Download Formation of Monoterpenes in Antirrhinum majus

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Formation of Monoterpenes in Antirrhinum majus and
Clarkia breweri Flowers Involves Heterodimeric
Geranyl Diphosphate Synthases
Dorothea Tholl,a,b,1 Christine M. Kish,c,1 Irina Orlova,c,1 Debra Sherman,c Jonathan Gershenzon,a
Eran Pichersky,b and Natalia Dudarevac,2
a Max
Planck Institute for Chemical Ecology, D-007745 Jena, Germany
of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109
c Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907
b Department
The precursor of all monoterpenes is the C10 acyclic intermediate geranyl diphosphate (GPP), which is formed from the C5
compounds isopentenyl diphosphate and dimethylallyl diphosphate by GPP synthase (GPPS). We have discovered that
Antirrhinum majus (snapdragon) and Clarkia breweri, two species whose floral scent is rich in monoterpenes, both possess
a heterodimeric GPPS like that previously reported from Mentha piperita (peppermint). The A. majus and C. breweri cDNAs
encode proteins with 53% and 45% amino acid sequence identity, respectively, to the M. piperita GPPS small subunit
(GPPS.SSU). Expression of these cDNAs in Escherichia coli yielded no detectable prenyltransferase activity. However, when
each of these cDNAs was coexpressed with the M. piperita GPPS large subunit (GPPS.LSU), which shares functional motifs
and a high level of amino acid sequence identity with geranylgeranyl diphosphate synthases (GGPPS), active GPPS was
obtained. Using a homology-based cloning strategy, a GPPS.LSU cDNA also was isolated from A. majus. Its coexpression in
E. coli with A. majus GPPS.SSU yielded a functional heterodimer that catalyzed the synthesis of GPP as a main product. The
expression in E. coli of A. majus GPPS.LSU by itself yielded active GGPPS, indicating that in contrast with M. piperita
GPPS.LSU, A. majus GPPS.LSU is a functional GGPPS on its own. Analyses of tissue-specific, developmental, and rhythmic
changes in the mRNA and protein levels of GPPS.SSU in A. majus flowers revealed that these levels correlate closely with
monoterpene emission, whereas GPPS.LSU mRNA levels did not, indicating that the levels of GPPS.SSU, but not GPPS.LSU,
might play a key role in regulating the formation of GPPS and, thus, monoterpene biosynthesis.
INTRODUCTION
Monoterpenes are low molecular weight volatile compounds that
play multiple roles in plant biology. They are often found in the
scent that many flowers emit to attract insects and other animal
pollinators (Knudsen et al., 1993; Dobson, 1994; Dudareva and
Pichersky, 2000; Pichersky and Gang, 2000; Pichersky and
Gershenzon, 2002). Monoterpenes also are often released from
vegetative tissues, where they function as toxic agents in direct
defense against microbes and animals or help to indirectly
protect the plant by attracting predators of the attacking
herbivores (Langenheim, 1994; Gershenzon and Kreis, 1999;
Kessler and Baldwin, 2001; Pichersky and Gershenzon, 2002).
All monoterpenes are derived from the same substrate,
geranyl diphosphate (GPP; C10), which is in turn synthesized in
a head-to-tail condensation reaction of isopentenyl diphosphate
(IPP) and dimethylallyl diphosphate (DMAPP). This reaction is
catalyzed by GPP synthase (GPPS; EC 2.5.1.1) (Poulter and
Rilling, 1981; Ogura and Koyama, 1998). GPPS belongs to the
1 These
authors contributed equally to this work.
whom correspondence should be addressed. E-mail dudareva@
hort.purdue.edu; fax 765-494-0391.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Natalia Dudareva
([email protected]).
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.020156.
2 To
family of short-chain prenyltransferases that also includes
farnesyl diphosphate (FPP) synthase (FPPS; EC 2.5.1.10) and
geranylgeranyl diphosphate (GGPP) synthase (GGPPS; EC
2.5.1.30). FPPS sequentially adds two molecules of IPP to
DMAPP to form the C15 diphosphate precursor of sesquiterpenes and triterpenes, and GGPPS adds three molecules of IPP
to DMAPP to form the C20 diphosphate precursor of diterpenes
and tetraterpenes (Ogura and Koyama, 1998; Koyama and
Ogura, 1999). These enzymes function at the branch points of
isoprenoid metabolism and can thus play a regulatory role in
controlling IPP flux into different terpenoid families (Gershenzon
and Croteau, 1993).
Characterization of FPPS and GGPPS activities, and in some
cases of the purified homodimeric enzymes, has been reported
from numerous plant sources as well as from animals and
microorganisms. Recently, genes encoding FPPS and GGPPS
have been isolated from a diverse range of plant species in which
they play a central role in both primary and secondary isoprenoid
metabolism (Poulter and Rilling, 1981; Gershenzon and Kreis,
1999). The sequences of these proteins are all related to each
other as well as to prenyltransferases from animals, fungi, and
bacteria (Chen et al., 1994), thus forming a family of short-chain
prenyltransferase proteins.
By contrast, GPPS activity has so far only been found in a few
plant species, mostly in tissues that produce abundant quantities
of monoterpenes, and has proven difficult to purify (Poulter and
Rilling, 1981; Croteau and Purkett, 1989; Heide and Berger,
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Figure 1. A Neighbor-Joining Tree Based on Degree of Sequence Similarity between Plant GPPS, FPPS, and GGPPS.
Sequence analysis was performed using ClustalX, and the nearest neighbor-joining method was applied to create trees. TreeView was used to visualize
the resulting trees. Only some of the known plant FPPS and GGPPS are shown.
1989; Suga and Endo, 1991; Clastre et al., 1993; Wise and
Croteau, 1999; Tholl et al., 2001). Consequently, the isolation and
functional characterization of cDNAs encoding GPPS have not
been described frequently. The first report was that of Burke et al.
(1999), who showed that GPPS exists as a heterodimer in
Mentha piperita (peppermint). One M. piperita subunit (designated as the large subunit, or GPPS.LSU) has high amino acid
sequence identity (56 to 75%) to plant GGPPS, whereas the
sequence of the second smaller subunit (GPPS.SSU) is related
to, but much more divergent from, the sequence of GGPPS (only
24 to 29% identity) (Burke et al., 1999). The two highly conserved
aspartate-rich motifs observed in other prenyltransferases and
determined to be involved in prenyl substrate binding (Marrero
et al., 1992; Song and Poulter, 1994; Tarshis et al., 1994) were
found in M. piperita GPPS.LSU but not in M. piperita GPPS.SSU.
Although both M. piperita subunits alone were catalytically
inactive, their coexpression led to the production of the heterodimeric enzyme that catalyzes the formation of GPP (Burke et al.,
1999). It was further shown that the small subunit is capable of
modifying the chain length specificity of its catalytic partner and
can bind to a variety of bona fide GGPPS enzymes (i.e., GGPPS
from Taxus canadensis [Canadian yew] and Abies grandis [grand
fir]) to form an active heterodimeric enzyme that could catalyze
GPP formation (Burke and Croteau, 2002a).
Subsequent to the report of a heterodimeric GPPS in M.
piperita, Bouvier et al. (2000) and Burke and Croteau (2002b)
identified homodimeric GPPS activities in Arabidopsis (Arabidopsis thaliana) and A. grandis, respectively. Although the
Arabidopsis and A. grandis GPPS proteins are members of the
prenyltransferase family, they are not particularly similar to each
other, and the A. grandis GPPS is closely related to A. grandis
GGPPS (Figure 1). Thus, the frequency of occurrence of the
heterodimeric form of GPPS in plant taxa has remained an open
question. Because monoterpene biosynthesis has been described in various lineages scattered throughout the plant kingdom, GPPS might have evolved from other prenyltransferases
on multiple occasions, resulting in a variety of different proteins
with this activity.
None of the previous studies of GPPS have been performed on
flowers, one of the major monoterpene-producing tissues in
Heterodimeric GPP Synthases
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Figure 2. Alignment of Deduced Amino Acid Sequences of A. majus and C. breweri GPPS.SSUs.
A. majus GPPS.SSU, C. breweri GPPS.SSU, and GPPS.SSU from M. piperita are included. Residues shaded in black indicate conserved residues
(identical in at least two out of three sequences shown), and residues shaded in gray are similar in at least two out of three sequences shown. Dashes
indicate gaps that have been inserted for optimal alignment. The horizontal line indicates the putative N-terminal transit peptide region. Arrowhead 1
indicates the truncation site, and arrowhead 2 indicates the intron position in A. majus GPPS.SSU.
plants. Here, we show that monoterpene-producing flowers of
two angiosperms, Anthirrinum majus (snapdragon, Scrophulariaceae) and Clarkia breweri (Onagraceae), whose floral scents
are rich in monoterpenes (Raguso and Pichersky, 1995;
Dudareva et al., 2000, 2003), possess heterodimeric GPPS.
The emission of monoterpenes from these flowers is developmentally regulated (Pichersky et al., 1994; Dudareva et al., 2003)
and in A. majus follows diurnal rhythms controlled by a circadian
clock (Dudareva et al., 2003). Analyses of tissue-specific,
developmental, and rhythmic expression of genes encoding
the small and large subunits of GPPS in A. majus flowers
revealed that the expression of the gene for the small subunit, but
not for the large subunit, closely correlates with monoterpene
emission, indicating that it might play a key role in regulating the
formation of GPP and, thus, monoterpene biosynthesis.
