Running head: Cistus creticus copal-8-ol diphosphate synthase · labd-7,13-dien-15-ol,...
Transcript of Running head: Cistus creticus copal-8-ol diphosphate synthase · labd-7,13-dien-15-ol,...
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Running head: Cistus creticus copal-8-ol diphosphate synthase
Corresponding author:
Angelos K. Kanellis
Department of Pharmaceutical Sciences
Aristotle University of Thessaloniki
541 24 Thessaloniki, Greece
Tel.: +30-2310-997656, Fax: +30-2310-997662, 997645
e-mail: [email protected]
Research area: Biochemical Processes and Macromolecular Structures – Associate
Editor John Ohlrogge
Plant Physiology Preview. Published on July 1, 2010, as DOI:10.1104/pp.110.159566
Copyright 2010 by the American Society of Plant Biologists
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A copal-8-ol diphosphate synthase from the angiosperm Cistus creticus subsp.
creticus is a putative key enzyme for the formation of pharmacologically active,
oxygen-containing labdane-type diterpenes
Vasiliki Falara, Eran Pichersky and Angelos K. Kanellis*
Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, 541 24
Thessaloniki, Greece (V.F., A.K.K.); and Department of Molecular, Cellular and
Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1048,
USA (V. F., E.P.)
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1This work was supported by Greek General Secretariat for Research and Technology
grants to A.K.K.: GR-USA-033, Greek-Spanish bilateral grant and PENED 2001-
01EΔ416, research projects, implemented within the framework of the
“Reinforcement Programme of Human Research Manpower” (PENED) and co-
financed by National and Community Funds (25% from the Greek Ministry of
Development-General Secretariat of Research and Technology and 75% from E.U.-
European Social Fund).
*Corresponding author; e-mail [email protected]
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ABSTRACT
The resin of Cistus creticus subsp. creticus, a plant native to Crete, is rich in
labdane-type diterpenes with significant antimicrobial and cytotoxic activities. The
full-length cDNA of a putative diterpene synthase was isolated from a C. creticus
trichome cDNA library. The deduced amino acid sequence of this protein is highly
similar (59 - 70% identical) to type B diterpene synthases from other angiosperm
species that catalyze a protonation–initiated cyclization. The affinity-purified
recombinant E. coli expressed protein used geranylgeranyl diphosphate as substrate,
and catalyzed the formation of copal-8-ol diphosphate. This diterpene synthase was
therefore named CcCLS (for Cistus creticus copal-8-ol diphosphate synthase). Copal-
8-ol diphosphate is likely to be an intermediate in the biosynthesis of the oxygen-
containing labdane-type diterpenes that are abundant in the resin of this plant. RNA
gel blot analysis revealed that CcCLS is preferentially expressed in the trichomes,
with higher transcript levels found in glands on young leaves than on fully expanded
leaves while CcCLS transcript levels increased after mechanical wounding. Chemical
analyses revealed that labdane-type diterpene production followed a similar pattern
with higher concentrations in trichomes of young leaves and increased accumulation
upon wounding.
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INTRODUCTION
Labdane-type diterpenes comprise a large group of plant compounds with a broad
range of biological activities (Chinou, 2005). Labdane-type diterpenes possess a
characteristic skeleton with a basic bicyclic structure and an additional C-6 skeleton
that might be either open or contributes three carbons to a six-member ring that may
or may not contain an oxygen atom (Fig. 1). Most research on biosynthesis of
labdane-type diterpenes has focused on the metabolic pathway leading to ent-kaurene,
the diterpenoid hydrocarbon precursor of gibberellins, and other labdane-type
diterpenes that like ent-kaurene do not contain oxygen in their skeleton. For example,
it has been shown that ent-kaurene is synthesized in a two-step reaction involving first
the cyclization of gereanylgeranyl diphosphate (GGDP) to ent-copalyl diphosphate
(ent-CDP) which is then converted to ent-kaurene. In angiospermous plants, the
enzyme catalyzing the first reaction is copalyl diphosphate synthase (CPS), which
performs a protonation-initiated cyclization and belongs to type B cyclases that
possess a characteristic DXDD motif. The second ionization-initiated cyclization of
CDP to ent-kaurene is catalyzed by kaurene synthase (KS), a type A cyclase that
possesses the aspartate-rich DDXXD motif (Sakamoto et al., 2004). Recently, distinct
CPS and KS enzymes apparently exclusively involved with gibberellin biosynthesis
were identified in gymnosperms (Keeling et al., 2010)
Labdane type diterpenes devoid of oxygen in their skeleton are produced as
defense compounds in rice (Oryza sativa), and it has been shown that their synthesis
goes through either ent-copalyl diphosphate or syn-copalyl diphosphate intermediates,
whose synthesis is catalyzed by two respective type B cyclases (Prisic et al., 2004; Xu
et al., 2004; Otomo et al., 2004a). These phosphorylated intermediates are then further
cyclized by type A cyclases to diterpene products such as oryzalexins, momilactones
and phytocassanes (Nemoto et al., 2004; Wilderman et al., 2004; Otomo et al., 2004b;
Kanno et al., 2006). In non-angiosperm plants, bifunctional type B/A terpene
synthases producing labdane-related diterpenes without skeletal oxygen have been
identified, for example abietadiene synthase from Abies grandis (Stofer Vogel et al.,
1996), levopimaradiene synthase from Ginkgo biloba (Schepmann et al., 2001), and
levopimaradiene/abietadiene from Picea abies (Martin et al., 2004). These
bifunctional enzymes catalyze both reactions, first cyclizing GGDP to CDP, and then
converting CDP to give the final skeleton, which could be further elaborated by
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oxidation (hydroxylation) (Ro et al., 2005; Hamberger and Bohlmann, 2006; Ro and
Bohlmann, 2006).
