Monoterpene synthase activities in leaves of Picea abies (L.) Karst. and Quercus ilex L.

Article abstract of DOI:10.1016/S0031-9422(00)00119-9

In addition to direct ecological functions in the interaction of plants with the environment, the emission of monoterpenes, especially from the foliage of evergreen trees, is of great importance for the production of ozone and photochemical oxidants in the troposphere. In the present work, we established a reproducible non-radioactive standard enzyme assay and characterized monoterpene synthase activities in needles of Norway spruce (Picea abies (L.) Karst.) and in leaves of holm oak (Quercus ilex L.). In Norway spruce, the dominant monoterpenes formed were α-pinene, camphene, and to a lesser extent β-pinene and limonene. In holm oak, α-pinene, sabinene, and β-pinene were the main products, while limonene was a minor component. Under optimum conditions, in both Norway spruce and holm oak, monoterpene formation remained constant up to 180 min and 90 min, respectively, and varied with the buffer and Mg²⁺ and Mn²⁺ concentrations used. Optimum temperature for monoterpene synthase activity was 40°C in both species; optimal pH ranged between 6.5 and 7.5 in both species. Apparent Michaelis- constants for the substrate GDP were ca. 17.9 ± 5.1 μM for Norway spruce and ca. 69.4 ± 22.1 μM for holm oak. Molecular weight determination by FPLC indicated that the monoterpene synthases in Norway spruce and holm oak have native molecular weights of ca. 59 and 50 kDa, respectively. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(00)00119-9

Phytochemistry 54 (2000) 257±265
www.elsevier.com/locate/phytochem
Monoterpene synthase activities in leaves of Picea abies (L.)
Karst. and Quercus ilex L.
Robert J. Fischbach, Ina Zimmer, Rainer Steinbrecher, Andreas P®chner,
Jorg-Peter Schnitzler*
Fraunhofer-Institut fur AtmosphaÈrische Umweltforschung, Kreuzeckbahnstr. 19, D-82467 Garmisch-Partenkirchen, Germany
È
Received 8 November 1999; received in revised form 17 March 2000
Abstract
In addition to direct ecological functions in the interaction of plants with the environment, the emission of monoterpenes,
especially from the foliage of evergreen trees, is of great importance for the production of ozone and photochemical oxidants in
the troposphere. In the present work, we established a reproducible non-radioactive standard enzyme assay and characterized
monoterpene synthase activities in needles of Norway spruce (Picea abies (L.) Karst.) and in leaves of holm oak (Quercus ilex
L.). In Norway spruce, the dominant monoterpenes formed were a-pinene, camphene, and to a lesser extent b-pinene and
limonene. In holm oak, a-pinene, sabinene, and b-pinene were the main products, while limonene was a minor component.
Under optimum conditions, in both Norway spruce and holm oak, monoterpene formation remained constant up to 180 min
and 90 min, respectively, and varied with the bu€er and Mg2+ and Mn2+ concentrations used. Optimum temperature for
monoterpene synthase activity was 408C in both species; optimal pH ranged between 6.5 and 7.5 in both species. Apparent
Michaelis-constants for the substrate GDP were ca. 17.9 2 5.1 mM for Norway spruce and ca. 69.4 2 22.1 mM for holm oak.
Molecular weight determination by FPLC indicated that the monoterpene synthases in Norway spruce and holm oak have
native molecular weights of ca. 59 and 50 kDa, respectively. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Picea abies; Pinaceae; Quercus ilex; Fagaceae; Evergreen leaves; Enzyme characterization; Monoterpene synthases
1. Introduction
against natural enemies, as well as allelopathic agents
and as attractants for pollinators (for overview see
Harborne, 1991). Monoterpenes are preferentially
released by species containing specialized organs such
as glandular trichomes, like the leaves of Lamiaceae,
or resin ducts like conifer needles (Lerdau, 1991; Tin-
gey et al., 1991). Such large storage pools in the nee-
dles of Norway spruce (Picea abies (L.) Karst.)
