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Plant Physiol. (1999) 120: 879-886
Limonene Synthase, the Enzyme Responsible for Monoterpene
Biosynthesis in Peppermint, Is Localized to Leucoplasts of Oil Gland
Secretory Cells1
Glenn Turner,
Jonathan Gershenzon2,
Erik E. Nielson,
John E. Froehlich, and
Rodney Croteau*
Institute of Biological Chemistry, and Department of Biochemistry
and Biophysics, Washington State University, Pullman, Washington
99164-6340 (G.T., J.G., R.C.); and Department of Energy Plant Research
Laboratory, Michigan State University, East Lansing, Michigan 48823 (E.E.N., J.E.F.)
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ABSTRACT |
Circumstantial
evidence based on ultrastructural correlation, specific labeling, and
subcellular fractionation studies indicates that at least the early
steps of monoterpene biosynthesis occur in plastids.
(4S)-Limonene synthase, which is responsible for the
first dedicated step of monoterpene biosynthesis in mint species, appears to be translated as a preprotein bearing a long plastidial transit peptide. Immunogold labeling using polyclonal antibodies raised
to the native enzyme demonstrated the specific localization of limonene
synthase to the leucoplasts of peppermint (Mentha × piperita) oil gland secretory cells during the period
of essential oil production. Labeling was shown to be absent from all
other plastid types examined, including the basal and stalk cell
plastids of the secretory phase glandular trichomes. Furthermore, in
vitro translation of the preprotein and import experiments with
isolated pea chloroplasts were consistent in demonstrating import of
the nascent protein to the plastid stroma and proteolytic processing to
the mature enzyme at this site. These experiments confirm that the
leucoplastidome of the oil gland secretory cells is the exclusive location of limonene synthase, and almost certainly the preceding steps
of monoterpene biosynthesis, in peppermint leaves. However, succeeding
steps of monoterpene metabolism in mint appear to occur outside the
leucoplasts of oil gland cells.
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INTRODUCTION |
Peppermint (Mentha × piperita),
spearmint (Mentha spicata), and other essential oil plants
of the Lamiaceae produce and accumulate monoterpenes in anatomically
specialized glandular trichomes (Fahn, 1979 ). The mints bear two types
of nonphotosynthetic glandular trichomes, a small capitate type with a
limited capacity to store secreted material, and a peltate type
containing a basal cell, a stalk cell, and eight secretory cells
arranged in a disc (Fig. 1) (Amelunxen,
1965; Amelunxen et al., 1969; Fahn, 1979). The latter type develops a
large oil-storage space at the apex of the glandular trichome, where
the thick cuticle separates from the secretory cells to produce a
subcuticular pocket and is therefore thought to be responsible for the
production of the bulk of the monoterpenoid essential oil (Amelunxen et
al., 1969).
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| Figure 1.
Schematic diagram of a peppermint leaf peltate
glandular trichome illustrating the placement of these epidermal
structures and the relationship of the disc of secretory cells to the
stalk and basal cells and to the subcuticular storage space.
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Biochemical studies with isolated peppermint peltate glandular
trichomes have revealed that the secretory cells are not only responsible for the secretion of monoterpenes into the oil-storage space, but also serve as the actual site of monoterpene biosynthesis (Gershenzon et al., 1992; McCaskill et al., 1992). Plant essential oil-
and resin-secreting glands often share a syndrome of specialized ultrastructural features, including numerous amoeboid leucoplasts and
abundant smooth ER (Schnepf, 1974; Dell and McComb, 1978; Fahn, 1988).
There have been relatively few ultrastructural studies of Lamiaceae oil
glands, and most of those that have been done have stressed the
possible role of the extensive ER or the densely staining cytoplasm in
monoterpene biosynthesis (Amelunxen, 1965; Schnepf, 1974; Bosabalidis
and Tsekos, 1982). Recent papers by Bourett et al. (1994) and Ascensao
et al. (1997) have discussed the possible role of leucoplasts. Oil
gland leucoplasts are nonpigmented plastids, often with few internal
membranes (Carde, 1984), that have been implicated in monoterpene
biosynthesis by a survey of the secretory structures of nearly 50 plant
species that demonstrated a close correlation between the presence of
such leucoplasts and the accumulation of monoterpenes (Cheniclet and
Carde, 1985).
Related evidence for the role of leucoplasts and other plastids in
monoterpene formation derives from studies showing that isolated
plastids are capable of monoterpene biosynthesis when supplied with
exogenous precursors (Gleizes et al., 1983; Pauly et al., 1986; Mettal
et al., 1988; Pérez et al., 1990; Soler et al., 1992). Finally,
the labeling patterns of several monoterpenes derived from exogenous
[13C]Glc are consistent with their origin via the
mevalonate-independent pathway (Eisenreich et al., 1997; Adam et al.,
1998), an isoprenoid biosynthetic pathway known to operate in some
Eubacteria and exclusively in the plastids of a variety of
phylogenetically divergent plants and green algae (Lichtenthaler et
al., 1997). Although there is considerable circumstantial evidence to
indicate that at least the early steps of monoterpene biosynthesis are
associated with gland cell plastids, there is as yet no direct evidence
for the localization of relevant biosynthetic enzymes to these
structures that would reveal the details of subcellular pathway
organization.
