Monoterpene Synthases from Grand Fir (Abies grandis)
cDNA ISOLATION, CHARACTERIZATION, AND FUNCTIONAL EXPRESSION OF MYRCENE SYNTHASE, (-)-(4S)-LIMONENE SYNTHASE, AND (-)-(1S,5S)-PINENE SYNTHASE*

(Received for publication, March 3, 1997, and in revised form, June 10, 1997)

Jörg Bohlmann Dagger , Christopher L. Steele § and Rodney Croteau

From the Institute of Biological Chemistry, and Department of Biochemistry and Biophysics, Washington State University, Pullman, Washington 99164-6340

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (-)-limonene and (-)-alpha - and (-)-beta -pinene are prominent components; (-)-limonene and (-)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression in Escherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (-)-limonene synthase, a myrcene synthase, and a (-)-pinene synthase that produces both alpha -pinene and beta -pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA blot hybridization using probes derived from the three monoterpene synthase cDNAs. The availability of cDNA species encoding these monoterpene synthases will allow an understanding of the regulation of oleoresin formation in conifers and will ultimately permit the transgenic manipulation of this defensive secretion to enhance resistance to insects. These cDNAs also furnish tools for defining structure-function relationships in this group of catalysts that generate acyclic, monocyclic, and bicyclic olefin products.


INTRODUCTION

Chemical defense of conifer trees against bark beetles and their associated fungal pathogens relies primarily upon constitutive and inducible oleoresin biosynthesis (1, 2). This defensive secretion is a complex mixture of monoterpene and sesquiterpene olefins (turpentine) and diterpene resin acids (rosin) that is synthesized constitutively in the epithelial cells of specialized structures, such as resin ducts and blisters or, in the case of induced oleoresin formation, in undifferentiated cells surrounding wound sites (3). The volatile fraction of conifer oleoresin, which is toxic to both bark beetles and their fungal associates (4), may consist of up to 30 different monoterpenes (5), including acyclic types (e.g. myrcene), monocyclic types (e.g. limonene), and bicyclic types (e.g. pinenes) (Fig. 1). Although the oleoresin is toxic, many bark beetle species nevertheless employ turpentine volatiles in host selection and can convert various monoterpene components into aggregation or sex pheromones to promote coordinated mass attack of the host (2, 6). In grand fir (Abies grandis), increased formation of oleoresin monoterpenes, sesquiterpenes, and diterpenes is induced by bark beetle attack (3, 7, 8), and this inducible defense response is mimicked by mechanically wounding sapling stems (3, 8, 9). Therefore, grand fir has been developed as a model system to study the biochemical and molecular genetic regulation of constitutive and inducible terpene biosynthesis in conifers (10).


Fig. 1. Mechanism for the conversion of geranyl diphosphate to myrcene, (-)-limonene, beta -phellandrene, (-)-alpha -pinene, and (-)-beta -pinene by monoterpene synthases from grand fir. Formation of the monocyclic and bicyclic products requires preliminary isomerization of geranyl diphosphate to linalyl diphosphate. The acyclic product could be formed from either geranyl diphosphate or linalyl diphosphate via carbocations 1 or 2. OPP denotes the diphosphate moiety.
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Most monoterpenes are derived from geranyl diphosphate, the ubiquitous C10 intermediate of the isoprenoid pathway, by synthases that catalyze the divalent metal ion-dependent ionization (to 1) and isomerization of this substrate to enzyme-bound linalyl diphosphate which, following rotation about C2-C3, undergoes a second ionization (to 2) followed by cyclization to the alpha -terpinyl cation, the first cyclic intermediate en route to both monocyclic and bicyclic products (11, 12) (Fig. 1). Acyclic monoterpenes, such as myrcene, may arise by deprotonation of carbocations 1 or 2, whereas the isomerization step to linalyl diphosphate is required in the case of cyclic types, such as limonene and pinenes, which cannot be derived from geranyl diphosphate directly because of the geometric impediment of the trans-double bond at C2-C3 (11, 12). Many monoterpene synthases catalyze the formation of multiple products, including acyclic, monocyclic, and bicyclic types, by variations on this basic mechanism (13-15). For example, (-)-limonene synthase, the principal monoterpene synthase of spearmint (Mentha spicata) and peppermint (Mentha × piperita), produces small amounts of myrcene, (-)-alpha -pinene and (-)-beta -pinene in addition to the monocyclic product (16, 17). Conversely, six different inducible monoterpene synthase activities have been demonstrated in extracts of wounded grand fir stem (18) indicating that formation of acyclic, monocyclic, and bicyclic monoterpenes in this species involves several genes encoding distinct catalysts. The inducible (-)-pinene synthase has been purified (19) and isotopically sensitive branching experiments employed to demonstrate that this enzyme synthesizes both (-)-alpha - and (-)-beta -pinene (20).

Deciphering the molecular genetic control of oleoresinosis and examining structure-function relationships among the monoterpene synthases of grand fir require isolation of the cDNA species encoding these key enzymes. Although a protein-based cloning strategy was recently employed to acquire a cDNA for the major wound-inducible diterpene synthase from grand fir, abietadiene synthase (9, 21, 22), all attempts at the reverse genetic approach to cloning of grand fir monoterpene synthases have failed (10). As an alternative, a similarity based PCR1 strategy was developed (10) that employed sequence information from terpene synthases of angiosperm origin, namely a monoterpene synthase, (-)-4S-limonene synthase, from spearmint (M. spicata, Lamiaceae) (17), a sesquiterpene synthase, 5-epi-aristolochene synthase, from tobacco (Nicotiana tabacum, Solanaceae) (23), and a diterpene synthase, casbene synthase, from castor bean (Ricinus communis, Euphorbiaceae) (24).

