Both for the molecular studies and the behavioral bioassays with parasitoids, plant material was taken from P. sylvestris trees growing in forests near Berlin, Germany. In the laboratory, cut pine twigs were subjected to standardized and controlled conditions during treatment (25°C, 18:6 h light/dark cycle, and approx. 2,000 lx). Twigs kept at these standardized conditions have been proven to provide reproducible results when studying the parasitoid’s response to oviposition-induced pine odor (Hilker et al. 2002b; Mumm et al. 2005). The inducibility of pine twigs by sawfly egg deposition was checked during the time when samples were taken for molecular studies. The twigs were considered inducible and used for molecular studies when the egg parasitoid C. ruforum responded positively to odor from samples 72 h after sawfly egg deposition. Branches of P. sylvestris used for analyses were cut from the middle part of 10- to 15-year-old trees and also from the lower part of 35- to 45-year-old trees. The lower part of a branch was cleaned, sterilized according to Moore and Clark (1968), and placed into water for treatment (see below). Plant material was taken in different seasons in 2004–2007.
The sawfly D. pini (Hymenoptera, Diprionidae) was reared in the laboratory on pine branches as described by Bombosch and Ramakers (1976) and Eichhorn and Pschorn-Walcher (1976) at 25 ± 1°C, 65% RH, and 18:6 h light/dark cycles. The egg parasitoid C. ruforum (Hymenoptera, Eulophidae) was collected in the field in southern Finland. Pine needles with parasitized host eggs were kept in Petri dishes at 5°C. To initiate parasitoid emergence from host eggs, parasitized eggs were transferred to a climate chamber with 25 ± 1°C, 65% RH, and 18:6 h light/dark cycles. Emerging adults were collected daily and transferred to 10 ± 1°C, 65% RH, and 16:8 h light/dark cycles until they were used for bioassays.
All types of twigs (egg-laden, artificially wounded, and untreated) were always cut from the same larger branch to minimize possible intra-tree-variation in terpenoid metabolism. For each experiment, pine twigs with 80–100 needles were cut, placed into a glass cylinder covered by a gauze lid, and supplied with water. An oviposition-induced twig is referred to here as test twig. For controls, two types of twigs were used: (a) a twig with an artificial wounding mimicking the ovipositional wounding, and (b) an untreated twig. Test and control twigs were always cut at the same time and kept at the same conditions. To obtain needle material for the molecular analyses, needles from control twigs were always removed at the same time points as needles from the respective test twigs and subjected to further analyses (see below).
Pine twig treatments
To obtain egg-laden pine twigs, three female and three male D. pini were added to a glass cylinder with a twig for a period of 16 h. During this time, the sawflies mated, and eggs were laid. When insects were removed from the cylinder, this was designated time zero (t0). The egg-laden twig (with about eight to ten needles carrying an egg mass) was then kept for 48, 72, or 96 h at the conditions described above. After these time periods, needles without eggs were removed from these twigs and subjected to further analyses. To obtain artificially wound-induced pine twigs, eight to ten needles of a pine twig were longitudinally slit at time point t0. The depth and length of the slit mimicked the wounding made by an ovipositing female D. pini with her ovipositor valves. The unwounded needles were removed from the wound-induced twigs 48, 72, or 96 h after treatment and used for further analyses. To obtain a branch-specific untreated twig control, needles from untreated control twigs were removed at the same time points as from the respective oviposition- or wound-induced twigs and subjected to further analyses. The time points when samples were collected for the molecular analyses reflected the times when behavioral studies were conducted.
