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Plant Physiol. (1999) 121: 173-180
Spider Mite-Induced (3S)-(E)-Nerolidol
Synthase Activity in Cucumber and Lima Bean. The First Dedicated Step
in Acyclic C11-Homoterpene Biosynthesis1
Harro J. Bouwmeester*,
Francel W.A. Verstappen,
Maarten A. Posthumus, and
Marcel Dicke
Research Institute for Agrobiology and Soil Fertility, P.O. Box 14, 6700 AA Wageningen, The Netherlands (H.J.B., F.W.A.V.); Laboratory of
Organic Chemistry, Wageningen Agricultural University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands (M.A.P.); and Laboratory of
Entomology, Wageningen Agricultural University, Binnenhaven 7, 6709 PD
Wageningen, The Netherlands (M.D.)
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ABSTRACT |
Many plant species respond to
herbivory with de novo production of a mixture of volatiles that
attracts carnivorous enemies of the herbivores. One of the major
components in the blend of volatiles produced by many different plant
species in response to herbivory by insects and spider mites is the
homoterpene 4,8-dimethyl-1,3(E),7-nonatriene. One study
(J. Donath, W. Boland [1995] Phytochemistry 39: 785-790) demonstrated that a number of plant species can convert the acyclic sesquiterpene alcohol (3S)-(E)-nerolidol
to this homoterpene. Cucumber (Cucumis sativus L.) and
lima bean (Phaseolus lunatus L.) both produce
4,8-dimethyl-1,3(E),7-nonatriene in response to
herbivory. We report the presence in cucumber and lima bean of a
sesquiterpene synthase catalyzing the formation of
(3S)-(E)-nerolidol from farnesyl
diphosphate. The enzyme is inactive in uninfested cucumber leaves,
slightly active in uninfested lima bean leaves, and strongly induced by
feeding of the two-spotted spider mite (Tetranychus
urticae Koch) on both plant species, but not by mechanical wounding. The activities of the
(3S)-(E)-nerolidol synthase correlated well with the levels of release of
4,8-dimethyl-1,3(E),7-nonatriene from the leaves of the
different treatments. Thus,
(3S)-(E)-nerolidol synthase is a good
candidate for a regulatory role in the release of the important
signaling molecule 4,8-dimethyl-1,3(E),7-nonatriene.
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INTRODUCTION |
Many plant species respond to arthropod herbivory with the
production of a blend of volatiles that attracts carnivorous enemies of
the herbivores, such as predators or parasitic wasps (Dicke et al.,
1990b ; Turlings et al., 1990, 1995; Vet and Dicke, 1992). For example,
upon infestation with two-spotted spider mites (Tetranychus urticae), lima bean (Phaseolus lunatus L.) plants
respond with the emission of a mixture of volatiles attracting the
predatory mite Phytoseiulus persimilis (Dicke et al.,
1990b), which effectively eliminates local populations of spider mites
(Dicke et al., 1990a). The induced production of carnivore-attracting
volatiles has been recorded for over 20 plant species in 13 families
(Dicke, 1999), and these volatiles are usually either not emitted or
emitted only in minor quantities in response to mechanical wounding.
Examples are lima bean (Dicke et al., 1990b), corn (Turlings et al.,
1990), and cucumber (Cucumis sativus L.) (Takabayashi et
al., 1994). Each plant species emits its own specific blend of
volatiles originating from several different biosynthetic pathways such
as the isoprenoid, shikimic acid, and lipoxygenase pathways.
One of the compounds derived from the isoprenoid biosynthetic pathway,
the homoterpene 4,8-dimethyl-1,3(E),7-nonatriene (Fig. 1), was shown to be an important
constituent of the volatile blend emitted by the leaves of a large
number of plant species in response to herbivory (e.g. lima bean
[Dicke et al., 1990b]; cucumber [Dicke et al., 1990a;
Takabayashi et al., 1994], corn [Turlings et al., 1990], cotton
[Loughrin et al., 1994], and apple [Takabayashi et al.,
1994]), but also of the fragrance produced by many flowers (Kaiser, 1991). The homoterpene is an important component of the volatile blend of lima bean and cucumber plants infested with the
spider mite T. urticae, and attracts the predatory mite
P. persimilis (Dicke et al., 1990a). A synthetic mixture of
volatiles, including this homoterpene, that mimics the blend of
volatiles that is emitted by corn plants infested with Spodoptera
exigua caterpillars, attracts the parasitoid Cotesia
marginiventris, which parasitizes the caterpillars (Turlings et
al., 1991).
