AU776161B2 - Genetic manipulation of isoflavonoids - Google Patents

Description

WO 00/53771 PCTIUSOO0/05915 GENETIC MANIPULATION OF ISOFLAVONOIDS TECHNICAL FIELD OF THE INVENTION The invention relates to gene manipulation in plants.

BACKGROUND OF THE INVENTION The flavonoids are a major class ofphenylpropanoid-derived plant natural products. Their fifteen carbon (C 6

-C

3

-C

6 backbone can be arranged as a 1,3diphenylpropane skeleton (flavonoid nucleus) or as a 1,2-diphenylpropane skeleton (isoflavonoid nucleus). Although 1,3-diphenylpropane flavonoid derivatives are almost ubiquitous among terrestrial plants, the 1,2-diphenylpropane isoflavonoids are restricted primarily to the Leguminosae, although they occur rarely in other families such as the Apocynaceae, Pinaceae, Compositae, and Moraceae (Tahara, S. and R. K.

Ibrahim, 1995, "Prenylated isoflavonoids an update," Phytochemistry 38: 1073- 1094).

The limited taxonomic distribution of the isoflavonoids is directly related to the occurrence of the enzyme complex isoflavone synthase (IFS), which catalyzes the aryl migration reaction leading to the formation of an isoflavone from a flavanone.

While flavanones are ubiquitous in higher plants, the IFS reaction, which is a two-step process specific for isoflavonoid biosynthesis (Kochs, G. and H. Grisebach, 1986, "Enzymic synthesis of isoflavones," European J Biochem 155: 311-318), is limited to the Leguminosae and the other diverse taxa in which isoflavonoids are occasionally found.

The presence of isoflavonoids provides several advantages to plants. One such advantage is provided by the function of isoflavonoids as antimicrobial phytoalexins in plant-microbe interactions. For example, the simple isoflavones daidzein and genistein act as initial precursors in the biosynthesis of various antimicrobial isoflavonoid phytoalexins in a wide variety of legumes (Dixon, R. A. and N. L. Paiva, 1995, "Stress-induced phenylpropanoid metabolism," Plant Cell 7: 1085-1097).

Isoflavonoid compounds have been shown to accumulate in infected plant cells to WO 00/53771 PCT/US00/05915 levels known to be antimicrobial in vitro. The temporal, spatial and quantitative aspects of accumulation are consistent with a role for these compounds in disease resistance (Rahe, J. 1973, "Occurrence and levels of the phytoalexin phaseollin in relation to delimitation at sites of infection of Phaseolus vulgaris by Colletotrichum lindemuthianum," Canadian J Botany 51: 2423-2430; Hadwiger, L. A. and D. M.

Webster, 1984, "Phytoalexin production in five cultivars of pea differentially resistant to three races of Pseudomonas syringae pv. pisi," Phytopathology 74: 1312-1314; Long, et al., 1985, "Further studies on the relationship between glyceollin accumulation and the resistance of soybean leaves to Pseudomonas syringae pv.

glycinea," Phytopathology 75: 235-239; Bhattacharyya, M. K. and E. W. B. Ward, 1987, "Biosynthesis and metabolism of glyceollin I in soybean hypocotyls following wounding or inoculation with Phytophthora megasperma f. sp. glycinea," Physiol and Mol Plant Pathology 31: 387-405). Moreover, it has been reported that many plant pathogens are much more sensitive to phytoalexins of non-host species than they are to the phytoalexins of their natural hosts, because they can often detoxify the host's phytoalexins. (VanEtten, et al., 1989, "Phytoalexin detoxification: importance for pathogenicity and practical implications," An Rev Phytopathology 27: 143-164).

Isoflavonoids also function in plant-microbe interactions in the establishment of bacterial or fungal symbioses with plants. Isoflavonoids have been reported to regulate bacterial nodulation genes, acting as a major nod gene inducer (Kosslak, et al., 1987, "Induction of Bradyrhizobiumjaponicum common nod genes by isoflavones isolated from Glycine max," Proc Natl Acad Sci USA 84: 7428-7432) and/or transcription activator (Dakora, et al., 1993, "Common bean root exudates contain elevated levels of daidzein and coumestrol in response to Rhizobiun inoculation," Mol Plant-Microbe Interact 6: 665-668). Isoflavonoids have also been shown to have a role on the establishment of the symbiotic vesicular arbuscular mycorrhizal (VAM) association of the fungus Glomus with legume roots. (Kape, et al., 1992, "Legume root metabolites and VA-mycorrhiza development," J Plant Physiol 141: 54-60). Xie et al have reported that the isoflavonoids coumestrol, daidzein and genistein have small but significant stimulatory effects on the degree of mycorrhizal colonization of soybean, and that one effect of isoflavonoids on the WO 00/53771 PCT/US00/05915 soybean mycorrhizal symbiosis could be via induction ofnodulation factors from cocolonizing Rhizobia, since nod-factors have also been shown to stimulate fungal colonization (Xie, et al., 1995, "Rhizobial nodulation factors stimulate mycorrhizal colonization of nodulating and nonnodulating soybeans," Plant Physiology 108: 1519- 1525).

In addition to the advantages that the presence of isoflavonoids confers to plants, a significant body of evidence indicates that dietary consumption of isoflavonoids can provide benefits to human health. Dietary isoflavones have been ascribed strong cancer chemopreventative activity in humans, and display a range of pharmacological activities suggestive of various other health promoting effects, including phytoestrogen activity as both estrogenic and anti-estrogenic agents (Coward, et al., 1993, "Genistein, daidzein, and their -glycoside conjugates: antitumor isoflavones in soybean foods from American and Asian diets," J Agricultural and Food Chemistry 41: 1961-1967; Martin, et al., 1996, "Interactions between phytoestrogens and human sex steroid binding protein," Life Sciences 58: 429-436); anticancer effects associated with phytoestrogenic activity (Lee, et al., 1991, "Dietary effects on breast-cancer risk in Singapore," Lancet 337: 1197-1200; Adlercreutz, et al., 1991, "Urinary excretion oflignans and isoflavonoid phytoestrogens in Japanese men and women consuming a traditional Japanese diet," Am J Clin Nutr 54: 1093-1100); anticancer effects associated with inhibition of several enzymes including DNA topoisomerase and tyrosine protein kinase (Akiyama, et al., 1987, "Genistein, a specific inhibitor of tyrosine-specific protein kinases," JBiol Chem 262: 5592-559; Uckun, et al., 1995, "Biotherapy of B-cell precursor leukemia by targeting genistein to CD19-associated tyrosine kinases," Science 267: 886- 8 9 1);suppression of alcohol consumption (Keung, W. M. and B. L. Vallee, 1993, "Daidzin: A potent, selective inhibitor of human mitochondrial aldehyde dehydrogenase," Proc Natl Acad Sci USA 90: 1247-1251; Keung, et al., 1995, "Daidzin suppresses ethanol consumption by Syrian golden hamsters without blocking acetaldehyde metabolism," Proc Natl Acad Sci USA 92: 8990-8993); antioxidant activity (Arora, et al., 1998, "Antioxidant activities of isoflavones and their biological metabolites in a lipsomal system," Arch Biochem Biophys 356: 133-141; Tikkanen, et WO 00/53771 PCT/US00/05915 al., 1998, "Effect of soybean phytoestrogen intake on low density lipoprotein oxidation resistance," Proc Natl Acad Sci USA 95: 3106-3110); effects on calcium metabolism, some of which may be linked to protective effects against osteoporosis (Tomonaga, et al., 1992, "Isoflavonoids, genistein, PSI-tectorigenin, and orobol, increase cytoplasmic free calcium in isolated rat hepatocytes," Biochem Biophys Res Com 182: 894-899; Draper, et al., 1997, "Phytoestrogens reduce bone loss and bone resorption in oophorectomized rats," JNutr 127: 1795-1799); and cardiovascular effects (Wagner, et al., 1997, "Dietary soy protein and estrogen replacement therapy improve cardiovascular risk factors and decrease aortic cholesteryl ester content in ovariectomized cynomolgus monkeys," Metabolism Clinical and Experimental 46: 698-705).

At present, the only dietary sources of isoflavonoids for humans are certain legumes such as soybean or chickpea. The development of methods to genetically manipulate isoflavonoids in plants, either to widen the source of dietary isoflavonoids for humans, or to exploit the biological activities of isoflavonoids for plant protection and improvement, is wholly dependent on the availability of cloned genes encoding the various enzymes ofisoflavonoid biosynthesis. Of these, the isoflavone synthase (IFS) complex constitutes the first committed reactions, and as such represents the means to introduce isoflavonoids into plants that do not possess the pathway.

In 1984, Hagmann and Grisebach provided the first evidence for the enzymatic conversion of flavanone to isoflavone (the IFS reaction) in a cell free system (Hagmann, M. and H. Grisebach, 1984, "Enzymatic rearrangement of flavanone to isoflavone," FEBS Letters 175: 199-202). They demonstrated that microsomes from elicitor-treated soybean cell suspension cultures could catalyze the conversion of 2(S)naringenin to genistein, or of 2 (S)-liquiritigenin to daidzein, in the presence of NADPH. The crude microsomal enzyme preparation, which was stable at -70 0 C but had a half-life of only 10 minutes at room temperature, was absolutely dependent on NADPH and molecular oxygen. It was subsequently shown that the reaction proceeded in two steps. The flavanone was converted in a cytochrome P450-catalyzed reaction requiring NADPH and 02 to the corresponding 2-hydroxyisoflavanone. This WO 00/53771 PCT/US00/05915 relatively unstable compound, which could, however, be identified by mass spectrometric analysis, then underwent dehydration to yield the isoflavone. The dehydration reaction appeared to be catalyzed by an enzyme present predominantly in the cytoplasmic supernatant, although it was not possible to remove all this activity from the microsomes. The corresponding 2-hydroxyisoflavanone spontaneously converted to genistein, for example, in methanol at room temperature. Kinetic analysis indicated that the 2-hydroxyisoflavanone was formed prior to genistein, consistent with its being an intermediate in isoflavone formation. (Kochs, G. and H.

Grisebach, 1986, "Enzymic synthesis of isoflavones," European J Biochem 155: 311- 318).

Involvement of cytochrome P450 in the 2-hydroxyisoflavanone synthase reaction was confirmed by inhibition by CO, replacing 02 with N 2 and examining the effects of a range of known P450 inhibitors of which ancymidol was the most effective. The enzyme co-migrated with the endoplasmic reticulum markers cinnamate 4-hydroxylase (another cytochrome P450) and cytochrome b5 reductase on Percoll gradients. The enzyme is stereoselective, and (2R)-naringenin is not a substrate. (Kochs, G. and H. Grisebach, 1986, "Enzymic synthesis of isoflavones," European J Biochem 155:311-318).

The origin of the 2-hydroxyl group was determined from studies on the IFS present in microsomes from elicited cell cultures of Pueraria lobata. 180 from 802 was incorporated into the 2-hydroxyl group, resulting in a 2-hydroxyisoflavanone with molecular ion shifted by two mass units, whereas there was no corresponding shift in the molecular ion of daidzein, consistent with the subsequent dehydration reaction (Hashim, et al., 1990, "Reaction mechanism of oxidative rearrangement of flavanone in isoflavone biosynthesis," FEBS Letters 271: 219-222). The currently accepted model for the reaction pathway of IFS as illustrated in Fig. 1, therefore, involves P450-catalyzed hydroxylation coupled to aryl migration, a reaction with mechanistic similarities to the well described proton migration mechanism of some P450 reactions (Hakamatsuka, et al., 1991, "P-450-dependent oxidative rearrangement in isoflavone WO 00/53771 PCT/US00/05915 biosynthesis: reconstitution of P-450 and NADPH:P450 reductase," Tetrahedron 47: 5969-5978).

