Phytochemistry 96 (2013) 4656

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

Isolation and characterization of terpene synthases in cotton (Gossypium

hirsutum)
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 for
Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
b
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 2013
Received in revised form 23 July 2013
Available online 24 September 2013
Keywords:
Cotton (Gossypium hirsutum)
Malvaceae
Terpene synthase
VIGS

a b s t r a c t
Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins
against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about
the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas
chromatographymass spectrometry (GCMS). By EST data mining combined with Rapid Amplication of
cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were
isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into b-caryophyllene and
a-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11diene 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 expressed
only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl
jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were
induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes
of cotton, at least of this Upland cotton.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Terpenoids constitute the largest family of natural products
with more than 30,000 structures (Degenhardt et al., 2009b),
which are grouped into different classes on the basis of the number
of 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), photosynthesis
pigments (carotenoids and chlorophylls), and membrane structural
components (sterols), terpenoids also have ecological functions in
mediating plant interactions with biotic and abiotic factors. Volatile terpenes may help plants to attract pollinators or predators
of herbivores (Degenhardt et al., 2003; Pichersky and Gershenzon,
2002), and terpenoid phytoalexins can participate in defense
against phytopathogens and herbivores (Balkema-Boomstra et al.,
2003; Nagegowda, 2010; Tan et al., 2000; Wang et al., 2004).

Corresponding author. Tel.: +86 21 54924034; fax: +86 21 54924015.

E-mail address: ljwang@sibs.ac.cn (L.-J. Wang).
0031-9422/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.09.009

In plant cells, precursors of terpenoids are synthesized via

either the cytosolic mevalonate pathway (MVA pathway) or the
plastidial 2-C-methyl-D-erythritol-4-phosphate pathway (MEP
pathway), and are then condensed into structural diverse terpenoids by the family of terpene synthases (TPSs). The TPS family
consists of isoprene synthases producing 5-carbon isoprene using
dimethylallyl pyrophosphate (DMAPP), monoterpene synthases
producing 10-carbon monoterpenes from geranyl pyrophosphate
(GPP), sesquiterpene synthases producing 15-carbon sesquiterpenes from farnesyl pyrophosphate (FPP), and diterpene synthases
producing 20-carbon diterpenes from geranylgeranyl pyrophosphate (GGPP) or copalyl pyrophosphate (CPP) (Nagegowda, 2010;
Tholl, 2006). Most sesquiterpene synthases are localized in the
cytosol, whereas monoterpene and diterpene synthases are usually
in the plastid and have a N-terminal plastid transit peptide
upstream of the RRX8W motif (Williams et al., 1998). Almost
all TPSs contain the DDXXD and the NSE/DTE motifs at the Cterminal region for the metal dependent (frequently Mg2+ or
Mn2+) ionization of the prenyl diphosphate substrate that are
essential for their catalytic activities (Chen et al., 2011; Tholl,

C.-Q. Yang et al. / Phytochemistry 96 (2013) 4656

2006). They constitute families with various members in plant genomes, ranging from only one functional and three pseudogenes (or
fragments) of TPSs in moss (Physcomitrella patens), to 152 TPSs in
grapevine (Vitis vinifera) (Hayashi et al., 2006; Martin et al.,
2010). Recent phylogenetic analysis of TPSs from gymnosperms
and angiosperms established presence of 7 subfamilies of TPS-a,
b, c, d, e/f, g and h, with most monoterpene and sesquiterpene synthases of angiosperms being distributed in TPS-a, b and g subfamilies (Chen et al., 2011).
Cotton (Gossypium spp.) is an important economic crop and a
major source of natural ber for the textile industry. Cotton plants
with epidermal pigment glands accumulate the sesquiterpene
aldehyde gossypol (1) and related sesquiterpene aldehydes (hemigossypol (2), hemigossypolone (3), heliocides H1 (4), H2 (5), H3 (6)
and H4 (7)) as major toxins against herbivorous insects, such as
Helicoverpa armigera, Heliothis virescens and Spodoptera exigua, of
these, hemigossypol (2) is a major phytoalexin that protects the
plant from pathogens such as Verticillium dahliae, Rhizoctonia solani, Xanthomonas campestris and Fusarium oxysporum. Together, these
pests 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

