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  • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
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  • C12N9/1007—Methyltransferases (general) (2.1.1.)

Definitions

• the invention relates to gene manipulation in plants.
• the isoflavonoids of the Leguminosae are among the most important biologically active classes of phenylpropanoid-derived plant natural products. 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. "Isoflavonoids: biochemistry, molecular biology and biological functions.” In U. Sankawa, eds, Comprehensive Natural Products Chemistry, Elsevier, pp 773-823), and the health promoting activity of high soy diets are believed to reside in their isoflavone components (Barnes, et al. 1990.
• Daidzein and genistein act as 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). Furthermore, due to their estrogenic activity, high levels of isoflavonoids such as the phytoestrogen formononetin can adversely affect the reproductive capacity of sheep grazing forage legumes (Shutt, D.A. 1976. "The effects of plant oestrogens on animal reproduction," Endeavour 75: 110-113).
• Isoflavonoids have been ascribed key roles in plant-pathogen interactions because many have quite strong antimicrobial activity.
• Antimicrobial isoflavonoids fall into two functional classes, the pre-formed “phytoanticipins” and the inducible “phytoalexins” (VanEtten, et al. 1994. "Two classes of plant antibiotics: phytoalexins versus phytoanticipins," The Plant Cell 6: 1191-1192).
• Examples of the former class include the prenylated isoflavones of lupin, which are synthesized in various organs of the plant during seedling development (Ingham, et al. 1983.
• Isoflavonoid compounds have been shown to accumulate in infected plant cells to 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,
• isoflavones for example formononetin (7-hydroxy-4'- methoxyisoflavone) and biochanin A (5, 7-dihydroxy-4'-methoxyisoflavone) have been shown to provide a nutraceutical benefit.
• biochanin A and formononetin are reported to be phytoestrogens, and biochanin A has been shown to be effective in animal cancer study models. (Yangihara, et al. 1993. "Antiproliferative effects of isoflavones on human cancer cell lines established from the gastrointestinal tract," Cancer Res 53:5815- 5821; and Zhou-Jin-Rong, et al. 1998.
• the biosynthetic branch pathway leading to isoflavones in plants involves a cytochrome P450 mediated 2-hydroxylation/aryl migration of a flavanone intermediate formed from phenylpropanoid- and acetate-derived precursors via the chalcone synthase and chalcone isomerase reactions (Fig. 1) (Kochs, G. and H. Grisebach. 1986. "Enzymic synthesis of isoflavones,” Eur J Biochem 155: 311-318; Hakamatsuka, et al. 1991.
• Genistein (4', 5, 7-trihydroxyisoflavone) is the product of aryl migration/dehydration of naringenin (4', 5, 7- trihydroxyflavanone), whereas daidzein (4', 7-dihydroxyisoflavone) is formed in a similar manner from liquiritigenin (4', 7-dihydroxyflavanone).
• SAM S-adenosyl- L-methionine
• ⁇ MT O-methyltransferase
• the isoflavone 7-OMT has been cloned from alfalfa and the recombinant enzyme converts daidzein exclusively to isoformononetin when expressed in E. coli (He, et al. 1998. "Stress responses in alfalfa (Medicago sativa L.) XXII. cDNA cloning and characterization of an elicitor-inducible isoflavone 7-O-methyltransferase," Plant Mol Biol 36: 43-54). This enzyme activity, and its corresponding transcripts, are strongly induced in elicited alfalfa cell cultures coordinately with other enzymes of medicarpin biosynthesis (Dalkin, et al. 1990.
• the enzymes exhibit strict regiospecificity; for example, a series of distinct, position-specific OMTs is involved in the synthesis of polymethylated flavonols in Chrysosplenium americanum (Ibrahim, et al. 1987. "Enzymology and compartmentation of polymethylated flavonol glucosides in Chrysosplenium americanum ⁇ Phytochemistry 26: 1237-1245; Gauthier, et al. 1996. "cDNA cloning and characterization of a 375 '-O-methyltransferase for partially methylated flavonols from Chrysosplenium americanum," Plant Mol Biol 32: 1163-1169).
• OMTs appear to be more versatile, acting on both flavonoids and hydroxycinnamic acids (Gauthier, et al. 1998. "Characterization of two cDNA clones which encode O-methyltransferases for the methylation of both flavonoid and phenylpropanoid compounds," Arch Biochem Biophys 351: 243-249), or are specific for more than one related substrate, such as the well-studied caffeic acid/5-hydroxyferulic acid OMTs of lignin biosynthesis (Bugos, et al. 1991.
