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The worldwide interest in sustainable agriculture has contributed to the development of alternative methods for the control of plant pests and diseases. This study aimed to assess the efficiency of Lachancea thermotolerans CCMA 0763 and its metabolites in controlling Meloidogyne incognita in soybean and their effects on plant defense enzymes and glyceollin synthesis. The following treatments were applied as foliar spray 4 days before nematode inoculation: fermentation broth, broth filtrate, yeast cells, sugarcane juice, acibenzolar-S-methyl (ASM). An inoculated untreated control and an absolute control were also included. The same treatments were used in the glyceollin assay. For analysis of resistance induction, the treatments were fermentation broth, broth filtrate, yeast cells, ASM, and water (control). Fermentation broth and yeast cells reduced total nematode number, population density and reproduction factor. Yeast-based treatments and ASM enhanced glyceollin synthesis compared with sugarcane juice and water. Peroxidase activity was highest at 4 and 10 days after treatment in plants treated with yeast-based treatmentss. Broth filtrate and yeast cells increased polyphenol oxidase activity at 4 days after treatment. Phenylalanine ammonia-lyase and glucanase activities were not influenced by treatment. L. thermotolerans stimulated phytoalexin synthesis in soybean cotyledons and defense enzyme activity in soybean roots, showing potential as a biopesticide.

Glyceollin isomers I, II, and III are the major pathogen-elicited secondary metabolites (i.e. phytoalexins) of soybean (Glycine max) that, collectively with other 5-deoxyisoflavonoids, provide race-specific resistance to Phytophthora sojae. The NAC-family transcription factor (TF) GmNAC42-1 is an essential regulator of some but not all glyceollin biosynthesis genes, indicating other essential TF(s) of the glyceollin gene regulatory network remain to be identified. Here, we conducted comparative transcriptomics on soybean hairy roots of the variety Williams 82 and imbibing seeds of Harosoy 63 upon treatment with wall glucan elicitor from P. sojae and identified two homologous R2R3-type MYB TF genes, GmMYB29A1 and GmMYB29A2, up-regulated during the times of peak glyceollin biosynthesis. Overexpression and RNA interference silencing of GmMYB29A2 increased and decreased expression of GmNAC42-1, GmMYB29A1, and glyceollin biosynthesis genes and metabolites, respectively, in response to wall glucan elicitor. By contrast, overexpressing or silencing GmMYB29A1 decreased glyceollin I accumulation with marginal or no effects on the expressions of glyceollin synthesis genes, suggesting a preferential role in promoting glyceollin turnover and/or competing biosynthetic pathways. GmMYB29A2 interacted with the promoters of two glyceollin I biosynthesis genes in vitro and in vivo. Silencing GmMYB29A2 in Williams 82, a soybean variety that encodes the resistance gene Rps1k, rendered it compatible with race 1 P. sojae, whereas overexpressing GmMYB29A2 rendered the susceptible Williams variety incompatible. Compatibility and incompatibility coincided with reduced and enhanced accumulations of glyceollin I but not other 5-deoxyisoflavonoids. Thus, GmMYB29A2 is essential for accumulation of glyceollin I and expression of Phytophthora resistance.

Aromatase inhibitors (AIs) are currently the standard treatment for postmenopausal women with estrogen-dependent metastatic breast cancer. While effective, patients develop resistance leading to tumor relapse, metastasis, and more aggressive phenotypes. Previous research suggested that AI resistance is due to upregulation of growth factor pathways (i.e., HER2 and EGFR) and increased cellular motility. Our lab previously demonstrated that a novel phytoalexin, glyceollins, inhibits proliferation, survival, and migration of hormone independent letrozole-resistant breast cancer cells (LTLT-Ca). However, many postmenopausal women with AI-resistance tumors remain hormone dependent. Therefore, there is a need to understand distinctions between estrogen receptor (ER+) positive and ER negative (ER-) AI resistant tumors and their response to therapy. We hypothesize that treating ER+ letrozole-resistant breast cancer using a combination approach of glyceollin and lapatinib (a dual EGFR/HER2 inhibitor) will reduce growth factor signaling, inhibit proliferation and motility. To test this hypothesis, T47Darom cells (T47D cells which stably overexpress aromatase) and T47DaromLR cells (T47Darom cells that are letrozole resistant) were treated with lapatinib, glyceollin, or the combination. To evaluate viability, resazurin assays were performed and results demonstrated that glyceollin and/or lapatinib had no effect on proliferation in the T47Darom cells. However, glyceollin alone and glyceollin + lapatinib inhibited proliferation of T47DaromLR cells by 46% and 59%, respectively. Cell survival was assessed by colony formation assays and glyceollin caused a 33% reduction in cell survival and glyceollin + lapatinib caused a 60% reduction in the colony formation of T47Darom cells. Interestingly, in T47DaromLR cells glyceollin and the combination significantly inhibited cell survival by 94% alone and 96%. To further evaluate changes that occur as AI sensitive cells transition to AI resistance, a quantitative proteomic analysis of the whole cell lysates of T47DaromLR (AI-resistant) versus T47Darom cells (AI-sensitive) was performed to identify significant protein expression changes. Results demonstrated a 3.195-fold-increase (p Citation Format: Rashidra R Walker, Jankiben Patel, A. Michael Davidson, Karen Gallegos, Syreeta L Tilghman. A novel phytoalexin, glyceollins, trigger anti-proliferative effects in aromatase inhibitor resistant breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-15.

Apart from the physiological functions of soybean phytoalexins, the production sites in soybeans remain unknown. In this study, the dynamic production of phytoalexins, glyceollins, in germinating soybeans inoculated with Aspergillus oryzae was visually investigated using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging. During a 3-day sensitization using a fungus, glyceollins I-III were produced in germinating soybeans (from 0.03 mg/g for glyceollin III to 0.96 mg/g for glyceollin I). Imaging analysis provided visual evidence that glyceollins were produced only in the regions of seed coat and germinated root of the soybeans, while no production was observed in other regions, including the cotyledons. In contrast, their precursor, isoflavone, was distributed throughout the soybean. The evidence that the inoculation of the inactivated fungi also caused glyceollin production at the seed coat led us to speculate that glyceollins could be produced in the region of soybean attached to the fungus body.

Root lesion nematodes, Pratylenchus penetrans, are major pests of legumes with little options for their control. We aimed to prime soybean cv. Primus seedlings to improve basic defense against these nematodes by root application of N-3-oxo-tetradecanoyl-L-homoserine lactone (oxo-C14-HSL). The invasion of soybean roots by P. penetrans was significantly reduced in plants that were pre-treated with the oxo-C14-HSL producing rhizobacterium Ensifer meliloti strain ExpR+, compared to non-inoculated plants or plants inoculated with the nearly isogenic strain E. meliloti AttM with plasmid-mediated oxo-C14-HSL degradation. The nematodes were more clustered in the root tissues of plants treated with the AttM strain or the control compared to roots treated with the ExpR+ strain. In split-root systems primed on one side with strain ExpR+, root invasion was reduced on the opposite side compared to non-primed plants indicating a systemic plant response to oxo-C14-HSL. No additional local effect was detected, when inoculating nematodes on the ExpR+ primed side. Removal of oxo-C14-HSL after root exposure resulted in reduced root invasion compared to non-primed plants when the nematodes were added 3, 7, or 15 days later. Thus, probably the plant memorized the priming stimulus. Similarly, the plants were primed by compounds released from the surface of the nematodes. HPLC analysis of the root extracts of oxo-C14-HSL treated and untreated plants revealed that priming resulted in enhanced phytoalexin synthesis upon P. penetrans challenge. Without root invading nematodes, the phytoalexin concentrations of primed and non-primed plants did not significantly differ, indicating that priming did not lead to a persistently increased stress level of the plants. Upon nematode invasion, the phytoalexins coumestrol, genistein, and glyceollin increased in concentration in the roots compared to control plants without nematodes. Glyceollin synthesis was significantly more triggered by nematodes in primed plants compared to non-primed plants. The results indicated that the priming of soybean plants led to a more rapid and strong defense induction upon root invasion of nematodes.

