Methods to increase the isoflavonoid levels in plants and plants producing increased levels of isoflavonoids

This application claims the benefit of U.S. Provisional Application No. 60/388,280, filed 13 Jun. 2002. The entire content of this provisional application is herein incorporated by reference.

This invention pertains to methods of increasing isoflavonoid production in isoflavonoid-producing plants by transforming plants with at least one construct comprising nucleic acid fragments encoding at least a portion of a flavanone 3-hydroxylase, a C1 myb transcription factor, and an R-type myc transcription factor.

Isoflavonoids represent a class of secondary metabolites produced in legumes by a branch of the phenylpropanoid pathway and include such compounds as isoflavones, isoflavanones, rotenoids, pterocarpans, isoflavans, quinone derivatives, 3-aryl-4-hydroxycoumarins, 3-arylcoumarins, isoflav-3-enes, coumestans, alpha-methyldeoxybenzoins, 2-arylbenzofurans, isoflavanol, coumaronochromone and the like. 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 (Dewick, P. M. (1993) in The Flavonoids, Advances in Research Since 1986, Harborne, J. B. Ed., pp. 117–238, Chapman and Hall, London). Isoflavonoid-derived compounds also are involved in symbiotic relationships between roots and rhizobial bacteria which eventually result in nodulation and nitrogen-fixation (Phillips, D. A. (1992) in Recent Advances in Phytochemistry . Vol. 26, pp 201–231, Stafford, H. A. and Ibrahim, R. K., Eds, Plenum Press, New York), and overall they have been shown to act as antibiotics, repellents, attractants, and signal compounds (Barz, W. and Welle, R. (1992) Phenolic Metabolism in Plants , pg 139–164, Ed by H. A. Stafford and R. K. Ibrahim, Plenum Press, New York).

Isoflavonoids have also been reported to have physiological activity in animal and human studies. For example, it has been reported that the isoflavones found in soybean seeds possess antihemolytic (Naim, M., et al. (1976) J. Agric. Food Chem . 24:1174–1177), antifungal (Naim, M., et al. (1974) J. Agr. Food Chem . 22:806–810), estrogenic (Price, K. R. and Fenwick, G. R. (1985) Food Addit. Contam . 2:73–106), tumor-suppressing (Messina, M. and Barnes, S. (1991) J. Natl. Cancer Inst . 83:541–546; Peterson, G., et al. (1991) Biochem. Biophys. Res. Commun . 179:661–667), hypolipidemic (Mathur, K., et. al. (1964) J. Nutr . 84:201–204), and serum cholesterol-lowering (Sharma, R. D. (1979) Lipids 14:535–540) effects. In addition, both epidemiological and dietary-intervention studies indicate that when isoflavones in soybean seeds and in subsequent protein products prepared from the seeds are part of the human dietary intake, those products provide many significant health benefits (Messina, M. J. (1999) Am. J. Clin. Nutr . 70:439S–450S).

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. During processing acetylglucoside forms are produced: 6′-O-acetyldaidzin, 6′-O-acetyl genistin, and 6′-O-acetyl glycitin. The content of isoflavonoids in soybean seeds is quite variable and is affected by both genetics and environmental conditions such as growing location and temperature during seed fill (Tsukamoto, C., et al. (1995) J. Agric. Food Chem . 43:1184–1192; Wang, H. and Murphy, P. A. (1994) J. Agric. Food Chem . 42:1674–1677). In addition, isoflavonoid content in legumes can be stress-induced by pathogen attack, wounding, high UV light exposure and pollution (Dixon, R. A. and Paiva, N. L. (1995) Plant Cell 7:1085–1097). The genistein isoflavonoid forms make up the most abundant group in soybean seeds and most food products, while the daidzein and the glycitein forms are present in lower levels (Murphy, P. A. (1999) J. Agric. Food Chem . 47:2697–2704).

The biosynthetic pathway for isoflavonoids in soybean and their relationship with several other classes of phenylpropanoids is presented in FIG. 1 . The enzyme flavanone 3-hydroxylase (F3H; EC 1.14.11.9) catalyzes the conversion of flavanones to dihydroflavonols, which are intermediates in the biosynthesis of flavonols, anthocyanidins, catechins and proanthocyanidins. This enzyme is also referred to as naringenin 3-dioxygenase, and naringenin, 2-oxoglutarate 3-dioxygenase, among others. In soybean, both flavanone 3-hydroxylase and isoflavone synthase (IFS) compete for naringenin as a substrate and it is not clear how this competition is regulated. Suppression of F3H has been shown to increase the resistance of cultivated plants to chemical stress (PCT publication WO 00/78979, published Dec. 28, 2000).

