STILBENOID PRENYLTRANSFERASES FROM PLANTS

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and is a continuation in part of U.S. Patent Application No. 62/351,117 filed on June 16, 2016 entitled "STILBENOID PRENYLTRANSFERASES FROM PEANUT."

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This work was supported by USDA-NIFA Grant 2014-67014-21701, National Sciences Foundation-EPSCoR Grant EPS 0701890 (Center for Plant-Powered Production-P3), the National Science Foundation Division of Biological Infrastructure Grant ABI-1062432 to Indiana University (National Center for Genome Analysis Support), the Arkansas ASSET Initiative, the Arkansas Science & Technology Authority, and the Arkansas Biosciences Institute.

REFERENCE TO A MICROFICHE APPENDIX

Not Applicable.

RESERVATION OF RIGHTS

A portion of the disclosure of this patent document contains material which is subject to intellectual property rights such as but not limited to copyright, trademark, and/or trade dress protection. The owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent files or records but otherwise reserves all rights whatsoever.

BACKGROUND OF THE INVENTION

The present invention relates generally to prenylated stilbenoids and the production of prenylated stilbenoids.

II. Description of the Known Art

A substantial part of non-host defense responses in many plants is the pathogen-induced production of secondary metabolites, generally termed phytoalexins, that locally restrict disease progression due to bioactivities toxic to the pathogen (reviewed in Ahuja et al., 2012). Peanut or groundnut (Arachis hypogaed) tissues mount a defense against infection by the soil fungus Aspergillus flavus and other pathogens by overproducing stilbene derivatives around sites of wounding and elicitor perception (Sobolev, 2013).

SUMMARY OF THE INVENTION

The present invention is related to prenylated stilbenoids and a method of producing the prenylated stilbenoids. Harnessing the inducible bioproduction capabilities of the peanut hairy root culture system, we have newly produced a prenylated stilbenoid, i.e., arachidin-5, and have demonstrated that the prenyl moiety on peanut prenylated stilbenoids is derived from a plastidic biosynthesis pathway. We have characterized for the first time plant membrane-bound stilbenoid-specific prenyltransferase activity from the microsomal fraction of peanut hairy roots. With multidisciplinary approaches, we have isolated stilbenoid prenyltransferase genes and comprehensively characterized their functionality as stilbenoid prenyltransferase via a transient expression system in Nicotiana benthamiana and stable expression in tobacco plants and hairy roots. Moreover, we have observed the enzymatic degradation of exogenous resveratrol by peanut hairy root tissue, an observation that will lead to elucidation of further mechanisms governing phytoalexin accumulation in plants.

Prenylated stilbenoids synthesized in some legumes exhibit plant pathogen defense properties and pharmacological activities with potential benefits to human health. Despite their importance, the biosynthetic pathways of these compounds remain to be elucidated. Peanut (Arachis hypogaea) hairy root cultures produce a diverse array of prenylated stilbenoids upon treatment with elicitors. Using metabolic inhibitors of the plastidic and cytosolic isoprenoid biosynthetic pathways, we demonstrated that the prenyl moiety on the prenylated stilbenoids derives from a plastidic pathway. We further characterized for the first time a membrane-bound stilbenoid-specific prenyltransferase from the microsomal fraction of peanut hairy roots. This microsomal fraction-derived prenyltransferase utilizes 3,3-dimethylallyl pyrophosphate as a prenyl donor. The microsomal fraction also prenylates pinosylvin to chiricanine A and piceatannol to arachidin-5, a prenylated stilbenoid produced for the first time in this study. Using a transcriptomics and targeted metabolomics approach, we have isolated stilbenoid prenyltransferase genes from peanut and confirmed their functionality as stilbenoid prenyltransferase via a transient expression system in Nicotiana benthamiana and stable expression in tobacco plants and hairy roots.

Defense responses of peanut (Arachis hypogaea) include the synthesis of prenylated stilbenoids as phytoalexins in response to biotic and abiotic stresses. Despite their importance, the biosynthetic pathways of prenylated stilbenoids remain to be elucidated and genes encoding stilbenoid-specific prenyltransferases have yet to be identified in any plant species. In this study, we combined targeted transcriptome and metabolome analyses to discover prenyltransferase genes from elicitor-treated peanut hairy roots. Transcripts encoding five enzymes were identified, and two of these were functionally characterized in a transient expression system of Agrobacterium- ^' filtrated leaves of Nicotiana benthamiana. One of the prenyltransferases, AhR4DT-l catalyzes a key reaction involved in the biosynthesis of prenylated stilbenoids, in which resveratrol is prenylated at its C-4 position to form arachidin-2, while another, AhR3'DT- 1, was able to add the prenyl group to C-3' of resveratrol. Each of these prenyltransferases has a high specificity for stilbenoid substrates, and their subcellular location in the plastid was confirmed by fluorescence microscopy. Structure analysis of the prenylated stilbenoids suggest that these two prenyltransferase activities represent the first committed steps in the biosynthesis of a large number of prenylated stilbenoids and their derivatives in peanut.

Stilbenoids are phenylpropanoid compounds that accumulate in response to biotic and abiotic stresses in a small number of higher plant families including grape (Vitaceae), pine (Pinaceae) and peanut (Fabaceae). These compounds serve as phytoalexins and provide protection to the host plant against various microbial pathogens (Ahuja et al., 2012). Resveratrol studied compound in the stilbene family has attracted great attention in the scientific community, not only because of its important role for disease defense in plants (Ahuja et al., 2012), but also because of numerous bioactivities including anticancer, cardioprotective, antioxidant, anti-inflammatory and neuroprotective properties in human cell culture and in vivo, although the extent of its bioavailability has been questioned (Gambini et al., 2015; Tome-Carneiro et al., 2013; Baur and Sinclair, 2006).

Interestingly, resveratrol is synthesized in peanut, along with stilbenoids conjugated to a prenyl group, a modification not common in other stilbene-producing plants (Aguamah et al., 1981; Cooksey et al., 1988; Sobolev et al., 2006). The prenylation of the stilbene backbone is the primary feature that contributes to the diversity of these peanut secondary metabolites.

Differences occur in the position of prenylation as well as in subsequent modifications of the prenyl moiety, such as cyclization and hydroxylation. To date, more than 45 prenylated stilbenoids and their derivatives have been detected in peanut tissues subjected to biotic or abiotic stress (Wu et al., 2011; Sobolev, 2013; Sobolev et al., 2010, 2009, 2006, 2016). The prenylation of stilbenoids increases their lipophilicities and membrane permeabilities, and may have additional impacts on bioactivities. Prenylated stilbenoids have shown equivalent or enhanced bioactivities relative to non-prenylated forms, such as resveratrol, in in vitro studies (Huang et al., 2010; Chang et al., 2006; Sobolev et al., 2011). The prenylated stilbenoids arachidin-1 and arachidin-3 showed favorable metabolic profiles when compared to their non- prenylated analogs piceatannol and resveratrol (Brents et al., 2012). Furthermore, arachidin-1 and arachidin-3 exhibited specific bioactivities not found in their non-prenylated forms, such as inhibiting the replication of rotavirus in HT29.f8 cells (Ball et al., 2015). Also, prenylated stilbenoids showed higher affinity to human cannabinoid receptors when compared to non- prenylated stilbenoids (Brents et al., 2012).

Prenyltransferase(s) responsible for these stilbenoid modifications is/are crucial for the biosynthesis of peanut bioactive compounds of interest. To date however, no gene encoding a stilbenoid prenyltransferase has been identified in plants. Previously, we developed a peanut hairy root culture system to serve as a platform for sustainable production of prenylated stilbenoids (Yang et al., 2015; Condori et al., 2010). Leveraging this system more recently, we characterized biochemically the first stilbenoid-specific prenyltransferase activity from the microsomal fraction of these elicited cultures (Yang et al., 2016). This prenyltransferase utilizes plastid-derived dimethylallyl pyrophosphate (DMAPP) to prenylate resveratrol or piceatannol into arachidin-2 or arachidin-5, respectively, and shares several features in common with flavonoid prenyltransferases reported in other legume species. For instance, all identified flavonoid and stilbenoid prenyltransferase enzymatic reactions require divalent cations as cofactors, and show maximum activity at basic pH (Sasaki et al., 2008; Akashi et al., 2008; Sasaki et al., 2011; Shen et al, 2012; Li et al., 2014; Chen et al., 2013; Yang et al., 2016). These characteristics and others guided cloning of the peanut prenyltransferase genes described here. We took a dual approach, combining parallel targeted transcriptome and metabolome analyses to isolate prenyltransferase activities. We generated extensive RNAseq data assemblies designed to capture elicitor-induced mRNAs, and used these sequences to clone potential prenyltransferase cDNAs. Here we describe the first transcripts to be identified as encoding stilbenoid-specific prenyltransferase enzymes. These catalyze two distinct dimethylallylation reactions in the biosynthesis of prenylated stilbenoids. Functionalities of the enzymes are uncovered using a transient expression system of Agrobacterium-mfiltr&ted Nicotiana benthamiana leaves, and their subcellular location is proposed from fluorescence microscopy imaging in particle- bombarded onion epidermal cells. We further use available genome assemblies of the two diploid progenitors of A. hypogaea to align the prenyltransferase transcripts and estimate their genomic structure.

It is an object of the present invention to isolate or purify nucleic acid molecules comprising a gene sequence that encodes a polypeptide having stilbenoid prenyltranferase activity.

It is another object of the present invention to produce a prenylated stilbenoid in an organism, cell or tissue, said method comprising the expression or over-expression of a gene that encodes for a stilbenoid prenyltransferase.

It is another object of the present invention to produce a prenylated stilbenoid in an organism wherein the organism is peanut, tobacco, grape, muscadine, Polygonum, pine, or yeast.

It is another object of the present invention to produce a prenylated stilbenoid in an organism wherein the method comprises expressing a gene sequence that encodes a polypeptide having stilbenoid prenyltransferase activity in an organism, including but not limited to, peanut, tobacco, grape, muscadine, Polygonum, pine, or yeast.

It is another object of the present invention to provide a new prenylated stilbenoid.

It is another object of the present invention to provide an improved method of producing a prenylated stilbenoid.

It is another object of the present invention to provide a method of producing the improved stilbenoid.

It is another object of the present invention to produce the stilbenoid in greater quantities. It is another object of the present invention to provide a more effective stilbenoid. It is another object of the present invention to provide a method of producing the more effective stilbenoid.

These and other objects and advantages of the present invention, along with features of novelty appurtenant thereto, will appear or become apparent by reviewing the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following drawings, which form a part of the specification and which are to be construed in conjunction therewith, and in which like reference numerals have been employed throughout wherever possible to indicate like parts in the various views:

Figure 1 shows A, Structure of stilbene backbone and main prenylation patterns found on peanut prenylated stilbenoids. B, Chemical structures of stilbenoids identified in elicited peanut hairy roots: (a) resveratrol; (b) piceatannol; (c) arachidin-2; (d) arachidin-5; (e) arachidin-3; (f) arachidin-1. All compounds are shown in their trans isomers.

Figure 2 shows a HPLC chromatogram (UV 320 nm) of ethyl acetate extract from peanut hairy root culture medium after 72 h post treatment with 100 μΜ methyl jasmonate and 9 g/L methyl- -cyclodextrin. a, arachidin-5 derivative; b, arachidin-2 derivative.

Figure 3 shows effects of mevastatin and clomazone on the production of stilbenoids in elicited peanut hairy root culture. A, HPLC chromatograms (UV 340 nm) of ethyl acetate extracts from peanut hairy root cultures after 48-hour treatment with (a) 100 μΜ methyl jasmonate (MeJA) and 9 g/L methyl-P-cyclodextrin (CD); (b) 100 μΜ MeJA, 9 g/L CD, and 10 μΜ mevastatin; (c) 100 μΜ MeJA, 9 g/L CD, and 10 μΜ clomazone and (d) 100 μΜ MeJA, 9 g/L CD, and 100 μΜ clomazone. B, Yields of resveratrol, arachidin-2, arachidin-3, piceatannol, arachidin-5, and arachidin-1 after 48 hour and 72 hour co-treatment of 10 μΜ mevastatin, 10 μΜ or 100 μΜ clomazone with elicitors (CD + MeJA). Values shown are the average mM yield of three replicates and error bars represent standard deviation. The asterisks above bars represent significant difference when compared to the group treated with MeJA and CD alone (*, j?<0.05; **, _p<0.01; ***, pO.001; ****, p< 0.0001; n.s., not significant; n.d., not detected ); and the asterisks above the connecting line represent significant difference between 48 hour and 72 hour treatments (*, p<0.05; ***, p<0.001; ****, p< 0.0001; n.s., not significant). Statistical analyses were performed by two-way ANOVA with Dunnett's and Sidak's multiple-comparisons test respectively. .

Figure 4 shows enzymatic degradation of resveratrol by crude cell-free protein extract from non-elicited peanut hairy root. HPLC chromatograms (UV 320 nm) of ethyl acetate extracts from the incubation mixtures contained 1 mM resveratrol with (A) 50 μg crude cell-free protein extract for 30 min; (B) 50 μg heat-denatured crude cell-free protein extract for 30 min; (C) 50 μg crude cell-free protein extract and additional 5 mM DTT for 120 min. All reactions were done in a pH 7.6 Tris-HCl buffer. (D) Close-up (3.92-7.19 min) of chromatogram shown in (A).

Figure 5 shows resveratrol prenyltransferase activity in microsomal fraction of elicited peanut hairy root. HPLC chromatograms (UV 320 nm) of (A) purified arachidin-2; (B) ethyl acetate extract of 500 μΐ reaction mixture containing 30 μg microsomal fraction, 100 μΜ resveratrol, 300 μΜ DMAPP, 10 mM MgCl ₂ and 5 mM DTT (C) ethyl acetate extract of 500 μΐ reaction mixture containing heat-denatured 30 μg microsomal fraction, 100 μΜ resveratrol, 300 μΜ DMAPP, 10 mM MgCl ₂ and 5 mM DTT. All reactions were done for 60 min in a pH 9.2 Tris-HCl buffer.

Figure 6 shows substrate specificity of resveratrol prenyltransferase in microsomal fraction of elicited peanut hairy root. A, Chemical structures of prenyl acceptors used for substrate specificity analysis and their prenylated products: stilbenoids (al, resveratrol, a2 arachidin-2; bl, piceatannol, b2, arachidin-5; cl, pinosylvin, c2, chiricanine-A, d, oxyresveratrol and e, pterostilbene), flavanone (e, naringenin), flavone (f, apigenin) and isoflavone (g, genistein). B, Prenylation activity of microsomal fraction with various prenyl acceptors. Values are the average of 2 replicates and error bars represent standard deviation (n.d., not detected). Figure 7 shows a table of detected stilbenoids.

Figure 8 shows a table of resveratrol prenyltransferase activity.

Figure 9 shows enzymatic characterization of AhR4DT transiently expressed in Nicotiana benthamiana leaf. HPLC chromato grams (UV 320 nm) of ethyl acetate extraction of 1000 μΐ reaction mixture of 200 μΜ resveratrol, 600 μΜ DMAPP, 10 mM MgCl ₂ and 10 mM DTT incubated with 50 μΐ crude protein of Nicotiana benthamiana leaf after vacuum infiltration with Agrobacterium tumefaciens LBA4404 harboring pBIBKan-9bl3 binary vector for (A) 48 hours and (B) 72 hours in a pH 9.2 Tris-HCl buffer for 40 min. Chromatograms of the reactions with (C) heated denatured protein) and (D) no DMAPP added for control.

Figure 10 shows enzymatic characterization of resveratrol prenyltransferase transiently expressed in Nicotiana benthamiana leaf. HPLC chromatograms (UV 320 nm) of ethyl acetate extraction of 1000 μΐ reaction mixture of 200 μΜ resveratrol, 600 μΜ DMAPP, 10 mM MgCl ₂ and 10 mM DTT incubated with 50 μΐ crude protein of Nicotiana benthamiana leaf after vacuum infiltration with Agrobacterium tumefaciens LBA4404 harboring (A) pBIBKan-lOkl ; (B) pBIBKan-4el; (C) pBIBKan-4elO and (D) pBIBKan-5m3 binary vectors in a pH 9.2 Tris-HCl buffer for 40 min.

Figure 11 shows the development of transgenic tobacco plants expressing a stilbenoid- specific prenyltransferase from peanut. A, B: Regenerated shoots from the Agrobacterium inoculation sites. C: Rooting of transgenic tobacco plant.

Figure 12 shows a sequence view of the gene and protein sequence of stilbenoid-specific prenyltransferase AhR4DT-9bl3 (renamed as AhR4DT-l; GenBank accession No. KY565244) involved in the biosynthesis of arachidin-2. Figure 13 shows a sequence view of the gene and protein sequence of stilbenoid prenyltransferase AhRPT-4el (Renamed as AhR3'DT-2; GenBank Accession No. KY565246).

Figure 14 shows a sequence view of the gene and protein sequence of stilbenoid prenyltransferase AhRPT-4elO (Renamed as AhR3'DT-3; GenBank Accession No. KY565247).

Figure 15 shows a sequence view of the gene and protein sequence of stilbenoid prenyltransferase AhRPT-5m3 (Renamed as AhR3'DT-4; GenBank AccesionNo. KY565248).

Figure 16 shows a sequence view of the gene and protein sequence of stilbenoid prenyltransferase AhRPT-lOkl (AC1) (Renamed as AhR3'DT-l; GenBank AccesionNo. KY565245).

Figure 17 shows 1H NMR analysis of arachidin-5. ^! H NMR was recorded at 400 MHz in acetone-d ₆ on a Bruker AV-400 NMR spectrometer.

Figure 18 shows ¹³ C NMR analysis of arachidin-5. ¹³ C NMR was recorded at 100 MHz in acetone-d ₆ on a Bruker AV-400 NMR spectrometer.

Figure 19 shows effect of mevastatin on the production of stilbenoids in elicited peanut hairy root culture. HPLC chromatograms (UV 340 nm) of ethyl acetate extracts from peanut hairy root cultures after 48-hour treatment with (A) 100 μΜ methyl jasmonate (MeJA) and 9 g/L methyl- -cyclodextrin (CD); (B) 100 μΜ MeJA, 9 g/L CD, and 1 μΜ mevastatin; (C) 100 μΜ MeJA, 9 g/L CD, and 10 μΜ mevastatin and (D) 100 μΜ MeJA, 9 g/L CD, and 100 μΜ mevastatin.

Figure 20 shows effect of clomazone on the production of stilbenoids in elicited peanut hairy root culture. HPLC chromatograms (UV 340 nm) of ethyl acetate extracts from peanut hairy root cultures after 48-hour treatment with (A) 100 μΜ methyl jasmonate (MeJA) and 9 g/L methyl- -cyclodextrin (CD); (B) 100 μΜ MeJA, 9 g/L CD, and 1 μΜ clomazone; (C) 100 μΜ MeJA, 9 g/L CD, and 10 μΜ clomazone and (D) 100 μΜ MeJA, 9 g/L CD, and 100 μΜ clomazone.

