Protoplasma DOI 10.1007/s00709-014-0735-8

ORIGINAL ARTICLE

Rice terpene synthase 20 (OsTPS20) plays an important role in producing terpene volatiles in response to abiotic stresses Gun Woong Lee & Sungbeom Lee & Moon-Soo Chung & Yeon Sim Jeong & Byung Yeoup Chung

Received: 24 September 2014 / Accepted: 17 November 2014 # Springer-Verlag Wien 2014

Abstract This study examined the volatile terpenes produced by rice seedlings in response to oxidative stress induced by various abiotic factors. Solid-phase microextraction–gas chromatography–mass spectrometry (SPME–GC–MS) analyses revealed that when exposed to UV-B radiation, rice seedlings emitted a bouquet of monoterpene mixtures in a timedependent manner. The mixtures comprised limonene, sabinene, myrcene, α-terpinene, β-ocimene, γ-terpinene, and α-terpinolene. Among them, (S)-limonene was the most abundant volatile, discriminated by chiral SPME–GC–MS. The volatile profiles collected from rice plants treated with both γ-irradiation and H2O2 were identical to those observed in the UV-B irradiated plants, thus indicating that the volatile mixtures were specifically produced in response to oxidative stress, particularly in the presence of H2O2. Using a reverse genetics approach, we isolated full-length rice terpene synthase 20 (OsTPS20, 599 amino acids, 69.39 kDa), which was further characterized as an (S)-limonene synthase by removing the N-terminal signal peptide (63 amino acids) of the protein. The recombinant OsTPS20 protein catalyzed the conversion of geranyl diphosphate to (S)-limonene and other minor monoterpenes, essentially covering all of the volatile compounds detected from the plant. Moreover, qRT-PCR revealed that the transcript levels of OsTPS20 were significantly induced in response to oxidative stress, thereby suggesting that Handling Editor: Peter Nick Gun Woong Lee and Sungbeom Lee contributed equally to this study. Electronic supplementary material The online version of this article (doi:10.1007/s00709-014-0735-8) contains supplementary material, which is available to authorized users. G. W. Lee : S. Lee : M.4-fold induction was observed (Fig. 4e). Similarly, an extended exposure to UV-B radiation (12 h) upregulated the transcript abundance by more than 3-fold (Fig. 4g).

For the enzyme assays, the in silico-predicted putative signal peptide (63 amino acids) at the N-terminal end was removed from the full-length OsTPS20. The recombinant protein (62.67 kDa, pI=5.53) was overproduced in E. coli, and the His-tagged soluble protein of interest was affinity purified prior to the in vitro assays. OsTPS20 could accept GPP (a precursor for a monoterpene synthase) as a substrate, thereby producing sabinene, myrcene, α-terpinene, β-ocimene, γterpinene, α-terpinolene, and limonene as major products. The volatile profiles of these products were identical to those released from the seedlings after they were exposed to oxidative abiotic stresses (Figs. 2 and 6a). The reaction products of the enzyme were, however, not detected when the enzymes were heat denatured, thus confirming the enzymatic catalyses of the reaction products (Fig. 6b). OsTPS20 did not show any enzymatic activities toward FPP (a precursor for a sesquiterpene synthase) or GGPP (a precursor for a diterpene synthase) (data not shown). We analyzed the chirality of the limonene collected from the rice seedlings after the three different oxidative stresses. Analyses by solid-phase microextraction–gas chromatography–mass spectrometry using a chiral column revealed that

Measurement of H2O2 content We assessed the H2O2 contents in the rice seedlings treated with oxidative abiotic stress-inducing factors to confirm elicitation of the oxidative pathways by the stress treatments. All the treatments elicited an increased amount of H2O2 in the seedlings, thus indicating that the seedlings responded to the oxidative stresses and that the treatments caused the seedlings to produce H2O2 (Fig. 5).

