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First published online October 1, 2004; 10.1104/pp.104.046805 Plant Physiology 136:3124-3133 (2004) © 2004 American Society of Plant Biologists
Overexpression of Constitutive Differential Growth 1 Gene, Which Encodes a RLCKVII-Subfamily Protein Kinase, Causes Abnormal Differential and Elongation Growth after Organ Differentiation in Arabidopsis1Division of Biological Sciences, Graduate School of Environmental Earth Science, and Division of Biological Sciences, Graduate School of Science (H.M., K.T.Y.), Hokkaido University, Sapporo 060–0810, Japan; Kihara Institute for Biological Research, Graduate School of Integrated Science, Yokohama City University, Yokohama 244–0813, Japan (N.Y., K.H.); and RIKEN, Wako, Saitama 351–0198, Japan (T.A.)
To better understand genetic regulation of differential growth of plant organs, a dominant and semidwarf mutant, constitutive differential growth 1-Dominant (cdg1-D), was isolated utilizing the technique of activation tagging. cdg1-D showed pleiotropic phenotype including dwarfism, exaggerated leaf epinasty, and twisted or spiral growth in hypocotyl, inflorescence stem, and petiole. Hypocotyls of cdg1-D were longer than those of wild type under light conditions. The phenotype was caused by activation tagging of CDG1 gene that encodes a receptor-like cytoplasmic kinase of RLCKVII subfamily. When treated with high concentrations of brassinolide, light-grown wild-type seedlings showed long hypocotyls and strong leaf epinasty as observed in cdg1-D seedlings. Treatment of cdg1-D with brassinazole, a specific inhibitor of brassinosteroid (BR) biosynthesis, did not rescue the mutant phenotype. Gene expression of CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM involved in BR biosynthesis and phyB ACTIVATION-TAGGED SUPPRESSOR1 that inactivates BR was repressed and induced, respectively, in cdg1-D plants, suggesting constitutive activation of BR signaling in the mutant. CDG1 was expressed at a very low level in all the organs of the wild type tested. We isolated two independent intragenic suppressors of cdg1-D. However, they showed normal morphology and responded to BR in a similar manner to wild type. Taken together, CDG1 gene may interfere with signal transduction of BR when overexpressed, but is not an essential factor for it in the wild type.
After differentiating in the apical meristem or embryo, plant organs continue to grow to reach their adult size, accompanying cell division and enlargement (Howell, 1998  ). Plant cells in an organ do not always grow uniformly, resulting in differential growth. Since plant cells are surrounded by cell walls, they do not change their position relative to neighbor cells. Thus, differential growth is one of the major means for plant organs to change their shape after cell division ceases. Plants change their form depending on their developmental stage and in response to environmental conditions. Consequently, plants often exhibit differential growth throughout their life. The process of differential growth is important for plant development and survival and represents a fundamental difference in how plants and animals react to environmental change.
Phototropism and gravitropism are the best characterized differential-growth responses induced by environmental conditions. Auxin plays a central role in these processes (Iino, 2001
Activation tagging with the enhancer from the cauliflower mosaic virus (CaMV) 35S transcript promoter is a technique in plant functional genomics that can create transgenic plants in which the T-DNA carrying 35S enhancer at its right border is spliced into the plant genome at random sites (Hayashi et al., 1992 In an attempt to determine factors involved in genetic regulation of differential growth of plant organs, we have carried out screening of gain-of-function mutants by use of activation-tagging methods in this study. From 10,000 activation-tagged lines, a novel, dominant, and semidwarf mutant, constitutive differential growth 1-Dominant (cdg1-D), was isolated. Although adult cdg1-D mutants have a semidwarf phenotype, cdg1-D hypocotyls are longer than wild-type hypocotyls under white light. In cdg1-D, the hypocotyl, stem, petiole, and fruit grow in a spiral or twisted shape and the leaves are small and show severe epinasty. Here we show that these defects were induced by activation of a gene for a Ser/Thr protein kinase and that the kinase may interfere with BR signaling.
Mutant Screening of Activation-Tagging Lines
We generated approximately 10,000 activation-tagged primary lines of Arabidopsis ecotype Columbia by using pPCVICEn4HPT (Hayashi et al., 1992 As a result, we isolated a dominant mutant that showed a twisted or spiral hypocotyl and an open-hook structure in the dark and excessively epinastic leaves when grown in the light condition. Three-quarters of the T2 progeny of this line showed the mutant phenotype and hygromycin resistance. F1 plants of the mutant allowed to backcross to Columbia also showed the mutant phenotype. These traits of the mutant are consistent with it having a single dominant allele. This dominant mutation was named cdg1-D after its leaf epinasty and twisted growth in the hypocotyl.
