Isoflavones have drawn much attention because of their benefits to human health. These compounds, which are produced almost exclusively in legumes, have natural roles in plant defense and root nodulation. Isoflavone synthase catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway. To identify the gene encoding this enzyme, we used a yeast expression assay to screen soybean ESTs encoding cytochrome P450 proteins. We identified two soybean genes encoding isoflavone synthase, and used them to isolate homologous genes from other leguminous species including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine, as well as from the nonleguminous sugarbeet. We expressed soybean isoflavone synthase in Arabidopsis thaliana, which led to production of the isoflavone genistein in this nonlegume plant. Identification of the isoflavone synthase gene should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes.

Metabolic engineering for production of isoflavones in non-legume plants may provide the health benefits of these phytoestrogens from consumption of more widely used grains. In legumes, isoflavones function in both the symbiotic relationship with rhizobial bacteria and the plant defense response. Expression of a soybean isoflavone synthase (IFS) gene in Arabidopsis plants was previously shown to result in the synthesis and accumulation of the isoflavone genistein in leaf and stem tissue (Jung et al., 2000). Here we further investigate the ability of the heterologous IFS enzyme to interact with the endogenous phenylpropanoid pathway, which provides the substrate for IFS, and produces genistein in several plant tissue systems. In tobacco (Nicotiana tabacum) floral tissue that synthesizes anthocyanins, genistein production was increased relative to leaves. Induction of the flavonoid/anthocyanin branch of the phenylpropanoid pathway through UV-B treatment also enhanced genistein production in Arabidopsis. In a monocot cell system, introduced expression of a transcription factor regulating genes of the anthocyanin pathway was effective in conferring the ability to produce genistein in the presence of the IFS gene. Introduction of a third gene, chalcone reductase, provided the ability to synthesize an additional substrate of IFS resulting in production of the isoflavone daidzein in this system. The genistein produced in tobacco, Arabidopsis, and maize (Zea mays) cells was present in conjugated forms, indicating that endogenous enzymes were capable of recognizing genistein as a substrate. This study provides insight into requirements for metabolic engineering for isoflavone production in non-legume dicot and monocot tissues.

A soybean [Glycine max (L.) Merr. I mutation conferring resistance to a wide range of sulfonylurea (SU) herbicides would greatly enhance the weed control options available to soybean farmers. This report describes the selection, characterization, and potential utility of such mutants. Seed mutagenesis (using N‐nitroso‐N‐methylurea and ethyl methanesulfonate) followed by selection for resistance to chlorsulfuron [2‐chloro‐N‐[(4‐methoxy‐6‐methyl‐l,3,5‐triazin‐2‐yl)aminocarbonyl] benzenesulfonamide] yielded a soybean mutant with a high degree of resistance to both postemergence and pre‐emergence applications of a variety of SU herbicides. Resistance was monogenic, semidominant, and not allelic to any of the previously identified recessive genes hs1, hs2, or hs3 that confer tolerance to SU herbicides. Biochemical tests indicate that the mechanism of resistance is reduced sensitivity of acetolactate synthase to SU inhibition. The SU resistance afforded by this mutation (designated Alsl) can be used to enhance soybean weed control options and can serve as a selectable marker for seed purification.