Download Two Oxidosqualene Cyclases Responsible for

Two Oxidosqualene Cyclases Responsible
for Biosynthesis of Tomato Fruit
Cuticular Triterpenoids1[C][W][OA]
Zhonghua Wang, Ortwin Guhling, Ruonan Yao, Fengling Li, Trevor H. Yeats,
Jocelyn K.C. Rose, and Reinhard Jetter*
Department of Botany (Z.W., O.G., R.Y., F.L., R.J.) and Department of Chemistry (R.J.), University of British
Columbia, Vancouver, British Columbia, Canada V6T 1Z1; and Department of Plant Biology, Cornell
University, Ithaca, New York 14853 (T.H.Y., J.K.C.R.)
The first committed step in triterpenoid biosynthesis is the cyclization of epoxysqualene into various triterpene alcohol
isomers, a reaction catalyzed by oxidosqualene cyclases (OSCs). The different OSCs have characteristic product specificities,
which are mainly due to differences in the numbers of high-energy intermediates the enzymes can stabilize. The goal of this
investigation was to clone and characterize OSCs from tomato (Solanum lycopersicum), a species known to accumulate d-amyrin
in its fruit cuticular wax, in order to gain insights into the enzymatic formation of this particular triterpenoid. We used a
homology-based approach to isolate two tomato OSCs and tested their biochemical properties by heterologous expression in
yeast as well as overexpression in tomato. One of the enzymes was found to be a product-specific b-amyrin synthase, while the
other one was a multifunctional OSC synthesizing 48% d-amyrin and six other products. The product spectra of both OSCs
together account for both the range and the relative amounts of the triterpenoids found in the fruit cuticle. Both enzymes were
expressed exclusively in the epidermis of the tomato fruit, indicating that their major function is to form the cuticular
triterpenoids. The relative expression levels of both OSC genes, determined by quantitative reverse transcription-polymerase
chain reaction, were consistent with product profiles in fruit and leaves of the tomato cultivar MicroTom. However, the
transcript ratios were only partially consistent with the differences in amounts of product triterpenoids between the tomato
cultivars MicroTom, M82, and Ailsa Craig; thus, transcriptional control of the two OSCs alone cannot explain the fruit
triterpenoid profiles of the cultivars.
Triterpenoids are a very diverse group of natural
products with wide distribution and particularly high
chemical diversity in plants. They include compounds
such as betulinic acid, the avenacins, and glycyrrhizin,
which have important biological functions and medicinal properties (Reichardt et al., 1984; Papadopoulou
et al., 1999; Hayashi et al., 2001). The biosynthetic
pathway toward triterpenoids proceeds by joining six
1
This work was supported by Natural Sciences and Engineering
Research Council of Canada Special Research Opportunity and
Strategic Grants, the Canada Foundation for Innovation, the British
Columbia Knowledge Development Fund, and the Canada Research
Chairs Program, by the National Science Foundation Plant Genome
Research Program (grant no. DBI–0606595 to J.K.C.R.), and by the
National Institutes of Health (chemistry/biology interface training
grant no. T32 GM008500 to T.H.Y.).
* Corresponding author; e-mail [email protected].
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.plantphysiol.org) is:
Reinhard Jetter ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
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The online version of this article contains Web-only data.
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Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.110.162883
540
isoprene units together to form the branched long-chain
hydrocarbon squalene (Eschenmoser et al., 1955). In
prokaryotes, squalene is directly cyclized into hopanoid
triterpenes, whereas in eukaryotes, it is first activated
into 2,3-epoxysqualene and then cyclized (Abe, 2007).
The cyclizations are highly regiospecific and stereospecific, establishing the final carbon structure of the
triterpenoid products.
The overall cyclization reaction comprises (1) an
initial protonation step, (2) a polyene addition cascade
forming the up to five carbon cycles, (3) a series of 1,2shifts of hydride and/or methyl groups, and (4) a final
deprotonation (Fig. 1; Xu et al., 2004; Phillips et al.,
2006). The entire sequence of steps is catalyzed by
single enzymes that are designated as triterpenoid
synthases after their preferred products or as oxidosqualene cyclases (OSCs) after their common substrate
(Abe et al., 1993). The great diversity of triterpenoid
structures, with more than 100 different carbon skeletons, is due to different OSCs and, in particular, to the
numbers of rearrangement steps the different enzymes
can catalyze in the third stage of the reaction (Xu et al.,
2004). Approximately 50 OSCs have been cloned from
various plant species and have been characterized,
typically using heterologous expression in yeast.
Many of the plant OSCs were found to form predominantly one triterpenoid product, but some were also
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Biosynthesis of Tomato Fruit Triterpenoids
Figure 1. Mechanism for the cyclization of epoxysqualene into pentacyclic triterpenoids. The reaction starts with the protonation of oxidosqualene (step 1), then involves a series of carbocationic intermediates that first undergo cyclization (step 2) and
various rearrangements (step 3), before deprotonation (step 4) yields the various natural products.
reported to be multifunctional (Kushiro et al., 2000;
Morita et al., 2000; Basyuni et al., 2006). For a few plant
species, more than one OSC has been characterized, so
that the suite of OSCs accounted for at least part of the
triterpenoid profile in those species (Morita et al., 1997,
2000; Hayashi et al., 2000, 2001; Iturbe-Ormaetxe et al.,
2003; Sawai et al., 2006a, 2006b). It is generally assumed that the OSC product specificities determined
in vivo in yeast would accurately reflect the true
enzyme activities; however, in planta data to corroborate the specificities have only rarely been reported
(Han et al., 2006) and in vitro results are missing,
probably due to problems with handling the lipophilic
substrate and the membrane-associated enzymes.
