Cloning and characterization of isoprenyl diphosphate synthases with farnesyl diphosphate and geranylgeranyl diphosphate synthase activity from Norway spruce (Picea abies) and their relation to induced oleoresin formation

Article abstract of DOI:10.1016/j.phytochem.2007.05.037

The conifer Picea abies (Norway spruce) employs terpenoid-based oleoresins as part of its constitutive and induced defense responses to herbivores and pathogens. The isoprenyl diphosphate synthases are branch-point enzymes of terpenoid biosynthesis leading to the various terpene classes. We isolated three genes encoding isoprenyl diphosphate synthases from P. abies cDNA libraries prepared from the bark and wood of methyl jasmonate-treated saplings and screened via a homology-based PCR approach using degenerate primers. Enzyme assays of the purified recombinant proteins expressed in Escherichia coli demonstrated that one gene (PaIDS 4) encodes a farnesyl diphosphate synthase and the other two (PaIDS 5 and PaIDS 6) encode geranylgeranyl diphosphate synthases. The sequences have moderate similarity to those of farnesyl diphosphate and geranylgeranyl diphosphate synthases already known from plants, and the kinetic properties of the enzymes are not unlike those of other isoprenyl diphosphate synthases. Of the three genes, only PaIDS 5 displayed a significant increase in transcript level in response to methyl jasmonate spraying, suggesting its involvement in induced oleoresin biosynthesis.

Full text of DOI:10.1016/j.phytochem.2007.05.037

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PHYTOCHEMISTRY
Phytochemistry 68 (2007) 2649–2659
www.elsevier.com/locate/phytochem
Cloning and characterization of isoprenyl diphosphate synthases
with farnesyl diphosphate and geranylgeranyl diphosphate
synthase activity from Norway spruce (Picea abies) and
their relation to induced oleoresin formation
*
Axel Schmidt , Jonathan Gershenzon
Max Planck Institute for Chemical Ecology, Department of Biochemistry, Hans-Kno¨ll-Str. 8, D-07745 Jena, Germany
Received 19 March 2007; received in revised form 24 May 2007; accepted 24 May 2007
Available online 10 July 2007
Abstract
The conifer Picea abies (Norway spruce) employs terpenoid-based oleoresins as part of its constitutive and induced defense responses
to herbivores and pathogens. The isoprenyl diphosphate synthases are branch-point enzymes of terpenoid biosynthesis leading to the
various terpene classes. We isolated three genes encoding isoprenyl diphosphate synthases from P. abies cDNA libraries prepared from
the bark and wood of methyl jasmonate-treated saplings and screened via a homology-based PCR approach using degenerate primers.
Enzyme assays of the purified recombinant proteins expressed in Escherichia coli demonstrated that one gene (PaIDS 4) encodes a far-
nesyl diphosphate synthase and the other two (PaIDS 5 and PaIDS 6) encode geranylgeranyl diphosphate synthases. The sequences have
moderate similarity to those of farnesyl diphosphate and geranylgeranyl diphosphate synthases already known from plants, and the
kinetic properties of the enzymes are not unlike those of other isoprenyl diphosphate synthases. Of the three genes, only PaIDS 5 dis-
played a significant increase in transcript level in response to methyl jasmonate spraying, suggesting its involvement in induced oleoresin
biosynthesis.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Picea abies; Pinaceae; Gymnosperm; Conifer defense; Prenyltransferase; Methyl jasmonate; Enzyme kinetics, Phylogenetic analysis
1. Introduction
evolutionary persistence indicate the presence of effective
defenses. The best known example of conifer defense is
oleoresin, a pungent, viscous mixture of terpenoids found
in specialized ducts, blisters and cells in stems and foliage.
Oleoresin is both a constitutive and an inducible defense.
For instance, some species of the Pinaceae, especially in
the subfamily Piceoideae and to some degree the Abietoi-
deae, form new (traumatic) resin ducts (TRDs) in response
to attack by stem-boring insects and their associated fungi.
These oleoresin-filled structures are found mainly in the
sapwood and bark of coniferous trees, and are believed
to help resist attack by augmenting the constitutive resin
flow, thus providing a strong barrier against most herbi-
vores and pathogens (Nagy et al., 2000; Martin et al.,
2002; Hudgins et al., 2004; Franceschi et al., 2005; Byun-
McKay et al., 2006; Keeling and Bohlmann, 2006).
Conifers of the family Pinaceae frequently come under
attack from herbivorous insects, such as the tree-killing
bark beetle, Ips typographus, and its fungal associate, Cer-
atocystis polonica (Phillips and Croteau, 1999; Trapp and
Croteau, 2001; Baier et al., 2002; Franceschi et al., 2005).
However, the long life spans of many conifers and their
Abbreviations: TRD, traumatic resin duct; IPP, isopentenyl diphos-
phate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate;
FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; IDS, is-
oprenyl diphosphate synthase; FPPS, farnesyl diphosphate synthase;
GGPPS, geranylgeranyl diphosphate synthase; MJ, methyl jasmonate.
*
Corresponding author. Tel.: +49 3641 571331; fax: +49 3641 571302.
E-mail address: aschmidt@ice.mpg.de (A. Schmidt).
0031-9422/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.05.037

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A. Schmidt, J. Gershenzon / Phytochemistry 68 (2007) 2649–2659
PLASTID
With more than 30,000 structural variants; terpenes are
+ IPP
GPPS
+ 3 IPP
GGPPS
the largest class of plant secondary metabolites. Oleoresin
consists mainly of monoterpenes (C10) and diterpene resin
acids (C20) as well as smaller amounts of sesquiterpenes
(C15) (Langenheim, 2003). The biosynthesis of all terpenoids
is initiated by the synthesis of isopentenyl diphosphate (IPP)
via the mevalonic acid pathway or the methylerythritol
phosphate pathway (Gershenzon and Kreis, 1999) (Fig. 1).