Figure 21 shows effect of clomazone on the production of stilbenoids in non-elicited peanut hairy root culture. HPLC chromatograms (UV 340 nm) of ethyl acetate extracts from peanut hairy root cultures after 48-hour treatment with (A) 100 μΜ methyl jasmonate and 9 g/L methyl-P-cyclodextrin; (B) 10 μΜ clomazone; (C) 50 μΜ clomazone and (D) 100 μΜ clomazone.

Figure 22 shows comparison of stilbenoid yields from the culture medium and root tissue. Ethyl acetate extracts were prepared from elicited peanut hairy root cultures. HPLC chromatograms (UV 340 nm) of ethyl acetate extract from (A) culture medium and (B) lyophilized root tissue. Cultures were elicited for 48 hours with 100 μΜ methyl jasmonate and 9 g L methyl- -cyclodextrin.

Figure 23 shows time course of resveratrol prenyltansferase assay without DTT and subsequent degradation of resveratrol and arachidin-2 in the reaction. Assays were done for 0 to 120 min. HPLC chromatograms (UV 320 nm) of ethyl acetate extracts from the reaction mixture of 30 μg microsomal fraction, 100 μΜ resveratrol, 300 μΜ DMAPP, and 10 niM MgCl ₂ . Assays were done for 0, 10, 20, 30 , 45, 60 or 120 min in a pH 9.2 Tris-HCl buffer.

Figure 24 shows biotransformation of arachidin-2 by protein extracts from elicited peanut hairy root culture. HPLC chromatograms (UV 320 nm) of ethyl acetate extracts from the 60 min reaction contained (a) 100 μΜ arachidin-2 with 30 μg heat-denatured crude cell-free protein extract as control; (b) 30 μg crude cell-free protein extract; (c) 30 μg microsomal fraction and (d) 30 μg 156,000 g supernatant. All reactions were done in apH 9.2 Tris-HCl buffer. Figure 25 shows resveratrol prenyltransferase activity. A, Concentrations of remaining resveratrol and generated arachidin-2 from the reaction mixtures with varying incubation times (30, 60, 90 and 120 min) were measured by HPLC. B, Concentrations of remaining resveratrol and generated arachidin-2 from the reaction mixtures with varying amounts of microsomal fraction (30, 60, 90 and 120 μg) were measured by HPLC.

Figure 26 shows pH dependency of resveratrol prenyltransferase activity. Resveratrol prenyltransferase activity at various pH values was measured using 100 mM Tris-HCl buffer at pH 7.0, 8.0, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6 and 10.

Figure 27 shows divalent cation requirement for resveratrol prenyltransferase activity. Resveratrol prenyltransferase activity was compared in the presence of various divalent cations: Mg ²⁺ ,' Mn ²⁺ , Fe ²⁺ , Ca ²⁺ , Co ²⁺ , Zn ²⁺ , Ni ⁺ and Cu ²⁺ . The activity in the presence of Mg ²⁺ is shown as 100%.

Figure 28 shows biochemical characterization of resveratrol prenyltransferase in microsomal fraction of elicited peanut hairy root. HPLC chromatogram (UV 320 nm) of ethyl acetate extract of a 60 min incubation mixture containing (A) standard reaction (30 μg microsomal fraction, 100 μΜ resveratrol, 300 μΜ DMAPP, 10 mM MgCl ₂ and 5 mM DTT in a pH 9.2 Tris-HCl buffer); (B) standard reaction without divalent cation added; (C) standard reaction with 300 μΜ IPP instead of DMAPP and (D) standard reaction with 30 g microsomal fraction isolated from non-elicited peanut hairy root instead of 30 μg microsomal fraction isolated from elicited peanut hairy root.

Figure 29 shows effects of (A) resveratrol and (B) DMAPP concentrations on resveratrol prenyltransferase activity. Enzymatic activity was measured with a microsomal fraction from peanut hairy roots. The apparent K _m and V _max values for resveratrol and DMAPP were determined with varying concentrations of resveratrol (10 - 640 μΜ) and of DMAPP (10 - 640 μΜ) respectively and calculated via nonlinear regression analysis with Michaelis-Menten equation by Graphpad Prism 6 software.

Figure 30 shows substrate specificity of resveratrol prenyltransferase in microsomal fraction of elicited peanut hairy root. HPLC chromatograms (UV 320 nm) of ethyl acetate extracts of a 60 min incubation mixture containing 30 μg microsomal fraction with 300 μΜ DMAPP, 10 niM MgCl ₂ and 5 mM DTT, and 100 μΜ prenyl acceptors (A, resveratrol; B, piceatannol; C, pinosylvin; D, pterostilbene; E, naringenin; F, apigenin and G, genistein) in a pH 9.2 Tris-HCl buffer.

Figure 31 shows biotransformation of arachidin-5 by protein fractions from elicited peanut hairy root culture. HPLC chromatograms (UV 320 nm) of ethyl acetate extracts from the 60 min incubation mixtures contained 40 μΜ arachidin-5 with (A) 30 μg heated microsomal fraction as control and (B) 30 μg microsomal fraction. All reactions were done in a.pH 9.2 Tris- HCl buffer.

Figure 32 shows substrate specificity of resveratrol prenyltransferase in microsomal fraction of elicited peanut hairy root. A, Chemical structures of oxyresveratrol and its prenylated product. B, HPLC chromatograms (UV 320 nm) of ethyl acetate extraction of reaction mixtures contained 100 μΜ oxyresveratrol, 300 μΜ DMAPP, 10 mM MgCl ₂ , 5 mM DTT and 30 μg microsomal fraction (above); heated denatured microsomal fraction (below) in a pH 9.2 Tris- HCl buffer for 60 min. C, HPLC-PDA-ESI-MS ³ analysis of oxyresveratrol, prenylated oxyresveratrol and its derivative.

Figure 33 shows enzymatic characterization of stilbenoid prenyltransferase genes (AhR4DT-9bl3, AhRPT-lOkl, AhRPT-4el, AhRPT-4elO, and AhRPT5m3) transiently expressed in Nicotiana benthamiana leaf. HPLC chromatograms (UV 320 nm) of ethyl acetate extraction of 1000 μΐ reaction mixture of 200 μΜ resveratrol, 600 μΜ DMAPP, 10 mM MgCl ₂ and 10 mM DTT incubated with 50 μΐ crude protein of Nicotiana benthamiana leaf after, vacuum infiltration with Agrobacterium tumefaciens LBA4404 harboring pBIBKan-9bl3 (A); pBIBKan- lOkl (B); pBIBKan-4el (C); pBIBKan-4elO (D); pBIBKan-5m3 (E) and pBIBKan (F) binary vectors in a pH 9.2 Tris-HCl buffer for 40 min.

Figure 34 shows resveratrol prenyltransferase activities in transgenic tobacco {Nicotiana tabacum) expressing AhRPT-lOkl gene cloned from peanut hairy roots. HPLC chromatograms (UV 320 nm) of ethyl acetate extraction of 500 μΐ reaction mixture of 200 μΜ resveratrol, 300 μΜ DMAPP, 10 mM MgCl ₂ and 10 mM DTT incubated with 50 g crude protein of transgenic N. tabacum leaf expressed lOkl gene (A) and 50 μg of microsomal fraction of transgenic N. tabacum hairy roots expressed lOkl gene (B) for 2 hours.

Figure 35 shows the enzymatic characterization of recombinant stilbenoid

prenyltransferases expressed in Nicotiana benthamiana. (A) AhR4DT-l and AhR'3DT-l/2/3/4 from peanut catalyzes the 4 and 3'-prenylation of resveratrol, respectively. (B) Enzymatic characterization of resveratrol prenyltransferase transiently expressed in Nicotiana benthamiana leaf. HPLC chromatograms (UV 320 nm) of ethyl acetate extract of 1 mL reaction mixture of 100 μΜ resveratrol, 300 μΜ DMAPP, 10 mM MgCl ₂ and 10 mM DTT incubated with 5 mg crude protein of N. benthamiana leaf after vacuum infiltration wit Agrobacterium tumefaciens LBA4404 harboring (B) pBIB-Kan-AhR4DT-l ; (C) pBIB-Kan-AhR3'DT-l; (D) pBIB-Kan- AhR3'DT-2; (E) pBIB-Kan-AhR3 'DT-3 ; (F) pB IB-Kan- AhR3 'DT-4 and (G) pBIB-Kan binary vectors in a pH 9.0 Tris-HCl buffer for 40 min. Figure 36 shows the primary structures of stilbenoid prenyltransferases. Two conserved NQXXDXXXD and KDXXDXEGD motifs are boxed.

Figure 37 shows the phylogenetic relationship between peanut stilbenoid

prenyltransferases and related prenyltransferases accepting aromatic substrates. Species abbreviations are: Aa, Allium ampeloprasum; Ah, Arachis hypogaea; At, Arabidopsis thaliana; CI, Citrus limon; Cp, Cuphea avigera var pulcherrima; Cr, Chlamydomonas reinhardtii; Ct, Cudrania tricuspidata; Gm, Glycine max; HI, Humulus lupulus; Hv, Hordeum vulgare; La, Lupinus albus; Le, Lithospermum erythrorhizon; Ma, Morus alba; Os, Oryza sativa; Pc, Petroselinum crispum; Sf, Sophora flavescens; Ta, Triticum aestivum; Zm, Zea mays.

Homogentisate phytyltransferases (VTE2-ls) and homogentisate geranylgeranyltransferases (HGGTs), homogentisate solanesyltransferases (VTE2-2s), _j p-hydroxybenzoate

geranyltransferase (PGT) and -hydroxybenzoate polyprenyltransferases (PPTs) are involved in the biosynthesis of vitamin E, plastoquinone, shikonin and ubiquinone, respectively. IDT is isoliquiritigenin dimethylallyltransferase. Accession numbers of these proteins are listed in Figure 69.

Figure 38 shows the substrate specificity of AhR4DT-l and AhR3'DT-l. A, Chemical structures of prenyl acceptors used for substrate specificity analysis and their prenylated products: stilbenoids (a, resveratrol, b, piceatannol, c, oxyresveratrol, d, pinosylvin, e, piceid and f, pterostilbene), flavanone (g, naringenin), flavone (h, apigenin) and isoflavone (i, genistein). B, Relative prenylation activity of AhR4DT-l (B) and AhR3'DT-l (C) with various prenyl acceptors were compared with that of resveratrol. The prenyl donor specificity of AhR4DT-l (D) and AhR3'DT-l (E) were tested using DMAPP, IPP, GPP, FPP, GGPP with resveratrol as a prenyl acceptor. All these reactions were performed in 100 mM Tric-HCl buffer (pH 9.0) at 28°C for 40 mins. Values are the average of triplicate and error bars represent standard deviation (n.d., not detected).

Figure 39 shows the microscopic analysis of subcellular localization of AhR4DT-l and AhR3'DT-l fused with GFP in onion epidermal cells upon particle bombardment. (A, D) Plasmids containing CaMV35S-TEV-GFP and RS-TP-mCherry, (B, E) AhR4DT-l-GFP and RS- TP-mCherry or (C, F) AhR3 'DT-l-GFP and RS-TP-mCherry were mixed and introduced into onion epidermal cells by particle bombardment.

Magnification: 20 x; Scale bars in A, B and C equal 100 μιη.

Al , B 1 , C 1 : Bright field images of onion cells.

A2, B2, C2: Expression pattern of mCherry from construct CaMV35S-RS-TP-mCherry

A3: Expression pattern of GFP from construct CaMV35S-TEV-GFP

B3: Expression pattern of GFP from construct AhR4DT-l-GFP

C3: Expression pattern of GFP from construct AhR3 'DT-l-GFP

A4: Merge of A2 and A3

B4: Merge of B2 and B3

C4: Merge of C2 and C3

Close up of the same cells shown. Scale bars in D, E and F equal 10 μπι

Dl, El, Fl : Expression pattern of mCherry from construct CaMV35 S-RS-TP -mCherry

D2: Expression pattern of GFP from construct CaMV35S-TEV-GFP

E2: Expression pattern of GFP from construct AhR4DT-l-GFP

F2: Expression pattern of GFP from construct AhR3'DT-l-GFP

D3: Merge of Dl and D2

E3: Merge of El and E2 F3: Merge ofFl and F2

CaMV35S: cauliflower mosaic virus 35S promoter; TEV: tobacco etch virus translational enhancer; GFP: green fluorescent protein; RS-TP: rubisco small subunit transit peptide.

Figure 40 shows the enzyme activities and transcript coexpression during elicitation time course in peanut hairy root cultures. (A) Prenyltransferase activities from crude cell-free extracts. (B) Relative transcript accumulation of AhR4DT-l and (C) AhR3 'DT-1 as determined by RT-qPCR. (D) Uniquely mapped RNA-Seq reads coverage over reference A. hypogaea transcripts AhR4DT-l, (E) AhR3 'DT-1, (F) AhR3 'DT-2/3 and (G) AhR3 'DT-4 as described. E9 and E72, 9 h and 72 h MeJA + CD treatment; C9 and C72, 9 h and 72 h control treatments.

Figure 41 shows the proposed pathway of prenylated stilbenoids in peanut. Stilbenoids identified from the medium of peanut hairy root culture upon elicitors treatment are bolded and their proposed pathway is highlighted in yellow. Other prenylated stilbenoids identified in fungal-challenged peanut seeds are divided into three groups based on the prenyl unit and hydroxyl groups on their stilbene backbone. Prenylation reactions catalyzed by AhR4DT-l and AhR3'DT-l identified in this study are labeled with red solid arrows. Enzymatic reactions confirmed in peanut are marked with black solid arrow, while other proposed reactions are labeled in black arrows with dashed lines. *: Pinosylvin, 3-methyl-2-butenyl-3'-resveratrol and the prenylation product of piceatannol by AhR3'DT-l have not been reported in peanut tissue.

Figure 42 shows the prenylation of resveratrol by AhR3'DT-l.

Prenyltransferase activity of AhR3'DT-l in microsomal fraction of Nicotiana benthamiana leaf after vacuum infiltration with Agrobacterium tumefaciens LBA4404 harboring pBIB-Kan- AhR3'DT-l was confirmed by HPLC and LC-MS" analysis. (A) Chemical structures of resveratrol and its prenylated product, 3-methyl-2-butenyl~3'- resveratrol.

(B) HPLC chromatograms (UV 320 nm) of ethyl acetate extraction of reaction mixtures contained 100 μΜ resveratrol, 300 μΜ DMAPP, 10 mM MgCl ₂ , 5 mM DTT and 30 μg microsomal fraction in a pH 9.0 Tris-HCl buffer for 40 min.

(C) HPLC-P AD-ESI-MS ³ analysis of prenylated product, 3 -methyl-2-butenyl-3 ' - resveratrol.

Figure 43 shows NMR spectra of the lmM 3 -methyl-2-butenyl-3' -resveratrol isolated from a large-scale enzymatic assay.

ID 1H NMR (A), ID ¹³ C NMR spectrum (B), 2D 1H- ¹³ C HMBC (C) and 2D ^l R- ^l3 C HSQC (D) spectra obtained on 700 MHz Bruker Avance spectrometer dissolved in d6-Acetone at 298K. The peaks circled in red are consistent in both ID and 2D experiments which represent protons on the specific locations of the scaffold.

Figure 44 shows Prenylation of resveratrol by AhR4DT-l.

Prenyltransferase activity of AhR4DT-l in microsomal fraction of Nicotiana

benthamiana leaf after vacuum infiltration with Agrobacterium tumefaciens LBA4404 harboring pBIB-Kan-AhR4DT- 1 was confirmed by HPLC and LC-MS ⁿ analysis.

(A) Chemical structures of resveratrol and its prenylated product, arachidin-2.

(B) HPLC chromatograms (UV 320 nm) of ethyl acetate extraction of reaction mixtures contained 100 μΜ resveratrol, 300 μΜ DMAPP, 10 mM MgCl ₂ , 5 mM DTT and 30 μg microsomal fraction in a pH 9.0 Tris-HCl buffer for 40 min.

(C) HPLC-PAD-ESI-MS ³ analysis of prenylated product, arachidin-2.

Figure 45 shows the Comparison of AhR4DT-l and AhRPT-9i2. (A) Alignment of AhR4DT-l with AhRPT-9i2 was performed using ClustalX.

(B) Potential transmembrane domains of AhR4DT-l and AhRPT-9i2 were predicted by TMHMM.

(C) Resveratrol prenylation activity of AhR4DT-l and AhRPT-9i2 were analyzed by using HPLC.

Figure 46 shows a comparison of AhR3'DT-l, AhRPT-10a4 and AhRPT-10d4.

(A) Alignment of AhR3'DT-l, AhRPT-10a4 and AhRPT-10d4 was performed using ClustalX.

(B) Potential transmembrane domains of AhR3'DT-l, AhRPT-10a4 and AhRPT-10d4 were predicted by TMHMM.

(C) Resveratrol prenylation activity of AhR3'DT-l, AhRPT-10a4 and AhRPT-10d4 were analyzed by using HPLC.

Figure 47 shows a comparison of AhR3'DT-2 and AhR3'DT-3.

(A) Alignment of AhR3'DT-2 with AhR3'DT-3 was performed using ClustalX.

(B) Potential transmembrane domains of AhR3'DT-2 and AhR3'DT-3 were predicted by TMHMM.

(C) Resveratrol prenylation activity of AhR3'DT-2 and AhR3'DT-3 were analyzed by using HPLC.

Figure 48 shows a structural analysis of AhR3'DT-4.

(A) Primary structure of AhR3'DT-4.

(B) Potential transmembrane domains of AhR3'DT-4 was predicted by TMHMM.

(C) Resveratrol prenylation activity of AhR3'DT-4 was analyzed by using HPLC. Figure 49 shows a temperature dependency of AhR4DT-l and AhR3'DT-l activity. AhR4DT-l (A) and AhR3'DT-l (B) activities were measured at various temperature (20, 25, 28, 30, 37, 40 and 50 °C) in 100 mM Tris-HCl buffer (pH 9.0) for 40 mins.

Figure 50 shows the incubation time and amounts of microsomal fraction dependency of AhR4DT-l and AhR3'DT-l activity.

(A) Concentrations of generated prenylated resveratrol from AhR4DT-l and AhR3'DT-l reaction mixtures with varying incubation times (30, 60, 90 and 120 min) were quantified by HPLC.

(B) Concentrations of generated prenylated resveratrol from AhR4DT-l and AhR3'DT-l reaction mixtures with varying amounts of microsomal fraction (25, 50, 75 and 100 μg) were quantified by HPLC.

Figure 51 shows a pH dependency of AhR4DT-l and AhR3'DT-l activity.