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| Figure 1.
Schematic representation of the release of
4,8-dimethyl-1,3(E),7-nonatriene, a volatile homoterpene
released by many plant species after herbivory. Donath and Boland
(1995) demonstrated the ability of leaves and flowers of several
species to convert nerolidol to
4,8-dimethyl-1,3(E),7-nonatriene (2). We
investigated the induction by herbivory in cucumber and lima bean of
nerolidol synthase (1), the enzyme that should precede the
sequence of reactions reported by Donath and Boland (1995).
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Studies on the signaling cascade leading to the production of
4,8-dimethyl-1,3(E),7-nonatriene have recently been
initiated, and it was demonstrated that herbivore oral secretions and
components thereof can induce its formation in plants (Turlings et al.,
1990; Alborn et al., 1997). Using
13CO2 pulse-labeling
experiments, Paré and Tumlinson (1997) demonstrated that, among
many other volatiles, 4,8-dimethyl-1,3(E),7-nonatriene is
biosynthesized de novo in response to herbivory. Using
2H-labeled precursors, Donath and Boland (1994,
1995) elegantly demonstrated the ability of several plant species, such
as lima bean, to produce
4,8-dimethyl-1,3(E),7-nonatriene from
(3S)-(E)-nerolidol. The ability to convert
(3S)-(E)-nerolidol to
4,8-dimethyl-1,3(E),7-nonatriene is not restricted to plants
infested by herbivores: Uninfested leaves and flowers of
several species convert exogenous nerolidol to
4,8-dimethyl-1,3(E),7-nonatriene (Gäbler et al., 1991;
Donath and Boland, 1995), showing that the biosynthesis of
4,8-dimethyl-1,3(E),7-nonatriene from nerolidol is not
regulated or induced by herbivory. Therefore, regulation of the release
of 4,8-dimethyl-1,3(E),7-nonatriene must occur in the
biosynthetic pathway upstream of the sesquiterpenoid nerolidol. The
enzyme catalyzing the formation of a sesquiterpene skeleton from the
ubiquitous precursor farnesyl diphosphate (FDP) is a good candidate for
playing such a regulatory role (Gershenzon and Croteau, 1990; McGarvey
and Croteau, 1995).
Although nerolidol, the sesquiterpene analog of the monoterpenoid
linalool, is a component of many essential oils and flower headspaces
(Bauer et al., 1990; Knudsen et al., 1993), enzymes catalyzing the
formation of nerolidol from FDP have not to our knowledge been
reported. The products of many of the enzymes catalyzing the formation
of a terpenoid skeleton from the respective diphosphate substrates are
mostly cyclic hydrocarbons (Bohlmann et al., 1998; Bouwmeester et al.,
1998; De Kraker et al., 1998), but there are some exceptions to this
rule. For example, Munck and Croteau (1990) purified a patchoulol
synthase from Pogostemon cablin. The enzyme catalyzing the
formation of the acyclic terpenol linalool from geranyl diphosphate was
purified and the corresponding gene cloned from Clarkia
breweri (Dudareva et al., 1996).
In the present study we investigated whether a nerolidol synthase
catalyzes the first dedicated step in the biosynthesis of 4,8-dimethyl-1,3(E),7-nonatriene in response to herbivory
(Fig. 1). To that end we infested lima bean and cucumber plants with T. urticae to induce volatile production and measured the
induction of sesquiterpene synthase activity.