Fig. 5 Hydrogen peroxide content in rice seedlings. a control, b γirradiation at 100 Gy, c γ- irradiation at 300 Gy, d 10 mM of H2O2, e 20 mM of H2O2, f UV-B irradiation for 6 h, g UV-B irradiation for 12 h. The values are the means (±standard deviation) of triplicate, representing three independent determinations

Fig. 6 SPME–GC–MS analyses of the reaction products collected from in vitro assays (m/z=93.1, extracted ion chromatograph). a OsTPS20, b boiled OsTPS20. 1 sabinene, 2 myrcene, 3 α-terpinene, 4 limonene, 5 βocimene, 6 γ-terpinene, 7 α-terpinolene, and *non-enzymatic products

Rice terpene synthase 20 (OsTPS20) plays an important role

the enantiomer of limonene was (S)-limonene (Rt =11.6) rather than (R)-limonene (Rt=11.8). The above finding was confirmed by comparing the retention times and the MS data of authentic isomeric standards (Fig. 7). Moreover, the limonene collected from the in vitro enzyme assay using recombinant OsTPS20 was shown to be an identical isomer (i.e., (S)limonene) of the molecule detected in planta (Fig. 7). Taken together, these findings indicated that OsTPS20 plays a major role in effectively producing most of the volatile terpenes emitted by rice seedlings in response to oxidative stress. Alignment of the deduced amino acid sequence of OsTPS20 The deduced amino acid sequence of OsTPS20 (599 amino acids) was compared with that of known monoterpene

Fig. 7 Chiral analysis of limonene. a (S)-/(R)-limonene standards, b UV-B irradiation, c γ-irradiation, d H2O2 treatment, e enzymatic products of OsTPS20. 1 (S)-limonene and 2 (R)-limonene

synthases. The sequence contained a DDXXD motif, which is a characteristic of metal ion-binding sites and is highly conserved among plant TPSs (Fig. 8). Notably, an RR(X8)W motif at the N terminus, which is another conserved domain necessary for the proper folding of monoterpene synthases, was not found in these sequences, which instead contained a KE(X8)W motif. The sequence homology of OsTPS20 with those of the known monoterpene synthases was unexpectedly low (less than 70 %) (Table S4). The Nterminal 63 amino acids were found to harbor a signal peptide sequence by the ChloroP (Emanuelsson et al. 1999) and TargetP (Emanuelsson et al. 2000) programs, which predicted the localization of OsTPS20 to chloroplasts, where GPP pools are available to be cyclized into monoterpenes such as limonene.

G.W. Lee et al.

Discussion Many plants release a bouquet of volatiles in response to high temperatures and UV irradiation (Loreto et al. 1998; Delfine et al. 2000; Duhl et al. 2008; Gil et al. 2012). In the current study, we delineated volatile mixtures; the production of which was inducibly increased in the rice seedlings in response to oxidative stress-inducing factors (UV-B, γ-irradiation, and H2O2). The mixtures comprised monoterpenes, including limonene, sabinene, myrcene, α-terpinene, βocimene, γ-terpinene, and α-terpinolene (Figs. 1 and 2b). Among these, limonene, which has also been reported to be an antioxidant in the oils extracted from various plants, was found to be the major volatile (Colby et al. 1993; Aggarwal et al. 2002; Wei and Shibamoto 2007). Therefore, it would be of interest to explore the physiological role(s) of limonene (or those of the volatile mixtures) in rice seedlings after exposure to oxidative stress. Limonene synthases catalyzing either the (R)- or (S)-enantiomer have been cloned and functionally characterized from diverse dicotyledonous plants (Rajaonarivony et al. 1992; Colby et al. 1993; Yuba et al. 1996; Bohlmann et al. 1997). However, to the best of our knowledge, a limonene synthase has not been reported from monocot plants, including rice. We screened OsTPS candidates for a putative limonene synthase by adopting a qualitative RT-PCR, using the mRNAs extracted from the rice seedlings after exposure to γ-irradiation, which significantly increased the production of limonene. Five OsTPSs showed steady-state induced transcript levels following the treatment, as compared with that of control treatment. We further narrowed down the five candidates by excluding the loci that had been previously characterized (i.e., OsTPS19 and OsTPS26), or those for which the gene transcript showed negligible induction patterns (i.e., OsTPS25 and OsTPS26). OsTPS30 was also excluded owing to the insolubility of the corresponding recombinant protein. In vitro biochemical assays using purified recombinant OsTPS20 revealed that OsTPS20 only accepts GPP as a substrate, and catalyzes the production of limonene and several other minor monoterpenes. The reaction products obtained from these assays were identical to the products whose volatile profiles were determined for rice plants exposed to oxidative stresses, thereby suggesting that a single locus, OsTPS20, plays a major role in generating oxidative stress-responsive terpene volatiles in rice seedlings. The emission of monoterpenes by rice seedling subjected to oxidative stress is probably mediated by H2O2. We confirmed H2O2 generation after UV-B irradiation, and the H2O2 content was increased after a longer exposure to the radiation. Similarly, the ionizing radiation (i.e., γ-rays) also initiated the production of H2O2, which was proportional to the amount of dose applied (Fig. 5). After exposure to abiotic stresses, the increase in H2O2 production in the seedlings was positively