• the invention is a method for increasing the level of at least one 4'-O- methylated isoflavonoid compound in a target plant comprising transforming the target plant with a DNA fragment comprising an isoflavone O-methyltransferase gene to form a transgenic plant and over-expressing the isoflavone O-methyltransferase gene in the transgenic plant under the control of a suitable constitutive or inducible promoter.
• the 4 '-O-methylated isoflavonoid compound can be a 4'-O-methylated isoflavonoid phytoalexin or a 4'-O-methylated isoflavonoid nutraceutical.
• the DNA fragment used to transform the plant comprises SEQ ID N ⁇ :l or a sequence exhibiting at least moderate hybridization with SEQ ID NO:l.
• the invention is a method for producing at least one 4'-O- methylated isoflavonoid compound in a target plant that does not produce the 4'-O- methylated isoflavonoid compound comprising transforming the target plant with a DNA fragment comprising an isoflavone O-methyltransferase gene to form a transgenic plant and expressing said isoflavone O-methyltransferase gene in said transgenic plant under the control of a suitable constitutive or inducible promoter, wherein the transgenic plant contains all the other necessary enzymes of isoflavonoid biosynthesis to produce the 4'-O- methylated isoflavonoid compound.
• the 4' -O-methylated isoflavonoid compound can be a 4' -O-methylated isoflavonoid phytoalexin or a 4 '-O-methylated isoflavonoid nutraceutical.
• the target plant can possess native DNA encoding the other necessary enzymes for isoflavonoid biosynthesis.
• the target plant if it lacks a necessary enzyme, it can be transformed with a DNA fragment encoding the deficient enzyme.
• the DNA fragment used to transform the plant comprises SEQ ID N ⁇ .T or a sequence exhibiting at least moderate hybridization with SEQ ID NO:l.
• the invention is a method for producing at least one 4'-O- methylated isoflavonoid nutraceutical in non-plant cell system by expression of a DNA fragment comprising an isoflavone O-methyltransferase gene under the control of a suitable constitutive or inducible promoter in cells that have been genetically transformed to contain all the other necessary enzymes of isoflavonoid biosynthesis to make the 4 '-O-methylated isoflavonoid nutraceutical.
• the DNA fragment used to transform the plant comprises SEQ ID N ⁇ :l or a sequence exhibiting at least moderate hybridization with SEQ ID NO: 1.
• the invention is a method for decreasing the levels of formononetin, at least one of its conjugates or mixtures thereof in a transgenic forage legume such as alfalfa comprising antisense expression or sense gene-mediated silencing using a DNA fragment comprising an isoflavone O-methyltransferase gene under the control of a suitable constitutive or inducible promoter.
• the isoflavone O- methyltransferase could be down-regulated by nucleic acid-mediated insertional inactivation.
• the DNA fragment used to transform the plant comprises SEQ ID NO:l or a sequence exhibiting at least moderate hybridization with SEQ ID NO.l.
• the invention is a method for decreasing the levels of at least one 4'-O-methylated isoflavonoid compound in a target plant having all the necessary enzymes for synthesizing said 4'-O-methylated isoflavonoid compound comprising transforming the target plant with a DNA fragment comprising an isoflavone O-methyltransferase gene to form a transgenic plant and inducing antisense expression, sense gene-mediated silencing, or nucleic acid-mediated insertional inactivation of the isoflavone O-methyltransferase gene under the control of a suitable constitutive or inducible promoter.
• the DNA fragment used to transform the plant comprises SEQ ID N ⁇ :l or a sequence exhibiting at least moderate hybridization with SEQ ID NO:l.
• the invention is a method for decreasing the level of at least one 4'-O-methylated isoflavonoid nutraceutical, at least one 4' -O-methylated isoflavonoid nutraceutical conjugate or mixtures thereof in a target plant having all the necessary enzymes for synthesizing the 4 '-O-methylated isoflavonoid nutraceutical or conjugate comprising transforming the target plant with a DNA fragment comprising an isoflavone O- methyltransferase gene to form a transgenic plant and inducing antisense expression or sense gene-mediated silencing of the isoflavone O-methyltransferase gene under the control of a suitable constitutive or inducible promoter, thereby increasing the level of the corresponding non-methylated precursor, its conjugate or mixture thereof.