47

has been made in characterizing enzymes and transcription factors

for gossypol (1) biosynthesis. The enzymes, farnesyl diphosphate
synthase (FPS) (Liu et al., 1999a), (+)-d-cadinene synthase (CDN
or CAD1) (Chen et al., 1995; Tan et al., 2000), and CYP706B1, a
cytochrome P450 monooxygenase that hydroxylates (+)-d-cadinene (8) at the 8-position (Luo et al., 2001), catalyze three consecutive steps of gossypol (1) biosynthesis. Other related enzymes and
proteins include P450 reductases (Yang et al., 2010), a peroxidase
(Benedict et al., 2006; Stipanovic et al., 1992), a laccase (Liu
et al., 2008), a dirigent protein (Liu et al., 2008) and a methyltransferase (Liu et al., 1999b). The transcription factor GaWRKY1 was
shown 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 compounds. For example, Arabidopsis owers emit a mixture of volatiles consisting of over 20 sesquiterpenes (Tholl et al., 2005); rice
(Oriza sativa) synthesizes 13 sesquiterpenes after methyl jasmonate (MeJA) treatment (Cheng et al., 2007); and Selaginella moellendorfi, a lycophyte, produces the monoterpene linalool (9) and the
sesquiterpenes b-elemene (10), germacrene D (11), b-sesquiphellandrene (12) and nerolidol (13) after elicitation with alamethicin,
a fungal antibiotic (Li et al., 2012). It has been reported that cotton

Fig. 1. Structures of monoterpenes and sesquiterpenes produced by cotton plants and in vitro activity assays of cotton terpene synthases.

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C.-Q. Yang et al. / Phytochemistry 96 (2013) 4656

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

Cotyledon

Leaf

Petal

Pericarp (5 DPA)

Pericarp (10DPA)

Pericarp (25 DPA)

11.2
3.3
8.1
1.5
5.2

281.8
54.4
192.6
48.4
179.1

334.5
54.7
178.9
27.6
148.9

93.6
21.1
28.0
25.0
118.3

0.4
0.3
17.6
7.5
0.9
3.2
0.7
4.3

23.9

9.1

4.1

210.3
91.7

113.7
51.7

53.1
26.1

262.4
342.2
27.6
42.5
20.7

213.3
282.6
20.0
33.2
12.2

107.8
120.3
9.6
17.5
8.7

1777.6

1480.4

633.2

Monoterpene

a-Pinene (14)
b-Pinene (15)
b-Myrcene (16)
b-phellandrene (22)
(E)-b-Ocimene (17)
Sesquiterpene
a-Copaene (23)
b-Elemene (10)
b-Caryophyllene (18)
a-Humulene (19)
Guaia-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)
Total

4.6
1.1
2.3
0.5
0.9

13.6
5.8

28.8

2.4

2.4

64.2

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

foliage produces at least 4 monoterpenes (a-pinene (14), b-pinene

(15), myrcene (16), b-ocimene (17)) and 4 sesquiterpenes (b-caryophyllene (18), a-humulene (19), c-bisabolene (20) and b-bisabolol
(21)), in addition to sesquiterpene aldehydes such as hemigossypolone (3) and heliocides H1H4 (47), and that herbivoral or
mechanical damage to older leaves could lead to increased
amounts of these terpenoids in younger leaves (ElZen et al.,
1985; Minyard et al., 1965, 1966; Opitz et al., 2008; Pare and Tumlinson, 1997). Up to now (+)-d-cadinene synthase (CDN), a sesquiterpene 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 gossypol (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-cadinenetype sesquiterpenes of cotton, but also identied the respective
TPSs (see Fig. 1).

