Identification and Characterization of an Auxin ...

Mol. Cells, Vol. 18, No. 3, pp. 346-352

Molecules and Cells KSMCB 2004

Identification and Characterization of an Auxin-inducible Protein Kinase, VrCRK1, from Mungbean Chian Kwon1,†, Hye Sup Yun1,†, Peter B. Kaufman2, Seong-Ki Kim3, Tae-Wuk Kim3, Bin Goo Kang1,*, and Soo Chul Chang4,* 1

Department of Biology, Yonsei University, Seoul 120-749, Korea; Molecular, Cellular and Developmental Biology Department, University of Michigan, Ann Arbor, Michigan 48109-1048, USA; 3 Department of Life Science, Chung-Ang University, Seoul 156-756, Korea; 4 University College, Department of Biology, Yonsei University, Seoul 120-749, Korea. 2

(Received July 23, 2004; Accepted September 3, 2004)

An auxin-inducible protein kinase, VrCRK1, was isolated by a differential reverse transcriptase-polymerase chain reaction, using mRNAs extracted from auxintreated mungbean hypocotyls. VrCRK1 exhibits high homology with plant CDPKs over catalytic domains, however, it does not have any calcium-binding EF-hand which is typically shown in plant CDPKs. Auxin treatment increased the expression level of VrCRK1. However, the increased level was reduced to basal level by treatment with PCIB, an auxin inhibitor. When extracts of mungbean hypocotyls were immunoprecipitated and the resultant immunoprecipitates were used as the enzyme source, kinase activity of VrCRK1 was found, and such activity was also increased by auxin treatment. In transgenic tobacco plants that express VrCRK1, the transcript levels of some auxin-dependent genes were elevated as much as those in wild type plants treated with auxin. These results indicate that gene expression of VrCRK1 is specifically induced by auxin, and that VrCRK1 may play a role in auxin signaling via protein phosphorylation. Keywords: Auxin Signaling; Protein Phosphorylation; Transgenic Tobacco; Vigna radiata; VrCRK1.

Introduction Auxin plays key roles in various aspects of plant growth †

These first two authors contributed equally to this work.

* To whom correspondences should be addressed. Tel: 82-2-2123-6044; Fax: 82-2-313-0328 E-mail: [email protected] (BGK)/ [email protected] (SCC)

and development including cell division, cell expansion, tropisms, abscission, senescence, ripening, and flowering (Davies, 1995; Park et al., 2004). Many workers have employed molecular and genetic approaches in order to explain the mechanism of these auxin responses. Among suggested models, one of the most probable mechanisms is that the auxin signaling involves a specific proteindegradation pathway (Gray and Estelle, 2000; Reed, 2001). This pathway is known to be mediated by ubiquitin and the breakdown of a repressor that inhibits expression of auxinresponsive genes. Several auxin mutants, such as axr1 and tir1 Arabidopsis plants, reveal that auxin signaling is mediated by a ubiquitin-proteolytic system (Leyser et al., 1993; Ruegger et al., 1998). AXR1 and TIR1 encode homologous proteins to the N-terminal half of ubiquitin-activating enzyme and F-box protein, a component of a ubiquitin protein ligase complex, respectively. It is proposed that the ubiquitin pathway, mediated by AXR1 and TIR1, degrades a repressor that inhibits the expression of auxinresponsive genes. Ulmasov et al. (1997) showed that an Aux/IAA protein repressed the expression of auxinresponsive genes in a transient assay and others have shown that the Aux/IAA proteins are subjected to ubiquitin-mediated protein degradation (Ouellet et al., 2001; Ramos et al., 2001; Worley et al., 2000; Zenser et al., 2001). Therefore, it has been suggested that Aux/IAA proteins are negative regulators in auxin signaling and that degradation of Aux/IAA proteins by ubiquitin-mediAbbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; CDPK, calcium-dependent protein kinase; CRK, CDPK-related kinase; IAA, indole-3-acetic acid; NAA, α-naphthaleneacetic acid; PCIB, p-chlorophenoxy isobutyric acid; RT-PCR, reverse transcription-polymerase chain reaction.

