How storytelling through the use of avatars could help Fukushima evacuees express themselves: digital archive for people in the diaspora.

The Memorytalk online platform allows users to upload animated human stories. They may also choose to display related photos/characters and construct scenarios. Memorytalk may thus constitute a useful collaborative format for survivors of the Great East Japan Earthquake, especially evacuees from the town of Namie, who are now relocated in different areas across Japan, particularly as they are hesitant to talk about where they are from. This study examined various narrated animations created by Namie high school students within the Memorytalk platform, specifically for the purpose of investigating their storytelling methods based on Erving Goffman's role-distance theory. Memorytalk not only allows avatars that provide users with anonymity when expressing a wide range of memories, it also creates (1) opportunities for face-to-face workshops in the context of computer-mediated communication and (2) the therapeutic benefits of being humorous. In sum, the platform may provide users with a form of role distancing in which people in the diaspora can more freely express themselves and also store valuable local memories.

Extracellular stimulation of lung fibroblasts with arachidonic acid increases interleukin 11 expression through p38 and ERK signaling.

Interleukin-11 (IL-11) is a pleiotropic cytokine that regulates proliferation and motility of cancer cells. Fibroblasts reside in the cancer microenvironment and are the primary source of IL-11. Activated fibroblasts, including cancer-associated fibroblasts that produce IL-11, contribute to the development and progression of cancer, and induce fibrosis associated with cancer. Changes in fatty acid composition or its metabolites, and an increase in free fatty acids have been observed in cancer. The effect of deregulated fatty acids on the development and progression of cancer is not fully understood yet. In the present study, we investigated the effects of fatty acids on mRNA expression and secretion of IL-11 in lung fibroblasts. Among the eight fatty acids added exogenously, arachidonic acid (AA) increased mRNA expression and secretion of IL-11 in lung fibroblasts in a dose-dependent manner. AA-induced upregulation of IL-11 was dependent on the activation of the p38 or ERK MAPK signaling pathways. Furthermore, prostaglandin E2, associated with elevated cyclooxygenase-2 expression, participated in the upregulation of IL-11 via its specific receptor in an autocrine/paracrine manner. These results suggest that AA may mediate IL-11 upregulation in lung fibroblasts in the cancer microenvironment, accompanied by unbalanced fatty acid composition.

A Cytosol-Localized Geranyl Diphosphate Synthase from Lithospermum erythrorhizon and Its Molecular Evolution.

Geranyl diphosphate (GPP) is the direct precursor of all monoterpenoids and is the prenyl source of many meroterpenoids, such as geranylated coumarins. GPP synthase (GPPS) localized in plastids is responsible for providing the substrate for monoterpene synthases and prenyltransferases for synthesis of aromatic substances that are also present in plastids, but GPPS activity in Lithospermum erythrorhizon localizes to the cytosol, in which GPP is utilized for the biosynthesis of naphthoquinone pigments, which are shikonin derivatives. This study describes the identification of the cytosol-localized GPPS gene, LeGPPS, through EST- and homology-based approaches followed by functional analyses. The deduced amino acid sequence of the unique LeGPPS showed greater similarity to that of farnesyl diphosphate synthase (FPPS), which generally localizes to the cytosol, than to plastid-localized conventional GPPS. Biochemical characterization revealed that recombinant LeGPPS predominantly produces GPP along with a trace amount of FPP. LeGPPS expression was mainly detected in root bark, in which shikonin derivatives are produced, and in shikonin-producing cultured cells. The GFP fusion protein in onion (Allium cepa) cells localized to the cytosol. Site-directed mutagenesis of LeGPPS and another FPPS homolog identified in this study, LeFPPS1, showed that the His residue at position 100 of LeGPPS, adjacent to the first Asp-rich motif, contributes to substrate preference and product specificity, leading to GPP formation. These results suggest that LeGPPS, which is involved in shikonin biosynthesis, is recruited from cytosolic FPPS and that point mutation(s) result in the acquisition of GPPS activity.

Cytochrome P450 CYP71BE5 in grapevine (Vitis vinifera) catalyzes the formation of the spicy aroma compound (-)-rotundone.

