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. 2010 Jul 1;154(1):301–310. doi: 10.1104/pp.110.159566

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 Diterpenes1,[OA]

Vasiliki Falara 1, Eran Pichersky 1, Angelos K Kanellis 1,*
PMCID: PMC2938158  PMID: 20595348

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 Escherichia coli-expressed protein used geranylgeranyl diphosphate as substrate and catalyzed the formation of copal-8-ol diphosphate. This diterpene synthase, therefore, was named CcCLS (for C. 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.

Labdane-type diterpenes constitute 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 contribute three carbons to a six-member ring that may or may not contain an oxygen atom (Fig. 1). Most research on the 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 geranylgeranyl 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, a type A cyclase that possesses the Asp-rich DDXXD motif (Sakamoto et al., 2004). Recently, distinct CPS and kaurene synthase enzymes apparently exclusively involved with gibberellin biosynthesis were identified in gymnosperms (Keeling et al., 2010)

Figure 1.

Figure 1.

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Labdane-type diterpenes. A, Basic labdane skeleton. B, Manoyl oxide and epi-manoyl oxide. C, Labd-13-ene-8α,15-diol.

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 (Otomo et al., 2004b; Prisic et al., 2004; Xu et al., 2004). These phosphorylated intermediates are then further cyclized by type A cyclases to diterpene products such as oryzalexins, momilactones, and phytocassanes (Nemoto et al., 2004; Otomo et al., 2004a; Wilderman et al., 2004; Kanno et al., 2006). In nonangiosperm 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 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 Nicotiana tabacum contain the diterpenes abienol, labdenediol, and sclareol, and protein extracts from these species supplied with GGDP were 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, and indeed the identity of the responsible enzymes, were 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, 2006; Matsingou et al., 2005, 2006). Moreover, recent studies indicated significant antitumor activity of sclareol, a labdane-type diterpene, on human colon cancer tumors (HCT116), which often develop 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 Escherichia coli-produced protein. We show that the enzyme encoded by CcCLS catalyzes the formation of copal-8-ol diphosphate and therefore is 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 with other parts of the plant. The high similarity in protein sequence among previously characterized diterpene synthases prompted us to design several degenerate primers corresponding to highly conserved sequences and to 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 the isolation of a full-length cDNA that we designated CcCLS (for C. creticus copal-8-ol diphosphate synthase). 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; accession no. BAD91286) and 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.

Figure 2.

Figure 2.

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Alignment of CcCLS with other diterpene synthases. The deduced amino acid sequence for CcCLS was compared with Stevia rebaudiana copalyl diphosphate synthase (SrCPS), Physcomitrella patens bifunctional kaurene synthase (PpCPS/KS), Abies grandis abietadiene synthase (AgAS), and Cucurbita maxima kaurene synthase (CmKS). The black and gray shading indicates identical and similar amino acids in properties, respectively. The conserved Asp-rich motifs are underlined.

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).

Figure 3.

Figure 3.

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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.

Heterologous Expression and Functional Characterization of CcCLS

The sequence similarities between CcCLS and other diterpene synthases and the common Asp-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 diterpene 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 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 chromatographic 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 was 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 farnesyl diphosphate. Finally, comparison of the assay products with authentic samples of labdane-type diterpenes isolated from C. creticus (Demetzos et al., 1994) revealed that peak 3 corresponded to labd-13-ene-8α,15-diol, based on both identical retention times (Fig. 5) and mass spectra (Fig. 6).

Figure 4.

Figure 4.

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Recombinant CcCLS protein. A, E. coli-expressed truncated version of CcCLS without the transit peptide. Lane 1, Soluble protein fraction (S, supernatant) from isopropyl thiogalactopyranoside (IPTG)-induced culture; lane 2, insoluble protein fraction (A, aggregate) from IPTG-induced culture; lane 3, soluble protein fraction (S) from noninduced culture; lane 4, insoluble protein fraction (A) from noninduced culture; lane 5, protein marker. B, Purified His-tagged CcCLS (lane 1) and protein marker (lane 2).