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MATERIALS AND METHODS |
Plant Material
Cucumber (Cucumis sativus L. cv Corona) plants were
grown from seeds in 1-L pots containing potting compost in a greenhouse at a 20°C/18°C, 12-h/12-h light/dark cycle. Plants of lima bean (Phaseolus lunatus L. cv Sieva) were grown from seeds in 1-L
plastic pots containing potting compost in a greenhouse at a 20°C to
30°C, 16-h/8-h light/dark cycle, with natural daylight supplemented with high-pressure mercury lights. Two-spotted spider mites
(Tetranychus urticae Koch) were reared on lima bean plants
under the same greenhouse conditions. Cucumber and lima bean plants
were infested with spider mites at 30 and 21 d after germination,
respectively, by placing leaves of spider mite-infested lima bean
plants on the leaves of plants to be infested. Mechanical damage to
cucumber plants was applied by rubbing the entire surface of all
expanded leaves with carborundum powder (180 grit) on a wet cotton wool
swab and spraying with water 0 and 5 d after infection of spider
mite-treated plants. The plants were kept under the same greenhouse
conditions until harvest. For each treatment two plants were used and
leaves were harvested at d 5 after infestation. One sample of leaves was enclosed in a 1-L jar for headspace analysis (see below); another
sample was frozen in liquid N2 and stored at
 80°C for analysis of sesquiterpene synthase activity (see below).
Headspace Analysis
Glass jars (1 L) containing the detached leaves of the respective
treatments were closed with a Teflon-lined lid equipped with an inlet
and an outlet, and placed upside down in a climate room at 25°C and a
light intensity of 210 µmol m 2
s1 provided by 400-W lights (HPI-T, Philips,
Eindhoven, The Netherlands). A vacuum pump was used to draw off air
through the glass jar at approximately 100 mL
min1, with the incoming air purified through a
glass cartridge (140 × 4 mm) containing 150 mg of Tenax TA
(20/35-mesh, Alltech). At the outlet the volatiles emitted by the
detached leaves were trapped on a similar cartridge. Volatiles were
sampled for 3 h. Cartridges were eluted using 3 × 1 mL of
re-distilled pentane:diethyl ether (4:1). Of the (non-concentrated)
samples, 2 µL was analyzed by GC-MS using a gas chromatograph (5890 series II, Hewlett-Packard) equipped with a 30-m × 0.25-mm i.d.,
0.25-µm film thickness column (5MS, Hewlett-Packard) and a
mass-selective detector (model 5972A, Hewlett-Packard). The GC was
programmed at an initial temperature of 45°C for 1 min, with a ramp
of 10°C min1 to 220°C and final time of 5 min. The injection port (splitless mode), interface, and MS source
temperatures were 250°C, 290°C, and 180°C, respectively, and the
He inlet pressure was controlled with an electronic pressure control to
achieve a constant column flow of 1.0 mL min1.
The ionization potential was set at 70 eV, and scanning was performed
from 30 to 250 atomic mass units.
Enzyme Isolation and Product Identification
During enzyme isolation and preparation of the assays, all
operations were carried out on ice or at 4°C. Frozen leaves (1.0 g)
were ground in a pre-chilled mortar and pestle in 8 mL of buffer containing 50 mM MOPSO (pH 6.8), 20% (v/v) glycerol, 50 mM sodium ascorbate, 50 mM
NaHSO3, 10 mM
MgCl2, and 5 mM DTT (buffer A) slurried with polyvinylpolypyrrolidone (0.1 g
g1 tissue) and a spatula tip of purified sea
sand. Polystyrene resin (0.5 g g1 tissue,
Amberlite XAD-4, Sigma) was added and the slurry was stirred carefully
for 10 min and then filtered through cheesecloth. The filtrate was
centrifuged at 20,000g for 20 min, the pellet discarded, and
the supernatant centrifuged at 100,000g for 90 min. Three
milliliters of the 100,000g supernatant were desalted to
buffer B containing 15 mM MOPSO (pH 7.0), 10%
(v/v) glycerol, 10 mM
MgCl2, 1 mM sodium
ascorbate, and 2 mM DTT. For experiments with the
phosphohydrolase inhibitor vanadate, buffer B was supplemented with 5 mM sodium orthovanadate (Janssen Chimica, Geels,
Belgium).
Depending on the experiment, 10 or 50 µM
[3H]FDP (at 100 or 20 Ci
mol1, respectively)
([3H]FDP from Amersham; unlabeled FDP from
Sigma; De Kraker et al., 1998) was added to 0.5 or 1 mL of the enzyme
preparations. Assays without MgCl2 (omitted from
buffer B) were run to test the effect of Mg2+.