Though the branch initiated by isoflavone synthase that leads to synthesis of isoflavonoids is mainly limited to the legumes, the remainder of the phenylpropanoid pathway occurs in other plant species. In maize , genes of the phenylpropanoid pathway are regulated by the transcription factor C1 in combination with an R-type factor. Together C1 and an R-type factor activate expression of a set of genes that leads to the synthesis and accumulation of anthocyanins in maize cells (Grotewold, E., et al. (1998) Plant Cell 10:721–740).

Maize C1 is a myb-type transcription factor that regulates expression of genes involved in anthocyanin production and accumulation in maize cells. However C1 cannot activate gene expression alone, and requires interaction with an R-type myc transcription factor for activation of target gene promoters. The R-type factors include, among others, alleles of R, alleles of the homologous B gene of maize , and alleles of the homologous Lc gene. These genes function similarly and make up the R/B gene family (Goff, S. A., et al. (1992) Genes Dev . 6:864–875). The various genes of the R/B gene family may be in turn each found as diverging alleles that fluctuate in expression pattern within the corn plant due to differences in their promoters. The members of this family encode proteins with very similar amino acid sequences and thus have comparable effects on the anthocyanin pathway structural genes. The specificity of the different promoters provides tissue specificity of anthocyanin biosynthesis (Radicella, J. P. et al. (1992) Genes Dev. 6:2152–2164; Walker, E. L. (1995) EMBO J . 14:2350–2363). The skilled artisan will recognize that the coding region of any functional gene of this large family could be used in conjunction with a promoter of choice to obtain R-gene function in the desired tissue or developmental stage. Examples of R/B family genes and alleles include, but are not limited to, Lc, R, R-S, R-P, Sn, B-Peru, and B-I. The coding regions of particular alleles of the Lc or B genes, especially the B-Peru allele, have been most commonly used in experiments in conjunction with C1.

Cell suspension lines of the maize inbred Black Mexican Sweet (BMS) that were transformed with an estradiol-inducible version of a fusion of C1 and R (CRC) were analyzed after the addition of estradiol. The cDNA fragments from the known flavonoid genes, except chalcone isomerase, were induced in the CRC-expressing line after hormone induction (Bruce et al. (2000) Plant Cell 12:65–80). Maize C1 and an R-type factor together can promote the synthesis of anthocyanins in Arabidopsis tissues that do not naturally express anthocyanins (Lloyd, A. M., et al. (1992) Science 258:1773–1775), and in petunia leaves (Quattrocchio, F., et al. (1993) Plant Cell 5:1497–1512). WO 99/37794, published Jul. 29, 1999, discloses that the expression of maize C1 and the Lc allele of R in tomato fruit results in increased levels of the flavonol kaempferol. Thus, it is known that C1 and an R-type factor can regulate expression of individual genes of the phenylpropanoid pathway in plants including Arabidopsis, petunia, tomato, and maize leading to production of anthocyanins or flavonols. These are all plants that do not produce isoflavonoids. Isoflavonoid production is almost exclusively limited to the legumes. An example of one of the few non-legume plants that does produce isoflavonoids is sugar beet.

WO 00/44909, published Aug. 3, 2000, discloses transformation of soybeans with maize C1 and R (as a CRC chimera) in conjunction with overexpression of the isoflavone synthase gene. Visual inspection, after transient expression of C1 and B-Peru in white clover and pea, which are legumes, shows production of anthocyanin in several tissues (Majnik, et al. (1998) Aust. J. Plant Phys . 25:335–343). Any possible effect of C1 and B-Peru on isoflavonoid levels was not assayed. Expression of a CRC chimera in an isoflavonoid-producing plant alters the levels of isoflavonoids in the transgenic plant. Soybean plants expressing the CRC chimera contain higher levels of daidzein and lower levels of genistein, with the total daidzein to total genistein ratios being increased, when compared to an untransformed plant (U.S. patent application 60/297981 cofiled with the present on Jun. 13, 2002).

The physiological benefits associated with isoflavonoids in both plants and humans make the manipulation of their contents in crop plants highly desirable. For example, increasing levels of isoflavonoids in soybean seeds increases the efficiency of extraction and lowers the cost of isoflavone-related products sold today for use in either reduction of serum cholesterol or in estrogen replacement therapy. Therefore there is a need to enhance the level of isoflavonoids in isoflavonoid-producing plants. Combining suppression of flavanone 3-hydroxylase (F3H) with activation of the phenylpropanoid pathway is one method to accomplish this goal.