2. Results
2.1. Glanded cotton plant produces multiple monoterpenes and
sesquiterpenes
To detect terpenes produced by cotton plants other than (+)-dcadinene (8), hypocotyls and cotyledons from seedlings, and leaf,
petal and pericarp tissues at different developmental stages of
the mature glanded cultivar Gossypium hirsutum cv. CCRI12 were
extracted with n-pentane and subjected to gas chromatography
mass spectrometry (GCMS) 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), guaia-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 identied (Table 1 and Fig. 2). Some terpenes,

Fig. 2. Volatile terpenes in true leaf of cotton (G. hirsutum cv. CCRI12). Leaves from the 60-day-old cotton plant were extracted with n-pentane for 1 h at room temperature,
and analyzed by GCMS. Peaks are: INS: Internal Standard, nonyl acetate; (a) a-pinene (14); (b) b-pinene (15); (c) b-myrcene (16); (d) b-phellandrene (22); (e) b-ocimene
(17); (f) b-caryophyllene (18); (g) guaia-1(5),11-diene (24); (h) a-humulene (19); (i) guaia-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) 4656

including a-pinene (14), b-pinene (15), b-myrcene (16) and

b-caryophyllene (18), were detected in all organs examined except
the cotyledon and petal, whereas others showed distinct distribution patterns. For example, guaia-1(5),11-diene (24) and guaia1(10),11-diene (25) were detected in leaf extracts only, whereas
a-bergamotene (27) and b-farnesene (28) were in pericarps at various developmental stages. The gossypol precursor (+)-d-cadinene
(8) was detected at low abundance in pericarp.
Of these samples, the pericarp contained the most abundant
and diverse monoterpenes and sesquiterpenes, with the highest
content up to 1,777 lg/g fresh weight (FW) or approximately
1.8 of the total FW at an early developmental stage of 5 days post
anthesis (DPA), and whose content decreased during development,
with 633 lg/g FW detected at 20 DPA. The leaf and hypocotyl tissues showed much lower levels of terpene production, with totals
of 64 and 28 lg/g FW, respectively. However, although at low
abundance, diverse terpenes were also present in leaf tissue,

49

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 terpenes were detected in cotyledons, and none were found in petals
at the owering stage (0 DPA) (Table 1).

2.2. Identication of terpene synthases from cotton

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

Fig. 3. Alignment of amino acid sequence of GhTPSs and other monoterpene synthases and sesquiterpene synthases of plants. Deduced protein sequences of GhTPSs were
aligned with selected terpene synthases with software clustalw, and edited with Jalview. Conserved domains of RRx8W and DDxxD are highlighted. GhCAD-C4: G. hirsutum
(+)-d-cadinene synthase, AAX44033; NtEAS: Nicotiana tabacum 5-epi-aristolochene synthase, Q40577; VaCar: Vitis vinifera (E)- b-caryophyllene synthase, ADR74192; CuOci:
Citrus unshiu (E)-b-ocimene synthase, BAD91046; AaPin: Artemisia annua ()-b-pinene synthase, AAK58723; MgTer: Magnolia grandiora a-terpineol synthase, ACC66282.

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C.-Q. Yang et al. / Phytochemistry 96 (2013) 4656

products by modication enzymes, such as the P450 monooxygenase CYP706B1.

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

(NM_117775), a monoterpene synthase, were used as bait to

search EST databases of G. hirsutum with Blastn program (http://
blast.ncbi.nlm.nih.gov/Blast.cgi). ESTs showing nucleotide sequen
ce identities >50% with either were collected and assembled into
12 contigs and 23 singlets using the CAP3 software (http://pbil.u-

Fig. 4. Product spectrum of GhTPS1, GhTPS2 and GhTPS3. Recombinant GhTPS1, GhTPS2 and GhTPS3 proteins were expressed in E. coli as fusion proteins and puried with
NiNTA resin. After incubation with FPP or GPP in the reaction buffer for 30 min at 37 C, the reaction mixture was extracted with n-pentane and analyzed by GCMS. Peaks
are: (a) b-caryophyllene (18); (b) a-humulene (19); (c) b-elemene (10); (d) guaia-1(5),11-diene (24); (e) alloaromadendrene (29); (f) guaia-1(10),11-diene (25); (g) a-pinene
(14); (h) b-pinene (15); (i) b-phellandrene (22); (j) c-terpinene (30).

