Phytochemistry 96 (2013) 46–56

Contents lists available at ScienceDirect

Phytochemistry

journal homepage: www.elsevier .com/locate /phytochem

Isolation and characterization of terpene synthases in cotton (Gossypiumhirsutum)

0031-9422/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.phytochem.2013.09.009

⇑ Corresponding author. Tel.: +86 21 54924034; fax: +86 21 54924015.E-mail address: ljwang@sibs.ac.cn (L.-J. Wang).

Chang-Qing Yang a, Xiu-Ming Wu a, Ju-Xin Ruan a, Wen-Li Hu a, Yin-Bo Mao a, Xiao-Ya Chen a,b,Ling-Jian Wang a,⇑a National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research, Institute of Plant Physiology and Ecology, Shanghai Institutes forBiological Sciences, Chinese Academy of Sciences, Shanghai 200032, Chinab Plant Science Research Center, Shanghai Chenshan Botanical Garden, Shanghai 201602, China

a r t i c l e i n f o

Article history:Received 24 April 2013Received in revised form 23 July 2013Available online 24 September 2013

Keywords:Cotton (Gossypium hirsutum)MalvaceaeTerpene synthaseVIGS

a b s t r a c t

Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexinsagainst pathogens and feeding deterrents to herbivorous insects. However, to date little is known aboutthe biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesqui-terpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gaschromatography–mass spectrometry (GC–MS). By EST data mining combined with Rapid Amplification ofcDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 wereisolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesqui-terpene synthases: the former converted farnesyl pyrophosphate (FPP) into b-caryophyllene anda-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced a-pinene,b-pinene, b-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate(GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant.GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressedonly in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyljasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes wereinduced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenesof cotton, at least of this Upland cotton.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Terpenoids constitute the largest family of natural productswith more than 30,000 structures (Degenhardt et al., 2009b),which are grouped into different classes on the basis of the numberof 5-carbon building blocks (Aharoni et al., 2005; Haagen-Smit,1953). In addition to their physiological roles as phytohormones(gibberellic acid, abscisic acid and strigolactone), photosynthesispigments (carotenoids and chlorophylls), and membrane structuralcomponents (sterols), terpenoids also have ecological functions inmediating plant interactions with biotic and abiotic factors. Vola-tile terpenes may help plants to attract pollinators or predatorsof herbivores (Degenhardt et al., 2003; Pichersky and Gershenzon,2002), and terpenoid phytoalexins can participate in defenseagainst phytopathogens and herbivores (Balkema-Boomstra et al.,2003; Nagegowda, 2010; Tan et al., 2000; Wang et al., 2004).

In plant cells, precursors of terpenoids are synthesized viaeither the cytosolic mevalonate pathway (MVA pathway) or theplastidial 2-C-methyl-D-erythritol-4-phosphate pathway (MEPpathway), and are then condensed into structural diverse terpe-noids by the family of terpene synthases (TPSs). The TPS familyconsists of isoprene synthases producing 5-carbon isoprene usingdimethylallyl pyrophosphate (DMAPP), monoterpene synthasesproducing 10-carbon monoterpenes from geranyl pyrophosphate(GPP), sesquiterpene synthases producing 15-carbon sesquiter-penes from farnesyl pyrophosphate (FPP), and diterpene synthasesproducing 20-carbon diterpenes from geranylgeranyl pyrophos-phate (GGPP) or copalyl pyrophosphate (CPP) (Nagegowda, 2010;Tholl, 2006). Most sesquiterpene synthases are localized in thecytosol, whereas monoterpene and diterpene synthases are usuallyin the plastid and have a N-terminal plastid transit peptideupstream of the ‘‘RRX8W’’ motif (Williams et al., 1998). Almostall TPSs contain the ‘‘DDXXD’’ and the ‘‘NSE/DTE’’ motifs at the C-terminal region for the metal dependent (frequently Mg2+ orMn2+) ionization of the prenyl diphosphate substrate that areessential for their catalytic activities (Chen et al., 2011; Tholl,

C.-Q. Yang et al. / Phytochemistry 96 (2013) 46–56 47

2006). They constitute families with various members in plant gen-omes, ranging from only one functional and three pseudogenes (orfragments) of TPSs in moss (Physcomitrella patens), to 152 TPSs ingrapevine (Vitis vinifera) (Hayashi et al., 2006; Martin et al.,2010). Recent phylogenetic analysis of TPSs from gymnospermsand angiosperms established presence of 7 subfamilies of TPS-a,b, c, d, e/f, g and h, with most monoterpene and sesquiterpene syn-thases of angiosperms being distributed in TPS-a, b and g subfam-ilies (Chen et al., 2011).

Cotton (Gossypium spp.) is an important economic crop and amajor source of natural fiber for the textile industry. Cotton plantswith epidermal pigment glands accumulate the sesquiterpenealdehyde gossypol (1) and related sesquiterpene aldehydes (hemi-gossypol (2), hemigossypolone (3), heliocides H1 (4), H2 (5), H3 (6)and H4 (7)) as major toxins against herbivorous insects, such asHelicoverpa armigera, Heliothis virescens and Spodoptera exigua, ofthese, hemigossypol (2) is a major phytoalexin that protects theplant from pathogens such as Verticillium dahliae, Rhizoctonia solan-i, Xanthomonas campestris and Fusarium oxysporum. Together, thesepests cause severe yield loss in cotton-producing areas of the world(Abraham et al., 1999; Bell, 1967; Liu et al., 1999a; Stipanovic et al.,2006, 2008; Turco et al., 2007; Wu and Baldwin, 2010). Progress

Fig. 1. Structures of monoterpenes and sesquiterpenes produced by cot

has been made in characterizing enzymes and transcription factorsfor gossypol (1) biosynthesis. The enzymes, farnesyl diphosphatesynthase (FPS) (Liu et al., 1999a), (+)-d-cadinene synthase (CDNor CAD1) (Chen et al., 1995; Tan et al., 2000), and CYP706B1, acytochrome P450 monooxygenase that hydroxylates (+)-d-cadin-ene (8) at the 8-position (Luo et al., 2001), catalyze three consecu-tive steps of gossypol (1) biosynthesis. Other related enzymes andproteins include P450 reductases (Yang et al., 2010), a peroxidase(Benedict et al., 2006; Stipanovic et al., 1992), a laccase (Liuet al., 2008), a dirigent protein (Liu et al., 2008) and a methyltrans-ferase (Liu et al., 1999b). The transcription factor GaWRKY1 wasshown to regulate one of the (+)-d-cadinene synthase gene,CDN-A (Xu et al., 2004).