( )-Limonene synthase catalyzes the cyclization of geranyl
diphosphate, the universal C10 precursor of the
monoterpenes, to ()-4S-limonene (Croteau, 1991, 1993)
(Fig. 2). This olefin serves as the
common progenitor of the characteristic monoterpenes in mints,
including menthol in peppermint and carvone in spearmint, following a
series of enzymatic redox transformations (Croteau and Gershenzon,
1994) (Fig. 2). The conversion of geranyl diphosphate to limonene is
one of the simplest of all terpenoid cyclization reactions, and this
synthase (cyclase) serves as a model for the general enzyme type (Wise
and Croteau, 1999). The operationally soluble
()-4S-limonene synthase from spearmint has been purified and characterized (Alonso et al., 1992; Rajaonarivony et al., 1992),
and has been employed to prepare polyclonal antibodies that were shown
to cross-react with the enzyme from other mint species (Alonso et al.,
1993).
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| Figure 2.
Pathway for the conversion of C5
isoprenoid units via geranyl diphosphate and limonene to the principal
essential oil components ()-menthol (peppermint) and ()-carvone
(spearmint). The responsible enzymes are: isopentenyl diphosphate
isomerase (1); geranyl diphosphate synthase (2);
4S-limonene synthase (3);
4S-limonene-3-hydroxylase (4); and
4S-limonene-6-hydroxylase (5). The broken arrow
indicates five enzymatic steps. OPP, Diphosphate moiety.
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A limonene synthase cDNA has been isolated from spearmint and
functionally expressed in Escherichia coli (Colby et al.,
1993); the limonene synthase cDNA from peppermint was recently isolated and shown to be 93% identical to the spearmint clone at the deduced amino acid level (B.M. Lange and R.B. Croteau, unpublished data). Evaluation of the encoded sequence of the 72.4-kD protein with respect
to the physical properties of the 65-kD native enzyme suggests that
limonene synthase is translated as a preprotein bearing what appears to
be a substantial N-terminal plastid-targeting peptide (Colby et al.,
1993). Deletion of the presumptive transit peptide by truncation and
heterologous expression of the corresponding cDNA yielded a fully
active, "pseudomature" form of the enzyme, which is consistent with
the concept of plastidial targeting and subsequent proteolytic
processing (Williams et al., 1998).
Because of the rigorous conditions required to disrupt oil gland cells
of mint, classical subcellular fractionation studies to locate the
operationally soluble enzyme in an intact organelle have not been
possible. In this paper, we confirm the leucoplastidial localization of
limonene synthase by immunogold cytochemical studies and by in vitro
synthesis of the labeled preprotein coupled to plastidial import and
processing experiments, thereby demonstrating that this committed step
of monoterpene biosynthesis in mint occurs exclusively at this
subcellular site.
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MATERIALS AND METHODS |
Materials
Peppermint (Mentha × piperita L. cv
Black Mitcham) plants were propagated and grown under controlled
conditions, as described previously (Alonso et al., 1992). Young,
folded leaves approximately 6 to 10 mm in length (about 2 weeks old)
were used in all experiments. Pea (Pisum sativum var Little
Marvel) seeds were obtained from the Olds Seed Company (Madison, WI)
and were grown as described previously (Bruce et al., 1994).
Polyclonal antibodies were generated in rabbits against the
SDS-denatured limonene synthase from spearmint (Mentha
spicata) as previously described (Alonso et al., 1993).
Gold-conjugated goat anti-rabbit secondary antibodies were obtained
from Goldmark Biologicals (Phillipsburg, NJ). Percoll and ATP were
purchased from Sigma, L-[35S]Met from
DuPont/NEN, and nuclease-treated rabbit reticulocyte lysate from
Promega.
Tissue Preparation
Several different fixation protocols were used that varied in the
quality of tissue preservation and retention of immunogenicity but that
gave similar overall patterns of labeling. Some specimens were fixed
for 3 h at 4°C with 1% (v/v) glutaraldehyde in 50 mM Pipes buffer, pH 7.2, prior to dehydration in a graded
ethanol series to 100% ethanol. The samples were then slowly
infiltrated with LR White resin through a graded series (resin:ethanol
1:3, 1:1, and 3:1) for 12 h each, followed by three 12-h
incubations in pure resin, prior to polymerization at 60°C. These
specimens retained good immunogenicity but cellular preservation was
relatively poor. Other specimens were fixed with 0.75% (v/v)
glutaraldehyde, 1% (v/v) paraformaldehyde, and approximately 0.02%
(w/v) osmium tetraoxide in 50 mM Pipes buffer, pH 7.3, for
2 h at 4°C. These specimens were then transferred to an
identical solution lacking the osmium tetraoxide, and fixed for an
additional 12 h at 4°C prior to dehydration and embedding as
before. These samples showed good immunogenicity, low levels of
background staining, and adequate tissue preservation.
Additionally, some specimens were rapidly fixed and dehydrated with a
microwave tissue-processing system (model 3450, Pelco, Clovis, CA;
Gibberson et al., 1997) prior to embedding. These samples were fixed
for 2.5 min at 37°C in buffered 1% (v/v) glutaraldehyde, followed by
rapid dehydration in a graded ethanol series (10% increments, 80 s for each step at 40°C). This procedure gave superior tissue
preservation (especially for postsecretory stage glands), but less
immunogenicity was retained.
