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Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes

Woosuk Jung1, Oliver Yu1, Sze-Mei Cindy Lau2, Daniel P. O'Keefe2, Joan Odell1, Gary Fader1, and Brian McGonigle1*

1Agricultural

Biotechnology, The Dupont Company, Experimental Station; PO Box 80402; Wilmington, DE 19880-0402. 2Central Research and Development, The DuPont Company, Experimental Station, P.O. Box 80402, Wilmington, DE 19880-0402. *Corresponding author ([email protected]). Received 24 July 1999; accepted 12 November 1999

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Isoflavones have drawn much attention because of their benefits to human health. These compounds, which are produced almost exclusively in legumes, have natural roles in plant defense and root nodulation. Isoflavone synthase catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway. To identify the gene encoding this enzyme, we used a yeast expression assay to screen soybean ESTs encoding cytochrome P450 proteins. We identified two soybean genes encoding isoflavone synthase, and used them to isolate homologous genes from other leguminous species including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine, as well as from the nonleguminous sugarbeet. We expressed soybean isoflavone synthase in Arabidopsis thaliana, which led to production of the isoflavone genistein in this nonlegume plant. Identification of the isoflavone synthase gene should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes.

Keywords: isoflavone, cytochrome P450, phenylpropanoid

The isoflavones genistein and daidzein are naturally occurring plant compounds that are being studied for their substantial health benefits. They are found almost exclusively in soybeans and other leguminous plants. The reported health benefits include relief of menopausal symptoms1,2, reduction of osteoporosis3,4, improvement in blood cholesterol levels5, and lowering risk of certain hormonerelated cancers6,7 and coronary heart disease8. The basis for these effects has not been established, but the weak estrogenic activity of isoflavones, which are sometimes referred to as phytoestrogens, may be a factor in conferring these properties. As a result, many food manufacturers are striving to provide products containing soy and/or isoflavones to consumers. Soybean seeds and protein products produced from seeds are the primary source of isoflavones in the human diet. The isoflavone content in soybean seeds varies depending on the variety and environmental conditions when grown9,10. Losses of isoflavones due to processing of seeds for traditional soy foods or protein products can reach 50% or more11. Together these factors can contribute to difficulties in reaching efficacious levels of isoflavones in soy products. This problem could be addressed by increasing isoflavone concentrations and reducing their variability in soybean seeds. Isoflavones have been shown to impart a negative taste component to foods12 and the reduction of isoflavone concentrations would be of value for other products. Another benefit of manipulating isoflavone synthase expression in legume and nonlegume crop species is that increased levels of isoflavones may increase resistance to various pathogens13. Developing other grain crops that can synthesize isoflavones would provide food manufacturers with alternatives to soy for use in their products. Isoflavones are synthesized by a branch of the phenylpropanoid pathway of secondary metabolism14. Other branches of this pathway (Fig. 1) produce lignin and anthocyanin pigments. In plants, isoflavones play major roles in the defense response to pathogen attack15­19 and in establishing the symbiotic relationships between the roots of leguminous plants and rhizobial bacteria, which lead to nodu208

lation and nitrogen fixation20. Genes encoding many enzymes active in the phenylpropanoid pathway have been isolated from many species. However, the gene encoding isoflavone synthase, the first step in the branch of the phenylpropanoid pathway that commits metabolic intermediates to the synthesis of isoflavones, has proved difficult to identify. Using a soybean EST collection, we have identified the cDNA encoding isoflavone synthase through functional expression in yeast. We have cloned homologs from several other related legume species as well as sugarbeet (a nonlegume) and shown that these genes are both structurally and functionally similar. We have expressed isoflavone synthase in Arabidopsis thaliana, a plant that does not normally make isoflavones, and shown that this transgenic plant is now able to produce genistein. Results and discussion Identification of isoflavone synthase cDNA. Isoflavone synthase (IFS) catalyzes the oxidation of 7,4'-dihyroxyflavanone (liquiritigenin) or 5,7,4'-trihydroxyflavanone (naringenin) to daidzein or genistein, respectively. Because earlier work suggested that the enzyme that catalyzes this reaction is a cytochrome P450 (refs 21,22), we therefore used homology to known cytochromes P450 as the criterion for selecting candidate cDNA clones from among libraries of soybean ESTs. Because IFS shows enhanced activity in fungally challenged tissue, we preferentially chose cDNAs with enhanced abundance in libraries constructed from leaves infected with the fungus Sclerotinia sclerotiorum. Included among this group of candidates were cDNAs homologous to those described by Schopfer and Ebel23. The candidate cDNAs were individually expressed in yeast to screen for functional activity of the encoded proteins. Cytochrome P450-dependent monooxygenases generally catalyze NADPH- and O2-dependent hydroxylation reactions but do not use NADPH directly, instead relying upon an interaction with a flavoprotein known as a P450 reductase that transfers electrons from the cofactor NADPH to the cytochrome P450 (ref. 24). The yeast strain used for

