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Plant Molecular Biology 51: 119–133, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands. 119
Traumatic resin defense in Norway spruce (Picea abies): Methyl
jasmonate-induced terpene synthase gene expression, and cDNA cloning
and functional characterization of (+)-3-carene synthase
Jenny Fäldt, Diane Martin, Barbara Miller, Suman Rawat and Jörg Bohlmann∗
Biotechnology Laboratory, Department of Botany and Department of Forest Sciences, University of British
Columbia, 6174 University Boulevard, Vancouver V6T 1Z3, B.C. Canada (∗author for correspondence; e-mail:
bohlmann@interchange.ubc.ca)
Received 20 January 2002; accepted in revised form 21 May 2002
Key words: 3-carene, chemical plant defense, conifer biotechnology, methyl jasmonate, monoterpene synthase,
Picea abies (Norway spruce)
Abstract
Picea abies (L.) Karst. (Norway spruce) employs constitutive and induced resin terpenoids as major chemical
and physical defense-shields against insects and pathogens. In recent work, we showed that a suite of terpenoids,
monoterpenoids and diterpenoids was induced in stems of Norway spruce after treatment of trees with methyl
jasmonate (MeJA) (Martin et al., 2002). Increase of enzyme activities of terpenoid biosynthesis and accumulation
of terpenoids was associated with MeJA-induced de novo differentiation of xylem resin ducts. The formation of
defense-related traumatic resin ducts was also found in Norway spruce after attack by stem boring insects or after
infestation with fungal pathogens. In the present study, we analyzed the traumatic resin response in Norway spruce
further at the molecular genetic level. Treatment of trees with MeJA induced transient transcript accumulation of
monoterpenoid synthases and diterpenoid synthases in stem tissues of Norway spruce. In screening for defense-
related terpenoid synthase (TPS) genes from Norway spruce, a full-length monoterpenoid synthase cDNA, PaJF 6 7 ,
was isolated and the recombinant enzyme expressed in E. coli and functionally characterized in vitro. The cloned
PaJF67 cDNA represents a new monoterpenoid synthase gene and the gene product was identified as 3-carene
synthase. The enzyme encoded by Pa J F 6 7 forms stereospecifically (+)-3-carene (78% of total product) together
with minor acyclic and cyclic monoterpenes, including the mechanistically closely related terpinolene (11% of total
product). (+)-3-Carene is a characteristic monoterpene of constitutive and induced oleoresin defense of Norway
spruce and other members of the Pinaceae.
Abbreviations: 2D-GC, two-dimensional gas chromatography; GC, gas chromatography; GPP, geranyl
diphospahte; GGPP, geranylgeranyl diphospahte; FPP, farnesyl diphosphate; LPP, linalyl diphosphate; MeJA,
methyl jasmonate; MS, mass spectroscopy; TPS, terpenoid synthase.
Introduction
Conifer resins are composed of a large suite of struc-
turally diverse terpenoid natural products that have
long been recognized for their many functions in the
chemical ecology of conifers and coniferophagus in-
sects (Berryman, 1972; Langenheim, 1994; Bohlmann
and Croteau, 1999; Seybold et al., 2000; Trapp
and Croteau, 2001), as well as for their industrial
value (Dawson, 1994; Schrader and Berger, 2001).
The oleoresin of Norway spruce (Picea abies) con-
sists mainly of monoterpenoids (10-carbon), sesquiter-
penoids (15-carbon), and diterpenoids (20-carbon).
Monoterpenoids and diterpenoids are equally abun-
dant and together account for more than 98% of
resin terpenoids in stems of Norway spruce (Martin
et al., 2002). The turpentine fraction of Norway spruce
resin includes more than 20 different monoterpenoids,
120
several of which exist as pairs of their respective
stereoisomers (Borg-Karlson et al., 1993; Persson
et al., 1993, 1996) (Figure 1).
Recent chemical, biochemical and histological
studies demonstrated that accumulation of monoter-
penoids and diterpenoids in sapling stems of Norway
spruce is elicited by treatment of trees with methyl
jasmonate (MeJA) (Martin et al., 2002). Induced ter-
penoid accumulation is the result of a xylem-specific
increase in enzyme activities of terpenoid biosynthe-
sis, namely prenyl transferases and terpenoid syn-
thases. These chemical and biochemical responses are
associated with defense-related de novo formation of
traumatic resin ducts in the cambium zone and in the
differentiating xylem. The MeJA-induced oleoresin
response described in Norway spruce is similar and
possibly identical to the defense response induced by
stem boring insects and microbial pathogens in sev-
eral species of spruce, including Norway spruce, Sitka
spruce (Picea sitchensis) and white spruce (Picea
glauca) (Alfaro, 1995; Alfaro et al., 2002; Nagy et al.,
2000; Tomlin et al., 1998, 2000).
Induced terpenoid defense in Norway spruce
xylem includes accumulation of 3-carene, a bicyclic
monoterpene hydrocarbon with an unusual cyclo-
propyl function (Figure 1). 3-Carene is a common
component of many conifer turpentines, however, pro-
portions of 3-carene relative to other resin monoter-
penes and absolute amounts of 3-carene can vary
drastically between different conifer species and be-
tween chemotypes of the same species (Hiltunen et al.,
1975; Persson et al., 1996; Plomion et al., 1996;
Sjödin et al., 1996, 2000). In some conifer-pest in-
teractions, quantitative variation of 3-carene has been
associated with resistance or susceptibility of trees.
For example, in lodgepole pine (Pinus contorta)re-
sistance to Douglas-fir pitch moth (Synanthedon no-
varoensis) correlates with high amounts of 3-carene
(Rocchini et al., 2000). In clones of Scots pine (Pinus
sylvestris) high levels of 3-carene were found to corre-
late with low larval survival of the sawfly Diprion pini
(Pasquier-Barre et al., 2001). As the major monoter-
pene induced in roots of Scots pine upon infection
with the ectomycorrhizal fungus, Boletus variegates,
3-carene was suggested to be involved in development
of the root-mycorrhizae symbiosis and in disease re-
sistance of the mycorrhizal system (Krupa and Fries,
1971). Infection of wound sites of lodgepole pine
stems with the fungus Ceratocystis clavigera resulted
in an increase of 3-carene (Croteau et al., 1987), a pat-
tern that was also found in Scots pine phloem infected
by Leptographium wingfieldii (Fäldt et al., 2002).
