JXB Advance Access first published online on February 28, 2008
This version published online on March 2, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern046
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Identification and characterization of isoflavonoid specific glycosyltransferase and malonyltransferase from soybean seeds
Southern Crop Protection and Food Research Center, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario, N5V 4T3, Canada
* To whom correspondence should be addressed. E-mail: dhaubhadels{at}agr.gc.ca
Received 26 November 2007; Revised 11 January 2008 Accepted 16 January 2008
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Abstract |
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Isoflavonoids are a diverse group of biologically active natural products that accumulate in soybean seeds during development. The majority of isoflavonoids are accumulated in the form of their glyco- and malonyl-conjugates in soybean seeds. The conjugation step confers stability and solubility to isoflavone aglycones enabling their compartmentalization to vacuoles or transport to the site of accumulation. A functional genomic approach was used to identify isoflavonoid specific glycosyltransferase (UGT) and malonyltransferase (MT) from soybean (Glycine max) seeds. An expressed sequence tag database for soybean was searched by key words to make a list of candidate genes. The full-length cDNAs for candidate UGTs and MTs were obtained and cloned into an expression vector for the production of recombinant enzymes. The in vitro enzymatic activity assays were conducted for recombinant UGTs and MTs using uridine diphosphate glucose and malonyl CoA, respectively, as donors with isoflavone substrates. Among several recombinant enzymes, UGT73F2 showed glycosylation activity towards all three soybean isoflavone aglycones and GmMT7 exhibited malonylation activity towards isoflavone glycosides. The subcellular localization study revealed both UGT73F2 and GmMT7 to be in the cytoplasm. The transcripts and protein accumulation patterns for UGT73F2 and GmMT7 genes have provided further support for their in planta function.
Key words: Glycosyltransferase, isoflavonoids, malonyltransferase, soybean
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Isoflavonoids are a group of secondary metabolites derived from the phenylpropanoid pathway. They are found predominantly in soybeans and other leguminous plants and are associated with a wide array of biological activities. Isoflavonoids play an important function as signal molecules for the induction of nod genes during symbiosis between legumes and rhizobia (Phillips, 1992; Ferguson and Mathesius, 2003) and also serve as precursors for the production of phytoalexins during plant microbe interactions (Aoki et al., 2000; Dixon and Ferreria, 2002). Clinical studies have suggested a positive effect of isoflavonoids in human health and nutrition, such as a reduction in the risks of hormonally dependent cancers, menopausal symptoms, osteoporosis, and cardiovascular disease (Messina and Barnes, 1991; Civitelli, 1997; Cornwell et al., 2004; Dixon, 2004). Due to these health-related benefits associated with isoflavonoids, there is great interest in soybean-based foods and purified soybean isoflavonoids as neutraceuticals. Since isoflavonoids have estrogenous activity, the presence of these compounds in some foods such as soy-based infant formulas may not be advantageous (Setchell et al., 1997). Recently, the isoflavonoid content of soybean seed has been a critical consideration in breeding programmes for developing soybean cultivars with specific levels of isoflavonoids that meets the requirements of different age groups.
Isoflavonoids are synthesized as a branch of the phenylpropanoid pathway. The isoflavone skeleton originates from the central flavanone intermediate naringenin and liquiritigenin. Naringenin is a product of chalcone isomerase and it is the core metabolite that leads to the production of anthocyanins, condensed tannins, flavones, and various other compounds; it is ubiquitously present in all plant species. In legumes, a branch point enzyme 2-hydroxyisoflavanone synthase (IFS), introduces the isoflavonoid biosynthetic pathway. Soybean possesses two IFS enzymes, IFS1 and IFS2, that catalyse a 2,3 aryl migration of flavanones to their corresponding isoflavone aglycones (Heller and Forkmann, 1994; Steele et al., 1999; Dhaubhadel et al., 2003). Soybean seeds contain three major isoflavone aglycones (daidzein, glycitein, and genistein) which may also be present as the corresponding 7-O-glycosides (daidzin, glycitin, and genistin); and malonyl glycosides (6''-O-malonylgenistin, 6''-O-malonyldaidzin, and 6''-O-malonylglycitin) (Kudou et al., 1991). Malonyl glycosides are thermally unstable during the processing of soybeans and soy foods and can be decarboxylated to produce acetyl glycosides (6''-O-acetylgenistin, 6''-O-acetyldaidzin, and 6''-O-acetylglycitin) (Griffith and Collison, 2001). In general, conjugation of glycosyl- and malonyl- groups to the aglycones provides the metabolites with increased water solubility and reduced chemical reactivity, ensuring their improved in vivo stability and altered biological activity (Jones and Vogt, 2001). Enzymes catalysing the conjugation processes belong to the group of transferases; uridine diphosphate glycosyltransferases (UGTs) for glycosylation and malonyltransferases (MTs) for malonylations. Glycosylation involves a UGT-catalysed transfer of a nucleotide diphosphate-activated sugar molecule to the acceptor aglycone (Vogt and Jones, 2000). UGTs constitute a superfamily of enzymes that catalyse conjugation of sugar moieties to proteins, lipids, oligosaccharides, and secondary metabolites. There are more than 12 000 glycosyltransferase encoding sequences in CAZy database (http://www.cazy.org/). Glycosylation is the final step in flavonoid biosynthesis in many plants (Heller and Forkmann, 1994). It reduces the cytotoxic effects of high levels of flavonols into less reactive forms (Pollak et al., 1993). In soybean, following the process of glycosylation, the isoflavone glycosides are further converted into their respective malonyl derivatives, as shown in Fig. 1. Malonylation involves a regiospecific malonyl group transfer from malonyl-CoA to the glycosyl moiety of the glycosides. Presumably, this process further enhances the metabolite solubility, protects glycosides from enzymatic degradation by glycosidases, and helps in their intracellular transport (Heller and Forkmann, 1994). Malonylation of anthocyanins has been studied in several plant species and diverse functional activities have been proposed for this chemical modification (Suzuki et al., 2003; D'Auria et al., 2007; Luo et al., 2007). The involvement of glycosyltransferase (Köster and Barz, 1981) and malonyltransferase (Köster et al., 1984) in isoflavonoid biosynthesis was demonstrated over two decades ago by using purified enzymes from roots of chick pea (Cicer arietinum L.). Despite the long-standing biochemical interest in these enzymes and their role in isoflavonoid biosynthesis, not much is known about the corresponding genes.
