A Bifunctional Geranyl and Geranylgeranyl Diphosphate Synthase Is Involved in Terpene Oleoresin Formation in Picea abies

Abstract

The conifer Picea abies (Norway spruce) defends itself against herbivores and pathogens with a terpenoid-based oleoresin composed chiefly of monoterpenes (C(10)) and diterpenes (C(20)). An important group of enzymes in oleoresin biosynthesis are the short-chain isoprenyl diphosphate synthases that produce geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)), and geranylgeranyl diphosphate (C(20)) as precursors of different terpenoid classes. We isolated a gene from P. abies via a homology-based polymerase chain reaction approach that encodes a short-chain isoprenyl diphosphate synthase making an unusual mixture of two products, geranyl diphosphate (C(10)) and geranylgeranyl diphosphate (C(20)). This bifunctionality was confirmed by expression in both prokaryotic (Escherichia coli) and eukaryotic (P. abies embryogenic tissue) hosts. Thus, this isoprenyl diphosphate synthase, designated PaIDS1, could contribute to the biosynthesis of both major terpene types in P. abies oleoresin. In saplings, PaIDS1 transcript was restricted to wood and bark, and transcript level increased dramatically after methyl jasmonate treatment, which induces the formation of new (traumatic) resin ducts. Polyclonal antibodies localized the PaIDS1 protein to the epithelial cells surrounding the traumatic resin ducts. PaIDS1 has a close phylogenetic relationship to single-product conifer geranyl diphosphate and geranylgeranyl diphosphate synthases. Its catalytic properties and reaction mechanism resemble those of conifer geranylgeranyl diphosphate synthases, except that significant quantities of the intermediate geranyl diphosphate are released. Using site-directed mutagenesis and chimeras of PaIDS1 with single-product geranyl diphosphate and geranylgeranyl diphosphate synthases, specific amino acid residues were identified that alter the relative composition of geranyl to geranylgeranyl diphosphate.

Full text

A Bifunctional Geranyl and Geranylgeranyl Diphosphate
Synthase Is Involved in Terpene Oleoresin Formation in
Picea abies1[W][OA]
Axel Schmidt, Betty Wa
¨chtler2, Ulrike Temp, Trygve Krekling, Armand Se
´guin, and Jonathan Gershenzon*
Max Planck Institute for Chemical Ecology, Department of Biochemistry, Beutenberg Campus, D–07745 Jena,
Germany (A. Schmidt, B.W., U.T., J.G); Department of Plant and Environmental Sciences, Norwegian
University of Life Sciences, N–1432 A
˚s, Norway (T.K.); and Natural Resources Canada, Canadian Forest
Service, Laurentian Forestry Centre, Quebec, Quebec, Canada G1V 4C7 (A. Se
´guin)
The conifer Picea abies (Norway spruce) defends itself against herbivores and pathogens with a terpenoid-based oleoresin
composed chiefly of monoterpenes (C10) and diterpenes (C20). An important group of enzymes in oleoresin biosynthesis are the
short-chain isoprenyl diphosphate synthases that produce geranyl diphosphate (C10), farnesyl diphosphate (C15), and
geranylgeranyl diphosphate (C20) as precursors of different terpenoid classes. We isolated a gene from P. abies via a
homology-based polymerase chain reaction approach that encodes a short-chain isoprenyl diphosphate synthase making an
unusual mixture of two products, geranyl diphosphate (C10) and geranylgeranyl diphosphate (C20). This bifunctionality was
confirmed by expression in both prokaryotic (Escherichia coli) and eukaryotic (P. abies embryogenic tissue) hosts. Thus, this
isoprenyl diphosphate synthase, designated PaIDS1, could contribute to the biosynthesis of both major terpene types in P. abies
oleoresin. In saplings, PaIDS1 transcript was restricted to wood and bark, and transcript level increased dramatically after
methyl jasmonate treatment, which induces the formation of new (traumatic) resin ducts. Polyclonal antibodies localized the
PaIDS1 protein to the epithelial cells surrounding the traumatic resin ducts. PaIDS1 has a close phylogenetic relationship to
single-product conifer geranyl diphosphate and geranylgeranyl diphosphate synthases. Its catalytic properties and reaction
mechanism resemble those of conifer geranylgeranyl diphosphate synthases, except that significant quantities of the
intermediate geranyl diphosphate are released. Using site-directed mutagenesis and chimeras of PaIDS1 with single-product
geranyl diphosphate and geranylgeranyl diphosphate synthases, specific amino acid residues were identified that alter the
relative composition of geranyl to geranylgeranyl diphosphate.
Conifers are frequently subject to attack by herbiv-
orous insects and fungal pathogens (Phillips and
Croteau, 1999; Trapp and Croteau, 2001; Franceschi
et al., 2005; Keeling and Bohlmann, 2006a). However,
the long life span and evolutionary persistence of these
trees suggest that they possess effective defense strat-
egies. The best known example of conifer chemical
defense is oleoresin, a viscous mixture of terpenoids
found in specialized ducts. Oleoresin may be both a
constitutive and an inducible defense. For example, in
Picea abies (Norway spruce), resin ducts are found
constitutively in bark and foliage. However, this spe-
cies also forms new (traumatic) resin ducts in the
wood in response to attack by stem-boring insects and
their associated fungi or after trees are sprayed with
methyl jasmonate (MJ). Traumatic ducts are believed
to help resist attack by augmenting the constitutive
resin flow to provide a stronger physical and chemical
barrier against herbivores 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, 2006a).
Terpenoids are the largest class of plant secondary
metabolites, with more than 30,000 structural variants.
Oleoresin consists mainly of monoterpenes (C10) and
diterpene resin acids (C20) as well as smaller amounts
of sesquiterpenes (C15; Langenheim, 2003). The bio-
synthesis of oleoresin, like all other terpenoids, begins
with the synthesis of isopentenyl diphosphate (IPP)
via the mevalonic acid pathway or the methylerythri-
tol phosphate pathway (Gershenzon and Kreis, 1999;
Fig. 1). IPP and its isomer, dimethylallyl diphosphate
(DMAPP), are the five-carbon building blocks of ter-
penoids that undergo successive condensation reactions
to form the larger intermediates geranyl diphosphate
(GPP; C10), farnesyl diphosphate (FPP; C15), and ger-
anylgeranyl diphosphate (GGPP; C20). These terpene
diphosphate intermediates are in turn the precursors
of monoterpenes, sesquiterpenes, and diterpenes, re-
spectively, as well as many larger products (Fig. 1).
1
This work was supported by the Max Planck Society.
2
Present address: Department of Microbial Pathogenicity Mech-
anisms, Leibniz Institute for Natural Product Research and Infection
Biology, D–07745 Jena, Germany.
* Corresponding author; e-mail gershenzon@ice.mpg.de.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Jonathan Gershenzon (gershenzon@ice.mpg.de).
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a sub-
scription.
www.plantphysiol.org/cgi/doi/10.1104/pp.109.144691
Plant PhysiologyÒ,February 2010, Vol. 152, pp. 639–655, www.plantphysiol.org Ó2009 American Society of Plant Biologists 639

