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Keywords:

  • Citrus limon;
  • functional expression;
  • (+)-limonene synthase;
  • (–)-β-pinene synthase;
  • γ-terpinene synthase

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the peel of young developing fruit, four monoterpene synthase cDNAs were isolated that appear to be new members of the previously reported tpsb family. Based on sequence homology and phylogenetic analysis, these sequences cluster in two separate groups. All four cDNAs could be functionally expressed in Escherichia coli after removal of their plastid targeting signals. The main products of the enzymes in assays with geranyl diphosphate as substrate were (+)-limonene (two cDNAs) (–)-β-pinene and γ-terpinene. All enzymes exhibited a pH optimum around 7; addition of Mn2+ as bivalent metal ion cofactor resulted in higher activity than Mg2+, with an optimum concentration of 0.6 mm. Km values ranged from 0.7 to 3.1 µm. The four enzymes account for the production of 10 out of the 17 monoterpene skeletons commonly observed in lemon peel oil, corresponding to more than 90% of the main components present.

Abbreviations
e.e.

enantiomeric excess (|%R − %S|)

MDGC-MS

multidimensional tandem GC-MS system.

Lemon, Citrus limon (L.) Burm. f., is a member of the large Rutaceae family containing 130 genera in seven subfamilies, with many important fruit and essential oil producers. Lemon essential oil has the highest import value of all essential oils imported to the USA and is widely used as flavouring agent in bakery, as fragrance in perfumery and also for pharmaceutical applications [1]. The essential oil is produced from the peel or flavedo of the fruit. This layer consists of the epidermis covering the exocarp consisting of irregular parenchymatous cells, which are completely enclosing numerous glands or oil sacs. Below this green layer in maturing fruits is the albedo layer (mesocarp), a thick spongy white mass of tissue, rich in pectins, surrounding the fleshy, juicy interior of the fruit. Aldehydes, such as citral are minor components present in the C. limon essential oil. However, they contribute more to the characteristic flavour than the bulk components which are the olefinic monoterpenes [1]. Monoterpenes are the C10 branch of the terpene family and consist of two head to tail coupled isoprene units (C5). They are beneficial for plants as they function in the defence against herbivores and plant pathogens or as attractants for pollinators. Sites for biogenesis of monoterpenes have been investigated extensively. In gymnosperms, such as grand fir, terpenes are produced in resin ducts [2,3]. Their biosynthesis is induced upon wounding [4–6], indicating their role in the defence against bark beetle infestation. For angiosperms many investigations have been carried out on Labiatae, especially on Mentha species, where monoterpenes are formed in the glandular trichomes, and on the umbelliferous caraway, where monoterpenes are produced in essential oil ducts of the fruits [7–12]. In Citrus, the specialized structures for the storage and accumulation of large amounts of terpenes are the glands in the flavedo, the so-called secretory cavities. Research on lemon showed that these cavities develop schizogenously on most aerial plant parts [3,13]. The cells lining these secretory cavities are thought to be responsible for the production of the terpenoids [13]. In cold pressed lemon peel oil from different origins, around 61% of the total monoterpene content consists of limonene together with lower levels of β-pinene (17%) and γ-terpinene (9%) [1]. Recently, the enantiomeric composition of some of the chiral terpene olefins present in the lemon oil was determined using a multidimensional tandem GC-MS system (MDGC-MS) [14]. The main chiral components of the cold pressed lemon oil were 4R-(+)-limonene with 96.6% enantiomeric excess (e.e.), and (–)-(1S,5S)-β-pinene with 88% e.e. [14].

The main monoterpenes of lemon can be obtained by heterologous expression of enzymes from several plant species that were isolated using a number of different strategies. cDNAs encoding (–)-limonene synthase were previously isolated from several Mentha species, Abies grandis and Perilla frutescens, using a PCR based approach, with sequence information obtained by protein sequencing of the purified enzyme [10], or by using the first cloned Mentha spicata cDNA as a probe [15]. For A. grandis homology-based cloning, degenerate PCR primers based on conserved domains of a number of terpene synthase genes were used [16]. So far only one cDNA encoding a (+)-limonene synthase has been isolated from Schizonepeta tenuifolia, a member of the Labiatae family [17].

(–)-(1S,5S)-β-Pinene was the major product of a β-pinene synthase cDNA from Artemisia annua submitted to GenBank (accession no. AF276072), and of a (–)-(1S,5S)-pinene synthase that was previously isolated from A. grandis[16]. This enzyme produces 58% (–)-(1S,5S)-β-pinene, but also 42% (–)-(1S,5S)-α-pinene. A cDNA encoding γ-terpinene synthase as its main activity has not been reported on yet.

Although the composition of lemon essential oil has had considerable attention and enzymes responsible for the production of monoterpenes in the peel of lemon have been partially purified [18], their corresponding cDNAs have never been isolated and characterized. So far only the cDNA of a sesquiterpene synthase producing (E)-β-farnesene as main product has been described from Citrus junos[19]. Here we report on the isolation of four new monoterpene synthase cDNAs by random sequencing of a flavedo-derived cDNA library of C. limon and their characterization by functional expression in Escherichia coli.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Plant material, substrate, and reagents

Lemon plants [C. limon (L.) Burm. f.], obtained from a nursery in Sicily, Italy, were grown in pots in the greenhouse in peat moss/clay mixture (50 : 50, v/v), under 18 h supplemental lighting provided by two 400-W high pressure sodium lamps (Philips, Eindhoven, the Netherlands), at 28 °C/20 °C (day/night) temperature cycle. Plants were watered as needed and fertilized weekly with a liquid fertiliser.

[1-3H]Geranyl diphosphate and [1-3H]farnesyl diphosphate were obtained from American Radiochemicals Inc. (St Louis, MO, USA) and Amersham Biosciences (Piscataway, NJ, USA), respectively. Unlabelled geranyl diphosphate and farnesyl diphosphate were purchased from Sigma–Aldrich (Sigma–Aldrich, Chemie b.v., Zwijndrecht, the Netherlands) and were used after a buffer change as described for farnesyl diphosphate [20].

Unless otherwise stated, reagents were obtained from Sigma–Aldrich. DNA sequences were assembled and analysed using mnastar software (DNASTAR, Inc., Madison, WI, USA). Sequencing primers were purchased from either Isogen Bioscience (Maarssen, the Netherlands) or Amersham Biosciences. Sequencing reagents were supplied by PerkinElmer (Foster City, CA, USA). Restriction enzymes, enzymes and buffers used were from Gibco BRL (Invitrogen corporation, Breda, the Netherlands). DNA fragments were isolated from Agarose gel by a GFX™ PCR DNA and Gel band purification kit (Amersham Biosciences). Amino-acid alignment was made using mnastar-mnastar 1.81, with Gonnet250 matrix and default settings.

Phylogenetic analysis was carried out using mnastar-mnastar 1.81, with PAM350 matrix [multiple alignment parameters: gap opening set at 10 (default), gap extension set at 2 (0.2 is default)] and the neighbour joining method for calculating the tree [21,22]. The bootstrapped tree was corrected for multiple substitutions as recommended by the program [23].