A glimpse of the biochemical diversity within OSCs
can be seen in the case of Arabidopsis (Arabidopsis
thaliana), where the genome was found to contain 13
OSC genes (Fazio et al., 2004). Six of the corresponding
gene products were characterized as monofunctional
enzymes forming cycloartenol, thalianol, marneral,
arabidiol, lanosterol, or b-amyrin (Corey et al., 1993;
Fazio et al., 2004; Suzuki et al., 2006; Xiang et al., 2006;
Xiong et al., 2006; Shibuya et al., 2009). Six other
Arabidopsis OSC genes were found to encode multifunctional enzymes (Herrera et al., 1998; Kushiro et al.,
2000; Segura et al., 2000; Husselstein-Muller et al.,
2001; Ebizuka et al., 2003; Lodeiro et al., 2007; Kolesnikova
et al., 2007; Shibuya et al., 2007), while the function of
one gene remains unknown. Even though the previous studies provide substantial information about the
sequence variability and biochemical specificity
within this large gene family, the information on the
cyclization mechanism is still fairly limited. This is
mainly due to the fact that the large majority of OSCs
characterized to date form lupeol and b-amyrin, the
two triterpenoids most commonly found throughout
the plant kingdom. Only rarely have OSCs forming
other pentacyclic triterpenoid products been described. That such OSCs with diverse product profiles exist can be seen from a recent report in which
three Kalanchoe daigremontiana OSCs were shown to
synthesize taraxerol, glutinol, and friedelin (i.e. products with carbon skeletons requiring relatively
many rearrangements in the course of the OSC reaction; Wang et al., 2010). For some of the known
triterpenoid products, including d-amyrin, no OSC
activities have been described to date. However,
information on a broad range of OSCs from various
plant species, and with varying product specificities,
would help us understand how the different enzymes
can catalyze specific numbers of rearrangement steps,
stabilize the high-energy intermediates involved, and
quench the reaction by deprotonation of a particular
carbocation to form specific end products.
A number of studies have reported that d-amyrin
accumulates to relatively high concentrations in the
lipid mixture coating the surface of tomato (Solanum
lycopersicum) fruit (Bauer et al., 2004b; Vogg et al., 2004;
Hovav et al., 2007; Saladié et al., 2007; Isaacson et al.,
2009). Besides, 10 other triterpenoids have also been
identified in the fruit cuticular waxes extracted from
the surfaces of various tomato cultivars that have been
studied in much detail in recent years (Baker et al.,
1982; Smith et al., 1996; Bauer et al., 2004a, 2004b; Vogg
et al., 2004; Hovav et al., 2007; Leide et al., 2007; Saladié
et al., 2007; Mintz-Oron et al., 2008; Adato et al., 2009;
Isaacson et al., 2009; Kosma et al., 2010). In particular,
the surface wax on mature fruit of the tomato variety
MicroTom contains approximately 25% triterpenoids,
and 36% of this fraction is d-amyrin (Vogg et al., 2004).
The relative portions of the triterpenoid fraction
within the waxes decrease in the course of fruit development, while the relative amounts of very-long-chain
aliphatics derived from fatty acid metabolism increase,
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Wang et al.
most importantly unbranched and methyl-branched
alkanes (Leide et al., 2007; Mintz-Oron et al., 2008).
Thus, the chemical data indicate that triterpenoids are
formed during early fruit expansion rather than ripening, so expression of OSC genes should peak relatively early. Various studies have further shown that
fruit waxes from different tomato cultivars differ in
the relative portions of triterpenoids, especially of
b-amyrin, with ratios of 3:3:2 between d-amyrin,
b-amyrin, and a-amyrin in MicroTom and ratios of
3:2:2 in most other cultivars, including M82 and Ailsa
Craig (Vogg et al., 2004; Mintz-Oron et al., 2008;
Isaacson et al., 2009). This suggests that more than
one OSC should be involved in formation of the
different triterpenoids and that the varying product
ratios should reflect different expression levels and/or
enzyme activities in the different cultivars. However, it
has not been determined how many OSCs are involved in forming the tomato fruit cuticular triterpenoids and which of those OSCs are product-specific or
multifunctional enzymes.
Based on all the previous evidence, the goal of this
investigation was to clone and characterize multiple
OSCs from tomato fruit. In particular, one or more
enzymes forming d-amyrin were targeted. The primary focus of the investigation was on cv MicroTom;
however, other cultivars, such as M82 and Ailsa Craig,
were also included in order to compare OSC sequences
and expression patterns. With this, we sought to answer
the question of which OSCs are responsible for the
formation of the triterpenoids accumulating in the
tomato fruit cuticle and, thus, contribute to the important ecophysiological functions of the fruit epidermis.
RESULTS
The goal of this investigation was to isolate and
characterize OSCs from tomato fruit and to test their
involvement in the formation of cuticular triterpenoids accumulating in the fruit skin. To this end, a set
of four experiments was carried out centered on the
tomato cv MicroTom. First, a homology-based PCR
approach was used to clone the gene(s), initially
targeting a core segment of the sequence and then
extending it by RACE experiments at the 5# and 3#
termini. Second, the biochemical characterization of
the enzyme(s) was carried out by heterologous expression in yeast and by overexpression in tomato.
Third, the expression of OSC enzyme(s) in the epidermis and/or in inner parts of the fruit was evaluated.
Finally, quantitative reverse transcription (qRT)-PCR
Figure 2. Amino acid sequences of the two OSCs, SlTTS1 and SlTTS2, isolated from the tomato cv MicroTom. The conserved
QW motifs thought to stabilize OSC structure are highlighted by single underlines, while the highly conserved DCTAE motif
involved in substrate binding and protonation is marked by a double underline. [See online article for color version of this figure.]
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Biosynthesis of Tomato Fruit Triterpenoids
Table I. Identity levels in pairwise comparisons between the SlTTS1 and SlTTS2 cDNAs and alleles
in the tomato cultivars MicroTom, M82, and Ailsa Craig
Nucleotide identities are given as percentages in normal font, and percentages of amino acid identity
are shown in italics.
cDNA
Cultivar
TTS1
MicroTom
M82
TTS2
Ailsa Craig
MicroTom
M82
Ailsa Craig
89.2
89.1
89.1
–
99.4
99.4
89.2
89.1
89.1
99.8
–
100
89.2
89.1
89.1
99.8
100
–
%
TTS1
TTS2
MicroTom
M82
Ailsa Craig
MicroTom
M82
Ailsa Craig
–
99.6
99.5
87.8
87.7
87.7
99.7
–
100
87.7
87.5
87.5
was used to determine the relative expression levels of
the OSC genes, both in the leaves and fruit of MicroTom. The expression studies were extended to the
tomato cultivars M82 and Ailsa Craig and complemented by chemical analyses of the triterpenoids in
the M82 and MicroTom cuticular waxes.
