D-galactopyranoside at 30 °C, and OD420 was recorded. The activities were expressed as Miller units.
Immunoblot analysis
For immunoblot analysis, yeast cells were grown in 50 ml YPD (yeast extract/peptone/dextrose) medium for 4–8 h (OD600=0.6), pelleted by centrifugation, and frozen in liquid nitrogen. The pellets were added to 400 µl extraction buffer (40 mM TRIS HCl pH 6.8, 8 M urea, 5% SDS, 0.1 mM EDTA, 0.4 mg ml–1 bromophenol blue, 0.8% 2-mercaptoethanol, 6.2 µg ml–1 pepstatin A, 1.86 µM leupeptin, 9.0 mM benzamidine, 23.0 µg ml–1 aprotinin, 0.77 mg ml–1 phenylmethylsulphonylfluoride), and homogenized. Crude cell extract (12 µl) was loaded onto each lane, and proteins were separated by SDS-PAGE and electrotransferred onto nylon membrane. Anti-myc or anti-HA antibodies was used to detect the proteins tested.
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Promoter activity of the LKP2 gene
The organ-specific LKP2 mRNA level had been examined previously: mRNA for LKP2 accumulated in rosette leaves, flowers, and siliques, and small amounts of mRNA were detected in roots, stems, cauline leaves, and seeds (Schultz et al., 2001
). To monitor the promoter activity of LKP2, transgenic Arabidopsis plants containing an LKP2 promoter:GUS construct (Fig. 1) were generated. GUS signals were detected in all parts of the seedlings, but they were restricted to rosette leaves and root tips in 2-week-old rosette plants. In flowers, GUS activity was detected in sepals. GUS signals were detected in young siliques, but they were undetectable in old siliques. These results agree with the LKP2 mRNA level detected by RT-PCR (Schultz et al., 2001), suggesting that the 1.6 kb promoter region has enough elements to regulate LKP2 expression.
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Intracellular localization of GFP–LKP2
To visualize the intracellular localization of LKP2, the CaMV 35S promoter-derived GFP–LKP2 fusion construct was introduced into Arabidopsis, and high-expression lines were selected by RNA gel blot analysis (data not shown). The transgenic plants showed elongated hypocotyl and late-flowering phenotype, as observed in 35S:LKP2 plants (Schultz et al., 2001
), suggesting that the introduced LKP2-GFP protein functions in the transgenic plants (data not shown). In those lines, GFP-associated fluorescence was distributed in nuclei but not in nucleoli (Fig. 2H), and very strong GFP signals were detected throughout the cells of 35S:GFP plants (Fig. 2B). These data suggest that LKP2 is a nuclear protein.
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LKP2 as an F-box protein
In Arabidopsis, more than 700 F-box proteins have been identified by genome analysis (Gagne et al., 2002
; Kuroda et al., 2002; Risseeuw et al., 2003). However, since these annotation data were mainly based on computer program analysis, proteins that have no F-box activity or have lost their activity during evolution may be included in these numbers. To examine whether LKP2 functions as an F-box protein, interaction tests were performed with Arabidopsis SKP1-like (ASK) proteins in a yeast two-hybrid system (Fig. 3A). Among 19 ASK proteins (ASK1-5, ASK7-14, ASK16-19, ASK20A, and ASK20B) tested, LKP2 interacted with 9 (ASK1-5, ASK11, ASK14, ASK20A, and ASK20B). The interactions of LKP2 with ASK proteins were similar to those of the other two with the ASK proteins, except with ASK3-5, ASK20A, and ASK20B. Since amino acid sequences of F-box regions of LKP1, LKP2, and FKF1 are similar to each other (LKP1 versus LKP2, 68.1%; LKP2 versus LKP3, 63.8%; LKP1 versus LKP3, 66.0% identity), the ASK preference (ASK1, ASK2, ASK11, and ASK14) of these proteins is not surprising. Furthermore, ASK1, ASK2, and ASK11 interact with a wide range of F-box proteins in Arabidopsis (Gagne et al., 2002; Risseeuw et al., 2003). The F-box regions of LKP1, LKP2, and FKF1 are sufficient to bind to ASK proteins (Fig. 3B–D).