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ated proteolysis transduce the auxin signal to physiological responses. As in other various plant responses, protein phosphorylation/dephosphorylation reactions are also involved in the regulation of auxin responses. The level of in vitro phosphorylation of an Arabidopsis MAP kinase in extracts from auxin-treated tobacco cultured cells is greater by 3- to 4fold over that of auxin-starved cultured cells (Mizoguchi et al., 1994). Auxin also induces mammalian ERK-like MAP kinase activity in Arabidopsis roots, and inhibitors of a mammalian MAPK kinase blocked the activity of an auxinresponsive promoter (Mockaitis and Howell, 2000). Kovtun et al. (1998) shows that overexpression of the catalytic domain of the tobacco MAPKK kinase, NPK1, blocked the activity of auxin-responsive promoter in maize protoplasts. Overexpression of PINOID, a serine/threonine protien kinase, results in phenotypes that are similar to those of auxininsensitive mutants in both shoots and roots of Arabidopsis (Christensen et al., 2000). When Arabidopsis rcn1, which encodes a protein phosphatase 2A regulatory subunit A, is inactivated, auxin transport is altered (Garbers et al., 1996). In this study, we identified a protein kinase gene, VrCRK1 (Vigna radiata CDPK-related kinase), from mungbean hypocotyls by use of a differential-display reverse transcription-polymerase chain reaction. VrCRK1 belongs to the family of plant CDPK-related kinases (CRKs) rather than to plant CDPKs, due to absence of an EF-hand (Furumoto et al., 1996; Lindzen and Choi, 1995). It has been reported that CRKs do not bind and are not activated by calcium (Farmer and Choi, 1999; Furumoto et al., 1996). However, the in vivo function of CRKs is not known yet. Here, we report that both transcript level and kinase activity of VrCRK1 were specifically induced by active auxin. In addition, the transcript level of tobacco auxin-dependent genes, Nt-gh3 and Nt-iaa4.3, is greatly increased when VrCRK1 is expressed in tobacco plants. These results suggest that VrCRK1 play a role at both gene and protein levels in response to auxin treatment.

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method (Sambrook et al., 1989). Poly (A)+ RNA was isolated by the use of the Poly A Tract mRNA Isolation system III (Promega) according to the manufacturer’s protocols. cDNA was synthesized from 5 µg of Poly (A)+ RNA by using oligo (dT)n primers and then amplified by PCR using degenerate primers corresponding to the amino acid sequence DLKPE/DN [5′-GAC(T)C(T)TA(G, C, T)AAG(A)CCA(G, C, T)GAA(G, C, T)AA-3′] used as a sense primer and YIAPEI/V [5′-AC(T)C (T)TCA(G, C, T)GGA(G, C, T)GCT(G, A)ATA(G)TA-3′] used as an antisense primer, conserved at catalytic subdomain VIb and VIII of serine/threonine protein kinases, respectively (Hanks and Quinn, 1991). The conditions for PCR were as follows: 1.5 min at 94°C, 1.5 min at 37°C, 2 min at 72°C for 5 cycles and 1.5 min at 94°C, 1.5 min at 45°C, 2 min at 72°C for 30 cycles. PCR products were fractionated on a 1.5% (w/v) agarose gel and ligated into the pGEMT-EASY vector (Promega). Construction and screening of a cDNA library A cDNA library was constructed from Poly (A)+ RNA extracted from auxin-treated mungbean hypocotyls, as described above, using a UniZAP cDNA library synthesis kit (Stratagene) according to the manufacturer’s protocols. The cDNA library was screened with a VrCRK1 PCR fragment as a probe according to the protocols. Six positive plaques were isolated and a clone which had the longest insert was finally selected. The nucleotide sequence was determined by using Sequenase version 2.0 (United States Biochemical) according to the manufacturer’s protocols.

Plant materials Seeds of mungbean (Vigna radiata L.) were soaked overnight in aerated tap water. Seedlings were grown on 0.5% (w/v) agar plates in the dark at 27°C with 100% relative humidity. For treatment with auxins and other chemicals, one cm-long hypocotyl segments were excised 0.5 cm below the seedling hooks and were incubated in 5 mM potassium phosphate buffer (pH 6.8) for the indicated time periods in the dark at 27°C. Ten segments were used for the incubation treatments unless otherwise indicated.