Immunolabeling
Thin sections (70-90 nm), collected on uncoated, 200-mesh nickel
grids, were first incubated for 1 h in a blocking solution of TBS
(10 mM Tris/NaOH, pH 7.8, with 0.5 M NaCl)
containing 0.1% (v/v) Tween 20 and 1% (w/v) BSA (TBST-BSA). For some
trials, 0.2% (w/v) PVP (Mr 10,000, Sigma) was added to the blocking solutions to reduce nonspecific
labeling of phenolic compounds. Sections were incubated for 6 h in
the TBST-BSA solution containing either a 1:30 dilution of rabbit
polyclonal antibodies raised against spearmint limonene synthase
(Alonso et al., 1993), or a comparable dilution of preimmune serum.
After washing with TBST-BSA, all sections were incubated for 1 h
in TBST-BSA containing a 1:50 dilution of gold (10 or 20 nm) labeled
goat anti-rabbit polyclonal antibodies. Sections were then rinsed in
TBST-BSA, then TBST, and then water. Sections were stained for 15 min
in a 1:4 mixture of 1% (w/v) potassium permanganate and 1% (w/v)
aqueous uranyl acetate prior to viewing with an electron microscope
(model JEM 1200EX, JEOL). Specimens were photographed with electron
microscopy film (Kodak).
Import Experiments
Intact chloroplasts were isolated from 8- to 12-d-old pea
seedlings and purified over a Percoll gradient as previously described (Bruce et al., 1994). Intact chloroplasts were re-isolated and resuspended in import buffer (50 mM Hepes/KOH, pH 8.0, containing 330 mM sorbitol) at a concentration of 1 mg
chlorophyll/mL, and stored in the dark on ice prior to use in import
experiments.
The plasmid pLC 5.2 encoding the presumptive limonene synthase
preprotein (Colby et al., 1993) was linearized and transcribed with T3
RNA polymerase. Limonene synthase was then translated in the presence
of [35S]Met using a nuclease-treated rabbit
reticulocyte lysate system according to the manufacturer's protocol
(Promega). The plasmid encoding the precursor to SSU (Olsen and
Keegstra, 1992) was linearized with PstI, transcribed with
SP6 RNA polymerase, and translated using a wheat germ system and
[35S]Met, as previously described (Bruce et
al., 1994). After translation, residual nucleotides were removed by gel
filtration (Olsen et al., 1989).
Each binding or import reaction (adapted from Bruce et al., 1994)
contained the following: 106 dpm of either
[35S]limonene synthase or
[35S]SSU as a control; either 0.1 mM ATP for
binding or 4 mM ATP for translocation (final
concentration); and intact chloroplasts corresponding to 25 µg of
chlorophyll in a final volume of 150 µL. Binding and translocation
reactions were run at room temperature for the times indicated in the
figure legends. Reactions were terminated by recovering intact
chloroplasts via sedimentation through a 40% Percoll cushion.
Recovered chloroplasts were then separated into a crude membrane and a
soluble fraction according to the method of Bruce et al. (1994). In a
similar fashion, protease protection assays were performed on recovered
chloroplasts according to the method of Cline et al. (1984). All
samples were solubilized in 2× SDS-PAGE sample buffer and analyzed by
SDS-PAGE (Laemmli, 1970), followed by fluorography and documentation by
exposure to film (Bruce et al., 1994).
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RESULTS AND DISCUSSION |
Limonene Synthase Is Localized to Leucoplasts of Secretory Cells
()-4S-Limonene synthase appears to originate as a
preprotein bearing an amino-terminal plastid-targeting sequence (Colby et al., 1993), which is consistent with considerable circumstantial evidence indicating that at least the early steps of monoterpene biosynthesis are localized to this organelle (Gershenzon and Croteau, 1990; McGarvey and Croteau, 1995; Wise and Croteau, 1999). The operationally soluble native enzyme cannot be directly localized by
classical subcellular fractionation techniques because of experimental limitations imposed by the walls and cuticle surrounding the oil gland
secretory cells (Gershenzon et al., 1991, 1992). This means that
approaches to enzyme localization are limited to immunocytochemical and
in vitro methods.
Immunoblotting analysis revealed that the polyclonal antibodies
previously prepared against the spearmint limonene synthase were very
specific for denatured limonene synthase from all Mentha sp., but these antibodies failed to recognize the native protein by
immunoprecipitation or Ouchterlony double-diffusion assay (Alonso et
al., 1993). However, preliminary immunohistochemistry indicated the
presence of an antigen target in only the glandular (not the nonglandular) cells. Further investigations showed dense, specific labeling with the anti-limonene synthase antibodies to the stroma of
leucoplasts of the disc cells from secretory-phase peltate glandular
trichomes (Fig. 3, A and B),
which was absent from the corresponding leucoplasts of the preimmune
serum controls (Fig. 3, C and D).
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| Figure 3.
A, Immunogold labeling against limonene synthase.
Colloidal gold particles strongly label leucoplasts (P) of a secretory
stage glandular cap cell, while cytoplasm and other organelles are
essentially unlabeled. Bar = 2 µm. B, High magnification of a
leucoplast from a secretory stage glandular cap cell showing
immunolabeling against limonene synthase. Bar = 1 µm. C,
Preimmune serum control. Leucoplasts (P) of this secretory stage
glandular cap cell are unlabeled after
treatment with preimmune serum and colloidal gold
labeled secondary antibodies. Bar = 1 µm. D, High magnification
of a leucoplast from a secretory stage glandular cap cell showing no
labeling when treated with preimmune serum and colloidal gold labeled
secondary antibodies. Bar = 500 nm. E, High magnification of a
plastid (P) from a stalk cell of a secretory stage peltate gland
immunolabeled against limonene synthase showing very little labeling.