The enzymes catalyzing the condensations of IPP
and DMAPP to GPP, FPP, and GGPP are referred to
collectively as short-chain isoprenyl diphosphate
synthases (IDSs), members of a large enzyme class
known as prenyltransferases (Kellogg and Poulter,
1997; Ogura and Koyama, 1998; Liang et al., 2002;
Liang, 2009). IDSs have been frequently studied be-
cause they direct flux into different branches of terpe-
noid biosynthesis and so control product distribution.
GPP, FPP, and GGPP are each formed by a specific,
short-chain IDS: GPP synthase (EC 2.5.1.1) condenses
DMAPP with one molecule of IPP; FPP synthase (EC
2.5.1.10) condenses DMAPP successively with two
IPP molecules; and GGPP synthase (EC 2.5.1.30) con-
denses DMAPP successively with three IPP molecules
(Gershenzon and Kreis, 1999; Fig. 1). Plant short-chain
IDSs have been the subject of much research in recent
years, but comparatively little attention has been paid
to the enzymes in conifers (Hefner et al., 1998; Tholl
et al., 2001; Burke and Croteau, 2002b; Martin et al.,
2002; Schmidt et al., 2005; Schmidt and Gershenzon,
2007, 2008).
In addition to the short-chain IDSs, there are other
types. Medium- and long-chain IDSs catalyze the
formation of products with more than 20 carbon
atoms, such as intermediates in ubiquinone, plasto-
quinone, dolichol, and rubber biosynthesis (Liang
et al., 2002; Kharel and Koyama, 2003; Liang, 2009).
IDS proteins can also be classified by the configuration
of the double bond formed. The major short-chain
prenyl diphosphate intermediates, such as GPP, FPP,
and GGPP, all have (E)-double bonds, and most short-
chain IDSs reported make products with (E)-double
bonds. However, two enzymes making (Z)-products
have recently been described (Sallaud et al., 2009;
Schilmiller et al., 2009). IDSs producing (Z)-products
show little sequence similarity to those making (E)-
products (Wang and Ohnuma, 2000; Liang et al., 2002;
Liang, 2009). The vast majority of (E)-product, short-
chain IDSs have a homodimeric architecture, but the
existence of heterodimeric GPP synthases has been
well documented (Burke et al., 1999; Tholl et al., 2004).
A recent study suggests that these enzymes may be
widespread in the plant kingdom (Wang and Dixon,
2009).
Most of the short-chain, homodimeric IDSs de-
scribed make only a single main product, usually
GPP, FPP, or GGPP. However, products with one more
or one fewer C5unit than the main product are
occasionally reported at low levels in in vitro assays.
For example, FPP synthase from maize (Zea mays)
produces up to 10% GGPP (Cervantes-Cervantes et al.,
2006), and a GPP synthase from the orchid Phalaenopsis
bellini produces nearly 20% FPP (Hsiao et al., 2008).
However, it is not clear if these enzymes have such a
broad product spectrum in vivo. And no example is
Figure 1. Outline of terpenoid biosynthesis leading to the major conifer oleoresin components, monoterpenes and diterpenes, as
well as to other classes of terpenes or compounds with terpene components. In the first phase of terpenoid biosynthesis, IPPand
DMAPP are formedvia the plastidial methylerythritol phosphate pathway and the cytosolic mevalonate pathway. The next phase
consists of the reactions catalyzed by short-chain IDSs, GPP synthase, FPP synthase, and GGPP synthase. GPP synthase
condenses one molecule of DMAPP and one molecule of IPP. FPP synthase condenses one molecule of DMAPP with two
molecules of IPP in succession. GGPP synthase condenses one molecule of DMAPP with three molecules of IPP in succession.
During these repeated condensations, the intermediate prenyl diphosphates are normally bound and not released by the
enzymes. The PaIDS1 protein is believed to act like a GGPP synthase, but it releases a significant portion of the GPP formed as an
intermediate. The remainder of the GPP is converted directly to GGPP without release of FPP. OPP indicates a diphosphate
group.
Schmidt et al.
640 Plant Physiol. Vol. 152, 2010

yet known of such a short-chain IDS making two
products that differ from each other in size by more
than one C5unit.
The amino acid sequences of short-chain plant
IDSs making products with (E)-double bonds show
significant amino acid similarity and contain two
highly conserved regions with numerous Asp resi-
dues, designated the first and second Asp-rich motifs,
respectively (Ashby et al., 1990; Fig. 2). Site-directed
mutagenesis studies found that most of the Asp res-
idues in these two highly conserved motifs are critical
for substrate binding and catalysis (Koyama et al.,
1995, 1996). The amino acid sequence features that
determine the chain length of the product have been
studied by random chemical mutagenesis and exper-
iments on an avian FPP synthase, from which an x-ray
structure has been obtained (Tarshis et al., 1994). Here,
it was found that the fifth amino acid residue before
the first Asp-rich motif is the key residue in determin-
ing product chain length. Replacement of the Phe at
this position with smaller amino acids resulted in the
product specificity shifting to GGPP or longer pro-
ducts. Conversely, replacement of a small amino acid
with a larger one shortened the chain length of the
product to GPP (Fernandez et al., 2000).
Here, we report the characterization of a new IDS
from P. abies that possesses the unique ability to
produce both GPP (C10) and GGPP (C20) but not FPP
(C15). The product spectrum of this enzyme, its cellular
location, and the expression pattern of the correspond-
ing gene all suggest the involvement of this enzyme in
oleoresin biosynthesis.
RESULTS
Isolation of a P. a b i e s I D S cDNA Clone and
Sequence Comparison
The isolation of IDS genes from P. abies was under-
taken by PCR using primers designed from conserved
regions of known plant GGPP synthases with P. abies
RNA as the template. To find IDSs with a role in
Figure 2. Alignment of deduced amino acid sequences of PaIDS1 (accession no. GQ369788) and selected additional conifer
IDSs, including PaIDS2 (EU432047), A. grandis GPP synthase1 (AgGPPS1; AAN01133), AgGPPS2 (AAN01134), AgGPPS3
(AAN01135), PaIDS5 (EU432050), PaIDS6 (EU432051), and AgGGPPS (AAL17614). Identical amino acids are boxed in black.
The artificial translation initiation site for PaIDS1 is indicated by a triangle. The Asp-rich motifs conserved among IDSs are
indicated by the black lines. The amino acid sequence used for the generation of peptide antibodies is marked by the dotted line.
Dashes indicate sequence gaps introduced to optimize the alignment.
Characterization of a Bifunctional IDS
Plant Physiol. Vol. 152, 2010 641

oleoresin formation, the fragments obtained were then
employed to screen cDNA libraries constructed with
mRNA isolated from saplings that had been treated
with MJ to induce oleoresin accumulation. A full-
length clone was obtained (PaIDS1) that was repre-
sented by four different sequences differing from each
other only in the 3#untranslated region, which con-
sisted of 66 to 132 nucleotides between the stop codon
and the poly(A)+tail. The protein encoded by PaIDS1
consisted of 383 amino acids and had a calculated
mass of 41.8 kD. The amino acid sequence had the
highest similarity to other conifer GPP and GGPP
synthase sequences (Table I), reaching 86% identity to
the GPP synthase 1 of Abies grandis and 84% and 82%
identity to GGPP synthases from P. abies (PaIDS5) and
A. grandis, respectively (Burke and Croteau, 2002b;
Schmidt and Gershenzon, 2007). Other conifer GPP
synthase sequences, like PaIDS2 and PaIDS6 from P.
abies and the GPP synthases 2 and 3 from A. grandis,
showed 55% to 70% identity to PaIDS1 (Burke and
Croteau, 2002b; Schmidt and Gershenzon, 2008). The
two Asp-rich motifs, DDxxxxDxDDxRRG and DDxxD,
found in other plant IDS proteins forming products
with (E)-configurations are both conserved in the gene
(Fig. 2). The TargetP 1.1 software (http://www.cbs.
dtu.dk/services/TargetP; Emanuelsson et al., 2000)
predicted a chloroplast signal peptide for PaIDS1 at
the 5#end of the cDNA (Fig. 3). When the PaIDS1
sequence was subjected to a phylogenetic analysis
with the known conifer IDS genes showing GPP or
GGPP synthase activity, it clustered closely together
with the GPP synthase1 gene of A. grandis and associ-
ated at a greater distance with the GGPP synthase
genes PaIDS5 and AgGGPP synthase (Fig. 3). The other
GPP and GGPP synthase gene sequences isolated from
P. abies and A. grandis clustered more distantly.
Heterologous Expression in Escherichia coli and
Characterization of PaIDS1
To study the enzymatic activity of the isolated clone,
the protein encoded by PaIDS1 was expressed in E. coli
after truncation of the signal sequence at the 5#end of
the coding region for directing chloroplast import.
Analysis of crude bacterial extracts of the expressing
cultures with SDS-PAGE revealed a recombinant pro-
tein band with an apparent molecular mass of ap-
proximately 40 kD. These extracts were subsequently
purified on a nickel-nitrilotriacetic acid agarose col-
umn (Supplemental Fig. S1). When incubated with
[1-14C]IPP and DMAPP as substrates, the purified
recombinant PaIDS1 protein exhibited both GPP and
GGPP synthase enzyme activity, whereas FPP was not
detected (Fig. 4). The ratio of GPP to GGPP produced
averaged approximately 9:1, taking into account the
fact that 1 mol of GGPP (which is formed from three
IPP units and one of DMAPP) should incorporate
three times as much radioactivity from [1-14C]IPP as
1 mol of GPP (formed from one IPP and one DMAPP).
Neither the empty vector controls nor heat-treated
assays showed any significant enzyme activity.
To explore the mechanism of GGPP (C20) formation,
the shorter allylic diphosphates, GPP (C10) and FPP
(C15), were offered as substrates. Like DMAPP (C5),
both GPP and FPP were incorporated into GGPP in the
presence of IPP (Fig. 5), suggesting that reaction pro-
ceeds by a sequential addition of IPP units as for other
IDS proteins. The kinetic constants of the recombinant
PaIDS1 (measured under linear conditions with re-
spect to time and protein concentration) showed an
apparent Kmvalue of 390 mMfor DMAPP, with the IPP
concentration constant at 250 mM, and 170 mMfor IPP,
with the DMAPP concentration constant at 800 mM
(for Lineweaver-Burk plots, see Supplemental Fig. S2).
The Vmax was 4.2 nkat mg21and the kcat was 1.4 s21.
Activity required MgCl2and reached its highest value
at 10 mMMgCl2. At 5 or 15 mMMgCl2, activity was
Figure 3. Phylogenetic tree of the deduced amino acid sequences of
PaIDS1 and other gymnosperm GPP synthase (GPPS) and GGPP
synthase (GGPPS) sequences isolated from P. abies and A. grandis.
The program DNA Lasergene (MegAlign) was used to create a neighbor-
joining tree. The accession numbers of the sequences used are listed in
Figure 2. Not all known conifer GPP synthase and GGPP synthase
sequences were included.
Table I. Sequence relatedness of spruce and fir IDSs
Sequence Relatedness
PaIDS1 100
PaIDS2 76.3 100
AgGPPS1 86.1 74.1 100
AgGPPS2 74.6 76.3 74.1 100
AgGPPS3 71.6 72.2 71.4 75.0 100
PaIDS5 83.6 75.4 83.2 75.8 74.4 100
PaIDS6 69.3 63.2 66.5 63.6 63.1 67.0 100
AgGGPPS 82.4 74.9 81.7 75.3 75.0 91.3 65.7 100
PaIDS1 PaIDS2 AgGPPS1 AgGPPS2 AgGPPS3 PaIDS5 PaIDS6 AgGGPPS
Schmidt et al.
642 Plant Physiol. Vol. 152, 2010