DNA and RNA gel blot analysis Genomic DNA was extracted by the cetyltrimethylammonium bromide (CTAB) method and purified by equilibrium sedimentation in a CsCl gradient. Genomic DNA (10 µg) was digested with restriction enzymes overnight, fractionated on a 0.7% (w/v) agarose gel, and blotted on Hybond H+ membranes (Amersham Pharmacia Biotech). Total RNA was extracted as described above. Total RNA (20 µg) was fractionated on a 1% (w/v) formaldehyde-agarose gel and blotted on Hybond N+ nylon membranes. Blots were hybridized with a 32P-labeled VrCRK1 probe corresponding to 1506−2636 bp (Fig. 2A) at 42°C in hybridization solution containing 50% (v/v) formamide, 6× SSPE, 0.5% (w/v) SDS, 5% (v/v) Irish cream and 100 µg⋅ml−1 denatured salmon sperm DNA. Blots were then washed three times at 65°C with 2× SSPE and 0.1% (w/v) SDS and analyzed by BAS-2500 (Fuji Film). For RNA gel blot analysis of Nt-gh3 and Nt-iaa4.3, cDNAs were obtained by RT-PCR using specific primers (5′-TTAAGGAATTCAACACT-3′ as a sense primer and 5′-CTGAACGGCGTCATTGA-3′ as an antisense primer for Nt-gh3; 5′-TCGTTGGAGATGTAC-3′ as a sense primer and 5′-ACTTTGTGTACAATC-3′ as an antisense primer for Nt-iaa4.3) (Dargeviciute et al., 1998; Roux and Perrot-Rechenmann, 1997), which were verified by DNA sequencing and used as probes.

Differential reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from untreated and auxintreated mungbean hypocotyl segments by the phenol-SDS

Determination of kinase activity of VrCRK1 For preparation of protein extracts, hypocotyl segments treated with or without auxin were ground in liquid nitrogen, homogenized in soluble

Materials and Methods

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protein extraction buffer [50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM PMSF and 0.1% (v/v) β-mercaptoethanol], and centrifuged at 30,000 × g for 30 min at 4°C. For immunoprecipitation of native VrCRK1 from mungbean hypocotyls, polycolonal antiserum against the C-terminal region of VrCRK fused to a maltose-binding protein (MBP-cVrCRK1), which has no catalytic domain and can immunoprecipitate VrCRK1 without any interference with kinase activity of the protein in the mixture, was generated in mice. Then, antibodies specific to MBP-cVrCRK1 were purified using CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) coupled with MBP-cVrCRK1 according to the manufacturer’s protocols. In order to detect VrCRK1 kinase activity, soluble proteins extracted from mungbean hypocotyls were immunoprecipitated with anti-MBP-cVrCRK1 antibody. Immunoprecipitation was performed essentially as described previously (Colon-Carmona et al., 2000). In brief, total protein extracts were first precleared with protein A-Sepharose CL-4B beads (Amersham Pharmacia Biotech) for 1 h. The beads were then removed by centrifugation. MBP-VrCRK1 antibodies were added to the supernatant and incubated with agitation for 6 h at 4°C followed by addition of protein A Sepharose CL-4B beads and incubation for 2 h at 4°C. After collection by centrifugation, the immunoprecipitates were washed and eluted with 50 mM Tris-HCl (pH 7.5). Five microliters of the immunoprecipitates were used for the enzyme reaction. The kinase assay was performed as follows: The kinase reaction mixtures contain 10 mM Tris-HCl (pH 7.5), 2 mM DTT, 15 mM MgCl2, and 1 mM EGTA, 20 µM ATP, 30 µg histone III-S (Sigma-Aldrich), and 10 µCi [γ-32P]ATP (Amersham Pharmicia Biotech). The reaction mixture was incubated for 20 min at 30°C after enzyme solution was added. Following this incubation, proteins were precipitated with TCA and eluted with SDS sample buffer. After boiling, proteins were separated on 10% (w/v) SDS-polyacrylamide gels and stained with Coomasie brilliant blue (Sigma-Aldrich). Radioactivity of the phosphorylated histones was analyzed by BAS-2500. Tobacco transformation For generation of VrCRK1-overexpressing tobaccos, the coding region of VrCRK1 was inserted downstream of a CaMV 35S promoter of the binary vector pBI121 (BD Biosciences Clontech) in which the GUS coding region was deleted (pBI-VrCRK1). Tobacco leaf discs were transformed with Agrobacterium tumefaciens strain LBA4404 carrying pBI-VrCRK1 according to methods of Horsch et al. (1988). Transgenic plants that contain the constructs were selected in the presence of 200 µg·ml−1 kanamycin. The third leaves of the transgenic tobaccos were used for RNA gel blot analysis.

