A cDNA clone for β-caryophyllene synthase from Artemisia annua

Article abstract of DOI:10.1016/S0031-9422(02)00265-0

An homology-based cloning strategy yielded a full-length cDNA from Artemisia annua that encoded a protein of 60.3 kDa which resembled a sesquiterpene synthase in sequence. Heterologous expression of the gene in Escherichia coli provided a soluble recombinant enzyme capable of catalyzing the divalent metal ion-dependent conversion of farnesyl diphosphate to β-caryophyllene, a sesquiterpene olefin found in the essential oil of A. annua. In reaction parameters and kinetic properties, β-caryophyllene synthase resembles other sesquiterpene synthases of angiosperms. The β-caryophyllene synthase gene is expressed in most plant tissues during early development, and is induced in mature tissue in response to fungal elicitor thus suggesting a role for β-caryophyllene in plant defense.

Full text of DOI:10.1016/S0031-9422(02)00265-0

Phytochemistry 61 (2002) 523–529
www.elsevier.com/locate/phytochem
A cDNA clone for b-caryophyllene synthase from Artemisia annua§
a
a
b
c
a
b,
Yu Cai , Jun-Wei Jia , John Crock , Zhi-Xin Lin , Xiao-Ya Chen , Rodney Croteau *
aNational Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, P.R. China
bInstitute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA
cCollege of Life Science and Biotechnology, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, P.R. China
Received9 May 2002; receivedin revisedform 28 June 2002
Abstract
An homology-basedcloning strategy yieldeda full-length cDNA from Artemisia annua that encoded a protein of 60.3 kDa which
resembleda sesquiterpene synthase in sequence. Heterologous expression of the gene in Escherichia coli provided a soluble recom-
binant enzyme capable of catalyzing the divalent metal ion-dependent conversion of farnesyl diphosphate to b-caryophyllene, a
sesquiterpene olefin foundin the essential oil of A. annua. In reaction parameters andkinetic properties, b-caryophyllene synthase
resembles other sesquiterpene synthases of angiosperms. The b-caryophyllene synthase gene is expressedin most plant tissues du r-
ing early development, and is induced in mature tissue in response to fungal elicitor thus suggesting a role for b-caryophyllene in
plant defense.
#
2002 Elsevier Science Ltd. All rights reserved.
Keywords: Artemisia annua; Asteraceae; Sweet wormwood; Homology-based cDNA cloning; Recombinant protein expression; Sesquiterpene syn-
thase; b-Caryophyllene synthase; Sesquiterpene; b-Caryophyllene; Gene expression; Elicitation
1
. Introduction
synthase (Hua andMatsu da , 1999; Mercke et al., 1999)
which catalyzes a very complex sesquiterpene cycli-
zation from the precursor farnesyl diphosphate. In this
paper we describe another sesquiterpene cyclase cDNA
from A. annua (AF472361) that encodes a b-caryophyllene
synthase.
b-Caryophyllene is a common sesquiterpene that is
quite widely distributed in plants (Knudsen et al., 1993;
Kubo et al., 1996). It possesses anti-inflammatory
(Martin et al., 1993; Tambe et al., 1996) andanti-
carcinogenic (Kubo et al., 1996; Zheng et al., 1992)
activities, andthis macrocyclic olefin andits de rivatives
also couldplay a role in plant de fense (Ulubelen et al.,
1994; Wadhams et al., 1999). Reported here are the
expression patterns of this caryophyllene synthase gene
during the development of A. annua andin response to
wounding and elicitation.
