Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate.
ABSTRACT We identified a cis-prenyltransferase gene, neryl diphosphate synthase 1 (NDPS1), that is expressed in cultivated tomato (Solanum lycopersicum) cultivar M82 type VI glandular trichomes and encodes an enzyme that catalyzes the formation of neryl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. mRNA for a terpene synthase gene, phellandrene synthase 1 (PHS1), was also identified in these glands. It encodes an enzyme that uses neryl diphosphate to produce beta-phellandrene as the major product as well as a variety of other monoterpenes. The profile of monoterpenes produced by PHS1 is identical with the monoterpenes found in type VI glands. PHS1 and NDPS1 map to chromosome 8, and the presence of a segment of chromosome 8 derived from Solanum pennellii LA0716 causes conversion from the M82 gland monoterpene pattern to that characteristic of LA0716 plants. The data indicate that, contrary to the textbook view of geranyl diphosphate as the "universal" substrate of monoterpene synthases, in tomato glands neryl diphosphate serves as a precursor for the synthesis of monoterpenes.
- SourceAvailable from: sciencedirect.com[show abstract] [hide abstract]
ABSTRACT: The enzyme dehydrodolichyl diphosphate (dedol-PP) synthase is a cis-prenyltransferase that catalyzes the synthesis of dedol-PP, the long-chain polyprenyl diphosphate used as a precursor for the synthesis of dolichyl phosphate. Here we report the cloning and characterization of a cDNA from Arabidopsis thaliana encoding dedol-PP synthase. The identity of the cloned enzyme was confirmed by functional complementation of a yeast mutant strain defective in dedol-PP synthase activity together with the detection of high levels of dedol-PP synthase activity in the transformed yeast mutant. The A. thaliana dedol-PP synthase mRNA was detected at high levels in roots but was hardly detected in flowers, leaves, stems and in A. thaliana suspension-cultured cells.FEBS Letters 08/2000; 477(3):170-4. · 3.58 Impact Factor
- Archives of Biochemistry and Biophysics 11/1976; 176(2):734-46. · 3.37 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Artemisia annua is an annual herb used in traditional Chinese medicine. A cDNA library was constructed from leaves of A. annua seedlings and target sequences were amplified by PCR using degenerate primers derived from a consensus sequence of angiosperm terpene synthases. Two clones, QH1 and QH5, with high sequence similarity to plant monoterpene synthases were ultimately obtained and expressed in Escherichia coli. These cDNAs encode peptides of 567 aa (65.7 kDa) and 583 aa (67.4 kDa), respectively, and display 88% identity with each other and 42% identity with Mentha spicata limonene synthase. The two recombinant enzymes yielded no detectable activity with isopentenyl diphosphate, dimethylallyl diphosphate, chrysanthemyl diphosphate, farnesyl diphosphate, (+)-copalyl diphosphate, or geranylgeranyl diphosphate, but were active with geranyl diphosphate in yielding (3R)-linalool as the sole product in the presence of divalent metal cation cofactors. QH1-linalool synthase displays a K(m) value of 64 microM for geranyl diphosphate, which is considerably higher than other known monoterpene synthases, and a K(m) value of 4.6 mM for Mg(+2). Transcripts of QH1 and QH5 could be detected by RT-PCR in the leaves and inflorescence of A. annua, but not in the stem stele or roots; transcripts of QH5 could also be detected in stem epidermis. Linalool could not be detected by GC-MS in the essential oil of A. annua, nor in acid or base hydrolysates of aqueous extracts of leaves. RT-PCR demonstrated a wound-inducible increase in QH1 and QH5 transcript abundance in both leaves and stems over a 3-day time course.Archives of Biochemistry and Biophysics 01/2000; 372(1):143-9. · 3.37 Impact Factor
Monoterpenes in the glandular trichomes of tomato
are synthesized from a neryl diphosphate precursor
rather than geranyl diphosphate
Anthony L. Schilmillera,1, Ines Schauvinholdb,1, Matthew Larsonc, Richard Xub, Amanda L. Charbonneaua,
Adam Schmidtb, Curtis Wilkersona,d, Robert L. Lasta,d, and Eran Picherskyb,2
aDepartments of Biochemistry and Molecular Biology anddPlant Biology, andcBioinformatics Core, Research Technology Support Facility,
Michigan State University, East Lansing, MI 48824-1319; andbDepartment of Molecular, Cellular, and Developmental Biology,
University of Michigan, Ann Arbor, MI 48109-1048
Communicated by Anthony R. Cashmore, University of Pennsylvania, Philadelphia, PA, April 20, 2009 (received for review December 19, 2008)