• the DNA fragment used to transform the plant comprises SEQ ID N ⁇ :l or a sequence exhibiting at least moderate hybridization with SEQ ID NO: 1.
• the invention is a method for the production of a 7-O-methylated isoflavonoid compound comprising contacting intact plants or cell suspension cultures with a non-methylated isoflavone precursor of the 7-O-methylated isoflavonoid compound, with the intact plants or cell suspension cultures transformed with a DNA fragment comprising an isoflavone O-methyltransferase gene under the control of a suitable constitutive or inducible promoter.
• the DNA fragment used to transform the plant comprises SEQ ID NO: 1 or a sequence exhibiting at least moderate hybridization with SEQ ID NO:l.
• the invention is a method for the production of a 7-O-methylated isoflavonoid compounds comprising contacting a soluble or immobilized isoflavone O- methyltransferase enzyme with a non-methylated isoflavone precursor to the 7-O- methylated isoflavonoid compound, wherein the enzyme is produced by expression of a DNA fragment comprising the corresponding isoflavone O-methyltransferase gene in transgenic plants or a heterologous system.
• the heterologous system is selected from the group consisting of transfected bacterial, yeast and insect cells.
• the DNA fragment used to transform the plant comprises SEQ ID NO: 1 or a sequence exhibiting at least moderate hybridization with SEQ ID NO: 1.
• the invention is an antiserum against 4 '-O-methyltransferase used as a reagent for determining transgene expression of said 4 '-O-methyltransferase in plants.
• the invention is a method of increasing disease resistance in a target plant by transforming the target plant with a DNA fragment comprising an isoflavone O-methyltransferase gene, wherein the transformed plant exhibits increased levels of 4'-O- methylated isoflavonoids when compared to levels of 4 '-O-methylated isoflavonoids in plants of the same species which do not comprise the DNA fragment.
• the DNA fragment used to transform the plant comprises SEQ ID N ⁇ :l or a sequence exhibiting at least moderate hybridization with SEQ ID NO: 1.
• the invention is a composition comprising at least one 4'-O- methylated isoflavonoid suitable for administration as a food stuff, a nutritional supplement, an animal feed supplement, a nutraceutical, or a pharmaceutical, wherein the 4'-O- methylated isoflavonoid is isolated from at least a portion of a transgenic plant transformed with a DNA fragment comprising an isoflavone O-methyltransferase gene and wherein the transgenic plant exhibits increased levels of 4'-O-methylated isoflavonoids when compared to levels of 4'-O-methylated isoflavonoids in plants of the same species which do not comprise the DNA fragment.
• the DNA fragment used to transform the plant comprises SEQ ID NO: 1 or a sequence exhibiting at least moderate hybridization with SEQ ID NO: 1.
• An exemplary plant is a legume.
• the invention is a transgenic plant comprising at least one recombinant DNA sequence encoding a portion of an isoflavone O-methyltransferase gene, wherein the plant upon expression of the gene exhibits increased levels of 4'-O-methylated isoflavonoid compounds when compared to levels of 4 '-O-methylated isoflavonoid compounds in plants of the same species which do not comprise the recombinant DNA sequence.
• the invention also includes seed, progeny, and progeny from the seed from this transgenic plant.
• the invention is a transgenic plant comprising at least one recombinant DNA sequence encoding a portion of an isoflavone O-methyltransferase gene, wherein the plant upon expression of the gene exhibits decreased levels of 4' -O-methylated isoflavonoid compounds when compared to levels of 4 '-O-methylated isoflavonoid compounds in plants of the same species which do not comprise the recombinant DNA sequence.
• the invention also includes seed, progeny, and progeny from the seed from this transgenic plant.
• Fig. 1 depicts pathways for the formation and O-methylation of isoflavones in plants.
• the flavanones naringenin and liquiritigenin are formed by the action of chalcone synthase and chalcone isomerase, with loss of the 5-hydroxyl in liquiritigenin occurring by co-action of chalcone reductase with chalcone synthase.
• Aryl migration of the B-ring is catalyzed by 2-hydroxyisoflavanone synthase leading, after dehydration, to daidzein or genistein.
• Fig. 2 depicts the nucleotide sequence for the alfalfa isoflavone 4'-O-methyl transferase (I ⁇ MT)8 cDNA (SEQ ID NO: 1 ).
