The potential health benefits of dietary isoflavones have generated considerable interest in engineering the synthesis of these phytoestrogens into plants. Genistein glucoside production (up to 50 nmol g 21 fresh weight) was engineered in alfalfa (Medicago sativa) leaves by constitutive expression of isoflavone synthase from Medicago truncatula (MtIFS1). Glucosides of biochanin A (4#-O-methylgenistein) and pratensein (3#-hydroxybiochanin A) also accumulated. Although MtIFS1 was highly expressed in all organs examined, genistein accumulation was limited to leaves. MtIFS1-expressing lines accumulated several additional isoflavones, including formononetin and daidzein, in response to UV-B or Phoma medicaginis, whereas the chalcone and flavanone precursors of these compounds accumulated in control lines. Enhanced accumulation of the phytoalexin medicarpin was observed in P. medicaginis-infected leaves of MtIFS1-expressing plants. Microarray profiling indicated that MtIFS1 expression does not significantly alter global gene expression in the leaves. Our results highlight some of the challenges associated with metabolic engineering of plant natural products, including tissue-specific accumulation, potential for further modification by endogenous enzyme activities (hydroxylation, methylation, and glycosylation), and the differential response of engineered plants to environmental factors.Isoflavonoids are a predominantly legume-specific subclass of flavonoid secondary metabolites, with roles in plant defense and nodulation (Dixon, 1999). The protective effects of the soy (Glycine max) isoflavones genistein and daidzein against hormonedependent cancers, cardiovascular disease, osteoporosis, and menopausal symptoms (for review, see Cornwell et al., 2004;Dixon, 2004) have generated considerable interest in engineering these isoflavones into more commonly consumed food crops . Isoflavones are synthesized from common flavanone intermediates (either liquiritigenin or naringenin; Fig. 1) by aryl migration catalyzed by the enzyme 2-hydroxyisoflavanone synthase (also referred to as isoflavone synthase or IFS). Cloning of IFS has allowed isoflavone synthesis to be engineered into plants that normally do not make isoflavones, as demonstrated in Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), and cell cultures of maize (Zea mays; Jung et al., 2000aJung et al., , 2000bYu et al., 2000;Liu et al., 2002). These studies have also highlighted some of the factors impacting isoflavone accumulation in plants. For example, in IFS-expressing Arabidopsis, accumulation of genistein was limited by competition from the flavonol pathway (Liu et al., 2002), whereas in IFSexpressing tobacco, it was correlated with the activity of the phenylpropanoid pathway leading to the accumulation of anthocyanins (Yu et al., 2000). In all cases, levels of genistein accumulating in transgenic plants were several orders of magnitude lower than levels of genistein in soybean.Engineering isoflavonoid biosynthesis in leguminous plants may provide enh...

In leguminous plants such as pea (Pisum sativum), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), and chickpea (Cicer arietinum), 49-O-methylation of isoflavonoid natural products occurs early in the biosynthesis of defense chemicals known as phytoalexins. However, among these four species, only pea catalyzes 3-O-methylation that converts the pterocarpanoid isoflavonoid 6a-hydroxymaackiain to pisatin. In pea, pisatin is important for chemical resistance to the pathogenic fungus Nectria hematococca. While barrel medic does not biosynthesize 6a-hydroxymaackiain, when cell suspension cultures are fed 6a-hydroxymaackiain, they accumulate pisatin. In vitro, hydroxyisoflavanone 49-O-methyltransferase (HI49OMT) from barrel medic exhibits nearly identical steady state kinetic parameters for the 49-O-methylation of the isoflavonoid intermediate 2,7,49-trihydroxyisoflavanone and for the 3-O-methylation of the 6a-hydroxymaackiain isoflavonoid-derived pterocarpanoid intermediate found in pea. Protein x-ray crystal structures of HI49OMT substrate complexes revealed identically bound conformations for the 2S,3R-stereoisomer of 2,7,49-trihydroxyisoflavanone and the 6aR,11aR-stereoisomer of 6a-hydroxymaackiain. These results suggest how similar conformations intrinsic to seemingly distinct chemical substrates allowed leguminous plants to use homologous enzymes for two different biosynthetic reactions. The three-dimensional similarity of natural small molecules represents one explanation for how plants may rapidly recruit enzymes for new biosynthetic reactions in response to changing physiological and ecological pressures.

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Previous studies have identified two distinct O-methyltransferases (OMTs) implicated in isoflavonoid biosynthesis in Medicago species, a 7-OMT methylating the A-ring 7-hydroxyl of the isoflavone daidzein and a 4'-OMT methylating the B-ring 4'-hydroxyl of 2,7,4'-trihydroxyisoflavanone. Genes related to these OMTs from the model legume Medicago truncatula cluster as separate branches of the type I plant small molecule OMT family. To better understand the possible functions of these related OMTs in secondary metabolism in M. truncatula, seven of the OMTs were expressed in E. coli, purified, and their in vitro substrate preferences determined. Many of the enzymes display promiscuous activities, and some exhibit dual regio-specificity for the 4' and 7-hydroxyl moieties of the isoflavonoid nucleus. Protein structure homology modeling was used to help rationalize these catalytic activities. Transcripts encoding the different OMT genes exhibited differential tissue-specific and infection- or elicitor-induced expression, but not always in parallel with changes in expression of confirmed genes of the isoflavonoid pathway. The results are discussed in relation to the potential in vivo functions of these OMTs based on our current understanding of the phytochemistry of M. truncatula, and the difficulties associated with gene annotation in plant secondary metabolism.

Metabolic profiling of elicited barrel medic (Medicago truncatula) cell cultures using high-performance liquid chromatography coupled to photodiode and mass spectrometry detection revealed the accumulation of the aurone hispidol (6-hydroxy-2-[(4-hydroxyphenyl)methylidene]-1-benzofuran-3-one) as a major response to yeast elicitor. Parallel, large-scale transcriptome profiling indicated that three peroxidases, MtPRX1, MtPRX2, and MtPRX3, were coordinately induced with the accumulation of hispidol. MtPRX1 and MtPRX2 exhibited aurone synthase activity based upon in vitro substrate specificity and product profiles of recombinant proteins expressed in Escherichia coli. Hispidol possessed significant antifungal activity relative to other M. truncatula phenylpropanoids tested but has not been reported in this species before and was not found in differentiated roots in which high levels of the peroxidase transcripts accumulated. We propose that hispidol is formed in cell cultures by metabolic spillover when the pool of its precursor, isoliquiritigenin, builds up as a result of an imbalance between the upstream and downstream segments of the phenylpropanoid pathway, reflecting the plasticity of plant secondary metabolism. The results illustrate that integration of metabolomics and transcriptomics in genetically reprogrammed plant cell cultures is a powerful approach for the discovery of novel bioactive secondary metabolites and the mechanisms underlying their generation.