(-)-Rotundone is a potent odorant molecule with a characteristic spicy aroma existing in various plants including grapevines (Vitis vinifera). It is considered to be a significant compound in wines and grapes because of its low sensory threshold and aroma properties. (-)-Rotundone was first identified in red wine made from the grape cultivar Syrah and here we report the identification of VvSTO2 as a α-guaiene 2-oxidase which can transform α-guaiene to (-)-rotundone in the grape cultivar Syrah. It is a cytochrome P450 (CYP) enzyme belonging to the CYP 71BE subfamily, which overlaps with the very large CYP71D family and, to the best of our knowledge, this is the first functional characterization of an enzyme from this family. VvSTO2 was expressed at a higher level in the Syrah grape exocarp (skin) in accord with the localization of (-)-rotundone accumulation in grape berries. α-Guaiene was also detected in the Syrah grape exocarp at an extremely high concentration. These findings suggest that (-)-rotundone accumulation is regulated by the VvSTO2 expression along with the availability of α-guaiene as a precursor. VvSTO2 expression during grape maturation was considerably higher in Syrah grape exocarp compared to Merlot grape exocarp, consistent with the patterns of α-guaiene and (-)-rotundone accumulation. On the basis of these findings, we propose that VvSTO2 may be a key enzyme in the biosynthesis of (-)-rotundone in grapevines by acting as a α-guaiene 2-oxidase.

Effect of light exposure on linalool biosynthesis and accumulation in grape berries.

Linalool is an important compound that contributes to the floral aroma in wines. This study showed the effect of light exposure on linalool accumulation in berries. The grape bunches were covered with films that block the full light spectrum (Shade) and the UV spectrum (UV-block), and a transparent film (Control). The linalool content was significantly higher in juice from Control-covered berries than in juice from Shade- and UV-block-covered berries, and the expression levels of the representative genes in linalool biosynthesis in Shade- and UV-block-covered berries were markedly lower than in Control-covered berries. These findings suggest that exposing berries to light is essential for linalool biosynthesis. To reflect sunlight onto grape clusters, reflective sheets were placed on the ground of a vineyard. The linalool content in berries exposed to sunlight reflected from the reflective sheets was higher than those in the control.

Molecular cloning and characterization of UDP-glucose: furaneol glucosyltransferase gene from grapevine cultivar Muscat Bailey A (Vitis labrusca × V. vinifera).

2,5-Dimethyl-4-hydroxy-3(2H)-furanone (furaneol) is an important aroma compound in fruits, such as pineapple and strawberry, and is reported to contribute to the strawberry-like note in some wines. Several grapevine species are used in winemaking, and furaneol is one of the characteristic aroma compounds in wines made from American grape (Vitis labrusca) and its hybrid grape. Furaneol glucoside was recently isolated as an important furaneol derivative from the hybrid grapevine cultivar, Muscat Bailey A (V. labrusca × V. vinifera), and this was followed by its isolation from some fruits such as strawberry and tomato. Furaneol glucoside is a significant 'aroma precursor of wine' because furaneol is liberated from it during alcoholic fermentation. In this study, a glucosyltransferase gene from Muscat Bailey A (UGT85K14), which is responsible for the glucosylation of furaneol was identified. UGT85K14 was expressed in the representative grape cultivars regardless of species, indicating that furaneol glucoside content is regulated by the biosynthesis of furaneol. On the other hand, furaneol glucoside content in Muscat Bailey A berry during maturation might be controlled by the expression of UGT85K14 along with the biosynthesis of furaneol. Recombinant UGT85K14 expressed in Escherichia coli is able to transfer a glucose moiety from UDP-glucose to the hydroxy group of furaneol, indicating that this gene might be UDP-glucose: furaneol glucosyltransferase in Muscat Bailey A.

Molecular characterization of a membrane-bound prenyltransferase specific for isoflavone from Sophora flavescens.

Prenylated isoflavones are secondary metabolites that are mainly distributed in legume plants. They often possess divergent biological activities such as anti-bacterial, anti-fungal, and anti-oxidant activities and thus attract much attention in food, medicinal, and agricultural research fields. Prenyltransferase is the key enzyme in the biosynthesis of prenylated flavonoids by catalyzing a rate-limiting step, i.e. the coupling process of two major metabolic pathways, the isoprenoid pathway and shikimate/polyketide pathway. However, so far only two genes have been isolated as prenyltransferases involved in the biosynthesis of prenylated flavonoids, namely naringenin 8-dimethylallyltransferase from Sophora flavescens (SfN8DT-1) specific for some limited flavanones and glycinol 4-dimethylallyltransferase from Glycine max (G4DT), specific for pterocarpan substrate. We have in this study isolated two novel genes coding for membrane-bound flavonoid prenyltransferases from S. flavescens, an isoflavone-specific prenyltransferase (SfG6DT) responsible for the prenylation of the genistein at the 6-position and a chalcone-specific prenyltransferase designated as isoliquiritigenin dimethylallyltransferase (SfiLDT). These prenyltransferases were enzymatically characterized using a yeast expression system. Analysis on the substrate specificity of chimeric enzymes between SfN8DT-1 and SfG6DT suggested that the determinant region for the specificity of the flavonoids was the domain neighboring the fifth transmembrane α-helix of the prenyltransferases.

