molecules Article Distinct Mechanisms of Biotic and Chemical Elicitors Enable Additive Elicitation of the Anticancer Phytoalexin Glyceollin I Kelli Farrell 1,†, Md Asraful Jahan 2,† and Nik Kovinich 2,* 1 Department of Biology, West Virginia University, Morgantown, WV 26506, USA; [email protected] 2 Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-304-293-9240 † These Authors contributed equally to this article. Received: 16 June 2017; Accepted: 25 July 2017; Published: 27 July 2017 Abstract: Phytoalexins are metabolites biosynthesized in plants in response to pathogen, environmental, and chemical stresses that often have potent bioactivities, rendering them promising for use as therapeutics or scaffolds for pharmaceutical development. Glyceollin I is an isoflavonoid phytoalexin from soybean that exhibits potent anticancer activities and is not economical to synthesize. Here, we tested a range of source tissues from soybean, in addition to chemical and biotic elicitors, to understand how to enhance the bioproduction of glyceollin I. Combining the inorganic chemical silver nitrate (AgNO3) with the wall glucan elicitor (WGE) from the soybean pathogen Phytophthora sojae had an additive effect on the elicitation of soybean seeds, resulting in a yield of up to 745.1 µg gt−1 glyceollin I. The additive elicitation suggested that the biotic and chemical elicitors acted largely by separate mechanisms. WGE caused a major accumulation of phytoalexin gene transcripts, whereas 00 AgNO3 inhibited and enhanced the degradation of glyceollin I and 6 -O-malonyldaidzin, respectively. Keywords: bioproduction; phytoalexin; isoflavonoid; glyceollin; soybean [Glycine max (L.) Merr.] 1. Introduction Plants, like other organisms, have metabolic pathways that remain silent until activated by stresses. Phytoalexins are defense metabolites biosynthesized in response to pathogens, but which for unknown reasons also accumulate in response to specific environmental stresses and inorganic chemicals, such as heavy metals [1–3]. Much of what is known about phytoalexin elicitation mechanisms comes from studies of the glyceollins in soybean, camalexins in Arabidopsis, diterpenoids and flavonoids in rice, stilbenes in grapevine, alkaloids in California poppy, and the 3-deoxyanthocyanidins, terpenoids, and phytodienoic acids in maize. However, very few studies have attempted to distinguish the elicitation mechanisms of biotic and chemical elicitors. Biotic elicitation begins when a microbial derived pathogen-associated molecular pattern (PAMP) or effector binds to a pattern recognition receptor at the plasma membrane of the plant cell. Mitogen-activated protein kinase (MAPK) or phospholipase signaling ultimately results in the expression of transcription factors (TFs) that directly activate the transcription of phytoalexin biosynthesis genes. MYB-, bHLH-, or WRKY-type TFs directly activate some or all of the phytoalexin biosynthesis genes in cotton, sorghum, rice, Arabidopsis, and grapevine [4–6]. In soybean, no phytoalexin TF has been identified, but transcription of glyceollin biosynthesis genes was coordinately induced in response to the pathogen Phytophthora sojae [7,8]. Heavy metals, such as silver nitrate (AgNO3), have elicited chemically diverse phytoalexins in many plant species. The molecular target(s) of these heavy metals remain(s) unknown. AgNO3 was Molecules 2017, 22, 1261; doi:10.3390/molecules22081261 www.mdpi.com/journal/molecules Molecules 2017, 22, 1261 2 of 13 Molecules 2017, 22, 1261 2 of 12 shown to inhibit developmental processes triggered by exogenous ethylene treatment, and thus has shown to inhibit developmental processes triggered by exogenous ethylene treatment, and thus has been considered a potent inhibitor of ethylene perception [9]. Some evidence has suggested that AgNO3 been considered a potent inhibitor of ethylene perception [9]. Some evidence has suggested that AgNO3 and pathogens elicit phytoalexins primarily by different mechanisms. The P. sojae-resistant soybean and pathogens elicit phytoalexins primarily by different mechanisms. The P. sojae-resistant soybean variety Harosoy 63 elicited glyceollins rapidly in response to race 1 P. sojae, but the susceptible variety variety Harosoy 63 elicited glyceollins rapidly in response to race 1 P. sojae, but the susceptible variety Harosoy did not, whereas both varieties responded similarly to AgNO3 [10]. Feeding AgNO3-elicited Harosoy did not, whereas both varieties responded similarly to AgNO3 [10]. Feeding AgNO3-elicited soybean cotyledons the radiolabeled intermediate phenylalanine did not result in radiolabeled soybean cotyledons the radiolabeled intermediate phenylalanine did not result in radiolabeled glyceollins, but