In this paper, we describe the successful application of this strategy to the amplification of specific hybridization probes and their use in the isolation of six new "terpene synthase-like" cDNAs. Three of the full-length clones were functionally expressed in Escherichia coli and thereby identified as myrcene synthase, (-)-limonene synthase, and a pinene synthase that produces both (-)-alpha - and (-)-beta -pinene (Fig. 1). This is the first report of the isolation of any cDNA encoding a monoterpene synthase from a gymnosperm, and the first report to describe the cloning of several different monoterpene synthases (for acyclic, monocyclic, and bicyclic products) from a single plant species. Sequence comparison revealed significantly greater conservation between the grand fir monoterpene synthases and other gymnosperm terpene synthases than with angiosperm terpene synthases, and targeted a number of highly conserved amino acid residues for further study. Additionally, Northern hybridization analysis demonstrated that induced oleoresinosis in grand fir is regulated at the level of monoterpene synthase RNA accumulation.


EXPERIMENTAL PROCEDURES

Substrates, Reagents, and cDNA Library

[1-3H]Geranyl diphosphate (250 Ci/mol) (25), [1-3H]farnesyl diphosphate (125 Ci/mol) (26), and [1-3H]geranylgeranyl diphosphate (120 Ci/mol) (21) were prepared as described previously. Terpenoid standards were from our own collection. All other biochemicals and reagents were purchased from Sigma or Aldrich, unless otherwise noted. Construction of the lambda ZAP II cDNA library, using mRNA isolated from wounded grand fir sapling stems, was described previously (22).

PCR-based Probe Generation

Based on comparison of sequences of limonene synthase from spearmint (17), 5-epi-aristolochene synthase from tobacco (23), and casbene synthase from castor bean (24), four conserved regions were identified for which a set of consensus degenerate primers (primers A-D) were synthesized. Primers A-C have been described previously (10); primer D (see Fig. 2) was designed based on the conserved amino acid sequence motif DD(T/I)(I/Y/F)D(A/V)Y(A/G) of the above noted terpene synthases (17, 23, 24). The sequence of sense primer D was 5'-GA(C/T) GA(C/T) III T(T/A)(T/C) GA(C/T) GCI (C/T)A(C/T) GG-3'. Each of the sense primers, A, B, and D, was used for PCR in combination with antisense primer C by employing a broad range of amplification conditions. PCR was performed in a total volume of 50 µl containing 20 mM Tris/HCl (pH 8.4), 50 mM KCl, 5 mM MgCl2, 200 µM each dNTP, 1-5 µM each primer, 2.5 units of Taq polymerase (Life Technologies, Inc.), and 5 µl of purified grand fir stem cDNA library phage as template (1.5 × 109 plaque-forming units/ml). Analysis of the PCR reaction products by agarose gel electrophoresis (27) revealed that only the combination of primers C and D generated a specific PCR product of approximately 110 bp. This PCR product was gel-purified, ligated into pT7Blue (Novagen), and transformed into E. coli XL1-Blue cells. Plasmid DNA was prepared from 41 individual transformants, and the inserts were sequenced (DyeDeoxy Terminator Cycle Sequencing, Applied Biosystems). Four different insert sequences were identified and were designated as probes 1, 2, 4, and 5. Subsequent isolation of four new cDNA species, encoding terpene synthases from grand fir corresponding to these probes, allowed the identification of three additional conserved sequence elements which were used to design a set of three new PCR primers.


Fig. 2. Alignment of deduced amino acid sequences of plant terpene synthases. 1, grand fir (-)-pinene synthase ag3.18; 2, grand fir myrcene synthase ag2.2; 3, grand fir (-)-limonene synthase ag10; 4, grand fir abietadiene synthase (22); 5, castor bean casbene synthase (24); 6, tobacco epi-aristolochene synthase (23); and 7, spearmint (-)-limonene synthase (17). The alignment was created with the PILEUP program. Amino acids 127-351 of the complete sequence of abietadiene synthase (22) reveal no significant similarity to other terpene synthases of this alignment and are, therefore, not shown (XXX in 4). The positions of primers employed for similarity based PCR cloning are marked by horizontal bars and designated by capital letters. Dots indicate residues that are highly or absolutely conserved between all plant terpene synthases (17, 22-24, 34, 35, 64-68).
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Degenerate primer E (designed to conserved element GE(K/T)(V/I)M(E/D)EA (see Fig. 2)) and degenerate primer F (designed to conserved element Q(F/Y/D)(I/L)(T/L/R)RWW) were based on comparison of the sequences of five cloned terpene synthases from grand fir as follows: a monoterpene synthase corresponding to probe 2, two sesquiterpene synthases2 corresponding to probe 4 and probe 5, respectively, a previously described diterpene synthase (22), and a truncated terpene synthase3 corresponding to probe 1. The sequence of sense primer E was 5'-GGI GA(A/G) A(A/C)(A/G) (A/G)TI ATG GA(A/G) GA(A/G) GC-3' and of sense primer F was 5'-GA(A/G) (C/T)TI CA(G/A) (C/T)TI (A/C/T)(C/G/T)I (A/C)GI TGG TGG-3'. Degenerate primer G (see Fig. 2) was designed according to the amino acid sequence DVIKG(F/L)NW obtained from a peptide generated by trypsin digestion of purified (-)-pinene synthase from grand fir.4 The sequence of antisense primer G was 5'-CCA (A/G)TT IA(A/G) ICC (C/T)TT IAC (A/G)TC-3'. Primers E and F were independently used for PCR amplification in combination with primer G, with grand fir stem cDNA library as template. The combination of primers E and G yielded a specific PCR product of approximately 1020 bp. This PCR product was ligated into pT7Blue and transformed into E. coli XL1-Blue. Plasmid DNA was prepared from 20 individual transformants, and inserts were sequenced from both ends. The sequence of this 1022-bp insert was identical for all 20 plasmids and was designated as probe 3.