We identified a cis-prenyltransferase gene, neryl diphosphate
synthase 1 (NDPS1), that is expressed in cultivated tomato (Sola-
num lycopersicum) cultivar M82 type VI glandular trichomes and
encodes an enzyme that catalyzes the formation of neryl diphos-
phate from isopentenyl diphosphate and dimethylallyl diphos-
phate. mRNA for a terpene synthase gene, phellandrene synthase
1 (PHS1), was also identified in these glands. It encodes an enzyme
that uses neryl diphosphate to produce ?-phellandrene as the
major product as well as a variety of other monoterpenes. The
profile of monoterpenes produced by PHS1 is identical with
the monoterpenes found in type VI glands. PHS1 and NDPS1 map
to chromosome 8, and the presence of a segment of chromosome
8 derived from Solanum pennellii LA0716 causes conversion from
the M82 gland monoterpene pattern to that characteristic of
LA0716 plants. The data indicate that, contrary to the textbook
view of geranyl diphosphate as the ‘‘universal’’ substrate of
monoterpene synthases, in tomato glands neryl diphosphate
serves as a precursor for the synthesis of monoterpenes.
plant biochemistry ? terpene synthases ? cis-prenyltransferases ?
biochemical diversity ? specialized metabolism
of structures known and a plethora of functions. The vast
majority of the terpenoids are synthesized from the C5 building
blocks isopentenyl diphosphate (IPP) and dimethyl allyl diphos-
phate (DMAPP). IPP and DMAPP are condensed, with the loss
of one diphosphate group, to form larger prenyl diphosphate
intermediates. These prenyl diphosphates serve as substrates for
terpene synthases (TPSs), the enzymes that elaborate the back-
bone of the final terpenoid products (1).
Mono (C10), sesqui (C15), and di (C20) terpenes are very
common in plants and serve various physiological and ecological
roles. In particular, those containing no oxygen functionalities or
only hydroxyl or carbonyl functionalities are generally volatile
and are often emitted from plants to attract pollinators or repel
herbivores. These also contribute to the aroma and flavor of
foods consumed by humans. Consequently, the biosynthesis of
these compounds has been extensively investigated in plants.
Early studies demonstrated that partially purified monoterpene
synthases preferentially used the prenyl diphosphate precursor
geranyl diphosphate (GPP), in which the two isoprene units are
joined in the trans (E) configuration, for the production of the
observed monoterpenes (2, 3). Taken together with labeling
studies showing that conversion of GPP to neryl diphosphate
(NPP), the cis-isomer of GPP, was not necessary before cycliza-
tion (4) and several studies showing use of GPP by recombinant
monoterpene synthases (5, 6), it has become widely accepted
Likewise, sesquiterpene synthases have been found to use
the C15-diphosphate intermediate E,E-farnesyl diphosphate
erpenoids constitute a very large class of compounds syn-
(FPP), and diterpene synthases use the C20-diphosphate inter-
mediate E,E,E-geranylgeranyl diphosphate (GGPP) (7). The
enzymes that synthesize these intermediates are designated as
trans-prenyltransferases, and they consist of a family of struc-
of life (8).
In contrast, the isoprene units of some long-chain plant
terpenoids such as rubber and dolichols (the latter group of
compounds is present in bacteria, fungi, and animals as well) are
linked to each other in the cis (Z) conformation (9, 10). They are
synthesized from Z-prenyl diphosphates, which are in turn
synthesized by cis-prenyltransferases directly from IPP and
DMAPP (although in the case of rubber, the starting substrate
may be E,E-FPP and IPP) (11). The cis-prenyltransferases are
also structurally related to each other but they are not homol-
ogous to the trans-prenyltransferases and appear to use a dif-
ferent mechanism (12). Several cis-prenyltransferases have been
characterized in bacteria and yeast (13–15). Bioinformatic anal-
ysis of plant sequences identified several proteins with sequence
similarity to these cis-prenyltransferases (10, 11). The protein
encoded by one Arabidopsis thaliana putative cis-prenyltrans-
ferase gene catalyzes the formation of C90 to C130 cis-prenyl
diphosphates in vitro (10). It was hypothesized that this enzyme
is involved in the biosynthesis of dolichols in vivo, but no direct
in planta evidence was presented. The A. thaliana genome
contains a total of nine genes with sequence similarity to
cis-prenyltransferases, but clear functional assignment for any of
them is lacking.