The invention concerns a method of increasing isoflavonoid production in an isoflavonoid-producing plant, the method comprising:

a) transforming a plant with (1) a first recombinant DNA construct comprising a polynucleotide selected from the group consisting of: (i) a polynucleotide encoding all or part of a flavanone 3-hydroxylase; (ii) a polynucleotide from 5′ and/or 3′ ends of an isolated nucleic acid fragment which encodes flavanone 3-hydroxylase; or (iii) a polynucleotide comprising (i) and (ii); and (2) at least one second recombinant DNA construct comprising a polynucleotide encoding a C1 myb transcription factor and a R myc-type transcription factor; and b) growing the transformed plant of (a); and c) evaluating the plant or plant part obtained from the transformed plant for an increased quantity of isoflavonoid in the transformed plant or plant part as compared plant or plant parts obtained from an untransformed plant.

In a second embodiment, the first recombinant DNA construct described above comprises a promoter operably linked, in a sense or anti-sense orientation, to a polynucleotide encoding all or part of a flavanone 3-hydroxylase, or a promoter operably linked to a stem-loop structure selected from the group consisting of: (1) a structure wherein the loop comprises the polynucleotide nucleotide encoding all or part of a flavanone 3-hydroxylase and (2) a structure wherein the stem comprises the polynucleotide nucleotide encoding all or part of a flavanone 3-hydroxylase.

In a third embodiment, the isoflavonoid-producing plant is selected from the group consisting of soybean, clover, mung bean, lentil, hairy vetch, alfalfa, lupine, sugar beet, and snow pea. Also of interest are seeds or plant parts of a plant transformed with a recombinant expression construct of the invention from which isoflavonoid-containing products can be obtained or extracted.

In a fourth embodiment, this invention concerns a food or beverage incorporating these isoflavonoid-containing products.

In a fifth embodiment, this invention concerns a method of producing an isoflavonoid-containing product which comprises: (a) cracking the seeds obtained from plants transformed with any of the recombinant expression constructs of the invention to remove the meats from the hulls; and (b) flaking the meats obtained in step (a) to obtain the desired flake thickness.

In a sixth embodiment, the invention concerns an isoflavonoid-producing plant comprising in its genome:

(a) a first recombinant DNA construct comprising a polynucleotide selected from the group consisting of: (i) a polynucleotide encoding all or part of a flavanone 3-hydroxylase; (ii) a polynucleotide from 5′ and/or 3′ ends of an isolated nucleic acid fragment which encodes flavanone 3-hydroxylase; or (iii) a polynucleotide comprising (i) and (ii); and

(b) at least one second recombinant DNA construct comprising a polynucleotide encoding a C1 myb transcription factor and a R myc-type transcription factor; wherein seeds obtained from the transformed plant have an increased quantity of isoflavonoid as compared to seeds obtained from an untransformed plant.

The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing which form a part of this application.

FIG. 1 depicts the soybean biosynthetic pathway from phenylalanine to glycitein, daidzein, genistein, and dihydroflavonol.

FIG. 2 depicts a representation of the seed-specific expression vector pKS151. The two copies of the 36-nucleotide sequence are indicated by “EL”, and the inverted repeat of the 36-nucleotide sequence are indicated by “(EL)”.

FIG. 3 depicts the total isoflavones as well as the genistin, daidzin, and glycitin levels observed for individual R1 seeds from transformed plants expressing the F3H construct.

FIG. 4 through FIG. 9 depict the total isoflavones as well as the genistin, daidzin, and glycitin levels observed for individual R1 seeds from transformed plants expressing the F3H construct together with the CRC chimeric construct.

FIG. 10 depicts the total isoflavones as well as the genistin, daidzin, and glycitin levels observed for individual R2 seeds from transformed plants expressing the F3H construct together with the CRC chimeric construct.

The following sequence descriptions and Sequences Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821–1.825. The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUB standards described in Nucleic Acids Research 13:3021–3030 (1985) and in the Biochemical Journal 219 (No. 2):345–373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO:1 is the nucleotide sequence of plasmid pKS151 used for the expression of a fragment of flavonone 3-hydroxylase.

SEQ ID NO:2 is the nucleotide sequence of primer flavanone 3-hydroxylase NotI sense, used to amplify a flavanone 3-hydroxylase fragment from clone sfl1.pk0040.g11.