Library Screening

For library screening, 100 ng of each probe (1 through 5) was amplified by PCR, gel purified, randomly labeled with [alpha -32P]dATP (28), and used individually to screen replica filters of 105 plaques of the wound-induced grand fir stem cDNA library plated on E. coli LE392. Hybridization with probes 1, 2, 4, and 5 was performed for 14 h at 65 °C in 3 × SSPE and 0.1% SDS. Filters were washed three times for 10 min at 55 °C in 3 × SSPE with 0.1% SDS and exposed for 12 h to Kodak XAR film at -70 °C (27). All of the lambda ZAPII clones yielding positive signals were purified through a second round of hybridization (probe 1 gave 25 positives, probe 2 gave 16 positives, probe 4 gave 49 positives, and probe 5 gave 12 positives). Hybridization with probe 3 was performed as before, but the filters were washed three times for 10 min at 65 °C in 3 × SSPE and 0.1% SDS before exposure. Approximately 400 lambda ZAPII clones yielded strong positive signals, and 34 of these were purified through a second round of hybridization at 65 °C. Approximately 400 additional clones yielded weak positive signals with probe 3, and 18 of these were purified through a second round of hybridization for 20 h at 45 °C. Purified lambda ZAP II clones isolated using all five probes were in vivo excised as Bluescript II SK- phagemids and transformed into E. coli XLOLR according to the manufacturer's instructions (Stratagene). The size of each cDNA insert was determined by PCR using T3 and T7 promoter primers, and selected inserts (>1.5 kb) were partially sequenced from both ends.

cDNA Expression in E. coli and Enzyme Assays

Except for cDNA clones pAG3.18 and pAG3.48, all of the partially sequenced inserts were either truncated at the 5'-end, or were out of frame, or bore premature stop codons upstream of the presumptive methionine start codon. For the purpose of functional expression, a 2001-bp insert fragment from plasmid pAG2.2 and a 1903-bp insert fragment from pAG3.18 were subcloned in frame into pGEX vectors (Pharmacia Biotech Inc.). A 2046-bp insert fragment from pAG10 was subcloned in frame into the pSBETa vector (29). To introduce suitable restriction sites for subcloning, fragments were amplified by PCR using primer combinations 2.2-BamHI (5'-CAA AGG GAT CCA GAA TGG CTC TGG-3') and 2.2-NotI (5'-AGT AAG CGG CCG CTT TTT AAT CAT ACC CAC-3') with pAG2.2 as template, 3.18-EcoRI (5'-CTG CAG GAA TTC GGC ACG AGC-3') and 3.18-SmaI (5'-CAT AGC CCC GGG CAT AGA TTT GAG CTG-3') with pAG3.18, and 10-NdeI (5-GGC AGG AAC ATA TGG CTC TCC TTT CTA TCG-3') and 10-BamHI (5'-TCT AGA ACT AGT GGATCC CCC GGG CTG CAG-3' with pAG10. PCR reactions were performed in volumes of 50 µl containing 20 mM Tris/HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 5 µg of bovine serum albumin, 200 µM each dNTP, 0.1 µM each primer, 2.5 units of recombinant Pfu polymerase (Stratagene), and 100 ng of plasmid DNA with the following program: denaturation at 94 °C, 1 min; annealing at 60 °C, 1 min; extension at 72 °C, 3.5 min; 35 cycles with final extension at 72 °C, 5 min. The PCR products were purified by agarose gel electrophoresis and used as template for a secondary PCR amplification with the identical conditions in total volumes of 250 µl each. Products from this secondary amplification were digested with the above indicated restriction enzymes, purified by ultrafiltration, and then ligated, respectively, into BamHI/NotI-digested pGEX-4T-2 to yield plasmid pGAG2.2, into EcoRI/SmaI-digested pGEX-4T-3 to yield plasmid pGAG3.18, and into NdeI/BamHI-digested pSBETa to yield plasmid pSBAG10; these plasmids were then transformed into E. coli XL1-Blue or E. coli BL21(DE3).

