Plant materials and treatments
Arabidopsis thaliana (L.) Heynh (ecotype Ler) plants were grown on soil in a climate-controlled growth chamber similar to that reported by Chen et al. (2003) Either glufosinate-ammonium- or kanamycin-resistant seedlings were transferred to soil and later the respective transgenes were confirmed by PCR. All treatments were pre-formed with 4–6-week-old rosette stage (non-bolting) plants of A. thaliana (ecotype Ler). For the mechanical wounding treatment, two lateral incisions to each side of the midvein of the leaves were made with a sterile razor blade. Eggs of S. exigua (Lepidoptera: Noctuidae) were obtained from Aventis (Frankfurt, Germany) and were reared on an artificial wheat-germ diet (Heliothis mix; Stonefly Industries, Bryan, TX, USA) for about 10–15 days at 22°C under an illumination of 750 μmol m−2 s−1. For the herbivory treatments, two-third instar larvae were placed on the rosette portion of each plant and were allowed to feed for the duration of the headspace collections.
The T-DNA insertion line SALK_025557 was obtained from the Arabidopsis Biological Resource Center (ABRC) and analyzed for resistance to kanamycin as described above (Alonso et al., 2003). In addition, a PCR-based assay was designed to detect the presence of the T-DNA insertion by utilizing a primer from the T-DNA left border, LBb1, 5′-GCGTGGACCGCTTGCTGCAACT-3′, in conjunction with an internal primer specific for At3g03480, 5′-CCACCCGTATGGAACCGACA-3′. In addition, a second primer specific for a region downstream of the T-DNA insertion was also used to test for zygosity, 5′-TCATCCTTTAGACACATTTAGCACTCC-3′, by performing a PCR reaction with both gene-specific primers. Plants homozygous for the T-DNA insert and segregating for kanamycin resistance were then backcrossed for seven generations to Ler plants. In every generation, 30 progeny were screened for the presence of the T-DNA insert and a minimum of five positive plants, based on the PCR assay, were used as the pollen donors for backcrossing. Plants positive for a T-DNA insert after the seventh generation of backcrossing were allowed to self-fertilize and were subsequently used for analysis.
All solvents and reagents were either molecular biology or reagent grade and were obtained from Fluka (Munich, Germany), Sigma-Aldrich (Munich, Germany), or Roth (Karlsruhe, Germany), unless otherwise noted.
Radiolabeled [1-14C]acetyl-CoA (57.5 mCi mmol−1) was purchased from Amersham Biosciences (Uppsala, Sweden). [7-14C]Benzoyl-CoA was enzymatically synthesized from [7-14C] benzoic acid (57 mCi mmol−1; Movarek Biochemicals, Brea, CA, USA) as previously described (Beuerle and Pichersky, 2002). Radiolabeled [2-14C]malonyl-CoA (53 mCi mmol−1) was purchased from Movarek.
Enzyme extraction and assay
Crude protein extracts were prepared and assayed as previously reported (D'Auria et al., 2002). For each time point, three replicates consisting of leaves from three different plants were combined, and assays were repeated at least three times. Assay samples were prepared by adding the following to a 0.6-ml microcentrifuge tube: 5.0 μl of crude extract, 10.0 μl of assay buffer (250 mm Bis-Tris propane, pH 7.7), 1.0 μl of 100 mm alcohol substrate dissolved in dimethyl sulfoxide, 1.0 μl of [1-14C]acetyl-CoA, and 33 μl of water to bring the assay volume to 50 μl.
The assays were carried out at room temperature (25°C) for 30 min and the radio-labeled acetate was counted as previously described (D'Auria et al., 2002). The raw data (disintegrations per minute) were converted to picokatals based on the specific activity of the radiolabeled substrate and the known efficiency of the scintillation counter used. Assays in which no alcohol substrate was added were performed to test background thioesterase activity, which was found to be less than 5 pkat mg protein−1. In addition, boiled enzyme extracts were substituted for intact enzyme to test the non-enzymatic breakdown of the CoA thioester via a reaction with reducing agents in the enzyme extract. The identities of the products were verified by GC-MS as described previously using authentic standards (Dudareva et al., 1998).