• Fig. 3A-3B depicts generation and molecular analysis of transgenic alfalfa altered in expression of IOMT.
• Fig. 3 A depicts the binary vector constructs harboring IOMT sequences in sense and antisense orientations.
• Fig. 3B depicts enzymatic activity of IOMT in leaf tissue from untransformed "C” and empty vector transformed "V” controls, and a range of IOMT8 sense transformants.
• Fig. 4A-4D depicts the metabolism of daidzein after infiltration into untreated alfalfa leaves.
• Fig. 4A and 4B depict high pressure liquid chromatography (HPLC) profiles of phenolic compounds from leaves of empty vector control plant #62C and IOMT sense transgenic line #69 24 hr after infiltration with Pi buffer, respectively.
• Fig. 4C and 4D depict HPLC profiles of leaf extracts from the same vector control and sense transgenic lines, respectively, 24 hr after feeding daidzein in Pi buffer by leaf infiltration.
• Fig. 4E and 4F show UV spectra of 2', 4, 4 '-trihydroxy chalcone and isoformononetin, respectively.
• Fig. 5A-5H depict abiotic elicitation of isoflavonoid compounds in control and IOMT over-expressing transgenic alfalfa.
• Fig. 5 A and 5B are HPLC profiles of leaf extracts from CuCl -treated plants of vector control line #9C and IOMT over-expressing line #67, respectively, 32 hr after exposure to 3 niM CuCl 2 . Peaks labeled 1-4 were identified as apigenin conjugate, 7, 4'-dihydroxyflavone, 7, 4'-dihydroxyflavanone and apigenin aglycone, respectively.
• Fig. 5A-5H depict abiotic elicitation of isoflavonoid compounds in control and IOMT over-expressing transgenic alfalfa.
• Fig. 5 A and 5B are HPLC profiles of leaf extracts from CuCl -treated plants of vector control line #9C and IOMT over-expressing line #67, respectively, 32 hr after exposure to 3
• 5C and 5D are HPLC profiles of leaf extracts from plants of vector control line #9C and IOMT over-expressing line #67, respectively, 32 hr after transfer of seedlings to water.
• Fig. 5E and 5G depict levels of formononetin and
• Fig. 5F and 5H depict levels of medicarpin in leaves of replicate vegetatively propagated vector control lines #9C and #62C (control lines) and IOMT over-expressing lines #67 and #69, 32 hr after exposure to H 2 O or 3 mM CuCl 2 .
• Fig. 6A-6F depict accumulation of isoflavonoid compounds in response to fungal infection in control and IOMT over-expressing transgenic alfalfa.
• Fig. 6A and 6B are HPLC profiles of extracts from Phoma medicaginis infected leaves of empty vector control line #64C and IOMT over-expressing line #67, respectively at 12 hr post-inoculation.
• Fig. 6C and 6D are HPLC profiles of extracts from uninoculated leaves of line #64C and #67, respectively.
• Fig. 6A-6F depict accumulation of isoflavonoid compounds in response to fungal infection in control and IOMT over-expressing transgenic alfalfa.
• Fig. 6A and 6B are HPLC profiles of extracts from Phoma medicaginis infected leaves of empty vector control line #64C and IOMT over-expressing line #67, respectively at 12 hr post-inoculation.
• Fig. 6C and 6D are HPLC profiles of extracts
• 6E and 6F depict levels of formononetin glucoside or medicarpin, respectively, in leaves of replicate vegetatively propagated vector control (line #64C) and IOMT over-expressing (lines #67 and #69) plants at various times after inoculation with P. medicaginis.
• Fig. 7A-7B depict accumulation of isoflavonoid compounds in control and putatively IOMT gene-silenced transgenic alfalfa.
• Levels of formononetin glucoside (Fig. 7A) and medicarpin (Fig. 7B) were measured in leaves from replicate cuttings of vector control line #56C and IOMT sense transformant #78 20hr after inoculation with spores of Phoma medicaginis.
• Fig. 8A-8B depict disease resistance of transgenic alfalfa modified in expression of isoflavone O-methyltransferase.
• Fig. 8A depicts sizes of 100 individual lesions on leaves of empty vector control line #64C and IOMT over-expressing line #69, measured 5 days post- inoculation. The average value for the size of the wounds produced by the tracing wheel alone for a parallel series of 100 wound sites has been subtracted.