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Figure 1. A simplified diagram of the phenylpropanoid pathway showing intermediates and enzymes involved in isoflavone synthesis. Branches for lignin and anthocyanin synthesis are marked. Dotted arrows represent multiple enzymatic steps.

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expression assays had previously been engineered to produce a plant cytochrome P450 reductase25. Because both the reductase and cytochromes P450 are generally membrane-associated enzymes, microsomal membranes were prepared from yeast transformed with candidate cDNA expression genes and used in enzyme activity assays. To assay for IFS activity, we measured the ability of the yeast microsomal membranes to carry out the NADPH-dependent conversion of naringenin or liquiritigenin to genistein and daidzein, respectively. Following reverse-phase high-pressure liquid chromatography (HPLC), the isoflavonoids were identified by three criteria: retention time, UV absorption spectra, and electrospray mass spectroscopy. Because the isoflavonoids have distinct UV absorption spectra when compared to the flavonoids, it is possible to enhance detection selectivity using different detector wavelengths (Figs 2, 3), as well as to provide a positive confirmation of identity. In electrospray mass spectrometry (MS), positive ionization of genistein yielded two prominent ions at m/z = 271 and 312, at a ratio of 4.5:1. These correspond to the singly protonated ion of genistein, [MH]+, and the commonly observed acetonitrile solvent adduct [M:CH3CN:H]+. With daidzein a single prominent ion was observed at m/z = 255, corresponding to [MH]+. For confirmation of the in vitro production of genistein and daidzein, we required that the retention time, ultraviolet spectrum, and mass spectrum were all consistent with authentic standards. Microsomes from a yeast strain expressing a cDNA designated IFS1 (GenBank accession number AF195798) had the ability to convert naringenin to genistein. All other microsome assays of expressed cDNAs were negative for this conversion. A time course of IFS1 activity in the microsome assay showed an increase in genistein over time with a corresponding decrease in naringenin (Fig. 2). Without the addition of NADPH no genistein was detected, indicating a need for reducing equivalents to drive the reaction (Fig. 2C). In the yeast microsome assay, IFS1 was able to convert liquiritigenin to daidzein (Fig. 3). The formation of both daidzein and genistein was linear for up to 10 h, with the amount of genistein converted from naringenin by IFS1 being 50% less than the amount of daidzein converted from liquiritigenin by IFS1 (Fig. 4A). An NADPH-dependent metabolite of naringenin, designated "peak 2" in Figure 2, was also detected in the HPLC profile of products from microsomes producing genistein. Kochs and Grisebach22 proposed that the oxidative aryl migration required to convert naringenin to genistein proceeds through a cytochrome P450 monooxygenase-mediated conversion of the 2S-flavanone to a 2-hydroxyisoflavanone, followed by dehydration to the isoflavone, possibly mediated by a soluble dehydratase22. The 2-hydroxyisoflavanone intermediate was described as unstable and could spontaneously convert to genisNATURE BIOTECHNOLOGY VOL 18 FEBRUARY 2000

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Figure 2. Genistein synthesis in assays of yeast microsomes containing IFS1. Each pair of panels presents chromatograms at 260 nm (top) and 280 nm (bottom). In each assay 75 µg of yeast microsomal proteins were used. Assays were incubated for (A) 0 h (B) 4 h in the presence of NADPH, and (C) 4 h without NADPH. Five minutes of isocratic separation using a LiChrosopher RP-C18 column (5 µm) and 65% methanol as mobile phase were employed. Peaks with retention times and molecular masses of naringenin and genistein standards are labeled. Peak 2 is described in the text.