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Interaction of LKP2 with ADO/FKF/LKP/ZTL family proteins
In fission yeasts and mammals, F-box proteins were reported to function as homo- and heterodimers (Kominami et al., 1998
; Suzuki et al., 2000). Furthermore, self-dimerization of WC-1 protein, a LOV domain containing a Zn-finger transcription factor involved in blue light sensing in Neurospora, was reported (Ballario et al., 1998). Therefore, the interactions of LKP2 with ADO/FKF/LKP/ZTL family proteins were examined by using the yeast two-hybrid system. Interactions were observed between LKP2 and LKP1, LKP2 and LKP2, and LKP2 and FKF1, but none were detected between LKP1 and LKP1, or LKP1 and FKF1 by yeast growth tests on restricted medium (Fig. 4A). The interactions were also supported by the ß-galactosidase activity (Fig. 4B). These results suggest that LKP2 may function in combination with LKP family proteins. The effects of light quality on yeast growth on restricted medium under different light conditions (red, far-red, blue, green, or white) were examined, but no growth differences were observed (data not shown).
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Interactions of LKP2 with circadian clock components
From the analysis using 35S:LKP2 plants, LKP2 was postulated to function either within or very close to the circadian oscillator (Schultz et al., 2001
). Since LKP2 is a nuclear protein (Fig. 2), an interaction analysis of LKP2 was performed with nuclear-localized clock-associated factors. In the growth test on SD medium lacking Ade, His, Leu, and Trp, a strong interaction of LKP2 and TOC1 was detected, and a weak interaction of LKP1 and TOC1 was detected (Fig. 5A). These interactions were also observed when the bait/prey configuration was inverted (data not shown). No interaction between FKF1 and TOC1 was observed. No interaction of LKP2 with CCA1 or LHY was detected. LKP1 and FKF1 also did not interact with CCA1 or LHY. The ß-galactosidase activities were tested to confirm these interactions. Strong galactosidase activities were detected in yeasts possessing LKP2 and TOC1 constructs, and weak activities were detected in yeasts possessing LKP1 and TOC1 constructs (Fig. 5B). No significant galactosidase activities were detected in yeasts possessing any other combination of constructs assayed. These results also suggest that TOC1 can interact with both LKP1 and LKP2, but not with FKF1. None of the interactions were affected by any light quality treatment that was performed (data not shown).
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The portion of the LKP2 polypeptide that binds to TOC1 was also localized by the yeast two-hybrid system. Six constructs that expressed either one or two domains of LKP2 were examined (Fig. 6). Interactions between the LOV domain-containing peptides and TOC1 were detected, but no interactions were detected between TOC1 and LKP2 peptides without the LOV domain in this two-hybrid system. These results indicate that the LOV domain of LKP2 is necessary and sufficient for the binding of TOC1.
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Interactions of LKP2 with TOC1 paralogues
TOC1 is a component of a central oscillator in Arabidopsis, and forms a feedback loop with the myb-like DNA-binding proteins CCA1 and LHY. On the other hand, there are four TOC1 paralogues in Arabidopsis, namely, APRR3, APRR5, APRR7, and APRR9. To analyse the specificity of LKP2 binding, APRR family proteins were used for two-hybrid analysis (Fig. 7A, B). As in the case of TOC1, a strong interaction was detected between LKP2 and APRR5, and a weak interaction was detected between LKP1 and APRR5. These interactions were also observed when the bait/prey configuration was inverted (data not shown). No other interactions were detected between LKP family proteins and any other APRR family proteins. Light quality did not affect the interaction between LKP2 and APRR5 in those examined (data not shown). The portion of LKP2 polypeptide that binds to APRR5 was also determined by the yeast two-hybrid system. As in the case of TOC1, the LOV domain of LKP2 is necessary and sufficient for the binding of APRR5 (Fig. 8).