Terpene synthase (TPS) enzyme activities respon-
sible for the formation of 3-carene have been charac-
terized in vitro in stems of Douglas fir (Pseudotsuga
menziesii) and in partially purified stem extracts of
lodgepole pine (Savage and Croteau, 1993). These
studies established both the regio- and stereochem-
istry of the formation of (+)-3-carene. 3-Carene is
formed from geranyl diphosphate (GPP), the com-
mon precursor of all monoterpenes (Wise and Croteau,
1998), by monoterpene synthase activity via a cou-
pled isomerization-cyclization mechanism that pro-
ceeds through linalyl diphosphate (LPP) and the α-
terpinyl cation (Savage and Croteau, 1993; Wise and
Croteau, 1998). The cyclopropyl function of 3-carene
is formed by an anti-1,3-elimination of a C-5 proton
in the α-terpinyl cation. Induced monoterpene syn-
thase activities that catalyze the cyclization of GPP
to 3-carene have been described for grand fir (Abies
grandis) (Gijzen et al., 1991; Katoh and Croteau,
1998) and for Norway spruce (P. abies) (Martin et al.,
2002).
In a combined molecular genetic and biochem-
ical approach, a group of constitutive and induced
monoterpene synthases was cloned as cDNAs from
grand fir and the recombinant enzymes were func-
tionally characterized (Bohlmann et al., 1997, 1999).
Although the grand fir TPS cDNAs account for many
of the monoterpenes characteristic of conifer oleo-
resin, a TPS gene or cDNA yielding 3-carene eluded
isolation. Genetic analysis of quantitative variation of
3-carene allowed mapping of a quantitative trait locus
(QTL) for this monoterpene in maritime pine (Pinus
pinaster)(Plomionet al., 1996), but also did not yet
result in the isolation of a gene that controls formation
of 3-carene.
This paper is part of a program that studies the
molecular genetics and biochemistry that underlie
the complex processes of traumatic resinosis in Nor-
way spruce. In the present study, we used molecular
probes for TPS gene expression analysis of the MeJA-
induced traumatic resin response and for full-length
TPS cDNA isolation. Functional characterization of a
new monoterpene synthase cDNA revealed for the first
time a gene for the formation of (+)-3-carene.
121
Figure 1. Characteristic monoterpenes of the turpentine fraction of Norway spruce oleoresin and their enantiomers. The 10-carbon monoter-
penes occur as acyclic, monocyclic, bicyclic or tricyclic structures. Several monoterpenes, such as α-pinene and β-pinene, can exist as pairs of
two enantiomers in the turpentine of conifers. Other monoterpenes are found only in the form of one of two possible enantiomers. For instance
only the (+)-enantiomer of 3-carene was found in Norway spruce.
Materials and methods
Plant materials
Norway spruce (Picea abies (L.) Karst.) trees of
clonal line 244-932 were from the Niedersächsis-
che Forstliche Versuchsanstalt, Escherode, Germany,
grown as described previously by Martin et al. (2002).
Substrates, Standards and Reagents
[1-3H]Geranyl diphosphate (GPP) (150 Ci/mol), [1-
3H]farnesyl diphosphate (FPP) (125 Ci/mol) and [1-
3H]geranylgeranyl diphosphate (GGPP) (120 Ci/mol)
were gifts of Dr Rodney Croteau, Washington State
University, Pullman WA, USA. Non-labeled GPP
(1 mg/mL) was from Echelon Research Laboratories
Inc. (Nevada, USA). Terpenoid standards, other chem-
icals and reagents were purchased from Sigma Chem-
ical Co., Fluka Chemical Co or Aldrich Chemical Co.,
unless otherwise noted.
Treatment of trees with methyl jasmonate (MeJA)
Two-year old trees were treated with MeJA as de-
scribed during the period of active growth approxi-
mately six weeks after bud burst (Martin et al., 2002).
Individual trees were sprayed with a solution of 0.01%
(v/v) or 415 µM methyl jasmonate (95% pure) dis-
solved in 0.1% (v/v) Tween 20 (Fisher Scientific).
Control trees were sprayed with 0.1% (v/v) Tween
20. Trees were placed in a ventilated fume hood and
each tree was sprayed with 150 mL of MeJA solu-
tion over a period of 30 min to obtain a complete
and even coating. Saplings were kept in fume hoods
for 1 h after treatment to allow evaporation of excess
MeJA solution prior to transferring to growth cham-
bers, where the photoperiod and ambient temperature
cycled from 1 h at 220 µmol/m−2s−1(20 ◦C),4hat
440 µmol m−2s−1(20 ◦C),3hat660µmol m−2s−1
[22 ◦C(2h),24◦C (1 h)], 7 h at 440 µmol m−2s−1
[24 ◦C(1h),22◦C(2h),20◦C(4h)],and1hat
220 µmol m−2s−1(18 ◦C). This was followed by
122
8 h of darkness (18 ◦C). The relative humidity was
maintained at 50% throughout the entire cycle. Trees
were harvested two, four and 16 days after spray-
ing by removing all branches and needles from stems
and cutting stem sections of second year growth into
10 cm fragments. All tissues were immediately frozen
in liquid nitrogen and stored at −80 ◦C.
cDNA library construction
Young developing shoots and foliage of Norway
spruce were harvested within four weeks of bud burst
and RNA was extracted according to a procedure of
Lewinsohn et al. (1994) with the following modifica-
tions. Triton X-100 (1% (v/v)), sodium deoxycholate
(1% (w/v)), and sodium N-lauryl sarcosine (1.5%
(w/v)) were added to the mRNA extraction buffer.