A four-arm airflow olfactometer (Pettersson 1970; Vet et al. 1983) was used to test whether the egg parasitoid C. ruforum is attracted to odor of egg-laden pine twigs with an induction time other than 72 h (Hilker et al. 2002b). Airflows of 155 ml min−1 were allowed to enter a walking arena from four sides, thus establishing four distinct odor fields. One field was supplied with odor from an oviposition-induced pine twig, while the other fields were supplied with charcoal-filtered, humidified air (for further details see Hilker et al. 2002b). The parasitoid’s response was tested to odor from twigs with 48, 72, and 96 induction time. Even though the parasitoid’s response to odor from induced twigs 72 h after oviposition was known to be positive, this bioassay was included as positive control. When starting the bioassay, a single parasitoid female was introduced into the arena. The time the parasitoid spent walking in each of the four odor fields was recorded during an observation period of 600 s using a software program, The Observer 3.0 (Noldus, Wageningen, The Netherlands). Only data from active parasitoids walking at least 300 s of the observation period were used for statistical analysis. The number of parasitoids used per treatment was 34–37 (Table 1). The number of odor sources (twigs) tested was 6–9 per bioassay (Table 1). Data were analyzed by Friedman ANOVA by comparing walking times within each of the four odor fields. Wilcoxon–Wilcox tests were used for post-hoc comparison (Köhler et al. 1995). The analysis was performed using StatSoft, Version 1999, STATISTIKA for Windows (Tulsa, OK, USA).
All chemicals and solvents were of analytical grade and were obtained from Merck, Serva, or Sigma. The substrates geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) were from Echelon Res. Lab. Inc. (Salt Lake City, UT, USA).
RNA isolation and cDNA synthesis
Needle tissue from treated P. sylvestris twigs (see above) harvested 48, 72, or 96 h after oviposition or artificial wounding treatments or from respective control twigs was ground in liquid nitrogen with a sterilized mortar and pestle. To isolate RNA, the Invisorb Spin RNA Mini Kit (Invitek, Berlin, Germany) protocol was followed. Approximately, 100 mg of plant tissue was used per extraction. The RNA was eluted with 30 μl of RNAse free deionized water. For qRT-PCR, an additional DNase treatment was added using the RNase-Free DNase Kit (Qiagen, Hilden, Germany). Total RNA was checked for integrity and purity by spectrophotometer and tested additionally with RNA Nano Chips (Regent kit guide, RNA 6000 Nano assay, Agilent Technologies) by using a Bioanalyzer Agilent 2100 (Agilent Technologies). The synthesis of single stranded cDNA was carried out using Superscript III reverse transcriptase (Invitrogen), 0.6–3 μg RNA and oligo (dT)20 primers (Invitrogen) according to the manufacturer’s instructions. For qRT-PCR analysis, identical amounts of total RNA were used for reverse transcription.
Isolation of pine terpene synthase cDNA clones
Conserved regions of gymnosperm sesquiterpene synthases sequences from the following species (listed with accession numbers) were used to design degenerate primers: Picea abies (AAC05727, AAK39129, AAS47695) and Abies grandis (AAK83561, AAC06728). Using these primers, cDNA fragments were amplified from pine twigs 72 h after egg deposition by PCR under the following conditions: 0.2 μl Taq DNA Polymerase (5 U/μl), 2.5 μl 10× PCR-buffer for Taq Polymerase, 1 μl dNTPs (10 mM), 1 μl primer 1 and 2 (10 pmol/μl; see Supplemental data, Table S1), 0.2–3 μl cDNA, and H2O (added up to 25 μl). The PCR was conducted with an initial denaturation at 94°C for 3 min, 40 cycles of denaturation at 94°C for 40 s, annealing at 55°C for 40 s, extension at 72°C for 70 s, and a final step at 72°C for 5 min. For ligation and cloning of PCR fragments, the TOPO TA cloning TM kit for sequencing was used (Invitrogen). To generate the full-length coding cDNA sequence of the corresponding cDNA fragments, the BD SMARTTM RACE cDNA Amplification Kit (Clontech) was used according to the manufacturer’s instructions. The resulting cDNA amplicons were cloned into vector pCR 4-TOPO (Invitrogen) and sequenced using an ABI 3100 automatic sequencer (Applied Biosystems).