Volatile collection and analysis
Volatile collections used for GC-MS analysis from rosette plants were performed in a climate-controlled growth chamber [22°C, 55% relative humidity, and 150 μmol m−2 s−1 photosynthetically active radiation (PAR)] in 1-l flat-flanged glass vessels (Schott, Mainz Germany). The dynamic headspace collection was performed by supplying compressed air (2 l min−1) that was purified by passage over a charcoal filter linked to a ZeroAir generator (Parker Hannifin Corp., Haverhill, MA, USA). The air was then pumped out of the glass vessel at a flow rate of 1 l min−1 and passed over a filter containing 1.5 mg of activated charcoal (CLSA-Filter; Le Ruisseau de Mont-brun, Daumazan sur Arize, France). The remaining air escaped through the opening around the base of the chamber providing a positive pressure barrier against the entrance of ambient air. Headspace was collected for 4 h and the filters were then eluted with 100 μl CH2Cl2 containing 100 ng nonyl acetate as an internal standard. The volatile headspace collections were analyzed on a Hewlett-Packard 6890 gas chromatograph coupled to a Hewlett-Packard 5973 quadrupole mass-selective detector. Separation was performed on a (5%-phenyl)-methylpolysiloxane (DB5) column (J&W Scientific, Folsom, CA, USA) of 30 m × 0.25 mm inner diameter × 0.25-m thickness. Helium was the carrier gas (flow rate 2 ml min−1), a splitless injection (2 μl) was used and a temperature gradient of 5°C min−1 from 40°C (3-min hold) to 240°C was applied. Parameters for electron impact ionization in the quadrupole mass detector were as follows: repeller, 30 V; emission, 34.6 μA; electron energy, 70 eV; quadrupole temperature, 150°C; source temperature, 230°C; transfer line temperature, 250°C. The mass spectrometer was run in the scan mode with an m/z ration of 40–350.
The identities of (Z)-3-hexen-1-yl acetate, (Z)-3-hexen-1-ol and (Z)-3-hexenal were determined by a comparison of retention times and mass spectra with those of authentic standards and with mass spectra in the National Institute of Standards and Technology (NIST) and Wiley (West Sussex, England) libraries. For quantification, response factors were computed using a seven-point standard curve made for each compound of interest using concentrations ranging from 897 pg μl−1 to 89 ng μl−1. The efficiency of the dynamic headspace collection system was tested by dissolving several different known concentrations of the three major A. thaliana GLVs in methanol. This solution (200 μl) was then placed in a 3-ml glass vial. Dynamic headspace collections were performed on duplicate samples under identical conditions as described for plant volatile collections. At the end of the experiment, the vials and filters were each extracted with 100 μl dichloromethane and analyzed alongside an unused portion of the same standard by GC-MS. After taking into account dilution factors, the efficiency of collection was determined by dividing the total quantity of compound collected on the charcoal filter by the total quantity of compound placed in the chamber subtracted from what was left over in the vial after the experiment. These factors were then taken into account and used as normalizers for the quantification of all GLVs collected from the dynamic system. For example, the efficiency of collection for (Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate were found to be 60 and 50%, respectively, in the range normally emitted by plants in this study.
Headspace analysis of wild-type plants, those expressing an At3g03480 RNAi hairpin construct and T-DNA knockout plants was accomplished using a zNose model 4200, a portable, rapidly-separating GC using an internal pump for vapor samples and a quartz, surface acoustic wave detector (SAW) (Electronic Sensor Technology, Newbury Park, CA, USA) in a similar fashion to that previously described by Kunert et al. (2002). For the analysis of At3g03480 RNAi and T-DNA knockout plants, one leaf was excised from each plant, weighed, squeezed four times with a surgical clamp, and then placed in a 2-ml teflon sealed glass vial. After 15 min, the leaf headspace was sampled by inserting a small syringe needle through the cap and inserting the needle-like entrance port of the zNose into the vial. Air was sampled from the vial for 15 s and adsorbed onto a stainless steel GC column (length 1 m; DB 5; film thickness 0.25 μm; inner diamater 0.25 mm). Helium was the carrier gas at a flow rate of 3 ml min−1 with the column temperature starting at 40°C and increasing to 180°C at a rate of 5°C sec−1, while keeping the SAW at a constant 40°C for the duration of the run. The quantification of compounds was achieved by making a seven-point standard curve using standards for (Z)-3-hexen-1-yl acetate, (Z)-3-hexen-1-ol and (Z)-3-hexenal. Standards were introduced into the zNose using a heated glass desorber tube connected to a model 3100 vapor calibrator (Electronic Sensor Technology,) set at 210°C. The zNose method used in the analysis of A. thaliana GLVs is suitable for the separation of the three most common GLV compounds found in the Ler ecotype. The detector response for all three compounds was found to be linear over a range of 1–1000 ng, which is within the physiological ranges observed in our headspace collections.