Currently, there have been no reports on purification to homogeneity or molecular cloning of the cytochrome P450 of the IFS complex because of the extreme lability of the enzyme. The 2-hydroxyisoflavanone synthase cytochrome P450 from Pueraria has been solubilized with Triton X-100, and partially purified by DEAE- Sepharose chromatography; the enzymatic reaction could be reconstituted by addition ofNADPH cytochrome P450 reductase that separated from the hydroxylase on the ion exchange column (Hakamatsuka, et al., 1991, Tetrahedron 47: 5969-5978). A 2hydroxyisoflavanone dehydratase has been purified from elicitor-treated P. lobata cells, and has been shown to be a soluble monomeric enzyme of subunit Mr 38,000 (Hakamatsuka, et al., 1998, "Purification of 2-hydroxyisoflavanone dehydratase from the cell cultures of Pueraria lobata," Phytochemistry 49: 497-505). It is not yet clear whether this enzyme physically associates with the P450 hydroxylase catalyzing the aryl migration, or even whether this activity is essential for isoflavone formation in planta in view of the spontaneous conversion of 2-hydroxyisoflavanone to isoflavone.

Flavanone is a potential substrate for more than one type of hydroxylation reaction at the 2-position. Thus, elicitor-treated cell cultures of alfalfa and Glycyrrhiza echinata have been shown to accumulate the dibenzoylmethane licodione (Kirikae, et al., 1993, "Biosynthesis of a dibenzoylmethane, licodione, in cultured alfalfa cells induced by yeast extract," Biosci Biotech Biochem 57: 1353-1354).

Licodione synthase is, by classical criteria, a cytochrome P450, the activity of which is induced by yeast elicitor in Glycyrrhiza cells (Otani, et al., 1994, "Licodione synthase, a cytochrome P450 monooxygenase catalyzing 2-hydroxylation of in cultured Glycyrrhiza echinata L. cells," Plant Physiol 105: 1427-1432). The reaction it catalyzes involves 2-hydroxylation of flavanone followed by hemiacetal opening instead of aryl migration, and the reaction was thought to have mechanistic similarities to the flavone synthase II enzyme previously characterized from soybean (Kochs, G. and H. Grisebach, 1987, "Induction and characterization of a NADPHdependent flavone synthase from cell cultures of soybean," Z. Naturforsch 42C: 343- 348). A gene encoding the flavone synthase II/licodione synthase from Glycyrrhiza has been cloned (Akashi, et al., 1998, "Identification of a cytochrome P450 cDNA encoding (2S)-flavanone 2-hydroxylase of licorice (Glycyrrhiza echinata Fabaceae) which represents licodione synthase and flavone synthase II," FEBS Letters 431: 287- 290), and a different cytochrome P450 gene encoding flavone synthase II has recently been cloned from Gerbera hybrida (Martens, S. and G. Forkmann, "Cloning and expression of flavone synthase II from Gerbera hybrids," Plant J20: 611-618).

Although the reactions catalyzed by IFS are critical for the formation of all isoflavonoids in plants, there have been no previous reports of the isolation of genes encoding components of isoflavone synthase, although genes encoding most of the other enzymes of the isoflavonoid pathway, including downstream enzymes converting simple isoflavones to antimicrobial phytoalexins, have been characterized (Dixon, et al., 1995, "The isoflavonoid phytoalexin pathway: from enzymes to genes to transcription factors," Physiologia Plantarum 93: 385-392). Thus, the unavailability of isoflavone synthase genes has made it heretofore impossible to utilize the downstream genes for regulating isoflavonoid concentrations in legumes and other plants that do have the isoflavonoid pathway, or for engineering antimicrobial and pharmacologically active isoflavonoids in transgenic plants of species that do not have the isoflavonoid pathway.

20 Genes encoding the enzyme catalyzing the first step of the isoflavone synthase reaction have now been isolated and purified from soybean and Medicago truncatula (barrel medic).

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a 25 context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Throughout this specification the word "comprise", or variations such as 30 "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

7a BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 depicts the currently accepted model for the reaction pathway of IFS wherein the flavanone is converted in a cytochrome P450-catalyzed reaction requiring NADPH and 02 to the corresponding 2-hydroxyisoflavanone which then undergoes dehydration to yield the isoflavone.

Fig. 2 depicts the nucleotide sequence of soybean CYP93Clv2.

g WO 00/53771 PCT/US00/05915 Fig. 3 depicts the amino acid sequence of soybean CYP93Clv2 compared to licorice CYP93B1.

Fig. 4 depicts the nucleotide sequence of Medicago truncatula mtIFSE3.

Fig. 5 depicts the amino acid sequence ofMedicago truncatula mtIFSE3 compared to soybean CYP93Clv2.

Fig. 6A and Fig. 6B depict HPLC traces of extracts from pooled tissues (leaves, shoots, flowers) ofArabidopsis thaliana ecotype Columbia harboring an empty tDNA vector (Fig. 6A) and Arabidopsis thaliana ecotype Columbia harboring the soybean CYP93C v2 cDNA sequence (Fig. 6B). The empty vector transformed line contains a number of flavonol glycosides and other phenolic compounds that are also present in the CYP93C v2 transformed line. These compounds were identified as rhamnose (Rha)- glucose (Glc)- quercetin uncharacterized conjugate of Q, Rha-Glc-Rha-Kaempferol Glu-Rha-Q, Rha-Rha-Q, Glc-Rha-K, (g) sinapic acid, Rha-Rha-K. Three additional compounds were observed in the CYP93Clv2 transformed line (Fig. 6B), and labeled and Fig. 6C depicts a total ion chromatogram of partially purified peaks 2 and 3, and the insets show the specific ions generated from these compounds. Peak 2 has a parental molecular mass ion of 579.5 consistent with genistein conjugated to a glucose-rhamnose disaccharide, and two further mass ions of 417.5 and 271.3, representing Rha-genistein and free genistein, respectively. Peak 3, which has a parental molecular ion of mass 417.5, is thereby identified as Rha-genistein.

Fig. 7A and Fig. 7B depict HPLC traces of the same extracts as shown in Fig.

6A (empty-vector transformed) and Fig. 6B, (CYP93Clv2 transformed), but following digestion with p-glucosidase. Peaks 2 and 3 remained at the same retention time as in Fig. 6A and 6B. However, Peak 1 disappeared, and was replaced with a new Peak 4 of much later retention time. Fig. 7C shows the total ion chromatograph of purified Peak 4, and the inset shows the parental molecular ion, with mass of 271.2, consistent with Peak 4 being free genistein. Fig. 7D shows a total ion chromatograph, and the parental molecular ion, of an authentic sample of genistein.

WO 00/53771 PCT/US00/05915 Fig. 8A, 8B, 8C and 8D are high performance liquid chromatography (HPLC) chromatograms depicting the presence of new peaks at RT 29.96 and 37.7 min representing the presence of the isoflavone daidzein formed from the flavanone liquiritigenin, or the isoflavone genistein formed from the flavanone naringenin, in insect cell microsomes expressing CYP93Clv2. Fig. 8A depicts the presence of NADPH during incubation with liquiritigenin. Fig. 8B depicts the absence of NADPH during incubation with liquiritigenin. Fig. 8C depicts the presence of NADPH during incubation with naringenin. Fig. 8D depicts the lack of a reaction when soybean CYP93E expressed in insect cells is incubated with liquiritigenin in the presence of

NADPH.

Fig. 9A and Fig. 9B are mass spectra ofBSTFA (N,O-bis(trimethylsilyl) trifluoroacetamide) derivatives. Fig. 9A depicts the mass spectrum of the BSTFA derivative of the product of the reaction catalyzed by CYP93Clv2 in insect cells using liquiritigenin as substrate, and Fig. 9B shows the mass spectrum of the BSTFA derivative of an authentic sample of daidzein SUMMARY OF THE INVENTION In one aspect, the invention is a method for introducing into a naturally nonisoflavonoid-producing plant species the enzyme catalyzing the aryl migration of a flavanone to form an isoflavanone intermediate or an isoflavone, comprising introducing a DNA segment encoding the enzyme into the plant to form a transgenic plant, wherein the transgenic plant expresses the DNA segment under the control of a suitable constitutive or inducible promoter when the transgenic plant is exposed to conditions which permit expression. The DNA segment can comprise isolated genomic DNA or recombinant DNA. Preferably, the DNA segment is a CYP93C gene. An exemplary DNA segment from a soybean CYP93C gene consists essentially of the sequence from about nucleotide 36 to about nucleotide 1598 of the sequence depicted in SEQ ID NO: 1. Another preferred DNA segment comprises a Medicago truncatula homolog of a CYP93C gene, more preferably, the sequence from about nucleotide 92 to about nucleotide 1657 of the sequence depicted in SEQ ID NO:4.

Plants transformed by this method may also preferably express chalcone synthase, WO 00/53771 PCT/US00/05915 chalcone reductase, and chalcone isomerase genes to cause in vivo formation of daidzein or a daidzein derivative, and the chalcone synthase, chalcone reductase, and chalcone isomerase genes may also be transgenes. Plants transformed by this method may also preferably further comprise downstream genes, for example, isoflavone 0methyltransferase, isoflavone 2'-hydroxylase, isoflavone reductase, and vestitone reductase, to metabolize a formed isoflavone to biologically active isoflavonoid derivatives or conjugates. The plant can comprise isoflavone 4'-O-methyl-transferase to cause formation of biochanin A or a biochanin A derivative from the isoflavanone intermediate. An exemplary flavanone substrate for this transformation method is liquiritigenin and/or naringenin.

In another aspect, the present invention is a method for increasing the level of isoflavonoid compounds in naturally isoflavonoid-producing plants comprising introducing a DNA segment encoding the enzyme catalyzing the aryl migration of a flavanone to yield an isoflavonoid to form a transgenic plant, wherein the transgenic plant expresses the DNA segment under the control of a suitable constitutive or inducible promoter when the transgenic plant is exposed to conditions which permit expression. With this method, the resulting isoflavonoid can be an isoflavanone intermediate, an isoflavone, an isoflavone derivative, and an isoflavone conjugate.

The DNA segment can comprise isolated genomic DNA or recombinant DNA.

Preferably, the DNA segment is a CYP93C gene. An exemplary DNA segment from a soybean CYP93C gene consists essentially of the sequence from about nucleotide 36 to about nucleotide 1598 of the sequence depicted in SEQ ID NO: 1. Another preferred DNA segment comprises a Medicago truncatula homolog of a CYP93C gene, more preferably, the sequence from about nucleotide 92 to about nucleotide 1657 of the sequence depicted in SEQ ID NO:4. An exemplary flavanone substrate for this transformation method is liquiritigenin and/or naringenin.

In another aspect, the invention is a method for synthesizing an isoflavanone intermediate or an isoflavone from a flavanone by expressing a recombinant CYP93C gene segment in a suitable bacterial, fungal, algal, or insect cell system. An exemplary gene segment consists essentially of the sequence from about nucleotide 36 WO 00/53771 PCT/US00/05915 to about nucleotide 1598 of the sequence depicted in SEQ ID NO: 1. Another exemplary gene segment consists essentially of the sequence from about nucleotide 92 to about nucleotide 1657 of the sequence depicted in SEQ ID NO:4.

In another aspect, the invention is a method of reducing the levels of isoflavonoid compounds in a naturally isoflavonoid-producing plant comprising introducing and expressing an antisense or gene silencing construct that contains an intact CYP93C gene or segments thereof into the plant. An exemplary gene consists essentially of the sequence from about nucleotide 36 to about nucleotide 1598 of the sequence depicted in SEQ ID NO: 1. Another exemplary gene consists essentially of the sequence from about nucleotide 92 to about nucleotide 1657 of the sequence depicted in SEQ ID NO:4.

In another aspect, the invention is a naturally non-isoflavonoid-producing plant cell transformed by introducing a DNA segment encoding the enzyme catalyzing the aryl migration of a flavanone to form an isoflavanone intermediate or an isoflavone, wherein the transformed plant cell expresses the DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression. The DNA segment can comprise isolated genomic DNA or recombinant DNA. Preferably, the DNA segment is a CYP93C gene. An exemplary DNA segment from a soybean CYP93C gene consists essentially of the sequence from about nucleotide 36 to about nucleotide 1598 of the sequence depicted in SEQ ID NO:1.