AgNO3 treatment reduced the degradation of radiolabeled glyceollins [11]. glyceollins, but AgNO3 treatment reduced the degradation of radiolabeled glyceollins [11]. GlyceollinsGlyceollins are are thethe major major phytoalexins phytoalexins of the of thesoyb soybean.ean. They belong They to belong the pterocarpan to the pterocarpan subclass subclassof isoflavonoids, of isoflavonoids, which possesses which great possesses potential great as scaffolds potential for pharmaceutical as scaffolds fordevelopment pharmaceutical [12]. developmentGlyceollins [are12]. biosynthesized Glyceollins are from biosynthesized the isoflavonoid from thedaidzein, isoflavonoid which can daidzein, result whichfrom de can novo result frombiosynthesis de novo biosynthesis beginning at beginningphenylalanine, at phenylalanine, or possibly from or possiblythe hydrolysis from of the preformed hydrolysis isoflavonoid- of preformed isoflavonoid-glycosideglycoside conjugates conjugates(Figure 1). (FigureGlyceollins1). Glyceollinshave broad-spectrum have broad-spectrum antiproliferative antiproliferative or antitumor or antitumoractivities activities against human against lung, human breast, lung, prostate, breast, prostate,ovary, skin, ovary, and skin,kidney and cancers. kidney Glyceollin cancers. Glyceollin I is the I ismost the potent, most potent, and directly and directlyantagonizes antagonizes human estrogen human receptor estrogen α (ER receptorα) andα ER(ERβ [13].α)and Glyceollin ERβ [ 13I ]. Glyceollinalso exhibits I also a rare exhibits ER-independent a rare ER-independent mode-of-action via mode-of-action a mechanism that via is anot mechanism yet fully understood that is not [14]. yet fullyIn understoodlight of the therapeutic [14]. In light potential of the therapeutic of glyceollin potential I, studies of have glyceollin attempted I, studies to produce have attemptedlarge-scale to produce(gram)large-scale amounts by (gram) chemical amounts synthesis by or chemical by the elic synthesisitation of orsoybean by the [15,16]. elicitation However, of soybean the yield [15 by,16 ]. However,chemical the synthesis yield by was chemical up to 12%, synthesis and required was up a high to 12%,ly concerted and required effort of a highlyspecialists concerted six months effort to of specialistscomplete, six rendering months toit complete,uneconomical rendering for commercial it uneconomical production for commercial[15]. Here, we production aimed to [identify15]. Here, wewhich aimed soybean to identify tissues which and soybean treatments tissues provide and optimal treatments glyceollin provide I bioproduction optimal glyceollin in vitro. I bioproduction Our study in vitroprovides. Our novel study insight provides into how novel biotic insight and chemical into how elicitation biotic and pathways chemical are elicitation distinct, and pathways how this are can be exploited to enhance the bioproduction of glyceollin I. distinct, and how this can be exploited to enhance the bioproduction of glyceollin I. Figure 1. Glyceollin I biosynthetic pathway. In addition to de novo biosynthesis, the constitutively Figure 1. Glyceollin I biosynthetic pathway. In addition to de novo biosynthesis, the constitutively 00 accumulatingaccumulating isoflavone isoflavone conjugate conjugate 66″--OO-malonyldaidzin-malonyldaidzin may may be be hydrolyzed hydrolyzed to to provide provide daidzein daidzein intermediatesintermediates for for glyceollin glyceollin I biosynthesis. biosynthesis. CHS, CHS, chalcone chalcone synthase; synthase; CHR, CHR,chalcone chalcone reductase; reductase; CHI, 0 0 CHI,chalcone chalcone isomerase; isomerase; IFS, isoflavo IFS, isoflavonene synthase; synthase; I2’H, isoflavone I2 H, isoflavone2′-hydroxylase; 2 -hydroxylase; G4DT, glycinol G4DT, 4- glycinoldimethylallyl 4-dimethylallyl transferase; transferase; G2DT, glycinol G2DT, 2-dimethylallyl glycinol 2-dimethylallyl transferase; transferase;GLS, glyceollin GLS, synthase; glyceollin 00 synthase;UF7GT UF7GT(UGT88E3) (UGT88E3) UDP-glucose:isoflavone-7- UDP-glucose:isoflavone-7-O-glucosyltransferase;O-glucosyltransferase; I6″OMT (GmMT7) I6 OMT isoflavone- (GmMT7) 00 isoflavone-7-7-O-glucoside-6O-glucoside-6″-O-methyltransferase.-O-methyltransferase. Molecules 2017, 22, 1261 3 of 13 Molecules 2017, 22, 1261 3 of 12 2.2. Results and DiscussionDiscussion 2.1.2.1. Imbibing SoybeanSoybean SeedsSeeds AreAre thethe MostMost AbundantAbundant SourceSource ofof GlyceollinGlyceollin II PriorPrior studiesstudies havehave foundfound thatthat glyceollinsglyceollins werewere readilyreadily elicitedelicited upup toto thethe first-true-leaffirst-true-leaf stagestage ofof development,development, whenwhen they they were were essential essential for for