HlPT-1, a membrane-bound prenyltransferase responsible for the biosynthesis of bitter acids in hops.

Female flowers of hop (Humulus lupulus L.) develop a large number of glandular trichomes called lupulin glands that contain a variety of prenylated compounds such as α- and ß-acid (humulone and lupulone, respectively), as well as xanthohumol, a chalcone derivative. These prenylated compounds are biosynthesized by prenyltransferases catalyzing the transfer of dimethylallyl moiety to aromatic substances. In our previous work, we found HlPT-1 a candidate gene for such a prenyltransferase in a cDNA library constructed from lupulin-enriched flower tissues. In this study, we have characterized the enzymatic properties of HlPT-1 using a recombinant protein expressed in baculovirus-infected insect cells. HlPT-1 catalyzed the first transfer of dimethylallyl moiety to phloroglucinol derivatives, phlorisovalerophenone, phlorisobutyrophenone and phlormethylbutanophenone, leading to the formation of humulone and lupulone derivatives. HlPT-1 also recognized naringenin chalcone as a flavonoid substrate to yield xanthohumol, and this broad substrate specificity is a unique character of HlPT-1 that is not seen in other reported flavonoid prenyltransferases, all of which show strict specificity for their aromatic substrates. Moreover, unlike other aromatic substrate prenyltransferases, HlPT-1 revealed an exclusive requirement for Mg(2+) as a divalent cation for its enzymatic activity and also showed exceptionally narrow optimum pH at around pH 7.0.

Characterization of coumarin-specific prenyltransferase activities in Citrus limon peel.

Coumarins, a large group of polyphenols, play important roles in the defense mechanisms of plants, and they also exhibit various biological activities beneficial to human health, often enhanced by prenylation. Despite the high abundance of prenylated coumarins in citrus fruits, there has been no report on coumarin-specific prenyltransferase activity in citrus. In this study, we detected both O- and C-prenyltransferase activities of coumarin substrates in a microsome fraction prepared from lemon (Citrus limon) peel, where large amounts of prenylated coumarins accumulate. Bergaptol was the most preferred substrate out of various coumarin derivatives tested, and geranyl diphosphate (GPP) was accepted exclusively as prenyl donor substrate. Further enzymatic characterization of bergaptol 5-O-geranyltransferase activity revealed its unique properties: apparent K(m) values for GPP (9 µM) and bergaptol (140 µM) and a broad divalent cation requirement. These findings provide information towards the discovery of a yet unidentified coumarin-specific prenyltransferase gene.

Tetrachloroaurate (III)-induced oxidation increases non-thermal plasma-induced oxidative stress.

Non-thermal plasma (NTP) devices have been explored for medical applications. NTP devices discharge electrons, positive ions, ultraviolet (UV), reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as the hydroxyl radical (•OH), singlet oxygen (1O2), superoxide (O2•-), hydrogen peroxide (H2O2), ozone, and nitric oxide, at near-physiological temperature. At preclinical stages or in human clinical trials, NTP promotes blood coagulation, eradication of bacterial, viral, and biofilm-related infections, wound healing, and cancer cell death. Here, we observed that ferric, vanadium, and gold(III) ions significantly elevated lipid peroxidation, which was measured by 2-thiobarbituric acid-reactive substances (TBARS) in combination with NTP exposure. Using 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) as a spin probe in electron paramagnetic resonance (EPR), we observed that tetrachloroaurate (III) yielded an M4PO-X spin adduct. Tetrachloroaurate-induced oxidation was attenuated efficiently by reduced (GSH) and oxidized glutathione (GSSG), while glycine (Gly), and L-glutamate (Glu), components of GSH, were ineffective. Furthermore, GSH and GSSG efficiently suppressed tetrachloroaurate-induced lipid peroxidation, while Gly and Glu were ineffective in suppressing TBARS elevation. These results indicate that tetrachloroaurate-induced oxidation is attenuated by GSH as well as GSSG. Further studies are warranted to elucidate the redox reactions between metal ions and biomolecules to advance the clinical application of NTP.

Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases.

Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7-O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner.

Monoterpene engineering in a woody plant Eucalyptus camaldulensis using a limonene synthase cDNA.