correlated with the induction patterns of OsTPS20 transcripts as well as with the induced amount of monoterpene compounds collected from identical seedlings (Fig. 4), indicating that limonene mixtures were mainly biosynthesized in the seedlings by OsTPS20 after being subjected to abiotic oxidative stress, possibly via the mediation of the signaling molecule H2O2. However, we cannot rule out the possibility that other signaling chemicals or unknown OsTPSs may also be involved in limonene biosynthesis. Of the 33 putative OsTPSs, 32 (we excluded OsTPS5 because it was predicted to be a transposon-like gene in an NCBI blast search) were annotated to encode terpene synthases (15 of which have been characterized to date, whereas the remaining 17 have not been reported) (Table S1) (Chen et al. 2011). Furthermore, the genomic structures (exon/intron) were analyzed using the MIPS Oryza sativa Database from the Institute of Bioinformatics and Systems Biology (IBIS, http://www.helmholtzmuenchen.de/en/ibis), indicating that they showed the general exon/intron structures, previously proposed for plant TPSs (Table S1) (Trapp and Croteau 2001). The signal peptide and its subcellular localization were analyzed by using prediction programs (TargetP and ChloroP). The analyses suggested that 12 loci, including OsTPS20, would be localized to the chloroplast, and four loci (i.e., OsTPS7, OsTPS13, OsTPS26, and OsTPS27) were predicted to be most likely targeted to the mitochondria (Table S1). OsTPS20 contains a well-conserved DDXXD catalytic motif but lacks the RR sequences in the arginine-rich domain (i.e., RR(X8)W) near the N-terminal signal peptide (Fig. 8). This unusual characteristic feature was revealed when the above-mentioned sequence was compared with the amino acid sequences of most monoterpene synthases, with the exception of the monoterpene synthases isolated from snapdragon flowers (Dudareva et al. 2003). Moreover, OsTPS20, a monoterpene synthase, was positioned in a clade of sesquiterpene synthases in the corresponding phylogenetic tree (Fig. S2). This could be the result of evolution through different lineages of monocots and dicots corresponding to ancestral plants. (S)-(−)-limonene synthases have previously been isolated from the herbaceous plants Perilla frutescens and Mentha spicata (Colby et al. 1993; Yuba et al. 1996). The biological effects of (S)-(−)-limonene and (R)-(+)-limonene have been documented elsewhere. Furthermore, enantiomers of the two limonenes isolated from the oils of Mentha spicata L. and Anethum sowa Roxb showed antimicrobial effects against human pathogenic fungi and bacteria (Aggarwal et al. 2002). Antioxidative roles of terpenes have been proposed by determining radical scavenging activity in a 2,2-diphenyl-1picrylhydrazyl assay of a mixture of citrus oil, in which limonene is a major volatile compound (Singh et al. 2010). This observation is consistent with in planta findings, where monoterpene fumigation resulted in a decrease in photo-

Rice terpene synthase 20 (OsTPS20) plays an important role

Fig. 8 A comparison of the deduced amino acid sequences of OsTPS20 with those of the known limonene synthases. AAM53944, Citrus limon; CCM43927, Coffea arabica; ABB73044, Lavandula angustifolia;