RESULTS
Isolation and Characterization of A. majus and C. breweri
cDNAs Encoding Proteins with Sequence Similarity to
M. piperita GPPS.SSU
We searched for potential GPPS sequences in small EST
collections (800 sequences each) made from floral tissue of
two species that accumulate monoterpenes in their flowers. The
A. majus ESTs were from the upper and lower petal lobes of 1- to
5-d-old flowers, the parts of the flower highly specialized for floral
scent biosynthesis (Negre et al., 2002; Dudareva et al., 2003),
whereas the C. breweri ESTs were from petals and stigmata of
flowers harvested at the peak stage of volatile emission (D’Auria
et al., 2002). In both databases, cDNAs were found that encoded
proteins with >45% amino acid sequence identity to the
GPPS.SSU from M. piperita (Figure 2) (Burke et al., 1999). A
full-length A. majus cDNA contained 1368 bp and encoded an
open reading frame of 891 bp, corresponding to a protein of 297
amino acids with a calculated molecular weight of 32,622 (Figure
2). The full-length C. breweri cDNA, 1000 bp in size, encoded an
open reading frame of 900 bp, corresponding to a protein of 300
amino acids with calculated molecular weight of 32,049 (Figure
2). Similar to M. piperita, the A. majus and C. breweri proteins lack
the conserved aspartate-rich motif DD(X)2-4D (where X is any
amino acid) that comprises an essential substrate binding element in all other short-chain prenyltransferases identified thus far
(Tarshis et al., 1994; Ogura and Koyama, 1998; Koyama and
Ogura, 1999; Wang and Ohnuma, 1999). Also similar to M.
piperita, both proteins appear to contain an N-terminal targeting
sequence. Screening of an A. majus genomic library with
a GPPS.SSU probe revealed that the coding region of GPPS.SSU
is interrupted by only one intron of 683 bp in size (Figure 2).
Functional Characterization of the GPPS.SSU Proteins
from A. majus and C. breweri
The possible function of the proteins expressed from the A.
majus and C. breweri cDNAs encoding GPPS.SSU-like proteins
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Isolation and Characterization of an A. majus
GPPS.LSU cDNA
Figure 3. Identification by Radio Gas Chromatograms of Labeled Dephosphorylated Reaction Products Generated by A. majus GPPS.SSU.
(A) and (B) The radioactivity detector responses to the dephosphorylated
compounds present in the reaction solution after incubation of [14C]-IPP
and DMAPP with recombinant A. majus GPPS.SSU (A) and with
recombinant A. majus GPPS.SSU coexpressed with GPPS.LSU from
M. piperita (B).
(C) The detector response to coinjected authentic standards: isopentenol (peak 1), dimethylallyl alcohol (peak 2), nerol (peak 3), geraniol (peak
4), cis- and trans-nerolidol (peaks 5 and 6, respectively), trans-farnesol
(peak 7), geranylgeraniol (peak 8), and monoterpenes (M).
was evaluated by transferring each open reading frame into
the pET-T7 expression system and producing the proteins in
Escherichia coli with and without a C-terminal polyhistidine
(6xHis) tag extension (see Methods). The proteins thus expressed had no detectable prenyltransferase activity, even after
partial purification (Figure 3A). Truncation of the signal peptides
also did not yield functional prenyltransferases. However, when
the A. majus cDNA with a His tag extension was coexpressed
with the M. piperita GPPS.LSU (accession number AF182828)
subcloned in the expression vector pSBET without a His tag
(Schenk et al., 1995), the E. coli protein crude extract exhibited
GPPS activity, and this activity could be purified by nickel-based
affinity chromatography. The active enzyme produced exclusively GPP from [14C]-IPP and DMAPP, as confirmed by radio
gas chromatographic analysis of the enzymatically dephosphorylated reaction products, thereby indicating the presence of
GPPS (Figure 3B). Coexpression of the C. breweri cDNA with the
M. piperita GPPS.LSU also resulted in mostly GPPS activity (data
not shown). We thus concluded that these A. majus and
C. breweri cDNAs encode bona fide GPPS.SSU proteins.
Some plant species contain multiple GGPPS and GGPPS-related
enzymes (Okada et al., 2000; Burke and Croteau, 2002b).
However, the A. majus floral EST database did not contain
sequences with close similarity to M. piperita GPPS.LSU or to any
other GGPPS proteins. To isolate cDNAs encoding a potential
GPPS.LSU from A. majus, we examined 10,000 nonredundant
ESTs from a normalized A. majus cDNA library constructed using
mRNAs isolated from roots, seedlings, young/old vegetative
apex, inflorescences, young buds, and flowers at different
developmental stages as well as fertilized and unfertilized
carpels. This search revealed three different GGPPS-like contigs.
Of these, we determined experimentally that one was not
expressed in petals (data not shown), and a second sequence
was expressed in petals but had no enzymatic activity in E. coli
when expressed alone or coexpressed with GPPS.SSU (data not
shown). The third cDNA encoded a protein with 75% amino acid
sequence identity to the M. piperita GPPS.LSU, and a full-length
cDNA was obtained by 59 rapid amplification of cDNA ends
(RACE). The complete cDNA contains 1286 nucleotides and
encodes an open reading frame of 1116 nucleotides corresponding to a predicted protein of 372 amino acids (Figure 4) with
a calculated molecular mass of 40,554. The N terminus of this
protein has characteristic features of a plastid-targeting peptide.
Functional characterization of the protein encoded by this A.
majus cDNA was obtained by subcloning the coding region into
the expression vector pSBET followed by expression in E. coli
BL21 (DE3). GGPP was the exclusive product of the resulting
enzyme as revealed by assays of the crude soluble fraction with
[1-14C]-IPP and its allylic cosubstrate DMAPP followed by radio
gas chromatographic analysis (Figure 5A). However, when this
cDNA was coexpressed with A. majus GPPS.SSU carrying a His
tag extension, the resulting recombinant protein, after purification by affinity chromatography, produced GPP with a small
amount (7%) of GGPP (Figure 5B). These results indicated that
catalytically active A. majus GPPS probably exists as a heterodimer, in which the small subunit protein alone is inactive in
prenyltransfer catalysis, and the large subunit protein alone
forms an active GGPPS enzyme that produces GGPP. Gel
filtration chromatography revealed the molecular mass of this
recombinant GPPS protein to be 75 kD (concordant with the
approximate sum of the two expressed preproteins, small
subunit of 32.4 kD and large subunit of 40.5 kD), further
indicating that the enzyme produced by coexpression of two
cDNAs is a functional heterodimer (Figure 6A).
Characterization of Native and Recombinant
A. majus GPPS
To verify the subunit architecture of native A. majus GPPS, the
protein was partially purified from upper and lower petal lobes of
4- to 6-d-old A. majus flowers (floral tissue with the highest
emission of monoterpenes, Dudareva et al., 2003) using a weak
anion-exchange diethylaminoethyl (DEAE)-cellulose column
followed by Mono-Q anion-exchange chromatography (Figure
7A). Radio gas chromatography (GC) analysis of the products
Heterodimeric GPP Synthases
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Figure 4. Comparison of the Predicted Amino Acid Sequences of A. majus GPPS.LSU and Related Proteins.
A. majus GPPS.LSU sequence was aligned with GPPS.LSU from M. piperita and GGPPS from Arabidopsis (At4g36810), A. grandis, and T. canadensis.
Residues shaded in black indicate conserved residues (identical in at least three out of six sequences shown), and residues shaded in gray are similar in
at least three out of six sequences shown. Dashes indicate gaps that have been inserted for optimal alignment.
formed from [1-14C]-IPP and DMAPP in the reaction catalyzed by
the purified plant protein revealed labeled geraniol exclusively
(Figure 5D), indicating that isolated GPPS was completely free of
FPPS and IPP isomerase activities. Phosphohydrolase activity
also was undetectable. Polyclonal antibodies raised against the
A. majus GPPS.SSU protein detected the small subunit in the
GPPS-active fractions with the GPPS.SSU level positively
correlated with enzymatic activity (Figure 7B). This SDS-PAGE
immunoblot analysis detected a band of 28 kD, which is close
to the molecular mass of 29,142 D predicted by the iPSORT
program for mature (processed) A. majus GPPS.SSU. The
molecular mass of the native protein determined by gel filtration
chromatography was 63 kD, suggesting that native A. majus
GPPS exists in the heterodimeric form comprised of the mature
28-kD small and the mature 37-kD large subunits (Figure 6B)
(mature A. majus GPPS.LSU is predicted by the iPSORT program
to have a molecular mass of 37,163 D). The presence of the 28kD GPPS.SSU in the GPPS active gel filtration chromatography
fractions was again confirmed by protein gel blot analysis using
polyclonal anti-GPPS.SSU antibodies (Figure 6C). No evidence
was found for the existence of active heterotetramers as
reported for M. piperita GPPS (Burke and Croteau, 2002a).
Kinetic characterization of the purified recombinant GPPS
revealed that apparent Km values for IPP (8.3 mM) and DMAPP
(24.1 mM) were very similar to those of the partially purified A.
majus GPPS (Table 1) and were within the range of Km values
previously reported for partially purified native GPPS from A.
grandis (Tholl et al., 2001) and for the recombinant truncated
version of M. piperita GPPS (Burke and Croteau, 2002a). The
apparent Km value for DMAPP is usually about twice that of IPP
(Burke and Croteau, 2002a, 2002b). In this case, A. majus native
and recombinant GPPS have an apparent Km value for DMAPP
that is approximately threefold higher than that for IPP (Table 1).
When enzyme assays were conducted with the recombinant
GPPS using [1-14C]-IPP and DMAPP at a ratio of 1:3, radio GC
analysis determined that only GPP was produced, without even
traces of GGPP (Figure 5C). Similar to recombinant GPPS from
M. piperita, the A. majus protein was unable to utilize GPP or FPP
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as allylic cosubstrates with IPP. GPPS activity was inhibited by
IPP concentrations >20 mM with an I50 value of 89 mM. There
was no inhibition by the allylic cosubstrate DMAPP up to
a concentration of 120 mM. We also found that GPPS activity
was sensitive to GPP concentrations with an I50 value of 59 mM.
