Production of the isoflavones genistein and daidzein in non-legume dicot and monocot tissues.

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.

Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes.

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.

Seed growth rate and carbohydrate pool sizes of the soybean fruit.

The relationships between various carbohydrate pools of the soybean (Glycine max [L.] Merrill) fruit and growth rate of seeds were evaluated. Plants during midpod-fill were subjected to various CO(2) concentrations or light intensities for 7 days to generate different rates of seed growth. Dry matter accumulation rates of seeds and pod wall, along with glucose, sucrose, and starch concentrations in the pod wall, seed coat, and embryo were measured in three-seeded fruits located from nodes six through ten. Seed growth rates ranged from 4 to 37 milligrams.day(-1).fruit(-1). When seed growth rates were greater than 12 milligrams.day(-1).fruit(-1), sucrose concentration remained relatively constant in the pod wall (1.5 milligrams.100 milligrams dry weight(-1)), seed coat (8.5 milligrams.100 milligrams dry weight(-1)), and embryo (5.0 milligrams.100 milligrams dry weight(-1)). However, sucrose concentrations decreased in all three parts of the fruit as growth rate of the seeds fell below 12 milligrams.day(-1).fruit(-1). This relationship suggests that at high seed growth rates, flux of sucrose through the sucrose pools of the fruit was more important than pool size for growth. Starch concentration in the pod wall remained relatively constant (2 milligrams.100 milligrams dry weight(-1)) at higher rates of seed growth but decreased as seed growth rates fell below 12 milligrams.day(-1).fruit(-1). This suggests that pod wall starch may buffer seed growth under conditions of limiting assimilate availability. There was no indication that carbohydrate pools of the fruit were a limitation to transport or growth processes of the soybean fruit.

Relationships between Respiration Rate and Adenylate and Carbohydrate Pools of the Soybean Fruit.

Relationships between respiration rate and adenylate and carbohydrate pools of the soybean (Glycine max L. Merrill) fruit during rapid seed growth were evaluated. Plants at mid pod-fill were subjected to different concentrations of CO(2) to alter the amount of photosynthate produced and, thus, available to the fruit. Respiration rate of the intact fruits was measured, along with glucose, sucrose, and starch concentrations, adenylate energy charge (AEC), and total adenylate pool (SigmaAdN) in the pod wall, seed coat, and cotyledons. The concentration of sucrose remained relatively constant in the pod wall (1.0 milligram per 100 milligrams dry weight), seed coat (6.5 milligrams per 100 milligrams dry weight), and cotyledons (4.5 milligrams per 100 milligrams dry weight) at moderate and high respiration rates. Furthermore, AEC remained relatively constant in the pod wall (0.55), seed coat (0.24), and cotyledons (0.44) during changes in respiration rate. This suggests that the amount of assimilate transported to the fruit, and its flux through the sucrose pools of the fruit parts, were important in the regulation of the respiration rate of the fruit. The average SigmaAdN in the seed coat (1300 picomoles per milligram dry weight) was significantly greater than in the cotyledons (750 picomoles per milligram dry weight) and pod wall (300 picomoles per milligram dry weight). In addition, the SigmaAdN in the seed coat and cotyledons increased with increasing respiration rate of the fruit. The high SigmaAdN in the seed coat and its increase with increases in respiration rate of the fruit suggest that an energy-requiring process is involved in the movement of sucrose through the seed coat.

Relationships between Carbon Assimilation, Partitioning, and Export in Leaves of Two Soybean Cultivars.

To evaluate leaf carbon balance during rapid pod-fill in soybean (Glycine max [L.] Merrill), measurements were made of CO(2) assimilation at mid-day and changes in specific leaf weight, starch, and sucrose concentrations over a 9-hour interval. Assimilate export was estimated from CO(2) assimilation and leaf dry matter accumulation. Chamber-grown ;Amsoy 71' and ;Wells' plants were subjected on the day of the measurements to one of six photosynthetic photon flux densities in order to vary CO(2) assimilation rates.Rate of accumulation of leaf dry matter and rate of export both increased as CO(2) assimilation rate increased in each cultivar.Starch concentrations were greater in Amsoy 71 than in Wells at all CO(2) assimilation rates. At low CO(2) assimilation rates, export rates in Amsoy 71 were maintained in excess of 1.0 milligram CH(2)O per square decimeter leaf area per hour at the expense of leaf reserves. In Wells, however, export rate continued to decline with decreasing CO(2) assimilation rate. The low leaf starch concentration in Wells at low CO(2) assimilation rates may have limited export by limiting carbon from starch remobilization.Both cultivars exhibited positive correlations between CO(2) assimilation rate and sucrose concentration, and between sucrose concentration and export rate. Carbon fixation and carbon partitioning both influenced export rate via effects on sucrose concentration.