SEQ ID NO:3 is the nucleotide sequence of primer flavanone 3-hydroxylase NotI antisense, used to amplify a flavanone 3-hydroxylase fragment from clone sfl1.pk0040.g11.

SEQ ID NO:4 is the nucleotide sequence of the cDNA insert in plasmid sfl1.pk0040.g11, encoding a flavanone 3-hydroxylase.

SEQ ID NO:5 is the nucleotide sequence of primer 3, used to detect the presence of the flavanone 3-hydroxylase construct in tranformed plants.

SEQ ID NO:6 is the nucleotide sequence of primer 4, used to detect the presence of the flavanone 3-hydroxylase construct in tranformed plants.

SEQ ID NO:7 is the nucleotide sequence of primer 5, used to detect the presence of the flavanone 3-hydroxylase construct in tranformed plants.

SEQ ID NO:8 is the nucleotide sequence of primer 6, used to detect the presence of the flavanone 3-hydroxylase construct in tranformed plants.

SEQ ID NO:9 is the nucleotide sequence of primer 1, used to detect the presence of the CRC chimeric construct in tranformed plants.

SEQ ID NO:10 is the nucleotide sequence of primer 2, used to detect the presence of the CRC chimeric construct in tranformed plants.

All patents, patent applications and publications cited are incorporated herein by reference in their entirety.

In the context of this disclosure, a number of terms shall be utilized.

The term “isoflavonoid(s)”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, as well as, the acetylglucoside forms: 6′-O-acetyldaidzin, 6′-O-acetyl genistin, and 6′-O-acetyl glycitin that are formed during processing.

The term “isoflavonoid-producing plant” refers to a plant in which isoflavonoids naturally occur. Examples of isoflavonoid-producing plants include, but are not limited to, soybean, clover, mung bean, lentil, hairy vetch, alfalfa, lupine, sugar beet, and snow pea. In a more preferred embodiment, the preferred isoflavonoid-producing plant would be soybean. Examples of other isoflavonoid-producing plants can be found in WO 93/23069, published Nov. 25, 1993, the disclosure of which is hereby incorporated by reference.

The term “flavanone 3-hydroxylase” refers to the polypeptide or enzyme that catalyzes the conversion of flavanones to dihydroflavonols.

The term “C1 myb transcription factor” refers to a protein encoded by a maize C1 gene and to any protein which is functionally equivalent to a C1 myb transcription factor.

The term “R myc-type transcription factor” refers to a protein with a basic helix-loop-helix domain encoded by a member of the R/B gene family and to any protein that is functionally equivalent to an R myc-type transcription factor.

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usually found in their 5′-monophosphate form) are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.

The terms “subfragment that is functionally equivalent” and “functionally equivalent subfragment” are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment in which the ability to alter gene expression or produce a certain phenotype is retained whether or not the fragment or subfragment encodes an active enzyme. For example, the fragment or subfragment can be used in the design of chimeric genes to produce the desired phenotype in a transformed plant. Chimeric genes can be designed for use in suppression by linking a nucleic acid fragment or subfragment thereof, whether or not it encodes an active enzyme, in the sense or antisense orientation relative to a plant promoter sequence.

The terms “homology”, “homologous”, “substantially similar” and “corresponding substantially” are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences.

Moreover, the skilled artisan recognizes that substantially similar nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize, under moderately stringent conditions (for example, 0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of preferred conditions involves a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions involves the use of higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions involves the use of two final washes in 0.1×SSC, 0.1% SDS at 65° C.

“Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure. An “allele” is one of several alternative forms of a gene occupying a given locus on a chromosome. When all the alleles present at a given locus on a chromosome are the same that plant is homozygous at that locus. If the alleles present at a given locus on a chromosome differ that plant is heterozygous at that locus.

“Coding sequence” refers to a DNA sequence that codes for a specific amino acid sequence. “Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

“Promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a DNA sequence which can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro, J. K., and Goldberg, R. B. (1989) Biochemistry of Plants 15:1–82.