Most plants produce and emit a large number of volatile com-pounds. For example, Arabidopsis flowers emit a mixture of vola-tiles consisting of over 20 sesquiterpenes (Tholl et al., 2005); rice(Oriza sativa) synthesizes 13 sesquiterpenes after methyl jasmo-nate (MeJA) treatment (Cheng et al., 2007); and Selaginella moellen-dorffii, a lycophyte, produces the monoterpene linalool (9) and thesesquiterpenes b-elemene (10), germacrene D (11), b-sesquiphel-landrene (12) and nerolidol (13) after elicitation with alamethicin,a fungal antibiotic (Li et al., 2012). It has been reported that cotton

ton plants and in vitro activity assays of cotton terpene synthases.

Table 1Terpenes in plant of G. hirsutum cv. CCRI12, a glanded cotton cultivar.

Hypocotyl Cotyledon Leaf Petal Pericarp (5 DPA) Pericarp (10DPA) Pericarp (25 DPA)

Monoterpenea-Pinene (14) 4.6 11.2 281.8 334.5 93.6b-Pinene (15) 1.1 3.3 54.4 54.7 21.1b-Myrcene (16) 2.3 8.1 192.6 178.9 28.0b-phellandrene (22) 0.5 1.5 48.4 27.6 25.0(E)-b-Ocimene (17) 0.9 5.2 179.1 148.9 118.3

Sesquiterpenea-Copaene (23) 0.4 23.9 9.1 4.1b-Elemene (10) 0.3b-Caryophyllene (18) 13.6 2.4 17.6 210.3 113.7 53.1a-Humulene (19) 5.8 7.5 91.7 51.7 26.1Guaia-1(5),11-diene (24) 0.9Guaia-1(10),11-diene (25) 3.2b-Himachalene (26) 0.7 262.4 213.3 107.8b-bisabolol (21) 4.3 342.2 282.6 120.3a-Bergamotene (27) 27.6 20.0 9.6b-Farnesene (28) 42.5 33.2 17.5(+)-d-Cadinene (8) 20.7 12.2 8.7

Total 28.8 2.4 64.2 1777.6 1480.4 633.2

Tissues of hypocotyl, cotyledon, leaf, petal and pericarp were individually extracted with n-pentane for 1 h and analyzed by GC–MS (lg/g fresh weight, FW). Cotton bolls(pericarps) were harvested at 5-, 15- and 25-days post-anthesis (DPA).

48 C.-Q. Yang et al. / Phytochemistry 96 (2013) 46–56

foliage produces at least 4 monoterpenes (a-pinene (14), b-pinene(15), myrcene (16), b-ocimene (17)) and 4 sesquiterpenes (b-caryo-phyllene (18), a-humulene (19), c-bisabolene (20) and b-bisabolol(21)), in addition to sesquiterpene aldehydes such as hemigossypo-lone (3) and heliocides H1–H4 (4–7), and that herbivoral ormechanical damage to older leaves could lead to increasedamounts of these terpenoids in younger leaves (ElZen et al.,1985; Minyard et al., 1965, 1966; Opitz et al., 2008; Pare and Tuml-inson, 1997). Up to now (+)-d-cadinene synthase (CDN), a sesqui-terpene synthase, is the only TPS characterized in cotton.However, CDN is basically a single product enzyme, and in cotton(+)-d-cadinene (8) serves as a precursor for the biosynthesis of gos-sypol (1) and related sesquiterpene phytoalexins (Chen et al.,1995; Liang et al., 2000; Meng et al., 1999; Tan et al., 2000). Thus,this study analyzed not only the monoterpenes and non-cadinene-type sesquiterpenes of cotton, but also identified the respectiveTPSs (see Fig. 1).

Fig. 2. Volatile terpenes in true leaf of cotton (G. hirsutum cv. CCRI12). Leaves from the 6and analyzed by GC–MS. Peaks are: INS: Internal Standard, nonyl acetate; (a) a-pinene(17); (f) b-caryophyllene (18); (g) guaia-1(5),11-diene (24); (h) a-humulene (19); (i) guaia

2. Results

2.1. Glanded cotton plant produces multiple monoterpenes andsesquiterpenes

To detect terpenes produced by cotton plants other than (+)-d-cadinene (8), hypocotyls and cotyledons from seedlings, and leaf,petal and pericarp tissues at different developmental stages ofthe mature glanded cultivar Gossypium hirsutum cv. CCRI12 wereextracted with n-pentane and subjected to gas chromatography–mass spectrometry (GC–MS) analysis. A total of 5 monoterpenes(a-pinene (14), b-pinene (15), b-myrcene (16), b-phellandrene(22) and (E)-b-ocimene (17)) and 11 sesquiterpenes (a-copaene(23), b-elemene (10), b-caryophyllene (18), a-humulene (19), gua-ia-1(5),11-diene (24), guaia-1(10),11-diene (25), b-himachalene(26), b-bisabolol (21), a-bergamotene (27), b-farnesene (28), (+)-d-cadinene (8)) were identified (Table 1 and Fig. 2). Some terpenes,

0-day-old cotton plant were extracted with n-pentane for 1 h at room temperature,(14); (b) b-pinene (15); (c) b-myrcene (16); (d) b-phellandrene (22); (e) b-ocimene-1(10),11-diene (25); (j) b-himachalene (26); (k) (+)-(4S, 8R)-8-epi-b-bisabolol (21).

C.-Q. Yang et al. / Phytochemistry 96 (2013) 46–56 49

including a-pinene (14), b-pinene (15), b-myrcene (16) andb-caryophyllene (18), were detected in all organs examined exceptthe cotyledon and petal, whereas others showed distinct distribu-tion patterns. For example, guaia-1(5),11-diene (24) and guaia-1(10),11-diene (25) were detected in leaf extracts only, whereasa-bergamotene (27) and b-farnesene (28) were in pericarps at var-ious developmental stages. The gossypol precursor (+)-d-cadinene(8) was detected at low abundance in pericarp.

Of these samples, the pericarp contained the most abundantand diverse monoterpenes and sesquiterpenes, with the highestcontent up to 1,777 lg/g fresh weight (FW) or approximately1.8‰ of the total FW at an early developmental stage of 5 days postanthesis (DPA), and whose content decreased during development,with 633 lg/g FW detected at 20 DPA. The leaf and hypocotyl tis-sues showed much lower levels of terpene production, with totalsof 64 and 28 lg/g FW, respectively. However, although at lowabundance, diverse terpenes were also present in leaf tissue,

Fig. 3. Alignment of amino acid sequence of GhTPSs and other monoterpene synthasesaligned with selected terpene synthases with software clustalw, and edited with Jalview(+)-d-cadinene synthase, AAX44033; NtEAS: Nicotiana tabacum 5-epi-aristolochene synthCitrus unshiu (E)-b-ocimene synthase, BAD91046; AaPin: Artemisia annua (�)-b-pinene s

including guaia-1(5),11-diene (24) and guaia-1(10),11-diene (25)(Table 1). In contrast to these organs, only trace amounts of terp-enes were detected in cotyledons, and none were found in petalsat the flowering stage (0 DPA) (Table 1).