For expression, bacterial strains E. coli XLOLR/pAG3.18, E. coli XLOLR/pAG3.48, E. coli XL1-Blue/pGAG2.2, E. coli XL1-Blue/pGAG3.18, and E. coli BL21(DE3)/pSBAG10 were grown to A600 = 0.5 at 37 °C in 5 ml of LB medium (27) supplemented with 100 µg of ampicillin/ml or 30 µg of kanamycin/ml as determined by the vector. Cultures were then induced by addition of 1 mM isopropyl-1-thio-beta -D-galactopyranoside and grown for another 12 h at 20 °C. Cells were harvested by centrifugation (2000 × g, 10 min) and resuspended in either 1 ml of monoterpene synthase assay buffer (50 mM Tris/HCl (pH 7.5), 500 mM KCl, 1 mM MnCl2, 5 mM dithiothreitol, 0.05% (w/v) NaHSO3, and 10% (v/v) glycerol), 1 ml of sesquiterpene synthase assay buffer (10 mM dibasic potassium phosphate, 1.8 mM monobasic potassium phosphate (pH 7.3), 140 mM NaCl, 10 mM MgCl2, 5 mM dithiothreitol, 0.05% (w/v) NaHSO3, and 10% (v/v) glycerol), or 1 ml of diterpene synthase assay buffer (30 mM Hepes (pH 7.2), 7.5 mM MgCl2, 5 mM dithiothreitol, 10 µM MnCl2, 0.05% (w/v) NaHSO3, and 10% (v/v) glycerol). Cells were disrupted by sonication (Braun-Sonic 2000 with microprobe at maximum power for 15 s at 0-4 °C); the homogenates were cleared by centrifugation (18,000 × g, 10 min), and 1 ml of the resulting supernatant was assayed for monoterpene synthase activity with 2.5 µM [1-3H]geranyl diphosphate, for sesquiterpene synthase activity with 3.5 µM [1-3H]farnesyl diphosphate, or for diterpene synthase activity with 5 µM [1-3H]geranylgeranyl diphosphate following standard protocols (11, 21, 26). In the case of the monoterpene synthase and sesquiterpene synthase assays, the incubation mixture was overlaid with 1 ml of pentane to trap volatile products. In all cases, after incubation at 31 °C for 2 h, the reaction mixture was extracted with pentane (3 × 1 ml), and the combined extract was passed through a 1.5-ml column of anhydrous MgSO4 and silica gel (Mallinckrodt 60 Å) to provide the terpene hydrocarbon fraction free of oxygenated metabolites. The columns were subsequently eluted with 3 × 1 ml of ether to collect any oxygenated products, and an aliquot of each fraction was taken for liquid scintillation counting to determine conversion rate.

Product Identification

To obtain sufficient product for analysis by radio-GLC, chiral capillary GLC, and GLC-MS, preparative-scale enzyme incubations were carried out. Thus, the enzyme was prepared from 50 ml of cultured bacterial cells by extraction with 3 ml of assay buffer as above, and the extracts were incubated with excess substrate overnight at 31 °C. The hydrocarbon fraction was isolated by elution through MgSO4-silica gel as before, and the pentane eluate was concentrated for evaluation by capillary radio-GLC as described (30), by chiral column capillary GLC (5), and by combined GLC-MS (Hewlett-Packard 6890 GC-MSD with cool (40 °C) on-column injection, detection via electron impact ionization (70 eV), helium carrier at 0.7 p.s.i., column: 0.25-mm inner diameter × 30-m fused silica with 0.25-µm film of 5MS (Hewlett-Packard) programmed from 35 °C (5 min hold) to 230 °C at 5 °C/min).

Sequence Analysis

Inserts of all recombinant bluescript plasmids, pAG1.28, pAG2.2, pAG3.18, pAG3.48, pAG4.30, pAG5.9, and pAG10, and inserts of all recombinant pGEX plasmids, pGAG2.2, pGAG3.18, and pSBAG10, were completely sequenced on both strands via primer walking and nested deletions (27) using the DyeDeoxy Terminator Cycle Sequencing method (Applied Biosystems). Sequence analysis was done using the Wisconsin Package version 9.0, Genetics Computer Group (GCG), Madison, WI.

RNA Extraction and Northern Blotting

Grand fir sapling stem tissue was harvested prior to wounding or 2 days after wounding by a standard procedure (18). Total RNA was isolated (31), and 20 µg of RNA per gel lane was separated under denaturing conditions (27) and transferred to nitrocellulose membranes (Schleicher and Schuell) according to the manufacturer's protocol. To prepare hybridization probes, cDNA fragments of 1.4-1.5 kb were amplified by PCR from ag2.2 with primer JB29 (5'-CTA CCA TTC CAA TAT CTG-3') and primer 2-8 (5'-GTT GGA TCT TAG AAG TTC CC-3'), from ag3.18 with primer 3-9 (5'-TTT CCA TTC CAA CCT CTG GG-3') and primer 3-11 (5'-CGT AAT GGA AAG CTC TGG CG-3'), and from ag10 with primer 7-1 (5'-CCT TAC ACG CCT TTG GAT GG-3') and primer 7-3 (5'-TCT GTT GAT CCA GGA TGG TC-3'). The probes were randomly labeled with [alpha -32P]dATP (28). Blots were hybridized for 24 h at 55 °C in 3 × SSPE and 0.1% SDS, washed at 55 °C in 1 × SSPE and 0.1% SDS, and subjected to autoradiography as described above at -80 °C for 24 h.