Cloning and Sequence Analysis of OSCs from Tomato
In order to obtain a core sequence, PCR was performed with a set of degenerate primers designed
using conserved OSC amino acid sequences and
cDNAs isolated from the epidermis of growing fruit.
The primary PCR product corresponded to the expected size of approximately 1,000 bp. A second PCR
99.6
100
–
87.5
87.4
87.4
was used to extend the fragment, the product was
isolated and cloned into Escherichia coli, and sequencing inserts from five bacterial colonies revealed the
presence of only two different cDNAs. Both had
substantial similarity to other OSC sequences and
were subjected to 5# and 3# RACE, resulting in two
full-length cDNAs that were designated as SlTTS1 and
SlTTS2.
The open reading frames (ORFs) of SlTTS1 and
SlTTS2 are predicted to encode proteins of 761 and 763
amino acids with masses of 89.7 and 88.1 kD, respectively (Fig. 2). The two protein sequences are 88%
identical, and both contain the DCTAE motif thought
to be involved in substrate binding and the four
QW motifs characteristic of the OSC superfamily.
Figure 3. Phylogenetic analysis comparing the two new OSC cDNAs
cloned from tomato cv MicroTom
with previously known OSCs from
other plant species. The gene names
and sequences as well as the full
names of species are given in “Materials and Methods.”
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Wang et al.
Figure 4. Gene structure of the tomato
cv MicroTom OSCs in comparison
with those of other plant species. Exons
are represented by boxes and introns
as lines. Nucleotide numbers of exons
are shown above the boxes, exons with
common length between most OSC
genes are shaded in light gray, and
exons with a characteristic length in
only a few species are highlighted in
dark gray.
Furthermore, SlTTS1 and SlTTS2 are highly similar to
previously reported b-amyrin synthases and multifunctional triterpene synthases (Table I). Phylogenetic
analysis using neighbor-joining methods showed that
SlTTS1 and SlTTS2 are more closely related to each
other than to any other OSC and that they, together
with the Panax ginseng b-amyrin synthases (Kushiro
et al., 1998a, 1998b), form a subclade within a group of
OSC enzymes that were all characterized as b-amyrin
synthases from various plant species (Fig. 3).
In order to analyze the gene structure of SlTTS1 and
SlTTS2, the tomato genome sequence database (www.
solgenomics.net) was queried using the two corresponding cDNA sequences. One bacterial artificial
chromosome clone, Hba0131G17, was found to contain both OSC genes; accordingly, SlTTS1 and SlTTS2
are arranged in tandem in a 23-kb region on chromosome 12. The intron patterns and exon lengths of the
two SlTTS genes are very similar to those of other
OSCs (Fig. 4), with SlTTS2 gene organization most
closely resembling OSC3 of Lotus japonicus (Sawai
et al., 2006b). However, SlTTS2 differs from L. japonicus OSC3 in the length of the first and last exons. The
two tomato genes differ from each other in that exons
Figure 5. Mass spectra of pentacyclic triterpenoids. Compounds 1, 2, and 4, formed by yeast strains heterologously expressing
the two tomato cv MicroTom OSCs (left), have fragmentation patterns identical to those of authentic standards of d-amyrin,
b-amyrin, and a-amyrin (right). Spectra are shown for the trimethylsilyl derivatives of the triterpenoid alcohols.
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Biosynthesis of Tomato Fruit Triterpenoids
16 and 17, which are distinct in SlTTS2, are fused into
a single exon 16 in SlTTS1, which indeed distinguishes SlTTS1 from all other OSC genes studied to
date.
Biochemical Characterization of the Tomato
SlTTS Proteins
In order to biochemically characterize SlTTS1 and
SlTTS2, two sets of experiments using heterologous
expression in yeast and overexpression in tomato fruit
were carried out. For yeast expression, the full-length
cDNAs of the two putative OSCs were cloned into the
expression vector pYES2, and this construct was transformed into the yeast mutant GIL77 (gal2 hem3-6 erg7
ura3-167). This host strain lacks lanosterol synthase
and so accumulates 2,3-oxidosqualene, which can serve
as a substrate for heterologously expressed OSCs
(Morita et al., 1997). Yeast transformants harboring
one of the pYES-TTS constructs or the empty pYES
vector were grown, gene expression was induced with
Gal for 24 h, and then lipophilic compounds were
extracted with hexane. After purification by thin-layer
chromatography (TLC), the triterpenoids were identified by comparing their gas chromatography-mass
spectrometry (GC-MS) characteristics with those
of authentic standards and literature data (Figs. 5
and 6).
Yeast cells expressing SlTTS1 cDNA were found to
produce a single triterpenoid product that was identified
as b-amyrin (Fig. 7). In contrast, heterologous expression
of SlTTS2 led to the formation of a mixture of triterpenoids, comprising 48% 6 0.3% d-amyrin, 13% 6 0.1%
b-amyrin, 18% 6 0.8% a-amyrin, and 3% to 7% each of
multiflorenol, C-taraxasterol, taraxasterol, and one unidentified triterpene alcohol isomer. The yeast expression experiments were repeated three times, always with
very similar results. Overall, they indicate that SlTTS1 is
a monofunctional b-amyrin synthase, while SlTTS2 is a
multifunctional OSC enzyme catalyzing the formation of
seven different triterpenoid isomers.
In a second experiment, the potential OSC activities
of the two tomato SlTTS proteins were further assessed
by overexpression in tomato and chemical analysis of
the resulting fruit surface waxes. Both SlTTS cDNAs
were cloned into the pGWB5 vector, transformed into
tomato cv MicroTom, and expressed under transcriptional control of the 35S promoter. Two sets of 20
independent lines constitutively expressing SlTTS1 or
SlTTS2 were recovered, and the surface materials of
fruit from the F1 generation were extracted for wax
analysis. The SlTTS1 overexpressor lines were found
Figure 6. Mass spectra of pentacyclic triterpenoids. Compounds 3 and 5 to 7, formed by yeast strains heterologously expressing
the two tomato cv MicroTom OSCs (left), have fragmentation patterns identical to those of triterpenoids found in the cuticular
wax of tomato fruit (right). Wax constituents 5 to 7 were identified as multiflorenol, C-taraxasterol, and taraxasterol in
accordance with the literature, while compound 3 remained unidentified. Spectra are shown for the trimethylsilyl derivatives of
the triterpenoid alcohols.