2.2. Identification of terpene synthases from cotton

Gossypol (1) and related sesquiterpene aldehydes are abundantand are distributed throughout the cotton plant (Benedict et al.,2004; Meng et al., 1999; Stipanovic et al., 2005). Interestingly, inour GC–MS analysis, (+)-d-cadinene (8) was almost undetectablein most cotton tissues, with only a trace amount in the pericarp,even though transcripts of (+)-d-cadinene synthase genes arepresent at high levels throughout the plant (Liang et al., 2000;Meng et al., 1999; Tan et al., 2000). This points to a highly efficientconversion of (+)-d-cadinene (8) to hemigossypol (2) and related

and sesquiterpene synthases of plants. Deduced protein sequences of GhTPSs were. Conserved domains of RRx8W and DDxxD are highlighted. GhCAD-C4: G. hirsutumase, Q40577; VaCar: Vitis vinifera (E)- b-caryophyllene synthase, ADR74192; CuOci:ynthase, AAK58723; MgTer: Magnolia grandiflora a-terpineol synthase, ACC66282.

50 C.-Q. Yang et al. / Phytochemistry 96 (2013) 46–56

products by modification enzymes, such as the P450 monooxygen-ase CYP706B1.

To isolate TPSs responsible for biosynthesis of monoterpenesand non-cadinene type sesquiterpenes, cotton CDN (U23206), asesquiterpene synthase, and Arabidopsis (E)-b-ocimene synthase

Fig. 4. Product spectrum of GhTPS1, GhTPS2 and GhTPS3. Recombinant GhTPS1, GhTPS2Ni–NTA resin. After incubation with FPP or GPP in the reaction buffer for 30 min at 37 �Care: (a) b-caryophyllene (18); (b) a-humulene (19); (c) b-elemene (10); (d) guaia-1(5),11(14); (h) b-pinene (15); (i) b-phellandrene (22); (j) c-terpinene (30).

(NM_117775), a monoterpene synthase, were used as bait tosearch EST databases of G. hirsutum with Blastn program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). ESTs showing nucleotide sequence identities >50% with either were collected and assembled into12 contigs and 23 singlets using the CAP3 software (http://pbil.u-

and GhTPS3 proteins were expressed in E. coli as fusion proteins and purified with, the reaction mixture was extracted with n-pentane and analyzed by GC–MS. Peaks-diene (24); (e) alloaromadendrene (29); (f) guaia-1(10),11-diene (25); (g) a-pinene

C.-Q. Yang et al. / Phytochemistry 96 (2013) 46–56 51

niv-lyon1.fr/cap3.php), of which 5 contigs and 16 singlets shared>90% identities with (+)-d-cadinene synthase and they were con-sidered members of the CDN family. The remaining 7 contigs and7 singlets were investigated further.

To obtain the full length cDNAs, 5 and 3 RACE were performed.Finally, three putative terpene synthases, destined GhTPS1(KC878726), GhTPS2 (KC878727) and GhTPS3 (KC878728), wereobtained, which have lengths of 1,943, 1,951 and 1940 bp, encod-ing proteins of 545, 559 and 595 amino acid residues, respectively.The deduced protein sequences of GhTPS1 and GhTPS2 are 65%identical to each other and �55% to members of (+)-d-cadinenesynthase. As the CDN family can be divided into four (A, B, C andD) subfamilies whose members share amino acid identities of>77% (Townsend et al., 2005), GhTPS1 and GhTPS2 were consid-ered candidates of new sesquiterpene synthases. GhTPS3 has ahigh sequence identity (59%) to (E)-b-ocimene/myrcene synthaseof V. vinifera (ADR74206). Like other terpene synthases, the threecotton TPSs contain the highly conserved RRX8W motif at N-termi-nal and the DDXXD domain at C-terminal (Fig. 3), which is essen-tial for covalent binding (Aharoni et al., 2005; Nagegowda, 2010).Analysis by SignalP (http://genome.cbs.dtu.dk/services/SignalP/)and ChloroP (http://www.cbs.dtu.dk/services/ChloroP/) softwareindicated that GhTPS3 has a N-terminal 38-amino acid peptide,whereas GhTPS1 and GhTPS2 do not. Phylogenetic analysis with

Fig. 5. Expression of GhTPS genes and terpene contents in leaf of the VIGS-treated cottonGhTPS1, GhTPS2, GhTPS3 or empty vector, as indicated. Total RNAs from true leaves wereMS. V-GhTPS1, V-GhTPS2 and V-GhTPS3 refer to plants with suppressed expression of Ghcaryophyllene (18), guaia-1(10),11-diene (25) and a-pinene (14) contents in true leaves

TPSs of the CDN family and those from other plant species estab-lished that, GhTPS1 and GhTPS2 could be classified with sesquiter-pene synthases in the clade TPS-a, closest to cotton (+)-d-cadinenesynthase, whereas GhTPS3 was with monoterpene synthases in theclade TPS-b (Supplementary Fig. 1).

2.3. Functional characterization of GhTPS1, GhTPS2 and GhTPS3

To determine enzyme activity, open reading frames (ORFs) ofGhTPS1, GhTPS2 and GhTPS3 (with a deletion of 35 amino acid res-idues at N-terminal) were expressed in Escherichia coli as fusionproteins. GC–MS analysis of the n-pentane extract of the reactionmixture indicated that both GhTPS1 and GhTPS2 converted FPPinto multiple sesquiterpene products (Fig. 4A and B). GhTPS1 pro-duced b-caryophyllene (18) and a-humulene (19) in a ratio of71:29 (Fig. 4A). Notably, in most cotton tissues examined theb-caryophyllene (18) to a-humulene (19) ratio was around 2.2:1(Table 1), close to that of GhTPS1. The products of GhTPS2 includedguaia-1(10),11-diene (25) (55%), guaia-1(5),11-diene (24) (14%), a-humulene (19) (10%), b-elemene (10) (10%), alloaromadendrene(29) (6%) and b-caryophyllene (18) (5%) (Fig. 4B), a compositionsimilar to the sesquiterpene spectrum in leaves (Table 1). TheGhTPS3 protein converted GPP into a-pinene (14) (77.2%), b-pinene (15) (14.7%) and b-phellandrene (22) (5.5%), with a trace

plant. Cotyledons of cotton plants were inoculated with TRV harboring fragments ofanalyzed by real-time PCR. n-Pentane extracts of true leaves were analyzed by GC–TPS1, GhTPS2 or GhTPS3 by VIGS. (A) Expression of GhTPS genes in true leaves; (B) b-of VIGS plants; Error bars indicate SD of three biological replicates.