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In this report, the promoter activity of the LKP2 gene is shown by using a fusion of the 1.6 kb promoter region and a GUS reporter gene. High expression of the LKP2 promoter–GUS fusion was observed in cotyledons and rosette plants (Fig. 1A, B), where higher levels of promoter activity or mRNA accumulation of LKP1 and FKF1 were reported (Kiyosue and Wada, 2000
; Nelson et al., 2000). LKP2 promoter–GUS activity was also detected in sepals (Fig. 1C), where activity of LKP1 and FKF1 promoters was reported (Nelson et al., 2000); in root tips (Fig. 1B), where FKF1 promoter–GUS activity was reported (Nelson et al., 2000); and in young siliques (Fig. 1D), where GUS activity was detected in LKP1 promoter–GUS plants (Kiyosue and Wada, 2000). Since LKP2 can interact with LKP1 and FKF1 (Fig. 4), LKP2 may function with LKP1 and/or FKF1 where they express in common. It was also shown that LKP2 can interact with TOC1 and APRR5 (Figs 5, 7). Although the organ-specific expression of APRR5 has not been examined yet, expression of TOC1 and APRR5 was detected in rosette leaves (Makino et al., 2000; Sato et al., 2002), which agrees with the activity of the LKP2 promoter and the expression of LKP2 in rosette leaves (Fig. 1) (Schultz et al., 2001).
In 35S:GFP–LKP2 plants, the GFP-associated signals were distributed in nuclei but not in nucleoli (Fig. 2H). This distribution pattern is the same for that of the GFP signals in 35S:GFP–LKP1 plants (Kiyosue and Wada, 2000) and that of the immunolocalization signals for ASK1 in different Arabidopsis tissues (Farras et al., 2001), which agrees with the protein–protein interaction of LKP2 with LKP1 and ASK1 (Figs 3, 4). TOC1, which is able to interact with LKP2 (Fig. 7), is located in the nucleus (Makino et al., 2000). A YFP–TOC1 signal in transiently transfected tobacco cells showed a speckled pattern in the nucleus, suggesting that TOC1 may function in transcriptomes, spliceosomes, or proteasomes; this is consistent with LKP2s probable function as an SCF complex that ubiquitinates target protein(s), in which ubiquitination triggers the transfer of target protein(s) to proteasomes for degradation (Strayer et al., 2000). Although the subcellular localization of APRR5 has not been reported so far, APRR5 seems to be a nuclear protein, as it possesses a CCT domain in its C-terminal region. The CCT domain is a common motif found in CONSTANS, CONSTANS-like, and APRR/TOC1 family proteins, and is postulated to be important for the nuclear localization of these proteins (Makino et al., 2000; Robson et al., 2001).
In Arabidopsis, there are 21 Skp1-like ASK genes, although ASK6 and ASK15 are postulated to be pseudogenes, because they have a frame shift (Risseeuw et al., 2003). From ASK20, two proteins, ASK20A and ASK20B, are generated from the same gene because of an alternative splicing (Risseeuw et al., 2003). These results show that LKP2 as well as LKP1 and FKF1 interacted with ASK1, ASK2, ASK11, and ASK14 (Fig. 3). The yeast two-hybrid interactions of LKP1 with ASK1, ASK2, and ASK11 and of FKF1 with ASK1 have also been reported so far (Kuroda et al., 2002; Risseeuw et al., 2003). Risseeuw et al. (2003) also reported the interactions of LKP1 with ASK3, ASK4, ASK7, ASK8, ASK9, ASK12, and ASK19, which could not be detected in this study's two-hybrid analysis, except with ASK4. These differences could be due to the culture conditions and/or the vector systems. The two-hybrid assays were performed here with a stringent selection condition at 30 °C, while their assay was performed at 20 °C. The yeast two-hybrid vectors that were used in this work are more reliable, because the three reporter genes (ADE2, HIS3, and MEL1/lacZ) are under the control of three completely heterologous promoters. It is known that Skp1 proteins in animals and yeasts bind to F-box regions of F-box proteins, as well as to Cul1 and Rbx1, to form SCF complexes that function as E3 ubiquitin ligases for the target proteins that are recognized by the F-box proteins (Skowyra et al., 1999; Kamura et al., 1999). The interactions of F-box regions of ADO/FKF/LKP/ZTL family proteins with Arabidopsis Skp1-like proteins (Fig. 3B–D) suggest that these proteins are components of SCF complexes and function as E3 ubiquitin ligases.
