ISOFLAVONOID PRODUCTION IN PLANTS FIELD OF INVENTION

[0001] This invention relates to isoflavone production. More specifically, this invention pertains to producing isoflavone conjugates in plants. BACKGROUND OF THE INVENTION

[0002] Isoflavonoids are an important biologically active classes of phenylpropanoid- derived plant natural products. They are found predominantly in soybeans and other leguminous plants. A wide array of biological activities associated with isoflavonoids suggests the role for isoflavonoids in human health and plant microbial interactions. [0003] In plants, these compounds are known to be involved in interactions with other organisms and to participate in the defense responses of legumes against phytopathogenic microorganisms. Isoflavonoid-derived compounds are also involved in symbiotic relationships between roots and rhizobial bacteria which eventually result in nodulation and nitrogen-fixation, and overall they have been shown to act as antibiotics, repellents, attractants, and signal compounds (Ebel, 1986). Several clinical studies have demonstrated a role for isoflavonoids in human health and nutrition, such as reducing risk of hormonally dependent cancers (Dixon, 2004), reducing menopausal symptoms (Cassidy and Bingham,1995), reduction in the risk of osteoporosis (Civitelli, 1997) and cardiovascular disease (Anderson et al., 1995). In particular, isoflavones such as daidzein, genistein and biochanin A exhibit a wide range of pharmacological effects including estrogenic, antiangiogenic, antioxidant and anticancer activities (Dixon, R. A. 1999.), and the health promoting activity of high soy diets are believed to reside in their isoflavone components (Barnes, et al. 1990). Due to these health related benefits associated with isoflavonoids, there is large interest in soybean foods and purified soybean isoflavonoids as neutraceuticals.

[0004] In soybean, the isoflavones daidzein, genistein , and glycitein are synthesized via the phenylpropanoid pathway and stored in the vacuole as glucosyl- and malonyl- glucose conjugates (Graham, 1991). The pathway to daidzein branches from the phenylpropanoid pathway following a chalcone synthase catalyzed reaction through a legume- specific enzyme, chalcone reductase (CHR). Glycitein synthesis appears to be derived from isoliquiritigenin. Genistein synthesis shares a naringenin intermediate

which is a product of chalcone with the flavonoid/anthocyanin branch of the phenylpropanoid pathway. Naringenin is a core metabolite that leads to the production of lignin, anthocyanins, condensed tannins, flavones and various other compounds, and is ubiquitously present in all plant species. In all cases, a unique aryl migration reaction to create isoflavones is mediated by isoflavone synthase (IFS). The gene encoding IFS has been identified (Jung et al., 2000).

[0005] Free isoflavones rarely accumulate to high levels in soybeans. Instead they are usually conjugated to carbohydrates or organic acids. Soybean seeds contain three types of isoflavones: aglycones, glucosides, and malonylglucosides. Each isoflavone type is found in three different forms: daidzein, genistein, and glycitein form the aglycones; daidzin, genistin, and glycitin form the glucosides; and 6"-O- malonyldaidzin, 6"-O-malonylgenistin and 6"-O-malonylglycitin form the malonylglucosides. Malonyl glucosides are thermally and chemically unstable and during the processing of soybeans and soy foods and they get converted into acetyl glucosides (6"-O-acetylgenistin, 6"-O-acetyldaidzin, and 6"-O-acetylglycitin).

Conjugation of glucosyl- and malonyl- groups to aglycones provide the metabolite with increased water solubility, reduced chemical reactivity, improved chemical stability and altered biological activity (Jones and Vogt, 2001). Enzymes catalyzing the conjugation processes belong to the group of transferases; uridine diphosphate glycosyltransferases (UGTs) for glycosylation and malonyltransfesrase (MTs) for malonylation. Glycosylation involves transfer of a nucleotide diphosphate activated sugar molecule to the acceptor aglycone that is catalysed by UGTs (Vogt and Jones, 2000). UGTs constitute a superfamily of enzymes that catalyze conjugation of sugar moieties to proteins, lipids, oligosaccharides and secondary metabolites. There are more than 12,000 UGT encoding sequences in CAZy database (see URL: cazy.org/).

[0006] Glycosylation is one of the final steps in flavonoid biosynthesis in majority of plants that alters cytotoxic effects of high levels of flavonols into less reactive form (Heller and Forkmann, 1994). Following glycosylation, the isoflavone glucosides get converted into their respective malonyl derivatives (Figure 1). Malonylation involves a regiospecific malonyl group transfer from malonyl-CoA to the glycosyl moiety of the glycosides. This process enhances the solubility of the gylcosides, protects glycosides from enzymatic degradation, and helps in the intracellular transport of the

glycosides (Heller and Forkmann, 1994). Studies on uptake of dietary genistein have suggested that the free aglycone is highly bioavailable (Sfakianos et al., 1997), and isoflavone glycosides are hydrolyzed to the aglycones by Lactobacilli, Bacteroides and Bifidobacteria in the intestinal flora. Anthocyanin that has been malonylated exhibit diverse functional activities (Suzuki et al., 2003). Isoflavinoids accumulate in soybean seeds mainly in the form of malonyl-glyco conjugates. Despite the detailed biochemical understanding of isoflavonoid biosynthesis pathway, the genes encoding these transferases have not been identified in soy.

[0007] Production of isoflavonoids into plants can theoretically be achieved by transformation with a single enzyme, isoflavone synthase (IFS). cDNAs encoding IFS have been cloned from soybean and other species, and IFS has been introduced into non-legumes (Chang- Jun Liu et al 2002) . However, only small amounts of isoflavones were observed to accumulate. Introducing soybean IFS into Arabidopsis thaliana (a plant in which isoflavones do not naturally occur) results in the accumulation of low levels of genistein glycosides. US 7,038, 113 discloses the introduction of soybean CYP93C, a gene encoding cytochrome P450, into Arabidopsis and the production of isoflavone intermediates. US 7,189,895 discloses increasing isoflavonoid production in a plant by transforming the plant with flavone 3- hydroxylase.

SUMMARY OF THE INVENTION

[0008] This invention relates to isoflavone production. More specifically, this invention pertains to producing isoflavone conjugates in plants.

[0009] In one aspect, the invention provides a method (A) for producing one or more than one isoflavonoid compound in a target plant that does not produce the isoflavonoid compound, the method comprising, i) transforming the target plant with a nucleotide sequence encoding isoflavone glycosyltransferase, isoflavone malonyltransferase, or both isoflavone glycosyltransferase and isoflavone malonyltransferase to form a transgenic plant, and

ii) expressing the nucleotide sequence in the transgenic plant, to produce the one or more than one isoflavonoid compound, wherein the target plant contains all the other necessary enzymes of isoflavonoid biosynthesis to produce the one or more than one isoflavonoid compound. [0010] The present invention also provides a method (B) for producing one or more than one isoflavonoid compound in a plant comprising, i) providing a production plant comprising one or more than one non-native nucleotide sequence encoding isoflavone glycosyltransferase, isoflavone malonyltransferase, or both isoflavone glycosyltransferase and isoflavone malonyltransferase, wherein in the absence of the one or more than one non- native nucleotide sequence, the production plant does not produce the isoflavonoid compound, and ii) expressing the one or more than one non-native nucleotide sequence in the production plant, to produce the one or more than one isoflavonoid compound, wherein the production plant contains all the other necessary enzymes of isoflavonoid biosynthesis to produce the one or more than one isoflavonoid compound.

[0011] The production plant used in method (B) described above, can possess native nucleotide sequences encoding the other necessary enzymes for isoflavonoid biosynthesis. Alternatively, if the production plant lacks one or more than one necessary enzyme for isoflavonoid biosynthesis, the desired enzyme can be introduced into the production plant via crossing the plant with another plant that comprises one or more than one nucleotide sequence that encodes the one or more than one deficient enzyme, or by transforming the plant with one or more than one nucleotide sequence that encodes the one or more than one deficient enzyme. [0012] The present invention also provides a method (C) for producing one or more than one 7-O-glycosylated isoflavonoid compound in a target plant that does not produce the 7-O-glycosylated isoflavonoid compound, the method comprising, i) transforming the target plant with a nucleotide sequence comprising an isoflavone glycosyltransferase gene, to form a transgenic plant, and

ii) expressing the isoflavone glycosyltransferase gene in the transgenic plant, to produce the one or more than one 7-0-glycosylated isoflavonoid compound, wherein the target plant contains all the other necessary enzymes of isoflavonoid biosynthesis to produce the one or more than one 7-O-glycosylated isoflavonoid compound.

[0013] For example, the nucleotide sequence encoding isoflavone glycosyltransferase comprises SEQ ID NO: 2.

[0014] The target plant used in the method described (C) above can possess native nucleotide sequences encoding the other necessary enzymes for isoflavonoid biosynthesis. Alternatively, if the production plant lacks one or more than one necessary enzyme for isoflavonoid biosynthesis, the desired enzyme can be introduced into the production plant via crossing the plant with another plant that comprises one or more than one nucleotide sequence that encodes the one or more than one deficient enzyme, or by transforming the plant with one or more than one nucleotide sequence that encodes the one or more than one deficient enzyme.

[0015] The present invention also provides a method (D) for producing one or more than one 7-O-glycosylated isoflavonoid compound in a plant comprising, i) providing a production plant comprising a non-native nucleotide sequence encoding an isoflavone glycosyltransferase, wherein in the absence of the non- native nucleotide sequence, the production plant does not produce the 7-O- glycosylated isoflavonoid compound, and ii) expressing the non-native nucleotide sequence encoding isoflavone glycosyltransferase in the production plant, to produce the one or more than one 7-O-glycosylated isoflavonoid compound, wherein the production plant contains all the other necessary enzymes of isoflavonoid biosynthesis to produce the one or more than one 7-O-glycosylated isoflavonoid compound.

