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Mining of Medicago truncatula EST databases and screening of a root cDNA library led to the identification of three cytochrome P450 81E subfamily members. Two were functionally characterized by expression in yeast. The recombinant enzymes in yeast microsomes utilized the same isoflavone substrates, but produced different products hydroxylated at the 2′ and/or 3′ positions of the B-ring. When transiently expressed in alfalfa leaves, green fluorescent protein (GFP) fusions of the isoflavone 2′- and 3′-hydroxylases localized to the endoplasmic reticulum. The isoflavone 2′-hydroxylase was functional when expressed in Arabidopsis. Differential tissue-specific and biotic/abiotic stress-dependent expression patterns were observed for the isoflavone 2′-hydroxylase and 3′-hydroxylase genes, suggesting differential involvement of 2′- and 3′-hydroxylated isoflavonoids in pathogen defense and insect-induced responses, respectively, in Medicago.
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Isoflavonoids are a subclass of phenylpropanoid metabolites distributed primarily in legumes (Dixon and Sumner, 2003). They possess a wide range of biological activities (Dixon, 1999), but most research has focused on their functions as pathogen-inducible antimicrobial compounds (phytoalexins; Dewick, 1993; Dixon, 1999; Ingham, 1982) or as dietary phytoestrogens implicated in human disease prevention (Adlercreutz and Mazur, 1997; Dixon and Ferreira, 2002). Different legume species produce different classes of isoflavonoid phytoalexins, of which, substituted pterocarpans, such as medicarpin from alfalfa and pisatin from pea, are the best known (Figure 1).
Figure 1. Biosynthetic pathways leading to complex isoflavonoids in legumes.
The compounds shown are found in different species, as indicated. The enzymes are: CYP73A, cinnamate 4-hydroxylase; CHS, chalcone synthase; CHR, chalcone reductase; CHI, chalcone isomerase; CYP93C2, 2-hydroxyisoflavanone synthase, also known as isoflavone synthase (IFS); IOMT, 2-hydroxyisoflavanone 4′-O-methyltransferase; DH, 2-hydroxyisoflavanone dehydratase; CYP81E1/7, isoflavone 2′-hydroxylase (I2′H); CYP81E9, isoflavone 3′-hydroxylase (I3′H); IFR, isoflavone reductase; VR, vestitone reductase, CYPX, P450 catalyzing methylenedioxy ring closure; CYP81E(X), P450 catalyzing 2′-hydroxylation of pseudobaptigenin. Dotted arrows indicate pathways that are yet to be fully characterized. Double arrows indicate two or more reactions. The numbering system for isoflavones is shown for formononetin and biochanin A. Note that the 2′- and 5′-, and 3′- and 6′-positions are synonymous because of rotation about the bond linking the aryl group to the 3′-position of the heterocyclic ring.
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Complex isoflavonoid derivatives such as the rotenoids rotenone, deguelin, and amorphigenin from Amorpha, Lonchocarpus, Derris, and Tephrosia species possess insecticidal and parasiticidal properties (Lambert et al., 1993; Nicholas et al., 1985). Maackiain (Figure 1), which accumulates along with medicarpin (the major phytoalexin in Medicago species) in red clover (Trifolium pratense), subterranean clover (T. subterraneum), and chickpea (Cicer arietinum; Dewick and Ward, 1978; Higgins, 1972; Ingham, 1982), has recently been shown to have larvicidal activity against caterpillars of Heliocoverpa armigera that attack chickpea (Simmonds and Stevenson, 2001).
The biosynthesis of isoflavonoids diverges from the ubiquitous flavonoid pathway and is shown in Figure 1, which also provides details of the A- and B-ring and position designations of isoflavonoid compounds. The 5-deoxyflavanone liquiritigenin is converted to an isoflavone, and then undergoes several steps of hydroxylation, methylation, reduction, and ring closure to form pterocarpans such as medicarpin and maackiain (Dewick and Martin, 1979; Dixon, 1999). The 5-hydroxyflavanone naringenin is also a starting point for synthesis of isoflavonoids such as biochanin A and pratensin in chickpea and red clover (Clemens et al., 1993; Dewick and Ward, 1978; Figure 1). Hydroxylations catalyzed by membrane-bound, NADPH-dependent cytochrome P450 monooxygenases are critical steps in the biosynthesis of complex isoflavonoids. For example, 2-position hydroxylation of liquiritigenin and naringenin accompanied by B-ring migration from the 2- to the 3-position occurs at the entry point into the isoflavonoid pathway (Kochs and Grisebach, 1986), whereas 2′- or 3′-position hydroxylation of the B-ring of isoflavones is essential for formation of pterocarpans and/or methylenedioxy-substituted compounds such as maackiain and pisatin (Clemens et al., 1993; Dewick and Ward, 1978; Gunia et al., 1991; Hinderer et al., 1987; Figure 1). 6a-Hydroxylation of pterocarpans occurs in the biosynthesis of the glyceollins in soybean (Kochs and Grisebach, 1989) and of pisatin in pea (Figure 1).
Isoflavone 2′-hydroxylase (I2′H) activity has been identified in microsomal fractions of elicited cells of soybean (Kochs and Grisebach, 1986), chickpea (Clemens et al., 1993; Gunia et al., 1991; Hinderer et al., 1987) and alfalfa (Medicago sativa; Choudhary et al., 1990), and an I2′H (CYP81E1) gene characterized from licorice (Glycyrrhiza echinata L.). Recombinant CYP81E1 catalyzed the 2′-hydroxylation of formononetin (7-hydroxy, 4′-methoxyisoflavone) and the 2′- and 3′-hydroxylation of daidzein (7,4′-dihydroxyisoflavone) in vitro in yeast microsomes (Akashi et al., 1998). Several cDNA clones with high sequence identity to I2′H have been isolated from elicited Lotus japonicus and chickpea cell suspension cultures by PCR strategies based on P450 conserved motifs (Overkamp et al., 2000; Shimada et al., 2000), but their functional characterization has not been reported.
Hydroxylation at the 3′-position of the B-ring of an isoflavone is a key step in the formation of the methylenedioxy bridge of maackiain (Clemens and Barz, 1996; Clemens et al., 1993; Dewick and Ward, 1978) and in the formation of rotenoids (Dixon, 1999). Isoflavone 3′-hydroxylase (I3′H) activities have been detected in the fungus Fusarium (Mackenbrock and Barz, 1983); in roots, leaves, and elicited cell suspension cultures of chickpea (Clemens et al., 1993; Hinderer et al., 1987); and, more recently, in human liver (Tolleson et al., 2002) in which P450 enzymes are presumably involved in isoflavone catabolism. However, genes encoding I3′H have not yet been identified.
In this report, we have utilized genomics resources available for the model legume Medicago truncatula (Bell et al., 2001; Cook, 1999; Oldroyd and Geurts, 2001) to identify three CYP81E subfamily members (CYP81E7, 8, and 9). Two of the three were functionally characterized by expression in yeast. They share high similarity at the amino acid level and utilize the same methylated isoflavone substrates, but encode distinct isoflavone 2′- (CYP81E7) and 3′- (CYP81E9) hydroxylases. The I2′H (CYP81E7) was transferred into Arabidopsis and was functional. We describe differential expression patterns of the I2′H and I3′H genes in response to a variety of biotic and abiotic stimuli, and discuss the results in terms of the known phytochemistry of Medicago species.