Moreover, the enzyme was sensitive to inorganic diphosphate,
the second product of the reaction, for which an I50 value of
810 mM was determined.
Subcellular Localization of the A. majus GPPS.SSU Protein
Biochemical localization studies have shown that GPPS is
a plastidial enzyme (Soler et al., 1992), which is consistent with
Figure 6. Determination of the Molecular Size of A. majus Recombinant
and Native GPPS.
Figure 5. Radio Gas Chromatograms of the Labeled Dephosphorylated
Reaction Products Generated by A. majus GPPS.LSU and Recombinant
and Native GPPS.
(A) to (D) The radioactivity detector responses to the dephosphorylated
compounds present in the reaction solution after incubation of [14C]-IPP
and DMAPP with recombinant A. majus GPPS.LSU (A), with recombinant
A. majus GPPS.SSU coexpressed with A. majus GPPS.LSU (B), with
recombinant A. majus GPPS.SSU coexpressed with A. majus GPPS.LSU
at [1-14C]-IPP to DMAPP ratio of 1:3 (C), and with GPPS isolated from A.
majus upper and lower lobes (D).
(E) The detector response to coinjected authentic standards: geraniol
(peak 1), cis-farnesol (peaks 2), trans-farnesol (peak 3), and geranylgeraniol (peak 4).
(A) and (B) Gel filtration chromatography of A. majus recombinant GPPS
(A) and partially purified native GPPS (B) on Superdex 200-HR.
(C) Immunodetection of GPPS.SSU in corresponding gel filtration
chromatography fractions with native GPPS activity using polyclonal
anti-GPPS.SSU antibodies. Crude petal protein extract was loaded in the
first (left) lane and used as a control (C).
(D) The elution behavior of the standards: b-amylase (200 kD), alcohol
dehydrogenase (150 kD), BSA (66 kD), and carbonic anhydrase (29 kD).
Ve/Vo is the ratio of elution volume to void volume.
Heterodimeric GPP Synthases
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gold labeling also was absent in the conical cells of the inner
epidermal layer of lower lobes of 1-d-old A. majus flowers
(Figure 8A), a floral developmental stage in which monoterpene
synthesis does not yet occur (Dudareva et al., 2003).
Tissue-Specific, Developmental, and Rhythmic
Expression of A. majus GPPS
Figure 7. Purification of Native GPPS from A. majus Upper and Lower
Petal Lobes.
(A) Anion exchange chromatography (Mono Q) of GPPS activity
containing fractions after initial DEAE-Sepharose chromatography.
Absorbance at 280 nm, prenyltransferase activities (in cpm), and the
NaCl gradient are indicated. Radio GC analysis of labeled dephospholylated reaction products revealed FPPS activity in peak area 1 (fractions 15
to 25) and GPPS activity in peak area 2 (fractions 26 to 31) (see Figure 5D).
(B) Immunodetection of GPPS.SSU in the corresponding fractions
separated by anion exchange chromatography (A) using the polyclonal
antibodies raised against the native GPPS.SSU protein. Crude extract
was loaded in the last (right) lane and used as a control (C).
the plastidial localization of monoterpene biosynthesis in plants
(Turner et al., 1999). Similar to the M. piperita GPPS.SSU, A. majus
GPPS.SSU also contains a transit peptide (Figure 2). Monoterpene synthesis and emission in A. majus flowers occurs mostly in
upper and lower petal lobes of A. majus flower (Dudareva et al.,
2003). To determine the intracellular distribution of the A. majus
GPPS.SSU protein, immunoelectron microscopy was performed
with transverse sections of the lower lobes of 7-d-old A. majus
flowers. Substantial specific immunogold labeling with antibodies made against the A. majus GPPS.SSU was found within
the nonpigmented plastids (leucoplasts) of the conical epidermal
cells (Figure 8B), which are extensively involved in scent
production (Kolosova et al., 2001b). No specific immunolabeling
was observed in control sections treated with preimmune serum
instead of anti-GPPS.SSU antibodies (Figure 8C). Immuno-
To test whether the genes encoding GPPS.SSU and GPPS.LSU
are expressed in spatial and temporal patterns that are correlated with monoterpene emission, total RNA was isolated
from leaves and different floral parts of 3-d-old A. majus flowers
(sepals, pistils, stamens, and different regions of the corolla,
including the upper petal lobes, the lower petal lobes, and the
tube). The level of gene expression of GPPS.SSU was analyzed
by RNA gel blot analysis. For GPPS.LSU, an RT-PCR method
with specific primers was used to exclude possible crosshybridization with related GGPPS sequences.
The highest level of transcript accumulation for GPPS.SSU
was found in the upper and lower lobes of petals (Figure 9A), the
parts of the flower that were previously shown to be primarily
responsible for monoterpene production and emission in A.
majus (Dudareva et al., 2003). A low level of transcripts was
detected in the tube and leaves. No detectable signals were
found in pistils, stamens, sepals, and ovaries. Whereas expression of GPPS.SSU was found to be petal specific (Figure 9A), the
gene encoding GPPS.LSU was constitutively expressed (Figure
9B). Moreover, when the steady state levels of mRNAs for
GPPS.SSU and GPPS.LSU were analyzed in upper and lower
petal lobes during flower development (from mature flower buds
1 d before anthesis to open flowers 12 d after anthesis), only the
expression of the gene encoding GPPS.SSU closely correlated
with the pattern of monoterpene emission as determined
previously (Dudareva et al., 2003). GPPS.SSU mRNA was first
detected in 1-d-old flowers, 1 d ahead of the beginning of monoterpene emission. Its level peaked on day 5 after anthesis, 1 d
ahead of maximum myrcene and (E)-b-ocimene emissions,
and remained relatively stable for the next 6 d, decreasing
thereafter (Figure 10A). Analysis of GPPS.LSU expression
revealed that although its expression was highest on day 4 after
anthesis, its expression levels changed very little during the
course of flower development and did not show strong correlation with monoterpene emission (Figure 10B).
Previously we have shown that the emission of myrcene and
(E)-b-ocimene from A. majus flowers is controlled by a circadian
clock and reaches a maximum level between 11 AM and 6 PM
(Dudareva et al., 2003). Accumulation of GPPS.SSU mRNA was
Table 1. Apparent Km Values for A. majus Recombinant and Native Plant GPPS
Enzyme
Km IPP (mM)a
Km DMAPP (mM)b
Km MgCl2 (mM)c
Recombinant GPPS
Plant GPPS
8.3 6 1.12
14.96 6 2.58
24.12 6 2.08
37.24 6 2.11
2.37 6 0.29
0.32 6 0.03
a At
80 mM DMAPP.
20 mM IPP.
c At 20 mM IPP and 80 mM DMAPP.
b At
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Figure 8. Intracellular Localization of GPPS.SSU in A. majus.
(A) Transmission electron microscopy (TEM) image of conical cells of 1-d-old A. majus lower petal lobe labeled with anti-GPPS.SSU antibodies and
gold-conjugated goat anti-rabbit antibodies. p, plastid.
(B) TEM image of conical cells of 7-d-old A. majus lower petal lobe labeled with anti-GPPS.SSU antibodies and gold-conjugated goat anti-rabbit
antibodies. m, mitochondria; p, plastid.
(C) TEM image of conical cells of 7-d-old A. majus lower petal lobe treated with preimmune serum and gold-conjugated goat anti-rabbit antibodies.
p, plastid.
examined in upper and lower petal lobes at nine time points
during a 27-h interval, and only slight variations in GPPS.SSU
mRNA levels were found during the daily light/dark cycle (Figure
11A). Levels of transcripts of GPPS.SSU increased during the
light period, with maximum accumulation detected around 3 to 6
PM, and then declined at least twofold during the dark period (12
AM), showing a weak diurnal oscillation pattern. The rhythmic
pattern of mRNA accumulation was retained under continuous
dark (Figure 11B), suggesting that the cyclic expression of the
gene encoding GPPS.SSU is under circadian control.
The levels of GPPS.SSU protein in different floral parts of 4-dold A. majus flowers, as well as in upper and lower petal lobes,
over flower development were determined by the chemiluminescence protein gel blotting technique using polyclonal antiGPPS.SSU antibodies. The polyclonal antibodies raised against
the native GPPS.SSU protein (see Methods) selectively recognized a single protein with an apparent molecular mass of 28 kD
in crude extracts separated by SDS-PAGE (Figure 12A). This
protein migrated slightly faster than the GPPS.SSU protein of
32 kD produced in E. coli, indicating that the plant preprotein
containing an N-terminal plastidial transit peptide went through
subsequent proteolytic processing to the mature form (Figure
12B). The GPPS.SSU protein was detected primarily in upper
and low petal lobes and in tubes (Figure 12A), which is consistent
with GPPS.SSU gene expression determined by RNA gel blot
analysis. During flower development, the GPPS.SSU protein was
first detected in 1-d-old flower petals, peaked on days 5 to 7 after
anthesis, and decreased afterwards (Figure 12C).
DISCUSSION
Heterodimeric Subunit Architecture of A. majus and
C. breweri GPPS
Until recently, floral scent research has mainly concentrated on
the isolation and characterization of enzymes and genes involved
in the final steps of scent biosynthesis (summarized in Dudareva,
2002). However, it has been shown that the level of scent
biosynthetic enzymes is not the only limiting factor and that the
level of supplied substrate in the cell also could contribute to the
regulation of scent production (Dudareva et al., 2000; Kolosova
et al., 2001a). Although monoterpenes are very common constituents of floral scent, the regulation of monoterpene biosynthesis has been mainly studied in vegetative tissue (Gershenzon
et al., 2000; McConkey et al., 2000), where they serve mostly as
defense compounds, and little is known about how floral tissues
regulate their developmental and rhythmic emission of monoterpenes.