[0016] For example, the non-native nucleotide sequence encoding isoflavone glycosyltransferase comprises SEQ ID NO: 2.

[0017] The production plant used in method (D) described above, can possess native nucleotide sequences encoding the other necessary enzymes for isoflavonoid biosynthesis. Alternatively, if the production plant lacks one or more than one necessary enzyme for isoflavonoid biosynthesis, the desired enzyme can be introduced into the production plant via crossing the plant with another plant that comprises one or more than one nucleotide sequence that encodes the one or more than one deficient enzyme, or by transforming the plant with one or more than one nucleotide sequence that encodes the one or more than one deficient enzyme.

[0018] In another aspect, the invention relates to a method (E) for producing one or more than one 6"-O-malonyl isoflavonoid compound in a target plant that does not produce the 6"-O-malonyl isoflavonoid compound, the method comprising, i) transforming the target plant with a nucleotide sequence comprising an isoflavone malonyltransferase gene, to form a transgenic plant, and ii) expressing the an isoflavone malonyltransferase gene in the transgenic plant, to produce the one or more than one 6"-O-malonyl isoflavonoid compound, wherein the target plant contains all the other necessary enzymes of isoflavonoid biosynthesis to produce the one or more than one 6"-O-malonyl isoflavonoid compound.

[0019] For example the nucleotide sequence encoding isoflavone malonyltransferase comprises SEQ ID NO: 1.

[0020] The target plant used in the method described (E) above can possess native nucleotide sequences encoding the other necessary enzymes for isoflavonoid biosynthesis. Alternatively, if the production plant lacks one or more than one necessary enzyme for isoflavonoid biosynthesis, the desired enzyme can be introduced into the production plant via crossing the plant with another plant that comprises one or more than one nucleotide sequence that encodes the one or more than one deficient enzyme, or by transforming the plant with one or more than one nucleotide sequence that encodes the one or more than one deficient enzyme.

[0021] The present invention also provides a method (F) for producing one or more than one 6"-O-malonyl isoflavonoid compound in a plant comprising,

i) providing a production plant comprising a non-native nucleotide sequence encoding an isoflavone malonyltransferase, wherein in the absence of the non- native nucleotide sequence, the production plant does not produce the 6"-O- malonyl isoflavonoid compound, and ii) expressing the non-native nucleotide sequence encoding isoflavone glycosyltransferase in the production plant, to produce the one or more than one 6"-O-malonyl isoflavonoid compound, wherein the production plant contains all the other necessary enzymes of isoflavonoid biosynthesis to produce the one or more than one 6"-O-malonyl isoflavonoid compound.

[0022] For example, the non-native nucleotide sequence encoding isoflavone malonyltransferase comprises SEQ ID NO: 2.

[0023] The production plant used in method (F) described above, can possess native nucleotide sequences encoding the other necessary enzymes for isoflavonoid biosynthesis. Alternatively, if the production plant lacks one or more than one necessary enzyme for isoflavonoid biosynthesis, the desired enzyme can be introduced into the production plant via crossing the plant with another plant that comprises one or more than one nucleotide sequence that encodes the one or more than one deficient enzyme, or by transforming the plant with one or more than one nucleotide sequence that encodes the one or more than one deficient enzyme.

[0024] In another aspect, the invention is a transgenic plant comprising one ore more than one nucleotide sequence encoding isoflavone glycosyltransferase, isoflavone malonyltransferase, or both isoflavone glycosyltransferase and isoflavone malonyltransferase, wherein upon expression of the one or more than one nucleotide sequence exhibits increased levels of isoflavone glyco-conjugates, isoflavone malonyl-conjugates, or both isoflavone glyco- conjugates and isoflavone malonyl- coηjugates when compared to levels of the same isoflavone conjugates in plants of the same species that do not comprise the nucleotide sequence.

[0025] The invention overcomes the deficiencies of the prior art by providing methods and compositions for producing plants with enhanced isoflavonoid biosynthesis. For example, transformation of Arabidopsis with soybean isoflavone

synthase (IFS), in the absence of an introduced 2-hydroxyisoflavanone dehydratase or soybean cytochrome P450 reductase results in production of low levels of genistein in the leaves. Arabidopsis appears to glycosylate the new natural product in a manner similar to endogenous flavonols. However, Arabidopsis expressing both a soybean IFS and an alfalfa CHI do not produce higher amounts of genistein than plants expressing the IFS alone. Therefore, other strategies are necessary to obtain additional improvements in isoflavone accumulation in plants, including plants that otherwise do not accumulate isoflavones.

[0026] In the present invention, high levels of isoflavonoid production may be achieved in plants through the expression of the uridine diphosphate glycosyltransferase (UGTs) and malonyltransferase (MTs). It has been found that a significant problem in previous attempts at engineering isoflavone biosynthesis is that aglycones may be a poor substrate for endogenous glycosyltransferases and the aglycones may be turned over. [0027] Glycosylation of isoflavones, malonylation of isoflavones, or both, is a favorable trait for engineered nutraceutical products. For example, glycosylation results in the storage of isoflavones in the vacuole, away from further potential metabolism, and produces a form that does not compromise bioavailability.

[0028] Increased isoflavone content in legumes is associated with beneficial health effects in humans.

[0029] This summary of the invention does not necessarily describe all features of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

[0031] FIGURE IA shows the pathway of isoflavone synthesis in soybean;. PAL (Phenylalanine ammonia-lyase), C4H (cinnamate 4-hydroxylase), 4CL (4- coumarate:CoA ligase), CHS (chalcone synthase), CHR (chalcone reductase), CHI (Chalcone isomerase), F6H (flavonone-6-hydroxylase), IFS (Isoflavone synthase), IMT (isoflavone methyl-transf erase), FIGURE IB shows the last two steps in the

biosynthesis of isoflavone conjugates in soybean. Isoflavone aglycones (daidzein, genistein or glycitein) undergoes a glycosylation process at its 7-0 position catalyzed by a glycosyltransferase to produce isoflavone-glyco conjugates (daidzin, genistin or glycitin), which subsequently undergoes a malonylation at its 6" position catalyzed by a malonyltransferase, leading to the production of isoflavone malonyl glycosides (6"-

O-malonylgenistin, 6"-O-malonyldaidzin, and 6"-O-malonylglycitin).

[0032] FIGURE 2 shows the analysis of enzymatic activity of recombinant glycosyltransferase and malonyltransferase. The isoflavone aglycones or glycosides were incubated with UGT73F2 or GmMT7 enzyme, respectively and the reaction products were separated by thin layer chromatography. The positions of the authentic standards are marked along the left side of the figure. D, daidzein; Ge, genistein; Gl, glycitein.

[0033] FIGURE 3 shows HPLC analysis of isoflavonoid composition of soybean seeds and reaction products of UGT73F2+isoflavone aglycones (daidzein, genistein or glycitein) (C-E) and GmMT7+isoflavone-glyco conjugates (daidzin, genistin or glycitin) (F-H). HPLC chromatogram of standard is shown in A. D, daidzein; Ge, genistein; Gl, glycitein; DG, daidzin; GlG, glycitin; GeG genistin; MD, malonyldaidzin; MGl, malonylglycitin; MGe, malonylgenistin.

[0034] FIGURE 4 shows the analysis of UGT73F2 and GmMT7 transcripts and protein accumulation in soybean tissues during development. A. Number of occurrences of soybean ESTs corresponding to UGT73F2 according to source cDNA library. B. Number of occurrences of soybean ESTs corresponding to GmMT7 according to source cDNA library. C. Transcript detection of UGT73F2 and GmMT7 by RT-PCR with gene-specific primers. Template for RT-PCR was total RNA (3 μg) isolated from reproductive organs (5-50 DAP), or from stems or leaves at early (E) to mid (M) stages of development. Shown as control is rRNA visualized by staining with ethidium bromide (EtBr). D. Accumulation of UGT73F2 and GmMT7 in soybean tissues as in B. Proteins (15 μg) were separated on SDS-PAGE and transferred to PVDF membrane by electroblotting. UGT73F2 or GmMT7 proteins were detected by sequential incubation of the blot with α-UGT73F2 or α-GmMT7 antibody and anti-

rabbit IgG conjugated to horshradish peroxidase, followed by chemiliminescent reaction (ECL system, Amersham Biosciences).

[0035] FIGURE 5 shows Phylogenetic analysis and alignment of the deduced amino acid sequence of UGT73F2 and GmMT7 with UGTs and malonyltransferases, respectively, from different species.

[0036] FIGURE 6 shows Subcellular localization of UGT73F2 and GmMT7. A. GFP alone, B. and C. GFP-KDEL, D. RFP-KDEL, E. overlay of GFP-KDEL/RFP-KDEL, F. UGT73F2+GFP, G. RFP-KDEL, H. overlay of UGT73F2-GFP/RFP-KDEL, I. GmMT7+GFP, J. RFP-KDEL, K. overlay of GmMT7+GFP/RFP-KDEL. The fusion constructs were infiltrated into tobacco epidermal cells and visualized by confocal microscopy.

[0037] FIGURE 7 shows analysis of UGT73F2 and GmMT7 transcripts and protein accumulation in developing embryos from soybean cultivars RCAT Angora and Harovinton. A. Transcript detection of UGT73F2 and GmMTV by RT-PCR with gene- specific primers. Template for RT-PCR was total RNA (3 μg) isolated from developing embryos from 25 to 70 days after pollination. Shown as control is rRNA visualized by staining with ethidium bromide (EtBr). B. Accumulation of UGT73F2 and GmMT7 in soybean developing embryos from 50 to 70 days after pollination. Proteins (15 μg) were separated on SDS-PAGE and transferred to PVDF membrane by electroblotting. UGT73F2 or GmMT7 proteins were detected by sequential incubation of the blot with α-rUGT73F2 or α-rGmMT7 antibody and anti-rabbit IgG conjugated to horshradish peroxidase, followed by chemiluminescent reaction (ECL system, Amersham Biosciences).

