JXB Advance Access originally published online on June 1, 2007
Journal of Experimental Botany 2007 58(10):2471-2478; doi:10.1093/jxb/erm104
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© 2007 The Author(s).
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RESEARCH PAPER |
Modulation of flower colour by rationally designed dominant-negative chalcone synthase
1Kumho Life and Environmental Science Laboratory, Gwangju 500-712, Korea
2Department of Biological Sciences, KAIST, Daejeon 305-701, Korea
* To whom correspondence should be addressed. E-mail: hanumappam{at}missouri.edu
Received 20 December 2006; Revised 4 April 2007 Accepted 17 April 2007
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Abstract |
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The intensity of flower colour, mainly determined by the amount of anthocyanin, is an important horticultural trait. To modulate flower colour intensity, post-transcriptional gene silencing (PTGS)-based technology has been widely used. The constraint of PTGS, however, is that it requires a high degree of conservation in the nucleotide sequences of the target and the silencer. Further, it is difficult to restrict PTGS to the desired tissue or organ due to its systemic spread. To overcome these problems, dominant-negative chalcone synthase (CHS) enzymes have been developed by mutating a cysteine that is essential for the catalytic activity and a methionine that protrudes into the adjoining CHS monomer, as shown through crystallography. The dominant-negative action of mutated CHS enzymes from Mazus japonicus are demonstrated using transgenic Arabidopsis. Also, the modulation of Petunia flower colour intensity by the dominant-negative CHS is shown. The data support the crystallography result showing the importance of the protruding methionine for the function of the adjoining CHS monomer. Furthermore, the modulation of anthocyanin production by the mutated Mazus CHS in Arabidopsis and petunia suggests that the dominant-negative CHS can be used even in distantly related species.
Key words: Anthocyanin, dominant-negative chalcone synthase, flavonoid, flower colour, metabolic engineering
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Flower colour is an important horticultural trait and is mainly produced by the flavonoid pigments, anthocyanins. Primarily produced to attract pollinators, flavonoids also protect the plant and its reproductive organs from UV damage, pests, and pathogens (Brouillard and Cheminat, 1988; Gronquist et al., 2001). Classical breeding methods have been extensively used to develop cultivars with flowers varying in both the colour and its intensity. The recent advance of knowledge on flower colouration at the biochemical and molecular level has made it possible to achieve this by genetic engineering (Tanaka et al., 1998).
Three different classes of anthocyanidins are responsible for the primary shade of flower colour in many angiosperms: pelargonidin (orange to brick red), cyanidin (red to pink), and delphinidin (purple to blue). The anthocyanidin biosynthetic pathway is well established and most of the enzymes involved in the synthesis have been identified (Holton and Cornish, 1995; Winkel-Shirley, 2001). It starts with the condensation of 4-coumaroyl-CoA and malonyl-CoA by chalcone synthase (CHS) to synthesize anthocyanidins that are then glycosylated by flavonoid 3-O-glucosyl transferase to produce anthocyanins. Further modification by rhamnosylation, methylation, or acylation results in a wide variety of anthocyanins (Kroon et al., 1994; Ronchi et al., 1995; Fujiwara et al., 1997; Yoshida et al., 2000; Yabuya et al., 2001). The spectral difference in flower colour is mainly determined by the ratio of different classes of anthocyanins and other factors such as vacuolar pH, co-pigmentation, metal ion complexation, and molecular stacking (Holton et al., 1993; Markham and Ofman, 1993; Mol et al., 1998; Tanaka et al., 1998; Aida et al., 2000). The final shade may be altered further by various factors including the shape of the epidermal cells or the presence of starch that gives creaminess (Markham and Ofman, 1993; Noda et al., 1994; Mol et al., 1998; van Houwelingen et al., 1998).