In contrast, the biosynthesis of labdane-type diterpenes with oxygen in their basic
skeleton is not as well understood. The trichomes of Nicotiana glutinosa and N.
tabacum contain the diterpenes abienol, labdenediol and sclareol, and protein extracts
from these species supplied with GGDP was able to support their synthesis (Guo et
al., 1994; Guo and Wagner, 1995). It was hypothesized that their synthesis involved a
copal-8-ol diphosphate intermediate, but whether one or more enzymes were
involved, or indeed the identity of the responsible enzymes, was not determined.
Cistus creticus subsp. creticus, a plant indigenous to the Mediterranean region,
secretes a characteristic resin - labdanum- remarkably rich in labdane-type diterpenes
(Fig. 1). Labd-13-ene-8α, 15-diol and its derivative labd-13-ene-8α-ol-15-yl acetate,
labd-7,13-dien-15-ol, labd-14-ene-8,13-diol (sclareol) and 3β-hydroxy-13-epi-manoyl
oxide are compounds isolated from the plant resin that have been found to be
pharmacologically active, for example having cytotoxic activity against human
leukemic and breast cancer cell lines (Dimas et al., 2001; Matsingou et al., 2005;
Dimas et al., 2006; Matsingou et al., 2006). Moreover, recent studies indicated
significant antitumor activity of sclareol, a labdane-type diterpene on human colon
cancer tumors (HCT116), which often develops in mice with Severe Combined
Immunodeficiency Disease (Hatziantoniou et al., 2006).
A trichome-specific cDNA library was recently constructed from C. creticus and
EST sequencing and bioinformatic analysis revealed one cDNA with significant
similarity to type B diterpene synthases (Falara et al., 2008). Here, we describe the
isolation of the full-length cDNA (CcCLS) and functional characterization of the
recombinant E. coli-produced protein. We show that the enzyme encoded by CcCLS
catalyzes the formation of copal-8-ol diphosphate and is therefore a putative key
enzyme in the biosynthesis of oxygen-containing labdane-type diterpenes.
RESULTS
cDNA Isolation and Sequence Analysis of CcCLS
Expression of genes encoding labdane-type diterpene synthases is expected to be
high in C. creticus trichomes compared to other parts of the plant. The high similarity
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in protein sequence among previously characterized diterpene synthases prompted us
to design several degenerate primers corresponding to highly conserved sequences,
and employ them to carry out PCR with DNA from the C. creticus trichome library as
template. These experiments resulted in the synthesis of an 870 bp long DNA that
encodes a protein fragment with high similarity to known diterpene synthases (Fig. 2).
In addition, one contig representing two ESTs from the same library had previously
been shown to encode a protein fragment with sequence similarity to the C- terminus
of a putative diterpene synthase (Falara et al., 2008). PCR amplification with gene-
specific primers revealed that the above partial cDNAs corresponded to the same gene
and ultimately led to isolation of a full-length cDNA that we designated CcCLS. This
cDNA contains an open reading frame (ORF) of 804 codons. The deduced amino acid
sequence of the encoded protein had the highest similarity to Scoparia dulcis copalyl
diphosphate synthase (50% identity, BAD91286) and it also highly resembled several
CPSs from other angiosperm species (38-50% identity) (Fig. 2). Sequence analysis
with TargetP 1.1 network-based methodology predicted a putative transit peptide of
50 amino acids at the N-terminus of CcCLS indicative of plastid localization for the
mature protein.
Multiple sequence alignment analyses of CcCLS with other type A, type B and
bifunctional plant diterpene synthases revealed the presence in CcCLS of the highly
conserved DXDD motif found in all type B synthases (Fig. 2). Protein-based
phylogenetic analysis of plant diterpene synthases further underlined the significant
similarity of CcCLS to angiosperm CPSs (Fig. 3).