C.-Q. Yang et al. / Phytochemistry 96 (2013) 4656

niv-lyon1.fr/cap3.php), of which 5 contigs and 16 singlets shared

>90% identities with (+)-d-cadinene synthase and they were considered members of the CDN family. The remaining 7 contigs and
7 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), were
obtained, which have lengths of 1,943, 1,951 and 1940 bp, encoding 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-cadinene
synthase. As the CDN family can be divided into four (A, B, C and
D) subfamilies whose members share amino acid identities of
>77% (Townsend et al., 2005), GhTPS1 and GhTPS2 were considered candidates of new sesquiterpene synthases. GhTPS3 has a
high sequence identity (59%) to (E)-b-ocimene/myrcene synthase
of V. vinifera (ADR74206). Like other terpene synthases, the three
cotton TPSs contain the highly conserved RRX8W motif at N-terminal and the DDXXD domain at C-terminal (Fig. 3), which is essential 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/) software
indicated that GhTPS3 has a N-terminal 38-amino acid peptide,
whereas GhTPS1 and GhTPS2 do not. Phylogenetic analysis with

51

TPSs of the CDN family and those from other plant species established that, GhTPS1 and GhTPS2 could be classied with sesquiterpene synthases in the clade TPS-a, closest to cotton (+)-d-cadinene
synthase, whereas GhTPS3 was with monoterpene synthases in the
clade TPS-b (Supplementary Fig. 1).
2.3. Functional characterization of GhTPS1, GhTPS2 and GhTPS3
To determine enzyme activity, open reading frames (ORFs) of
GhTPS1, GhTPS2 and GhTPS3 (with a deletion of 35 amino acid residues at N-terminal) were expressed in Escherichia coli as fusion
proteins. GCMS analysis of the n-pentane extract of the reaction
mixture indicated that both GhTPS1 and GhTPS2 converted FPP
into multiple sesquiterpene products (Fig. 4A and B). GhTPS1 produced b-caryophyllene (18) and a-humulene (19) in a ratio of
71:29 (Fig. 4A). Notably, in most cotton tissues examined the
b-caryophyllene (18) to a-humulene (19) ratio was around 2.2:1
(Table 1), close to that of GhTPS1. The products of GhTPS2 included
guaia-1(10),11-diene (25) (55%), guaia-1(5),11-diene (24) (14%), ahumulene (19) (10%), b-elemene (10) (10%), alloaromadendrene
(29) (6%) and b-caryophyllene (18) (5%) (Fig. 4B), a composition
similar to the sesquiterpene spectrum in leaves (Table 1). The
GhTPS3 protein converted GPP into a-pinene (14) (77.2%), bpinene (15) (14.7%) and b-phellandrene (22) (5.5%), with a trace

Fig. 5. Expression of GhTPS genes and terpene contents in leaf of the VIGS-treated cotton plant. Cotyledons of cotton plants were inoculated with TRV harboring fragments of
GhTPS1, GhTPS2, GhTPS3 or empty vector, as indicated. Total RNAs from true leaves were analyzed by real-time PCR. n-Pentane extracts of true leaves were analyzed by GC
MS. V-GhTPS1, V-GhTPS2 and V-GhTPS3 refer to plants with suppressed expression of GhTPS1, GhTPS2 or GhTPS3 by VIGS. (A) Expression of GhTPS genes in true leaves; (B) bcaryophyllene (18), guaia-1(10),11-diene (25) and a-pinene (14) contents in true leaves of VIGS plants; Error bars indicate SD of three biological replicates.

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C.-Q. Yang et al. / Phytochemistry 96 (2013) 4656

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 RNAs
from 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 from
which seeds were removed) were harvested at 5 and 10 days post anthesis (DPA), respectively. (B) Expression of GhTPSs in leaves treated with mechanical wounding, fungal
elicitor 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 three
biological replicates.