Hydro distillation of C. limon peel

Samples of lemon flavedo (0.5 g) from green fruits (2 × 1 cm) were ground in liquid N2 and used for hydro distillation with ethyl acetate as a keeper as previously described [24]. After a 1 : 200 dilution, 2 µL of the ethylacetate phase was injected into a GC-MS using an HP5890 series II gas chromatograph (Hewlett Packard, Agilent Technologies, Alpharetta, GA, USA) and an HP 5972A Mass Selective Detector essentially as described previously [25]. The GC was equipped with an HP-5MS column (30 m × 0.25 mm internal diameter, film thickness = 0.25 µm) and programmed at an initial temperature of 45 °C for 1 min, with a ramp of 10 °C·min−1 to 280 °C, and final time of 10 min. Products were identified by comparison of retention times and mass spectra with authentic reference compounds. The α-thujene standard was purchased from Indofine (Indofine Chemical Company Inc., Hillsborough, NJ, USA).

RNA isolation, cDNA library construction, random sequencing and library screening

Plant material from a fruit bearing C. limon plant was harvested and frozen directly in liquid N2. Total RNA for cDNA library construction was isolated from the flavedo layer of 2 × 1 cm young green fruits, according to a slightly modified RNA isolation protocol for recalcitrant plant tissues [26], by using maximally 2.5 g of tissue per 30 mL RNA extraction buffer. mRNA was extracted from the total RNA using a mRNA purification kit according to manufacturers recommendations (Amersham Biosciences). Of this amount 15 µg was used to construct a custom cDNA UNI-ZAP XR™ library (Stratagene Europe, Amsterdam Zuidoost, the Netherlands).

Mass excision

The E. coli strains XL1-MRF′ and SOLR were used for mass excision according to the manufacturers recommendations (Stratagene). One-hundred and fifty microliters of the primary unamplified library was mixed with 150 µL of XL-1 MRF′ cells (D600 = 1), with 20 µL of helper phage (Stratagene). The mix was grown for only 2.5 h in order to minimize disturbance of the clonal representation. Finally, for 100 single colonies to be picked 1–3 µL of the resulting phagemids was used each time to infect 200 µL of SOLR cells and the next day single colonies were picked from Luria–Bertani plates.

DNA isolation and sequencing

Plasmid DNA was isolated from overnight grown bacterial cultures using a Qiaprep 96 Turbo kit on a Qiagen Biorobot 9600 according to the manufacturers recommendations (Qiagen GmbH, Hilden, Germany). Between 0.5 and 3 µL of plasmid DNA was used for sequencing isolated clones using Ready Reaction Dye Terminator Cycle mix (PerkinElmer) and 100 ng of pBluescript SK primer (5′-CGC TCTAGAACTAGTGGATC-3′). Sequencing PCR was performed according to the manufacturers recommendations (PerkinElmer) in a MJ research PTC Peltier thermal cycler (MJ Research Inc., Watertown, MC, USA). After precipitation and dissolving in TSR buffer (PerkinElmer), the samples were sequenced on an ABI 310 capillary sequencer (PerkinElmer). A total of 960 clones were sequenced and analysed for homology to known genes by using the mnastar and mnastar programs of the NCBI (http://www.ncbi.nlm.nih.gov/blast/blast.cgi).

Full length sequencing and cloning

After sequencing, nine putative terpene synthase genes were identified, representing three different clones. These clones, B93, C62 and D85 were full length sequenced by designing sequence specific overlapping primers based on the obtainedsequence information. On the basis of sequence alignments, sequences that were most distant to each other were selected for further screening of the cDNA library.

Using clones B93 and C62 as 32P-labelled probes, 75 µL of the custom unamplified cDNA library (Stratagene) from lemon was screened by plaque lifts using Hybond N+ nylon membranes according to the manufacturers recommendations (Amersham Biosciences). Hybridization was performed at 55 °C in buffer containing 10% dextran sulfate (Amersham Biosciences), 1 mnastar NaCl and 1% (w/v) SDS. Filters were washed three times at 55 °C, once in 4 × NaCl/Cit and 0.1% (w/v) SDS and twice in 2 × NaCl/Cit and 0.1% (w/v) SDS. Plaques that were radioactively labelled were picked and using the single clone excision protocol, separate E. coli SolR colonies were obtained from the cDNA library as described in the Unizap-XR manual (Stratagene). After growth and subsequent DNA isolation the clones were sequenced as described above.

cDNA expression in E. coli

For putative targeting signal prediction the computer programs mnastar and mnastarr were used, which gave scores for the most likely localization of the proteins. A description of the interpretation is given on the websites (http://www.inra.fr/servlets/WebPredotar;http://www.cbs. dtu.dk/services/TargetP/)

The four clones were subcloned in truncated form in order to exclude the putative plastid-targeting signal from being expressed, because this can lead to the formation of inclusion bodies [27]. The conserved N-terminal amino-acid sequence of the RR motif was shown not to be required for functional expression of monoterpene synthases in E. coli. Removing this sequence drastically improved the activity of the isolated enzymes [27]. The clones were truncated and religated in the pBluescript SK vector in frame with the LacI promoter for induced expression by isopropyl thio-β-mnastarr-galactoside as previously described [28]. Primers for truncation were designed on the 5′ end of the sequences to include a methionine preceding the RR motif and a restriction site for in-frame cloning with the LacI promoter. PCR amplification was carried out using Pfu polymerase with the T7 primer and a gene specific restriction site containing primer on an MJ research PTC Peltier thermal cycler (94 °C, 30 s; 50 °C, 30 s; 72 °C, 2 min; 30 cycles). The sense primer for B93 contained a PstI restriction site 5′-GCCAACTGCAGAATGAGGCGATCTGCCGATT ACG-3′. The sense primer for C62 and M34 was 5′-GCCAGGATCCAATGAGGAGATCAGCAAACTA CC-3′, containing a BamHI restriction site. The sense primer for D85 contained a BamHI restriction site 5′-GCCAGGATCCAATGAGGCGATCTGCTGATTA CG-3′. PCR products were digested using the restriction sites introduced by the sense primers and restriction sites in the 3′ multiple cloning site of pBluescript, that was included in the PCR fragment by amplification with the T7 primer. The pBluescript expression vectors with the truncated cDNA clones were obtained using standard molecular biological techniques [29]. The clones were fully resequenced after subcloning to check for unwanted changes in the ORF.

For cloning the monoterpene synthases including a His-tag for easy purification, the expression vector pRSET B (Invitrogen corporation) was used for the expression of the four putative full-length monoterpene synthases in E. coli (Stratagene: BL21-CodonPlus™-RIL strain), using the original pRSET B vector as negative control for the experiments. For all four clones, primers for amplification of the truncated cDNAs including the RRX8W motif were designed. PCR amplification was performed for all clones using Pfu turbo DNA polymerase (Stratagene) and the same programme on a MJ research PTC Peltier thermal cycler (94 °C, 30 s; 55 °C, 30 s; 72 °C, 2 min; 30 cycles). For clone B93 a sense primer including a BglII restriction site, named B93HISFBGL (5′-AGAGTCAGATCTTAGGCG ATCTGCCGATTACG-3′) was designed. The clone was amplified using this primer and a T7 primer (5′-GTAAT ACGACTCACTATAGGGC-3′). In the 3′ UTR of the gene another BglII site was present, providing a PCR fragment after digestion that could be directly ligated to a BamHI digested pRSET B vector after dephosphorylation using calf intestinal alkaline phosphatase.

In the 3′ UTR of the C62 clone, a SalI site was introduced to facilitate cloning, by the Quickchange Site Directed Mutagenesis PCR method (Stratagene) according to the manufacturers recommendations and the following program (95 °C, 30 s; 55 °C, 1 min; 68 °C, 10 min; 14 cycles). The complementary primers used were C62FOR (5′-GCAGTTTCAGTCGACGTTGGCCTCCAC-3′) and C62REV (5′-GTGGAGGCCAACGTCGACTGAAACT GC-3′). Only the two underlined nucleotides were altered. The resulting 3′ UTR modified pBluescript C62 clone was used as template for cloning into the PRSET B vector. A sense primer including a BglII restriction site, named C62HISFBGL (5′-CTTGACAGATCTTAGGAGATCA GCAAACTAC-3′) was used together with the T7 primer to amplify the cDNA. After purification from the gel, the PCR fragment was digested with BglII and SalI and ligated to a pRSET B vector fragment digested with compatible BamHI and XhoI sites.