(Schonwitz et al., 1990) constitute a permanent poten-
tial emission source which complements a light-depen-
dent monoterpene emission that is closely linked to
photosynthetic activity of the needles (Schurmann et
al., 1993). Recent observations indicate that leaves of
some oak species, e.g. the evergreen sclerophyllous
holm oak (Quercus ilex L.), also emit large amounts of
monoterpenes despite the absence of storage pools
(Loreto et al., 1996a; Steinbrecher et al., 1997; Staudt
Emissions from trees provide a major source of reac-
tive volatile organic compounds (VOC) which are
essential for the production of ozone and photochemi-
cal oxidants in the troposphere (Fehsenfeld et al.,
1992). Isoprene and monoterpenes are the most abun-
dant biogenic VOC emitted from vegetation (Guenther
et al., 1995) and thus contribute signi®cantly to ozone
production in areas with high anthropogenic NOx
emissions (Carter, 1994). In addition to their contri-
bution to atmospheric chemistry, monoterpenes play
important ecological roles as defense compounds
* Corresponding author. Tel.: +49-8821-183131; fax: +49-8821-
73573.
E-mail address: schnitzler@ifu.fhg.de (J.-P. Schnitzler).
0031-9422/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00119-9

258
R.J. Fischbach et al. / Phytochemistry 54 (2000) 257±265
and Bertin, 1998). This monoterpene emission is lar-
gely associated with photosynthesis, since it is stimu-
lated by light, declines in dark, and is inhibited in the
absence of CO2 in air (Loreto et al., 1996a, 1996b).
Despite the information available on the physiologi-
cal control of monoterpene emission from evergreen
leaves and needles, information on the biochemical
properties of the monoterpene synthases that catalyze
the ®nal steps leading from geranyl diphosphate
(GDP) to the various monoterpene products in these
species is still lacking.
Several monoterpene synthases of various plant
species have been isolated and characterized (Alonso
and Croteau, 1993; Colby et al., 1993; Savage et al.,
1995; Bohlmann et al., 1998). The monoterpene
synthases (or cyclases) responsible for generation of
various acyclic, bicyclic or tricyclic terpenes have quite
similar properties and all operate by electrophilic reac-
tion mechanisms (Gershenzon and Croteau, 1993) as
do other enzymes using prenyl-diphosphate. Di€erent
cyclization processes catalyzed by synthases lead to
various monoterpenes (Gershenzon and Croteau,
1993). With native molecular weights Mr of ca. 50±120
kDa (either monomers or homodimers), rather low pI
values, and signi®cant hydrophobicity, the properties
of these soluble enzymes are quite similar. The pH
optima of the enzymes range from 6 to 7, and bivalent
metal ions (usually Mg2+ or Mn2+) are required as
cofactor. Previously reported Michaelis-constants (Km)
for GDP are in the 10±20 mM range (Alonso and Cro-
teau, 1993).
on the gas chromatographic analysis of head space
containing the synthesized monoterpenes released
during the assay. Using this method, monoterpene
synthase activities (Fig. 1) were detected in cell-free
extracts of Norway spruce and holm oak leaves. In
Norway spruce (Fig. 1a), the dominant monoterpenes
produced were a-pinene, camphene, and to a lesser
extent b-pinene and limonene. In extracts of holm oak
(Fig. 1b), a-pinene, sabinene, b-pinene, and limonene
were formed. GC-MS analysis additionally revealed
the formation of traces of a-thujene (9.8 min) and
myrcene (11.6 min) as well as E-ocimene (13.1 min), b-
phellandrene (13.2 min), and Z-ocimene (13.4 min) in
holm oak. Boiling protein extracts resulted in complete
loss of enzyme activity (see Fig. 1). Enzyme assays
with native protein extracts without the substrate GDP
showed low monoterpene background levels of 0.1±
4% compared to the enzymatically formed monoter-
In the present work, we describe the extraction of
monoterpene synthase activities and the development
of a non-radioactive enzyme assay in evergreen leaves
of Norway spruce and holm oak. In addition, kinetic
properties of the enzyme activities of both species are
also reported.
2. Results and discussion
2.1. Determination of monoterpene synthase activity by
head space analysis
This is the ®rst report on monoterpene synthase ac-
tivities in Norway spruce needles and holm oak leaves.
Monoterpene synthase activities in other species have
been previously tested with 3H-labeled GDP, by
extracting the in vitro formed monoterpenes with or-
ganic solvents and measuring the monoterpenes
directly by scintillation counting (Lewinsohn et al.,
1992a, 1992b) or after separation of the products on
TLC or GC (for review see Alonso and Croteau,
1993). In the present work, we adapted a non-radio-
active assay from the measurement of isoprene
synthase activity (Lehning et al., 1999) which is based
Fig. 1. Gaschromatographic analysis of monoterpenes formed in leaf
extracts of (a) needles of Norway spruce and (b) holm oak leaves.