C.-Q. Yang et al. / Phytochemistry 96 (2013) 4656

amount of c-terpinene (30) (2.6%) (Fig. 4C). The apparent Km was

determined 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 rattle
virus (TRV)-based virus induced gene silencing (VIGS) system
was employed to silence their expression in cotton. The 456-,
486- and 444-bp fragments of GhTPS1, GhTPS2 and GhTPS3 were
used, and the corresponding Agrobacteria were inltrated into cotton seedlings. Thirty days after inoculation, expression of target
genes in true leaves was analyzed by quantitative real-time
RT-PCR (qRT-PCR). In each VIGS-treated plant selected, transcripts
of target genes decreased by more than 85%, whereas non-target
TPS gene expressions were largely unaffected (Fig. 5 A). Consistent
with target gene suppression, production of b-caryophyllene (18)
and a-humulene (19) was decreased by 90% in GhTPS1-silenced
plants (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) were
undetectable in GhTPS2-suppressed leaves (Fig. 5B, Supplementary
Fig. 2A and C). In the GhTPS3-silenced plant, a-pinene (14),
b-pinene (15) and b-phellandrene (22) contents decreased by more
than 80%, whereas b-myrcene (16) was unchanged (Fig. 5B, Supplementary Fig. 2D and E). Such a correlation between suppressed TPS
gene expression and decreased contents of terpene products suggest precise silencing of target genes, and conrms activities of
the three GhTPSs in planta.
2.4. Expression Patterns of GhTPS1, GhTPS2 and GhTPS3
Spatial expression patterns of the three TPS genes were analyzed by qRT-PCR. Transcripts of GhTPS1 were detected in hypocotyl, cotyledon, leaf, pericarp and ovule (20-DPA) tissues, with
the highest expression level in pericarp tissue and the lowest in
cotyledons; transcripts were also undetectable in petals (Fig. 6A).
This expression pattern is consistent with the distribution of bcaryophyllene (18) and a-humulene (19) (Table 1). For GhTPS2,
the transcript level was high in leaf, low in pericarp, and hardly
detectable in other organs, this being in agreement with the
leaf-specic detection of guaia-1(5),11-diene (24) and guaia1(10),11-diene (25) (Fig. 6A and Table 1). GhTPS3 showed a similar
expression pattern as GhTPS1 (Fig. 6A).
Gossypol (1) accumulation and expression of the biosynthesis
genes can be strongly induced by mechanical wounding, V. dahliae
(Vde) elicitor and MeJA treatments (Luo et al., 2001; Xu et al.,
2004). To nd out whether the production of these terpenes in cotton was also inducible, true leaves of glanded cotton plants were
treated with these stimuli. Expression levels of the three TPS genes
were drastically induced, ranging from 3- to 9-fold (Fig. 6B).
GCMS analysis of the n-pentane extracts of these plants established an increased accumulation of both monoterpenes and sesquiterpenes in response to the treatments (Fig. 6C), consistent
with 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 sesquiterpene aldehydes, cotton plants also produce a mixture of monoterpenes and sesquiterpenes. Plant volatile terpenes play
important roles in attracting pollinators, mediating direct and indirect 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 (Diospyros kaki L.) infested by Japanese wax scales (Ceroplastes japonicus) is a cue to attract the ladybeetle predator (Chilocorus kuwanae)
(Zhang et al., 2009b); the volatile (E)-b-farnesene (28) helps to
defend plants against aphid infestation as it is also the principal