For D85 a sense primer including a BglII site (5′-AGA GTCAGATCTTAGGCGATCTGCTGATTACG-3′) was used together with the T7 primer to amplify the cDNA. After gel purification of the PCR product it was digested with BglII and AflIII restriction enzymes, AflIII cuts in the 3′ UTR of the cDNA. The digested fragment was ligated to thecompatible sites of pRSET B digested with BamHI and NcoI.

For subcloning the M34 clone the sense primer C62HIS FBGL and the antisense primer M34HISXHO (5′-TGAT CACTCGAGGAATTCGCAACGCATCG-3′), annealing in the 3′ UTR of the cDNA introducing an XhoI site, were used. After PCR the product isolated from the gel was digested with BglII and XhoI and ligated to PRSET B vector digested with BamHI and XhoI.

All the ligations were transformed to E. coli strain XL1-blue MRF′ supercompetent cells (Stratagene). Isolated DNA from bacterial colonies was fully resequenced in order to check for orientation, mutations and if the gene was integrated in the right frame, resulting in a fusion protein at the N-terminus with a peptide that included an ATG translation initiation codon, a series of six histidine residues (His-tag), and an anti-Xpress (Invitrogen) epitope. Plasmid DNA of the four pRSET B clones and the control (original pRSET B vector) were transformed to BL21-CodonPlus™-RIL competent cells according to the manufacturers recommendations (Stratagene).

Protein expression

The pBluescript expression vectors were induced for protein expression and after centrifugation, the bacterial pellets were dissolved in assay buffer exactly as described previously [28].

For induction of protein expression of the His-tag vectors, single colonies were picked from the Luria–Bertani 100 mg·L−1 ampicillin plates with the BL21 transformations containing the putative terpene synthases and the original pRSET vector. They were transferred to 5 mL Luria–Bertani broth supplemented with 100 mg·L−1 ampicillin and grown overnight. Aliquots of 0.5 mL were used to inoculate 250 mL concial flasks containing 50 mL Luria–Bertani broth with ampicillin (50 µg·mL−1) and chloramphenicol (37 µg·mL−1). This was grown at 37 °C with vigorous agitation to D600 = 0.6. For induction of expression isopropyl thio-β-mnastarr-galactoside was added to a final concentration of 1 mmnastarr and the cultures were grown at 20 °C overnight with agitation at 250 r.p.m. Proteins were isolated using His-tag purification by passing the lysate over Ni-nitrilotriacetatic acid spin columns according to the manufacturers recommendations (Qiagen). After washing, the bound protein was eluted using the buffer recommended by the manufacturer containing 50 mmnastarr NaH2PO4, 300 mmnastarr NaCl and 250 mmnastarr imidazole pH 8, and the eluted protein was supplemented with glycerol to 30% and stored at −70 °C. For protein concentration measurement the proteins were first precipitated in 10% trichloroacetic acid on ice for 15 min, followed by centrifugation for 10 min. The resulting pellet was washed twice with acetone and after drying dissolved in 5 mmnastarr Tris, pH 6.8, 0.2% (w/v) SDS and 1% glycerol. Protein concentration was determined using the BCA Protein assay kit using BSA as protein standard reference, according to the manufacturers recommendations (Pierce, Rockford, IL, USA).

Enzymatic characterization of the four recombinant citrus clones

Enzyme assay.  Ten microlitres or less of the eluted His-tagged purified protein was used in each assay to check for enzymatic activity. In most cases it was necessary to dilute the enzyme further to guarantee linearity. The assay buffer was a 15 mmnastarr Mopso buffer (pH 7) containing 10% glycerol, 1 mmnastarr ascorbic acid and 2 mmnastarr dithiothreitol. The putative synthases were tested for activity with 2 µmnastarr[1-3H]geranyl diphosphate (740 GBq·mmol−1) or 20 µmnastarr[1-3H]farnesyl diphosphate (555 GBq·mmol−1). For geranyl diphosphate they were incubated with varying concentrations of either 0.05–1.5 mmnastarr MnCl2 or 2.5–15 mmnastarr MgCl2 as cofactors to check their specific bivalent metal ion preference, for farnesyl diphosphate only 10 mmnastarr MgCl2 was used. The synthases were also tested without addition of metal ions. The reaction was performed in a total volume of 100 µL and before incubation for 30 min at 30 °C with gentle shaking, the assay was overlaid with 1 mL of hexane. To investigate the linearity of the assays with time the enzymes were incubated for 0, 10, 20, 30, 45 and 60 min at 30 °C. For testing the pH optimum of the enzymes they were incubated in Mopso buffer with a pH ranging from 6.4 to 7.6, with intervals of 0.3 pH units. Also the affinity for the monovalent ion K+ was tested at different concentrations of KCl ranging from 0 to 150 mmnastarr. All assays were performed in duplicate. After incubation the assays were vigorously mixed and after a short centrifugation step to separate phases, 500 µL of the hexane phase from each sample was added to 4.5 mL Ultima Gold cocktail (Liquid scintillation solution) (Packard Bioscience, Groningen, the Netherlands) for liquid scintillation counting. For Km determination the enzymes were incubated with geranyl diphosphate concentrations ranging from 1 µmnastarr to 180 µmnastarr for β-pinene and γ-terpinene synthase, or 0.1–100 µmnastarr for both limonene synthases, at 0.6 mmnastarr MnCl2 and pH 7. For some concentrations of [1-3H]geranyl diphosphate buffer controls were used to estimate background levels of hexane soluble radioactivity. After the assays the hexane phase was removed and mixed with about 20 mg of silica to remove any nonspecific polar compounds. After centrifugation at 10 000 g for 10 min, 500 μL of the hexane phase was used for scintillation counting as described above. For the analysis of product formation the same procedure was followed, but in larger volumes. Two hundred microliters of enzyme was used in a total reaction volume of 1 mL, including 10 mmnastarr MgCl2, or 0.6 mmnastarr MnCl2. For analysis on GC-MS 50 µmnastarr geranyl diphosphate, and for analysis using radio-GC 20 µmnastarr[1-3H]geranyl diphosphate (740 GBq·mmol−1) was used as a substrate. After the addition of a 1-mL redistilled pentane overlay, the tubes were carefully mixed and incubated for 1 h at 30 °C. Following the assay, the tubes were vortexed, the organic layer was removed and passed over a short column of aluminium oxide (Al2O3) overlaid with anhydrous Na2SO4. The assay mixture was re-extracted with 1 mL of pentane: diethyl ether (80 : 20), which was also passed over the aluminium oxide column, and the column washed with 1.5 mL of diethyl ether. 100 µL from each sample was added to 4.5 mL Ultima Gold cocktail for scintillation counting.

Samples of the pentane/ether fraction were analysed using GC-MS as described above and on a radio-GC consisting of a Carlo-Erba 4160 Series gas chromatograph (Carlo-Erba, Milano, Italy) equipped with a RAGA-90 radioactivity detector (Raytest, Straubenhardt, Germany) essentially as described previously [30].