For comparison, chromatograms of enzyme assays with active and
heat-denaturated protein are shown. One ml samples of head space
were focussed on a precolumn at room temperature. By heating the
precolumn to 2408C the compounds were injected onto a capillary
column and separated with a temperature program starting at 358C
and ending at 1608C. Monoterpenes were detected by ¯ame ioniz-
ation (FID) and identi®ed by co-injection with authentic standards
as well as by GC-MS analysis. Traces of a-thujene (9.8 min), myr-
cene (11.6 min), E-ocimene (13.1 min), b-phellandrene (13.2 min),
and Z-ocimene (13.4 min) were detected.

R.J. Fischbach et al. / Phytochemistry 54 (2000) 257±265
259
penes in the presence of GDP, indicating that small
amounts of monoterpenes or GDP had passed the size
exclusion chromatography on the PD-10 columns.
To ensure the reproducibility of the head space
analysis, mixing experiments with monoterpene stan-
dards, protein extracts, various bu€ers under assay
conditions at di€erent incubation temperatures were
performed (Fig. 2). Addition of 1 ml monoterpene stan-
dard mixture (0.1 mM) to Norway spruce (Fig. 2a) or
holm oak leaf extracts (Fig. 2b) under standard assay
conditions at 10, 40, and 608C gave additive amounts
of monoterpenes when the enzyme substrate GDP was
added compared to assays without substrate. Variation
of the incubation temperature from 10 to 608C had no
signi®cant in¯uence ( pr0.05 ) on the recovery of the
monoterpene standards from the head space. Compari-
son of enzyme assays co-incubated with monoterpene
standard to normal assays showed that in both cases
the enzyme activities were statistically similar. In ad-
dition, replacing the protein extracts by bu€er also
had no in¯uence ( pr0.05) on the recovery of mono-
terpene standards (data not shown), indicating that
under the test conditions evaporation of monoterpenes
from the assay mixture is not hampered by protein
extract.
Nevertheless, for calibration of the assay (Fig. 3) 1
ml of monoterpene standard mixtures (10 mM to 1 mM
in pentane) was incubated in leaf extracts at 408C
without the enzyme substrate GDP under standard
assay conditions (see Section 2.4 and Table 1) for 60
min prior to the analysis of the head space. Under
these conditions, the detection of monoterpenes in the
head space was highly linear (r2 > 0.99) for about 2
orders of magnitude. Compared to the initially men-
tioned procedures (Lewinsohn et al., 1992a, 1992b;
Alonso and Croteau, 1993), which include several
extraction and puri®cation steps, the capillary GC
analysis of the head space containing the synthesized
monoterpenes is more convenient, and facilitates pro-
cessing a large number of samples with a detection
limit of ca. 1.5 ppmv and an accuracy of 5.2%.
2.2. Selection of optimal extraction and assay bu€ers
The presence of phenolic compounds, oil and resins
which inactivate monoterpene synthases and other
plant enzymes is a particulary severe problem in ever-
green leaves of tannin-rich and essential oil bearing
plants, like holm oak and Norway spruce, and has
been discussed in detail by Gegenheimer (1990). There-
fore, an initial objective of the present work was to
maximize the level of extractable activity in order to
adequately de®ne reaction parameters and permit sub-
sequent protein puri®cation. Monoterpene synthase ac-
Fig. 2. Co-incubation of Norway spruce (a) and holm oak (b) leaf
extracts and monoterpene standards. Leaf extracts with (1) monoter-
pene standards (1 ml of 0.1 mM solution in pentane) and (2) stan-
dard mixture plus enzyme substrate GDP were incubated under
standard assay conditions at 10, 40, and 608C. After 60 min, the
liquid phase was removed with a gas-tight syringe and 1.0 ml
samples of the head space were analyzed as described in Section 3.
ꢀn ˆ 5; 2sd).
Fig. 3. Calibration curve of monoterpenes by the head space analy-
sis. Increasing amounts of monoterpene standard mixture (ranging
from 10 mM to 1 mM) were added in 1 ml pentane to 100 ml Norway
spruce leaf extracts and incubated at 408C in gas-tight vials (2 ml).
After 60 min, the liquid phase was removed with a gas-tight syringe
and 1.0 ml samples of the head space were analyzed as described in
Section 3. ꢀn ˆ 5; 2sd; r2-values of the linear regressions were ca.
0.99).