[0038] FIGURE 8 shows the analysis of enzymatic activity of recombinant glycosyltransferase GT4 in comparison with UGT73F2 (GT6). The isoflavone aglycones were incubated with glycosyltransferase enzyme and [ ¹⁴ C] UDP- glucose and the reaction products were separated by thin layer chromatography. D, daidzein; Ge, genistein; Gl, glycitein. No enzymatic activity was detected with GT4 by thin layer chromatography.

[0039] FIGURES 9-11 show the analysis of enzymatic activity of recombinant malonyltransferase enzymes MTl, MT2, and MT5. The isoflavone glycosides were incubated with MT enzyme and [ ¹⁴ C] malonyl CoA and the reaction products were separated by thin layer chromatography. The positions of the expected size is marked along the right side of the figure. DG, daidzin; GeG, genistin; GlG, glycitin. No enzymatic activity was detected with MTl, MT2, and MT5 by thin layer chromatography.

[0040] FIGURE 12 shows the nucleotide sequence of GmMT7and UGT73F2

DETAILED DESCRIPTION [0041] This invention relates to isoflavone production. More specifically, this invention pertains to producing isoflavone conjugates in plants.

[0042] The following description is of a preferred embodiment.

[0043] The present invention provides a method for producing one or more than one isoflavonoid compound in a target plant that otherwise does not produce the isoflavonoid compound. The method may comprise transforming the target plant with a nucleotide sequence encoding isoflavone glycosyltransferase, isoflavone malonyltransferase, or both isoflavone glycosyltransferase and isoflavone malonyltransferase to form a transgenic plant, and expressing the nucleotide sequence in the transgenic plant, to produce the one or more than one isoflavonoid compound. An example of a nucleotide sequence encoding isoflavone glycosyltransferase is SEQ

E) NO:2. An example of a nucleotide sequence encoding isoflavone malonyltransferase is SEQ ID NO:1.

[0044] The target plant may contain all the other necessary enzymes of isoflavonoid biosynthesis to produce the one or more than one isoflavonoid compound. If the target plant lacks one or more than one necessary enzyme for isoflavonoid biosynthesis, the desired one or more than one necessary enzyme can be introduced into the production plant via crossing the plant with another plant that comprises one or more than one nucleotide sequence that encodes the one or more than one necessary enzyme, or by transforming the plant with one or more than one nucleotide sequence that encodes the one or more than one necessary enzyme. Examples of other

necessary enzymes of isoflavonoid biosynthesis include one or more than one of the enzymes indicated in Figure IA. For example, the one or more than one necessary enzyme may be selected from the group comprising: phenylalanine ammonia lyase, cinnamic acid hydroxylase, coumarate:CoA ligase, caffeic acid 3-O-methyltransferase, caffeoyl-CoA 3-O-methyltransferase, chalcone synthase, chalcone reductase, chalcone isomerase, isoflavone synthase. Additional enzymes may also be modulated to effect isoflavonoid synthesis for example Flavone synthase (FS) and Flavanone 3- hydroxylase (F3H); while not involved in isoflavonoid biosysthetic pathway silencing of these gene products can enhance isoflavonoid production by metabolic channeling. [0045] The present invention also provides an alternate method for producing one or more than one isoflavonoid compound in a plant. This alternate method comprises providing a production plant comprising one or more than one non-native nucleotide sequence encoding isoflavone glycosyltransferase, isoflavone malonyltransferase, or both isoflavone glycosyltransferase and isoflavone malonyltransferase, wherein in the absence of the one or more than one non-native nucleotide sequence, the production plant does not produce the isoflavonoid compound, and expressing the one ore more than one non-native nucleotide sequence in the production plant, to produce the one or more than one isoflavonoid compound. An example of a nucleotide sequence encoding isoflavone glycosyltransferase is SEQ ED NO:2. An example of a nucleotide sequence encoding isoflavone malonyltransferase is SEQ ID NO:1.

The production plant may contain all the other necessary enzymes of isoflavonoid biosynthesis to produce the one or more than one isoflavonoid compound. If the target plant lacks one or more than one necessary enzyme for isoflavonoid biosynthesis, the desired one ore more than one necessary enzyme can be introduced into the production plant via crossing the plant with another plant that comprises one or more than one nucleotide sequence that encodes the one or more than one necessary enzyme, or by transforming the plant with one or more than one nucleotide sequence that encodes the one or more than one necessary enzyme. Examples of other necessary enzymes of isoflavonoid biosynthesis include one or more than one of the enzymes indicated in Figure IA. For example, the one or more than one necessary enzyme may be selected from the group comprising: phenylalanine ammonia lyase, cinnamic acid hydroxylase, coumarate:CoA ligase, caffeic acid 3-O-methyltrasnferase,

caffeoyl-CoA 3-O-methyltrasnferase, chalcone synthase, chalcone reductase, chalcone isomerase, isoflavone synthase. Additional enzymes may also be modulated to effect isoflavonoid synthesis for example Flavone synthase (FS) and Flavanone 3- hydroxylase (F3H); while not involved in isoflavonoid biosysthetic pathway silencing of these gene products can enhance isoflavonoid production by metabolic channeling.

By "glycosylation", it is meant the process or result of addition of a saccharide unit to a molecule.

[0046] The term "glycosyltransferase" (GT) or "uridine diphosphate glycosyltransferase" (UGT), as used herein refers to an enzyme that transfers monosaccharide unit from an activated sugar phosphate to an acceptor molecule.

[0047] The term "glucosyltransferase" used herein refers to a type of glycosyltransferase which enables the transfer of glucose.

[0048] The term "malonytransferase" (MT), as used herein refers to an enzyme transferring malonyl from malonyl-CoA to an acceptor molecule. [0049] The term "isoflavone" is a general term for isoflavone compounds and isoflavone derivatives, and is used interchangeably with the term "isoflavonoid(s)". Isoflavones or isoflavonoids refers to a large group of polyphenolic compounds, based on a common diphenylpropane skeleton, which occur naturally in plants. This term, as used herein, includes, but is not limited to, the three types of isoflavones in three different forms: the aglycones, daidzein, genistein and glycitein; the glucosides, daidzin, genistin and glycitin; and the malonylglucosides, 6"-O-malonyldaidzin, 6"-O- malonylgenistin and 6"-O-malonylglycitin that are formed during processing. An example of the biosynthetic pathway leading to isoflavone or isoflavonoid production is shown in Figure 1. [0050] The term "aglycone", as used herein, refers to the non glycosylated isoflavone, for example but not limited to daidzein, genistein and glycitein.

[0051] The term "glucoside" or "glyco conjugate", as used herein, refers to isoflavone with a sugar molecule bound to it, for example but not limited to daidzin, genistin and glycitin.

[0052] The terms "malonylglucoside" and "malonyl-glyco conjugate", as used herein, refers to a glucoside with a malonyl group bound to the sugar molecule for example but not limited to 6"-O-malonyldaidzin, 6"-O-malonylgenistin and 6"-O- malonylglycitin. [0053] The term isoflavone synthase (IFS) which is also known as 2- hydroxyisoflavanone synthase (2-HIS), as used herein, refers to the enzyme that catalyzes migration of B -ring to 3 -position, followed by hydroxylation at the 2- position.

[0054] The term "chalcone isomerase" (CHI) is also known as chalcone-flavanone isomerase and is used herein to refer to the enzyme that catalyzes the cyclization of chalcone into naringenin.

[0055] In accordance with the present invention, high levels of isoflavonoid accumulation may be achieved in plants through the expression of uridine diphosphate glycosyltransferase (UGT), malonyltransferase (MT), or both UGT and MT. Without wishing to be bound by theory, a problem in previous attempts at engineering isoflavone biosynthesis is that aglycones may be a poor substrate for endogenous glycosyltransferases and the aglycones may be turned over. By ectopically expressing UGT, MT or both UGT and MT the newly synthesized aglycones are exposed to substrate specific enzymes thereby increasing their stability and enhancing proper compartmentalization in the cell. These events lead to increased isoflavonoid accumulation. UGT, MT or both UGT and MT may be ectopically expressed in plants that comprise the enzyme complement required for isoflavone biosynthesis, for example soybean, alfalfa, or pea, resulting in an increase in isoflavone production. Alternatively, UGT, MT or both UGT and MT may be introduced into plants that do not naturally comprise the complement of enzymes required for isoflavone synthesis.

In this case, the required enzymes would also be introduced into the plant either prior to, or along with, the introduction of UGT, MT or both UGT and MT into the same plant. As one of skill in the art would realize, the required enzymes can be introduced into the plant by transformation, or by crossing plants comprising the desired enzymes together using standard breeding techniques.

[0056] The nucleotide sequences of the present invention may be used to create transgenic plants where the UGT and MT levels are altered with respect to non- transgenic plants which would result in plants with an altered phenotype.

[0057] Furthermore, overexpression of UGT and MT may result in an increase of isoflavonoid-glycosyl conjugates and isoflavonoid malonyl conjugates. More specifically, UGT and MT overexpression may result in an increase in levels of malony- glycosides (see Figure IB).

[0058] The one or more than one 7-O-glycosylated isoflavonoid compound can be produced in non-plant systems (including but not limited to transfected bacterial, yeast or insect cells which have been genetically transformed to contain all the other necessary enzymes of isoflavonoid biosynthesis) by expression of the UTG gene under the control of a suitable constitutive or inducible promoter using methods known in the art (Frick, S., Kutchan, T. M. 1999. "Molecular cloning and functional expression of O-methyltransferases common to isoquinoline alkaloid and phenylpropanoid biosynthesis," Plant J 17: 329-339; Batard, et al. 1998. "Molecular cloning and functional expression in yeast of CYP76B1, a xenobiotic-inducible 7- ethoxycoumarin O-de-ethylase from Helianthus tuberosus," Plant J 14: 111-120 all of which are herein incorporated by reference).