Results and Discussion Isolation of an auxin-inducible VrCRK1 (Vigna radiata CDPK-related kinase) from mungbean hypocotyls In

order to isolate a protein kinase gene whose expression is induced by auxin, RT-PCRs were carried out. The RT-PCR was performed using degenerate primers corresponding to highly conserved sequences of serine/threonine protein kinases, the subdomains VIb and VIII (Hanks and Quinn, 1991). A 147 bp cDNA fragment whose amplification was increased to a greater extent in auxin-treated tissues than in control plants was identified (data not shown). Sequence analysis showed that the cDNA fragment was highly homologous to the corresponding regions of protein kinases including another conserved subdomain VII. RNA gel blot analysis shows that expression of the gene containing the fragment was increased at 5 h after auxin treatment (Fig. 1A). A mungbean cDNA library prepared from auxin-treated hypocotyls was screened with the cDNA fragment, and a full-length clone, VrCRK1, was isolated. The VrCRK1 cDNA is 2755 bp long and contains an open reading frame of 1398 bp encoding a 51.3 kDa polypeptide of 465 amino acids. The predicted protein encoded by VrCRK1 has all the catalytic subdomains of serine/threonine protein kinases and shares amino acid identity with maize CDPK (53%; accession number, T03271), Arabidopsis putative CDPK (48%; accession number, NP_191312), and tobacco CDPK (49%; accession number, CAC82998), over the catalytic domain. The C-terminus of VrCRK1, however, does not show any significant similarity to the calmodulin-like domains and does not even have a calcium-binding motif, EF-hand, which is typically shared in the family of plant CDPKs. We have also found two homologues that are highly similar to VrCRK1, designated as AtCRK1 (accession number, NP_172719) of Arabidopsis and OsCRK1 (accession number, BAA90814) of rice (Oryza sativa) (Fig. 1B). Interestingly, four patches of serine/phenylalanine-rich region have been identified in the C-termini of VrCRK1, AtCRK1, and OsCRK1 (Fig. 1B) although their function is not known. Based on the completed Arabidopsis genome sequence (Arabidopsis Genome Initiative, 2000), eight CRK genes are contained in Arabidopsis genome. To estimate the genomic complexity of VrCRK1 gene in mungbean, DNA gel blot analysis was performed using the C-terminal region of VrCRK1 as a gene-specific probe (Fig. 2A). As shown in Fig. 2B, HindIII- or XbaI-restriction digests of genomic DNA exhibited a single DNA fragment that hybridized with the probe. Taken together, these results indicate that VrCRK1 is present as a single copy gene in the mungbean genome. Gene expression of VrCRK1 When the expression of VrCRK1 gene was investigated by the gene-specific probe (Fig. 2A), the transcript level of VrCRK1 was increased by IAA (Fig. 3A), however, IAA treatment in the presence of PCIB resulted in a decrease in the level. Further, the transcript level of VrCRK1 was increased by the active

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Fig. 2. Restriction map of VrCRK1 cDNA and genomic DNA gel blot analysis. A. Restriction map of VrCRK1 cDNA. Lines are untranslated regions and a black box is a coding region. The arrow matches the region used for a gene specific probe. B. The DNA gel blot analysis of VrCRK1 performed with a gene specific probe shown in Fig. 1A. Equal amounts of restricted mung bean genomic DNA were loaded and separated on a 1% (w/v) agarose gel.

Fig. 1. Identification of VrCRK1. A. RNA gel blot analysis was performed using total RNAs extracted from mungbean hypocotyls. Hypocotyl segments were harvested from mungbean plants grown for 3 d and incubated for 0 h (the control) or for 5 h with 10 µM IAA (+ IAA), or without IAA (- IAA) in the dark. Blots were probed with 32P-labeled RT-PCR fragment. B. Comparison of deduced amino acid sequence of VrCRK1 with corresponding proteins of Arabidopsis (AtCRK1) and rice (Oryza sativa) (OsCRK1). Alignment was performed using amino acids deduced from isolated (VrCRK1) and previously reported cDNAs (AtCRK1 and OsCRK1). Roman numbers and lines above amino acids indicate conserved catalytic subdomains for serine/threonine protein kinases and conserved serine/phenylalanine patches in CRKs, respectively. Letters in black and gray boxes denote identical amino acids.