Figure 5.

Figure 5.

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GC-MS chromatograph of the labdane-type diterpene product of CcCLS. Detection of hexane-extracted compounds obtained after the following. A, Alkaline phosphatase treatment of GGDP. B, Alkaline phosphatase treatment after purified CcCLS was incubated with GGDP as substrate for 1 h. C, No alkaline phosphatase treatment after purified CcCLS was incubated with GGDP as substrate for 1 h. D, Labd-13-ene-8α,15-diol authentic standard. Peak 1 corresponds to tetradecane (internal control), peak 2 to geranylgeraniol, and peak 3 to labd-13-ene-8α,15-diol. Multiple ion chromatogram[81] was used for detection. RT, Retention time.

Figure 6.

Figure 6.

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Comparison of the mass spectra of the product of the CcCLS-catalyzed reaction and labd-13-ene-8α,15-diol authentic standard. The mass spectrum of peak 3 (A) matches with that of labd-13-ene-8α,15-diol (B).

The purified enzyme showed sigmoid kinetics, producing a sigmoid velocity 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 μm, with a Hill number of 3.57, an indication of a positive cooperativity of the GGDP binding.

Figure 7.

Figure 7.

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Kinetic analysis of CcCLS. Velocity (Vi) of CcCLS for different concentrations of GGDP was measured (mean values and sd of at least two replicates are shown). Data were mathematically fitted to the Hill equation. A fitted curve is shown with the dashed line.

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, 0.5–1 cm; S2, 1–2 cm; S3, 2–3 cm; S4, 3–4 cm), with CcCLS transcripts being more abundant in young leaves (stages S1, S2, and S3), and especially in stage S2, in comparison with fully expanded leaves (stage S4; Fig. 8B).

Figure 8.

Figure 8.

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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 (see text). C, Labdane-type diterpene accumulation in isolated trichomes at different leaf developmental stages. 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 sd given.

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 gradually 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 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).

Figure 9.

Figure 9.

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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, and 1, 3, 6, and 12 h after wounding. B, Labdane-type diterpene accumulation in leaf trichomes 3 h after 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 sd given.

Trichomes isolated from wounded leaves as well as nonwounded 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. 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 C. creticus subsp. creticus. We show 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-ene-8α,15-diol, which is an abundant compound in the resin of C. creticus (Demetzos et al., 1994; Anastasaki et al., 1999). Therefore, it is 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 cyclized to the several manoyl oxide isomers observed in the resin of this plant by an as yet unidentified type A diterpene synthase (Fig. 10).

Figure 10.

Figure 10.

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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 oxide isomers, while labd-13-ene-8α,15-diol could be formed either by phosphatase activity or type A diterpene synthase activity.

Similar to the formation of CDP, the formation of copal-8-ol diphosphate 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 Guo 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 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 are at least partly regulated at the transcription level of terpene synthase 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 nonvolatile 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 especially to 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. The ovipositional response of tobacco budworm moths to labdane-type diterpenes from N. 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 (N. tabacum ‘Samsun NN’), 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).

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 an E-36L growth chamber (Percival Scientific) with a 16/8-h photoperiod at 120 μmol m−2 s−1 and a 23°C/18°C (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 (approximately 1–2 cm), and tips. Mechanical wounding was performed by cutting the leaves in uniform strips with scissors. Leaf material was sampled 15 min, 30 min, and 1, 2, 3, 6, and 12 h after 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 nos. 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. An 870-bp fragment was obtained when the 5′-GAYACIGCDTGGGTAGC-3′ and 5′-GAIACATYRTABCCGT-3′ primers were used under certain amplification conditions (94°C for 2 min; 10 cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 1 min; an additional 30 cycles of 94°C for 30 s, touchdown temperature of 65°C to 55°C for 30 s, and 72°C for 1 min; 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′-TGATCGCCTACAACGTCTAGG-3′) and one antisense primer based on the library EST (5′-TAGGGGCAATTTCGTAAGATC-3′) revealed that the above partial cDNAs were fragments of the same gene. Amplification of the missing 5′ end was performed using one antisense gene-specific primer based on the sequence of the PCR-derived fragment (5′-CTTGAGGAGACGATCGTATGCG-3′) and one universal primer (SP6, 5′-ATTTAGGTGACACTATA-3′). The full-length cDNA (CcCLS) was obtained by PCR amplification on library-extracted DNA as template using the Pfx50 proofreading polymerase (Invitrogen).

