Insect Attack and Wounding Induce Traumatic Resin Duct Development and Gene Expression of (—)-Pinene Synthase in Sitka Spruce

cDNA Cloning and Functional Expression of (—)-Pinene Synthase

To obtain a homologous tps probe for characterization of insect- and wound-induced traumatic resinosis in Sitka spruce, oligonucleotide primers were designed based on conserved sequences of grand fir monoterpene synthases (Bohlmann et al., 1997, 1999). A 534-bp partial tps-like cDNA fragment, PsTPS1 (GenBank accession no. AW287755), was isolated by reverse-transcriptase (RT)-PCR using RNA isolated from the leaders of lateral branches of Sitka spruce trees harvested 7 d after drill wounding. A high degree of similarity exists between PsTPS1 and the published sequences of conifer monoterpene synthases. The highest percentage of deduced amino acid sequence identity were shared with (—)-pinene synthase (76%), (—)-camphene synthase (74%), and terpinolene synthase (74%) from grand fir (Bohlmann et al., 1997, 1999), a monoterpene synthase-like protein (74%; GenBank accession no. AA061229) and (—)-α-pinene synthase from loblolly pine (72%; Phillips et al., 2003), and a putative β-phellandrene synthase-like protein (72%) from Norway spruce (GenBank accession no. AF369918).

A full-length cDNA related to the partial tps cDNA fragment, PsTPS1, was cloned by RACE and was designated PsTPS2 (GenBank accession no. AY237645; Fig. 1). PsTPS2 shares 81% nucleotide sequence identity with PsTPS1. Despite several attempts, we could not isolate a full-length cDNA with an exact nucleotide sequence match for PsTPS1. Based on overall sequence relatedness, PsTPS2 was classified as a member of the tps-d subfamily of plant terpene synthases (Bohlmann et al., 1998b). This subfamily contains monoterpene synthases, sesquiterpene synthases, and diterpene synthases from gymnosperms. PsTPS2 is most closely related to conifer monoterpene synthases, (—)-α-pinene synthase from loblolly pine (82% amino acid identity and 91% amino acid similarity), (—)-pinene synthase from grand fir (80% amino acid identity and 88% amino acid similarity), α-terpineol synthase from loblolly pine (75% amino acid identity and 86% amino acid similarity), and (—)-camphene synthase from grand fir (73% amino acid identity and 83% amino acid similarity; Figs. 1 and 2). The deduced open reading frame of PsTPS2 is 627 amino acids long and contains the conserved RRX₈W and DDXXD motifs (Bohlmann et al., 1998b; Davis and Croteau, 2000; Aubourg et al., 2002; Fig. 1).

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Figure 1.

Multiple sequence alignment of the deduced amino acid sequences of PsTPS2 and the partial cDNA PsTPS1 from Sitka spruce with (—)-α-pinene synthase (AAO61225) and α-terpineol synthase (AAO61227) from loblolly pine, and (—)-pinene synthase (U87909), (—)camphene synthase (U87910), terpinolene synthase (AF139206), (—)-β-phellandrene synthase (AF139205), (—)-limonene synthase (AF006193), and myrcene synthase (U87908) from grand fir. Identical amino acids are shown as white letters on a black background; similar amino acids are shaded gray. The RRX₈W and DDXXD motifs are indicated below the alignment. The number above the alignment represents amino acid position.

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Figure 2.

Relatedness of deduced amino acid sequences of selected conifer monoterpene synthases from grand fir and Sitka spruce sequence PsTPS2. The dendrogram includes (—)-α-pinene synthase and α-terpineol synthase from loblolly pine, and (—)-pinene synthase, (—)-limonene synthase, myrcene synthase, terpinolene synthase, (—)-camphene synthase, and (—)-β-phellandrene synthase from grand fir. All methods of phylogenetic analyses converged upon an identical tree topology that suggests PsTPS2 is most closely related to loblolly pine (—)-α-pinene synthase, loblolly pine α-terpineol synthase and grand fir (—)-pinene synthase. The above figure shows the single parsimony tree obtained from PAUP^* (tree length = 1,524, CI = 0.82, RI = 0.46, RC = 0.37). Numbers below branches are bootstrap values based on 1,000 heuristic replicates. Branch lengths (numbers above branches) reflect number of synapomorphies.