Another preferred DNA segment comprises a Medicago truncatula homolog of a CYP93C gene, more preferably, the sequence from about nucleotide 92 to about nucleotide 1657 of the sequence depicted in SEQ ID NO:4. Plants transformed by this method may also preferably express chalcone synthase, chalcone reductase, and chalcone isomerase genes to cause in vivo formation of daidzein or a daidzein derivative, and the chalcone synthase, chalcone reductase, and chalcone isomerase genes may also be transgenes. Plants transformed by this method may also preferably further comprise downstream genes, for example, isoflavone O-methyltransferase, isoflavone 2'-hydroxylase, isoflavone reductase, and vestitone reductase, to metabolize a formed isoflavanone intermediate to biologically active isoflavonoid WO 00/53771 PCT/US00/05915 derivatives or conjugates. The plant can comprise isoflavone 4'-O-methyl-transferase to cause formation of biochanin A or a biochanin A derivative from the isoflavanone intermediate.

In another aspect, the invention is a naturally isoflavonoid-producing plant cell transformed by introducing a DNA segment encoding the enzyme catalyzing the aryl migration of a flavanone to yield an isoflavonoid to form a transformed plant cell, wherein the transformed plant cell expresses the DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression. With this method, the resulting isoflavonoid can be an isoflavanone intermediate, an isoflavone, an isoflavone derivative, and an isoflavone conjugate.

The DNA segment can comprise isolated genomic DNA or recombinant DNA.

Preferably, the DNA segment is a CYP93C gene. An exemplary DNA segment from a soybean CYP93C gene consists essentially of the sequence from about nucleotide 36 to about nucleotide 1598 of the sequence depicted in SEQ ID NO:1. Another preferred DNA segment comprises a Medicago truncatula homolog of a CYP93C gene, more preferably, the sequence from about nucleotide 92 to about nucleotide 1657 of the sequence depicted in SEQ ID NO:4.

In another aspect, the invention is a transgenic plant cell having reduced levels of isoflavonoid compounds, the plant cell transformed by introducing an antisense or gene silencing construct that contains an intact CYP93C gene or segments thereof into the plant cell. An exemplary gene consists essentially of the sequence from about nucleotide 36 to about nucleotide 1598 of the sequence depicted in SEQ ID NO:1.

Another exemplary gene consists essentially of the sequence from about nucleotide 92 to about nucleotide 1657 of the sequence depicted in SEQ ID NO:4.

In another aspect, the invention is an isolated gene or DNA segment comprising a portion which encodes a cytochrome P450 that can catalyze the aryl migration ofa flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the portion consists essentially of about nucleotide 36 to about nucleotide 1598 of the sequence depicted in SEQ ID NO: 1. An exemplary gene is the soybean gene encoding the enzyme catalyzing the aryl migration of liquiritigenin. Another 12 WO 00/53771 PCT/US00/05915 exemplary gene is the soybean gene encoding the enzyme catalyzing the aryl migration of naringenin.

In another aspect, the invention is a protein encoded by a portion of an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the portion consists essentially of about nucleotide 36 to about nucleotide 1598 of the sequence depicted in SEQ ID NO:1.

In another aspect, the invention is an isolated gene or DNA segment comprising a portion which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the portion is a Medicago truncatula homolog of a CYP93C gene. An exemplary gene or DNA segment consists essentially of about nucleotide 92 to about nucleotide 1657 of the sequence depicted in SEQ ID NO:4. An exemplary gene is the Medicago truncatula gene encoding the enzyme catalyzing the aryl migration of liquiritigenin. Another exemplary gene is the Medicago truncatula gene encoding the enzyme catalyzing the aryl migration of naringenin.

In another aspect, the invention is a protein encoded by a portion of an isolated gene or a DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the portion is a Medicago truncatula homolog of a CYP93C gene.

In yet another aspect, the invention is a food comprising edible transgenic plant material capable of being ingested for its nutritional value, wherein the transgenic plant has been transformed with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, and wherein the transgenic plant exhibits increased levels of an isoflavonoid when compared to the level of the isoflavonoid in plants of the same species which do not comprise the isolated gene or DNA segment.

In yet another aspect, the invention is a food comprising at least one isoflavonoid, wherein the isoflavonoid is isolated from a transgenic plant transformed 13 WO 00/53771 PCT/US00/05915 with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, and wherein the transgenic plant exhibits increased levels of an isoflavonoid when compared to the level of the isoflavonoid in plants of the same species which do not comprise the isolated gene or DNA segment.

In yet another aspect, the invention is a composition comprising at least a portion of a transgenic plant transformed with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the transgenic plant exhibits increased levels of an isoflavonoid when compared to the level of the isoflavonoid in plants of the same species which do not comprise the isolated gene or DNA segment, and wherein the composition is suitable for ingestion as a food stuff, a nutritional supplement, an animal feed supplement, or a nutraceutical.

In yet another aspect, the invention is a composition comprising an isoflavonoid suitable for administration as a food stuff, a nutritional supplement, an animal feed supplement, a nutraceutical, or a pharmaceutical, wherein the isoflavonoid is isolated from at least a portion of a transgenic plant transformed with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, and wherein the transgenic plant exhibits increased levels of an isoflavonoid when compared to the level of the isoflavonoid in plants of the same species which do not comprise the isolated gene or DNA segment.

In yet another aspect, the invention is a method of increasing the nutritional value of a plant by transforming the plant with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration ofa flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the transgenic plant exhibits increased levels of an isoflavonoid when compared to the level of the isoflavonoid in plants of the same species which do not comprise the isolated gene or DNA segment.

WO 00/53771 PCT/US00/05915 In yet another aspect, the invention is a method of using a transgenic plant transformed with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the transgenic plant exhibits an increased level of an isoflavonoid when compared to the level of the isoflavonoid in plants of the same species which do not comprise the isolated gene or DNA segment, to provide a nutraceutical benefit to a human or animal administered the isoflavonoid. The isoflavonoid can be administered by ingestion of at least a portion of the plant. The isoflavonoid can also be administered by ingestion of a composition comprising an isoflavonoid isolated from the plant.

In yet another aspect, the invention is a method of using an isoflavonoid isolated from a transgenic plant transformed with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration ofa flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the transgenic plant exhibits an increased level of an isoflavonoid when compared to the level of the isoflavonoid in plants of the same species which do not comprise the isolated gene or DNA segment, to provide a pharmaceutical benefit to a patient administered the isoflavonoid.

In yet another aspect, the invention is a method of increasing disease resistance in a plant by transforming the plant with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the transgenic plant exhibits an increased level of an isoflavonoid when compared to the level of the isoflavonoid in plants of the same species which do not comprise the isolated gene or DNA segment.

In yet another aspect, the invention is a method of increasing nodulation efficiency of a leguminous plant by transforming the plant with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the transgenic plant exhibits an increased level of an isoflavonoid released from the roots WO 00/53771 PCT/US00/05915 when compared to the level of the isoflavonoid released from the roots of plants of the same species which do not comprise the isolated gene or DNA segment.

In yet another aspect, the invention is a transgenic leguminous plant exhibiting increased nodulation efficiency transformed with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the transgenic plant exhibits an increased level of an isoflavonoid released from the roots when compared to the level of the isoflavonoid released from the roots of plants of the same species which do not comprise the isolated gene or DNA segment.

In yet another aspect, the invention is a method of increasing bacterial or fungal symbiosis in a plant by transforming the plant with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the transgenic plant exhibits an increased level of an isoflavonoid when compared to the level of the isoflavonoid in plants of the same species which do not comprise the isolated gene or DNA segment.

In yet another aspect, the invention is a transgenic plant exhibiting increased bacterial or fungal symbiosis transformed with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration ofa flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the transgenic plant exhibits an increased level of an isoflavonoid when compared to the level of the isoflavonoid in plants of the same species which do not comprise the isolated gene or DNA segment.

In yet another aspect, the invention is a transgenic plant comprising at least one recombinant DNA sequence encoding a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the transgenic plant exhibits an increased level of an isoflavonoid when compared to the level of the isoflavonoid in plants of the same species which do not comprise the recombinant DNA sequence.

WO 00/53771 PCT/USO/05915 In yet another aspect, the invention is seed from a transgenic plant comprising at least one recombinant DNA sequence encoding a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the transgenic plant exhibits an increased level of an isoflavonoid when compared to the level of the isoflavonoid in plants of the same species which do not comprise the recombinant DNA sequence.

In yet another aspect, the invention is progeny from a transgenic plant comprising at least one recombinant DNA sequence encoding a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the transgenic plant exhibits an increased level of an isoflavonoid when compared to the level of the isoflavonoid in plants of the same species which do not comprise the recombinant DNA sequence.

In yet another aspect, the invention is progeny from seed of a transgenic plant comprising at least one recombinant DNA sequence encoding a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the transgenic plant exhibits an increased level of an isoflavonoid when compared to the level of the isoflavonoid in plants of the same species which do not comprise the recombinant DNA sequence.

In yet another aspect, the invention is use of a transgenic plant transformed with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the transgenic plant exhibits an increased level of an isoflavonoid when compared to the level of the isoflavonoid in plants of the same species which do not comprise the isolated gene or DNA segment, for the preparation of a nutraceutical preparation for achieving a nutritional effect.

In yet another aspect, the invention is use of a transgenic plant transformed with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein the transgenic plant exhibits an increased level of an isoflavonoid WO 00/53771 PCT/US00/05915 when compared to the level of the isoflavonoid in plants of the same species which do not comprise the isolated gene or DNA segment, for the preparation of a pharmaceutical preparation for achieving a therapeutic effect.

DETAILED DESCRIPTION One aspect of the present invention is an isolated gene which encodes the first step of the isoflavone synthase reaction: a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavone. Genes and corresponding cDNA of the soybean or Medicago truncatula CYP93 family have been isolated. The enzymes encoded by the genes of the present invention are isoflavone synthases (IFS) and can catalyze the aryl migration of a flavanone to yield an isoflavone either directly or through the intermediacy of a 2-hydroxyisoflavanone. One isolated soybean gene is classified as CYP93Cv2.

Cytochrome P450 enzymes belong to a large superfamily of enzymes that are abundant in every living organism. The P450 nomenclature committee has determined that each P450 should carry a "CYP" designation and arbitrarily divided the superfamily into families (alphabetical designation), subfamilies (numerical designation) and allelic variants plus numerical designation) based on amino acid identity of and respectively (Nelson, et al. 1993. "The P450 superfamily update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature," DNA Cell Biol 12:1). Thus, CYP93Clv2 is a variant of the first described P450 belonging to the third subfamily of the ninetythird P450 family.

Utilizing the procedures presented herein, any plant known to produce isoflavonoids may also serve as sources of suitable DNA, or coding sequences may be synthesized in vitro based on the sequences for the IFS genes of the present invention.

CYP93 family members can also be obtained from other plant species by polymerase chain reaction amplification methods known to those skilled in the art, using primer sequences corresponding to regions of nucleotide conservation between CYP93 family members. Furthermore, the genes of the present invention are defined by their WO 00/53771 PCT/US00/05915 catalytic activity: the aryl migration of a flavanone to yield an isoflavone. The gene sequences presented as SEQ ID NO: 1 and SEQ ID NO:4 are exemplary, and it is understood that modifications to these genes which do not alter the catalytic activity of its encoded protein fall within the scope of the present invention. While a preferred IFS gene contains the entire open reading frame, portions of or the entire 5' and 3' untranslated regions as well as portions of the vector sequence can also be present.

With the isolation and functional identification of these isoflavone synthase (IFS) genes that encode the first key step in isoflavone formation, the aryl migration reaction, it is now possible to introduce the isoflavonoid pathway into all plant species, including those that do not naturally possess this pathway.