Details of the PTR-MS instrument have been described in detail previously (de Gouw et al., 2003; Lindinger et al., 1998). The PTR-MS was operated at an E/N of 90 Td. The transmission of masses was calibrated and included in the calculated count rate per second measured for each mass, given here in arbitrary units (ncps; normalized counts per second). The detection limit of the PTR-MS was given by the background signal measured from contaminant-free air and the count rate per second, and was lower than 1 ncps.
The GLVs (Z)-3-hexenal, (Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate fragment in proton-transfer reactions using H3O+ under the settings described giving the following pattern of mass fragments. Because only singly charged ions play a role in PTR-MS, ions are referred to by their mass rather than by their mass-to-charge ratio. (Z)-3-Hexenal: m99 (31%, parent ion [RH+]), m81 (51%) and m57 (18%); (Z)-3-hexen-1-ol: m101 (1%, RH+), m83 (76%), m81 (1%), m67 (1%) and m55 (22%); (Z)-3-hexen-1-yl acetate: m143 (2 %, RH+), m83 (73%), m61 (4%), m57 (1%) and m55 (18%). As a result of an overlap of fragment masses at m83 and m55, the abundance of (Z)-3-hexenal, (Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate were corrected by the respective factor contributing to the sum of all fragments. This method for correction of the fragmentation pattern of (Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate was validated in a test experiment where four mixtures of a known ratio of (Z)-3-hexen-1-ol:(Z)-3-hexen-1-yl acetate were measured with the PTR-MS. The measured ratio was calculated by the correction factor as described above. In all ratios tested, the deviation from the expected outcome was less than 5%.
Quadruplicate experiments were carried out using five plants of the Ler ecotype of A. thaliana per experiment. For simultaneous measurements of PTR-MS on-line and zNose GC, a 2 l glass desiccator supplied with volatile organic compound (VOC)-free, ozone-scrubbed air at a flow rate of 4 l min−1 was used. The desiccator had one outlet line connected to the PTR-MS and one outlet for sampling with the z-Nose. The plants were set in the desiccator, wounded and immediately measured. The time resolution of the PTR-MS data was 9.2 s and data was integrated according to the time of air exchange of the desiccator to 27 s.
Isolation and cloning of At3g03480 (CHAT) cDNA and expression in E. coli and A. thaliana (ecotype Ler)
The cDNA for At3g03480 (accession AF500201) was isolated as previously described (D'Auria et al., 2002). The coding region of At3g03480 was subcloned into the pET-T7 (28a) vector (Novagen, Madison, WI, USA) by introducing an NdeI site on the 5′ end using the forward primer 5′-ACATATGGACCATCAAGTGTCTCTGCCAC-3′ and a BamHI site on the 3′ end after the stop codon with the backward primer 5′-TGGATCCTCATCCTTTAGACACATTTAGCACTCC-3′, which introduced a 6×His N-terminal tag. Expression and harvesting of the recombinant protein in BL21(DE3) E. coli cells was accomplished as previously described (D'Auria et al., 2002).
The open reading frame of CHAT was subcloned into the binary vector pJML5 (courtesy of Jianming Li, University of Michigan, Ann Arbor, MI, USA), a derivative of the binary vector pCGN1547 (McBride and Summerfelt, 1990). This vector contains a CaMV 35S promoter, followed by a pBluescriptSK multicloning site (Stratagene, La Jolla CA, USA), and is flanked by the RbcSE9 terminator (accession no. M21375). The At3g03480 cDNA was subcloned into the vector by insertion between the XbaI and BamHI restriction sites. The 5′ end forward primer used was 5′-CTCTAGAATGGACCATCAAGTGTCTCTGCCAC-3′, as well as the BamHI reverse primer mentioned above. Sequencing of all subcloned products in this study revealed that no PCR errors were obtained. The resulting plasmid, CHAT-pJML5, was subsequently transformed into Agrobacterium tumefaciens strain ASE101, which was used to transform A. thaliana ecotype Ler using the floral vacuum infiltration method (Bechtold et al., 1993). Several independent lines were maintained as single-insertion homozygous lines based on their segregation of kanamycin resistance, increased enzyme activity and RT-PCR for the presence of the corresponding transgene.