Because the exact biochemical function of enzymes encoded by tps genes cannot be predicted based on primary structure, PsTPS2 was expressed in the form of a truncated protein without the predicted plastid target sequence in E. coli using the pET101/d-TOPO expression vector system. The truncated, “pseudomature” (Williams et al., 1998) protein was designed by introducing a starting Met immediately upstream of the tandem Arg residues of the conserved RRX₈W-motif (Fig. 1), which defines the approximate truncation site of the plastid target sequence of nuclear-encoded monoterpene synthases (Williams et al., 1998). Recombinant PsTPS2 protein was active with geranyl diphosphate (GPP) as substrate and produced the monoterpene olefins (—)-α-pinene and (—)-β-pinene in a ratio of approximately 35:10 (Fig. 3). The monoterpene products were identified by gas chromatography-mass spectrometry (GC-MS) using authentic standards for comparison (Fig. 3).

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Figure 3.

GC and MS of the monoterpene products derived from GPP by PsTPS2 (—)-pinene synthase. A, Total ion chromatogram of products of PsTPS2 enzyme activity formed from GPP and separated on a HP-5 capillary column. The major products are by order of retention time: (—)-α-pinene (62.5% of total product, peak 1) and (—)-β-pinene (17.8% of total product, peak 2). Enantiomers of α-pinene and β-pinene were separated on a Cyclodex-B capillary column, and (—)-α-pinene and (—)-β-pinene were identified by comparison of retention times with enantiomerically pure standards. B, Mass spectra of enzyme products peak 1 (—)-α-pinene and peak 2 (—)-β-pinene. Smaller inserts show mass spectra of authentic (—)-α-pinene and (—)-β-pinene.

Traumatic Resin Duct Development in Sitka Spruce

Before testing the effect of wounding and insects on tps gene expression, we established that trees of this study could respond to a mechanical wounding event with formation of TRD in the developing xylem. For these experiments, leaders of lateral branches were drill wounded and subsequently examined by light microscopy for the presence of TRD. Branches were harvested 4 months after treatment so that the traumatic resinosis response was well established and embedded within the current years wood. TRD were clearly evident in the xylem tissue of drill-wounded lateral branches (Fig. 4A). Induction of traumatic resinosis was a significant event in leaders of wounded lateral branches, and xylem resin ducts were rare in nonwounded controls being detected in just one sample (Fisher's exact test, P = 0.01, n = 18; Fig. 4B).

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Figure 4.

Induced TRD in Sitka spruce developing xylem. A, Representative cross-sections through nonwounded (score = 0; i) and drill-wounded (score = 2.0; ii) lateral branches. Branches were harvested for histology 4 months after treatment. B, Mean histochemical scores for drill-wounded and nonwounded lateral branches. The criteria used to establish the scores are described in “Materials and Methods.” The numbers within the bars represent the number of samples used for analyses.

SE bars are indicated.

Mono-tps Gene Expression in Sitka Spruce Is Responsive to Insect Attack and Mechanical Wounding

Leaders of lateral branches of Sitka spruce trees were subjected to weevil attack by caging three males and three females in crinoline bags for 1, 2, 4, and 7 d. These branches were subsequently harvested and RNA was extracted from combined bark and wood tissue samples. Mono-tps gene expression was assessed using the partial PsTPS1 probe. Weevil responsive mono-tps gene expression was assessed in four or five separate trees for each time point examined, and expression data were pooled and compared with that present in nontreated trees (Fig. 5). In addition, expression was monitored in branches harvested from trees that were dormant. Constitutive mono-tps expression was low in the leaders of lateral branches derived from dormant trees and was somewhat higher in the nontreated controls harvested at the time of experimental treatments. Weevil attack elicited a 2.0-fold induction of mono-tps transcripts 1 d after attack. By d 2 and 4, a significant 2.3-fold increase in transcript accumulation was observed (means contrast, P < 0.05). Transcript levels declined by d 7.

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Figure 5.

Mono-tps gene expression in weevil-attacked lateral branches. RNA was isolated from a combined bark/xylem tissue sample derived from the terminal growth of lateral branches 1, 2, 4, and 7 d after onset of exposure to weevils, and from branches to which no weevils were caged (dormant and control). RNA was applied to dot blots and relative expression levels were obtained by dividing the log-transformed dot intensity areas obtained after hybridization with the mono-tps probe PsTPS1 by that obtained using the 16S rRNA probe. The numbers within each bar represent the number of pooled branch samples; for each sample, an individual tree was used. SEs are shown for each treatment.