Tissues were extracted in a ratio of 1 g per 5 mL
extraction buffer. Poly-A mRNA was purified using
Streptavidin MagneSphereParamagnetic Particles
(Promega, Madison WI, USA). A cDNA library was
constructed using the λ-ZAPII system (Stratagene, La
Jolla CA, USA) and recombinant phage DNA pack-
aged using the Gigapack Gold system (Stratagene).
Probe isolation
A probe for monoterpene synthase screening and
northern analysis was generated by two rounds of
PCR from the Norway spruce cDNA library us-
ing two pairs of nested primers derived from align-
ments of grand fir monoterpene synthases (Bohlmann
et al., 1997, 1999) and partial TPS-like expressed se-
quence tags (ESTs) of other conifers identified in the
NCBI dbest database (http://www.ncbi.nlm.nih.gov).
PCRs were performed each in a total volume of
50 µL containing 20 mM Tris/HCl (pH 8.4), 50 mM
KCl, 5 mM MgCl2, 200 µM of each dNTP,
0.1 µM each of a forward primer and a reverse
primer (Operon technologies, Inc., Alameda CA,
USA), 1 U of Ta q polymerase (Gibco BRL, Burling-
ton, Canada) and 5 µL of template. As tem-
plate in the primary PCR, 5 µLoftheNorway
spruce cDNA library (5 ×108pfu/mL) was used
in combination with the primary forward primer (5-
GCGCTGGATTACGTGTACAGTTATTGG-3)and
the primary reverse primer (5-CCGGTTTCTTTC
CACCATCTGGAG-3). Five µL of the completed
primary PCR served as template in a secondary am-
plification with the secondary forward primer (5-
GTGGGAGAGATAGTGTTGTTGCTG-3)andthe
secondary reverse primer (5-CTGTTACAAGGAGTG
AAAGATATTGAACTC-3). The temperature pro-
gram for primary and secondary PCR was: denaturing
at 95 ◦C for 2 min., 35 cycles: 30 s at 95 ◦C, 30 s
at 45 ◦C (for the primary reaction) or at 60 ◦C (for
the secondary PCR reaction), 1 min at 72 ◦C. PCR re-
actions were analyzed by agarose gel electrophoresis.
The amplicon of the secondary reaction was ligated
into pCR2.1-TOPO vector (Invitrogen, Burlington,
Canada), and transformed into E. coli TOP10F’ cells
(Invitrogen). Plasmid DNA was prepared from indi-
vidual transformants and the inserts were sequenced
(BigDye Terminator Cycle Sequencing kit, Perkin
Elmer Applied Biosystems, Streetsville, Canada). A
481-bp TPS-like insert sequence was identified and
was designated as probe PaTPS13.
Northern analysis
RNA isolation was performed following the proce-
dure of Wang et al. (2000). Samples of 10 µg
of total RNA were separated under denaturing con-
ditions using formaldehyde in a 1.2% agarose gel
at 4–5 V/cm for 5–6 h according to the protocol
described in Sambrook and Russel (2001). Ribo-
somal RNA was visualized (ChemiImagerTM 5500
with AlphaEaseFCTM software, Alpha Innotech Cor-
poration, San Leandro CA, USA) and the RNA
was transferred overnight by capillary action to pos-
itively charged nitrocellulose (Hybond-N+,Amer-
sham Pharmacia Biotech,) using standard protocols
(Sambrook and Russel, 2001). Gene fragments for
hybridization probes were generated by PCR using
1UTaq DNA-polymerase (Gibco BRL), 1×PCR
buffer (Gibco BRL), 4.0 mM MgCl2,0.2mMof
each dNTP, 50 pmol of each primer (Operon tech-
nologies, Inc.), and 1 ng plasmid-DNA in a volume
of 50 µL. Primers for amplification of monoterpene
synthase probe PaTPS13 were as described above. A
diterpene synthase probe was generated fromgrand fir
abietadiene synthase (Stofer Vogel et al., 1996) using
primers 5-GCCATGCCTTCCTCTTCATTG-3and
5-AGGCAACTGGTTGGAAGAGGC-3.Thetem-
perature program for PCR was: 36 cycles: 30 s at
94 ◦C, 1 min. at 55–65 ◦C, 1.5 min at 72 ◦C. The
PCR products were purified using QIAquick system
(Qiagen). DNA labeling of purified PCR products was
performed following instruction of the Strip-EZTM
DNA Kit (Ambion Inc., Austin, USA) using (α-
32P)dATP (3000 Ci/mmol, 10 mCi/mL, Perkin Elmer).
Unincorporated label was removed on a gel filtra-
tion column (MicrospinTM S-300, Amersham Phar-
123
macia Biotech). After one hour of prehybridization
at 65 ◦C in hybridization buffer (0.05 M Na4P2O7
×10 H2O, 0.115 M NaH2PO4,7%SDS,1mM
EDTA, 100 µg/mL salmon sperm DNA) blotted mem-
branes were incubated in hybridization buffer with
heat denatured terpene synthase probes for 16 h at
65 ◦C. Membranes were rinsed in 10 ml prewarmed
wash buffer (0.05 M Na4P2O7×10 H2O, 0.115 M
NaH2PO4, 1% SDS, 1 mM EDTA) and washed twice
in 40 mL wash bufferat 65 ◦C and once in 40 mL wash
buffer at 68 ◦C. To detect the hybridization signals the
membranes were exposed to autoradiography (Kodak
BioMax MS film, Kodak BioMax intensifyingscreen)
for 2–20 h at −80 ◦C.
Library screening
50 ng of probe PaTPS13 was purified by agarose
gel electrophoresis and gel extraction (Qiagen gel
extraction kit, Mississauga, Canada), labelled with
[α-32P]dCTP (Easytide, Perkin Elmer) using the
Readiprime II random prime labelling system (Amer-
sham Pharmacia Biotech Inc., Piscataway, USA), and
used to screen replica filters of 3 ×105plaques of the
Norway spruce cDNA library plated on E. coli XL1
Blue MRF’. Hybridization was performed for 18 h
at 55 ◦Cin3×SSPE and 0.1% SDS. Filters were
washed three times for 10 min. each at 55 ◦Cin3×
SSPE with 0.1% SDS and exposed for 17 h to Kodak
BioMax-MS film at −80 ◦C. Of 274 clones yielding
positive signals, all of which were purified through a
second round of hybridization, 72 pBluescript SK(−)
phagemids were excised in vivo in E. coli XL1 Blue
MRF’ and transformed into E. coli SOLR. Plasmid
DNA was prepared from individual transformants ac-
cording to standard protocols (Sambrook and Russel,
2001).