Bar = 1 µm. F, Leucoplasts from a presecretory stage glandular
cap cell immunolabeled against limonene synthase showing relatively
little labeling. Bar = 1 µm. G, Leucoplasts (P) from a
microwave-fixed, postsecretory-stage glandular cap cell immunolabeled
against limonene synthase showing relatively little labeling.
(Generally, microwave-fixed specimens showed less labeling than
specimens prepared by other methods.) Bar = 1 µm. H, Plastid
from a capitate glandular trichome immunolabeled against limonene
synthase showing relatively little labeling. Bar = 1 µm.
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Immunogold labeling was absent from mesophyll chloroplasts, from basal
cell plastids, and from stalk cell plastids of secretory phase peltate
glands (Fig. 3E). Intense immunogold labeling was observed in the
stroma of leucoplasts only during the relatively short (approximately
20 h) secretory phase of gland development, in which filling of
the subcuticular storage cavity with the essential oil occurs. Plastids
of pre-secretory peltate gland cells (Fig. 3F), cells of postsecretory
glands (Fig. 3G), and cells of capitate trichomes (Fig. 3H) exhibited
little labeling above background levels. However, substantial labeling
was observed to occur along cell walls, especially secondary walls of
tracheary elements, but this labeling represented a nonspecific
affinity for wall material, since both the anti-limonene synthase
antibodies and the preimmune serum controls exhibited cell wall
labeling of equal intensity. These observations indicate that limonene
synthase accumulates in the stroma of secretory cell leucoplasts and
only during the secretory phase of oil gland filling.
Limonene Synthase Is Imported into Chloroplasts and Is Localized to
the Stroma
Most chloroplastic proteins are nuclear encoded, synthesized on
cytosolic ribosomes, and imported posttranslationally across the two
membranes of the chloroplast envelope (Chua and Schmidt, 1978;
Highfield and Ellis, 1978). An amino-terminal extension called a
transit peptide targets these precursor proteins to chloroplasts (Schmidt et al., 1979). During or after translocation, the transit peptide is cleaved by the stromal processing peptidase (Oblong and
Lamppa, 1992; VanderVere et al., 1995; Richter and Lamppa, 1998). The
newly imported proteins are then folded or further directed to the
thylakoid membrane.
The limonene synthase cDNA encodes a predicted protein with a molecular
mass of 72.4 kD, and analysis of the amino acid sequence suggests the
presence of a potential transit peptide at the amino terminus (Colby et
al., 1993). To determine if limonene synthase can be targeted to and
subsequently imported into chloroplasts, in vitro import assays with
the in vitro-translated preprotein were performed using isolated pea
chloroplasts. Figure 4A demonstrates that
limonene synthase binds to and is imported into pea chloroplasts in a
time-dependent manner. Five minutes after the initiation of the assay,
the radiolabeled full-length preprotein (72.4 kD) was associated
entirely with the chloroplast pellet, the membrane fraction. However,
at 10 min, a smaller product (65 kD) began to appear in the
supernatant, and by 20 min only this mature protein was observed
exclusively in the supernatant. These changes are consistent with
initial binding of the preprotein to the surface of the chloroplast,
followed by transport across the envelope membranes, processing to
remove the amino-terminal transit peptide, and eventual release of the
mature protein into the stroma.
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| Figure 4.
Analysis of import and localization of limonene
synthase. A, Time course of import of 35S-labeled limonene
synthase into pea chloroplasts. Following confirmation of binding at
0°C to 4°C in the dark in the presence of 0.1 mM Mg-ATP
(data not shown), import assays were performed at room temperature in
the presence of 4 mM Mg-ATP. Aliquots of the chloroplast
preparation were removed at 0, 5, 10, and 20 min, sedimented through a
40% Percoll cushion, lysed, and then separated into a crude pellet (P)
and a supernatant (S) fraction. All samples were analyzed by SDS-PAGE
and fluorography. Tr, Unmodified limonene synthase translation product
at about 72 kD. The protein band at about 65 kD is diffuse, likely due
to imprecise proteolytic processing to mature forms of limonene
synthase (Williams et al., 1998). B, Protease treatment and
fractionation of imported limonene synthase. Limonene synthase was
imported into pea chloroplasts for 20 min at room temperature as above
and the intact chloroplasts, recovered by sedimentation through a 40%
Percoll cushion, were incubated in the absence () or presence (+) of
thermolysin for 30 min on ice in the dark. Proteolysis was terminated
by the addition of EDTA, and the intact chloroplasts were recovered by
sedimentation through a 40% Percoll cushion containing 5 mM EDTA. Chloroplasts were lysed and separated into a crude
membrane fraction (P) and a soluble fraction (S). MW, Molecular-mass
standards (in kilodaltons); Tr, limonene synthase translation product;
SSU, positive control experiment conducted with the precursor to the
SSU.
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The localization of limonene synthase within the chloroplasts was
investigated by employing import assays with the protease thermolysin
added at a concentration that would degrade any externally exposed
proteins but would not cause lysis of the chloroplast (Cline et al.,
1984). Thermolysin treatment did not affect the appearance of the
mature limonene synthase in the supernatant (Fig. 4B), indicating that
the processed protein must reside in the stroma, where it is protected
from protease action. A stromal location is consistent with the soluble
nature of the native limonene synthase activity observed in cell-free
extracts of peppermint (Alonso et al., 1992; Rajaonarivony et al.,
1992).