Heterodimeric GPP Synthases
9 of 16
All monoterpenes are formed from GPP, which is synthesized
from DMAPP and IPP by GPPS. We searched for GPPS genes in
A. majus and C. breweri, whose flowers produce a scent rich in
myrcene and (E)-b-ocimene (Dudareva et al., 2003) and linalool
(Pichersky et al., 1994), respectively. From an A. majus petalspecific cDNA library, cDNAs were isolated (Figures 2 and 4) that
were very similar to those encoding the large (GPPS.LSU) and
small (GPPS.SSU) subunits of M. piperita GPPS (Burke et al.,
1999) (Figures 1, 2, and 4). The A. majus cDNAs showed 53% and
75% amino acid sequence identity with GPPS.SSU and
GPPS.LSU from M. piperita, respectively, and were able to form
GPP when coexpressed in E. coli (Figure 5B). A. majus
GPPS.SSU is inactive when expressed alone, similar to the M.
piperita GPPS.SSU (Figure 3A). Conversely, A. majus GPPS.LSU
is a functional GGPPS on its own (Figure 5A), in contrast with M.
piperita GPPS.LSU. A. majus GPPS.SSU could interact productively not only with its GGPPS (GPPS.LSU) partner in A.
majus, but like the M. piperita GPPS.SSU, also with GPPS.LSU
from M. piperita (Figure 3B) and GGPPS from Arabidopsis
(At4g36810, Figure 4) (data not shown) to form active GPPS.
From C. breweri, a cDNA encoding GPPS.SSU also was isolated,
which shows 45% identity to the M. piperita GPPS.SSU (Figure 2)
Figure 10. Developmental Changes in Steady State GPPS.SSU and
GPPS.LSU mRNA Levels in Upper and Lower Lobes of A. majus Petals.
Figure 9. Tissue Specificity of A. majus GPPS.SSU and GPPS.LSU
Gene Expression.
(A) RNA gel blot of total RNA (5 mg per lane) isolated from leaves, upper
and lower petal lobes, tubes, pistils, stamens, and ovaries of 3-d-old A.
majus flowers. The top gel represents the results of hybridization with
a coding region of the GPPS.SSU gene as a probe. Autoradiography was
performed overnight. The blot was rehybridized with an 18S rDNA probe
(bottom gel) to standardize samples.
(B) RT-PCR with GPPS.LSU gene-specific primers was performed on
RNA isolated from leaves, upper and lower petal lobes, tubes, pistils,
stamens, and ovaries of 3-d-old A. majus flowers. The amplified
products were run on 1.2% agarose gel, blotted onto nitrocellulose
membrane, and hybridized with the GPPS.LSU gene as a probe. The
RT-PCR products for rRNA are shown at the bottom.
(A) Representative RNA gel blot hybridization with mRNA isolated from
upper and lower petal lobes at different stages of development, from
mature flower buds to day 12 after anthesis. Each lane contained 5 mg of
total RNA. A coding region of the GPPS.SSU gene was used as a probe.
Autoradiography was performed overnight. The blots were rehybridized
with an 18S rDNA probe (bottom gel) to standardize samples. RNA gel
blots were scanned with a Storm 860 PhosphorImager, and the values
were corrected by standardizing for the amounts of 18S rRNA measured
in the same runs. The maximum transcript level was taken as 1. Each
point is the average of six different experiments (including the one shown
in [A]). Standard error values are indicated by vertical bars.
(B) Representative RT-PCR with GPPS.LSU gene-specific primers on RNA
isolated from upper and lower petal lobes at different stages of development, from mature flower buds to day 12 after anthesis. The amplified
products were run on 1.2% agarose gel, blotted onto nitrocellulose
membrane, and hybridized with the GPPS.LSU gene as a probe. The RTPCR products for rRNA are shown at the bottom. Hybridization signals
were analyzed using a Storm 860 PhosphorImager and ImageQuant
software (Molecular Dynamics). The maximum transcript level was taken
as 1. Each point is the average of four different experiments (including the
one shown in [B]). Standard error values are indicated by vertical bars.
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The Plant Cell
sequences very similar to those of the M. piperita GPPS.SSU
protein and appear to be closely related (Figure 1). The presence
of these GPPS.SSU proteins in three plant families (Lamiaceae,
Onagraceae, and Scrophulariaceae) representing different angiosperm clades suggests that the heteromeric GPPS may be
widespread in the angiosperms.
When such a heteromeric protein arose during plant evolution
remains to be determined. Not all angiosperm species contain
heteromeric GPPS proteins. A search of the complete genome
sequence of Arabidopsis failed to identify such a gene. Instead, it
was shown experimentally that Arabidopsis has a divergent
protein in the prenyltransferase family that is capable of forming
a functional homodimeric GPPS (Bouvier et al., 2000). Together
with the identification of a homodimeric GPPS in the gymnosperm tree A. grandis, whose protein sequence closely
resembles that of GGPPS (Burke and Croteau, 2002b), these
observations suggest that GPPS enzymatic activities have
evolved independently multiple times during plant evolution.
GPPS.SSU Is Localized to Leucoplasts of Conical Cells
of A. majus Epidermis
Figure 11. GPPS.SSU mRNA Expression in Petal Tissue (Upper and
Lower Lobes) of 3-d-old A. majus Flowers during a Normal Light/Dark
Cycle and in Continuous Dark.
(A) RNA gel blot analysis of steady state GPPS.SSU mRNA levels in A.
majus petals during a normal light/dark cycle. Total RNA was isolated
from upper and lower petal lobes of 3-d-old flowers at time points
indicated at the top of the figure, and 5 mg of total RNA was loaded in
each lane. The top gel represents the results of hybridization with
a GPPS.SSU probe. Autoradiography was performed overnight. The blot
was rehybridized with an 18S rDNA probe (bottom gel) to standardize
samples. RNA gel blots were scanned with a PhosphorImager and
corrected by standardizing for the amounts of 18S rRNA measured in the
same runs. The maximum transcript level was taken as 1. Each point is
the average of four different experiments (including the one shown in [A]).
Standard error values are indicated by vertical bars. Shaded and light
areas correspond to dark and light, respectively.
(B) RNA gel blot analysis of steady state GPPS.SSU mRNA levels in A.
majus flowers exposed to continuous dark. Total RNA was isolated from
upper and lower petal lobes of 5-d-old flowers exposed to the second
constant dark cycle at time points indicated at the top of the figure, and
5 mg of total RNA was loaded in each lane. The blot was rehybridized
with an 18S rDNA probe (bottom gel) to standardize samples.
and could form GPP when coexpressed with M. piperita
GPPS.LSU but was inactive by itself.
Heteromeric GPPS Is Found in Multiple Angiosperm
Species
Our results indicate that both A. majus and C. breweri flowers
contain a heteromeric GPPS enzyme, demonstrating that
heteromeric GPPS activities are not restricted to M. piperita.
Moreover, the small subunits of A. majus and C. breweri have
The biosynthesis of IPP proceeds in higher plants via two parallel
pathways located in different subcellular compartments, the
acetate/mevalonate pathway in the cytoplasm (Qureshi and
Porter, 1981; Newman and Chappell, 1999) and the pyruvate/
Figure 12. GPPS.SSU Protein Levels in Different Floral Tissues and in
Petals during Flower Development.
(A) Expression of GPPS.SSU protein in sepals, upper and lower petal
lobes, tubes, pistils, stamens, and ovaries of 4-d-old A. majus flowers.
Representative protein gel blot shows the 30-kD protein recognized by
anti-GPPS.SSU antibodes. Proteins were extracted from different floral
tissues, and 20 mg of protein was loaded in each lane. The blot shown
represents a typical result of two independent experiments.
(B) Immunodetection of GPPS.SSU in the recombinant GPPS and in
crude petal extract.
(C) Expression of the GPPS.SSU protein in upper and lower petal lobes
at different stages of development. Proteins were extracted from upper
and lower petal lobes at different stages of development, and 20 mg of
protein was loaded in each lane. The blot shown represents a typical
result of six independent experiments.
Heterodimeric GPP Synthases
glyceraldehyde-3-phosphate pathway in plastids (RodriguezConcepcion and Boronat, 2002). The cytosolic pool of IPP serves
as a precursor of FPP and, ultimately, the sesquiterpenes and
triterpenes, whereas the plastidial pool of IPP provides the
precursor of GPP and GGPP and, ultimately, the monoterpenes,
diterpenes, and tetraterpenes. Although it is generally accepted
that GGPP is formed in the plastids, recent investigations of the
GGPPS gene family in Arabidopsis revealed five isozymes of
GGPPS, which are differentially expressed in a variety of organs
and localized in three subcellular compartments: chloroplasts,
endoplasmic reticulum, and mitochondria (Okada et al., 2000).
Similarly, it has long been considered that FPPS is a cytosolic
enzyme. However, recently it has been shown that Arabidopsis
FPPS1 is a bifunctional gene encoding cytosolic and mitochondrial FPPS isoforms (Cunillera et al., 1997). Moreover, immunocytochemical staining using anti-FPPS1 antibodies followed
by electron microscopy suggested chloroplast localization of
FPPS1 in rice (Oryza sativa) mesophyll cells (Sanmiya et al.,
1999). FPPS also was localized in the chloroplast fraction in
Triticum aestivum (wheat) and Nicotiana tabacum (tobacco)
using cellular fractionation followed by immunoblot analysis
(Sanmiya et al., 1999).