260
R.J. Fischbach et al. / Phytochemistry 54 (2000) 257±265
tivities in holm oak leaves were extracted best with a
700 mM Mops bu€er (pH 7.3) containing antioxidants
(200 mM ascorbic acid, 50 mM 2-mercaptoethanol)
and a mixture of additives for adsorption of phenolic
compounds (8.3% (w/v) Dowex 1 Â 2, 1% (w/v) PVP-
30, 1.5% (w/v) PEG-1500). Omission of PVP-30 or
Dowex 1 Â 2 during extraction, or lowering the mo-
larity of the bu€er (to 100 mM) caused nearly com-
plete loss of enzyme activity. Omission of PEG-1500
lowered the enzyme activity by ca. 13%. PVP-30 could
not be replaced by insoluble polyvinylpyrrolidone
polymer PVPP (at 1% (w/v) of the bu€er). Screening
for the best assay bu€er showed that with 50 mM KPi
as bu€er reagent monoterpene synthase activity could
be enhanced by 85 2 3% compared to other bu€ers
(Tris, Hepes, Mops, each 50 mM) tested.
Measurements of monoterpene synthase activities in
Norway spruce needles were based on the extraction
procedure of monoterpene synthases from wood or
bark tissue of conifers (Lewinsohn et al., 1992a). With
this extraction bu€er (50 mM Hepes pH 7.3 containing
10% (v/v) glycerol, 2% (w/v) PVP-30), antioxidants (5
mM each of Na-ascorbate, Na-disul®te, DTT) mono-
terpene synthase activity was detected in Norway
spruce needles. Replacement of Hepes as extraction
bu€er by Tris, KPi, Mops or Bis±Tris did not signi®-
cantly enhance enzyme activity. Among the assay buf-
fers tested, highest enzyme activity was found with 50
mM Mops.
Mg2+ and/or Mn2+) (Alonso and Croteau, 1993;
Bohlmann et al., 1998). Presence of these cations at
concentrations of 0.1±20 mM was also found to be
essential for monoterpene synthase activity in Norway
spruce and holm oak. From work on other terpene
synthases, it is proposed that the function of bivalent
cations may be to neutralize the negative charge of the
allylic diphosphate moiety by complex formation (Cro-
teau, 1988; Alonso and Croteau, 1993). In Norway
spruce, enzyme activity was stimulated by Mg2+ and
Mn2+ with di€erent preferences for Mg2+ or Mn2+ as
cofactor for the synthesis of a-pinene/camphene and b-
pinene/limonene, respectively. Maximum monoterpene
synthase activities were achieved by the following com-
bination of Mg2+ and Mn2+ in the assays: addition of
Mn2+ up to a concentration of 2.5 mM stimulated
formation of a-pinene and camphene by 50%, while at
higher Mn2+ concentrations a-pinene and camphene
synthesis was inhibited. Formation of b-pinene and
limonene was maximally stimulated at 5 mM Mn2+ by
a factor of ca. 3, compared to tests with Mg2+ alone.
In holm oak, monoterpene synthase activity strongly
depended on the presence of Mg2+ in the assay.
Replacement of Mg2+ by Mn2+ reduced enzyme ac-
tivities by ca. 80%. When both cations were added,
increasing amounts of Mn2+ up to 20 mM strongly
inhibited the synthesis of a-pinene, sabinene, b-pinene,
and limonene. Since the bivalent cation requirement
di€ered between Norway spruce and holm oak leaves,
standard enzyme assays of Norway spruce extracts
were performed with 2.5 mM Mn2+ and 20 mM
Mg2+, while monoterpene synthase activities of holm
oak leaves were measured in the presence of 20 mM
Mg2+ (see Table 1).
Addition of the proteinase inhibitors phenylmethyl-
sulfonyl ¯uoride (PMSF) and benzamidine hydrochlo-
ride (BA) (each 1 mM) as well as the presence of
molybdate (1 mM) as inhibitor of phosphatases in the
extraction and assay bu€er did not in¯uence monoter-
pene synthase activities.