53

component of the alarm pheromone of many aphid species, which

is released when aphids are attacked by enemies (Kunert et al.,
2010; Thomson et al., 1973). These defensive terpenes were also
detected in tissues of the cotton plant. Unlike snapdragon and
Arabidopsis, as well as many other plants, whose owers synthesize and emit terpenes (Dudareva et al., 2003; Tholl et al., 2005),
the cotton plant produces the most abundant (up to 0.2% fresh
weight) and diverse (5 monoterpenes and 8 sesquiterpenes) terpenes in pericarp tissue, which encloses seeds that produce cotton
ber. Moreover, production of these terpenes and expression of
the TPS genes were inducible by mechanical wounding and elicitation. These data imply a protective role of these terpenes in cotton.
b-caryophyllene (18) and a-humulene (19) have been detected
in many plant species and they seem to be present throughout
seed plants. Both are usually produced by a single enzyme (Cai
et al., 2002; Irmisch et al., 2012; Kollner et al., 2008), and have been
reported to participate in plant-insect interactions (Degenhardt
et al., 2009a; Rasmann et al., 2005). However, the guaia-1(5),11diene (24) and guaia-1(10),11-diene (25), detected here in cotton
leaves, are rare in the plant kingdom. There is no report of any
plant-derived guaia-1(5),11-diene (24), and guaia-1(10),11-diene
(25) has been reported only in two plant species, Peucedanum tauricum of Apiaceae and Ocimum basilicum of Lamiaceae (Tesso et al.,
2005; Zhang et al., 2009a). To our knowledge, GhTPS2 is the rst
reported enzyme which synthesizes guaia-1(5),11-diene (24) and
guaia-1(10),11-diene (25).
The cadinene-type sesquiterpene aldehydes are major defensive
compounds found in cotton. They are constitutively synthesized
and stored in pigment glands, and in addition, their biosynthesis
is inducible (Baksha et al., 2006; Tan et al., 2000; Xu et al., 2004).
In this investigation, it was found that formation of the non-cadinene-type volatile terpenes and expression of the three TPS genes are
also both constitutive and inducible. The similar expression pattern
between gossypol pathway genes and the three TPSs reported here
raises a question that biosynthesis of mono- and sesquiterpenes in
cotton share a common regulatory network. Isolation of key regulators in this network will be important in not only understanding
terpenoid metabolism regulation in the plant, but also facilitating
cotton breeding for enhanced pest and disease control.
4. Concluding remarks
Cotton plants produce large amount of volatile terpenes, but to
date little is known about their biosynthesis. In this research, two
sesquiterpene synthases and one monoterpene synthase were isolated and characterized by both in vitro enzyme assays and in vivo
VIGS assays. They showed distinct and inducible expression patterns, and account for the biosynthesis of the most volatile terpenes in cotton.
5. Experimental
5.1. Plant material
Cotton (G. hirsutum cv. CCRI12) plants were grown in a phytotron at 28 0.5 C. Leaf, petal and pericarp tissues at various developmental stages were collected from 5-month-old plants;
hypocotyls and cotyledons were collected from 10-day-old seedlings. Fresh plant samples were used directly for chemical analysis
and RNA extraction.
5.2. Terpene identication by GCMS
Plant tissues (200 mg) were frozen and ground in liquid N2 into
a ne powder and extracted with n-pentane (2 mL) at 28 C for 1 h

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C.-Q. Yang et al. / Phytochemistry 96 (2013) 4656

with constant shaking. Nonyl acetate (standard) was added to the

extracts to a nal concentration of 5 lg/mL. The solution was centrifuged at 18,000 g for 10 min, and supernatants were dried with
anhyde Na2SO4 before GCMS analysis. All extractions were performed in triplicate.
Terpenes and enzyme products were identied by gas chromatographymass spectrometry (GCMS) on an Agilent 6890 Series
GC System coupled to an Agilent 5973 Network Mass Selective
Detector using an HP 5 (Agilent Technologies) capillary column
(0.25 mm i.d.  30 m with 25-lm lm). The injector was operated
splitless at a temperature of 200 C with a column ow of 1 mL He/
min. The following temperature program was used: initial temperature of 40 C (5-min hold), increase to 230 C at 5 C/min, then to
280 C at 30 C/min (10 min hold). Terpenes were identied by
mass spectra comparison using the Enhanced Chemstation (version E.02.00.493, Agilent Technologies) software and NIST (National Institute of Standards and Technology) library, and product
peaks were quantied by integration of peak areas and calibration
with internal standard nonyl acetate.
5.3. Total RNA isolation and cDNA synthesis
Tissue samples (100 mg) were ground into a ne powder and
suspended with extraction buffer (1 mL, 0.1 M Tris, pH 8.0,
50 mM EDTA, 1 M NaCl, 1% cetyltrimethyl ammonium bromide
and 1% b-mercaptoethanol) at 65 C for 30 min. After extraction
with an equal volume of CHCl3 and centrifugation at 15,000g, the
supernatant was precipitated by LiCl (nal concentration of 2 M)
at 20 C for 8 h. After centrifugation at 18,000 g for 10 min, the
total RNA pellet was washed with 1 mL EtOHH2O (3:1) and dissolved in RNase free H2O.
Total RNAs was treated with DNase I and reverse transcribed
using PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa, China)
according to the manufacturers protocol.