• the solid lines show the means for the control lines and the dashed lines show the means for the IOMT over- expressing lines. The bars show the standard deviations.
• Fig. 8B depicts sizes of 100 individual lesions on leaves measured 5 days post-inoculation as described above, but showing lesions on IOMT over-expressing line #67 and the empty vector control line #64C infected in parallel.
• transgenic reduction of isoflavone 4'-O-methylation can also be used to increase the levels of the immediate precursors of this reaction, daidzein and genistein, which are important nutraceuticals. It is shown herein that isoflavone OMT functions operationally as a rate- limiting enzyme for production of the antimicrobial phytoalexin medicarpin in infected alfalfa leaves, such that over-expression of isoflavone OMT in transgenic alfalfa causes a significant increase in the levels of medicarpin following infection by a pathogenic fungus, leading to strongly increased disease resistance.
• the functional identification of the alfalfa IOMT has now made possible methods for increasing or decreasing the levels of formononetin or other 4'-O-methylated isoflavonoid nutraceuticals such as biochanin A, texasin, afrormosin, and pseudobaptigenin in plants that produce these compounds, and for increasing the levels of medicarpin or related 4'-O-methylated isoflavonoid phytoalexins such as maackiain or pisatin and thereby improving disease resistance.
• transgenic expression of isoflavone ⁇ MT in legumes can be used to engineer both phytoalexin levels for improved disease resistance and health promoting nutraceutical phytochemicals.
• IOMT can also be used to engineer isoflavone 4'- O-methylation in plants, or other organisms, that do not naturally produce isoflavonoids, but have been engineered to do so by introduction of the necessary isoflavone synthase.
• IOMT isoflavone O-methyltransferase
• the full-length alfalfa IOMT8 cDNA (SEQ ID NO:l and Fig. 2) was placed in the sense and antisense orientations under control of the cauliflower mosaic virus 35S promoter for constitutive expression in alfalfa. All recombinant DNA techniques were performed as described in Sambrook et al. (Sambrook, et al. 1989. Molecular Cloning. A Laboratory Manual (2nd Ed), Cold Spring Harbor Laboratory Press, New York). For sense vector construction, the expression plasmid pET15b / IOMT8, which contains the full-length IOMT8 cDNA (He, et al. 1998. Plant Mol Biol 36: 43-54), was digested with BamHl and Ncol.
• the full length IOMT8 cDNA was amplified by polymerase chain reaction (PCR) using the following primers: 5'-GGGTACCTGGATAGATCTCAATAAGAGA-3' (SEQ ID NO:2) and 5'- CGCGGATCCATGGCTTCATCAATTAATGG-3' (SEQ ID NO:3), with added Kpnl and BamHl restriction sites, and the PCR product was digested with Kpnl and BamHl prior to cloning into pRTL2.
• PCR polymerase chain reaction
• Plasmids containing IOMT sequences in pRTL2 were digested with Hindl ⁇ l, and 2.2 kb fragments containing the cauliflower mosaic virus 35S promoter, tobacco etch virus 5' untranslated leader sequence, IOMT sequence and CaMV terminator, were isolated and ligated into the H dIII site of the binary vector pGA482 (An, G. 1986. "Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco cells," Plant Physiol 81: 86- 91).
• Alfalfa (Medicago sativa L. cv. Regen SY) was transformed and regenerated through somatic embryogenesis using kanamycin (25 mg/L) as selectable marker (Thomas, et al. 1990. "Selection of interspecific somatic hybrids of Medicago by using Agrobacterium - transformed tissue," Plant Sci 69: 189-198).
• Trifoliate leaves were surface-sterilized for 10 sec in 70% ethanol and for 15 min in 1% (v/v) sodium hypochlorite solution containing 0.1% Tween 20 and then immersed for 10-15 min in a suspension of Agrobacterium LBA4404 harboring the IOMT constructs or empty vector (control).
• tissues were transferred to the above medium but containing 2 ⁇ M BAP, 500 ⁇ g/ml carbenicillin, 25 ⁇ g/ml kanamycin and no acetosyringone (A2 medium).
• A2 medium 500 ⁇ g/ml carbenicillin, 25 ⁇ g/ml kanamycin and no acetosyringone
• calli were transferred to A2 medium with addition of 30 mM L-proline but no BAP (A3 medium) for embryo development. Somatic embryos were transferred to A3 medium without 2,4 D (A4 medium) for embryo germination. Seedlings that developed were finally transferred to A4 medium without casein hydrolysate and proline for rooting. Potted kanamycin-resistant plantlets were maintained in the greenhouse.