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Wang et al.
Figure 7. GC analysis of triterpenoids
in transgenic yeast. Yeast was transformed with the vectors indicated in
the various panels, grown, and extracted. Triterpenoid alcohols were separated from other neutral lipids by TLC
and converted into trimethylsilyl ethers
prior to GC analysis. In the empty
vector control, no triterpenoids were
detected. In contrast, the yeast strains
harboring SlTTS1 and SlTTS2 constructs were found to contain the triterpenoid compound 2 and a series of
compounds, 1 to 7, respectively. All
seven compounds 1 to 7 were also
detected in the MicroTom fruit cuticular wax, together with very-longchain fatty acid derivatives a to e (a,
n-nonacosane; b, n-triacontane; c,
n-hentriacontane; d, n-dotriacontane;
e, n-tritriacontane).
to have amounts of d-amyrin and a-amyrin similar to
those on wild-type tomato fruit surfaces (Fig. 8). In
contrast, the amounts of b-amyrin were doubled from
0.7 mg cm22 in the wild type to 1.4 mg cm22 in SlTTS1
overexpressors. All the non-amyrin triterpenoids remained unchanged after SlTTS1 overexpression (data
not shown).
The transgenic lines harboring the SlTTS2 overexpression construct showed highly variable triterpenoid amounts in their fruit cuticular waxes, while the
very-long-chain fatty acid derivatives were present at
constant levels identical to those in the wild type. The
transgenic fruit could be classified into three distinct
categories with significantly different triterpenoid profiles (Student’s t test, P , 0.01). One of them, found for
four of the lines, had triterpenoid amounts very similar to wild-type levels. Three of the lines showed a
gain-of-function phenotype, characterized by significant increases of all seven fruit wax triterpenoids.
Notably, the amounts of d-, b-, and a-amyrin were all
approximately doubled while the ratios between them
were unchanged in comparison with the wild type
(Fig. 8). The remaining four lines recovered in the
course of the SlTTS2 overexpression experiment had
fruit surface waxes containing increased levels of
b-amyrin and drastically reduced levels of d-amyrin
and a-amyrin when compared with the wild type (Fig.
8). Similarly, the quantities of the non-amyrin triterpenoids were also decreased, in these cases to trace levels
that were detectable only by single ion monitoring GCMS (data not shown).
Investigation of SlTTS Expression Patterns in
Tomato Fruit
RT-PCR analyses were employed to test the tissue
specificity of OSC transcription within the green tomato fruit. To this end, two pairs of primers were
designed based on the SlTTS1 and SlTTS2 sequences
and proved to be gene specific when tested with
plasmid templates harboring either of the two OSC
cDNAs (data not shown). To determine whether the
SlTTS genes were expressed differentially between
inner parts of the tomato fruit and the epidermis layer,
total RNA was isolated from both tissues. RT-PCR
analysis using subsequently derived cDNA templates
showed that both SlTTS transcripts are expressed in
the epidermal cells but not in the inner tissues of the
fruit (Fig. 9). Parallel metabolite analyses revealed that
the major part of the triterpenoids in the tomato fruit
reside in the cuticle, with 74%, 79%, and 74% of the
total d-amyrin, b-amyrin, and a-amyrin aglycone
amounts present in the surface wax, respectively,
and the remaining 21% to 26% in the underlying
epidermal cell layer and the internal tissues of fruit.
Relative Expression Levels of SlTTS1 and SlTTS2 in Fruit
and Leaves of Different Tomato Cultivars
Finally, qRT-PCR analyses were carried out to determine the relative expression levels of SlTTS1 and
SlTTS2 within leaves and fruit. Since the ratios of
different triterpenoid products vary not only between
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Biosynthesis of Tomato Fruit Triterpenoids
Figure 8. Relative amounts of amyrin isomers in the fruit cuticular waxes
of various lines of MicroTom. The percentages of d-amyrin, b-amyrin, and
a-amyrin are given for the wild type (WT), transgenic fruit overexpressing
the OSCs [SlTTS1(+) and SlTTS2(+)], and overexpressors with cosuppressed SlTTS2 [SlTTS2(2)]. Amounts of each triterpenoid were quantified as percentages relative to the level of wild-type d-amyrin and are
given as averages of three independent lines and analyses with SD.
Asterisks denote significant differences from the wild type.
these two organs but also between fruit of different
tomato cultivars (Vogg et al., 2004; Mintz-Oron et al.,
2008; Isaacson et al., 2009), this experiment was to also
compare expression patterns of both SlTTS genes
between MicroTom and the cultivars M82 and Ailsa
Craig. The same primers originally designed for the
cloning of the full-length sequence from MicroTom
were used to amplify corresponding cDNAs from M82
and Ailsa Craig. One allele each of SlTTS1 and SlTTS2
was cloned and sequenced from both cultivars, and
the ORFs of the orthologous genes were found to be
very similar or identical between M82 and Ailsa Craig
(Supplemental Fig. S1). However, both SlTTS1 and
SlTTS2 differed between these cultivars and MicroTom, with up to eight nucleotide differences between
cultivars resulting in up to four amino acid changes
and more than 99.6% DNA and more than 99.4%
protein sequence identity (Table I). The similarities
between both genes were approximately 89% on the
nucleotide level across cultivars and 87% to 88% on the
amino acid level.
The expression levels of SlTTS1 and SlTTS2 were
found to be similar to each other in the leaves and fruit
of cv MicroTom (Fig. 10). Both genes were also expressed at similar levels to each other in leaves of M82,
whereas in the fruit of this cultivar, SlTTS2 was expressed at a much higher level than SlTTS1. Ailsa
Craig differed further, with higher expression levels of
SlTTS2 in fruit and leaves.
Terpenoid Profiles in Fruit and Leaves of Different
Tomato Cultivars
The terpenoid profiles of the cuticular wax mixtures
of leaves and fruits from the MicroTom, M82, and
Ailsa Craig cultivars were assessed, with particular
focus on the relative compositions of amyrin isomers.