Fig. 6. Expression patterns of GhTPS genes in cotton plant and induction. (A) Relative expression levels of GhTPSs in hypocotyl, cotyledon, leaf, petal and pericarps. Total RNAsfrom hypocotyl and cotyledon of the 10-day-old seedlings, and from leaf, petal and pericarp of the 5-month-old plants were analyzed by qRT-PCR. Pericarps (cotton bolls fromwhich seeds were removed) were harvested at 5 and 10 days post anthesis (DPA), respectively. (B) Expression of GhTPSs in leaves treated with mechanical wounding, fungalelicitor and MeJA. True leaves of 20-day-old plants were sprayed with Verticillium dahliae (Vde) elicitor, or 100 lM MeJA for 5 h, or cut into 0.5-cm fragments, respectively;total RNAs were then extracted for qRT-PCR analysis. (C) Volatile terpene contents in true leaves subjected to treatments as described in B. Error bars indicate SD of threebiological replicates.

52 C.-Q. Yang et al. / Phytochemistry 96 (2013) 46–56

C.-Q. Yang et al. / Phytochemistry 96 (2013) 46–56 53

amount of c-terpinene (30) (2.6%) (Fig. 4C). The apparent Km wasdetermined to be 28.93 and 15.38 lM FPP for GhTPS1 and GhTPS2,respectively, and 79.27 lM GPP for GhTPS3.

To investigate in vivo functions of these GhTPSs, tobacco rattlevirus (TRV)-based virus induced gene silencing (VIGS) systemwas employed to silence their expression in cotton. The 456-,486- and 444-bp fragments of GhTPS1, GhTPS2 and GhTPS3 wereused, and the corresponding Agrobacteria were infiltrated into cot-ton seedlings. Thirty days after inoculation, expression of targetgenes in true leaves was analyzed by quantitative real-timeRT-PCR (qRT-PCR). In each VIGS-treated plant selected, transcriptsof target genes decreased by more than 85%, whereas non-targetTPS gene expressions were largely unaffected (Fig. 5 A). Consistentwith target gene suppression, production of b-caryophyllene (18)and a-humulene (19) was decreased by �90% in GhTPS1-silencedplants (Fig. 5B, Supplementary Fig. 3A and B), and the b-elemene(10), guaia-1(5),11-diene (24) and guaia-1(10),11-diene (25) wereundetectable in GhTPS2-suppressed leaves (Fig. 5B, SupplementaryFig. 2A and C). In the GhTPS3-silenced plant, a-pinene (14),b-pinene (15) and b-phellandrene (22) contents decreased by morethan 80%, whereas b-myrcene (16) was unchanged (Fig. 5B, Supple-mentary Fig. 2D and E). Such a correlation between suppressed TPSgene expression and decreased contents of terpene products sug-gest precise silencing of target genes, and confirms activities ofthe three GhTPSs in planta.

2.4. Expression Patterns of GhTPS1, GhTPS2 and GhTPS3

Spatial expression patterns of the three TPS genes were ana-lyzed by qRT-PCR. Transcripts of GhTPS1 were detected in hypo-cotyl, cotyledon, leaf, pericarp and ovule (20-DPA) tissues, withthe highest expression level in pericarp tissue and the lowest incotyledons; transcripts were also undetectable in petals (Fig. 6A).This expression pattern is consistent with the distribution of b-caryophyllene (18) and a-humulene (19) (Table 1). For GhTPS2,the transcript level was high in leaf, low in pericarp, and hardlydetectable in other organs, this being in agreement with theleaf-specific detection of guaia-1(5),11-diene (24) and guaia-1(10),11-diene (25) (Fig. 6A and Table 1). GhTPS3 showed a similarexpression pattern as GhTPS1 (Fig. 6A).

Gossypol (1) accumulation and expression of the biosynthesisgenes can be strongly induced by mechanical wounding, V. dahliae(Vde) elicitor and MeJA treatments (Luo et al., 2001; Xu et al.,2004). To find out whether the production of these terpenes in cot-ton was also inducible, true leaves of glanded cotton plants weretreated with these stimuli. Expression levels of the three TPS geneswere drastically induced, ranging from 3- to 9-fold (Fig. 6B).GC–MS analysis of the n-pentane extracts of these plants estab-lished an increased accumulation of both monoterpenes and ses-quiterpenes in response to the treatments (Fig. 6C), consistentwith enhanced expression of the TPS genes.

3. Discussion

In agreement with other researchers, it was demonstrated that,in addition to the cadinene-type gossypol (1) and related sesqui-terpene aldehydes, cotton plants also produce a mixture of mono-terpenes and sesquiterpenes. Plant volatile terpenes playimportant roles in attracting pollinators, mediating direct and indi-rect defense reactions (Aros et al., 2012; Lucas-Barbosa et al., 2011;Majetic et al., 2009; Nagegowda, 2010; Pichersky and Gershenzon,2002). For example, a-pinene (14) from the persimmon tree (Dio-spyros kaki L.) infested by Japanese wax scales (Ceroplastes japoni-cus) is a cue to attract the ladybeetle predator (Chilocorus kuwanae)(Zhang et al., 2009b); the volatile (E)-b-farnesene (28) helps todefend plants against aphid infestation as it is also the principal

component of the alarm pheromone of many aphid species, whichis released when aphids are attacked by enemies (Kunert et al.,2010; Thomson et al., 1973). These defensive terpenes were alsodetected in tissues of the cotton plant. Unlike snapdragon andArabidopsis, as well as many other plants, whose flowers synthe-size and emit terpenes (Dudareva et al., 2003; Tholl et al., 2005),the cotton plant produces the most abundant (up to 0.2% freshweight) and diverse (5 monoterpenes and 8 sesquiterpenes) terp-enes in pericarp tissue, which encloses seeds that produce cottonfiber. Moreover, production of these terpenes and expression ofthe TPS genes were inducible by mechanical wounding and elicita-tion. These data imply a protective role of these terpenes in cotton.

b-caryophyllene (18) and a-humulene (19) have been detectedin many plant species and they seem to be present throughoutseed plants. Both are usually produced by a single enzyme (Caiet al., 2002; Irmisch et al., 2012; Kollner et al., 2008), and have beenreported to participate in plant-insect interactions (Degenhardtet al., 2009a; Rasmann et al., 2005). However, the guaia-1(5),11-diene (24) and guaia-1(10),11-diene (25), detected here in cottonleaves, are rare in the plant kingdom. There is no report of anyplant-derived guaia-1(5),11-diene (24), and guaia-1(10),11-diene(25) has been reported only in two plant species, Peucedanum taur-icum of Apiaceae and Ocimum basilicum of Lamiaceae (Tesso et al.,2005; Zhang et al., 2009a). To our knowledge, GhTPS2 is the firstreported enzyme which synthesizes guaia-1(5),11-diene (24) andguaia-1(10),11-diene (25).

