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

www.plantphysiol.orgon February 10, 2020 - Published by Downloaded from Copyright © 2010 American Society of Plant Biologists. All rights reserved.

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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.



15

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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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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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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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.



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

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.



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 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.



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.



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.



Running head: Cistus creticus copal-8-ol diphosphate synthase · labd-7,13-dien-15-ol,...

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