Mdgc-ms

The enantiomeric distribution of the main and the side products produced by the monoterpene synthases, with the cold assays, were analysed using MDGC-MS. The MDGC-MS analyses were performed with a Fisons 8160 GC connected to a Fisons 8130 GC and a Fisons MD 800 quadrupole mass spectrometer and using Fisons mnastarr v1.3 (Fisons, Manchester, UK). The system setup was as described previously although the settings were different [31]. The fused silica capillary column in GC1 (J & W, Folsom, CA, USA) DB-Wax 20 M (25 m × 0.25 mm internal diameter; film thickness = 0.25 µm) was maintained at 40 °C then programmed to 240 °C at 1 °C·min−1 (sabinene and pinene preseparation) and at 50 °C then programmed to 240 °C at 3 °C·min−1 (limonene preseparation) with He gas flow at 3 mL·min−1. The fused silica capillary column in GC2 (J & W Cyclodex B (30 m × 0.25 mm internal diameter; film thickness = 0.25 µm) was maintained at 45 °C (12 min) then programmed to 200 °C at 5 °C·min−1 with He gas flow at 3 mL·min−1. The compounds of interest were transferred from GC1 to GC2 from 6.6 min to 7.1 min (α-pinene) and 10.2 min to 10.4 min (β-pinene). The fused silica capillary column in GC2 (30% 2,3-diethyl-6-tert-butyl-dimethyl-β-cyclodextrin/PS086 (25 m × 0.25 mm internal diameter; film thickness = 0.15 µm) was maintained at 60 °C (15 min) then programmed to 200 °C at 0.5 °C·min−1 with He gas flow at 3 mL·min−1. The compounds of interest were transferred from GC1 to GC2 from 9.3 min to 9.7 min (limonene) and 11.1 min to 11.5 min (sabinene). The MS operating parameters were ionization voltage, 70 eV (electron impact ionization); ion source and interface temperature, 230 °C and 240 °C, respectively.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Monoterpene content of lemon fruits

The monoterpene content of young lemon fruits was analysed using GC-MS. The major monoterpene was identified as limonene (75%), followed by γ-terpinene (11%) and β-pinene (4%); some p-cymene (2%), α-pinene (1%) and myrcene (1%) were also detected. Trace levels (below 1%) were found of the monoterpenoids α-thujene, sabinene, α-terpinene (E)-β-ocimene, terpinolene, linalool and α-terpineol.

cDNA isolation and sequencing

Random sequencing of a cDNA library made from mRNA isolated from the peel of young lemon fruits resulted in the identification of nine putative monoterpene synthase genes. mnastarr searches using the first 500 bp of the 5′ side of the ESTs showed significant sequence homology (all with Expect score below 1 × 10−9) with other monoterpene synthases reported in the GenBank mnastarr database (NCBI; http://www.ncbi.nlm.nih.gov/BLAST/) [32]. The nine ESTs all proved to be full-length cDNAs and were found to represent three different clones, designated B93, C62 and D85. The cDNA library was rescreened with the two most divergent clones as probe under low stringency, and the positive plaques were sequenced. This rescreening yielded one additional putative monoterpene synthase, designated as M34, with a high level of identity to one of the already isolated cDNAs. The nucleotide sequences of B93, C62, D85 and M34 have been submitted to GenBank and are available under accession nos AF51486, AF514287, AF514288 and AF514289, respectively.

Sequence analysis

The cDNAs all encoded full-length putative monoterpene synthases from 600 to 606 amino acids long with a calculated molecular mass of around 70 kDa. According to targeting signal prediction programs mnastarr and mnastarr they all had a cleavable transit peptide for plastid localization. The scores of the mnastarr program for chloroplast transit peptide, were in all cases higher than scores for targeting to other cell compartments. The lengths of the preproteins were predicted to be 22–40 amino acids. mnastarr gave significantly higher scores for plastid localization than for mitochondrial localization.

The deduced amino-acid sequences of the four lemon cDNAs were aligned with their closest homologues in GenBank: St(+)LIMS (Schizonepeta tenuifolia (+)-limonene synthase: (Q9FUW5) [17]), QiMYRS (Quercus ilex myrcene synthase: (Q93×23) [33]) and Aa(–)βPINS (Artemisia annua (–)-β-pinene synthase: (Q94G53) (Fig. 1). The alignment illustrates many conserved regions between these seven monoterpene synthases from different plant species. The previously reported conserved amino acids for terpene synthases are all found in the four new sequences and they are indicated with an asterisk [34]. The levels of identity to the lemon monoterpene synthases range from 42 to 60%, when the sequences are aligned from the RRX8W motif onwards, from where significant similarity starts (Table 1). This RRX8W motif, located at the N-terminus, is conserved amongst all the monoterpene synthases depicted in Fig. 1. The sequences of the lemon monoterpene synthases cluster into two separate groups. One group consists of B93 and D85, showing 84% identity. The other group consists of C62 and M34 that show 97% identity. Between the groups the identity is not higher than 51%. For the putative targeting signals there is a clear relation between B93 and D85. The identity of the sequences of B93 and D85 up to the RRX8W motif is 90%. They are very different from the targeting signals of C62 and M34 (16% identity), which are again very similar to each other (91% identity).

image

Figure 1. Alignment of deduced amino-acid sequences of monoterpene synthases of the tpsb family to the lemon monoterpene synthases. Cl(+)LIMS1 (C62, lemon (+)-limonene synthase 1), Cl(+)LIMS2 (M34, lemon (+)-limonene synthase 2), St(+)LIMS (Schizonepeta tenuifolia (+)-limonene synthase, accession number: Q9FUW5 [17]), QiMYRS (Quercus ilex myrcene synthase, accession number: Q93×23 [33]), ClγTS (B93, lemon γ-terpinene synthase), Cl(–)βPINS (D85, lemon (–)-β-pinene synthase), Aa(–)βPINS (Artemisia annua (–)-β-pinene synthase, accession number: Q94G53). The alignment was created with the mnastarrx program using the Gonnet matrix. Shading indicates conserved identity for the aligned amino acids: black background shading indicates 100% conservation, dark grey shading indicates 80% conservation, and light grey shading indicates 60% conservation. Asterisks indicate residues that are highly or absolutely conserved between all plant terpene synthases [34]. The highly conserved RRx8W motif, directly after the supposed plastid targeting signal, and the metal ion-binding motif DDxxD are indicated below the sequence alignments.

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Table 1.  Analysis of sequence identity levels (%) between cDNAs of C. limon and some other monoterpene synthases. Swiss-Prot accession numbers: QiMYRS (Quercus ilex myrcene synthase): Q93×23. Aa(–)βPINS (Artemisia annua (–)β-pinene synthase): Q94G53, St(+)LIMS (Schizonepeta tenuifolia (+)-limonene synthase): Q9FUW5. In the alignments up to the DDXXD motif, the targeting signal was not taken into account.
  B93D85C62M34
  1. a Truncated cDNA is the cDNA without the supposed targeting signal. Targeting signal is considered as the N-terminal sequence until the RRX 8W motif.

Truncated cDNAaB93 845051
D85  4849
C62   97
St(+)LIMS42424546
QiMYRS60605555
Aa(–)βPINS49494445
Targeting signalaB93 901616
D85  1618
C62   91
Up to DDXXD motifB93 894850
D85  4950
C62   96
From DDXXD motifB93 785454
D85  4950
C62   98

In a phylogenetic analysis the separate clustering within the tpsb family of C62 and M34 from B93 and D85 is clear (Fig. 2). The B93 and D85 sequences group together with the myrcene synthase from Q. ilex and the A. annua monoterpene synthases while the limonene synthases from C. limon form a distinct branch.

image

Figure 2. Phylogram of mnastarrx alignment of dicotyledonous C5 to C15 terpene synthases using PAM350 matrix and the neighbour joining method. The tree was corrected for multiple substitutions. The sesquiterpene synthases (tpsa) were defined as outgroup and the tree was rooted with the outgroup. The lemon synthases are located in the tpsb family. Scale bar: 0.1 is equal to 10% sequence divergence. Bootstrap values are given for nodes, and are considered as a value for significance of the branches. Values higher than 850 are likely to be significant.