only 65% of wild-type PaIDS1, and no change of the
product profile was observed (data not shown).
PaIDS1 Expression in Transgenic P. a b i e s
Embryogenic Tissue
To confirm the surprising bifunctional GPP and
GGPP synthase activity of PaIDS1 in an appropriate
eukaryotic expression system, the full-length PaIDS1
gene was introduced into embryogenic tissue of P.
abies under the control of the ubi1 promoter via Agro-
bacterium tumefaciens transformation. Four transgenic
lines were obtained that tested positive for the pres-
ence of both the nptII and uidA genes (data not shown).
The relative abundance of the PaIDS1 gene in pooled
samples from these four lines was about 10-fold higher
than in either the vector control or untransformed
plants, and PaIDS1 mRNA transcript levels in embry-
ogenic tissue were around 3,000-fold higher than in
either the vector control or untransformed controls,
indicating the success of the transformation (Fig. 6).
Enzyme assays performed with crude protein extracts
of PaIDS1-transformed embryogenic tissue exhibited
both GPP and GGPP synthase enzyme activity when
incubated with IPP and DMAPP as substrates,
whereas only traces of FPP were observed, just as for
the assay of PaIDS1 protein expressed in E. coli (Fig. 7).
Crude protein extracts of empty vector control lines
and untransformed embryogenic cultures of P. abies
exhibited mainly FPP synthase enzyme activity, with
only traces of GPP and GGPP.
Comparison of PaIDS1 Transcript Levels among Organs
and after MJ Treatment
Terpene oleoresin is synthesized in ducts found
in wood, bark, and needles. To determine whether
PaIDS1 is localized in these organs, transcript levels
were measured in P. abies saplings by quantitative real-
time PCR (qRT-PCR). The highest transcript level was
found in wood (Fig. 8). PaIDS1 transcripts in bark were
only present at about 20% of the level of the wood,
whereas no transcripts were detectable in needles.
To determine whether the PaIDS1 enzyme had a role
in induced oleoresin formation in P. abies stems, tran-
script levels were measured over a 10-d time course
after saplings had been sprayed with MJ. In bark (and
attached cambium), transcripts of the IDS1 gene in-
creased continuously and reached a maximum at day
8, representing a 350-fold increase from the first time
point (Fig. 9). In wood, the PaIDS1 genes also showed
a transcript accumulation upon MJ spraying; however,
the magnitude was less than in bark. Here, maximum
induction, with a relative abundance of almost 14-fold
relative to the first time point, occurred 2 d after MJ
treatment (Fig. 9).
Figure 4. Catalytic activities of recombinant PaIDS1 protein heterologouslyexpressed in E. coli and assayed with [1-14C]IPP and
DMAPP. Reaction products were hydrolyzed enzymatically, and the resulting alcohols were analyzed by radio-gas chroma-
tography. The main hydrolysis products detected were geraniol (G) and geranylgeraniol (GG), indicating that GPP and GGPP
were the principal enzyme products. No release of FPP (detected as farnesol [F]) was measured(third panel from top). The ratio of
GPP to GGPP was calculated as approximately 9:1, taking into account that 1 mol of GGPP incorporates three times as much
radioactivity from IPP (three units) as GPP does (one IPP unit). The substrate IPP was hydrolyzed to some extent, but the resulting
isopentenol eluted close to the solvent front and is not shown. Purified protein extracts of bacteria expressing the empty vector or
boiled protein extracts of bacteria expressing PaIDS1 did not show any measurable activity (two top panels). Compounds were
identified by coinjection of standards, as depicted in the thermal conductivity detector trace (bottom panel). Standards included
geraniol (23 min), nerol (27 min), linalool (29 min), (E,E)-farnesol (33 min), and (E,E,E)-geranylgeraniol (40.5 min). At least five
different replicates of each sample were analyzed, and the variance was below 5%.
Characterization of a Bifunctional IDS
Plant Physiol. Vol. 152, 2010 643

Localization of PaIDS1 Protein in Resin Duct
Epithelial Cells
Antiserum raised against PaIDS1 gave a single
strong band on western blots with extracts of E. coli
heterologously expressing the recombinant protein,
but it did not yield signals with extracts expressing
PaIDS2 or PaIDS5, the most similar isolated IDS en-
zymes from P. abies (Schmidt and Gershenzon, 2007,
2008). The molecular mass of the detected band was
approximately 42 kD on SDS-PAGE (data not shown),
consistent with the predicted molecular mass of the
PaIDS1 protein. In sections of P. abies stems treated
with the fungus Ceratocystis polonica to induce oleo-
resin formation, immunogold labeling of the PaIDS1
antiserum appeared extensively in epithelial cells that
line the inner surface of traumatic resin ducts (Fig. 10).
These cells are thought to synthesize the oleoresin
components and secrete them into the ducts. There
was no reproducible immunogold labeling above the
background signal in any other cell type or in sections
treated with preimmune serum only. For comparison,
antiserum raised against a P. abies FPP synthase
(PaIDS4) that is not involved in induced oleoresin
defense reactions (Schmidt and Gershenzon, 2007) was
also tested. This did not show any signals, either in
stem cells in C. polonica-treated tissue or in the un-
treated controls (data not shown).
Alteration of PaIDS1 Product Specificity by Site-Directed
Mutagenesis and the Creation of Chimeric IDS Proteins
To explore the contribution of individual amino acid
residues to the unusual product spectrum of PaIDS1,
site-directed mutagenesis was performed on selected
positions in or around the regions previously shown to
be important in the product length determination
of IDS proteins (Ohnuma et al., 1996a, 1996b, 1997;
Tarshis et al., 1996; Fernandez et al., 2000). In several
cases, residues were changed to those found in previ-
ously characterized single-product GPP or GGPP syn-
thases from conifers to see if this would make PaIDS1 a
single-product enzyme.
The majority of the mutated proteins showed no
difference in product composition from that of the
wild-type enzyme (Table II). However, changes in
residues at some positions within the first Asp-rich
motif, known to be involved in substrate binding, did
cause significant changes in the products of PaIDS1
catalysis. When a Met residue at position 175 (Fig. 11)
was changed to Ile, as found in the sequence of the P.
abies GPP synthase PaIDS2, the product distribution
shifted toward more GPP. While the wild-type enzyme
produced 90% GPP and 10% GGPP, the M175I mutant
formed 95% GPP plus small amounts of both GGPP
and FPP (Fig. 12). The immediately adjacent residue
174 is a Pro in the wild-type PaIDS1 and a Cys in
PaIDS2. Mutation of Pro to Cys (P174C) caused no
change in product composition, but when combined
with the previously mentioned mutation (P174C and
M175I), the product composition shifted completely to
100% GPP (Fig. 12).
Previous studies on IDS enzymes had shown that
the fifth amino acid prior to the first Asp-rich motif
(position 165 in PaIDS1) is particularly critical in
determining product size (Fig. 11). Larger amino acids
block extension of the chain, leading to a decrease in
product length. Consistent with this trend, mutation of
Figure 5. Catalytic activities of recombinant PaIDS1
protein heterologously expressed in E. coli and
assayed with combinations of radioactive and non-
radioactive IPP, GPP, and FPP. Substrates and reaction
products were hydrolyzed enzymatically, and the
resulting alcohols were analyzed by radio-gas chro-
matography (for details of compound identification,
analysis, and abbreviations, see Fig. 4). No formation
of GGPP was found after using only [1-3H]GPP as a
substrate (top panel). However, GPP or FPP together
(radioactive or nonradioactive) with IPP were ac-
cepted as substrates for PaIDS1 and led to the forma-
tion of radioactively labeled GGPP (bottom panels),
indicating that catalysis proceeds by sequential ad-
dition of IPP units. At least three biological replicates
of each sample were analyzed, and the variance was
below 5%.
Schmidt et al.
644 Plant Physiol. Vol. 152, 2010