Also depicted in Figure 4B are the results of an import assay conducted
with an aliquot from the same chloroplast preparation using the SSU, a
nuclear-encoded protein known to be targeted to the plastid (Keegstra
et al., 1989). The protease-protected import of SSU into the stroma and
its concurrent reduction in size shows that these chloroplasts were
fully competent in protein uptake and processing. Taken together, the
plastid-import experiments and immunocytochemical studies demonstrate
that the amino-terminal region of the limonene synthase preprotein can
serve as a targeting sequence, and that this transit peptide is
sufficient to direct the import of limonene synthase into
plastids.
Within the peppermint secretory cell leucoplasts, limonene synthase
appeared to be distributed throughout the stroma and was not associated
with any particular region of the stromal compartment. Other
plastid-localized enzymes of terpenoid biosynthesis have been reported
to have more restricted distributions within this organelle. For
instance, in developing chromoplasts of pepper fruits, the
prenyltransferase geranylgeranyl diphosphate synthase is concentrated
at certain sites in the stroma in association with plastoglobuli
(Cheniclet et al., 1992). In chloroplasts, some steps of isoprenoid
formation are associated with the envelope membrane (Biggs et al.,
1990; Joyard et al., 1991) or the thylakoids (Linden et al., 1993).
The successful import of limonene synthase into pea chloroplasts not
only confirms the plastidial location of this enzyme, but also attests
to the basic similarities between the protein import machinery of
chloroplasts and that of secretory cell leucoplasts. Early work on
plastid protein import suggested that various transit peptides could be
recognized by all types of plastids (Halpin et al., 1989; Klosgen et
al., 1989). However, more recently, two isoforms of plastid pyruvate
kinase have been described whose import characteristics with
leucoplasts and chloroplasts differ as a consequence of differences in
their transit peptides (Wan et al., 1995). The present study also hints
that differences may exist between chloroplast and leucoplast import,
because the broad, indistinct nature of the mature protein band shown
in Figure 4A (panel S, 10- and 20-min lanes) indicates that processing
of leucoplast-targeted limonene synthase by pea chloroplasts is
imprecise. However, the processing of limonene synthase in planta by
peppermint secretory cell leucoplasts is also inexact, as MS analysis
of the purified native enzyme has revealed a heterogeneous population
of modified forms (Williams et al., 1998).
Several lines of evidence suggest that, in addition to limonene
synthase, all of the preceding steps of monoterpene biosynthesis in
peppermint are localized in the leucoplasts of the glandular trichome
secretory cells. First, the isotopic labeling pattern of monoterpenes
derived from applied [13C]Glc (Eisenreich et
al., 1997) indicates that these metabolites originate from isopentenyl
diphosphate via the glyceraldehyde-3-P/pyruvate pathway, a
mevalonate-independent sequence of reactions that is plastid localized
in higher plants (Lichtenthaler et al., 1997). Second, two specific
monoterpene biosynthetic enzymes, deoxyxylulose-5-P synthase, which
catalyzes the first step of the glyceraldehyde-P/pyruvate pathway
(Lange et al., 1998), and geranyl diphosphate synthase, which mediates
the condensation of isopentenyl diphosphate and dimethylallyl
diphosphate to geranyl diphosphate (M. Wildung, C. Burke, J. Gershenzon, and R. Croteau, unpublished results) (Fig. 2), both possess
plastid-targeting peptides. However, the limonene hydroxylases
responsible for the conversion of limonene to
()-trans-isopiperitenol and to
()-trans-carveol (Fig. 2) do not bear plastidial transit
peptides, but rather amino-terminal membrane insertion sequences more
typical of this class of Cyt P450 monooxygenases (Lupien et al., 1995).
Additionally, these Cyt P450 hydroxylases have been localized to the
microsomal fraction of oil gland cell homogenates (Karp et al., 1990),
further suggesting that these enzymes reside in the extensive smooth ER
characteristic of these secretory cells.
Preliminary evidence (B.M. Lange and R. Croteau, unpublished results)
indicates that the catalysts responsible for the redox metabolism of
trans-isopiperitenol and trans-carveol are
cytosolic, suggesting that enzymes from multiple subcellular
compartments participate in monoterpene biosynthesis in mint.
Additional immunocytochemical studies are in progress to examine
pathway organization in greater detail.
Plastids have long been recognized as a major subcellular site of
terpenoid metabolism, a fact underscored by the recent discovery that
this organelle possesses a novel mevalonate-independent pathway (Lichtenthaler et al., 1997) for the production of the universal terpenoid precursor isopentenyl diphosphate (McCaskill and Croteau, 1999). The biosynthesis of hemiterpenes (isoprene), diterpenes, tetraterpenes (carotenoids), and a variety of prenylated quinones occurs in plastids (Gray, 1987; Kleinig, 1989; Wildermuth and Fall,
1996). The localization of limonene synthase described in the present
study directly confirms that monoterpenes are also of plastidial
origin. Several branches or segments of terpenoid metabolism are found
outside the plastids in the cytosol, ER, and mitochondria (Gray, 1987;
Kleinig, 1989; Disch et al., 1998). For example, sesquiterpenes and
triterpenes are synthesized in the cytosol/ER compartment (Chappell,
1995), and microsomal Cyt P450 oxygenases and cytosolic redox enzymes
are largely responsible for synthesizing derivatives of the various
terpenoid structural types of plastidial origin (Gershenzon and
Croteau, 1993; McGarvey and Croteau, 1995; Wise and Croteau, 1999). The
organization of terpenoid metabolism involving several different
subcellular compartments may be important in regulating the production
of this large and diverse group of natural products.