Genetic engineering to alter flower colour has been attempted using various genes. Some species lack a particular colour due to the absence of a biosynthetic gene or the substrate specificity of an enzyme in the pathway. For example, carnation lacks blue/purple-coloured flowers due to the absence of flavonoid 3'5'-hydroxylase (F3'5'H), while petunia lacks orange and brick-red flowers due to the inability of its dihydroflavonol 4-reductase (DFR) to reduce dihydrokaempferol (Gerats et al., 1982; Forkmann and Ruhnau, 1987). Spontaneous mutations of the flavonoid 3'-hydroxylase (F3'H) gene confer reddish flowers in blue- and purple-flowered morning glory species (Hoshino et al., 2003). Genetic engineering of blue/purple-coloured carnation was achieved by introducing the petunia F3'5'H gene and orange-coloured petunia was developed by introducing DFR from other species (Meyer et al., 1987; Brugliera et al., 2000; Johnson et al., 2001). The modulation of colour intensity has been another target for genetic engineering. Expression of biosynthetic genes such as CHS, F3H, and DFR in sense or antisense directions has been the most exploited method (van der Krol et al., 1990; Courtney-Gutterson et al., 1994; Jorgensen et al., 1996; Tanaka et al., 1998). The resulting sense suppression or antisense inhibition is collectively called post-transcriptional gene silencing (PTGS). Alternatively, transcription factors that can either activate or repress the transcription of anthocyanin biosynthetic genes have been shown to be useful in regulating colour intensity in model plants such as Arabidopsis, tobacco, and petunia (Lloyd et al., 1992; Mol et al., 1998; Borevitz et al., 2000; Aharoni et al., 2001). The overexpression of transcription factors, however, generally alters the expression of many genes, thus the commercial viability of such transgenic flowers is yet to be determined (Lloyd et al., 1994; Bruce et al., 2000).
The biochemical and structural characterization of CHS suggests the possibility of designing a dominant-negative CHS that can be used to regulate flower colour intensity. CHS is the first enzyme in the synthesis of various flavonoids including anthocyanins. It functions as a homodimer and carries out a series of decarboxylation, condensation, cyclization, and aromatization reactions at a single active site (Tropf et al., 1995). The enzyme condenses a molecule of 4-coumaroyl-CoA with three of malonyl-CoA and folds the tetraketide intermediate into an aromatic ring structure to yield chalcone (Ferrer et al., 1999; Schroder, 1999). Site-directed mutagenesis and inhibitor studies have identified the conserved cysteine and histidine residues that are important for the catalytic function of CHS (Lanz et al., 1991; Suh et al., 2000). The mutation of this cysteine to either serine or alanine has been shown to inactivate the CHS (Lanz et al., 1991; Tropf et al., 1995; Jez et al., 2000). The crystal structure of alfalfa CHS confirms that the conserved Cys164, Phe215, His303, and Asn336 form the catalytic active site (Ferrer et al., 1999). The structure also revealed that CHS functions as a homodimer, forming a symmetric dimer with each monomer where the N-terminal helices entwine with each other. The crystal structure also shows that Met137 from the adjoining monomer extends into the cyclization pocket of CHS. Using substrate and product analogues, Ferrer et al. (1999) confirm that each monomer consists of two structural domains and two functionally independent active sites as previously reported (Tropf et al., 1995). Point mutations confirm the role of Cys164 as an active site nucleophile, elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction, and suggest that Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate (Jez et al., 2000). Three interconnected cavities, a CoA binding tunnel, a coumaroyl binding pocket, and a cyclization pocket, intersect with these four residues to form the active site architecture of CHS. Each active site consists of residues from a single monomer, with the only exception being Met137 which comes from the adjoining monomer. This suggests that a CHS monomer requires the methionine from the adjoining monomer for its activity. Based on this structural information by Ferrer et al. (1999), CHS has been generated that has alanine instead of cysteine at the active site and either glycine or lysine instead of the methionine. The mutation of cysteine to alanine will result in the inactive form of CHS, while the mutation of methionine to glycine or lysine is expected to inactivate the function of an adjoining CHS if the methionine is really important, as suggested by the crystal structure. Using transgenic Arabidopsis, it is demonstrated that the mutated CHS is indeed dominant-negative. The present results confirm the importance of the methionine residue and demonstrate the utility of the dominant-negative CHS in modulating flower colour intensity even in a distantly related species.