[0059] 7-O-glycosylated Isoflavonoid compounds may also have nutraceutical activity, and the soy UTG can be used for making these compounds by feeding non- glucosylated isoflavone precursors to intact plants, plant cell suspension cultures, or non-plant systems (including but not limited to transfected bacterial, yeast or insect cells which have been genetically transformed to contain all the other necessary enzymes of isoflavonoid biosynthesis) which have been transformed with an UTG gene under the control of a suitable constitutive or inducible promoter. Alternatively, the 7-O-glycosylated isoflavonoid compounds can be produced using in vitro processes by contacting non-glycosylated isoflavone precursors with isolated soluble or immobilized UTG enzyme which has been produced and isolated from transgenic plants, plant cell suspension cultures or a non-plant system (including but not limited to transfected bacterial, yeast or insect cells).

[0060] The one or more than one 6"malonyl isoflavone glucosides can be produced in non-plant systems (including but not limited to transfected bacterial, yeast or insect cells which have been genetically transformed to contain all the other necessary enzymes of isoflavonoid biosynthesis) by expression of the UTG gene and the MT gene under the control of a suitable constitutive or inducible promoter using methods known in the art (Frick, S., Kutchan, T. M. 1999. "Molecular cloning and functional expression of O-methyltransferases common to isoquinoline alkaloid and phenylpropanoid biosynthesis," Plant J 17: 329-339; Batard, et al. 1998. "Molecular cloning and functional expression in yeast of CYP76B1, a xenobiotic- inducible 7- ethoxycoumarin O-de-ethylase from Helianthus tuberosus," Plant J 14: 111-120 all of which are herein incorporated by reference).

[0061] 6"malonyl isoflavone glucosides may also have nutraceutical activity, and the soy UTG and MTG can be used for making these compounds by feeding non- glucosylated isoflavone precursors to intact plants, plant cell suspension cultures, or non-plant systems (including but not limited to transfected bacterial, yeast or insect cells which have been genetically transformed to contain all the other necessary enzymes of isoflavonoid biosynthesis) which have been transformed with an UTG gene under the control of suitable constitutive or inducible promoters and MT gene under the control of suitable constitutive or inducible promoters. Alternatively, the 6"malonyl isoflavone glucosides can be produced using in vitro processes by contacting non-glycosylated isoflavone precursors with isolated soluble or immobilized UTG enzyme which has been produced and isolated from transgenic plants, plant cell suspension cultures or a non-plant system (including but not limited to transfected bacterial, yeast or insect cells) and isolated soluble or immobilized MT enzyme which has been produced and isolated from transgenic plants, plant cell suspension cultures or a non-plant system (including but not limited to transfected bacterial, yeast or insect cells).

[0062] By "operatively linked" it is meant that the particular sequences interact either directly or indirectly to carry out their intended function as described herein. The interaction of operatively linked sequences may for example be mediated by proteins that in turn interact with the sequences. A transcriptional regulatory region and a plant optimized nucleic acid molecule encoding a UTG polypeptide or fragment or

variant thereof are "operably linked" when the sequences are functionally connected so as to permit transcription of the UTG nucleotide sequence to be mediated or modulated by the transcriptional regulatory region. A transcriptional regulatory region and a plant optimized nucleic acid molecule encoding a MT polypeptide or fragment or variant thereof are "operably linked" when the sequences are functionally connected so as to permit transcription of the MT nucleotide sequence to be mediated or modulated by the transcriptional regulatory region.

[0063] By "regulatory region" "regulatory element" or "promoter" it is meant a portion of nucleic acid typically, but not always, upstream of the protein coding region of a gene, which may be comprised of either DNA or RNA, or both DNA and RNA.

When a regulatory region is active, and in operative association, or operatively linked, with a gene of interest, this may result in expression of the gene of interest. A regulatory element may be capable of mediating organ specificity, or controlling developmental or temporal gene activation. A "regulatory region" includes promoter elements, core promoter elements exhibiting a basal promoter activity, elements that are inducible in response to an external stimulus, elements that mediate promoter activity such as negative regulatory elements or transcriptional enhancers. "Regulatory region", as used herein, also includes elements that are active following transcription, for example, regulatory elements that modulate gene expression such as translational and transcriptional enhancers, translational and transcriptional repressors, upstream activating sequences, and mRNA instability determinants. Several of these latter elements may be located proximal to the coding region.

[0064] In the context of this disclosure, the term "regulatory element" or "regulatory region" typically refers to a sequence of DNA, usually, but not always, upstream (5') to the coding sequence of a structural gene, which controls the expression of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at a particular site. However, it is to be understood that other nucleotide sequences, located within introns, or 3 ¹ of the sequence may also contribute to the regulation of expression of a coding region of interest. An example of a regulatory element that provides for the recognition for RNA polymerase or other transcriptional factors to ensure initiation at a particular site is a promoter element.

Most, but not all, eukaryotic promoter elements contain a TATA box, a conserved

nucleic acid sequence comprised of adenosine and thymidine nucleotide base pairs usually situated approximately 25 base pairs upstream of a transcriptional start site. A promoter element comprises a basal promoter element, responsible for the initiation of transcription, as well as other regulatory elements (as listed above) that modify gene expression.

[0065] There are several types of regulatory regions, including those that are developmentally regulated, inducible or constitutive. A regulatory region that is developmentally regulated, or controls the differential expression of a gene under its control, is activated within certain organs or tissues of an organ at specific times during the development of that organ or tissue. However, some regulatory regions that are developmentally regulated may preferentially be active within certain organs or tissues at specific developmental stages, they may also be active in a developmentally regulated manner, or at a basal level in other organs or tissues within the plant as well. Examples of tissue-specific regulatory regions, for example see- specific a regulatory region, include the napin promoter, and the cruciferin promoter

(Rask et al., 1998, J. Plant Physiol. 152: 595-599; Bilodeau et al., 1994, Plant Cell 14: 125-130). An example of a leaf-specific promoter includes the plastocyanin promoter (Figure Ib or SEQ ID NO:23; US 7,125,978, which is incorporated herein by reference).

[0066] An inducible regulatory region is one that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer the DNA sequences or genes will not be transcribed. Typically the protein factor that binds specifically to an inducible regulatory region to activate transcription may be present in an inactive form, which is then directly or indirectly converted to the active form by the inducer. However, the protein factor may also be absent. The inducer can be a chemical agent such as a protein, metabolite, growth regulator, herbicide or phenolic compound or a physiological stress imposed directly by heat, cold, salt, or toxic elements or indirectly through the action of a pathogen or disease agent such as a virus. A plant cell containing an inducible regulatory region may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating or

similar methods. Inducible regulatory elements may be derived from either plant or non-plant genes (e.g. Gatz, C. and Lenk, I.R.P., 1998, Trends Plant Sci. 3, 352-358; which is incorporated by reference). Examples, of potential inducible promoters include, but not limited to, tetracycline-inducible promoter (Gatz, C, 1997, Ann. Rev. Plant Physiol. Plant MoI. Biol. 48, 89-108; which is incorporated by reference), steroid inducible promoter (Aoyama, T. and Chua, N.H.,1997, Plant J. 2, 397-404; which is incorporated by reference) and ethanol-inducible promoter (Salter, M.G., et al, 1998, Plant Journal 16, 127-132; Caddick, M.X., et al,1998, Nature Biotech. 16, 177-180, which are incorporated by reference) cytokinin inducible IB6 and CKIl genes (Brandstatter, I. and Kieber, JJ.,1998, Plant Cell 10, 1009-1019; Kakimoto, T.,

1996, Science 274, 982-985; which are incorporated by reference) and the auxin inducible element, DR5 (Ulmasov, T., et al., 1997, Plant Cell 9, 1963-1971; which is incorporated by reference).

[0067] A constitutive regulatory region directs the expression of a gene throughout the various parts of a plant and continuously throughout plant development.

Examples of known constitutive regulatory elements include promoters associated with the CaMV 35S transcript. (Odell et al., 1985, Nature, 313: 810-812), the rice actin 1 (Zhang et al, 1991, Plant Cell, 3: 1155-1165), actin 2 (An et al, 1996, Plant J., 10: 107-121), or tms 2 (U.S. 5,428,147, which is incorporated herein by reference), and triosephosphate isomerase 1 (Xu et. al., 1994, Plant Physiol. 106: 459-467) genes, the maize ubiquitin 1 gene (Cornejo et al, 1993, Plant MoI. Biol. 29: 637-646), the Arabidopsis ubiquitin 1 and 6 genes (Holtorf et al, 1995, Plant MoI. Biol. 29: 637- 646), and the tobacco translational initiation factor 4A gene (Mandel et al, 1995 Plant MoI. Biol. 29: 995-1004). The term "constitutive" as used herein does not necessarily indicate that a gene under control of the constitutive regulatory region is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types even though variation in abundance is often observed.

[0068] The one or more than one nucleotide sequence of the present invention may be expressed in any suitable plant host that is transformed by the nucleotide sequence, or constructs, or vectors of the present invention. Examples of suitable hosts include, but are not limited to, agricultural crops including alfalfa, canola, Brassica spp., maize,

Nicotiana spp., alfalfa, potato, ginseng, pea, oat, rice, soybean, wheat, barley, sunflower, and cotton.

[0069] The one or more chimeric genetic constructs of the present invention can further comprise a 3' untranslated region. A 3' untranslated region refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by effecting the addition of polyadenylic acid tracks to the 3' end of the mRNA precursor. Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon. One or more of the chimeric genetic constructs of the present invention can also include further enhancers, either translation or transcription enhancers, as may be required. These enhancer regions are well known to persons skilled in the art, and can include the ATG initiation codon and adjacent sequences. The initiation codon must be in phase with the reading frame of the coding sequence to ensure translation of the entire sequence.