In addition to the strict dependency on bivalent cat-
ions, Bohlmann et al. (1997) reported a further
requirement of monoterpene synthases for the mono-
valent cation K+ from grand ®r (Abies grandis L.). In
the present work, additional supplementation of K+
of up to 600 mM has no e€ect on monoterpene
synthase activities in both species. Moreover, the repla-
2.3. Bivalent cation requirement of monoterpene
synthase activities
In general, monoterpene synthase activities are
strongly dependent on bivalent metal ions (usually
Table 1
Kinetic properties of monoterpene synthase activities in leaf extracts from Norway spruce needles and holm oak leaves
Norway spruce
Holm oak
pH optimum
Temperature optimum
Activation energy Ea (Arrhenius plot)
Broad optimum between pH 6.5 and 7.5
408C; activities measurable up to 508C
Sharp optimum at pH 6.7
408C; activities measurable up to 608C
1
1
a-Pinene: 60.5 kJ mol
Camphene: 78.2 kJ mol
a-Pinene: 62.6 kJ mol
Sabinene: 78.2 kJ mol
1
1
1
1
b-Pinene: 39.8 kJ mol
Limonene: 120.5 kJ mol
b-Pinene: 70.3 kJ mol
Limonene: 114.2 kJ mol
1
1
Apparent Michaelis-constants (Km) according to Hanes
a-Pinene: 17.7 mM
Camphene: 13.2 mM
b-Pinene: 26.2 mM
Limonene: 14.6 mM
a-Pinene: 55.0 mM
Sabinene: 57.3 mM
b-Pinene: 57.8 mM
Limonene: 107.6 mM

R.J. Fischbach et al. / Phytochemistry 54 (2000) 257±265
261
cement of the bivalent cations by the monovalent cat-
ion K+ in the assay caused a complete loss of enzyme
activity in both species.
monoterpene synthase activities dropped rapidly and
reached zero at about 508C. The temperature optima
of ca. 408C for monoterpene synthases from Norway
spruce needles were similar to the previously described
temperature optimum for the ( )-1S, 5S-pinene
synthase that was isolated from the hard wood of
grand ®r (Lewinsohn et al., 1992b).
2.4. Kinetic properties of monoterpene synthase
activities
In contrast to the sharp decline of enzyme activities
at temperatures higher than the optimum temperature
in Norway spruce, the temperature response curves of
monoterpene synthases in holm oak were bell-shaped
between 20 and 608C with maximal enzyme activities
at ca. 408C. This high temperature optimum of mono-
terpene synthases in holm oak leaves of ca. 408C is
likely to trigger the in vivo temperature optimum for
monoterpene emission between 35 and 458C (Loreto et
al., 1996a; Ciccioli et al., 1997; Staudt and Bertin,
1998) of this species.
Based on these results, standard enzyme assays were
run for 60 min with substrate concentrations (corre-
sponding to 5-times apparent Michaelis-constants) of
100 mM GDP for Norway spruce and 250 mM GDP
for holm oak. In addition, the protein concentrations
in the assay were kept to ca. 10 and 30 mg for Norway
spruce and holm oak, respectively. Measurement of
phosphatase activities with [1 3H]-GDP performed in
parallel to the non-radioactive enzyme assays showed
that under standard assay conditions approx. 13% and
3% of GDP was converted to oxygenated products
(i.e. geraniol), while approx. 6% and 8% of GDP was
found in the volatile monoterpene fraction in Norway
spruce and holm oak, respectively. These data clearly
show that under standard monoterpene assay con-
ditions no substrate limitation for the monoterpene
synthases can occur by competing phosphatase activi-
ties, as indicated by the time linearity of the monoter-
pene formation (see Table 1), and the fact that
phosphatase inhibition has no in¯uence as mentioned
above (see. Section 2.2).
Monoterpene synthase activity proceeded linearly
for 180 min in Norway spruce and 90 min in holm
oak leaf extracts (Table 1). With increasing protein
contents in the assay mixture, a linear increase in
monoterpene synthase activity up to 20 and 60 mg pro-
tein per assay in Norway spruce and holm oak was
observed, respectively.
The monoterpene synthase activities of Norway
spruce and holm oak leaves showed properties similar
to those of enzymes from other species (Croteau, 1988;
Alonso and Croteau, 1993; Lewinsohn et al., 1992a,
1992b). In Norway spruce (Table 1), apparent Michae-
lis-constants (Km), calculated from Hanes plots (Biss-
wanger, 1994), ranged between 13.2 mM for camphene
and 26.2 mM for b-pinene, while in leaf extracts of
holm oak higher Km-values were measured ranging
from 54.3 mM for sabinene to 107.6 mM for limonene.
Nevertheless, the bu€er system chosen in¯uenced the
apparent Michaelis-constants measured here. When
Hepes was used as assay bu€er for Norway spruce leaf
extracts, slightly lower constants were found ranging
from 7.1 mM for limonene to 14.6 mM for b-pinene.
Especially, in holm oak leaf extracts, the use of Hepes
as assay bu€er considerably lowered the apparent Km
to values ranging from 5.5 mM for limonene to 8.0 mM
for b-pinene, but on the other hand the maximum
enzyme velocity was reduced by 50% compared to KPi
as assay bu€er.