Heterologous Expression and Functional Characterization of CcCLS
The sequence similarities between CcCLS and other diterpene synthases and the
common aspartate-rich motif in these proteins indicated that CcCLS is likely to act as
a diterpene synthase. Since CcCLS, like other diterpene synthases, is predicted to
contain a transit peptide of 50 amino acids at its N-terminus and previous work has
shown that better expression of ditepene synthases in a bacterial system is achieved
when the transit peptide coding region is eliminated, we constructed and expressed a
truncated version of CcCLS that lacks the transit peptide-encoding sequence (Fig. 4A)
in E. coli. Protein extracts from E. coli cells expressing the truncated CcCLS in fusion
with an N-terminal His-tag were used for affinity purification of the recombinant
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CcCLS (Fig. 4B). The affinity purified enzyme was incubated with GGDP in the
presence of magnesium ions. The products were extracted with hexane and analyzed
by gas chromatography/mass spectrometry (GC-MS) (Fig 5). Mass chromatograph
monitoring of the eluting peaks and comparison with the National Institute of
Standards and Technology spectral databases, NIST21 and NIST107, revealed one
major labdane-type diterpene product (labeled as peak 3 in Fig. 5; peak 2 corresponds
to geranylgeraniol, the hydrolytic product of the remaining GGDP substrate). When
the enzyme assay was not followed by alkaline phosphatase treatment, neither peak 3
nor geranylgeraniol were detected. Peak 3 was also not obtained when the boiled
enzyme was supplied with GGDP substrate (data not shown). No products were
identified when the enzyme was supplemented with GDP or FPP. Finally, comparison
of the assay products with authentic samples of labdane–type diterpenes isolated from
C. creticus revealed that peak 3 corresponded to labd-13-en-8α, 15-diol, based on
both identical retention times (Fig. 5) and mass spectra (Fig. 6A-B).
The purified enzyme showed sigmoid kinetics, producing a sigmoid velocity (V)
plot (Fig. 7). The rate of the reaction in response to increasing substrate
concentrations can be described by the Hill equation. Therefore the sigmoid fit of the
curve allowed the determination of the Hill constant for GGDP substrate which was
calculated to be Ki=54.36 μΜ with a Hill number of n=3.57, an indication of a
positive cooperativity of the GGDP binding.
Expression of CcCLS and Diterpene Production during Development and upon
Wounding
It was previously shown that CcCLS EST was present in a trichome-specific
cDNA library (Falara et al., 2008). This prompted us to perform RNA gel-blot
analysis of C. creticus organs and tissues to explore the possibility of an organ- or
tissue-specific expression pattern of the gene. Transcripts of CcCLS were detected in
stems, leaves and tips of C. creticus plants, whereas non-trichome bearing organs such
as roots and seeds did not accumulate CcCLS messages (Fig. 8A). Conversely, CcCLS
exhibited higher levels of transcript accumulation in trichomes than in whole leaves
bearing trichomes (Fig. 8B). Moreover, a change in the level of expression was
observed at different leaf developmental stages (S1: 0.5-1cm, S2:1-2cm, S3: 2-3cm,
S4: 3-4cm), with CcCLS transcripts being more abundant in young leaves (stages S1,
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S2 and S3), and especially in stage S2, in comparison to fully expanded leaves (stage
S4) (Fig. 8B).
Chemical analysis of isolated trichomes from leaves of the same developmental
stages revealed that indeed young leaves (stage 2) displayed the highest levels of most
of the hexane-extracted labdane-type diterpenes whereas trichomes from older leaves
exhibited gradual decreased concentrations (Fig. 8C). The labdane-type diterpenes of
known structures that were identified included 8,13-epoxy-15, 16 dinorlabd-12-ene,
13-epi-manoyl oxide, 3β-hydroxy-13-epi-manoyl oxide, labd-7, 13- diene-15-ol, labd-
7, 13-diene-15-yl acetate, 3β-acetyl-13-epi-manoyl oxide, labd-13-ene-8α, 15-diol and
labd-13-ene-8α, 15-yl acetate. The most abundant compounds identified in all
developmental stages were 3β-hydroxy-13-epi-manoyl oxide and 3β-acetyl-13-epi-
manoyl oxide, while labd-7,13-diene-15-yl acetate was the most abundant compound
in young leaves of stage 2 (Fig. 8C).
To gain an insight into the expression of CcCLS in response to stresses, profiling
of transcript levels was undertaken using total RNA isolated from leaves subjected to
wounding. Wounding caused a gradual increase in CcCLS mRNA levels in C. creticus
leaves with message being elevated 1 h after the treatment and maintained at high
levels for the time period studied (Fig. 9A).
Trichomes isolated from wounded leaves as well as non-wounded control leaves
3 h after treatment were extracted with hexane. Quantitative analysis of the extracted
labdane-type diterpenes revealed higher concentrations for most of the compounds
identified in the trichomes of the wounded leaves than in the control leaves. The
increase for some compounds reached 45% (Fig. 9B). Again, 3β-hydroxy-13-epi-
manoyl oxide and 3β-acetyl-13-epi-manoyl oxide were the most abundant compounds
extracted from both control and wounded leaves (Fig. 9B).
DISCUSSION
CcCLS Encodes an Enzyme that Converts GGDP to Copal-8-ol Diphosphate
The specific set of reactions and the identity of the enzymes involved in the
synthesis of oxygen-containing labdane-type diterpenes have remained unsolved.