After the addition of a 1-mL redistilled pentane overlay, the assays
were incubated for 30 or 60 min at 30°C. Controls that had been
boiled for 5 min showed no enzymatic activity. After the assay, the
tubes were vortexed and the pentane layer was removed and passed over a
short column of grade III aluminum oxide overlaid with anhydrous
MgSO4. The assay was extracted again with 1 mL of
redistilled diethyl ether, which was also passed over the aluminum oxide column, and the column was washed with 1.5 mL of diethyl ether.
The total volume of the pentane/diethyl ether extract was determined
and 50 or 100 µL of the extract was removed for liquid-scintillation counting in 4.5 mL of scintillation cocktail (Ultima Gold, Packard Bioscience, Groningen, The Netherlands). The distribution of the radiolabel over different products in the pentane/diethyl ether extract
was determined using radio-GLC (i.e. the ratio between the peak areas
in the radiosignal).
Before radio-GLC analysis, unlabeled reference compounds
(Z)- and (E)-nerolidol and
(E,E)-farnesol were added to the extract, which was then
slowly concentrated under a stream of N2.
Radio-GLC was performed on a gas chromatograph (4160 series,
Carlo-Erba, Milano, Italy) equipped with a radioactivity detector
(RAGA-90, Raytest, Straubenhardt, Germany). Sample components eluting
from the column were quantitatively reduced before radioactivity was measured by passage through a conversion reactor filled with platinum chips at 800°C. Samples of 1 µL were injected in the cold,
on-column mode. The GC was equipped with a 30-m × 0.32-mm i.d.,
0.25-µm film thickness EC-WAX column (EconoCap,
Alltech) or an enantioselective column coated with
heptakis(6-O-tert-butyldimethylsilyl-2,3-di-O-methyl) - -cyclodextrin
(50% in OV17, w/w) (25-m × 0.25-mm i.d.; König et al.,
1994) and operated with a He flow of 1.2 mL
min1. For use with the EC-WAX column, the oven
temperature was programmed to 70°C for 5 min, followed by a ramp of
5°C min1 to 210°C and a final time of 5 min. For use with the enantioselective column, the oven temperature was
programmed to 116°C for 2 min, followed by a ramp of 0.5°C
min1 to 125°C and a final time of 10 min.
About 20% of the column effluent was split with an adjustable splitter
to a flame ionization detector (310°C). The remainder was directed to
the conversion reactor and radiodetector. H2 was
added prior to the reactor at 3 mL min1, and
CH4 as a quench gas prior to the radioactivity
detector (5-mL-counting tube) to give a total flow of 36 mL
min1. A sample containing the four isomers of
nerolidol was a kind gift of W.A. König, who also determined
their elution order on the enantioselective column (König et al.,
1992).
GC-MS identification of the products was carried out as described above
in "Headspace Analysis," but scanning was performed in both the
scanning mode and the selected ion monitoring mode: m/z 93, 107, 136, and 161.
Linearity of the enzymatic reaction was tested on 100-µL enzyme
preparations (undiluted and 2-fold diluted) in buffer B supplemented with 5 mM sodium orthovanadate in Eppendorf tubes to which
50 µM [3H]FDP was added. The
reaction mixture was overlaid with 1 mL of hexane to trap volatile
products, and the contents were carefully mixed and centrifuged briefly
to separate phases. After incubation at 30°C, the vials were
immediately cooled, vigorously mixed, and centrifuged to separate
phases. A 750-µL portion of the hexane phase was removed for liquid
scintillation counting in 4.0 mL of scintillation cocktail. All assays
were performed in duplicate; controls that had been boiled for 5 min
and incubated for 45 min were used to determine nonenzymatic
hexane-soluble products. The ratio between nerolidol and farnesol
produced was determined in 1.0-mL enzyme assays run for 30, 60, and 90 min in buffer B under the same conditions as the 100-µL assays. These
assays were worked up and analyzed by GLC as described above. The ratio
between nerolidol and farnesol in the 1-mL assays was used to calculate
the contribution of nerolidol to the total hexane-soluble radiolabeled
products of the 100-µL assays.