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The purpose of this study was to identify and characterize isoflavonoid-specific UGT and MT from soybean seeds, and demonstrate their specificity towards isoflavonoids. A functional genomics approach based upon the analysis of expressed sequence tag (EST) data was undertaken, since this has been shown to be a powerful tool for initiating gene discovery in natural product pathways (Richman et al., 2005; Modolo et al., 2007). Several candidate UGTs and MTs were identified from sequence databases, isolated, and expressed in heterologous systems for recombinant protein production. The enzymatic activity of the recombinant proteins towards isoflavonoid substrates was tested by functional assays. This resulted in the identification of soybean glycosyltransferase (UGT73F2) able to glycosylate isoflavone aglycones to glycosides; and a malonyltransferase (GmMT7) able to malonylate isoflavone glycosides to malonyl-glycosides. The characterization of these enzymes, their subcellular localizations, and their transcript and protein accumulation patterns has provided insight into their in vivo function.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Identification of ESTs and isolation of full-length cDNAs
The DFCI soybean gene index database was searched by key words such as ‘glycosyltransferase’ for UGTs and ‘malonyltransferase’ or ‘acyltransferase’ for MTs. Several candidate ESTs were selected on the basis of their putative functions for both UGTs and MTs. The deduced amino acid sequences of selected ESTs for UGT were again searched for the presence of a PSPG box (WAPQVEVLAHPAVGCFVTHCGWNSTLESISAGVPMVAWPFFADQ). A total of six TCs for UGT and 10 TCs for MT that were annotated as genes involved in the phenylpropanoid pathway, and were previously uncharacterized, were selected for further analysis. A 5' and 3' Rapid Amplification of cDNA Ends (RACE) was performed to obtain full-length cDNA using First Choice RNA Ligase Mediated (RLM)-RACE kit (Ambion Inc., Austin, TX, USA) according to the manufacturer's instructions. The RLM-RACE products were gel purified using a gel purification kit (Qiagen Inc., Mississauga, ON, Canada), cloned in pGEM®-T Easy Vector (Promega Corporation, Madison, WI, USA) and sequenced. The sequence information from RLM-RACE and an EST sequence were used to design primers to isolate full-length cDNA from soybean embryos. PCR was performed using platinum Taq polymerase (Invitrogen) and the products were cloned into pGEM®-T Easy Vector (Promega Corporation, Madison, WI, USA). The clones for both UGTs and MTs were sequenced and compared with the original TC and RLM-RACE products for any mismatch. Full-length cDNAs were obtained for three UGTs and partial sequences for three UGTs. The complete sequences for four MTs and partial sequence for six MTs were also obtained. The full-length sequences were searched for signal peptides using the signalP program.
Heterologous expression of recombinant UGTs and MTs
The fragments containing the coding region of UGTs and MTs were PCR amplified to incorporate XhoI/EcoRV sites for cloning in pET30a expression vector (Novagen, Ancaster, ON, Canada) and transformed in E. coli BL21 (DE3) cells. For heterologous expression of each candidate gene with an N-terminal His tag attached and vector control, bacteria from a fresh colony were grown overnight at 37 °C in Luria-Bertani (LB) medium containing 100 µg ml–1 kanamycin. The cultures were grown at 37 °C to an OD600 of 0.4–0.6. Recombinant proteins were induced by the addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.4 mM for UGTs and 1 mM for MTs. The cultures were grown at 15 °C for 18 h with shaking. Bacterial cells were collected by centrifugation and resuspended in lysis buffer. Lysis buffer for UGTs consisted of 500 mM potassium phosphate pH 7.2 and 0.1% Triton X-100. Similarly, the lysis buffer for MTs included 200 mM TRIS–HCl pH 8.0, 5 mM 2-mercaptoethanol and 0.1% Triton X-100. Lysozyme was added to a final concentration of 100 µg ml–1 and the cells were incubated at 30 °C for 20 min followed by sonication on ice. Total protein concentrations were measured according to Bradford (1976).
Glycosyltransferase and malonyltransferase enzyme activity assay
The activity assay for UGT was conducted as described by Richman et al. (2005) with some modifications. The amount of recombinant protein in the bacterial crude cell extract was quantified by Western blotting using anti-His antibody against a His-tagged SC24 standard (Dhaubhadel et al., 2005). A total of 112 ng of recombinant protein contained in a crude cell lysate was used to initiate the reaction. The reaction mixture was incubated at 30 °C for 16 h and terminated by adding 200 µl of water-saturated 1-butanol. The samples were extracted three times with water-saturated 1-butanol. The pooled butanol extracts were concentrated using vacufuge concentrator (Eppendorf, Mississauga, ON, Canada).
For MT activity, 100 ng of recombinant proteins in a total cell lysate was added to the reaction mixture containing 200 mM TRIS–HCl pH 8.0, 30 mM 2-mercaptoethanol, 2 mg ml–1 BSA, 120 µM of isoflavone glucoside substrates, 60 µM malonyl CoA, and 3 µM of [14C] malonyl CoA (Amersham Biosciences, Buckinghamshire, UK) to a total volume of 100 µl. The reaction mixture was incubated at 30 °C for 30 min and terminated by adding 100 µl of ice-cold 0.5% trifluoroacetic acid. Equal volume of reaction products were spotted on a TLC plate and the samples were separated using chloroform:methanol:acetic acid as solvents at the ratio of 18.5:10.0:0.28 by vol., followed by exposure of the TLC plate to an imaging screen and visualization on a Molecular Imager Fx phosphoimager (Bio-Rad laboratories, Hercules, CA, USA). Standards were run under the same condition and visualized under UV light.
To determine apparent K m values, UGT and MT reactions were performed as described above with substrate concentrations ranging from 5–200 µM with no radiolabelled donor. The reaction mixtures were incubated at 30 °C for 16 h for UGTs and 30 min for MTs, followed by HPLC analysis. K m values were determined from Lineweaver-Burk plots of initial rate data.