In this study, the presence of N-terminal signal peptides on
the A. majus GPPS.SSU and GPPS.LSU suggested a plastidial
location for GPPS in this species. However, the GPPS.SSU
sequence contains two in-frame AUG codons, indicating the
possibility of more than one subcellular location. Previous
studies on GPPS are also equivocal. Biochemical localization
studies suggested that GPPS from Vitis vinifera is a plastidial
enzyme (Soler et al., 1992), whereas cell fractionation, marker
enzyme assays, and immunodetection showed that GPPS from
Lithospermum erythrorhizon is cytosolic (Somer et al., 1995).
Early labeling experiments with rose petals indicated that floral
tissues may synthesize monoterpenes using the acetatemevalonate pathway (Francis and O’Connell, 1969) and so
support a cytoplasmic localization of GPPS in petal tissue. Thus,
it was important to determine the location of GPPS in A. majus.
The immunogold labeling results presented here show that
GPPS.SSU is confined to the leucoplasts of the conical cells of
the inner epidermal layer of 7-d-old A. majus petals (Figure 8B),
a floral developmental stage in which monoterpene emission is at
its highest (Dudareva et al., 2003). These results also suggest
that monoterpenoid biosynthesis in A. majus takes place predominantly in plastids where the substrate GPP is synthesized
because the terpene synthases converting GPP to myrcene and
(E)-b-ocimene also posses N-terminal signal peptides (Dudareva
et al., 2003). Subcellular compartmentation may be crucial in the
regulation of GPPS activity and hence monoterpene formation
in A. majus cells.
Expression Levels of the GPPS.SSU mRNA and GPPS.SSU
Protein Correlate with Monoterpene Biosynthesis
and Emission in A. majus Flowers
Previously we showed that production and emission of the two
major monoterpenoid floral scent compounds myrcene and
(E)-b-ocimene occur in upper and lower petal lobes, where
high levels of monoterpene synthase gene expression have
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been found (Dudareva et al., 2003). Here, we have shown that
expression of GPPS.SSU, but not GPPS.LSU, displays a tissuespecific profile with highest levels in upper and lower petal lobes
(Figure 9A). Moreover, during flower development and the daily
light/dark cycle, the steady state levels of GPPS.SSU mRNA
correlate with monoterpene emission (Figures 10A and 11A). In
contrast with GPPS.SSU, GPPS.LSU mRNA expresses constitutively in leaves and all floral tissues examined (Figure 9B) and
its level changes only slightly in petals during flower development (Figure 10B). These expression studies coupled with the
biochemical characterization of GPPS suggest that levels of
GPPS.SSU alone control the rate of production of GPP.
Because some plant species contain multiple genes encoding
GGPPS and GGPPS-related enzymes (Okada et al., 2000; Burke
and Croteau, 2002b), we cannot exclude the possibility that the
A. majus GPPS.LSU sequence reported here, which can clearly
serve as a large subunit of GPPS, might not be the only one.
However, as described above, an extensive search of 10,000
nonredundant ESTs from a normalized A. majus cDNA library
failed to identify additional GPP.LSU sequences. Moreover, the
recombinant GPPS containing this GPPS.LSU protein and the
partially purified plant GPPS enzyme have very similar Km values
with both IPP and DMAPP (Table 1), suggesting that the
GPPS.LSU cDNA reported here has been correctly identified.
The level of GPPS.SSU protein is tightly correlated with the
steady state levels of GPPS.SSU mRNA (Figures 9A, 10A, 11A,
12A, and 12C), indicating that the developmental and rhythmic
regulation of GPPS could reside largely at the level of RNA.
However, we also found that A. majus GPPS displays significant
substrate inhibition that can add an additional level of regulation.
An IPP concentration of 89 mM reduces GPPS maximum activity
by 50%, and GPPS also is inhibited by its products, GPP and
inorganic diphosphate. Such feedback regulation together with
substrate inhibition could contribute to the regulatory control of
the flux to GPP and subsequently to monoterpene production.
METHODS
Plant Material
A. majus Maryland True Pink cultivar (Ball Seed, West Chicago, IL) was
grown under standard greenhouse conditions as described previously
(Dudareva et al., 2000).
Chemicals and Radiochemicals
[1-14C]-IPP (55 mCi/mmol) was purchased from American Radiolabeled
Chemicals (St. Louis, MO). Unlabeled IPP, DMAPP, GPP, and FPP were
from Echelon Research Laboratories (Salt Lake City, UT). Authentic
C5-C20 prenylalcohol standards were from Sigma Chemical (St. Louis,
MO), Aldrich Chemical (Milwaukee, WI), and CTC Organics (Atlanta, GA).
Alkaline phosphatase, apyrase, and protein molecular weight standards
were purchased from Sigma Chemical as well as all other chemicals,
unless otherwise noted.
EST Sequencing, RACE, and cDNA Library Screening
A. majus and C. breweri EST databases were constructed previously by
sequencing random clones from corresponding petal-specific cDNA
libraries (D’Auria et al., 2002; Dudareva et al., 2003). These ESTs can be
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The Plant Cell
found at https://sativa.biology.lsa.umich.edu/blast/blast.html. To isolate
a full-length cDNA clone encoding A. majus GPPS.LSU, a cDNA library
screening and 59-RACE were performed as described previously
(Dudareva et al., 2003).
Sequence Analysis
Amino acid sequence alignments were generated using the ClustalX
computer program (http://www-igbmc.u-strasbg.fr/BioInfo/ClustalX/
Top.html) (Human Genome Sequencing Center, Houston, TX). Sequence
relatedness was analyzed using the neighbor-joining method, and an
unrooted tree was visualized using TreeView (http://taxonomy.zoology.
gla.ac.uk/rod/treeview.html).
Expression of Small and Large Subunits of GPPS in E. coli
The coding region of A. majus GPPS.SSU was amplified by PCR using the
forward primer 59-CATATGGCCCACGGCCTCAC-39, which introduced
an NdeI site at the initiating ATG codon, in combination with the reverse
primer 59-AGATCTTTAAACACCAGCAAGACTATGC-39, which introduced a BglII site downstream of the stop codon and subcloned into
the expression vector pET 11a (Novagen, Madison, WI). For subcloning
into the pET 32a expression vector, the reverse primer 59-GATATCGCAACACCAGCAAGACTATGC-39, containing an EcoRV site, was used
that allowed a translational fusion to a C-terminal His6 tag. The coding
region of C. breweri GPPS.SSU was amplified by PCR using the forward
primer 59-CATATGCTGCTCGTACCTC-39, containing an NdeI site, and
the reverse primer 59-AGATCTTTAGTAGCTCTTGATAAG-39, containing
a BglII site, for subcloning into the pET 11a (and 59-CATATGCTGCTCGTACCTC-39 and 59-GAATTCGGGTAGCTCTTGATAAGTG-39
for subcloning into pET 32a) expression vector. A truncated version of
the A. majus small subunit with a deletion of the N-terminal plastidial
targeting sequence was obtained using the forward primer 59-AATGCCCGTGATTCTCACCATGACC-39, which introduced a starting Met in
place of Arg-29, and the reverse primer 59-CTTTAAACACCAGCAAGACTATGC-39 for subcloning into the pCR-T7/CT-TOPO vector (Invitrogen,
Carlsbad, CA). A similar truncated version of the C. breweri small subunit
was prepared and subcloned into the same vector using the forward
primer 59-AATGCCCACCGCTACGATGGCCAC-39, introducing a starting
Met in place of Ser-22, and the reverse primer 59-ATGCATAATATCTTTAGTAGCTCTTG-39. The coding region of A. majus GPPS.LSU was
cloned into the pSBET expression vector (Schenk et al., 1995) after PCR
amplification using the forward primer 59-CATATGAGCCTGGTAAATCCCATTAC-39, containing an NdeI site, and the reverse primer 59-GGATCCTTAGCCAACGCGATCGAC-39, introducing a BamHI site downstream
of the stop codon. The coding region of the M. piperita large subunit was
amplified from genomic DNA of M. piperita by repetitive PCR using the
forward primer 59-AATGAGTGCTCTTGTTAATCCTGTGG-39 and the
reverse primer 59-ATCAATTGTCCCTATAAGCAATATAATTGG-39 as first
primer pair and the forward primer 59-CATATGAGTGCTCTTGTTAATCCTG-39, containing an NdeI site, and reverse primer 59-GGATCCTCAATTGTCCCTATAAGC-39, with a BamHI site downstream of the stop
codon, as second primer pair. The amplified gene product was subcloned
into the pSBET vector. Sequencing revealed that no errors had been
introduced during PCR amplifications.
For functional expression, E. coli BL21 (DE3) cells were transformed
with the resulting recombinant plasmids as well as pSBET and pET
vectors without insert as controls. For coexpression, E. coli BL21 (DE3)
cells were cotransformed with two plasmids (e.g., small subunit and
GPPS.LSU or M. piperita large subunit) in a single transformation event.
Single positive bacterial colonies were used to inoculate 5 mL of LuriaBertani medium with 50 mg/mL of chloramphenicol and 100 mg/mL of
ampicillin (for pET11a, pET32a, and pCR-T7/CT-TOPO constructs) or
50 mg/mL of kanamycin (for pSBET constructs) or with both antibiotics
(for coexpression of both pET11a/pET32a and pSBET constructs), which
were grown overnight at 378C. Two milliliters of these cultures were
transferred to 100 mL of the same medium and continued to grow at 378C
until an OD600 of 0.5 was reached. Cultures were then induced by the
addition of isopropyl-1-thio-b-D-galactopyranoside to a final concentration of 1 mM and grown for an additional 18 h at 188C.