GhTPS1-R (5-TTCTAGCAAGCCTTTGACAT-3) for GhTPS1, GhTPS2-F

(5-TTGGGCATCTAACAATCCTA-3) and GhTPS2-R (5-TGGCCCTGGAG
TAACTTTG-3) for GhTPS2, and GhTPS3-F (5-ATCGGGTCCTGTGCTACTTG-3) and GhTPS3-R (5-CGGCCATACGATCTCTGTTC-3) for
GhTPS3.
5.6. Prokaryotic expression and purication of recombinant TPS
proteins
ORFs of the TPSs were amplied by pfu DNA polymerase using
primers of GhTPS1-F-SacI (5-GCGAGCTCATGTCTTCCATGTCCAAC
GT-3) and GhTPS1-R-NotI (5-TCGCGGCCGCTCATCAAATCG GCACC
GGAT-3) for GhTPS1; GhTPS2-F-SacI (5-GCGAGCTCATGTCTT CATCAAAGCTTCC-3) and GhTPS2-R-NotI (5-TCGCGGCCGC TTAATA AG
AGGCTGAAATTG-3) for GhTPS2; GhTPS3-F-KpnI (5-GCGGTACC
ATGGCTAGTCTGCTTACTTC-3) and GhTPS3-F-KpnI (5-GCGGTACCAGTCCCTCACAATC TGAAGC-3) and GhTPS3-R-XhoI (5-GCCTCGA
GTTATTTTGGCGATGGAATTG-3) for GhTPS3 (with 35 amino acid
truncation at N-terminal). PCR products were digested with SacI
and NotI (GhTPS1 and GhTPS2), or KpnI and XhoI (GhTPS3) and ligated into the expression vector pET32a and transformed into E.
coli BL21 (DE3).
E. coli cells harboring expression vectors were grown overnight
in LuriaBertani (LB) medium (contain 100 mg/L ampicillin) at
37 C in a shaking incubator, then diluted at 1:100 into LB medium
for growth at 37 C till OD600 = 0.5. Prokaryotic expression was
started by addition of 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) and shaking at 28 C for 12 h. Bacterial cells were
collected by centrifugation at 2000g for 5 min, washed and re-suspended in MOPS buffer (100 mM MOPS, 10% glycerol, 0.2 mM DTT,
1 mM EDTA, adjusted to pH 7.3 with NaOH). After sonication on
ice, the recombinant protein was puried with NiNTA resins
according to manufacturers manual (Novagen, Madison, USA).
5.7. Enzyme assay