Analysis of isoflavone-glycosides and malonyl glycosides by LC-MS
For the determination of kinetic parameters for UGT73F2 and GmMT7 proteins, the reaction mixtures were frozen immediately after the reaction was completed and stored at – 20 °C. The LC-UV/MS system consisted of an Alliance 2690 HPLC/autoinjector (Waters, Mississauga, ON), a model SPD-M6A Shimadzu diode array UV detector (Mandel Scientific, Guelph, ON), and a model LCT orthogonal time-of-flight mass spectrometer (Waters, Mississauga, ON). Samples of reaction mixtures (20 µl) were injected into a 100 µl loop attached to a Valco C10W 10-port valve (Chromatographic Specialties, Brockville, ON) configured to permit on-line trapping and washing of the sample on a 4x2 mm ID SecurityGuard C18 cartridge (Phenomenex, Torrance, CA) prior to chromatography on a 150x2 mm, 5 µm particle Prodogy ODS (3) column (Phenomenex, Torrance, CA) with solvent flowing at 0.2 ml min–1. After switching the valve to connect the loop and the cartridge, the sample was transported to the cartridge and the cartridge washed with 100:0.1 (v/v) water-formic acid for 10 min at 0.2 ml min–1. The valve was then switched to connect the cartridge and the analytical column and the system was eluted with a binary gradient. Solvent A was 90:10:0.1 (by vol.) water-acetonitrile-formic acid and Solvent B was 10:90:0.1 (by vol.) water-acetonitrile-formic acid and the initial conditions were 100% A. The isoflavonoids of interest were eluted during a linear increase to 40% B over 20 min. The solvent composition was then linearly increased to 100% B by 25 min, held at this composition for 5 min and decreased to 0% B by 35 min. The UV and MS detectors were connected in series to the column and separated by a second Valco C10W 10-port valve configured to permit diversion of the column flow from the MS and its replacement with 50:50:0.1 (by vol.) water-acetonitrile-formic acid at 0.2 ml min–1. UV data (200–450 nm) was collected during the entire 35 min. MS data (85–1500 m/z) was collected for 25 min. The column flow was diverted from the MS after the components of interest had been detected to avoid unnecessarily contaminating the ion source. The MS ion source was equipped with a standard nebulizer-assisted electrospray probe, which was operated in positive ion mode with nitrogen as desolvation gas at 350 °C flowing at 460 l h–1 and a potential of 2.7 kV applied to the capillary with the sample cone at 20 V. The MS was calibrated using a mixture of polyethylene glycols.
Transcript profiling using EST database, RNA isolation, and RT-PCR analysis
The UGT73F2 and GmMT7 sequence was used to search DFCI soybean gene index (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/Blast/index.cgi) by BLASTN. Transcripts matching UGT73F2 and GmMT7 were categorized according to the source cDNA library and the tissue type. The frequency of ESTs matching the UGT73F2 and GmMT7 cDNA sequences was calculated separately for their corresponding source cDNA library.
Total RNA was isolated from soybean tissues using the protocol of Wang and Vodkin (1994). RNA samples were treated with DNase I (Promega, Madison, WI) at 37 °C for 30 min prior to RT-PCR. DNase I-treated RNA samples were further purified by phenol:chloroform extraction 3:1 (v/v), precipitated with ethanol and checked on ethidium bromide gel to ensure equal loading. RT-PCR reactions were performed using ThermoscriptTM RT-PCR system (Invitrogen) according to the manufacturer's instructions. Primer sequences for PCR were as follows: For UGT73F2, GT6F 5'-CCCCGATATCATGGATCTTCAACAACGAC-3' and GT6R 5'-CCCCCTCGAGGTTAGTAAGTAAATCTGT-3'; for GmMT7, MT7F1 5'-CCCCGAATTCATGGCAGAGACACCAACC-3' and MT7R1 5'- CCCCCTCGAGGTTCCTCTCGTGACACAC-3'.
Antibody production and western blot analysis
Since both rUGT73F2 and rGmMT7 proteins were found predominantly in inclusion bodies, these proteins were gel purified for the purpose of antibody production in 20 mM TRIS–HCl pH 7.5 and 150 mM NaCl. The protein concentration determined by the Bradford (1976) assay and checked for their purity by running an aliquot of protein on SDS-PAGE.
Antibodies were raised in two rabbits for each of the recombinant proteins. The rabbits were bled to collect control serum prior to immunization. Purified rUGT73F2 and rGmMT7 with His-tag (200 µg) was mixed 1:1 (v/v) with complete Freund's adjuvant and injected into rabbits. Booster injections of both the recombinant proteins with incomplete Freund's adjuvant were performed 14, 45, and 60 d after the first injection. Antiserums to rUGT73F2 and rGmMT7 (
-UGT73F2 and -GmMT7) were collected on days 24, 45, and 60, and stored at –80 °C.
For western blot analysis, total soluble protein extraction and quantitation from soybean tissues were carried out as described in Dhaubhadel et al. (2005). For microsome isolation, leaf and embryo (60 DAP) tissues (3 g) were ground to a fine powder with 0.5% polyvinylpyrrolidine and mixed with ice-cold extraction buffer containing 0.1 M potassium phosphate buffer pH 7.5, 0.4 M sucrose and 14 mM β-mercaptoethanol. The extract was centrifuged at 10 000 g for 15 min at 4 °C. Supernatant was filtered through Miracloth (Calbiochem, EMD Biosciences, USA) and the filtrate was subjected to ultracentrifugation at 1 000 000 g for 1.5 h at 4 °C. The pellet was washed with a buffer containing 0.1 M potassium phosphate buffer pH 8.0, 0.4 M sucrose, and 3.5 mM β-mercaptoethanol and resuspended in the same buffer. The protein concentration in the pellet and supernatant fractions were determined by the Bradford assay. Equal amount of proteins (15 µg) in each lane were separated on 7.5% SDS-PAGE according to Laemmli (1970) and transferred on to a PVDF membrane using a semi-dry electroblotting device (Bio-Rad Ltd, Mississauga, Canada). Pre-immune serum was used to check if there was any cross reactivity. UGT73F2 and GmMT7 proteins were detected by sequential incubation of the blot with -UGT73F2 or -GmMT7 and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG, with a dilution of 1:1000 and 1:10000, respectively, followed by the chemiluminiscent detection (ECL system, Amersham Biosciences).
Plasmid construction
GFP fusions with UGT73F2 and GmMT7 encoding regions were created using bridging PCR with partially overlapping primers in a two step PCR reaction. A forward primer containing a restriction site was designed at the 5' end of the primers to assist cloning into plant transformation vectors. UGT73F2 and GmMT7 cDNA sequences were amplified using the 5' gene specific primer and a reverse primer containing the first 15 nt of smGFP coding region fused in frame with the last 15 nt of UGT73F2 or GmMT7 coding regions. In a separate PCR reaction, smGFP was amplified using a forward primer containing the last 15 nt of UGT73F2 or GmMT7 coding region followed by the first 15 nt of smGFP coding region sequence with a reverse primer containing an EcoRI restriction site at the 3' end of smGFP. A final PCR reaction using the products from both of the above PCR reactions along with the BamH1 forward primers and the EcoRI reverse primers created the fragments containing UGT73F2 or GmMT7 fused with GFP in frame. Overlapping primers were used in high fidelity PCR to add the 5' ER signal and the 3' KDEL ER retention signal to the GFP sequence. The GFP sequence was amplified from the psmGFP vector (Davis and Vierstra, 1998) using a forward primer introducing a BamHI restriction site at the 5' end and an EcoRI restriction site at the 3' end.