Figure 4 shows that LKP2 interacts with LKP family proteins. LKP2 may recognize a different variety of substrates with the combination of LKP1, LKP2, or FKF1; and/or LKP2 may function in the degradation of these family proteins. Kim et al. (2003) reported that the ZTL(LKP1) protein level was controlled by a different circadian phase-specific proteasome-dependent degradation. Since LKP2 can interact with ZTL(LKP1), LKP2 may cause the ubiquitination of ZTL(LKP1).
The LKP2 LOV domain construct was sufficient for the interactions with APRR1/TOC1 and APRR5 (Figs 6, 8). The involvement of a LOV-domain-containing region for protein–protein interaction was also demonstrated in PHOT1 (NPH1) versus NPH3 (Motchoulski and Liscum, 1999). In WC-1, a blue-light-sensing transcription factor of Neurospora, the LOV domain was shown to be involved in its self-dimerization (Ballario et al., 1998). Since LOV domains are found in both blue light receptors, there is a possibility that the interaction activity of the LOV domain of LKP2 is modulated by light. However, as far as those tested, light quality did not affect the protein–protein interactions via the LKP2 LOV-domain-containing region. The possibility cannot be excluded that a GAL4 activation domain or a DNA-binding domain attached to the N-terminal side of the LKP2 LOV domain affects its light sensitivity, or a certain factor lacking in yeast is needed for the light sensing.
In the yeast two-hybrid system, LKP1 and LKP2 interacted with both TOC1 and APRR5, but no interactions were detected between FKF1 and APRR/TOC1 family proteins (Fig. 7). Although the amino acid sequences of LKP1, LKP2, and FKF1 are similar overall (LKP1 versus LKP2, 74.1%; LKP2 versus FKF1, 61.9%; LKP1 versus FKF1, 66.1%) and they all have a LOV domain, an F-box, and a kelch repeat region, the gene expression profile for FKF1 was different from those for LKP1 and LKP2. The mRNA level for FKF1 is rhythmic, whereas those for LKP1 and LKP2 are constant throughout a day (Kiyosue and Wada, 2000; Nelson et al., 2000; Schultz et al., 2001). These differences of FKF1 from LKP1 and LKP2 imply a functional distinction between FKF1 and other LKP family proteins.
The fact that both LKP1 and LKP2 interacted with TOC1 and APRR5 seems to reflect the possibility of functional redundancy of LKP1 and LKP2. Since 35S:LKP1 and 35S:LKP2 plants showed similar phenotypes, such as long hypocotyls and late flowering under long-day condition (Kiyosue and Wada, 2000; Schultz et al., 2001), and their amino acid sequences are similar (73.0% identity), it is quite natural to think that their functions are redundant. However, a plant line with a T-DNA insert in ADO1/ZTL/LKP1 that knocked out the expression of ADO1/ZTL/LKP1 showed a delayed rhythm of CCR2 expression and cotyledon tip movement (Jarillo et al., 2001), suggesting that the redundancy of LKP1 and LKP2 functions is only partial.
There are at least two possible explanations for the interactions of LKP2 with TOC1 and APRR5. One is that TOC1 and APRR5 are the targets for the ubiquitination of the SCFLKP2 complex, or that LKP2 is involved in the stability of TOC1 and APRR5. TOC1 is a well-characterized probable component of a circadian oscillator (Schultz and Kay, 2003). APRR5 was reported to affect the amplification of the circadian signals to clocks and to regulate the circadian outputs. T-DNA disruption of APRR5 shortened the period of clock-mediated leaf movement (Michael et al., 2003), and shortened the period of the expressions of CCA1 and CCR2 under blue light (Eriksson et al., 2003). In APRR5-overexpressing plants, the magnitude of mRNA levels for clock-controlled genes was decreased, whereas their rhythmicities seemed to be unchanged (Sato et al., 2002). Therefore, it is possible that in 35S:LKP2 plants, a large amount of LKP2 decreases both TOC1 and APRR5 protein levels, resulting in the arrhythmic phenotypes.