AAG31438, Perilla frutescens; AAS47694, Picea abies; ABD77416, Rosmarinus officinalis; AAG01140, Schizonepeta tenuifolia; and AAB70907, Abies grandis. *signal peptides and **DDXXD motif

damage, which is normally associated with an oxidative burst in plants (Peñuelas and Llusià 2002). Moreover, the protective roles of monoterpenes and a volatile C5 hemiterpene isoprene against ozone-induced oxidative stress have been confirmed in Quercus ilex leaves (Loreto et al. 1998; Loreto et al. 2001; Loreto et al. 2004). Loreto and his colleagues have demonstrated that monoterpene and isoprene-emitting leaves maintained photosynthetic capacity and developed no visible damage when subjected oxidative stress. However, in fosmidomycin-treated leaves, which emitted no isoprene, there was an inhibition of photosynthesis and an accumulation of H2O2 and malondialdehyde. These authors speculated that volatile isoprenoids, including monoterpenes and isoprene, probably have similar antioxidative activities. Similarly, the high emission of (S)-(−)-

limonene in rice seedlings following oxidative stress leads us to the assumption that the limonene produced is directly associated with antioxidant mechanisms, or that compound(s) derived from limonene, such as carveol and carvone, could possibly be involved in this mechanism. It would also be of great interest to determine whether limonene stabilizes cell membrane structures under heat stress in rice, as proposed in the fumigation study using Quercus ilex (Loreto et al. 1998). Given that a rice plant undergoes abiotic oxidative stresses during its growth, our findings pertaining to the increase in volatile monoterpene emissions, and the associated discovery of the major TPS player, could provide key information for further investigations of the physiological and biological roles of volatile monoterpenes during oxidative stress in rice.

G.W. Lee et al. Acknowledgments This research was supported by the Nuclear R & D Program of the Ministry of Science, ICT and Future Planning (MSIP), Republic of Korea. Conflict of interest The authors declare that they have no conflict of interest.

References Abel C, Clauss M, Schaub A, Gershenzon J, Tholl D (2009) Floral and insect-induced volatile formation in Arabidopsis lyrata ssp. petraea, a perennial, outcrossing relative of A. thaliana. Planta 230:1–11 Aggarwal K, Khanuja S, Ahmad A, Santha Kumar T, Gupta VK, Kumar S (2002) Antimicrobial activity profiles of the two enantiomers of limonene and carvone isolated from the oils of Mentha spicata and Anethum sowa. Flavour Fragr J 17:59–63 Arimura G, Garms S, Maffei M, Bossi S, Schulze B, Leitner M, Mithöfer A, Boland W (2008) Herbivore-induced terpenoid emission in Medicago truncatula: concerted action of jasmonate, ethylene and calcium signaling. Planta 227:453–464 Attaran E, Rostás M, Zeier J (2008) Pseudomonas syringae elicits emission of the terpenoid (E, E)-4,8,12-trimethyl-1,3,7,11tridecatetraene in Arabidopsis leaves via jasmonate signaling and expression of the terpene synthase TPS4. Mol Plant-Microbe Interact 21:1482–1497 Barta C, Kálai T, Hideg K, Vass I, Hideg É (2004) Differences in the ROS-generating efficacy of various ultraviolet wavelengths in detached spinach leaves. Funct Plant Biol 31:23–28 Berli FJ, Moreno D, Piccoli P, Hespanhol-Viana L, Silva MF, BressanSmith R, Cavagnaro JB, Bottini R (2010) Abscisic acid is involved in the response of grape (Vitis vinifera L.) cv. Malbec leaf tissues to ultraviolet-B radiation by enhancing ultraviolet-absorbing compounds, antioxidant enzymes and membrane sterols. Plant Cell Environ 33:1–10 Bohlmann J, Steele CL, Croteau R (1997) Monoterpene synthases from grand fir (Abies grandis): cDNA isolation, characterization, and functional expression of myrcene synthase, (−)-(4S)-limonene synthase, and (−)-(1S,5S)-pinene synthase. J Biol Chem 272:21784– 21792 Bohlmann J, Meyer-Gauen G, Croteau R (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc Natl Acad Sci U S A 95:4126–4133 Boland W, Feng Z, Donath J, Gäbler A (1992) Are acyclic C11 and C16 homoterpenes plant volatiles indicating herbivory? Naturwissenschaften 79:368–371 Calogirou A, Larsen B, Kotzias D (1999) Gas-phase terpene oxidation products: a review. Atmos Environ 33:1423–1439 Chappell J (1995) Biochemistry and molecular biology of the isoprenoid biosynthetic pathway in plants. Annu Rev Plant Physiol Plant Mol Biol 46:521–547 Chen F, Tholl D, Bohlmann J, Pichersky E (2011) The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J 66:212–229 Cheng AX, Xiang CY, Li JX, Yang CQ, Hu WL, Wang LJ, Lou YG, Chen XY (2007) The rice (E)-β-caryophyllene synthase (OsTPS3) accounts for the major inducible volatile sesquiterpenes. Phytochemistry 68:1632–1641 Colby SM, Alonso WR, Katahira EJ, McGarvey DJ, Croteau R (1993) 4S-limonene synthase from the oil glands of spearmint (Mentha spicata). cDNA isolation, characterization, and bacterial expression of the catalytically active monoterpene cyclase. J Biol Chem 268: 23016–23024