The compositions of fruit waxes from the three cultivars (Vogg et al., 2004; Mintz-Oron et al., 2008; Isaacson
et al., 2009) and also the leaf wax composition of
MicroTom had been reported before (Vogg et al., 2004).
However, exact data on the leaf triterpenoid profiles of
all three cultivars were missing. Therefore, a detailed
study of the leaf wax from M82 and MicroTom was
carried out. It was found that the M82 leaf was covered
by 4.8 6 0.5 mg cm22 cuticular wax and that the wax
mixture contained 3.0% 6 1.0% triterpenoids, 64.3% 6
12.5% very-long-chain fatty acid derivatives, and
32.7% 6 13.5% unidentified compounds (Fig. 11).
n-Alkanes (C27–C33) were the dominating compound
class, at 45.6% 6 8.9%, accompanied by 16.6% 6 3.1%
branched alkanes (total carbon nos. C29–C33) and
20.5% 6 7.4% alcohols (C28 and C32). The triterpenoid
fraction was dominated by amyrin isomers, comprising 29%, 33.4%, and 14.4% d-amyrin, b-amyrin, and
a-amyrin, respectively. Other triterpenoid alcohols
were detected in the form of lupeol (23.3%) and trace
amounts of multiflorenol, C-taraxasterol, and taraxasterol. MicroTom leaf wax had a similar composition,
with a conspicuous ratio of 2:3:1 between d-amyrin,
b-amyrin, and a-amyrin, respectively.
DISCUSSION
In this study, a homology-based approach was used
to clone two tomato OSCs, SlTTS1 and SlTTS2, which
were found to encode highly similar amino acid sequences. Our principal goal was to characterize the
product spectrum of the enzymes, with the expectation that a d-amyrin synthase might be uncovered.
Furthermore, we aimed to elucidate the role of the
enzymes in the formation of the triterpenoids accumulating in the fruit cuticular wax.
A phylogenetic analysis showed that SlTTS1 and
SlTTS2 fall into a clade consisting entirely of b-amyrin
Figure 9. RT-PCR analysis of the expression patterns of the two OSC
genes in MicroTom fruit. Transcripts corresponding to the SlTTS1 and
SlTTS2 enzymes generating pentacyclic triterpenoids were found expressed only in the epidermis layers but not in the internal tissues of
the fruit.
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Figure 10. qRT-PCR analysis of the two OSC genes in fruit and leaves of
the three tomato cultivars MicroTom (MT), M82, and Ailsa Craig (AC).
The relative expression levels were determined in green immature fruit
and normalized for each mRNA sample (n = 3; error bars indicate SD).
synthases. Thus, the primary sequence of the new
OSCs is in accordance with the prediction that the
tomato enzymes catalyze the formation of amyrins.
However, based on genetic information alone, it was
not possible to predict the product specificities of the
tomato enzymes. It should also be noted that, in
contrast to the similarity of exon sequences with
amyrin synthases, the intron pattern of SlTTS2 was
identical to that of OSC3 from L. japonicus, a lupeol
synthase that had fairly dissimilar sequence (Sawai
et al., 2006b).
The product profiles of SlTTS1 and SlTTS2 were
determined by heterologous expression in yeast. It
was found that SlTTS1 forms b-amyrin as its sole
product, while SlTTS2 catalyzes the formation of seven
different triterpenoids, with d-amyrin as the major
product. The in planta overexpression of SlTTS1 and
SlTTS2 led to increased accumulation of cuticular
triterpenoids, in both cases of the same products that
had also been found in yeast. This finding is very
important, since most previous reports on biochemical
characterizations of OSCs had relied entirely on yeast
expression systems, and it had only rarely been tested
whether the heterologous environment might alter the
enzyme specificity (Han et al., 2006). The match between yeast and in planta expression results in this
study confirms the validity of the yeast expression
system, at least for the two tomato OSCs studied.
Overall, we conclude that the two enzymes are a
single-product b-amyrin synthase and a multifunctional OSC, respectively. SlTTS2, to our knowledge, is
the first enzyme reported to synthesize predominantly
d-amyrin.
Interestingly, some of the tomato lines harboring the
SlTTS2 construct exhibited a wax triterpenoid phenotype opposite to that of the SlTTS2 overexpressors,
suggesting that in these lines the native gene was
down-regulated by cosuppression. The observed increase in b-amyrin may be the result of additional
2,3-oxidosqualene substrate availability for SlTTS1
upon silencing of SlTTS2. The SlTTS2 loss-of-function
lines thus not only confirm the biochemical function of
the enzyme but also indicate that this enzyme must
play a central role in the formation of the tomato fruit
wax triterpenoids. We conclude that SlTTS2 is crucial
for the biosynthesis of six of the seven triterpenoid
isomers found in the wax and partially also contributes to the formation of the seventh component,
b-amyrin. Another part of the latter compound is
formed by the closely related enzyme SlTTS1. Both
enzyme product profiles taken together completely
match the wax triterpenoid composition; therefore,
SlTTS1 and SlTTS2 are likely the two major enzymes
forming the triterpenoids found in the tomato fruit
cuticular wax. The involvement of additional OSCs cannot be excluded at this point, but it seems unlikely that
any OSC other than SlTTS1 and SlTTS2 would contribute
substantially to the triterpenoid amounts or spectrum.
Two apparently orthologous OSCs were identified
from each of the tomato cultivars M82 and Ailsa Craig,
with a high degree of sequence identity to those from
MicroTom. The differences between the two genes
within one cultivar were much greater than the differences between the alleles in the cultivars. As differences between orthologous proteins were restricted to
changes in three or four amino acids, it is very likely
that both SlTTS1 and SlTTS2 were functional enzymes
in all three cultivars and that their biochemical functions should be very similar in MicroTom, M82, and
Ailsa Craig. We conclude that all three cultivars have
at least one b-amyrin synthase (SlTTS1) and one
multifunctional OSC producing d-amyrin and other
isomers (SlTTS2).
The relative expression levels of the SlTTS1 and
SlTTS2 genes varied between fruit of the three tomato
cultivars investigated, with transcripts of both genes
showing similar abundance in MicroTom but SlTTS2
Figure 11. Chemical composition of cuticular wax on leaves of tomato
cultivars M82 and MicroTom. The absolute amounts of all identified
compounds are given as averages of three independent parallel experiments with SD.