The cadinene-type sesquiterpene aldehydes are major defensivecompounds found in cotton. They are constitutively synthesizedand stored in pigment glands, and in addition, their biosynthesisis inducible (Baksha et al., 2006; Tan et al., 2000; Xu et al., 2004).In this investigation, it was found that formation of the non-cadin-ene-type volatile terpenes and expression of the three TPS genes arealso both constitutive and inducible. The similar expression patternbetween gossypol pathway genes and the three TPSs reported hereraises a question that biosynthesis of mono- and sesquiterpenes incotton share a common regulatory network. Isolation of key regula-tors in this network will be important in not only understandingterpenoid metabolism regulation in the plant, but also facilitatingcotton breeding for enhanced pest and disease control.

4. Concluding remarks

Cotton plants produce large amount of volatile terpenes, but todate little is known about their biosynthesis. In this research, twosesquiterpene synthases and one monoterpene synthase were iso-lated and characterized by both in vitro enzyme assays and in vivoVIGS assays. They showed distinct and inducible expression pat-terns, and account for the biosynthesis of the most volatile terp-enes in cotton.

5. Experimental

5.1. Plant material

Cotton (G. hirsutum cv. CCRI12) plants were grown in a phyto-tron at 28 ± 0.5 �C. Leaf, petal and pericarp tissues at various devel-opmental stages were collected from 5-month-old plants;hypocotyls and cotyledons were collected from 10-day-old seed-lings. Fresh plant samples were used directly for chemical analysisand RNA extraction.

5.2. Terpene identification by GC–MS

Plant tissues (200 mg) were frozen and ground in liquid N2 intoa fine powder and extracted with n-pentane (2 mL) at 28 �C for 1 h

54 C.-Q. Yang et al. / Phytochemistry 96 (2013) 46–56

with constant shaking. Nonyl acetate (standard) was added to theextracts to a final concentration of 5 lg/mL. The solution was cen-trifuged at 18,000 g for 10 min, and supernatants were dried withanhyde Na2SO4 before GC–MS analysis. All extractions were per-formed in triplicate.

Terpenes and enzyme products were identified by gas chroma-tography–mass spectrometry (GC–MS) on an Agilent 6890 SeriesGC System coupled to an Agilent 5973 Network Mass SelectiveDetector using an HP 5 (Agilent Technologies) capillary column(0.25 mm i.d. � 30 m with 25-lm film). The injector was operatedsplitless at a temperature of 200 �C with a column flow of 1 mL He/min. The following temperature program was used: initial temper-ature of 40 �C (5-min hold), increase to 230 �C at 5 �C/min, then to280 �C at 30 �C/min (10 min hold). Terpenes were identified bymass spectra comparison using the Enhanced Chemstation (ver-sion E.02.00.493, Agilent Technologies) software and NIST (Na-tional Institute of Standards and Technology) library, and productpeaks were quantified by integration of peak areas and calibrationwith internal standard nonyl acetate.

5.3. Total RNA isolation and cDNA synthesis

Tissue samples (100 mg) were ground into a fine powder andsuspended with extraction buffer (1 mL, 0.1 M Tris, pH 8.0,50 mM EDTA, 1 M NaCl, 1% cetyltrimethyl ammonium bromideand 1% b-mercaptoethanol) at 65 �C for 30 min. After extractionwith an equal volume of CHCl3 and centrifugation at 15,000g, thesupernatant was precipitated by LiCl (final concentration of 2 M)at �20 �C for 8 h. After centrifugation at 18,000 g for 10 min, thetotal RNA pellet was washed with 1 mL EtOH–H2O (3:1) and dis-solved in RNase free H2O.

Total RNAs was treated with DNase I and reverse transcribedusing PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa, China)according to the manufacturer’s protocol.

5.4. 5 and 3 RACE and full length cDNA amplification

Rapid Amplification of cDNA Ends (RACE) was performed as de-scribed (Scotto-Lavino et al., 2006). For 5 RACE, first strand cDNAwas synthesized for each gene with specific primers of TPS1-RT(5-ACCCATCAGGAGCCACCATAAG-3), TPS2-RT (5-AGTCTCGAGAGTTTGGTCATCCT-3) or TPS3-RT (5-AAGGCATCAATAAGCTCAAG-3),and then amplified with gene specific primers of TPS1-5R (5-CTATTTTCATTTTGGAGGGACATG-3), TPS2-5R (5-GTAGAGCAACTCATCATCCATCACT-3) or TPS3-5R (5-CACTTCTTCCTTCAGTTTGC-3), in combi-nation with adaptor primer (5-GAGGACTCGAGCTCAAGC-3), respectively. 3- RACE was performed with 3-Full RACE Core Set (TaKaRa,China), with gene specific primers of TPS1-3F (5-ATACAG GGTCCAATACGC-3), TPS2-3F (5-TCATGTCAAATATGCGAAA-3) or TPS3-3F(5-GGATTGCAGACCTTTGAGGA-3), respectively.

Based on assembly of fragments obtained by 5 and 3 RACE, fulllength cDNAs were amplified with primer sets of TPS1-F (5-CAC-AACAAGAAGATATCGTCTCA-3) and TPS1-R (5-AACATGCCGAGAT-GA ACCAC-3) for GhTPS1, TPS2-F (5-TCATTTCGTAGTGTTTCCCA-3)and TPS2-R (5-GGTGTTAACACCTCAAACATA-3) for GhTPS2, andTPS3-F (5-TTTTACTCCATCCCTTTGGA-3) and TPS3-R (5-TTGAA-ACTTATTTGAGACAACAC-3) for GhTPS3.

5.5. Real-Time PCR analysis

Real-time PCR was performed using SYBR Premix Ex Taq re-agent (TaKaRa, China) on a Mastercycler system (Eppendorf, Ger-many). Cotton histone3 gene (AF024716) with primers of his3-F(5-GAAGCCTCATCGATACCGTC-3) and his3-R (5-CTACCACTACCAT-CATGG-3) was used for normalization. Primers for GhTPSs expres-sion analysis are: GhTPS1-F (5-ACTCGACAAGAATACGAAGA-3) and

GhTPS1-R (5-TTCTAGCAAGCCTTTGACAT-3) for GhTPS1, GhTPS2-F(5-TTGGGCATCTAACAATCCTA-3) and GhTPS2-R (5-TGGCCCTGGAGTAACTTTG-3) for GhTPS2, and GhTPS3-F (5-ATCGGGTCCTGTGC-TACTTG-3) and GhTPS3-R (5-CGGCCATACGATCTCTGTTC-3) forGhTPS3.