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Although Guo et al. (1994) and Guo and Wagner (1995) have hypothesized that
copal-8-ol diphosphate is an intermediate in this pathway, no enzyme capable of
synthesizing this compound has been reported. Here we report the characterization of
a cDNA of the gene CcCLS, which is expressed in the labdane-producing trichomes
of Cistus creticus subsp. creticus. We showed that the protein encoded by CcCLS
catalyzes the formation of stable copal-8-ol diphosphate from GGDP. Alkaline
phosphatase treatment of this phosphorylated intermediate resulted in the formation of
labd-13-en-8α, 15-diol, which is an abundant compound in the resin of C. creticus
(Demetzos et al., 1994; Anastasaki et al., 1999). It is therefore possible that copal-8-ol
diphosphate is indeed the precursor of this compound in vivo as well. In addition, it is
also possible that copal-8-ol diphosphate can be further cyclised to the several manoyl
oxide isomers observed in the resin of this plant by as yet unidentified type A
diterpene synthase (Fig. 10).
Similar to the formation of CDP, the formation of copal-8-ol diphosphate
formation is likely initiated by protonation of the terminal double bond of GGDP and
the formation of a bicyclic carbocation followed by capture of a hydroxyl anion, as
discussed by Guo and Wagner (1995). The sigmoidal kinetics observed for CcCLS is
similar to what has been observed for other enzymes that use allylic diphosphates
possibly due to the interactions between the substrate and the divalent metal ion
cofactor (Scott et al., 2003). Our demonstrated activity of CcCLS provides
experimental evidence to the conjecture of Gao and Wagner (1995) that the hydroxyl
group at C8 is derived directly from the cyclization process rather than from the
catalytic activity of a cytochrome P450. A second ionization-initiated cyclization of
this intermediate diphosphate ester is expected to lead to the formation of a mixture of
the C13 manoyl oxide epimers. This biosynthetic route is further supported by the in
vitro formation of manoyl oxides from copal-8-ol diphosphate when the latter was
incubated with recombinant abietadiene synthase from A. grandis (Ravn et al., 2000).
Enhanced CcCLS Gene Expression is correlated with Increased Labdane-type
Diterpene Production
The expression pattern of CcCLS suggests involvement of the encoded enzyme
in labdane diterpene biosynthesis, since the preferential expression of CcCLS in the
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trichomes is consistent with the observed accumulation of manoyl oxide derivatives in
trichome exudates of C. creticus (Anastasaki et al., 1999). Moreover, the elevated
CcCLS transcript levels observed in trichomes isolated from young leaves are also
consistent with the higher labdane-type diterpene concentrations in that
developmental stage than in fully expanded leaves. In several species both
transcriptional data and terpene analysis support the fact that changes in biosynthesis
of volatile terpenes that occur during the development of plant organs is at least partly
regulated at the transcription level of terpene synthases genes. To date, the majority of
studies have focused on the synthesis and emission of volatile monoterpenes and
sesquiterpenes, and their results have suggested that such terpenes have a role in plant
defense (for vegetative tissues) or reproductive success (in the case of inflorescence
and fruit) (McConkey et al., 2000; Chen et al., 2003; Dudareva et al., 2003; Aharoni
et al., 2004; Guitton et al., 2010). On the other hand, the labdane-type diterpenes
described in this work are non-volatile terpenes that were shown to accumulate in
higher concentrations in the resin of young leaves. It appears that this is an example
whereby the plant invests the energy cost of producing these specialized compounds
to especially protect young tissues that are more susceptible to feeding by insects and
invasion by pathogens.
To further explore the ecological role of Cistus labdane-type diterpenes, leaves
were subjected to wounding and both transcriptional and chemical profiling of the
wounded tissues were processed. Mechanical wounding has been widely used to
simulate insect feeding. Enhanced transcript accumulation of CcCLS upon mechanical
wounding was accompanied by an increase in labdane-type diterpene concentration in
the trichomes of the wounded leaves. These results suggest a putative role of labdane-
type diterpenes in direct and indirect defenses against herbivores. Ovipositional
response of tobacco budworm moths to labdane-type diterpenes from Nicotiana
glutinosa has implicated labdenediol and manool in this indirect defense mechanism
that caused reduced infestation levels of tobacco budworm larvae (Jackson et al.,
1999). Furthermore, in tobacco, labda-11, 13-diene-8α, 15-diol is suggested to act as
an endogenous signal, responsible for the activation of defense responses (Seo et al.,
2003). An additional protective role against plant pathogens on the wound site could
be attributed to the antimicrobial properties of C. creticus labdane-type diterpenes
(Demetzos et al., 1997; Bouamama et al., 2006).