Purification of Recombinant Proteins and Antibody Preparation
Cells containing recombinant protein were harvested by centrifugation,
resuspended in either 5 mL of assay buffer (25 mM Mopso, pH 7.0, 10 mM
MgCl2, 10% glycerol, and 2 mM DTT) or in 4 mL of His tag lysis buffer
(50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10% glycerol, and 1 mM
phenylmethanesulfonyl fluoride). Cells containing nontagged proteins
were disrupted by sonication using a Bandelin Sonopuls (Bandelin
Electronic, Berlin, Germany) at 50% power with two interrupted 4-min
bursts at 48C. Lysates were cleared by centrifugation at 20,000g (15 min),
and the resulting supernatants containing the soluble enzyme were run
through an Econo-Pac10DG column (Bio-Rad Laboratories, Hercules,
CA) equilibrated with assay buffer.
For the purification of His6 tag recombinant proteins, the initial extracts
(4 mL) were lysed with lysozyme (1 mg/mL) for 30 min on ice followed by
4-min sonication. After removal of the cellular debris by centrifugation for
15 min at 20,000g, the supernatant was combined with 1 mL of nickelnitrilotriacetic acid agarose (Qiagen, Hilden, Germany), mixed on a rotary
shaker for 1 h at 48C, and poured into a 5-mL disposable polypropylene
column (Qiagen) to gravity drain. The nickel-nitrilotriacetic acid agarose
was washed with 6 mL of washing buffer containing 50 mM NaH2PO4, pH
8.0, 300 mM NaCl, 10% glycerol, and 20 mM imidazole, followed by
elution with 2 mL of the same buffer containing 250 mM imidazole. The
protein fractions of 0.5 mL were collected and desalted on NAP 5 columns
(Amersham Pharmacia Biotech, Uppsala, Sweden) into assay buffer.
The His-tagged purified recombinant protein corresponding to the
small subunit of A. majus GPPS was used to generate polyclonal
antibodies in rabbits (Eurogentec, Cologne, Germany). Preimmune sera
and antisera with the highest antibody titer of one rabbit were used for
immunodetection of the GPPS.SSU in crude protein extracts and
fractions of partially purified GPPS of A. majus flower petals.
Prenyltransferase Assays and Product Analysis by Radio GC
Prenyltransferase assays were performed in a final volume of 100 mL
containing 40 mM [1-14C]-IPP (55 mCi/mmol) and 40 mM DMAPP in assay
buffer (25 mM Mopso, pH 7.0, 10% [v/v] glycerol, 2 mM DTT, and 10 mM
MgCl2). After the reaction was initiated by addition of an aliquot of the
enzyme, the assay mixture was immediately overlaid with 1 mL of hexane
and incubated for 40 min at 308C. Assays were stopped by adding 10 mL
of 3 N HCl and then incubated for an additional 20 min at 308C to
hydrolyze the acid-labile allylic diphosphates formed during the assay to
their corresponding alcohols. Hydrolysis products (and alcohols produced by endogenous phosphohydrolase activity during the assay) were
extracted into the hexane phase by vigorous mixing for 15 s. After
centrifugation for 2 min, 500 mL of the hexane phase were counted
in a liquid scintillation counter (Liquid Scintillation Analyzer Tri-Carb
2300TR; Canberra-Packard, Dreieich, Germany). Chromatographic fractions were routinely assayed with reduced substrate concentrations
(10 mM [1-14C]-IPP and 10 mM DMAPP). Controls included assays with
boiled protein extracts and without substrate, and background radioactivity produced in such assays was subtracted from all results.
To determine endogenous phosphohydrolase activity, assays were
performed as described above but with [1-14C]-IPP as the only substrate
and without acid treatment. After incubation at 308C, samples were
cooled on ice and the radiolabeled isopentenol was directly extracted into
the hexane layer.
Heterodimeric GPP Synthases
For the identification of reaction products, larger scale assays were
performed in a final volume of 500 mL. Assays were incubated for several
hours at 308C. To stop the assay and hydrolyze all diphosphate esters
(including unreacted substrate as well as products), 500 mL of a solution
containing 2 units of bovine intestine alkaline phosphatase (18 units/mg;
Sigma) and 2 units of potato apyrase (25.2 units/mg; Sigma) in 0.2 M TrisHCl, pH 9.5, were added, and incubation of samples at 308C continued for
8 to 12 h. After enzymatic hydrolysis, the resulting prenyl alcohols were
further extracted into 2 mL of diethylether and after addition of a standard
prenyl alcohol mixture, the organic extract was prepared for radio GC as
described previously (Burke et al., 1999).
Radio GC analysis was performed on an Agilent 6890 gas chromatograph (splitless, injector temperature 2208C) (Hewlett-Packard, Palo Alto,
CA). Hydrogen was used as the carrier gas (2 mL minÿ1), and the outlet of
the column (DB-5MS, 30 m 3 0.25 mm with 0.25-mm phase coating; J&W
Scientific, Folsom, CA) was coupled to a thermal conductivity detector at
2508C followed by a Raga radiodetector (Raga 92; Raytest, Straubenhardt,
Germany). One microliter of concentrated organic phase was injected, and
the separation was achieved with a temperature program of 3 min at 708C,
followed by a gradient from 70 to 2408C at 68C minÿ1 with increase to
3208C at 408C minÿ1 afterwards. Both mass and radioactivity traces were
monitored simultaneously. Radioactive compounds were identified by
comparison of retention times with those of coinjected authentic unlabeled
standards recoded by the thermal conductivity detector.
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recombinant protein, an apparent Km value for IPP was determined at
saturating concentrations of DMAPP (80 mM), whereas the apparent Km
value for DMAPP was determined at a saturating concentration of IPP
(20 mM). For determination of the Km value for MgCl2, both substrates
were at saturating concentrations. Kinetic data were evaluated using
Lineweaver-Burk, Eadie-Hofstee, and Hanes plots.
DNA Isolation, RNA Isolation, and RNA Gel Blot Analysis
Genomic DNA from M. piperita was isolated from young leaves according
to the method of Rogers and Bendich (1985). Total RNA from A. majus
floral tissues and petals at different stages of flower development were
isolated and analyzed as previously described (Dudareva et al., 1996,
1998, 2000, 2003; Wang et al., 1997; Kolosova et al., 2001a), with nine
time points during a daily light/dark cycle and eight time points during
continuous dark conditions. A 1-kb EcoRI fragment containing the coding
region of GPPS.SSU was used as a probe in RNA gel blot analysis. Five
micrograms of total RNA was loaded in each lane. Hybridization signals
were quantified using Storm 860 phosphor screen (Molecular Dynamics,
Sunnyvale, CA), and mRNA transcript levels were normalized to rRNA
levels to overcome errors in RNA quantitation by spectrophotometry.
Multiple independent RNA gel blots were prepared and analyzed for each
time point, and a representative time-course profile is presented.
RT-PCR
Partial Purification of GPPS from A. majus Petals
Freshly excised upper and lower petal lobes of 4- to 6-d-old A. majus
flowers were frozen in liquid N2 and ground to a fine powder using a mortar
and pestle. The frozen powder was immediately slurried with freshly
prepared extraction buffer (4:1 [v/w], buffer:tissue) containing 50 mM BisTris-HCl, pH 6.9, 5 mM DTT, 10 mM MgCl2, 5 mM Na2S2O5, 1% (w/v)
polyvinylpyrrolidone (PVP-40), 1 mM phenylmethanesulfonyl fluoride, and
10% (v/v) glycerol. The slurry was passed through two layers of Miracloth
(Calbiochem, CA) and centrifuged for 30 min at 20,000g. The pellet was
discarded and the supernatant was loaded onto a DEAE-cellulose
column (10 mL of DE53; Whatman, Clifton, NJ) preequilibrated with
a solution containing 50 mM Bis-Tris-HCl, pH 6.9, 10% glycerol, 10 mM
MgCl2, and 5 mM DTT (buffer A) at a flow rate of 1 mL minÿ1. After washing
off unabsorbed material from the column with 30 mL of buffer A, elution
of the enzyme was achieved with a linear gradient (100 mL) from 0 to
800 mM NaCl in buffer A. Fractions (2 mL) were collected and assayed
for prenyltransferase activity. Fractions with the highest activity in the
180 to 280 mM NaCl range were combined (total of 40 mL) and desalted
on Econo-Pac10DG columns equilibrated with buffer A.
The desalted eluent (50 mL) was loaded at a flow rate of 0.5 mL minÿ1
onto a MonoQ anion exchange column (gel bed volume 8 mL) (Amersham
Pharmacia Biotech) preequilibrated with buffer A. After washing the
column with 25 mL of buffer A, protein was eluted with a linear gradient of
0 to 600 mM NaCl (100 mL, flow rate 1 mL minÿ1) in buffer A. Fractions
(1 mL) were tested for prenyltransferase activity, and product identification was performed by radio GC analysis. GPPS eluted from the column
at 170 to 190 mM NaCl. Active fractions were combined and desalted
into assay buffer as described previously and used for enzyme characterization.
Quantitative RT-PCR was performed as previously described (Dudareva
et al., 2003). One microgram of total RNA was used to produce cDNA
using random hexamer primers with the Advantage RT-for-PCR kit
according to the manufacturer’s instructions (CLONTECH, Palo Alto, CA).