AhR4DT-l (A) and AhR3'DT-l (B) activities at various pH values were measured in three different buffers: 100 mM Tris-HCl buffer at pH 7.0, 8.0, 8.6 and 9.0; 100 mM Glycine-NaOH buffer at pH 8.6, 9.0, 9.4, 10.0 and 10.6; and 100 mM NaHC0 ₃ -Na ₂ C0 ₃ buffer atpH 9.2, 9.7, 10.2 and 10.7. All the reactions were performed at 28 °C for 40 min.

Figure 52 shows a divalent cation dependency of AhR4DT-l and AhR3'DT-l activity. AhR4DT-l (A) and AhR3'DT-l (B) activity with various divalent cation were measured with 10 mM MnCl ₂ , FeCl ₂ , CaCl ₂ , CoCl ₂ , ZnC12, NiCl ₂ , or CuCl and the enzyme activity was compared with the reaction of 10 mM MgCl ₂ . Reactions without divalent cation and 10 mM EDTA instead of MgCl ₂ were used as controls. All the reactions were performed in 100 mM Tris-HCl buffer (pH 9.0) at 28 °C for 40 min. (t.a., trace amount (<0.5%), n.d., Not detected.). Means and the standard deviation (error bars) were calculated from three replicates. Figure 53 shows the kinetic values of AhR4DT-l and AhR3'DT-l.

Dependency of AhR4DT-l (left) and AhR3'DT-l (right) on the concentration of resveratrol (A&D), piceatannol (B&E) and DMAPP (C&F) measured with a microsomal fraction from leaves of Nicotiana benthamiana. The apparent K _m and V _max values for resveratrol and piceatannol were determined with varying concentrations (10-640 μΜ) using 640 μΜ DMAPP as prenyl donor, while that for DMAPP were determined with varying concentrations (10-640 μΜ) using 640 μΜ resveratrol as prenyl acceptor. All the values were calculated from nonlinear regression analysis with Michaelis-Menten equation by Graphpad Prism 6 software. Means and the standard deviation (error bars) were calculated from three replicates.

Figure 54 shows the prenylation of piceatannol by AhR4DT-l .

Substrate specificity of AhR4DT-l in microsomal fraction of Nicotiana benthamiana leaf after vacuum infiltration with Agrobacterium tumefaciens LBA4404 harboring pBIB-Kan-AhR4DT-l was analyzed by HPLC and LC-MS".

(A) Chemical structures of resveratrol and its prenylated product.

(B) HPLC chromatograms (UV 320 nm) of ethyl acetate extraction of reaction mixtures contained 100 μΜ piceatannol, 300 μΜ DMAPP, 10 mM MgCl ₂ , 5 mM DTT and 30 μg microsomal fraction in a pH 9.0 Tris-HCl buffer for 40 min.

(C) HPLC-PAD-ESI-MS ³ analysis of prenylated product, arachidin-5.

Figure 55 shows the prenylation of pinosylvin by AhR4DT-l .

Substrate specificity of AhR4DT-l in microsomal fraction of Nicotiana benthamiana leaf after vacuum infiltration with Agrobacterium tumefaciens LBA4404 harboring pBIB-Kan- AhR4DT-l was analyzed by HPLC and LC-MS".

(A) Chemical structures of resveratrol and its prenylated product. (B) HPLC chromatograms (UV 320 nm) of ethyl acetate extraction of reaction mixtures contained 100 μΜ pinosylvin, 300 μΜ DMAPP, 10 mM MgCl ₂ , 5 mM DTT and 30 μg microsomal fraction in a pH 9.0 Tris-HCl buffer for 40 min.

(C) HPLC-PAD-ESI-MS ³ analysis of prenylated product, chiricanine A.

Figure 56 shows the prenylation of oxyresveratrol by AhR4DT-l.

Substrate specificity of AhR4DT-l in microsomal fraction of Nicotiana benthamiana leaf after vacuum infiltration with Agrobacterium tumefaciens LBA4404 harboring pBIB-Kan-AhR4DT-l was analyzed by HPLC and LC-MS ⁿ .

(A) Chemical structures of resveratrol and its prenylated product.

(B) HPLC chromatograms (UV 320 nm) of ethyl acetate extraction of reaction mixtures contained 100 μΜ oxyresveratrol, 300 μΜ DMAPP, 10 mM MgCl ₂ , 5 mM DTT and 30 μg microsomal fraction in a pH 9.0 Tris-HCl buffer for 40 min.

(C) HPLC-PAD-ESI-MS ³ analysis of prenylated product.

Figure 57 shows the prenylation of piceatannol by AhR3'DT-l.

Substrate specificity of AhR3'DT-l in microsomal fraction of Nicotiana benthamiana leaf after vacuum infiltration with Agrobacterium tumefaciens LBA4404 harboring pBIB-Kan- AhR3'DT-l was analyzed by HPLC and LC-MS".

(A) Chemical structures of resveratrol and its prenylated product, arachidin-2.

(B) , HPLC chromatograms (UV 320 nm) of ethyl acetate extraction of reaction mixtures contained 100 μΜ piceatannol, 300 μΜ DMAPP, 10 mM MgCl ₂ , 5 mM DTT and 30 μg microsomal fraction in a pH 9.0 Tris-HCl buffer for 40 min.

(C) HPLC-PAD-ESI-MS ³ analysis of prenylated product.

Figure 58 shows the prenylation of oxyresveratrol by AhR3'DT-l. Substrate specificity of AhR3'DT-l in, microsomal fraction of Nicotiana benthamiana leaf after vacuum infiltration vAthAgrobacterium tumefaciens LBA4404 harboring pBIB-Kan- AhR3'DT-l was analyzed by HPLC and LC-MS".

(A) Chemical structures of resveratrol and its prenylated product, arachidin-2.

(B) HPLC chromatograms (UV 320 nm) of ethyl acetate extraction of reaction mixtures contained 100 μΜ oxyresveratrol, 300 μΜ DMAPP, 10 mM MgCl ₂ , 5 mM DTT and 30 μg microsomal fraction in a pH 9.0 Tris-HCl buffer for 40 min.

(C) HPLC-PAD-ESI-MS ³ analysis of prenylated product.

Figure 59 shows the cloning strategy of binary vectors for peanut prenyltransferase genes screening.

Figure 60 shows the cloning strategy of binary vectors for subcellular localization of AhR4DT-l or AhR3'DT-l.

Figure 61 shows the cloning strategy of binary vectors containing GFP for subcellular localization control.

Figure 62 shows the primer efficiencies for real-time qPCR.

The efficiency of AhR4DT-l (A) was tested in 5x serial dilutions of peanut hairy root cDNA (range of 2 to 0.00032 ng). The efficiencies of AhR3 'DT-1 (B) was calculated in the range of 10 to 0.0064 ng cDNA, and that of ATC7 (C) and EFal (D) were performed with 10 to 0.00032 ng cDNA.

Figure 63 shows a table of Prenyltransferase transcripts described in this application. Activities of expressed protein products and loci of best alignment in diploid Arachis reference genomes are shown. Figure 64 shows a table of AhR4DT-l and AhR3'DT-l activity from N. benthamiana leaves fractions. The preparation of fractions and the resveratrol prenyltransferase assay are described in "Materials and Methods." Values are the mean ± standard deviation for three replicates.

Figure 65 shows a table of comparison of kinetic values of AhR4DT-l, AhR3'DT-l and prenyltransferase activity identified from peanut hairy root. The apparent K _m and V _max values of AhR4DT-l and AhR3'DT-l for resveratrol, piceatannol and DMAPP were measured using the microsomal fraction of N. benthamiana leaves transiently expressing these two enzymes. Values are the mean ± standard deviation of three replicates. The apparent K _m and F _max values of prenyltransferase activity identified from the microsomal fraction of peanut hairy root for resveratrol and DMAPP were previously reported by Yang et al. (2016).

Figures 66A-66B show a list of primers used in this study. The restriction site on each primer is underlined.

Figure 67 shows substrates used for specificity assay and the prenylated products from reaction mixtures catalyzed by AhR4DT-l or AhR3'DT-l . Analysis was done by HPLC-PDA- electrospray ionization-MS3.

Figure 68 shows a list of plasmids used in this study. For details of the binary vectors, see Materials and Methods.

Figures 69A-69B show accession numbers of proteins used for phylogenetic analysis.

Figure 70 shows a primer design for analysis of transgenic plants expressing peanut stilbenoid-specific prenyltransferases (AhR4DT-l and AhR3'DT-l). Targeting position of primers, SubLoc-F-pBIBKan and SubLoc-R-pBIBKan on (A) pBIBKan, (B) pBIB-Kan- ^' AhR4DT- 1 and (C) pBIB-Kan-AhR3'DT- 1. Figure 71 shows a characterization of transgenic tobacco plants expressing peanut stilbenoid prenyltransferase AhR4DT-l gene.

(A) AhR4DT-l from peanut catalyzes the 4-C prenylation of resveratrol.

(B) PCR Analyses of transgenic tobacco plants expressing AhR4DT-l gene. Genomic DNA was isolated from transgenic lines 1, 2, 3, 4, 5 and 6. Plasmid pBIB-Kan-AhR4DT-l was used as positive control (+). Genomic DNAs of the transgenic plant transformed with pBIB-Kan empty vector and wild type plant were used as empty vector control (-) and negative control, respectively. Positive amplicon (2673 bp); negative amplicon (523 bp).

(C) Enzymatic characterization of AhR4DT-l activity in the leaf of transgenic tobacco plants. HPLC chromatograms of ethyl acetate extract of 1 mL reaction mixture of 100 μΜ resveratrol, 300 μΜ DMAPP, 10 n M MgCl ₂ and 5 mM DTT incubated with 50 μΐ of crude protein (approximately 75 μg) from the leaf of transgenic tobacco plants in a pH 9.0 Tris-HCl buffer for 40 min. The crude protein from the leaf of transgenic plant transformed with pBIB- Kan vector was used as empty vector control.

Figure 72 shows a characterization of transgenic tobacco plants expressing peanut stilbenoid prenyltransferase AhR3 'DT-1 gene.

(A) AhR3'DT-l from peanut catalyzes the 3'-C prenylation of resveratrol.

(B) PCR Analyses of transgenic tobacco plants expressing AhR3 'DT-1 gene. Genomic DNA was isolated from transgenic lines 1, 3, 4, 5, 6, 7, 8, 10, 12 and 14. Plasmid pBIB-Kan- AhR3'DT-l was used as positive control (+). Genomic DNAs of the transgenic plant transformed with pBIB-Kan vector was used as empty vector control (-). Positive amplicon (2619 bp); negative amplicon (523 bp). (C) Enzymatic characterization of AhR3'DT-l activity in the leaf of transgenic tobacco lines. HPLC chromatograms of ethyl acetate extract of 1 mL reaction mixture of 200 μΜ resyeratrol, 300 μΜ DMAPP, 10 mM MgCl ₂ and 5 mM DTT incubated with 50 μΐ

(approximately 75 μg) of crude protein from the leaf of transgenic tobacco lines in a pH 9.0 Tris- HC1 buffer for 120 min. The crude protein from the leaf of transgenic plant transformed with pBIB-Kan vector was used as empty vector control.

Figure 73 shows a phenotype of transgenic hairy roots of tobacco expressing peanut stilbenoid prenyltransferase AhR3 'DT-1.

From left to right, hairy root lines developed from wild type of tobacco (WT), transgenic tobacco transformed with pBIB-Kan vector (pBIBKan - control), and AhR3 'DT-1 -expressing line 12 (Line 1 and Line 2). Hairy roots were developed via Agrobacterium rhizogenes- ediated transformation.

DETAILED DESCRIPTION

A substantial part of non-host defense responses in many plants is the pathogen-induced production of secondary metabolites, generally termed phytoalexins, that locally restrict disease progression due to bioactivities toxic to the pathogen (reviewed in Ahuja et al., 2012). Peanut or groundnut (Arachis hypogaea) tissues mount a defense against infection by the soil fungus Aspergillus flavus and other pathogens by overproducing stilbene derivatives around sites of wounding and elicitor perception (Sobolev, 2013). Resveratrol (3,5,4'-trihydroxy-stilbene), one of the most studied phytoalexin stilbenoids, has attracted great attention because of its bioactive properties shown through in vitro and in vivo assays to benefit human health. These include antiinflammatory (Das and Das, 2007) and antioxidant properties, as well as antitumor and favorable cardiovascular effects (Gambini et al., 2015). However, the limited oral bioavailability of resveratrol due to its rapid absorption and metabolism restricts the future of this potentially valuable drug in clinical trials (Tome-Carneiro et al., 2013; Gambini et al., 2015).

Prenylated stilbenoids naturally produced as phytoalexins in the peanut plant possess one or two isoprenyl moieties bound to the aromatic ring of the stilbene molecule (Figure 1). When compared to resveratrol, these compounds exhibit similar or enhanced bioactivity in in vitro experiments. For instance, arachidin-1 and resveratrol showed similar anti-inflammatory activity in lipid polysaccharide-treated RAW 264.7 macrophages and this correlated with the inhibition of prostaglandin E ₂ production (Chang et al., 2006; Djoko et al., 2007). Arachidin-1, arachidin-2 and arachidin-3, also applied to macrophages, were more effective than resveratrol in inhibiting inducible nitric oxide production (Sobolev et al., 2011). In other antioxidant activity assays, arachidin-1 inhibited lipid oxidation more effectively than resveratrol (Abbott et al., 2010), and arachidin-2 and arachidin-3 showed greater potency over resveratrol in inhibiting production of intracellular reactive oxygen species (Sobolev et al., 2011). Aracbidin-1 further showed higher cytotoxicity than resveratrol to leukemia HL-60 cells (Huang et al., 2010) and other cancer cells (SK-MEL, KB, BT-549, and SK-OV-3) (Sobolev et al., 2011). Interestingly, arachidin-1 and arachidin-3 were shown to bind to human cannabinoid receptors 2 (hCBR2s), while the affinity of their non-prenylated analogous, piceatannol and resveratrol for hCB2Rs was 5- to 10-fold lower. Molecular modeling studies with hCBR2s indicated that the prenyl moiety of the arachidins improved the binding affinity to the receptors (Brents et al., 2012).

In addition to the above-mentioned stilbenoids (arachidin-1, arachidin-2 and arachidin-3), more than 20 other prenylated stilbenoids have been described in peanut tissues (Sobolev et al., 2006; Wu et al., 2011; Sobolev, 2013; Sobolev et al, 2016). The biosynthesis of stilbenoids derives from both the phenylpropanoid and acetate pathways. These merge to produce resveratrol by the action of resveratrol synthase which catalyzes the cyclization of 4-coumaroyl- CoA and malonyl-CoA (Schoppner and Kindl, 1984). The prenylation step, in which either of two prenyl patterns (3,3-dimethylallyl or 3-methyl-but-l-enyl) are introduced to various positions of the stilbene backbone (Figure 1), along with the oxidation, methylation and cyclization steps plays a major role in the diversification of peanut prenylated stilbenoids. Although the enzymes involved in resveratrol biosynthesis have been elucidated (Chong et al., 2009), the enzymes involved in the prenylation steps of resveratrol or any other stilbenoid have not been described.

For other prenylated aromatic compounds, a prenyltransferase was found to be the critical activity for coupling the aromatic compound biosynthesis and terpenoid biosynthesis, the latter leading to the formation of the prenyl unit (Yazaki et al., 2009). Two pathways are known for the biosynthesis of prenylated compounds in plants, the mevalonic acid (MVA) pathway in the cytosol and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in the plastid (Lohr et al., 2012). Many studies have shown that dimethylallyl pyrophosphate (DMAPP) derived from the MEP pathway is used as prenyl donor to form prenylated flavonoids or prenylated isoflavonoids in the plastid (Yamamoto et al., 2000; Yazaki et al, 2009). To determine the biosynthetic origin of these terpenoids, distinct metabolic inhibitors were applied to inhibit the key rate limiting enzymes involved in either the MVA or MEP pathway. For instance, mevastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase involved in the MVA pathway was used in hairy root cultures of ginseng to study the biosynthesis of ginsenosides (Zhao et al., 2014); while clomazone, a herbicide that inhibits l-deoxy-D-xylulose-5-phosphate synthase (DXS) during early steps of DMAPP biosynthesis in the plastid was used to investigate the synthesis of monoterpenes in Catharanthus roseus (Han et al., 2013).

In order to elucidate the biosynthesis of peanut prenylated stilbenoids, we established hairy root cultures of peanut (Condori et al., 2010) and recently demonstrated that a sustainable production of the prenylated stilbenoids arachidin-1 and arachidin-3 can be achieved upon co- treatment of these cultures with methyl jasmonate (MeJA) and cyclodextrin (CD) (Yang et al., 2015a). In the current study, we took advantage of this bioproduction system and produced arachidin-2 and a new prenylated stilbenoid, named arachidin-5, as the prenylated products of the hairy root microsomal fraction using resveratrol and piceatannol as substrates, respectively. To determine the biosynthetic origin of the prenyl moiety of these prenylated stilbenoids, two metabolic inhibitors, mevastatin and clomazone were selected and applied to peanut hairy root cultures co-treated with MeJA and CD as elicitors. In the process, we identified and characterized a resveratrol 4-dimethylallyltransferase (AhR4DT) from the microsomal fraction of elicited peanut hairy roots. To our knowledge, this enzyme is the first stilbenoid-specific prenyltransferase that prenylates resveratrol and other specific stilbenoids at the 4-C position of the aromatic ring (Figure 1).

RESULTS

Purification and structural elucidation of a new prenylated stilbenoid from peanut hairy root culture

Hairy roots of peanut have the capability to produce and secrete resveratrol (3,5,4'- trihydroxy-trara-stilbene), piceatannol (3,5,3',4'-tetrahydroxy-tra¾y-stilbene) and their prenylated analogs, arachidin-3 and arachidin-1, in the medium upon co-treatment with MeJA and CD as elicitors (Yang et al., 2015a). In addition, in the current study we also identified arachidin-2 in the ethyl acetate extract of the culture medium by comparing with the HPLC retention time, characteristic UV spectrum (λ _1ΜΧ =323 mn) (Figure 2) and mass spectrometric analysis of the purified arachidin-2 isolated from fungus-infected peanut seeds. The structure of arachidin-2 purified from peanut hairy root culture was subsequently confirmed by 1H- and ¹³ C- NMR spectra (data not shown).

A new prenylated stilbenoid with 327 nm in the HPLC mobile phase was also isolated from the peanut hairy root culture medium (Figure 2). We named it arachidin-5. Mass spectrometry analysis (Table 1 shown in FIG. 7) of arachidin-5 (m/z 313 [M+H] ⁺ ) gave a main

_ _| _ 2

fragment with a m/z 257 [M+H-56] in MS which suggested the presence of a prenyl moiety. Arachidin-5 and arachidin-1 share very similar MS, MS ² and MS ³ spectra (Table 1 shown in FIG. 7) indicating that the structural difference between these two compounds might be only in the position of olefinic bond on their prenylated moieties.