The glandular trichomes found on the surface of the leaves
and stems of the cultivated tomato (Solanum lycopersicum) were
previously shown to contain a mixture of volatile terpenoids
consisting mostly of monoterpenes and sesquiterpenes (16). To
date, however, only two monoterpene synthases, designated
MTS1 and MTS2, have been characterized in tomato (17). The
expression of MTS1 in trichomes was shown to be low under
normal conditions but highly induced by the application of
methyl jasmonate (12). MTS1 protein was shown to catalyze the
formation of linalool from GPP in vitro (17), but consistent with
its low level of expression under normal conditions, no linalool
Author contributions: A.L.S., I.S., C.W., R.L.L., and E.P. designed research; A.L.S., I.S., R.X.,
A.L.S., I.S., R.L.L., and E.P. wrote the paper.
The authors declare no conflict of interest.
database (accession nos. SlNDPS1, FJ797956, SlPHS1, and FJ797957).
See Commentary on page 10402.
1A.L.S. and I.S. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
June 30, 2009 ?
vol. 106 ?
no. 26 ?
is observed in tomato trichomes under such conditions (16).
?-myrcene, and sabinene from GPP (17).
It was recently reported that the major sesquiterpenes pro-
duced in Solanum habrochaites trichomes are synthesized from
IPP and DMAPP via a cis-prenyltransferase that makes Z,Z-
FPP. This, in turn, is a substrate for a novel sesquiterpene
synthase with highest similarity to diterpene synthases in the
TPSe subfamily (18). Here, we show that tomato trichomes
express mRNA for a gene encoding a cis-prenyltransferase,
which catalyzes the formation of NPP. NPP in turn is a substrate
for a monoterpene synthase, whose gene is highly expressed in
the trichome, to make several monoterpenes including 2-carene,
?-phellandrene, ?-terpinene, ?-phellandrene, limonene, and
?-terpinene. Contrary to the prevailing view of monoterpene
biosynthesis, our results indicate that GPP is not the only
substrate used by terpene synthases to produce monoterpenes.
Tomato Type VI Glandular Trichomes Make a Number of Monoter-
penes, and Gene(s) on Chromosome 8 Are Involved. Previous reports
indicated that trichomes on the surface of tomato leaves, stems,
and green fruit produce monoterpenes and sesquiterpenes (16,
19–21). We analyzed terpene content by using gas chromatog-
raphy–mass spectrometry (GC-MS) in leaf trichomes by briefly
dipping detached leaflets in tert-butyl methyl ether (MTBE). S.
lycopersicum cultivar M82 produces a mixture of several mono-
terpenes, with ?-phellandrene being the main volatile (Fig. 1A,
of monoterpenes, with ?-phellandrene as the main volatile (Fig.
1A, Table S1). To determine the genetic basis for differences in
monoterpene biosynthesis between M82 and LA0716, introgres-
sion lines (ILs) were screened for changes in terpene accumu-
S. pennellii DNA at the top of chromosome 8 (IL8-1-1) accu-
mulated the same monoterpenes as LA0716 (Fig. 1A), whereas
ILs from elsewhere in the genome consistently displayed the
M82 monoterpene profile (e.g., IL1-4) (Fig. 1A).
IL8-1-1 as well as from other types of glandular trichomes (type
I in M82, type IV in LA0716) was also profiled by using pulled
Pasteur pipettes to selectively collect individual glands into
MTBE followed by GC-MS analysis. Terpenes were observed
only in type VI glands (Fig. 1B).
Type VI Glandular Trichomes of M82 also Make a Number of Sesquit-
erpenes. GC-MS analysis of the terpenes found on the surface of
the leaf and in isolated glands also showed small amounts of
sesquiterpenes. ?-Caryophyllene, ?-humulene, and ?-elemene
were identified in extracts from M82, IL8-1-1, and IL1-4 but not
in LA0716 (Table S1). Based on previous work, ?-elemene is
probably a decay product of germacrene C produced under
elevated temperature of the GC inlet (23). Previous reports
indicated that two sesquiterpene synthases genes on chromo-
some 6, designated SSTLE1 and SSTLE2, are responsible for the
synthesis of sesquiterpenes in type VI glands (20, 21).