Metabolic engineering aimed at monoterpene production has become an intensive research topic in recent years, although most studies have been limited to herbal plants including model plants such as Arabidopsis. The genus Eucalyptus includes commercially important woody plants in terms of essential oil production and the pulp industry. This study attempted to modify the production of monoterpenes, which are major components of Eucalyptus essential oil, by introducing two expression constructs containing Perilla frutescens limonene synthase (PFLS) cDNA, whose gene products were designed to be localized in either the plastid or cytosol, into Eucalyptus camaldulensis. The expression of the plastid-type and cytosol-type PFLS cDNA in transgenic E. camaldulensis was confirmed by real-time polymerase chain reaction (PCR). Gas chromatography with a flame ionization detector analyses of leaf extracts revealed that the plastidic and cytosolic expression of PFLS yielded 2.6- and 4.5-times more limonene than that accumulated in wild-type E. camaldulensis, respectively, while the ectopic expression of PFLS had only a small effect on the emission of limonene from the leaves of E. camaldulensis. Surprisingly, the high level of PFLS in Eucalyptus was accompanied by a synergistic increase in the production of 1,8-cineole and alpha-pinene, two major components of Eucalyptus monoterpenes. This genetic engineering of monoterpenes demonstrated a new potential for molecular breeding in woody plants.

Two solanesyl diphosphate synthases with different subcellular localizations and their respective physiological roles in Oryza sativa.

Long chain prenyl diphosphates are crucial biosynthetic precursors of ubiquinone (UQ) in many organisms, ranging from bacteria to humans, as well as precursors of plastoquinone in photosynthetic organisms. The cloning and characterization of two solanesyl diphosphate synthase genes, OsSPS1 and OsSPS2, in Oryza sativa is reported here. OsSPS1 was highly expressed in root tissue whereas OsSPS2 was found to be high in both leaves and roots. Enzymatic characterization using recombinant proteins showed that both OsSPS1 and OsSPS2 could produce solanesyl diphosphates as their final product, while OsSPS1 showed stronger activity than OsSPS2. However, an important biological difference was observed between the two genes: OsSPS1 complemented the yeast coq1 disruptant, which does not form UQ, whereas OsSPS2 only very weakly complemented the growth defect of the coq1 mutant. HPLC analyses showed that both OsSPS1 and OsSPS2 yeast transformants produced UQ9 instead of UQ6, which is the native yeast UQ. According to the complementation study, the UQ9 levels in OsSPS2 transformants were much lower than that of OsSPS1. Green fluorescent protein fusion analyses showed that OsSPS1 localized to mitochondria, while OsSPS2 localized to plastids. This suggests that OsSPS1 is involved in the supply of solanesyl diphosphate for ubiquinone-9 biosynthesis in mitochondria, whereas OsSPS2 is involved in providing solanesyl diphosphate for plastoquinone-9 formation. These findings indicate that O. sativa has a different mechanism for the supply of isoprenoid precursors in UQ biosynthesis from Arabidopsis thaliana, in which SPS1 provides a prenyl moiety for UQ9 at the endoplasmic reticulum.

Prenylation of flavonoids by biotransformation of yeast expressing plant membrane-bound prenyltransferase SfN8DT-1.

Prenylated flavonoids are natural products that exhibit diverse biological effects and often represent the active components of various medicinal plants. This study demonstrated the production of prenylated naringenin by biotransformation using transgenic yeast expressing naringenin 8-dimethylallyltransferase, a membrane-bound enzyme, without feeding of prenyl donors. This method provides the possibility of generating prenylated flavonoids that occur rarely in nature.

Impact of Lactobacillus plantarum on thiol precursor biotransformation leading to production of 3-sulfanylhexan-1-ol.

3-Sulfanylhexan-1-ol (3SH) is an important contributor to the fruity notes of wine. 3SH exists as odorless precursors in grape and its release from the precursors is generally mediated by yeast during alcoholic fermentation. Here, the impact of lactic acid bacteria on 3SH production was investigated. Among the species tested, only Lactobacillus plantarum released 3SH from S-3-(hexan-1-ol)-l-cysteine (3SH-S-cys) and S-3-(hexan-1-ol)-l-cysteinylglycine (3SH-S-cysgly) in the whole-cell biotransformation assay. The conversion yields of 3SH from 3SH-S-cysgly by L. plantarum were always higher than those from 3SH-S-cys, suggesting that the direct cleavage of 3SH-S-cysgly to yield 3SH predominantly occurred. L. plantarum biotransformed the 3SH precursors, including 3SH-S-glut, to release 3SH in fermented grape juice. The results indicate that L. plantarum induces the release of 3SH from the 3SH precursors. To the best of our knowledge, this is the first study showing the impact of L. plantarum on thiol precursor biotransformation.