tein. In electrospray liquid chromatography/mass spectrometry (LC/MS) the most prominent peak in the spectrum of "peak 2" is at m/z = 289, consistent with it being the [MH]+ form of the proposed hydroxylated intermediate. A similar intermediate (at m/z = 273) was also detected in the conversion of liquiritigenin to daidzein (Fig. 3). [A discussion of the relationship of IFS1 to other cytochromes P450 can be found at the Nature Biotechnology Web Extras site26. Activity of a closely related soybean cytochrome P450. The nucleotide sequence of the IFS1 coding region has 92.5% similarity to the sequence of the soybean cytochrome P450 cyp93c1 (GenBank) accession number AF022462; ref. 27), whereas the encoded proteins have 96.7% similarity in their amino acid sequences. No enzymatic activity has been reported for cyp93c1. Because of the high degree of similarity to IFS1, we hypothesized that the cyp93c1 protein may also exhibit IFS activity. To test this possibility RT-PCR was used to generate a DNA fragment containing the coding region of cyp93c1, which was then expressed in yeast, and the activity of the encoded protein was assayed in the microsome system. Conversion of naringenin to genistein in the assay confirmed that cyp93c1 also encodes a protein with IFS activity (Fig. 4B). Similarly to IFS1, the second IFS enzyme (IFS2) converts both naringenin to genistein and liquiritigenin to daidzein in an NADPH-dependent, time-dependent manner. IFS2 also has a substrate preference similar to IFS1, with the amount of genistein converted from naringenin being 50% less than the amount of daidzein converted from liquiritigenin (Fig. 4B). It is important to note that the activities cannot be directly compared in this system as microsome preparations are a complex mixture of proteins and membranes and the levels of IFS1 and IFS2 proteins present in each microsome preparation may not be equivalent because of differences in the yeast expression. During the preparation of this manuscript, Steele and colleagues28 reported the identification of a soybean IFS cDNA through functional screening of cytochrome P450 candidate cDNAs using an insect cell

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Figure 4. Comparison of daidzein and genistein synthesis by IFS1 and IFS2. Synthesis of daidzein and genistein was monitored at 260 nm in assays of yeast microsomes containing: (A) IFS1 and (B) IFS2. Assays were repeated three times and vertical lines represent error bars. Figure 3. Daidzein synthesis in assays of yeast microsomes containing IFS1. Each pair of panels presents chromatograms at 260 nm (top) and 280 nm (bottom). For these assays, 30 mg of yeast microsomal proteins were used. Assays were incubated with NADPH for (A) 0 h, and (B) 10 h. Five minutes of isocratic separation using a Phenomenex Luna 3u C18 (2) column (3 µm) and 65% methanol as mobile phase were employed. Peaks with retention times and molecular masses of liquiritigenin and daidzein are labeled.

expression system. This IFS differs in three amino acids from IFS2 presented in this paper, and the amino acids in those positions are those found in IFS1. Although we find the amount of conversion of naringenin to genistein to be 50% less than the conversion of liquiritigenin to daidzein for both IFS1 and IFS2, the IFS identified by Steele et al. was reported to convert naringenin much less efficiently than liquiritigenin. The differences may be attributable to variations in the assay conditions or to different cytochrome P450 reductases in the two different expression systems. Isoflavone synthase gene characterizations. To determine the gene structures of IFS1 and IFS2, genomic DNA fragments containing the two soybean IFS cDNA sequences were produced from PCR using primers within the coding region for IFS1 and in the 5 and 3 untranslated regions of IFS2. Sequencing of these fragments indicated that each gene (GenBank accession numbers AF195818 and AF195819, respectively) contains a single intron located in the same position within the coding region, the splice junction occurring within the codon for amino acid 300. The IFS1 intron of 218 bp and the IFS2 intron of 135 bp have 46% sequence similarity. Isolation of isoflavone synthases from other legumes. Using the two IFS sequences from soybean, we designed a set of PCR primers for RT-PCR amplification of IFS cDNAs from several other legume species. DNA fragments were generated, cloned, and sequenced confirming the isolation of multiple homologs to IFS from mung bean (GenBank accession numbers AF195806, AF195807, AF195808, and AF195809), red clover (AF195810 and AF195811), and snow pea (AF195812). The proteins encoded by these sequences showed greater than 95% amino acid similarity to both IFS1 and IFS2. Yeast overexpressing a selection of the cDNAs, AF195807 (mung bean), AF195811 (red clover), and AF195812 (snow pea), were prepared and assays of the encoded proteins using naringenin as a substrate verified that these genes encode IFS enzymes. It should be noted that the first eight amino acids and the last eight amino acids of the encoded proteins are not necessarily representative of the endogenous gene, but instead are derived from the primers used in cloning. The additional coding sequences of the nonsoybean cDNAs allowed us to design another set of nested PCR primers and, using these primers, we were able to clone multiple variants of IFS from white clover (GenBank accession numbers AF195814 and AF195815), lentil (AF195804 and AF195805), hairy vetch (AF195803), alfalfa