Another aspect of the present invention is a genetically modified plant which has been transformed with a gene of the present invention. For example, when the CYP93Clv2 gene is transferred into the model plant Arabidopsis thaliana, which does not naturally produce isoflavonoids, the isoflavone genistein accumulates as a series of glycoconjugates (Example This demonstrates that the genes of the present invention can be genetically engineered into plants which do not naturally contain the isoflavonoid pathway, and the transgenic plants can then produce isoflavonoids, resulting in plants with improved disease resistance and/or value added health benefits for humans. In the present invention, unless otherwise stated, as used herein, the term "plant" or "progeny" includes plant parts, plant tissue, plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, explants, plant cells that are intact in plants, or parts of plants, such as embryos, pollen, ovules, flowers, capsules, stems, leaves, seeds, roots, root tips, and the like. Furthermore, the present invention includes the IFS genes expressed in various parts of the plant, in aerial portions of the plant useful for increasing disease resistance or production of health promoting isoflavonoid nutraceuticals, in seeds useful for increasing levels of isoflavones and their conjugates, or in roots useful for increasing disease resistance or production of nodulation gene inducing isoflavones.

WO 00/53771 PCT/US00/05915 In another aspect, the present invention is a method of improving disease resistance and a transgenic plant with increased disease resistance. By transforming a plant which does not naturally make isoflavones with an IFS gene of the present invention, disease resistance can be genetically engineered into the plant by providing the necessary enzyme to convert its natural flavanones into isoflavonoids. The introduction and subsequent expression of an IFS gene of the present invention into a crop species which naturally possesses the isoflavonoid pathway results in increased levels of the isoflavonoid defense compounds.

In another aspect, the present invention is a method of increasing levels of isoflavonoids that might be beneficial to the establishment of bacterial or fungal symbioses with plants and a transgenic plant with an increased capacity for symbiotic association with bacteria or fungi. Bacterial nodulation can be stimulated in transgenic leguminous plants by expression of an IFS gene of the present invention and decreased by expression of antisense constructs or constructs designed to promote gene silencing that contain an intact IFS gene or segments thereof. Mycorrhizal colonization of leguminous plants can also be increased through the introduction and expression of an IFS gene of the present invention.

In yet another aspect, the present invention is a method of producing isoflavonoid compounds in plants or any other organism to be used in nutraceuticals or pharmaceuticals to confer human or animal health benefits. Edible transgenic plants high in isoflavonoids can be utilized as food for humans and animals. Edible compositions high in isoflavonoids can also be made by incorporation of the transgenic plants or plant materials, or by incorporation of isoflavonoids isolated from the transgenic plants. Compositions useful for administration as a food stuff, a nutritional supplement, an animal feed supplement, a nutraceutical, or a pharmaceutical can be made by incorporation of the transgenic plants or plant materials, or by incorporation of isoflavonoids isolated from the transgenic plants.

The nutritional value of a plant can be increased by transforming the plant with an IFS gene of the present invention and, as a result, accumulating high amounts of isoflavonoids in the plant.

WO 00/53771 PCT/US00/05915 The soybean IFS gene of the present invention was isolated and purified according to the detailed procedures outlined in Example 2. The DNA sequence is shown in SEQ ID NO:1 and Fig. 2, and the encoded protein sequence of the isolated soybean CYP93C clone is shown in SEQ ID NO:2 and Fig. 3. For comparison, Fig. 3 also shows the protein sequence alignment between the isolated CYP93C clone (SEQ ID NO:2) and CYP93B (SEQ ID NO:3), the licorice licodione synthase.

The DNA and protein sequences of the soybean CYP93C 1 open reading frame were deposited in the Genbank data base under accession AF022462. The deposition was made by Siminszky, Dewey and Corbin, and the sequence described as representing a gene induced in soybean in response to herbicide safeners. However, the function of the gene was not known and there was no understanding that it could be involved in isoflavonoid biosynthesis at the time the deposit was made (Siminszky, Corbin, Ward, Fleischmann, T.J. and Dewey, R.E. ,1999, "Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast and tobacco enhances the metabolism of phenylurea herbicides." Proc. Natl. Acad. Sci. USA 96: 1750-1755).

The sequence of the clone characterized herein differs from CYP93C1 in three nucleotide substitutions in the open reading frame that change proline 140 to leucine, threonine 156 to isoleucine, and glutamate 295 to lysine. Thus, the soybean gene identified herein has been classified as CYP93Clv2.

The cDNA insert from CYP93Clv2 was used to probe 240,000 phage plaques from a Medicago truncatula root cDNA library (van Buuren, I.E. Maldonado- Mendoza, A.T. Trieu, L.A. Blaylock, and M.J. Harrison, 1999, "Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis formed between Medicago truncatula and Glomus versiforme," Mol. Plant-Microbe Interact. 12, 171-181). Five positive plaques were purified, in vivo excised, and sequenced. A full length clone designated mtIFSE3 was completely sequenced on both strands, and shown to encode the Medicago truncatula homolog of soybean CYP93C1. The nucleotide sequence of mtIFSE3 is shown in SEQ ID NO:4 and Fig. 4, and the protein sequence, in SEQ ID An alignment between the protein sequences of mtIFSE3 and CYP93Clv2 is shown in Fig. WO 00/53771 PCTIUSOO/05915 An IFS gene of the soybean or Medicago truncatula CYP93C subfamily or corresponding cDNA sequence, the open reading frame of which encodes a cytochrome P450 that can catalyze the aryl migration ofa flavanone to yield an isoflavone, either directly or through the intermediacy of a 2-hydroxyisoflavanone, can be used to introduce the isoflavonoid pathway into any plant species that does not naturally possess this pathway. Soybean CYP93Clv2 acts on the flavanones liquiritigenin to yield daidzein, and naringenin to yield genistein. Liquiritigenin is only formed in plants that possess the enzyme chalcone reductase (CHR) (Welle, R.

and Grisebach, 1989, "Phytoalexin synthesis in soybean cells: elicitor induction of reductase involved in biosynthesis of 6'-deoxychalcone." Arch Biochem Biophys 272: 97-102), and a form of chalcone isomerase that is active against trihydroxychalcone, the product of the co-action of chalcone synthase (CHS) with CHR (Dixon, Blyden, Robbins, van Tunen, A.J. and Mol, J.N.M., 1988, "Comparative biochemistry of chalcone isomerases." Phytochemistry 27: 2801- 2808). Such genes are common in legumes, but not in most other plant families.

Thus, to form daidzein in transgenic plants that do not possess the isoflavonoid pathway, it would be necessary to introduce three new genes, namely CHR, to co-act with CHS to form 2',4,4'-trihydroxychalcone, a suitable CHI to convert trihydroxychalcone to liquiritigenin, and IFS, assuming that the 2hydroxyisoflavanone intermediate can spontaneously dehydrate in planta, a phenomenon that is demonstrated below. Without CHR present, no liquiritigenin would be formed, and IFS would only be able to act on naringenin to yield, assuming spontaneous dehydration of the 2-hydroxyisoflavanone, genistein.

The IFS genes of the present invention can be introduced into non-leguminous plants such as by standard Agrobacterium-based or biolistic transformation procedures (Horsch, et al., 1985, "A simple and general method for transferring genes into plants," Science 227:1229-1231; and Klein, et al., 1988, "Stable genetic transformation of intact Nicotiana cells by the particle bombardment process," Proc Natl Acad Sci USA 85:8502-8505). Both procedures require the construction of a plasmid vector containing a desirable transcriptional promoter driving expression of the gene of interest (in this case IFS), followed by a transcriptional terminator and a WO 00/53771 PCT/US00/05915 selectable marker gene for resistance, such as to an antibiotic or a herbicide. The biolistic procedure coats metal particles with plasmid DNA containing the gene of interest and places them on a micro carrier disk. Using the biolistic apparatus, the particles are physically propelled into plant tissue. The plant tissue is then put under selection antibiotic or herbicide) followed by regeneration. The two Agrobacterium-based procedures are "in planta" and "ex-planta", respectively. Both procedures require the above gene construct to be placed into a T-DNA vector, which is then transferred into Agrobactrium tumefaciens. The in planta procedure places the transformed Agrobacterium in the presence of plant material (flower or meristem) and the plants are allowed to seed followed by selection antibiotic or herbicide) during germination. The ex-planta procedure also places Agrobacterium in the presence of plant material (callus, cell culture, leaf disk, hypocotyl) which is placed directly under selection antibiotic or herbicide) followed by regeneration.

Thus, the isoflavonoid pathway can be introduced into any plant species that does not possess the enzyme catalyzing the IFS reaction by expressing the IFS gene in transgenic plants under the control of a suitable constitutive or inducible promoter.

Example 1: Transformation ofArabidopsis thaliana with Soybean CYP93C1V2 Soybean CYP93C1v2 cDNA was placed in the binary plant transformation vector pCHF3, in which it is under control of the cauliflower mosaic virus promoter, using standard recombinant DNA methods (Sambrook, et al. 1989.

Molecular Cloning. A Laboratory Manual, 2nd Ed, Cold Spring Harbor Laboratory Press, New York). The gene was then transformed into plants of the crucifer, Arabidopsis thaliana ecotype Columbia, using Agrobacterium tumefaciens and a standard floral infiltration procedure (Clough, S.J. and Bent, 1998, "Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana". Plant J 16: 735-743). Transgenic plants were selected by germinating the seedlings on kanamycin, and those surviving selection were allowed to set seed. T 2 seedlings expressing CYP93C1v2 were identified by standard DNA and RNA gel blot analysis (Sambrook, et al. 1989. Molecular cloning. A Laboratory Manual, 2nd Ed, WO 00/53771 PCTIUSOO/05915 Cold Spring Harbor Laboratory Press, New York), and analyzed for accumulation of genistein in leaves by HPLC analysis, according to a method developed to profile the flavonoid components of Arabidopsis leaves (Graham, 1998, "Flavonoid and flavonol glycoside metabolism in Arabidopsis". Plant Physiol Biochem 36: 135-144).

Figure 6A shows a typical HPLC trace of a leaf extract from an untransformed plant. The major components are glycosides (containing glucose and rhamnose) of the flavonols kaempferol and quercetin. Plants harboring the soybean CYP93C1v2 gene showed an additional three peaks on HPLC analysis (Fig. 6B), indicated by the arrows labeled as and No free genistein, free 2-hydroxyisoflavanone or 2hydroxyisoflavanone conjugates were observed. However, following treatment of extracts with almond p-glucosidase (Fig 7B), one of the new peaks disappeared, and free genistein was now observed, consistent with the peak being a glucoside of genistein. LC-MS analysis confirmed the identities of the new compounds as a glucoside of genistein, glucose-rhamnose-genistein, and rhamnose-genistein (Figs. 6C and 7C and 7D, insets). Therefore, expression of CYP93C1v2 in transgenic Arabidopsis leads to formation of genistein with no requirement for an enzyme to catalyze the dehydration of the presumed 2-hydroxyisoflavanone intermediate.

Arabidopsis plants then modify the genistein by exactly the same chemistry they use to conjugate their endogenous flavonols, namely by conjugation to glucose and rhamnose. Transgenic production of conjugates of genistein are suitable for nutraceutical applications, because genistein is also glycosylated in soybean, its natural dietary source (Graham, 1991, Flavonoid and isoflavonoid distribution in developing soybean seedling tissues and in seed and root exudates." Plant Physiol 594-603).

In addition to introducing the isoflavonoid pathway into plants that do not possess this pathway, the level of isoflavonoid compounds can be controlled in plants that do possess the pathway by manipulating the level of expression of the IFS gene.

Increasing the levels of isoflavonoid compounds in leguminous plants by expression of the IFS gene of the present invention in transgenic plants under the control of a suitable constitutive or inducible promoter can be accomplished by standard methods WO 00/53771 PCT/US00/05915 such as Agrobacterium-based or biolistic transformation methods known in the art.

Alternatively, the level of isoflavonoid compounds in plants can be reduced by expression of antisense constructs or constructs designed to promote gene silencing that contain an intact IFS gene, or segments thereof, in transgenic plants using methods known in the art. (Bourque, 1995, "Antisense strategies for genetic manipulations in plants," Plant Science 105:125-149; and Angell, S. M. and D. C.