Because a search of 10,000 nonredundant ESTs from a normalized A.
majus cDNA library constructed using mRNAs isolated from roots,
seedlings, young/old vegetative apex, inflorescences, young buds, and
flowers at different developmental stages and fertilized and unfertilized
carpels, available at Max Planck Institute, revealed three types of closely
related GGPPS genes, including GPPS.LSU isolated via a screening of
the petal-specific cDNA library with the M. piperita large subunit of GPPS,
gene-specific primers were generated to analyze the expression pattern
of the isolated GPPS.LSU. These primers amplified a fragment of 490 bp
in size of the coding region and were as follows: 59-AGCCAGCTCTGTTAACAAAGC-39 (forward) and 59-CTCCAAATGCTCCAATCCAAC-39 (reverse). Reactions were performed in duplicates for 30 cycles with the
annealing temperature at 528C. Analysis of products obtained with
different amounts of cDNA in PCR reactions (5, 10, 15, and 20 mL) showed
a linear increase of the reaction product. Fifteen microliters of cDNA was
chosen as optimal for further PCR reactions. To ensure that an equal
amount of RNA was used for all samples and that RT reactions were
equally effective, PCR with the 18S rRNA gene-specific primers (59GATAAAAGGTCGACACGGGCTCTGC-39, forward, and 59-AACGGAATTAACCAGACAAATCGCTCC-39, reverse) was performed. For rDNA, PCR
reactions were performed for 15 cycles with the annealing temperature at
608C. The amplified products were run on 1.2% agarose gel, blotted onto
nitrocellulose membrane (Trans-Blot transfer medium; Bio-Rad), and
hybridized with the corresponding DNA probe, a 1.2-kb EcoRI fragment
of A. majus GGPPS or 1-kb BamHI-EcoRI fragment of rDNA. Quantitation
was performed using a Storm 860 PhosphorImager and ImageQuant
software (Molecular Dynamics).
Kinetic Properties
For kinetic analysis, an appropriate enzyme concentration was chosen so
that the reaction velocity was proportional to the enzyme concentration
and was linear with respect to incubation time for at least 30 min. Protein
concentrations were determined by the Bradford method (Bradford,
1976) using the Bio-Rad protein reagent and BSA as a standard. For the
Immunoblots
Crude extracts were prepared from the upper and lower petal lobes of
A. majus flowers and Petunia hybrida (petunia) petals as described
previously (Dudareva et al., 2000). Immunodetection was performed
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The Plant Cell
using rabbit anti-GPPS.SSU polyclonal antibodies (1:2500 dilution), with
goat anti-rabbit IgG horseradish peroxidase conjugate (1:30,000 dilution)
as a secondary antibody. Antigen bands were visualized using chemiluminescence reagents (DuPont–New England Nuclear Life Science
Products, Boston, MA) for protein gel blots according to the manufacturer’s protocols and exposed on Eastman Kodak X-OMAT AR film
(Rochester, NY).
(N.D.). We thank Glenn Turner and Rodney Croteau for antibodies
against the M. piperita GPPS.LSU, Zsuzsanna Schwarz-Sommer for A.
majus genomic library, Josh Blakeslee for assistance in screening of
cDNA library, and Bettina Raguschke for technical assistance. This
paper is contribution 17251 from Purdue University Agricultural
Experimental Station.
Received December 17, 2003; accepted February 5, 2004.
Immunogold Labeling
Sections from lower petal lobes of 1- and 7-d-old A. majus flowers were
prepared for immunogold labeling as described previously (Kolosova
et al., 2001b). In brief, the tissue was fixed in 4% paraformaldehyde and
0.5% glutaraldehyde, dehydrated in ethanol, and embedded in LR White
resin (Electron Microscopy Science, Fort Washington, PA). Ultrathin
section (80 to 100 nm) were cut and mounted on formvar and carboncoated 100-mesh nickel grids. Sections on grids were incubated for 20
min at room temperature with Tris-buffered saline (TBS)-TB (20 mM TrisHCl, pH 7.4, with 150 mM NaCl containing 0.3% [v/v] Tween 20 and 1%
[w/v] BSA) blocking solution, followed directly by incubation for >16 h at
48C with preimmune serum or rabbit polyclonal antibodies raised against
the small subunit of GPPS from A. majus diluted 1:500 with TBS-B
solution. After washing with TBS-T buffer three times for 10 min each,
sections on grids were incubated for 1 h at room temperature with goat
anti-rabbit IgG conjugated to 10-nm gold (Ted Pella, Redding, CA) diluted
1:50 in TBS-TB buffer. Sections were then washed with TBS-T buffer
followed by distilled water. Lastly, they were air-dried, stained with
aqueous 2% uranyl acetate for 3 min, and observed using a Philips CM-10
Biotwin electron microscope (FEI, Hillsboro, OR). To increase visibility of
small gold particles used to visualize the GPPS.SSU protein, selected
electron micrographs were scanned into an Apple Macintosh G4
computer at a resolution of 600 dots per inch using an Epson Expression
1600 scanner (Epson American, Longbeach, CA) and digitally enhanced
by adjusting levels in Adobe Photoshop 7.0 (Mountain View, CA). Gold
particles were enhanced to facilitate viewing by overlaying a black dot of
similar size. The image files were printed on a dye sublimation/thermal
printer Codonics NP-1660 (Codonics, Middleburg Heights, OH).
Accession Numbers
The GenBank accession numbers for the sequences mentioned in the
article are as follows: A. majus GPPS.SSU, AY534686; C. breweri
GPPS.SSU, AY534745; A. majus GPPS.LSU, AY534687; M. piperita
GPPS.SSU, AF182827; M. piperita GPPS.LSU, AF182828; Arabidopsis
GGPPS1 (At4g36810), NP_195399; Capsicum annuum GGPPS, X80267;
Lupinus albus GGPPS, U15778; A. grandis GGPPS, AF425235; A. grandis
GPPS1, AF513111; T. canadensis GGPPS, AF081514; Arabidopsis
GPPS, Y17376; Citrus sinensis GPPS, AJ243739; L. albus FPPS,
U15777; Artemisia annua FPPS, U36376; Arabidopsis FPPS1,
NM_124151; C. annuum FPPS, X84695; Zea mays FPPS, L39789;
O. sativa FPPS, NM_192229.
Sequence data from this article have been deposited with the
EMBL/GenBank data libraries under accession numbers AY534686,
AY534745, and AY534687.
ACKNOWLEDGMENTS
This work is supported by National Science Foundation Grants
MCB-0212802 (N.D.) and IBN0211697 (E.P.) and by grants from Fred
Gloeckner Foundation and Deutscher Akademischer Austauschdienst
REFERENCES
Bouvier, F., Suire, C., d’Harlingue, A., Backhaus, R.A., and Camara,
B. (2000). Molecular cloning of geranyl diphosphate synthase and
compartmentation of monoterpene synthesis in plant cells. Plant J.
24, 241–252.
Bradford, M.M. (1976). A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 72, 248–254.
Burke, C.C., and Croteau, R. (2002a). Interactions with the small
subunit of geranyl diphosphate synthase modifies the chain length
specificity of geranylgeranyl diphosphate synthase to produce geranyl
diphosphate. J. Biol. Chem. 277, 3141–3149.
Burke, C.C., and Croteau, R. (2002b). Geranyl diphosphate synthase
from Abies grandis: cDNA isolation, functional expression, and
characterization. Arch. Biochem. Biophys. 405, 130–136.
Burke, C.C., Wildung, M.R., and Croteau, R. (1999). Geranyl diphosphate synthase: Cloning, expression, and characterization of this
prenyltransferase as a heterodimer. Proc. Natl. Acad. Sci. USA 96,
13062–13067.
Chen, A., Kroon, P.A., and Poulter, C.D. (1994). Isoprenyl diphosphate
synthases: Protein sequence comparisons, a phylogenetic tree, and
predictions of secondary structure. Protein Sci. 3, 600–607.
Clastre, M., Bantignies, B., Feron, G., Soler, E., and Ambid, C. (1993).
Purification and characterization of geranyl diphosphate synthase
from Vitis vinifera L. cv Muscat de Frontignan cell cultures. Plant
Physiol. 102, 205–211.
Croteau, R., and Purkett, P.T. (1989). Geranyl pyrophosphate
synthase: Characterization of the enzyme and evidence that this
chain-length specific prenyltransferase is associated with monoterpene biosynthesis in sage (Salvia officinalis). Arch. Biochem.
Biophys. 271, 524–535.
Cunillera, N., Boronat, A., and Ferrer, A. (1997). The Arabidopsis
thaliana FPS1 gene generates a novel mRNA that encodes a mitochondrial farnesyl diphosphate synthase isoform. J. Biol. Chem. 272,
15381–15388.
D’Auria, J.C., Chen, F., and Pichersky, E. (2002). Characterization of
an acyltransferase capable of synthesizing benzylbenzoate and other
volatile esters in flowers and damaged leaves of Clarkia breweri. Plant
Physiol. 130, 466–476.
Dobson, H.E.M. (1994). Floral volatiles in insect biology. In Insect-Plant
Interactions, Vol. 5, E. Bernays, ed (Boca Raton, FL: CRC Press), pp.
47–81.
Dudareva, N. (2002). Molecular control of floral fragrance. In Breeding
for Ornamentals: Classical and Molecular Approaches, A. Vainstein,
ed (Dordrecht, Germany: Kluwer Academic Publishers), pp. 295–309.
Dudareva, N., Cseke, L., Blanc, V.M., and Pichersky, E. (1996).
Evolution of floral scent in Clarkia: Novel patterns of S-linalool
synthase gene expression in the C. breweri flower. Plant Cell 8,
1137–1148.
Dudareva, N., D’Auria, J.C., Nam, K.H., Raguso, R.A., and Pichersky,
E. (1998). Acetyl CoA:benzyl alcohol acetyltransferase—An enzyme
Heterodimeric GPP Synthases
involved in floral scent production in Clarkia breweri. Plant J. 14,
297–304.
Dudareva, N., Martin, D., Kish, C.M., Kolosova, N., Gorenstein, N.,
Faldt, J., Miller, B., and Bohlman, J. (2003). (E)-b-Ocimene and
myrcene synthase genes of floral scent biosynthesis in snapdragon:
Function and expression of three terpene synthase genes of a new
TPS-subfamily. Plant Cell 15, 1227–1241.