Identification of Putative Prenyltransferases and Monoterpene Syn-
thases in Tomato Trichome Expressed Sequence Tag (EST) Databases.
To examine the profile of genes expressed in tomato trichomes,
a cDNA library was made from isolated stem and petiole
trichomes and sequenced by massively parallel pyrosequencing
(24). Reads from the GS20 sequencer were assembled into
contigs that were searched against various databases at the
National Center for Biotechnology Information, by using the
BLASTX algorithm. All contig sequence data and BLAST
results are stored in a searchable database (http://bioinfo.bch.m-
su.edu/M82/). Using this database, a search was conducted to
find candidate prenyltransferase and monoterpene synthase
genes. No genes with significant similarity to GPP synthase
(GDPS) subunits were found; therefore, the search was ex-
panded to look for any prenyltransferase sequences. Nineteen
sequences (0.0078% of library reads) were found that represent
a putative tomato FPP synthase (FPS1; AF048747). Expression
of FPS1 in trichomes is consistent with the presence of sesquit-
sequences with similarity to a GGPP synthase (GGDPS) were
A large collection of ESTs (1632 reads; 0.67% of the library)
were found in a contig encoding a protein that we designated as
NDPS1 (for neryl diphosphate synthase 1) (see below) (Figs. S1
and S2). The inferred protein is highly similar (95.4% amino acid
sequence identity) to the recently reported Z,Z-FPP synthase
from S. habrochaites, as well as to several verified and putative
dehydrodolichyl diphosphate synthases (DDPSs) from A. thali-
ana (Fig. S1). To date, two DDPS genes have been investigated
in A. thaliana. At2g23410 was shown to complement a Saccha-
romyces cereviseae (baker’s yeast) rer2 DDPS structural gene
mutation, indicating that this A. thaliana protein could catalyze
the production of dolichols in yeast (10). At1g11755/lew1 was
identified in a mutant screen for plants with a leaf-wilting
phenotype under normal growth conditions (25). The lew1
mutation resulted in reduced levels of dolichols in A. thaliana.
NDPS1 shows 41.7% and 16.0% amino acid identity with
At2g23410 and At1g11755, respectively, and 32.4% and 40.7%
identity to DDPS proteins from yeast and Escherichia coli
A BLAST search of the database identified several contigs
that encode proteins with similarity to known TPSs. Fifty-one
sequences (0.021% of total reads) matched to MTS1 (GenBank
accession no. AY840091). Two other contigs with a total of 719
sequences (0.29% of total reads) were identical with the tomato
sesquiterpene synthase SSTLE1/2 (GenBank accession no.
AF279453) from chromosome 6 (20). Finally, a second abundant
transcript (741 reads; 0.30%) represented a gene that we des-
ignated as PHS1 (for phellandrene synthase 1) (see below) (Fig.
(LA0716) plants and two isogenic chromosomal substitution lines and ana-
lyzed by GC-MS. (A) A leaf was dipped briefly in MTBE and the extract
analyzed. (B) Type VI trichomes were collected by hand and placed into a
vial containing MTBE. Numbered peaks are as follows: 1, ?-2-carene; 2, ?-
phellandrene; 3, ?-terpinene; 4, limonene; 5, ?-phellandrene; 6, ?-terpinene.
No linalool was detected in any of the samples. Chromatograms show the
detection of m/z ? 93. Rt, retention time.
Monoterpenes obtained from S. lycopersicum (M82) and S. pennellii
www.pnas.org?cgi?doi?10.1073?pnas.0904113106Schilmiller et al.