Although lateral branches and apical leaders are attacked by weevils during feeding, apical leaders are the primary targets for egg deposition. To compare the response elicited by mechanical wounding with that induced by insect attack in these stem samples, apical leaders and leaders of lateral branches were drill wounded. Constitutive levels of mono-tps transcripts were lower in apical leaders than in leaders of lateral branches (Fig. 6). Mono-tps transcript levels increased 2 d after drill wounding in both stem samples, although the increase in apical leaders was greater than that in the lateral branches relative to the corresponding undrilled controls. A significant 2.9- and 2-fold increase in mono-tps expression 4 d after wounding in the apical leaders and leaders of lateral branches, respectively, was observed (Tukey-Kramer α = 0.05; Fig. 6). This suggests that results obtained for the leaders of lateral branches may be indicative of a comparable albeit weaker induction than that which occurs in apical leaders under similar treatment.

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Figure 6.

Expression of mono-tps in drill-wounded leaders and lateral branches of Sitka spruce. RNA was isolated from leaders and branches 2 and 4 d after drill wounding, and from nonwounded leaders and branches (control). All other information is as found in the legend for Figure 5.

Given the high degree of sequence similarity between various mono-tps genes, it was anticipated that the partial PsTPS1 probe would hybridize to a pool of related transcripts. PsTPS1 shares 81% nucleotide sequence identity with PsTPS2, raising the possibility that the PsTPS1 probe used for expression analyses may hybridize to a different pool of mono-tps transcripts than that recognized by the (—)-pinene synthase probe. To establish whether this was the case, identical blots containing RNA isolated from drill-wounded Sitka spruce leaders were hybridized with the partial cDNA PsTPS1 or full-length PsTPS2 probes. A similar pattern of hybridization to RNA isolated from drill-wounded leaders was consistently obtained for both probes. Representative data derived from two distinct Sitka spruce genotypes is presented for which a similar wound-responsive pattern of mono-tps transcript accumulation from 12 h to 4 d after drilling was observed (Fig. 7). Southern analyses with both probes hybridized against each other and to other putative di- and sesqui-tps cDNAs revealed maximum hybridization between the PsTPS probes and their corresponding cDNAs (data not shown). Approximately 60% cross-hybridization was observed between PsTPS1 and PsTPS2. No cross-hybridization was detected with di- and sesqui-tps cDNAs (data not shown). These data suggest that results obtained with PsTPS1 and PsTPS2 are equivalent for monitoring overall mono-tps transcript accumulation without discriminating gene-specific signals.

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Figure 7.

Mono-tps transcript level in drill-wounded leaders detected by northern hybridization analyses with PsTPS1 and PsTPS2. Data presented in A and B are derived from two different Sitka spruce genotypes. RNA was isolated from leaders harvested 12 to 72 h after drill wounding and from a nonwounded leader (control). A single tree was used for each time point.

Plant Material

Six-year-old Sitka spruce (Picea sitchensis Bong. Carr.) clonal trees derived from somatic embryos outplanted at the University of British Columbia Malcolm Knapp research forest (Maple Ridge, British Columbia, Canada) were used in 1997 for wound induction to obtain RNA for isolation of a partial tps cDNA. The Sitka spruce experimental plantation at the North Arm Substation of the British Columbia Forest Service's Cowichan Lake Experiment Station (Lake Cowichan, British Columbia, Canada) was used for weevil (Pissodes strobi Peck.) induction experiments in 1998 and wound induction experiments in 1999. This plantation was previously described in Alfaro et al. (2000). Trees at the experimental plantation were 7 to 8 years old at the time of treatment. Wounding studies were also conducted in 2001 on 8-year-old Sitka spruce trees as part of a clonal progeny trial established at Armishaw Road (Sayward, British Columbia, Canada) by the Ministry of Forests. Two-year-old Sitka spruce trees (clone 898) from the British Columbia Ministry of Forests breeding program was used to obtain RNA for isolation of a full-length cDNA.

Plant Treatment

All wounding treatments were conducted in the spring to coincide with maximal weevil activity on the British Columbia coast. Leaders of lateral branches and apical leaders of Sitka spruce were mechanically wounded to simulate an insect attack using a minimite drill (Dremel, Racine, WI) equipped with a 0.95-mm drill bit. Each branch was drill wounded to a depth of 2 to 4 mm (depending on stem diameter) so that the bark was just penetrated. Wounding proceeded in two straight lines from the apex to the base along two sectors of the circumference with each wound spaced approximately 1 cm apart. Wounded branches and leaders were harvested at 1, 2, 4, and 7 d, frozen on dry ice, and subsequently stored at —80°C. A branch from a nonwounded tree was similarly harvested to serve as a control. Four months after wounding, branches were harvested from a wounded and nonwounded tree for histology. For weevil exposure, three female and three male weevils were caged within crinoline bags for a period of 1, 2, 4, or 7 d on a leader of a lateral branch of Sitka spruce. Tissue was harvested as described above. In addition, a dormant lateral branch sample was harvested 3 months before weevil exposure treatments.