[0070] Non-limiting examples of suitable 3' regions are the 3' transcribed non- translated regions containing a polyadenylation signal of Agrobacterium tumor inducing (Ti) plasmid genes, such as the nopaline synthase (Nos gene) and plant genes such as the soybean storage protein genes and the small subunit of the ribulose-1, 5- bisphosphate carboxylase (ssRUBISCO) gene.

[0071] To aid in identification of transformed plant cells, the constructs of this invention may be further manipulated to include plant selectable markers. Useful selectable markers include enzymes that provide for resistance to chemicals such as an antibiotic for example, gentamycin, hygromycin, kanamycin, or herbicides such as phosphinothrycin, glyphosate, chlorosulfuron, and the like. Similarly, enzymes providing for production of a compound identifiable by colour change such as GUS (beta-glucuronidase), or luminescence, such as luciferase or GFP, may be used.

[0072] Also considered part of this invention are transgenic plants, plant cells or seeds containing the chimeric gene construct of the present invention. Methods of

regenerating whole plants from plant cells are also known in the art. In general, transformed plant cells are cultured in an appropriate medium, which may contain selective agents such as antibiotics, where selectable markers are used to facilitate identification of transformed plant cells. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be used to establish repetitive generations, either from seeds or using vegetative propagation techniques. Transgenic plants can also be generated without using tissue cultures.

[0073] The regulatory elements of the present invention may also be combined with coding region of interest for expression within a range of host organisms that are amenable to transformation, or transient expression. Such organisms include, but are not limited to plants, both monocots and dicots, for example but not limited to corn, cereal plants, wheat, barley, oat, Nicotiana spp, Brassica spp, soybean, bean, pea, alfalfa, potato, tomato, ginseng, and Arabidopsis.

[0074] Methods for stable transformation, and regeneration of these organisms are established in the art and known to one of skill in the art. The method of obtaining transformed and regenerated plants is not critical to the present invention.

[0075] By "transformation" it is meant the interspecific transfer of genetic information (nucleotide sequence) that is manifested genotypically, phenotypically, or both. The interspecific transfer of genetic information from a chimeric construct to a host may be heritable and the transfer of genetic information considered stable, or the transfer may be transient and the transfer of genetic information is not inheritable.

[0076] The present invention further includes a suitable vector comprising the chimeric construct suitable for use with either stable or transient expression systems.

The genetic information may be also provided within one or more than one construct. For example, a nucleotide sequence encoding a protein of interest may be introduced in one construct, and a second nucleotide sequence encoding a protein that modifies glycosylation of the protein of interest may be introduced using a separate construct. These nucleotide sequences may then be co-expressed within a plant. However, a

construct comprising a nucleotide sequence encoding both the protein of interest and the protein that modifies glycosylation profile of the protein of interest may also be used. In this case the nucleotide sequence would comprise a first sequence comprising a first nucleic acid sequence encoding the protein of interest operatively linked to a promoter or regulatory region, and a second sequence comprising a second nucleic acid sequence encoding the protein that modifies the glycosylation profile of the protein of interest, the second sequence operatively linked to a promoter or regulatory region.

[0077] By "co-expressed" it is meant that two or more than two nucleotide sequences are expressed at about the same time within the plant, and within the same tissue of the plant. However, the nucleotide sequences need not be expressed at exactly the same time. Rather, the two or more nucleotide sequences are expressed in a manner such that the encoded products have a chance to interact. For example, the protein that modifies glycosylation of the protein of interest may be expressed either before or during the period when the protein of interest is expressed so that modification of the glycosylation of the protein of interest takes place. The two or more than two nucleotide sequences can be co-expressed using a transient expression system, where the two or more sequences are introduced within the plant at about the same time under conditions that both sequences are expressed. Alternatively, a platform plant comprising one of the nucleotide sequences, for example the sequence encoding the protein that modifies the glycosylation profile of the protein of interest, may be transformed, either transiently or in a stable manner, with an additional sequence encoding the protein of interest. In this case, the sequence encoding the protein that modifies the glycosylation profile of the protein of interest may be expressed within a desired tissue, during a desired stage of development, or its expression may be induced using an inducible promoter, and the additional sequence encoding the protein of interest may be expressed under similar conditions and in the same tissue, to ensure that the nucleotide sequences are co-expressed.

[0078] The constructs of the present invention can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, micro- injection, electroporation, etc. For reviews of such techniques see for example

Weissbach and Weissbach, Methods for Plant Molecular Biology, Academy Press, New York VIII, pp. 421-463 (1988); Geierson and Corey, Plant Molecular Biology, 2d Ed. (1988); and Miki and Iyer, Fundamentals of Gene Transfer in Plants. In Plant Metabolism, 2d Ed. DT. Dennis, DH Turpin, DD Lefebrve, DB Layzell (eds), Addison Wesly, Langmans Ltd. London, pp. 561-579 (1997). Other methods include direct

DNA uptake, the use of liposomes, electroporation, for example using protoplasts, micro-injection, microprojectiles or whiskers, and vacuum infiltration. See, for example, Bilang, et al. (Gene 100: 247-250 (1991), Scheid et al. (MoI. Gen. Genet. 228: 104-112, 1991), Guerche et al. (Plant Science 52: 111-116, 1987), Neuhause et al. (Theor. Appl Genet. 75: 30-36, 1987), Klein et al., Nature 327: 70-73 (1987);

Howell et al. (Science 208: 1265, 1980), Horsch et al. (Science 227: 1229-1231, 1985), DeBlock et al., Plant Physiology 91: 694-701, 1989), Methods for Plant Molecular Biology (Weissbach and Weissbach, eds., Academic Press Inc., 1988), Methods in Plant Molecular Biology (Schuler and Zielinski, eds., Academic Press Inc., 1989), Liu and Lomonossoff (J. Virol Meth, 105:343-348, 2002,), U.S. Pat. Nos.

4,945,050; 5,036,006; and 5,100,792, U.S. patent application Ser. Nos. 08/438,666, filed May 10, 1995, and 07/951,715, filed Sep. 25, 1992, (all of which are hereby incorporated by reference).

[0079] As described below, transient expression methods may be used to express the constructs of the present invention (see Liu and Lomonossoff, 2002, Journal of

Virological Methods, 105:343-348; which is incorporated herein by reference). Alternatively, a vacuum-based transient expression method, as described by Kapila et al. (1997, which is incorporated herein by reference) may be used. These methods may include, for example, but are not limited to, a method of Agro- inoculation or Agro-infiltration, however, other transient methods may also be used as noted above.

With either Agro-inoculation or Agro-infiltration, a mixture of Agrobacteria comprising the desired nucleic acid enter the intercellular spaces of a tissue, for example the leaves, aerial portion of the plant (including stem, leaves and flower), other portion of the plant (stem, root, flower), or the whole plant. After crossing the epidermis the Agrobacteria infect and transfer t-DNA copies into the cells. The t-

DNA is episomally transcribed and the mRNA translated, leading to the production of

the protein of interest in infected cells, however, the passage of t-DNA inside the nucleus is transient.

[0080] If the nucleotide sequence of interest encodes a product that is directly or indirectly toxic to the plant, then by using the method of the present invention, such toxicity may be reduced throughout the plant by selectively expressing the gene of interest within a desired tissue or at a desired stage of plant development. In addition, the limited period of expression resulting from transient expression may reduce the effect when producing a toxic product in the plant. The coding region of interest or the nucleotide sequence of interest may be expressed in any suitable plant host which is either transformed or comprises the nucleotide sequences, or nucleic acid molecules, or genetic constructs, or vectors of the present invention. Examples of suitable hosts include, but are not limited to, Arabidopsis, agricultural crops including for example canola, Brassica spp., maize, Nicotiana spp., alfalfa, potato, ginseng, pea, oat, rice, soybean, wheat, barley, sunflower, and cotton. [0081] One aspect of the present invention relates to an isolated nucleotide sequence that encodes uridine diphosphate glycosyltransferase from soybean. This enzyme, 7- O-glycosyltransferase, is classified as isoflavonoid glycosyltransferase enzyme (UGT73F2) and catalyzes the 7-0 glycosylation of aglycones to yield to isoflavone- glyco conjugates. [0082] hi another aspect the present invention pertains to a nucleotide sequence encoding malonyltransferase from soybean. This enzyme catalyzes malonylation of isoflavone-glyco conjugates at 6" position leading to the production of isoflavone malonyl glycosides. The enzyme is classified as isoflavonoid malonyltransferase enzyme (GmMT7). [0083] The nucleotide sequences encoding soybean UGT73F2 and GmMT7 were isolated and purified according to the procedure outlined in Example 1. The nucleotide sequence of GmMT7 is shown in SEQ ID NO:1 (Figure 12), and the amino acid sequence is shown in SEQ ID NO: 3 (Figure 5). The nucleotide sequence of UGT73F2 is shown in SEQ ID NO:2 (Figure 12), and the amino acid sequence is shown in SEQ ID NO: 4 (Figure 5).

[0084] The nucleotide sequence of the UGT73F2 characterized herein differs from GT4 (accession number: DQ278439) in two nucleotide substitutions in the open reading frame that changes the unidentified amino acid (X) 210 to Glutamic acid (E), and Glycine (G) 281 to Glutamic acid (E). The GT4 glycosyltransferase does not have enzymatic activity (Figure 8), however UGT73F2 exhibits activity.