Delfine S, Csiky O, Seufert G, Loreto F (2000) Fumigation with exogenous monoterpenes of a non-isoprenoid-emitting oak (Quercus suber): monoterpene acquisition, translocation, and effect on the photosynthetic properties at high temperatures. New Phytol 146: 27–36 Dolzhenko Y, Bertea CM, Occhipinti A, Bossi S, Maffei ME (2010) UVB modulates the interplay between terpenoids and flavonoids in peppermint (Mentha x piperita L.). J Photochem Photobiol B 100: 67–75 Dudareva N, Martin D, Kish CM, Kolosova N, Gorenstein N, Faldt J, Miller B, Bohlmann J (2003) (E)-β-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily. Plant Cell 15:1227–1241 Duhl T, Helmig D, Guenther A (2008) Sesquiterpene emissions from vegetation: a review. Biogeosciences 5:761–777 Emanuelsson O, Nielsen H, Heijne GV (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978–984 Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1016 Esnault M-A, Legue F, Chenal C (2010) Ionizing radiation: advances in plant response. Environ Exp Bot 68:231–237 Fares S, Loreto F, Kleist E, Wildt J (2008) Stomatal uptake and stomatal deposition of ozone in isoprene and monoterpene emitting plants. Plant Biol (Stuttg) 10:44–54 Gil M, Pontin M, Berli F, Bottini R, Piccoli P (2012) Metabolism of terpenes in the response of grape (Vitis vinifera L.) leaf tissues to UV-B radiation. Phytochemistry 77:89–98 Goff SA, Ricke D, Lan T-H, Presting G, Wang R, Dunn M, Glazebrook J, Sessions A, Oeller P, Varma H, Hadley D, Hutchison D, Martin C, Katagiri F, Lange BM, Moughamer T, Xia Y, Budworth P, Zhong J, Miguel T, Paszkowski U, Zhang S, Colbert M, W-l S, Chen L, Cooper B, Park S, Wood TC, Mao L, Quail P, Wing R, Dean R, Yu Y, Zharkikh A, Shen R, Sahasrabudhe S, Thomas A, Cannings R, Gutin A, Pruss D, Reid J, Tavtigian S, Mitchell J, Eldredge G, Scholl T, Miller RM, Bhatnagar S, Adey N, Rubano T, Tusneem N, Robinson R, Feldhaus J, Macalma T, Oliphant A, Briggs S (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296:92–100 Jansen MA, Gaba V, Greenberg BM (1998) Higher plants and UV-B radiation: balancing damage, repair and acclimation. Trends Plant Sci 3:131–135 Jenkins GI (2009) Signal transduction in responses to UV-B radiation. Annu Rev Plant Biol 60:407–431 Kappers IF, Aharoni A, van Herpen TW, Luckerhoff LL, Dicke M, Bouwmeester HJ (2005) Genetic engineering of terpenoid metabolism attracts bodyguards to Arabidopsis. Science 309:2070–2072 Lee S, Badieyan S, Bevan DR, Herde M, Gatz C, Tholl D (2010) Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis. Proc Natl Acad Sci U S A 107:21205–21210 Lichtenthaler HK (1999) The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 50:47–65 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408 Loreto F, Förster A, Dürr M, Csiky O, Seufert G (1998) On the monoterpene emission under heat stress and on the increased thermotolerance of leaves of Quercus ilex L. fumigated with selected monoterpenes. Plant Cell Environ 21:101–107 Loreto F, Mannozzi M, Maris C, Nascetti P, Ferranti F, Pasqualini S (2001) Ozone quenching properties of isoprene and its antioxidant role in leaves. Plant Physiol 126:993–1000