Fusion products and GFP alone were cloned into pGEMT-Easy (Promega, USA) and sequenced to confirm the sequence integrity, followed by cloning into a binary vector pCAMterX to produce pCAMter-GFP, pCAMter-ER-GFP, pCAMter-UGT73F2-GFP, and pCAMter-GmMT7-GFP. Each plasmid DNA was transformed into Agrobacterium tumefaciens strain EHA105 via electroporation.
Subcellular localization of UGT73F2 and GmMT7
The transient expression of GFP fusion proteins was performed in tobacco plants via leaf disc infiltration. A single colony of A. tumefaciens containing the construct of interest was selected and grown at 28 °C in a shaker to a stationary phase in liquid LB media containing 50 µg ml–1 kanamycin and 10 µg ml–1 rifampicin. For inoculation, 1 ml of the bacterial cells was pelleted and resuspended in the infiltration medium (0.5% glucose w/v, 50 mM MES pH 5.6, 2 mM Na3PO4, 100 µM acetosyringone) bringing the OD600 to 0.6–0.7. The Agrobacterium cells containing different constructs were infiltrated by injecting the bacterial cells into the abaxial epidermal surface of a tobacco leaf with a syringe. The plant was then incubated at room temperature for 3–4 d. For observation, a small piece of the infected leaf tissue was cut out and mounted in water followed by confocal microscopy. Imaging of GFP and GFP fusion proteins was performed by a Leica TCS SP2 inverted confocal microscope using a 63x water immersion objective and Leica Confocal software. Serial optical sections of 0.5–2 µm were obtained using an excitation wavelength of 488 nm, and emisions were collected between 500 nm to 560 nm.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Isolation of candidate UGT and MT cDNAs
The DFCI Soybean Gene Index database contains more than 330 000 ESTs and 31 000 tentative contigs (TC) was used as a source to identify potential isoflavonoid specific UGTs and MTs (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=soybean). A keyword search of the database was used to identify 175 TCs for UGTs and 43 TCs for MTs as candidate sequences (see Materials and methods). A total of six candidate TCs for UGTs and 10 candidate sequences for MTs that were previously uncharacterized, were selected based on their annotations as phenylpropanoid UGTs or MTs. Two of the TCs for MT had full-length cDNA sequences. The full-length cDNAs for three candidate UGTs and four MTs genes could be obtained using RLM-RACE (Table 1).
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Recombinant protein production and enzyme activity assay
The full-length candidate UGTs and MTs were cloned into expression vectors and expressed heterologously in Escherichia coli cells. A large amount of the recombinant protein for both groups of transferases accumulated in insoluble fractions. However, a small amount of recombinant protein was detected in the soluble fraction by western blotting analysis using an anti-histidine tag antibody (data not shown). The concentration of recombinant protein in the soluble fraction was determined by comparing the signal obtained by western blotting analysis of recombinant protein with a known concentration of histidine-tagged SC24 protein as a standard (Dhaubhadel et al., 2005). The activities of the recombinant UGT proteins were tested using UDP-[14C] glucose as the sugar donor and daidzein, glycitein, and genistein as acceptor aglycones. Similarly, the activities of the recombinant MT proteins were tested using malonyl CoA as the malonyl donor and daidzin, glycitin, and genistin as acceptor molecules. Incorporation of radiolabel into the product was determined qualitatively by TLC. These results are shown in Fig. 2. Among several UGT and MT recombinant proteins used in our study, only recombinant UGT6-1 showed activity towards isoflavone aglycones and was able to transfer radiolabelled sugar to isoflavone aglycones; this was re-named as UGT73F2, to follow the standard UGT nomenclature. A substitution mutation at amino acid 280 from glutamic acid to glycine completely abolished UGT73F2 enzymatic activity (data not shown). Among MTs, only recombinant GmMT7 (rGmMT7) exhibited activity towards isoflavone glyco-conjugates. The reaction products were compared with the authentic standards.
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HPLC and LC-MS analysis of reaction products
The TLC results of the rUGT73F2 and rGmMT7 reaction products were further verified by HPLC and LC-MS analysis. Measurement of the reaction products from rUGT73F2 and isoflavone aglycones by HPLC was identical to the authentic standards with regard to retention time and UV spectra (Fig. 3C–E). Soybean seed extracts were also analysed by HPLC and the peak identities of the malonyl derivatives present were confirmed by hydrolysis to their glycosides (Dhaubhadel et al., 2003) as well as mass determination by LC-MS analysis. The identified isoflavone malonyl glyco-conjugates in these extracts were then used as standards to identify the GmMT7 reaction products by HPLC. Among four candidate rMTs tested, only rGmMT7 showed malonyltransferase activity with isoflavone glycosides converting daidzin, glycitin, and genistin to malonyldaidzin, malonylglycitin, and malonylgenistin, respectively (Fig. 3F–H). The molecular masses of both rUGT73F2 and rMT7 reaction products were verified by LC-MS analysis.
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The substrate specificity and kinetic properties of rUGT73F2 and rGmMT7 were also determined using HPLC. A wide range of phenolic acceptors were tested, together with donor molecules and the recombinant proteins as shown in Table 2. The substrate preference for rUGT73F2 was as follows: genistein > glycitein > biochanin A > daidzein> liquiritigenin > naringenin > apigenin > formononetin. No reaction products were detected with quercetin or with salicylic acid as acceptors. The specific activity for genestein was 15 nkatal mg–1 protein. Both donor and substrate specificity was determined for rGmMT7. Three different acyl CoAs were tested for their donor specificity using daidzin as an acyl acceptor. The results demonstrated a high specificity of rGmMT7 towards its acyl donor, since only malonyl CoA was able to function in this capacity. Among the various glycoside substrates, rGmMT7 was most specific to daidzin with the specific catalytic activity of 270 nkatal mg–1 protein, followed by glycitin and genistin. Less than 40% relative activity was detected with the other flavonoid glycosides tested for rGmMT7 activity.
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The kinetic parameters for rUGT73F2 and rGmMT7 were determined using UDP-glucose as the sugar donor, and daidzein, glycitein or genistein as sugar acceptors and malonyl CoA as the acyl donor, and daidzin, glycitin or genistin as acyl acceptors, respectively. The V max was not calculated since saturating conditions for some of the reactions could not be attained due to limited substrate solubility. The apparent K m values were identified based on the Lineweaver-Burk plot (Table 2). The results showed that rUGT73F2 and rGmMT7 had the highest affinity for glycitein and glycitin, respectively.
