Biosynthesis of White Lupin Isoflavonoids from [U-¹⁴C]l-Phenylalanine and Their Release into the Culture Medium

Abstract

Pulse-labeling experiments of white lupin (Lupinus albus L.) cell cultures with [U-14C1L-phenylalanine for 72 h resulted in the incorporation of the radioactivity into the isoflavone aglucones, glucosides, and prenylated derivatives. Both the aglucones genistein and 2'-hydroxygenistein and their 7-0-glucosides accounted for 85% of the total isoflavonoids identified in the cultured cells and contained 35% of the radioactivity, whereas the prenylated derivatives comprised 15 and 65%, respectively. Almost 20% of the labeled isoflavones of the cellular pool was recovered from the culture medium, 90% of which were monoprenylated and diprenylated derivatives containing 80% of the radioactivity. These results clearly demonstrate the release into the culture medium of a substantial amount of the endogenously synthesized isoflavonoids, especially the prenylated derivatives.With few exceptions, most plant cell cultures fail to produce the secondary metabolites/natural products characteristic of the intact plants from which they were derived (13,17). Furthermore, of the limited variety of natural products that have been produced by cultured cells, flavonoid compounds are the least represented (4, 9). Among the few examples that have been reported in the literature are isoflavonoids formed in response to biotic or abiotic elicitors (1,3,11) or those produced by cell suspension and hairy root cultures transformed with Agrobacterium tumefaciens and A. rhizogenes, respectively (2).Cell cultures of white lupin (Lupinus albus L.) accumulate a complex pattern of isoflavones ( Fig. 1) [3-5, 3a-5a], and 6,3'-diprenyl [6, 6a] The isoflavonoid pattern is quite similar to that of the intact lupin roots (8,10,18,19). However, it was observed that lupin cell cultures release a portion of their metabolites into the culture medium, the latter eventually turning reddishbrown in color. Microscopic examination revealed that this phenomenon was not due to lysis of the cultured cells because the latter remained viable until the end of the culture period. It was considered important, therefore, to investigate the biosynthesis of lupin isoflavones, using "4C-labeled L-phenylalanine as a precursor, and their release into the culture medium. MATERIALS AND METHODS Cell CultureA cell-suspension culture derived from the radicles of white lupin (Lupinus albus L. cv Kievskij Mutant) was maintained on B5 nutrient medium (6) containing 2% sucrose and supplemented with 2,4-D (1 ppm) and kinetin (0.1 ppm). Preparation of Cell and Medium ExtractsAt the end of the metabolic period, the cultured cells were separated from the media by centrifugation at 2000g and washed twice with distilled water. The cells were extracted twice by homogenization with 80% aqueous MeOH4 (5:1, w/ v) at room temperature. The combined extracts were evaporated to an aqueous residue in a rotary evaporator at 300C, and the latter extracted twice with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate before evaporation, and the residue dissolved in 8...

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“…Besides, the concentration of Cu found in root apoplasm of root epidermis and root cortex was nearly 2-fold higher than the concentration of Cu localised in symplasm. Previous works showed that genistein is located in the cell wall of white lupin (Ferrer et al, 1990;Gagnon et al, 1992a) and that β-glucosidase and isoflavone glucosides are coexistent in lupin cell walls (Pislewska et al, 2002). Hence, our results suggest that a substantial fraction of Cu had been immobilized onto genistein and its glucosides in the cell wall.…”

Cell wall‐associated isoflavonoids andβ‐glucosidase activity inLupinus albusplants responding to environmental stimuli

“…1991). Preliminary data on the biosynthesis of lupin isoflavonoids have been revealed (Gagnon et al . 1992; Laflamme et al .…”

Characterization of an Isoflavonoid-Specific Prenyltransferase from Lupinus albus

“…LaPT1 was also unable to produce wighteone or lupiwighteone, although these are naturally occurring isoflavonoid compounds in white lupin (Schröder et al, 1979;Ingham et al, 1983;Gagnon et al, 1992;Shibuya et al, 1992;Laflamme et al, 1993). It has been reported that a crude microsomal membrane pellet from lupin radicle extracts can catalyze the prenylation of genistein at the 39-, 6-, and 8-positions, but the microsomal fraction from suspension cultures only catalyzes prenylation at the 39-and 6-positions (Laflamme et al, 1993), and an enzyme extract from hypocotyls of white lupin produces only 6-prenylated genistein (Schröder et al, 1979).…”

“…In contrast to the typical microbially induced synthesis of isoflavonoid defense compounds in many plant species, Lupinus species constitutively produce various monoprenylated and diprenylated isoflavonoids, with the major components being genistein derivatives such as wighteone (6-prenylgenistein), isowighteone (39-prenylgenistein), and lupiwighteone (8-prenylgenistein; Fig. 1), in addition to minor amounts of cyclized pyrano derivatives (Harborne et al, 1976;Schröder et al, 1979;Tahara et al, 1984Tahara et al, , 1989Gagnon et al, 1992;Katagiri et al, 2000;Bednarek et al, 2001). Lupinus species, therefore, are good model plants for studying the biosynthesis of prenylated aromatic compounds.…”

“…In addition to S. flavescens and soybean, prenylated isoflavonoids are commonly found in several legume species, for example white lupin (Lupinus albus) and other Lupinus species (Harborne et al, 1976;Schröder et al, 1979;Tahara et al, 1984Tahara et al, , 1989Gagnon et al, 1992;Katagiri et al, 2000;Bednarek et al, 2001). In contrast to the typical microbially induced synthesis of isoflavonoid defense compounds in many plant species, Lupinus species constitutively produce various monoprenylated and diprenylated isoflavonoids, with the major components being genistein derivatives such as wighteone (6-prenylgenistein), isowighteone (39-prenylgenistein), and lupiwighteone (8-prenylgenistein; Fig.…”