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Functional expression of the putative monoterpene synthases in E. coli

The putative monoterpene synthases were expressed without the plastid targeting signals in order to prevent inclusion bodies of the expressed protein [27]. Although the precise cleavage site is not yet known for terpene synthase preproteins, truncation of monoterpene synthases upstream of the conserved tandem arginine motif (RRX8W) has been demonstrated to result in fully active enzymes [27,35,36]. Enzyme activity was verified using radio-GC. Although the pentane fractions of the assays showed the main nonalcoholic products of the synthases, the high activity of aspecific phosphohydrolases in the crude E. coli lysates also resulted in production of large amounts of geraniol (data not shown), competing for the radiolabeled substrate. Therefore the cloning of the synthases truncated at the RRX8W motif was repeated in the pRSET vector (Invitrogen), which contains a His-tag for purification of the expressed protein. The pRSET vectors were expressed in E. coli Bl21-DE3-RIL cells. This strain contains the RIL plasmid for expression of tRNA codons that are rare in E. coli, to give better expression and accumulation of the protein. In small scale assays, the His-tag purified enzymes were analysed for activity by scintillation counting using [1-3H]geranyl diphosphate and [1-3H]farnesyl diphosphate as substrates. The enzymes all proved to be active with geranyl diphosphate and not with farnesyl diphosphate (data not shown).

GC-MS analysis

GC-MS analysis demonstrated that the cDNA-encoded enzymes produced three different major products (Fig. 3). B93 produced γ-terpinene and is therefore designated ClγTS (Fig. 3B), C62 and M34 both produced limonene and are designated Cl(+)LIMS1 and Cl(+)LIMS2, respectively (Fig. 3C,E) and D85 produced β-pinene and is designated Cl(–)βPINS (Fig. 5D). The chirality of the products was determined using MDGC-MS, as described in the next section. Also side products and their abundance were determined for each synthase (Fig. 3, Table 2). Concentration of the samples showed additional side product traces. No monoterpene products were detected in the pRSET empty vector control (Fig. 3A). The major product of ClγTS was γ-terpinene (71.4%), with lower amounts of limonene (9.1%), α-pinene (5.6%), β-pinene (4.7%), α-terpinolene (3.7%), α-thujene (2.5%), α-terpinene (1.7%), myrcene (0.9%), sabinene (0.4%) and a trace of p-cymene (Fig. 3B, Table 2). Both Cl(+)LIMS1 and Cl(+)LIMS2 produced almost exclusively limonene (99.15%), with a small amount of β-myrcene (0.85%) and a trace of α-pinene (Fig. 3C,E, Table 2). The major product of the Cl(–)βPINS enzyme was β-pinene (81.4%), with sabinene (11%), α-pinene (4.1%), limonene (3.5%) and a trace of γ-terpinene as side products (Fig. 3D, Table 2).

image

Figure 3. GC-MS profiles of products formed by the four heterologously expressed monoterpene synthases. (A) Empty pRSET vector control (B) B93 (C) C62 (D) D85 and (E) M34. B93 mainly produces γ-terpinene, C62 and M34 produce limonene and D85 mainly produces β-pinene. Peak identities were confirmed using standards, whose mass spectra and retention times exactly matched these products. The mass spectra of the main products and their standards are depicted next to each chromatogram. Monoterpenes are numbered: 1,α-thujene; 2, α-pinene; 3, sabinene; 4, β-pinene; 5, myrcene; 6, α-terpinene; 7,p-cymene; 8, limonene; 9, γ-terpinene; 10, terpinolene.

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image

Figure 5. Cl (–)βPINS enzyme activity curves. Enzyme activities were measured with substrate concentrations up to 180 µmnastarrx geranyl diphosphate. A Michaelis–Menten curve (featuring a Km of 3.1 µmnastarrx and an apparent Vmax of 28.49 µmol·h−1·mg−1) and a substrate inhibition curve (featuring a Km of 13.5 µmnastarrx, an apparent Vmax of 89.47 µmol·h−1·mg−1 and a Ksi of 5.65 µmnastarrx) were fitted to the values obtained.

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Table 2.  Ratios of products formed by the monoterpene synthases as determined by GC-MS and their corresponding enantiomeric composition as determined by MDGC-MS. The percentages of the products formed by each synthase were determined on the GC-MS without concentrating the samples. –, not detected; ND, not determined.
 ClγTS (B93)Cl(–)βPINS (D85)Cl(+)LIMS1 (C62), Cl(+)LIMS2 (M34)
 (%)(–) : (+)(%)(–) : (+)(%)(–) : (+)
  • a

    The sabinene in this sample coeluted with the myrcene on the MDGC-MS preventing accurate determination of the enantiomeric composition.

α-Thujene  2.5ND    
α-Pinene  5.662 : 38  4.193 : 713 : 87
Sabinene  0.4 a 11.087 : 13  
β-Pinene  4.72 : 9881.499.5 : 0.5  
β-Myrcene  0.9     0.85 
α-Terpinene  1.7     
p-Cymene     
Limonene  9.180 : 20  3.589 : 1199.150 : 100
γ-Terpinene71.4    
Terpinolene  3.7     

Enantiomeric analysis by MDGC-MS

The chirality of the monoterpene products was analysed on a multidimensional GC-MS (MDGC-MS) (Table 2). Both Cl(+)LIMS1 and Cl(+)LIMS2 produced exclusively R-(+)-limonene, in contrast to ClγTS and Cl(–)βPINS that produced mainly S-(–)-limonene as a side product and only a small amount of R-(+)-limonene (Fig. 4, Table 2). Cl(–)βPINS produced almost exclusively (–)-β-pinene, and 86% e.e. (|%R − %S|) of (–)-α-pinene. The sabinene side product of Cl(–)βPINS was determined to be 74% e.e. of (–)-sabinene (Table 2). ClγTS produced (–)-α-pinene as a side product with an e.e. of 24%, but (+)-β-pinene was produced with an e.e. of 96%. The chirality of the side product sabinene of ClγTS could not be determined with certainty since it coeluted with the side product myrcene. The α-pinene trace of Cl(+)LIMS2 consisted mainly of the (+)-enantiomer (Table 2).

image

Figure 4. GC-MS profiles of enantiomers of limonene formed by the different synthases. (A) shows separation of the reference limonene enantiomers. (B) and (C) show that M34 and C62 (Cl(+)LIMS1 and CL(+)LIMS2) produce R-(+)-limonene. (D) and (E) show that B93 (ClγTS) and D85 (Cl(–)βPINS) produce predominantly S-(–)-limonene as a side product.