GmMYB176 is an R1 MYB transcription factor that regulates multiple genes in the isoflavonoid biosynthetic pathway, thereby affecting their levels in soybean roots. While GmMYB176 is important for isoflavonoid synthesis, it is not sufficient for the function and requires additional cofactor(s). The aim of this study was to identify the GmMYB176 interactome for the regulation of isoflavonoid biosynthesis in soybean. Here, we demonstrate that a bZIP transcription factor GmbZIP5 co-immunoprecipitates with GmMYB176 and shows protein–protein interaction in planta. RNAi silencing of GmbZIP5 reduced the isoflavonoid level in soybean hairy roots. Furthermore, co-overexpression of GmMYB176 and GmbZIP5 enhanced the level of multiple isoflavonoid phytoallexins including glyceollin, isowighteone and a unique O-methylhydroxy isoflavone in soybean hairy roots. These findings could be utilized to develop biotechnological strategies to manipulate the metabolite levels either to enhance plant defense mechanisms or for human health benefits in soybean or other economically important crops., Anguraj Vadivel et al. show that a bZIP transcription factor GmbZIP5 interacts with an R1 MYB transcription factor GmMYB176 to regulate the biosynthesis of isoflavonoid in soybean. This study suggests biotechnological strategies to manipulate the levels of metabolites for plant defense or human health benefits in economically important crops.

Glyceollins are a class of antimicrobial prenylated pterocarpans produced in soybean seedlings upon fungus elicitation. Priming with reactive oxygen species (ROS) prior to elicitation with Rhizopus oligosporus/oryzae (R) was investigated for its potential to enhance glyceollin production. ROS-priming prior to R-elicitation (ROS + R) increased glyceollin production (8.6 ± 0.9 µmol/g dry weight (DW)) more than 4-fold compared to elicitation without priming (1.9 ± 0.4 µmol/g DW). Furthermore, ROS-priming was superior to two physical primers which were used as benchmark primers, namely slicing (5.0 ± 0.6 µmol glyceollins/g DW) and sonication (4.8 ± 1.0 µmol glyceollins/g DW). Subsequently, the robustness of ROS + R was assessed by applying it to another soybean cultivar, where it also resulted in a significantly higher glyceollin content than R-elicitation without priming. ROS-priming prior to elicitation provides opportunities for improving the yield in large-scale production of natural antimicrobials due to the ease of application and the robustness of the effect across cultivars.

Terpenes produced by plants comprise a diverse range of secondary metabolites, including volatile organic compounds (VOCs). Terpene VOC production may be altered after damage or by biological stimuli such as bacterial, fungal and insects, and subsequent triggering of plant defense responses. These VOCs originate in plants from two independent pathways: the mevalonate and the methylerythritol phosphate pathways, which utilize dimethylallyl and isopentenyl diphosphates to form the terpenoidal precursors. Phakopsora pachyrhizi fungi causes Asian soybean rust, limiting soybean production and resulting in losses of up to 80% if no control strategies are applied. By using a transcriptome datasets, we investigated the regulation of genes of the mevalonate pathway under different biotic stresses. We studied the impact of P. pachyrhizi infection in vivo expression profile of genes involved in terpenoid and glyceollin biosynthesis in genotypes harboring different resistance genes (Rpp), and across the infection cycle. In addition, we used UPLC and UPGC analysis to evaluate glyceollin and VOC production, respectively, to identify metabolites associated with soybean responses to pathogen infection. The regulation of soybean genes involved in terpene production was influenced by genotypes, depending on the Rpp gene, while glyceollin was induced in all genotypes. Furthermore, a sesquiterpene was identified as a potential marker associated with rust symptoms on soybean.

Secondary metabolites may play a number of roles, among them, is the activation of plant defense mechanisms, such as phytoalexins and the synthesis of antioxidative enzymes. This study aimed to test the eliciting activity of phytoalexins of Lippia sidoides Cham. and six endophytic fungi associated with it and to verify the synthesis of the enzymes, as a response to the antioxidative and antifungal system. The assays were performed using spectrophotometric methods. Of the concentrations of essential oil used, 7.5 mg mL-1 was 66.48% higher than the Acorda® positive control for the induction of glyceollin. The endophytic fungus, Verticillium sp. showed the best result for 3-deoxythianocyanidine production. Excellent results were demonstrated by the LS-14 fungus for superoxide dismutase enzyme activity (130 U g-1 E-1 min-1); by L. sidoides for the activity of ascorbate peroxidase enzyme (3821 μmol ASA g-1 E-1 min-1); by L. sidoides for the activity of catalase enzyme (2429 μmol H2O2 g-1 E-1 min-1); by Fusarium sp. 2 for the activity of phenol peroxidase (87 μmol H2O2 g-1 E-1 min-1) and by Fusarium sp. 1 for the activity of chitinase (0.214 U min-1). Both the essential oil of L. sidoides and the extracts of the endophytic fungi had a positive response to the induction of phytoalexin and presence of enzymes related to antioxidative activity were verified and shown. Key words: Catalase, superoxide dismutase, peroxidase phenols, ascorbate peroxidase, chitinase.

Prenylated pterocarpans are valuable natural products that play significant roles in plant defence and possess diverse biological activities. However, structural diversity of prenylated pterocarpans is still limited. Prenyltransferases (PTs) could catalyze the transfer of prenyl moieties to acceptor molecules and increase the structural diversity and biological activity of natural products. Up to date, only two pterocarpan PTs have been identified from plants. In this study, a new pterocarpan prenyltransferase gene, designated as PcM4DT, was identified from Psoralea corylifolia. The deduced polypeptide is predicted to be a membrane-bound protein with eight transmembrane regions. Functional characterization of recombinant PcM4DT demonstrated this enzyme could catalyze C-4 prenylation of pterocarpans, and exhibited strict substrate specificity to maackiain and 3-hydroxy-9-methoxy-pterocarpan. It also showed a strict donor specificity to DMAPP. Furthermore, removal of the putative transit peptide of PcM4DT obviously increased the catalytic activity (up to 90%). PcM4DT represents the first PT identified from the Psoralea genus.

Summary Glyceollins, stress-induced phytoalexins from parent soya bean isoflavones, were elicited with Aspergillus oryzae. This solid-state fermentation facilitated the conversion of isoflavone glycosides into aglycones and glyceollins, which could mainly enrich in soya bean protein isolate (SPI) during protein preparation due to protein–polyphenol interactions. Glyceollin-enriched SPI exhibited less flavour volatiles, higher solubility, lower whiteness and higher antioxidant activity than unfermented SPI. Fermented SPI was more easily to be digested during in vitro pepsin–trypsin digestion. This may be attributed to partial degradation of protein, especially α′ and α subunits of β-conglycinin and acidic subunit of glycinin. The antioxidant activity of digestive products derived from fermented SPI was obviously enhanced with increasing digestion time due to simultaneous release of antioxidant peptides and glyceollins involved in the interior of protein molecule. These results suggest an effective technique for producing a nutrient-enhanced SPI as novel functional ingredients applied in food industry.

The rampant use of antibiotics in the swine industry has caused an increase in antibiotic-resistant microorganisms. Thus, investigating novel natural antimicrobial compounds that can be supplemented into swine feed is of great importance. Glyceollins are members of a class of inducible phytochemicals that are naturally produced by soybeans and have antimicrobial and antifungal properties. Previous research has concluded that 3 mg glyceollins per g soybean is the target production that would enable glyceollins to be used as an antimicrobial in swine feed. In this study, the optimal soybean germination conditions for glyceollin production were elucidated and utilized. Also, 8 fungal strains were evaluated for their ability to stimulate glyceollin production in soybeans. The fungal strains were inoculated on soybeans that were either manually de-hulled and halved or still encased by the hull. It was determined that Trichoderma reesei NRRL 3653 inoculated on de-hulled soybeans yielded significantly higher glyceollin production (3.763 mg/g) than all other treatments investigated in this study, which was a ∼3-fold increase in production compared to a previously published study. This study strongly suggests that T. reesei can stimulate glyceollin production from soybeans at a high enough concentration to become a novel natural antimicrobial in swine feed.