[0085] The UGT73F2 and GmMT7 genes of the present invention can be introduced into leguminous and non-leguminous plants by any method of transformation, for example, Agrobacterium-based, or biolistic transformation procedures (Horsch, et al., 1985 and Klein, et al., 1988). Both procedures may involve the construction of a plasmid vector containing a desirable transcriptional promoter driving expression of the coding region of interest, for example UGT73F2, or GmMT7, followed by a transcriptional terminator and a selectable marker sequence encoding for example an enzyme that can be used for a colorimetric (for example but not limited to GUS) fluorimetric (for example but not limited to GFP, green fluorescence protein), antibiotic (for example but not limited to kanamycin) or a herbicide (for example but not limited to Basta).

[0086] 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 (e.g., antibiotic or herbicide) followed by regeneration.

Agrobacterium-based procedures require the construct of the present invention to be placed into a T-DNA vector, which is then transferred into Agrobαctrium tumefαciens.

[0087] The present invention will be further illustrated in the following examples.

Example 1: Identification and Isolation of Glycosyltransferase and

Mάlonyltransf erase Enzymes

[0088] The DFCI Soybean Gene Index database contains more than 330,000 expressed sequence tags (ESTs) and 31,000 tentative contigs (TC); see URL: compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=soyb ean. This database was screened to identify potential isoflavonoid specific UGTs and MTs. The key word search of the database identified 175 tentative contigs for UGTs and 43 TCs for MTs

as candidate sequences. A total of 6 candidate TCs for UGTs and 10 candidate sequences for MTs were selected based on their annotations. Two of the TCs for MT had full length cDNA sequences. The full length cDNAs for 3 candidate UGTs and 4 MTs genes were obtained using RLM-RACE (see methods below). Identification of ESTs and Isolation of Full Length cDNAs

[0089] The DFCI soybean gene index database (former TIGR soybean gene index) was searched by key words such as 'glucosyltransferase' for UGTs and 'malonyltransf erase' or 'acyltransferase' for MTs. Several candidate ESTs were selected on the basis of their putative functions for both UGTs and MTs. The deduced amino acid sequences of selected ESTs for UGT were searched for the presence of a

PSPG box:

WAPQVEVLAHPAVGCFVTHCGWNSTLESISAGVPMVAWPFFADQ (SEQ ID

NO: 14).

[0090] A total of 6 TCs for UGT and 10 TCs for MT were selected for further analysis. A 5' and 3' Rapid Amplification of cDNA Ends (RACE) was performed to obtain full length cDNA using First choice RNA Ligase Mediated RACE (RLM Race) kit (Ambion Inc., Austin, TX, USA) according to manufacturer's instructions. The RLM Race products were gel purified using gel purification kit (Qiagen Inc., Mississauga, ON, Canada), cloned in pGEM ^® -T Easy Vector (Promega Corporation, Madison, WI, USA) and sequenced. The sequence information from RLM-RACE and the EST sequences were used to design primers to isolate full length cDNA from soybean embryos. PCR was performed using platinum Taq polymerase (Invitrogen) and the products were cloned into pGEM ^® -T Easy Vector (Promega Corporation, Madison, WI, USA). The clones for both UGTs and MTs were sequenced and compared with the original TC and RLM-RACE products for any mismatch. Full length cDNAs for 3 UGTs and partial sequence for 3 UGTs were obtained. The complete sequences for 4 MTs and partial sequence for 6 MTs were obtained. The full length sequences were searched for signal peptides using signalP prog 2iram (Nielsen et al., 1997) and signature motifs for UGT and MT specific motifs. Recombinant Protein Production and Enzyme Activity Assay

[0091] The full length candidate UGTs and MTs were cloned into pET30a expression vector and expressed heterologously in Escherichia coli BL21 (DE3) cells (see methods below). Recombinant proteins were identified in soluble fractions by Western blotting analysis using anti-his antibody (data not shown). [0092] The activities of the recombinant UGT proteins were tested using UDP-[ ¹⁴ C] glucose as the sugar donor and daidzein, glycitein and genistein as acceptor aglycones. Similarly, the activity of the recombinant MT proteins were tested using [ ¹⁴ C]- malonyl CoA as the malonyl donor and daidzin, glycitin and genistin as acceptor molecules, followed by TLC analysis. [0093] Only UGT6-1 showed activity towards isoflavone aglycones and was able to transfer radiolabeled sugar to isoflavone aglycones. This UGT and was named UGT73F2 as per standard UGT nomenclature. Among the MTs tested, only recombinant GmMT7 exhibited activity towards isoflavone glyco-conjugates (Figure 2). The reaction products were compared with the authentic standards where possible Standards for malonyl derivatives are not commercially available.

Heterologous Expression of Recombinant UGTs and MTs in E. coli

[0094] The fragments containing the coding region of UGTs and MTs were PCR amplified to incorporate XhollEcόRN sites for cloning in pET30a expression vector (Novagen, Ancaster, ON, Canada) and transformed in E. coli BL21 (DE3) cells. For heterologous expression of each candidate gene and vector control, bacteria from a fresh colony were grown overnight at 37 ⁰ C in Luria-Bertani (LB) medium containing 100 μg/mL kanamycin. An aliquot of overnight grown culture was used to inoculate 50 mL of fresh LB medium containing kanamycin. The cultures were grown at 37 C to an OD _6OO of 0.4-0.6. Recombinant proteins were induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM for

UGTs and 1 mM for MTs. The cultures were grown at 15 ⁰ C for 18 h with shaking. Bacterial cells were collected by centrifugation and resuspended in lysis buffer. Lysis buffer for UGTs consisted of 500 mM potassium phosphate pH 7.2 and 0.1% Triton X-100. Similarly, the lysis buffer for MTs included 200 mM Tris-HCl pH 8.0, 5 mM 2-mercaptoethanol and 0.1% Triton X-100. Lysozyme was added to a final concentration of 100 μg/mL and the cells were incubated at 30 ⁰ C for 20 min followed

by sonication on ice. Total protein concentrations were measured according to Bradford (1976) using Bio-Rad protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin (BSA) as the standard. The recombinant proteins were visualised using anti His antibody (Amersham Biosciences, Buckinghamshire, UK) following manufacturer's instructions.

Glucosyltransferase and Malonyltransferase Enzyme Activity Assay of Recombinant UGT73F2 and GmMU

[0095] The activity assay for UGT was conducted as described by Richman et al. (2005) with some modifications. A total of 112 ng of recombinant protein contained in a crude cell lysate was used to initiate the reaction. The reaction mixture was incubated at 30 ⁰ C for 16 h and terminated by adding 200 μL of water saturated 1- butanol. The samples were extracted three times with water saturated 1-butanol. The pooled butanol extracts were concentrated using vacufuge.

[0096] For MT activity, 100 ng of recombinant proteins in a total cell lysate was added to the reaction mixture containing 200 mM Tris-HCl pH 8.0, 30 mM 2- mercaptoethanol, 2 mg/mL BSA, 120 μM of isoflavone glucoside substrates, 60 μM malonyl CoA and 3 μM of [ ¹⁴ C] malonyl CoA (Amersham Biosciences, Buckinghamshire, UK) to a total volume of 100 μL. The reaction mixture was incubated at 30 ⁰ C for 30 min and terminated by adding 100 μL of ice cold 0.5% trifluroacetic acid. The reaction products were identified by thin layer chromatography (TLC) using chloroform:methanol:acetic acid at the ratio of 18.5: 10:0.28, followed by exposure of the TLC plate to imaging screen and visualization on a Molecular Imager Fx phosphoimager (Bio-Rad laboratories, Hercules, CA, USA). Standards were run under the same condition and visualized under UV light.

[0097] To determine apparent K _m values, UGT and MT reactions were performed as described above with substrate concentrations ranging from 5-200 μM with no radiolabeled donor. The reaction mixtures were incubated at 30 ⁰ C for 16 h for UGTs and 30 min for MTs, followed by HPLC analysis. K _m values were determined from Lineweaver-Burk plots of initial rate data.

HPLC and LC-MS Confirmation

[0098] The TLC results of the rUGT73F2 and rGmMT7 reaction products were further verified by HPLC and LC-MS analysis. Analysis of the reaction product from rUGT73F2 and isoflavone aglycons by HPLC detected peaks with identical retention time and spectra to the authentic standards (Figures 3c-3e). The soybean seed extracts were also run in HPLC and the peak identities of malonyl derivatives were confirmed by hydrolysis to their glycosides (Dhaubhadel et al., 2003) as well as mass determination by LC-MS analysis. The retention time of soybean seed isoflavone malonyl glyco-conjugates were then used as standards to identify the GmMT7 reaction products. Only rGmMT7 showed malonyltransf erase activity against isoflavone glycosides converting daidzin, glycitin and genistin to malonyldaidzin, malonylglycitin and malonylgenistin, respectively (Figures 3f-3h). Analysis of rGmMT7 reaction products using mass spectrometry further confirmed the identity of the products.

[0099] The substrate specificity and kinetic properties of rUGT73F2 and rGmMT7 were also determined using HPLC. A wide range of phenolic acceptors were used together with donor molecules and the recombinant proteins as shown in Table 1. The substrate preference for rUGT73F2 was in the order as follows: glycitein, genistein, biochanin, daidzein, liquiritigenin, naringenin, apigenin, followed by formononetin. No reaction products were detected with quercetin and salicylic acid as acceptors. [00100] Both donor and substrate specificity was determined using rGmMT7.

Three different acyl CoAs were tested for their donor specificity using daidzin as an acyl acceptor. The results revealed that rGmMT7 was very specific to its acyl donor and only malonyl CoA was able to donate acyl group to the daidzin. Among various glycoside substrates, rGmMT7 was most specific to daidzin, followed by glycitin and genistin. Less than 40% relative activity was detected with other flavonoid glycosides tested for rGmMT7 activity.