DNA sequencing and sequence analysis
Inserts of all recombinant plasmids were com-
pletely sequenced on both strands via primer walk-
ing using the cycle sequencing dideoxy chain ter-
mination reaction with BigDye terminators (Perkin
Elmer Applied Biosystems). Sequence analysis was
done using programs from the Lasergene DNA-Star
Package version 5, ClustalX (http://www-igbmc.u-
strasbg.fr/BioInfo/ClustalX/Top.htm) and Genedoc
(http://www.psc.edu/biomed/genedoc). Sequence re-
latedness was analyzed with ClustalX using the Neigh-
bor Joining method and an unrooted tree was visual-
ized using Treedraw (http://taxonomy.zoology.gla.
ac.uk/rod/treeview.html).
cDNA expression in E. coli and enzyme assays
The full-length Pa J F 6 7 cDNA insert of plas-
mid pBluescript-PaJ F 6 7 was subcloned into the
pET100/D-TOPO expression vector (Invitrogen) fol-
lowing the manufacturer’s instructions. The Pa J F 6 7
insert was amplified by PCR using forward primer (5-
CACCATGTCTGTTATTTCCATTTTGCCG-3)in
combination with reverse primer (5-CTTACATAGG
CACAGGTTCAAGAAC-3). PCRs were performed
in volumes of 100 µL containing 20 mM Tris/HCl
(pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4,2mM
MgSO4, 0.1% Triton X-100, 10 µg BSA, 200 µM
of each dNTP, 0.1 µM of each primer, 2.5 U of high
fidelity Turbo Pfu polymerase (Stratagene) and 2 ng
plasmid pBluescript-PaJ F 6 7 . After a denaturing step
at 95 ◦C for 2 min, 30 cycles of amplification were
performed employing the following temperature pro-
gram in a MJ PTC100-thermocycler: 30 s at 95 ◦C,
30 s at 59 ◦C, 2 min at 72 ◦C, followed by 10 min
final extension at 72 ◦C. The amplified segment was
cloned into pET100/D-TOPO vector and recombinant
plasmids were transformed into E. coli TOP10 Fcells
according to the protocol for ligation and transfor-
mation (Invitrogen). The plasmid pET-PaJF6 7 was
purified and transformed for expression into E. coli
BL21-CodonPlus(DE3) (Stratagene). Positive clones
were analyzed by PCR using insert and/or vector
based primers.
For functional expression, bacterial strain E. coli
BL21-CodonPlus(DE3)/pET-Pa J F 6 7 was grown to
A600 =0.5at37◦Cin5mlofLBmediumsup-
plemented with 20 µg/mL ampicillin. Cultures were
then induced by addition of isopropyl-1-thio-β-D-
galactopyranoside to a final concentration of 1 mM
and grown for another 12 h at 20 ◦C. Cells were
harvested by centrifugation, resuspended in 1 mL
monoterpene synthase buffer (25 mM HEPES, pH 7.2,
100 mM KCl, 10 mM MnCl2, 10% glycerol, 5 mM
DTT) and disrupted by sonication using a Branson
Sonifier 250 (Branson Ultrasonic Corporation, Dan-
bury CT, USA) at constant power (5W) for 10 s.
Lysates were cleared by centrifugation and the result-
ing supernatants containing the soluble enzyme were
assayed for monoterpene, sesquiterpene and diterpene
synthase activity using the appropriate radio-labeled
substrates as described previously (Bohlmann et al.,
1997, 1999). In the case of the monoterpene synthase
124
and sesquiterpene synthase assay, the assay mixture
(1 mL) was overlaid with 1 mL of pentane to trap
volatile products. In all cases, after incubation at
30 ◦C for 1 h, the reaction mixture was extracted with
pentane (3 ×1 mL) and the combined extract was
passed through a 400 µL column of equal amounts
of anhydrous MgSO4and silica gel (60 Å) to obtain
the terpenoid hydrocarbon fraction free of oxygenated
metabolites. To collect possible oxygenated products
and to control for the possibility the products were a
result of rearrangements (caused by the presence of
silica gel), an independent set of assay mixtures were
extracted with hexane (3 ×1 mL). The hexane frac-
tions were combined, extracted with water and the
hexane fraction was dried with anhydrous MgSO4.
Aliquots of each fraction were taken for liquid scin-
tillation counting to determine activity. The counter
efficiency was estimated to be 57% by using [1-
3H]toluene as standard (2.24 ×106dpm/mL, ARC, St.
Louis, USA). To obtain product for analysis by GC-
FID and GC-MS, we used unlabeled GPP at a final
concentration of 137 µM. Controls for product for-
mation independent of Pa J F 6 7 cDNA were performed
using extracts of E. coli BL21-CodonPlus(DE3) trans-
formed with pET100/D-TOPO plasmid without the
PaJF67 insert.
Monoterpene product identification by gas
chromatography (GC) and mass spectrometry (MS)
Monoterpenes were identified and quantified by GC-
MS analysis on an Agilent 6890 Series GC System
coupled to an Agilent 5973 Network Mass Selective
Detector (70 eV) using a DB-WAX (J&W Scientific,
Palo Alto CA, USA) capillary column (0.25 mm i.d.