to the S. habrochaites sesquiterpene synthase that uses Z,Z-FPP
as a substrate (88.9%), to an uncharacterized tobacco terpene
synthase (GenBank accession no. AY528645; 60.7%), and to
ent-kaurene synthases from various plants (Figs. S4 and S5). All
these sequences belong to the TPSe subfamily of terpene
synthases (Fig. S5). Compared with most monoterpene syn-
thases, which range from 600 to 650 aa in length, PHS1 and
other related TPSs are significantly larger, with PHS1 having
778 aa. This is due to an insertion of ?100 aa that is related to
the ancestral internal element typically found in diterpene
Both NDPS1 and PHS1 Map to Chromosome 8.Todeterminewhether
NDPS1 and PHS1 are involved in specifying the monoterpene
profiles seen in M82 and LA0716, we determined the map
position of each gene. Surprisingly, the nucleotide sequences of
the coding region of the NDPS1 gene in M82 and LA0716
(amplified by PCR) were identical, precluding the use of poly-
morphisms within the gene for mapping. As an alternative,
restriction fragment length polymorphism analysis was per-
formed by using a probe derived from NDPS1 with digested
DNA from M82, LA0716, as well as introgression lines IL1-4 and
IL8-1-1. The restriction fragment pattern was consistent with
NDPS1 being located on the top of chromosome 8 within the
IL8-1-1 introgression (Fig. S6). For mapping PHS1, the 3? end of
the gene was amplified and sequenced. The IL8-1-1 sequence
was identical with that of LA0716, but different from the
sequenced amplified from M82 and other ILs (Fig. S7), indicating
that this TPS gene is also located on the top of chromosome 8.
NDPS1 and PHS1 Are Highly Expressed in S. lycopersicum Trichomes.
Only 9 ESTs (0.003%) for NDPS1 and 13 ESTs (0.005%) for
PHS1 were found out of the 249,138 ESTs in the TIGR tomato
transcript assembly (release 5; http://plantta.jcvi.org/cgi-bin/
plantta?release.pl), which does not include any trichome-specific
cDNA libraries. The abundance of NDPS1 and PHS1 ESTs from
our S. lycopersicum trichome database, combined with the very
low frequency of these ESTs in databases of other tissues,
suggests that NDPS1 and PHS1 expression is specific to
trichomes. Quantitative RT-PCR measurements indicated that
respectively, in isolated M82 stem trichomes than in M82 stems
from which trichomes have been removed (Fig. 2). In addition,
although NDPS1 and PHS1 transcripts were present in LA0716
stem trichomes, the expression levels were 324- and 250-fold
higher in M82 stem trichomes compared with LA0716 for
NDPS1 and PHS1, respectively.
NDPS1 Catalyzes the Formation of NPP from IPP and DMAPP. The
predicted NDPS1 ORF encodes a protein of 303 aa. Comparison
with the S. cereviseae and E. coli homologs (Fig. S2) show that
tomato NDPS1, as well as some A. thaliana homologs (e.g.,
At2g23410, but not At1g11755) have an N-terminal extension
sequence of ?50 aa likely to serve as a transit peptide directing
the proteins into the plastids, after which the transit peptide
would be removed. Attempts to express the full-length ORF in
E. coli resulted in insoluble protein. We therefore constructed an
ORF of NDPS1 that began at Ser-45 (with an initiating Met
codon in front) and a His-tag extension at the C terminus and
expressed this protein in E. coli. The affinity-purified protein
(Fig. S8) was assayed for activity with IPP and DMAPP. The
product of the reaction was isolated, hydrolyzed with alkaline
phosphatase or with HCl, and the resulting prenyl alcohols
(prenols) were analyzed by GC-MS. After alkaline hydrolysis,
only nerol was detected (Fig. 3 A and B), indicating that the
product of the condensation of IPP and DMAPP catalyzed by
NDPS1 was neryl diphosphate. As a control, commercially
obtained GPP was hydrolyzed in similar conditions, and the
product obtained was geraniol (Fig. 3 C and D). In addition,
because it was previously reported that acid hydrolysis of GPP
produces mostly linalool (26), but acid hydrolysis of neryl
diphosphate results in linalool, ?-terpineol, and nerol (27), we
hydrolyzed the product of the reaction catalyzed by NDPS1 and
observed linalool, ?-terpineol, and nerol (Fig. S9).
Kinetic analysis of the purified NDPS1 showed a Kmvalue for
IPP of 152 ?M, a Km value for DMAPP of 177 ?M, and a
turnover rate of 0.2 s?1(Table 1).
PHS1 Catalyzes the Formation of five Monoterpenes from NPP. The
complete ORF of PHS1 encodes a protein of 778 aa. It has
M82 and LA0716 trichomes. Expression of NDPS1 and PHS1 was measured by
qRT-PCR and normalized to expression of EF-1? in total trichomes isolated
from stem and petiole tissue and from stem and petiole tissue after removal
of the trichomes.