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FOOTNOTES |
1
This work was supported in part by the U.S.
Department of Energy, Division of Energy Biosciences, the National
Science Foundation Cell Biology Program, the Mint Industry Research
Council, and Project 0268 from the Agricultural Research Center,
Washington State University.
2
Present address: Max-Planck-Institut für
Chemische Ökologie, D-07745 Jena, Germany.
*
Corresponding author; e-mail croteau{at}mail.wsu.edu; fax
1-509-335-7643.
Received February 8, 1999;
accepted March 28, 1999.
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ABBREVIATIONS |
Abbreviation:
SSU, small subunit of Rubisco.
| |
ACKNOWLEDGMENTS |
We thank John Crock and Vincent Franceschi for helpful
discussions, Thom Koehler for raising the plants, and Joyce Tamura for
typing the manuscript.
| |
LITERATURE CITED |
Adam K-P,
Theil R,
Zapp J,
Becker H
(1998)
Involvement of the mevalonic acid pathway and the glyceraldehyde-pyruvate pathway in terpenoid biosynthesis of the liverworts Ricciocarpos natans, and Conocephalum conicum.
Arch Biochem Biophys
354:
181-187
[CrossRef][Medline]
Alonso WR,
Crock JE,
Croteau R
(1993)
Production and characterization of polyclonal antibodies in rabbits to 4S-limonene synthase from spearmint (Mentha spicata).
Arch Biochem Biophys
301:
58-63
[Medline]
Alonso WR,
Rajaonarivony JIM,
Gershenzon J,
Croteau R
(1992)
Purification of 4S-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha × piperita) and spearmint (M. spicata).
J Biol Chem
267:
7582-7587
[Abstract/Free Full Text]
Amelunxen F
(1965)
Electromikroskopishe Untersuchungen an den Drüsenschuppen von Mentha piperita L.
Planta Med
13:
457-473
Amelunxen F,
Wahlig T,
Arbeiter H
(1969)
Über den Nachweis des atherischen Öls in isolierten Drüsenhaaren und Drüsenschuppen von Mentha piperita L.
Z Pflanzenphysiol
61:
68-72
Ascensao L,
Marques N,
Pais MS
(1997)
Peltate glandular trichomes of Leonotis leonurus leaves: ultrastructure and histochemical characterization of secretions.
Int J Plant Sci
158:
249-258
[CrossRef]
Biggs DR,
Welle R,
Grisebach H
(1990)
Intracellular localization of prenyltransferases of isoflavonoid phytoalexin biosynthesis in bean and soybean.
Planta
181:
244-248
Bosabalidis A,
Tsekos I
(1982)
Glandular scale development and essential oil secretion in Origanum dictamnus.
Planta
156:
496-504
[CrossRef]
Bourett TM,
Howard RJ,
O'Keefe DP,
Hallahan DL
(1994)
Gland development on leaf surfaces of Nepeta racemosa.
Int J Plant Sci
155:
623-632
[CrossRef]
Bruce BD,
Perry S,
Froehlich J,
Keegstra K
(1994)
In vitro import of protein into chloroplasts.
In
SB Gelvin,
RB Schilperoort,
eds, Plant Molecular Biology Manual.
Kluwer Academic Publishers, Boston, pp 1-15
Carde J-P
(1984)
Leucoplasts: a distinct kind of organelles lacking typical 70S ribosomes and free thylakoids.
Eur J Cell Biol
34:
18-26
[Medline]
Chappell J
(1995)
Biochemistry and molecular biology of the isoprenoid biosynthetic pathway in plants.
Annu Rev Plant Physiol Plant Mol Biol
46:
521-547
[CrossRef][Web of Science]
Cheniclet C,
Carde J-P
(1985)
Presence of leucoplasts in secretory cells and of monoterpenes in the essential oil: a correlative study.
Isr J Bot
34:
219-238
Cheniclet C,
Rafia F,
Saint-Guily A,
Verna A,
Carde J-P
(1992)
Localization of the enzyme geranylgeranylpyrophosphate synthase in Capsicum fruits by immunogold cytochemistry after conventional chemical fixation or quick-freezing followed by freeze-substitution. Labelling evolution during fruit ripening.
Biol Cell
75:
145-154
Chua NH,
Schmidt GW
(1978)
Post-translational transport into intact chloroplasts of a precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase.
Proc Natl Acad Sci USA
75:
6110-6114
[Abstract/Free Full Text]
Cline K,
Werner-Washburne M,
Andrews J,
Keegstra K
(1984)
Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts.
Plant Physiol
75:
675-678
[Abstract/Free Full Text]
Colby SM,
Alonso WR,
Katahira EJ,
McGarvey DJ,
Croteau R
(1993)
4S-Limonene synthase from the oil glands of spearmint (Mentha spicata): cDNA isolation, characterization and bacterial expression of the catalytically active monoterpene cyclase.