residue 165 from Met to the bulkier Tyr eliminated all
GGPP formation, triggering the formation of 10% FPP
with 90% GPP (Fig. 12). When the Met at 165 was
mutated to the smaller Cys instead, there was no effect
on product distribution. Two other sequence changes
based on differences between PaIDS1 and other P. abies
or A. grandis IDS, I235T and L273F, completely elimi-
nated the formation of the longer GGPP, leaving only
GPP as the product.
Mutations to residues found in single-product
GGPP synthases of the conifers P. abies and A. grandis
gave mixed results. When Leu at position 180 was
changed to Phe, there was a significant increase in GPP
and a decrease in GGPP. However, the double mutant,
L180F and P174C, gave a significant increase in GGPP
from 10% to 35% at the expense of GPP (Fig. 12).
As another approach to investigating the structure-
function relationships of PaIDS1, we created chimeric
proteins assembled from PaIDS1 and two other P. abies
IDS enzymes, the GPP synthase, PaIDS2, and the GGPP
synthase, PaIDS5. Dividing the proteins between the
two Asp-rich motifs at residues 213 to 216 (Fig. 2), we
produced chimeras in which either the N-terminal or
C-terminal portion of PaIDS1 was exchanged for the
corresponding portion of PaIDS2 or PaIDS5. The pro-
teins in which the PaIDS1 N-terminal sequence had
been exchanged, PaIDS2-1 and PaIDS5-1, had product
spectra according to the origin of their N-terminal
sequences. PaIDS2-1, which contained the N-terminal
sequence of PaIDS2 (a pure GPP synthase) and the
C-terminal sequence of PaIDS1, produced 95% GPP
and 5% GGPP (Fig. 13). Meanwhile, PaIDS5-1, which
contained the N-terminal sequence of PaIDS5 (a pure
GGPP synthase) and the C-terminal sequence of PaIDS1,
produced 40% GPP and 60% GGPP. However, the
PaIDS1-2 and PaIDS1-5 proteins, which contained the
N-terminal sequence of PaIDS1 and the C-terminal
sequence of either PaIDS2 or PaIDS1-5, did not pro-
duce any GGPP, but only 80% GPP and 20% FPP (Fig.
13). All of the chimeric proteins had only 10% to 15%
of the activity of wild-type PaIDS1.
DISCUSSION
PaIDS1 Is an Unusual Short-Chain IDS That Produces
Both GPP and GGPP
The short-chain IDSs combine the basic C5units of
the terpenoid pathway into C10 (GPP), C15 (FPP), and
C20 (GGPP) intermediates and so control the levels of
precursors for different classes of terpenes (Fig. 1). We
have been investigating the genes encoding the short-
chain IDSs of P. abies to learn more about the control
of terpene oleoresin formation in conifers, which is a
mixture of monoterpenes (C10) and diterpenes (C20).
In previous investigations, we have isolated IDSs with
GPP synthase, FPP synthase, and GGPP synthase ac-
tivities (Schmidt and Gershenzon, 2007, 2008). Here,
we report an unusual IDS (PaIDS1) that produces both
GPP and GGPP in substantial amounts, but no FPP.
This novel activity was first demonstrated by heterol-
ogous expression in E. coli (Fig. 4) and then confirmed
by expression in embryonic tissue of P. abies that was
transformed via A. tumefaciens with the PaIDS1 gene
under the control of a constitutive promoter (Figs. 6
and 7). To our knowledge, this is the first report of A.
tumefaciens-mediated gene transfer in conifers involv-
ing the overexpression of a characterized endogenous
gene. Previously, the overexpression of an endogenous
cinnamoyl alcohol dehydrogenase gene was achieved
in Pinus radiata by particle bombardment, but this
transformation caused cosuppression, resulting in
lower activity of the encoded enzyme (Mo
¨ller et al.,
2003). In another study, even though overexpression of
an endogenous peroxidase-like gene in P. abies showed
a higher level of total peroxidase activity, no corre-
lation to lignin polymer composition was found
(Elfstrand et al., 2001). The ratio of GPP to GGPP
formed by PaIDS1 was approximately 9:1 (Fig. 4).
PaIDS1 is, to our knowledge, the first short-chain,
homodimeric IDS described that makes two major
products with such a large difference in size. Reports
on other bifunctional IDSs indicate that enzymes mak-
Figure 6. Relative abundance of PaIDS1 gene copies and mRNA
transcripts in embryogenic tissue transformed with PaIDS1.Geneabun-
dance was measured by quantitative genomic PCR (top), and transcript
abundance of the IDS1 gene was measured by qRT-PCR (bottom), both of
which employed SYBR Green for detection and the ubiquitin gene for
normalization (for details, see text). Samples consisted of embryogenic
tissue transformed with PaIDS1, tissue transformed with the empty vector,
and wild-type tissue. Abundance of the vector control was set to 1.0.
Each value is the average of four biological replicates, each of which is
represented by at least four technical replicates.
Characterization of a Bifunctional IDS
Plant Physiol. Vol. 152, 2010 645

ing predominantly a single product in in vitro assays
can also catalyze the formation of lower amounts of
products with one additional or one fewer C5unit. For
example, there are GPP synthases that also make low
amounts of FPP (Burke and Croteau, 2002b; Hsiao et al.,
2008), FPP synthases that also make GGPP (Cervantes-
Cervantes et al., 2006), and GGPP synthases that pro-
duce minor amounts of FPP (Takaya et al., 2003).
Interestingly, bifunctionality is not limited to plants.
For example, Vandermoten et al. (2008) described a GPP
synthase from Myzus persicae producing significant
amounts of FPP. Other dual FPP/GGPP synthases
have been reported for a protozoan and a hyperther-
mophilic archaeon (Chen and Poulter, 1994; Ling et al.,
2007). However, there are no other reports of a homo-
dimeric, short-chain IDS that makes multiple products
differing by 10 or more carbon atoms.
Bifunctional activities observed for short-chain IDSs
in general may be examples of “catalytic promiscuity,”
an expression that was coined to describe the ability
of some enzymes to display an adventitious second-
ary activity at the active site responsible for the primary
activity (Copley, 2003). If this adventitious secondary
activity becomes useful to the organism at some point,
the enzyme may be recruited to provide more of the
secondary product (Vandermoten et al., 2009). The
existence of a bifunctional protein, such as PaIDS1,
that produces precursors for both monoterpene and
diterpene formation is not surprising, as monoterpenes
and diterpenes occur together as principal constituents
of oleoresin.
PaIDS1 Has the Properties of a GGPP Synthase That
Releases a Substantial Portion of the Intermediate GPP
The enzymological properties of PaIDS1 are gener-
ally similar to those of single-product GPP and GGPP
synthases characterized from P. abies and other coni-
fers. All of these enzymes share a requirement for the
same divalent metal ion, Mg2+. While the Kmvalues of
PaIDS1 for the substrates IPP and DMAPP are some-
what greater than those determined for other short-
chain conifer IDSs, the ratio between the Kmfor
DMAPP and that for IPP is approximately the same.
The substrate specificity of PaIDS1, its ability to use
other allylic diphosphates (Fig. 5), also parallels that
of GGPP synthases from conifers and other species
(Hefner et al., 1998; Ogura and Koyama, 1998; Takaya
et al., 2003), suggesting that reaction also involves
successive condensation of IPP units (Fig. 1). These
successive condensations usually proceed without
significant release of the intermediates GPP and FPP.
PaIDS1, therefore, may be simply a GGPP synthase in
Figure 8. Relative abundance of PaIDS1 mRNA transcripts in different
plant organs. Transcript abundance was measured by qRT-PCR using
SYBR Green for detection and the ubiquitin gene for normalization (for
details, see text). Each value is the average of three biological repli-
cates, each of which is represented by three technical replicates. Values
for wood and needles are expressed relative to bark, whichwas set to 1.
n.d., Not detectable.
Figure 7. IDS activity of crude protein extract of embryogenic tissue transformed with a full-length construct of PaIDS1 and
assayed in vitro with [1-14C]IPPand DMAPP. Reaction products were enzymatically hydrolyzed, and the resulting alcohols were
analyzed by radio-gas chromatography. For details and abbreviations, see Figure 4. The main products of the PaIDS1 reaction
were GPP (G) and GGPP (GG; bottom panel). Only traces of FPP (F) were detected. An extract of embryogenic tissue transformed
with the empty vector control showed only FPP synthase activity (top panel). Compounds were identified by coinjection of
standards, as depicted in the thermal conductivity detector trace (for details, see Fig. 4). At least four biological replicates of each
sample were analyzed, and the variance was below 5%.
Schmidt et al.
646 Plant Physiol. Vol. 152, 2010