Defects of the cdg1-D mutant were pleiotropic. cdg1-D plants were dwarf and had small and remarkably epinastic leaves (Fig. 1, a–c). Mature leaves of the mutant were smaller than those of the wild type. Rosettes of the mutant plants showed a pineal shape because of its small, curly leaves and shorter petioles (Fig. 1c). Mutant plants bore only a small number of seeds. These phenotypes were more severe in homozygous cdg1-D plants (Fig. 1c) than heterozygous plants (Fig. 1b), indicating that cdg1-D is a semidominant mutation.
cdg1-D hypocotyls grew in a twisted manner (Fig. 1e) or sometimes spirally in the dark with a vertical screw axis showing normal gravitropic responses. The hypocotyls were as long as those of the wild type. Twisted growth was also seen in various organs of adult cdg1-D plants such as inflorescence stem, petiole, and fruit (Fig. 1d). Petioles of cdg1-D also sometimes showed spiral growth (Fig. 1g, arrowhead). All leaves of cdg1-D, including cotyledons, rosette, and cauline leaves, showed exaggerated epinasty (Fig. 1, c and f). cdg1-D leaves were bent at the junction of the blade and petiole in a longitudinal direction before the leaf blade began to expand (Fig. 1g). Then they started to expand and curl in both longitudinal and latitudinal directions. Microscopic observation was performed to examine cellular basis of abnormal differential growth such as leaf epinasty and twisted growth of hypocotyl and inflorescence stem (Fig. 2). The overall anatomy of the mutant organs appeared unaltered, suggesting that the mutation did not affect organ differentiation. But tissue development in each organ was disordered in the mutant. Cortex and epidermal cells of cdg1-D hypocotyl expanded randomly (Fig. 2, d and h). They became uneven in shape and size. cdg1-D tissue had extensive intercellular spaces in comparison with wild type. Consequently, the surface of cdg1-D hypocotyls was not as smooth as that of the wild type. Longitudinal observations of cdg1-D hypocotyls after immersion in India ink also showed a distortion of epidermal cell files and rough surface, which was dented and bulged from place to place (Fig. 2, b and f).
Epidermal and cortex cells of cdg1-D were also enlarged randomly in inflorescence stems, while those of the wild type were small and organized in order (Fig. 2, i and j). In contrast, the central stele of the mutant was relatively normal, with a smooth and round shape (Fig. 2j). These observations suggest that abnormal radial growth of the cortex and epidermal cells in cdg1-D disrupts epidermal cell files and causes twisted or spiral growth in both hypocotyls and inflorescence stems. Leaf epidermal cells on the adaxial side were generally larger than those on the abaxial side in wild-type plants (Fig. 2k). In cdg1-D, epidermal cells on the adaxial side were larger than those of wild type, while those on the abaxial side were much smaller than those of wild type, especially in the region of the leaf margin (Fig. 2l). Namely, the difference in cell size between the adaxial and abaxial epidermal cells was expanded by the cdg1-D mutation. The amplified differential expansion of leaf cells appeared to be the cause of leaf epinasty in the latitudinal direction displayed in the mutant. Leaf parenchyma cells of cdg1-D, like the adaxial epidermal cells, also swelled. Epinasty in the longitudinal direction was also increased in a similar manner (data not shown). Underdevelopment of vascular tissues, another prominent characteristic of cdg1-D, was observed in all organs examined (Fig. 2, d, h, j, and l).
Hypocotyls of cdg1-D were about three times longer than those of the wild type under white-light condition (Fig. 1f). cdg1-D hypocotyls had longer epidermal cells than those of wild type (Fig. 2, e and f). Therefore, the longer cdg1-D hypocotyls probably resulted from an increase in length rather than an increase in the number of epidermal cells. Since plant hormones are often involved in the long-hypocotyl phenotype, effects of growth regulators were examined on the growth of hypocotyls under white-light conditions. Seedlings were grown for 1 week on agar plates that contained various substances, including gibberellin (1 µM GA3), BR (1 µM brassinolide [BL]), inhibitors of ethylene synthesis (1 µM aminoethoxyvinylglycine) and perception (1 µM AgNO3), ethylene precursor (10 µM 1-aminocyclopropane-1-carboxylic acid [ACC]), and a synthetic auxin (0.1 µM 2,4-dichlorophenoxyacetic acid; Fig. 3a). GA treatment increased hypocotyl length about 2-fold in both wild-type and cdg1-D seedlings, showing that GA exhibited an additive interaction with the cdg1-D mutation. GA treatment did not affect leaf morphology in either the wild type or cdg1-D (Fig. 3, d and g).