The structure of the prenylated moiety on the arachidin-5 was further determined by 1H- and ¹³ C- NMR. The presence of a five-carbon unit of the 3,3-dimethylallyl structure was evident from the peak at 5.30 ppm (t, 7.1 Hz) that is coupled to the peak at 3.36 ppm (d, 7.1), as well as the peaks at 1.77 and 1.65 ppm for the two methyl groups (H-5" and H-4", respectively) (Figure 17). These proton resonances are the same as those published for arachidin-2 (Park et al., 2011). The 3,3- dimethylallyl structure is also supported by the presence of a quaternary carbon peak at 130.9 (C- 3") in the ¹³ C-NMR spectrum, as well as peaks at 124.4 (C-2"), 26.0 (C-4"), 23.1 (C-Γ) and 17.9 (C-5") (Figure 18). These resonances are in agreement with published resonances for the isoprene tail of arachidin-2 (Park et al., 2011). The NMR results showed that arachidin-5 has the same 3,3-dimethylallyl moiety as arachidin-2 but an additional hydroxyl group at the 3' position, while arachidin-1 and arachidin-3 have the 3-methyl-but-l-enyl moieties instead.

In addition to arachidin-5, arachidin-1, arachidin-2 and arachidin-3, HPLC analysis of the culture medium showed the presence of 2 other compounds with similar characteristic of arachidin-5 and arachidin-2 based on UV and MS, MS ² and MS ³ spectra (Figure 2 and Table 1 shown in FIG. 7). These compounds were later designated as arachidin-5 derivative and arachidin-2 derivative respectively.

Effects of metabolic inhibitors on yield of prenylated stilbenoids in peanut hairy root culture

As expected for peanut phytoalexins, non-prenylated and prenylated stilbenoids were only present in the peanut hairy root cultures after elicitor treatment (Medina-Bolivar et al., 2007; Yang et al., 2015a). To study the metabolic origin of the prenyl moiety of the prenylated stilbenods, mevastatin or clomazone was added to 9-day peanut hairy root cultures co-treated with 100 μΜ MeJA and 9 g/L CD. In preliminary experiments we found that mevastatin at 1, 10, or 100 μΜ did not affect the levels of prenylated stilbenoids (Figure 19). On the other hand, the effects on the accumulation of resveratrol and inhibition of prenylated stilbenoids accumulation were increased with the concentration of clomazone (1 μΜ to 100 μΜ) (Figure 20). Hence, to quantify these effects, we determined yields of resveratrol and prenylated stilbenoids after 48 h and 72 h of elicitor treatment together with mevastatin (10 μΜ), or clomazone (10 μΜ and 100 μΜ). The yields of stilbenoids were expressed in micromoles to assess the molar contribution of resveratrol as a precursor of the prenylated stilbenoids.

Mevastatin had no significant effect on the yields of resveratrol, piceatannol and prenylated stilbenoids with the exception of arachidin-5 and arachidin-1, which showed a 27% and 41% increase in yield, respectively, after a 72 -h treatment (Figure 3). During the 24-h interval between 48 and 72-h treatments, non-inhibitor and mevastatin treated groups had significant increases in the yields of arachidin-5, arachidin-1, arachidin-2, and arachidin-3 indicating that mevastatin did not inhibit the accumulation of these prenylated stilbenoids (Figure 3).

With an inherent limited production (about hundred fold lower than that of resveratrol) upon elicitor treatment, piceatannol concentration increased only slightly in the 100 μΜ clomazone group after 72 h of treatment (Figure 3). However, in both 10 μΜ and 100 μΜ clomazone treated groups, there were significantly higher yields of resveratrol and significantly lower yields of prenylated stilbenoids when compared to the non-inhibitor group. In particular, the accumulation of arachidin-5 and arachidin-1 was almost completely inhibited and only trace amounts were observed in the 48-hour treated groups containing 100 μΜ clomazone (Figure 3). There were no significant increases in the levels of all prenylated stilbenoids during the 24-h interval between the 48- and 72-h time points indicating that clomazone has an inhibitory effect on the accumulation of prenylated stilbenoids in peanut hairy root cultures. Since each of the inhibitors was applied to 9-day-old hairy root cultures together with elicitors, DMAPP or IPP might have been already synthesized and stored in the tissue prior to the inhibition. Thus, even if the biosynthesis of DMAPP and IPP was blocked when clomazone was added into the medium, small amounts of arachidin-2 and arachidin-3 could be detected in the 48- and 72 -h clomazone treated samples (Figure 3). Moreover, when different concentrations of clomazone were applied to the peanut hairy root culture without elicitors, none of these stilbenoids was detected in the ethyl acetate extracts of the culture medium suggesting that clomazone was not able to induce the production of stilbenoids by itself (Figure 21). However, in the co-treated group of 100 μΜ clomazone with elicitors, the yield of resveratrol reached up to 830.73 ± 25.83 μΜ after 72 h which was 6.3 fold higher than that in the non-inhibitor group. The micromolar increase of resveratrol was about 1.54 fold greater than the overall decrease in the accumulation of arachidin-5, arachidin-1, arachidin-2, and arachidin-3, suggesting that these prenylated stilbenoids may have been derived from resveratrol.

Degradation of exogenous resveratrol in peanut hairy roots

Upon co-treatment of peanut hairy root cultures with MeJA and CD as elicitors, most of the total resveratrol and prenylated stilbenoids produced were secreted into the culture medium and only trace amounts were found in the ethyl acetate extracts of the root tissue (Figure 22). Based on previous observations regarding loss of resveratrol accumulation in medium of non- elicited peanut hairy root cultures (Yang et al., 2015a), we speculated that resveratrol could be taken up by the hairy roots and metabolized by one or multiple enzymatic mechanisms in the roots. To confirm this hypothesis, 1 mM resveratrol was co-incubated with a crude cell-free extract from non-elicited peanut hairy roots. After 30-min incubation, the concentration of resveratrol declined to 53% of that in the control group co-incubated with heat-denatured crude cell-free extract. Meanwhile, piceatannol and other unidentified compound with 327 nm referred to as a resveratrol derivative, were detected as reaction products (Figure 4a and Figure 4d). In fact, according to our preliminary experiments, not only was resveratrol metabolized, but the accumulation of prenylated stilbenoids like arachidin-2 was likewise affected. Consequently, after 120-min incubation, neither resveratrol nor its prenylation product, arachidin-2 was detected in the ethyl acetate extracts of the reaction mixtures (Figure 23). To prevent the oxidation of resveratrol, dithiothreitol (DTT) as reducing agent was added to the reaction mixture of the crude cell-free extract from non-elicited peanut hairy root using 1 mM resveratrol as substrate. After 120-min co-incubation with 5 mM DTT, more than 95% of the amount of resveratrol remained and no piceatannol was detected in the mixture (Figure 4c), suggesting that DTT was able to prevent the oxidation of resveratrol.

Detection of resveratrol prenyltransferase (AhR4DT) activity in peanut hairy roots

All flavonoid-specific prenyltransferases reported to date from other legume plants have been localized to plastid membranes, and results described above indicate that the accumulation of prenylated stilbenoids in our hairy root culture system can be blocked in the presence of plastid MEP pathway inhibitor. We therefore hypothesized that the stilbenoid-specific prenyltransferase(s) in peanut might also be membrane localized and use DMAPP or IPP originating from the MEP pathway as the prenyl donor. Furthermore, our clomazone inhibitor experiments suggested resveratrol as a prenyl acceptor and a precursor of prenylated stilbenoids. We therefore prepared a microsomal fraction using ultracentrifugation from 48-h elicited peanut hairy root treated with resveratrol, DMAPP, along with DTT to reduce the degradation of resveratrol and its prenylated product. Two enzymatic products were synthesized in the reaction mixture; the predominant one was identified as arachidin-2 ,by comparison of its retention time, UV spectrum, mass spectra and fragmentation patterns obtained by tandem mass spectrometry

9

(MS ^Z and MS ^J ), each of which were identical to that of arachidin-2 purified from peanut hairy root culture (Figure 5b). Another product (Figure 5b) shared identical retention time, UV

9 ^

spectrum, mass spectra, MS and MS with that of unidentified compound found in peanut hairy root culture (Table 1 shown in FIG. 7). Due to the presence in the enzymatic reaction with 100 μΜ arachidin-2 and crude cell-free extract (Figure 24), this product is considered as an enzymatic derivative of arachidin-2. None of the prenylated products were detected in the sample incubated with the microsomal fraction from non-elicited hairy root tissue (Figure 28), indicating the inducibility of this resveratrol prenyltransferase in peanut hairy roots. Moreover, the specific activity of resveratrol prenyltransferase in the microsomal fraction (421.16 ± 16.25 pkat mg ^"1 of protein, based on the production of arachidin-2) was 9.3 -fold higher than that in the crude cell- free extracts, suggesting that resveratrol prenyltransferase in peanut was bound to the membrane fraction of the root cells (Table 2 shown in FIG. 8).

Biochemical characterization of resveratrol prenyltransferase AhR4DT

In the resveratrol prenyltransferase reaction, the accumulation of the prenylated product arachidin-2 followed a linear relationship with input between 30 μg and 120 μg of microsomal protein. The reaction was also linear over the 120 min incubation time (Figure 25). The optimum pH for this prenyltransferase was 9.2 in a Tris-HCl buffer (Figure 26), which was close to the optimum pH values of 9-10 for flavonoid specific prenyltransferases (Yamamoto et al., 2000). Divalent cations were absolutely required for resveratrol prenyltransferase activity and Mg was the most effective these (100%), followed by Mn ²⁺ (71.8%), Fe ^z+ (14.6%) and Ca ⁺ (0.9%) (Figure 27). No prenyltransferase activity was detected when a divalent cation was absent from the reaction (Figure 28). Isoprenoid precursor isopentenyl diphosphate (IPP) was tested as a prenyl donor for the prenyltransferase with resveratrol as the prenyl acceptor and no activity was detected in the assay (Figure 28). The apparent k _m values for resveratrol and DMAPP were calculated as 111.1 ± 40.44 μΜ and 91.89 ± 7.032 μΜ, respectively (Figure 29).

Substrate specificity of resveratrol prenyltransferase AhR4DT

To analyze the substrate specificity of the resveratrol prenyltransferase from peanut hairy root, stilbenoids (piceatannol, pinosylvin, pterostilbene and oxyresveratrol), flavanone (naringenin), flavone (apigenin) and isoflavone (genistein) were incubated with a microsomal fraction using DMAPP as a prenyl donor. When piceatannol was used as a prenyl acceptor, the microsomal fraction catalyzed the synthesis of arachidin-5 as the predominant prenylated product (Figure 30). Similar to the arachidin-2 derivative, another product considered as an enzymatic derivative of arachidin-5, was also detected in the reaction (Figure 31). In addition, the microsomal fraction catalyzed pinosylvin into chiricanine A, confirmed by comparison of its retention time, UV spectrum, mass spectra and fragmentation patterns with those of chiricanine- A standard purified from fungus-challenged peanut seeds (Figure 6 and Figure 30). Using this microsomal fraction, an isoprene unit was also transferred to oxyresveratrol. Mass spectroscopic analysis of its reaction product (m/z 313 [M+H] ⁺ ) showed a main fragment with a m/z 257 [M+H-56] ⁺ in MS ² which suggested the presence of a prenyl moiety (Figure 32). However, the position of the prenyl moiety on this prenylated oxyresveratrol remains undetermined due to the insufficient amount of compound for further structure elucidation. Interestingly, pterostilbene which has methoxy groups at C-3 and C-5 positions, respectively, did not produce any product within the microsomal fraction (Figures 6 and Figure 30), suggesting that the additional methyl group on its structure might block prenylation and that either or both of the hydroxyl groups at C-3 and C-5 position might be required for the prenylation reaction. Furthermore, neither prenylated flavanone, prenylated flavone nor prenylated isoflavone was detected in the reaction mixtures (Figures 6 and Figure 30), indicating that this peanut prenyltransferase may be a stilbenoid-specific prenyltransferase.

DISCUSSION

AhR4DT, a membrane-bound prenyltransferase specific for stilbenoids from peanut hairy root

As the limiting enzyme involved in the biosynthesis of prenylated flavonoids, flavonoid prenyltansferases have been a focus of previous research particularly in plants of the Leguminosae family known to accumulate these type of specialized metabolites. The first flavonoid prenyltansferase, SfN8DT, was identified in Sophora flavescens cell cultures by Sasaki et al. in 2008. Subsequently, SfG6DT and SfiLDT, which prenylate genistein to produce wighteone (6-dimethylallylgenistein) and isoliquiritigenin to dimethyllylisoliquiritigenin respectively, were identified in the same species (Sasaki et al., 2011). While SfG6DT prenylates genistein on the A-ring of the isoflavone, LaPTl isolated from another legume, white lupin (Lupinus albus), catalyzes the prenylation of the B-ring of genistein and 2'-hydroxygenistein to form isowighteone (3'-dimethylallylgenistein) and luteone (2'-hydroxy-3'-dimethylallyl- genistein), respectively (Shen et al., 2012). As a crucial prenyltransferase involved in glyceollin biosynthesis, GmG4DT was identified and characterized in soybean {Glycine max). This enzyme catalyzes the dimethylallylation of glycinol at position 4 to produce the precursor of the phytoalexin, glyceollin I (Akashi et al., 2008). More recently, GuA6DT, a flavone prenyltransferase was identified in another legume species, liquorice (Glycyrrhiza uralensis) (Li et al., 2014).

In this study, we have described the first plant stilbenoid prenyltransferase activity, and named this enzyme as resveratrol 4-dimethylallyltransferase (AhR4DT). AhR4DT catalyzes the dimethylallylation of resveratrol at C-4 and is derived from the microsomal fraction of elicited peanut hairy roots. AhR4DT shows several common features with other prenyltransferases, such as those described above. For instance, all prenylation activities mentioned above and demonstrated here require a divalent cation as cofactor and a basic buffer for optimal reaction rate (except for GmG4DT which reaction rate optimum is pH 7.5 buffer for the reaction, (Akashi et al., 2008). Because the activity of AhR4DT was concentrated in the microsomal fraction and the accumulation of prenylated stilbenoid in peanut hairy roots was inhibited by clomazone, we suggest that this peanut resveratrol prenyltransferase is a membrane-bound protein located in the plastid. This further suggests that the DMAPP used in the prenylation reaction was derived from the MEP plastidic pathway. Our hypothesis is in agreement with the plastidic subcellular location of flavonoid prenyltransferases identified in other legume species.

During the biosynthesis of stilbenoids and flavonoids, both resveratrol synthase and chalcone synthase use 4-coumaroyl-CoA as a substrate and perform three condensation reactions with malonyl-CoA to form a linear tetraketide, which is later folded into new ring structures. These two enzymes are distinguished by a special property of stilbene synthases, which looses the terminal carboxyl group as C0 , resulting in release of 4 C0 ₂ . In contrast, each reaction catalyzed by chalcone synthase releases 3 C0 ₂ molecules. It has been suggested that stilbene synthase may have developed from chalcone synthase via gene duplication and mutation rendering new and improved functions (Tropf et al., 1994).

Among the prenylation products of resveratrol, piceatannol, pinosylvin, and oxyresveratrol catalyzed by the microsomal fraction containing AhR4DT, only arachidin-2 and arachidin-5 were detected in the spent medium of peanut hairy root cultures co-treated with Me J A and CD. Interestingly, oxyresveratrol and prenylated oxyresveratrol reported in the bark of Artocarpus dadah (Su et al., 2002) along with pinosylvin isolated from pine hardwood, have not been identified in any peanut tissue, while the prenylated product of pinosylvin, chiricanine A that was first described in the roots of Lonchocarpus chiricanus (loset et al., 2001), has been described in peanut seeds after infection with the fungus Aspergillus flavus (Sobolev et al., 2009).

Biosynthesis of prenylated stilbenoids in peanut hairy root cultures

The use of abiotic elicitors to induce the biosynthesis of stilbenoid in hairy root cultures provides for an axenic sustainable and controlled production platform for these specialized metabolites which can be leveraged to study their biosynthetic pathway. The metabolic steps to produce resveratrol from phenylalanine including phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate: coenzyme A ligase (4CL), and resveratrol synthase (RS) have already been elucidated (Watts et al., 2006). In the current study, a stilbenoid prenyltransferase, AhR4DT, which prenylates resveratrol to form arachidin-2 was characterized from the microsomal faction of elicited peanut hairy roots. Since resveratrol accumulated in the medium while clomazone blocked the plastid MEP pathway in our inhibitor feeding experiments, it may be that prenylated stilbenoids in addition to arachidin-2 also derive from resveratrol. A study of phytoalexins induced in peanut kernels by soil fungal exposures also suggests that resveratrol might be the precursor for other prenylated stilbenoids in peanut (Sobolev, 2008).

The main prenylated stilbenoids produced by peanut hairy root can be categorized into two groups according to the structure of their prenyl side chains. One group includes arachidin-2 and arachidin-5 (identified in this study) having a 3,3-dimethylallyl moiety. This is the most common type of prenylation found in prenylated stilbenoids from various plant families. For instance, longistylines C, longistylines D and chiricanine A found in Lonchocarpus chiricanus (Leguminosae) (Ioset et al., 2001) also have the same dimethylallyl moiety. Artoindonesianin N with one dimethylallyl moiety and 4-dimethylallyl-oxystilbene were reported in Artocarpus integer (Moraceae) (Boonlaksiri et al., 2000) and A. gomezianus (Moraceae) (Hakim et al., 2002) respectively. Mappain found in Macaranga mappa (Euphorbiaceae) (Van Der Kaaden et al., 2001) has one dimethylallyl moiety and one geranyl moiety, while schweinfurthin C with two geranyl moieties was isolated from M. alnifolia {Euphorbiaceae) (Yoder et al., 2007). Moreover, the dimethylallyl moiety is also the most common prenylation pattern present in prenylated flavonoids, which are mostly found in the following families: Cannabaceae, Guttiferae, Leguminosae, Moraceae, Rutaceae, and Umbelliferae. The longer form of the dimethylallyl moiety, geranyl and lavandulyl moiety, has also been reported in prenylated flavonoids (Botta et al., 2005; Yang et al., 2015b). During the biosynthesis of these prenylated stilbenoids and prenylated flavonoids, prenyltransferases are responsible for these dimethylallylation and geranylation attaching DMAPP and GPP, respectively, to different positions of the stilbenoid and flavonoid skeletons. One major group of prenylated stilbenoids in peanut hairy roots is represented by arachidin-1 and arachidin-3 which harbor a 3-methyl-but-l-enyl moiety. When compared with the dimethylallyl moieties commonly present in other prenylated stilbenoids and flavonoids, this unique prenylated form has been reported in peanut and very few other species. To our knowledge, the 3-methyl-but-l-enyl-oxystilbene isolated from A. integer (Moraceae) (Boonlaksiri et al, 2000) is the only prenylated stilbenoid with this moiety reported in a species other than peanut. Due to the difference in the position of the olefinic bond on the prenylated moieties, 3-methyl-but-l-enyl stilbenoids such arachidin-3 and arachidin-1 have higher lipophilicity as evident from a later retention time in reverse-phase HPLC chromatograms when compared with their 3,3-dimethylallyl analogs, arachidin-2 and arachidin-5 respectively. The max of UV absorbance of 3-methyl-but-l-enyl stilbenoids has an apparent shift to 335-340 nm when compared with that shown at 323-327 nm for 3,3-dimethylallyl stilbenoids (Table 1 shown in FIG. 7). However, the effects of these two moieties on the bioactivity of peanut stilbenoids is still unclear because arachidin-1 and arachidin-3 are not commercially available limiting their studies and the relatively low yields of arachidin-2 and arachidin-5 produced in peanut hairy roots.