RESULTS AND DISCUSSION

Similarity Based Cloning of Grand Fir Terpene Synthases

Grand fir has been developed as a model system for the study of induced oleoresin production in conifers in response to wounding and insect attack (1, 2, 7, 10, 32). The chemistry and biosynthesis of the oleoresin monoterpenes, sesquiterpenes, and diterpenes have been well defined (5, 8, 9, 18, 19, 21, 33); however, structural analysis of the responsible terpene synthases as well as studies on the regulation of oleoresinosis require the isolation of cDNA species encoding the terpene synthases. Protein purification from conifers, as the basis for cDNA isolation, has been of limited success (22) and thus far has not permitted cloning of any of the monoterpene synthases from these species (10).

As a possible alternative to protein-based cloning of terpene synthases, a homology-based PCR strategy was recently proposed (10) that was founded upon the three terpene synthases of plant origin then available, a monoterpene synthase, (-)-(4S)-limonene synthase, from spearmint (M. spicata, Lamiaceae) (17), a sesquiterpene synthase, 5-epi-aristolochene synthase, from tobacco (N. tabacum, Solanaceae) (23), and a diterpene synthase, casbene synthase, from castor bean (Ricinus communis, Euphorbiaceae) (24). Despite the taxonomic distances between these three angiosperm species and the differences in substrate utilized, reaction mechanism, and product type of the three enzymes, a comparison of the deduced amino acid sequences identified several conserved regions that appeared to be useful for the design of degenerate PCR primers (see Fig. 2 and "Experimental Procedures"). Using cDNA from a wound-induced grand fir stem library as template, one set of primers (C and D) PCR-amplified products corresponding to four distinct sequence groups, all of which showed significant similarity to sequences of cloned terpene synthases of plant origin. The four different inserts were designated as probes 1, 2, 4, and 5 and were employed for isolation of the corresponding cDNA clones by plaque hybridization.

Screening of 105 cDNA phage plaques from the wounded grand fir stem library, with each of the four probes, yielded a 4-fold difference in the number of positives (see "Experimental Procedures"), most likely reflecting different levels of expression of the corresponding genes. Size-selected inserts (>1.5 kb) of purified and in vivo excised clones were partially sequenced from both ends and were shown to segregate into four distinct groups corresponding to the four hybridization probes. Since all cDNAs corresponding to probes 1, 4, and 5 were truncated at their 5'-ends, only inserts of the largest representatives of each group, clone ag1.28, clone ag2.2 (apparently full length), clone ag4.30, and clone ag5.9, were completely sequenced. The sequences of clone ag1.28 (2414 bp, with an ORF of 2350 nt encoding 782 amino acids), clone ag2.2 (2196 bp, with an ORF of 1881 nt encoding 627 amino acids), clone ag4.30 (2979 bp, with an ORF of 1731 nt encoding 577 amino acids), and clone ag5.9 (1394 bp, with an ORF of 1194 nt encoding 398 amino acids) were compared pairwise with each other and with other cloned plant terpene synthases (Fig. 3). Truncated clone ag1.28 resembled most closely in size and sequence (72% similarity, 49% identity) a diterpene cyclase, abietadiene synthase, from grand fir (22). Clones ag4.30 and ag5.9 share approximately 80% similarity (60% identity) at the amino acid level and are almost equally distant from both clone ag1.28 and full-length clone ag2.2 (range of 65-70% similarity and 45-47% identity); the amino acid sequence similarity between ag1.28 and ag2.2 is 65% (41% identity). Considering the high level of homology between ag4.30 and ag5.9, these comparisons suggest that the four new cDNAs, ag1.28, ag2.2, ag4.30, and ag5.9, represent the three major subfamilies of grand fir terpene synthase genes (Fig. 3) encoding monoterpene synthases, sesquiterpene synthases, and diterpene synthases. Isolation of full-length clones corresponding to ag1.28, ag4.30, and ag5.9, by employing PCR-based rapid amplification of cDNA ends, and functional identification of ag4.30 and ag5.9 as two new sesquiterpene synthases will be described elsewhere.2,3


Fig. 3. Sequence comparison of plant terpene synthases. A three-letter designation (tps) for the gene family is proposed with sub-groups (tpsa through tpsf) defined by a minimum of 40% amino acid identity between members. The numbers in parentheses are the references to the published sequences.
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Identification of cDNA Clone ag2.2 as Myrcene Synthase

The pAG2.2 insert appeared to be a full-length clone encoding a protein of molecular weight 72,478 with a calculated pI at 6.5 (Fig. 2). The size of the translated protein encoded by ag2.2 (627 residues) is in the range of the monoterpene synthase preproteins for limonene synthase from spearmint (17) and Perilla frutescens (34) but is about 240 amino acids shorter than the two gymnosperm diterpene synthase preproteins for abietadiene synthase (22) and taxadiene synthase (35). Monoterpene and diterpene biosyntheses are compartmentalized in plastids, whereas sesquiterpene biosynthesis is cytosolic (reviewed in Refs. 36-38); thus, monoterpene and diterpene synthases are encoded as preproteins bearing an amino-terminal transit peptide for import of these nuclear gene products into plastids where they are proteolytically processed to the mature forms (39, 40). Both the size of the deduced protein and the presence of an amino-terminal domain (of 60-70 amino acids) with features characteristic of a targeting sequence (rich in serine residues (16-18%) and low in acidic residues (four Asp or Glu) (39, 40)) suggest that ag2.2 encodes a monoterpene synthase rather than a sesquiterpene synthase or a diterpene synthase.