5.4. 5 and 3 RACE and full length cDNA amplication

Rapid Amplication of cDNA Ends (RACE) was performed as described (Scotto-Lavino et al., 2006). For 5 RACE, rst strand cDNA
was synthesized for each gene with specic primers of TPS1-RT
(5-ACCCATCAGGAGCCACCATAAG-3), TPS2-RT (5-AGTCTCGAGAGT
TTGGTCATCCT-3) or TPS3-RT (5-AAGGCATCAATAAGCTCAAG-3),
and then amplied with gene specic primers of TPS1-5R (5-CTATT
TTCATTTTGGAGGGACATG-3), TPS2-5R (5-GTAGAGCAACTCATCATC
CATCACT-3) or TPS3-5R (5-CACTTCTTCCTTCAGTTTGC-3), in combination with adaptor primer (5-GAGGACTCGAGCTCAAGC-3), respec
tively. 3- RACE was performed with 3-Full RACE Core Set (TaKaRa,
China), with gene specic primers of TPS1-3F (5-ATACAG GGTCCA
ATACGC-3), TPS2-3F (5-TCATGTCAAATATGCGAAA-3) or TPS3-3F
(5-GGATTGCAGACCTTTGAGGA-3), respectively.
Based on assembly of fragments obtained by 5 and 3 RACE, full
length cDNAs were amplied with primer sets of TPS1-F (5-CACAACAAGAAGATATCGTCTCA-3) and TPS1-R (5-AACATGCCGAGATGA ACCAC-3) for GhTPS1, TPS2-F (5-TCATTTCGTAGTGTTTCCCA-3)
and TPS2-R (5-GGTGTTAACACCTCAAACATA-3) for GhTPS2, and
TPS3-F (5-TTTTACTCCATCCCTTTGGA-3) and TPS3-R (5-TTGAAACTTATTTGAGACAACAC-3) for GhTPS3.
5.5. Real-Time PCR analysis
Real-time PCR was performed using SYBR Premix Ex Taq reagent (TaKaRa, China) on a Mastercycler system (Eppendorf, Germany). Cotton histone3 gene (AF024716) with primers of his3-F
(5-GAAGCCTCATCGATACCGTC-3) and his3-R (5-CTACCACTACCATCATGG-3) was used for normalization. Primers for GhTPSs expression analysis are: GhTPS1-F (5-ACTCGACAAGAATACGAAGA-3) and

For enzyme assays of GhTPSs, recombinant protein (1 lg) was

added into 100 lL reaction buffer containing 25 mM HEPES, 10%
glycerol, 5 mM DTT, 5 mM MgCl2 and 50 lM FPP (GhTPS1 and
GhTPS2) or GPP (GhTPS3), and incubated at 37 C for 30 min. Reactions were stopped by addition of 0.5 M EDTA (10 lL) and the
whole was then extracted with n-pentane (200 lL), which was
then evaporated and a portion subjected to GCMS analysis. The
GCMS analysis procedure was the same as that for terpene identication from plant tissue extracts.
5.8. Cotton VIGS
Tobacco rattle virus (TRV)-based silencing system was used for
cotton VIGS assay (Gao et al., 2011). Fragments of GhTPSs were
amplied with primers of VGhTPS1-F-BamHI (5-GCGGATCCATACAGGGTCCAATACGC-3) and VGhTPS1-R-XbaI (5-GCTCTAGAAACGCAAGTAAGGACTGGAA-3) for GhTPS1, VGhTPS2-F-BamHI (5-GCGG
ATCCTCATGTCAAATATGCGAAAG-3) and VGhTPS2-R-XbaI (5-GCTC
TAGACGAGGACCCTTGTAAGAT-3) for GhTPS2, VGhTPS3-F-BamHI
(5-GCGGATCCGGATTGCAGACCTTTGAGGA-3) and VGhTPS3-R-XbaI
(5-GCTCTAGACCATGTCGCATCAATCAACT-3) for GhTPS3, followed
by insertion into BamHI-XbaI sites of pTRV2.
Plasmids of pTRV1 and pTRV2 or pTRV2 with fragments of
GhTPSs were introduced into Agrobacterium tumefaciens strain
LBA4404. Overnight grown Agrobacterial cells were collected and
suspended in 10 mM MES, 10 mM MgCl2 and 200 lm acetylsyringone solution to a nal OD600 = 2.0, and then left at room temperature for 4 h. A. tumefaciens suspensions of pTRV1 was mixed with
an equal volume of pTRV2 or its derivatives and inltrated into
cotyledons of 10-day-old cotton seedlings. Thirty days after inocu-

C.-Q. Yang et al. / Phytochemistry 96 (2013) 4656

lation, true leaves were collected for analysis of target gene expression and volatile terpene components analysis.
Acknowledgments
This work was supported by State Key Basic Research Program
of China (2013CB127000), the Chinese Academy of Sciences
(KSCX2-EW-N-03), and the National Natural Science Foundation
of China (30630008 and 90917021).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2013
.09.009.
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