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(AF195800, AF195801, and AF195802), and lupine (AF195813). The sequences cloned using the nested primers represent 92% of the fulllength coding regions. The nucleotide sequences show a high level of identity to each other with a majority of the differences encoding silent amino acid changes. The encoded proteins show a remarkably high amino acid similarity (95­99%) to both IFS1 and IFS2. The high degree of conservation may reflect the functional constraints necessary for the enzyme to be capable of carrying out an aryl migration, a unique reaction. (The encoded IFS proteins from all of the legumes analyzed show a high degree of similarity, as shown in an alignment figure at the Nature Biotechnology Web Extras site26.) Isolation of isoflavone synthases from sugarbeet, a nonlegume. Although isoflavones are found predominantly in legumes they are also found in several other diverse families of plants29. Sugarbeet (Beta vulgaris), a member of the family Chenopodiaceae, has been shown to produce isoflavones in pathogen-infected tissue30. Two IFS cDNAs from sugarbeet were cloned using RT-PCR and nested primers. Their sequences (GenBank accession numbers AF195816 and AF195817) indicate that both encoded proteins have greater then 95% similarity to the soybean IFS1 protein. To verify these results, we used the soybean IFS1 cDNA as a probe for Southern blot analysis against sugarbeet DNA. After high-stringency hybridization and washing, bands appear that are consistent with the presence of two genes in sugarbeet that are closely related to the soybean genes. The high degree of similarity between the soybean and sugarbeet IFS sequences is surprising because of the relatively distant relationship of these two species, and suggests a stringent requirement for the sequence of the protein capable of performing this reaction. Expression of soy IFS1 in transgenic Arabidopsis. Neither Southern blot analysis nor the analysis of the Arabidopsis EST and genomic sequence database reveals a gene with high homology to IFS. Though isoflavones are not synthesized in Arabidopsis, the naringenin substrate of IFS is an intermediate in the anthocyanin biosynthetic pathway that is present in Arabidopsis (Fig. 1). Naringenin may be accessible to an introduced foreign IFS enzyme, creating the possibility of synthesizing the isoflavone genistein in Arabidopsis. To test the function of the soybean IFS gene in a nonlegume plant, a chimeric gene including the 35S promoter of CaMV, the soybean IFS1 coding region, and the Nos 3 was introduced into Arabidopsis. Extracts of kanamycin-selected transformants were assayed by HPLC to look for the presence of genistein. A peak corresponding to the position of the genistein standard was detected in samples from five independent primary transformants, while no corresponding peak was detected in samples from control plants (Fig. 5A,B). The identity of the peak in the transformant extracts was verified by GC/MS to be the isoflavone genistein (Fig. 5C,D). These results indicate that the soybean IFS gene expresses an enzyme that is active in Arabidopsis, and