Purification of recombinant proteins
All work with harvesting and purification of recombinant proteins was performed either on ice or in a refrigerated chamber operating at 8°C. Partial purification of CHAT from A. thaliana plants harboring the CHAT-pJML5 transgene construct was achieved by the use of DE53 anion exchange chromatography, followed by MonoQ, and finally by gel sizing on Superdex 75 (Amersham Pharmacia Biotech, Uppsala, Sweden) as previously described (D'Auria et al., 2002). Fractions were tested by radioactive enzyme assay and those fractions containing peak activity were pooled and used for subsequent purification steps.
CHAT protein containing a 6×His tag was harvested in His-Tag lysis buffer (50 mm Bis-Tris, pH 7.0, 500 mm NaCl, 10 mm imidazole and 10% w/v glycerol). BL21(DE3) cells containing heterologously expressed CHAT protein were sonicated twice with a Sonopuls HD2070 sonicator (Bandelin, Berlin, Germany) with the microprobe attachment at 60% full power for a 5-min 20% cycle. Cell debris was removed by centrifugation at 20 000 g. The supernatant was used for FPLC chromatography using a 1-ml HisTrap Ni Sepharose column (Amersham Biosciences). The elution of protein was performed in a three-step gradient consisting of 100, 250 and 500 mm imidazole. Fractions containing peak CHAT activity were pooled and concentrated using an Amicon 8200 concentrator with a 63.55-mm polyethersulfone filter with a 10-kDa molecular weight cut-off (Millipore, Bedford, MA, USA). The enzyme was then desalted into assay buffer (50 mm Bis-Tris, pH 7.0, 5 mm DTT and 10% w/v glycerol) using an Econo-Pac 10DG desalting column (Bio-Rad, Hercules, CA, USA). All purification steps were analyzed by SDS-PAGE gel electrophoresis followed by either Coomassie Brilliant Blue or silver staining of the gel. CHAT protein concentration was determined as previously described (Bradford, 1976).
Construction of the At3g03480 RNAi vector
In order to clone the At3g03480 RNAi construct, the binary vector pFGC5941 for dsRNA production was obtained from the Arabidopsis Biological Resource Center (Columbus, OH, USA; stock no. CD3-447). A 452-bp segment from the 3′ end of the CHAT cDNA (position 914–1365) was amplified by PCR using the forward primer, 5′-AATCTAGAGGCGCGCCAGAAACCCACCGCTAGAGCC-3′, and backward primer, 5′-AAGGATCCATTTAAATTCATCCTTTAGACACATTTAGCACTC-3′. The resulting fragment was cloned and placed into the TOPO-4 vector (Invitrogen, Carlsbad, CA, USA) for maintenance and sequencing. The vector containing the RNAi fragment was digested with AscI and SwaI and ligated into pFGC5941 to generate the plasmid CHAT-447.5. The vector was also digested with BamHI and XbaI, and ligated into CHAT-447.5 to generate the plasmid CHAT-447. The resulting product has a copy of the 452-bp fragment inserted between the AscI and SwaI sites, and an inverted repeat of the fragment inserted between the BamHI and XbaI sites. A spacer consisting of the chalcone synthase intron from Petunia hybrida lies between the two inserted sequences. CHAT-447 was transformed into A. tumefaciens strain GV3850, and was then used for the transformation of A. thaliana (ecotype Ler) via floral vacuum infiltration (Bechtold et al., 1993). Several independent lines were maintained as single insertion homozygous stocks based on segregation with glufosinate ammonium, and the presence of the transgene.