Terpenoidbiosynthesis in Artemisia annua L. (Aster-
aceae) has receivedconsi de rable attention because this
plant (sweet wormwood; qinghao in traditional Chinese
medicine) is the source of the endoperoxide sesqui-
terpene lactone antimalarial drug artemisinin (Klay-
man, 1985). Several probable steps of the pathway have
been described (Bouwmeester et al., 1999; Dhingra and
Narasu, 2001; Wallaart et al., 1999), anda number of
groups have reportedthe molecular cloning of the ses-
quiterpene cyclase amorpha-4,11-diene synthase
responsible for catalyzing the committedstep in the
biosynthesis of artemisinin (Chang et al., 2000; Mercke
et al., 2000; Wallaart et al., 2001). Searches by us and
others have revealeda number of cDNAs from A. annua
that encode other terpenoid synthases (Jia et al., 1999;
Van Geldre et al., 2000), including that for epi-cedrol
2
. Results and discussion
§
The nucleotide sequence reported in this article has been depos-
itedwith the GenBank/EMBL Database un ed r accession number
AF472361
2.1. Isolation of a ꢀ-caryophyllene synthase cDNA
*
Corresponding author. Fax: +1-509-335-7643.
E-mail addresses: xychen@iris.sipp.ac.cn (X.-Y. Chen), croteau@
mail.wsu.edu (R. Croteau).
To isolate cDNA clones encoding terpene synthases
of A. annua, a comparison of angiosperm terpene
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00265-0

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Y. Cai et al. / Phytochemistry 61 (2002) 523–529
synthase sequences was first conducted. This search
revealeda consensus pept ie d sequence, GVYXEP,
foundonly in angiosperm sesquiterpene synthases.
Using a primer ed signedto this sequence andan
anchored-PCR strategy with A. annua library cDNA as
target yielded a 900 bp DNA fragment that showed
significant homology to plant sesquiterpene synthases.
Subsequent screening of the same cDNA library with
this probe ledto the isolation of a clone, de signated
QHS1, which containedan insert of 1902 bp with an
apparent open reading frame of 1644 bp. A stop codon
immediately upstream of the first ATG codon con-
firmedthe latter as the translation initiation site, from
which a molecular mass of 60.3 kD was calculatedfor
the translatedprotein; this size is consistent with that of
other enzymes of this general class (Cane, 1999).
Heterologous expression of this sequence from the
pET28 vector in Escherichia coli afforded a 60 kD pro-
tein in the resulting soluble enzyme fraction, as deter-
minedby SDS-PAGE, that was absent in control
preparations derived from the empty vector similarly
expressed. Evaluation of the soluble enzyme prepara-
tion by the standard sesquiterpene synthase assay using
3
[
1
1- H]farnesyl diphosphate as substrate (Crock et al.,
997; Mercke et al., 1999) revealedthe pro du ction of
both sesquiterpene olefins andoxygenatedpro ud cts.
Ten-foldscale-up of the recombinant enzyme prepara-
tion allowedmore than sufficient conversion of farnesyl
diphosphate to the biosynthetic products for capillary
GC–MS analysis (Crock et al., 1997; Steele et al., 1998)
which revealedthe presence of essentially a single pro-
duct (97% of the olefin fraction) with a spectral library
match to that of b-caryophyllene. Subsequent analysis
confirmedthe biosynthetic olefin to possess a GC
retention time andmass spectrum i de ntical to those of
an authentic trans-b-caryophyllene standard (Fig. 1);
the presence of about 2% a-humulene was also con-
firmed. Similar examination of the oxygenated sesqui-
terpenoids revealed very low levels of farnesol and
nerolidol, resulting from the action of endogenous
phosphatases and/or nonenzymatic solvolysis during
the course of the assay, anda trace of humulene oxi de ,
likely derived by oxidation of a-humulene by the bac-
terial host. Only farnesol and nerolidol were produced
from farnesyl diphosphate in control assays using
extracts from bacteria containing empty vector. Steam
distillation of A. annua leaves (6-week-oldplants) by
standard protocol (Gershenzon et al., 2000) yielded an
essential oil in which the presence of b-caryophyllene
(
30%) and a-humulene (2%) was confirmedby similar
analytical procedures; this observation is consistent with
a previous analysis (Tellez et al., 1999). Thus, a cDNA
encoding the b-caryophyllene synthase of A. annua had
been isolated. The cyclization mechanism of b-caryo-
phyllene synthase (Fig. 2) from Salvia officinalis has
been described previously (Dehal and Croteau, 1988).