Expression analysis of UGT73F2 and GmMT7
As an initial step to investigate the expression patterns of soybean UGT73F2 and GmMT7 genes, ESTs present in the DFCI Soybean Gene Index database were probed for sequences revealing similarities to UGT73F2 and GmMT7. Nucleotide sequences corresponding to these two transferases were used separately in BLASTN searches (Altschul et al., 1997) of 330 436 soybean ESTs (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/Blast/index.cgi) originating from more than 80 different cDNA libraries (Shoemaker et al., 2002). The search resulted in four matching ESTs for UGT73F2 and 21 matching ESTs for GmMT7 from 18 different source cDNA libraries. These EST data were pooled according to the tissue type of the source cDNA library. The results shown in Fig. 4A and B demonstrate that UGT73F2 was most represented in the cDNA libraries constructed from immature cotyledons, infected tissues, and roots, whereas GmMT7 was most prevalent in the cDNA libraries constructed from stressed tissues and roots. A detailed transcript analysis using an RT-PCR approach with gene-specific primers from RNA isolated from the soybean cultivar Harosoy63 showed that UGT73F2 transcripts accumulated in pods, embryos, flower buds, flowers, pod walls, early leaf, and early to mid-seed coat tissues. No UGT73F2 transcript accumulation was detected in seed coat tissue 50 d after pollination (DAP) and root tissues. Similarly, GmMT7 transcript accumulated to a high level in embryos, flowers and pod wall tissues (Fig. 4C).
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To measure UGT73F2 and GmMT7 protein accumulation in different soybean tissues and during seed development, polyclonal antibodies raised against rUGT73F2 and rGmMT7 were used to assay equal amounts of soluble protein extracts from different soybean plant parts by western blotting analysis. The results indicated that UGT73F2 was present in comparable levels in embryo tissues from early to late seed maturity stages (before seed desiccation). The UGT73F2 protein was present in all soybean tissues examined except for flowers and leaves at the late stages of development. There was a high accumulation of UGT73F2 in pods, flower buds, and younger leaf tissues. By contrast, the accumulation of GmMT7 was most pronounced in embryos throughout their development. A low amount of GmMT7 was present in leaf tissue, but it was undetectable in other tissues studied as shown in Fig. 4D. Both the antibodies recognized proteins of slightly higher molecular masses. This cross-reactivity could be due to a structural similarity between UGT73F2 and GmMT7 with other UGT or MT, respectively, or there may be some smaller proteins interacting with these transferases.
Sequence analysis and phylogeny of UGT73F2 and GmMT7
The full-length UGT73F2 cDNA sequence of 1569 bp was predicted to encode a protein of 476 amino acid residues with a calculated molecular mass of 53.2 kDa and a pI of 6.42. The carboxyl terminal of the protein contained the plant secondary product glycosyltransferase (PSPG) box signature motif (Fig. 5A). The UGT73F2 protein showed 63% amino acid identity with UGT73F1 from Glycyrrhiza echinata (Nagashima et al., 2004) and 24% amino acid identity with GmIF7GT (UGT88E3) isolated from roots of soybean seedlings (Noguchi et al., 2007). A phylogenetic analysis of UGT73F2 in comparison with UGTs from other plant species also grouped UGT73F2 together with the UGT73F1 (Fig. 5B).
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The full-length cDNA sequence for GmMT7 (1404 bp) encoded an open reading frame comprising 467 amino acid residues with a calculated molecular mass of 51.737 kDa and a pI of 5.98. The primary protein structure analysis revealed the presence of an N terminus HXXXDG motif and a C terminus DFGWGKP motif, suggesting that GmMT7 is a member of the BAHD family of acyltransferases (St-Pierre and De Luca, 2000). A third conserved motif specific to the anthocyanin acyltransferase (YFGNC motif) was also found in the middle of the GmMT7 protein sequence. The deduced amino acid sequence of GmMT7 showed the highest sequence similarities to two GmIF7MaT from soybean roots (99% identity, accession no. BAF73620 [GenBank] ; 86% identity, accession no. BAF73621 [GenBank] ), two unknown sequences from Medicago truncatula (42% and 41% identity, accession no. ABE91262 and ABE91277, respectively), and an anthocyanin acyltransferase-like protein from Arabidopsis (38% identity, accession no. BAB10831 [GenBank] ). Phylogenetic analysis showed that GmMT7 is a member of clade I of the BAHD superfamily (Fig. 6).
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Analysis of UGT73F2 and GmMT7 sequences for the N-terminal targeting signal or the C terminal membrane anchor signal using SignalP and TMHMM web-based programs predicted both the proteins to be non-secretory with an absence of predicted signal peptides or transmembrane signals (Nielsen et al., 1997; Krogh et al., 2001). Nucleotide sequence analysis of genomic DNA revealed the presence of a single intron (437 bp) in GmMT7 (accession no. EU192928); whereas UGT73F2 DNA sequence had no introns. Southern blotting analysis of the restriction fragments from soybean genome DNA using full-length UGT73F2 or GmMT7 cDNA as a probe indicated a single copy of UGT73F2 and more than one copy of GmMT7 in the soybean genome (data not shown).
Both UGT73F2 and GmMT7 are cytosolic proteins
The in vivo subcellular localization of UGT73F2 and GmMT7 were determined by creating GFP fusion proteins and monitoring their transient expression in tobacco epidermal cells. Both UGT73F2-GFP and GmMT7-GFP fusions showed characteristics of a cytoplasmic location. Comparison of UGT73F2-GFP and GmMT7-GFP fusions with the ER targeted GFP control verified that these transferases may not be localized to the ER. Although it is known that GFP can accumulate in the nucleus by crossing through the nuclear pores (Grebenok et al., 1997), the much larger size of the fusion proteins were not expected to pass through the nuclear pores. Our results show that untargeted GFP produced extremely bright fluorescing nuclei compared to fluorescence of the cytoplasm, whereas the UGT73F2-GFP and GmMT7-GFP fusions showed relatively lower or similar intensity of the fluorescence in the nucleus compared to the cytoplasm (Fig. 7A–E).