Baulcombe, 1997, "Consistent gene silencing in transgenic plants expressing a replicating potato virus X RNA," EMBO J 16:3675-3684). Antisense constructs for gene silencing are constructed by placing the whole or part of the cDNA in a three prime to five prime orientation behind a desirable transcriptional promoter and ahead of a transcriptional terminator in a plasmid vector. The vector may be used for biolistic transformation or the new antisense gene may be transferred to a T-DNA vector for Agrobacterium-based transformation. The actual mechanism of silencing by antisense constructs is unknown. Homology-dependent gene silencing or cosuppression requires the over-expression of a homologous gene; therefore, to achieve co-suppression a construct is made using a strong promoter, the gene of interest (in this case IFS) and a transcriptional terminator. The gene should be transferred to plants as described above. Gene silencing is an epigenetic phenomenon that may or may not occur with a particular gene construct. When it does occur, the inhibition of gene expression can be greater than with the antisense approach.

Isoflavones can be synthesized from flavanones, utilizing recombinant IFS expressed in any suitable bacterial, fungal, algal, or insect cell system. For example, naringenin is extracted in large amounts from grapefruits. A CYP93C1 enzyme can be used convert naringenin to 2,5,7,4'-tetrahydroxyisoflavanone, which spontaneously converts to the valuable nutraceutical genistein under weak acid conditions.

Furthermore, daidzin can be synthesized from liquiritigenin utilizing recombinant CYP93C1 and an isoflavone glucosyltransferase (Kister, J. and W. Barz, 1981, "UDP-Glucose: isoflavone 7-O-glucosyltransferase from roots of chick pea (Cicer arietinum Arch Biochem Biophys 212: 98-104).

WO 00/53771 PCT/US00/05915 Example 2: Methodology Used to Isolate and Identify IFS cDNA Clones In an attempt to obtain cDNA clones encoding IFS, a functional genomics approach was followed. IFS activity is present in soybean seeds, which accumulate daidzein and genistein. Furthermore, IFS activity can be induced in soybean tissues in response to infection with Phytophthora infestans, associated with the accumulation of the isoflavonoid phytoalexin glyceollin (Bhattacharyya, M. K. and E. W. B. Ward, 1987, "Biosynthesis and metabolism of glyceollin I in soybean hypocotyls following wounding or inoculation with Phytophthora megasperma f. sp. glycinea," Physiol Mol Plant Path 31: 387-405). It was also known that an enzyme catalyzing a similar reaction to IFS, namely the 2-hydroxylation of flavanone but without aryl migration, belongs to the CYP93B1 subclass ofcytochrome P450s (Akashi, et al, 1998, "Identification of a cytochrome P450 cDNA encoding (2S)-flavanone 2-hydroxylase of licorice (Glycyrrhiza echinata Fabaceae) which represents licodione synthase and flavone synthase II," FEBS Letters 431: 287-290). We therefore searched an expressed sequence tag (EST) database of partial soybean sequences obtained by mass sequencing of two cDNA libraries: a Phytophthora-infected hypocotyl cDNA library (48 hours after infection) and a mid to late developmental stage seed library. Nine candidate P450 sequences were identified, of which three belonged to the CYP93 family. DNA probes were made from the EST clones of the three CYP93 candidates and were used to probe an RNA blot of transcripts from alfalfa suspension cells at various times after exposure to yeast elicitor, a treatment known to induce IFS activity at the onset of isoflavonoid phytoalexin accumulation (Kessmann, et al., 1990, "Stress responses in alfalfa (Medicago sativa III. Induction of medicarpin and cytochrome P450 enzyme activities in elicitor-treated cell suspension cultures and protoplasts," Plant Cell Reports 9: 38-41). One P450 probe cross-hybridized and detected alfalfa transcripts that were strongly induced by elicitation. This probe was derived from a clone with high homology to soybean CYP93C1 as described below, and the insert in the EST clone was full length. The insert was excised and then cloned into the baculovirus expression system for functional identification by heterologous expression in insect cells (Pauli, H. H. and T. M. Kutchan, 1998, "Molecular cloning and functional heterologous expression of two alleles encoding (S)-N-methylcoclaurine 3'- WO 00/53771 PCT/US00/05915 hydroxylase (CYP80B a new methyl jasmonate-inducible cytochrome P-450dependent mono-oxygenase of benzylisoquinoline alkaloid biosynthesis," The Plant J 13: 793-801).

The carbon monoxide difference spectrum ofmicrosomes isolated from insect cells expressing the soybean CYP93C clone indicated the presence of expressed cytochrome P450, as seen from an absorption peak at 450 nm that was not present in similar spectra from insect microsomes originating from cells transformed with a control vector. Unlabeled liquiritigenin was then fed to the microsomes in the presence of NADPH. The substrate remained unconverted in microsomes from cells harboring the control vector. However, in microsomes expressing the CYP93C clone, a new peak of RT 29.96 min was observed by high performance liquid chromatography (Fig 8A). The amount of this peak was reduced 10-fold if NADPH was omitted from the incubations (Fig. 8B). The UV spectrum of the product, obtained by diode array detection, was identical to that of authentic daidzein (hmax 248 nm, sh 302 nm, Xmin 222 nm). The product was collected, derivatized, and analyzed by GC-MS. The mass spectrum of the BSTFA derivative was identical to that of an authentic sample of daidzein (Fig. Microsomes containing the CYP93C clone also metabolized naringenin to yield genistein, although somewhat less efficiently than the reaction with liquiritigenin (Fig. 8C). Insect cell microsomes expressing a different soybean cytochrome P450 cDNA, CYP93E, did not convert liquiritigenin to daidzein when incubated in the presence of NADPH (Fig. 8D). These results indicate that the soybean CYP93C encodes IFS.

Example 3: Method of Increasing Dietary Isoflavonoid Intake Transgenic tomato plants are produced by the introduction of CYP93Clv2 via standard Agrobacterium-based procedures. In a preferred embodiment, the CYP93C v2 coding sequence is under control of a gene promoter giving specific expression in the fruit. Progeny containing the coding region of the CYP93Clv2 gene are selected at the seedling stage by standard polymerase chain reaction and/or DNA blot analysis known to those skilled in the art. Plants scoring positive for possession of the transgene are grown to fruiting, and fruit analyzed for the presence of WO 00/53771 PCT/US00/05915 isoflavones by the HPLC methods shown in Fig. 7 and Fig. 8 of the present invention.

Fruit harvested from the transgenic tomato plants are ingested to increase the dietary intake of isoflavonoids.

It is to be understood that the above description is of preferred exemplary embodiments of the invention and is intended to be illustrative of the invention, but is not to be construed to limit the scope of the invention in any way. Modifications may be made in the structural features of the invention without departing from the scope of the invention.

In summary, isoflavones can now be genetically engineered to provide potential human health benefits of dietary isoflavones and to increase disease resistance in plants. Isoflavones can now be produced in transgenic plants species in which isoflavones do not naturally occur, in species other than legumes. For example, engineering constitutive production of daidzein and/or genistein or their conjugates into tomato, potato, corn, or other popular components of the human diet, leads to human health benefits, such as reduced cancer risk, reduced incidence of osteoporosis, and treatment for alcoholism. Alternatively, introducing infectioninducible isoflavonoid biosynthesis into non-legumes qualitatively complements these plants' phytoalexin defenses against microbial pathogens, whereas over-expression of the isoflavonoid pathway in legumes quantitatively increases this defense response.

Finally, modifying the extent of production of isoflavonoids in legume roots positively impacts nodulation efficiency and therefore plant yield.

EDITORIAL NOTE APPLICATION NUMBER 37287/00 The following Sequence Listing pages 1 to 11 are part of the description. The claims pages follow on pages "29" to "37".

WO 00/53771 PCTIUSOO/05915 SEQUENCE LISTING <110> THE SAMUEL ROBERTS NOBLE FOUNDATION, INC.

STEELE, Christopher L.

DIXON, Richard A.

<120> GENETIC MANIPULATION OF ISOFLAVONOIDS <130> 11137/05002 <140> <141> <150> 60/123,267 <151> 1999-03-08 <160> <170> PatentIn Ver. 2.1 <210> <211> <212> <213> <220> <221> <222> 1 1717