IPP and its isomer, dimethylallyl diphosphate (DMAPP),
are the five-carbon building blocks that undergo sequential
condensation reactions to form geranyl diphosphate (GPP,
C10), farnesyl diphosphate (FPP, C15), and geranylgeranyl
diphosphate (GGPP, C20), the precursors of monoterpenes,
sesquiterpenes and diterpenes, respectively. The enzymes
catalyzing these sequential condensations are a group of
prenyltransferases referred to collectively as isoprenyl
diphosphate synthases. GPP, FPP, and GGPP are each
believed to be formed by a specific isoprenyl diphosphate
synthase (IDS) named for its product: geranyl diphosphate
synthase (GPPS), FPP synthase (FPPS), and GGPP syn-
thase (GGPPS) (Gershenzon and Kreis, 1999) (Fig. 2). In
subsequent steps, these linear diphosphates serve as sub-
strates for terpene synthases, which form a complex variety
of mono-, sesqui-, and diterpene carbon skeletons (Martin
et al., 2004; Keeling and Bohlmann, 2006). An important
feature of terpenoid biosynthesis is the way this process is
partitioned among different cell compartments. The forma-
tion of GPP and GGPP and their subsequent conversion to
OPP
Geranyl diphosphate
OPP
DMAPP
OPP
Geranylgeranyl diphosphate
+ 2 IPP
FPPS
OPP
OPP
DMAPP
Farnesyl diphosphate
CYTOSOL
Fig. 2. Scheme for IDS catalysis in terpenoid biosynthesis. Each enzyme
catalyzes 1, 2 or 3 sequential condensations to give a single product
without significant release of intermediates. GPPS and GGPPS occur in
the plastid, whereas FPPS is localized in the cytosol.
mono- and diterpenes occur in the plastids, whereas the bio-
synthesis of FPP and its conversion to sesquiterpenes are
localized in the cytosol (Fig. 2).
We are currently attempting to characterize the molecu-
lar and biochemical basis of conifer defenses using Norway
spruce (Picea abies) L. Karst as a model. Norway spruce
stems are constitutively defended by oleoresin, but also
possess a suite of induced defenses. The induced defense
response of this species to bark beetle attack can be mim-
icked by spraying trees with methyl jasmonate (MJ) (Martin
et al., 2002; Byun-McKay et al., 2006; Erbilgin et al., 2006;
Zeneli et al., 2006). This treatment triggers the formation of
Pyruvate
Glyceraldehyde-3-phosphate
Acetyl-CoA
MEVALONATE PATHWAY
METHYLERYTHRITOL PHOSPHATE PATHWAY
OPP
OPP
IPP (C5)
DMAPP (C5)
1xIPP
Terpene
synthases
2xIPP
OPP
OPP
OPP
MONOTERPENES (C10)
Isoprenyl
diphosphate
synthases
α
( )- -and ß-pinene
β
3xIPP
GPP (C10)
(-)-limonene
(-)- ß-phellandrene
β
SESQUITERPENES (C15)
(E)-α-bisabolene
FPP (C15)
(E)-ß-farnesene
β
DITERPENES (C20)
levopimaric acid
abietic acid
GGPP (C20)
Fig. 1. Outline of terpenoid oleoresin biosynthesis in conifers showing the formation of IPP and DMAPP, the condensations to make larger prenyl
diphosphates and the subsequent cyclizations to form the major products in P. abies. The isoprenyl diphosphate synthases catalyze the branch-point
reactions leading to the different terpene classes.

A. Schmidt, J. Gershenzon / Phytochemistry 68 (2007) 2649–2659
2651
TRDs in the developing sapwood and is associated with an
accumulation of mono- and diterpenes (Franceschi et al.,
2002; Martin et al., 2002; Zeneli et al., 2006). Numerous
genes and enzymes involved in conifer defense have been
characterized recently, but attention has focused on later
steps, including terpene synthases (Fa¨ldt et al., 2003; Hiet-
ala et al., 2004; Martin et al., 2004; Huber et al., 2005;
Byun-McKay et al., 2006; Ro and Bohlmann, 2006) and
cytochrome P 450 mono-oxygenases (Hamberger and
Bohlmann, 2006). Very little information is available about
the isoprenyl diphosphate synthases (IDSs) of the Pinaceae
(Hefner et al., 1998; Tholl et al., 2001; Burke and Croteau,
2002; Schmidt et al., 2005) despite the fact that these
enzymes catalyze the branch-point reactions leading to
GPP, FPP and GGPP, and so may channel flux among
the different terpene classes present in oleoresin.
IDSs have been extensively studied at the molecular
level in angiosperms, which generally produce much lower
amounts of terpenes than conifers. FPPS and GGPPS
activity have been investigated in Arabidopsis thaliana, rub-
ber tree (Hevea brasiliensis), banana (Musa acuminata),
escobilla (Scoparia dulcis) and maize (Zea mays) (Kojima
et al., 2000; Okada et al., 2000; Masferrer et al., 2002;
Takaya et al., 2003; Cervantes-Cervantes et al., 2006).
The pool size of GPP, FPP, and GGPP has been suggested
to limit terpenoid biosynthesis in these plants (Aharoni
et al., 2003; Han et al., 2006), thus implicating IDSs as
primary regulators of terpenoid formation.