5.6. Prokaryotic expression and purification of recombinant TPSproteins

ORFs of the TPSs were amplified by pfu DNA polymerase usingprimers of GhTPS1-F-SacI (5-GCGAGCTCATGTCTTCCATGTCCAACGT-3) and GhTPS1-R-NotI (5-TCGCGGCCGCTCATCAAATCG GCACCGGAT-3) for GhTPS1; GhTPS2-F-SacI (5-GCGAGCTCATGTCTT CAT-CAAAGCTTCC-3) and GhTPS2-R-NotI (5-TCGCGGCCGC TTAATA AGAGGCTGAAATTG-3) for GhTPS2; GhTPS3-F-KpnI (5-GCGGTACCATGGCTAGTCTGCTTACTTC-3) and GhTPS3-F-KpnI (5-GCGGTAC-CAGTCCCTCACAATC TGAAGC-3) and GhTPS3-R-XhoI (5-GCCTCGAGTTATTTTGGCGATGGAATTG-3) for GhTPS3 (with 35 amino acidtruncation at N-terminal). PCR products were digested with SacIand NotI (GhTPS1 and GhTPS2), or KpnI and XhoI (GhTPS3) and li-gated into the expression vector pET32a and transformed into E.coli BL21 (DE3).

E. coli cells harboring expression vectors were grown overnightin Luria–Bertani (LB) medium (contain 100 mg/L ampicillin) at37 �C in a shaking incubator, then diluted at 1:100 into LB mediumfor growth at 37 �C till OD600 = 0.5. Prokaryotic expression wasstarted by addition of 1 mM isopropyl b-D-1-thiogalactopyrano-side (IPTG) and shaking at 28 �C for 12 h. Bacterial cells werecollected by centrifugation at 2000g for 5 min, washed and re-sus-pended in MOPS buffer (100 mM MOPS, 10% glycerol, 0.2 mM DTT,1 mM EDTA, adjusted to pH 7.3 with NaOH). After sonication onice, the recombinant protein was purified with Ni–NTA resinsaccording to manufacturer’s manual (Novagen, Madison, USA).

5.7. Enzyme assay

For enzyme assays of GhTPSs, recombinant protein (1 lg) wasadded into 100 lL reaction buffer containing 25 mM HEPES, 10%glycerol, 5 mM DTT, 5 mM MgCl2 and 50 lM FPP (GhTPS1 andGhTPS2) or GPP (GhTPS3), and incubated at 37 �C for 30 min. Reac-tions were stopped by addition of 0.5 M EDTA (10 lL) and thewhole was then extracted with n-pentane (200 lL), which wasthen evaporated and a portion subjected to GC–MS analysis. TheGC–MS analysis procedure was the same as that for terpene iden-tification from plant tissue extracts.

5.8. Cotton VIGS

Tobacco rattle virus (TRV)-based silencing system was used forcotton VIGS assay (Gao et al., 2011). Fragments of GhTPSs wereamplified with primers of VGhTPS1-F-BamHI (5-GCGGATCCATA-CAGGGTCCAATACGC-3) and VGhTPS1-R-XbaI (5-GCTCTAGAAACG-CAAGTAAGGACTGGAA-3) for GhTPS1, VGhTPS2-F-BamHI (5-GCGGATCCTCATGTCAAATATGCGAAAG-3) and VGhTPS2-R-XbaI (5-GCTCTAGACGAGGACCCTTGTAAGAT-3) for GhTPS2, VGhTPS3-F-BamHI(5-GCGGATCCGGATTGCAGACCTTTGAGGA-3) and VGhTPS3-R-XbaI(5-GCTCTAGACCATGTCGCATCAATCAACT-3) for GhTPS3, followedby insertion into BamHI-XbaI sites of pTRV2.

Plasmids of pTRV1 and pTRV2 or pTRV2 with fragments ofGhTPSs were introduced into Agrobacterium tumefaciens strainLBA4404. Overnight grown Agrobacterial cells were collected andsuspended in 10 mM MES, 10 mM MgCl2 and 200 lm acetylsyrin-gone solution to a final OD600 = 2.0, and then left at room temper-ature for 4 h. A. tumefaciens suspensions of pTRV1 was mixed withan equal volume of pTRV2 or its derivatives and infiltrated intocotyledons of 10-day-old cotton seedlings. Thirty days after inocu-

C.-Q. Yang et al. / Phytochemistry 96 (2013) 46–56 55

lation, true leaves were collected for analysis of target gene expres-sion and volatile terpene components analysis.

Acknowledgments

This work was supported by State Key Basic Research Programof China (2013CB127000), the Chinese Academy of Sciences(KSCX2-EW-N-03), and the National Natural Science Foundationof China (30630008 and 90917021).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.phytochem.2013.09.009.

References

Abraham, K.J., Pierce, M.L., Essenberg, M., 1999. The phytoalexinsdesoxyhemigossypol and hemigossypol are elicited by Xanthomonas inGossypium cotyledons. Phytochemistry 52, 829–836.

Aharoni, A., Jongsma, M.A., Bouwmeester, H.J., 2005. Volatile science? Metabolicengineering of terpenoids in plants. Trends Plant Sci. 10, 594–602.

Aros, D., Gonzalez, V., Allemann, R.K., Muller, C.T., Rosati, C., Rogers, H.J., 2012.Volatile emissions of scented Alstroemeria genotypes are dominated byterpenes, and a myrcene synthase gene is highly expressed in scentedAlstroemeria flowers. J. Exp. Bot. 63, 2739–2752.

Baksha, R., Mavlanov, G.T., Nasirova, G.B., Djataev, S.A., 2006. Gossypolaccumulation and morphogenesis in cotton (G. hirsutum L.) callus culture. J.Biol. Sci. 6, 1126–1129.

Balkema-Boomstra, A.G., Zijlstra, S., Verstappen, F.W., Inggamer, H., Mercke, P.E.,Jongsma, M.A., Bouwmeester, H.J., 2003. Role of cucurbitacin C in resistance tospider mite (Tetranychus urticae) in cucumber (Cucumis sativus L.). J. Chem. Ecol.29, 225–235.

Bell, A.A., 1967. Formation of gossypol in infected or chemically irritated tissues ofGossypium species. Phytopathology 57, 759–764.

Benedict, C.R., Liu, J., Stipanovic, R.D., 2006. The peroxidative coupling ofhemigossypol to (+)- and (�)-gossypol in cottonseed extracts. Phytochemistry67, 356–361.

Benedict, C.R., Martin, G.S., Liu, J., Puckhaber, L., Magill, C.W., 2004. Terpenoidaldehyde formation and lysigenous gland storage sites in cotton: variant withmature glands but suppressed levels of terpenoid aldehydes. Phytochemistry65, 1351–1359.