Cytochrome P450, CYP93A1, is involved in the synthesis of the phytoalexin glyceollin in soybean (Glycine max L. Merr). The gene encoding CYP93A1 has been used as defense marker in soybean cell cultures, however, little is known regarding how this gene is expressed in the intact plant. To further understand the tissue-specific role of CYP93A1 in soybean defense, we analyzed the expression of this gene in mechanically damaged leaves and stems. In leaves, CYP93A1 was constitutively expressed; its expression did not change in response to mechanical damage. In stems, however, expression of CYP93A1 was induced as quickly as 4 h after mechanical damage and remained upregulated for at least 48 h. The induction of CYP93A1 was associated with the synthesis of glyceollins. In comparison to several other defense-related genes encoding cysteine protease inhibitors L1 and R1 and storage proteins vspA and vspB, CYP93A1 was the most strongly induced by stem wounding. The induction of CYP93A1 was observed only locally, not systemically. Similar stem expression patterns were consistently observed among three different soybean genotypes. The strong induction of CYP93A1 in mechanically damaged stems suggests an important role in the soybean stem defense response; therefore, this study expands the use of CYP93A1 as a defense response marker to stems, not just soybean cell cultures.

Glyceollins are phytoalexins produced in soybeans under stressful growth conditions. On the basis of prior evaluations, they show potential to treat multiple diseases, including certain cancers, Type 2 diabetes, and cardiovascular conditions. The aim of the present study was to expand on recent studies designed to initially characterize the intestinal disposition of glyceollins. Specifically, studies were undertaken in Caco-2 cells to evaluate glyceollins' effects on apical efflux transporters, namely, MRP2 and BCRP, which are the locus of several intestinal drug-drug and drug-food interactions. 5- (and 6)-carboxy-2',7'-dichloroflourescein (CDF) was used to provide a readout on MRP2 activity, whereas BODIPY-prazosin provided an indication of BCRP alteration. Glyceollins were shown to reverse MRP2-mediated CDF transport asymmetry in a concentration-dependent manner, with activity similar to the MRP2 inhibitor, MK-571. Likewise, they demonstrated concentration-dependent inhibition of BCRP-mediated efflux of BODIPY-prazosin with a potency similar to that of Ko143. Glyceollin did not appreciably alter MRP2 or BCRP expression following 24 h of continuous exposure. The possibility that glyceollin mediated inhibition of genistein metabolite efflux by either transporter was evaluated. However, results demonstrated an interaction at the level of glyceollin inhibition of genistein metabolism rather than inhibition of metabolite transport.

Aromatase inhibitors (AIs) are the standard endocrine treatment for postmenopausal women with estrogen-dependent metastatic breast cancer. While effective, some patients develop resistance leading to tumor relapse, metastasis, and more aggressive phenotypes. Previous research suggested that AI resistance arises as a result of increased growth factor pathways (i.e., HER2 and EGFR) and is associated with increased cellular motility. Our lab previously demonstrated that a novel phytoalexin, glyceollins, inhibits proliferation, survival, and migration of hormone independent letrozole-resistant breast cancer cells. However, many postmenopausal women with AI-resistant tumors remain hormone dependent. Therefore, there is a need to understand distinctions between estrogen receptor positive (ER+) and ER negative (ER-) AI resistant tumors and their response to therapy. We hypothesize that treating ER+ letrozole-resistant breast cancer with a combination of glyceollin and lapatinib (a dual EGFR/HER2 inhibitor) will reduce growth factor signaling, inhibit proliferation, and induce apoptosis. T47Darom cells (T47D cells which stably express the aromatase gene) and T47DaromLR cells (letrozole-resistant T47Darom cells) were characterized by measuring protein expression. As cells transition from letrozole-sensitive to letrozole-resistance ERα and aromatase expression were slightly decreased while HER2 was increased. A receptor tyrosine kinase phospho-antibody array was performed to evaluate changes in protein expression between the two cell lines. While many proteins were altered, two cell cycle regulators (p38 and p53) were significantly downregulated by in the T47DaromLR cells suggesting that T47DaromLR cells have acquired the ability to progress uncontrollably through the cell cycle, thereby causing enhanced proliferation and cell survival. To interrogate the hypothesis, both cell lines were treated with lapatinib, glyceollin, or the combination and cell viability was measured. Results demonstrated that drug treatment had no effect on the proliferation of T47Darom cells, however, glyceollin alone and glyceollin + lapatinib inhibited T47DaromLR cell viability by 46% and 59%, respectively. Glyceollin alone or the combination caused a 33% and 60% reduction in T47Darom colony formation, respectively. Interestingly, glyceollin alone or the combination of glyceollin + lapatinib significantly inhibited T47DaromLR colony formation by greater than 90%. Apoptosis studies were conducted to measure caspase 3/7 activity in both cell lines. Lapatinib alone had no effect on apoptosis, however glyceollin and the combination significantly induced apoptosis in both cell lines; the most dramatic effect was observed in T47Darom cells, suggesting that additional mechanisms may contribute to the anti-cancer effect observed in T47DaromLR cells. Taken together, these results suggest that dual inhibition using glyceollins and lapatinib may have potential as a novel combination therapy approach for postmenopausal patients with hormone-dependent, letrozole-resistant breast cancer. This work was funded by an NIH grant awarded to SL Tilghman (1SC1GM125617). Citation Format: Rashidra R. Walker, Jankiben Patel, A. Michael Davidson, Syreeta L. Tilghman. A novel phytoalexin, glyceollins, trigger anti-proliferative and apoptotic effects in aromatase inhibitor resistant breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4116.

Background Glyceollins are isoflavonoid-derived pathogen-inducible defense metabolites (phytoalexins) from soybean (Glycine max L. Merr) that have important roles in providing defense against pathogens. They also have impressive anticancer and neuroprotective activities in mammals. Despite their potential usefulness as therapeutics, glyceollins are not economical to synthesize and are biosynthesized only transiently and in low amounts in response to specific stresses. Engineering the regulation of glyceollin biosynthesis may be a promising approach to enhance their bioproduction, yet the transcription factors (TFs) that regulate their biosynthesis have remained elusive. To address this, we first aimed to identify novel abiotic stresses that enhance or suppress the elicitation of glyceollins and then used a comparative transcriptomics approach to search for TF gene candidates that may positively regulate glyceollin biosynthesis. Results Acidity stress (pH 3.0 medium) and dehydration exerted prolonged (week-long) inductive or suppressive effects on glyceollin biosynthesis, respectively. RNA-seq found that all known biosynthetic genes were oppositely regulated by acidity stress and dehydration, but known isoflavonoid TFs were not. Systemic acquired resistance (SAR) genes were highly enriched in the geneset. We chose to functionally characterize the NAC (NAM/ATAF1/2/CUC2)-family TF GmNAC42–1 that was annotated as an SAR gene and a homolog of the Arabidopsis thaliana (Arabidopsis) indole alkaloid phytoalexin regulator ANAC042. Overexpressing and silencing GmNAC42–1 in elicited soybean hairy roots dramatically enhanced and suppressed the amounts of glyceollin metabolites and biosynthesis gene mRNAs, respectively. Yet, overexpressing GmNAC42–1 in non-elicited hairy roots failed to stimulate the expressions of all biosynthesis genes. Thus, GmNAC42–1 was necessary but not sufficient to activate all biosynthesis genes on its own, suggesting an important role in the glyceollin gene regulatory network (GRN). The GmNAC42–1 protein directly bound the promoters of biosynthesis genes IFS2 and G4DT in the yeast one-hybrid (Y1H) system. Conclusions Acidity stress is a novel elicitor and dehydration is a suppressor of glyceollin biosynthesis. The TF gene GmNAC42–1 is an essential positive regulator of glyceollin biosynthesis. Overexpressing GmNAC42–1 in hairy roots can be used to increase glyceollin yields > 10-fold upon elicitation. Thus, manipulating the expressions of glyceollin TFs is an effective strategy for enhancing the bioproduction of glyceollins in soybean. Electronic supplementary material The online version of this article (10.1186/s12864-019-5524-5) contains supplementary material, which is available to authorized users.