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Biosynthesis of Tomato Fruit Triterpenoids
Table II. Accession data for the OSCs used in the phylogenetic analyses
Accession No.
At4g15340
At4g15370
At5g48010
At5g42600
At1g78500
At3g45130
At1g78970
At1g78960
At1g78950
At1g66960
At2g07050
AB263204
AB257507
AB037203
AB181244
AB034802
AJ430607
AF478455
AB034803
AB289585
AB014057
AB009030
AB263203
AB206469
AB181245
AB116228
AB181246
AB244671
Species
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Rhizophora stylosa
Kandelia candel
Glycyrrhiza glabra
Lotus japonicus
Pisum sativum
Medicago truncatula
L. japonicus
P. sativum
Bruguiera gymnorhiza
P. ginseng
P. ginseng
R. stylosa
Euphorbia tirucalli
L. japonicus
G. glabra
L. japonicus
L. japonicus
accumulating to higher levels than SlTTS1 in M82 and
Ailsa Craig. This expression pattern is in accord with
the amyrin profiles in the corresponding fruit waxes,
where the latter two cultivars had lower levels of the
SlTTS1 product b-amyrin than MicroTom (Vogg et al.,
2004; Mintz-Oron et al., 2008; Isaacson et al., 2009).
However, it should be noted that the differences in
triterpenoid profiles between cultivars may further be
due to factors other than differential gene expression,
possibly including differences in enzyme activities
and/or additional OSCs being present.
Our qRT-PCR results showed that the SlTTS1 and
SlTTS2 genes are expressed not only in tomato fruit
but also in the leaves of MicroTom, M82, and Ailsa
Craig. Therefore, it seems likely that both genes also
contribute to the formation of leaf cuticular triterpenoids. However, it must be noted that the leaf waxes
have triterpenoid profiles differing from those in the
fruit in quantitative and in qualitative terms, as shown
by our detailed analyses of M82 leaf wax in comparison with literature data. The most prominent quantitative difference is the shift in amyrin ratios from 3:2:2
for d-amyrin, b-amyrin, and a-amyrin in the fruit to
2:2:1 in the leaf wax. This trend correlates with the
differences in expression levels of SlTTS1 and SlTTS2
between both organs of M82 and can thus, at least in
part, be explained by transcriptional control. It should
be noted that Ailsa Craig leaves showed a very conspicuous expression pattern with particularly low
Function
Arabidiol synthase PEN1
Baruol synthase BARS1/PEN2
Thalianol synthase PEN4
Marneral synthase PEN5
Multifunctional triterpene synthase
Lanosterol synthase LAS1/PEN7
Multifunctional triterpene synthase
Multifunctional triterpene synthase
b-Amyrin synthase AtBAS LUP4
Multifunctional triterpene synthase
Cycloartenol synthase CAS1
Multifunctional triterpene synthase
Multifunctional triterpene synthase
b-Amyrin synthase GgbAS1
b-Amyrin synthase OSC1
b-Amyrin synthase PSY
b-Amyrin synthase AMY1
Multifunctional triterpene synthase
Multifunctional triterpene synthase
b-Amyrin synthase BgbAS
b-Amyrin synthase PgbAS/PNY2
b-Amyrin synthase PNY
Multifunctional triterpene synthase
b-Amyrin synthase EtbAS
Lupeol synthase OSC3
Lupeol synthase GgLUS1
Cycloartenol synthase QSC5
Lanosterol synthase OSC7
PEN6
LUP1
LUP2
LUP5
RsM2
KcMS
LjAMY2
PSM
RsM1
levels of SlTTS2 transcript. Based on this finding, it
may be expected that the leaf waxes of this cultivar
should contain relatively little b-amyrin; however, the
amyrin profiles of the leaf wax of Ailsa Craig have not
been determined to date.
One outstanding qualitative difference between the
triterpenoid compositions of fruit and leaves, at least
of the cultivars MicroTom and M82, is the presence of
lupeol in the latter organ, a product that is not formed
by either SlTTS1 or SlTTS2. Therefore, one or more
other OSCs must be involved in the formation of leaf
cuticular triterpenoids. More detailed investigations
are needed to determine the contribution of SlTTS1
and SlTTS2 to the biosynthesis of tomato leaf cuticles.
Our results on the tissue-specific expression of
SlTTS1 and SlTTS2 showed that these enzymes are
localized exclusively in the epidermis of tomato fruit.
We further found that the triterpenoid products are
also restricted to the fruit skin, as they accumulate to
high concentrations in the cuticular waxes coating the
epidermis but not in the internal parts of the fruit. The
close match in localization of transcripts and metabolites makes it very likely that SlTTS1 and SlTTS2 are
dedicated entirely to making the triterpenoids destined for the cuticular wax of the fruit surface. With
this, a major biological function can be assigned to
these two OSCs, as the cuticular triterpenoids contribute significantly to the chemical composition and to
the ecophysiological properties of the fruit cuticle
Plant Physiol. Vol. 155, 2011
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Wang et al.
(Vogg et al., 2004; Isaacson et al., 2009). It should be
noted that similar biological functions had previously
been attributed to a few other OSCs, for example, a
glutinol synthase and a friedelin synthase from K.
daigremontiana (Wang et al., 2010).
The sequences of SlTTS1 and SlTTS2 are relatively
similar to each other, containing only 92 amino acid
changes. On the other hand, the two enzymes were
found to have fairly distinct product profiles, with
SlTTS1 yielding exclusively b-amyrin whereas SlTTS2
is catalyzing the formation of d-amyrin (among other
products). It has been predicted that the formation of
d-amyrin requires only one rearrangement step less
than b-amyrin in the course of the OSC-catalyzed
cyclization (Fig. 1; Xu et al., 2004; Phillips et al., 2006).
This relatively subtle difference in the mechanism of
formation of both triterpenoids must be due to small
differences in protein architecture, possibly involving
specific changes in amino acids lining the active site
cavity. The sequence differences between SlTTS1 and
SlTTS2 give the first information on the candidate
residues that may be involved in determining the
amyrin isomer specificity.
