Comprehensive RNA sequencing and co-expression network analysis to complete the biosynthetic pathway of coumestrol, a phytoestrogen.

Coumestrol (CMS), a coumestan isoflavone, plays key roles in nodulation through communication with rhizobia, and has been used as phytoestrogens for hormone replacement therapy in humans. Because CMS content is controlled by multiple genetic factors, the genetic basis of CMS biosynthesis has remained unclear. We identified soybean genotypes with consistently high (Daewonkong) or low (SS0903-2B-21-1-2) CMS content over 2 years. We performed RNA sequencing of leaf samples from both genotypes at developmental stage R7, when CMS levels are highest. Within the phenylpropanoid biosynthetic pathway, 41 genes were tightly connected in a functional co-expression gene network; seven of these genes were differentially expressed between two genotypes. We identified 14 candidate genes involved in CMS biosynthesis. Among them, seven were annotated as encoding oxidoreductases that may catalyze the transfer of electrons from daidzein, a precursor of CMS. Two of the other genes, annotated as encoding a MYB domain protein and a MLP-like protein, may increase CMS accumulation in response to stress conditions. Our results will help to complete our understanding of the CMS biosynthetic pathway, and should facilitate development of soybean cultivars with high CMS content that could be used to promote the fitness of plants and human beings.

The Stimulatory Effect of Strontium Ions on Phytoestrogens Content in Glycine max (L.) Merr.

The amount of secondary metabolites in plants can be enhanced or reduced by various external factors. In this study, the effect of strontium ions on the production of phytoestrogens in soybeans was investigated. The plants were treated with Hoagland's solution, modified with Sr(2+) with concentrations ranging from 0.5 to 3.0 mM, and were grown for 14 days in hydroponic cultivation. After harvest, soybean plants were separated into roots and shoots, dried, and pulverized. The plant material was extracted with methanol and hydrolyzed. Phytoestrogens were quantified by HPLC. The significant increase in the concentration of the compounds of interest was observed for all tested concentrations of strontium ions when compared to control. Sr(2+) at a concentration of 2 mM was the strongest elicitor, and the amount of phytoestrogens in plant increased ca. 2.70, 1.92, 3.77 and 2.88-fold, for daidzein, coumestrol, genistein and formononetin, respectively. Moreover, no cytotoxic effects were observed in HepG2 liver cell models after treatment with extracts from 2 mM Sr(2+)-stressed soybean plants when compared to extracts from non-stressed plants. Our results indicate that the addition of strontium ions to the culture media may be used to functionalize soybean plants with enhanced phytoestrogen content.

Induction of the soybean phytoalexins coumestrol and glyceollin by Aspergillus.

Several isoflavonoid phytoalexins produced by soybeans are known to be estrogenic, with potential beneficial health effects in humans. Increased production of phytoalexins by the soybean plant will facilitate research efforts in this area. In this study, phytoalexin induction and accumulation in soybean cotyledon tissue was observed using four species of Aspergillus: A. sojae, A. oryzae, A. niger, and A. flavus. All four Aspergillus species tested elicited phytoalexin accumulation in living soybean cotyledons. Results from a time course study indicated that maximum concentrations of the phytoalexin glyceollin, 955 microg/g fresh weight (fw), occurred at day 3 in soybean cotyledon tissue inoculated with A. sojae. Other Aspergillus species caused an accumulation of glyceollin at significantly lower levels. A maximum concentration of coumestrol of 27.2 microg/g fw was obtained from soybean cotyledons inoculated with A. niger. Soybean phytoalexins induced by food-grade A. sojae and A. oryzae allowed the collection of higher concentrations of phytoalexins for further examination in several in vitro and in vivo biological studies conducted to determine potential estrogenic activities.