The e€ect of pH in the assay mixture was examined
using di€erent bu€er systems (Mes/KOH pH range
5.4±6.7; Hepes/KOH pH range 6.5±7.3; Tris/HCl pH
range 6.9±8.4). In Norway spruce (Table 1), a broad
pH optimum between pH 6.5 and 7.5 was observed for
monoterpene synthase activities. The pH optimum of
monoterpene synthase activities in holm oak was shar-
per with an optimum value at pH 6.7. Enzyme activi-
ties decreased drastically at pH values higher or lower
than 6.7 (see Table 1).
2.5. Molecular weight determination
Monoterpene synthases are soluble, although most
of them are associated with plastids in vivo (Colby et
al., 1993; Bohlmann et al., 1997; Turner et al., 1999).
To date, the vast majority of monoterpene synthases
characterized, e.g. the ( )-1S, 5S-pinene synthase from
grand ®r (Lewinsohn et al., 1992b), the ( )-4S-limo-
nene synthase from Mentha spicata L. (Colby et al.,
1993), or the 1,8-cineol synthase from Salvia ocinalis
L. (Wise et al., 1998), are monomers with native mol-
ecular weights Mr of ca. 56±72 kDa, although dimeric
forms are also found, e.g. the g-terpinene synthase (Mr
ca. 96 KDa) from Thymus vulgaris L. (Alonso and
Croteau, 1991) or the bornyldiphosphate synthase (Mr
ca. 135 KDa) from S. ocinalis (Wise et al., 1998).
Among several environmental variables tested, tem-
perature had a dominant e€ect on monoterpene emis-
sion of plants (Tingey et al., 1991). Monoterpene
synthase activities of both species also responded
strongly to assay temperature (Table 1). In Norway
spruce, the enzyme activities responsible for a-pinene
and camphene formation increased exponentially
between 20 and 408C by a factor of 5.1 and 7.5, re-
spectively, while synthesis of b-pinene and limonene
was stimulated in this temperature range by a factor of
2.1 and 2.3, respectively. At higher temperatures,

262
R.J. Fischbach et al. / Phytochemistry 54 (2000) 257±265
Calibrated gel permeation chromatography with leaf
protein extracts. Nevertheless, depending on di€erent
temperature response curves and di€erent preferences
for Mg2+ ions in Norway spruce, two groups of
enzymes, i.e. (a) forming a-pinene and camphene and
(b) forming b-pinene and limonene, can be distin-
guished in this species. In holm oak, the biochemical
characteristics of the limonene-forming enzyme di€ered
from those of the a-pinene, sabinene, and û-pinene
producing activities and therefore also give indications
for the presence of several monoterpene synthases.
Future work on Norway spruce and holm oak will
focus on the isolation of the di€erent monoterpene
synthase proteins and genes, which are prerequisites
for studying the regulation of enzyme activities and
gene expression in order to understand the contri-
bution of monoterpene synthases to the observed
monoterpene emission of plants.
extracts of Norway spruce and holm oak (after a
100,000 g centrifugation to remove microsomal mem-
branes) revealed single peaks of activity at elution
volumes corresponding to Mr of ca. 59 and 50 kDa for
Norway spruce (Fig. 4a) and holm oak (Fig. 4b), re-
spectively, indicating that the native proteins are also
functional monomers.
Since many monoterpene synthases generate mul-
tiple products from GPD (Bohlmann et al., 1997; Wise
et al., 1998), as e.g. the ( )-4S-limonene synthase
found in the wood of grand ®r generates ( )-limonene
and to a lesser extent ( )-a-pinene, ( )-b-pinene and
( )-b-phellandrene (Bohlmann et al., 1997), our
measurements re¯ect the overall enzymatic synthesis of
acyclic (myrcene), monocyclic (limonene) and bicyclic
monoterpenes (e.g. a-pinene), and do not allow any
conclusions on the distinct number of monoterpene
synthases present in holm oak or in Norway spruce
3. Experimental
3.1. Chemicals
Bu€er reagents and other chemicals were obtained
commercially and were of analytical grade. The syn-
thesis of geranyl-diphosphate (GDP) was carried out
according to Keller and Thompson (1993). The pro-
1
duct was identi®ed by H- and 31P-NMR spectroscopy
(AC-400 FT-NMR spectrometer, Bruker, Karlsruhe,
Germany) with D2O as solvent.
3.2. Plant material, growth and irradiation conditions
Norway spruce seeds were purchased from the
Staatliche Samenklenge Laufen (Bavaria, Germany).