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 the ClustalW program (Chenna et al., 2003). Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4 (Tamura et al., 2007). Subcellular localization of CcCLS and cleavage site prediction were performed with TargetP (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). The construct was introduced into the Escherichia coli strain BL21 (DE3), and liquid cultures of the bacteria harboring the expression construct were grown at 37°C to an optical density at 600 nm of 0.5. At that time, isopropyl thiogalactopyranoside 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, and 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) 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 dithiothreitol, 5% glycerol, and 60 μm GGDP (Echelon Biosciences). 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) 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 farnesyl diphosphate or 60 μm 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 was used, and reactions were stopped by adding 5 μL of 2 n HCl.

After hexane extraction, identification of the enzymatic products was conducted by GC-MS using a Shimadzu QP-2010 system fitted with an HP-5MS column (0.25 mm diameter, 30 m long, 0.25 μm film thickness) and equipped with an AOC20i-AOC20s autosampler. Samples were injected onto the column at 250°C in the splitless mode. After a 2-min isothermal hold at 50°C, the column temperature was increased by 10°C min−1 to 275°C with a 10-min isothermal hold at 275°C. The flow rate of the helium carrier gas was 1.36 mL min−1.

Metabolite Profile Analysis

Glandular and nonglandular trichomes isolated from leaves at different developmental stages and from wounded (after 3 h) and nonwounded (control) leaves were extracted in n-hexane for 18 h. Extracts were evaporated to dryness and subjected to GC-MS analysis. An HP-5MS capillary column (30 m × 0.25 mm, 0.25 μm film thickness) was used. The column was temperature programmed as follows: 110°C for 4 min, increased to 230°C at a rate of 3.3°C min−1, and increased up to 250°C at a rate of 10°C min−1 (splitless mode). MS conditions were as follows: ion source temperature of 230°C, ionization energy of 70 eV, electron current of 1,453 μα.

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 described previously (Pateraki and Kanellis, 2004), electrophoretically analyzed on denaturing formaldehyde agarose gels, blotted onto nylon membranes (Schleicher and Schuell), and hybridized with [α-32P]dCTP-labeled 3′ untranslated fragment of the full-length cDNA (Church and Gilbert, 1984).

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number HM537017.

Acknowledgments

The isolated labdane-type diterpenes from C. creticus used as standards were a kind gift from Dr. Alexios-Leandros Skaltsounis (manoyl oxide and 13-epi-manoyl oxide) and Dr. Constantinos Demetzos (labd-13-ene-8α,15-diol; National and Kapodistrian University of Athens). The company Vioryl SA, Athens, kindly provided sclareol, and syn- and ent-copalol were a gift from Dr. Robert M. Coates (University of Illinois). We thank Dr. Irene Pateraki, Ms. Anastasia Yupsani, and Ms. Fani Chatzopoulou (Aristotle University of Thessaloniki) for the plant treatments and RNA preparations and Dr. Ioannis Poulakakis (Princeton University) for curve fitting of the biochemical data. Lastly, we appreciate the help of Dr. Constantinos Demetzos in the diterpene determination shown in Figure 8.