The IDS activities of gymnosperms are only poorly
known. An FPPS from Abies grandis has been purified
(Tholl et al., 2001), and GGPPSs genes were characterized
from A. grandis and Canadian yew (Taxus canadensis)
(Hefner et al., 1998; Burke and Croteau, 2002). However,
T. canadensis contains no oleoresin and A. grandis pro-
duces resin only on induction. Our model species, P. abies,
possesses both constitutive and inducible resin duct systems
in its wood, and so may require much more complex con-
trols over terpene biosynthesis. Here we report the cloning
and expression of three IDS genes from P. abies. The
expressed clones possessed FPPS and GGPPS activity are
useful for understanding the molecular regulation of oleo-
resin biosynthesis in conifers.
treated with methyl jasmonate (MJ) to induce terpene oleo-
resin biosynthesis. Positive clones were isolated and sub-
cloned into pTriplEx2 yielding three P. abies (Pa) IDS
cDNA clones, designated PaIDS 4, PaIDS 5 and PaIDS 6.
Of the three clones, PaIDS 4 had the highest similarity
to other plant FPPS sequences based on comparisons at
the amino acid level. Seven different PaIDS 4 sequences
were actually found that differed from each other only in
the 30-untranslated region, consisting of 330–699 nucleo-
tides between the stop codon and the poly (A)+ tail. The
protein encoded by PaIDS 4 consisted of 347 amino acids
and had a calculated mass of 39.9 kDa. The highest homol-
ogy of the deduced amino acid sequence (Fig. 3, Table 1)
was to an FPPS of the maidenhair tree (Ginkgo biloba) with
86% identity and an FPPS from banana (M. acuminata)
with 77% identity. The two active site, aspartate-rich
DDxxD motifs, found in other plant FPPSs are both con-
served in the P. abies gene. Neither the PaIDS 4 nor any
other isolated plant FPPS contains a putative transit pep-
tide at the 50-end of the cDNA (Fig. 3). When the PaIDS
4 sequence was subjected to a phylogenetic analysis with
all known plant IDSs, it clustered with other plant FPPS
genes apart from the plant GPPS and GGPPS genes.
Because of limited sequence information, gymnosperm
and angiosperm FPPSs cannot be differentiated (Fig. 4).
Seven Norway spruce IDS clones had high similarity to
other plant GGPPSs sequences based on comparisons at
the amino acid level. These fell into two groups that shared
only 60% identity in the deduced amino acid sequences of
their coding regions. Within each group, differences among
the amino acid sequences were only 1-3%, with more sub-
stantial differences in the 110–325 nucleotides 30-untrans-
lated region. Two representative cDNA clones of each
class, designated PaIDS 5 and PaIDS 6, with lengths of
1146 bp and 1155 bp in the coding region, respectively,
were selected for further study. The proteins encoded by
PaIDS 5 and PaIDS 6 contain 382 and 385 amino acids,
respectively, and have calculated masses of 42.0 and
41.7 kDa (Fig. 3). The highest similarity of the deduced
amino acid sequence of PaIDS 5 was to the sequences of
gymnosperm GGPPSs (90% identity) and to the A. grandis
GPPS (82% identity). In contrast, the deduced amino acid
sequence of PaIDS 6 showed the highest identity (72%) to
the angiosperm Adonis palaestina GGPPS and the large
subunit of snapdragon (Antirrhinum majus) GPPS (64%
identity) (Table 1). Unlike PaIDS 4, both PaIDS 5 and
PaIDS 6 contain a putative transit peptide for plastid tar-
geting and possess the same conserved aspartate-rich
DDxxxxDxDD and DDxxD motifs as other plant
GGPPSs (Fig. 3). When sequences of GPPS and GGPPS
are analyzed phylogenetically, there is no distinction
between the two, but an almost complete separation
between angiosperm and gymnosperm sequences appears.
A cluster of gymnosperm GPPS and GGPPS genes is evi-
dent in which PaIDS 5 is present, while a second cluster
contains angiosperm GGPPS genes along with PaIDS 6
(Fig. 4).
2. Results
2.1. Isolation and sequence comparisons of cDNA clones
encoding Norway spruce isoprenyl diphosphate synthases
(IDSs)
To isolate Norway spruce IDS genes, cDNA fragments
were obtained by PCR using primers designed to conserved
regions of known plant farnesyl diphosphate synthases
(FPPS) and geranylgeranyl diphosphate synthases
(GGPPS) with Norway spruce RNA as a template. These
fragments were then employed to screen cDNA libraries
constructed with mRNA isolated from Norway spruce

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A. Schmidt, J. Gershenzon / Phytochemistry 68 (2007) 2649–2659
Fig. 3. Alignment of PaIDS 4, PaIDS 5 and PaIDS 6 with the plant IDS sequences of highest deduced amino acid sequence identity. The listed sequences
are FPPS from Ginkgo biloba (AY389818), GGPPS from Adonis palaestina (AY661707) and GGPPS from Abies grandis (AF425235). Amino acid residues
with pair-wise identity between PaIDS 4 and GGPPS G. biloba, PaIDS 5 and GGPPS A. palaestina and PaIDS 6 and GGPPS A. grandis are indicated by
black boxes. Dashes indicate sequence gaps introduced to optimize the alignment. The conserved, aspartate-rich motifs are indicated by the black
underline.
Table 1
Sequence similarity among PaIDS 4, PaIDS 5 and PaIDS 6, and other IDS genes, Ginkgo biloba FPPS, Adonis palaestina GGPPS and Abies grandis
GGPPS, in percent
FPPS G. biloba
PaIDS 5
86.5
32.3
30.5
GGPPS A. palaestina
PaIDS 6
29.7
30.3
28.5
28.2
60.5
62.3
71.4
GGPPS A. grandis
31.7
30.0
91.1
62.6
61.3
PaIDS 4
FPPS G. biloba
PaIDS 5
GGPPS A. palaestina
PaIDS 6
The analysis was performed using ClustalX and DNAstar. The accession numbers of the non-P. abies genes are given in the legend of Fig. 4.
