Metabolomics, with the aim at qualitative and quantitative analysis of the full complement of metabolites in biological samples, has truly established itself as a valuable tool for plant functional genomics and studies of plant biochemical composition. However, its potential to find new natural products remains untapped due to the diversity and complexity of natural products. Optimized metabolomics strategies for the discovery of new natural products are needed. Therefore, we proposed a series of simple operations for new natural products discovery based on metabolomic studies. The main steps include (1) LC-ESI-MS profiling analysis, (2) peak detection, supervised retention time alignment and peak matching for multiple groups of samples, (3) selection of the metabolites that are unknown induced natural products, (4) purification of target metabolites by preparative HPLC and finally, (5) structure elucidation based on NMR. This strategy was applied to the metabolite profiling and systematic identification of defense-response-induced secondary metabolites in soybean cotyledons. We were able to simultaneously detect and identify 13 isoflavones, 2 coumestans and 6 pterocarpans. Totally, 5 compounds were discovered as natural products for the first time in soybean. Comparative metabolite profiling of soybean cultivars resistant and susceptible to Phytophthora sojae (Kauf. and Gerde.) was performed. Principal component analysis clearly demonstrated a separation of elicitor-activated resistant and susceptible soybean cultivars. 3’-prenylisoafrormosin, glyceocarpin, glyceofuran, 3'-prenylgenistein and phaseol were identified as the key secondary metabolites accounting for the separation. The metabolite profiling results present the most complete analysis of soybean induced secondary metabolites to date, which can be further utilized to evaluate chemical components of soybean samples for plant biology, food science and pharmaceutical studies. Our results also provide additional knowledge of the soybean secondary metabolite pathways involved in defense. Moreover, the proposed strategy demonstrates a promising future approach for novel compound discovery even in relatively well studied plants.
In soybean, phenylpropanoids play critical roles in defense responses. They regulate certain aspects of oxidative stress and hypersensitive cell death and act as phytoalexins, which directly inhibit pathogen growth. Interestingly, some of phenylpropanoids from soybean also have many reported activities in animal cells. In particular, genistein is one of the most potent phytoestrogens and glyceollin has been reported a good lead for anticancer activity. Due to these important activities, an in depth study of how phenylpropanoid pathways are regulated was conducted. Over 25 compounds were examined, including exogenous elicitors, signal molecules and signal transduction regulators for their effects on wound-, light- and glucan defense elicitor-induced phenylpropanoid responses in soybean. As for exogenous elicitors, many of the Pathogen Associated Molecular Patterns (PAMPs) tested (chitin oligomers, LPS, wall glucan elicitor, mycolaminaran) induce the production of glyceollin as expected. Interestingly, all chemicals that are H+ and/or K+ ion effectors (vanadate, monensin, valinomycin, nigericin and fusicoccin) cause a massive accumulation of inducible secondary metabolites. Additionally, 2-methoxy-3,9-dihydroxycoumestone, 1-methoxy-3,9-dihydroxy coumestone, 8-methoxy-3,9-dihydroxycoumestone, and 7,4’-dihydroxy-5’-methoxycoumaronochromone were discovered as natural products for the first time in soybean in this study. Overall, the results of this study suggest that phenylpropanoid metabolism may be controlled by manipulating the transmembrane potential.