• Leaf tissues of transformants were extracted in 200 mM Tris-HCl, pH7.5, 250 mM NaCl, 25 mM ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS), centrifuged for 5 min in a microfuge, and supernatants transferred to fresh Eppendorf tubes to which equal amounts of 2-propanol were added. After incubation for 2 min at room temperature, DNA was precipitated by centrifugation. Pellets were resuspended in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
• EDTA ethylenediaminetetraacetic acid
• SDS sodium dodecyl sulfate
• PCR reactions were performed in 50 ⁇ l volumes containing 10 mM Tris-HCl pH 8.3, 50 mM KC1, 1.25 mM MgCl 2 , 200 ⁇ M dNTPs, 0.5 units of Taq polymerase, 0.4 ⁇ M oligonucleotide primers and 200 ng of plant DNA.
• PCR amplification of IOMT sequences was performed with 5' (5'-GGCCATATGGCTTCATCAATTATGGC-3') (SEQ ID NO:4) and 3' (5'- CGGGATCCTTATGGATAGATCTCAA-3') (SEQ ID NO:5) sequences of IOMT8 as primers.
• a Perkin Elmer Cetus 480 thermocycler was used for the amplification with 35 cycles of denaturation (94 °C for 1 min), annealing (55 °C for 1 min), and extension (72 °C for 1 min with a 5 min extension for the last cycle).
• Controls included DNA from empty vector transformed and non-transformed plants. More than 70% of the transformants contained the full length IOMT cDNA insert (data not shown). All the transgenic lines produced were phenotypically normal. Genomic integration of the transgene was confirmed in a subset of the transformants by Southern-blot analysis. To perform this, genomic DNA was isolated from leaf tissues (Edwards, et al. 1991.
• Nucleic Acids Res 19: 1359 digested with EcoRI or H dIII, subjected to electrophoresis through a 0.8% agarose gel and transferred to a ⁇ ybond-N membrane (Amersham, Piscataway, NJ) by capillary blotting.
• the membrane was hybridized with P-labeled 800 bp IOMT probe (from nucleotide 249 to 1035 of S ⁇ Q ID NO:l and Fig. 2) and washed in 0.2 x SSC, 0.1% SDS at 42 °C for 20 min and 3 times for 30 min at 65 °C.
• Sense transformants #52, #61, #62, #67, #69 and #78 had a higher transgene copy number than the other lines analyzed.
• IOMT transcripts are not expressed in healthy alfalfa leaves (He, et al. 1998. Plant
• Leaf tissue extracts from representative sense transformants and vector control transgenic alfalfa plants were, therefore, subjected to western blot analysis, using a polyclonal antiserum against alfalfa IOMT expressed in E. coli, to assess levels of ectopic expression of the enzyme in leaves.
• Polyclonal antiserum against IOMT was produced by immunizing rabbits (Covance Research Products Inc., Denver, PA) with the protein antigen expressed from E. coli (He, et al. 1998. Plant Mol Biol 36: 43-54).
• Protein extracts from leaf tissues were solubilized in sample buffer (25 mM Tris-HCl, pH 6.8, 1% SDS, 2.5% 2-mercaptoethanol, 5% glycerol). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham, Piscataway, NJ) by electrophoretic blotting in transfer buffer (25 mM Tris-HCl, pH 8.3, 150 mM glycine, 20% v/v methanol). Blots were probed using polyclonal antiserum against purified alfalfa IOMT as primary antibody, with anti-(rabbit IgG)-peroxidase conjugate as secondary antibody.
• the IOMT signal was detected by exposing the blots to X-ray film shortly after incubation with ECL reagent (Amersham, Piscataway, NJ). While all of the sense transformants exhibited the presence of IOMT protein, Lines #67 and #69 exhibited the highest IOMT protein levels.