Functional expression of PsTPS 1, PsTPS 2, and PsTPS 3
The complete open reading frames of the full length cDNA of PsTPS 1, PsTPS 2, and PsTPS 3 clones and the signal peptide truncated version of PsTPS 2 were used for functional expression. PCRs were performed with primers (see Supplemental data Table S1) using the Expand High Fidelity Plus PCR System (Roche) as directed by the manufacturer. The amplification products were cloned into the pET-100D TOPOTM expression vector (Invitrogen). The expression vector was transformed into the E. coli strain Top 10F′ and its sequence verified. Mutation-free plasmids were transformed into the BL21 (DH3) pLysS strain of E. coli (Invitrogen).
For bacterial expression, a starter culture (10 ml Luria–Bertani medium with 35 μg/ml chloramphenicol and 100 μg/ml of carbenicillin) was grown for 3 days at 18°C; 5 ml of starter culture in 100 ml LB medium (with 35 μg/ml of chloramphenicol and 100 μg/ml carbenicillin) was induced with 2 mM isopropyl-β-galactoside (IPTG) at an OD = 0.6 and kept at 18°C for at least 15 h. The cells were centrifuged for 20 min at 9,000g. The pellets were resuspended in 3 ml of extraction buffer (Martin et al. 2004) and disrupted by sonication (Bandelin Sonopuls HD 2070, Berlin, Germany) for 4 min, cycle 2, power 60%. After freezing (10 min at −20°C, 10 min at −80°C), the cell fragments were collected by centrifugation.
The supernatant containing the total bacterial crude protein extract was assayed. Each assay was performed in a 1 ml volume with 69.9 μM FPP, overlaid with 1 ml pentane, and incubated at 30°C. For control assays, substrate concentrations of 99.5 μM GPP and 37.0 μM GGPP were used. One hour after pentane addition, the assay was stopped by vigorous vortexing with the pentane overlay for 30 s and separation of the aqueous and organic fractions by centrifugation at 2,500g for 2 min. The pentane fraction was removed, and the residue was overlaid again with 1 ml pentane. In total, three consecutive pentane extractions were conducted. Finally, the pentane fractions were combined, dried over a silica/MgSO4 column, and evaporated to 50–100 μl. These samples were subjected to GC–MS analyses (see below, product identification).
Enzyme concentrations were measured according to Bradford (1976) by using the BioRad reagent with bovine serum albumin (BSA) as standard. The protein concentration used in each assay was adjusted prior to a range of 0.5–2.5 μg/ml.
Sesquiterpene extraction from needle tissue
To investigate whether the expression levels of the sesquiterpene synthases in differently treated pine twigs were reflected by different amounts of the major products of these synthases, we analyzed (a) oviposition-induced pine twigs 72 h after egg deposition and (b) artificially wounded pine twigs 72 h after treatment for their sesquiterpene contents. For terpene extraction, 200 mg ground needles (see above, RNA isolation and cDNA synthesis) were used. The extraction procedure was based on a method described by Martin et al. (2002). All steps were carried out in 2 ml vials tightly closed with a teflon-coated screw cap (Hewlett-Packard, Palo Alto, CA, USA). The needle samples were submerged into 1.0 ml of tert-butyl methyl ether containing 150 μg/ml isobutylbenzene as internal standard and extracted 14 h overnight with constant shaking at room temperature. The ethereal supernatant was transferred to a fresh vial and washed with 0.3 ml of 0.1 M (NH4)2CO3 (pH 8.0). This sample was filtered through a Pasteur pipette column filled with 0.3 g of silica gel (Sigma 60 Å) overlaid with 0.2 g of anhydrous MgSO4. The column was washed with 1 ml of diethyl ether. The eluate was evaporated to an approximate volume of 100 μl and used for further GC–MS analyses of terpenoids (see below, product identification).