Since pAG2.2 contained the terpene synthase insert in reversed orientation, the ORF was subcloned in frame with glutathione S-transferase, for ultimate ease of purification (41, 42), into pGEX-4T-2, yielding plasmid pGAG2.2. The recombinant fusion protein was expressed in E. coli strain XL1-Blue/pGAG2.2 and then extracted and assayed for monoterpene synthase, sesquiterpene synthase, and diterpene synthase activity using tritium-labeled geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate as the respective substrate. Enzymatic production of a terpene olefin was observed only with geranyl diphosphate as substrate, and the only product was shown to be myrcene (Fig. 1) by radio-GLC and GLC-MS comparison to an authentic standard (Fig. 4). Bacteria transformed with pGEX vector containing the ag2.2 insert in antisense orientation did not afford detectable myrcene synthase activity when induced, and the protein was isolated and assayed as above. A myrcene synthase cDNA has not been obtained previously from any source, although myrcene is a minor co-product (2%) of the native and recombinant limonene synthase from spearmint (16, 17) and of several enzymes from sage (15). cDNA cloning and functional expression of myrcene synthase, which is one of several wound-inducible monoterpene synthase activities of grand fir (18), demonstrates that this acyclic monoterpene is formed by a distinct enzyme and is not a co-product of another synthase.


Fig. 4. GLC-MS analysis of the products of the recombinant protein encoded by ag2.2. The GLC profile of the total pentane-soluble products generated from geranyl diphosphate when incubated with a cell-free extract of E. coli XL1-Blue/pGAG2.2 is illustrated (a), as are the mass fragmentation patterns for the monoterpene product with Rt = 12.22 min (b), and for authentic myrcene (c).
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Identification of cDNA Clone ag3.18 as (-)-Pinene Synthase and cDNA Clone ag10 as (-)-Limonene Synthase

Alignment of the four new terpene synthase cDNA sequences (ag1.28, ag2.2, ag4.30, and ag5.9) and that for abietadiene synthase (22) allowed the identification of several conserved sequence motifs among this enzyme family from grand fir, which provided the foundation for an extended similarity based cloning approach. Two new sense primers E and F were designed according to conserved sequence elements, whereas a degenerate antisense primer G was designed based upon very limited amino acid sequence information from pinene synthase4 (see Fig. 2 and "Experimental Procedures"). Only the combination of primers E and G amplified a specific product of 1022 bp, which was designated as probe 3.

Hybridization of 105 grand fir lambda ZAP II cDNA clones with probe 3 yielded two types of signals comprised of about 400 strongly positive clones and an equal number of weak positives, indicating that the probe recognized more than one type of cDNA. Thirty-four of the former clones and 18 of the latter were purified, the inserts were selected by size (2.0-2.5 kb), and the in vivo excised clones were partially sequenced from both ends. Those clones that afforded weak hybridization signals were shown to contain inserts that were either identical to myrcene synthase clone ag2.2 or exhibited no significant sequence similarity to terpene synthases. Clone pAG3.48 contained the myrcene synthase ORF in the correct orientation and in frame for expression from the Bluescript plasmid vector. This cDNA was functionally expressed in E. coli, and the resulting enzyme was shown to accept only geranyl diphosphate as the prenyl diphosphate substrate and to produce myrcene as the exclusive reaction product. This finding with pAG3.48 confirms that expression as the glutathione S-transferase fusion from pGAG2.2 does not influence substrate utilization or product outcome of the myrcene synthase.

Clones that gave strong hybridization signals segregated into distinct sequence groups represented by clone ag3.18 (2018-bp insert with ORF of 1884 nt; encoded protein of 628 residues at 71,505 Da and pI of 5.5) and ag10 (2084-bp insert with ORF of 1911 nt; encoded protein of 637 residues at 73,477 Da and pI of 6.4) (Fig. 2). ag3.18 and ag10 form a subfamily together with the myrcene synthase clone ag2.2 that is characterized by a minimum of 79% pairwise similarity (64% identity) at the amino acid level (Fig. 3). Like myrcene synthase, both ag3.18 and ag10 encode amino-terminal sequences of 60-70 amino acids that are rich in serine (19-22 and 11-15%, respectively) and low in acidic residues (4 and 2 residues, respectively) (Fig. 2) characteristic of plastid transit peptides (39, 40).

Plasmid pAG3.18 contained the presumptive terpene synthase ORF in frame for direct expression from the Bluescript plasmid, whereas the ag10 ORF was in reversed orientation. Both ag3.18 and ag10 were subcloned into expression vectors yielding plasmids pGAG3.18 and pSBAG10. Recombinant proteins were expressed in bacterial strain E. coli XLOLR/pAG3.18, E. coli XL1-Blue/pGAG3.18, and E. coli BL21(DE3)/pSBAG10. When extracts of the induced cells were tested for terpene synthase activity with all of the potential prenyl diphosphate substrates, only geranyl diphosphate was utilized. Extracts from E. coli BL21(DE3)/pSBAG10 converted geranyl diphosphate to limonene as the major product with lesser amounts of alpha -pinene, beta -pinene, and beta -phellandrene, as determined by radio-GLC and combined GLC-MS (Fig. 5). Chiral phase capillary GLC on beta -cyclodextrin revealed the limonene product to be the (-)-(4S)-enantiomer and the pinene products to be the related (-)-(1S,5S)-enantiomers. Although optically pure standards were not available for the analysis, stereochemical considerations suggest that the minor product beta -phellandrene is also the mechanistically related (-)-(4S)-antipode (13, 14, 20, 43, 44). Similar analysis of the monoterpene products generated from geranyl diphosphate by cell-free extracts of E. coli XLOLR/pAG3.18 and E. coli XL1-Blue/pGAG3.18 demonstrated the presence of a 42:58% mixture of alpha -pinene and beta -pinene (Fig. 6), the same product ratio previously described for the purified, native (-)-pinene synthase from grand fir (19). Chiral phase capillary GLC confirmed the products of the recombinant pinene synthase to be the (-)-(1S,5S)-enantiomers, as expected. No other monoterpene co-products were detected with the recombinant (-)-pinene synthase, as observed previously for the native enzyme (19).