2.2. Heterologous expression of Norway spruce isoprenyl
diphosphate synthase genes
the 50-end of the coding region for directing plastidial
import were first truncated. Analysis of the crude bacterial
extracts of the expressed proteins with SDS–PAGE
revealed recombinant protein bands with an apparent
molecular mass of approximately 40 kDa for PaIDS 4,
and 37 kDa for both the truncated PaIDS 5 and PaIDS
6. The proteins were subsequently purified with Ni-NTA
To confirm that PaIDS 4, PaIDS 5, and PaIDS 6 are
functional isoprenyl diphosphate synthase, we expressed
full length cDNA in E. coli in a vector adding a C-terminal
His tag. For PaIDS 5 and PaIDS 6, the signal sequences at

A. Schmidt, J. Gershenzon / Phytochemistry 68 (2007) 2649–2659
2653
PaIDS5
GGPPS A. grandis
GPPS1 A. grandis
Gymnosperm
GGPPS and GPPS
GPPS2 A. grandis
GPPS3 A. grandis
GGPPS G. biloba
GGPPS T. canadiensis
GGPPS L. albus
GGPPS C. annuum
Angio- / gymnosperm
GGPPS
GGPPS C. sublyratus
GGPPS S. alba
PaIDS6
FPPS A. annua
Angio- / gymnosperm
FPPS
FPPS M. acuminata
FPPS G. biloba
PaIDS4
FPPS A. thaliana
20 0
125.6
120
100
80
60
40
Nucleotide Substitutions (x100)
Fig. 4. Phylogenetic tree of the deduced amino acid sequences of PaIDS 4, PaIDS 5 and PaIDS 6 and other gymnosperm and angiosperm GPPS, GGPPS
and FPPS sequences. Analysis was performed using Clustal X and DNAstar. The accession number of the sequences are as follows: GGPPS Abies grandis
AF425235, GPPS1 A. grandis AF513111, GPPS2 A. grandis AF513112, GPPS3 A. grandis AF513113, GGPPS Ginkgo biloba AY371321, GGPPS Taxus
canadensis AF081514, GGPPS Lupinus albus U15778, GGPPS Capsicum annuum X80267, GGPPS Croton sublyratus AB034249, GGPPS S. alba X98795,
FPPS Artemisia annua AF112881, FPPS M. acuminata AAL82595, FPPS G. biloba AAR27053, FPPS Arabidopsis thaliana Q43315. Not all known plant
FPPS and GGPPS sequences were included in the tree.
GPP, while no side products were observed in the PaIDS
5 and PaIDS 6 assays (Fig. 6). Neither the empty vector
controls nor heat-treated assays showed any significant
enzyme activity. No change of enzyme activity was
observed in control assays without 250 mM imidazole in
the assay buffer (data not shown). Thus the expected prod-
uct specificities, FPP for PaIDS 4 and GGPP for PaIDS 5
and PaIDS 6, were confirmed.
2.3. Kinetic properties of Norway spruce isoprenyl
diphosphate synthases
To compare the biochemical properties of the P. abies
enzymes to other plant IDSs, the kinetic constants of the
recombinant P. abies FPPS (PaIDS 4) and GGPPS (PaIDS
Fig. 5. SDS–PAGE analysis of recombinant IDS proteins expressed in
E. coli and visualized by Coomassie staining. M, molecular mass markers;
BL21, bacterial extract expressing empty vector; C, crude bacterial
extracts; P, purified recombinant proteins after Ni-NTA agarose chro-
matography. The expression and purification of PaIDS 5 was similar to
that of PaIDS 6.
6) were measured under linear conditions with respect to
time and protein concentration (Table 2). The data are sim-
ilar to those measured for other conifer IDSs, such as
native A. grandis FPPS (Tholl et al., 2001) and recombi-
nant T. canadensis GGPPS (Burke and Croteau, 2002).
PaIDS 4 had an apparent Km value of 71 lM for DMAPP
and 230 lM for IPP, while PaIDS 6 had a Km value of
181 lM for DMAPP and 72 lM for IPP. Maximum
velocity VMax of 0.95 ng/mg for PaIDS 4 and 0.18 ng/mg
for PaIDS 6 was calculated. As for A. grandis FPPS
(Tholl et al., 2001) and T. canadensis GGPPS (Burke and
agarose columns (Fig. 5). The purified PaIDS 4 protein
exhibited FPPS enzyme activity, while both PaIDS 5 and
PaIDS 6 showed GGPPS enzyme activity when incubated
with [1-14C] IPP and DMAPP as substrates. PaIDS 4 was
also able to catalyze the synthesis of a small amount of

2654
A. Schmidt, J. Gershenzon / Phytochemistry 68 (2007) 2649–2659
70
Bq
G
F
GG
Empty vector control
150
PaIDS 4
PaIDS 5
140
70
PaIDS 6
40
5
10
15
20
20
25
25
30
30
35
35
40
20
µV
Standard
40
5
10
15
40
time in min
Fig. 6. Catalytic activities of recombinant PaIDS 4, PaIDS 5 and PaIDS 6 proteins heterologously expressed in E. coli after assay with [1-14C] IPP and
DMAPP. Reaction products were enzymatically hydrolyzed and the resulting alcohols were analyzed by radio-gas chromatography. The four upper panels
show the output of the radioactivity detector. The main product of the PaIDS 4 reaction was farnesol (F) with minor amounts of geraniol (G), and the
products of the PaIDS 5 and the PaIDS 6 reactions were geranylgeraniol (GG). The empty vector control showed no activity. Compounds were identified
by coinjection of standards as depicted in the thermal conductivity detector trace (bottom panel).