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GATTGATAGTTTATAGTAGG-3. The product was cloned into pCR2.1 and sequenced on a PE 3700 using dye terminator technology using a combination of vector and insert-specific primers. Sequence editing was done using DNAStar (DNASTAR, Madison, WI). The sequence generated represents coverage at least two times in both directions. Examination of sequence showed it to be identical to cyp93c1 (GenBank accession number AF022462). The coding region was amplified and cloned into pRS315-gal using "gap repair" as described above. Isoflavone synthase activity assay. Yeast microsomes were prepared from control WHT1 and strains expressD C ing a cytochrome P450 cDNA according to the methods of Pompon and colleagues25. The protein content of each microsome preparation was assayed using the Bradford protein micro assay (Bio-Rad, Hercules, CA). From 30 to 150 µg of microsomal proteins were incubated at room temperature in 80 mM K2HPO4, 0.5 mM glutathione, 20% (wt/vol) sucrose, pH 8.0, with 100 µM naringenin or 100 µM liquiritigenin substrate, and 40 nmol of NADPH added per each 100 µl of final reaction volume. Following incubation, reactions were Figure 5. Assays of Arabidopsis plant extracts. (A and B) HPLC assay of extracts from (A) extracted with ethyl acetate. Samples were assayed on a wild-type and (B) transgenic IFS plant, showing absorbance at 260 nm. The peak with Hewlett-Packard 1100 series HPLC system using either retention time and spectrum corresponding to genistein is labeled. Five minutes of isocratic a LiChrospher RP-C18 column (5 m; 250 × 3 mm) or a separation using a Phenomenex Luna 3u C18 (2) column (3 µm) and 65% methanol as mobile Phenomenex Luna C18 (2) column (3 u; 150 × 4.6 phase were employed. (C and D) GC-MS assay of extracts from (C) wild-type, and (D) mm). On the first column, samples in ethyl acetate of transgenic IFS plant, showing selected ion monitoring at 414 m/z. The 414 m/z peak candidate cDNA assays were isocratically separated for characteristic of the genistein standard is labeled. 5 min employing 65 % methanol as the mobile phase. For the second column, samples were evaporated and that the naringenin intermediate of the anthocyanin pathway is availresuspended in 80% methanol and then were separated using a 10 min linear able as a substrate for the introduced foreign enzyme. Naringenin was gradient from 20% methanol/80% 10 mM ammonium acetate, pH 8.3 to not detected in the control plants or in IFS transformants, suggesting 100% methanol at a flow rate of 1 ml min-1 or using 65% methanol as mobile that IFS is competing with other enzymes of the phenylpropanoid phase for isocratic elution. Genistein and daidzein were monitored by the pathway for a limited or transient amount of this intermediate. The absorbance at 260 nm, and naringenin and liquiritigenin were monitored by amount of genistein produced is 2 ng µg-1 of fresh weight, as deterthe absorbance at 280 nm. Using authentic naringenin, liquiritigenin, genismined by comparison with a quantitated genistein standard. tein, and daidzein (Indofine Chemical, Somerville, NJ) dissolved in ethanol as standards for calibration, peak areas were converted to nanograms. These results pave the way for manipulating the expression of IFS To confirm the identity of genistein, samples were evaporated and resusin legumes for improving pathogen and stress responses, and propended in 25% acetonitrile in water and assayed on a Hewlettducing valuable plant products or nutritionally enhanced foods. The Packard/Micromass LC/MS by running 25 µl on a Zorbax Eclipse XDB-C8 soybean IFS1 gene produces a functional enzyme in a plant species reverse-phase column (3 × 150 mm, 3.5 µm) isocratically with 25% solvent B that does not naturally produce isoflavones, producing genistein, (0.1% formic acid in acetonitrile), in solvent A (0.1% formic acid in water). demonstrating the potential for producing isoflavones in nonMass spectrometry was done by electrospray scanning from 200 to 400 m/e, isoflavone-producing crop species. using +60 volt cone voltage. The diode array signals were monitored between 200 and 400 nm in both instruments. Experimental protocol Preparation of IFS genomic clones. Soybean genomic DNA was prepared Candidate cDNA expression in yeast. Soybean ESTs generated in the DuPont from Glycine max cv. Wye following standard protocols (DNeasy Plant Maxi Genomics Program (S.V. Tingey, G.H. Miao, and M. Dolan, personal comKit, Qiagen, Valencia, CA). Using this DNA as template, a fragment containing munication) were screened by BLAST searching 31 of the NCBI database to IFS1 was produced by PCR with the primers: 5-TGCTGGAACTTGCACTTGidentify cDNA clones with homology to cytochromes P450. For each candiGT-3 and 5-GTATATGATGGGTACCTTAATTAAGAAAGGAG-3. A DNA date cDNA sequence to be tested, specific 5 primers were made that include fragment containing IFS2 was produced with the primers: 5-AAAATTAGC5 coding sequence and extensions of 24 bp that are homologous to the CTCACAAAAGCAAAG-3 and 5-GCAAACGAAGACAAATGGGAGATGApRS315-gal vector. A general 3 primer with homology to the cDNA cloning TA-3. These PCR fragments were cloned into the pCR2.1 vector (Invitrogen) vector linked to sequences homologous to the pRS315-gal vector was used to and sequenced on the PE 3700 sequencer as described above. PCR amplify the cDNA. The pRS315 (ref. 32) vector had previously been Isolation of additional isoflavone synthase cDNAs. Mung bean sprouts modified by the insertion of a bidirectional gal1/10 promoter33 between XhoI and snow pea sprouts were obtained from the grocery store. Seeds for alfalfa, and HindIII sites. Each coding region was amplified using the Advantage-GC red clover, white clover, hairy vetch, and lentil were obtained from Pinetree cDNA polymerase kit (Clontech, Palo Alto, CA), hybridized to the digested Garden Seeds (New Gloucester, ME), seeds for lupine cv. Russell Mix were vector, and transformed into yeast strain WHT1 where the expression plasobtained from Botanical Interests (Boulder, CO), and seeds for sugarbeet were mid is formed by gap repair34. WHT1 was constructed in the same manner as obtained from a commercial source. Seedlings were grown and RNA prepared described by Pompon25 but substituting the Helianthus tuberosum NADPHusing TRIzol Reagent (Gibco BRL) and first-strand cDNA was prepared as ferrihemoprotein reductase (GenBank accession number Z26250; ref. 35) for described above. OligodT was used as the reverse transcription primer in all the Arabidopis thaliana NADPH-ferrihemoprotein reductase (Daniele cases except with white clover for which random hexamers were used as the Werck-Reichhart personal communication). reverse transcription primer. Polymerase chain reaction amplification was The coding region of cyp93c1 was isolated by reverse transcription (RT)carried out using Advantage-GC cDNA polymerase mix (Clontech) using PCR from RNA prepared using TRIzol Reagent (Gibco BRL, Rockville, MD) primer set one: 5-ATGTTGCTGGAACTTGCACTT-3 and 5-TTAAGAAAGfrom soybean leaf tissue infected with Sclerotinia sclerotiorum. The first-strand GAGTTTAGATGCAACG-3 or the nested primer set two: 5-TGTTTCTGcDNA was synthesized from an oligo-dT primer using the Superscript preampliCACTTGCGTCCCAC-3 and 5-CCGATCCTTGCAAGTGGAACAC-3 as fication system (Gibco BRL, Grand Island, NY). The cyp93c1 coding region was follows. Mung bean and red clover PCR products amplified using primer set amplified using Advantage-GC cDNA polymerase mix (Clontech) with the one were cloned directly into pCR2.1. For white clover, lentil, hairy vetch, primers: 5-AAAATTAGCCTCACAAAAGCAAAG-3 and 5-ATATAAGalfalfa, lupine, and beet a first PCR with primer set one was followed by a sec-