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The presence of these transferases in the soluble fraction was further confirmed by cellular fractionation of total proteins from leaf and embryo (60 DAP) tissues into soluble and membrane fractions followed by western blot analysis. The results revealed that both UGT73F2 and GmMT7 were only present in the soluble fraction (Fig. 7F).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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To manipulate the level of isoflavonoid accumulation in plants an in-depth knowledge of the biosynthesis of these metabolites is necessary. In addition, it is clear that the proper conjugation and compartmentalization of natural products requires special attention. In soybean seeds, most of the isoflavonoids are accumulated in the form of their glycosyl and malonyl derivatives. The introduction of isoflavonoids into any plant species can theoretically be achieved by transformation with a single enzyme, IFS. The cDNAs encoding IFS have been cloned from soybean and other species (Akashi et al., 1999; Steele et al., 1999; Jung et al., 2000), and have been introduced into Arabidopsis thaliana, a plant that normally does not produce isoflavonoids (Liu et al., 2002). Introducing soybean IFS into A. thaliana resulted in the accumulation of low levels of genistein glycosides. The formation of isoflavone glycosides in transgenic Arabidopsis probably arose from the activity of non-specific UGTs, since these enzymes may exhibit general regio-specificity rather than tight substrate specificity (Lim et al., 2003). Since the catalytic activity of these UGTs varies depending upon the available substrate, a more specific UGT is required for the accumulation of particular glycosides. In the present study, a functional genomic approach was followed to identify isoflavonoid specific UGT73F2 and GmMT7 cDNAs from soybean seeds. By tracing the mRNA and protein accumulation of UGT73F2 and GmMT7 in various soybean organs throughout development, it is shown that both of these genes are expressed abundantly in seeds. The enzymatic activity of heterologously expressed UGT73F2 and GmMT7 provided evidence demonstrating the biochemical function and substrate specificity of the recombinant enzymes.
The soybean UGT73F2 protein belonged to glycosyltransferase family 1, as do most of the UGTs involved in plant secondary metabolism. This protein possessed a PSPG box with conserved sequence of 
45 amino acid residues and it showed specificity towards isoflavone aglycones. Two other cDNAs encoding isoflavonoid specific UGTs, UGT73F1 and UGT88E3, have recently been identified from G. echinata cell suspension cultures (Nagashima et al., 2004) and from the roots of soybean seedlings (Noguchi et al., 2007), respectively. Sequence analysis showed UGT73F2 is most closely related to UGT73F1 from G. echinata and belongs to the same clade of the phylogenetic tree (Fig. 5B). However, these enzymes differ considerably in their substrate specificities. UGT73F2 demonstrated highest activity towards isoflavonoid genistein followed by glycitein. Formononetin was a poor substrate for UGT73F2. In contrast, formononetin and daidzein were the most suitable substrates for UGT73F1 (Nagashima et al., 2004). Although UGT88E3 was isolated from soybean, its relative activity towards formononetin and daidzein was similar to UGT73F1. By contrast, the phylogenetic analysis revealed that UGT88E3 was distantly related to both UGT73F1 and UGT73F2. This observation is consistent with past suggestions that UGT function and specificity are not predictable based on sequence information alone (Modolo et al., 2007). Prediction of UGT function could be aided by tissue-specific expression data together with metabolite profiles. Analysis of in vitro substrate specificity obviously provides strong evidence for functionality (Modolo et al., 2007). A comparative study of isoflavonoid content in various tissues from soybean has previously been conducted and it was found that mature embryos accumulate the highest level of isoflavonoids compared to other tissues (Dhaubhadel et al., 2003). The in vitro substrate specificity of UGT73F2 towards isoflavone aglycones, the abundance of its corresponding transcript, and the protein accumulation in reproductive tissues support the hypothesis that the enzyme functions in vivo as an important component of the isoflavonoid biosynthetic pathway in soybean.
The subcellular localization study of UGTs from various plant species have suggested cytosolic (Yazaki et al., 1995; Achnine et al., 2005), vacuolar (Anhalt and Weissenböck, 1992), endomembrane (Ibrahim, 1992) or cytochrome P450s-associated ER (Jones and Vogt, 2001) location. Since IFS is the preceding enzyme in the isoflavonoid biosynthetic pathway, and IFS is assumed to be a membrane-bound cytochrome P450, it was speculated that UGT73F2 may be fully or weakly associated with the membrane as well. Based on the analysis of UGT73F2-GFP fusion protein localization, it appears that UGT73F2 is a soluble cytoplasmic enzyme. The detection of UGT73F2 accumulation in the soluble protein fraction provided further supporting evidence for a soluble enzyme (Fig. 7).
The mechanism and specificity of PSPG catalysed effects has been illustrated by recent crystallographic studies of PSPGs from Medicago truncatula (Shao et al., 2005) and Vitis vinifera (Offen et al., 2006), along with the work on site-directed mutagenesis of these enzymes (He et al., 2006). These studies have pointed out an N-terminal histidine residue that is highly conserved among UGTs that may act as a key catalytic residue. The His residue is proposed to activate the hydroxyl group of the glycosyl acceptor to facilitate glycosydic linkage formation. Another well-conserved Asp residue, downstream to the His residue, is hydrogen bonded with the His residue and is believed to help during catalysis. The UGT73F2 protein possesses each of these conserved residues and may follow the proposed mechanism of catalysis. During the cloning of UGT73F2, a sequence-altered version was isolated from PCR reaction products in which the glutamic acid residue at position 280 of the ORF was changed to glycine, thus producing an E280G mutant. This single amino acid substitution completely abolished the enzymatic activity of UGT73F2. These observations indicate that Glu-280 plays a critical role for maintaining functionality of the enzyme. This Glu-residue is well conserved among UGTs, however, its role in UGT73F2 catalysis is not yet clear.
During the preparation of this manuscript, Suzuki and colleagues (2007) independently reported the cloning of isoflavone 7-O-glucoside-6''-O-malonyltransferase (GmIF7MaT) from soybean roots using an homology-based strategy. The GmIF7MaT ORF differs by a single amino acid (W240R) from GmMT7 presented in this paper and could be an allele of the same gene. GmMT7 exhibited highest activity towards daidzin (270 nkatal mg–1 protein) and demonstrated very high specificity towards soybean isoflavone glycosides (Table 2). The apparent K m values were comparable (in µM range) to other flavonoid glycoside specific malonyltransferases (Suzuki et al., 2002; Luo et al., 2007), however, the differed from GmIF7MaT by one order of magnitude. The differences may be due to differences in the assay conditions, as well as differences in the activities of two enzymes. Similar to GmIF7MaT, GmMT7 showed very narrow selectivity for its acyl donor, accepting nothing except malonyl CoA. The results from both cell fractionation studies and GFP fusions strongly suggested that GmMT7 is a cytoplasmic enzyme. This is consistent with the fact that GmMT7 accumulation was detected in total soluble protein extracts using antibody raised against GmMT7. These results are congruent with previous findings that BAHD family members are localized to the cytosol (D'Auria, 2006). Taken together with results from past studies (Yu and McGonigle, 2005), the current understanding of the spatial organization of the isoflavonoid pathway in soybean is summarized in Fig. 8.