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MATERIALS AND METHODS
Plant Material
Cistus creticus subsp. creticus plants were grown in pots containing a mixture of
soil and perlite (3:1, v/v) under controlled environmental conditions in a E-36L
growth chamber (PERCIVAL SCIENTIFIC, Perry, IA, USA) with a 16/8 h
photoperiod at 120 μmol m-2 s-1 and a 23oC/18oC (day/night) temperature cycle. For
gene expression studies, total RNA was isolated from trichomes and leaves at
different developmental stages determined by the leaf length (S1: 0.5-1 cm, S2: 1-2
cm, S3: 2-3 cm, S4: 3-4 cm) and from various organs such as seeds, roots, stems,
young leaves (~1-2 cm ) and tips. Mechanical wounding was performed by cutting the
leaves in uniform stripes with scissors. Leaf material was sampled 15 min, 30 min, 1-
3-, 6- and 12 hours upon wounding. Total trichomes and RNA were isolated
according to Falara et al., (2008).
Isolation and Sequence Analysis of CcCLS
Previously, an EST analysis of a C. creticus trichome cDNA library revealed two
poly-A containing cDNA clones (Accession numbers FF404867 and FF405049) with
similarity to plant diterpene synthases (Falara et al., 2008). Moreover, PCR on the
above cDNA library was carried out with several combinations of sense and antisense
degenerate primers based on short conserved sequence elements present in plant
diterpene synthases. A 870 bp fragment was obtained when the 5΄- GAY ACI GCD
TGG GTA GC- 3΄ and 5΄-GAI ACA TYR TAB CCG T- 3΄set of primers were used
under certain amplification conditions (94°C for 2 min, 10 cycles of 94°C for 30 sec,
65°C for 30 sec, and 72°C for 1 min and additional 30 cycles of 94°C for 30 sec,
touch down temperature 65°C to 55οC for 30 sec, and 72°C for 1min and a final
extension step of 5 min at 72°C).
Sequence information from the ESTs and the PCR-derived fragment was used to
design gene specific primers. The amplification of a cDNA segment with one sense
gene specific primer based on the PCR derived fragment (5΄- TGA TCG CCT ACA
ACG TCT AGG-3΄) and one antisense primer based on the library EST (5΄- TAG
GGG CAA TTT CGT AAG ATC-3΄) revealed that the above partial cDNAs were
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fragments of the same gene. Amplification of the missing 5 ́ prime-end was
performed using one antisense gene-specific primer based on the sequence of the PCR
derived fragment (5΄- CTT GAG GAG ACG ATC GTA TGC G-3΄) and one universal
primer (SP6: 5΄-ΑΤ ΤΤΑ GGT GAC ACT ATA-3΄). The full length cDNA (CcCLS)
was obtained by PCR amplification on library extracted DNA as template using the
Pfx50 proofreading polymerase (Invitrogen, Carlsbad, CA, USA).
The complete sequence of the ORF of CcCLS was deposited in GenBank
(http://www.ncbi.nlm.nih.gov) with the accession number HM537017. Multiple
sequence alignments were generated with CLUSTALW program (Chenna et al.,
2003). Phylogenetic and molecular evolutionary analysis was conducted using MEGA
version 4 (Tamura et al., 2007). Subcellular localization of CcCLS and cleavage site
prediction were performed with Target P (Nielsen et al., 1997; Emanuelsson et al.,
2000).
Heterologous Expression and Protein Extraction of CcCLS
The ORF of CcCLS was cloned and inserted into the bacterial expression vector
pEXP5-NT/TOPO (INVITROGEN, Carlsbad, CA, USA). The construct were
introduced into the E. coli strain BL21 (DE3) and liquid cultures of the bacteria
harbouring the expression construct were grown at 37°C to an OD600 of 0.5. At that
time, isopropyl-thiogalactopyranoside (IPTG) was added to a final concentration of
0.4 mM and the cultures were incubated for 20 h at 18°C. The cells were collected by
centrifugation and disrupted by treatment with sonication in chilled extraction buffer
(50 mM Hepes pH 8.0, 300 mM KCl, 5% (v/v) glycerol, 5 mM Dithiothreitol (DTT),
5 mM imidazole). After centrifugation at 14,000g for cellular debris removal, the
supernatant was rescued. Affinity purification was carried out using Talon Metal
Affinity resin (CLONTECH, Palo Alto, California, USA), according to the
manufacturer’s instructions.
Enzyme Assays and GC-MS Analysis of Enzymatic Products
For product identification the enzyme reaction contained 5 μg of affinity-purified
protein in assay buffer containing 50 mM Hepes pH 8.0, 100 mM KCl, 7.5 mM
MgCl2, 0.5 mM DTT, 5% glycerol and 60 µM GGDP (ECHELON BIOSCIENCES,
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Salt Lake City, UT, USA). Assay mixtures were incubated at 30°C for 1 h and the
resultant mixture was either directly extracted with n-hexane or subjected to
dephosphorylation by incubation with bacterial alkaline phosphatase (40 units, Takara
Bio, Otsu, Japan) at 37°C for 1 h and subsequently hexane extracted. Negative
controls included assays with affinity-purified protein in the absence of substrate and
assays with 60 µM of farnesyl diphosphate or 60 µM of geranyl diphosphate as
substrate, as well as boiled enzyme with 60 µM GGDP. For kinetic studies, 2 μg of
affinity-purified protein in assay buffer were used and reactions were stopped by
adding 5μL of 2N HCl.