• IOMT enzyme activity was measured in extracts from leaf tissues taken from plants maintained in the greenhouse. IOMT activity was analyzed as previously described (He, X- Z and R. A. Dixon. 1996. Arch Biochem Biophys 336: 121-129). Leaf tissues were ground into powder in liquid nitrogen, and extracted in enzyme assay buffer (200 mM Tris-HCl, pH 8.3, 5 mM EDTA, 14 mM 2-mercaptoethanol, 10% PVPP). After centrifugation, supernatants were used for enzyme activity assay. The reaction mixtures contained 100
• FMG formononetin malonyl glucoside
• MMG medicarpin malonyl glycoside
• isoformononetin was produced on feeding unlabeled daidzein to leaves of IOMT over-expressing transgenic alfalfa (Fig. 4D), whereas the daidzein remained unconverted when fed to leaves of control plants (Fig. 4C).
• the regiospecificity of IOMT in planta is identical to that displayed in vitro if the substrate is exogenously supplied to unstressed tissues, again consistent with the designation of the enzyme as an isoflavone 7-O-methyltransferase.
• Trifoliate leaves were placed on 2 layers of moist filter paper in a petri dish and incubated for a further 24 hours. Levels of induced isoflavonoids were then determined by HPLC as described above. Leaves that had not been exposed to copper contained no detectable formononetin or medicarpin, as depicted in Fig. 5C and 5D. Copper treatment led to a modest induction of formononetin and medicarpin in the various control lines, but to much stronger induction in the IOMT over-expressing lines (Fig. 5A and 5B).
• CuCl2 itself does not affect the regiospecificity of the enzyme. No isoformononetin was observed in any of the elicited leaf samples.
• Sense transformant # 78 had a high transgene copy number but did not express IOMT protein in the leaves (Fig. 3B). It appeared to exhibit epigenetic gene silencing (Matzke, M.A. and A.J. M. Matzke. 1995. "How and why do plants inactivate homologous (trans)genes?" Plant Physiol 107: 679-685). Analysis of replicate cuttings of line #78 and empty vector control line #56C indicated that, following infection with P. medicaginis, induction of formononetin glucoside and medicarpin in line #78 was significantly less than in the control plants (Fig. 7A and 7B).
• Phoma medicaginis is a successful pathogen of alfalfa, leading to disease symptoms on the cultivar (Regen SY) used for the genetic transformation.
• IOMT over-expressing and control plants were inoculated on the leaves using a pin wheel to produce a line of small wounds, into which fungal spores would enter to initiate infection on the susceptible alfalfa cultivar.
• the sizes of the brown Phoma lesions were then measured at 5 days post-infection. The results showed that the lesion size was much reduced in the IOMT over-expressing lines #67 and #69 compared to the controls.
• transgenic over-expression of IOMT is a viable strategy for engineering disease resistance in alfalfa, and, by analogy, in other plants in which 4'-O-methylated isoflavonoids serve as phytoalexins.
• RNA gel blot analysis of isoflavonoid pathway transcripts in Phoma infected leaves of empty vector control (line #64C) and IOMT over-expressing (line #69) alfalfa using labeled cDNA for various enzymes in the isoflavonoid pathway is given in Table I.
• Total RNA was isolated from leaves at 6, 24 and 48 hours after infection with P.
• chalcone isomerase CHI
• I2'OH isoflavone 2'-hydroxylase
• Table I RNA Gel Blot Analysis of Isoflavonoid Pathway Transcripts in Phoma Infected Leaves of Empty Vector Control (Control) a and IOMT Over-expressing (Line #69) Alfalfa Over Time b
• RNA gel blots are reported in terms of relative intensity of bands.
• c Enzymes alfalfa PAL (L-phenylalanine ammonia-lyase); alfalfa C4H (cinnamate 4-hydroxylase); alfalfa ACC (cytosolic acetyl CoA carboxylase); alfalfa CHI (chalcone isomerase); Medicago truncatula IFS (isoflavone synthase); alfalfa IOMT (isoflavone-O- methyltransferase); Medicago truncatula I2' ⁇ H (isoflavone 2'-hydroxylase); and alfalfa IFR (isoflavone reductase).
• IOMT genes can be introduced into plants by standard plant transformation strategies including, but not limited to, Agrobacte ⁇ um-mediated transformation (Horsch, B. et al. 1985. "A simple and general method for transferring genes into plants," Science 227: 1229-1231) or particle bombardment (Klein, et al. 1988. “Stable genetic transformation of intact Nicoti ⁇ n ⁇ cells by the particle bombardment process," Proc N ⁇ tlAc ⁇ dSci USA 85: 8502-8505).
• any polynucleotide encoding IOMT having the property to induce the production of 4-O-methylated phytoalexins, nutraceuticals, their conjugates or mixtures thereof can be utilized.