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The transcript accumulation analysis revealed that although GmMT7 was expressed in most of the tissues in soybean including roots, its expression was highest in embryos and flowers. In contrast, GmIF7MaT was reported to be expressed in lower amounts in seeds and pod tissues as compared to root, stem, and leaf tissues. A large amount of GmMT7 protein accumulated in the embryo tissues throughout seed development. Previous work has shown that IFS expression and isoflavonoid product accumulation are comparatively low in the early stages of embryo development and reach maximum levels during the final stages of seed maturity (Dhaubhadel et al., 2003, 2007). Thus, the expression of UGT73F2 and GmMT7 somewhat preceded IFS expression and isoflavonoid accumulation, but the significance or reason for these differences is not clear. The accumulation of mRNA for both UGT73F2 and GmMT7 did not show direct correlation with the corresponding protein in a particular tissue type. These results suggest a tissue-specific post-transcriptional regulation of these genes. However, the possibility of these transferases being more stable in certain soybean tissues compared to other tissues can not be ruled out.
In conclusion, UGT73F2 and GmMT7 are the two transferases involved in glycosylation and malonylation of soybean isoflavonoids, respectively. The results obtained in the present study clearly demonstrate the specificity of these two enzymes towards isoflavonoid substrates. The enzymes and the corresponding genes may serve as a valuable tool in manipulating isoflavonoid accumulation in soybean seeds or metabolic engineering of the pathway leading to isoflavonoid production in non-legumes. Further work is required to determine whether naturally occurring variation in the sequence or regulation of UGT73F2 or GmMT7 may constitute a mechanism for controlling isoflavonoid synthesis and accumulation. Regardless, the positive identification of the genes controlling the final two steps of the isoflavonoid biosynthetic pathway is an important advance, given that malonylated products are the most abundant forms of isoflavonoids in soybean seeds.
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Acknowledgements |
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We thank Vaino Poysa (Agriculture and Agri-Food Canada, Harrow) for soybean seeds, Alexandra Reid for help with GFP work, Mark Gijzen and Frederic Marsolais for critical review of the manuscript as well as much helpful discussions, and Alex Molnar for the art work. This work was supported by Agriculture and Agri-Food Canada's Canadian Crop Genomics Initiative grant to SD.
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References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Achnine L, Huhman DV, Tarag MA, Sumner LW, Blount JW, Dixon RA. Genomics-based selection and functional characterization of triterpene glycosyltransferases from the model legume Medicago truncatula . The Plant Journal (2005) 41:875–887.[CrossRef][ISI][Medline]
Akashi T, Aoki Y, Ayabe S. Cloning and functional expression of a cytochrome P450 encoding 2-hydroxyisoflavanone synthase involved in biosynthesis of the isoflavonoid skeleton in licorice. Plant Physiology (1999) 121:821–828.
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of proteins database search programs. Nucleic Acids Research (1997) 25:3389–3402.
Anhalt S, Weissenböck G. Subcellular localization of luteolin glucuronides and related enzymes in rye mesophyll. Planta (1992) 187:83–88.[ISI]
Aoki T, Akashi T, Ayabe S. Flavonoids of leguminous plants: structure, biological activity, and biosynthesis. Journal of Plant Research (2000) 113:475–488.[CrossRef][ISI]
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry (1976) 72:248–254.[CrossRef][ISI][Medline]
Civitelli R. In vitro and in vivo effects of ipriflavone on bone formation and bone biomechanics. Calcified Tissue International (1997) 61:12s–14s.[CrossRef]
Cornwell T, Cohick W, Raskin I. Dietary phytostrogens and health. Phytochemistry (2004) 65:995–1016.[CrossRef][ISI][Medline]
D'Auria JCD. Acyltransferases in plants: a good time to be BAHD. Current Opinion in Plant Biology (2006) 9:331–340.[CrossRef][ISI][Medline]
D'Auria JCD, Reichelt M, Luck K, Svatos A, Gershenzon J. Identification and characterization of the BAHD acyltransferase malonyl CoA: anthocyanidin 5-O-glucoside-6''-O-malonyltransferase (At5MAT) in Arabidopsis thaliana . FEBS Letters (2007) 561:872–878.
Davis SJ, Vierstra RD. Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants. Plant Molecular Biology (1998) 36:521–528.[CrossRef][ISI][Medline]
Dhaubhadel S, Gijzen M, Moy P, Farhangkhoee M. Transcriptome analysis reveals a critical role of CHS7 and CHS8 genes for isoflavonoid synthesis in soybean seeds. Plant Physiology (2007) 143:326–338.
Dhaubhadel S, Kuflu K, Romero MC, Gijzen M. A soybean seed protein with carboxylate-binding activity. Journal of Experimental Botany (2005) 56:2335–2344.
Dhaubhadel S, McGarvey B, Williams R, Gijzen M. Isoflavonoid biosynthesis and accumulation in soybean seeds. Plant Molecular Biology (2003) 53:733–743.[CrossRef][ISI][Medline]
Dixon RA. Phytoestrogens. Annual Review of Plant Biology (2004) 55:225–261.[CrossRef][Medline]
Dixon RA, Ferreria D. Genestein. Phytochemistry (2002) 60:205–211.[CrossRef][ISI][Medline]
Ferguson JA, Mathesius U. Signaling interactions during nodule development. Journal of Plant Growth Regulator (2003) 22:47–72.[CrossRef]
Grebenok RJ, Pierson E, Lambert GM, Gong FC, Afonso CL, Haldeman-Cahill R, Carrington JC, Galbraith DW. Green fluorescent protein fusions for efficient characterization of nuclear targeting. The Plant Journal (1997) 11:573–586.[CrossRef][ISI][Medline]
Griffith AP, Collison MW. Improved methods for the extraction and analysis of isoflavones from soy-containing foods and nutritional supplements by reverse-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry. Journal of Chromatography (2001) 913:397–413.[CrossRef][Medline]
He XZ, Wang X, Dixon RA. Mutational analysis of the Medicago glycosyltransferase UGT71G1 reveals residues that control regioselectivity for (iso)flavonoid glycosylation. Journal of Biological Chemistry (2006) 281:34441–34447.
Heller W, Forkmann G. Biosynthesis of flavonoids. In: The flavonoids: advances in research since 1986—Harborne JB, ed. (1994) London: Chapman and Hall. 499–535.
Ibrahim RK. Immunolocatozation of flavonoid conjugates and their enzymes. In: Phenolic metabolism in plants—Stafford HA, Ibrahim RK, eds. (1992) NY: Plenum Press. 25–61.