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ond PCR with primer set two, and the resulting fragments cloned. For snow pea, a first PCR with primer set one was followed by a second PCR with high annealing temperature (60°C) using primer set one. The expected size product was gel purified and used as the template in a third PCR with the high annealing temperature and primer set one. The resulting product was cloned into pCR2.1. All PCR fragments in pCR2.1 were sequenced on the PE 3700 sequencer as previously described. All alignments were carried out using DNAStar software and the Clustal algorithm set to default parameters. The coding regions for accession numbers AF195807 (mung bean), AF195811 (red clover), and AF195812 (snow pea) were amplified and cloned into pRS315-gal using "gap repair," and microsomes were produced and assayed as described. Construction of isoflavone synthase plant expression vector. A soybean IFS1 coding region DNA fragment starting with the second codon and continuing through the stop codon, with the addition of a KpnI site, was produced by PCR amplification using the Pfu DNA polymerase (Stratagene, La Jolla, CA) using primers: 5-TTGCTGGAACTTGCACTTGGT-3 and 5-GTATATGATGGGTACCTTAATTAAGAAAGGAG-3, and the cDNA clone as template. The resulting 1.6 kb fragment was cloned as a blunt-KpnI fragment between CaMV 35S promoter36 and Nos 3 region37 fragments in pUC18. Blunt ligation of the filled in NcoI site at the 3 end of the promoter fragment to the IFS1 PCR fragment results in the coding region falling in frame with the ATG initiation codon within the NcoI site. From this clone, the 4.3 kb HindIIISalI fragment containing the chimeric 35S-soyIFS1-Nos 3 gene was transferred (as HindIII-PstI and PstI-SalI fragments) between HindIII and SalI sites of the pPZP211 binary vector that contains a kanamycin resistance marker gene for plant selection38, producing plasmid pOY204. Transformation of Arabidopsis. pOY204 was transformed into Agrobacterium tumefaciens strain GV3101 and introduced into Arabidopsis thaliana ecotype WS by in planta vacuum infiltration following standard procedures39. Plants recovered from seed germinated on 75 mg/L kanamycin containing plates were transferred to soil and grown for two weeks before analysis. Analysis of Arabidopsis transformants. Samples were prepared from 12 randomly selected individual transformants and control wild-type plants. The entire above-ground portion of the plant was ground in liquid nitrogen, extracted with 10 ml of 80% ethanol at room temperature for 20 min, and passed through an Acrodisc CR-PTFE syringe filter (Gelman Sciences, Ann Arbor, MI). Extraction solutions were concentrated by evaporation under nitrogen and three volumes of 1 N HCl were added, followed by incubation at 95°C for 2 h to hydrolyze any conjugated forms of isoflavones. After hydrolysis, samples were extracted once with ethyl acetate, dried under nitrogen, and resuspended in 80% methanol. Samples were assayed as described above on the Phenomenex Luna 3 µm C18 (2) column. Samples were analyzed by gas chromatography-mass spectrometry (GC-MS) to confirm the identity of genistein. The samples were converted to trimethylsilyl (TMS) ether derivatives by adding N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) (Supelco, Bellefonte, PA), and incubating at 37°C for 1 h. Samples were then dried under nitrogen, and dissolved in chloroform. GC-MS analysis was conducted using a Hewlett-Packard 6890 GC interfaced with a Hewlett-Packard 5973 mass selective detector. Samples were resolved using a 15 m × 0.25 mm i.d. DB-1ht column (J&W Scientific, Mid Glamorgan, UK). The oven temperature was programmed from 200°C (3.5 min hold) to 300°C at a rate of 5°C min-1 with a column flow rate of 7.5 ml min-1 He. The ionization potential of the mass selective detector was 70 eV. Genistein-TMS derivatives in plant extract samples were identified by comparison of retention times and mass spectra with that of a genistein standard derivatized with BSTFA. Diagnostic ions included 414 and 399 m/z that arise from the fragmentation of partially derivatized genistein, which is the form obtained using the above procedure.