The effect of herbicide applications to V3 soybeans on the reproductive ability of race 3 soybean cyst nematode (SCN) was measured. The hatching response of SCN eggs to soybean root exudates collected after postemergence herbicide application, the viability of SCN eggs formed on treated plants and the levels of glyceollin in roots of treated soybeans was also measured. Soybeans were treated postemergence with IX and 2X label rates of herbicides plus a recommended adjuvant and the adjuvants alone. None of the treatments tested were found to stimulate SCN reproduction. Acifluorfen, bentazon, lactofen, crop oil concentrate (COC) and nonionic surfactant (NIS) treatments reduced SCN populations by 50 to 60% at four and eight weeks after application when compared with the untreated control. Fluazifop-P, sethoxydim and imazethapyr treatments had SCN reproduction that was similar to the untreated control. Adjuvant treatments were as effective as some herbicide treatments in reducing SCN reproduction. However, no additive effect for adjuvant-herbicide combinations was observed nor did herbicide rate affect SCN reproduction The treatments reduced SCN reproduction only when applied to soybean plants and exhibited no effect when applied to the soil. All root exudate solutions (RES) increased SCN hatching more than deionized water, but less than ZnS04 solutions. However, no statistical differences due to herbicide treatment were observed. Filter sterilization of RES increased SCN hatching when compared with the use of non-sterile exudates. Acifluorfen, bentazon and lactofen treatments negatively affected the viability of SCN eggs in adult females that developed on treated plants. The acifluorfen, bentazon, COC and NIS treatments also increased root glyceollin levels of

Phytoalexin glyceollins are soybean-specific antimicrobial compounds that are derived from the isoflavonoid pathway. They are synthesized by soybean in response to extrinsic stress such as pathogen attack or injury, thereby conferring partial resistance if synthesized rapidly at the site of infection and at the required concentration. Soybean produces multiple forms of glyceollins that result from the differential prenylation reaction catalyzed by prenyltransferases (PTs) on either the C-2 or C-4 carbon of a pterocarpan glycinol. The soybean genome contains 77 PT-encoding genes (GmPTs) where at least 11 are (iso)flavonoid-specific. Transcript accumulation of five candidates GmPTs was increased in response to Phytophthora sojae infection, suggesting their role in phytoalexin synthesis. The induced GmPTs localize to plastids and display tissue-specific expression. We have in this study identified two additional GmPTs: an isoflavone dimethylallyltransferase 3 (IDT3); and a glycinol 2-dimethylallyl transferase GmPT01. GmPT01 prenylates (-)-glycinol at the C-2 position, localizes in the plastid, and exhibits root-specific gene expression. Furthermore, its expression is induced rapidly in response to stress, and is associated with a quantitative trait loci linked with resistance to P. sojae. Based on these results, we conclude that GmPT01 are possibly one of the loci involved in conferring partial resistance against stem and root rot disease in soybean.

Soybean (Glycine max [L.] Merr) is one of the main grain legumes worldwide. Soybean farmers lose billions of dollars’ worth of yield annually due to root and stem rot disease caused by the oomycete Phytophthora sojae. Many strategies have been developed to combat the disease however these methods have proven ineffective in the long term. A more cost effective and durable approach is to select a trait naturally found in soybean that can increase resistance. One such trait is the increased production of phytoalexin glyceollins in soybean. Glyceollins are isoflavonoids, synthesized via the legume-specific branch of general phenylpropanoid pathway. The first key enzyme exclusively involved in glyceollin synthesis is chalcone reductase (CHR) which coacts with chalcone synthase for the production of isoliquirigenin chalcone, the precursor for glyceollin biosynthesis. Here we report the identification of 14 putative CHR genes in soybean where 11 of them are predicted to be functional. Our results show that GmCHRs display tissue-specific gene expression, and that only root-specific GmCHRs are induced upon P. sojae infection. Among 4 root-specific GmCHRs, GmCHR2A is located near a QTL that is linked to P. sojae resistance suggesting GmCHR2A as a novel locus for partial resistance that can be utilized for resistance breeding.

An estimated 70% of breast cancer tumors utilize estrogen receptor (ER) signaling to maintain tumorigenesis and targeting of the estrogen receptor is a common method of treatment for these tumor types. However, ER-positive (+) breast cancers often acquire drug resistant or altered ER activity in response to anti-estrogens. Here we demonstrate glyceollin, an activated soy compound, has anti-estrogen effects in breast cancers. We demonstrate through estrogen response element luciferase and phosphorylation-ER mutants that the effects of glyceollin arise from mechanisms distinct from conventional endocrine therapies. We show that glyceollin suppresses estrogen response element activity; however, it does not affect ER-alpha phosphorylation levels. Additionally we show that glyceollin suppresses the phosphorylation of proteins known to crosstalk with ER-alpha signaling, specifically we demonstrate an inhibition of Ribosomal Protein S6 Kinase, 70kDa (p70S6) phosphorylation following glyceollin treatment. Our data suggests a mechanism for glyceollin inhibition of ER through the induced suppression of p70S6 and demonstrates novel mechanisms for ER inhibition.

Soybean (Glycine max) 5-deoxyisoflavonoids (daidzein and its conjugates) are precursors of glyceollin phytoalexins. They are also converted to equol by microbes in the human intestine, resulting in health benefits. 5-Deoxyisoflavonoids accumulate in the roots (93% mol/mol of the total root isoflavonoids) and seeds of unstressed soybean plants. Chalcone reductase (CHR) is a key enzyme mediating 5-deoxyisoflavonoid biosynthesis because it catalyzes the production of 6'-deoxychalcone through its effects on the chalcone synthase (CHS)-catalyzed reaction. The soybean genome encodes at least 11 CHR-related homologs, but it is unclear which ones are functionally important for daidzein accumulation in unstressed plants. Among the CHR homologs, the temporal and spatial expression patterns of GmCHR5 were the most correlated with the distribution patterns of 5-deoxyisoflavonoids. The CHR activity of GmCHR5 was confirmed in vitro and in planta. In the in vitro assays, the ratio of CHR products (6'-deoxychalcone) to total CHS products (R value) was dependent on GmCHR5 and CHS concentrations, with higher concentrations resulting in higher R values (i.e. approaching 90%). Subcellular localization analyses revealed that GmCHR5 was present in the cytoplasm and nucleus. Protein-protein interaction assays indicated that GmCHR5, but not GmCHR1 and GmCHR6, interacted with 2-hydroxyisoflavanone synthase (IFS) isozymes. The CHS isozymes also interacted with IFS isozymes but not with GmCHR5. The proposed micro-compartmentalization of isoflavone biosynthesis through the formation of an IFS-mediated metabolon is probably involved in positioning GmCHR5 close to CHS, resulting in an R value that is high enough for the accumulation of abundant 5-deoxyisoflavonoids in soybean roots.

Glyceollins synthesized in soybeans that are exposed to biotic or abiotic stress have been reported to have health benefits. Considering that glyceollins are de novo synthesized from daidzein via several enzymatic steps and that isoflavone concentration widely varies among soybean varieties, the abilities of 60 soybean cultivars to synthesize glyceollins were compared under different elicitation conditions. Soybeans accumulated glyceollins differentially depending upon the cultivar when elicited with Aspergillus sojae. Contrary to our hypothesis that high isoflavone varieties may accumulate glyceollins more efficiently upon elicitation, glyceollin accumulation in response to fungal elicitation was not related with the concentration of either total isoflavones or daidzein in soybeans. Rather the glyceollin levels were significantly affected by soybean cultivar and most effectively increased by fungal infection. The data suggest that the selection of a strong fungal elicitor and a soybean cultivar with genotype that highly expresses the genes involved in glyceollin biosynthesis is essential for efficient glyceollin production.

Excessive consumption of fat and fructose leads to obesity due to lipid accumulation. The excessive lipid causes hypertrophy in the adipocytes which lead to cell death. Consequently, dead adipocytes will produce adipokines, which cause macrophages and lymphocytes to infiltrate into the adipose tissue, elevating pro-inflammatory cytokines, thus triggering the production of pro-inflammatory cytokines through NFκB activity. Elicited soybeans extract (ESE) with bacteria and light contain Glyceollin and Isoflavones, which inhibit the activation of NFКB and reduce plasma cholesterol levels by upregulating cholesterol metabolism. This study aimed to analyze the effect of ESE against the relative number of CD4+ NFκB+ cells in BALB/c mice spleen after administrated by high-fat diet food and fructose (HFD) for 20 weeks. Mice were given orally with ESE after administrated by HFD at dose 78 mg/kgBW (D1), 104 mg/kgBW (D2), and 130 mg/kgBW (D3) for 4 weeks. This study also used positive control (HFD mice model without ...