DNA

Glycine max

CDS

(36)..(1598) <400> 1 gagcaaagat caaacaaacc aaggacgaga acacg atg ttg ctt gaa ctt gca Met Leu Leu Glu Leu Ala ctt ggt tta Leu Gly Leu act gca aaa Thr Ala Lys ttg Leu gtt ttg gct ctg Val Leu Ala Leu ctg cac ttg Leu His Leu cgt ccc aca ccc Arg Pro Thr Pro cca cca agc cca Pro Pro Ser Pro 101 149 tca aaa gca ctt Ser Lys Ala Leu cgc Arg 30 cat ctc cca aac His Leu Pro Asn aag cct Lys Pro cgt ctt ccc ttc ata gga cac ctt cat Arg Leu Pro Phe Ile Gly His Leu His tta aaa gac aaa Leu Lys Asp Lys ctc cac tac gca Leu His Tyr Ala ctc Leu atc gac ctc tcc Ile Asp Leu Ser aaa cat ggt ccc Lys His Gly Pro ttc tct ctc tac Phe Ser Leu Tyr ttt Phe ggc tcc atg cca Gly Ser Met Pro acc Thr 80 gtt gtt gcc tcc Val Val Ala Ser aca cca Thr Pro gaa ttg ttc Glu Leu Phe aag Lys ctc ttc ctc caa Leu Phe Leu Gin cac gag gca act His Glu Ala Thr tec ttc aac Ser Phe Asn 100 WO 00/53771 WO 0053771PCT/USOO/0591 aca agg ttc Thr Arg Phe 105 caa acc tca gcc Gin Thr Ser Ala aga cgc ctc acc Arg Arg Leu Thr gat agc tca Asp Ser Ser gtg gcc Val Ala 120 atg gtt ccc ttc Met Val Pro Phe gga Gly 125 cct tac tgg aag Pro Tyr Trp Lys ttc Phe 130 gtg agg aag ctc Val Arg Lys Leu atc Ile 135 atg aac gac ctt Met Asn Asp Leu aac gcc acc act Asn Ala Thr Thr gta Val1 145 aac aag ttg agg Asn Lys Leu Arg cct Pro 150 437 485 533 ttg agg acc caa Leu Arg Thr Gin atc cgc aag ttc Ile Arg Lys Phe agg gtt atg gcc Arg Val Met Ala caa ggc Gin Gly 165 gca gag gca Ala Giu Ala acc aac agc Thr Asn Ser 185 cag Gin 170 aag ccc ctt qac Lys Pro Leu Asp ttg Leu 175 acc gag gag ctt Thr Giu Giu Leu ctg aaa tgg Leu Lys Trp 180 gag gag atc Glu Giu Ile acc atc tcc atg Thr Ile Ser Met atg Met 190 atg ctc ggc gag Met Leu Giy Giu gct Ala 195 aga gac Arg Asp 200 atc gct cgc gag Ile Aia Arg Giu ctt aag atc ttt Leu Lys Ile Phe ggc Gi y 210 gaa tac agc ctc Giu Tyr Ser Leu act Th r 215 gac ttc atc tgg Asp Phe Ile Trp cca Pro 220 ttg aag cat ctc Leu Lys His Leu gtt gga aag tat Vai Gly Lys Tyr aaq agg atc gac Lys Arg Ile Asp gac Asp 235 atc ttg aac aag Ile Leu Asn Lys ttc Phe 240 gac cct gtc gtt Asp Pro Val Vai gaa agg Giu Arg 245 gtc atc aag Vai Ile Lys gtt gtt gag Val Val Giu 265 cgc cgt gag atc Arg Arg Giu Ile gtg Val1 255 agg agg aga aag Arg Arg Arg Lys aac gga gag Asn Giy Giu 260 ttg ctt gaa Leu Leu Giu ggt gag gtc agc Gly Giu Vai Ser ggg Giy 270 gtt ttc ctt gac Vai Phe Leu Asp ttc gct Phe Aia 280 gag gat gag acc Giu Asp Giu Thr atg Met 285 gag atc aaa atc Giu Ile Lys Ile acc Thr 290 aag gac cac atc Lys Asp His Ile aag Lys 295 ggt ctt gtt gtc Gly Leu Vai Val ttt ttc tcg gca Phe Phe Ser Ala aca gac tcc aca Thr Asp Ser Thr gcg Aia 310 917 965 1013 gtg gca aca gag Vai Ala Thr Giu t gg Trp 315 gca ttg gca gaa Ala Leu Aia Glu ctc Le u 320 atc aac aat cct Ile Asn Asn Pro aag gtg Lys Vai 325 ttg gaa aag Leu Giu Lys gct Al a 330 cgt gag gag gtc Arg Giu Giu Val tac Tyr 335 agt gtt gtg gga Ser Vai Val Giy aag gac aga Lys Asp Arg 340 1061 WO 00/53771 WO 00/377 1PCT/USOO/0591 ctt gtg gac Leu Val Asp 345 qaa gtt gac act Glu Val Asp Thr caa Gl1 350 aac ctt cct tac Asn Leu Pro Tyr at t Ile 355 aga gca atc Arg Ala Ile 1109 gtg aag Val Lys 360 gag aca ttc cgc Giu Thr Phe Arg cac ccg cca ctc His Pro Pro Leu cca Pro 370 gtg gtc aaa aga Val Val Lys Arg tgc aca gaa gag Cys Thr Giu Glu tgt C ys 380 gag att aat gga Glu Ile Asn Gly gtg atc cca gag Val Ile Pro Giu gga Gly 390 1157 1205 1253 gca ttg att ctc Ala Leu Ile Leu ttc Phe 395 aat gta tgg caa Asn Val Trp Gin gga aga gac ccc Giy Arg Asp Pro aaa tac Lys Tyr 405 tgg gac aga Trp Asp Arg gct gaa ggg Ala Giu Gly 425 cca Pro 410 tcg gag ttc cgt Ser Giu Phe Arg cc t Pro 415 gag agg ttc cta Giu Arg Phe Leu gag aca ggg Giu Thr Giy 420 cat ttt caa His Phe Gin 1301 1349 gaa gca ggg cct Giu Ala Gly Pro ctt Leu 430 gat ctt agg gga Asp Leu Arg Gly ca a Gin 435 ctt ctc Leu Leu 440 cca ttt ggg tct Pro Phe Giy Ser ggg Gly 445 agg aga atg tgc Arg Arg Met Cys gga gtc aat ctg Gly Vai Asn Leu gct Al a 455 act tcg gga atg Thr Ser Giy Met gca Ala 460 aca ctt ctt gca Thr Leu Leu Ala tct Ser 465 ctt att cag tgc Leu Ile Gin Cys tt c Phe 470 1397 1445 1493 gac ttg caa gtg Asp Leu Gin Val ggt cca caa gga Gly Pro Gin Gly ata ttg aag ggt Ile Leu Lys Giy ggt gac Gly Asp 485 gcc aaa gtt Ala Lys Vai cat agt ctt His Ser Leu 505 ctc ctt tct Leu Leu Ser 520 agc Se r 490 atg gaa gag aga Met Giu Giu Arg gcc Al a 495 ggc ctc act gtt Gly Leu Thr Vai cca agg gca Pro Arg Ala 500 gca tct aaa Aia Ser Lys 1541 1589 gtc tgt gtt cca Vai Cys Val Pro ctt Leu 510 gca agg atc ggc Ala Arg Ile Gly gtt Vali 515 taattaagat catcgtcatc atcatcatat gtaatattta 1638 ctttttgtgt gttgataatc atcatttcaa taaggtctca ttcatctact ttttatgaag 1698 tatataagcc cttccatgc <210> 2 <211> 521 <212> PRT <213> Glycine max 1717 WO 00/53771 <400> 2 Met Leu 1 His Leu Pro Asn His Leu Lys Lys Val Val Glu Ala Leu Thr Lys Phe 130 Val Asn 145 Arg Val Glu Glu Gly Glu Phe Gly 210 Lys Val 225 Asp Pro Arg Arg Leu Asp Ile Thr 290 Gly Thr 305 PCT/USOO/05915 Leu Glu Leu Ala Leu Gly Leu Leu Val Leu Ala Leu Phe Leu 5 10 Arg Pro Thr Pro Thr Ala Lys Ser Lys Ala Leu Arg His Leu 25 Pro Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly His Leu 40 Leu Lys Asp Lys Leu Leu His Tyr Ala Leu Ile Asp Leu Ser 55 His Gly Pro Leu Phe Ser Leu Tyr Phe Gly Ser Met Pro Thr 70 75 Ala Ser Thr Pro Glu Leu Phe Lys Leu Phe Leu Gin Thr His 90 Thr Ser Phe Asn Thr Arg Phe Gin Thr Ser Ala Ile Arg Arg 100 105 110 Tyr Asp Ser Ser Val Ala Met Val Pro Phe Gly Pro Tyr Trp 115 120 125 Val Arg Lys Leu Ile Met Asn Asp Leu Leu Asn Ala Thr Thr 135 140 Lys Leu Arg Pro Leu Arg Thr Gin Gin Ile Arg Lys Phe Leu 150 155 160 Met Ala Gin Gly Ala Glu Ala Gin Lys Pro Leu Asp Leu Thr 165 170 175 Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser Met Met Met Leu 180 185 190 Ala Glu Glu Ile Arg Asp Ile Ala Arg Glu Val Leu Lys Ile 195 200 205 Glu Tyr Ser Leu Thr Asp Phe Ile Trp Pro Leu Lys His Leu 215 220 Gly Lys Tyr Glu Lys Arg Ile Asp Asp Ile Leu Asn Lys Phe 230 235 240 Val Val Glu Arg Val Ile Lys Lys Arg Arg Glu Ile Val Arg 245 250 255 Lys Asn Gly Glu Val Val Glu Gly Glu Val Ser Gly Val Phe 260 265 270 Thr Leu Leu Glu Phe Ala Glu Asp Glu Thr Met Glu Ile Lys 275 280 285 Lys Asp His Ile Lys Gly Leu Val Val Asp Phe Phe Ser Ala 295 300 Asp Ser Thr Ala Val Ala Thr Glu Trp Ala Leu Ala Glu Leu 310 4 315 320 WO 00/53771 PCT/US00/05915 Ile Asn Asn Pro Lys Val Leu Glu Lys Ala Arg Glu Glu Val Tyr Ser 325 330 335 Val Val Gly Lys Asp Arg Leu Val Asp Glu Val Asp Thr Gin Asn Leu 340 345 350 Pro Tyr Ile Arg Ala Ile Val Lys Glu Thr Phe Arg Met His Pro Pro 355 360 365 Leu Pro Val Val Lys Arg Lys Cys Thr Glu Glu Cys Glu Ile Asn Gly 370 375 380 Tyr Val Ile Pro Glu Gly Ala Leu Ile Leu Phe Asn Val Trp Gin Val 385 390 395 400 Gly Arg Asp Pro Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg Pro Glu 405 410 415 Arg Phe Leu Glu Thr Gly Ala Glu Gly Glu Ala Gly Pro Leu Asp Leu 420 425 430 Arg Gly Gin His Phe Gin Leu Leu Pro Phe Gly Ser Gly Arg Arg Met 435 440 445 Cys Pro Gly Val Asn Leu Ala Thr Ser Gly Met Ala Thr Leu Leu Ala 450 455 460 Ser Leu Ile Gin Cys Phe Asp Leu Gin Val Leu Gly Pro Gin Gly Gin 465 470 475 480 Ile Leu Lys Gly Gly Asp Ala Lys Val Ser Met Glu Glu Arg Ala Gly 485 490 495 Leu Thr Val Pro Arg Ala His Ser Leu Val Cys Val Pro Leu Ala Arg 500 505 510 Ile Gly Val Ala Ser Lys Leu Leu Ser 515 520 <210> 3 <211> 523 <212> PRT <213> Glycyrrhiza echinata <400> 3 Met Glu Pro Gin Leu Val Ala Val Ser Val Leu Val Ser Ala Leu Ile 1 5 10 Cys Tyr Phe Phe Phe Arg Pro Tyr Phe His Arg Tyr Gly Lys Asn Leu 25 Pro Pro Ser Pro Phe Phe Arg Leu Pro Ile Ile Gly His Met His Met 40 Leu Gly Pro Leu Leu His Gin Ser Phe His Asn Leu Ser His Arg Tyr 55 WO 00/53771 Gly Pro Leu Ser Thr Pro Phe Asn Cys Ser Ser Leu 115 Lys Leu Ser 130 Gin His Leu 145 Asn Arg Ala Lys Leu Thr Glu Ala Arg 195 Asn Val Ser 210 Phe Gly Lys 225 Glu Arq Ile Asn Gly Lys Leu Asp Ile 275 Ile Gin Arg 290 Giy Thr Asp 305 Vai Lys Lys Val Val Gly Pro Tyr Leu 355 Val Pro Met 370 Phe His Arg 100 Al a Met Arg Ar g As n 180 Asp Asp Arg Ile Lys 260 Leu Vali Thr Pro Lys 340 Gin ValI Ser Phe Ile Phe Asn Ala Ala 165 As n Val1 Phe Ile Se r 245 Giy Le u His Thr Ser 325 Asp Ala Thr As n Lys Se r Pro Leu 135 Giu Glu Ile Arg Trp 215 Asp Arg Gin Cys Lys 295 Ile Leu Leu Leu Arg 375 Phe Gin Thr Tyr 120 Leu Thr Ala Ser Asp 200 Leu Leu Gi u Gly Thr 280 Ala Se r Gin Val1 Lys 360 Cys Gi y Leu Al a 105 Gi y Gi y His Val1 Ile 185 Val Phe Phe Gin Ser 265 Glu Leu Thr Lys Glu 345 Glu Val1 Ser Le u 90 Val1 Asp Ser Gin As n 170 Met Thr Lys Gin Thr 250 Gly Asp I le Giu Val 330 Giu Th r Al a Val1 75 Gin Lys Tyr Arg Leu 155 Ile Met Giu Lys Arg 235 Arq Asp Glu Met T rp 315 Arg Ser Phe Glu C ys As n Leu Arg 125 Ile Arg Glu Gly Phe 205 Asp Asp Asp Ile Se r 285 Phe Leu Giu Cys Le u 365 Thr Val1 Glu Thr 110 Phe As n Leu Glu Glu 190 Gly Leu Thr Arg Arg 270 Giu Phe Val1 Ile Pro 350 His Val1 PCT/USOO/05915 Val Ala Leu Ala Tyr Giu Ile Lys Asn Phe Leu Ser 160 Leu Leu 175 Ala Glu Giu Phe Gin Gly Leu Val 240 Arg Arg 255 Asp Phe Ile Lys Thr Ala Glu Leu 320 Asp Asn 335 Asn Leu Pro Pro Giu Asn WO 00/53771 PCT/US00/05915 Tyr Val Ile Pro Glu Asp Ser Leu Leu Phe Val Asn Val Trp Ser Ile 385 390 395 400 Gly Arg Asn Pro Lys Phe Trp Asp Asn Pro Leu Glu Phe Arg Pro Glu 405 410 415 Arg Phe Leu Lys Leu Glu Gly Asp Ser Ser Gly Val Val Asp Val Arg 420 425 430 Gly Ser His Phe Gin Leu Leu Pro Phe Gly Ser Gly Arg Arg Met Cys 435 440 445 Pro Gly Val Ser Leu Ala Met Gin Glu Val Pro Ala Leu Leu Gly Ala 450 455 460 Ile Ile Gin Cys Phe Asp Phe His Val Val Gly Pro Lys Gly Glu Ile 465 470 475 480 Leu Lys Gly Asp Asp Ile Val Ile Asn Val Asp Glu Arg Pro Gly Leu 485 490 495 Thr Ala Pro Arg Ala His Asn Leu Val Cys Val Pro Val Asp Arg Thr 500 505 510 Ser Gly Gly Gly Pro Leu Lys Ile Ile Glu Cys 515 520 <210> 4 <211> 1811 <212> DNA <213> Medicago truncatula <220> <221> CDS <222> (92)..(1657) <400> 4 caacacctaa gagtaactaa taagaacttt ctttctactt cttagtatac ttaacaactt aagtaaatat actacaaaga agctatacac c atg ttg gtg gaa ctt gca gtt 112 Met Leu Val Glu Leu Ala Val 1 act cta ttg ctc att get ctc ttc tta cac ttg cgt cca aca cct act 160 Thr Leu Leu Leu Ile Ala Leu Phe Leu His Leu Arg Pro Thr Pro Thr 15 gca aaa tca aag gct ctt cgc cac ctt cca aat cca cca age cct aaa 208 Ala Lys Ser Lys Ala Leu Arg His Leu Pro Asn Pro Pro Ser Pro Lys 30 cca cgt ctt cca ttc ata ggt cat ctt cac ctt ttg gat aac cca ctt 256 Pro Arg Leu Pro Phe Ile Gly His Leu His Leu Leu Asp Asn Pro Leu 45 50 ctt cac cac act ctt atc aag tta gga aag cgt tat ggc cct ttg tac 304 Leu His His Thr Leu Ile Lys Leu Gly Lys Arg Tyr Gly Pro Leu Tyr 65 7 WO 00/53771 PCTUSO/05915 act ctt tac ttt ggt Thr Leu Tyr Phe Gly too atg cct acc gtt gtt gca tcc act cct gac Pro Asp Ser Met Pro Val Val Ala Ser ttg ttt aaa Leu Phe Lys ctt ttc ctt caa Leu Phe Leu Gin acc Thr 95 cat gaa got act His Glu Ala Thr tcc Ser 100 ttt aac aca Phe Asn Thr aga ttc Arg Phe 105 caa acc tct gct Gin Thr Ser Ala att Ile 110 agt cgt ctt acc tat gac aac tct gtt Ser Arg Leu Thr Tyr Asp Asn Ser Val 115 448 gct atg gtt Ala Met Val 120 cca ttt gca Pro Phe Ala 125 cct tat tgg aag Pro Tyr Trp Lys ttt Phe 130 att aga aag ctt Ile Arg Lys Leu atc Ile 135 496 atg aac gac ttg Met Asn Asp Leu aac gcc acc act Asn Ala Thr Thr aac aaa ttg agg Asn Lys Leu Arg cca ttg Pro Leu 150 agg agc cga Arg Ser Arg gaa act caa Glu Thr Gin 170 gaa Glu 155 atc ctt aag gtt Ile Leu Lys Val ctt Leu 160 aag gtc atg gct Lys Vai Met Ala aat agt gct Asn Ser Ala 165 aag tgg aca Lys Trp Thr cag cca ctt gat Gin Pro Leu Asp gtc Val 175 act gag gag ctt Thr Giu Giu Leu ctc Leu 180 aac agc Asn Ser 185 aca atc tct acc Thr Ile Ser Thr atg Met 190 atg ttg ggt gag Met Leu Gly Glu gcc Ala 195 gaa gag gtt aga Glu Glu Val Arg att got cgt gat Ile Ala Arg Asp gtt Val 205 ott aag ato ttt Leu Lys Ile Phe gga Gly 210 gaa tat agt gtt Glu Tyr Ser Val aca Thr 215 688 736 784 aac ttt att tgg Asn Phe Ile Trp ttg aac aag ttt Leu Asn Lys Phe ttt gga aac tat Phe Gly Asn Tyr gat aag Asp Lys 230 aga act gag Arg Thr Glu ato aag aaa Ile Lys Lys 250 gag Glu 235 att ttc aat aag Ile Phe Asn Lys tat Tyr 240 gat cot atc att Asp Pro Ile Ile gaa aag gtt Glu Lys Val 245 gga gaa ato Gly Giu Ile oga caa gag att Arg Gin Giu Ile gtg Val 255 aac aaa aga aaa Asn Lys Arg Lys gta gaa Val Glu 265 ggc gag cag aat Gly Giu Gin Asn gtt Val 270 gtt ttt ott gao Val Phe Leu Asp act Thr 275 ttg ott gaa ttt Leu Leu Glu Phe caa gat gag aco Gin Asp Giu Thr gag ato aaa att Glu Ile Lys Ile aca Thr 290 aag gaa caa ato Lys Giu Gin Ile aag Lys 295 928 976 1024 ggt ott gtt gtg Gly Leu Val Val gat Asp 300 ttt ttc tot gca Phe Phe Ser Ala aca gao too aco Thr Asp Ser Thr gc gtg Ala Val 310 WO 00/53771 tct aca gaa Ser Thr Glu aag aaa gct Lys Lys Ala 330 PCTIUSOO/05915 tgg act tta tea gag ctc atc aat aat cct aga gtg ttg Val Leu 1072 Trp 315 Thr Leu Ser Glu Leu 320 Ile Asn Asn Pro Arg 325 cga gag gag att Arg Giu Glu Ile tct gtt gtg gga Ser Val Val Gly aaa Lys 340 gat aga ctg Asp Arg Leu 1120 gtt gat Val Asp 345 gaa tca gat gtt Glu Ser Asp Val aat ctt cct tac Asn Leu Pro Tyr att Ile 355 aaa gcc atc gta Lys Ala Ile Val aaa Lys 360 gaa gca ttt cgc Glu Ala Phe Arg ttg Leu 365 cac eca cca eta His Pro Pro Leu gta gtc aaa aga Val Val Lys Arg 1168 1216 1264 tgt aca caa gaa Cys Thr Gin Glu gag atc gac ggg Glu Ile Asp Gly tat Tyr 385 gtg gtt cca gaa Vai Val Pro Glu gga gca Gly Ala 390 cta ata ctt Leu Ile Leu gta aag cca Val Lys Pro 410 ttc Phe 395 aat gtc tgg gca Asn Val Trp Ala gtg Va1 400 gga aga gac cca Gly Arg Asp Pro aaa tat tgg Lys Tyr Trp 405 aat gtt ggt Asn Val Gly ttg gaa ttt cgt Leu Giu Phe Arg cca Pro 415 gag agg ttc ata Glu Arg Phe Ile gaa Glu 420 gaa ggt Glu Gly 425 gaa gca get tca Glu Ala Ala Ser gat ctt agg ggt Asp Leu Arg Gly caa Gin 435 eat ttc aca ett His Phe Thr Leu 1312 1360 1408 1456 1504 eta Leu 440 cca ttt gg tct Pro Phe Gly Ser gga Gly 445 aga agg atg tgt Arg Arg Met Cys gga gte aat ttg Gly Val Asn Leu act gca gga atg Thr Ala Gly Met aca atg att gca Thr Met Tie Ala tct Ser 465 att ate eaa tgc Ile Ile Gin Cys ttc gat Phe Asp 470 etc eaa gta Leu Gin Val aag gtt age Lys Vai Ser 490 ect Pro 475 ggt caa eat gga Gly Gin His Gly gaa Glu 480 ata ttg aat ggt Ile Leu Asn Gly gat tat gct Asp Tyr Ala 485 agg gca cat Arg Ala His 1552 1600 atg gaa gag aga Met Giu Giu Arg ect Pro 495 ggt ctc aca gtt Gly Leu Thr Val cca Pro 500 aat etc Asn Leu 505 atg tgt gtt ect Met Cys Val Pro ctt Leu 510 gca aga get ggt Ala Arg Ala Gly gte Val 515 gca gat aaa ctt Ala Asp Lys Leu 1648 ett tec tee Leu Ser Ser 520 taaaatatet tgagaggatg aateaceaac atatagccte 1697 tctttggtae tacaaaatta tgatgtaatt ttettatttt ttetgteaca aaggaagtgt 1757 tgtaaettgt aattgeatac aaaatctata aattttatca tectatteat tatt 1811 WO 00/53771 PCT/US0010591 <210> <211> 522 <212> PRT <213> Medicaqo truncatula <400> Met Leu Val Glu Leu Ala Val Thr Leu Leu Leu Ile Ala Leu Phe Leu 1 His Pro His Lys Val Glu Leu Lys ValI 145 Lys Glu Gly Phe Lys 225 Asp Lys Leu Leu As n Leu Arg Val Ala Thr Phe 130 Asn Val1 Glu Giu Gly 210 Phe Pro Arg %.sp Arc Pro Leu Tyr Ala Thr Tyr 115 I le Lys Met Leu Ala 195 Giu Gly Ile Lys Thr 275 Pro Pro Asp Gly Ser Ser 100 Asp Arq Leu Ala Leu 180 Glu Tyr Asn Ile Asn 260 Leu 5 Thr Ser Asn Pro Thr Phe As n Lys Arg Asn 165 Lys Giu Ser Tyr Giu 245