Among the most commonly used promoters are the nopaline synthase (NOS) promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci. U.S.A . 84:5745–5749), the octapine synthase (OCS) promoter, caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al. (1987) Plant Mol Biol . 9:315–324), the CaMV 35S promoter (Odell et al. (1985) Nature 313:810–812), and the figwort mosaic virus 35S promoter, the light inducible promoter from the small subunit of rubisco, the Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. U.S.A . 84:6624–66280, the sucrose synthase promoter (Yang et al. (1990) Proc. Natl. Acad. Sci. U.S.A . 87:4144–4148), the R gene complex promoter (Chandler et al. (1989) Plant Cell 1:1175–1183), the chlorophyll a/b binding protein gene promoter, etc. Other commonly used promoters are, the promoters for the potato tuber ADPGPP genes, the sucrose synthase promoter, the granule bound starch synthase promoter, the glutelin gene promoter, the maize waxy promoter, Brittle gene promoter, and Shrunken 2 promoter, the acid chitinase gene promoter, and the zein gene promoters (15 kD, 16 kD, 19 kD, 22 kD, and 27 kD; Perdersen et al. (1982) Cell 29:1015–1026). A plethora of promoters is described in WO 00/18963, published on Apr. 6, 2000, the disclosure of which is hereby incorporated by reference.

The “translation leader sequence” refers to a polynucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, G. D. (1995) Mol. Biotech . 3:225–236).

The “3′ non-coding sequences” refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht, I. L., et al. (1989) Plant Cell 1:671–680.

“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript. An RNA transcript is referred to as the mature RNA when it is an RNA sequence derived from post-transcriptional processing of the primary transcript. “Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into protein by the cell. “cDNA” refers to a DNA that is complementary to and synthesized from a mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into the double-stranded form using the Klenow fragment of DNA polymerase I. “Sense” RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA, and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes. The terms “complement” and “reverse complement” are used interchangeably herein with respect to mRNA transcripts, and are meant to define the antisense RNA of the message.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another example, the complementary RNA regions of the invention can be operably linked, either directly or indirectly, 5′ to the target mRNA, or 3′ to the target mRNA, or within the target mRNA, or a first complementary region is 5′ and its complement is 3′ to the target mRNA.

The term “expression”, as used herein, refers to the production of a functional end-product e.g., a mRNA or a protein (precursor or mature).

“Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. “Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.

“Stable transformation” refers to the transfer of a nucleic acid fragment into a genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance. In contrast, “transient transformation” refers to the transfer of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. Preferred methods of cell transformation for rice, corn, and other monocots are the particle-accelerated or “gene gun” transformation technology (Klein et al., (1987) Nature ( London ) 327:70–73; U.S. Pat. No. 4,945,050), and an Agrobacterium -mediated method using an appropriate Ti plasmid containing the transgene (Ishida Y. et al., 1996 , Nature Biotech . 14:745–750).

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual ; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989. Transformation methods are well known to those skilled in the art and are described above.

“PCR” or “Polymerase Chain Reaction” is a technique for the synthesis of large quantities of specific DNA segments, consists of a series of repetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.). Typically, the double stranded DNA is heat denatured, the two primers complementary to the 3′ boundaries of the target segment are annealed at low temperature and then extended at an intermediate temperature. One set of these three consecutive steps is referred to as a cycle.

The term “recombinant” refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.

The terms “recombinant construct”, “expression construct”, “chimeric construct”, “construct” and “recombinant DNA construct” are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not found together in nature. For example, a chimeric construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such construct may be used by itself or may be used in conjunction with a vector. If a vector is used then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the invention. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., (1985) EMBO J . 4:2411–2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78–86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, among others.

“Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. “Co-suppression” refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020). Co-suppression constructs in plants previously have been designed by focusing on overexpression of a nucleic acid sequence having homology to an endogenous mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (see Vaucheret et al. (1998) Plant J 16:651–659; and Gura (2000) Nature 404:804–808). The overall efficiency of this phenomenon is low, and the extent of the RNA reduction is widely variable. Recent work has described the use of “hairpin” structures that incorporate all, or part, of an mRNA encoding sequence in a complementary orientation that results in a potential “stem-loop” structure for the expressed RNA (PCT Publication WO 99/53050 published on Oct. 21, 1999). This increases the frequency of co-suppression in the recovered transgenic plants. Another variation describes the use of plant viral sequences to direct the suppression, or “silencing”, of proximal mRNA encoding sequences (PCT Publication WO 98/36083 published on Aug. 20, 1998). Both of these co-suppressing phenomena have not been elucidated mechanistically, although recent genetic evidence has begun to unravel this complex situation (Elmayan et al. (1998) Plant Cell 10:1747–1757).