The yields of arachidin-1 and arachidin-3 in our peanut hairy root culture system were much higher than those of their dimethylallyl analogs, arachidin-2 and arachidin-5. As described in the activity assays above, both arachidin-2 and arachidin-5 were further modified to other derivatives, suggesting that dimethylallyl stilbenoids are important intermediates for the biosynthesis of other peanut prenylated stilbenoids. As a substrate recognized by AhR4DT, piceatannol was considered to be the putative precursor of arachidin-5 in peanut. However, the yield of piceatannol in our peanut hairy root culture is very limited and far less than that of resveratrol. Even after blocking the biosynthesis of the prenylated moiety by 100 μΜ clomazone, the cultures only produced 9.02 μΜ of piceatannol compared with 845.7 μΜ of resveratrol (Figure 3). These observations suggests that arachidin-5 and arachidin-1 might not be synthesized from piceatannol due to its limited amounts, instead arachidin-1 may derive from arachidin-3, which is relatively abundant in the medium, by hydroxylation at C-3' position.

Metabolism of prenylated stilbenoids in peanut hairy root

Under treatment with MeJA and CD, arachidin-1, arachidin-2, arachidin-3, and arachidin- 5, are the major prenylated stilbenoids secreted and accumulated in the spent culture together with resveratrol. When CD is not added in the hairy root cultures, only limited amounts of resveratrol and stilbenoids are detected in the medium. These observations were reported previously when peanut hairy roots were treated with NaOAc, ¾0 ₂ or MeJA alone as elicitors (Yang et al., 2015a). Importantly, when resveratrol was added in non-elicited peanut hairy root cultures, 47% and 97 % reductions of the initial resveratrol concentration in the culture medium were observed after 0.5 h and 1 h co-incubation, respectively (Yang et al., 2015a). In the current study, we confirmed that the degradation of extrinsic resveratrol in peanut hairy root culture occurs due to activities of the root tissue. Unlike resveratrol synthase, which is induced and involved in stilbenoid biosynthesis, the enzymes involved in the degradation of resveratrol appear to be constitutively expressed in non-elicited root tissue, leading to the dramatic decline of extrinsic resveratrol. Results from experiments described here suggest that peanut enzymes present initiate this process by oxidizing resveratrol into piceatannol and then secondly by converting this into other derivatives. This enzymatic degradation process may also apply to other prenylated stilbenoids, such as arachidin-2 (Figure 23), and may explain the observation that only trace amounts of stilbenoids are detected in ethyl acetate extracts of root tissue (Figure 22). The constitutive degradation of stilbenoids may provide the plant with the ability to manage potential toxic effect of stilbenoids when they accumulate at high levels within the cell. Interestingly, other species such as grape accumulate resveratrol as glucosides (i.e. piceid). However, this conjugate was not identified in the peanut hairy roots and has not been described in fungal elicited peanut kernels. Altogether, these findings suggest that peanut may have evolved a distinct mechanism to metabolize resveratrol-type phytoalexins after they are produced.

CONCLUSION OF ARACHIDIN-5 PRODUCTION

Harnessing the inducible bioproduction capabilities of the peanut hairy root culture system, we have newly produced a prenylated stilbenoid, i.e., arachidin-5, and have demonstrated that the prenyl moiety on peanut prenylated stilbenoids is derived from a plastidic biosynthesis pathway. We have characterized for the first time a plant membrane-bound stilbenoid-specific prenyltransferase, i.e., AhR4DT, from the microsomal fraction of peanut hairy roots. The characteristics of AhR4DT provide important information for subsequent cloning and comprehensive definition of the prenyltransferase gene(s) of peanut. Moreover, we have observed the enzymatic degradation of exogenous resveratrol by peanut hairy root tissue, an observation that will lead to elucidation of further mechanisms governing phytoalexin accumulation in plants.

MATERIALS AND METHODS

Chemical reagents

Authentic standards of resveratrol and piceatannol were obtained from Biophysica and Axxora, respectively; arachidin-1, arachidin-2, arachidin-3, and arachidin-5 standards were purified from elicited peanut hairy root as described below. Pinosylvin, oxyresveratrol, pterostilbene, naringenin, apigenin, and genistein used in this study were purchased from Sigma- Aldrich. Chiricanine-A was purified from fungal-challenged peanut seeds as described below. DMAPP was obtained from Isoprenoids, LC. Stock solutions of inhibitors, mevastatin (100 mM, Sigma- Aldrich) and clomazone (100 mM, Sigma- Aldrich), were prepared in ethanol and stored at 4 °C.

Isolation and purification of chiricanine-A from fungal challenged peanut seeds

One kg of seeds of the 31-1314 peanut runner breeding line from the National Peanut Research Laboratory (Dawson, GA), stored at 4°C for two years after harvest, were allowed to imbibe distilled water for 18 h at 4°C. They were then chopped with a custom-made hand cutter into 3-6 mm pieces, thoroughly washed with distilled water, blotted with a paper towel, air-dried to the condition where sliced peanuts did not leave water spots on filter paper, and placed on aluminum trays so that the thickness of the layer did not exceed 1 cm. The trays were evenly sprayed with the fungal spores of Aspergillus caelatus NRRL 25528 (lOVmL), placed into autoclave bags, and incubated at 30 °C for 96 h. The bags were opened every 24 h to allow fresh air to the peanuts and growing fungus.

The incubated peanut seeds were extracted with 3.0 L of MeOH overnight at room temperature without agitation. This procedure was repeated two more times. The combined mixture was filtered through a paper filter in a Biichner type funnel under reduced pressure. The combined filtrates were defatted three times with 0.5 L of n-hexane. The MeOH layer was evaporated to dryness. The residue was redissolved in CHCI ₃ and applied to a chromatographic column (34 mm i.d.) packed with silica gel (silica gel 60, 0.063-0.200 mm; EM Science, Gibbstown, NJ) to the height of 400 mm. The column was subsequently eluted with 0.5 L of CHC1 ₃ , 1.0 L of EtOAc, 1.0 L of acetone, and 1.0 L of MeOH (all solvents were purchased from Fisher). Eight fractions were collected from the column and analyzed by , HPLC. Fractions containing chiricanine A were combined, evaporated to dryness with a rotary evaporator, and subjected to further purification on a similar silica gel column. The column was subsequently eluted with 0.4 L of CHC1 ₃ , 1.5 L of CHCl ₃ EtOAc (1 : 1), 1.3 L of EtOAc, and 1.0 L of acetone. Twenty four fractions were collected. Combined fractions containing chiricanine A were evaporated to dryness on a rotary evaporator, redissolved in MeOH, filtered, and subjected to final purification with a preparative 100 mm ^χ 19 mm i.d., 5 μτη XTerra Prep RP18 OBD HPLC column (Waters). The flow rate was 9.5 mL/min, and column temperature was maintained at 40 °C. The following mobile phase was used: 73% CH ₃ CN, 3% of 1% HCOOH in H ₂ 0, and 24% of H ₂ 0. Pure fractions of chiricanine A obtained from HPLC were evaporated with a rotary evaporator to a point where almost all of the organic solvent was removed. Then the target compound was extracted four times with EtOAc (H ₂ 0/EtOAc ratio 1 :1, v/v). The combined EtOAc layers were evaporated nearly to dryness with a rotary evaporator. The residue was transferred into a 15 mL vial with EtOAc and evaporated nearly to dryness with a stream of N ₂ . The residue was redissolved in EtOAc, filtered, and transferred into 4 mL vials and evaporated to dryness with a stream of N ₂ . Then the vial was placed into a lyophilizer for 2 h at room temperature to remove traces of the solvents. Chiricanine A was obtained as a slightly yellowish oil (6.5 mg).

Analyses of stilbenoids from peanut hairy root cultures

Hairy root cultures of peanut (Arachis hypogaea) cv. Hull line 3 established by the Medina-Bolivar laboratory (Condori et al., 2010) were maintained in 50 mL modified Murashige and Skoog medium (MSV) (Condori et al., 2010) with 3% sucrose in 250 ml flasks. In order to induce synthesis and secretion of stilbenoids, the spent medium of nine-day-old hairy root cultures was discarded and replaced with 50 mL fresh MSV medium containing 3% sucrose with 100 μΜ methyl jasmonate (MeJA) and 9 g/L methyl-P-cyclodextrin (CD; Cavasol™) as elicitors and incubated in the dark at 28 °C for an additional 72 hours as previously described (Yang et al., 2015a). After elicitation. period, one milliliter spent medium was partitioned with ethyl acetate twice. The combined organic phase was evaporated under nitrogen gas and dissolved in methanol for subsequent HPLC and mass spectrometry analyses.

Quantitative analysis of stilbenoids in peanut hairy root culture extracts was performed in an UltiMate 3000 LC system (Dionex, Thermo Scientific), equipped with a photodiode array detector. The separation was performed on a SunFire™ C ₁₈ , 5 μηι, 4.6 x 250 mm column (Waters) at 40 °C with a flow rate at 1.0 ml/min. The mobile phase consisted of 2% formic acid in water (A) and methanol (B). The column was initially equilibrated with 100% A for 1 min. Then a linear gradient was performed from 40% A and 60% B to 35% A and 65% B (1 to 20 min), followed by a linear gradient from 35% A and 65% B to 100% B (20 to 25 min). Then the column was washed with 100% A for 5 min (25 to 30 min). Calibration curves of various stilbenoids were established using absorbance at 320 nm for resveratrol, piceatannol, arachidin-2 and arachidin-5 and at 340 nm for arachidin-1 and arachidin-3.

For LC-mass spectrometry qualitative analysis of stilbenoids, an UltiMate 3000 rapid separation LC system (Dionex, Thermo Scientific) was used for the chromatographic separation. The separation method was similar to the HPLC conditions described above with the following modification, 0.02% formic acid in water was used instead 2% in the mobile phase A. The LTQ XL™ linear ion trap mass spectrometer (Thermo Scientific) with an electrospray ionization (ESI) source was used to obtain structural information of stilbenoids and followed the method described previously (Marsh et al., 2014). Briefly, all mass spectra were performed in the positive ion mode with ion spray voltage at 4 kV, sheath gas (high purity nitrogen) at 45 arbitrary units (AU), auxiliary gas (high purity nitrogen) at 15 AU, capillary voltage at 9 V, capillary temperature at 300 °C, and tube lens offset at 45 V. Full mass scan was recorded in the range m/z 100-2000. Ultrahigh pure helium (He) was used as the collision gas and 35% of collision energy was applied in collision-induced dissociation (CID). The data were recorded and analyzed by Xcalibur software (Thermo Scientific).

Purification and characterization of arachidin-5 from peanut hairy root cultures

For purification of arachidin-5 and other prenylated stilbeniods, one liter of spent medium obtained from a pool of about 20 flasks of 72 h-elicited peanut hairy root cultures was collected and extracted with an equal volume of ethyl acetate twice in a 2 L separatory funnel. The organic phase was recovered and dried in a rotavapor (Buchi) and the crude extract (-800 mg) was stored at 4 °C for subsequent high performance counter current chromatography (HPCCC) fractionation. A two-phase HPCCC solvent system (hexane: ethyl acetate: methanol: water (4:5:3:3, v/v/v/v) was equilibrated at room temperature in a 2 L separatory funnel. The upper phase of this solvent mixture was used as stationary phase and the lower one was used as mobile phase for preparative HPCCC (Dynamic Extractions) system. The multilayer coil was filled with the stationary phase at a flow rate of 8 mL/min while spinning at 1600 rpm.. Then the hydrodynamic equilibrium was established by pumping 6 mL/min of mobile phase into the column until a clear mobile phase was eluted at the outlet. Crude extract (300 mg) was dissolved in 5 mL of the two-phase solvent and manually injected. The effluent was monitored at UV 340 nm. The fractions were collected every 30 s, dried in a SpeedVac, and analyzed by HPLC as described above. According to the HPLC profiles, the fractions collected between 27 to 32 min contained arachidin-1 and arachidin-5, whereas those collected between 53 to 62 min contained arachidin-2 and arachidin-3. Fractions containing arachidin-1 and arachidin-5 were combined as one sample, whereas those containing arachidin-2 and arachidin-3 were combined as a separate sample. Then, the organic solvent in the samples was removed using rotavapor. The remaining aqueous mixtures containing the target compounds were extracted with ethyl acetate (1:1, v/v), evaporated nearly to dryness with a rotavapor and redissolved in methanol. Then these combined fractions were applied to TLC plates (TLC silica gel 60 RP-18 F ₂ 5 ₄ s, Millipore) and separated using as developing solvent a solvent system composed of methanol: water: acetic acid (15:45:40, v/v/v). After separation, the purified prenylated stilbenoids on the adsorbent were scraped off, re-dissolved in methanol and dried under nitrogen gas for subsequent 1H and ¹³ C NMR spectra analysis. In summary, 4.6 mg of arachidin-5, 20.3 mg of arachidn-1, 5.2 mg of arachidin-2 and 17.8 mg of arachidin-3 were purified from the peanut hairy root culture medium using the HPCCC and preparative TLC method.

For NMR analysis of purified arachidin-5 and arachidin-2, 1H NMR was recorded at 400 MHz, ¹³ C NMR was recorded at 100 MHz in acetone- d ₆ on a Bruker AV-400 NMR spectrometer.

Inhibitor feeding experiment

Nine-day-old peanut hairy root cultures were used in the inhibitor feeding experiment. Prior to treatment, the spent medium from each culture was removed and replaced with 50 mL fresh MSV medium containing 3% sucrose and 100 μΜ MeJA and 9 g/L CD as elicitors. Then, mevastatin (10 μΜ) or clomazone (10 μΜ and 100 μΜ) was applied to the culture medium. For the control group, 50 \L of absolute ethanol (solvent of mevastatin and clomazone) was added to the medium. All treatments were performed at 28 °C and continuous darkness with three biological replicates per treatment. One milliliter of spent medium was collected from each treatment at 48 h and 72h after treatment. The 1 mL medium aliquots were extracted with 1 mL ethyl acetate, and then the organic phase was collected and dried under nitrogen gas and re- dissolved in methanol for HPLC analysis as described above.

Enzyme preparation

Enzyme solutions from peanut hairy roots for prenyltransferase assay were prepared using nine-day-old roots elicited with 100 μΜ MeJA and 9 g/L CD for 48 h. Ten grams (fresh weight) of elicited hairy root tissues were grinded and homogenized in 20 mL of extraction buffer composed of 100 mM Tris-HCL buffer (pH 7.6), 10 mM dithiothreitol (DTT) and 2.5% (w/v) polyvinylpyrrolidone (average mol wt 40,000; PVP-40, Sigma) using a mortar and pestle. The homogenate was centrifuged at 12,000xg for 15 min at 4 °C to remove the cell debris. Crude cell -free extracts were obtained by passing 2.5 mL of the 12,000 xg supernatant through a PD-10 desalting column (GE Healthcare), using 100 mM Tris-HCL (pH 9.2) containing 10 mM DTT as equilibration buffer. About 13.5 mL of the remaining 12,000 xg supernatant were subsequently centrifuged at 156,000 xg for 45 min at 4 °C. The 156,000 xg supernatant was collected and cleaned up through a PD-10 desalting column (GE Healthcare) equilibrated with 100 mM Tris- HC1 buffer (pH 9.2) containing 10 mM DTT. The microsomal pellet was resuspended in 100 mM Tris-HCl buffer (pH 9.2) containing 10 mM DTT, re-centrifugated (156,000xg for 45 min), and finally resuspended in 1 mL of the same buffer for subsequent enzyme reaction.

For the degradation of resveratrol assay, crude cell-free extract was prepared from 12-day-old non-elicited peanut hairy roots following a similar procedure to that described for elicited roots above except that no DTT was included in the extraction and equilibration buffers.

Protein quantification Protein contents in the various enzyme solutions were determined using the Coomassie protein assay (Thermo Scientific) using bovine serum albumin as standard.

Degradation of resveratrol assay

The standard assay condition contained 1 mM resveratrol with 50 μg crude cell-free extract from 12-day-old non-elicited peanut hairy root in 500 μΐ, of 100 mM Tris-HCl buffer (pH 7.6). To study the effect of reducing agent on the degradation of resveratrol, additional 5 mM DTT was added to the standard assay mixture. After 30 min incubation at 28 °C, the reaction mixture was extracted with 500 μΐ, ethyl acetate and the amount of resveratrol remaining in the mixture was analyzed by HPLC. i the control group, crude cell-free extract was heated at 99 °C for 20 min.

Prenyltransferase assay

The standard assay was performed in a total volume of 500 μΐ, containing 100 μΜ resveratrol, 300 μΜ DMAPP as a prenyl donor, 10 mM MgCl ₂ , 5 mM DTT and 30 μg microsomal fractions in 100 mM Tris-HCl buffer (pH 9.2). After 60 min incubation at 28 °C, the reaction mixture was terminated by adding 20 μΐ, of 6 M HCl and extracted with 500 μΐ, of ethyl acetate. The extracts were dried under nitrogen gas, dissolved in methanol and the reaction product was quantified by HPLC analysis. The prenylation activity for each reaction was quantified by the molar concentration of generated prenylated product per second with one milligram microsomal fractions (kat/mg).