Sudden death syndrome (SDS) is caused by the fungal pathogen, Fusarium virguliforme, and is a major threat to soybean production in North America. There are two major components of this disease: (i) root necrosis and (ii) foliar SDS. Root symptoms consist of root necrosis with vascular discoloration. Foliar SDS is characterized by interveinal chlorosis and leaf necrosis, and in severe cases by flower and pod abscission. A major toxin involved in initiating foliar SDS has been identified. Nothing is known about how root necrosis develops. In order to unravel the mechanisms used by the pathogen to cause root necrosis, the transcriptome of the pathogen in infected soybean root tissues of a susceptible cultivar, ‘Essex’, was investigated. The transcriptomes of the germinating conidia and mycelia were also examined. Of the 14,845 predicted F. virguliforme genes, we observed that 12,017 (81%) were expressed in germinating conidia and 12,208 (82%) in mycelia and 10,626 (72%) in infected soybean roots. Of the 10,626 genes induced in infected roots, 224 were transcribed only following infection. Expression of several infection-induced genes encoding enzymes with oxidation-reduction properties suggests that degradation of antimicrobial compounds such as the phytoalexin, glyceollin, could be important in early stages of the root tissue infection. Enzymes with hydrolytic and catalytic activities could play an important role in establishing the necrotrophic phase. The expression of a large number of genes encoding enzymes with catalytic and hydrolytic activities during the late infection stages suggests that cell wall degradation could be involved in root necrosis and the establishment of the necrotrophic phase in this pathogen.

Soybean hairy roots transformed with the resveratrol synthase and resveratrol oxymethyl transferase genes driven by constitutive Arabidopsis actin and CsVMV promoters were characterized. Transformed hairy roots accumulated glycoside conjugates of the stilbenic compound resveratrol and the related compound pterostilbene, which are normally not synthesized by soybean plants. Expression of the non-native stilbenic phytoalexin synthesis in soybean hairy roots increased their resistance to the soybean pathogen Rhizoctonia solani. The expression of the AhRS3 gene resulted in 20% to 50% decreased root necrosis compared to that of untransformed hairy roots. The expression of two genes, the AhRS3 and ROMT, required for pterostilbene synthesis in soybean, resulted in significantly lower root necrosis (ranging from 0% to 7%) in transgenic roots than in untransformed hairy roots that had about 84% necrosis. Overexpression of the soybean prenyltransferase (dimethylallyltransferase) G4DT gene in soybean hairy roots increased accumulation of the native phytoalexin glyceollin resulting in decreased root necrosis.

Glyceollins are the major pathogen- and stress-inducible natural products (phytoalexins) of soybean that possess broad-spectrum anticancer and neuroprotective properties. Yet like other phytoalexins, glyceollins are difficult to obtain because they are typically biosynthesized only transiently and in low amounts in plant tissues. We recently identified acidity stress (pH 3.0 growth medium) as an elicitor that exerted prolonged (week-long) inductive effects on glyceollin biosynthesis and identified the NAC family TF gene GmNAC42-1 that activates glyceollin biosynthesis in response to acidity stress or WGE from the soybean pathogen Phytophthora sojae. GmNAC42-1 was annotated as an SAR gene and SAR genes were statistically overrepresented in the transcriptomic response to acidity stress suggesting that acidity stress triggers the systemic elicitation of glyceollin biosynthesis. Here, we demonstrate that acidity stress acts as a systemic elicitor when provided to soybean roots. Acidity stress preferentially elicited specific glyceollins in different soybean organs with exceptionally high yields of glyceollin I in root tissues.

Cutaneous secretions produced by amphibians of the family Bufonidae are rich sources of bioactive compounds that can be useful as new chemical templates for agrochemicals. In crop protection, the use of elicitors to induce responses offers the prospect of durable, broad-spectrum disease control using the plant’s own resistance. Therefore, we evaluated the potential of methanolic extracts of cutaneous secretions of two species of amphibians of the family Bufonidae found in the Amazon biome—Rhaebo guttatus (species 1) and Rhinella marina (species 2)—in the synthesis of phytoalexins in soybean cotyledons, bean hypocotyls, and sorghum mesocotyls. Additionally, changes in the enzyme activity of β-1,3-glucanase, peroxidase (POX), and polyphenol oxidase (PPO) and in the total protein content of soybean cotyledons were determined. In the soybean cultivar ‘TMG 132 RR’, our results indicated that the methanolic extract of R. guttatus cutaneous secretions suppressed glyceollin synthesis and β-1,3-glucanase activity and increased POX and PPO activities at higher concentrations and total protein content at a concentration of 0.2 mg/mL. On the other hand, the methanolic extract of R. marina cutaneous secretions induced glyceollin synthesis in the soybean cultivars ‘TMG 132 RR’ and ‘Monsoy 8372 IPRO’ at 0.1–0.2 mg/mL and 0.2 mg/mL, respectively. The methanolic extract of R. marina cutaneous secretions also increased the specific activity of POX and PPO in ‘Monsoy 8372 IPRO’ and ‘TMG 132 RR’, respectively, and decreased the activity of β-1,3-glucanases in ‘Monsoy 8372 IPRO’. At 0.3 mg/mL, it stimulated phaseolin synthesis. The extracts did not express bioactivity in the synthesis of deoxyanthocyanidins in sorghum mesocotyls. The study in soybean suggests that the bioactivity in defense responses is influenced by cultivar genotypes. Therefore, these results provide evidence that extracts of cutaneous secretions of these amphibians species may contribute to the bioactivity of defense metabolites in plants.

The glyceollins are soybean-derived phytoalexins that accumulate in the seeds in response to various extrinsic factors. Several elicitors such as fungi, UV irradiation and physical injury have been demonstrated to alter the secondary metabolome of soybeans via induction of glyceollin synthesis and accumulation. The pterocarpan phytoalexins have exhibited antiestrogenic activity against estrogen receptor-mediated cellular processes. As a result, the glyceollins display anticancer activities against breast and ovarian cancers through inhibition of tumor growth and progression, and also by inducing cell cycle arrest in the cancer cells. Moreover, glyceollins were found to exhibit antimicrobial activity against pathogenic fungi, cellular antioxidant activities against endotoxin-induced oxidative stress, and regulatory effects on glucose metabolism and endotoxin-induced inflammatory response. The soybean-derived compounds warrant further studies to evaluate detailed molecular mechanisms during aberrant cellular processes, safety and efficacy in animal disease models and human subjects, and potential use for food preservation. The bioactivity of glyceollins can result in the enrichment of soybean with these phytoalexins for improved health functions and other novel applications.

The different isoflavonoid phytoalexins produced by soybeans are known to possess antioxidant and estrogenic effects, with potential health benefits in humans. Enhanced production of phytoalexins by soybean plant will help in research efforts. In this study, the effects of stress induced by Aspergillus oryzae Lu brewing 3042 on the composition of wounded surface of Nigerian soybean seeds were analyzed. Glyceollin, one of the inducible phytoalexins produced by plants, were induced in the Nigerian soybeans variety through fungal infection. Glyceollins I, II, and III were separated by preparative high performance liquid chromatography (Prep-HPLC) from other phytochemicals and had purity of 97.5%. Results from the time course study indicated that the maximum concentration of glyceollins, 0.74 mg/g, occurred at day 3 in the soybean cotyledons inoculated with A. oryzae Lu brewing 3042. The soybean seeds soak time in water was examined and a suitable soak time of 4 h was found best in the accumulation of glyceollins after the soak time study was conducted. Cut or wounded surface of soybean cotyledons produced glyceollins upon fungal treatment as elicitor and the glyceollins were confirmed by the ultra performance liquid chromatography–mass spectrometry (UPLC–MS). Analysis of elicitor-treated Nigerian soybean extract showed that A. oryzae Lu brewing 3042 treatments achieved maximum concentration of phytoalexin glyceollins at 1.32 mg/g extract dry weight. These results indicate that using food-grade A. oryzae Lu brewing 3042 elicits the biosynthesis of phytoalexins, alters the secondary metabolite profiles of the soybeans and offers enhanced bioactivity of soybean as a functional food ingredient.