Table 2
Kinetic constants for the recombinant enzymes PaIDS 4 and PaIDS 6, calculated by Lineweaver–Burk plotting
Apparent saturation IPP (lM)
Apparent saturation DMAPP (lM)
Km DMAPP (lM)
Km IPP (lM)
VMax (nkat/mg)
PaIDS 4
PaIDS 6
800
250
200
800
71 5.2
181 7.2
230 8.4
72 4.2
0.95 0.35
0.18 0.02
Each value is the average of three independent biological replicates.
Croteau, 2002), the P. abies enzymes were both dependent
on MgCl2. PaIDS 4 activity was optimal with 5 mM
MgCl2 and PaIDS 5 activity with 10 mM MgCl2.
these increases were much less than that of PaIDS 5 in
the bark. The maximum induction of PaIDS 4 transcript
occurred 1 day after MJ treatment whereas induction of
PaIDS 5 was at its maximum at 2 days and the induction
of PaIDS 6 occurred much more slowly, over a period of
8 days (Fig. 7). No significant transcript accumulation
for the investigated genes was found in control saplings
(data not shown).
2.4. Expression of Norway spruce isoprenyl diphosphate
synthase genes after oleoresin induction
To determine the potential role of PaIDS 4, PaIDS 5,
and PaIDS 6 in the induced oleoresin formation of
P. abies, transcript levels were measured over a 10 day time
course after saplings had been sprayed with MJ. In bark
(and attached cambium), the only transcript that increased
in abundance in response to MJ treatment was from PaIDS
5. Transcripts of this GGPPS gene increased continuously
and reached a maximum at day 8 representing a 120-fold
increase in transcript. In contrast, PaIDS 4 and PaIDS 6
transcript levels remained relatively constant after spray-
ing. In wood, each of the PaIDS genes showed some tran-
script increase upon MJ spraying, but the magnitudes of
3. Discussion
The isoprenyl diphosphate synthases (IDS) catalyze the
branch-point reactions leading to the different classes of
terpenes. To learn more about how conifers control pro-
duction of the major constituents of their defensive oleo-
resin, we cloned three genes encoding IDSs from Norway
spruce (P. abies) using a homology-based, nested PCR
approach. One of the genes, PaIDS 4, encodes a farnesyl

A. Schmidt, J. Gershenzon / Phytochemistry 68 (2007) 2649–2659
2655
PaIDS 4
PaIDS 5
PaIDS 6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
140
120
100
80
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Bark
60
40
20
0
0
0.5
1
2
4
6
8
10
0
0.5
1
2
4
6
8
10
0
0.5
1
2
4
6
8
10
2.0
1.5
1.0
0.5
0.0
8
6
4
2
0
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Wood
0
0.5
1
2
4
6
8
10
0
0.5
1
2
4
6
8
10
0
0.5
1
2
4
6
8
10
days after MJ spraying
days after MJ spraying
days after MJ spraying
Fig. 7. Relative abundance of mRNA transcripts of PaIDS 4, PaIDS 5 and PaIDS 6 genes in bark and wood of MJ-treated Norway spruce saplings.
Transcript abundance of each IDS gene was measured by quantitative RT-PCR using SYBR-Green for detection and normalized against ubiquitin. The
transcript abundance at day 0 was set to 1.0. Each value is the average of two independent biological replicates, each of which is represented by three
technical replicates.
diphosphate synthase (FPPS). This is the first FPPS gene to
be isolated from a conifer, although there are many exam-
ples known from angiosperms (Cunillera et al., 1996, 1997;
Hemmerlin et al., 2003; Cervantes-Cervantes et al., 2006;
Han et al., 2006). The other two genes, PaIDS 5 and
PaIDS 6, both encode geranylgeranyl diphosphate syn-
thases (GGPPS). Multiple GGPPS genes have previously
been described from H. brasiliensis and A. thaliana (Okada
et al., 1999; Takaya et al., 2003). All three P. abies genes
contain two aspartate-rich motifs that are thought to be
responsible for substrate binding and are found in all
IDS described to date (Chen et al., 1994).
A phylogenetic analysis of a range of known plant IDS
genes, excluding heterodimeric forms, shows that FPPS
sequences cluster separately from GPPS and GGPPS
sequences (Fig. 4). In this gene tree, the P. abies FPPS
and GGPPS genes cluster with others of their respective
types. However, there are two groups of GPPS/GGPPS
sequences, each of which contains one of the P. abies
GGPPS genes. The upper cluster (Fig. 4) contains other
gymnosperm GPPS and GGPPS plus PaIDS 5, while
the lower contains angiosperm GGPPS plus PaIDS 6.
These relationships suggest that PaIDS 5 may have a
specific function only in gymnosperm, like the active
formation of oleoresin and other terpene secondary
metabolites, while PaIDS 6 has a more general function
in all plants, perhaps in making GGPP for the synthesis
of gibberellins, carotenoids or the phytol side chain of
chlorophyll.
In keeping with the compartmentalization of terpenoid
metabolism in the plant cell, the encoded IDS proteins
are localized to different sites. FPPSs are located princi-
pally in the cytosol. However, for the A. thaliana FPPS1
gene, it has been shown that the use of an alternative trans-
lation start site yields a longer pre-protein which is targeted
to the mitochondria (Cunillera et al., 1997a). Nevertheless,
the P. abies IDS 4 enzyme is likely targeted to the cytosol
since no plastidial transit peptide could be detected by
the available algorithms (see Section 4).