RNA extraction and determination of CHAT, HPL1 and APT1 gene expression by quantitative RT-PCR
Plant RNA was either extracted as previously described (D'Auria et al., 2002) or via an LiCl precipitation method. Regardless of the isolation method, all RNA was subjected to an on-column DNAse treatment (Qiagen, Hilden, Germany) to remove traces of genomic DNA. An aliquot of the RNA was measured by a spectrophotometer in the range of 230–300 nm. Based on the spectrophotometer reading, a dilution was made to 100 ng ml−1 and the RNA was further analyzed on an Agilent 2100 Bioanalyzer RNA 6000 Nano Lab kit (Agilent Technologies, Palo Alto, CA, USA). The RNA concentration for all subsequent steps was based on the Bioanalyzer estimate.
Standardization of cDNA qRT-PCR templates was carried out according to a standardized protocol (Phillips M. and D'Auria J., manuscript in preparation). For reverse transcription, Superscript III (Invitrogen) was used according to the manufacturer's instructions. RNA was removed after the reaction by a 30-min digestion with 5 μl Rnase A (10 μg ml−1) at 37°C. The resulting first-strand cDNA was purified on a Qiagen QIAquick PCR column by the addition of 10× volume of PB buffer. The purified cDNA was analyzed with the Bioanalyzer on a RNA 6000 Pico labchip (Agilent Technologies) without any dilution. Using smear analysis software (Agilent Technologies), the cDNA fragments in the range of approximately 500 bp–6 kb were used to estimate the effective cDNA concentration. All cDNA samples were diluted to 100 pg μl−1 with nuclease-free water and used as templates in subsequent quantitative real time PCR experiments.
All quantitative RT-PCR experiments were performed on a Stratagene Mx3000P qRT-PCR machine using SYBR® green I with 6-carboxyl-X-rhodamine (ROX) as an internal standard according to the manufacturers protocol. Each 20-μl reaction contained 100 pg purified cDNA as a template with the exception of non-RT controls, in which the cDNA was substituted with 100 pg total RNA. The thermal program was run as follows: 96°C for 10 min followed by 40 cycles of 30 s at 96°C, 30 s at 60°C and 30 s at 72°C. Fluorescence was read following both the primer annealing and elongation phases. All runs were followed by a melting curve analysis in which the temperature range was 55–95°C with a 1°C change per second. In all cases, the primers were optimized for efficiency by performing a series of five, fourfold dilutions on the purified cDNA template of known concentration. In this way, the linearity of the cycle times (Ct) values was also tested over three orders of magnitude against the log of template concentrations. R2 values and primer efficiencies were calculated using the MxPro software (Stratagene, Amsterdam, The Netherlands). For each primer pair tested, three independent biological replicates were tested with similar template concentrations. In addition, three technical replicates were performed for each biological replicate.
The primers used in this study were all designed by Beacon Designer version 4.0 software (Premier Biosoft, Palo Alto, CA, USA). All primer pairs incorporated an intron-spanning region so that a larger gene product would be found in the presence of genomic DNA. Among the criteria for primer design was the rejection of any potential primers displaying either high secondary structure or cross homology with all known similar sequences. The primers designed for At3g03480 are 5′-AGCTTCTCTTTGATGTGGAAGGC-3′ for the forward primer, and 5′-GAAGCGGAGTGCGAAGATAAATC-3′ for the backward primer. The subsequent cDNA amplicon is 107 bp in length. The primers for HPL1 At4g15440 are 5′-GGCGTTCGTGTTGGAGTTTATC-3′ for the forward primer, and 5′-GGATTCGATTGTTCCCCAGAA-3′ for the backward primer. The subsequent cDNA amplicon is 190 bp in length. The primers designed for APT1 At1g27450 are 5′-GTTGCAGGTGTTGAAGCTAGAGGT-3′ for the forward primer, and 5′-TGGCACCAATAGCCAACGCAATAG-3′ for the backward primer. The subsequent cDNA amplicon is 64 bp in length. All primers were HPLC purified and have efficiencies between 90–100%. In addition, the products of each primer combination were cloned and sequenced a minimum of three independent times to confirm the sequence of the amplicon.
Comparative quantification experiments included a minimum of two biological replicates with three technical replicates for each sample unless otherwise stated. The ΔΔCt method of comparative quantification employing efficiency correction was used to judge the relative quantification of the starting template for all genes of interest (Pfaffl, 2001). Data were not used further when the non-template (H2O) controls had an amplified product within five Ct values of the highest Ct for the true biological samples.