auxins, 2,4-D and α-NAA but not by β-NAA, an inactive auxin analog (Fig. 3B). These results indicate that gene expression of VrCRK1 is specifically induced by auxin. The elevated level of VrCRK1 gene is maintained throughout a 10 h period (data not shown). In order to examine the tissue-specific pattern of VrCRK1 gene expression, RNA gel blot analysis was performed with mRNAs extracted from different organs (cotyledons, leaves, hypocotyls, stems and roots) obtained from light-grown versus dark-grown mungbean seedlings. Expression of the VrCRK1 gene is clearly evident in cotyledons, hypocotyls, and roots of dark-grown seedlings, and to a lesser extent, in stems and roots of light-grown plants (Fig. 4). The expression level was significantly greater in hypocotyls than that in roots and cotyledons of dark-grown plants. No significant signal was detected in leaves and cotyledons of light-grown plants. VrCRK1 has kinase activity, which is affected by auxin treatment In order to determine whether VrCRK1 encodes a functional protein kinase, gene products were made by in vitro transcription and translation of the coding region of the gene in reticulocyte extracts. When the translated products were separated on a 10% SDS-PAGE gel, a single band of about 51 kDa was obtained, which corresponds to the molecular weight (51.3 kDa) of the predicted VrCRK1 protein (data not shown). In addition,

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Fig. 3. Expression of VrCRK1 gene. A. Effects of PCIB and/or IAA on changes in transcript level of VrCRK1. Hypocotyl segments were incubated in the presence or absence of 1 mM PCIB or 10 µM IAA for 5 h. In the fourth lane, the segments were pretreated with 1 mM PCIB for 3 h and subsequently treated with 10 µM IAA for 5 h. After the treatment, total RNA was extracted and subjected to RNA gel blot analysis. B. Effects of various auxins on transcript levels of VrCRK1. For treatment, hypocotyl segments were incubated in a solution containing 10 µM IAA, 2,4-D, α-NAA, or β-NAA for 5 h.

Fig. 4. Expression of VrCRK1 in different mungbean organs. Total RNAs were extracted from different organs of 3-day old dark-grown (Dark-grown) or 3-week old light grown mungbean plants (Light-grown) and subjected to RNA gel blot analysis. C, cotyledon; H, hypocotyl; L, leaf; R, root; S, stem.

histone III-S was phosphorylated only in a mixture containing the in vitro translated products (data not shown), indicating that VrCRK1 encodes an active kinase. In order to determine whether or not the kinase activity of VrCRK1 is also affected by auxin, immunoprecipitation was performed using soluble proteins extracted from auxin-treated hypocotyls and anti-MBP-VrCRK1 antibody.

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Fig. 5. Activity of native VrCRK1 extracted from mungbean hypocotyls. A. Specificity of anti-MBP-VrCRK1 antibody. Competition was performed by adding various amounts of inactive recombinant MBP-cVrCRK1 protein to the immunoprecipitation mixtures. Kinase assays were carried out using the immunoprecipitates as described in Materials and Methods. The amounts of the competitors are indicated above the gel photos. B. Timecourse for the auxin effect on the kinase activity of VrCRK1. Kinase assays were performed using immunoprecipitates extracted from hypocotyl segments incubated with 10 µM IAA for the different time periods indicated above the gel photos. C. Changes in relative histone kinase activity of VrCRK1. Assay of the kinase activity was performed using the immunoprecipitates extracted from hypocotyls segments untreated (- IAA) or treated with 10 µM IAA for 30 min (+ IAA). Relative amounts of radiolabeled histone III-S were detected and analyzed by use of BAS2500 (Fuji film). The intensity of histone bands under autoradiography reveals that the same amounts of histones were loaded on the gel regardless of the treatment conditions.