Scope Endothelial progenitor cells (EPCs) are derived from hematopoietic stem cells, and have the ability to differentiate into mature endothelial cells and contribute to neovascularization. Glyceollins are a type of phytoalexin produced in soybeans under stress conditions. The aim of this study is to determine the effect of glyceollin treatment on EPCs during early tumor vasculogenesis. Methods and results We found that glyceollin treatment significantly decreased the number of EPC colony-forming units in human cord blood-derived AC133+ cells and mouse bone-marrow-derived c-Kit+/Sca-1+/Lin− cells. Glyceollin treatment diminished the number of lineage-committed EPC cells in a dose-dependent manner (1–20 μM). Glyceollin treatment inhibited EPC migration, tube formation and the mRNA expression of angiopoietin-1 (Ang-1), Tie-2, stromal-derived factor-1 (SDF-1), C-X-C-chemokine receptor-4 (CXCR4), and endothelial nitric oxide synthase (eNOS) in cultured EPCs. Glyceollin treatment suppressed activation of Akt, Erk, and eNOS induced by SDF-1α or vascular endothelial growth factor (VEGF). Treatment with 10 mg/kg glyceollins significantly reduced the number of tumor-induced circulating EPCs and the incorporation of EPCs into neovessels in bone marrow transplanted mice. Conclusion These results suggest that glyceollins inhibit the function of EPCs in tumor neovascularization. Glyceollins from soybean elicitation could be beneficial in prevention of cancer development via vasculogenesis.

Glyceollin has been shown to have antidiabetic properties, although its molecular mechanism is not known. Here, we have investigated the metabolic effects of glyceollin in animal models of insulin resistance and in endoplasmic reticulum (ER) stress-responsive muscle cells. db/db mice were treated with glyceollin for 4weeks and triglycerides, total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) levels were measured. Glyceollin reduced serum insulin and triglycerides and increased HDL levels in db/db mice. Furthermore, glyceollin caused a significant improvement in glucose homeostasis without altering body weight and food intake in db/db mice. In muscle cells, glyceollin increased the activity of AMP-activated protein kinase (AMPK) as well as cellular glucose uptake. Fatty acid oxidation was also increased. In parallel, phosphorylation of acetyl-CoA carboxylase (ACC) at Ser-79 was increased, consistent with decreased ACC activity. An insulin-resistant state was induced by exposing cells to 5μg/ml of tunicamycin as indicated by decreased insulin-mediated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and glucose uptake. Inhibition of insulin-mediated tyrosine phosphorylation of IRS-1 and glucose uptake under ER stress was prevented by glyceollin. Strikingly, glyceollin reduced ER stress-induced, c-Jun NH2-terminal kinase activation and subsequently increased insulin signaling via stimulation of AMPK activity in L6 myotubes. Pharmacologic inhibition or knockdown of Ca(2+)/calmodulin-dependent protein kinase kinase blocked glyceollin-increased AMPK phosphorylation and insulin sensitivity under ER stress conditions. Taken together, these results indicate that glyceollin-mediated enhancement of insulin sensitivity under ER stress conditions is predominantly accomplished by activating AMPK, thereby having beneficial effects on hyperglycemia and insulin resistance.

The isoflavonoid profile of soybean was altered in different ways by stimulation of defense response upon germination. The combination of simultaneous germination and induction by Rhizopus oryzae increased the total isoflavonoid content of soybeans over 2-fold. Pterocarpans became the predominant isoflavonoids, up to 50% (w/w) of total isoflavonoids. To modulate both isoflavonoid content and composition further, the treatment was extended with wounding or light stimuli. The total isoflavonoid content could be increased over 3-fold compared to untreated beans by growing fungus-elicited soybean seedlings in light, whereas wounding was less effective. Interestingly, light altered the composition of prenylated pterocarpans by mediating the position of prenylation. The 2-prenylated pterocarpan level increased 2-fold, whereas that of 4-prenylated pterocarpan remained similar. Taken together, fungus was the most effective elicitor to alter the isoflavonoid content and composition of soybean seedlings, the impact of which can be further enhanced and mediated by additional stimuli, particularly light.

Objective Our previous in vitro study demonstrated that glyceollins help normalize glucose homeostasis by potentiating β-cell function and survival in insulinoma cells as well as improving glucose utilization in adipocytes. Here, we investigated whether fermented soybeans containing glyceollins had an antidiabetic action in type 2 diabetic animals. Methods The diabetic mice, their diabetes induced by intraperitoneal injections of streptozotocin (20 mg/kg bw), were administered a high fat diet with no soybeans (control), 10% unfermented soybeans and 10% fermented soybeans containing glyceollins, respectively, (FSG) for 8 weeks. As positive controls, rosiglitazone (20 mg/kg/bw) was given to diabetic mice fed a no soybean diet and non-diabetic mice were also placed on the same diet. Results Among the diabetic mice, FSG-treated mice exhibited the lowest peak for blood glucose levels with an elevation of serum insulin levels during the first part of oral glucose tolerance testing. FSG also made blood glucose levels drop quickly after the peak and it decreased blood glucose levels more than the control during insulin tolerance testing. This improvement was associated with increased hepatic glycogen accumulation and decreased triglyceride storage. The phosphorylation of Akt, AMP-kinase, and acetyl-CoA carboxylase in the liver was potentiated by FSG, whereas phosphoenolpyruvate carboxykinase expression decreased. The enhancement of glucose homeostasis was comparable to the effect induced by rosiglitazone, a commercial peroxisome proliferator-activated receptor-γ agonist, but it did not match the level of glucose homeostasis in the non-diabetic mice. Conclusion Glyceollin-containing FSG improves glucose homeostasis, partly by enhancing hepatic insulin sensitivity in type 2 diabetic mice.

Glyceollins, produced to induce disease resistance responses against specific species, such as an incompatible pathogen Phytophthora sojae in soybeans, have the potential to exhibit anti-inflammatory activity in RAW 264.7 cells. To investigate the anti-inflammatory effects of elicited glyceollins via a signaling pathway, we studied the glyceollin signaling pathway using several assays including RNA and protein expression levels. We found that soybean glyceollins significantly reduced LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production, as well as the expression of inducible ΝΟ synthase (iNOS) and cyclooxygenase-2 (COX-2) via the suppression of NF-κB activation. Glyceollins also inhibited the phosphorylation of IκBα kinase (IKK), the degradation of IκBα, and the formation of NF-κB-DNA binding complex in a dose-dependent manner. Furthermore, they inhibited pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-18, but increased the generation of the anti-inflammatory cytokine IL-10. Collectively, the present data show that glyceollins elicit potential anti-inflammatory effects by suppressing the NF-κB signaling pathway in RAW 264.7 cells.

The goal of the present study was to determine the effects of glyceollins on intestinal ABCC2 (ATP Binding Cassette C2, multidrug resistance protein 2, MRP2) and ABCG2 (ATP Binding Cassette G2, breast cancer resistance protein, BCRP) function using the Caco-2 cell intestinal epithelial cell model. Glyceollins are soy-derived phytoestrogens that demonstrate anti-proliferative activity in several sources of cancer cells. 5 (and 6)-carboxy-2′,7′-dichloroflourescein (CDF) was used as a prototypical MRP2 substrate; whereas BODIPY-prazosin provided an indication of BCRP function. Comparison studies were conducted with genistein. Glyceollins were shown to inhibit MRP2-mediated CDF transport, with activity similar to the MRP2 inhibitor, MK-571. They also demonstrated concentration-dependent inhibition BCRP-mediated efflux of BODIPY-prazosin, with a potency similar to that of the recognized BCRP inhibitor, Ko143. In contrast, genistein did not appear to alter MRP2 activity and even provided a modest increase in BCRP efflux of BODIPY-prazosin. In particular, glyceollin inhibition of these two important intestinal efflux transporters suggests the potential for glyceollin to alter the absorption of other phytochemicals with which it might be co-administered as a dietary supplement, as well as alteration of the absorption of pharmaceuticals that may be administered concomitantly.