×30 m with 25-µm film). A Cyclodex B (perme-
thylated β-cyclodextrin in DB-1701, J&W Scientific)
capillary column (0.25 mm i.d. ×30 m with 25-µm
film) was used for chiral separation of monoterpene
products. An injector (6 sec. splitless) was used at
200 ◦C and a column flow of 1 mL He min−1.Using
the DB-WAX column the following temperature pro-
gram was used: initial temperature of 40 ◦C(4min
hold) which was then increased to 150 ◦Cat4◦C
min−1followed by a 20 ◦Cmin
−1ramp until 230 ◦C
(5 min hold). For chiral separations (Cyclodex B) of
α-pinene, sabinene, limonene, β-phellandrene, the
following temperature program was used: initial tem-
perature was 55 ◦C (1 min hold) which was then
increased to 100 ◦Cat1◦Cmin
−1followed by a
10 ◦Cmin
−1ramp until 230 ◦C (10 min hold). For
Figure 2. MeJA-induced monoterpene synthase and diterpene syn-
thase transcript accumulation in stems of Norway spruce. Total
RNA was extracted from stems of control trees and from trees
treated with 0.01% (v/v) MeJA (415 µM). Northern blots were
hybridized with class-specific monoterpene synthase and diterpene
synthase probes, PaTPS13 (this study) and grand fir abietadiene
synthase (Stofer Vogel et al., 1996). Conifer monoterpene synthases
and diterpene synthases are less than 30–40% identical at the nu-
cleic acid level. No cross hybridization was found between the two
probes under the conditions of our experiments. Equal loading of
total RNA in each lane was monitored by ethidium bromide staining
of ribosomal RNA (not shown) and hybridization to ribosomal RNA
probe.
assignment of the stereochemistry of 3-carene, the en-
zyme product was co-injected with pure (+)-3-carene
(Sigma-Aldrich) or with a (−)-3-carene standard pre-
pared from black pepper (Anna-Karin Borg-Karlson,
personal communication). Enantiomers of 3-carene
were separated (baseline separation) on a Lipodex E
column (dipentylbuturyl-γ-cyclodextrin, 0.25 mm i.d.
×25 m) (Macherey Nagel, Switzerland) using a two-
dimensional GC (2D-GC) system coupled to a flame
ionization detector (FID) (Borg-Karlson et al., 1993;
Sjödin et al., 2000) with the following temperature
program: first GC (DB-WAX): initial temperature of
40 ◦C (1 min hold) which was then increased to 180 ◦C
at 6 ◦Cmin
−1followed by a 15 ◦Cmin
−1ramp to
230 ◦C (5 min hold); second GC (Lipodex E): 30 ◦C
isothermal for 50 min. Compounds were identified by
comparing mass spectra using Agilent Technologies
software and Wiley126 MS-library, as well as by com-
paring retention times and mass spectra with those of
authentic standards. Stereochemistry of products was
assigned based on matching of retention times with
enantiomerically pure standards.
125
Results and discussion
MeJA-induced TPS gene expression in Norway spruce
In recent work, Martin et al. (2002) demonstrated
that treatment of Norway spruce saplings with MeJA
induced a multi-layered resinosis response in stem tis-
sues. The MeJA-induced response involves a series
of histological, biochemical and chemical changes in
the developing xylem that include (i) de novo dif-
ferentiation of traumatic resin duct cells and resin
canals, (ii) induced activities of prenyl transferases
(geranylgeranyl diphosphate synthase) and terpene
synthases (monoterpene synthases and diterpene syn-
thases), and (iii) strongly increased accumulation of
monoterpenoids and diterpenoids in the newly formed
resin ducts. This MeJA-induced response of Norway
spruce saplings closely resembles the traumatic resin
response evoked by stem boring insects and microbial
pathogens observed in several species of spruce, such
as Norway spruce, Sitka spruce and white spruce (Al-
faro, 1995; Alfaro et al., 2002; Tomlin et al., 1998,
2000; Nagy et al., 2000). To further characterize the
resinosis response of spruce stem tissues at the level of
transcript accumulation, northern hybridizations were
performed for transcripts of monoterpene synthases
and diterpene synthases. A 481-bp monoterpene syn-
thase cDNA fragment, PaTPS13 (GenBank acces-
sion AF461459) was isolated from Norway spruce
and used to evaluate monoterpene synthase gene ex-
pression under constitutive and induced conditions.
Monoterpene synthases exist in species of conifers as
small gene families of members that are from 65 to
99% identical at the nucleotide level (Bohlmann et al.,
1998b, 1999). Therefore, we used probe PaTPS13
at hybridization conditions that could reveal possible
expression of all membersof such a monoterpene syn-
thase family in Norway spruce. PaTPS13 is in the
range of approximately 65–80% identical with pre-
viously characterized conifer monoterpene synthase
of grand fir. Since it is known that grand fir cDNA
abietadiene synthase, a conifer diterpene synthase
(Stofer Vogel et al., 1996), does not cross-hybridize
with monoterpene synthase transcripts (Steele et al.,
1998b), this gene was employed as a heterologous
probe to measure diterpene synthase transcripts in
Norway spruce. Northern analysis revealed strong ac-
cumulation of monoterpene synthase transcripts in
stems of Norway spruce within two days after treat-
ment with MeJA applied at a concentration of 0.01%
(v/v) (415 µM) as a surface spray. Lower levels
Figure 3. Constitutive monoterpene synthase transcript accumula-
tion in foliage and stems of Norway spruce. RNA was extracted
from combined bark and xylem of one-year old stems (lane 1),
from young shoots and foliage four to six weeks after bud burst
(lane 2), or from one-year old foliage (lane 3). Northern blots were
hybridized with monoterpene synthase probe PaTPS13. Equal load-
ing of total RNA in each lane was monitored by ethidium bromide
staining of ribosomal RNA (not shown). Constitutive accumulation
of monoterpene synthase transcripts was low in one-year old stems
and foliage of Norway spruce, but was higher in young developing
needles and shoots within four to six weeks of bud burst.
of monoterpene transcripts were detected four days
and 16 days after treatment (Figure 2). The time
course of rapid monoterpene transcript accumulation
clearly precedes MeJA-induced increase in monoter-
pene synthase enzyme activities (maximum ten to
15 days after treatment) and induced accumulation
of monoterpenoids in Norway spruce (maximum be-
tween days 15 to 20 after treatment) (Martin et al.,
2002). Although weaker signals were found for diter-
pene synthase transcripts (Figure 2), possibly due to
the heterologous origin of the hybridization probe,
similar time courses are followed for monoterpene
synthase and diterpene synthase transcripts in stems
in response to MeJA treatment. This finding is also
consistent with time courses of induced diterpene syn-
thase enzyme activities and diterpenoid accumulation
in MeJA treated stem tissues (Martin et al., 2002).