The present invention concerns a method of increasing isoflavonoid production in an isoflavonoid-producing plant, said method comprising: a) transforming a plant with (1) a first recombinant DNA construct comprising a polynucleotide selected from the group consisting of: (i) a polynucleotide encoding all or part of a flavanone 3-hydroxylase; (ii) a polynucleotide from 5′ and/or 3′ ends of an isolated nucleic acid fragment which encodes flavanone 3-hydroxylase; or (iii) a polynucleotide comprising (i) and (ii); and (2) at least one second recombinant DNA construct comprising a polynucleotide encoding a C1 myb transcription factor and a R myc-type transcription factor; and b) growing the transformed plant of (a); and c) valuating the plant or plant parts obtained from the transformed plant for an increased quantity of isoflavonoid in the transformed plant or plant part as compared plant or plant part obtained from an untransformed plant.

The first recombinant DNA construct that may be used to transform plants of the present invention may comprise:

(i) a polynucleotide encoding all or part of a flavanone 3-hydroxylase;

(ii) a polynucleotide from 5′ and/or 3′ ends of an isolated nucleic acid fragment which encodes flavanone 3-hydroxylase; or

(iii) a polynucleotide comprising (i) and (ii).

The polynucleotide mentioned above in (i), (ii), or (iii) may be operably linked to a promoter in a sense orientation or an antisense orientation, or may be forming part of a stem loop structure with the polynucleotide forming either the loop or the stem.

The second recombinant DNA construct, comprising a polynucleotide encoding a C1 myb transcription factor and an R myc-type transcription factor, which can be used to transform an isoflavonoid-producing plant, may comprise:

(a) the polynucleotide encoding a C1 myb transcription factor and the polynucleotide encoding an R myc-type transcription factor can be entirely separate, e.g., one polynucleotide may comprise a promoter operably linked to an isolated nucleic acid fragment encoding a C1 myb transcription factor and another separate polynucleotide may comprise a promoter operably linked to an isolated nucleic acid fragment encoding an R-myc type transcription factor;

(b) the polynucleotide encoding a C1 myb transcription factor and the polynucleotide encoding an R myc-type transcription factor can be in a single construct comprising at least one promoter operably linked to an isolated nucleic acid fragment encoding a C1 myb transcription factor and at least one promoter operably linked to an isolated nucleic acid fragment encoding an R-myc type transcription factor; and

(c) the polynucleotide encoding a C1 myb transcription factor and the polynucleotide encoding an R myc-type transcription factor can be in a single construct comprising a promoter operably linked to an isolated nucleic acid fragment encoding all or a part of a C1 myb transcription factor and an isolated nucleic acid fragment encoding all or a part of an R-myc type transcription factor such that a fusion protein combining the two encoded proteins is produced.

The polynucleotide comprising a polynucleotide encoding at least a portion of a flavanone 3-hydroxylase and the polynucleotide comprising a C1 myb transcription factor and an R-myc type factor may each form part of at least one recombinant DNA construct, or may form part of one single recombinant DNA expression construct.

The transformed plant is then grown under conditions suitable for the expression of the recombinant DNA construct or constructs. Expression of the recombinant DNA construct or constructs alters the quantity of isoflavonoid of the transformed plant compared to the quantity of isoflavonoid of an untransformed plant.

In a more preferred, embodiment, an isoflavonoid-producing plant can be transformed with a recombinant expression construct comprising a promoter operably linked to a flavanone 3-hydroxylase and a recombinant expression construct comprising a promoter operably linked to an isolated nucleic acid fragment encoding a chimeric transcription factor comprising the maize R coding region situated between the C1 DNA binding domain and the C1 activation domain.

The regeneration, development and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, In.: Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc., San Diego, Calif. (1988)). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign, exogenous gene that encodes a protein of interest is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.

There are a variety of methods for the regeneration of plants from plant tissue.

The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated.

Methods for transforming dicots, primarily by use of Agrobacterium tumefaciens , and obtaining transgenic plants have been published for cotton (U.S. Pat. Nos. 5,004,863, 5,159,135, 5,518,908); soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011, McCabe et. al., BiolTechnology 6:923 (1988), Christou et al., Plant Physiol . 87:671–674 (1988)); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al., Plant Cell Rep . 15:653–657 (1996), McKently et al., Plant Cell Rep . 14:699–703 (1995)); papaya; and pea (Grant et al., Plant Cell Rep . 15:254–258, (1995)).

Assays for gene expression based on the transient expression of cloned nucleic acid constructs have been developed by introducing the nucleic acid molecules into plant cells by polyethylene glycol treatment, electroporation, or particle bombardment (Marcotte et al., Nature 335:454–457 (1988); Marcotte et al., Plant Cell 1:523–532 (1989); McCarty et al., Cell 66:895–905 (1991); Hattori et al., Genes Dev. 6:609–618 (1992); Goff et al., EMBO J. 9:2517–2522 (1990)).