J Biol Chem
268:
23016-23024
[Abstract/Free Full Text]
Croteau R (1991) Metabolism of monoterpenes in mint
(Mentha) species. Planta Med 57 (Suppl): 10-14
Croteau R (1993) Biosynthesis of limonene in Mentha
species. In P Schreier, P Winterhalter, eds, Progress in
Flavor Precursor Studies: Analysis, Generation, Biotechnology. Allured
Publishing Co, Wheaton, IL, pp 113-122
Croteau R, Gershenzon J (1994) Genetic control of monoterpene
biosynthesis in mints (Mentha:Lamiaceae). In BE
Ellis, G Kuroki, HA Stafford, eds, Genetic Engineering of Plant
Secondary Metabolism. Plenum Press, New York, pp 193-229
Dell B,
McComb AJ
(1978)
Plant resins: their formation, secretion, and possible functions.
Adv Bot Res
6:
277-316
Disch A, Hemmerlin A, Bach TJ, Rohmer M (1998) Mevalonate-derived
isopentenyl diphosphate is the biosynthetic precursor of ubiquinone
prenyl side chain in tobacco BY-2 cells. Biochem J 331 (part
2): 615-621
Eisenreich W,
Sagner S,
Zenk MH,
Bacher A
(1997)
Monoterpenoid essential oils are not of mevalonoid origin.
Tetrahedron Lett
38:
3889-3892
[CrossRef]
Fahn A (1979) Secretory Tissues in Plants. Academic Press, London,
pp 162-164
Fahn A
(1988)
Secretory tissues in vascular plants.
New Phytol
108:
229-257
[CrossRef]
Gershenzon J, Croteau R (1990) Regulation of monoterpene
biosynthesis in higher plants. In GHN Towers, HA Stafford,
eds, Biochemistry of the Mevalonic Acid Pathway to Terpenoids. Plenum
Press, New York, pp 99-160
Gershenzon J, Croteau R (1993) Terpenoid biosynthesis: the basic
pathway and formation of monoterpenes, sesquiterpenes and diterpenes.
In TS Moore Jr, ed, Lipid Metabolism in Plants. CRC Press,
Boca Raton, FL, pp 340-388
Gershenzon J, McCaskill D, Rajaonarivony JIM, Mihaliak C,
Karp F, Croteau R (1991) Biosynthetic methods for plant natural
products: new procedures for the study of glandular trichome
constituents. In NH Fischer, MB Isman, HA Stafford, eds,
Modern Phytochemical Methods. Plenum Press, New York, pp 347-370
Gershenzon J,
McCaskill D,
Rajaonarivony JIM,
Mihaliak C,
Karp F,
Croteau R
(1992)
Isolation of secretory cells from plant glandular trichomes and their use in biosynthetic studies of monoterpenes and other gland products.
Anal Biochem
200:
130-138
[CrossRef][Web of Science][Medline]
Gibberson RT,
Demaree RS Jr,
Nordhausen RW
(1997)
Four-hour processing of clinical/diagnostic specimens for electron microscopy using microwave techniques.
J Vet Diagn Invest
9:
61-67
[Abstract/Free Full Text]
Gleizes M,
Pauly G,
Carde J-P,
Marpeau A,
Bernard-Dagan C
(1983)
Monoterpene hydrocarbon biosynthesis by isolated leucoplasts of Citrofortunella mitis.
Planta
159:
373-381
[CrossRef]
Gray JC
(1987)
Control of isoprenoid biosynthesis in higher plants.
Adv Bot Res
14:
25-91
Halpin C,
Musgrove JE,
Lord JM,
Robinson C
(1989)
Import processing of proteins by castor bean leucoplasts.
FEBS Letts
258:
32-34
[CrossRef]
Highfield PE,
Ellis RJ
(1978)
Synthesis and transport of the small subunit of chloroplastic ribulose bisphosphate carboxylase.
Nature
271:
420-424
[CrossRef][Medline]
Joyard J,
Block MA,
Douce R
(1991)
Molecular aspects of plastid envelope biochemistry.
Eur J Biochem
199:
489-509
[Web of Science][Medline]
Karp F,
Mihaliak CA,
Harris JL,
Croteau R
(1990)
Monoterpene biosynthesis: specificity of the hydroxylations of ()-limonene by enzyme preparations from peppermint (Mentha piperita), spearmint (Mentha spicata), and perilla (Perilla frutescens) leaves.
Arch Biochem Biophys
276:
219-226
[CrossRef][Web of Science][Medline]
Keegstra K,
Olsen LJ,
Theg SM
(1989)
Chloroplastic precursors and their transport across the envelope membranes.
Annu Rev Plant Physiol Plant Mol Biol
40:
471-501
[CrossRef][Web of Science]
Kleinig H
(1989)
The role of plastids in isoprenoid biosynthesis.
Annu Rev Plant Physiol Plant Mol Biol
40:
39-59
[CrossRef][Web of Science]
Klosgen RB,
Saedler H,
Weil J-H
(1989)
The amyloplast-targeting transit peptide of the waxy protein of maize also mediates protein transport in vitro into chloroplasts.
Mol Gen Genet
217:
155-161
[CrossRef][Medline]
Laemmli UK
(1970)
Cleavage of structural protein during the assembly of the head of bacteriophage T4.
Nature
227:
680-685
[CrossRef][Medline]
Lange BM,
Wildung MR,
McCaskill DG,
Croteau R
(1998)
A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway.