Fig. 1. GC–MS analysis of the products generated from farnesyl di-
phosphate by the recombinant b-caryophyllene synthase. The spectra
illustratedcorrespondto
b-caryophyllene (a), a-humulene (b) and
humulene oxide (c); the latter is almost certainly formed by secondary
oxidation of a-humulene. The total ion chromatogram (d) is provided
to illustrate the relative amounts of b-caryophyllene (peak 1,
t t
R =15.38 min) and a-humulene (peak 2, R 15.81 min).

Y. Cai et al. / Phytochemistry 61 (2002) 523–529
525
The deduced sequence of b-caryophyllene synthase is
nearly identical (98.5%) to that of a terpene synthase
sequence (cASC34; accession no. AJ271793) of unde-
finedfunction previously reportedby Van Gel dr e et al.
efficient at a saturating concentration of 0.1 mM. The
Km value with farnesyl diphosphate as substrate was
ꢁ1
about 1.8 mM, with estimated kcat of 0.04 s . b-Car-
yophyllene synthase was not active with geranylgeranyl
diphosphate (C ) as substrate, andgeranyl diphosphate
(
2000), andabout 60% i de ntical to the epi-cedrol syn-
2
0
thase (Hua andMatsuda, 1999; Mercke et al., 1999) and
amorpha-4,11-diene synthase (Chang et al., 2000;
Mercke et al., 2000; Wallaart et al., 2001) of A. annua.
The sequence of b-caryophyllene synthase clearly
resembles (40–60% identity) those of other sesqui-
terpene synthases of angiosperm origin, with which it
shares a common reaction mechanism (Cane, 1999), and
is more distantly related to extant monoterpene syn-
thases and di terpene synthases (Bohlmann et al., 1998).
b-Caryophyllene synthase shares less than 30% deduced
sequence identity with the monoterpene synthase (3R)-
linalool synthase of A. annua (Jia et al., 1999).
The b-caryophyllene synthase bears the GVYXEP
element common to angiosperm sesquiterpene syn-
thases, andthe DDXXD di valent metal ion-substrate
binding motif that is highly conserved in terpene syn-
thases (Davis andCroteau, 2000). The sequence lacks
any N-terminal organelle targeting information, con-
sistent with the assumedcytosolic location of sesqui-
terpene biosynthesis, and is devoid of the diterpene
synthase insertion element; b-caryophyllene synthase
(C ) was an inefficient substrate (K >25 mM;
1
0
m
ꢁ
1
kcatꢀ0.004 s ) which afforded principally limonene
(42%), along with terpinolene (30%), b-pinene (16%),
g-terpinene (9%), a-terpinene (3%), andminor amounts
of linalool andgeraniol as pro ud cts. Surprisingly,
b-caryophyllene synthase readily accepted (+)-chrys-
anthemyl diphosphate as substrate (with kinetics similar
to that of farnesyl diphosphate) and afforded two irre-
gular monoterpenes, yomogi alcohol (63%) andarte-
misia alcohol (37%), along with solvolysis products.
Most of these monoterpenes have been previously
reportedin the essential oil of A. annua (Tellez et al.,
1999); however, because of the compartmentalization of
monoterpene andsesquiterpene biosynthesis (Bohlmann
et al., 1998), it seems unlikely that b-caryophyllene syn-
thase is the source of these monoterpenoidcompounds
in planta. In principal reaction parameters andgeneral
properties, b-caryophyllene synthase of A. annua
resembles b-caryophyllene synthase of Salvia officinalis
(Dehal andCroteau, 1988), andis typical of the general
class of sesquiterpene synthases from angiosperms
(Cane, 1999).
(
accession no. AF472361) falls within the Tpsa sub-
family of terpenoidsynthases (Bohlmann et al., 1998;
Trapp andCroteau, 2001).