Quantitative RT-PCR analysis of NDPS1 and PHS1 gene expression in
10 1112 13
Purified NDPS1 was incubated with IPP and DMAPP for 30 min at 30 °C, after
which alkaline phosphatase was added to the reaction and the solution
incubated overnight and then analyzed. (B) Authentic nerol standard. (C)
Authentic geraniol standard. (D) Commercial GPP was treated with alkaline
phosphatase as in A. GC-MS chromatograms show the detection of m/z ? 93.
In vitro assay for identification of NDPS1 product using GC-MS. (A)
Schilmiller et al.PNAS ?
June 30, 2009 ?
vol. 106 ?
no. 26 ?
significant similarity to several plant proteins including plastid-
localized (28) kaurene synthase (Figs. S4 and S5). The compar-
ison with kaurene synthase strongly suggests that the complete
ORF of PHS1 also encodes a plastid transit peptide. We
included a His-tag extension at the N terminus, and expressed
this protein in E. coli.
The affinity-purified protein (Fig. S8) was assayed with NPP
and the reaction products analyzed by GC-MS. The profile of the
the profile of the monoterpenes extracted from the surface of
the leaves and specifically from type VI trichomes, with ?-
phellandrene as the main product (Figs. 1 and 4 A and B). The
Kmvalue of PHS1 for NPP was determined to be 9.1 ?M, with
a turnover rate of 4.1 s?1(Table 1). A coupled reaction in which
NDPS1, PHS1, and DMAPP and IPP were incubated together
also gave the same pattern (Fig. 4C). In contrast, when incubated
with GPP, PHS1 catalyzed the formation of the acyclic myrcene
and ocimene as major products in addition to ?-phellandrene
(Fig. 4D), but the Kmvalue with this substrate was considerably
higher and the turnover rate extremely low (Table 1). As a
control, MTS1 gave only linalool when incubated with GPP,
confirming a previous report (17), and did not catalyze the forma-
tion of any monoterpenes when incubated with NPP (Fig. S10).
We show here that the trichomes of S. lycopersicum express a
gene, NDPS1, which encodes an enzyme catalyzing NPP forma-
tion (Fig. 3). NDPS1 produces a prenyl diphosphate with a cis
configuration, rather than a trans configuration as does GDPS
(Fig. 5), possibly because it extracts a different proton from
carbon 4 than GDPS does (12, 29). We also show that NPP can
be used by the monoterpene synthase PHS1 in vitro to catalyze
the formation of the same monoterpenes also found, and in
similar ratios, in the glands of S. lycopersicum (Fig. 4 A–C),
suggesting that these compounds are produced in the glands by
the action of PHS1 on NPP. Although PHS1 can also use GPP
as a substrate in vitro, there is apparently little GPP in the
trichomes, PHS1 has a very poor affinity to this substrate (Table
1), and the mixture and ratio of the products resulting from the
action of PHS1 on GPP are very different from those seen in
planta (Fig. 4 A and D). In particular, the major product of PHS1
whereas the major portion of the products with GPP are acyclic
(i.e., myrcene, ocimene, and linalool).
The differences in products formed by PHS1 with NPP and
GPP can be explained by the reaction mechanism of terpene
synthases (30). As illustrated in Fig. 6, NPP most likely ionizes
to a neryl cation, which can further isomerize to an ?-terpinyl
cation. Further transformations of this cyclic intermediate (30)
can eventually yield all observed products of PHS1. When GPP
is used as the substrate (albeit a very poor one) of PHS1, the
linalyl cation intermediate is formed first (Fig. 6). Although
the linalyl cation can undergo trans-cis isomerization, leading to
the neryl cation (30), it appears that substantial amounts of the
acyclic terpenes myrcene, ocimene, and some linalool, are
formed directly from the linalyl cation. Our results also suggest
that with PHS1, the neryl cation can be converted only to the
?-terpinyl cation but not to the linalyl cation, because no acyclic
monoterpenes are obtained in vitro when NPP is used as the
substrate, nor are they detected in planta.