The kinase activity of VrCRK1 increased about 1.5-fold within 30 min of auxin treatment and reached a maximum level after 1 h; after that, it returned to the basal level (Fig. 5B). This result indicates that the kinase activity is auxininducible and the enzyme reaction occurs rapidly and transiently. However, such changes in the enzyme activity were not found when extracts of non-auxin treated hypocotyls were used as the enzyme source (Fig. 5C). When the kinase activity of the immunoprecipitates was exam-

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Fig. 6. Overexpression of VrCRK1 in tobacco. A. Transcript level of VrCRK1 in transgenic tobaccos overexpressing VrCRK1 (35S::VrCRK1). Total RNAs were extracted from wild type (WT) or selected transgenic tobaccos independently and used for RNA gel blot analysis. RNA gel blot analysis was carried out using the gene specific probe of VrCRK1 as in Fig. 2A. B. Transcript level of Nt-gh3 and Nt-iaa4.3 in VrCRK1-expressing transgenic tobaccos (35S::VrCRK1). Total RNAs were extracted from leaves of wild type tobaccos treated with (IAA) or without (Cont) 10 µM IAA for 5 h, or from those of VrCRK1overexpressing transgenic tobaccos (1, 9, 14, and 16).

ined in the presence of various concentrations of the recombinant MBP-cVrCRK1 proteins which do not have the catalytic domains of protein kinases, the kinase activity of the resultant immunoprecipitates decreased in proportion to the amount of the competitors added, indicating that the antibodies are specific to VrCRK1 (Fig. 5A). When the immunoprecipitates were tested by in-gel kinase assay without any substrate, there was no band showing evidence of phosphorylation. This indicates that VrCRK1 has no autophosphorylation activity (data not shown). Similar transient activation of protein kinases by continuous stimuli has been reported in barley and tobacco plants (Knetsch et al., 1996; Sessa et al., 1996; Suzuki and Shinshi, 1995). Taken together, it is likely that auxin affects the expression of VrCRK1 in two ways: one is by activation of VrCRK1 kinase activity for a short timeperiod, and the other is by elevation and maintenance of VrCRK1 transcript level for a long time-period. It is also likely that auxin may exert its role on both the fine tuning and effectiveness of the auxin response via these two possible pathways in mungbean hypocotyls. Overexpression of VrCRK1 in tobacco In order to fur-

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ther investigate the role of VrCRK1 in auxin responses, VrCRK1 was expressed in tobacco plants under the control of the cauliflower mosaic virus 35S promoter. Four independent kanamycin-resistant plants (lines 1, 9, 14, and 16) that express the VrCRK1 gene were selected (Fig. 6A). We examined the expression levels of Nt-gh3 and Nt-iaa4.3, tobacco versions of GH3 and Aux/IAA genes, respectively (Dargeviciute et al., 1998; Roux and PerrotRechenmann, 1997), in these transformants. RNA gel blot analysis shows that the transcript levels of Nt-gh3 and Ntiaa4.3 increased in response to auxin (Fig. 6B, lanes 1 and 2), confirming the previous results that those genes are auxin-responsive respectively (Dargeviciute et al., 1998; Roux and Perrot-Rechenmann, 1997). As shown in Fig. 6B, in all the VrCRK1-overexpressing tobacco lines, the transcript levels of Nt-gh3 were greater than that of untreated wild type tobacco, however the levels were lesser than or similar to that of auxin-treated one. Furthermore, the transcript levels of Nt-iaa4.3 increased to levels that were similar to (line 1) or higher than (lines 9, 14, and 16) that of auxin-treated wild type tobacco as well as untreated one. These results propose that VrCRK1 protein positively regulates the transcription of auxinresponsive genes in tobacco. In the present work, we report that the transcript level and kinase activity of VrCRK1 are specifically induced by auxin, and that the increased kinase activity becomes rapidly reduced in mungbean hypocotyls. In addition, the transcript level of tobacco auxin-dependent genes, Nt-gh3 and Nt-iaa4.3, is greatly increased when VrCRK1 is expressed in tobacco plants. These results suggest that VrCRK1 plays a role at both gene and protein levels in response to auxin treatment. Auxin signal is proposed to be transduced by a specific proteolytic pathway and a protein kinase(s) is suggested to be involved in targeting a repressor to be degraded by the pathway (Gray and Estelle, 2000; Reed, 2001). To gain a further understanding of involvement of VrCRK1 in auxin signaling, it will be required to determine whether or not VrCRK1 can phosphorylate an Aux/IAA protein doomed to be degraded via ubiquitin-mediated proteolysis.

Acknowledgments This work was supported in part by Grant R01-2002-000-00367-0 from Korea Science and Engineering Foundation, by Korea Research Foundation Grant KRF-2000041-D00267, and by the Postdoctoral Research Program of Chung-Ang University 2003 year (Hye Sup Yun).

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