BR also promoted hypocotyl elongation in both wild type and cdg1-D (Fig. 3a). Dose response of the BR effects in Figure 3b showed that BL started to increase hypocotyl length of the wild type at 10 nM and that the promotive effects reached a plateau at 1 µM. In contrast, growth of cdg1-D hypocotyls was not affected as much as the wild type. Consequently, cdg1-D hypocotyls were as long as those of the wild type at BL concentrations higher than 1 µM. Since BL treatment also induced leaf epinasty in wild type (Fig. 3e), wild-type plants grown in the presence of 1 µM BL looked very similar to cdg1-D grown in the same condition (Fig. 3, e versus h) and somewhat resembled cdg1-D grown in the absence of the hormone (Fig. 3, e versus f). However, treatment of cdg1-D with 1 µM brassinazole, a specific inhibitor of BR biosynthesis (Asami and Yoshida, 1999 ; Min et al., 1999), did not restore the aberrant phenotype of cdg1-D (Fig. 3, i–k). Thus, BL treatment appeared to mimic the cdg1-D phenotype in wild-type seedlings under white-light conditions, and the mutant phenotype was not likely due to overproduction of BR in it. Neither aminoethoxyvinylglycine nor AgNO3 had any effects on hypocotyl elongation (Fig. 3a) or leaf epinasty (data not shown) in the wild type or cdg1-D. On the other hand, ACC induced hypocotyl elongation in the wild type. In the presence of ACC, hypocotyls of the wild type were more than twice as long as those of the control. They were, however, still shorter than those of cdg1-D (Fig. 3a). ACC also induced leaf epinasty in the wild type (data not shown), but its effects were smaller in extent than those observed by either the cdg1-D mutation (Fig. 3f) or BR treatment (Fig. 3e). These results suggest that ethylene is not a major factor for the expression of the cdg1-D phenotype, although sensitivity to ethylene was reduced in cdg1-D. Similarly, 2,4-dichlorophenoxyacetic acid had only a slight promotive effect on hypocotyl elongation in both the wild type and cdg1-D (Fig. 3a).
The CDG1 gene was cloned by plasmid rescue. Nucleotide sequences adjacent to both ends of T-DNA perfectly matched sequences in a P1 genomic clone, MOJ10, which is mapped on chromosome 3. The tetramerized CaMV 35S enhancer sequences were perfectly conserved in the fragment. A hypothetical gene (GI, 9279618, At3g26940) with similarity to Ser/Thr protein kinases was downstream of the enhancer sequences. Since no expressed sequence tags were available for the predicted gene, cDNA was isolated by the PCR after reverse transcription of RNA (RT-PCR) using primers designed to amplify the fragment from the putative start to stop codons of this gene. Results of 3' and 5' RACE showed that polyadenylation occurred 146 bp downstream of the putative stop codon and that the 5' initiation site was located 332 bp upstream of the putative start codon. These results indicate that the CDG1 gene is transcribed into a sequence of 1,754 nucleotides and consists of 5 exons (Fig. 4a). T-DNA was inserted at 47 bp upstream of the determined 5' end of the CDG1 transcript. No canonical TATA box was found upstream of the initiation site. The CDG1 transcript contained two open reading frames (ORFs) in addition to the protein kinase ORF at the 5' end (Fig. 4a, indicated by asterisks). Start codons of these upstream ORFs (uORFs) were 242 and 79 bp upstream of the start codon of the protein kinase ORF, respectively. The uORFs can be translated into short peptides, 11 and 8 amino acid residues, respectively, that exhibit no similarity to any of the protein sequences in the GenBank database.