895­898 (1999). 2. Murkies, A.L. et al. Dietary flour supplementation decreases post- menopausal hot flushes: effect of soy and wheat. Maturitas 21, 189­195 (1995). 3. Civitelli, R. In vitro and in vivo effects of ipriflavone on bone formation and bone biomechanics. Calcif. Tissue Int. 61, Suppl: S12­14 (1997). 4. Gennari, C. et al. Effects of ipriflavone--a synthetic derivative of natural isoflavones-- on bone mass in early years after menopause. Menopause 5, 9­15 (1998). 5. Sharma, R.D. Isoflavones and hypercholesterolemia in rats. Lipids 14, 535­540. (1979). 6. Peterson, G. & Barnes, S. Genistein inhibition of the growth of human breast cancer cells: independence from estrogen receptors and the multi-drug resistance gene. Biochem. Biophys. Res. Commun. 179, 661­667 (1991). 7. Messina, M. & Barnes, S. The role of soy products in reducing cancer risk. J. Natl. Cancer Inst. 83, 541­546 (1991). 8. Food labeling: health claims; soy protein and coronary heart disease; final rule. Federal Register 64 FR 57699, October 26, 1999. (http://www.fda.gov/). 9. Tsukamoto, C. et al. Factors affecting isoflavone content in soybean seeds: changes in isoflavones, saponins and composition of fatty acids at different temperatures during seed development. J. Agric. Food Chem. 43, 1184­1192 (1995). 10. Eldridge, A.C. & Kwolek, W.F. Soybean isoflavones: effect of environment and variety on composition. J. Agric. Food Chem. 31, 394­396 (1983). 11. Wang, H.-J. & Murphy, P.A. Mass balance study of isoflavones during soybean processing. J. Agric. Food Chem. 44, 2377­2383 (1996). 12. Okubo, K., et al. Components responsible for the undesirable taste of soybean seeds. Bioscience. Biotechnol. Biochem. 56, 99­103 (1992). 13. Padmavati, M. & Reddy, A.R. Flavonoid biosynthetic pathway and cereal defence response: an emerging trend in crop biotechnology. Plant Biochem. Biotechnol. 8, 15­20 (1999). 14. Dixon, R.A. & Pavia, N.L. Stress-induced phenylpropanoid metabolism. Plant Cell 7, 1085­1097 (1995). 15. Blount, J.W., Dixon, R.A. & Paiva, N.L. Stress response in alfalfa (Medicago sativa L.). XVI. Antifungal activity of medicarpin and its biosynthetic precursors: implications for the genetic manipulation of stress metabolites. Physiol. Mol. Plant Pathol. 41, 333­349 (1992). 16. Graham, T.L. in Handbook of phytoalexins metabolism and action (eds Daniel, M.& Purkayastha, R.P.) 85­116 (Marcel Dekker, New York; 1995). 17. Ebel, J. Phytoalexin synthesis: the biochemical analysis of the induction process. Annu. Rev. Phytopathol. 24, 235­264 (1986). 18. Rivera-Vargas, L.I., Schmitthenner, A.F. & Graham, T.L. Soybean flavonoid effects on and metabolism by Phytophthora sojae. Phytochemistry 32, 851­857 (1993). 19. Graham, T.L. & Graham, M.Y. in Plant­microbe interactions. (eds Keen, N. & Stacey, G.) (APS Press, St. Paul; 2000), in press. 20. Pueppke, J.L. The genetics and biochemical basis for nodulation of legumes by rhizobia. Crit. Rev. Biotechnol. 16, 1­51 (1996). 21. Hashim, M.F., Hatkamatsuka, T., Ebizuka, Y. & Sankawa, U. Reaction mechanism of oxidative rearrangement of flavanone in isoflavone biosynthesis. FEBS Lett. 271, 219­222 (1990). 22. Kochs, G. & Griesbach, H. Enzymic synthesis of isoflavones. Eur. J. Biochem. 155, 311­318 (1986). 23. Schopfer, C.R. & Ebel, J. Identification of elicitor-induced cytochrome p450s of soybean (Glycine max L.) using differential display of mRNA. Mol. Gen. Genet. 258, 315­322 (1998). 24. Bolwell, G.P., Bozac, K. & Zimmerlin, A. Plant cytochrome P450. Phytochemistry 37, 1491­1506 (1994). 25. Pompon, D., Louerat, B., Bronne, A. & Urban, P. Yeast expression of animal and plant p450s in optimized redox environments. Methods Enzymol. 272, 51­64 (1996). 26. Nature Biotechnology Web Extras site (http://biotech.nature.com/web_extras/). 27. Siminszky, B., Corbin, F.T., Ward, E.R., Fleischmann, T.J. & Dewey, R.E. Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast and tobacco enhances the metabolism of phenylurea herbicides. Proc. Natl. Acad. Sci. USA 96, 1750­1755 (1999). 28. Steele, C.L., Gijzen, M., Qutob, D. & Dixon, R.A. Molecular characterization of the enzyme catalyzing the aryl migration reaction of isoflavonoid biosynthesis in soybean. Arch. Biochem. Biophys. 367, 146­150 (1999). 29. Dewick, P.M. in The flavonoids: advances in research. (eds Harborne, J.B.& Mabry, T.J.) 535­640 (Chapman and Hall, New York; 1982). 30. Geigert, J., Stermitz, F.R., Johnson, G., Maag, D.D. & Johnson, D.K. Two phytoalexins from sugarbeet (Beta vulgaris) leaves. Tetrahedron 29, 2703­2706 (1973). 31. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 215, 403­410 (1990). 32. Sikorski, R.S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 1, 19­27(1989). 33. Johnston, M. & Davis, R.W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol. Cell. Biol. 4, 1440­1448 (1984). 34. Hua, S.B., Qiu, M., Chan, E., Zhu, L. & Luo, Y. Minimum length of sequence homology required for in vivo cloning by homologous recombination in yeast. Plasmid 38, 91­96 (1997). 35. Hasenfratz, M.P. Clonage de la NADPH-cytochrome P450 reductase et d'une proteine calnexine-like chez Helianthus tuberosus. (Universite Louis Pasteur, Strasbourg, France; 1992). 36. Odell, J.T., Nagy, F. & Chua, N.-H. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313, 810­812 (1985). 37. Depicker, A., Stachel, S., Dhaese, P., Zambryski, P. & Goodman, H.M. Nopaline synthase: transcript mapping and DNA sequence. J. Mol. Appl. Genet. 1, 561­573 (1982). 38. Hajdukiewicz, P., Svab, Z. & Maliga, P. The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25, 989­994 (1994). 39. Bechtold, N., Ellis, J. & Pelletier, G. In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. Paris, Life Sci. 316, 1194­1199 (1993). NATURE BIOTECHNOLOGY VOL 18 FEBRUARY 2000

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Acknowledgments

Thanks to Alfred A. Ciuffetelli, Tina Henry-Smith and June Shi for technical help; Edgar Cahoon, Sean J. Coughlan, and David Styles for analytical help; and Mike Hanafey and Mike Ramaker for bioinformatics support. Scott Tingey, Guo-Hua Miao, and Maureen Dolan are responsible for the DuPont Genomics Program, which provided invaluable source material. Thanks also to Wolfgang Shuh (Pioneer Hibred International) for the fungally treated soybean tissue, Pal Maliga (Waksman Institute, Rutgers University) for the binary vector and Daniele Werck-Reichhart (CNRS- Strasbourg) for construction of WHT1. Thanks to Enno Krebbers and Bill Hitz for thoughtful discussion.

1. Nestel, P.J., et al. Isoflavones from red clover improve systemic arterial compliance but not plasma lipids in menopausal women. J. Clin. Endocrinol. Metab. 84,

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