Histology

Approximately 4.0 cm of the base of a drill-wounded lateral branch was fixed in 7% (v/v) formalin, 3% (v/v) glacial acetic acid, and 70% (v/v) ethanol), sectioned to 30 μm with a sliding microtome, and stained with 1% (w/v) aqueous safranin (Tomlin et al., 1998). Xylem of the current year's growth was examined for TRD using a light microscope. The wound response was quantified based on the extent of TRD formation and the relative quantity of red stained cells in the xylem at the approximate site of wounding. The scoring system was based on a modification of the method described by Alfaro et al. (1996) and Tomlin et al. (1998). Because the TRD response in leaders of lateral branches of Sitka spruce was less vigorous than that reported for leaders of white spruce, the scoring system was as follows: a) no apparent response in early wood was equal to a score of zero; b) red stain restricted to a few cells was equal to a score of 0.5; c) a red stained ring in the early wood was equal to a score of 1.0; d) a red stained ring associated with scattered TRD was equal to a score of 1.5; and e) an intense red stained ring with a ring of TRD was equal to a score of 2.0.

Isolation of a Sitka Spruce tps Partial cDNA Probe

Total RNA was isolated from 5 to 7 g of a combined bark/wood tissue sample derived from the leaders of lateral branches of drill-wounded Sitka spruce somatic embryo-derived clonal trees using a LiCl precipitation protocol optimized for conifer tissues (Wang et al., 2000). First strand cDNA was synthesized at 37°C for 1.5 h in an 80-μL reaction volume containing 140 μg of total RNA, 8 mm dithiothreitol (DTT), 0.28 mm oligo (dT)₁₆, 140 units of human placental ribonuclease inhibitor (Pharmacia Biotech, Mississauga, Canada), 0.63 mm dNTPs, 1,000 units of M-MLV reverse transcriptase (Invitrogen, Burlington, Canada), 50 mm Tris-HCl (pH 8.3), 75 mm KCl, and 3 mm MgCl₂. PCR was conducted using degenerate primers (sense primer: 5′-GCIYGAYTAYGTITAY-3′ and antisense primer: 5′-ACCACCTTYCTYWSICCI-3′) corresponding to conserved regions of various tps [taxadiene synthase from Taxus brevifolia; and (E)-α-bisabolene, abietadiene, myrcene, (-)-pinene, (-)-limonene, δ-selinene, and γ-humulene synthases from Abies grandis] and 150 ng of cDNA, 20 mm Tris-HCl (pH 8.4), 50 mm KCl, 0.1 mm dNTPs, 0.5 μm each sense and antisense primer, 3 mm MgCl₂, and 2.5 units of Taq polymerase (Invitrogen, Burlington, Canada). PCR was carried out for 45 cycles using the following steps: denaturation at 94°C for 1 min, annealing at 45°C for 1 min, and extension at 72°C for 2 min. Each reaction was initially denatured at 94°C for 4 min and received a final extension at 72°C for 10 min. PCR products were cloned into the pCR 2.1 TA plasmid (Invitrogen, Carlsbad, CA) and the nucleotide sequence was determined.