References

  1. Aharoni A, Giri AP, Verstappen FW, Bertea CM, Sevenier R, Sun Z, Jongsma MA, Schwab W, Bouwmeester HJ. (2004) Gain and loss of fruit flavor compounds produced by wild and cultivated strawberry species. Plant Cell 16: 3110–3131 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anastasaki T, Demetzos C, Perdetzoglou D, Gazouli M, Loukis A, Harvala C. (1999) Analysis of labdane-type diterpenes from Cistus creticus (subsp. creticus and subsp. eriocephalus) by GC and GC-MS. Planta Med 65: 735–739 [DOI] [PubMed] [Google Scholar]
  3. Bouamama H, Noel T, Villard J, Benharref A, Jana M. (2006) Antimicrobial activities of the leaf extracts of two Moroccan Cistus L. species. J Ethnopharmacol 104: 104–107 [DOI] [PubMed] [Google Scholar]
  4. Chen F, Tholl D, D’Auria JC, Farooq A, Pichersky E, Gershenzon J. (2003) Biosynthesis and emission of terpenoid volatiles from Arabidopsis flowers. Plant Cell 15: 481–494 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD. (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31: 3497–3500 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chinou I. (2005) Labdanes of natural origin: biological activities (1981–2004). Curr Med Chem 12: 1295–1317 [DOI] [PubMed] [Google Scholar]
  7. Church GM, Gilbert W. (1984) Genomic sequencing. Proc Natl Acad Sci USA 81: 1991–1995 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Demetzos C, Katerinopoulos H, Kouvarakis A, Stratigakis N, Loukis A, Ekonomakis C, Spiliotis V, Tsaknis J. (1997) Composition and antimicrobial activity of the essential oil of Cistus creticus subsp. eriocephalus. Planta Med 63: 477–479 [DOI] [PubMed] [Google Scholar]
  9. Demetzos C, Mitaku S, Couladis M, Harvala C, Kokkinopoulos D. (1994) Natural metabolites of ent-13-epi-manoyl oxide and other cytotoxic diterpenes from the resin “LADANO” of Cistus creticus. Planta Med 60: 590–591 [DOI] [PubMed] [Google Scholar]
  10. Dimas K, Demetzos C, Vaos V, Ioannidis P, Trangas T. (2001) Labdane type diterpenes down-regulate the expression of c-Myc protein, but not of Bcl-2, in human leukemia T-cells undergoing apoptosis. Leuk Res 25: 449–454 [DOI] [PubMed] [Google Scholar]
  11. Dimas K, Papadaki M, Tsimplouli C, Hatziantoniou S, Alevizopoulos K, Pantazis P, Demetzos C. (2006) Labd-14-ene-8,13-diol (sclareol) induces cell cycle arrest and apoptosis in human breast cancer cells and enhances the activity of anticancer drugs. Biomed Pharmacother 60: 127–133 [DOI] [PubMed] [Google Scholar]
  12. Dudareva N, Martin D, Kish CM, Kolosova N, Gorenstein N, Faldt J, Miller B, Bohlmann J. (2003) (E)-Beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily. Plant Cell 15: 1227–1241 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Emanuelsson O, Nielsen H, Brunak S, von Heijne G. (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300: 1005–1016 [DOI] [PubMed] [Google Scholar]
  14. Falara V, Fotopoulos V, Margaritis T, Anastasaki T, Pateraki I, Bosabalidis AM, Kafetzopoulos D, Demetzos C, Pichersky E, Kanellis AK. (2008) Transcriptome analysis approaches for the isolation of trichome-specific genes from the medicinal plant Cistus creticus subsp. creticus. Plant Mol Biol 68: 633–651 [DOI] [PubMed] [Google Scholar]
  15. Guitton Y, Nicole F, Moja S, Valot N, Legrand S, Jullien F, Legendre L. (2010) Differential accumulation of volatile terpene and terpene synthase mRNAs during lavender (Lavandula angustifolia and L. x intermedia) inflorescence development. Physiol Plant 138: 150–163 [DOI] [PubMed] [Google Scholar]