For quantitative comparison of enzymatic activities between treatments,
duplicate 0.5-mL assays were run under the conditions giving a linear
assay, i.e. with undiluted enzyme, for 60 min, as well as in the
presence of 50 µM [3H]FDP and 5 mM sodium orthovanadate. The assays were worked up and
analyzed by GLC as described above.
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RESULTS AND DISCUSSION |
Headspace Analysis
Undamaged leaves of cucumber emitted only traces of volatiles
(Fig. 2A). Spider mite infestation caused
a dramatic change in the production of volatile compounds by cucumber
leaves (Fig. 2B), with the major compounds being
(Z)-3-hexen-1-yl acetate (1), (E)--ocimene (2), and the C11 homoterpene
4,8-dimethyl-1,3(E),7-nonatriene (3). These
compounds have been reported previously to be induced in cucumber by
spider mite infestation (Takabayashi et al., 1994), and are also
important constituents of the herbivore-induced blends of volatiles in
corn and cotton (Turlings et al., 1991; Röse et al., 1996).
Mechanical wounding of cucumber leaves did not induce these volatiles
(Fig. 2C).
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| Figure 2.
Headspace analysis of cucumber and lima bean
leaves. A, Undamaged cucumber leaves; B, cucumber leaves infested with
spider mites for 5 d; C, cucumber leaves mechanically damaged with
carborundum powder at 0 and 5 d before sampling; D, uninfested
lima bean leaves; and E, lima bean leaves infested with spider mites
for 5 d. Headspace samples were analyzed by GC-MS. The peaks are:
(Z)-3-hexen-1-yl acetate (1);
(E)--ocimene (2);
4,8-dimethyl-1,3(E),7-nonatriene (3); methyl
salicylate (4); and
4,8,12-trimethyl-1,3(E),7(E),11-tridecatetraene
(5).
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Uninfested lima bean leaves produced traces of several volatiles,
including some normally associated with herbivore induction, such as
(E)--ocimene (2) and
4,8-dimethyl-1,3(E),7-nonatriene (3) (Fig. 2D).
However, spider mite infestation dramatically increased the production
of these volatiles by lima bean leaves and induced the formation of a
number of compounds not detectable in the headspace of control leaves,
such as methyl salicylate (4) and the C16 homoterpene
4,8,12-trimethyl-1,3(E),7(E),11-tridecatetraene (5) (Fig. 2E), which have been reported previously to
be induced by spider mites in lima bean (Dicke et al.,
1990b). The C16 homoterpene
4,8,12-trimethyl-1,3(E),7(E),11-tridecatetraene has been suggested to be derived from the diterpene analog of nerolidol, geranyllinalool (Gäbler et al., 1991), and is also a
constituent of herbivore-induced volatile blends of a number of other
plant species such as corn and cotton (Turlings et al., 1991;
Paré and Tumlinson, 1997). Methyl salicylate attracts the predatory mite P. persimilis to spider mite-infested lima
bean plants (Dicke et al., 1990b). In addition, methyl
salicylate is supposed to play an important role in the activation of
plant defense responses (Yang et al., 1997), such as the induction in tobacco plants of systemic acquired resistance against tobacco mosaic
virus reported by Shulaev et al. (1997).
Sesquiterpene Synthase Activity and Product Identification
In both cucumber and lima bean, the C11 homoterpene
4,8-dimethyl-1,3(E),7-nonatriene showed a strong induction
after spider mite infestation (Fig. 2; Dicke et al., 1990a,
1990b; Takabayashi et al., 1994). To investigate whether
this coincides with an increase in nerolidol synthase activity,
undamaged, mechanically wounded (cucumber only) and spider
mite-infested leaves were assayed for nerolidol synthase activity.
Enzyme assays on control and mechanically damaged cucumber leaves
showed only the formation of (E,E)-farnesol, produced from
[3H]FDP by phosphohydrolase activity, and some
unknown minor compounds (Fig. 3, B and
D). In assays on spider mite-infested
leaves, a second radiolabeled product was detected in addition to
(E,E)-farnesol, which co-eluted on radio-GLC with
(E)-nerolidol (Fig. 3C). The product also co-eluted with an
authentic standard of (E)-nerolidol on the more apolar
column used for GC-MS analysis, and had the same mass spectrum as the
standard (data not shown). In lima bean, (E)-nerolidol
synthase activity was also detected in control leaves (Fig.