Northern and Dot-Blot Hybridization Analyses

For northern-blot analyses, total RNA (30 μg) was size-separated in a denaturing formaldehyde 1% (w/v) agarose gel (Sambrook et al., 1989), transferred to a positively charged nylon membrane (Boehringer Mannheim Biomedicals, Laval, Canada), and fixed by UV-crosslinking (UV Stratalinker 2400; Stratagene, La Jolla, CA) followed by baking at 80°C for 30 min. Even loading of RNA samples was established by inspecting the ethidium bromide-stained gel for the major ribosomal RNAs and probing the blot with a 16S Sitka spruce rRNA probe. For dot-blot analyses, RNA and DNA samples were denatured and applied to a positively charged nylon membrane, and fixed to the membrane by UV-crosslinking followed by baking. Even loading of RNA was established by probing the dot blot with the 16S Sitka spruce rRNA probe. DNA probes for hybridization were prepared using 25 ng of DNA as template with 1.85 MBq [α-³²P]dCTP (Amersham, Baie d'Urfé, Canada) using a random labeling kit (Amersham Pharmacia, Piscataway, NJ). Membranes were prehybridized in 5% (w/v) SDS, 0.1% (w/v) bovine serum albumin, 1 mm EDTA, 500 mm NaH₂PO₄, and 50% (v/v) formamide for 1 h at 42°C, and were then hybridized overnight at the same temperature with ³²P-labeled DNA probe. After hybridization, membranes were washed at 42°C in 1× SSPE buffer containing 0.3% (w/v) SDS, and then in 1× SSPE/0.5% (w/v) SDS, and finally in 0.2× SSPE/1% (w/v) SDS. X-ray film (X-Omat blue XB-1; Eastman-Kodak, Toronto) was exposed to membranes with an intensifying screen. Dot intensity was determined using Scion image version 1.62c (macrofunction gel plot 2) and was subsequently normalized by dividing the hybridization signal obtained for the mono-tps probe by that of the rRNA probe. For dot-blot analyses, resulting data were log transformed to correct for heteroscedasticity and were analyzed using analysis of variance (generalized linear model) and either the Tukey-Kramer multiple means comparison test or a means contrast analysis (α = 0.05) using JPM4.0.3 (SAS Institute, Cary, NC).

Isolation of a Full-Length Sitka Spruce tps cDNA by 5′- and 3′-RACE

Two-year-old greenhouse-grown Sitka spruce trees (clone 898) were mechanically wounded as described above, sprayed with 0.01% (v/v) MeJA (Sigma Aldrich, Oakville, Canada) as described (Martin et al., 2002; Fȧldt et al., 2003), and loosely covered with clear plastic bags. The leader and the previous year's internode were harvested 2 d later, frozen in liquid nitrogen, and stored at —80°C. RNA was isolated (Wang et al., 2000) and used for 5′-and 3′-RACE. Based on the partial tps probe PsTPS1 sequence, RACE primers were designed: 5′ Outer (5′-AGAAACTTTTAGAACTAGC-3′) and 5′ Inner (5′-GGCATTGGGTGTGGGAGAGAGAGTGTTATTACGGA-3′). To obtain the 3′ end, one tps primer was used: 3′Outer (5′-CATCGACGTATTTGGACAGGAC-3′). 5′- and 3′-cDNA ends were amplified from RT reactions according to instructions outlined by the manufacturer (RLM-RACE; Ambion, Austin, TX), cloned into pCR 2.1 TOPO (Invitrogen, Burlington, Canada), and sequenced. Full-length cDNAs were obtained using primers whose design was based on the sequences of 5′-and 3′-RACE products. Amplification was performed in a 50-μL volume containing 10 mm Tris-HCl (pH 8.3), 50 mm KCl, 1.5 mm MgCl₂, 5 units of SuperTaq Plus (Ambion), 0.5 μm each primer, 50 μm dNTPs, and 1 μL of the 5′ RT reaction. The following cycling conditions were used: 30 cycles of 94°C for 1 min, 55°C for 35 s, and 68°C for 2 min preceded by an initial denaturation at 94°C for 3 min and followed by a 7-min extension at 68°C. Full-length cDNAs were cloned into pCR2.1TOPO, purified using the Qiaprep spin miniprep kit (Qiagen, Mississauga, Canada), and the nucleotide sequence of the inserts was determined. Sequences were aligned using CLUSTAL W (v1.4) in MacVector 7.1.1 using a 7.0 open gap penalty, 40% delay divergent, and Blosum similarity matrix.