G

1 y Leu Pro Pro Pro Leu 70 Pro As n Ser Leu Pro 150 S er Trp Val Val1 Asp 230 Lys Glu Glu Thr Lys Leu 55 Tyr Asp Th r Vai Ile 135 Leu Ala Th r Arg Thr 215 Lys Val Ile Phe Ala Pro 40 Leu Thr Le u Ar g Ala 120 Met Arg Giu As n Asp 200 As n PArg Ile 1a 1 kl a Lys 25 Arg His Leu Phe Phe 105 Met Asn Ser Thr Ser 185 Ile Phe Th r Lys Glu 265 Gin Ser Leu His Tyr Lys 90 Gin Val1 Asp Arg Gin 170 Thr Al a Ile Glu Lys 250

G

1 y Asp Lys Pro Thr Phe 75 Le u Thr Pro Leu Glu 155 Gin Ile Arg Trp Giu 235 Arg Glu Glu Al a Phe Leu Gly Phe Ser Phe Leu 140 Ile Pro Ser Asp Pro 220 Ile Gln Gin Thr Leu *Ile Ile Ser Leu Ala Ala 125 As n Leu Leu Thr Val1 205 Leu Phe Giu Asn Met Arg Giy Lys Met Gin Ile 110 Pro Al a Lys Asp Met 190 Le u Asn As n Ile Val1 270 Glu His His Leu Pro Thr Ser Tyr Thr Val1 Val1 175 Met Lys Lys Lys Val1 255 Val1 Ile Leu Leu Gly Thr His Arg Trp Thr Leu 160 Thr Leu Ile Phe Tyr 240 Asn Phe Lys 285 WO 00/53771 WO 0053771PCTIUSOO/05915 Ile Gi y 305 Ile Val Pro Leu Tyr 385 Gly Arg Arg Cys Ser 465 Ile Leu Thr 290 Thr Asn Val1 Tyr Pro 370 Val Arg Phe Gly Pro 450 Ile Leu Thr Lys Asp As n Giy Ile 355 Val1 Val1 Asp Ile Gin 435 Gly Ile Asn Val Glu Gin Ile Lys Giy Leu Val Vai 295 Ser Pro Lys 340 Lys Vali Pro Pro Giu 420 His Val1 Gin Giy Pro 500 Thr Arg 325 Asp Al a Lys Glu Lys 405 As n Phe Asn Cys Asp 485 Arg Aia 310 Val1 Arg Ile Arg Gly 390 Tyr Vali Thr Le u Phe 470 Tyr Ala Vali Le u Leu Vali Lys 375 Al a Trp Giy Leu Aia 455 Asp Aia His Ser Lys Val Lys 360 Cys Leu Vali Giu Leu 440 Thr Leu Lys As n Thr Lys Asp 345 Gi u Thr Ile Lys Giy 425 Pro Ala Gin Val1 Leu 505 Giu Aia 330 Giu Ala Gin Leu Pro 410 Giu Phe Gi y Val1 Ser 490 M-et Trp 315 Arq Ser Phe Giu Phe 395 Leu Al a Gly Met Pro 475 Met Cys Asp 300 Thr Glu Asp Arg Cys 380 Asn Glu Al a Ser Ala 460 Gi y Giu Val1 Phe Phe Ser Ala Leu Giu Val Leu 365 Giu Val1 Phe Ser Giy 445 Thr Gin Giu Pro Se r Ile Gin 350 His Ile Trp Arg Ile 430 Arg Met His Arg Leu 510 Gi i Asp 335 Asn Pro Asp Ala Pro 415 Asp Arg Ile Gly Pro 495 Ala Leu 320 Ser Leu Pro Gly Val 400 Giu Leu Met Aia Giu 480 Gly Arg Ala Gly Vai 515 Ala Asp Lys Leu Leu Ser Ser 520

Claims (62)

1. A method for introducing into a naturally non-isoflavonoid-producing plant species the enzyme catalyzing the aryl migration of a flavanone to form an isoflavanone intermediate or an isoflavone, comprising: introducing a DNA segment encoding said enzyme into said plant to form a transgenic plant, wherein said transgenic plant expresses said DNA segment under the control of a suitable constitutive or inducible promoter when said transgenic plant is exposed to conditions which permit expression.