which a large portion of the intermediate GPP is
released, while the remainder continues along the
reaction path toward GGPP. Unlike GPP, the interme-
diate FPP is not released.
To assess what structural features of PaIDS1 might
be responsible for the simultaneous formation of GPP
and GGPP, a program of site-directed mutagenesis
was undertaken. This effort relied on modeling of
PaIDS1 with reference to an avian FPP synthase (Tarshis
et al., 1994), the only short-chain IDS for which a
crystal structure is available. The sequence of this
avian FPP synthase shares 30% amino acid identity
to PaIDS1. Its structure is composed of 13 a-helices,
of which 10 form a large central cavity (Fig. 11). The
active site is located in the central cavity with two
conserved Asp-rich motifs facing each other on oppo-
site walls. These motifs have been demonstrated to
be involved in substrate binding and catalysis via
coordination with Mg2+, hence the requirement of this
avian FPP synthase and other IDSs for this cofactor
(Marrero et al., 1992; Joly and Edwards, 1993; Song
and Poulter, 1994; Koyama et al., 1994, 1995, 1996). In
PaIDS1, the first Asp-rich region spans amino acid
residues 170 to 183 and is located in helix D and an
adjacent loop, while the second region spans residues
309 to 313 and is located in helix H. These two helices,
as well as helix F1, form a significant portion of the
active site (depicted in Fig. 11).
In site-directed mutagenesis, selected residues of
PaIDS1 were changed to those present in other conifer
IDSs that form either GPP or GGPP as a single product.
A double mutant at P174C and M175I that changed
these residues to those present in single-product GPP
synthases (Burke and Croteau, 2002b; Schmidt and
Gershenzon, 2008) could catalyze only the formation
of GPP and no GGPP. The residues at positions 174
and 175 are thought to have been inserted in the IDS
sequence during evolution from bacteria to plants
(Wang and Ohnuma, 1999) and are clearly important
in determining product specificity. Mutation of the
fifth amino acid prior to the first Asp-rich motif
(M165Y) also caused the production of shorter prod-
ucts. This residue, which projects into the active site
cavity (Fig. 11), has been shown to influence product
length in other short-chain IDSs, with large, bulky R
groups appearing to block additional condensation
with IPP and thus to give shorter chain products
(Ohnuma et al., 1996b; Fernandez et al., 2000; Soderberg
et al., 2001). Here, mutagenesis from Met to Tyr gave
Figure 9. Relative abundance of PaIDS1 mRNA transcripts in MJ-
treated P. abies saplings. Transcript abundance of the IDS1 gene was
measured by qRT-PCR using SYBR Green for detection and the ubiq-
uitin gene for normalization (for details, see text). The time points
measured were 0, 0.5, 1, 2, 4, 6, 8, and 10 d after the onset of treatment.
Bark samples included the cambium. The time-zero measurement is the
untreated control; its abundance was set to 1.0. Each value is the
average of four biological replicates, each of which is represented by at
least three technical replicates.
Figure 10. Immunogold labeling using a PaIDS1 polyclonal antibody
applied to stem sections of P. abies that had been treated with the
fungus C. polonica. Gold particles were observed only in epithelial
cells (ec) lining the inner surface of traumatic resin ducts (TRD), which
contain induced oleoresin, and not in the remaining xylem cells,
including the ray channel (rc), a site of primary oleoresin secretion
(top). The antibody serum did not generate any labeling above the
background signal in tissue treated with preimmune serum only
(bottom picture).
Characterization of a Bifunctional IDS
Plant Physiol. Vol. 152, 2010 647

an increased proportion of shorter chain products,
while mutagenesis of Met to Cys had no effect on
product distribution. Interestingly, there were two
other residues where mutation (I235T and L273F)
increased the proportion of the short-chain product
GPP to 100%. These project into the central cavity from
helix F, immediately opposite Met-165, and so may
also prevent additional condensations by steric effects.
Mutagenesis of PaIDS1 sequence sites to those pres-
ent in sequences of similar, single-product GGPP
synthases was largely unsuccessful at altering product
composition. The one exception was the double mu-
tant, P174C and L180F, which gave a 65% GPP, 35%
GGPP distribution compared with 90% GPP, 10%
GGPP in the wild-type enzyme. Residues 174 and
180 are located in the first Asp-rich region in the loop
attached to helix D and so are likely to be part of the
active site. However, it is not obvious how modifica-
tion at these positions can result in a greater percent-
age of additional IPP condensations to give more
GGPP and less GPP.
The residues in and around the first Asp-rich region
(residues 170–183) of PaIDS1 are clearly important in
chain length determination. This conclusion is sup-
ported by experiments involving chimeras formed by
exchanging the N- or C-terminal portion of PaIDS1
with corresponding portions of similar single-product
GPP or GGPP synthases. When the N terminus (in-
cluding the first Asp-rich region) was replaced by the
N terminus of a single-product P. abies GPP synthase or
a single-product GGPP synthase, the product distri-
bution shifted accordingly to either GPP or GGPP (Fig.
13). Replacement of the C terminus in the same way
did not have such an effect, but the fact that mutagen-
esis of residues 235 and 273 also affected the length of
the product suggests that control of chain length
resides at many places in the protein, not all of which
are clearly known. Other open questions about PaIDS1
Figure 11. Three-dimensional ribbon model of the
complete structure of wild-type PaIDS1 (A) and the
substrate-binding region (B) constructed with refer-
ence to the avian FPP synthase crystal structure
(Tarshis et al., 1994). Depicted are the a-helices D
(amino acids 154–181), F1 + F2 + G (amino acids
222–277), and H (amino acids 309–313). Helices
that are part of the predicted substrate-binding pocket
are colored in blue (helices D + F), and the DDxxD
motifs (amino acids 170–176 and 309–313) are
indicated in red. Residues used for mutagenesis that
are visible in this diagram are shown in turquoise.
The model was displayed with the program UCSF
Chimera (National Centre for Research Resources).
Table II. Effects of site-directed mutagenesis on the reaction rate and product composition of PaIDS1
Mutation Effect on
Reaction Rate Effect on Product Compositiona
M159I Minor None
I161M Minor None
M165C Minor None
M165Y Minor 90% GPP, 10% FPP
P173 and P174 deletion Lower activitybNone
P174S Minor None
P174C Minor None
M175I Minor 95% GPP, 3% FPP, 2% GGPP
M175I and P174C Minor 100% GPP, 0% GGPP
M175I and P174S Minor None
L180F Minor 98% GPP, 2% GGPP
L180F and P174C Minor 65% GPP, 35% GGPP
V227M Lower activitybNone
I235T Lower activityb100% GPP, 0% GGPP
V240L Lower activitybNone
G257D and P174C Minor None
L273F Lower activityb100% GPP, 0% GGPP
aWild-type PaIDS1 produces approximately 90% GPP and 10% GGPP upon heterologous expression.
bAbout 20% of the activity of wild-type PaIDS1.
Schmidt et al.
648 Plant Physiol. Vol. 152, 2010

concern why a significant proportion of GPP, the C10
intermediate formed by the enzyme, is released but the
next larger intermediate, FPP, remains bound. It is
conceivable that as the product elongates from C10 to
C15 in the active site, it twists in the cavity in a way that
prevents release until it reaches C20 and cannot elon-
gate further. The residues responsible for preventing
release might be localized in the C-terminal domain of
PaIDS1, because site-directed mutagenesis at positions
235 and 273 and chimeras with replacements of the
C-terminal portion of PaIDS1 (residues 213–383) pro-
duced FPP as their final product instead of GGPP,
albeit with a much lower activity. More work is needed
to understand how PaIDS1 creates its unique bifunc-
tional product spectrum.
PaIDS1 Transcript and Protein Are Associated with
Oleoresin Biosynthesis
The ability of PaIDS1 to produce both GPP and
GGPP suggests that this enzyme could have a biolog-
ical role in both monoterpene and diterpene produc-
tion. Since the major components of conifer oleoresin
are monoterpenes and diterpenes, we looked for a
connection between the occurrence of PaIDS1 in P.
abies and oleoresin biosynthesis. The formation of
oleoresin in this species can be induced by treatment
with MJ, which mimics attack by bark beetles or
pathogenic fungi (Franceschi et al., 2002; Martin
et al., 2002; Hudgins et al., 2003; Byun-McKay et al.,
2006; Erbilgin et al., 2006; Zeneli et al., 2006). MJ
Figure 12. Catalytic activities of mutated recombi-
nant PaIDS1 proteins heterologously expressed in E.
coli and assayed with [1-14C]IPP and DMAPP. Reac-
tion products were enzymatically hydrolyzed, and
the resulting alcohols were analyzed by radio-gas
chromatography. For details of compound identifica-
tion, analysis, and abbreviations, see Figure 4. At
least three replicate assays of each mutant were
analyzed, and the variance was below 5%.
Figure 13. Catalytic activities of chimeric recombi-
nant PaIDS1 proteins heterologously expressed in E.
coli. Either the N-terminal or C-terminal half of
PaIDS1 was exchanged for the corresponding portion
of the known GPP synthase, PaIDS2, or the known
GGPP synthase, PaIDS5, and the resulting proteins
were assayed with [1-14C]IPP and DMAPP. Reaction
products were enzymatically hydrolyzed, and the
resulting alcohols were analyzed by radio-gas chro-
matography. For details of compound identification,
analysis, and abbreviations, see Figure 4. At least
three replicate assays of each mutant were analyzed,
and the variance was below 5%.
Characterization of a Bifunctional IDS
Plant Physiol. Vol. 152, 2010 649