Cai, Y., Jia, J.W., Crock, J., Lin, Z.X., Chen, X.Y., Croteau, R., 2002. A cDNA clone for b-caryophyllene synthase from Artemisia annua. Phytochemistry 61, 523–529.

Chen, F., Tholl, D., Bohlmann, J., Pichersky, E., 2011. The family of terpene synthasesin plants: a mid-size family of genes for specialized metabolism that is highlydiversified throughout the kingdom. Plant J. 66, 212–229.

Chen, X., Chen, Y., Heinstein, P., Davisson, V.J., 1995. Cloning, expression, andcharacterization of (+)-d-cadinene synthase: a catalyst for cotton phytoalexinbiosynthesis. Arch. Biochem. Biophys. 324, 255–266.

Cheng, A.X., Xiang, C.Y., Li, J.X., Yang, C.Q., Hu, W.L., Wang, L.J., Lou, Y.G., Chen, X.Y.,2007. The rice (E)-b-caryophyllene synthase (OsTPS3) accounts for the majorinducible volatile sesquiterpenes. Phytochemistry 68, 1632–1641.

Degenhardt, J., Gershenzon, J., Baldwin, I.T., Kessler, A., 2003. Attracting friends tofeast on foes: engineering terpene emission to make crop plants more attractiveto herbivore enemies. Curr. Opin. Biotechnol. 14, 169–176.

Degenhardt, J., Hiltpold, I., Kollner, T.G., Frey, M., Gierl, A., Gershenzon, J., Hibbard,B.E., Ellersieck, M.R., Turlings, T.C., 2009a. Restoring a maize root signal thatattracts insect-killing nematodes to control a major pest. Proc. Natl. Acad. Sci.USA 106, 13213–13218.

Degenhardt, J., Kollner, T.G., Gershenzon, J., 2009b. Monoterpene and sesquiterpenesynthases and the origin of terpene skeletal diversity in plants. Phytochemistry70, 1621–1637.

Dudareva, N., Martin, D., Kish, C.M., Kolosova, N., Gorenstein, N., Faldt, J., Miller, B.,Bohlmann, J., 2003. (E)-b-ocimene and myrcene synthase genes of floral scentbiosynthesis in snapdragon: function and expression of three terpene synthasegenes of a new terpene synthase subfamily. Plant Cell 15, 1227–1241.

ElZen, G.W., Williams, H.J., Bell, A.A., Stipanovic, R.D., Vinson, S.B., 1985.Quantification of volatile terpenes of glanded and glandless Gossypiumhirsutum L. cultivars and lines by gas chromatography. J. Agric. Food Chem.33, 1079–1082.

Gao, X., Britt Jr., R.C., Shan, L., He, P., 2011. Agrobacterium-mediated virus-inducedgene silencing assay in cotton. J. Vis. Exp..

Haagen-Smit, A.J., 1953. The biogenesis of terpenes. Annu. Rev. Plant Physiol. 4,305–324.

Hayashi, K., Kawaide, H., Notomi, M., Sakigi, Y., Matsuo, A., Nozaki, H., 2006.Identification and functional analysis of bifunctional ent-kaurene synthase fromthe moss Physcomitrella patens. FEBS Lett. 580, 6175–6181.

Irmisch, S., Krause, S.T., Kunert, G., Gershenzon, J., Degenhardt, J., Kollner, T.G., 2012.The organ-specific expression of terpene synthase genes contributes to theterpene hydrocarbon composition of chamomile essential oils. BMC Plant Biol.12, 84.

Kollner, T.G., Held, M., Lenk, C., Hiltpold, I., Turlings, T.C., Gershenzon, J.,Degenhardt, J., 2008. A maize (E)-b-caryophyllene synthase implicated inindirect defense responses against herbivores is not expressed in mostAmerican maize varieties. Plant Cell 20, 482–494.

Kunert, G., Reinhold, C., Gershenzon, J., 2010. Constitutive emission of the aphidalarm pheromone, (E)-b-farnesene, from plants does not serve as a directdefense against aphids. BMC Ecol. 10, 23.

Li, G., Kollner, T.G., Yin, Y., Jiang, Y., Chen, H., Xu, Y., Gershenzon, J., Pichersky, E.,Chen, F., 2012. Nonseed plant Selaginella moellendorffi has both seed plant andmicrobial types of terpene synthases. Proc. Natl. Acad. Sci. USA 109, 14711–14715.

Liang, W., Tan, X., Chen, X., Hashimoto, T., Yamada, Y., Heinstein, P., 2000. Isolationof a (+)-d-cadinene synthase gene CAD1-A and analysis of its expression patternin seedlings of Gossypium arboreum L. Sci. China C: Life Sci. 43, 245–253.

Liu, C.J., Heinstein, P., Chen, X.Y., 1999a. Expression pattern of genes encodingfarnesyl diphosphate synthase and sesquiterpene cyclase in cotton suspension-cultured cells treated with fungal elicitors. Mol. Plant Microbe Interact. 12,1095–1104.

Liu, J., Benedict, C.R., Stipanovic, R.D., Bell, A.A., 1999b. Purification andcharacterization of s-Adenosyl-L-methionine: desoxyhemigossypol-6-O-methyltransferase from cotton plants. An enzyme capable of methylating thedefense terpenoids of cotton. Plant Physiol. 121, 1017–1024.

Liu, J., Stipanovic, R.D., Bell, A.A., Puckhaber, L.S., Magill, C.W., 2008. Stereoselectivecoupling of hemigossypol to form (+)-gossypol in moco cotton is mediated by adirigent protein. Phytochemistry 69, 3038–3042.

Lucas-Barbosa, D., van Loon, J.J., Dicke, M., 2011. The effects of herbivore-inducedplant volatiles on interactions between plants and flower-visiting insects.Phytochemistry 72, 1647–1654.

Luo, P., Wang, Y.H., Wang, G.D., Essenberg, M., Chen, X.Y., 2001. Molecular cloningand functional identification of (+)-d-cadinene-8-hydroxylase, a cytochromeP450 mono-oxygenase (CYP706B1) of cotton sesquiterpene biosynthesis. PlantJ. 28, 95–104.

Majetic, C.J., Raguso, R.A., Ashman, T.-L., 2009. The sweet smell of success: floralscent affects pollinator attraction and seed fitness in Hesperis matronalis. Funct.Ecol. 23, 480–487.

Martin, D.M., Aubourg, S., Schouwey, M.B., Daviet, L., Schalk, M., Toub, O., Lund, S.T.,Bohlmann, J., 2010. Functional annotation, genome organization and phylogenyof the grapevine (Vitis vinifera) terpene synthase gene family based on genomeassembly, FLcDNA cloning, and enzyme assays. BMC Plant Biol. 10, 226.