The leaves of soybean ( Glycine max ) were extracted into four different polar solvents: ethylacetate, butanol, ethanol and water. The ethylacetate extract (EE) showed the lowest IC 50 value against α-glucosidase (70.1 μg/ml). To investigate the compounds responsible for this effect, activity guided fractionation of soybean leaves by chromatography yielded seven phenolic compounds which were identified as formononetin ( 1 ), afromosin ( 2 ), coumestrol ( 3 ), isotrifoliol ( 4 ), phaseol ( 5 ), glyceofuran ( 6 ) and a new compound, glyceollin V ( 7 ). Importantly, coumestrol 3 was not only the most potent component with IC 50 = 6.0 μM, but also the most abundant polyphenol in soybean leaves. There was shown to be an increasing inhibition across the developmental stage of the plant, which correlated strongly with an increase in compound 3 in the leaves. In fact, we have shown it can comprise up to 65% of the polyphenols in the leaves by HPLC analysis.

Numerous antioxidants have been reported to cause transcriptional activation of several antioxidant enzymes through binding antioxidant-response element on their promoter region. We, therefore, attempted to examine whether glyceollins, which share common structural features with many phase 2 enzyme inducers and antioxidant activity, could induce detoxifying/antioxidant enzymes. Glyceollins induced NAD(P)H:quinone oxidoreductase activity in a dose-dependent manner in both mouse hepatoma Hepa1c1c7 and its mutant BPRc1 cells. The compounds also increased the expression of some representative antioxidant enzymes, such as heme oxygenase 1,gamma-glutamylcysteine synthase, and glutathione reductase, by promoting nuclear translocation of the NF-E2-related factor-2 (Nrf2). Furthermore, phosphorylation of Akt and antioxidant response element-mediated reporter gene expression were enhanced by glyceollins but suppressed by LY294002, an inhibitor of phosphoinositide 3-kinases (PI3K). This suggests that glyceollins may cause Nrf2-mediated phase 2 enzyme induction through activation of the PI3K signaling pathway as well as interaction with Keap1. Our molecular docking simulations also suggest that the glyceollin isomers tightly bind into the binding pocket around Cys151, preventing Nrf2 from docking to Keap1. In conclusion, the current data suggest that glyceollins induced phase 2 detoxifying enzymes likely through promoting nuclear translocation of Nrf2, which is known to be regulated by phosphorylation of Nrf2 and/or disrupting Keap1-Nrf2 complex formation.

Phytoalexins from soya are mainly characterised as prenylated pterocarpans, the glyceollins. Extracts of non-soaked and soaked soya beans, as well as that of soya seedlings, grown in the presence of Rhizopus microsporus var. oryzae, were screened for the presence of prenylated flavonoids with a liquid chromatography/mass spectrometry (LC/MS)-based screening method. The glyceollins I-III and glyceollidins I-II, belonging to the isoflavonoid subclass of the pterocarpans, were tentatively assigned. The formation of these prenylated pterocarpans was accompanied by that of other prenylated isoflavonoids of the subclasses of the isoflavones and the coumestans. It was estimated that approx. 40% of the total isoflavonoid content in Rhizopus-challenged soya bean seedlings were prenylated pterocarpans, whereas 7% comprised prenylated isoflavones and prenylated coumestans. The site of prenylation (A-ring or B-ring) of the prenylated isoflavones was tentatively annotated using positive-ion mode MS by comparing the (1,3) A(+) retro-Diels-Alder (RDA) fragments of prenylated and non-prenylated isoflavones. Furthermore, the fragmentation pathways of the five pterocarpans in negative-ion (NI) mode were proposed, which involved the cleavage of the C-ring and/or D-ring. The absence of the ring-closed prenyl (pyran or furan) gave exclusively -H(2) O(x,y) RDA fragments, whereas its presence gave predominantly the common RDA fragments.

The extract of soybean exposed to biotic elicitors such as food-grade fungus is known to have antioxidant activity. Glyceollins were major bioactive compounds present in soybean elicited by fungi and shown to have antifungal and anticancer activities. The purpose of present study was to evaluate the antioxidant activities of glyceollins by measuring ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, singlet oxygen quenching, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, hydroxyl radical scavenging activity, and lipid peroxidation inhibition. In addition, the antioxidant potential of glyceollins were measured by a fluorescent probe, 2',7'-dichlorofluorescin diacetate (DCFDA), and dihydroethidium (DHE) in mouse hepatoma hepa1c1c7 cells in which they were insulted with H2O2 to generate reactive oxygen species (ROS). Glyceollins showed a strong reducing power and inhibited lipid peroxidation, with significant scavenging activities of radicals including singlet oxygen, superoxide anion, ABTS, and DPPH. We also found that glyceollins significantly suppressed H2O2-induced ROS production in hepa1c1c7 cells. Therefore, glyceollins deserve further study as natural antioxidants and nutraceuticals.

Plants recognize invading pathogens and respond biochemically to prevent invasion or inhibit colonization in plant cells. Enhancing this response in crop plants could improve sustainable methods to manage plant diseases. To enhance disease resistance in soybean, the soybean phytoalexin glyceollin was assessed in soybean hairy roots of two soybean genotypes, Spencer and PI 567374, transformed with either soybean isoflavone synthase (IFS2) or chalcone synthase (CHS6) genes that were inoculated with the soybean pathogens Diaporthe phaseolorum var. meridionales, Macrophomina phaseolina, Sclerotinia sclerotiorum, and Phytophthora sojae. The hairy-root-transformed lines had several-fold decreased levels of isoflavone daidzein, the precursor of glyceollin, and considerably lower concentrations of glyceollin induced by pathogens measured 5 days after fungal inoculation compared with the nontransformed controls without phenolic transgenes. M. phaseolina, P. sojae, and S. sclerotiorum grew much more on IFS2- and CHS6-transformed roots than on control roots, although there was no significant difference in growth of D. phaseolorum var. meridionales on the transformed hairy-root lines. In addition, glyceollin concentration was lower in D. phaseolorum var. meridionales-inoculated transformed and control roots than roots inoculated with the other pathogens. Glyceollin inhibited the growth of D. phaseolorum var. meridionales, M. phaseolina, P. sojae, S. sclerotiorum, and three additional soybean pathogens: Cercospora sojina, Phialophora gregata, and Rhizoctonia solani. The most common product of glyceollin conversion or degradation by the pathogens, with the exception of P. sojae, which had no glyceollin degradation products found in the culture medium, was 7-hydroxyglyceollin.

The aim of this study was to examine the efficacy of glyceollins isolated from soybean seeds elicited with Aspergillus sojae for its antifungal potential. Glyceollins I, II, and III were determined by HPLC-MS analysis. The glyceollins (200 and 600 microg/disk) revealed remarkable antifungal effect against Fusarium oxysporum , Phytophthora capsici, Sclerotinia sclerotiorum, and Botrytis cinerea, within the growth inhibition range of 10.9-61.0%, along with their respective MIC values ranging from 25 to 750 microg/mL. The glyceollins also had a strong detrimental effect on spore germination of all tested plant pathogens along with concentration- as well as time-dependent kinetic inhibition of P. capsici. Thus, the results obtained in this study demonstrate that glyceollins derived from soybean seeds elicited with A. sojae possess a wide range of fungicidal activity and could become an alternative to synthetic fungicides for controlling certain important fungal diseases.

Glyceollins, which are synthesized from daidzein in soybeans cultured with fungi, have been shown to have antifungal effects and cancer preventive properties. Several studies have proposed that isoflavones and their metabolites act as a mixed agonist/antagonist for estrogen. Although glyceollins were reported to suppress some cancer cells via anti-estrogenic activity, it is not clear whether the compounds possess estrogenic potential. In contrast to the anti-estrogenic action reported thus far, we observed estrogenic effects of glyceollins using E-screen assay and pS2 expression, whereas glyceollins showed higher affinity for estrogen receptor (ER) beta than ERalpha. We also found that glyceollins were more efficiently produced de novo in minced than in half-sliced soybean, following infection with Aspergillus sojae. In conclusion, glyceollins may be useful in the prevention or amelioration of postmenopausal complications because they had strong estrogenic activity, and their production could be variable depending upon processing prior to fungal inoculation.