Functional Expression of PsTPS2 cDNA in Escherichia coli

A pseudomature, truncated version of the PsTPS2 cDNA starting with a Met directly upstream of the RRX₈W motif was generated by PCR. PCR was performed in a 50-μL volume containing 10 ng of template pCR2.1-PsTPS19-12, 3 units of high-fidelity Pwo DNA polymerase (Roche Diagnostics, Laval, Canada), and 5 μL of the provided PCR buffer with MgSO₄ [100 mm Tris-HCl, pH 8.85, 250 mm KCl, 50 mm (NH₄)₂SO₄, and 20 mM MgSO₄], 10 mm dNTPs, and 20 pmol of each primer (5′-CACCATGTCTGATGATGGTGTAC-3′ and 5′-TTACAAAGTCACAGGATCAATCACG-3′). PCR conditions were 3 min at 95°C, followed by 30 cycles of 30 s at 95°C,30 s at 55°C, 2 min at 72°C, and ending with a 7-min final extension at 72°C. PCR products were cloned into the pET101/d-TOPO expression vector (Invitrogen, Burlington, Canada) following the manufacturer's instructions, and recombinant plasmid was transformed into E. coli TOP 10 F' cells (Invitrogen, Burlington, Canada). Plasmid DNA pET101/d-TOPO-PsTPS2 was purified and transformed into E. coli BL21-CodonPlus (DE3) cells (Stratagene, La Jolla, CA) for expression. Positive clones were screened by PCR (same conditions as above) using T7 Forward and T7 Reverse vector-based primers (Invitrogen, Burlington, Canada).

Monoterpene Synthase Enzyme Assays

Bacterial strain E. coli BL21-CodonPlus (DE3) containing plasmid pET101/ d-TOPO-PsTPS2 was grown at 37°C in 100 mL of Luria-Bertani broth supplemented with 20 μg mL^—1 ampicillin (Amp) to A₆₀₀ = 0.5. The culture was transferred to 20°C for 30 min and was then induced with 1 mm isopropylthio-β-galactoside and left to grow overnight. Cells were harvested by centrifugation, and were resuspended and disrupted in 1 mL of monoterpene synthase buffer (25 mm HEPES, pH 7.2, 100 mm KCl, 10 mm MnCl₂, 10% [v/v] glycerol, and 5 mm DTT) by sonication (Branson Sonifier 250; AmTech, Shelton, CT) at 5W for 10 s. Lysates were cleared by centrifugation, and 5 mm DTT and 164 mm GPP (Echelon Research Laboratories, Salt Lake City, UT) were added to the extract and overlaid with 1 mL of pentane. Assay mixtures were then incubated at 30°C for 2 h. Products were collected with three consecutive pentane extractions (3 × 1 mL) and were combined over water. The pentane fractions were dried over MgSO₄ and were concentrated under nitrogen. Products were analyzed by GC-MS.

Monoterpene Product Identification by GC-MS

Products of monoterpene synthase assays were identified on an GC System (Agilent 6890 Series; Agilent Technologies, Mississauga, Canada) coupled to an Network Mass Selective Detector (Agilent 5973; Agilent Technologies). Monoterpenes were initially identified using a HP-5 capillary column (0.25 mm i.d. × 30 m with 0.25-μm film; Agilent Technologies) with an initial temperature of 40°C (2-min hold), which was then increased 3°C min^—1 up to 140°C, followed by a 20°C ramp until 300°C (10-min hold). For differentiating enantiomers, a Cyclodex-B capillary column (0.25 mm i.d. × 30 m with 0.25-μm film; J & W Scientific, Folsam, CA) was used with an initial temperature of 60°C (15-min hold), which was then increased 0.5°C min^—1 up to 200°C, followed by a 200°C ramp (10-min hold). Compounds were identified by using Agilent Technologies software and Wiley 126 MS-library, as well as by comparing retention time with those of the appropriate enantiomerically pure standards (Aldrich Chemical Company, Milwaukee, WI).

Phylogenetic Analysis of Sitka Spruce TPS

Maximum parsimony (Eck and Dayoff, 1966; Fitch, 1977) and bioneighbour-joining (Gascuel, 1997) unrooted trees were generated in PAUP^* (Swofford, 2000) using full-length amino acid sequences (PsTPS2 from Sitka spruce), (—)-α-pinene and α-terpineol synthases from loblolly pine (Phillips et al., 2003), and (—)-pinene, (—)-limonene, (—)-camphene, (—)-β-phellandrene, terpinolene, and myrcene synthases from grand fir (Bohlmann et al., 1997, 1999) that contained the N-terminal transit peptide. Sequences of a grand fir sesquiterpene synthase (γ-humulene synthase U92267; Steele et al., 1998a) and a grand fir diterpene synthase (abietadiene synthase U50768; Stofer Vogel et al., 1996) were used as outgroups. A bioneighbour-joining tree was generated using pair-wise distances calculated using absolute distances. Maximum parsimony of equally weighted and unordered character state transformations were used to generate the most parsimonious trees based on a branch and bound search. Initial tree(s) were constructed using random stepwise addition. Branch swapping was implemented through tree bisection and reconnection and steepest descent.

For both types of trees, data was resampled using 1,000 heuristic bootstrap replicates.