In the linearity study, the prenyltransferase activities under varying incubation times (30, 60, 90 and 120 min) with 30 μg microsomal fraction and varying mount of microsomal faction (30, 60, 90 and 120 μg) incubated for 60 min were measured. For pH dependency study, the activities of prenyltransferase were measured using 100 mM Tris-HCl buffer at pH 7.0, 8.0, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, and 10.0. For divalent cation dependency study, 10 mM MnCl ₂ , FeCl ₂ , CaCl ₂ , CoCl ₂ , ZnCl ₂ , NiCl ₂ or CuCl ₂ was added to the reaction mixture instead of MgCl ₂ as described above, and the enzyme activity was compared with that reaction containing MgCl ₂ . For kinetic studies, varying concentration (10, 20, 40, 80, 160, 320, and 640 μΜ) of resveratrol with a fixed concentration of DMAPP (640 μΜ) and varying concentration (10, 20, 40, 80, 160, 320, and 640 μΜ) of DMAPP with a fixed concentration of resveratrol (640 μΜ) were incubated with microsomal fractions of peanut hairy root in a total volume of 250 μΐ _^ at 28 °C for 60 min. These reactions were used to calculate V _m and K _m values using nonlinear regression analysis of Michaelis-Menten equation using Graphpad Prism 6 software. For substrate specificity assay, 100 μΜ stilbenoids (resveratrol, piceatannol, pinosylvin, pterostilbene, and oxyresveratrol), flavanone (naringenin), flavone (apigenin), and isoflavone (genistein) with 300 μΜ DMAPP as a prenyl donor were incubated with microsomal fractions of peanut hairy root in a total volume of 250 μΐ, at 28 °C for 60 min. Arachidin-2 and arachidin-5 purified from peanut hairy root culture and chiricanine-A purified from fungus-challenged peanut seed were diluted to various concentration and calibration curves of their absorbance at 320 nm were used for quantitative analysis.

Identification of Resveratrol Prenyltransferase Activities from the A. hypogaea Hairy Root Transcriptome

The first flavonoid-specific prenyltransferase, SfN8DT-l, was cloned from a cDNA (EST) library of Sophora flavescens cell cultures and its enzymatic activity was characterized using the microsomal fraction of recombinant yeast (Sasaki et al., 2008). Sequence homology to SfN8DT-l was the basis for discovery of several other flavonoid prenyltransferases, such as SfiLDT and SfG6DT in S. flavescens (Sasaki et al., 2011) and LaPTl in Lupinus albus (Shen et al., 2012). Our previous work had indicated that resveratrol prenyltransferase(s) in peanut are membrane-bound proteins that utilize DMAPP from the plastidic terpenoid pathway as the prenyl donor (Yang et al., 2016). These two key features are also observed in flavonoid-specific prenyltransferases identified from other legume species (Sasaki et al., 2008; Akashi et al., 2008; Sasaki et al, 2011; Shen et al., 2012; Li et al., 2014; Chen et al, 2013), suggesting that the sequence of stilbenoid prenyltransferase(s) may share similarity with flavonoid

prenyltransferases genes.

To discover and clone peanut prenyltransferase genes, therefore, we first built a transcript sequence reference from RNA of our elicited hairy root culture system, and annotated likely candidate stilbenoid prenyltransferase transcripts by alignment to well-characterized flavonoid prenyltransferases. An RNA sequencing experiment was designed to capture rnRNA temporally associated with stilbene accumulation in the hairy root cultures. We assembled and evaluated a variety of transcript sequences from RNA-Seq (Mortazavi et al., 2008) reads sets (see Methods) and initially considered any whose translated product aligned to characterized flavonoid prenyltransferase sequences listed above. 224 transcripts from our set of 2,591,753 transcript assemblies encoded full-length protein sequences of 101 to 432 amino acid residues that aligned to the set of flavonoid prenyltransferase sequences over a length of at least 100, with >80% sequence identity. As derived from a tetraploid, we anticipated that these sequences would represent transcripts of unique enzyme genes, as well as homeologs, alleles and potential assembly errors. The cultivated peanut genome is allotetraploid thought to have been formed as the result of a single hybridization between two closely-related diploid species, Arachis duranensis and Arachis ipaensis. Reference sequence assemblies of the latter diploid genomes were reported recently (Bertioli et al., 2016), and these provided a draft proxy reference we used to evaluate and potentially reduce our transcriptome to unique genie loci.

Our previous work had implicated prenyltransferase activities of interest occurred in the plastid (Yang et al., 2016). We therefore reduced the candidate transcript sequences to ten whose encoded proteins contain predicted chloroplast targeting peptide sequences according to analyses with both ChloroP (Emanuelsson et al., 1999) and iPSORT (Bannai et al., 2002). All ten transcripts showed expression in the 9-hour elicitor-treated hairy root transcriptome, and therefore we used 9-hour elicited hairy roots to clone their cDNAs. PCR using primers designed against the assembled transcript sequences resulted in amplification of three full-length cDNAs (AhR3 'DT-1, AhRPT-10a4 and AhRPT-10d4) encoding stilbenoid prenyltransferase candidates (Figure 63). We subcloned these candidates into yeast expression vector pPICZ, for

heterogeneous expression in Pichia pastoris. The microsomal fractions of the yeast cultures were employed for prenyltransferase assays. However, prenylation activity using resveratrol as substrate was not detected in any of the recombinant yeast cultures assayed (data not shown). In previous studies, researchers failed to detect flavonoid prenyltransferase activity when the full- length open reading frame (ORF) of GmG4DT was expressed in yeast, while the truncated form of GmG4DT without its N-terminus transit peptide showed genistein prenyltransferase activity (Yazaki et al., 2009). Likewise, the microsomal fraction of yeast expressing a truncated form of LaPTl, in which the first 44 amino acids were deleted, showed 6-fold higher activity than that of the full-length protein (Shen et al., 2012). Reasons for these observations may be low tolerance of plant transit peptides in yeast, correlated with incorrect folding and decreased stability of the prenyltransferases, resulting in low enzymatic activity. Rather than removing N-terminal putative transit peptide sequences and reexamining the yeast expression system as in the flavonoid prenyltransferase studies mentioned, we shifted to a heterologous plant expression system. We subcloned the full length cDNAs of the three candidate stilbenoid prenyltransferase into binary vectors under the control of CaMV35S-TEV promoter and transiently expressed these by Agrobacterium-infiltration oiNicotiana benthamiana leaves. The crude cell extract of N.

benthamiana leaves was incubated with DMAPP and resveratrol to test prenyltransferase activities. One of the cDNA products (amplified with primers PT-lO-FW-Notl/PT-k-RV-iQwI, Figure 66) showed resveratrol dimethylallyltransferase activity (Figure 35). No activity was observed in the crude cell extract of N. benthamiana control leaves that were infiltrated with Agrobacterium harboring an empty binary vector (Figure 35). Mass spectrometry analysis of the reaction product (m/z 297 [M+H] ⁺ ) gave a primary fragment with a m/z 241 [M+H-56] ⁺ in MS ² which suggested the presence of a prenyl moiety (Figure 42; Figure 67). After recovery of the reaction product from a large-scale enzymatic assay, it was purified by semi-preparative HPLC and its structure was further elucidated by NMR analysis.

Because the purified compound was available in extremely low quantities, we continued performing each experiment ( ¹ H- ¹³ C FfMBC & HSQC) over longer time intervals with an increased number of scans. Data obtained showed well-resolved peaks, forming the basis for our unambiguous assignment of both ¹ H and ¹³ C chemical shifts. In both the ^! H and ¹³ C spectra, peaks assigned were in agreement with the original scaffold of resveratrol. To determine the position of the prenyl group on the resveratrol scaffold, NMR predict tool was used to generate multiple 1H and ¹³ C spectra for with various combinations of prenyl positions, with reference to the 4-hydroxyphenyl ring (Banfi and Patiny, 2008; Castillo et al., 2011). Two possible prenyl positions appear to be of highest probability and matched with the experimentally obtained 1H and C NMR spectra (Figures 43 A and 43 B). We further narrowed down to the final conformation by examining the 1H signals at 6.12 ppm, 6.38 ppm, 6.95 ppm, 7.04 ppm , 7.25 ppm, 7.49 ppm and were found to be consistent with the resveratrol scaffold. 1H peaks at 3.21 ppm and 5.75 ppm with matched well with the prenyl side chain. (Figure 43 A). The experimental NMR data strongly support the predicted location of the prenyl at the ortho position of the 4- hydroxyphenyl ring. The conformation of resveratrol is further corroborated by the 2D NMR data which show 13 C peaks at 104.5 ppm, 122.8 ppm and 129.0 ppm (Figure 43B). From the 1 H- ¹³ C HSQC and 1H- ¹³ C HMBC spectrum, the 1H signal at 7.25 ppm, 6.95 ppm and 6.38 ppm correlate with protons at the ortho position on 1,3-benzenediol, ethenyl proton and ortho- positioned proton on 4-hydroxyphenyl ring, respectively. Their corresponding C signals were at 127.0, 124.2 ppm, and 125.3 ppm, respectively (Figures 43C and 43D). Analysis of the NMR data suggest that the prenyl moiety is attached to the C-3' position of the resveratrol backbone to produce 3 -methyl-2-butenyl-3' -resveratrol. Thus, the enzyme that catalyzed this reaction was named as AhR3'DT-l (A. ^j5og ea resveratrol-3J _. -dimethylallyltransferase).

Interestingly, this prenylated resveratrol was not the arachidin-2 we had expected, given our previous work identifying prenylated products in the reaction using microsomal fraction of elicited peanut hairy root (Yang et al., 2016). Moreover, the 3 -methyl-2-butenyl-3 '-resveratrol reaction product described here was not observed in the elicited hairy root culture of peanut. Our results indicate then that there are other prenyltransferase(s) responsible for prenylating resveratrol to arachidin-2 in peanut. To further search for additional resveratrol

prenyltransferase(s) from the peanut hairy root transcriptome, other primer pairs were designed based on the alignment of the putative prenyltransferase transcripts (Figure 66). Using this approach, additional PCR amplicons were amplified from the cDNA of 9-hour-elicited peanut hairy roots and five were subsequently subcloned into binary vectors and transiently expressed in N. benthamiana leaves for prenyltransferase activity assays using DMAPP and resveratrol as substrates. Four additional cDNAs were identified as resveratrol prenyltransferase genes. One cDNA clone (amplified with primers PT-9-FW-NotI/PT-b-RV- p«I, Figure 66) showed a clear dimethylallyltransferase activity for resveratrol and prenylated it at the C-4 position to form arachidin-2 (Figure 35). This product was confirmed by comparison of its retention time, UV light spectrum, mass spectra, and fragmentation patterns obtained by tandem mass spectrometry (MS ² and MS ³ ) with arachidin-2 purified from peanut hairy root culture (Figure 44). Hence, this enzyme was designated as AhR4DT-l (A hypogaea resveratrol-4-dimethylallyltransferase). The other three cDNA clones (two amplified with primers and one amplified with primers VTS-FW-NotlfPT-m-KV-Kpnl, Figure 66) exhibited the same catalytic activities as AhR3'DT-l, converting resveratrol into 3 -methyl-2-butenyl-3' -resveratrol using DMAPP as prenyl donor. Hence, we named these AhR3'DT-2, AhR3'DT-3 and AhR3'DT-4. Among these four isoenzymes, AhR3'DT-l exhibited the highest activity. AhR3'DT-2 and AhR3'DT-3 exhibited activity levels that were 18% and 17% of AhR3'DT-l respectively, while AhR3'DT-4 reached only 5% that of AhR3'DT-l (Figure 35).

Genomic and Phylogenetic Relationships of the Prenyltransferases

We report here five active resveratrol prenyltransferases identified from biochemical analyses of eight peanut transcripts. Transcripts assembled from RNA-Seq reads and cloned as cDNAs each independently evidence that these encode polypeptides of 389-414 amino acids (Figure 36). All possess nine transmembrane a-helices as predicted by TMHMM 2.0 (Krogh et al., 2001) (Figures 4 to 7), as well as two aspartate-rich motifs, NQXXDXXXD in loop 2 and KD(I/L)XDX(E/D)GD in loop 6, that are also conserved in flavonoid prenyltransferases (Figure 36).

The gene structure of peanut prenyltransferases was estimated by aligning the transcripts . to available Arachis diploid progenitor genome sequence references (Bertioli et al., 2016). Four loci in each became apparent as candidate origins. Three of these were on pseudochromosome 8 in each genome, contained within a span containing notable gaps and described by Bertioli et al. (2016) as effected by a genomic reduction during polyp loidization. Only AhR3'DT-4 aligned completely to pseudochromosome 1 (Figure 63). Eliminating poor alignments in either progenitor, we estimated two loci in A. duranensis and two in A. ipaensis could explain the origin of the set of prenyltransferases characterized in this study (Figure 63). Transcript alignments showed that AhR3'DT-2 and -3 differ by a deletion in AhR3'DT-2 that encodes 16 amino acid residues in AhR3'DT-3 (Figure 36). Although the genomic reference contained gaps in this region that prevented gene structure validation, we expect that these two transcripts may be expressed from the same gene in A. hypogaea as alternatively spliced forms (Figure 63).

Among the eight peanut transcripts tested in N. benthamiana transient assays, three cDNAs failed to show resveratrol prenyltransferase activity. One appears to be alternative splice form or allele of AhR4DT-l, and two of Ah 3'DT-l. Observed variation among these may provide preliminary insights into structural requirements for the active forms characterized here. Interestingly, each of the three inactive cDNAs vary in length and sequence at the C-terminal end (45 and 46). A nine amino acid residue deletion at the C-terminus of the inactive AhR4DT- 1 transcript, AhRPT-9i2 likely reduces its encoded protein structure to eight transmembrane spans (Figure 45), suggesting that the integrity of the nine transmembrane domains in AhR4DT-l are essential for its activity. Two inactive transcripts of AhR3'DT-l encode a C-terminal extension that does not appear to have transmembrane properties (Figure 46). Each furthermore harbors several coding SNPs and an eight amino acid residue deletion (Δ41-48) that disrupts a region conserved in both active AhR4DT-l and AhR3'DT-l (Figure 36). Interestingly, although not fully deleted in AhR3'DT-2, -3 and -4, this sequence is highly variable in these three less active forms, suggesting this uncharacterized region may play a role in influencing the

prenyltransferase active site.

Phylogenetic analysis of these characterized prenyltransferase enzymes together showed that the peanut stilbenoid prenyltransferases form their own monophyletic group (Figure 37). This clade is notably distinct from the flavonoid prenyltransferases, as well as from

homogentisate (HG) prenyltransferases involved in ubiquinone and shikonin biosynthesis, and p- hydroxybenzoate (PHB) prenyltransferases involved in the tocotrienol, tocopherol and plastoquinone biosynthetic pathways (Figure 37).

Biochemical Characterization of AhR4DT-l and AhR3'DT-l

AhR3'DT-l, the enzyme with the highest activity among its group, along with AhR4DT- 1 were selected for further biochemical characterization. Similar to the previous resveratrol prenyltransferase activity characterized from peanut hairy roots (Yang et al., 2016), the specific activity in the microsomal fraction of N. benthamiana leaves expressing AhR4DT-l or

AhR3'DT-l were 8.9-fold and 13.9-fold higher than that in the crude cell-free extracts, respectively (Figure 64). Therefore, we used the microsomal fraction enriched with AhR4DT-l or AhR3'DT-l for subsequent enzymatic assays. Reactions were incubated with resveratrol, DMAPP and Mg ²⁺ as cofactor. Although the optimum activities of AhR4DT-l and AhR3'DT-l were observed at 37 °C and 30 °C, respectively, in the microsomal fraction of N. benthamina, all further prenylation assays were performed at 28 °C which corresponds to the culture temperature of peanut hairy roots in order to observe the actual behavior of AhR4DT-l and AhR3'DT-l during the elicitor-induced production of prenylated stilbenoids (Figure 49). The accumulation of the prenylated product produced by AhR4DT-l or AhR3'DT-l showed a linear relationship with the amount of the microsomal fraction (25-75 μg) and with incubation time (30-120 min) (Figure 50).

The effects of pH on AhR4DT-l and AhR3'DT-l activities were investigated using three buffers which spanned a pH range from 7.0 to 10.7. The optimum pH of AhR4DT-l activity was 9.4 in Glycine-NaOH buffer (relative activity of each prenyltransferase, 100%), 9.0 in Tris-HCl buffer (99.8%) and 9.7 (92.1%) in NaHC0 ₃ -Na ₂ C0 ₃ buffer (Figure 51A). AhR3'DT-l exhibited optimal activities at pH 9.0 in both Tris-HCl (relative activity, 100%) and Glycine-NaOH (92.8%) buffers, but at pH 9.7 in NaHC0 ₃ -Na ₂ C0 ₃ buffer (50.6%) (Figure 5 IB). In summary, for these two prenyltransferases, the optimum pH was approximately 9.0, which is consistent with the basic pH of the stroma (Hauser et al, 1995). Therefore 100 mM of Tris-HCl (pH 9.0) was used as a standard reaction buffer in subsequent prenyltransferase reactions.

A variety of divalent cations other than Mg ²⁺ were tested to determine their effects on AhR4DT-l and AhR3'DT-l activities. Mg ²⁺ was the most effective (100%) for AhR4DT-l, followed by Mn ²⁺ (83.9%) and Fe ²⁺ (6.4%) (Figure 52A). Interestingly, in AhR3'DT-l reactions, Mn ²⁺ (210.3%) provided 2.1 -fold higher efficiency than Mg ²⁺ (100%). Mg ²⁺ forms a bidentate complex with DMAPP which gets stabilized for an efficient transferase reaction. A possible reason why Mn ²⁺ exhibited a higher efficiency could be due to the larger ionic radius facilitating a stronger interaction with the pyrophosphate and the neighboring residues in the DMAPP binding pocket. Fe ²⁺ (34.8%) also appeared to contribute to AhR3'DT-l activity (Figure 52B). Trace amounts of AhR4DT-l and AhR3'DT-l activities (<0.5 %) were also detected with all other divalent cations (Ca , Co , Zn , Ni , and Cu ) and in the control group in which no divalent cation was added, while no activity was detected in the EDTA treated group (Figure 52). The trace activities observed are likely due to presence of low levels of Mg ²⁺ in the leaf microsomal fractions, released from chlorophyll-containing plastids.

The apparent R values of AhR4DT-l for both resveratrol (99.52 ± 15.11 μΜ) and DMAPP (153.7 ± 27.28 μΜ) were somewhat similar, and comparable with those of resveratrol prenyltransferase identified from peanut hairy root (Figure 65). In contrast, AhR3'DT-l exhibited a notably lower K _m for resveratrol (17.67 ± 1.601 μΜ), but a much higher K _m for DMAPP (691.4 ± 48.46 μΜ) (Figure 65; Figure 53). Piceatannol, a compound detected in peanut hairy root culture as another putative substrate for peanut prenyltransferase, provided another contrast. The AhR4DT-l K _m for piceatannol was 311.4 ± 54.83 while that of AhR3'DT-l was 50.3 ± 5.509 μΜ 153.7 ± 27.28 μΜ (Figure 65; Figure 53). Importantly, AhR4DT-l and

AhR3'DT-l each exhibited a higher V _max /K _m value for resveratrol than piceatannol, suggesting that both of these prenyltransferases prefer resveratrol over piceatannol as substrate.