The present study was undertaken to investigate the molecular mechanisms by which glyceollin I inhibits vascular contraction in rat aorta. Rat aortic rings were treated with either glyceollin I or vehicle when vascular contraction reached plateaus. We measured the activity of GTP-RhoA and Rho GTPase-activating protein (RhoGAP) and the phosphorylation level of the myosin light chain (MLC(20)), myosin phosphatase targeting subunit 1 (MYPT1), and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI17). Glyceollin I reduced vascular contraction whether endothelium is present or denuded. Glyceollin I reduced vascular contraction induced by NaF, U46619, phenylephrine, or PDBu. Blockers of K(+) channels did not affect the vasorelaxation induced by glyceollin I. Glyceollin I reduced activation of RhoA as well as phosphorylation level of MLC(20). Glyceollin I also reduced phosphorylation of MYPT1 and CPI17 induced by NaF but not PDBu. However, glyceollin I had no direct effect on RhoGAP activation in vitro. Glyceollin I reduced vascular contraction, at least in part, through inhibition of the RhoA/Rho-kinase signaling pathway.

Glyceollins are a category of phytoalexins that are produced by soybeans under fungal stress, but their effects on glucose homeostasis remain unknown. We hypothesized that glyceollins play an important role in glucose homeostasis by regulating glucose utilization in adipocytes and improving beta-cell function and survival. Glyceollins improved insulin-stimulated glucose uptake in 3T3-L1 adipocytes without activating the peroxisome proliferator-activated receptor-gamma agonist. They decreased triacylglycerol accumulation in adipocytes. In addition, glyceollins slightly improved glucose-stimulated insulin secretion without palmitate treatment in Min6 cells, and they potentiated insulinotropic actions when 500 microM palmitate was used to induce beta-cell dysfunction. This was associated with decreased beta-cell apoptosis because of the attenuation of endoplasmic reticulum stress, as determined by mRNA levels of XBP-1, ATF-4, ATF-6, and CHOP. Glyceollins also potentiated GLP-1 secretion to enhance insulinotropic actions in enteroendocrine cells. In conclusion, glyceollins help normalize glucose homeostasis by potentiating beta-cell function and survival and improving glucose utilization in adipocytes.

Understanding the metabolic responses of the plant to a devastating foliar disease, soybean rust, caused by Phakopsora pachyrhizi, will assist in development of cultivars resistant to soybean rust. In this study, differences in phenolic metabolism were analyzed between inoculated and noninoculated plants using two susceptible and three resistant soybean genotypes with known resistance genes. Rust infection resulted in increased accumulation of isoflavonoids and flavonoids in leaves of all soybean genotypes tested. Although the soybean phytoalexin glyceollin was not detected in leaves of uninfected plants, accumulation of this compound at marked levels occurred in rust-infected leaves, being substantially higher in genotypes with a red-brown resistant reaction. In addition, there was inhibition of P. pachyrhizi spore germination by glyceollin, formononetin, quercetin, and kaempferol. However, there was no correlation between concentrations of flavonoids quercetin and kaempferol and rust-induced isoflavonoid formononetin in soybean leaves and rust resistance. Lignin synthesis also increased in all inoculated soybean genotypes whereas there was no significant difference in all noninoculated soybean genotypes. Cell wall lignification was markedly higher in inoculated resistant lines compared with inoculated susceptible lines, indicating a possible protective role of lignin in rust infection development.

Prenylation plays a major role in the diversification of aromatic natural products, such as phenylpropanoids, flavonoids, and coumarins. This biosynthetic reaction represents the crucial coupling process of the shikimate or polyketide pathway providing an aromatic moiety and the isoprenoid pathway derived from the mevalonate or methyl erythritol phosphate (MEP) pathway, which provides the prenyl (isoprenoid) chain. In particular, prenylation contributes strongly to the diversification of flavonoids, due to differences in the prenylation position on the aromatic rings, various lengths of prenyl chain, and further modifications of the prenyl moiety, e.g., cyclization and hydroxylation, resulting in the occurrence of ca. 1000 prenylated flavonoids in plants. Many prenylated flavonoids have been identified as active components in medicinal plants with biological activities, such as anti-cancer, anti-androgen, anti-leishmania, and anti-nitric oxide production. Due to their beneficial effects on human health, prenylated flavonoids are of particular interest as lead compounds for producing drugs and functional foods. However, the gene coding for prenyltransferases that catalyze the key step of flavonoid prenylation have remained unidentified for more than three decades, because of the membrane-bound nature of these enzymes. Recently, we have succeeded in identifying the first prenyltransferase gene SfN8DT-1 from Sophora flavescens, which is responsible for the prenylation of the flavonoid naringenin at the 8-position, and is specific for flavanones and dimethylallyl diphosphate (DMAPP) as substrates. Phylogenetic analysis showed that SfN8DT-1 has the same evolutionary origin as prenyltransferases for vitamin E and plastoquinone. A prenyltransferase GmG4DT from soybean, which is involved in the formation of glyceollin, was also identified recently. This enzyme was specific for pterocarpan as its aromatic substrate, and (-)-glycinol was the native substrate yielding the direct precursor of glyceollin I. These enzymes are localized to plastids and the prenyl chain is derived from the MEP pathway. Further relevant genes involved in the prenylation of other types of polyphenol are expected to be cloned by utilizing the sequence information provided by the above studies.

Glyceollins, a group of novel phytoalexins isolated from activated soy, have recently been demonstrated to be novel antiestrogens that bind to the estrogen receptor (ER) and inhibit estrogen-induced tumor progression. Our previous publications have focused specifically on inhibition of tumor formation and growth by the glyceollin mixture, which contains three glyceollin isomers (I, II, and III). Here, we show the glyceollin mixture is also effective as a potential antiestrogenic, therapeutic agent that prevents estrogen-stimulated tumorigenesis and displays a differential pattern of gene expression from tamoxifen. By isolating the individual glyceollin isomers (I, II, and III), we have identified the active antiestrogenic component by using competition binding assays with human ERalpha and in an estrogen-responsive element-based luciferase reporter assay. We identified glyceollin I as the active component of the combined glyceollin mixture. Ligand-receptor modeling (docking) of glyceollin I, II, and III within the ERalpha ligand binding cavity demonstrates a unique type II antiestrogenic confirmation adopted by glyceollin I but not isomers II and III. We further compared the effects of glyceollin I to the antiestrogens, 4-hydroxytamoxifen and ICI 182,780 (fulvestrant), in MCF-7 breast cancer cells and BG-1 ovarian cancer cells on 17beta-estradiol-stimulated expression of progesterone receptor and stromal derived factor-1alpha. Our results establish a novel inhibition of ER-mediated gene expression and cell proliferation/survival. Glyceollin I may represent an important component of a phytoalexin-enriched food (activated) diet in terms of chemoprevention as well as a novel therapeutic agent for hormone-dependent tumors.

The primary induced isoflavones in soybean, the glyceollins, have been shown to be potent estrogen antagonists in vitro and in vivo. The discovery of the glyceollins' ability to inhibit cancer cell proliferation has led to the analysis of estrogenic activities of other induced isoflavones. In this study, we investigated a novel isoflavone, glycinol, a precursor to glyceollin that is produced in elicited soy. Sensitive and specific in vitro bioassays were used to determine that glycinol exhibits potent estrogenic activity. Estrogen-based reporter assays were performed, and glycinol displayed a marked estrogenic effect on estrogen receptor (ER) signaling between 1 and 10 microM, which correlated with comparable colony formation of MCF-7 cells at 10 microM. Glycinol also induced the expression of estrogen-responsive genes (progesterone receptor and stromal-cell-derived factor-1). Competitive binding assays revealed a high affinity of glycinol for both ER alpha (IC(50) = 13.8 nM) and ER beta (IC(50) = 9.1 nM). In addition, ligand receptor modeling (docking) studies were performed and glycinol was shown to bind similarly to both ER alpha and ER beta. Taken together, these results suggest for the first time that glycinol is estrogenic and may represent an important component of the health effects of soy-based foods.