The longest ORF of the CDG1 gene can be translated into a sequence of 431 amino acid residues (Fig. 4b) that contains all the 11 conserved subdomains of eukaryotic protein kinases. All the invariant amino acid residues are conserved in their proper positions (Hanks and Quinn, 1988 ). The putative kinase domain is flanked by short nonkinase domains on both sides. Since this protein contains no membrane-spanning regions or extra membrane domains, it is likely to be either a cytoplasmic or nuclear kinase.
An RNA gel-blot analysis using the CDG1 kinase ORF as a probe revealed overexpression of this gene in the cdg1-D mutant (Fig. 5a). In the wild type, no signal of CDG1 was detectable in 10 µg poly(A)+ RNA, while a signal was observed in cdg1-D using 0.1 µg poly(A)+ RNA. Therefore, CDG1 mRNA appeared to accumulate more than 100-fold in cdg1-D. This result suggests that the abnormalities of cdg1-D plants result from activation tagging of the CDG1 protein kinase gene. Expression of the CDG1 gene was higher in homozygous cdg1-D than in heterozygous cdg1-D (data not shown), suggesting that the severity of the cdg1-D phenotype depends on the dose of the CDG1 gene. Expression of the CDG1 gene was not detected in the wild type by RNA gel-blot analysis, but it was observed by RT-PCR in various organs of the wild type, such as root, stem, leaf, flower, and fruit (Fig. 5b). The amounts of CDG1 messages seem to be slightly higher in the flower and fruit. These results show that CDG1 mRNA accumulates at a very low level in all organs of wild-type plants.
Under control of the CaMV 35S promoter in the sense orientation, kinase ORF of the CDG1 mRNA was introduced into the genome of the wild type by the use of Agrobacterium tumefaciens. Of the nine independent T1 lines we generated, five lines showed dwarfism and epinastic leaves as observed in cdg1-D mutants. Their phenotype was heritable in T2 generation, linking with selection markers of T-DNA. Twelve-day-old plants of these transgenic lines were almost identical to cdg1-D plants with respect to their morphology (Fig. 6). They showed all the phenotypes seen in cdg1-D including dwarfism, twisted stem and fruit, and frizzy growth in dark condition and long hypocotyls under white-light condition (data not shown). Therefore, all the phenotypes seen in cdg1-D were reproduced in the CDG1 overexpressors. These results indicate that the cdg1-D phenotype is caused by overexpression of the protein kinase encoded by the CDG1 gene.
On the other hand, one of the nine transgenic lines showed the wild-type phenotype, and the other three lines exhibited weaker phenotype. Rosette leaves of the latter lines were as large as those of wild type; they displayed epinastic and twisted growth that was often seen in BR-treated plants, but their petioles were not as elongated as BR-treated ones (data not shown). These results clearly indicate that overexpression of CDG1 activates signaling pathway that is overlapping, but not identical with BR signaling.
Recently BR biosynthesis has been shown to be controlled in a feedback fashion (Fujioka and Yokota, 2003
Isolation and Characterization of cdg1, a Loss-of-Function Mutation of CDG1 By screening 10,000 M2 seeds, we isolated two independent suppressor mutants of cdg1-D that showed hygromycin resistance and suppressed all the phenotypes of cdg1-D. F1 plants of both mutants that were allowed to backcross with Columbia also showed the wild-type phenotype and hygromycin resistance. These traits of the suppressors suggest two possibilities: the mutations occurred at dominant loci, or they were intragenic suppressors that had mutations within the CDG1 gene downstream of the enhancer sequences in the T-DNA. Genomic sequencing of each line revealed a single-base substitution in CDG1 gene that results in a premature stop codon in exon 4 or disruption of a splice site junction between intron 3 and exon 4 (Fig. 4a). These findings mean that the mutated and overexpressed CDG1 mRNA in each allele should give rise to a nonfunctional protein that contains only subdomains I to V (Fig. 4b). Therefore, homozygotes of these suppressors must be loss-of-function mutants of CDG1 and were named cdg1-1 and 2, respectively. In contrast to the pleiotropic effects of overexpression of the CDG1 gene, cdg1-1 and 2 showed normal morphology in all organs (data not shown). We also examined effects of BR and GA on growth of these mutant alleles and found that they responded to the hormones essentially in the same fashion as the wild type (data not shown). These effects suggest that CDG1 gene possibly interferes with signal transduction of BR when overexpressed, but is not an essential factor for it in the wild type.