The other explanation is that TOC1 and APRR5 function in the signal transduction to LKP2 for the ubiquitination of its target protein(s). Plants that possess a point mutation of the kelch repeat region of ZTL (LKP1), which results in amino acid exchange of Asp to Asn, showed slow circadian rhythm phenotypes (Somers et al., 2000). The kelch repeat is predicted to form a beta-propeller and is postulated to function in protein–protein interactions for the circadian clock-related substrate for ubiquitination. Steve Kay's group has isolated DOF (DNA binding with one finger) transcription factors that interact with the kelch repeat region of LKP2, but not its mutated version (Asp to Asn). The overexpression of one of the factors resulted in the arrhythmic phenotype (Kay, 2002; Schultz et al., 2003). Therefore, it is possible that TOC1 and/or APRR5 modulate the interactions between such kelch repeat interacting factors and LKP2, and regulate the function of LKP2.
The interactions of LKP1 and LKP2 with TOC1 and APRR5 were shown in the yeast two-hybrid assay. To confirm these interactions, supporting data were needed, such as from co-immunoprecipitation, pull-down assay, or in vivo interactions. Although a preliminary co-immunoprecipitate assay was performed, strong interactions among those proteins were not observed (data not shown). It is considered that the interactions may be affected by the existence of some light-receiving chromophore(s), as the interaction of PHOT1(NPH1) with NPH3 requires the flavin mononucleotide (FMN) to bind to the LOV domain(s) of PHOT1 (Motchoulski and Liscum, 1999). Recently, it was reported that the LOV domain of FKF1 binds to FMN, and that the LOV domains of LKP1(ZTL1) and LKP2 possess spectral properties similar to that of FKF1 (Imaizumi et al., 2003). Therefore, the interactions of LKP1 and LKP2 with TOC1 and APRR5 via LOV domains may require the binding of suitable flavins to the LOV domains.
While this manuscript was under review, Mas et al. (2003) showed that a targeted degradation of TOC1 by ZTL modulates circadian function in Arabidopsis; they demonstrated a physical interaction of TOC1 with ZTL(LKP1), which was abolished by ztl mutations, resulting in constitutive levels of TOC1 protein expression. They also showed that the dark-dependent degradation of TOC1 protein requires functional ZTL(LKP1), and is prevented by inhibiting the proteasome pathway. Since LKP2 can recognize ZTL(LKP1) and TOC1 in two-hybrid assays (Fig. 4), it is possible that LKP2 functions in combination with LKP1 in TOC1 degradation. Further studies are needed to examine this possibility. Mas et al. (2003) showed a stronger two-hybrid interaction of ZTL and TOC1 and a weaker interaction of LKP2 and TOC1, while a stronger interaction between LKP2 and TOC1 has been shown here (Fig. 5). This is probably due to the difference between the GAL4 system used here and their LexA system. It is well known that some protein–protein interactions are not detectable in GAL4-based systems, but are detectable using a LexA-based two-hybrid system, and vice versa (Gyuris et al., 1993; Golemis et al., 1996; Mendelsohn and Brent, 1994).
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Acknowledgements |
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We thank Drs N Mizushima (NIBB) and K Ichimura (RIKEN) for helpful technical suggestions on yeast two-hybrid analysis, Ms K Sato for technical assistance, the Arabidopsis Biological Resource Center for psmRS-GFP, and RIKEN BRC for cDNA clones. This work was partly supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by a grant from TORAY Science Foundation to TK.
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Footnotes |
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* These authors contributed equally to this work.
Abbreviations: ADO, ADAGIO; APRR; Arabidopsis pseudo-response regulator; ASK, Arabidopsis Skp1-like protein; CaMV, cauliflower mosaic virus; CCA1, circadian clock associated 1; CO: CONSTANS; CRY, cryptochrome; FKF1, flavin-binding kelch repeat F-box 1; FKL, FKF-like; GFP, green fluorescent protein; GUS, ß-glucuronidase; LHY, late elongated hypocotyl-1; LKP2, LOV kelch protein 2; PHOT, phototropin; PHY, phytochrome; SCF, Skp1-Cullin-F-box protein; TOC1, timing of cab expression 1; YFP, yellow fluorescent protein; ZTL, ZEITLUPE.
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