Proc Natl Acad Sci USA
95:
2100-2104
[Abstract/Free Full Text]
Lichtenthaler HK,
Rohmer M,
Schwender J
(1997)
Two independent biochemical pathways for isopentenyl diphosphate and isoprenoid biosynthesis in higher plants.
Physiol Plant
101:
643-652
[CrossRef]
Linden H,
Lucas MM,
Rosario de Felipe M,
Sandmann G
(1993)
Immunogold localization of phytoene desaturase in higher plant chloroplasts.
Physiol Plant
88:
229-236
[CrossRef]
Lupien S,
Karp F,
Ponnamperuma K,
Wildung M,
Croteau R
(1995)
Cytochrome P450 limonene hydroxylases of Mentha species.
Drug Metab Drug Interact
12:
245-260
[Medline]
McCaskill DG,
Croteau R
(1999)
Isopentenyl diphosphate is the terminal product of the deoxyxylulose-5-phosphate pathway for terpenoid biosynthesis in plants.
Tetrahedron Lett
40:
653-656
[CrossRef]
McCaskill DG,
Gershenzon J,
Croteau R
(1992)
Morphology and monoterpene biosynthetic capabilities of secretory cell clusters isolated from glandular trichomes of peppermint (Mentha piperita L.).
Planta
187:
445-454
[Web of Science]
McGarvey D,
Croteau R
(1995)
Terpenoid metabolism.
Plant Cell
7:
1015-1026
[CrossRef][Web of Science][Medline]
Mettal U,
Boland W,
Beyer P,
Kleinig H
(1988)
Biosynthesis of monoterpene hydrocarbons by isolated chromoplasts from daffodil flowers.
Eur J Biochem
170:
613-616
[Medline]
Oblong JE,
Lamppa GK
(1992)
Identification of two structurally related proteins involved in proteolytic processing of precursors targeted to the chloroplast.
EMBO J
11:
4401-4409
[Web of Science][Medline]
Olsen LJ,
Keegstra K
(1992)
The binding of precursor proteins to chloroplast requires nucleotide triphosphates in the intermembrane space.
J Biol Chem
267:
433-439
[Abstract/Free Full Text]
Olsen LJ,
Theg SM,
Selman BR,
Keegstra K
(1989)
ATP is required for the binding of precursor proteins to chloroplasts.
J Biol Chem
264:
6724-6729
[Abstract/Free Full Text]
Pauly G,
Belingheri L,
Marpeau A,
Gleizes M
(1986)
Monoterpene formation by leucoplasts of Citrofortunella mitis and Citrus unshiu: steps and conditions of biosynthesis.
Plant Cell Rep
5:
19-22
Pérez LM,
Pauly G,
Carde J-P,
Berlingheri L,
Gleizes M
(1990)
Biosynthesis of limonene by isolated chromoplasts from Citrus sinensis fruits.
Plant Physiol Biochem
28:
221-229
Rajaonarivony JIM,
Gershenzon J,
Croteau R
(1992)
Characterization and mechanism of 4S-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha × piperita).
Arch Biochem Biophys
296:
49-57
[CrossRef][Web of Science][Medline]
Richter S,
Lamppa GK
(1998)
A chloroplastic processing enzyme functions as the general stromal processing peptidase.
Proc Natl Acad Sci USA
95:
7463-7468
[Abstract/Free Full Text]
Schmidt GW,
Devillers-Thiery A,
Desruisseaux H,
Blobel G,
Chua NH
(1979)
NH2-terminal amino acid sequences of precursor and mature forms of the ribulose-1,5-bisphosphate carboxylase small subunit from Chlamydomonas reinhardtii.
J Cell Biol
83:
615-622
[Abstract/Free Full Text]
Schnepf E
(1974)
Gland cells.
In
AW Robards,
eds, Dynamic Aspects of Plant Ultrastructure.
McGraw-Hill, Berkshire, UK, pp 331-357
Soler E,
Feron G,
Clastre M,
Dargent R,
Gleizes M,
Ambid C
(1992)
Evidence for a geranyl-diphosphate synthase located within plastids of Vitis vinifera L. cultivated in vitro.
Planta
187:
171-175
VanderVere PS,
Bennett TM,
Oblong JE,
Lamppa GK
(1995)
A chloroplast processing enzyme involved in precursor maturation shares a zinc-binding motif with a recently recognized family of metalloendopeptidases.
Proc Natl Acad Sci USA
92:
7177-7181
[Abstract/Free Full Text]
Wan J,
Blakeley SD,
Dennis DT,
Ko K
(1995)
Import characteristics of a leucoplast pyruvate kinase are influenced by a 19-amino-acid domain within the protein.
J Biol Chem
270:
16731-16739
[Abstract/Free Full Text]
Wildermuth MC,
Fall R
(1996)
Light-dependent isoprene emission. Characterization of a thylakoid-bound isoprene synthase in Salix discolor chloroplasts.
Plant Physiol
112:
171-182
[Abstract]
Williams DC,
McGarvey DJ,
Katahira EJ,
Croteau R
(1998)
Truncation of limonene synthase preprotein provides a fully active 'pseudomature' form of this monoterpene cyclase and reveals the function of the amino-terminal arginine pair.
Biochemistry
37:
12213-12220
[CrossRef][Medline]
Wise ML,
Croteau R
(1999)
Monoterpene biosynthesis.
In
DE Cane,
eds, Comprehensive Natural Products Chemistry: Isoprenoids.
Elsevier Science, Oxford, UK, pp 97-153
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Abstract
Introduction