Jones P, Vogt T. Glycosyltransferases in secondary plant metabolism: transquilizers and stimulant controllers. Planta (2001) 213:164–174.[CrossRef][ISI][Medline]
Jung W, Yu O, Lau SC, O'Keefe DP, Odell J, Fader G, McGonigle B. Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes. Nature Biotechnology (2000) 18:208–212.[CrossRef][ISI][Medline]
Köster J, Barz W. UDP-glucose:isoflavone 7-O-glycosyltransferase from roots of chick pea (Cicer arietinum L.). Archives Biochemistry and Biophysics (1981) 212:98–104.[CrossRef][Medline]
Köster J, Bussmann R, Barz W. Malonyl-coenzyme A:isoflavone 7-O-glucoside-6''-O-malonyltransfearse from roots of chick pea (Cicer arietinum L.). Archives of Biochemistry and Biophysics (1984) 234:513–521.[CrossRef][ISI][Medline]
Krogh A, Larsson B, von Heijne G, Sonnhammer ELL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. Journal of Molecular Biology (2001) 305:567–580.[CrossRef][ISI][Medline]
Kudou S, Fleury Y, Welt D, Magnolato D, Uchida T, Kitamura K. Malonyl isoflavone glycosides in soybean seeds (Glycine max Merrill). Agricultural and Biological Chemistry (1991) 55:2227–2233.[ISI]
Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (1970) 227:680–685.[CrossRef][Medline]
Lim EK, Baldauf S, Li Y, Elias L, Worrall D, Spencer SP, Jackson RG, Taguchi G, Ross J, Bowles DJ. Evolution of substrate recognition across a multigene family of glycosyltransferase in Arabidopsis. Glycobiology (2003) 13:139–145.
Liu C-J, Blount JW, Steele CL, Dixon RA. Bottlenecks for metabolic engineering of isoflavone glycoconjugates in Arabidopsis. Proceedings of the National Academy of Sciences, USA (2002) 99:14578–14583.
Luo J, Nishiyama Y, Fuell C, et al. Convergent evolution in the BAHD family of acyltransferases: identification and characterization of anthocyanin acyltransferases from Arabidopsis thaliana . The Plant Journal (2007) 50:678–695.[CrossRef][ISI][Medline]
Messina M, Barnes S. The role of soy products in reducing cancer risk. Journal of the National Cancer Institute (1991) 83:541–546.
Modolo LV, Blount JW, Achnine L, Naoumkina MA, Wang X, Dixon RA. A functional genomics approach to (iso)flavonoid glycosylation in the model legume Medicago truncatula . Plant Molecular Biology (2007) 64:499–518.[CrossRef][ISI][Medline]
Nagashima S, Inagaki R, Kubo A, Hirotani M, Yoshikawa T. cDNA cloning and expression of isoflavonoid-specific glycosyltransferase from Glycyrrhiza echinata cell-suspension cultures. Planta (2004) 218:456–459.[CrossRef][Medline]
Nielsen H, Engelbrencht J, Brunak S, von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering (1997) 10:1–6.
Noguchi A, Saito A, Homma Y, Nakao M, Sasaki N, Nishino T, Takahashi S, Nakayama T. A UDP-glucose:isoflavone 7-O-glycosyltransferase from the roots of soybean (Glycine max) seedlings: purification, gene cloning, phylogenetics, and an implication for an alternative strategy of enzyme catalysis. Journal of Biological Chemistry (2007) 282:23581–23590.
Offen W, Martinez-Fleites C, Yang M, Kiat-Lim E, Davis BG, Tarling CA, Ford CM, Bowles DJ, Davies GL. Structure of a flavonoid glycosyltransferase reveals the basis for plant natural product modification. EMBO Journal (2006) 25:1396–1405.[CrossRef][ISI][Medline]
Page RDM. TREEVIEW: An application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences: CABIOS (1996) 12:357–358.[Medline]
Phillip DA. Flavonoids: plant signals to soil microbes. In: Recent advances in phytochemistry: phenolic metabolism in plants—Stafford HA, Ibrahim RK, eds. (1992) Vol. 26. NY: Plenum Press. 201–223.
Pollak PE, Vogt T, Mo Y, Taylor LP. Chalcone synthase and flavonol accumulation in stigmas and anthers of Petunia hybrida . Plant Physiology (1993) 102:925–932.[Abstract]
Richman A, Swanson A, Humphrey T, Chapman R, McGarvey B, Pocs R, Brandle J. Functional genomics uncovers three glycosyltransferases involved in the synthesis of the major sweet glycosides of Stevia rebaudiana . The Plant Journal (2005) 41:56–67.[CrossRef][ISI][Medline]
Setchell KDR, Zimmer-Nechemias L, Cai J, Heubi JE. Exposure of infants to phytoestrogens from soy-based infant formula. The Lancet (1997) 350:23–27.
Shao H, He XZ, Achnine L, Blount JW, Dixon RA, Wang X. Crystal structures of a multifunctional triterpene/flavonoid glycosyltransferase from Medicago truncatula . The Plant Cell (2005) 17:3141–3154.
Shoemaker R, Keim P, Vodkin LO, et al. A compilation of soybean ESTs: generation and analysis. Genome (2002) 45:329–338.[Medline]
Steele CL, Gijzen M, Qutob D, Dixon RA. Molecular characterization of the enzyme catalysing the aryl migration reaction of isoflavonoid biosynthesis in soybean. Archives of Biochemistry and Biophysics (1999) 367:146–150.[CrossRef][ISI][Medline]
St-Pierre B, De Luca V. Evolution of acyltransferase genes: origin and diversification of the BAHD superfamily of acyltransferases involved in secondary metabolism. In: Evolution of metabolic pathways. Recent advances in phytochemistry—Romeo JT, Ibrahim R, Varin L, De Luca V, eds. (2000) Vol. 34. Oxford: Elsevier Science Ltd. 285–315.
Suzuki H, Nakayama T, Yonekura-Sakakibara Y, Fukui Y, Nakamura N, Yamaguchi M, Tanaka Y, Kusumi T, Nishino T. cDNA cloning, heterologous expression, and functional characterization of malonyl-Coenzyme A:anthocyanidin 3-O-glucoside-6-O-malonyltransferase from dahlia flowers. Plant Physiology (2002) 130:2142–2151.
Suzuki H, Nakayama T, Nishino T. Proposed mechanism and functional amino acid residues of malonyl-CoA:anthocyanin 5-O-glucoside-6''-O-malonytransferase from flowers of Salvia splendens, a member of the versatile plant acyltransferase family. Biochemistry (2003) 130:2142–2151.