The list of candidate residues for defining d-amyrin
synthases can be somewhat narrowed down based on
comparisons between the two OSCs characterized
here in three different tomato cultivars and with
b-amyrin synthases from other species. A second observation might further help select candidate residues:
many of the amino acid changes between both enzymes are clustered together, and many involve aromatic residues. Since it is well established that such
aromatic amino acids play important roles in stabilizing the positive charge of high-energy intermediates of
the cyclization reaction, it has been speculated that the
number and exact positions of aromatic residues lining
the active site might limit the number of rearrangement steps and, therefore, determine the product
outcome of OSC-catalyzed reactions (Christianson,
2006; Phillips et al., 2006). Consequently, the changes
between SlTTS1 and SlTTS2 involving aromatic amino
acids at positions 48/49, 103, 318, 388, 399, 412, 421,
478, 496, 560, 675, 742, and 760 make these residues
especially interesting candidates for defining the OSC
specificity of d-amyrin and b-amyrin synthases. It
appears very promising to use site-directed mutagenesis experiments to test this hypothesis in order to
further our detailed understanding of the enzyme
mechanisms involved in triterpenoid biosynthesis.
MATERIALS AND METHODS
Plant Material and Surface Wax Analysis
Tomato (Solanum lycopersicum ‘MicroTom’) plants were grown in standard
soil under ambient conditions in a greenhouse at the University of British
Columbia. Fruits and leaves were harvested and immediately immersed in
CHCl3 for 1 min at room temperature to extract the cuticular waxes. For direct GC-MS analyses, the wax mixtures were derivatized using bis-N,O(trimethylsilyl)trifluoroacetamide (in pyridine at 70°C for 60 min), dried
under N2, and dissolved in CHCl3. The qualitative composition was studied
using a 6890N gas chromatograph (Agilent) equipped with a mass spectrometric detector (Agilent 5973N) and an HP-1 capillary column (Agilent; length
30 m, i.d. 320 mm, 1-mm film thickness). One microliter of each sample was
injected on column into a flow of helium gas held constant at 1.4 mL min21.
The oven temperature was programmed for 2 min at 50°C, followed by a 40°C
min21 ramp to 200°C, held at 200°C for 2 min, increased by 3°C min21 to
320°C, and held at 320°C for 30 min. Triterpenoids were identified by
comparison with authentic compounds (d-amyrin, b-amyrin, a-amyrin,
lupeol) and with literature data. The quantitative composition was studied
using a similar GC system equipped with a flame ionization detector under
the same GC conditions as above, but H2 carrier gas inlet pressure was
programmed for 2 mL min21. A known amount of n-tetracosane was added to
the solvent prior to extraction and used as an internal standard for quantifying
compound amounts. The extracted surface areas of leaves were determined
using digital images and ImageJ software.
For the analysis of triterpenoids contained in internal tissues, fruits were
surface extracted with CHCl3 to remove cuticular waxes, frozen in liquid
nitrogen, and then ground to powder. The powder was extracted with CHCl3,
and the resulting lipid mixture was fractionated by TLC (20 3 20 cm, silica gel,
0.5 mm; Merck) using CHCl3 as the mobile phase. After the plate was stained
with primuline and viewed under UV light, distinct bands were scratched off,
extracted with CHCl3, and analyzed by GC-MS and GC-flame ionization
detection (FID) for identification and quantification of the triterpenoids,
respectively. GC conditions were as described above for wax analyses.
Cloning of OSC Genes from Tomato
Green tomato fruits were harvested, immediately plunged into liquid
nitrogen, and ground thoroughly with a mortar and pestle. Total RNA was
extracted from the powder using the Trizol Reagent (Invitrogen). The RNA
was used for cDNA synthesis by SuperScript II reverse transcriptase (Invitrogen) following standard protocols. The resulting cDNA mixture served
directly as a template for the following PCRs.
Sequence alignments of previously characterized plant OSCs revealed
conserved regions, which were used to design degenerate oligonucleotide
primers for the specific amplification of the core fragments of OSCs from the
cDNA mixture. The antisense primer AMYT was derived from the EST clone
TC160438 (The Institute for Genomic Research tomato EST database) suspected
to encode part of an OSC sequence. To obtain the triterpenoid synthase
core sequence, PCR was performed with the degenerate sense primer OGA1S
(5#-TTYGGHAGYCAARMRTGGGAT-3#) combined with antisense primer
AMYT (5#-CGGTATTCAGCCAAACCCCA-3#) with recombinant Taq DNA
Polymerase (Invitrogen) under the following cycling conditions: 2 min at
94°C, 30 cycles of 20 s at 94°C, 40 s at 54°C, and 70 s at 72°C, and a 10-min final
extension at 72°C. The resulting PCR products were separated by gel electrophoresis (1% agarose) and extracted using the QIAquick Gel Extraction Kit
(Qiagen). A DNA band of 1 kb was recovered, cloned into the pGEM-T vector
(Promega), and transformed into Top10 Competent Cells. Plasmid DNA was
purified from transformed cells using the QIAprep Spin Miniprep Kit
(Qiagen) and sequenced. All further PCR products mentioned below were
subcloned and sequenced by the same procedure.
The core fragment was extended in a second PCR (conditions as above)
using the primers OGT4S (5#-CAYCAGAAYGAAGATGGW-3#) and genespecific primer OGAS1A (5#-CATCATTCATCTCACTGGC-3#) synthesized
according to the obtained core sequences. Two different core sequences were
obtained, one named SlTTS1 and found to be identical to the database
sequence TC160438, and another one named SlTTS2. For 3#-end amplification
of the SlTTS2 cDNA, first-strand synthesis was carried out for 1 h at
42°C using 5 mg of total RNA, AP primer (5#-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3#), and reverse transcriptase in 20 mL. The
product served as template in a PCR (conditions as above except annealing
temperature of 55°C) with the adapter primer AUAP (5#-GGCCACGCGTCGACTAGTAC-3#) and the specific primer LEASB1S (5#-AGGGTTGTGGTAGTCAATC-3#). The 5#-ends of both cDNAs were amplified by the 5#
RACE system of Invitrogen (version 2.0) with two nested gene-specific
antisense primers, OGAS6A (5#-AGAATCCATTTCCTTGCTCTA-3#) and
OGAS7A (5#-CACGCATTATTTACACCGCC-3#).