2.3. Expression and induction patterns
2
.2. Properties of the recombinant enzyme
To determine the specificity of b-caryophyllene syn-
thase expression, total RNA was isolatedfrom several
tissues of 6-week-oldsee dl ings, as well as from inflor-
escences of mature plants, andwas analyzedby RT-
PCR. Synthase transcripts were observedin most tis-
sues, except roots (Fig. 3). In leaves, caryophyllene
transcripts were readily detectable, but expression levels
were seemingly much lower in petioles. Transcripts were
observedat a particularly high level in stem cortext and
epidermis, and were also detected in the stem stele
(Fig. 3). By contrast, message for the monoterpene syn-
thase, linalool synthase, couldnot be de tectedin the
stele, consistent with prior observation (Jia et al., 1999).
Comparedwith the abun da nce of b-caryophyllene syn-
thase transcripts in inflorescences, monoterpene syn-
thase transcript abundance was very low (data not
shown).
The recombinant b-caryophyllene synthase was par-
tially purifiedby anion-exchange chromatography and
gel permeation chromatography to remove competing
phosphatases (ꢀ40% pure by SDS–PAGE and de nsi-
tometry). Gel permeation chromatography on a cali-
bratedSepha de x S200 column in di cateda molecular
weight of about 60,000 which, along with the results of
SDS–PAGE, confirmedthe enzyme to be monomeric.
This synthase exhibiteda fairly broadpH optimum,
with maximum near pH 7.7, andrequireda di valent
metal ion for catalysis. The preferredmetal ion was
Mg2+ (saturation at 0.5 mM); Mn2+ was only half as
To assess temporal expression patterns, 4-, 6- and8-
week-oldsee dl ings were investigatedby similar means.
These results showedthat the level of b-caryophyllene
synthase transcripts in the respective tissues decreased
gradually with time (Fig. 4). In 8-week-old seedlings,
these transcripts were present at only low levels in stem
and were barely detected in leaves, indicating sub-
stantial diminution of expression with development. In
contrast to the present results, Van Geldre et al. (2000)
Fig. 2. Cyclization of farnesyl diphosphate to b-caryophyllene by
metal ion-dependent ionization with ring closure to the macrocyclic
humulyl carbocation (in brackets), followedby formation of the
cyclobutane ring and de protonation. a-Humulene is formedby di rect
deprotonation of the carbocationic intermediate.

526
Y. Cai et al. / Phytochemistry 61 (2002) 523–529
observedthat cASC34 (b-caryophyllene synthase) tran-
scripts were readily detectable in leaves late in develop-
ment. This minor difference in developmental
expression pattern may result from chemotypic varia-
tion in A. annua (AhmadandMisra, 1994).
to caryophyllene synthase, for which induction was
rapidbut transient, the transcriptional in du ction of the
monoterpene synthase linalool synthase was substantial
andof longer du ration (Fig. 5).
Wounding of basal leaves of 8 week-old plants, in
which the level of transcription of caryophyllene syn-
thase was low, resultedin observable gene activation.
During the 3-day interval following abrasive wounding,
caryophyllene transcripts accumulatedto a higher level
on the first day after wounding, then decreased sharply
on the secondandthird da y, yet to a level still some-
what higher than before wounding (Fig. 5). By contrast
Fig. 5. RT-PCR analysis of b-caryophyllene synthase transcripts and
linalool synthase transcripts in wounded basal leaves of 8-week-old A.
annua seedlings. (a) amplification with b-caryophyllene synthase-spe-
cific primers (750 bp); (b) amplification with linalool synthase-specific
primers (560 bp); (c) amplification with ubiquitin-specific primers (300
bp). For all panels, the lanes are: 1000, 750, 500 and250 bp markers
(1); RNA from leaves of non-wounded controls (2), and from woun-
ded leaves after 1 day (3), 2 days (4) and 3 days (5) post-treatment; the
plus sign denotes the positive control with b-caryophyllene synthase
(a) or linalool synthase (b) plasmidDNA; the minus sign de notes the
negative control without template.