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Materials and methods |
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Cloning and characterization of CHS from Mazus japonicus
A fragment of a putative CHS gene was cloned from Mazus japonicus, a common garden plant belonging to the Scrophulariaceae family, using the degenerate primers (5'-TAY CAR CAR GCN TGY TTY GCN GG-3', 5'-NAG DAT NGC NGG NCC NCC-3'). The full-length cDNA was cloned using the Marathon RACE kit with specific primers (5'-GTT GTC TGC TCC GAG ATC ACT-3' for 3' RACE; 5'-AGT GAT CTC GGA GCA GAC AAC-3' for 5' RACE) and the AP2 primer provided by the manufacturer (Clontech, Palo Alto, CA, USA).
To determine if the putative CHS gene encodes a functional CHS, the cDNA was amplified with primers (5'-GAG ATC TAG AAA AAT GAC GCC GAC CGT CGA GGA G-3' and 5'-GAG ATC TAG ATC AAT TCA TGA AGG GCA CAC T-3'), and the amplified coding sequence cut with XbaI was cloned into the XbaI site of GUS-deleted pBI121 vector, driven by the CaMV 35S promoter. The gene cloned into the vector was introduced into Agrobacterium strain GV3101 and transformed into Arabidopsis thaliana wild-type Landsberg erecta (Ler) or the tt4 mutant. Of the several independent homozygous lines established, three lines were chosen randomly for further analysis.
To determine the ability of the putative CHS to complement the tt4 mutation, the transgenic tt4 lines were grown for 5 d on water agar plates containing 3% sucrose (0.05% MES, 0.8% phytoagar, 3% sucrose, pH 5.7).
Construction and characterization of dominant-negative CHS
The coding sequence of MjCHS was also cloned into pTOPOII vector (Invitrogen, Carlsbad, CA, USA). To mutate the cysteine at the 165th residue to alanine (Cys165Ala), the MjCHS was amplified in pTOPOII with Pfu polymerase (Stratagene, La Jolla, CA, USA) using the appropriate primer set (5'-TTC GCC CGC GGG ACG GTC CTC-3', 5'-AGC ACC CTG CTG GTA CAT CAT-3'). The amplified product was phosphorylated by polynucleotide kinase and ligated by T4 DNA ligase. The ligated product was transformed into Escherichia coli. The mutated clone was confirmed by sequencing. To mutate Met138 to either lysine (mCHSK) or glycine (mCHSG) together with the Cys165Ala mutation, the Cys165Ala-mutated CHS clone was amplified in pTOPOII with either the K-primer set (5'-CCC GGT GCC GAC TAC CAG CTC-3', 5'-CTT GTC GAC CCC GCT GGT GGT-3') or the G-primer set (5'-CCC GGT GCC GAC TAC CAG CTC-3', 5'-GCC GTC GAC CCC GCT GGT GGT-3'). The amplified products were phosphorylated by polynucleotide kinase and ligated by T4 DNA ligase. The mutated genes were sequenced to confirm the mutations. The mutated full-length MjCHS genes, driven by the CaMV 35S promoter, were cloned into GUS-deleted pBI121 vector, and introduced into Arabidopsis. The homozygous lines were renumbered after quantitating anthocyanin.
Quantitation of anthocyanin
To determine anthocyanin levels in the transgenic plants, cold-imbibed seeds were sown on MS-agar plates containing 2% sucrose (1x MS salts, 0.05% MES, 0.8% phytoagar, 2% sucrose, pH 5.7) and grown for 5 d under continuous white light (2 mW cm–2). The quantitation was done as described before (Shirley et al., 1995). Briefly, 50 seedlings were picked and soaked in 0.5 ml of the extraction solution (100% methanol+0.5% HCl) overnight at 4 °C. The next morning, samples were centrifuged briefly and the supernatant was used for spectrophotometric assay. Anthocyanin was quantitated by absorbance at OD530. The OD530 values of tt4 were subtracted by the OD530 value of samples as an indicator of anthocyanin content. Each experiment was run in triplicate.