Similar overall induction of transcript accumulation
for monoterpene synthases and diterpene synthases
was previously found in grand fir stems as the result of
mechanical wounding (Steele et al., 1998b). However,
tissue damage associated with mechanical wounding
makes it difficult to study the molecular and biochem-
ical processes of induced de novo differentiation of
traumatic resin ducts. Application of MeJA provides
a useful, non-destructive method for studies of gene
expression of induced defense in spruce stems.
Isolation of full-length monoterpene synthase cDNA
PaJF67
Although several monoterpene synthase genes were
previously isolated from grand fir (Bohlmann et al.,
1997, 1999), additional monoterpene synthase genes
of unique biochemical functions must exist for the for-
mation of some typical conifer resin components such
126
Figure 4. Amino acid sequence alignment of Norway spruce PaJF67 predicted protein and grand fir monoterpene synthases. Sequences are
Norway spruce PaJF67 (AF461460, this study) [1], and the grand fir monoterpene synthases myrcene synthase (U87908) [2], (−)-pinene
synthase (U87909) [3], (−)-camphene synthase (U87910) [4], β-phellandrene synthase (AF139205) [5], terpinolene synthase (AF139206)
[6], (−)-limonene synthase (AF006193) [7], and (−)-limonene/(−)-α-pinene synthase (AF139207) [8]. Residues shaded in black are highly
conserved (100% similarity) in all sequences of the comparison. Other residues shaded in grey are also conserved although at lower level (above
80% similarity (dark grey) or above 60% similarity (light grey)). Conserved motifs RRx8W and DDxxD of known function in TPS reaction
mechanism are indicated (Bohlmann et al., 1998a). The alignment was created using ClustalX and visualized using GeneDoc software.
127
as 3-carene. Because Norway spruce possesses high
constitutive monoterpene synthase enzyme activity
(Fischbach et al., 2000) and high constitutive monoter-
pene synthase gene expression in young foliage and
developingshoots (Figure 3), these tissues are an ideal
source for cDNA isolation of new monoterpene syn-
thases. Partial cDNA PaTPS13 clone was employed
as a probe for screening for monoterpene synthases
in Norway spruce. This effort resulted in the isolation
of a new full-length cDNA clone, PaJ F 6 7 (GenBank
accession AF461460), which encodes for a deduced
protein of 627 amino acids with a predicted molecular
weight of 71,912 Da and predicted pI of 6.17 (Fig-
ure 4). The deduced amino acid sequence of cDNA
clone PaJF67 resembles typical monoterpene syn-
thases (Bohlmann et al., 1998b) and most closely
grand fir monoterpene synthases of the gymnosperm
TPSd group (Bohlmann et al., 1997, 1999) (Figure 4).
Multiple sequence alignment of the predicted PaJF67
protein with grand fir monoterpene synthases showed
that these sequences are, in many regions, highly
conserved (Figure 4). PaJF67 protein shares from
63% identity (78% similarity) with grand fir (−)-4S-
limonene synthase to 66% identity (80% similarity)
with grand fir terpinolene synthase (Bohlmann et al.,
1997, 1999), but shares less identity with angiosperm
monoterpene synthases such as mint limonene syn-
thase (25% identity, 44% similarity) (Colby et al.,
1993) or Arabidopsis thaliana myrcene/ocimene syn-
thase (22% identity, 43% similarity) (Bohlmann et al.,
2000b). The PaJF67 protein is also less similar to
the known sesquiterpene synthases of grand fir, 37%
identity with γ-humulene synthase (59% similarity)
and δ-selinene synthase (58% similarity) (Steele et al.,
1998a) and 26% identity (44% similarity) with E-α-
bisabolene synthase (Bohlmann et al., 1998a). PaJF67
protein is only 26% identical (39% similar) with abi-
etadiene synthase, a diterpene synthase from grand
fir (Stofer Vogel et al., 1996). These data suggest
that PaJF6 7 encodes indeed a monoterpene synthase,
rather than a sesquiterpene synthase or a diterpene
synthase.
The predicted PaJF67 protein contains the twin-
arginine/tryptophan motif RRx8W in position 64 to 74
from the N-terminus (Figure 4). This motif is char-
acteristic of all monoterpene synthases (Bohlmann
et al., 1998b, 2000a) and was shown to be involved
in the initial diphosphate migration step of the cou-
pled isomerization-cyclization reaction mechanism of
monoterpene synthases (Williams et al., 1998). The
region upstream of the twin-arginine residues bears
typical features of a plastid transit peptide characteris-
tic of nuclear-encoded monoterpene synthase prepro-
teins (Bohlmann et al., 1998b; Williams et al., 1998).
A conserved DDxxD motif previously identified to be
involved in divalent metal ion assisted substrate bind-
ing (reviewed in Davis and Croteau, 2000) was also
located in PaJF67 (Figure 4).
Functional expression of PaJF67 in E. coli
Monoterpene synthases are members of a family of
TPS genes in plants that apparently evolved from
a common ancestor by repeated gene duplication,
functional diversificationand functional specialization
(Bohlmann et al., 1998b, 2000a). Members of the TPS
family cluster into six subfamilies by sequence sim-
ilarity. However, specific functions of TPS enzymes
cannot be predicted based on sequence relatedness,
because similar functions did evolve independently
in separate subfamilies, and functions of members of
the same subfamily can be quite diverse (Bohlmann
et al., 1998b). Many monoterpene synthases also
yield multiple products from the same substrate as a
consequence of a cationic reaction mechanism that al-
lows for the formation of a suite of similar products
from common intermediates (Wise and Croteau, 1998;
Davis and Croteau, 2000). It was therefore necessary
to express and biochemically characterize the PaJF67
protein to obtain correct functional identification of
the gene product and to provide precise annotation of
gene function.
cDNA PaJ F 6 7 was subcloned into pET100/D-
TOPO vector for T7-RNA polymerase directed ex-
pression of the active protein in E. coli BL21-
CodonPlus(DE3) cells. Expressed recombinant PaJF67
protein was tested in cell-free extracts of trans-
formed E. coli for monoterpene synthase, sesquiter-
pene synthase and diterpene synthase activities using
the corresponding prenyl diphosphate substrates ger-
anyl diphosphate (GPP), farnesyl diphosphate (FPP)
and geranylgeranyl diphosphate (GGPP). Of these
substrates, only GPP was efficiently converted into
monoterpene hydrocarbons by enzyme activity of
PaJF67 protein. Extract prepared from E. coli BL21-
CodonPlus(DE3) transformed with pET100/D-TOPO
without PaJ F 67 insert served as a control for product
formation independent of recombinant TPS enzyme.