Unlike FPPSs, most GGPPSs are localized to the plas-
tids although some are found in mitochondria or in the
endoplasmic reticulum (Cunillera et al., 1997b; Okada
et al., 2000; Bick and Lange, 2003). The PaIDS 5 and
PaIDS 6 cDNA sequences appear to have plastidial target-
ing signals. To successfully express them in E. coli, it was
necessary to truncate the first 79 amino acids from the open
reading frame of each clone.
The catalytic properties of the recombinant P. abies
FPPS and GGPPS measured in vitro are similar to those
that have been reported for other plant enzymes of these
types (e.g., Cunillera et al., 1996; Hefner et al., 1998; Tholl
et al., 2001; Burke and Croteau, 2002; Hemmerlin et al.,
2003). Product specificity of both recombinant proteins is
usually quite high. As in our study small amounts of the
intermediate product GPP was occasionally observed for
FPPS.
The kinetic constants of the recombinant P. abies
enzymes are also of the same magnitude as previously

2656
A. Schmidt, J. Gershenzon / Phytochemistry 68 (2007) 2649–2659
reported in the literature, in keeping with the high level of
amino acid identity with these groups.
4.2. Plant material
FPP and GGPP are central intermediates in plant terpe-
noid metabolism which each have a diversity of metabolic
fates. For example, FPP is a precursor of sterols, ubiqui-
none and dolichols, as well as sesquiterpenes. GGPP is a
precursor of phytol, gibberellins and carotenoids, as well
as diterpenes. To determine the possible involvement of
the isolated P. abies FPPS and GGPPS genes in forming
the sesquiterpenes and diterpenes of defensive oleoresin,
we followed changes in their transcript levels after spraying
of saplings with MJ. Treatment with MJ mimics the
response of P. abies to bark beetle attack by inducing trau-
matic resin ducts (TRDs), which leads to increased oleo-
resin biosynthesis and accumulation (Martin et al., 2002;
Byun-McKay et al., 2006; Erbilgin et al., 2006; Zeneli
et al., 2006). The transcript levels of one of the GGPPS
genes (PaIDS 5) were increased by MJ, but there was much
less effect on the other GGPPS gene (PaIDS 6) and the
FPPS gene (PaIDS 4).
Four-year-old Norway spruce (P. abies) monoclonal
saplings were used that had been purchased from the
Samenklenge und Pflanzgarten Laufen (Germany) and
are members of clone 3369-Schongau. Saplings were grown
in standard soil under a 21 ꢁC day/16 ꢁC night temperature
cycle, controlled light conditions (16 h per day at 150–
250 lE, mixture of cool white fluorescent and incandescent
light) and a humidity of 70% in a climate chamber (Vo¨tsch,
Germany). Saplings used for experiments were grown for 3
weeks under these conditions, and induced by spraying
them with 100 lM methyl jasmonate (MJ) in 0.5% (v/v)
Tween 20 as detergent. Control trees were sprayed only
with Tween 20. After various time intervals, bark (includ-
ing the cambium layer) and wood were peeled off from
the uppermost internode (representing the previous year’s
growth) and frozen in liquid nitrogen in preparation for
RNA isolation. For each time point and treatment at least
two saplings were used, respectively.
The increase of PaIDS 5 transcripts in bark (and
attached cambium) in response to MJ was over three
orders of magnitude (Fig. 7), a clear suggestion that this
gene has an important role in the induced formation of
oleoresin diterpenes. This conclusion is in agreement with
the phylogenetic analysis (Fig. 4). Here PaIDS 5 was
shown to be most closely related to other gymnosperm
sequences, and so may be assumed to share a role in typical
gymnosperm functions, like diterpene oleoresin produc-
tion. The large increase in PaIDS 5 transcript level
observed corresponds with the large increases in GGPP
synthase activity and diterpene accumulation seen after
sapling treatment with MJ (Martin et al., 2002). Even
though TRDs are located in the wood, strictly speaking,
and not in the bark, they begin developing as cambium ini-
tials. Since the bark samples used in the present study also
contain cambium tissue, the increased PaIDS 5 expression
seen in both bark and wood samples is not surprising. All
three IDS genes show at least slight activation in the wood
which may reflect the general activation of terpenoid
metabolism in TRD formation, including formation not
only of oleoresin components, but also of common cell
components, such as sterols. Given the large variety of
terpenoids produced in P. abies, there are clearly additional
IDS genes in this species. The cloning and characterization
of these genes is ongoing.
4.3. Amplification and cloning of IDS sequences
Using FPPS sequences from A. thaliana, Z. mays, H.