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Characterization of the heterologously expressed enzymes

The bivalent metal ion cofactor dependence of each synthase was tested with Mn2+ and Mg2+. All synthases had around 30 times higher activity with Mn2+. The optimal Mn2+ concentration was about 0.6 mmnastarr for all four enzymes and higher concentrations inhibited enzyme activity. Mg2+ dependency was less pronounced and did not result in inhibition at concentrations up to 15 mmnastarr. K+ has been reported to strongly enhance the activity of monoterpene synthases from different plant families [37], but for the lemon monoterpene synthases, it appeared to be an inhibitor. Maximum inhibition was found for concentrations above 100 mmnastarr KCl, when ClγTS was incubated with increasing KCl concentrations (data not shown). The pH dependence was tested for all four enzymes and enzymatic activity was found to be maximal around pH 7 (data not shown). Kinetic properties of the enzymes were determined by incubating with a range of geranyl diphosphate concentrations from 0.1 to 180 µmnastarr. The monoterpene synthase enzymes all showed substrate inhibition characteristics, because the activity decreased with substrate concentrations above 10 µmnastarr.

K m values for the cyclases were determined ignoring substrate inhibition using an mnastarr template anemona.xlt [38] (available from http://genamics.com/software). Km values were 0.7 µmnastarr for both Cl(+)LIMS1 and Cl(+)LIMS2, 2.7 µmnastarr for ClγTS and 3.1 µmnastarr for Cl(–)βPINS. When the anemona mnastarr template was used to calculate substrate inhibition kinetics, the Km for Cl(–)βPINS was 13.5 µmnastarr(Fig. 5).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The four monoterpene synthase cDNAs that have been isolated and characterized here account for the formation of more than 90% of the content of lemon essential oil. Most of the monoterpenoids that were found in the young lemon peel are either main or side products of the monoterpene synthases isolated and characterized in the present paper. Only the origin of the trace amounts of linalool, α-terpineol and (E)-β-ocimene that are also present in the lemon extract remain unexplained, as they are not a product of any of the synthases presented in this paper.

To isolate these monoterpene synthases from lemon, we used a random sequencing approach on a cDNA library from young lemon flavedo. This method has previously been proven to be successful for the isolation of full length cDNAs, particularly if the source tissue of the library is highly specialized with regard to the process to be studied [39–41]. The levels of identity of the lemon monoterpene synthases indicate that they should be grouped within the tpsb clade of the angiosperm monoterpene synthases (Fig. 1, and Table 1) [34]. Although the four lemon cDNAs cluster in the same clade, they clearly form two distinct classes, one containing B93 and D85 and the other C62 and M34, because there are large differences both in the putative plastid targeting signals (only 16–18% identity) and the coding sequences (only 48–51% identity), suggesting that they have evolved separately.

This is confirmed by the phylogenetic analysis (Fig. 2). The separate clustering of the lemon genes B93, D85, Q. ilex myrcene synthase and the A. annua monoterpene synthases from the limonene synthases C62 and M34, suggests that the two groups of lemon synthases diverged in ancient times, even before Quercus and Artemisia separated from Citrus.

Monoterpene biosynthesis has been shown to be localized in the plastids in plants [9,42], and this is in accordance with the fact that all monoterpene synthases published to date bear an N-terminal transit peptide [10,15–17,28,33,35, 36,43,44]. Monoterpene synthases are nuclear encoded preproteins that are destined to be imported in the plastids, where they are proteolytically processed into their mature forms. Plastid targeting signals are typically rich in serines and threonines and low in acidic and basic amino acids and about 45–70 amino acids long. Usually they show only little homology.

The predictions using mnastarr and mnastarr indicate that all the four putative monoterpene synthases contain plastid targeting sequences. The lengths of the predicted targeting signals are rather short but the distance to the RRX8W motif, common to monoterpene synthases of the tpsb clade, from where significant homology starts with other monoterpene synthases is 52 or 55 amino acids long. The RRX8W motif is supposed to be required to give a functional mature protein and could have a function in the diphosphate migration step accompanying formation of the intermediate linalyl diphosphate before the final cyclization step catalysed by the monoterpene synthases [27]. The DDXXD motif, present in all terpene synthases, is supposed to bind the bivalent metal ion cofactor, usually Mn2+ or Mg2+ and is responsible for the ionization of the diphosphate group of geranyl diphosphate [34,45,46]. The active site domain of sesquiterpene synthases and probably also other terpene synthases is located on the C-terminal part of these proteins starting shortly before the DDXXD motif [47]. Therefore it was suggested that the C-terminal part of the terpene synthase proteins determines the final specific product outcome [35]. Less than 10% overall sequence divergence has been shown to result in a significantly different product composition [35]. Table 1 shows that the identity level before the DDXXD motif between the B93 and D85 proteins (ClγTS and Cl(–)βPINS) is higher (89%) than after the DDXXD motif (78%), suggesting that these two enzymes, although they are very homologous, are likely to catalyse the formation of two different products.

For the other two homologous protein sequences encoded by C62 and M34 (Cl(+)LIMS1 and Cl(+)LIMS2), the identity before the DDXXD motif was almost the same as from the DDXXD motif onwards. This makes it likely that these proteins catalyse the formation of identical products.

The characterization of product specificity by functional expression in E. coli of the monoterpene synthases of lemon confirmed that both C62 and M34 (Cl(+)LIMS1 and Cl(+)LIMS2) encode enzymes that specifically form a single product (+)-limonene, with only small traces of myrcene and (+)-α-pinene. Myrcene and α-pinene are trace products that were also described for (–)-limonene synthase from spearmint, but with undetermined stereochemistry [10]. Although both limonene synthase enzymes produce exclusively (+)-limonene as a main product, the stereoselectivity for the trace coproduct α-pinene is less strong.

The other two monoterpene synthases encoded by B93 and D85, which show less sequence identity, indeed produce different main products, γ-terpinene and (–)-β-pinene, respectively. Furthermore these are much less specific in their product formation, leading to formation of a number of side products (up to 11% of total). It is a common feature of many monoterpene synthases that they are able to form multiple products from geranyl diphosphate as was shown by functional expression of synthases from several species such as spearmint, sage and grand fir [10,16,35,43]. The (–)-β-pinene synthase produces almost exclusively the (–)-enantiomer, and its side products show a similar enantiomeric composition, but with less stereoselectivity than the main product.

Considering the high sequence homology of the γ-terpinene synthase, producing an achiral product, to the (–)-β-pinene synthase, it would be expected that all side products would give similar enantiomers. However, the data show that although the most prevalent side products above 5% have an e.e. for the (–)-enantiomer, there is also a side product with an e.e. of the opposite enantiomer [(+)-β-pinene]. Furthermore, the stereoselectivity for most of the side products is even weaker than for the other lemon clones. Remarkably, the (+)-enantiomer of the β-pinene side product is formed in very high e.e. (96%). Other monoterpene synthases have been described that have low stereoselectivity for some of their side products, such as 1,8-cineole synthase and bornyl diphosphate synthase from common sage. The 1,8-cineole synthase produces for most side products an e.e. of the (+)-enantiomers, but for β-pinene an e.e. of the (–)-enantiomer [43]. As an explanation, Croteau and coworkers suggested that the E. coli host could proteolytically process the enzyme to a form that could compromise substrate and intermediate binding conformations.

In an investigation where monoterpene synthase activity from lemon was partially purified, the preference for Mn2+ as a cofactor instead of Mg2+ was reported [18]. The heterologously expressed enzymes from lemon show the same cofactor preference.

Lemon monoterpene synthases apparently do not prefer Mg2+ as the other cloned angiosperm synthases, but Mn2+ like the gymnosperm synthases [34]. These latter enzymes also require a monovalent ion, preferably K+ for activity [34,37], while the lemon enzymes are inhibited by potassium ions. The pH optimum of the lemon synthases is close to pH 7 like other angiosperm synthases, while the gymnosperm synthases show a pH optimum that is generally higher, such as pH 7.8 for the grand fir and lodgepole pine synthases [34,37,48].