Our results may help explain anomalous results into the
function of monoterpene synthases from various species. For
example, there are reports in which no enzymatic activity was
detected when GPP was used as a substrate. In addition, there
are cases in which monoterpene products not found in the
species under investigation were detected when the enzyme was
Table 1. Kinetic parameters of NDPS1 and PHS1
IPP152 ? 68*
177 ? 42
9.1 ? 0.8
2,900 ? 300
0.2 ? 0.07
0.2 ? 0.03
4.1 ? 0.2
1.39 ? 0.2
1.04 ? 0.06
451.5 ? 18.5
3.4 ? 10?10? 0.26 ? 10?10
GPP 9.9 ? 10?10? 0.38 ? 10?10
substrates. (A) S. lycopersicum (M82) monoterpene profile obtained by dip-
of the in vitro-coupled reaction catalyzed by NDPS1 and PHS1 and using IPP
by PHS1 with NPP as the substrate. (D) GC analysis of products of the reaction
catalyzed by PHS1 with GPP as the substrate. Labeled peaks are as follows: 7,
myrcene; 8 and 9, ocimene isomers. A small linalool peak (equivalent to the
retention time (Rt) 17.4 min (not shown here). GC-MS chromatograms show
the detection of m/z ? 93. Heights of peaks are not comparable between
In vitro assays with purified recombinant PHS1 using different
molecule during the condensation reaction (based on ref. 29).
www.pnas.org?cgi?doi?10.1073?pnas.0904113106 Schilmiller et al.
tested with GPP in vitro (31). The observation that PHS1
converts NPP to the expected monoterpene products and the
prevalence of NDPS1-like sequences in plant genomes together
suggest that some previously negative or anomalous in vitro
results with putative monoterpene synthases could be explained
if these enzymes use NPP rather than GPP as the in vivo
substrate. Indeed, a report from 1976 (27) presented evidence
that cell-free extracts of sage (Salvia officinalis) can convert NPP
to several monoterpenes, but to our knowledge no specific
enzymes that carry out this reaction have been identified.
S. pennellii LA0716 glands make a somewhat different set of
monoterpenes (Fig. 1). We found that S. pennellii and S. lycop-
ersicum NDPS are identical in sequence. We have not yet
determined the complete sequence of S. pennellii PHS1, al-
though the partial sequence obtained indicates that it encodes a
protein that is not identical with S. lycopersicum. However,
because neither NDPS1 nor PHS1 are highly expressed in glands
of S. pennellii (Fig. 2), at present we do not know which enzyme
is responsible for the production of monoterpenes in the glands
of this species, nor whether it uses NPP or GPP.
Recently, Sallaud et al. (18) reported the sequences of the
orthologs of S. lycopersicum NDPS1 and PHS1 in S. habrochaites.
Both proteins were shown to be highly expressed in the glands
and localized in the plastids. Interestingly, although NDPS1 and
its S. habrochaites ortholog are 95% identical (Fig. S2), the S.
habrochaites protein has a different activity, catalyzing the
formation of Z,Z-FPP from IPP and DMAPP. Likewise, al-
protein are 89% identical (Fig. S4), they too have divergent
enzymatic activities: the S. habrochaites protein catalyzes the
formation of the sesquiterpenes bergamotene and santalene
from Z,Z-FPP, rather than monoterpenes from NPP.
Our results, together with those of Sallaud et al. (18), under-
line the remarkable capacity of plant TPSs, which are believed
to be monophyletic (1, 7), to evolve the ability to use different
substrates and to synthesize new products. Our results also bring
to the fore the evolution of new functions that occurs among
prenyltransferases in plants, which belong to at least two distinct
families. The identification of reactions in such well-studied
specialized metabolic pathways indicates that there is still much
to be learned about the diverse biosynthetic strategies of plants.
Materials and Methods
Plant Growth and Conditions. Tomato seed, S. lycopersicum cv. M82 and S.
pennellii LA0716, was obtained from the Tomato Genetic Resource Center
(http://tgrc/ucdavis.edu). Seedlings were grown in Jiffy peat pots (Hummert
dark at 20 °C.
Terpene and Prenyl Alcohols Analyses. For analysis of total trichome terpenes,
leaflets from the second leaf after the newly emerging leaf of 3-week-old
type VI glands from 3-week-old plants (M82, IL8-1-1, IL1-4) and greenhouse
grown plants (LA0716) were picked by using a pulled Pasteur pipette into 100
?L of MTBE containing tetradecane internal standard. GC-MS analysis was
performed as described in SI Materials and Methods.
cDNA Library Construction and Sequencing. Isolation of RNA from glandular
trichomes, construction of cDNA libraries, and sequence determination and
assembly are described in SI Materials and Methods.