brasiliensis, and Lupinus albus, and GGPPS sequences from
T. canadensis, Helianthus annus, C. sublyratus, Mentha x
piperita, and L. albus, degenerate primers were designed
to different conserved regions. For the amplification of
Norway spruce FPPS cDNA fragments, the upstream pri-
mer was (50-GG(C/T)TGGTG(C/T)ATTGAATGGCT(C/
T)CA(A/G)GC-30) and the downstream primer was (50-
CTA(C/T)TT(C/T)TGCCTCTT(A/G)TAIAT(C/T)TTII
(G/C)-30). For the amplification of GGPPS fragments, the
primer combination (50-CIATG(A/C)G(G/T)TA(C/T)TCI
CTICTIGCIGG-30) and (50-CTTATCIG(C/T)(A/C)AICA
AATC(C/T)TT(C/T)CC-30) was used. Reverse transcrip-
tion was carried out with pooled total RNA from MJ-trea-
ted spruce bark harvested at various time points after
treatment (6, 12 and 24 h; 2, 4, 6, 10 and 20 days) and
oligo-(dT) primers using SuperscriptTM II (Invitrogen),
according to the manufacturer’s instructions. Subse-
quently, PCR was performed with 35 cycles of 94 ꢁC for
1 min, annealing for 1 min, and then 72 ꢁC for 1 min in a
thermocycler (Stratagene). To identify the optimal anneal-
ing temperature, a temperature gradient was used with 2 ꢁC
intervals ranging from 42 ꢁC to 64 ꢁC. The resulting PCR
products were used as templates in nested PCR with (50-
GTTGGI(A/C)TG(A/G)TTGCII(C/T)IAA(C/T)GA(C/T)-
GG-30) and (50-CC(A/T)ATCTTICC(A/C)A(G/T)I(A/
G)I(C/T)TCIGGA(G/T)CA(G/C)C-30) as up- und down-
stream primers, respectively, to amplify FPPS fragments
and (50-GA(A/G)ATGATICA(C/T)ACIATGTC-30) and
(50-CATC(A/C)ACIAC(C/T)TGAAACA(A/G)(A/C)A(A/G)
(C/T)CC-30) to amplify GGPPS fragments. A 655-bp FPPS
fragment and a 430-bp GGPPS fragment were generated at
44 ꢁC and subcloned into pCRTM 4-TOPOTM (Invitrogen)
according to the manufacturer’s instructions.
4. Experimental
4.1. Chemicals
All chemicals and solvents were of analytical grade and
were obtained from Merck (Germany), Serva (Germany)
or Sigma (Germany). The substrate [1-14C] IPP
(55 Ci molꢀ1) was purchased from Biotrend (Germany),
while unlabeled DMAPP and IPP were obtained from Ech-
elon Res. Lab. Inc. (Salt Lake City, USA).

A. Schmidt, J. Gershenzon / Phytochemistry 68 (2007) 2649–2659
2657
4.4. Isolation of full length IDS cDNA clones
containing 250 mM imidazole, proteins were checked for
purity by SDS–PAGE and their enzyme activity measured.
Total RNA was isolated according to a method devel-
oped by Wang (Wang et al., 2000) from P. abies bark har-
vested at various time points (as above) after being sprayed
with 100 lM MJ. From total pooled RNA, poly (A)+
RNA was isolated using DynabeadsTM (Dynal). cDNA
was synthesized using the SMARTTM cDNA-Library-Con-
struction Kit (Clontech). In vitro packaging with Giga-
packTM III Gold (Stratagene) yielded a library of 8 · 107
plaque-forming units, which was subsequently screened
with the 32P-labeled 655 bp FPPS and 430 bp GGPPS frag-
ments previously amplified by PCR. Single plaques were
isolated from positive colonies and kDNA was converted
into pDNA by subcloning into pTriplEx2 vector and trans-
formation in E. coli BM25.8. Sequence analysis was carried
out using an ABI 3100 automatic sequencer (Applied Bio-
systems). One sequence named PaIDS 4 was obtained from
screening with the FPPS fragment, and two others named
PaIDS 5 and PaIDS 6 from screening with the GGPPS
fragment.
4.6. Assay of recombinant PaIDS 4, PaIDS 5 and PaIDS 6
Small-scale IDS assays were carried out in a final vol-
ume of 50 ll containing 20 mM Mopso (pH 7.0), 5 mM
MgCl2 (for PaIDS 4) or 10 mM MgCl2 (for PaIDS 5),
10% (v/v) glycerol, 2 mM DTT, and [1-14C] IPP (2 MBq/
lmol) and DMAPP at saturating concentrations (800 lM
IPP and 200 lM DMAPP for PaIDS 4, and 800 lM IPP
and 250 lM DMAPP for both PaIDS 5 and PaIDS 6).
The reaction was initiated by addition of recombinant pro-
tein, and the assay mixture was overlaid with 1 ml hexane
and incubated for 20 min (PaIDS 4) or 40 min (PaIDS 5
and PaIDS 6) at 30 ꢁC. Assays were stopped by addition
of 10 ll of 3 N HCl and incubated for an additional
20 min at 30 ꢁC to solvolyze the acid-labile allylic diphos-
phates formed during the assay to their corresponding
alcohols. Solvolysis products were extracted into the hex-
ane phase by vigorous mixing for 15 s. After centrifuga-
tion, 300 ll of the hexane phase were used for total
radioactivity determination by liquid scintillation counting.
Protein concentration was measured according to Bradford
(Bradford, 1976) using the BioRad reagent with bovine
serum albumin (BSA) as standard. The PaIDS 4 reaction
was linear for up to 60 min and the PaIDS 5 reaction for
up to 30 min at protein concentrations of 40 lg/ml. To cal-
culate kinetic parameters, the Sigma plot-Enzyme kinetics
Module (SPSS, Chicago, IL, USA) was used. All assays
were carried out in triplicate.
For the identification of reaction products of for PaIDS
4, PaIDS 5 and PaIDS 6, larger scale assays were carried
out in a final volume of 500 ll. The assay mixture was as
described above but concentrations of 40 lM [1-14C] IPP,
40 lM DMAPP and a 1 ml layer of pentane were used.