Transient expression systems may be used to functionally dissect gene constructs (see generally, Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995)). It is understood that any of the nucleic acid molecules of the present invention can be introduced into a plant cell in a permanent or transient manner in combination with other genetic elements such as vectors, promoters, enhancers etc.

In addition to the above discussed procedures, practitioners are familiar with the standard resource materials which describe, specific conditions and procedures for the construction, manipulation and isolation of macromolecules (e.g., DNA molecules, plasmids, etc.), generation of recombinant DNA fragments and recombinant expression constructs and the screening and isolating of clones, (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989); Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995); Birren et al., Genome Analysis: Detecting Genes, 1, Cold Spring Harbor, N.Y. (1998); Birren et al., Genome Analysis: Analyzing DNA, 2, Cold Spring Harbor, N.Y. (1998); Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer, New York (1997)).

Any promoter can be used in accordance with the method of the invention. Thus, the origin of the promoter chosen to drive expression of the coding sequence is not critical as long as it has sufficient transcriptional activity to accomplish the invention by expressing translatable mRNA for the desired nucleic acid fragments in the desired host tissue. The promoter for use in the present invention may be selected from the group consisting of a seed-specific promoter, root-specific promoter, vacuole-specific promoter, and an embryo-specific promoter.

Examples of a seed-specific promoter include, but are not limited to, the promoter for β-conglycinin (Chen et al. (1989) Dev. Genet . 10:112–122), the napin promoter, and the phaseolin promoter. Other tissue-specific promoters that may be used to accomplish the invention include, but are not limited to, the chloroplast glutamine synthase (GS2) promoter (Edwards et al. (1990) Proc. Natl. Acad. Sci. U.S.A . 87:3459–3463), the chloroplast fructose-1,6-biophosphatase promoter (Lloyd et al. (1991) Mol. Gen. Genet . 225:209–2216), the nuclear photosynthetic (ST-LS1) promoter (Stockhaus et al. (1989) EMBO J . 8:2445–2451), the serine/threonine kinase (PAL) promoter, the glucoamylase promoter, the promoters for the Cab genes (cab6, cab-1, and cab-1R, Yamamoto et al. (1994) Plant Cell Physiol . 35:773–778; Fejes et al. (1990) Plant Mol. Biol . 15:921–932; Lubberstedt et al. (1994) Plant Physiol . 104:997–1006; Luan et al. (1992) Plant Cell 4:971–981), the pyruvate orthophosphate dikanase promoter (Matsuoka et al. (1993) Proc. Natl. Acad. Sci. U.S.A . 90:9586–9590), the LhcB promoter (Cerdan et al. (1997) Plant Mol. Biol . 33:245–255), the PsbP promoter (Kretsch et al. (1995) Plant Mol. Biol . 28:219–229), the SUC2 sucrose H+ symporter promoter (Truernit et al. (1995) Planta 196:564–570), and the promoters for the thylakoid membrane genes (psaD, psaF, psaE, PC, FNR, atpC, atpD), etc.

Also within the scope of this invention are seeds or plant parts obtained from such transformed plants. Plant parts include differentiated and undifferentiated tissues, including but not limited to, roots, stems, shoots, leaves, pollen, seeds, tumor tissue, and various forms of cells and culture such as single cells, protoplasts, embryos, and callus tissue. The plant tissue may be in plant or in organ, tissue or cell culture.

In another aspect, this invention concerns an isoflavonoid-containing product high in total isoflavonoids obtained from the seeds or plant parts obtained from the transformed plants described herein. Examples of such an isoflavonoid-containing product include, but are not limited to, protein isolate, protein concentrate, meal, grits, full fat and defatted flours, textured proteins, textured flours, textured concentrates and textured isolates. In still another aspect, this invention concerns an isoflavonoid-containing product high in isoflavonoids extracted from the seeds or plant parts obtained from the transformed plants described herein. An extracted product could then used in the production of pills, tablets, capsules or other similar dosage forms made to contain a high concentration of isoflavonoids.

Methods for obtaining such products are well known to those skilled in the art. For example, in the case of soybean, such products can be obtained in a variety of ways. Conditions typically used to prepare soy protein isolates have been described by [Cho, et al, (1981) U.S. Pat. No. 4,278,597; Goodnight, et al. (1978) U.S. Pat. No. 4,072,670]. Soy protein concentrates are produced by three basic processes: acid leaching (at about pH 4.5), extraction with alcohol (about 55–80%), and denaturing the protein with moist heat prior to extraction with water. Conditions typically used to prepare soy protein concentrates have been described by Pass [(1975) U.S. Pat. No. 3,897,574] and Campbell et al. [(1985) in New Protein Foods, ed. by Altschul and Wilcke, Academic Press, Vol. 5, Chapter 10 , Seed Storage Proteins , pp 302–338].