Products of sesquiterpene synthase assays and extracts of pine needles were analyzed on a GC system (Agilent Hewlett-Packard 6890, Agilent Technologies) coupled to a Network Mass Selective Detector (Agilent Hewlett-Packard 5973, Agilent Technologies). For analyses, 1 μl concentrated pentane phase (assays) or ether phase (needle extract) was injected at an injector temperature of 220°C on a HP-5 capillary column (30 m × 0.25 mm with a 0.25 μm phase coating; Agilent Technologies). The temperature program started with 40°C for 2 min, raised to 210°C (5°C min−1), and raised further to 300°C (60°C min−1, 2 min hold; helium flow: 2 ml min−1). For identification of compounds, the MS detector was operated using the total ion mode at a temperature of 230°C. The products were identified by comparing mass spectra and retention times with those in the literature and in the Wiley 275.L or NIST 98.1 MS libraries. The identity of (E)-β-caryophyllene, α-humulene, longipinene, and longifolene was further verified by comparison with authentic standards.
Those sesquiterpenes that were the major products of the sesquiterpene synthases were quantified in pine needle extracts with 150 μg/ml isobutylbenzene as internal standard (compare above). Mean ± SE of the relative quantities of these compounds were calculated from three independent biological pine needle samples of each treatment with each biological sample analyzed three times.
Real time quantification of gene transcription was performed using SYBR green QPCR Master Mix from Stratagene (La Jolla, CA, USA) in order to address the following questions: (a) do transcript levels of PsTPS 1-3 differ between oviposition-induced samples and artificially wounded ones?; (b) how do transcript levels of these sequences change over time after treatment?
QRT-PCRs were performed as described in the operator’s manual using a Stratagene MX3000PTM. Gene-specific PCR primers were designed (see Supplemental data Table S1) using criteria including predicted melting temperature of at least 58°C, primer length of 22–24 nucleotides, guanosine–cytosine content of at least 48%, and an amplicon length of 120–150 bp. Primer specificity was confirmed by melting curve analysis, by an efficiency of product amplification of 1.0 ± 0.1, and by sequence verification of at least eight cloned PCR amplicons for each gene. Reactions with water instead of cDNA template were run with each primer pair as control. The standard thermal profile of 95°C for 10 min, then 60 cycles of 95°C for 30 s, 53°C for 30 s and 72°C for 30 s was used. The fluorescence signal was captured at the end of each cycle, and a melting curve analysis was performed from the annealing temperature to 95°C with data capture every 0.2°C during a 1 s hold.
The quantity of each transcript is the average of four (48 h), five (72 h), and three (96 h) independent biological replicates, each of which is represented by at least three technical replicates. All amplification plots were analyzed with the MX3000PTM software to obtain threshold cycle (C
t) values. Transcript abundance was normalized to the transcript abundance of ubiquitin (GenBank accession number EF681766). Relative transcript values were obtained by calibration first against the transcript abundance of the respective untreated twig control, and, second, against the transcript abundance of the artificial wounding control 72 h after treatment.
A two-way ANOVA was performed on qRT-PCR raw data to test the significance of differences in changes of PsTPS 1, PsTPS 2, and PsTPS 3 transcript levels in course of time (independent samples; between-subject factor time) and due to treatment (dependent, paired samples; within-subject factor treatment). Also the combined effect of time × treatment was statistically tested. Normal distribution of qRT-PCR raw data was found for PsTPS 1 and PsTPS 3. PsTPS 2 data were arctan-transformed prior to ANOVA (Sokal and Rohlf 1995). All analyses were performed using StatSoft, Version 1999, STATISTICA for Windows (Tulsa, OK, USA) (see Supplemental data Table S2).
Sequence and phylogenetic analyses
DNASTAR Lasergene program version 7.0 (Meg AlignTM) was used to align and to calculate the deduced amino acid sequences of each full-length P. sylvestris cDNA and of known sequences from gymno- and angiosperms. The amino acid alignment was assembled by use of ClustalW (gonnet 250 matrix, gap penalty: 10.00, gap length penalty: 0.20, delay divergent sequences: 30%, gap length 0.10, DNA transition weight 0.5). The same software was used to visualize the phylogenetic tree.