treatment increases the accumulation of resin mono-
terpenes and diterpenes and stimulates the formation
of traumatic resin ducts in stems in the newly formed
wood (xylem) just inside the vascular cambium. In this
work, MJ treatment also dramatically increased the
transcript abundance of PaIDS1 in wood and bark,
compared with untreated controls, indicating that the
encoded protein is involved in oleoresin formation. In
untreated tissue, PaIDS1 transcript is found in wood
and bark, but not needles, consistent with a role in
resin formation only in traumatic ducts. Although
traumatic resin ducts are located in wood, their de-
velopment is initiated in cells that are still part of the
vascular cambium (Martin et al., 2002; Franceschi
et al., 2005). Since the cambium layer is usually in-
cluded with the bark during sampling, it is not sur-
prising to observe expression of a gene involved in
traumatic resin duct formation in both the bark and
wood.
In previous studies on P. abies short-chain IDSs, we
have demonstrated that a single-product GPP syn-
thase (PaIDS2) and a single-product GGPP synthase
(PaIDS5) are also associated with oleoresin biosynthe-
sis in stems (Schmidt and Gershenzon, 2007, 2008).
While these single-product enzymes could suffice for
the formation of the major resin terpenes, it is inter-
esting that PaIDS1 transcript is induced much more
significantly than those of PaIDS2 or PaIDS5 after MJ
treatment and thus may have an especially important
function in induced resin formation. Additional evi-
dence for the role of PaIDS1 in induced oleoresin
formation comes from our studies that localized this
protein to epithelial cells lining the traumatic resin
ducts in stems treated with the fungus C. polonica.
Epithelial cells are thought to synthesize the resin and
secrete it into the lumen of the ducts (Bannan, 1936).
Antibodies to PaIDS1 did not react with antigens in
any other type of cell in the stem.
At the subcellular level, PaIDS1 appears to be local-
ized in the plastid by virtue of its predicted chloro-
plast signal peptide. A plastidial location is also
consistent with current concepts on the intracellular
compartmentalization of GPP- and GGPP-producing
or -utilizing enzymes in plant cells (Parmryd et al.,
1999; Welsch et al., 2000; Bouvier et al., 2002, 2005; Bick
and Lange, 2003; Martin et al., 2004; Tholl et al., 2004;
Keeling and Bohlmann, 2006b; Ro and Bohlmann,
2006).
PaIDS1 Has Strong Sequence Identity to Other Conifer
GPP and GGPP Synthases and Is Likely to Have Evolved
from a Single-Product Enzyme
A phylogenetic analysis of gene sequences for char-
acterized plant short-chain IDSs shows that PaIDS1
clusters closely with most previously reported, single-
product GPP and GGPP synthase genes from conifers.
However, PaIDS1 shares no significant sequence sim-
ilarity to a gene encoding one conifer GPP synthase
that clusters most closely with several angiosperm
GPP synthases thought to function in gibberellin bio-
synthesis (Bouvier et al., 2002; Van Schie et al., 2007;
Schmidt and Gershenzon, 2008). It also possesses no
significant similarity to the family of heterodimeric
GPP synthases known from angiosperms (Burke and
Croteau, 2002a; Tholl et al., 2004; Wang and Dixon,
2009).
Given its demonstrated ability to produce both GPP
and GGPP, it is not surprising that PaIDS1 has highest
sequence similarity to single-product GPP and GGPP
synthases from conifers. Its kinetic properties and
reaction mechanism also do not distinguish it from
other members of this group. PaIDS1 may be consid-
ered a GGPP synthase that has the ability to release a
substantial portion of the intermediate GPP, giving it
a novel product spectrum. Our site-directed mutagen-
esis experiments showing facile shifts of GPP and
GGPP after modification of one or two residues sug-
gest that the evolutionary conversion from single-
product GPP and GGPP synthases to an enzyme like
PaIDS1 may require only a few steps. Having a single
short-chain IDS that makes both the GPP and GGPP
precursors for conifer resin in a fixed proportion may
have been selected to allow better control of the ratio
between monoterpenes and diterpenes and to facili-
tate increased synthesis of both products simulta-
neously. Other plant species that produce mixtures
of terpene natural products may have similar dual-
function short-chain IDSs.
MATERIALS AND METHODS
Chemicals
All chemicals and solvents were analytical grade and were obtained from
Merck, Serva, or Sigma. [1-14C]IPP (55 Ci mol21) was purchased from
Biotrend, [1-3H]GPP (15 Ci mmol21), [1-3H]FPP (20 Ci mmol21), and [a-32P]
dATP were obtained from Hartmann Analytic, and unlabeled DMAPP and
IPP were obtained from Echelon Research.
Plant Material
The clonal 4-year-old Picea abies (Norway spruce) saplings used (Samenklenge
und Pflanzgarten Laufen) are members of clone 3369-Schongau. Saplings
were grown in standard soil in a climate chamber (Vo
¨tsch) under a 21°C-day/
16°C-night temperature cycle, controlled light conditions (16 h per day at
150–250 mmol, obtained from a mixture of cool-white fluorescent and incandes-
cent lamps), and a relative humidity of 70% in a climate chamber (Vo
¨tsch). For
induction experiments, saplings were sprayed with 100 mMMJ in 0.5% (v/v)
Tween 20 as detergent. When stem tissue was harvested, the uppermost
internode (representing the previous year’s growth) was used. Bark and wood
from this zone were separated and frozen in liquid nitrogen in preparation for
RNA isolation. The bark included the cambium layer that peeled off from the
wood during separation.
The embryogenic culture of P. abies (line 186/3c VIII; kindly provided by
Harald Kvaalen, Norwegian Forest and Landscape Institute) was initiated
from mature zygotic embryos, dissected from seeds, and cultured on modified
Litvay’s (MLV) medium (Klimaszewska et al., 2001). Casein amino acids
(Becton Dickinson) were added at 1 g L21,L-Gln (Duchefa) at 0.5 g L21, Suc at
10 g L21, and Gelrite (Duchefa) at 4 g L21when a semisolid medium was
required. The medium was supplemented with 10 mM2,4-dichlorophenoxy-
acetic acid and 5.0 mM6-benzyladenine. The pH of the medium was adjusted
with KOH to 5.7 prior to sterilization in the autoclave. The pH of the L-Gln
solution was adjusted to 5.7, and the solution was filter sterilized and added to
Schmidt et al.
650 Plant Physiol. Vol. 152, 2010