“Isoflavonoid-containing protein products” can be defined as those items produced from seed of a suitable plant which are used in feeds, foods and/or beverages. For example, “soy protein products” can include, but are not limited to, those items listed in Table 1. “Soy protein products”.

“Processing” refers to any physical and chemical methods used to obtain the products listed in Table 1 and includes, but is not limited to, heat conditioning, flaking and grinding, extrusion, solvent extraction, or aqueous soaking and extraction of whole or partial seeds. Furthermore, “processing” includes the methods used to concentrate and isolate soy protein from whole or partial seeds, as well as the various traditional Oriental methods in preparing fermented soy food products. Trading Standards and Specifications have been established for many of these products (see National Oilseed Processors Association Yearbook and Trading Rules 1991–1992). Products referred to as being “high protein” or “low protein” are those as described by these Standard Specifications. “NSI” refers to the Nitrogen Solubility Index as defined by the American Oil Chemists' Society Method Ac4 41. “KOH Nitrogen Solubility” is an indicator of soybean meal quality and refers to the amount of nitrogen soluble in 0.036 M KOH under the conditions as described by Araba and Dale [(1990) Poult. Sci . 69:76–83]. “White” flakes refer to flaked, dehulled cotyledons that have been defatted and treated with controlled moist heat to have an NSI of about 85 to 90. This term can also refer to a flour with a similar NSI that has been ground to pass through a No. 100 U.S. Standard Screen size. “Cooked” refers to a soy protein product, typically a flour, with an NSI of about 20 to 60. “Toasted” refers to a soy protein product, typically a flour, with an NSI below 20. “Grits” refer to defatted, dehulled cotyledons having a U.S. Standard screen size of between No. 10 and 80. “Soy Protein Concentrates” refer to those products produced from dehulled, defatted soybeans by three basic processes: acid leaching (at about pH 4.5), extraction with alcohol (about 55–80%), and denaturing the protein with moist heat prior to extraction with water. Conditions typically used to prepare soy protein concentrates have been described by Pass [(1975) U.S. Pat. No. 3,897,574; Campbell et al., (1985) in New Protein Foods, ed. by Altschul and Wilcke, Academic Press, Vol. 5, Chapter 10 , Seed Storage Proteins , pp 302–338]. “Extrusion” refers to processes whereby material (grits, flour or concentrate) is passed through a jacketed auger using high pressures and temperatures as a means of altering the texture of the material. “Texturing” and “structuring” refer to extrusion processes used to modify the physical characteristics of the material. The characteristics of these processes, including thermoplastic extrusion, have been described previously [Atkinson (1970) U.S. Pat. No. 3,488,770, Horan (1985) In New Protein Foods , ed. by Altschul and Wilcke, Academic Press, Vol. 1A, Chapter 8, pp 367–414]. Moreover, conditions used during extrusion processing of complex foodstuff mixtures that include soy protein products have been described previously [Rokey (1983) Feed Manufacturing Technology III , 222–237; McCulloch, U.S. Pat. No. 4,454,804].

Also, within the scope of this invention are food, food supplements, food bars, and beverages which have incorporated therein an isoflavonoid-containing product of the invention. The beverage can be in a liquid or in a dry powdered form.

The foods to which the isoflavonoid-containing product of the invention can be incorporated/added include almost all foods/beverages. For example, there can be mentioned meats such as ground meats, emulsified meats, marinated meats, and meats injected with an isoflavonoid-containing product of the invention; beverages such as nutritional beverages, sports beverages, protein fortified beverages, juices, milk, milk alternatives, and weight loss beverages; cheeses such as hard and soft cheeses, cream cheese, and cottage cheese; frozen desserts such as ice cream, ice milk, low fat frozen desserts, and non-dairy frozen desserts; yogurts; soups; puddings; bakery products; and salad dressings; and dips and spreads such as mayonnaise and chip dips. The isoflavonoid-containing product can be added in an amount selected to deliver a desired dose to the consumer of the food and/or beverage.

In still another aspect this invention concerns a method of producing an isoflavonoid-containing product which comprises: (a) cracking the seeds obtained from transformed plants of the invention to remove the meats from the hulls; and (b) flaking the meats obtained in step (a) to obtain the desired flake thickness.

The present invention is further defined in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.