Northern analysis
Northern analysis was done as described before (Shin et al., 2002). Briefly, total RNA was extracted from 5-d-old seedlings grown on MS-agar plates containing 2% sucrose under continuous white light as for anthocyanin extraction. Fifteen micrograms of total RNA was loaded into each lane and transferred to a nylon membrane. The membrane was probed with 32P-labelled coding sequence of Arabidopsis CHS or Mazus CHS.
Petunia transformation
Petunia (Petuniaxhybrida cv. Blue) was transformed with vector containing the mCHSK gene as described by Johnson et al. (1999). Transformants were grown in regular potting medium until flowering. As a control, transformants having the vector alone were generated and grown side by side.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Cloning and functional characterization of Mazus japonicus CHS
A putative CHS was cloned from Mazus japonicus, a common garden plant belonging to the Scrophulariaceae (Genbank accession no. AY131328). Mazus bears bilaterally symmetrical white flowers with a lavender-shaded corolla tube. Phylogenetic analysis indicated that the putative CHS from Mazus is very similar to other known CHS enzymes, especially to snapdragon (Antirrhinum majus) and torenia (Torenia hybrida), which also belong to the Scrophulariaceae (Fig. 1). Though it is likely that the gene encodes a real CHS, further experimental proof was required as stilbene synthase (STS) has been reported phylogenetically to group with CHS from related plants, suggesting that it has evolved from CHS (Tropf et al., 1994). Known STS enzymes have high sequence similarity to CHS and use the same precursor molecules and reaction mechanism to form a common tetraketide intermediate. However, while CHS catalyses a Claisen condensation to form chalcone, STS modulates an aldol condensation to yield resveratrol (Schroder et al., 1988; Lanz et al., 1991; Ferrer et al., 1999).
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To determine if the putative CHS isolated from Mazus encodes a functional CHS, the gene was expressed both in the wild-type Arabidopsis ecotype Landsberg erecta (Ler) and the chs mutant (tt4; Koornneef, 1990; Shirley et al., 1995; Saslowski et al., 2000) backgrounds, driven by the CaMV 35S promoter. Several independent homozygous lines were established and three lines of each were selected randomly for further analysis. To determine if the putative CHS can complement the tt4 mutation, the seedlings were grown on water agar plates containing 3% sucrose. tt4 has yellow cotyledons in contrast to the purple cotyledons of the wild-type plants (Koornneef, 1990; Shirley et al., 1995). As seen in Fig. 2, wild-type Ler plants and all three transgenic lines in tt4 background expressing the putative CHS (tc1, 2, and 3) showed purple cotyledons while tt4 showed yellow cotyledons. Consistently, both wild-type and transgenic seeds showed a brown coat colour unlike the yellow seed coat colour of the tt4 mutant. The recovery of anthocyanin production in the transgenic lines indicated that the putative CHS from Mazus encodes a functional CHS. Henceforth, this gene is referred to as MjCHS.
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To test if the overexpression of MjCHS can increase anthocyanin in Arabidopsis, both wild-type and transgenic Arabidopsis plants were grown on MS-agar plates containing 2% sucrose and anthocyanin content was quantitated using a spectrophotometer. Though it was difficult to distinguish visually, two out of the three randomly chosen homozygous lines expressing MjCHS in Ler background (LC1, 2, and 3) accumulated more anthocyanin compared with the non-transgenic wild type (Fig. 3A). To investigate if this increase reflects the expression of MjCHS, northern analysis was carried out. As seen in Fig. 3B, the two lines that showed a higher amount of anthocyanin expressed MjCHS, while the third line that showed an amount similar to that of the wild type did not express the transgene. The results indicate that the overexpression of MjCHS can increase the anthocayanin levels in Arabidopsis. No significant phenotypic difference was observed in adult plants overexpressing the MjCHS enzyme either in tt4 background or in Ler background.
