Metabolic Engineering of Isoflavonoid Biosynthesis in Alfalfa

Abstract

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...

A transcript profiling approach reveals an epicatechin-specific glucosyltransferase expressed in the seed coat of Medicago truncatula

“…Flavonoid profiles of various organs and developing seeds were analyzed by liquid chromatography-mass spectrometry. Conjugates of apigenin, luteolin, and quercetin (quercetin-3-Oglucoside) were found in all organs examined (data not shown), as previously shown in alfalfa (24). In contrast, a compound with the same HPLC retention time, and UV-and mass-spectral characteristics as epi-glc, was found only in developing seeds (Fig.…”

Secondary Metabolite Profiling in Various Parts of Tomato Plants

“…Plants produce secondary metabolites under biotic or abiotic stress because the compounds have antimicrobial, anti-herbivory, and allopathic effects (Dixon, 2001). In addition, various secondary metabolites show other beneficial properties such as color, scent, and flavor attractants (Frydman et al, 2004;Rohloff and Bones, 2005;Verdonk et al, 2003), UV-protectants, antioxidants, signaling factors, and nutraceutical and pharmacological efficacies (Aerts et al, 1999;Bagchi et al, 2000;Deavours and Dixon, 2005;D'Haeze and Holsters, 2002;Gidley, 2004;Manach et al, 2004;Merz-Demlow et al, 2000;Oldroyd, 2001;Relić et al, 1994;Setchell and Cassidy, 1999).…”

Metabolomics of Disease Resistance in Crops

“…Overexpression of isoflavone synthase (IFS) Increased formononetin and medicarpin accumulation in IFS overexpression lines (Deavours and Dixon, 2005).…”