The enzyme activity curves show that the activity decreases dramatically when the substrate concentration increases above 10–50 µmnastarr as shown for Cl(–)βPINS (Fig. 5). This cannot be caused by product inhibition as the products of the synthases will migrate to the hexane phase used in the assays and are therefore not expected to be interfering with the enzyme. The enzymes show substrate inhibition characteristics, a feature not previously reported for other cloned monoterpene synthases. The observation that the partially purified native monoterpene synthase enzyme fraction from lemon flavedo also showed substrate inhibition at higher substrate concentrations than five times the Km rules out the possibility that this phenomenon is the consequence of changes to the protein due to cloning artefacts [18]. An explanation could be that at higher concentrations, the allylic diphosphates start forming enzymatically inactive 2 : 1 complexes with metal ions, bound to the enzyme. Recent crystallographic work has shown that both epi-aristolochene and trichodiene synthase contain three Mg2+ ions in their active site, two of which are chelated by the DDXXD motif of the active site and a third which is liganded by a triad of active site residues [47,49].

The Km values determined for the monoterpene synthases from C. limon as determined by Michaelis–Menten kinetics are in a similar range as the values for other monoterpene synthases cloned thus far. The limonene synthases have a lower Km value than the β-pinene and the γ-terpinene synthase. Although no data are available about relative expression ratios of the four genes, the difference in Km may explain in part why the level of limonene compared to the other main products in the lemon peel is so much higher.

This report describes the first cloned monoterpene synthase that forms γ-terpinene as a major product. A homodimeric γ-terpinene synthase enzyme, purified from T. vulgaris produced in addition to the main product also small amounts of α-thujene and lesser quantities of myrcene, α-terpinene, limonene, linalool, terpinen-4-ol, and α-terpineol [50]. However the gene encoding this enzyme has so far not been isolated. In addition this is the first report on a (–)-β-pinene synthase cDNA.