Rice terpene synthase 20 (OsTPS20) plays an important role Loreto F, Pinelli P, Manes F, Kollist H (2004) Impact of ozone on monoterpene emissions and evidence for an isoprene-like antioxidant action of monoterpenes emitted by Quercus ilex leaves. Tree Physiol 24:361–367 Obara N, Hasegawa M, Kodama O (2002) Induced volatiles in elicitortreated and rice blast fungus-inoculated rice leaves. Biosci Biotechnol Biochem 66:2549–2559 Peñuelas J, Llusià J (2002) Linking photorespiration, monoterpenes and thermotolerance in Quercus. New Phytol 155:227–237 Prisic S, Xu M, Wilderman PR, Peters RJ (2004) Rice contains two disparate ent-copalyl diphosphate synthases with distinct metabolic functions. Plant Physiol 136:4228–4236 Rajaonarivony JIM, Gershenzon J, Croteau R (1992) Characterization and mechanism of (4S)-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha × piperita). Arch Biochem Biophys 296:49–57 Schnee C, Kollner TG, Held M, Turlings TC, Gershenzon J, Degenhardt J (2006) The products of a single maize sesquiterpene synthase form a volatile defense signal that attracts natural enemies of maize herbivores. Proc Natl Acad Sci U S A 103:1129–1134 Singh P, Shukla R, Prakash B, Kumar A, Singh S, Mishra PK, Dubey NK (2010) Chemical profile, antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima Burm. and Citrus sinensis (L.) Osbeck essential oils and their cyclic monoterpene, DL-limonene. Food Chem Toxicol 48:1734–1740 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Taniguchi S, Miyoshi S, Tamaoki D, Yamada S, Tanaka K, Uji Y, Tanaka S, Akimitsu K, Gomi K (2014) Isolation of jasmonate-induced sesquiterpene synthase of rice: product of which has an antifungal activity against Magnaporthe oryzae. J Plant Physiol 171:625–632

Tholl D (2006) Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Curr Opin Plant Biol 9: 297–304 Tholl D, Lee S (2011) Terpene specialized metabolism in Arabidopsis thaliana. The Arabidopsis Book: e0143 Tholl D, Sohrabi R, Huh JH, Lee S (2011) The biochemistry of homoterpenes—common constituents of floral and herbivoreinduced plant volatile bouquets. Phytochemistry 72:1635–1646 Trapp SC, Croteau RB (2001) Genomic organization of plant terpene synthases and molecular evolutionary implications. Genetics 158: 811–832 Van Poecke RMP, Posthumus MA, Dicke M (2001) Herbivore-induced volatile production by Arabidopsis thaliana leads to attraction of the parasitoid Cotesia rubecula: chemical, behavioral, and geneexpression analysis. J Chem Ecol 27:1911–1928 Wei A, Shibamoto T (2007) Antioxidant activities and volatile constituents of various essential oils. J Agric Food Chem 55:1737–1742 Wilderman PR, Xu M, Jin Y, Coates RM, Peters RJ (2004) Identification of syn-pimara-7,15-diene synthase reveals functional clustering of terpene synthases involved in rice phytoalexin/allelochemical biosynthesis. Plant Physiol 135:2098–2105 Wu S, Schalk M, Clark A, Miles RB, Coates R, Chappell J (2006) Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants. Nat Biotechnol 24:1441–1447 Xu M, Wilderman PR, Morrone D, Xu J, Roy A, Margis-Pinheiro M, Upadhyaya NM, Coates RM, Peters RJ (2007) Functional characterization of the rice kaurene synthase-like gene family. Phytochemistry 68:312–326 Yuan JS, Kollner TG, Wiggins G, Grant J, Degenhardt J, Chen F (2008) Molecular and genomic basis of volatile-mediated indirect defense against insects in rice. Plant J 55:491–503 Yuba A, Yazaki K, Tabata M, Honda G, Croteau R (1996) cDNA cloning, characterization, and functional expression of 4S-(−)-limonene synthase from Perilla frutescens. Arch Biochem Biophys 332:280–287