BR Signaling May Be Interfered within cdg1-D at the Seedling Stage
In this study we showed that activation of the Arabidopsis CDG1 gene by the use of CaMV 35S enhancers produced a dominant mutant, cdg1-D, which displayed pleiotropic defects in organ growth regulation after organogenesis. These defects included dwarfism, exaggerated differential growth in multiple organs such as hypocotyls, leaves, and inflorescence stems, and long-hypocotyl phenotype under white-light conditions. Treatment of wild-type seedlings with only BL phenocopied the long-hypocotyl phenotype of cdg1-D. Application of BL also induces epinasty of cotyledons, resulting in a cdg1-D-like morphology. It has been well known that BR biosynthesis is controlled by an elaborate feedback regulation (Fujioka and Yokota, 2003
Interestingly, CDG1 encodes a receptor-like kinase (RLK), and a few kinases have been identified that constitute the signal transduction pathway of BR (Thummel and Chory, 2002
Although cdg1-D seedlings look like BL-treated wild-type seedlings, mature cdg1-D plants are dwarf. In contrast, transgenic plants overexpressing BRI1-green fluorescent protein fusion protein (Wang et al., 2001
Based on its amino acid sequence similarity, GDG1 was judged to encode a Ser/Thr kinase of the RLK subfamily. CDG1 kinase is closely related to PBS1 of Arabidopsis (Swiderski and Innes, 2001
Based on the kinase domain phylogeny, Shiu and Bleeker (2001)
Though several genes of the subfamily have been described in the literature, physiological significance is well characterized for only one of them, PBS1. Limited information is available on three others: ARSK1 may be involved in osmotic response of root (Hwang and Goodman, 1995
The CDG1 transcript, in addition to a major ORF for protein kinase, contains two uORFs in the 5' leader sequence (Fig. 4a). uORFs occur in about 7% to 10% of plant genes and may have a role in reducing the translation efficiency of the downstream major ORFs (for review, see Futterer and Hohn, 1996 The presence of two uORFs in CDG1 gene raises the possibility that its expression is also regulated translationally. Although an RNA gel-blot analysis revealed more than a 100-fold accumulation of CDG1 gene in cdg1-D plants (Fig. 5a), CDG1 expression may be modified by translational repression in cdg1-D plants because transcripts of CDG1 contain intact uORFs in cdg1-D (Fig. 4a). On the other hand, overexpressors of CDG1 created in this study by the use of 35S promoter, contained only the kinase ORF of CDG1 mRNA without uORFs but still had the cdg1-D phenotype (Fig. 6). Thus, it is apparent that the uORFs do not contribute to abnormal growth in transgenic plants, and this is probably also the case in cdg1-D. In wild-type plants, however, the CDG1 mRNA level is quite low, and translation of the downstream ORF may be repressed by uORF. This suggests that activity of CDG1 is tightly repressed by both transcriptional and translational control in the wild type. This is probably the reason why CDG1 overexpression causes severe neomorphological abnormalities in cdg1-D mutants. To better understand the function of CDG1 gene in BR signaling, we have isolated loss-of-function mutants of CDG1. However, we could not detect any alterations of phenotype, including responses to BR, indicating that CDG1 is not an essential factor for BR in Arabidopsis. Redundancy of RLCKVII kinases in Arabidopsis might explain no aberrant phenotype of loss-of-function mutants of CDG1, though it is rather an isolated gene in the subfamily as described above. cdg1-D, at least, could be a useful tool for dissecting cellular processes involved in differential and elongation growth caused by BR.
Plant Materials
Transformation of Arabidopsis ecotype Columbia with activation-tagging vector, pPCVICEn4HPT (Hayashi et al., 1992
Seeds were surface sterilized with 1.5% (v/v) sodium hypochlorite and 0.02% Triton X-100 for 5 min with vigorous shaking, washed several times with sterile water, chilled in water at 4°C for 2 to 4 d, and plated onto half-strength Murashige and Skoog medium (Murashige and Skoog, 1962
Plants that had been stored frozen were ground with mortar and pestle with liquid N2. Ten milliliters of cetyltrimethylammonium bromide buffer that consisted of 140 mM sorbitol, 220 mM Tris-HCl, pH 8.0, 22 mM EDTA, 800 mM NaCl, 1% sarkosyl, and 0.8% hexadecyltrimethylammonium bromide were added to the powdered tissue. The homogenate was heated at 65°C for 15 min, and the same volume of chloroform was added. The mixture was mixed gently by a rotator for 15 min and centrifuged at 3,000 rpm for 10 min. The upper aqueous phase was taken, and the same volume of chloroform was added to it again. The sample was mixed and centrifuged as described above. After centrifugation, DNA was recovered by ethanol precipitation. Then DNA was dissolved in 10 mM Tris-HCl, pH 7.6, containing 1 mM EDTA (Tris-EDTA) buffer, and centrifuged at 90,000 rpm for 16 h in 1 mg/mL CsCl (CS100GX; Hitachi, Tokyo). After centrifugation, DNA was recovered and purified and finally dissolved in Tris-EDTA buffer.