the medium after sterilization. Unless stated otherwise, all cultures were kept
in the dark at 24°C.
Amplification and Cloning of the PaIDS1 Sequence
Degenerate primers were designed for conserved regions of GGPP syn-
thase sequences from Taxus canadensis,Helianthus annuus,Croton sublyratus,
and Lupinus albus for different conserved regions: 5#-CIATG(A/C)G(G/T)TA
(C/T)TCICTICTIGCIGG-3#and 5#-CTTATCIG(C/T)(A/C)AICAAATC(C/T)
TT(C/T)CC-3#. RT was carried out with pooled total RNA from MJ-treated P.
abies bark harvested at various time points after treatment (6, 12, and 24 h and
2, 4, 6, 10, and 20 d) and oligo-(dT) primers using SuperScript II (Invitrogen),
according to the manufacturer’s instructions. Subsequently, PCR was per-
formed with 35 cycles of 94°C for 1 min, annealing for 1 min, and then 72°Cfor
1 min in a thermocycler (Stratagene). To identify the optimal annealing
temperature, a temperature gradient was used with 2°C intervals ranging
from 42°Cto64°C. A 430-bp fragment was generated at 44°C and subcloned
into pCR 4-TOPO (Invitrogen) according to the manufacturer’s instructions.
Isolation of the PaIDS1 Clone
Total RNA was isolated according to a method developed by Wang et al.
(2000) from P. abies bark harvested at various time points (as above) after being
sprayed with 100 mMMJ. From total pooled RNA, poly(A)+RNA was isolated
using Dynabeads (Invitrogen). cDNA was synthesized using the SMART
cDNA Library Construction Kit (Clontech). In vitro packaging with Gigapack
III Gold (Stratagene) yielded a library of 8 3107plaque-forming units, which
was subsequently screened with the [a-32P]dATP-labeled 430-bp fragment
previously amplified by PCR. Single plaques were isolated from positive
colonies, and lDNA was converted into pDNA by subcloning into pTriplEx2
vector and transformation in Escherichia coli BM25.8. Sequence analysis was
carried out using an ABI 3100 automatic sequencer (Applied Biosystems). One
sequence was obtained that was named PaIDS1.
Sequence and Phylogenetic Analyses
The DNAStar Lasergene program version 7.0 (MegAlign) was used to
align and to calculate the deduced amino acid sequences of each full-length
cDNA or of known sequences from other gymnosperms and angiosperms.
The amino acid alignment was conducted by use of ClustalW (gonnet 250
matrix, gap penalty 10.00, gap length penalty 0.20, delay divergent sequences
30%, gap length 0.10, DNA transition weight 0.5). The same software was used
to visualize the phylogenetic tree.
Functional Expression of PaIDS1
The entire coding sequence of clone PaIDS1 lacking the predicted signal
peptide for chloroplast transport (based on analysis at http://www.cbs.dtu.
dk/services) was amplified with primers that included the start and stop
codons for translation. The primer combination 5#-ATGTGCTCAAACAC-
AAATGCCCAG-3#and 5#-GTTCTGTCTTTGTGCAATGTAATG-3#was used
to express PaIDS1. The amplification was carried out with the Expand High
Fidelity PCR System (Roche Applied Science). PCR was performed for 2 min
at 94°C initial denaturation, 10 cycles of 15 s at 94°C, 30 s at 55°C, and 55 s at
72°C, 20 cycles of 15 s at 94°C, 30 s at 55°C, and 55 s plus 5 s of elongation
for each cycle at 72°C, and a final extension for 7 min at 72°C in a Tpersonal
thermocycler (Biometra). The resulting cDNA fragments were cloned into the
expression vector pCR-T7 CT TOPO (Invitrogen), which adds a tag containing
six His 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 of 100 mL were grown to an optical density
(OD600nm) of 0.6, and transformants were induced with 2 mMisopropylthio-
b-galactoside as described by the manufacturer’s instructions, except that they
were grown exclusively at 18°C. Bacterial pellets were resuspended in 3 mL of
buffer containing 20 mM3-(N-morpholino)-2-hydroxypropanesulfonic acid
(MOPSO), pH 7.0, 10% (v/v) glycerol, and 10 mMMgCl2and sonicated using a
Sonopuls HD 2070 (Bandelin) for 4 min, cycle 2, power 60%. The PaIDS1
protein was purified over nickel-nitrilotriacetic acid agarose columns (Qiagen)
according to the manufacturer’s instructions. Protein was eluted with 20 mL of
250 mMimidazole in the assay buffer, and 1.5-mL fractions were collected.
After adding 2 mMdithiothreitol, each fraction was checked for purity by SDS-
PAGE. Fraction 2, which contained the highest amount of recombinant
protein, was used for the assay. The added 250 mMimidazole was not
removed before the assay, as it did not affect the activity.
Assay of Recombinant PaIDS1 and Product Identification
Small-scale PaIDS1 assays were carried out in a final volume of 50 mL
containing 20 mMMOPSO (pH 7.0), 10 mMMgCl2(for PaIDS5), 10% (v/v)
glycerol, 2 mMdithiothreitol, and 800 mMIPP or 250 mMDMAPP at saturated
concentrations. For radioactive detection of products, 10 mM[1-14C]IPP (55 Ci
mol21) was added to each assay. The reaction was initiated by the addition of
recombinant protein, and the assay mixture was overlaid with 1 mL of hexane
and incubated for 20 min at 30°C. Assays were stopped by the addition of
10 mLof3NHCl and incubated for an additional 20 min at 30°C to solvolyze
the acid-labile allylic diphosphates formed during the assay to their corre-
sponding alcohols. Solvolysis products were extracted into the hexane phase
by vigorous mixing for 15 s. After centrifugation, 300 mL of the hexane phase
was used for total radioactivity determination by liquid scintillation counting.
Protein concentration was measured according to Bradford (1976) using the
Bio-Rad reagent with bovine serum albumin (BSA) as standard. The reaction
was linear for up to 30 min at protein concentrations of 40 mgmL
21. Kinetic
parameters were calculated with Michaelis-Menten kinetics and Lineweaver-
Burk plotting using the enzyme kinetics program EkI 3 (Wiley-VCH). All
assays were carried out in triplicate.
For the identification of reaction products of PaIDS1, larger scale assays
were carried out in a final volume of 500 mL. The assay mixture was as
described above, but concentrations of 40 mM[1-14C]IPP, 40 mMDMAPP, 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 of potato apyrase
(Sigma) in 0.2 MTris-HCl, pH 9.5, was added to each assay and followed by
overnight incubation at 30°C. After enzymatic hydrolysis, the resulting
isoprenyl alcohols were extracted into 2 mL of diethyl ether. Then, following
the addition of a standard terpene mixture, the organic extracts were evap-
orated under N2and used for radio-gas chromatography (GC) measurements.
Radio-GC analysis was performed on a Hewlett-Packard HP 6890 gas chro-
matograph (injector at 220°C, thermal conductivity detector at 250°C) in
combination with a Raga radioactivity detector (Raytest) using a DB 5-MS
capillary column (J&W Scientific; 30 m 30.25 mm with a 0.25-mm phase
coating). Separation of the injected concentrated organic phase (1 mL) was
achieved under an H2flow rate of 2 mL min21with a temperature program of
3 min at 70°C, followed by a gradient from 70°Cto240°Cat6°Cmin
21with a
3-min hold at 240°C. Products measured by radio-GC were identified by
comparison of retention times with those of coinjected, nonradioactive,
authentic terpene standards monitored via a thermal conductivity detector.
To study the reaction mechanism, substrates were used as follows in the
assays depicted in Figure 5: 600 mM[1-3H]GPP (15 Ci mmol21); 400 mM[1-3H]
GPP (15 Ci mmol21) and 400 mMIPP; 400 mMGPP, 400 mMIPP, and 40 mM[1-14C]
IPP (55 Ci mol21); 200 mM[1-3H]FPP (20 Ci mmol21) and 600 mMIPP; and
200 mMFPP, 600 mMIPP, and 40 mM[1-14C]IPP (55 Ci mol21).
Agrobacterium tumefaciens Strain and
Culture Preparation
The disarmed A. tumefaciens strain C58/pMP90 (Koncz and Schell, 1986)
containing either the binary vector pCAMGW::IDS1 or pCAMBIA2301 (as a
control) was used in the transformation experiment. For construction of the
transformation vector, attR recombination sites (Gateway Technology, Invi-
trogen) were cloned into the vector pMJM (McKenzie et al., 1994), creating
pHJMGW. A 4-kb-long cassette from pMJMGW containing the maize (Zea
mays) ubiquinone promoter (ubi1) and the attR recombination sites was cloned
into pCAMBIA2301. The resulting plasmid pCAMGW carries the neomycin
phosphotransferase gene (nptII), which confers kanamycin resistance under
the control of the double 35S cauliflower mosaic virus promoter and termi-
nator and the GUS reporter gene (uidA), coding for GUS, under the control of
the 35S cauliflower mosaic virus promoter and the nopaline synthase termi-
nator. The complete open reading frame of IDS1 was cloned into pCAMGW
upstream of the ubi1 promoter using Gateway Technology.
The bacterial culture was grown in yeast extract peptone liquid medium
containing 50 mgmL
21rifampicin, 50 mgmL
21kanamycin sulfate, and 20 mg
mL21gentamicin sulfate (all Duchefa) on a shaker at 250 rpm at 28°C for 16 h.
Subsequently, the bacterial cells were pelleted by centrifugation and resus-
pended in liquid MLV medium to an OD600nm of 0.6.
Characterization of a Bifunctional IDS
Plant Physiol. Vol. 152, 2010 651