• a polynucleotide encoding IOMT useful in the present invention includes the sequence in SEQ ID NO:l and Fig. 2, polynucleotides encoding dominant negative forms of IOMT, and nucleic acid sequences complementary to the sequence in SEQ ID NO:l and Fig. 2.
• a complementary sequence may include an antisense nucleotide.
• the sequence is RNA
• the deoxynucleotides A, G, C, and T of the sequence in SEQ ID NO:l and Fig. 2 are replaced by ribonucleotides A, G, C, and U, respectively.
• fragments of the above-described nucleic acid sequences which are of sufficient nucleotide length to permit the fragment to selectively hybridize to DNA that encodes the protein of the sequence in SEQ ID NO:l and Fig. 2 under physiological conditions or a close family member of IOMT.
• Degenerate variants of the sequence in SEQ ID NO:l and Fig. 2, the sequence in SEQ ID NO:l and Fig. 2 where T can also be a U, and fragments of these sequences that will hybridize to DNA or RNA which encodes IOMT are also included.
• the term "selectively hybridize" refers to hybridization under moderately or highly stringent conditions which excludes non-related nucleotide sequences.
• Rate control by IOMT allows for manipulation of IOMT expression to drive increased accumulation of antimicrobial phytoalexins as a mechanism for improved plant disease resistance, or to increase or decrease the levels and/or methylation status of isoflavones.
• Isoflavones are valuable as nutraceuticals (Adlercreutz, H. and W. Mazur. 1997. "Phyto-estrogens and western diseases," Ann Med 29: 95-120), and are naturally limited to the leguminosae, where they occur with the 4'-hydroxyl group methylated (formononetin and biochanin A) or free (daidzein and genistein).
• Modification of IOMT expression in vivo can be used to alter the 4 '-O-methylation of a plant's isoflavones.
• 4 '-O-iso flavonoid phytoalexins include but are not limited to medicarpin, maackiain, pisatin, their respective conjugates, or mixtures thereof; and 4 '-O-methylated isoflavonoid nutraceuticals include but are not limited to formononetin, biochanin A, texasin, afromosin, pseudobaptigenin, their respective conjugates, or mixtures thereof.
• Increases in levels of 4'-O-isoflavonoid phytoalexins and 4'-O-methylated isoflavonoid nutraceuticals can be made by over-expression of the IOMT gene in transgenic plants which naturally make these compounds and by expression of the IOMT gene in transgenic plants which naturally do not make these compounds.
• 4'-O-methylated isoflavonoid nutraceuticals 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 IOMT 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).
• the present invention also includes a composition comprising at least one 4'-O- methylated isoflavonoid suitable for administration as a food stuff, a nutritional supplement, an animal feed supplement, a nutraceutical, or a pharmaceutical, wherein the source of the 4 '-O-methylated isoflavonoid is a transgenic plant expressing an isoflavone O- methyltransferase gene of the present invention.
• a portion of the transgenic plant comprising the 4 '-O-methylated isoflavonoid or 4 '-O-methylated isoflavonoid extracted from the transgenic plant can be utilized.
• IOMT gene sequences can be transformed in the antisense orientation (Bourque, J. E. 1995. "Antisense strategies for genetic manipulations in plants," Plant Sci 105: 125-149) or expressed from vectors designed to activate gene silencing (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).
• IOMT sequences used can be the alfalfa IOMT8 designated in SEQ ID NO: 1 and Fig. 2, or any other IOMT gene with in vivo 4'-O-methylation specificity that could be isolated by hybridization techniques using IOMT8 as a probe.
• Isoflavonoid compounds methylated at the 7-position may also have nutraceutical activity, and the alfalfa IOMT can be used for making these compounds by feeding non-methylated 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 IOMT gene under the control of a suitable constitutive or inducible promoter.
• non-methylated 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 IOMT gene under the control of a suitable constitutive or inducible promoter.
• the 7-O-methylated isoflavonoid compounds can be produced using in vitro processes by contacting non-methylated isoflavone precursors with isolated soluble or immobilized isoflavone-O-transferase 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 which have been genetically transformed to contain all the other necessary enzymes of isoflavonoid biosynthesis).
• 7-O- methylated isoflavonoid compounds include but are not limited to isoformononetin, prunetin, their respective conjugates, or mixtures thereof.

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