The corresponding full-length cDNAs were amplified using tomato cDNA
as a template and the specific N- and C-terminal primers, respectively:
LeTTS1F (5#-TTGGAGCTCAGGATGTGGAAATTGAAAATTGCTG-3#; SacI
site underlined), LeTTS1R (5#-CCCGAATTCTTAGTTGTTTTCTAATGGTAATAC-3#; EcoRI site underlined), LeTTS2F (5#-TTGGAGCTCAAGATGTGGAAGTTGAAGATTGCAA-3#; SacI site underlined), and LeTTS2R
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Biosynthesis of Tomato Fruit Triterpenoids
(5#-CCCGAATTCCTATATGTAGTTGTGTTTTAATGGT-3#; EcoRI site underlined). The PCRs were conducted with Phusion High-Fidelity DNA polymerase (New England Biolabs) in a final volume of 50 mL (1 mM of each primer and
1 mL of cDNA) under the following conditions: 30 s at 98°C, 28 cycles of 10 s at
98°C, 30 s at 55°C, and 75 s at 72°C, and 10 min at 72°C. The resulting 2.3-kb
PCR products were purified by gel electrophoresis and cloned for sequencing.
Functional Expression of OSC cDNAs in Yeast and
Product Analysis
The full-length cDNAs of putative triterpenoid synthases were double
digested with SacI and EcoRI enzymes and ligated into the yeast expression
vector pYES2 (Invitrogen) under the control of the GAL1 promoter. The
constructs were transformed into Top10 Competent Cells. Plasmid DNAs
were prepared and used to transform the mutant yeast strain GIL77 by the
lithium acetate/single-stranded carrier DNA/polyethylene glycol method
(Gietz and Woods, 2002). After Gal induction and 24 h of incubation in 0.1 M
potassium phosphate-containing Glc and hemin, cells were collected, refluxed
for 5 min in 20% KOH/50% ethanol, and extracted twice with hexane. Both
hexane solutions were combined, the solvent was removed under a gentle
stream of N2, and the residue was redissolved in 0.2 mL of CHCl3. The extracts
were either directly derivatized using bis-N,O-(trimethylsilyl)trifluoroacetamide at 70°C for 60 min and then analyzed by GC-FID/MS as described
above or further purified by TLC plate (20 3 20 cm, silica gel 60 F254, 0.25 mm;
Merck) for GC-FID/MS analysis. Plates were developed using a sandwich
technique and chloroform as the mobile phase, then stained with primuline
and viewed under UV light. The bands potentially containing cyclization
products were scratched from the plates, extracted with CHCl3, filtered, and
prepared for GC analysis.
Tomato Overexpression
SlTTS1 and SlTTS2 cDNAs were cloned into the pGWB5 vector under the
control of a 35S promoter using the Gateway cloning system (Invitrogen). The
vectors were transferred to Agrobacterium tumefaciens GV3301. Plant transformation was carried out as described by Dan et al. (2006). Transgenic plants
were grown in a greenhouse until the fruits turned to mature red, then
harvested for wax extraction and GC-FID/MS analysis as described before.
RT-PCR Analysis
The epidermal cell layers were peeled from the green fruit surface of
MicroTom, and the remaining inner tissue as well as the epidermis preparations were immediately frozen in liquid nitrogen, ground into powder using a
mortar and pestle, and used to extract RNA with Trizol Reagent (Invitrogen).
The RNA samples were used for cDNA synthesis by SuperScript II reverse
transcriptase (Invitrogen) following standard protocols. Gene-specific primers
(SlTTS1, 5#-CGTCGAATGCACTGCCTCAT-3# and 5#-GGACCAAATTGCACTCTATATC-3#; SlTTS2, 5#-TGTTGAGTGCACTAGCTCGG-3# and 5#-ACGGACAACTCGATTCACTAAGC-3#) were designed to amplify fragments of
the two OSCs. Additionally, the actin gene fragment was amplified as a
positive control using the primers actinF (5#-CAAGTCATCATCCGTTTG-3#)
and actinR (5#-ATACCAGTGGTACGACC-3#). PCR cycle numbers and template amounts were optimized to yield products in the linear range of the
reaction. PCR conditions were as follows: denaturing at 94°C for 2 min,
followed by 28 cycles of 94°C for 20 s, 55°C for 30 s, and 72°C for 60 s.
Reactions were maintained at 72°C for 2 min before separation of PCR
products by electrophoresis on a 1% agarose gel.
qRT-PCR Analysis
The RNA samples from tomato leaf or green fruits were used for cDNA
synthesis by SuperScript II reverse transcriptase (Invitrogen) following standard protocols. Gene-specific primers were designed to amplify a fragment of
SlTTS1 by TTS1F (5#-CGTCGAATGCACTGCCTCAT-3) and T1R2 (5#-TACCATGAACCATCAGGCATT-3#) and a fragment of SlTTS2 by TTS2F
(5#-TGTTGAGTGCACTAGCTCGG-3#) and T2R2 (5#-TACCATGAACCGTCAGGCTCC-3#). Quantitative PCR was carried out using the SYBR GreenER
qPCR SuperMix Universal Kit (Invitrogen). The qPCRs were programmed
at 95°C for 9 min, followed by 40 cycles of 94°C for 30 s, 55°C for 30 s, and
72°C for 30 s on a MJ Mini Opticon real-time PCR system.
Phylogenetic Analyses
Sequence alignments and phylogenetic analyses based on a neighborjoining method were carried out with the ClustalX program version 1.83
(Thompson et al., 1997) using the amino acid sequences of cloned and
characterized plant OSCs. A phylogenetic tree was created with the MEGA3.1
program. The number of bootstrap replications was 1,000.
Sequence information for the putative triterpenoid synthases SlTTS1 and
SlTTS2 has been deposited in GenBank with accession numbers HQ266579
and HQ266580, respectively. The GenBank accession numbers of the sequences used in the analysis are summarized in Table II.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Amino acid sequences of the SlTTS1 and SlTTS2
alleles of tomato cultivars MicroTom and M82.
ACKNOWLEDGMENTS
We thank Bangjun Wang for technical help and Dr. Y. Ebizuka for
providing yeast strain GIL77.
Received July 15, 2010; accepted October 18, 2010; published November 8,
2010.
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