These extracts did not yield detectable amounts of
enzymatic terpenoid products.
128
Figure 5. Gas chromatography (GC) and mass spectroscopy (MS) of the monoterpene products derived from geranyl diphosphate by PaJF67
(+)-3-carene synthase. (A) Total ion chromatogram of products of PaJF67 enzyme activity formed from GPP and separated on a DB-WAX
column. The most abundant products (more than 1% of total product) are by order of retention time: sabinene (5% of total product, tR
11.82 min.), 3-carene (78% of total product, tR12.86 min), myrcene (3% of total product, tR13.48 min), and terpinolene (11% of total product,
tR17.90 min). Low abundance products (1% or less of total product) are: α-pinene (0.9%, peak 1), α-terpinene (0.6%, peak 2), limonene (0.4%,
peak 3), β-phellandrene (0.7%, peak 4), and γ-terpinene (1%, peak 5). (B) Total ion chromatogram of monoterpene products of PaJF67 enzyme
separated on a Cyclodex B column. Enantiomers of chiral components were identified by co-injection with enantiomerically pure standards. (C)
Mass spectrum of enzyme product tR12.86 min. (D) Mass spectrum of authentic 3-carene. (E) Mass spectrum of enzyme product tR17.90 min.
(F) Mass spectrum of authentic terpinolene. (G) Mass spectrum of enzyme product tR11.82 min. (H) Mass spectrum of authentic sabinene. (I)
Mass spectrum of enzyme product tR13.48. (J) Mass spectrum of authentic myrcene.
129
Product identification of PaJF67 enzyme activity
Analysis of the monoterpene hydrocarbon product
fraction of recombinant PaJF67 enzyme by GC
and GC/MS revealed 3-carene as the major prod-
uct (77.7%) with GPP as substrate (Figure 5).
Additional cyclic and acyclic monoterpene prod-
ucts were identified as terpinolene (11%), sabinene
(5%), myrcene (3%), γ-terpinene (1%), α-pinene
(0.9%), β-phellandrene (0.7%), α-terpinene (0.6%)
and limonene (0.4%). It is clear from previous work
on P. abies (Martin et al., 2002) that the major as well
as the minor products of PaJF67 3-carene synthase are
indeed present in this plant. Thus, the product profile
seen in in vitro assays of the recombinant, heterolo-
gously expressed PaJF67 protein is likely to be the
same as that produced in vivo. The co-occurrence of
3-carene and terpinolene as the two most abundant
products can be rationalized based on similar reaction
mechanisms of their formation involving, respectively,
proton elimination at C-5 and C-4 of the α-terpinyl
cation intermediate (Figure 6) (Savage and Croteau,
1993; Wise and Croteau, 1998). The multiple cyclic
products of 3-carene synthase relate to a highly reac-
tive α-terpinyl cation intermediate that can, within the
constraints of the enzyme active site, undergo a variety
of transformations prior to ultimate quenching of the
carbocation (Wise and Croteau, 1998). No oxygenated
or phosphorylated monoterpenes were found in de-
tectable amounts in the product fraction of PaJF67
enzyme assays.
The PaJF67 enzyme produces enantiomerically
pure (+)-3-carene, with no detectable amounts of
the (−)-enantiomer as determined by 2D-GC. The
stereochemistry of (+)-3-carene is consistent with the
stereochemistry of all other chiral products of this
enzyme activity, (−)-sabinene, (−)-α-pinene, (−)-β-
phellandrene, and (−)-limonene, because all of these
products relate mechanistically to the 4S-configuration
of the intermediate α-terpinyl cation (Figure 6) (Wise
and Croteau, 1998). Interestingly, this multi-product
enzyme does not catalyze the biosynthesis of con-
stituents such as (−)-β-pinene and (−)-α-thujene,
which are structurally and mechanistically similar to
(−)-α-pinene and (−)-sabinene, respectively. cDNA
cloning, functional expression and biochemical iden-
tification of (+)-3-carene synthase from spruce pro-
vides new means for future evaluation of possible
active site residues responsible for the formation of
a monoterpene cyclopropyl function. A comparison
of spruce 3-carene synthase and grand fir terpino-
lene synthase is of particular interest, because these
enzymes provide a pair of conifer monoterpene syn-
thases that are closely related in their primary struc-
tures (Figure 5 and Figure 7) and enzyme mechanisms
(Figure 6).
Previously, a group of monoterpene synthases was
cloned from grand fir (Bohlmann et al., 1999). How-
ever, these cloned synthases did not produce 3-carene
as a major nor a minor product and therefore, could
not account for the formation of 3-carene and the
quantitative variability of 3-carene relative to other
monoterpenes in chemotypes of grand fir (Katoh and
Croteau, 1998). Our findings in Norway spruce prove
the existence of a gene for a highly specialized 3-
carene synthase in conifers and provide genetic and
biochemical mechanisms to explain for previously ob-
served quantitative variation of 3-carene relative to
other monoterpenes in species and chemotypes of
conifers (Hiltunen et al., 1975; Persson et al., 1996;
Plomion et al., 1996; Sjödin et al., 1996, 2000).