Dudareva, N., Murfitt, L.M., Mann, C.J., Gorenstein, N., Kolosova,
N., Kish, C.M., Bonham, C., and Wood, K. (2000). Developmental
regulation of methyl benzoate biosynthesis and emission in snapdragon flowers. Plant Cell 12, 949–961.
Dudareva, N., and Pichersky, E. (2000). Biochemical and molecular
aspects of floral scents. Plant Physiol. 122, 627–634.
Francis, M.J.O., and O’Connell, M. (1969). The incorporation of
mevalonic acid into rose petal monoterpenes. Phytochemistry 8,
1705–1708.
Gershenzon, J., and Croteau, R. (1993). Terpenoid biosynthesis: The
basic pathway and formation of monoterpenes, sesquiterpenes, and
diterpenes. In Lipid Metabolism in Plants, T.C.J. Moore, ed (Boca
Raton, FL: CRC Press), pp. 339–388.
Gershenzon, J., and Kreis, W. (1999). Biochemistry of terpenoids:
Monoterpenes, sesquiterpenes, diterpenes, sterols, cardiac
glycosides and steroid saponins. In Biochemistry of Plant
Secondary Metabolism, M. Wink, ed (Boca Raton, FL: CRC Press),
pp. 222–299.
Gershenzon, J., McConkey, M.E., and Croteau, R.B. (2000).
Regulation of monoterpene accumulation in leaves of peppermint.
Plant Physiol. 122, 205–213.
Heide, L., and Berger, U. (1989). Partial purification and properties of
geranyl pyrophosphate synthase from lithospremum erythrorhizon cell
cultures. Arch. Biochem. Biophys. 273, 331–338.
Kessler, A., and Baldwin, I.T. (2001). Defensive function of
herbivore-induced plant volatile emissions in nature. Science 291,
2142–2143.
Knudsen, J.T., Tollsten, L., and Bergstrom, G. (1993). Floral
scents—A checklist of volatile compounds isolated by head-space
techniques. Phytochemistry 33, 253–280.
Kolosova, N., Gorenstein, N., Kish, C.M., and Dudareva, N. (2001a).
Regulation of circadian methyl benzoate emission in diurnally and
nocturnally emitting plants. Plant Cell 13, 2333–2347.
Kolosova, N., Sherman, D., Karlson, D., and Dudareva, N. (2001b).
Cellular and subcellular localization of S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase, the enzyme responsible for
biosynthesis of the volatile ester methyl benzoate in snapdragon
flowers. Plant Physiol. 126, 956–964.
Koyama, T., and Ogura, K. (1999). Isopentenyl diphosphate isomerase
and prenyltransferases. In Comprehensive Natural Product Chemistry: Isoprenoids Including Carotenoids and Steroids, Vol. 2, D.E.
Cane, ed (Oxford: Pergamon Press), pp. 69–96.
Langenheim, J.H. (1994). Higher plant terpenoids: A phytocentric
overview of their ecological roles. J. Chem. Ecol. 20, 1223–1280.
Marrero, P.F., Poulter, C.D., and Edwards, P.A. (1992). Effect of sitedirected mutagenesis of the highly conserved aspartate residues in
domain II of farnesyl diphosphate synthase activity. J. Biol. Chem.
267, 21873–21878.
McConkey, M.E., Gershenzon, J., and Croteau, R.B. (2000). Developmental regulation of monoterpene biosynthesis in the glandular
trichomes of peppermint. Plant Physiol. 122, 215–223.
Negre, F., Kolosova, N., Knoll, J., Kish, C.M., and Dudareva, N.
(2002). Novel S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, an enzyme responsible for biosynthesis of methyl
salicylate and methyl benzoate, is not involved in floral scent production in snapdragon flowers. Arch. Biochem. Biophys. 406, 261–270.
15 of 16
Newman, J.D., and Chappell, J. (1999). Isoprenoid biosynthesis in
plants: Carbon partitioning within the cytoplasmic pathway. Crit. Rev.
Biochem. Mol. Biol. 34, 95–106.
Ogura, K., and Koyama, T. (1998). Enzymatic aspects of isoprenoid
chain elongation. Chem. Rev. 98, 1263–1276.
Okada, K., Saito, T., Nakagawa, T., Kawamukai, M., and Kamiya, Y.
(2000). Five geranylgeranyl diphosphate synthases expressed in
different organs are localized into three subcellular compartments in
Arabidopsis. Plant Physiol. 122, 1045–1056.
Pichersky, E., and Gang, D.R. (2000). Genetics and biochemistry of
secondary metabolites in plants: An evolutionary perspective. Trends
Plant Sci. 5, 439–445.
Pichersky, E., and Gershenzon, J. (2002). The formation and function
of plant volatiles: Perfumes for pollinator attraction and defense. Curr.
Opin. Plant Biol. 5, 237–243.
Pichersky, E., Raguso, R.A., Lewinsohn, E., and Croteau, R. (1994).
Floral scent production in Clarkia (Onagraceae). I. Localization and
developmental modulation of monoterpene emission and linalool
synthase activity. Plant Physiol. 106, 1533–1540.
Poulter, C.D., and Rilling, H.C. (1981). Prenyl transferases and
isomerase. In Biosynthesis of Isoprenoid Compounds, Vol. 1, J.W.
Porter and S.L. Spurgeon, eds (New York: John Wiley & Sons), pp.
161–224.
Qureshi, N., and Porter, W. (1981). Conversion of acetyl-Coenzyme A
to isopentenyl pyrophosphate. In Biosynthesis of Isoprenoid Compounds, Vol. 1, J.W. Porter and S.L. Spurgeon, eds (New York: John
Wiley & Sons), pp. 47–94.
Raguso, R.A., and Pichersky, E. (1995). Floral volatiles from Clarkia
breweri and C. concinna (Onagraceae): Recent evolution of floral
scent and moth pollination. Plant Syst. Evol. 194, 55–67.
Rodriguez-Concepcion, M., and Boronat, A. (2002). Elucidation of the
methylerythritol phosphate pathway for isoprenoid biosynthesis in
bacteria and plastids. A metabolic milestone achieved through
genomics. Plant Physiol. 130, 1079–1089.
Rogers, S.O., and Bendich, A.J. (1985). Extraction of DNA from
milligram amounts of fresh, herbarium and mummified plant-tissues.
Plant Mol. Biol. 5, 69–76.
Sanmiya, K., Ueno, O., Matsuoka, M., and Yamamoto, N. (1999).
Localization of farnesyl diphosphate synthase in chloroplasts. Plant
Cell Physiol. 40, 348–354.
Schenk, P.M., Baumann, S., Mattes, R., and Steinbiss, H.H. (1995).
Improved high-level expression system for eukaryotic genes in
Escherichia coli using T7 RNA-polymerase and rare (ARG)tRNAs.
Biotechniques 19, 196–200.
Soler, F., Feron, G., Clastre, M., Dargent, R., Gleizes, M., and
Ambid, C. (1992). Evidence for a geranyl-diphosphate synthase
located within the plastids of Vitis vinifera L. cultivated in vitro. Planta
187, 171–175.
Somer, S., Severin, K., Camara, B., and Heide, L. (1995). Intracellular
localization of geranylpyrophosphate synthase from cell cultures of
Lithospermum erythrorhizon. Phytochemistry 38, 623–627.
Song, L., and Poulter, C.D. (1994). Yeast farnesyl-diphosphate
synthase: Site-directed mutagenesis of residues in highly conserved
prenyltransferase domains I and II. Proc. Natl. Acad. Sci. USA 91,
3044–3048.
Suga, T., and Endo, T. (1991). Geranyl diphosphate synthase in leaves
of Pelargonium roseum. Phytochemistry 30, 1757–1761.
Tarshis, L.C., Yan, M., Poulter, C.D., and Sacchettini, J.C. (1994).
Crystal structure of recombinant farnesyl diphosphate synthase at
2.6-A resolution. Biochemistry 33, 10871–10877.
Tholl, D., Croteau, R., and Gershenzon, J. (2001). Partial
purification and characterization of the short-chain prenyltransferases, geranyl diphosphate synthase and farnesyl diphosphate
16 of 16
The Plant Cell
synthase, from Abies grandis (grand fir). Arch. Biochem. Biophys. 386,
233–242.
Turner, G., Gershenzon, J., Nielson, E.E., Froehlich, J.E., and
Croteau, R. (1999). Limonene synthase, the enzyme responsible for
monoterpene biosynthesis in peppermint, is localized to leucoplasts
of oil gland secretory cells. Plant Physiol. 120, 879–886.
Wang, J., Dudareva, N., Bhakta, S., Raguso, R.A., and Pichersky, E.
(1997). Floral scent production in Clarkia breweri (Onagraceae). II.
Localization and developmental modulation of the enzyme
S-adenosyl-L-methionine:(iso)eugenol O-methyl transferase and phenylpropanoid emission. Plant Physiol. 114, 213–221.
Wang, K., and Ohnuma, S.-I. (1999). Chain-length determination
mechanism of isoprenyl diphosphate synthases and implications for
molecular evolution. Trends Biochem. Sci. 24, 445–451.
Wise, M.L., and Croteau, R. (1999). Monoterpene biosynthesis. In
Comprehensive Natural Product Chemistry: Isoprenoids Including
Carotenoids and Steroids, Vol. 2, D.E. Cane, ed (Oxford: Pergamon
Press), pp. 97–153.
Formation of Monoterpenes in Antirrhinum majus and Clarkia breweri Flowers Involves
Heterodimeric Geranyl Diphosphate Synthases
Dorothea Tholl, Christine M. Kish, Irina Orlova, Debra Sherman, Jonathan Gershenzon, Eran Pichersky
and Natalia Dudareva
Plant Cell; originally published online March 18, 2004;
DOI 10.1105/tpc.020156
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