4B), but the activity increased strongly
upon spider mite feeding (Fig. 4C).
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| Figure 3.
Radio-GLC analysis of radiolabeled products formed
from 10 µM [3H]FDP (60-min assay in buffer
B) by enzyme preparations of cucumber leaves. A, Flame ionization
detection (FID) signal of co-injected, unlabeled standards of
(Z)-nerolidol (1),
(E)-nerolidol (2), and
(E,E)-farnesol (3). B to D,
Radiotraces showing radiolabeled products of assays with undamaged
cucumber leaves (B), cucumber leaves infested with spider mites for
5 d (C), and cucumber leaves mechanically damaged with carborundum
powder 0 and 5 d before sampling (D).
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| Figure 4.
Radio-GLC analysis of radiolabeled products formed
from 50 µM [3H]FDP (30-min assay in buffer
B) by enzyme preparations of lima bean leaves. A, FID signal of
co-injected, unlabeled standards of (Z)-nerolidol
(1), (E)-nerolidol (2), and
(E,E)-farnesol (3). B to C,
Radiotraces showing radiolabeled products of assays with undamaged lima
bean leaves (B) and lima bean leaves infested with spider mites for
5 d (C).
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For both plant species, assays in the presence of vanadate and 50 µM [3H]FDP on enzyme extracts of
spider mite-induced leaves were linear with protein concentration and
time (for over 60 min) (Fig. 5). In
cucumber, (E)-nerolidol synthase activity increased from
being undetectable in control leaves to 10.7 nmol
h1 g1 (fresh weight) in
spider mite-infested leaves (Table I). In the absence of vanadate, phosphohydrolase activity increased 10- to
15-fold in cucumber and lima bean, whereas (E)-nerolidol
synthase activity slightly decreased (Table I). This small decrease was probably due to the much higher substrate utilization by
phosphohydrolases in the absence of vanadate and the corresponding
lower substrate availability for (E)-nerolidol synthase
(Table I).
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| Figure 5.
Time-dependent formation (dpm per 100-µL assay)
of nerolidol ( , right y axis) and total
hexane-soluble products (left y axis) for undiluted
( ) and 2-fold-diluted ( ) enzyme preparations of spider
mite-infested cucumber (A) and lima bean (B). Enzyme extracts of spider
mite-infested leaves were desalted to assay buffer B supplemented with
5 mM sodium orthovanadate, and assayed with 50 µM [3H]FDP as a substrate. Total
hexane-soluble products were determined in duplicate 100-µL assays,
and nerolidol formation was analyzed by radio-GLC on 1-mL enzyme assays
run for 30, 60, and 90 min.
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Table I.
Nerolidol synthase and phosphohydrolase (production
of [3H]-(E,E)-farnesol) activities in leaves of lima bean
and cucumber with and without spider mite infestation
Results are means ± SD of two replicates. Enzyme
extracts were prepared as described in ``Materials and Methods''.
Duplicate 0.5-mL enzyme preparations (in buffer B) were incubated for
60 min at 30°C with 50 µM [3H]FDP (20 Ci
mol1) with or without 5 mM sodium
orthovanadate and with or without 10 mM MgCl2.
Total radioactivity in the pentane/diethyl ether extracts of the assays
(containing nerolidol, if present, as well as farnesol) was determined
on a 50-µL subsample using liquid-scintillation counting. The
distribution of the radiolabel over nerolidol and farnesol was
determined using radio-GLC (ratio of peak areas in radiotrace). For
further details, see ``Materials and Methods''.
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In lima bean, (E)-nerolidol synthase activity was detected
in both control and spider mite-infested leaves (Fig. 4, Table I), but
the activity of (E)-nerolidol synthase increased
approximately 6-fold upon spider mite infestation (Table I). In both
species the absence of Mg2+ in the assay buffer
decreased (E)-nerolidol synthase activity by 65% to 85%
(Table I). The requirement for Mg2+ is in line
with reports on other sesquiterpene synthases (Munck and Croteau, 1990;
De Kraker et al., 1998). Furthermore, the lack of an effect of vanadate
on nerolidol synthase activity, in contrast to the strong inhibition of
farnesol formation, supports the involvement of a specific
sesquiterpene synthase. The absence of (E)-nerolidol synthase activity in control and mechanically wounded cucumber leaves,
the low background activity in lima bean leaves, and the strong
induction of the enzyme upon spider mite infestation in both species
correlate well with the emission of
4,8-dimethyl-1,3(E),7-nonatriene, as shown in the
corresponding headspace profiles in Figure 2.
