Biotransformation of an exogenously supplied isoflavonoid by transgenic tobacco cells expressing alfalfa isoflavone reductase

Abstract

Isoflavone reductase (IFR) from alfalfa catalyzes the NADPH-dependent reduction of 2′-hydroxy isoflavones to 2′-hydroxyisoflavanones such as vestitone, thereby performing a key step in isoflavonoid phytoalexin biosynthesis. Tobacco (Nicotiana tabacum L. cv. Xanthi) was transformed with an alfalfa cDNA encoding IFR regulated by an enhanced cauliflower mosaic virus 35S promoter and used to generate cell suspension cultures. Transformed cells expressed high levels of IFR activity and could take up 2′-hydroxyformononetin (2′OHF; IFR substrate) and convert it to vestitone. Both 2′OHF and vestitone were each converted to two sugar conjugates. The structure of the most abundant vestitone conjugate was determined using NMR spectroscopic analysis to be a novel vestitone 2′-O-glucoside. Liquid chromatography-mass spectroscopy (LC-MS) indicated that the molecular weights of the three less abundant conjugates were consistent with hexose conjugates of vestitone and 2′OHF. The IFR reduction and conjugation reactions were complete within 4 h, with the net percentage conversion of 2′OHF to vestitone ranging as high as 60%.

Priming defense genes and metabolites in hybrid poplar by the green leaf volatile cis‐3‐hexenyl acetate

“…Our microarray analysis provided further support for the priming of KTI. Proteinase inhibitors incur large production costs (Zavala et al ., 2004); based on our results, we would expect that similar costs are also associated with the products of IFR ‐like genes if they serve a bona fide defensive function (Cooper et al ., 2002). In theory, priming should be a more favorable strategy than induced defenses when induced defenses are costly; our results regarding KTI and IFRL expression support that prediction.…”

“…4c). Isoflavone reductases catalyze the production of potent isoflavonoid phytoalexins, which are antifungal agents in Fabaceae (Paiva et al ., 1991; Cooper et al ., 2002) and homologs may have similar defensive functions in other plants (Ralph et al ., 2006). As with PdnKTI1 , PdnIFRL1 levels were very low before herbivore feeding, although the expression was nonetheless higher (107%) in the z 3HAC‐treated leaves ( t 3 = 3.27, P = 0.047).…”

Overexpression of Soybean Isoflavone Reductase (GmIFR) Enhances Resistance to Phytophthora sojae in Soybean

“…A huge variety of enzymes are thought to be involved in their biosynthetic pathways (Somerville and Somerville, 1999 ). Isoflavone reductase (IFR) is identified as a crucial enzyme involved in the synthesis of the glyceollins from daidzein (Graham et al, 1990 ; Oliver et al, 2003 ), and catalyzes a stereo-specific NADPH-dependent reduction to (3R)-isoflavanone (Guo et al, 1994 ; Cooper et al, 2002 ). In addition, IFR is a monomeric, cytosolic reductase, and the enzyme can use 2′-hydroxydaidzein, 2′-hydroxyformononetin, and 2′-hydroxygenistein as substrates in soybean (Wang et al, 2006 ).…”

Phytochemical and antioxidant activity studies on Ononis angustissima L. aerial parts: isolation of two new flavonoids

“…On the basis of accurate molecular weight determination, and taking into account the main fragments observed in the MS/MS spectra, the structure of further fourteen derivatives was proposed (Table S3). Particularly, nine other flavones (compounds a-e, g, h, j and l) and four isoflavones (f, k, m and n) were detected: the latter were distinguished by the loss of 56 amu fragment typically observed only in the MS/MS spectra of isoflavones (Cooper et al 2002). The hydroxylation pattern on A and B rings could be recognised taking into account the different ions 1,3 A + related to C-ring fragmentation (Wu et al 2004).…”

“…The 1 H NMR spectrum of 1(Table S2)showed signals for an oxygenated methine double of doublets at δ 3.85 (1H, dd, J = 11.0, 5.0 Hz, H-3), two methylene double of doublets at δ 4.61 (1H, dd, J = 13.0, 5.0 Hz, H-2a) and 3.49 (1H, dd, J = 12.5, 11.0 Hz, H-2b). The proton signals of ring A were observed at δ 7.75 (1H, d, J = 8.0 Hz, H-5), 6.48 (1H, br d, J = 8.0 Hz, H-6) and 6.31 (1H, br s, H-8), while in the B-ring, a 1,3,4-trisubstitution was evident from the three signals at δ 7.11 (1H, d, J = 8.0 Hz, H-5′), 6.95 (1H, dd, J = 8.0, 1.5 Hz, H-6′) and 6.97 (1H, d, J = 1.5 Hz, H-2′)(Cooper et al 2002).…”