After hexane extraction, identification of the enzymatic products was conducted
by GC/MS using a Shimadzu QP-2010 system (SHIMADZU, Kyoto, Japan) fitted
with a HP-5MS column (0.25 mm in diameter, 30 m long, 0.25 μm film thickness)
and equipped with an AOC20i-AOC20s autosampler. Samples were injected onto the
column at 250oC in the splitless mode. After a 2 min isothermal hold at 50oC, the
column temperature was increased by 10oC/min to 275oC with a 10 min isothermal
hold at 275oC. The flow rate of the helium carrier gas was 1.36 mL min-1.
Metabolite profile analysis
Glandular and non glandular trichomes isolated from leaves at different
developmental stages: stage 1 (~0.5 cm), stage 2 (1-2 cm), stage 3 (2-3 cm) and stage
4 (3-4 cm) and from wounded (after 3h) and not wounded (control) leaves were
extracted in n-hexane for 18 h. Extracts were evaporated to dryness and subjected to
gas chromatography/mass spectrometry (GC/MS) analysis. A HP-5MS capillary
column (30 m x 0.25 mm, 0.25 μm film thickness) was used. The column was
temperature programmed as follows: 110oC for 4 min, temperature increased to 230oC
at a rate of 3.3oC/min and up to 250oC at a rate of 10oC/min (splitless mode). Mass
spectrometry conditions were as follows: temperature of ion source 230oC, ionization
energy 70eV, electron current 1453 μΑ.
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Gene Expression Analysis of CcCLS
Analysis of the expression of the gene encoding CcCLS was conducted during
leaf development in various organs and upon wounding. Total RNA was isolated as
previously described (Pateraki and Kanellis, 2004), electrophoretically analyzed on
denaturing formaldehyde agarose gels, blotted onto Nylon membranes
(SCHLEICHER AND SCHUELL, GmbH, Dassel, Germany) and hybridized with
[a32P]dCTP-labeled 3 ́ untranslated fragment of the full length cDNA (Church and
Gilbert, 1984).
ACKNOWLEDGMENTS
The isolated labdane-type diterpenes from C. creticus used as standards was a
kind gift from Dr. Alexios-Leandros Skaltsounis (manoyl oxide and 13-epi-manoyl
oxide) and Dr. Constantinos Demetzos (labd-13-en-8α,15 diol) from the National and
Kapodistrian University of Athens and VIORYL SA (sclareol) while syn- and ent-
copalol from Dr. Robert M. Coates from the University of Illinois. We would like to
thank Dr. Irene Pateraki, Ms. Anastasia Yupsani and Ms. Fani Chatzopoulou for the
plant treatments and RNA preparations and Dr. Ioannis Poulakakis for curve fitting of
the biochemical data. Lastly, we appreciate the help of Dr. Constantinos Demetzos
in diterpene determination shown in Figure 8.
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16
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Legends to Figures Figure 1. Labdane-type diterpenes. A, Basic labdane skeleton. B, Manoyl oxide and
epi-manoyl oxide. C, Labd-13en-8α,15 diol.
Figure 2. Alignment of CcCLS with other diterpene synthases. The deduced amino-
acid sequence for CcCLS was compared to Stevia rebaudiana copalyl
diphosphate synthase (SrCPS), Physcomitrella. patens bifunctional
kaurene synthase (PpCPS/KS), Abies grandis abietadiene synthase (AgAS)
and Curcubita maxima kaurene synthase (CmKS). The black and grey
shading indicates identical and similar amino-acids in properties,
respectively. The conserved aspartate-rich motifs are underlined.
Figure 3. Phylogenetic relatedness of CcCLS to other plant diterpene synthases.
Deduced amino acid sequences were analyzed by minimum evolution
using MEGA 4.0. The cladogram was generated from an alignment of
Abies grandis abietadiene synthase (AgAS, Q38710), Arabidopsis thaliana
copalyl diphosphate synthase (AtCPS, NP_192187) and kaurene synthase
(AtKS, AAC39443), Cistus creticus manoyl oxide synthase (CcCLS),
Cucurbita maxima copalyl diphosphate synthase (CmCPS, AAD04292)
and kaurene synthase (CmKS, Q39548), Ginkgo biloba levopimaradiene
synthase (GbLS, AAL09965), Oryza sativa copalyl diphosphate synthase 1
(OsCPS1, BAD42449), copalyl diphosphate synthase 2 (OsCPS2,
AAS98158), pimara-7,15-diene synthase (OsPS, AAU05906) and stemer-
13-ene synthase (OsSS, NP_001067887), Physcomitrella patens kaurene
synthase (PpKS, BAF61135), Picea abies isopimaradiene synthase (PaPS,
AAS47690) and levopimaradiene/abietadiene synthase (PaLS/AS,
AAS47691), Pisum sativum copalyl diphosphate synthase (PsCPS,
O04408), Scoparia dulcis copalyl diphosphate synthase (SdCPS,
BAD91286), Stevia rebaudiana copalyl diphosphate synthase (SrCS,
AAB87091) and kaurene synthase (SrKS, AAD34294), Taxus brevifolia
taxadiene synthase (TbTS, AAK83566) and Zea mays copalyl diphosphate
synthase (ZmCPS, NP_001105257). Bootstrap values are shown next to
the branches.