Transformation Procedure for Embryogenic Tissue
Prior to the transformation experiment, embryogenic tissue was prepared
and cultured on filter papers as described by Klimaszewska and Smith (1997).
For the transformation experiments, the embryogenic tissue was harvested
from the filter papers 7 d after subculture and resuspended in liquid MLV
medium. Subsequently, an equal volume of A. tumefaciens culture (OD600nm =
0.6) in MLV was added to the cell suspension, resulting in 100 mg (fresh
weight) of embryogenic tissue suspended in 1 mL of bacterial culture at
OD600nm of 0.3 in MLV medium in a 100-mL Erlenmeyer flask. Control
embryogenic tissue was treated the same way except that A. tumefaciens was
omitted.
From the culture, 1 mL (100 mg of fresh material) was poured over a 5.5-cm
sterile filter paper (Whatman no. 2) in a Bu
¨chner funnel. A short vacuum pulse
(5 s, 45.7 cm of mercury) was applied to drain the liquid, and the filter was
placed on a semisolid MLV medium in a 100-mm 315-mm petri dish. A total
of 10 petri dishes were used for each transformation experiment and the
controls.
After 2 d of coculture on the semisolid medium supplemented with 50 mM
acetosyringone, filter papers with cells from five petri dishes were placed in an
Erlenmeyer flask (250 mL) with 50 mL of liquid MLV medium. The cells were
then dislodged by manual shaking and collected on the new filter papers in a
Bu
¨chner funnel. The filters papers with cells were then placed on fresh MLV
medium with 250 mg L21cefotaxime (Duchefa). At the first signs of embry-
ogenic tissue growth (approximately 2 d), the filters with cells were trans-
ferred to selective medium. Selective medium contained cefotaxime and 35
mg L21kanamycin. For maintenance, the transgenic embryogenic cultures of
P. abies were subcultured every 14 d and cultivated as described above.
Quantitative Genomic PCR
For quantitative genomic PCR of embryogenic tissue, genomic DNA was
isolated using the Plant DNA Isolation Kit (Qiagen) according to the manu-
facturer’s instructions. Genomic PCR was performed with Brillant SYBR
Green QPCR Master Mix (Stratagene) as described in the section on qRT-PCR
below, but with the following changes. The denaturation step at the beginning
was elongated to 13 min at 95°C. Gene abundance was normalized to the
abundance of the ubiquitin gene, amplified with the forward primer
(5#-CCCTCGAGGTAGAGTCATCG-3#) and the reverse primer (5#-CCAGA-
GTTCTCCCATCCTCC-3#). This quantity was the average of three indepen-
dent biological replicates, each represented by four measurements on three
technical replicates. Relative gene abundance was obtained by comparison
with the gene abundance of the pCAMGW vector control.
Assay of PaIDS1 in Transgenic Embryogenic Tissue
Embryogenic tissue culture was dried on a filter paper, resuspended in
assay buffer (see above), and treated with an Ultraturrax with maximal speed
for 20 s at 4°C. After centrifugation at 20,000gfor 10 min at 4°C, supernatant
containing crude protein extract of embryogenic tissue was assayed. Protein
concentration was measured according to Bradford (1976) using the Bio-Rad
reagent with BSA as standard. Identical amounts of protein extracts were used
in the larger scale assays as described for the assay of recombinant PaIDS1 and
product identification.
qRT-PCR
Total RNA from plants at different time points after MJ treatment and from
different organs was isolated according to the method of 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 was generated using SuperScript III (Invitrogen) accord-
ing to the manufacturer’s instructions. qRT-PCR was performed with Brillant
SYBR Green QPCR Master Mix (Stratagene). Specific qRT-PCR primers,
forward (5#-GACATCTGGTATCATCACTC-3#) and reverse (5#-GTGACCT-
TCCCCTTTACTTG-3#), were designed using the following criteria: predicted
melting temperature of at least 58°C, primer length of 22 to 24 nucleotides,
guanosine-cytosine content of at least 48%, and an amplicon length of 120 to
150 bp. Primer specificity was confirmed by melting curve analysis, by an
efficiency of product amplification of 1.0 60.1, and by sequence verification of
at least eight cloned PCR amplicons for each PCR procedure. Reactions with
water instead of cDNA template were run with each pri mer pair as controls. A
standard thermal profile of 95°C for 10 min, then 45 cycles of 95°C for 30 s,
53°C for 30 s, and 72°C for 30 s, was used. The fluorescence signal was
captured at the end of each cycle, and a melting curve analysis was performed
from 53°Cto95°C with data capture every 0.2°C during a 1-s hold. qRT-PCR
was performed as described in the operator’s manual using a Stratagene
MX3000P. Transcript abundance was measured as the average of three
biological replicates; each is represented by three determinations of three
technical replicates. All amplification plots were analyzed with the MX3000P
software to obtain threshold cycle values. Transcript abundance was normal-
ized with the transcript abundance of the ubiquitin gene (GenBank accession
no. EF681766), amplified with the forward primer (5#-GTTGATTTTTGCTGG-
CAAGC-3#) and reverse primer (5#-CACCTCTCAGACGAAGTAC-3#). Rela-
tive transcript abundance was obtained by calibration against the transcript
abundance at the onset of treatment.
For qRT-PCR of embryogenic tissue, RNA was isolated using the Plant
RNA Isolation Kit (Invitek) according to the manufacturer’s instructions,
including an additional DNA digestion step (RNase Free DNase Set; Qiagen).
Further steps were carried out as above. Relative transcript abundance was
calibrated against the transcript abundance of pCAMGW vector control
plants.
Microscopy and Immunogold Labeling of PaIDS1
Stems of P. abies trees (5 years old) were inoculated with Ceratocystis
polonica as described by Christiansen et al. (1999). Bark samples (2 33 mm,
including phloem, vascular cambium, and xylem with traumatic resin ducts)
were collected 36 d after inoculation. All samples were isolated from areas 5
cm above the inoculation site, well above the edge of necrotic lesions (Nagy
et al., 2000). Fixation, dehydration, and embedding were performed as
described previously (Nagy et al., 2000). Semithin sections (1 mm) were cut
using a LKB 2128 Ultratome (Leica Microsystems) and dried onto Superfrost
Plus slides (Menzel-Gla
¨zer) for light microscopy.
Immunogold labeling was performed as described previously (Nagy et al.,
2000). Polyclonal peptide antibodies for PaIDS1 were synthesized against
amino acids 285 to 300 with the sequence 5#-GASDDEIERVRKYARC-3#(Fig.
2) and affinity purified (Eurogentec). Polyclonal peptide antibodies for PaIDS4
were synthesized against the amino acids 172 to 187 with the sequence
5#-HEGATDLSKYKMPT-3#(Schmidt and Gershenzon, 2007). The primary
antibodies were diluted 1:4,000 in TBST/BSA buffer (10 mMTris, pH 7.2, 0.15 M
NaCl, 0.1% [w/v] Tween 20, and 2% [w/v] BSA), and the secondary antibody
goat anti-rabbit IgG conjugated to 10-nm gold (British Biocell International)
was diluted 1:4,000 in TBST/BSA buffer. The sections were counterstained
with 0.5% Safranin O, air dried, mounted with Biomount (British Biocell
International) for evaluation by a Leitz Aristoplan light microscope, and
documented with a Leica DC300 digital camera. To ensure the specificity of
the antibody labeling, controls included (1) incubation with the rabbit
preimmune serum instead of the primary antibody and (2) incubation with
the secondary antibody omitting the primary antibody step.
Three-Dimensional Structural Modeling of PaIDS1
The ribbon model of PaIDS1 was created with the program Swiss-Model
(version 36.0003). The crystallographic structure of avian FPP synthase was
used as a template to build the PaIDS1 model structure. The PaIDS1 sequence
was entered under the Web address http://swissmodel.expasy.org and cre-
ated as an image with DeepView SwissPdbViewer 3.7 (Sp5). Pairwise align-
ments were made between the PaIDS1 model structure and avian crystal
structure to establish positions and nearby contacts of analogous catalytic
residues located near the substrate-binding site. The image depicted (Fig. 11)
was created with the program UCSF Chimera (National Centre for Research
Resources).
Construction of Mutated PaIDS1
Site-directed mutagenesis was performed with the QuikChange XL Site-
Directed Mutagenesis Kit (Stratagene) following the manufacturer’s instruc-
tions. The cloned PaIDS1 in the expression vector pCR-T7 CT TOPO served as
a template for PCR amplification. Sequences of the primers used are listed in
Supplemental Table S2. PCR was performed for 60 s at 95°C initial denatur-
ation, 18 cycles of 60 s at 95°C, 60 s at 55°C, and 4 min at 68°C, and a final
annealing for 7 min at 68°C in a Tpersonal thermocycler (Biometra). After
amplification, the parental (methylated) DNA was digested with DpnI
Schmidt et al.
652 Plant Physiol. Vol. 152, 2010

(10 units mL21) for 1 h at 37°C. The DpnI-treated DNA was transformed in
XL10-Gold Ultracompetent Cells (Stratagene). All mutations were confirmed
by DNA sequencing, and the plasmid was transformed into E. coli BL21 (DE3)
pLysS (Invitrogen).
Generation of Chimeric IDS
Chimeric versions of PaIDS1 assembled with the N-terminal or C-terminal
portion of the coding sequence exchanged for the corresponding portion of
the coding sequence of PaIDS2 or PaIDS5 were generated by separate PCR
amplification followed by ligation. Amplification of the partial PaIDS2 and
PaIDS5 sequences was performed using primers that included the BssSI
restriction site at nucleotide position 652 for PaIDS2,theBanII site at position
717 for PaIDS5, and primers with the added appropriate restriction site for
amplification of the PaIDS1 partial cDNA (Supplemental Table S2). The PCR
conditions are as described for the functional expression of PaIDS1. Ampli-
cons were restricted with the appropriate restriction enzyme, gel purified
using of Wizard SV Gel and PCR Clean-Up System (Promega) following the
manufacturer’s instructions, and ligated using of T4 DNA Ligase (Invitrogen).
The chimeric constructs were cloned into the vector pCR-T7 CT TOPO
(Invitrogen) and transformed into One Shot Top 10 (Invitrogen) chemically
competent cells. After sequencing, the constructs were transformed into E. coli
BL21 (DE3)pLysS (Invitrogen).
The PaIDS1 sequence was deposited in GenBank with accession number
GQ369788.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. SDS-PAGE analysis with Coomassie Brilliant
Blue staining of PaIDS1 recombinant protein expressed in E. coli and
partially purified.
Supplemental Figure S2. Lineweaver-Burk plots of partially purified
recombinant PaIDS1 protein expressed in E. coli for determination of Km
and Vmax.for DMAPP and IPP.
Supplemental Table S1. Sequences of primers used for site-directed
mutagenesis, including the melting temperatures and the positions of
the changed amino acids.
Supplemental Table S2. Sequences of primers used for the construction of
chimeric IDS, including their melting temperatures.
ACKNOWLEDGMENTS
We thank Marion Sta
¨ger, Andrea Bergner, and Beate Rothe for excellent
technical assistance, Mike Phillips for providing the pCAMGW plasmid,
Harald Kvaalen for kindly providing the embryogenic culture of P. abies, Elin
Ørmen and Nina Nagy for technical assistance with the immunocytochem-
istry, and Almuth Hammerbacher and Kimberly Falk for critical reading of
the manuscript.
Received July 15, 2009; accepted November 19, 2009; published November 25,
2009.
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