For plasmid rescue, 2 µg of DNA were digested overnight with EcoRI (Takara, Kusatsu, Japan). After phenol-chloroform extraction, digested DNA was ligated at 16°C overnight with T4 DNA ligase (Takara), precipitated with ethanol, and was used for transformation of Escherichia coli DH5
RT-PCR was performed with the Access RT-PCR system (Promega, Madison, WI), according to the manufacturer's directions. The primers for amplification of CDG1 cDNA were 5'-TTTACTTCACTTTCATGGAGTCGGAGT-3' and 5'-ATGGTTAGTTGCTTGTGTTTTCGT-3'. For amplification of CPD, BAS1, and 18S ribosomal RNA, primers were designed according to Shimada et al. (2003)
Seeds were imbibed in water in the dark at 4°C for 4 d, surface sterilized as described above, incubated for 1 d at 23°C under continuous white light in liquid medium that contained half-strength Murashige and Skoog salt, half-strength B5 vitamins, and 1% Suc, transferred onto 1% agar medium that consisted of the above nutrients supplemented with various growth regulators, and grown at 23°C under continuous white light thereafter. Hypocotyl length was measured 1 week after the transfer.
RT-PCR products of the CDG1 kinase ORF were cloned into a binary vector pBI121 (CLONTECH Laboratories, Palo Alto, CA) under the control of the CaMV 35S promoter in the sense orientation. Then, the plasmid was introduced into Agrobacterium tumefaciens strain GV3101 by electroporation. Arabidopsis wild-type plants, ecotype Columbia, were transformed with Agrobacterium by the floral dip method (Clough and Bent, 1998
About 500 mg of seeds were harvested from heterozygous cdg1-D plants, treated with 0.1% ethyl methanesulfonate for 12 h at room temperature, extensively washed with water, vernalized at 4°C for 2 d, and sown in the soil. Plants showing the wild-type phenotype were discarded from the M1 population. The M1 plants that showed heterozygous or homozygous cdg1-D phenotype were self-pollinated, and M2 seeds were collected in five pools. About 10,000 M2 seeds from each pool were plated onto agar medium containing 40 µg/mL hygromycin and grown under white light for 2 weeks. Plants that showed suppression of cdg1-D phenotype were selected and grown in the soil.
Light microscopic observations were carried out according to the method of Kurata and Yamamoto (1998) Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank data libraries under accession number AB099698.
We are grateful to Ms. N. Nishizaki for her technical assistance. This study was partly carried out in the Laboratory of Genetic Research, Center for Advanced Science and Technology, Hokkaido University. Received May 26, 2004; returned for revision July 31, 2004; accepted July 31, 2004.
1 This work was supported in part by a Grant-in-Aid for Scientific Research (B) (to K.T.Y. and N.Y.), and by Grants-in-Aid for Scientific Research in Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology, and from the National Space Development Agency of Japan and the Japan Space Forum (Ground Research for Space Utilization; to K.T.Y.). 
2 Present address: Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060–0810, Japan.
3 Present address: Laboratory of Plant Science, Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113–0033, Japan. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.046805. * Corresponding author; e-mail kty{at}sci.hokudai.ac.jp; fax 81–11–706–2739.
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TOP
ABSTRACT
RESULTS
et al., 2002
competent cells (Toyobo, Tokyo) by heat shock at 42°C. Plasmid DNA extracted from ampicillin-resistant clones was sequenced using a Thermo Sequencing Primer Cycle sequencing kit 7-deaza dGTP (Amersham, Buckinghamshire, UK) with the primer 5'-TGGACGTGAATGTAGACACGTCGA-3', which was designed from the nucleotide sequence of the T-DNA left border. Denaturing gel electrophoresis was run by a DNA sequencer (4000 L; LI-COR, Lincoln, NE).