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21
Figure 4. Recombinant CcCLS protein. A, E. coli expressed truncated version of
CcCLS without the transit peptide. Soluble protein fraction (S,
supernatant) from IPTG induced culture (Lane 1); insoluble protein
fraction (A, aggregate) from IPTG induced culture (Lane 2); soluble
protein fraction (S) from non-induced culture (Lane 3); insoluble protein
fraction (A) from non-induced culture (Lane 4); protein marker (Lane 5).
B, Purified His-tagged CcCLS (Lane1); protein marker (Lane 2).
Figure 5. GC-MS chromatograph of the labdane-type diterpene product of CcCLS.
Detection of hexane-extracted compounds obtained after: A, Alkaline
phosphatase treatment of GGDP. B, Alkaline phosphatase treatment after
purified CcCLS was incubated with GGDP as substrate for 1 hour. C, No
alkaline phosphatase treatment after purified CcCLS was incubated with
GGDP as substrate for 1 hour. D, Labd-13-en-8α, 15-diol authentic
standard. Peak 1 corresponds to tetradecane (internal control), peak 2 to
geranylgeraniol, and peak 3 to labd-13-en-8α, 15-diol. MIC[81] was used
for detection.
Figure 6. Comparison of the mass spectra of the product of the CcCLS-catalyzed
reaction with those of labd-13-en-8α, 15-diol authentic standard. The mass
spectrum of peak 3 (A) matches with that of labd-13-en-8α, 15 diol (B).
Figure 7. Kinetic analysis of CcCLS. Velocity (Vi) of CcCLS for different
concentrations of GGDP was measured (the mean value and standard
deviation of at least 2 replicates is used). Data are mathematically fitted to
the Hill equation. Fitted curve is shown in dashed line.
Figure 8. Change in CcCLS RNA levels and labdane-type diterpenes during leaf
development. A, RNA gel-blot analysis for CcCLS for RNA extracted
from seeds, roots, stems, leaves and shoot tips. B, RNA gel-blot analysis
for CcCLS for RNA extracted from isolated trichomes and leaves at
different developmental stages (S1: 0.5-1 cm, S2:1-2 cm, S3: 2-3 cm, S4:
3-4 cm). C, Labdane-type diterpene accumulation in isolated trichomes at
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22
different leaf developmental stages (S1: 0.5-1 cm, S2:1-2 cm, S3: 2-3 cm,
S4: 3-4 cm). Methylene blue staining of the ribosomal RNA was used to
demonstrate equal loading of the northern blot. Each bar represents the
average of three independent experiments with the standard deviation
given.
Figure 9. Change in CcCLS RNA levels and labdane-type diterpenes in wounded
leaves. A, RNA gel blot analysis for CcCLS transcripts for RNA extracted
from leaves of C. creticus plants 15 min, 30 min, 1 h, 3 h, 6 h and 12 h
after wounding. B, Labdane-type diterpene accumulation in leaf trichomes
3 h upon wounding. Methylene blue staining of the ribosomal RNA was
used to demonstrate equal loading of the northern blot. Each bar represents
the average of three independent experiments with the standard deviation
given.
Figure 10. Proposed pathway to labdane-type diterpenes predominant in C. creticus
resin. A protonation-initiated cyclization catalyzed by CcCLS converts
GGDP to the stable bicyclic intermediate copal-8-ol diphosphate. A
second ionization-initiated cyclization of copal-8-ol diphosphate is
hypothesized to result in the formation of manoyl oxides isomers while
labd-13-en-8α,15-diol could be formed either by phosphatase activity or
type A diterpene synthase activity.
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Figure 1. Labdane-type diterpenes. A, Basic labdane skeleton. B, Manoyl oxide and epi-manoyl oxide.
C, Labd-13en-8α,15 diol.
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Figure 2. Alignment of CcCLS with other diterpene synthases. The deduced amino-acid sequence for CcCLS was compared to Stevia rebaudiana
copalyl diphosphate synthase (SrCPS), Physcomitrella patens bifunctional kaurene synthase (PpCPS/KS), Abies grandis abietadiene synthase
(AgAS) and Curcubita maxima kaurene synthase (CmKS). The black and grey shading indicates identical and similar amino-acids in properties,
respectively. The conserved aspartate-rich motifs are underlined.