The absolute configuration of the (E)-nerolidol produced was
established by radio-GLC using an enantioselective column. Figure 6 shows that for both species the
enzymatically produced nerolidol was
(3S)-(E)-nerolidol. For a number of plant
species, it has been shown that leaves and flowers, which have also
been shown to emit 4,8-dimethyl-1,3(E),7-nonatriene (Kaiser,
1991; Knudsen et al., 1993), were able to convert exogenous nerolidol
to 4,8-dimethyl-1,3(E),7-nonatriene (Gäbler et al.,
1991; Donath and Boland, 1995). The conversion in plant leaves occurred
with a high degree of enantioselectivity, and in all species
investigated (3S)-(E)-nerolidol was the preferred substrate compared with (3R)-(E)-nerolidol
(Donath and Boland, 1995).
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| Figure 6.
Radio-GLC analysis of sesquiterpene synthase
activities in spider mite-infested cucumber leaves using
heptakis(6-O-tert-butyldimethylsilyl-2,3-di-O-methyl)--cyclodextrin
(50% in OV17, w/w) as the enantioselective stationary phase. A, FID
signal of co-injected, unlabeled (3R)- and
(3S)-(Z)-nerolidol (1 and
2, elution order of the two enantiomers is unknown),
(3R)-(E)-nerolidol (3), and
(3S)-(E)-nerolidol (4).
B and C, Radiotraces showing radiolabeled product formed from 50 µM [3H]FDP (30-min assay in buffer B) by
enzyme preparations from cucumber leaves infested with spider mites for
5 d (B) and lima bean leaves infested with spider mites for 5 d (C).
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In all of the plant species tested in the above studies, the
conversion to 4,8-dimethyl-1,3(E),7-nonatriene was achieved
without herbivory or elicitor treatment, suggesting that the enzymes
required for this conversion are constitutively expressed.
Therefore, the specific release of
4,8-dimethyl-1,3(E),7-nonatriene after herbivory must be
regulated upstream of nerolidol (Paré and Tumlinson, 1997). This
could be achieved either by release of nerolidol from a stored
intermediate or by de novo biosynthesis, but Paré and Tumlinson
(1997) demonstrated that 4,8-dimethyl-1,3(E),7-nonatriene, among other volatiles, is synthesized de novo after herbivory in
cotton, excluding release from a stored intermediate as a possibility for regulation.
Our results show that in cucumber and lima bean,
(3S)-(E)-nerolidol synthase is induced upon
spider mite infestation, which supports the de novo biosynthesis of
nerolidol. An interesting aspect of the regulation of
4,8-dimethyl-1,3(E),7-nonatriene formation is that the
volatile nerolidol is not released from cucumber and lima bean leaves,
whereas both (E)-nerolidol and
4,8-dimethyl-1,3(E),7-nonatriene are important constituents
of the volatile blend produced in corn upon feeding by beet army worm
larvae (Turlings et al., 1990) and in gerbera in response to feeding by
spider mites (Krips et al., 1999). Furthermore, in the headspace of
several flowers, nerolidol is an important constituent, often together
with 4,8-dimethyl-1,3(E),7-nonatriene (Kaiser, 1991; Knudsen
et al., 1993). It seems likely that there are differences between plant
species and organs in the relative activities of the enzymes involved
in the 4,8-dimethyl-1,3(E),7-nonatriene pathway up- and
downstream of nerolidol that determine how much of each compound is
released. However, regulation through a controlled release of the two
compounds cannot be excluded.