Unique Solid-State Emission Behavior of Aromatic Difluoroboronated ß-Diketones as an Emitter in Organic Light-Emitting Devices.

Aromatic difluoroboronated ß-diketone (BF2 DK) derivatives are a widely known class of luminescent organic materials that exhibit high photoluminescent quantum efficiency and unique aggregation-dependent fluorescence behavior. However, there have been only a few reports on their use in solid-state electronic devices, such as organic light-emitting devices (OLEDs). Herein, we investigated the solid-state properties and OLED performance of a series of π-extended BF2 DK derivatives that have previously been shown to exhibit intense fluorescence in the solution state. The BF2 DK derivatives formed exciplexes with a carbazole derivative and exhibited thermally activated delayed fluorescence (TADF) behavior to give orange electroluminescence with a peak external quantum efficiency of 10 % that apparently exceeds the theoretical efficiency limit of conventional fluorescent OLEDs (7.5 %), assuming a light out-coupling factor of 30 %.

Gene expression and characterization of isoprene synthase from Populus alba.

Isoprene synthase cDNA from Populus alba (PaIspS) was isolated by RT-PCR. This PaIspS mRNA, which was predominantly observed in the leaves, was strongly induced by heat stress and continuous light irradiation, and was substantially decreased in the dark, suggesting that isoprene emission was regulated at the transcriptional level. The subcellular localization of PaIspS protein with green fluorescent protein fusion was shown to be in plastids. PaIspS expressed in Escherichia coli was characterized enzymatically: it had an optimum pH of approximately 8.0, and an optimum temperature 40 degrees C. Its preference for divalent cations for its activity was also studied.

Novel method to reduce fishy aftertaste in wine and seafood pairing using alcohol-treated yeast cells.

"Fishy aftertaste" is sometimes perceived in wine consumed with seafood. Iron in wine has been reported to be a key compound that produces fishy aftertaste. However, cost-effective methods to remove iron from wine have not been developed. Here, we describe a cost-effective and safe iron adsorbent consisting of alcohol-treated yeast (ATY) cells based on the observation that nonviable cells adsorbed iron after completion of fermentation. Treatment of cells with more than 40% (v/v) ethanol killed them without compromising their ability to adsorb iron. Drying the ATY cells did not reduce iron adsorption. Use of ATY cells together with phytic acid had a synergistic effect on iron removal. We term this means of removing iron the "ATY-PA" method. Sensory analysis indicated that fishy aftertaste in wine-seafood pairings was not perceived if the wine had been pretreated with both ATY cells and phytic acid.

Prenylation of aromatic compounds, a key diversification of plant secondary metabolites.

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

Molecular cloning and characterization of a cDNA for pterocarpan 4-dimethylallyltransferase catalyzing the key prenylation step in the biosynthesis of glyceollin, a soybean phytoalexin.

Glyceollins are soybean (Glycine max) phytoalexins possessing pterocarpanoid skeletons with cyclic ether decoration originating from a C5 prenyl moiety. Enzymes involved in glyceollin biosynthesis have been thoroughly characterized during the early era of modern plant biochemistry, and many genes encoding enzymes of isoflavonoid biosynthesis have been cloned, but some genes for later biosynthetic steps are still unidentified. In particular, the prenyltransferase responsible for the addition of the dimethylallyl chain to pterocarpan has drawn a large amount of attention from many researchers due to the crucial coupling process of the polyphenol core and isoprenoid moiety. This study narrowed down the candidate genes to three soybean expressed sequence tag sequences homologous to genes encoding homogentisate phytyltransferase of the tocopherol biosynthetic pathway and identified among them a cDNA encoding dimethylallyl diphosphate: (6aS, 11aS)-3,9,6a-trihydroxypterocarpan [(-)-glycinol] 4-dimethylallyltransferase (G4DT) yielding the direct precursor of glyceollin I. The full-length cDNA encoding a protein led by a plastid targeting signal sequence was isolated from young soybean seedlings, and the catalytic function of the gene product was verified using recombinant yeast microsomes. Expression of the G4DT gene was strongly up-regulated in 5 to 24 h after elicitation of phytoalexin biosynthesis in cultured soybean cells similarly to genes associated with isoflavonoid pathway. The prenyl part of glyceollin I was demonstrated to originate from the methylerythritol pathway by a tracer experiment using [1-(13)C]Glc and nuclear magnetic resonance measurement, which coincided with the presumed plastid localization of G4DT. The first identification of a pterocarpan-specific prenyltransferase provides new insights into plant secondary metabolism and in particular those reactions involved in the disease resistance mechanism of soybean as the penultimate gene of glyceollin biosynthesis.