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Figure 3. Phylogenetic relatedness of CcCLS to other plant diterpene synthases. Deduced amino acid
sequences were analyzed by minimum evolution using MEGA 4.0. The cladogram was generated from
an alignment of Abies grandis abietadiene synthase (AgAS, Q38710), Arabidopsis thaliana copalyl
diphosphate synthase (AtCPS, NP_192187) and kaurene synthase (AtKS, AAC39443), Cistus creticus
copal-8-ol diphosphate synthase (CcCLS), Cucurbita maxima copalyl diphosphate synthase (CmCPS,
AAD04292) and kaurene synthase (CmKS, Q39548), Ginkgo biloba levopimaradiene synthase (GbLS,
AAL09965), Oryza sativa copalyl diphosphate synthase 1 (OsCPS1, BAD42449), copalyl diphosphate
synthase 2 (OsCPS2, AAS98158), pimara-7, 15-diene synthase (OsPS, AAU05906) and stemer-13-ene
synthase (OsSS, NP_001067887), Physcomitrella patens kaurene synthase (PpKS, BAF61135), Picea abies
isopimaradiene synthase (PaPS, AAS47690) and levopimaradiene/abietadiene synthase (PaLS/AS,
AAS47691), Pisum sativum copalyl diphosphate synthase (PsCPS, O04408), Scoparia dulcis copalyl
diphosphate synthase (SdCPS, BAD91286), Stevia rebaudiana copalyl diphosphate synthase (SrCS,
AAB87091) and kaurene synthase (SrKS, AAD34294), Taxus brevifolia taxadiene synthase (TbTS,
AAK83566) and Zea mays copalyl diphosphate synthase (ZmCPS, NP_001105257). Bootstrap values are
shown next to the branches.
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Figure 4. Recombinant CcCLS protein. A, E. coli expressed truncated version of
CcCLS without the transit peptide. Soluble protein fraction (S, supernatant) from
IPTG induced culture (Lane 1); insoluble protein fraction (A, aggregate) from IPTG
induced culture (Lane 2); soluble protein fraction (S) from non-induced culture (Lane
3); insoluble protein fraction (A) from non-induced culture (Lane 4); protein marker
(Lane 5). B, Purified His-tagged CcCLS (Lane1); protein marker (Lane 2).
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Figure 5. GC-MS chromatograph of the labdane-type diterpene product of
CcCLS. Detection of hexane-extracted compounds obtained after: A, Alkaline
phosphatase treatment of GGDP. B, Alkaline phosphatase treatment after purified
CcCLS was incubated with GGDP as substrate for 1 hour. C, No alkaline phosphatase
treatment after purified CcCLS was incubated with GGDP as substrate for 1 hour. D,
Labd-13-en-8α, 15-diol authentic standard. Peak 1 corresponds to tetradecane
(internal control), peak 2 to geranylgeraniol, and peak 3 to labd-13-en-8α, 15-diol.
MIC[81] was used for detection.
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Figure 6. Comparison of the mass spectra of the product of the CcCLS-catalyzed
reaction with those of labd-13-en-8α, 15-diol authentic standard. The mass spectrum
of peak 3 (A) matches with that of labd-13-en-8α, 15 diol (B).
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Figure 7. Kinetic analysis of CcCLS. Velocity (Vi) of CcCLS for different concentrations
of GGDP was measured (the mean value and standard deviation of at least 2
replicates is used). Data are mathematically fitted to the Hill equation. Fitted curve is
shown in dashed line.
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Figure 8. Change in CcCLS RNA levels and labdane-type diterpenes during leaf development. A, RNA gel-
blot analysis for CcCLS for RNA extracted from seeds, roots, stems, leaves and shoot tips. B, RNA gel-blot
analysis for CcCLS for RNA extracted from isolated trichomes and leaves at different developmental
stages (S1: 0.5-1 cm, S2:1-2 cm, S3: 2-3 cm, S4: 3-4 cm). C, Labdane-type diterpene accumulation in
isolated trichomes at different leaf developmental stages (S1: 0.5-1 cm, S2:1-2 cm, S3: 2-3 cm, S4: 3-4
cm). Methylene blue staining of the ribosomal RNA was used to demonstrate equal loading of the RNA
gel-blot. Each bar represents the average of three independent experiments with the standard deviation
given.
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Figure 9. Change in CcCLS RNA levels and labdane-type diterpenes in wounded leaves. A,
RNA gel blot analysis for CcCLS transcripts for RNA extracted from leaves of C. creticus plants
15 min, 30 min, 1 h, 3 h, 6 h and 12 h after wounding. B, Labdane-type diterpene
accumulation in leaf trichomes 3 h upon wounding. Methylene blue staining of the
ribosomal RNA was used to demonstrate equal loading of the RNA gel-blot. Each bar
represents the average of three independent experiments with the standard deviation
given.
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Figure 10. Proposed pathway to labdane-type diterpenes predominant in C. creticus resin.
A protonation-initiated cyclization catalyzed by CcCLS converts GGDP to the stable bicyclic
intermediate copal-8-ol diphosphate. A second ionization-initiated cyclization of copal-8-ol
diphosphate is hypothesized to result in the formation of manoyl oxides isomers while labd-
13-en-8α, 15-diol could be formed either by phosphatase activity or type A diterpene
synthase activity.
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