Substrate Specificity of AhR4DT-l and AhR3'DT-l

In addition to resveratrol, various other stilbenoids (piceatannol, oxyresveratrol, pinosylvin, pterostilbene and piceid) and flavonoids (naringenin, apigenin and genistein) were used as potential substrates to analyze the prenyl acceptor specificity of AhR4DT-l and

AhR3'DT-l . The results (Figure 38; Figures 54 to 58; Figure 67) showed that AhR4DT-l can selectively catalyze piceatannol, pinosylvin and oxyresveratrol into arachidin-5, chiricanine A and prenylated oxyresveratrol (the position of the prenyl moiety on the prenylated oxyresveratrol remains undetermined), respectively. In the reactions of AhR3'DT-l, the prenylated products of piceatannol and prenylated oxyresveratrol were identified by HPLC-PDA/ESI-MS ⁿ analysis (Figures 57 to 58; Figure 67), although the positions of their prenyl moiety have not been confirmed due to the insufficient amount of these products for further structural elucidation. Pterostilbene which has two methoxy groups at the C-3 and C-5 positions along with piceid, a resveratrol glucoside with a glycosidic group at C-3 position, were not prenylated by either AhR4DT-l or AhR3'DT-l (Figure 38), suggesting that either or both hydroxyl groups on the C- 3 and C-5 of stilbene backbone might be crucial for substrate recognition by AhR4DT-l and AhR3'DT-l. Interestingly, no prenylated pinosylvin was produced in the AhR3'DT-l reaction (Figure 38C), suggesting that other than the necessary 3- and 5-hydroxyl groups, a hydroxyl group at C-4' position might be an additional requirement for AhR3'DT-l activity. Moreover, neither prenylated flavanone, prenylated fiavone, nor prenylated isoflavone was detected in either AhR4DT-l or AhR3'DT-l reactions when flavonoid was used as substrate, indicating that both of these peanut prenyltransferases may be stilbenoid-specific prenyltransferases.

To address the prenyl donor specificity of AhR4DT-l and AhR3'DT-l, in addition to DMAPP, other prenyl diphosphates, including isopentenyl pyrophosphate (IPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) were examined with resveratrol as a prenyl acceptor. Neither AhR4DT-l nor AhR3'DT-l showed any detectable activity when these prenyl diphosphates were used as prenyl donor, suggesting that both of these prenyltransferases had strict specificity for DMAPP (Figures 4D and 4E).

Subcellular Localization of AhR4DT-l and AhR3'DT-l

In evaluating primary structures using available software, we found conflicting predictions for the subcellular localization of these enzymes. The iPSORT program predicted a chloroplast transit peptide (cTP) in AhR4DT-l and a mitochondrial targeting peptide (mTP) in AhR3'DT-l. However, ChloroP and TargetP predictions suggested AhR4DT-l contained neither cTP nor mTP, while AhR3'DT-l contained an N-terminal cTP. To confirm their subcellular localizations experimentally, AhR4DT-l-GFP and AhR3'DT-l-GFP gene fusion constructs driven by the CaMV35S-TEV promoter were expressed transiently in onion epidermal cells via particle bombardment. As a positive control for plastid localization, we co-expressed a construct created by Nelson et al. (2007) that features the transit peptide (first 79 amino acids) of tobacco uBisCO small subunit fused to red fluorescent protein (RS-TP-mCherry). The green fluorescence signals of AhR4DT-l-GFP and AhR3'DT-l-GFP appeared in punctate patterns against organelles in the onion epidermal cells (Figure 39), patterns highly similar to that of the red fluorescence derived from RS-TP-mCherry (Figure 39). In contrast, control GFP driven by the Ca35S-TEV promoter was localized throughout the cytosol and nucleus (Figure 39). These results strongly suggest that AhR4DT-l and AhR3'DT-l in peanut are localized to plastids, similar to flavonoid prenyltransferases such as SfN8DT-l, GmG4DT and LaPTl characterized in other plant species (Yazaki et al., 2009; Shen et al., 2012; Sasaki et al., 2008).

Expression olAhR4DT-l and AhR3'DT-l in Peanut Hairy Roots during Elicitation

Our previous studies have shown that the accumulation of prenylated stilbenoids in peanut hairy root culture and the prenyltransferase activities from crude cell-free extracts of peanut hairy roots were upregulated by elicitor treatments and increased with incubation time (Yang et al., 2015, 2016). We therefore hypothesized that the mRNA of enzymes involved in the prenylation of stilbenoids, i.e. AhR4DT-l dAhR3 'DT-1, may accumulate upon elicitation in peanut hairy roots. To test this hypothesis, transcript levels of AhR4DT-l and AhR3 'DT-1 during co-treatment of hairy root cultures with MeJA and CD were quantified using real-time PCR. A rapid up-regulation of AhR4DT-l was observed after 0.5-hour post-elicitation, and its expression pattern was consistent with the prenyltransferase activities assayed in the same system (Figures 40 A and 40B). Despite an apparent delay in accumulation as compared to AhR4DT-l, levels of AhR3 'DT-1 mRNA increased as well, after 48-hour of elicitation in these experiments (Figure 40C). Whereas qPCR detects a short, sequence-unique portion of the target mRNA, comparative mapping of RNA-Seq reads can provide a more complete picture of differential expression. Assessment of differential expression of individual transcripts in this case is however confounded by the complexity of this tetraploid transcriptome, the large target enzyme family, and lack of A. hypogaea genome sequence reference. Employing available sequence references of the peanut diploid progenitors (above), we mapped all assembled A. hypogaea transcripts, both to confirm singularity of genomic loci and to select transcript sequence of each

prenyltransferase that is most sequence-inclusive to use as reference for quantification (Figure 65). Figures 6D-G represent sample-normalized counts of reads that mapped unambiguously to these transcript references. Not surprisingly, mock treatments (assayed at 9 h and 72 h) that mechanically stimulate the hairy root cultures resulted in increases in all transcripts. AhR4DT-l mRNA stood out, however, as accumulating to 2-3x over control levels early in response to addition of MeJA + CD (Figure 40D). AhR3 'DT-1, on the other hand, among treatments assayed, reached its highest levels only 72 h post-elicitation (Figure 40E). AhR3 'DT-2/3 and -4 transcripts were clearly detectable by RNA-Seq across the time course, but did not appear to change in response to elicitation (Figure 40F-G). These results indicate the activation of AhR4DT-l and AhR3 'DT-1 genes correlates with stress elicitation in peanut hairy root tissue, and that the accumulation of mRNA encoding these two enzymes correlates temporally with their prenyltransferase activities observed and with their catalyzed product accumulation. In contrast, niRNA of similar enzymes we show to exhibit activities that are not relevant, or less relevant, to arachidin-2 and 3 -methyl-2-butenyl-3' -resveratrol production in peanut are not noticeably transcriptionally responsive to the elicitation. As products attributable to AhR4DT-l and AhR3'DT-l activities do not accumulate in peanut hairy root cultures in response to the control treatments used here (Yang et al., 2015), protein expression and/or localization are likely to be controlled by mechanisms beyond the transcript accumulation observed.

DISCUSSION

Stilbenoid-Specific Prenyltransferases from Peanut

Prenylation of aromatic compounds plays an important role in the diversification of plant secondary metabolites and contributes to the enhancement of the biological activity of these polyphenolic compounds (Yazaki et al., 2009). To date only a few flavonoid prenyltransferase genes have been identified, including SJN8DT-1, SfiLDT, S/G6DT, SfFPT, GmG4DT, GuA6DT and LaPTl cloned from legume species (Sasaki et al., 2008, 2011; Chen et al., 2013; Yazaki et al., 2009; Li et al., 2014; Shen et al., 2012), along with MalDT and CtlDT&om non-legume species Morus alba and Cudrania tricuspidata, respectively (Wang et al., 2014). In the study reported here, we tested eight potential resveratrol prenyltransferase transcripts and five of them encoded enzymes with two distinct prenylation activities. The eight transcripts derived from four or more genes expressed in peanut hairy root cultures. Two of these, AhR4DT-l and AhR3 'DT-1, were characterized as stilbenoid-specific prenyltransferases.

The stilbenoid prenyltransferases described in this study share several common features with flavonoid prenyltransferases. First, each of these enzymes is a membrane-bound protein containing several putative transmembrane a-helices. The subcellular localization of the two stilbenoid prenyltransferases described here are primarily or exclusively in the plastid, as are the five flavonoid prenyltransferases previously characterized. Secondly, each enzyme contains two conserved aspartate-rich motifs. The observed divalent cation dependency of our prenylation reactions corroborates the proposed role of this structure in the active site, where the divalent cation and the prenyl diphosphate bind (Huang et al., 2014). Interestingly, with the exception of

2_ _| _

AhR3 'DT-1 in which Mn is most effective, all other flavonoid enzymes and the stilbenoid

9-1- enzyme AhR4DT-l show highest activity in the present of Mg . Lastly, most flavonoid prenyltransferases and the stilbenoid prenyltransferases identified in this study exhibit strict substrate specificity with respect to their prenyl acceptor and prenyl donor, a feature that contrasts sharply with the catalytically promiscuous aromatic prenyltransferases of fungi and bacteria.

Despite sharing key features with flavonoid prenyltransferases, the stilbenoid prenyltransferases are monophyletic to other plant prenyltransferases accepting aromatic substrates (Figure 40).

Involvement of AhR4DT-l and AhR3'DT-l in the Biosynthesis of Prenylated Stilbenoids in Peanut

AhR4DT-l specifically transfers a 3,3-dimethylallyl group to the A-ring at the C-4 position of resveratrol, piceatannol and pinosylvin. This enzyme exhibits biochemical properties that match well with the prenyltransferase activity identified from elicited peanut hairy roots (Yang et al., 2016), including Km values of resveratrol/DMAPP and identical preferences for prenyl acceptors and divalent cations. The consistency of all biochemical characteristics, along with our demonstration of transcript accumulation that is temporally correlated with C-4 prenylated stilbenoid (arachidin-l, arachidin-2, arachidin-3 and arachidin-5) accumulation, lead us to propose AhR4DT-l is responsible for the prenylation activity in the microsomal fraction of peanut hairy roots identified in our previous study (Yang et al., 2016).

The second stilbenoid specific prenyltransferase characterized here was AhR3'DT-l, that recognizes 3,5,4'-trihydroxystilbene and adds a 3,3-dimethylallyl group to C-3' of the B-ring. Notably, none of the prenylation products of resveratrol and piceatannol catalyzed by AhR3'DT- 1 were detected in peanut hairy root culture or peanut hairy root tissue. When compared to AhR4DT-l, AhR3'DT-l showed a lower Km for resveratrol and piceatannol, indicating a higher affinity for these prenyl acceptor substrates. In contrast, its affinity for DMAPP was much lower than that of AhR4DT-l. Furthermore, the F _max values of resveratrol, piceatannol and DMAPP for AhR4DT-l were 9.9-fold, 6.9-fold and 5.8-fold higher than that of AhR3'DT-l, respectively (Figure 65), indicating that the catalytic efficiency of AhR4DT-l, especially for utilizing DMAPP, is much higher than that in AhR3'DT-l. This might be a reason that no prenylation product of AhR3'DT-l was found in the peanut hairy root culture, even when the substrate for AhR3'DT-l was present.

Under the co-treatment of 100 μΜ MeJA and 9 g/L CD for 72 hours, the peanut hairy root cultures secrete into the medium large amounts of resveratrol (44.64 ± 10.26 mg/L), arachidin-l (77.85 ± 21.47 mg/L) and arachidin-3 (184.65 ± 22.29 mg/L), relatively moderate levels of arachidin-5 (15.54 ± 5.58 mg/L) and arachidin-2 (25.57 ± 2.98 mg/L), and even less piceatannol (4.02 ± 0.67 mg/L) (Yang et al., 2015, 2016). These observations suggest that arachidin-l and arachidin-3 may be end products during the tested period of elicitation. Differing from arachidin-5, arachidin-2, and most other prenylated flavonoids which harbor a 3,3- dimethylallyl moiety, arachidin-l and arachidin-3 have a unique 3-methyl-but-l-enyl moiety (Figure 41). Until now, the biosynthesis pathway(s) of arachidin-1 and arachdin-3 have not been fully elucidated, however several biosynthetic routes leading to their production could be proposed when considering results from our previous and current studies.

In our previous study, it was demonstrated that exogenous resveratrol could be oxidized to piceatannol by an extract from the peanut hairy root tissue through a very efficient enzymatic reaction (Yang et al., 2016). With the abundance of resveratrol in the culture medium of peanut hairy root, piceatannol generated from the oxidation of resveratrol could serve as a precursor, alternative to resveratrol, for prenylated stilbenoids in peanut. It appeared that this compound could be further metabolized into other derivatives, resulting in a relative low yield of piceatannol in the peanut hairy root culture.

AhR4DT-l identified here initiates the first step in the biosynthesis of prenylated stilbenoids in peanut by catalyzing the prenylation of resveratrol and piceatannol to form arachidin-2 and arachidin-5, respectively. Other than derived that from the prenylation of piceatannol, which is limited in the culture medium, it remains possible that arachidin-5 is also formed via hydroxylation of arachidin-2 by a stilbenoid monooxygenase (P450). Flavonoid 3'- monooxygenases, for example, are known to catalyze 3 '-hydroxylation of the flavonoid backbone (Tanaka and Brugliera, 2013). Similarly, arachidin-3 might be hydroxylated by a monooxygenase to produce arachidin-1 (Figure 41). Further enzyme discovery and testing are needed to explore these possibilities.

In the reactions with peanut hairy root microsomes, arachidin-2 and arachidin-5 prenylated from resveratrol and piceatannol, respectively, could be further converted into derivatives of arachidin-2 derivative and arachidin-5 that were detected in the medium upon the elicitor treatment (Yang et al., 2016). Nonetheless, these derivatives were not detected in the AhR4DT-l reactions (Figure 41), indicating other enzyme(s) that modify arachidin-2 and arachidin-5 are present downstream of AhR4DT-lT

In one proposed pathway for arachidin-l and arachidin-3, arachidin-2 and arachidin-5 might be directly converted to arachidin-3 and arachidin-l by an isomerase which could shift the olefmic bond position on their prenylated moieties (Figure 41). Alternatively, arachidin-2 or arachidin-5 might be converted into an intermediate product which is further modified into arachidin-3 or arachidin-l through multiple enzymatic steps (Figure 41). It is still unclear whether arachidin-2 derivative and arachidin-5 derivative found in the peanut hairy root culture were one of these intermediates involved in the biosynthesis of arachidin-3 and arachidin-l, which are the predominant compounds in the culture (Figure 41). Alternatively, it may still be a slight possibility that arachidin-3 and arachidin-l were directly synthesized from resveratrol and piceatannol catalyzed by AhR4DT-l or another specific prenyltransferase utilizing 3-methyl-but- 1-enyl pyrophosphate as prenyl donor. Although, as far as we know, this kind pyrophosphate has never been described in plants.

To date, over 45 prenylated stilbenoids and derivatives, including monomers and dimers, have been identified in peanut. Many of these chemical structures have been confirmed by NMR (Figure 41) or predicted by mass spectrometry. Interestingly, all these stilbenoids can be divided into two groups, one showing a prenyl unit or a derivative at the C-4 and a second group showing a prenyl unit or derivative at the C-3' position (Figure 41). These observations strongly suggest that the prenyltransferases encoded by AhR4DT-l and AhR3 'DT-1 act in the first committed steps that channel the diversification of non-prenylated stilbenoids into prenylated stilbenoids. As the first stilbenoid-specific prenylated transferases identified in a plant, these findings advance our understanding of this specialized gene family and the biosynthesis of important bioactive compounds in plant stress responses.

METHODS

Plant Materials and Chemical Reagents

Hairy root of peanut cv. Hull line 3 was was previously established by transforming peanut cotyledonary leaves with Agrobacterium rhizogenes strain ATCC 15834 and maintained in modified Murashige and Skoog (MSV) medium under continuous darkness at 28 °C as described before (Condori et al., 2010). The procedure of elicitation for stilbenoids production in peanut hairy root culture was performed according to Yang et al., (2015).

Authentic standards of resveratrol and piceatannol were obtained from Biophysica and Axxoram, respectively. Arachidin-1, arachidin-2, arachidin-3 and arachidin-5 standards were purified from elicited peanut hairy root cultures as described previously (Yang et al., 2016). Pinosylvin, oxyresveratrol, pterostilbene, naringenin, apigenin, genistein, and IPP, GPP, FPP and GGPP were purchased from Sigma- Aldrich. DMAPP used in this study was obtained from Isoprenoids.

RNA Preparation

A co-treatment time-course of 100 μΜ methyl jasmonate (MeJA; sigma) and 9 g/L (6.87 mM) methyl- -cyclodextrin (CD; Cavasal W7M) was applied to nine-day-old peanut hairy roots to induce the expression of genes involved in the biosynthesis of stilbenoids. Total RNA was extracted from elicited root tissue at 0.5, 3, 9, 18, 24 and 72 hours using TRIzol reagent (Life Technologies), according to the manufacturer's instructions. For controls, RNA was likewise extracted from cultures prior to treatment (t=0) and from non-elicited cultures, collected 9 and 72 hours after mock treatment, being refreshed with fresh MSV medium. Transcript Sequencing and Assembly

Strand-aware, polyA-enriched R A libraries were prepared using Illumina TruSeq Stranded m A Sample Preparation reagents with sequence-indexed adaptors, with inputs of 4 micrograms total RNA per sample. Average insert size of indexed libraries was 300 bp according to Bioanalyzer (Agilent) evaluation. Libraries were sent to the Roy J. Carver

Biotechnology Center / W.M. Keck Center at the University of Illinois at Urbana-Champaign, where they were quantified by qPCR and pooled together for sequencing of 2 x 101 paired cycles on an Illumina HiSeq2500 using TruSeq SBS sequencing kits version 3. Number of read pairs ranged from 34.8 to 58.9 M per sample. Data were processed and demultiplexed using Casava 1.8.2 (Illumina).

Reads were trimmed using Trimmomatic v 0.32 (Bolger et al., 2014) with headcrop 12, sliding window 5, minimum quality 25. Parallel assemblies of each sample-specific reads set as well as a combination of all reads were generated using Trinity v 2013-11-10 (Haas et al., 2013), TransABySS 1.5.5 (Robertson et al., 2010), Velvet-Oases 1.2.10 (Schulz et al., 2012) and SOAPtrans 1.03 (Li et al., 2009), yielding a total of 2.6 M putative complete and partial transcript sequences. Coding sequences (CDS) were predicted and translated using CD-Hit (Li and Godzik, 2006) through scripts of the Evigenes pipeline (Don Gilbert, Indiana University) to build the transcriptome BlastP and BLASTN databases.