The seeds were germinated as described by Rosemann
et al. (1991) for Scots pine, and were cultivated in a
growth chamber at 24/158C day/night temperature,
and a 14 h light period from 6 a.m. to 8 p.m. (Sylvania
VHO F195W cool white, Erlangen, Germany) at a
photosynthetic photon ¯ux density [PPFD] of 60 mmol
2
m
s
1. After thirty days of cultivation, the primary
needles of the seedlings were harvested, shock frozen
in liquid nitrogen and stored at 808C until use.
Current year leaves of naturally grown holm oak
trees were sampled on August 4, 1998 in the plant gar-
den of the Centre d'Ecologie Functionelle et Evolutive
du CNRS, Montpellier (France). Leaves were cut from
the twigs, shock frozen in liquid nitrogen, transferred
to Garmisch-Partenkirchen on dry ice, and stored at
808C until use.
Fig. 4. Molecular weight determination of monoterpene synthase ac-
tivities in Norway spruce (a) and holm oak (b) by FPLC on Super-
dex-200 HR. Monoterpene synthase activities were determined by
incubating aliquots of 88 ml under standard conditions (*±* a-
pinene, Q±Q camphene, R±R b-pinene, W±W limonene, T±T sabi-
nene; w±w protein). Calibration of the column (c) was performed
with the molecular weight markers (1) ferritin: 440 kDa, (2) catalase:
232 kDa, (3) aldolase: 158 kDa, (4) bovine serum albumine: 67 kDa,
(5) ovalbumine: 43 kDa, (6) thyroglobulin: 25 kDa ꢀn ˆ 2; 2sd).
3.3. Preparation of protein extracts
Leaf material (250 mg) of holm oak or Norway
spruce was homogenized under liquid nitrogen with a

R.J. Fischbach et al. / Phytochemistry 54 (2000) 257±265
263
mortar and pestle and suspended in 6 ml protein
extraction bu€er [for Norway spruce: 50 mM Hepes/
KOH pH 7.3 containing 10% (v/v) glycerol, 2% (w/v)
PVP-30, antioxidants (5 mM each of Na-ascorbate,
Na-disul®te, DTT); for holm oak: 700 mM Mops/HCl
pH 7.3 containing 1.5% (w/v) PEG-1500, 1% (w/v)
PVP-30, 8.3% (w/v) Dowex 1 Â 2, 20 mM MgCl2,
antioxidants (200 mM ascorbic acid, 50 mM 2-mercap-
toethanol)] and stirred in an ice bath for 20 min. Cell
debris were sedimented by centrifugation (18,000 g for
20 min) at 48C. Aliquots of 2.5 ml of the supernatant
were desalted on PD-10 columns [Pharmacia, Uppsala,
Sweden; equilibration and elution bu€er for Norway
spruce 50 mM Mops/HCl pH 7.3 or for holm oak 50
mM KPi, pH 7.3, each bu€er containing 10% (v/v)
glycerol and antioxidants (10 mM Na-ascorbate, Na-
disul®te, DTT, each)] resulting in a ®nal volume of 3.5
ml which was stored at 808C. Protein was determined
according to Bradford (1976) with BSA as a standard.
ruhe, Germany) and by GC-MS analysis (ATD 400,
Autosystem XL GC, Turbo Mass MS spectrometer,
Perkin Elmer, Beacons®eld, UK). The amounts of
monoterpenes formed in vitro were calculated using
the relative response factor obtained from standard
mixtures of known concentrations (see Fig. 3). Protein
extracts without the substrate GDP were carried out in
parallel in order to correct for non-enzymatic back-
ground.
3.5. GDP phosphatase assay
Measurement of phosphatase activity was performed
according to the method of Alonso et al. (1992). Tests
with [1 3H]-GDP (3.7 kBq nmol 1, Biotrend, Koln,
Germany) were done in Eppendorf tubes under similar
conditions and compared to the non-radioactive
monoterpene synthase assay (see Section 3.4). Follow-
ing the extraction of monoterpenes (in 2 Â 0.5 ml hex-
ane) and of oxygenated products (i.e. geraniol
liberated from the substrate by endogenous phospha-
tases) (in 2 Â 0.5 ml diethyl ether) 250 ml aliquots of
hexane and diethyl ether eludates were transferred to
scintillation vials containing 15 ml Quicksafe A (Zins-
ser Analytik, Frankfurt, Germany) for determination
of [3H] content (Beckman LS 3801, Munchen,
Germany; [3H] eciency = 51.5%).