Gene Expression Analysis. Steady-state levels of specific transcripts in tomato
trichome and leaves were determined by the quantitative RT-PCR (qRT-PCR)
method as described in SI Materials and Methods.
of NDPS1 and PHS1 without the transit peptides were spliced into the expres-
sion vector pEXP5-CT/TOPO (Invitrogen), mobilized into E. coli, and protein
produced and purified as described in SI Materials and Methods.
NDPS Enzyme Assay. For product identification, the reaction was initiated by
adding 2.5 ?g of affinity-purified His-tagged enzyme (10 ?L) in 50 mM Hepes,
pH 8, 5% vol/vol glycerol, 5 mM DTT, 100 mM KCl, and 7.5 mM MgCl2,
incubated for 30 min at 30 °C. The reaction was stopped by heating and then
treated by either adding 2 units of calf intestinal alkaline phosphatase (alka-
(acid hydrolysis). After hydrolysis (either acid or alkaline), glass vials contain-
ing the reaction were placed at 42 °C, the gas phase was extracted with a
solid-phase microextraction (SPME) fiber (polymethylsiloxane; 100 ?m; Su-
pelco) for 15 min and was then injected in GC-MS. Blanks with assay buffer
without enzyme and substrates, but with commercial standards, were per-
formed to confirm identity of peaks.
For kinetic studies, a similar protocol was followed, but 1.7 ?g of protein
were used and [1-14C]IPP (50 ?Ci/?mol, initial concentration 0.1 mCi/mL and
mixed in appropriate amounts with cold IPP) was used as substrate together
with DMAPP. The Kmvalue for IPP was determined by using 200 ?M DMAPP,
whereas the Kmvalue for DMAPP was determined with 200 ?M IPP. Reactions
were stopped by adding 5 ?L of 2 M HCl. Prenols were extracted with 200 ?L of
ethyl acetate and the14C counts in the organic phase was analyzed in a scintil-
no nonenzymatic IPP hydrolysis. IPP and DMAPP were obtained from Echelon
Biosciences. [1-14C]IPP was obtained from American Radiolabeled Chemicals.
PHS Enzyme Assay. For product identification, 2 ?g of affinity-purified His-
tagged enzyme (10 ?L) were incubated in 50 mM Hepes, pH 8, 5% vol/vol
glycerol, 5 mM DTT, 100 mM KCl, and 7.5 mM MgCl2, containing 3.5 ?M NPP
or 10 ?M GPP in a final volume of 50 ?L. Assays were incubated for 30 min at
30 °C, after which glass vials containing the reaction were placed at 42 °C, the
gas phase was extracted with a SPME fiber for 15 min and was then injected
in GC-MS. For coupled assay with NDPS1, the assay was first performed as
was added after 30 min of incubation in the reaction mixture and was
incubated for 30 min longer at the same temperature. Monoterpenes were
Commercial standards were spiked in assay buffer without enzyme or sub-
strates, extracted with SPME, and analyzed by using GC-MS.
For kinetic studies, similar conditions were followed but using 0.5 ?g of
purified enzyme and NPP or GPP as a substrate. Products were quantified by
using SPME. A calibration curve was performed by using the same SPME fiber,
to evaluate the linearity range for 2-carene and for tetradecane (used as an
internal standard). NPP was a gift from Charles Waechter and Jeffrey Rush,
and GPP was purchased from Echelon Biosciences.
ACKNOWLEDGMENTS. We thank Drs. Charles Waechter (University of Ken-
tucky, Lexington) and Jeffrey Rush (University of Kentucky, Lexington) for
generously provided by Dr. Dani Zamir (Hebrew University, Rehovot, Israel).
This work was supported by National Science Foundation Grant DBI-0604336.
uses GPP or NPP as substrates. A minor peak of ?-pinene is occasionally seen
in the in vitro assay of PHS1 with NPP, and a correspondingly small portion of
?-pinene is also occasionally seen in the profile of monoterpenes extracted
from S. lycopersicum leaves.
Proposed mechanism for the synthesis of monoterpenes when PHS1
Schilmiller et al.PNAS ?
June 30, 2009 ?
vol. 106 ?
no. 26 ?