Limonene is widely used in beverages and the cosmetics industry, and (+)-limonene also has anticarcinogenic properties [51]. The previously isolated (+)-limonene synthase from S. tenuifolia produces, apart from (+)-limonene, also a substantial amount of a nonidentified monoterpene side product [17]. The lemon cDNA encoding (+)-limonene synthase however, produces more than 99% pure and exclusively (+)-limonene. Such a pure compound synthesized by a heterologously expressed enzyme could perhaps be a more natural alternative than chemical synthesis and possibly a cheaper alternative than purification from plants.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We like to thank B. Weckerle for the MDGC-MS analyses and Dr Maurice Franssen for critical reading and helpful remarks on the manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Weiss, E.A. (1997) Essential Oil Crops, CAB International, Wallingford, UK.
  • 2
    Lewinsohn, E., Gijzen, M., Savage, T.J. & Croteau, R. (1991) Defense mechanism of conifers: relationship of monoterpene cyclase activity to anatomical specialization and oleoresin monoterpene content. Plant Physiol. 96, 3843.
  • 3
    Fahn, A. (1979) Secretory Tissues in Plants. Academic Press, London.
  • 4
    Lewinsohn, E., Gijzen, M., Muzika, R.M., Barton, K. & Croteau, R. (1993) Oleoresinosis in grand fir (Abies grandis) saplings and mature trees: modulation of this wound response by light and water stresses. Plant Physiol. 101, 10211028.
  • 5
    Funk, C., Lewinsohn, E., Vogel, B.S., Steele, C.L. & Croteau, R. (1994) Regulation of oleoresinosis in grand fir (Abies grandis). Coordinate induction of monoterpene and diterpene cyclases and two cytochrome P450-dependent diterpenoid hydroxylases by stem wounding. Plant Physiol. 106, 9991005.
  • 6
    Steele, C.L., Katoh, S., Bohlmann, J. & Croteau, R. (1998) Regulation of oleoresinosis in grand fir (Abies grandis): differential transcriptional control of monoterpene, sesquiterpene, and diterpene synthase genes in responses to wounding. Plant Physiol. 116, 14971504.
  • 7
    McConkey, M.E., Gershenzon, J. & Croteau, R.B. (2000) Developmental regulation of monoterpene biosynthesis in the glandular trichomes of peppermint. Plant Physiol. 122, 215223.
  • 8
    Gershenzon, J., McConkey, M.E. & Croteau, R.B. (2000) Regulation of monoterpene accumulation in leaves of peppermint. Plant Physiol. 122, 205213.
  • 9
    Turner, G., Gershenzon, J., Nielson, E.E., Froehlich, J.E. & Croteau, R. (1999) Limonene synthase, the enzyme responsible for monoterpene biosynthesis in peppermint, is localised to leucoplasts of oil gland secretory cells. Plant Physiol. 120, 879886.
  • 10
    Colby, S.M., Alonso, W.R., Katahira, E.J., McGarvey, D.J. & Croteau, R. (1993) 4S-Limonene synthase from the oil glands of spearmint (Mentha spicata): cDNA isolation, characterization, and bacterial expression of the catalytically active monoterpene cyclase. J. Biol. Chem. 268, 2301623024.
  • 11
    McCaskill, D., Gershenzon, J. & Croteau, R. (1992) Morphology and monoterpene biosynthetic capabilities of secretory cell clusters isolated from glandular trichomes of peppermint (Mentha piperita L.). Planta 187, 445454.
  • 12
    Bouwmeester, H.J., Gershenzon, J., Konings, M. & Croteau, R. (1998) Biosynthesis of the monoterpenes limonene and carvone in the fruit of caraway. I. Demonstration of enzyme activities and their changes with development. Plant Physiol. 117, 901912.
  • 13
    Turner, G.W., Berry, A.M. & Gifford, E.M. (1998) Schizogenous secretory cavities of Citrus limon (L.) Burm. f. and a reevaluation of the lysigenous gland concept. Int. J. Plant Sci. 159, 7588.
  • 14
    Mondello, L., Catalfamo, M., Cotroneo, A., Dugo, G., Dugo, G. & McNair, H. (1999) Multidimensional capillary GC-GC for the analysis of real complex samples: part IV. Enantiomeric distribution of monoterpene hydrocarbons and monoterpene alcohols of lemon oils. J. High Resoln. Chromatogr. 22, 350356.
  • 15
    Yuba, A., Yazaki, K., Tabata, M., Honda, G. & Croteau, R. (1996) cDNA cloning, characterization, and functional expression of 4S-(–)-limonene synthase from Perilla frutescens. Arch. Biochem. Biophys. 332, 280287.DOI: 10.1006/abbi.1996.0343
  • 16
    Bohlmann, J., Steele, C.L. & Croteau, R. (1997) Monoterpene synthases from grand fir (Abies grandis). cDNA isolation, characterization, and functional expression of myrcene synthase, (–)-(4S)-limonene synthase, and (–)-(1S,5S)-pinene synthase. J. Biol. Chem. 272, 2178421792.
  • 17
    Maruyama, T., Ito, M., Kiuchi, F. & Honda, G. (2001) Molecular cloning, functional expression and characterization of d-limonene synthase from Schizonepeta tenuifolia. Biol. Pharm. Bull. 24, 373377.
  • 18
    Chayet, L., Rojas, C., Cardemil, E., Jabalquinto, A.M., Vicuna, R. & Cori, O. (1977) Biosynthesis of monoterpene hydrocarbons from [1-3H]neryl pyrophosphate and [1-3H]geranyl pyrophosphate by soluble enzymes from Citrus limonum. Arch. Biochem. Biophys. 180, 318327.
  • 19
    Maruyama, T., Ito, M. & Honda, G. (2001) Molecular cloning, functional expression and characterization of (E)-β-farnesene synthase from Citrus junos. Biol. Pharm. Bull. 24, 11711175.
  • 20
    de Kraker, J.W., Franssen, M.C.R., de Groot, Æ., König, W.A. & Bouwmeester, H.J. (1998) (+)-Germacrene A biosynthesis: the committed step in the biosynthesis of bitter sesquiterpene lactones in chicory. Plant Physiol. 117, 13811392.
  • 21
    Saitou, N. & Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406425.
  • 22
    Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24, 48764882.
  • 23
    Kimura, M. (1983) The Neutral Theory of Molecular Evolution, pp.75. Cambridge University Press, Cambridge.
  • 24
    Helsper, J.P.F.G., Davies, J.A. & Verstappen, F.W.A. (2001) Analysis of rhythmic emission of volatile compounds of rose flowers. In Analysis of Taste and Aroma (Jackson, J.F. & Linskens, H.F., eds), pp. 269. Springer, Berlin.
  • 25
    Bouwmeester, H.J., Verstappen, F.W.A., Posthumus, M.A. & Dicke, M. (1999) Spider mite-induced (3S)-(E)-nerolidol synthase activity in cucumber and lima bean. The first dedicated step in acyclic C11-homoterpene biosynthesis. Plant Physiol. 121, 173180.
  • 26
    Schultz, D.J., Craig, R., Cox-Foster, D.L., Mumma, R.O. & Medford, J.I. (1994) RNA isolation from recalcitrant plant tissue. Plant Mol. Biol. Report 12, 310316.
  • 27
    Williams, D.C., McGarvey, D.J., Katahira, E.J. & Croteau, R. (1998) Truncation of limonene synthase preprotein provides a fully active ‘pseudomature’ form of this monoterpene cyclase and reveals the function of the amino-terminal arginine pair. Biochemistry 37, 1221312220.
  • 28
    Jia, J.W., Crock, J., Lu, S., Croteau, R. & Chen, X.Y. (1999) (3R)-linalool synthase from Artemisia annua L. cDNA isolation, characterization, and wound induction. Arch. Biochem. Biophys. 372, 143149.
  • 29
    Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring. Harbor Laboratory Press, Cold Spring Harbor, New York.
  • 30
    Bouwmeester, H.J., Wallaart, T.E., Janssen, M.H., van Loo, B., Jansen, B.J., Posthumus, M.A., Schmidt, C.O., de Kraker, J.W., Konig, W.A. & Franssen, M.C. (1999) Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis. Phytochemistry 52, 843854.
  • 31
    Lücker, J., Bouwmeester, H.J., Schwab, W., Blaas, J., van der Plas, L.H.W. & Verhoeven, H.A. (2001) Expression of Clarkia S-linalool synthase in transgenic petunia plants results in the accumulation of S-linalyl-β-mnastarr-glucopyranoside. Plant J. 27, 315324.
  • 32
    Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J.H., Zhang, Z., Miller, W. & Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 33893402.
  • 33
    Fischbach, R.J., Zimmer, W. & Schnitzler, J.P. (2001) Isolation and functional analysis of a cDNA encoding a myrcene synthase from holm oak (Quercus ilex L.). Eur. J. Biochem. 268, 56335638.
  • 34
    Bohlmann, J., Meyer Gauen, G. & Croteau, R. (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc. Natl Acad. Sci. 95, 41264133.
  • 35
    Bohlmann, J., Phillips, M., Ramachandiran, V., Katoh, S. & Croteau, R. (1999) cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies grandis). Arch. Biochem. Biophys. 368, 232243.
  • 36
    Bohlmann, J., Martin, D., Oldham Neil, J. & Gershenzon, J. (2000) Terpenoid secondary metabolism in Arabidopsis thaliana: cDNA cloning, characterization, and functional expression of a myrcene/(E)-beta-ocimene synthase. Arch. Biochem. Biophys. 375, 261269.
  • 37
    Savage, T.J., Hatch, M.W. & Croteau, R. (1994) Monoterpene synthases of Pinus contorta and related conifers: a new class of terpenoid cyclase. J. Biol. Chem. 269, 40124020.
  • 38
    Hernandez, A. & Ruiz, M.T. (1998) An EXCEL template for calculation of enzyme kinetic parameters by non-linear regression. Bioinformatics 14, 227228.
  • 39
    Lange, M.B., Wildung, M.R., Stauber, E.J., Sanchez, C., Pouchnik, D. & Croteau, R. (2000) Probing essential oil biosynthesis and secretion by functional evaluation of expressed sequence tags from mint glandular trichomes. Proc. Natl Acad. Sci. 97, 29342939.
  • 40
    Gang, D.R., Wang, J., Dudareva, N., Nam, K.H., Simon, J.E., Lewinsohn, E. & Pichersky, E. (2001) An investigation of the storage and biosynthesis of phenylpropenes in sweet basil. Plant Physiol. 125, 539555.
  • 41
    Aharoni, A., Keizer, L.C.P., Bouwmeester, H.J., Sun, Z., Alvarez, H.M., Verhoeven, H.A., Blaas, J., van Houwelingen, A.M.M.L., De Vos, R.C.H., van der Voet, H. et al. (2000) Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays. Plant Cell 12, 647661.
  • 42
    Bouvier, F., Suire, C., d'Harlingue, A., Backhaus, R.A. & Camara, B. (2000) Molecular cloning of geranyl diphosphate synthase and compartmentation of monoterpene synthesis in plant cells. Plant J. 24, 241252.
  • 43
    Wise, M.L., Savage, T.J., Katahira, E. & Croteau, R. (1998) Monoterpene synthases from common sage (Salvia officinalis): cDNA isolation, characterization, and functional expression of (+)-sabinene synthase, 1,8-cineole synthase, and (+)-bornyl diphosphate synthase. J.Biol. Chem. 273, 1489114899.
  • 44
    Cseke, L., Dudareva, N. & Pichersky, E. (1998) Structure and evolution of linalool synthase. Mol. Biol. Evol. 15, 14911498.
  • 45
    Lesburg, C.A., Zhai, G., Cane, D.E. & Christianson, D.W. (1997) Crystal structure of pentalenene synthase: Mechanistic insights on terpenoid cyclization reactions in biology. Science 277, 18201824.
  • 46
    Tarshis, L.C., Yan, M., Poulter, C.D., Sacchettini, J. & , C. (1994) Crystal structure of recombinant farnesyl diphosphate synthase at 2.6-Å resolution. Biochemistry 33, 1087110877.
  • 47
    Starks, C.M., Back, K., Chappell, J. & Noel, J.P. (1997) Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase. Science 277, 18151820.
  • 48
    Lewinsohn, E., Gijzen, M. & Croteau, R. (1992) Wound-inducible pinene cyclase from grand fir: purification, characterization, and renaturation after SDS-PAGE. Arch. Biochem. Biophys. 293, 167173.
  • 49
    Rynkiewics, M.J., Cane, D.E. & Christianson, D.W. (2001) Structure of trichodiene synthase from Fusarium sporotrichioides provides mechanistic inferences on the terpene cyclisation cascade. Proc. Natl Acad. Sci. USA 98, 1354313548.
  • 50
    Alonso, W.R. & Croteau, R. (1991) Purification and characterization of the monoterpene cyclase gamma-terpinene synthase from Thymus vulgaris. Arch. Biochem. Biophys. 286, 511517.
  • 51
    Crowell, P.L. & Gould, M.N. (1994) Chemoprevention and therapy of cancer by d-limonene. Crit. Rev. Oncogen. 5, 122.