3.4. Monoterpene synthase standard enzyme assay
For standard enzyme assays, 91 ml of protein
extracts were transferred into gas-tight 2 ml crimp seal
vials (Supelco, Bellefonte, USA) after addition of 4 ml
0.5 M MgCl2 (®nal concentration: 20 mM) for holm
oak and 4 ml 0.5 M MgCl2/ 62.5 mM MnCl2 (®nal
concentration: 20 mM MgCl2, 2.5 mM MnCl2) for
Norway spruce. The enzyme reaction was started by
the addition of 5 ml of a 2 mM or 5 mM geranyl-
diphosphate (GDP) solution (®nal concentration: 100
mM for Norway spruce and 250 mM for holm oak)
and incubated for 60 min at 408C. The enzyme reac-
tion was terminated by removing the reaction mixture
from the closed vial with a syringe, and washing the
vial with 150 ml assay bu€er. Samples of the head
space (1.0 ml) were trapped at 278C on a pre-column
®lled with Tenax TA 60/80 (Alltech, Unterhaching,
Germany), and reaction products were separated by
capillary gas chromatography (GC) after heating the
precolumn to 2408C. The GC system (Chrompack CP
9000, Chrompack, Frankfurt/M., Germany) was
equipped with a head space-volume-autosampler (HSS,
3.6. FPLC gel permeation chromatography
The native molecular weight of monoterpene
synthase proteins from Norway spruce and holm oak
were determined from 100,000 g centrifugation (1 h)
supernatants of crude leaf protein extracts, concen-
trated by ultra®ltration (Microcon-10, Amicon, Wit-
ten, Germany) to ®nal protein concentrations of c. 3
mg ml 1. 200 ml of theses fractions were separated on
a Superdex-200 HR column (Pharmacia, Uppsala,
Sweden) by FPLC (controller LCC-500, pumps P-500,
fraction collector Frac-100, Pharmacia, Uppsala, Swe-
den). The proteins were eluted in 50 mM Mops/HCl
pH 7.3 (for Norway spruce) and 50 mM KPi, pH 7.3
(for holm oak), each bu€er containing 10% (v/v) gly-
cerol and antioxidants (10 mM Na-ascorbate, Na-
disul®te, DTT, each) at a ¯ow rate of 0.4 ml bu€er
min 1. The protein concentration in each fraction
(0.25 ml) was determined according to Bradford
(1976). Monoterpene synthase activities were deter-
mined as described in Section 3.4. Calibration of the
Superdex-200 HR column was performed with the
high and low molecular weight protein kits from Phar-
macia (Uppsala, Sweden).
Gerstel, Mulheim, Germany),
a temperature-pro-
grammed-injection system (KAS 3, Gerstel, Mulheim,
Germany) and a capillary column (DB 1701, 30m, ID
0.25 mm, ®lm thickness 1 mm; J&W Scienti®c, Folsom,
CA, USA). Separation was performed with a tempera-
ture program starting at 358C, increasing after 0.5 min
1
at a rate of 308C min to 788C, remaining for 4 min
at this condition, the temperature was then raised at a
1
rate of 98C min to 1608C, and then at a rate of 358C
1
min to the ®nal temperature of 2508C. The products
were detected with a ¯ame ionization detector (FID;
temperature: 2708C), and monoterpenes were identi®ed
by co-injection with authentic standards (Roth, Karls-
3.7. Statistical analysis
Statistical analysis was performed with SPSS version

264
R.J. Fischbach et al. / Phytochemistry 54 (2000) 257±265
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8.0 for Windows. Di€erences between mean values of
the samples were compared using a one way analysis
of variance (ANOVA).
Gegenheimer, P., 1990. Preparation of extracts from plants. Methods
in Enzymology 182, 174±193.
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pathway and formation of monoterpenes, sesquiterpenes, and
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Acknowledgements
The authors thank Prof. H. Rennenberg, University
of Freiburg, Dr. W. Zimmer, Fraunhofer-Institut fur
Atmospharische Umweltforschung, Garmisch-Parten-
kirchen, and Dr. W. Heller, GSF-Institut fur Bioche-
mische P¯anzenpathologie, Neuherberg for critical
reading of the manuscript, and Dr. P. Haug for helpful
linguistic improvements. The work was supported by
the European Community in the frame of the
ECOVOC project (Parameterization of Environmental
and Physiological Controls of Volatile Organic Com-
pound Emissions from European Forests) as part of
the 4th Framework Programme on Environment and
Climate 1994±1998.
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Graedel, T., Harley, P., Klinger, L., Lerdau, M., McKay, W.A.,
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J., Zimmerman, P., 1995. A global model of natural volatile or-
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