Meng, Y.L., Jia, J.W., Liu, C.J., Liang, W.Q., Heinstein, P., Chen, X.Y., 1999. Coordinatedaccumulation of (+)-d-cadinene synthase mRNAs and gossypol in developingseeds of Gossypium hirsutum and a new member of the cad1 family from G.arboreum. J. Nat. Prod. 62, 248–252.

Minyard, J.P., Tumlinson, J.H., Hedin, P.A., Thompson, A.C., 1965. Constituents of thecotton bud. terpene hydrocarbons. J. Agric. Food Chem. 13, 599–602.

Minyard, J.P., Tumlinson, J.H., Thompson, A.C., Hedin, P.A., 1966. Constituents of thecotton bud. sesquiterpene hydrocarbons. J. Agric. Food Chem. 14, 332–336.

Nagegowda, D.A., 2010. Plant volatile terpenoid metabolism: biosynthetic genes,transcriptional regulation and subcellular compartmentation. FEBS Lett. 584,2965–2973.

Opitz, S., Kunert, G., Gershenzon, J., 2008. Increased terpenoid accumulation incotton (Gossypium hirsutum) foliage is a general wound response. J. Chem. Ecol.34, 508–522.

Pare, P.W., Tumlinson, J.H., 1997. De novo biosynthesis of volatiles induced byinsect herbivory in cotton plants. Plant Physiol. 114, 1161–1167.

Pichersky, E., Gershenzon, J., 2002. The formation and function of plant volatiles:perfumes for pollinator attraction and defense. Curr. Opin. Plant Biol. 5, 237–243.

Rasmann, S., Kollner, T.G., Degenhardt, J., Hiltpold, I., Toepfer, S., Kuhlmann, U.,Gershenzon, J., Turlings, T.C., 2005. Recruitment of entomopathogenicnematodes by insect-damaged maize roots. Nature 434, 732–737.

Scotto-Lavino, E., Du, G., Frohman, M.A., 2006. 50 end cDNA amplification usingclassic RACE. Nat. Protoc. 1, 2555–2562.

Stipanovic, R.D., Lopez Jr., J.D., Dowd, M.K., Puckhaber, L.S., Duke, S.E., 2006. Effect ofracemic and (+)- and (�)-gossypol on the survival and development ofHelicoverpa zea larvae. J. Chem. Ecol. 32, 959–968.

Stipanovic, R.D., Lopez Jr., J.D., Dowd, M.K., Puckhaber, L.S., Duke, S.E., 2008. Effect ofracemic, (+)- and (�)-gossypol on survival and development of Heliothisvirescens larvae. Environ. Entomol. 37, 1081–1085.

Stipanovic, R.D., Mace, M.E., Bell, A.A., Beier, R.C., 1992. The role of free radicals inthe decomposition of the phytoalexin desoxyhemigossypol. J. Chem. Soc., PerkinTrans. 1, 3189–3192.

Stipanovic, R.D., Puckhaber, L.S., Bell, A.A., Percival, A.E., Jacobs, J., 2005. Occurrenceof (+)- and (�)-gossypol in wild species of cotton and in Gossypium hirsutumVar. marie-galante (Watt) Hutchinson. J. Agric. Food Chem. 53, 6266–6271.

Tan, X.P., Liang, W.Q., Liu, C.J., Luo, P., Heinstein, P., Chen, X.Y., 2000. Expressionpattern of (+)-d-cadinene synthase genes and biosynthesis of sesquiterpenealdehydes in plants of Gossypium arboreum L. Planta 210, 644–651.

Tesso, H., Konig, W.A., Kubeczka, K.H., Bartnik, M., Glowniak, K., 2005. Secondarymetabolites of Peucedanum tauricum fruits. Phytochemistry 66, 707–713.

Tholl, D., 2006. Terpene synthases and the regulation, diversity and biological rolesof terpene metabolism. Curr. Opin. Plant Biol. 9, 297–304.

56 C.-Q. Yang et al. / Phytochemistry 96 (2013) 46–56

Tholl, D., Chen, F., Petri, J., Gershenzon, J., Pichersky, E., 2005. Two sesquiterpenesynthases are responsible for the complex mixture of sesquiterpenes emittedfrom Arabidopsis flowers. Plant J. 42, 757–771.

Thomson, K.S., Gall, J.G., Coggins, L.W., 1973. Nuclear DNA contents of coelacantherythrocytes. Nature 241, 126.

Townsend, B.J., Poole, A., Blake, C.J., Llewellyn, D.J., 2005. Antisense suppression of a(+)-d-cadinene synthase gene in cotton prevents the induction of this defenseresponse gene during bacterial blight infection but not its constitutiveexpression. Plant Physiol. 138, 516–528.

Turco, E., Vizzuso, C., Franceschini, S., Ragazzi, A., Stefanini, F.M., 2007. The in vitroeffect of gossypol and its interaction with salts on conidial germination andviability of Fusarium oxysporum sp. vasinfectum isolates. J. Appl. Microbiol. 103,2370–2381.

Wang, J.Y., Cai, Y., Gou, J.Y., Mao, Y.B., Xu, Y.H., Jiang, W.H., Chen, X.Y., 2004. VdNEP,an elicitor from Verticillium dahliae, induces cotton plant wilting. Appl. Environ.Microbiol. 70, 4989–4995.

Williams, D.C., McGarvey, D.J., Katahira, E.J., Croteau, R., 1998. Truncation oflimonene synthase preprotein provides a fully active ‘pseudomature’ form of

this monoterpene cyclase and reveals the function of the amino-terminalarginine pair. Biochemistry 37, 12213–12220.

Wu, J., Baldwin, I.T., 2010. New insights into plant responses to the attack frominsect herbivores. Annu. Rev. Genet. 44, 1–24.

Xu, Y.H., Wang, J.W., Wang, S., Wang, J.Y., Chen, X.Y., 2004. Characterization ofGaWRKY1, a cotton transcription factor that regulates the sesquiterpenesynthase gene (+)-d-cadinene synthase-A. Plant Physiol. 135, 507–515.

Yang, C.Q., Lu, S., Mao, Y.B., Wang, L.J., Chen, X.Y., 2010. Characterization of twoNADPH: cytochrome P450 reductases from cotton (Gossypium hirsutum).Phytochemistry 71, 27–35.

Zhang, J.W., Li, S.K., Wu, W.J., 2009. The main chemical composition and in vitroantifungal activity of the essential oils of Ocimum basilicum Linn. var. pilosum(Willd.) Benth. Molecules 14, 273–278.

Zhang, Y., Xie, Y., Xue, J., Peng, G., Wang, X., 2009b. Effect of volatile emissions,especially alpha-pinene, from persimmon trees infested by Japanese wax scalesor treated with methyl jasmonate on recruitment of ladybeetle predators.Environ. Entomol. 38, 1439–1445.