  16. Guo Z, Severson RF, Wagner GJ. (1994) Biosynthesis of the diterpene cis-abienol in cell-free extracts of tobacco trichomes. Arch Biochem Biophys 103: 103–108 [DOI] [PubMed] [Google Scholar]
  17. Guo Z, Wagner G. (1995) Biosynthesis of labdenediol and sclareol in cell-free extracts from trichomes of Nicotiana glutinosa. Planta 197: 627–632 [Google Scholar]
  18. Hamberger B, Bohlmann J. (2006) Cytochrome P450 mono-oxygenases in conifer genomes: discovery of members of the terpenoid oxygenase superfamily in spruce and pine. Biochem Soc Trans 34: 1209–1214 [DOI] [PubMed] [Google Scholar]
  19. Hatziantoniou S, Dimas K, Georgopoulos A, Sotiriadou N, Demetzos C. (2006) Cytotoxic and antitumor activity of liposome-incorporated sclareol against cancer cell lines and human colon cancer xenografts. Pharmacol Res 53: 80–87 [DOI] [PubMed] [Google Scholar]
  20. Jackson M, Severson RF, Sisson VA, Stephenson MG. (1999) Ovipositional response of tobacco budworm moths (Lepidoptera: Noctuidae) to cuticular labdanes and sucrose esters from the green leaves of Nicotiana glutinosa L. (Solanaceae). J Chem Ecol 17: 2489–2506 [DOI] [PubMed] [Google Scholar]
  21. Kanno Y, Otomo K, Kenmoku H, Mitsuhashi W, Yamane H, Oikawa H, Toshima H, Matsuoka M, Sassa T, Toyomasu T. (2006) Characterization of a rice gene family encoding type-A diterpene cyclases. Biosci Biotechnol Biochem 70: 1702–1710 [DOI] [PubMed] [Google Scholar]
  22. Keeling CI, Dullat HK, Yuen M, Ralph SG, Jancsik S, Bohlmann J. (2010) Identification and functional characterization of monofunctional ent-copalyl diphosphate and ent-kaurene synthases in white spruce reveal different patterns for diterpene synthase evolution for primary and secondary metabolism in gymnosperms. Plant Physiol 152: 1197–1208 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Martin DM, Faldt J, Bohlmann J. (2004) Functional characterization of nine Norway spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily. Plant Physiol 135: 1908–1927 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Matsingou C, Dimas K, Demetzos C. (2006) Design and development of liposomes incorporating a bioactive labdane-type diterpene: in vitro growth inhibiting and cytotoxic activity against human cancer cell lines. Biomed Pharmacother 60: 191–199 [DOI] [PubMed] [Google Scholar]
  25. Matsingou C, Hatziantoniou S, Georgopoulos A, Dimas K, Terzis A, Demetzos C. (2005) Labdane-type diterpenes: thermal effects on phospholipid bilayers, incorporation into liposomes and biological activity. Chem Phys Lipids 138: 1–11 [DOI] [PubMed] [Google Scholar]
  26. McConkey ME, Gershenzon J, Croteau RB. (2000) Developmental regulation of monoterpene biosynthesis in the glandular trichomes of peppermint. Plant Physiol 122: 215–224 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nemoto T, Cho EM, Okada A, Okada K, Otomo K, Kanno Y, Toyomasu T, Mitsuhashi W, Sassa T, Minami E, et al. (2004) Stemar-13-ene synthase, a diterpene cyclase involved in the biosynthesis of the phytoalexin oryzalexin S in rice. FEBS Lett 571: 182–186 [DOI] [PubMed] [Google Scholar]
  28. Nielsen H, Engelbrecht J, Brunak S, von Heijne G. (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10: 1–6 [DOI] [PubMed] [Google Scholar]
  29. Otomo K, Kanno Y, Motegi A, Kenmoku H, Yamane H, Mitsuhashi W, Oikawa H, Toshima H, Itoh H, Matsuoka M, et al. (2004a) Diterpene cyclases responsible for the biosynthesis of phytoalexins, momilactones A, B, and oryzalexins A-F in rice. Biosci Biotechnol Biochem 68: 2001–2006 [DOI] [PubMed] [Google Scholar]