Using GMAP 2016-04-04 (Wu and Watanabe, 2005), transcript assemblies were aligned independently to the Arachis duranensis and Arachis ipaensis genomes available in PeanutBase (www.peanutbase.org and Bertioli et al., 2016), which confirmed clustering of highly similar forms and allowed us to approximate a reduced^, hypogaea sequence reference against which we could quantify reads coverage attributable to the enzyme transcripts under study. Reads mapped to the resulting four genomic loci were then isolated using Samtools 1.3.1 (Li et al., 2009b) and re-mapped to A. hypogaea transcript references, determined here. Mapping of RNA- Seq reads was performed using Tophat2, v 2.0.7 (Kim et al., 2013) using the genome guided option. Uniquely mapped read counts from each sample were assessed using HTSeq v 0.6. lpl (Anders et al., 2015).

Cloning oiAhR4DT-l and AhR3'DT-l cDNA

The amino acid sequences of flavonoid prenyltransferases SfN8DT-l (accession number: BAG12671.1), SfG6DT (BAK52291.1), SfFPT (AHA36633.1), GmG4DT (BAH22520.1), GuA6DT (AITl 1912.1) and LaPTl (AER35706.1) were used as input to run BlastP (Altschul et al., 1990) against our translated peanut hairy root transcriptome sequence database. Predictions of chloroplast transit peptides (cTP) were made using both ChloroP

(http://www.cbs.dtu.dk/services/ChloroP/) and iPSORT (http://ipsort.hgc.jp/). To clone the full- length cDNA of these candidates, RNA of 9-hour-elicited peanut hairy roots was prepareded by TRIzol reagent and the cDNA was synthesized using iScript™ Select cDNA Synthesis kit (Bio- Rad) using oligo (dT) primer. One N-terminal primer and three C-terminal primers were synthesized with flanking Notl and Kpnl restriction sites, respectively (Figure 66) and PCR using these primers was performed with ExTaq DNA Polymerase (Takara) following the program below: initial denaturation (3 min, 94°C); 30 cycles (30s, 94°C; 30s, 52°C; 1 min 30s, 72 °C); and a final extension step (10 min, 72 °C). Three amplicons (including AhR3 'DT-1) were subcloned into a pGEM-T vector (Promega) and multiple clones from each amplicon were sequenced at University of Chicago Comprehensive Cancer Center (UCCCC). In the second screening, according to all the 108 candidate sequences, four N-terminal primers and five C- terminal primers with Notl/ Kpnl flanking restriction sites (Figure 66) were designed for prenyltransferase cloning and another four amplicons (including AhR4DT-l) obtained from the same cDNA template were cloned into pGEM-T vector for sequencing validation.

Phylogenetic Analysis

Protein sequences were aligned using MUSCLE (Edgar 2004), and a neighbor-joining phylogenetic tree computed with PhyML (Guindon and Gascuel 2003) using the Dayhoff substitution model and 100 bootstrapped replicates.

Construction of Binary Vectors

The cloning strategy of binary vectors is showed in Figure 59. In detail, the sequence of the double enhanced cauliflower mosaic virus 35S promoter (CaMV35S) fused to the translational enhancer from tobacco etch virus (TEV) was amplified from plasmid pR8-2 (constructed by Medina-Bolivar and Cramer, 2004) and subcloned into pGEM-T vector using Ca35S-FW-SM-l/TEV-RW-iV primers (Figure 66) with SaWNotl flanking restriction sites. After validation of the sequences, the pGEM-CaMV35S-TEV and pGEM-T vectors containing putative prenyltransferase gene (PT) were digested with SalllNotl and NotllKpnl, respectively. A high copy number vector, pBC KS(-) digested with KpnVSall was used as a transition vector. Two fragments of full-length cDNA and CaMV35S-TEV promoter were ligated into the transition vector in a 16 °C overnight reaction with T4 ligase (NEB). Then the fragment of CaMV35S-TEV-PT from the transition vector digested by Kpnl/Sall was subcloned into a binary vector, pBIB-Kan which was created by Becker (1990). Eventually, the constructed binary vector with the putative prenyltransferase gene under the control of CaMV35S-TEV chimeric promoter and three prime untranslated region (3'-UTR) of nopaline synthase from the original pBIB-Kan vector was transformed into A. tumefaciens LBA4404 for stilbenoid prenylation activity screening. Screening for Stilbenoid Prenylation Activity

The engineered A. tumefaciens was grown in 5 mL of YEP medium, containing 50 mg/L of kanamycin (Sigma- Aldrich) and 30 mg/L of streptomycin (Sigma- Aldrich) for antibiotic selection, at 28°C on an orbital shaker at 200 rpm. After cultivation for 2 days, 5 mL of bacterial suspension was inoculated into 50 mL of fresh YEP medium containing the antibiotics and allowed to grow for one additional day under the same conditions. Bacteria were pelleted by centrifugation, resuspended in 500 mL of induction medium containing 10 mM MgCl ₂ with 1 mM acetosyringone and incubated for 4 h at 28°C under 200 rpm orbital shaking until their OD ₆ oo reached to a range from 0.5 to 0.6. Before the infiltration, 0.005% of Tween-20, 0.005% of Triton 100 and 0.005%» of Silwet L-77 were added to bacterial cultures to enhance the efficiency of transformation in JV. benthamiana leaves. Agrobacterium-mediated vacuum infiltration was performed on 4-week-old JV. benthamiana following the methodology described previously (Medrano et al., 2009).

After 48 hours of post-infiltration, the "middle tier" of JV. benthamiana leaves were harvested for prenylation activity screening. Five grams of leaf tissue (fresh weight) were grounded and homogenized using a mortar with pestle in 10 mL of extraction buffer containing 100 mM Tris-HCl (pH 7.6) and 10 mM dithiothreitol (DTT). After removing the cell debris by centrifugation at 12,000 ^χ g for 20 min at 4 °C, the crude cell-free extract was obtained by passing the 12,000 ^χ g supernatant through a PD-10 desalting column (GE Healthcare) equilibrated with 100 mM Tris-HCl (pH 9.0) containing 10 mM DTT. The total protein concentration was determined by coomassie protein assay (Thermo Scientific) using bovine serum albumin as standard. The prenylation reactionscontained resveratrol (100 μΜ), DMAPP (300 μΜ), MgCl ₂ (10 mM) and DTT (5 mM) in a Tris-HCl buffer (100 mM, pH 9.0). After incubation (28°C, 40 min) with the crude cellrfree extract of N. benthamiana leaves (5 mg of total protein) in a total volume of 1 mL, the enzyme reaction was terminated by addition of HC1 (6 M, 20 μί,) and then the reaction mixture was extracted with ethyl acetate (1 mL). The ethyl acetate extract was dried under nitrogen gas and dissolved in 300 μΐ, of methanol. The reaction product was identified and quantified using HPLC ESI-MS" analysis as previously described (Yang et al., 2016). The reaction of crude cell-free extract of N. benthamiana leaves infiltrated with A. tumefaciens harboring the empty pBIB-Kan vector was used as control.

Enzymatic Characterization of AhR4DT-l and AhR3'DT-l

In preliminary experiments focused to study the effect of post-infiltration period on the prenylation activity in N. benthamiana, leaf tissues of plants which transiently expressed

AhR4D-l were harvested 24, 48, 72 and 96 hours post-infiltration. Among these reactions, the AhR4D-l activity in the crude cell-free extract of N. benthamiana leaves increased with the post- infiltration period from 24 to 72 hours, while the activity in the leaves harvested from 96 hours post-infiltration was similar to 72 hours (data not shown). In addition, since both AhR4DT-l and AhR3'DT-l were predicted as membrane bound proteins by TMHMM 2.0, the microsomal fraction of N. benthamiana leaves at 72 hours post-infiltration was used to study the biochemical properties of AhR4DT-l or AhR3'DT-l . Ten grams of fresh leaves were homogenized using a mortar with pestle in a 20 mL of extraction buffer (100 mM Tris-HCL pH 7.6 containing 10 mM DTT). The homogenate was centrifuged at 12,000 ^χ g for 20 min at 4 °C, and about 13.2 mL of the supernatant was centrifuged at 156,000 ^χ g for 45 min at 4 °C to pellet the microsomal fraction, while the 156,000 x g supernatant was prepared by using a PD-10 desalting column (GE Healthcare) equilibrated with Tris-HCl buffer (100 mM, pH 9.0) containing DTT (10 mM). The microsomal fraction was washed twice with Tris-HCl buffer (100 mM, pH 9.0) containing DTT (10 mM) and resuspended in 1 mL of the same buffer.

The basic reaction and measurement for AhR4DT-l and AhR3'DT-l activity were the same as that for prenylation activity screening with exception of using 30 μg of microsomal fraction of N. benthamiana leaves as enzyme instead of crude cell-free extract of N. benthamiana leaves in a 500 of reaction. To investigate the optimal pH, the enzymatic reactions were performed in Tris-HCl buffer (100 mM, pH 7.0 to 9.0), glycine-NaOH buffer (100 mM pH 8.6 to 10.6) andNaHC0 ₃ -Na ₂ C0 ₃ buffer (100 mM, pH 9.2 to 10.7). The optimal reaction temperatures for AhR4DT-l and AhR3'DT-l were tested at 20, 25, 28, 30, 37, 40, and 50 °C in Tris-HCl buffer (100 mM, pH 9.0). For the divalent cation dependency study, 10 mM MnCl ₂ , FeCl ₂ , CaCl ₂ , CoCl ₂ , ZnCl ₂ , NiCl ₂ , or CuCl ₂ was added to the reaction mixture instead of MgCl ₂ , and the enzyme activity was compared with the reaction containing MgCl ₂ . The reactions without divalent cation and 10 mM EDTA instead of MgCl ₂ were used as controls.

For the kinetic study, varying concentrations (10, 20, 40, 80, 160, 320, and 640 μΜ) of resveratrol or piceatannol with a fixed concentration of DMAPP (640 μΜ) and varying concentrations (10, 20, 40, 80, 160, 320, and 640 μΜ) of DMAPP with a fixed concentration of resveratrol (640 μΜ) were incubated with 30 μg of microsomal fractions of N. benthamiana leaves expressing AhR4DT-l or AhR3'DT-l to calculate the apparent K _m and V _max values by nonlinear regression analysis of the Michaelis-Menten equation using GraphPad Prism 6 software. The prenyl acceptor specificity of AhR4DT-l and AhR3'DT-l were tested using 100 μΜ of each stilbenoid (resveratrol, piceatannol, oxyresveratrol, pinosylvin, pterostilbene and piceid), flavanone (naringenin), flavone (apigenin), and isoflavone (genistein) with 300 μΜ DMAPP as a prenyl donor, while the prenyl donor specificities of these two enzymes were tested using 300 μΜ prenyl diphosphates (DMAPP, IPP, GPP, FPP, GGPP) with 100 μΜ resveratrol as a prenyl acceptor. All these reactions were performed in a total volume of 500 μΐ, with 100 mM Tris-HCl buffer (pH 9.0) at 28°C for 40 min.

NMR spectra

All NMR measurements were performed on a Bruker Avance 700MHz and spectrometers at 298 K. The 1H- ¹³ C HMBC and 1H- ¹³ C HSQC spectra were collected in d ₆ -acetone. The concentration of the sample was ~1 mM. For 1H NMR analysis, 16 transients were acquired with a 1 second relaxation delay using 32 K data points. The 90° pulse was 9.7 μ8 with a spectral width of 16 ppm. ID ¹³ C NMR spectra were obtained with a spectral width of 30 ppm collected with 64 K data points. Two-dimensional spectra were acquired with 2048 data points for t2 and 256 for t\ increments. All NMR data were analyzed using Topspin v 2.0 and SPARKY v 3.0 software. Peaks were integrated and overlaid with the simulated spectra for different versions of the prenyl chain attached on the resveratrol compound

Construction of GFP Fusion Proteins

The nucleotide sequence of modified green fluorescence protein (mGFP5) was amplified from pR8-2 (Medina-Bolivar and Cramer, 2004) using primers mgfp5-FW-5awHI/mgfp5-RW- Kpnl (Figure 66) and cloned into pGEM-T vector to give pGEM-mGFP5-l. PT-9M3-RV- BamHI or PT-lOkl-RV-BamHI reverse primer with Ca35S-FW-5'fl/I-2 forward primer were used to amplify the full-length of AhR4DT-l or AhR3'DT-l with CaMV35S-TEV promoter region from pBC-CaMV35S-TEV-9bl3 and pBC-CaMV35S-TEV-10kl vector, which were created during the construction of the binary vector (Figure 60; Figure 68). The PCR products were then cloned into pGEM-T vector for sequencing validation. After SaWBamRl digestion, CaMV35S- TEV-AhR4DT-l and CaMV35S-TEV-AhR3 'DT-1 fragments were isolated and inserted into pGEM-mGFP5-l to yield pGEM-CaMV35S-TEV-AhR4DT-l-GFP and pGEM-CaMV35S- ,TEV-AhR3 'DT-l-GFP, respectively. Lastly, the fragments of CaA V35S-TEV-AhR4DT-l -GFP and CaMV35S-TEV-AhR3 'DT-l-GFP were excised with SaWKpnl and ligated into binary vector pBIB-Kan to yield pBIBKan-AhR4DT- 1 -GFP and pBIBKan-AhR3 'DT-l-GFP, respectively (Figure 61). For the GFP control construct, mGFP5 gene was amplified from pR8-2 using primers mgfp5-FW-NotI/mgfp5-RW-Xp«I and cloned into pGEM-T vector to give pGEM- mGFP5-2. Two fragments CaMV35S-TEV digested from pGEM-CaMV35S-TEV by SaWNotl and mGFP5 digested from pGEM-mGFP5-2 by NotVKpnl were inserted into pBC KS(-) vector to form pBC-CaMV35S-TEV-GFP. The fragment of CaMV35 S-TEV-GFP was eventually subcloned into binary vector pBIB-Kan to form pBIB-Kan-GFP (Figure 61).

Particle Bombardment and Microscopy

To investigate the subcellular localization of AhR4DT-l and AhR3'DT-l, pBIB-Kan- AhR4DT-l-GFP, pBIB-Kan-AhR3 'DT-l-GFP and pBIB-Kan-GFP were co-bombarded with binary vector pt-rk (ABRC stock numbers: CD3-999, Nelson et al., 2007) containing a plastid marker fused with red fluorescent protein into the onion epidermal peel cells by PDS-1000/He™ systems (Bio-Rad) following the manufacturer's recommendations. In brief, 5 μg of target plasmid and 5 g of pt-rk plasmid were together coated on 50 of 60 mg/mL tungsten particles (Ml 7, 1 μπι; Bio-Rad) in the presence of 1 M CaCl ₂ and 15 mM spermidine. After several ethanol washes, plasmid-coated particles were dried on plastic discs and accelerated with a helium burst at 1100 psi in a bombardment chamber. Bombarded onion epidermal peels were kept on plates containing MS medium for 60 hours in the dark. The localization of the expressed proteins in the transformed cell was visualized with a Nikon Eclipse E800 microscope with a 20x/0.5W Fluor water immersion objective. Confocal fluorescence images were obtained by using Nikon digital eclipse CI microscope system with 488 nm laser illumination and 525/50 nm filter for GFP fluorescence and 543 nm laser with 595/50 nm filter for RFP fluorescence.

Quantitative Real-time PCR oiAhR4DT-l and AhR3 'DT-1

Total RNA was isolated from 100 μΜ MeJA and 9 g/L CD co-treated peanut hairy roots at 0.5, 3, 9, 18, 24 and 72 hours using TRIzol reagent, and cDNA was synthesized using iScript™ Select cDNASynthesis Kit (Bio-Rad) with oligo (dt) primers following the manufacturer's instructions. Primers for AhR4DT-l and AhR3 'DT-1 were designed using Allele ID (PREMIER Biosoft). Two reference genes, ACT7 (encoding actin 7) and EFal (encoding elongation factor al) were selected previously (Condori et al., 2011) and used to normalize the expression of AhR4DT-l and AhR3 'DT-1 in peanut hairy roots. Efficiencies of primers for these targets are shown in Figure 62. Quantitative real-time PCR (qPCR) reactions were carried out using iQ SYBR Green Supermix (Bio-Rad) as previously described (Yang et al., 2015) and the expression of AhR4DT-l and AhR3 'DT-l were analyzed by qbase+ (Biogazelle).

Accession Numbers

The nucleotide sequences of AhR4DT-l, AhR3'DT-l, AhR3'DT-2, AhR3'DT-3,

AhR3'DT-4 have been deposited in the GenBank™ database under the accession numbers

KY565244, KY565245, KY565246, KY565247 and KY565248, respectively.

Stable expression of peanut stilbenoid-specific prenyltransferase in tobacco plants and hairy roots.

To establish transgenic tobacco (Nicotiana tabacum) plants expressing peanut stilbenoid prenyltransferase genes, pBIB-Kan binary vectors containing either the AhR4DT-l or AhR3'DT-l cDNA under the control of the constitutive 35S promoter were transformed into Agrobacterium tumefaciens LBA4404. Leaf blade, petiole and stem explants of N. tabacum were wounded and inoculated with above engineered A. tumefaciens. After 48 hours of inoculation, explants were placed in regeneration medium (modified MS' medium containing 1 mg/L BAL and 0.1 mg/L NAA), 600 mg/L cefotaxime and 200 mg/L kanamycin for selection of transgenic plants. After 2-3 weeks, transgenic callus that developed at the inoculation site were harvested and transferred to medium containing 600 mg/1 cefotaxime and 200 mg/L kanamycin, and maintained at 24 °C for another 2 weeks for shoot development. Newly developed shoots were transferred to antibiotic-free medium for roots of whole plants (Figure 11).

To confirm that the prenyltransferase gene was integrated into the plant genome, genomic DNA was isolated from transgenic lines using DNeasy Plant Mini Kit (QIAGEN). Primer pairs that targeted the outside of prenyltransferase gene on pBIB-Kan vectors were used for molecular characterization of transgenic lines. The transgenic plants transformed with empty pBIB-Kan vector showed an amplicon of 523 bp, while the AhR4DT-l and AhR3'DT-l transgenic lines gave amplicons of 2673 bp and 2619 bp, respectively. The AhR4DT-l or AhR3'DT-l activity in these transgenic lines was further confirmed via enzymatic assay (Figures 71C and 72C). In summary, five AhR4DT-l -expressing tobacco plants and nine AhR3'DT-l -expressing tobacco plants of N. tabacum were established (Figures 71 and 72).

Two hairy root lines, line 1 and line 2, were developed from the AhR3'DT-l -expressing tobacco plant #12 via Agrobacterium rhizogenes 15834 infection and the activity of the AhR3'DT-l was detected from both of these transgenic hairy root lines (Figure 73).

These results indicate that the peanut stilbenoid-specific transferases (AhR4DT-l or AhR3'DT-l) were actively functional in the transgenic tobacco plants and hairy roots. Statistical analysis

Two-way ANOVA with multiple-comparisons tests was conducted for the data in Figure 3. Analyses were done with GraphPad Prism 6, version 6.02 software. LITERATURE CITED

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It will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims.

As many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.