Fig. 5. GLC-MS analysis of the products of the recombinant protein encoded by ag10. The GLC profile of the total pentane-soluble products generated from geranyl diphosphate when incubated with a cell-free extract of E. coli BL21(DE3)/pSBAG10 is illustrated (a), as are the mass fragmentation patterns for the principal monoterpene product with Rt = 13.93 min (b), and for authentic limonene (c).
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Fig. 6. GLC-MS analysis of the products of the recombinant protein encoded by ag3.18. The GLC profile of the total pentane-soluble products generated from geranyl diphosphate when incubated with a cell-free extract of E. coli XL1-Blue/pGAG3.18 is illustrated (a), as are the mass fragmentation patterns (selected ion mode) for the monoterpene products with Rt = 11.34 min (b), and Rt = 13.37 min (d), and for authentic alpha -pinene (c) and authentic beta -pinene (e).
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Evidence for the formation of both alpha - and beta -pinene by a single enzyme has been previously provided through co-purification studies, and differential inhibition and inactivation studies, as well as by isotopically sensitive branching experiments (13, 20, 45, 46). The cDNA cloning of pinene synthase provides the ultimate proof that a single enzyme forms both products. The calculated molecular weight of the (-)-pinene synthase deduced from ag3.18 is approximately 64,000 (excluding the putative transit peptide), which agrees well with the molecular weight of 63,000 established for the native enzyme from grand fir by gel permeation chromatography and SDS-polyacrylamide gel electrophoresis (19).

A limonene synthase cDNA has thus far been cloned only from two very closely related angiosperm species (17, 34), and the isolation of a pinene synthase cDNA has not been reported before. Pinene synthase has previously received considerable attention as a major defense-related monoterpene synthase in conifers (18, 19). In the grand fir cDNA library, which was synthesized from mRNA obtained from wound-induced sapling stems, clones corresponding to pinene synthase are at least 10 times more abundant than clones for myrcene synthase. This finding reflects the relative proportions of the induced levels of activities of these enzymes in grand fir saplings; pinene synthase and limonene synthase are the major monoterpene synthase activities, whereas the induced level of myrcene synthase activity is relatively low (18). The cDNAs for inducible monoterpene synthases provide probes for genetic and molecular analysis of oleoresin-based defense in conifers. Northern blots (Fig. 7) of total RNA extracted from non-wounded sapling stems and from stems 2 days after wounding (when enzyme activity first appears)5 were probed with cDNA fragments for ag2.2, ag3.18, and ag10 and thus demonstrated that increased mRNA accumulation for monoterpene synthases is responsible for this induced, defensive response in grand fir. The availability of cloned, defense-related monoterpene synthases presents several possible avenues for transgenic manipulation of oleoresin composition to improve tree resistance to bark beetles and other pests. For example, altering the monoterpene content of oleoresin may chemically disguise the host and decrease insect aggregation by changing the levels of pheromone precursors or predator attractants, or lower infestation by increasing toxicity toward beetles and their pathogenic fungal associates (1, 2, 6).


Fig. 7. Northern blot analysis of total RNA isolated from wounded and control stem tissue of grand fir saplings. Grand fir stem tissue was harvested for RNA extraction prior to wounding (C) and 2 days after wounding (W). Northern blots of 20 µg of total RNA per gel lane were probed with 32P-labeled cDNA fragments of ag2.2, ag3.18, and ag10.
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Properties of the Recombinant Monoterpene Synthases

All three recombinant enzymes require Mn2+ for activity, and Mg2+ is essentially ineffective as the divalent metal ion cofactor. This finding confirms earlier results obtained with the native monoterpene synthases of grand fir and lodgepole pine (Pinus contorta) (19, 47). All terpene synthases and prenyltransferases are thought to employ a divalent metal ion, usually Mg2+ or Mn2+, in the ionization steps of the reaction sequence to neutralize the negative charge of the diphosphate leaving group (12, 48, 49), and all relevant sequences thus far obtained bear a conserved aspartate-rich element (DDXXD) considered to be involved in divalent metal ion binding (22, 50-54). In addition to this strict, general dependence on a divalent metal ion, the monoterpene synthases of conifers are unique in their further requirement for a monovalent cation (K+), a feature that distinguishes the gymnosperm monoterpene synthases from their counterparts from angiosperm species and implies a fundamental structural and/or mechanistic difference between these two families of catalysts (47). All three recombinant monoterpene synthases depend upon K+, with maximum activity achieved at approximately 500 mM KCl. A requirement for K+ has been reported for a number of different types of enzymes, including those that catalyze phosphoryl cleavage or transfer reactions (55) such as Hsc70 ATPase (56). The crystal structure of bovine Hsc70 ATPase indicates that both Mg2+ and K+ interact directly with phosphate groups of the substrate and implicates three active site aspartate residues in Mg2+ and K+ binding (56), reminiscent of the proposed role of the conserved DDXXD motif of the terpene synthases and prenyltransferases in divalent cation binding, a function also supported by recent site-directed mutagenesis (57-60) and by x-ray structural analysis (52) of farnesyl diphosphate synthase.