Although nerolidol is a constituent of many essential oils and the
headspace of a large number of flowers, this is the first report (to
our knowledge) of an enzyme catalyzing the formation of the acyclic
sesquiterpene alcohol nerolidol from FDP. Ionization of FDP to the
farnesyl cation is the first step in the biosynthesis of a large number
of sesquiterpenes. This cation can attack either of the two double
bonds, leading to cyclic sesquiterpenoids such as germacranes,
eudesmanes, and caryophyllanes (McCaskill and Croteau, 1997). For a
large number of other sesquiterpenoids, the enzymatic reaction is
initiated by isomerization of FDP to the isomer nerolidyl diphosphate,
which is ionized to generate the nerolidyl cation (Fig.
7). This enzyme-bound carbocation can undergo electrophilic cyclizations, rearrangements, hydride shifts, and
deprotonation to yield cyclic sesquiterpenoid constituents such as the
cadinanes, bergamotanes, and bisabolanes (McCaskill and Croteau, 1997).
In the case of nerolidol formation, the nerolidyl carbocation is
quenched by the addition of water, which is analogous to the formation
of patchoulol described by Munck and Croteau (1990). In principle, the
formation of the enzyme-bound nerolidyl diphosphate is not even
required (Fig. 7), and whether this intermediate is formed or not
probably depends on the evolution of the enzyme.
The induction of terpene cyclases in response to attacking organisms is
widespread. Croteau and coworkers demonstrated a dramatic increase in
mono- and diterpene cyclase activity upon wounding of grand fir (Funk
et al., 1994). These enzymes are involved in the production of
oleoresin, a mixture of mono- and diterpenes produced to deter
attacking insects and fungi. The induction partly represented an
enhancement of constitutive activities, and partly the appearance of
new activities. The latter is also reported for glandless cotton in
response to inoculation with the bacterial pathogen Xanthomonas
campestris cv malvacearum (Davis and Essenberg, 1995).
The glandless cotton line lacks the constitutive terpene production of
glanded cotton, but does respond to the pathogen with a strong
induction of a sesquiterpene synthase catalyzing the formation of
(+)- -cadinene, an intermediate in the biosynthesis of cotton
phytoalexins. In tobacco and potato, microorganisms and elicitors
induce the activity of sesquiterpene synthases catalyzing the first
committed step in the biosynthesis of phytoalexins (Vögeli et
al., 1990; Zook et al., 1992).
For biosynthetic pathways that are not induced by pathogens or insects,
a regulatory role for terpene synthases has also been suggested
(Gershenzon and Croteau, 1990; McCarvey and Croteau, 1995). For
example, in Clarkia breweri there is a close correlation between linalool synthase activity and the production of linalool and
linalool oxides (Pichersky et al., 1994). Croteau and coworkers demonstrated the enzymatic cyclization of geranylgeranyl diphosphate to
taxa-4(5),11(12)-diene and suggested that this enzyme catalyzes the
regulatory step in the biosynthesis of taxol in Taxus
brevifolia (Koepp et al., 1995). The first dedicated step in the
biosynthesis of the antimalarial drug artemisinin in Artemisia
annua also has the typical characteristics of a regulatory role
(e.g. no accumulation of the enzyme's direct product) (Bouwmeester et
al., 1999).
The results of the present study suggest that the enzyme
(3S)-(E)-nerolidol synthase plays an important
role in regulating the formation of
4,8-dimethyl-1,3(E),7-nonatriene, a key signal molecule in
induced plant defense mediated by the attraction of enemies of
herbivores. The identification of this enzyme is an important step
forward in the elucidation of the signaling cascade that leads from
herbivory to the release of plant volatiles that attract the natural
enemies of herbivores.
| |
FOOTNOTES |
1
This work was supported by the Dutch Ministry of
Agriculture, Nature Management, and Fisheries (H.J.B., F.W.A.V.) and
the Uyttenboogaart-Eliasen Foundation (M.D.).
*
Corresponding author; e-mail h.j.bouwmeester{at}ab.dlo.nl; fax
31-317-423110.
Received January 15, 1999;
accepted May 21, 1999.
| |
ACKNOWLEDGMENTS |
The authors thank Wilfried König for the gift of the
nerolidol isomers, Unifarm (Wageningen, The Netherlands) for
growing the plants, Herman Dijkman for rearing spider mites, Jan-Willem de Kraker for helpful suggestions, Jacques Davies for technical assistance, and Hans Helsper for helpful comments concerning the manuscript.
| |
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Abstract
Introduction