  30. Otomo K, Kenmoku H, Oikawa H, Konig WA, Toshima H, Mitsuhashi W, Yamane H, Sassa T, Toyomasu T. (2004b) Biological functions of ent- and syn-copalyl diphosphate synthases in rice: key enzymes for the branch point of gibberellin and phytoalexin biosynthesis. Plant J 39: 886–893 [DOI] [PubMed] [Google Scholar]
  31. Pateraki I, Kanellis AK. (2004) Isolation of high-quality nucleic acids from Cistus creticus ssp. creticus and other medicinal plants. Anal Biochem 328: 90–92 [DOI] [PubMed] [Google Scholar]
  32. Prisic S, Xu M, Wilderman PR, Peters RJ. (2004) Rice contains two disparate ent-copalyl diphosphate synthases with distinct metabolic functions. Plant Physiol 136: 4228–4236 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ravn MM, Coates RM, Flory JE, Peters RJ, Croteau R. (2000) Stereochemistry of the cyclization-rearrangement of (+)-copalyl diphosphate to (−)-abietadiene catalyzed by recombinant abietadiene synthase from Abies grandis. Org Lett 2: 573–576 [DOI] [PubMed] [Google Scholar]
  34. Ro DK, Arimura G, Lau SY, Piers E, Bohlmann J. (2005) Loblolly pine abietadienol/abietadienal oxidase PtAO (CYP720B1) is a multifunctional, multisubstrate cytochrome P450 monooxygenase. Proc Natl Acad Sci USA 102: 8060–8065 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ro DK, Bohlmann J. (2006) Diterpene resin acid biosynthesis in loblolly pine (Pinus taeda): functional characterization of abietadiene/levopimaradiene synthase (PtTPS-LAS) cDNA and subcellular targeting of PtTPS-LAS and abietadienol/abietadienal oxidase (PtAO, CYP720B1). Phytochemistry 67: 1572–1578 [DOI] [PubMed] [Google Scholar]
  36. Sakamoto T, Miura K, Itoh H, Tatsumi T, Ueguchi-Tanaka M, Ishiyama K, Kobayashi M, Agrawal GK, Takeda S, Abe K, et al. (2004) An overview of gibberellin metabolism enzyme genes and their related mutants in rice. Plant Physiol 134: 1642–1653 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schepmann HG, Pang J, Matsuda SP. (2001) Cloning and characterization of Ginkgo biloba levopimaradiene synthase which catalyzes the first committed step in ginkgolide biosynthesis. Arch Biochem Biophys 392: 263–269 [DOI] [PubMed] [Google Scholar]
  38. Scott DJ, da Costa BM, Espy SC, Keasling JD, Cornish K. (2003) Activation and inhibition of rubber transferases by metal cofactors and pyrophosphate substrates. Phytochemistry 64: 123–134 [DOI] [PubMed] [Google Scholar]
  39. Seo S, Seto H, Koshino H, Yoshida S, Ohashi Y. (2003) A diterpene as an endogenous signal for the activation of defense responses to infection with tobacco mosaic virus and wounding in tobacco. Plant Cell 15: 863–873 [PMC free article] [PubMed] [Google Scholar]
  40. Stofer Vogel B, Wildung MR, Vogel G, Croteau R. (1996) Abietadiene synthase from grand fir (Abies grandis): cDNA isolation, characterization, and bacterial expression of a bifunctional diterpene cyclase involved in resin acid biosynthesis. J Biol Chem 271: 23262–23268 [DOI] [PubMed] [Google Scholar]
  41. Tamura K, Dudley J, Nei M, Kumar S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596–1599 [DOI] [PubMed] [Google Scholar]
  42. Wilderman PR, Xu M, Jin Y, Coates RM, Peters RJ. (2004) Identification of syn-pimara-7,15-diene synthase reveals functional clustering of terpene synthases involved in rice phytoalexin/allelochemical biosynthesis. Plant Physiol 135: 2098–2105 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Xu M, Hillwig ML, Prisic S, Coates RM, Peters RJ. (2004) Functional identification of rice syn-copalyl diphosphate synthase and its role in initiating biosynthesis of diterpenoid phytoalexin/allelopathic natural products. Plant J 39: 309–318 [DOI] [PubMed] [Google Scholar]

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