Assays were incubated overnight at 30 ꢁC. To stop the
assay and hydrolyze all diphosphate esters, a 1 ml solution
of 2 units of calf intestine alkaline phosphatase (Sigma)
and 2 units potato apyrase (Sigma) in 0.2 M Tris–HCl,
pH 9.5, was added to each assay and followed by overnight
incubation at 30 ꢁC. After enzymatic hydrolysis, the result-
ing isoprenyl alcohols were extracted into 2 ml of diethyl
ether and, after addition of a standard terpene mixture;
the organic extracts were evaporated under N2 and used
for radio-GC measurements. Radio-GC analysis was per-
formed on a Hewlett Packard HP 6890 gas chromatograph
(injector at 220 ꢁC, TCD at 250 ꢁC) in combination with a
Raga radioactivity detector (Raytest, Straubenhardt, Ger-
many) using a DB 5-MS capillary column (J&W scientific)
(30 m · 0.25 mM with a 0.25 lM phase coating). Separa-
tion of the injected concentrated organic phase (1 ll) was
achieved under a H2 flow rate of 2 ml/min with a tempera-
ture program of 3 min at 70 ꢁC, followed by a gradient
from 70 to 240 ꢁC at 6 ꢁC/min with a 3 min hold at
240 ꢁC. Products measured by radio-GC, were identified
by comparison of retention times with those of co-injected
4.5. Expression of IDS sequences in E. coli
The entire coding sequence of cDNA clone PaIDS 4 and
truncated sequences of clones PaIDS 5 and PaIDS 6 lack-
ing the predicted signal peptide for plastidial transport
(based on analysis at http://www.cbs.dtu.dk/services) were
amplified with primers that included the start and stop
codon for translation. The primer combination (50-
ATGGCTTCAAACGGCATCGTCGACG-30) and (50-
CTTCTGCCGCTTGTATATCTTCCC-30) was used to
express PaIDS 4. For amplifying PaIDS 5 and PaIDS 6,
the primer pairs (50-ATGGAAGAAGTAAAGGAGGT
C-30) and (50-GTTTTGTCTGAATGCAATGTAATCTG
C-30), and (50-ATGAATGAATCAGAAAACAAAGAT
C-30) and (50-GTTCTGTCTGTGGGCAATGTAATC-30)
were used, respectively. The amplification was carried out
with the Expand High Fidelity PCR System (La Roche),
and the resulting cDNA fragments were cloned into the
expression vector pCRTM-T7 CT TOPOTM (Invitrogen)
which adds a tag containing six histidine residues to the
C-terminus. Positive clones were first transferred into
E. coli strain TOP10F’ (Invitrogen) and then into strain
BL21 (DE3)pLysS (Invitrogen). Bacterial cultures were
grown to an OD of 0.6 and transformants were induced
with 2 mM IPTG as described by the manufacturer’s
instructions, except that they were grown exclusively at
18 ꢁC. Bacterial pellets were resuspended in a buffer con-
taining 20 mM Mopso, pH 7.0, 10% (v/v) glycerol and
10 mM MgCl2, and sonified by using a Sonopuls HD
2070 (Bandelin, Berlin, Germany) for 4 min, cycle 2, power
60%. PaIDS 4, PaIDS 5 and PaIDS 6 proteins were indi-
vidually purified over Ni-NTA agarose columns (Qiagen)
according to the manufacturer’s instructions. The recombi-
nant proteins were eluted with 250 mM imidazole in the
assay buffer. After adding of 2 mM DTT to the assay buffer

2658
A. Schmidt, J. Gershenzon / Phytochemistry 68 (2007) 2649–2659
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plastidial pathways of isoprenoid biosynthesis: unidirectional trans-
port of intermediates across the chloroplast envelope membrane. Arch.
Biochem. Biophys. 415, 146–154.
authentic non-radioactive terpene standards, monitored via
a thermal conductivity detector.
4.7. Measurement of PaIDS 4, PaIDS 5, and PaIDS 6
transcripts by quantitative PCR
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of microgram quantities of protein utilizing the principle of protein-
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Total RNA from each time points and tissues was iso-
lated according to the method of Wang, (Wang et al.,
2000) except that an additional DNA digestion step was
included (RNase Free DNase Set, Qiagen). Using identical
amounts of total RNA, template cDNA for subsequent
PCR reactions was generated using SuperscriptTM III
(Invitrogen) according to the manufacturer’s instructions.
Quantitative PCR was performed with Brillantꢂ SYBR
Green QPCR Master Mix (Stratagene) and a ubiquitin
fragment of P. abies to normalize transcripts of interest.
The primers used were as follows: ubiquitin forward (50-
GTTGATTTTTGCTGGCAAGC-30) and reverse (50-
CACCTCTCAGACGAAGTAC-30), PaIDS 4 forward
(50-GTCTGTAATAGACAGCTACAGG-30) and reverse
(50-CCAGCCAAGCACACATCC-30), PaIDS 5 forward
(50-CATTTCTGGTATCATCATCTAG-30) and reverse
(50-GTCCTCCTTTACTTCTTCACG-30), and PaIDS 6
forward (50-GTTGGTTCTCTTTATCAGAC-30) and
reverse (50-CTAATGGTGTCGTTACACTG-30). At least
8 amplicons from each primer pair were cloned and
sequenced to confirm primer specificity. Transcript abun-
dance was quantified with a Mx3000P Real Time PCR
Thermocycler (Stratagene) using a program with a maxi-
mum of 45 cycles of 95 ꢁC for 30 s, 55 ꢁC (or 52 ꢁC for
PaIDS 4 and PaIDS 6) for 30 s and 72 ꢁC for 30 s, followed
by a melting curve analysis of transcripts. The transcript
abundance of each PaIDS gene was normalized to ubiqui-
tin, and the relative amount of transcript was calculated
using the software of the thermocycler. The relative
amount of transcript at the onset of treatment was used
as calibrator. Each measurement was repeated with two
independent biological replicates, each of which was repre-
sented by at least three technical replicates.
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Anatomical and chemical defenses of conifer bark against bark beetles
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Acknowledgements
We thank Marion Sta¨ger, Andrea Bergner, and Beathe
Rothe for excellent technical assistance, Kimberly Falk
for critical reading of the manuscript and the Max–
Planck-Society for financial support.
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