Phylogenetic relatedness of (+)-3-carene and TPSd
genes
Norway spruce 3-carene synthase is a new member
of the gymnosperm TPSd group of the TPS family
that includes monoterpene synthases, sesquiterpene
synthases and diterpene synthases of plant origin (Fig-
ure 7) (Bohlmann et al., 1998b). 3-Carene synthase
is most closely related to other conifer monoterpene
synthases and shares a common origin in a puta-
tive ancestral gene with the mechanistically similar
grand fir terpinolene synthase (Bohlmann et al., 1999).
Norway spruce 3-carene synthase is only distantly
related to angiosperm monoterpene synthases. It is
known that specific functions of plant monoterpene
synthases evolved independently in angiosperms and
gymnosperms (Bohlmann et al., 1998b). To date,
an orthologue 3-carene synthase has not been re-
ported from any other species, including the conifer
grand fir. Therefore, it is currently not possible to
conclude whether specific biochemical functions of
conifer monoterpene synthases, such as 3-carene syn-
thase, evolved prior to speciation of extant members
of the Pinaceae. Alternatively, it is possible that func-
tional specialization of conifer monoterpene synthases
occurred independently in different genera or species
of this plant family. To test these models, additional
monoterpene synthases of members of the Pinaceae
will be cloned and characterized in future work.
130
Figure 6. Proposed mechanism for monoterpene formation by (+)-3-carene synthase (PaJF67). The proposed isomerization-cyclization scheme
accounts for all of the major and minor regio-chemically different monoterpenes of the multi-product 3-carene synthase. The stereochemistry
determined for all products can be rationalized via the intermediates of (+)-3S-linalyl diphosphate (LPP) and 4S-α-terpinyl cation. The two
most abundant products, 3-carene (78%) and terpinolene (11%) are derived by closely related mechanisms involving, respectively, proton
elimination at C-5 and C-4 of the 4S-α-terpinyl cation intermediate (Savage and Croteau, 1993; Wise and Croteau, 1998). OPP denotes the
diphosphate moiety.
131
Figure 7. Sequence relatedness of Norway spruce (+)-3-carene synthase (PaJF67) and grand fir monoterpene synthases, sesquiterpene syn-
thases and diterpene synthases. Deduced amino acid sequences were compared using ClustalX and trees were visualized using Treedraw.
Norway spruce 3-carene synthase is a member of the gymnosperm TPSd family (Bohlmann et al., 1998b) and is most closely related to
the mechanistically similar terpinolene synthase from grand fir (Bohlmann et al., 1999). Except for (+)-3-carene synthase from Norway
spruce, all other sequences of this dendrogram are from grand fir: monoterpene synthases (myrcene synthase (U87908), (−)-pinene synthase
(U87909), (−)-camphene synthase (U87910), β-phellandrene synthase (AF139205), terpinolene synthase (AF139206), (−)-limonene synthase
(AF006193), (−)-limonene/(−)-α-pinene synthase (AF139207)), sesquiterpene synthases (γ-humulene synthase (U92267), δ-selinene synthase
(U92266), E-α-bisabolene synthase (AF006195)), and a diterpene synthase (abietadiene synthase (U50768)).
Conclusions
This paper is a continuation of our previous histo-
logical, chemical, and biochemical characterization of
induced traumatic resinosis in Norway spruce (Martin
et al., 2002). Using homologous and heterologous TPS
probes, that are class-specific for conifer monoterpene
synthases or diterpene synthases, we demonstrated
that the multi-layered traumatic resin defense response
in Norway spruce involves increased transcript accu-
mulation for two classes of TPS genes, monoterpene
synthases and diterpene synthases. Because different
monoterpene synthases in conifers can be more than
90% identical (Bohlmann et al., 1999), gene-specific
expression analysis can only be performed in future
132
work when the complete monoterpene synthase gene
family of Norway spruce is known.
Screening for Norway spruce monoterpene syn-
thases resulted in the discovery and functional charac-
terization of a new TPS cDNA that is highly special-
ized for the stereospecific formation of (+)-3-carene,
a common component of constitutive and induced
conifer resins. In earlier studies, 3-carene was associ-
ated with defense and resistance of conifers to a suite
of insect pests and pathogens (Krupa and Fries, 1971;
Rocchini et al., 2000; Pasquier-Barre et al., 2001;
Fäldt et al., 2002). Isolation of a 3-carene synthase
cDNA and efficient methods for conifer transforma-
tion (Peña and Seguin, 2001) provide a means for
future directed manipulation of 3-carene synthesis and
the evaluation of proposed roles of 3-carene in conifer
chemical ecology.
The cloned 3-carene synthase forms additionalmi-
nor products, a characteristic feature of many plant
terpene synthases (Bohlmann et al., 1998b; Davis and
Croteau, 2000). Terpinolene is the second most abun-
dant product of Norway spruce 3-carene synthase and
is mechanistically closely related to the major prod-
uct, 3-carene (Figure 6). Interestingly, the deduced
protein sequence of 3-carene synthase from Norway
spruce is most similar to grand fir terpinolene synthase
(Figure 4 and Figure 7). Future work will address the
structure-function relationships of the formation of the
bicyclic 3-carene and monocyclic terpinolene and will
be guided by structure and sequence comparison of the
closely related conifer monoterpene synthases.
Acknowledgements
This research was supported by grants to J.B. from the
Natural Sciences and Engineering Research Council
of Canada (NSERC), the Canadian Foundation for In-
novation (CFI), and the Human Frontied Science Pro-
gram (HFSP). J.F. received postdoctoral fellowships
from the Bengt Lundqvist Minne Foundation (Swe-
den) and the Swedish Foundation for International
Cooperation in Research and Higher Education. D.M.
was supported, in part, by a Max Planck fellowship
provided by J. Gershenzon (MPICE, Jena, Germany).
We thank Anna-Karin Borg-Karlson (Royal Institute
of Technology, Stockholm, Sweden) for access to
the 2D-GC facilities, Michael Phillips for technical
advice, and Rodney Croteau (Washington State Uni-
versity, Pullman, USA) for the grand fir abietadiene
synthase probe and substrates.
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