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Development of two dominant-negative MjCHS
The crystal structure of alfalfa CHS suggests that a conserved methionine from a monomer is required for the function of an adjoining monomer (Ferrer et al., 1999). It was hypothesized that if the protruding methionine is functionally important, its alteration to other amino acids would inhibit the function of the adjoining CHS, thus the mutation will be dominant-negative. To test this, two mutated MjCHS genes were generated by site-directed mutagenesis (Fig. 4A). One mutated MjCHS (mCHSG) has glycine instead of methionine at the 138th residue. Since glycine has a shorter side chain compared with that of methionine, the substrate-binding pocket of the adjoining monomer will be altered. The other mutated MjCHS (mCHSK) has lysine instead of methionine at the 138th residue. Since lysine is positively charged, the electrochemical property of the substrate-binding pocket of the adjoining monomer will be altered. To eliminate the catalytic activity of the mutated MjCHS, the catalytically important 165th cysteine was also changed to alanine.
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To determine if the mutated MjCHS enzymes behave in a dominant-negative way, several transgenic Arabidopsis expressing the mutated mCHSG and mCHSK were generated, and three homozygous lines were chosen randomly for further analysis. For the analysis, both wild-type and the transgenic lines were grown on MS-agar plates containing 2% sucrose. Anthocyanin content was quantitated by a spectrophotometer (Fig. 4B). Both mCHSG and mCHSK transgenic lines showed significant reduction in anthocyanin levels. This reduction was not due to the lower expression of endogenous Arabidopsis CHS in the transgenic lines (Fig. 4C). No phenotypic differences were observed in plants overexpressing the mutated enzyme compared with Ler.
The degree of dominant-negativity depends on the amount of dominant-negative protein. Therefore, anthocyanin content in the transgenic plants is expected to be inversely correlated with the expression level of the mutated MjCHS enzymes. To test this hypothesis, a northern analysis of MjCHS mRNA was carried out. However, as shown in Fig. 4C, a direct correlation between anthocyanin content and the level of MjCHS mRNA was not detected. The lack of correlation could be due to the amount of the mutant proteins actually translated.
Modulation of flower colour by the dominant-negative MjCHS
To test further if the dominant-negative MjCHS can be used to modulate the flower colour intensity in a heterologous system, Petuniaxhybrida cv. Blue was transformed with mCHSK. Several transformants were obtained and all of them showed a similar phenotype. As shown in Fig. 5, petunia transformants expressing mCHSK showed reduced flower colour intensity compared with wild type, indicating that the mutated MjCHS can also inhibit petunia CHS. The empty vector control was similar to wild type. Unlike the various colour patterns observed in the sense suppression lines or antisense inhibition lines, all transgenic petunia lines expressing the dominant-negative CHS showed an even decrease in flower colour intensity. The dominant-negative action of the mutated MjCHS enzymes depends on their ability to heterodimerize with the intrinsic CHS enzyme. The inhibition of anthocyanin production indicated that MjCHS can heterodimerize with both Arabidopsis and petunia CHS. The ability to regulate anthocyanin production in two different species suggests further utility of the dominant-negative MjCHS to modulate flower colour intensity in distantly related horticultural species.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The rational design of dominant-negative CHS enzymes is reported and their utility in modulating flower colour intensity demonstrated. A CHS gene was cloned from Mazus japonicus and was functionally characterized by complementing the Arabidopsis chs mutant (tt4). To develop the dominant-negative CHS, both the cysteine, that has been shown to be important for catalysis, and the methionine, that is hypothesized to participate in the function of the adjoining monomer by extending into its cyclization pocket, were mutated. The dominant-negative action of the mutated CHS enzymes was confirmed by the decrease in anthocyanin production in transgenic Arabidopsis lines expressing the mutated CHS genes. Further, it was shown that the colour intensity of petunia flowers can be modulated by the dominant-negative CHS.