2. The method of claim 1, wherein chalcone synthase, chalcone reductase, and chalcone isomerase genes are also expressed in said plant to cause in vivo formation of daidzein or a daidzein derivative.

3. The method of claim 2, wherein said plant is further transformed to comprise said chalcone synthase, chalcone reductase, and chalcone isomerase genes.

4. The method of claim 1 or 2, wherein said plant further comprises downstream 0: genes to metabolize said formed isoflavanone intermediate or isoflavone to .o 20 biologically active isoflavonoid derivatives or conjugates.

5. The method of claim 4, wherein said downstream gene is selected from the group consisting of isoflavone O-methyltransferase, isoflavone 2'-hydroxylase, isoflavone reductase, and vestitone reductase.

6. The method of claim 5, wherein said plant comprises downstream gene methyltransferase to form biochanin A or a biochanin A derivative.

7. A method for increasing the level of isoflavonoid compounds in naturally 30 isoflavonoid-producing plants comprising: introducing a DNA segment encoding the enzyme catalyzing the aryl migration of a flavanone to yield an isoflavonoid to form a transgenic plant, wherein said transgenic plant expresses said DNA segment under the control of a suitable constitutive or inducible promoter when said transgenic plant is exposed to conditions which permit expression.

8. The method of claim 7, wherein said isoflavonoid is selected from the group consisting of an isoflavonone intermediate, an isoflavone, an isoflavone derivative, and an isoflavone conjugate.

9. The method of any preceding claim, wherein said DNA segment comprises isolated genomic DNA. The method of any one of claims 1 to 8, wherein said DNA segment comprises recombinant cDNA.

11. The method of any one of claims 7 to 10, wherein said DNA segment comprises CYP93C gene.

12. The method of any one of claims 7 to 10, wherein said DNA segment is a Medicago truncatula homolog of a CYP93C gene.

13. The method of any one of claims 1 to 12, wherein said flavanone is liquiritigenin. go o• 20 14. The method of any one of claims 1 to 12, wherein said flavanone is naringenin.

15. A method for synthesizing an isoflavanone intermediate or an isoflavone from a flavanone by expressing a recombinant CYP93C gene segment in a suitable bacterial, fungal, algal, or insect cell system.

16. A method of reducing the levels of isoflavonoid compounds in a naturally isoflavonoid-producing plant comprising introducing and expressing an antisense or gene silencing construct that contains an intact CYP93C gene or segments thereof into said plant. **30 Se" S17. The method of any one of claims 1, 11, 15 or 16, wherein said gene comprises a nucleotide sequence encoding the polypeptide of SEQ ID NO:2.

18. The method of any one of claims 1, 12, 15 or 16, wherein said gene comprises a nucleotide sequence encoding the polypeptide of SEQ ID

19. A naturally non-isoflavonoid producing plant cell transformed by introducing a DNA segment encoding the enzyme catalyzing the aryl migration of a flavanone to form an isoflavanone intermediate or an isoflavone, wherein said transgenic plant cell expresses said DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression. The plant cell of claim 19, wherein chalcone synthase, chalcone reductase, and chalcone isomerase genes are also expressed in said plant to cause in vivo formation of daidzein or a daidzein derivative.

21. The plant cell of claim 20, wherein said plant cell is further transformed to comprise said chalcone synthase, chalcone reductase, and chalcone isornerase genes.

22. The plant cell of claim 19 or claim 20, wherein said plant cell further comprises downstream genes to metabolize said formed isoflavanone intermediate or ***isoflavone to biologically active isoflavonoid derivatives or conjugates. *20 23. The plant cell of claim 22, wherein said downstream gene is selected from the group consisting of isoflavone O-methyltransferase, isoflavone 2'-hydroxylase, isoflavone reductase, and vestitone reductase.

24. The plant cell of claim 23, wherein said plant cell comprises downstream gene 25 4'-O-methyltransferase to form biochanin A or a biochanin A derivative.

25. A naturally isoflavonoid-producing plant cell transformed by introducing a DNA segment encoding the enzyme catalyzing the aryl migration of a flavanone to yield an isoflavonoid to form a transformed plant cell, wherein said 30 transformed plant cell expresses said DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression.

26. The plant cell of claim 25, wherein said isoflavonoid is selected from the group consisting of an isoflavonone intermediate, an isoflavone, an isoflavone derivative, and an isoflavone conjugate. 32

27. The plant cell of any one of claims 19, 25 or 26, wherein said DNA segment comprises isolated genomic DNA.

28. The plant cell of any one of claims 19, 25 or 26, wherein said DNA segment comprises recombinant cDNA.

29. The plant cell of any one of claims 19 or 25 to 28, wherein said DNA segment comprises CYP93C gene.

30. The plant cell of claim 19 or 25-28, wherein said DNA segment is a Medicago truncatula homolog of a CYP93C gene.

31. A transgenic plant cell having reduced levels of isoflavonoid compounds, said plant cell transformed by introducing an antisense or gene silencing construct that contains an intact CYP93C gene or segments thereof into said plant cell.

32. The plant cell of claim 29 or claim 31, wherein said gene comprises a nucleotide sequence encoding the polypeptide of SEQ ID NO:2. 20 33. The plant cell of claim 30 or claim 31, wherein said gene comprises a nucleotide Ssequence encoding the polypeptide of SEQ ID

34. An isolated gene or DNA segment comprising a portion which encodes a cytochrome P450 of the CYP93 family that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein said portion comprises a nucleotide sequence encoding the polypeptide of SEQ ID NO:2. o*

35. The gene or DNA segment of claim 34, wherein said gene is the soybean gene encoding the enzyme catalyzing the aryl migration of liquiritigenin.

36. The gene or DNA segment of claim 34, wherein said gene is the soybean gene encoding the enzyme catalyzing the aryl migration of naringenin.

37. A protein encoded by a portion of an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein said portion comprises a nucleotide sequence encoding the polypeptide of SEQ ID NO:2.

38. An isolated gene or DNA segment comprising a portion which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein said portion is a Medicago truncatula homolog of a CYP93C gene.

39. The gene or DNA segment of claim 38 comprising a nucleotide sequence encoding the polypeptide of SEQ ID The gene or DNA segment of claim 38 or claim 39, wherein said gene is the Medicago truncatula gene encoding the enzyme catalyzing the aryl migration of liquiritigenin.

41. The gene or DNA segment of claim 38 or claim 39, wherein said gene is the Medicago truncatula gene encoding the enzyme catalyzing the aryl migration of naringenin. 20 42. A protein encoded by a portion of an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein said portion is a Medicago truncatula homolog of a CYP93C gene.

43. A transgenic plant cell transformed with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein said transgenic plant cell exhibits increased levels of an isoflavonoid when compared to the level of said isoflavonoid in plant cells of the same species which do not comprise said isolated gene or DNA segment.

44. A food comprising edible transgenic plant material capable of being ingested for its nutritional value, wherein said transgenic plant comprises plant cells according to claim 43. 34 A method of preparing a food comprising at least one isoflavonoid comprising: transforming a plant according to the method of any one of claims 1 to 12, wherein said transgenic plant exhibits increased levels of an isoflavonoid when compared to the level of said isoflavonoid in plants of the same species which do not comprise said DNA segment, isolating said isoflavonoid and incorporating into said food.

46. A composition comprising at least a portion of a transgenic plant according to claim 43, wherein said composition is suitable for ingestion as a food stuff, a nutritional supplement, an animal feed supplement, or a nutraceutical.

47. A method of preparing a composition comprising an isoflavonoid suitable for administration as a food stuff, a nutritional supplement, an animal feed supplement, a nutraceutical, or a pharmaceutical, comprising: transforming a plant according to the method of any one of claims 1 to 12, wherein said transgenic plant exhibits increased levels of an isoflavonoid when compared to S. *the level of said isoflavonoid in plants of the same species which do not comprise said DNA segment, isolating said isoflavonoid and incorporating into S*i •said compositions.

48. A method of using a transgenic plant according to claim 43 to provide a nutraceutical benefit to a human or animal administered said isoflavonoid.

49. The method of claim 48, wherein said isoflavonoid is administered by ingestion of at least a portion of said plant. **o

50. The method of claim 48, wherein said isoflavonoid is administered by ingestion of a composition comprising an isoflavonoid isolated from said plant.

51. A method for making a pharmaceutical preparation, comprising: transforming a plant according to the method of any one of claims 1 to 12, wherein said transgenic plant exhibits increased levels of an isoflavonoid when compared to the level of said isoflavonoid in plants of the same species which do not comprise said DNA segment, isolating said isoflavonoid and formulating said isoflavonoid to form a pharmaceutical preparation.

52. A method of transforming a plant with an isolated gene or DNA segment which encodes a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone comprising introducing the isolated gene or DNA segment into the plant, wherein said transgenic plant exhibits increased levels of an isoflavonoid when compared to the level of said isoflavonoid in plants of the same species which do not comprise said isolated gene or DNA segment.

53. The method of claim 52, wherein the nutritional value of said plant is increased.

54. The method of claim 52, wherein the disease resistance in said plant is increased. The method of claim 52, wherein bacterial or fungal symbiosis in said plant is increased.

56. The method of claim 52, wherein said plant is a leguminous plant.

57. The method of claim 56, wherein the nodulation efficiency of said plant is 20 increased.

58. A leguminous transgenic plant exhibiting increased nodulatlon efficiency, wherein said transgenic plant is transformed according to the method of claim 52. S* 59. The transgenic plant of claim 43 exhibiting an increased level of bacterial or fungal symbiosis.

60. A transgenic plant comprising at least one recombinant DNA sequence encoding a cytochrome P450 that can catalyze the aryl migration of a flavanone to yield an isoflavanone intermediate or an isoflavone, wherein said transgenic plant exhibits an increased level of an isoflavonoid when compared to the level of said isoflavonoid in plants of the same species which do not comprise said recombinant DNA sequence.

61. The transgenic plant according to claim 60, wherein the recombinant DNA sequence comprises a nucleotide sequence encoding the polypeptide of SEQ ID NO:2.

62. The transgenic plant according to claim 60, wherein the recombinant DNA sequence comprises a nucleotide sequence encoding the polypeptide of SEQ ID

63. Seed from a transgenic plant according to claim

64. Progeny from a transgenic plant according to claim Progeny from seed of a transgenic plant according to claim

66. Use of a transgenic plant according to claim 43 for the preparation of a nutraceutical preparation for achieving a nutritional effect. o

67. Use of a transgenic plant according to claim 43 for the preparation of a pharmaceutical preparation for achieving a therapeutic effect.

68. A method according to any one of claims 1, 7, 15. 16, 45, 47, 51 or 52 substantially as hereinbefore described with reference to the examples.

69. A plant cell according to any one of claims 19, 25, 31 or 43 substantially as hereinbefore described with reference to the examples. 9°

70. An isolated gene or DNA segment according to claim 34 or claim 38 substantially as hereinbefore described with reference to the examples. 9

71. A protein according to claim 42 substantially as hereinbefore described with reference to the examples.

72. A food according to claim 44 substantially as hereinbefore described with reference to the examples.

73. A composition according to claim 46 substantially as hereinbefore described with reference to the examples.

74. A transgenic plant according to claim 58 or claim 60 substantially as hereinbefore described with reference to the examples.