Table 1 Substrate specificity of recombinant UGT73F2 and GmMT7

Enzyme Substrates Relative activity Km (μM)

Daidzein 54.4 28.33 ± 8.69

Glycitein 77.61 6.3 ± 0.69

Genistein 100.0 164.37 ±44.38

Biochanin 71.8 -

1-UGT73F2 Apigenin 3.11 -

Formononetin 3.08 -

Liquiritigenin 22.04 -

Naringenin 8.18 -

Querecitin nd -

Salicylic acid nd -

Acyl acceptor

Daidzin 100.0 68.13± 3.2

Glycitin 98.3 12.76 ± 3.0

Genistin 92.9 39.16 ± 6.5 rGmMT7 Eriodictyol 7-O-glucoside 26.8 -

Apigenin 7-O-glucoside 34.2 -

Isorhamnetin 3-O-glucoside 0.85 -

Luteolin 4'-O-glucoside 39.0 -

Luteolin 7-O-glucoside 0.27 -

Naringenin 7-O-Glucoside <0.1 -

Acyl donors

Malonyl CoA 100.0

Methylmalonyl CoA <0.1

Acetyl CoA <0.1

[00101] The kinetic parameters of the substrates of rUGT73F2 were determined using UDP-glucose as sugar donor, and daidzein, glycitein and genistein as sugar acceptors and for rGmMT7, malonyl CoA was used as acyl donor, and daidzin, glycitin and genistin as acyl acceptors. The apparent Km values were identified based on the Lineweaver-Burk plot (Table 1). The results showed that glycitein and glycitin were the most preferred substrates for rUGT73F2 and rGmMT7, respectively.

Analysis of Isoβavone-Glucosides and Malonyl Glucosides by LC-MS

[00102] For the determination of kinetic parameters for UGT73F2 and

GmMT7 proteins, the reaction mixtures were frozen immediately after the reaction was completed and stored at - 20 ⁰ C. Each sample was thawed just prior to LC-MS analysis. The LC-UWMS system consisted of an Alliance 2690 HPLC/autoinjector

(Waters, Mississauga, ON), a model SPD-M6A Shimadzu diode array UV detector (Mandel Scientific, Guelph, ON) and a model LCT orthogonal time-of-flight mass spectrometer (Waters, Mississauga, ON). Samples of reaction mixtures (20 μL) were injected into a 100 μL loop attached to a Valco ClOW 10-port valve (Chromatographic Specialties, Brockville, ON) configured to permit on-line trapping and washing of the sample on a 4 x 2 mm ID SecurityGuard C18 cartridge (Phenomenex, Torrance, CA) prior to chromatography on a 150 x 2 mm, 5 μm particle Prodogy ODS(3) column (Phenomenex, Torrance, CA) with solvent flowing at 0.2 mL/min. After switching the valve to connect the loop and the cartridge, the sample was transported to the cartridge and the cartridge washed with 100:0.1 (v/v) water-formic acid for 10 min at 0.2 mL/min. The valve was then switched to connect the cartridge and the analytical column and the system was eluted with a binary gradient. Solvent A was 90:10:0.1 (v/v/v) water-acetonitrile-formic acid and Solvent B was 10:90:0.1 (v/v/v) water-acetonitrile-formic acid and the initial conditions were 100% A. The isoflavonoids of interest were eluted during a linear increase to 40% B over 20 min. The solvent composition was then linearly increased to 100% B by 25 min, held at this composition for 5 min and decreased to 0% B by 35 min. The UV and MS detectors were connected in series to the column and separated by a second Valco ClOW 10-port valve configured to permit diversion of the column flow from the MS and its replacement with 50:50:0.1 (v/v/v) water-acetonitrile-formic acid at

0.2 mL/min. UV data (200-450 nm) was collected during the entire 35 min. MS data (85-1500 m/z) was collected for 25 min. The column flow was diverted from the MS after the components of interest had been detected to avoid unnecessarily contaminating the ion source. The MS ion source was equipped with a standard nebulizer assisted electrospray probe, which was operated in positive ion mode with nitrogen as desolvation gas at 350 ⁰ C flowing at 460 L h ^"1 and a potential of 2.7 kV applied to the capillary with the sample cone at 20V. The MS was calibrated using a mixture of polyethylene glycols.

Example 2: Expression Analysis of UGT73F2 and GmMU [00103] As an initial step to investigate the expression patterns of soybean

UGT73F2 and GmMT7 genes, ESTs present in the DFCI Soybean Gene Index database were probed for sequences revealing similarities to UGT73F2 and GmMTJ.

Nucleotide sequences corresponding to these two transferases were used separately in BLASTN searches of 330,436 soybean ESTs (see URL: compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/Blast/index.cgi) originating from more than 80 different cDNA libraries (Shoemaker et al., 2002). The search resulted in 4 matching ESTs for UGT73F2 and 21 matching ESTs for GmMT7 from 18 different source cDNA libraries. These EST data were pooled together according to the tissue type of source cDNA library (Figures 4a and 4b). The results indicated that UGT73F2 was most represented in the cDNA libraries constructed from immature cotyledon, infected tissues and root, whereas GmMT7 was most prevalent in the cDNA libraries constructed from stressed tissues and root. A detailed transcript analysis using RT-

PCR approach with gene specific primers from RNA isolated from soybean cv Harosoy63 showed that UGT73F2 transcripts accumulated in pod, embryo, flower buds, flower, pod wall and early to mid seed coat tissues during various stages of development. No UGT73F2 transcript accumulation was detected in seed coat tissue 50 days after pollination (DAP) and very low amount of transcripts were detected in leaf and mature stem. Similarly, GmMT7 transcript accumulated in most of the tissues included under the study including leaf and stem. However, no transcript was detected in seed coat at 30 DAP (Figure 4c).

Transcript Profiling Using EST Database, RNA Isolation and RT-PCR Analysis [00104] The UGT73F2 and GmMT7 sequence was used to search DFCI soybean gene index (see URL: compbio.dfci.harvard.edu/tgi/cgi- bin/tgi/Blast/index.cgi) by BLASTN. Transcripts matching UGT73F2 and GmMTl were categorized according to source cDNA library and tissue type. The frequency of ESTs matching UGT73F2 and GmMT7 cDNA sequence was calculated separately for their corresponding source cDNA library.

[00105] Total RNA was isolated from soybean tissues using the protocol of

Wang and Vodkin (1994). RNA samples were treated with DNase I (Promega, Madison, WI) at 37 ⁰ C for 30 min prior to RT-PCR. DNase I treated RNA samples were further purified by phenol: chloroform extraction 3:1 (v/v), precipitated with ethanol and checked on ethidium bromide gel to ensure equal loading. RT-PCR reactions were performed using Thermoscript™ RT-PCR system (Invitrogen) according to manufacturer's instructions. Primer sequences for PCR were as follows:

For UGT73F2, GT6F:

5'-CCCCGATATCATGGATCTTCAACAACGAC-S' (SEQ ID NO:5);

GT6R:

5'-CCCCCTCGAGGTTAGTAAGTAAATCTGT-S' (SEQ YD NO:6); GmMT7, MT7F1:

5'-CCCCGAATTCATGGCAGAGACACCAACC-3'(SEQ ID NO:7);

MT7R1: 5'-CCCCCTCGAGGTTCCTCTCGTGACACAC-S' (SEQ ID NO:8)

Example 3: Sequence Analysis and Phylogeny of UGT73F2 and GmMT?

[00106] The full length UGT73F2 cDNA sequence (1730 bp) was shown to encode a protein of 476 amino acid residues with a calculated molecular mass of 53.2 kDa and a pi of 6.42. The carboxyl terminal of the protein contained the signature motif (PSPG box) for plant glucosyltransferase (Figure 5a). UGT73F2 showed 62% amino acid identity with UGT73F1 from Glycyrrhiza echinata (Nagashima et al.,

2004). A phylogenetic analysis of UGT73F2 with several other UGTs from other plant species also grouped UGT73F2 together with the UGT73F1 (Figure 5b).

[00107] The full length cDNA sequence for GmMT7 (2180 bp) encoded an open reading frame comprised of 462 amino acid residues with a calculated molecular mass of 51.1 kDa and a pi of 6.4. The primary protein structure analysis revealed presence of an N terminus HXXXDG motif and C terminus DFGWGKP motif suggesting GmMT7 to be a member of BAHD family of acyltransf erases. A third conserved motif specific to the anthocyanin acyltransferase (YFGNC motif) was also found at the middle of the GmMT7 protein sequence (Figure 5b). The deduced amino acid sequence of GmMT7 showed highest sequence similarity to two unknown sequences from Medicago truncatula (42% and 41% identity, GenBank accession no. ABE91262 and ABE91277, respectively) and anthocyanin acyltransferase like protein from Arabidopsis (38% identity, GenBank accession no. BAB 10831). Phylogenetic analysis of GmMT7 is shown in Figure 5b.

[00108] Analysis of UGT73F2 and GmMT7 sequence for N-terminal targeting signal or C terminal membrane anchor signal using SignalP and TMHMM web based programs predicted both the proteins to be non-secretory with absence of predicted signal peptides or transmembrane signals (Nielsen et ah, 1997). Nucleotide sequence analysis of coding region revealed the presence of a single intron (437 bp) in GmMU

DNA, nevertheless, UGT73F2 DNA sequence had no intron in it. Southern blotting analysis of the restriction fragments from soybean genome DNA using full length UGT73F2 or GmM T7 cDNA as a probe indicated a single copy of UGT73F2 and more than one copy of GmMT7 in soybean genome (data not shown).

Example 4: UGT73F2 and GmMT7 are Cytosolic Proteins

[00109] The in vivo subcellular localization of UGT73F2 and GmMT7 were determined by creating a GFP fusion protein and monitoring the transient expression in tobacco epidermal cells. Both UGT73F2-GFP and GmMT7-GFP fusions showed characteristics of a cytoplasmic location and the pattern was identical to the untargeted soluble GFP. The co-expression of UGT73F2-GFP and GmMT7-GFP with RFP-

KDEL confirmed their cytosolic localisation (Figure 6).