The dominant-negative CHS provides an alternative tool to modulate flower colour intensity by genetic engineering. Currently, the most widely used method is to suppress anthocyanin biosynthetic genes either by antisense overexpression or by sense suppression, which is based on the PTGS phenomenon (Tanaka et al., 1998). Though the method has been very successful in manipulating flower colour intensity and inducing striking patterns, it has some limitations. First, it is necessary to clone the gene of interest from the same or closely related species. However, due to the relatively high functional conservation of CHS enzymes from different species, the dominant-negative CHS is a useful tool in a wider range of species. Therefore, the source does not necessarily have to be closely related. Secondly, it is difficult to down-regulate a gene in a tissue-specific manner using PTGS. Several studies have shown that the small-sized RNA molecules generated by PTGS can travel into different tissues and result in systemic gene silencing (Palauqui et al., 1997; Voinnet and Baulcombe, 1997; Vaucheret et al., 2001). Therefore, the resulting patterns and intensities cannot be rationally designed. Since a dominant-negative CHS requires simple overexpression, tissue-specific promoters can be used to achieve a rationally designed pattern. Thirdly, because the establishment of stable PTGS over generations is more difficult than simple overexpression, a dominant-negative strategy has an advantage in species that have low transformation efficiency. Additionally, though overexpression of maize CHS in Arabidopsis did not increase anthocyanin production (Dong et al., 2001), the present results show that the overexpression of MjCHS can increase the anthocyanin levels in Arabidopsis. Regardless of the ability of introduced CHS to increase anthocyanin production, a dominant-negative enzyme can reduce anthocyanin content simply by dimerizing with the native enzyme.
The dominant-negative function of the mutated CHS enzymes that have either glycine or lysine instead of Met138, indicates that the methionine is functionally important. The crystal structure indicates that the methionine protrudes and is positioned at the substrate binding pocket of the adjoining monomer (Ferrer et al., 1999). Each monomer in a CHS dimer can function independently and a mutation in the cysteine residue abolishes enzyme activity of the monomer (Lanz et al., 1991) while not affecting the activity of the adjoining monomer (Tropf et al., 1995). Therefore, the reduced anthocyanin level in the mCHSG and mCHSK transgenic lines indicates that the mutated MjCHS enzymes behave dominant-negatively. As a corollary, the methionine which extends into the cyclization pocket of the dimerizing partner is functionally important for the activity of the adjoining monomer. The dominant-negative action of both mCHSG and mCHSK further suggests that altering the substrate binding pocket either by changing the side chain length or altering the electrochemical property can inhibit CHS activity.
The rapid accumulation of biochemical and structural information on enzymes provides an opportunity to engineer the functions of various enzymes through a rational approach (Shao and Arnold, 1996; Regan, 1999; Cedrone et al., 2000). Specific amino acids can be substituted, added, or deleted by site-directed mutagenesis to confer the desired functional properties. The engineering of flavonoid biosynthetic enzymes by rational design has also been reported. CHS enzymes engineered to have different substrate specificity or to generate different condensation products have been developed by mutating amino acid residues in the substrate-binding pocket (Jez et al., 2000, 2001, 2002; Lukacin et al., 2001). The conversion of acridone synthase to CHS has been achieved by site-directed mutagenesis (Lukacin et al., 2001). Further downstream of CHS, the substrate preference of gerbera DFR has been changed by mutating an amino acid in the putative substrate-binding region (Johnson et al., 2001). The current development of dominant-negative CHS by rational design based on the available structural information and its use in modulating flower colour demonstrate the utility of this approach for the in planta metabolic engineering of flavonoid biosynthesis.
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Acknowledgements |
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We thank Dr Pill-Soon Song, former laboratory members, Sean Blake, Dr In-Jeong Cho, and Dr Fakruddin Bashasab for helpful discussions. The work was partially supported by Plant Metabolism Research Center (Kyung Hee University, Korea).
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Footnotes |
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Present address: 25 Ag Building, Division of Plant Sciences, University of Missouri-Columbia, Columbia, MO 65211, USA. 
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