cDNA cloning and functional expression of the myrcene, limonene, and pinene synthases from grand fir represent the first example of the isolation of multiple synthase genes from the same species and provide tools for evaluation of structure-function relationships in the construction of acyclic, monocyclic, and bicyclic monoterpene products and for detailed comparison to catalysts from phylogenetically distant plants that carry out ostensibly identical reactions (13, 16, 17, 61). The recent acquisition of cDNA isolates encoding sesquiterpene synthases2 and diterpene synthases (22) from grand fir should, together with the monoterpene synthases, also permit addressing the structural basis of chain length specificity for prenyl diphosphate substrates in this family of related enzymes.

Sequence Comparison and a Proposed Gene Nomenclature

Previous studies based on substrate protection from inactivation with selective amino acid modifying reagents have implicated functionally important cysteine, histidine, and arginine residues in a range of different monoterpene synthases (16, 19, 47, 62, 63). Sequence alignment of 21 terpene synthases of plant origin (17, 22-24, 34, 35, 64-68) reveals two absolutely conserved arginine residues, corresponding to Arg184 and Arg365 of pinene synthase (Fig. 2), one highly conserved cysteine residue (pinene synthase Cys543), and one highly conserved histidine residue (pinene synthase His186). The DDXXD sequence motif (pinene synthase Asp379, Asp380, and Asp383) (Fig. 2) is absolutely conserved in all relevant plant terpene synthases, as are several other amino acid residues corresponding to Phe198, Leu248, Glu322, Trp329, Trp460, and Pro467 of pinene synthase.

Amino acid sequences of the plant terpene synthases were compared with each other and with the deduced sequences of several sesquiterpene synthases cloned from microorganisms (54, 69, 70). As with all other plant terpene synthases, no significant conservation in primary sequence exists between the monoterpene synthases from grand fir and the terpene synthases of microbial origin, except for the DDXXD sequence motif previously identified as a common element of all terpene synthases, and prenyltransferases that employ a related electrophilic reaction mechanism (45, 51, 71). The evidence is presently insufficient to determine whether extant plant and microbial terpene synthases represent divergent evolution from a common ancestor, which may also have given rise to the prenyltransferases, or whether these similar catalysts evolved convergently.

Comparative levels of identity among the known plant terpene synthases (17, 22-24, 34, 35, 64-68) are indicated in Fig. 3. This family of genes, which we suggest be denoted by the three-letter designation tps (terpene synthase), currently consists of six sub-groups with at least 40% amino acid identity between members. The sub-groups are ordered tpsa through tpsf corresponding to the priority of publication of a member sequence. Interestingly, the monoterpene synthases, sesquiterpene synthases, and diterpene synthases from gymnosperms (tpsd), including taxadiene synthase from Pacific yew (Taxus brevifolia) (35), are more closely related to each other than to their respective counterparts from angiosperms (Fig. 3). This pattern of segregation implying limited evolutionary change in these gymnosperm catalysts is noteworthy since fossilized oleoresin (amber) dating from the carboniferous period (72, 73) indicates that gymnosperm terpene synthases had undergone functional specialization over 300 million years ago.


FOOTNOTES

*   This investigation was supported in part by United States Dept. of Agriculture NRI Grant 94-37302-0614, National Institutes of Health Grant GM-31354, and Project 0268 from the Agricultural Research Center, Washington State University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U87908, U87909, and AF006193.


Dagger    Feodor Lynen Fellow of the Alexander von Humboldt Foundation.
§   Present address: Plant Biology Division, S. R. Noble Foundation, Ardmore, OK 73402.
   To whom correspondence should be addressed. Tel.: 509-335-1790; Fax: 509-335-7643; E-mail: croteau{at}mail.wsu.edu.
1   The abbreviations used are: PCR, polymerase chain reaction; GLC, gas liquid chromatography; MS, mass spectrum/spectrometry; bp, base pair(s); nt, nucleotide(s); ORF, open reading frame; I, inosine.
2   C. L. Steele, J. Bohlmann, J. E. Crock, and R. Croteau, submitted for publication.
3   J. Bohlmann, J. E. Crock, R. Jetter, and R. Croteau, manuscript in preparation.
4   C. L. Steele, E. Lewinsohn, and R. Croteau, unpublished results.
5   C. L. Steele, S. Katoh, J. Bohlmann, and R. Croteau, manuscript in preparation.

ACKNOWLEDGEMENTS

We thank Eva Katahira, Hiroko Ichii, David Williams, Michael Phillips, and Thomas Savage for technical assistance; Gerhard Munske of the Washington State University Laboratory for Bioanalysis and Biotechnology for primer synthesis and nucleotide sequencing; Douglas J. McGarvey for guidance in developing the gene nomenclature; and Joyce Tamura-Brown for typing the manuscript.


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