Plasmid Construction

[00110] GFP fusions with UGT73F2 and GmMT7 encoding regions were done using bridging PCR with partially overlapping primers in two steps PCR reaction. A forward primer containing a restriction site was designed at the 5 'end of the gene specific primers to assist cloning into plant transformation vectors. UGT73F2 and GmMT7 cDNA sequences were amplified using 5 ^V gene specific forward primer (βαmHMJGTF 5' -TGAGG ATCC ATGGAT CTTC AAC AACG ACC A-3' (SEQ ID No:9) and BamHI-MTlF 5'-ATATATGGATCCATGGCA GAGACACCAACC-3' (SEQ ID No: 10)) with a reverse primer containing the first 15 nt of smGFP (Davis and Vierstra, 1998) coding region fused in frame with the last 15 nt of UGT73F2 or GmMT7 coding regions (GFP-UGTR 5'-TTCTCC

TTTACTC ATGGTGGCCGACTT AG A-3' (SEQ ID No: 11) and (GFP-MT7R 5'- TTCTCCTTT ACTC ATAGTCCTCTGAGC AAA-3' (SEQ ID No: 12)). In a separate PCR reaction, smGFP was amplified using a forward primer containing the last 15 nt of UGT73F2 or GmMT7 coding region followed by the first 15 nt of smGFP coding

region sequence with a reverse primer containing an EcoRI restriction site at the 3 'end of smGFP. A final PCR reaction using the products from both of the above PCR reactions along with the Bamϋl gene specific forward primers (as shown above) and the EcoRI reverse primers (EcoRI-GFPR 5'-GCGGGGGCGGAATTCTTATTT GTATAGTTCATC-B' (SεQ ID NO: 13)) were used to create the fragments containing UGT73F2 or GmMT7 fused with GFP in frame. Overlapping primers were used in high fidelity PCR to add 5' ER signal and 3' KDEL ER retention signal to GFP sequence. The GFP sequence was amplified from the psmGFP vector (Davis and Vierstra, 1998; Arabidopsis Biological Resource Center, OH) using a forward primer introducing a BamHl restriction site at the 5 'end and an EcoRI restriction site at the 3'end as above (EcoRZ-GFPR GCGGGGGCGGAATTCTTATTT GTATAGTTCATC (SεQ ID No: 13)).

[00111] Fusion products and GFP alone were cloned into pGεMT-εasy

(Promega, USA) and sequenced to confirm the sequence integrity, followed by cloning into a binary vector pCAMter X to produce pCAMter-GFP, pCAMter-εR-

GFP, pCAMter-UGT73F2-GFP and pCAMter- GmMT7-GFP. Each plasmid DNA was transformed into Agrobacterium tumefaciens strain EHA105 via electroporation.

Transient Expression of GFP Fusion Proteins for UGT73F2 and GmMT7 localization [00112] The transient expression of GFP fusion proteins was performed in tobacco plants via leaf disc infiltration. A single colony of A. tumefaciens containing the construct of interest was selected and grown at 28° C shaker to a stationary phase in liquid LB media containing 50 μg/mL kanamycin and 10 μg/mL rifampicin. For inoculation, 1 mL of the bacterial cells was pelleted and resuspended in the infiltration medium (0.5% glucose w/v, 50 niM MES pH 5.6, 2 mM Na ₃ PO ₄ , 100 μM acetosyringone) bringing the OD ₆ oo to 0.6 to 0.7. For co-inoculation, the

Agrobacterium cells containing different constructs with same concentrations were mixed in equal volumes and infiltrated by injecting the bacterial cells into the abaxial epidermal surface of tobacco leaf with a syringe. The plant was then incubated at room temperature for 3 to 4 days. For observation, a small piece of the infected leaf tissue was cut out and mounted in water followed by confocal microscopy. Imaging of

GFP and GFP/RFP fusion proteins were performed by a Leica TCS SP2 inverted confocal microscope using a 63X water immersion objective and Lieca Confocal

software. Serial optical sections of 0.5-2 μm were obtained using an excitation wavelength of 488 nm, and emisions were collected between 500 nm to 560 nm. RFP imaging was performed at an exitation wavlenght of 543 nm and the emission was collected between 595 nm to 665 nm

Example 5: Accumulation of UGT73F2 and GmMTJ in RCAT Angora and

Harovinton developing embryos

[00113] To determine the accumulation pattern of native UGT73F2 and

GmMTJ gene transcripts and their corresponding proteins, two soybean cultivars that differ in seed isoflavonoid content were used. Total RNA and proteins were extracted from RC AT- Angora (high isoflavonoid cultivar) and Harovinton (low isoflavonoid cultivar) developing embryos and analysed by RT-PCR and Western blotting, respectively. The RT-PCR analysis revealed that both UGT73F2 and GmMTJ transcripts were accumulated at higher level in RCAT Angora compared to Harovinton during embryo development. UGT73F2 transcript was detected in the embryos only until 40 DAP in Harovinton while it was present until 60 DAP in RCAT

Angora embryos. Both the cultivars accumulated much higher level of GmMTJ transcripts compared to UGTJ3F2 and the level of accumulation was much higher in RCAT Angora compared to Harovinton during the later stages of development (Figure 7a).

[00114] UGT73F2 and GmMT7 protein accumulation followed the same pattern as of their corresponding transcripts in both the cultivars. The level of protein accumulation decreased as the seed progressed towards maturity, however, RCAT Angora showed higher levels of UGT73F2 and GmMT7 compared to Harovinton developing embryos (Figure 7b). A protein of slightly higher mass than GmMT7 was also detected using the antibody raised against rGmMT7. The identity of this protein is not known.

Antibody Production and Western Blot Analysis

[00115] Since both rUGT73F2 and rGmMT7 proteins were found predominantly in inclusion bodies, these proteins were gel purified for the purpose of antibody production. The inclusion bodies were solubilzed in 20 mM Tris pH 7.5

containing 1% SDS and run on SDS-PAGE with a pre-stained marker. The area of the gel where the recombinant proteins were expected, were cut from the gel and frozen under liquid nitrogen and stored at -20 ⁰ C. Frozen gel pieces were ground to powder and proteins were extracted from gel with 20 mM Tris-HCl pH 7.5 and 150 πiM NaCl. The protein samples were concentrated using a centrifugal filter device with 10 kDa molecular weight cut off (Amicon, Millipore). The protein concentration determined by Bradford assay and checked for their purity by running an aliquot of protein on SDS-PAGE.

[00116] Antibodies were raised in two rabbits for each of the recombinant proteins. The rabbits were bled to collect control serum prior to immunization.

Purified rUGT73F2 and rGmMT7 with His-tag (200 μg) was mixed 1:1 (v/v) with complete Freund's adjuvant and injected into rabbits. Booster injections of both the recombinant proteins with incomplete Freund's adjuvant were performed 14, 45 and 60 d after the first injection. Antiserums to rUGT73F2and rGmMT7 (α- UGT73F2 and α-GmMT7) were collected on days 24, 45 and 60, and stored at -80 ⁰ C.

[00117] For western blot analysis, protein extraction and quantitation from soybean tissues were carried out as described in Dhaubhadel et al. (2005). Proteins (30 μg from each sample) were separated on 10 % SDS-PAGE according to Laemmli (1970) and transferred onto to PVDF membrane using a semi dry electroblotting device (Bio-Rad Ltd, Mississauga, Canada). GT6-1 and MT7 proteins were detected by sequential incubation of the blot with α-UGT73F2 or α-GmMT7 and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG, with a dilution of 1:1000 and 1:5000, respectively, followed by the chemilumiscent detection (ECL system , Amersham Biosciences). Example 6: Expression of G. max UGT73F2 and GmMT7 in Arabidopsis thaliana

[00118] Transgenic Arabidopsis IFS/CHI lines (obtained from R. Dixon,

Samuel Roberts Noble Foundation, OK; Chang- Jun Liu et al 2002) that contain isoflavone synthase (IFS) and chalcone isomerase (chalcone-flavanone isomerase; CHI) are used as a platform for further modification in order to determine if isoflavone synthesis can be increased in a non-legume. The UGT73F2 (UGT) and

GmM T7 (MT) nucleotide sequences are introduced into the plant transformation

vector, pCB 302-3, carrying bar resistance marker gene (Plant Molecular Biology, 1999, 40, 711-717) and transformed into Agrobacterium tumefacien strain LBA4404. Arabidopsis IFS/CHI lines are grown in growth chamber and transformed by floral dip method (Clough and Bent, 1998, Plant J. 16, 735-743). The plants are grown to maturity and seeds harvested. Homozygous recombinant lines comprising IFS, CHI, UGT, and MT are obtained, leaves harvested and tested for total isoflavonoid production by HPLC analysis as described above (Dhaubhadel et al, 2003, Plant Molecular Biology, 53, 733-743). The production of isoflavones in the IFS/CHI lines are compared to lines comprising IFS, CHI, UGT, and MT.

[00119] The same plants are also transformed with nuclear acid sequence encoding CHR using Agrobacterium tumefacien strain LB A4404 and the floral dip method as described above. The plants are grown to maturity and seeds harvested. Homozygous recombinant lines comprising IFS, CHI, UGT, MT and CHR are obtained, leaves harvested and tested for total isoflavonoid production by HPLC analysis as described above (Dhaubhadel et al, 2003, Plant Molecular Biology, 53, 733-743). The production of isoflavones in the IFS/CHI lines are compared to lines comprising IFS, CHI, UGT, MT and CHR.

Sequence Listing

[00120] All citations are hereby incorporated by reference.

[00121] The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.

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