Fig. 3. RT-PCR analysis of b-caryophyllene synthase transcripts in
tissues of A. annua. (a) amplification with b-caryophyllene synthase-
specific primers (750 bp); (b) amplification with ubiquitin-specific pri-
mers (300 bp) as internal standard. For both panels, the lanes are:
1
000, 750, 500 and250 bp markers (1); RNA from 6-week-oldseedling
basal leaves (2), petioles (3), whole stem (4), stem epidermis and cortex
5), stem stele (6), root (7), andinflorescences of mature plants (8); the
(
plus sign denotes the positive control with b-caryophyllene synthase
plasmidDNA; the minus sign de notes the negative control without
template.
Fig. 4. RT-PCR analysis of b-caryophyllene synthase transcripts in 4-, 6- and8-week-old A. annua seedlings. (a and b) amplification with b-car-
yophyllene synthase-specific primers (750 bp); (c and d) amplification with ubiquitin-specific primers (300 bp) as internal standard. Total RNA was
isolatedfrom basal leaves (a andc) andwhole stem (b and d) . For all panels, the lanes are: 1000, 750, 500 and250 bp markers (1); RNA from 4-
week-old (2), 6-week-old (3), and 8-week-old (4) seedlings; the plus sign denotes the positive control with b-caryophyllene synthase plasmidDNA;
the minus sign denotes the negative control without template.

Y. Cai et al. / Phytochemistry 61 (2002) 523–529
527
Extracts of the phytopathogenic fungus Verticillium
3. Experimental
dahliae often serve as non-specific elicitors of oxidative
burst andphytoalexin biosynthesis in plant cells (Davis
et al., 1992). When intact basal leaves of 8-week-old A.
annua seedlings were treated with such an elicitor pre-
paration, transcription levels of caryophyllene synthase
dramatically increased, with a maximum on the second
day post-elicitation; at the same time, the transcription
levels of linalool synthase apparently decreased (Fig. 6).
Thus, ‘fungal’ elicitation seemingly resultedin simulta-
neous induction of the sesquiterpene synthase and sup-
pression of transcription of the monoterpene synthase.
These preliminary results, indicating differential expression
of the monoterpene synthase andsesquiterpene synthase
genes after elicitation, in addition to their differential
induction patterns by wounding, suggest that A. annua
may rely on terpenoid-based defenses, and that indepen-
dent signaling pathways may operate in regulating the
induced biosynthesis of monoterpenes in response to insect
attack (wounding) and the induced biosynthesis of ses-
quiterpenes in response to fungal infection (elicitation).
3.1. cDNA isolation
A. annua was grown as described previously (Jia et al.,
1999) andplants from 4 to 8 weeks oldwere usedfor
RT-PCR. A PCR-basedstrategy was employedto iso-
late sesquiterpene synthase cDNA fragments using a
primer designed by comparing extant terpene synthases,
from which a peptide sequence GVPFEP was found to
occur only in angiosperm sesquiterpene synthases.
0
Thus, degenerate reverse primer tGVY [5 -
GG(TC)TC(AG)TA(AG)AA(AGCT)AC(AGCT)CC-3]
andvector-specific primer T3 (Stratagene) were usedto
amplify sequences from the previously described A.
annua l-ZAP cDNA library (Jia et al., 1999). The pro-
ducts were reamplified with previously described reverse
primer 9315 (Chen et al., 1995) andanother vector-spe-
0
cific primer TP1 [5 -CTGCAGGTTCGACACT-
0
AGTGG-3 ]. The resulting amplicon of about 900 bp
was subcloned and sequenced, and shown to encode a
DNA fragment bearing 45.5% identity to that of d-
cadinene synthase from Gossypium arboreum (Chen et
0
al., 1995). Specific forwardprimer P320 [5 -ATGGT-
0
GATGACTGGAAAGGC-3 ] andreverse primer M760
0 0
[
5 -TGGAAACTTCGCTAAGCTCC-3 ] were employed
to generate a 446 bp fragment usedas a labeledprobe to
screen the cDNA library as previously outlined(Jia et
al., 1999). Following five rounds of purification, the
clone designated QHS1 (AF472361) was obtained and
fully sequenced.
3.2. cDNA expression
Full length QHS1 was amplifiedby PCR using Pfu
DNA polymerase (Promega) with vector-specific reverse
0
primer T7 (Stratagene) andforwardprimer QHSB1 [5 -
0
CGGATCCCATGTCTGTTAAAG-3 ] designed to
0
introduce a 5 -BamHI site. Following digestion with
BamHI and XhoI, the sequence verifiedDNA was liga-
ted into similarly digested pET28b (yielding pETQHS1)
andtransformedinto E. coli BL21(DE3). Cells harbor-
ing pETQHS1 or the empty vector as control were cul-
tured in LB medium as described previously (Jia et al.,
1999; Mercke et al., 1999) except that kanamycin was
usedfor selection. These bacterial cultures were rea di ly
scaledfrom 50 to 500 ml.
Fig. 6. RT-PCR analysis of b-caryophyllene synthase transcripts and
linalool synthase transcripts in elicitor-treatedbasal leaves of 8 week-
old A. annua seedlings. (a) amplification with ꢀ-caryophyllene syn-
thase-specific primers (750 bp); (b) amplification with linalool syn-
thase-specific primers (560 bp); (c) amplification with ubiquitin-specific
primers (300 bp). For all panels, the lanes are: 1000, 750, 500 and250
bp markers (1); RNA from leaves of non-elicitedcontrols (2), sterile
water-treatedcontrols (3), andfrom elicitor-treatedleaves after 1 da y
3.3. Enzyme isolation and characterization
Enzyme isolation procedures and methods for partial
purification by anion-exchange chromatography have
been described (Mercke et al., 1999). Each preparation
was monitoredby assay andby SDS–PAGE. The stan-
dard assay for sesquiterpene synthases was employed
(Mercke et al., 1999) in which the conversion of
(
4), 2 days (5) and 3 days (6) post-treatment; the plus sign denotes the
positive control with b-caryophyllene synthase (a) or linalool synthase
b) plasmidDNA; the minus sign denotes the negative control without
template.
(

528
Y. Cai et al. / Phytochemistry 61 (2002) 523–529
3
[
7
1- H]farnesyl diphosphate (10 mM, 125 Ci/mol), at pH
.0 in the presence of 10 mM MgCl and20 mM MnCl ,
to sesquiterpene olefins andalcohols is de terminedby
PCR) were repeatedthree times (three individual plants)
with similar results.
2
2
pentane extraction andsilica gel separation, with scin-
tillation counting of the product classes. Scale-up of the
preparation allowedgeneration of sufficient pro du ct for
identification by established GC–MS procedures (Crock
et al., 1997; Steele et al., 1998). Establishedprotocols
for determination of size, divalent metal ion require-
ment, pH optimum, kinetic constants, andsubstrate
selectivity have also been described elsewhere (Crock et
al., 1997; Mercke et al., 1999). Authentic b-caryophyllene
Acknowledgements
This work was supportedby the National Natural
Science Foundation of China (Grants 3992505 and
30030020), the Shanghai Science andTechnology
Development Foundation (Grant 003912077) and
by a grant from the US National Institutes of Health
(GM-31354).
(
97%) was from Fluka.
3
.4. RNA analysis
References
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0
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stele) androots from 6-week-oldsee ldi ngs, andthe
inflorescences from mature plants, were analyzed.
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parallel for comparison of the temporal expression pat-
terns in basal leaves andstem. Eight-week-oldsee dl ings
were wounded by a described protocol (Lewinsohn et
al., 1992). The elicitor preparation was obtainedfrom
the phytopathogenic fungus V. dahliae as previously
described (Heinstein, 1985; Liu et al., 1999). This pre-
paration, at a final con1centration of 1mg sucrose
equivalent/ml in sterilized dd water, was usedto paint
unwounded leaves with a pen brush. Total RNA was
isolatedfrom woun de dandelicitor-treated(unwoun-
ded) leaves of individual plants over a 3-day time
course, andRT–PCR was carriedout as before. All
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(
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