Petunia × hybrida‘Mitchell Diploid’ (MD) was utilized as the control for all experiments and served as the genetic background for the production of CaMV 35S:etr1-1 line 44568 (Wilkinson et al., 1997) and PhCFAT RNAi transgenic petunias. Plants were grown in a glass greenhouse without artificial lighting and an average temperature of 20 ± 5°C, dependent on the time of day. Plants were grown in 1.2-l pots with Fafard 2B potting material (Fafard Inc., Apopka, FL, USA), and fertilized four times a week with 150 mg l−1 nitrogen in Scott’s Excel 15-5-15 (Scotts Co., Marysville, OH, USA).
Generation of PhCFAT RNAi transgenic petunia
To determine the in vivo function of PhCFAT, RNAi-induced gene-silencing technology was utilized. Two fragments of the PhCFAT cDNA (corresponding to bases 1010-1344 and 1010-1671) were amplified using PCR and ligated end to end in a sense/antisense orientation. The resulting construct was ligated downstream of the pFMV (Richins et al., 1987) constitutive promoter and upstream of the nopaline synthase (nos) 3′ terminator sequence. The subsequent RNAi construct was subcloned into a binary transformation vector containing the kanamycin resistance gene, neomycin phosphotransferase (NPTII), and mated with Agrobacterium tumefaciens strain ABI. Six-week-old MD leaves were transformed as previously described (Jorgensen et al., 1996), and 16 primary transformants were recovered, transferred to soil, grown to maturity, and screened for an altered volatile profile using flame-ionization gas chromatography and reduced PhCFAT transcript abundance using quantitative RT-PCR (see below). Plants showing reduced isoeugenol emission and PhCFAT transcript levels when compared to MD were self-pollinated and T1 progeny grown. T1 lines were screened for segregation of the transgene using PCR (verifying the presence of the NPTII gene), reduced PhCFAT transcript and reduced isoeugenol emission. Flowers from lines exhibiting 3:1 segregation were again self-pollinated and T2 seeds produced. Screening was repeated as above, and two 3:1 segregating lines (lines 6 and 16) and one homozygous line (line 15) were identified and used for subsequent research reported here.
PhCFAT expression analysis by real-time RT-PCR
For spatial transcript analysis, total RNA was isolated from stem, root, leaf, whole flower (at anthesis), petal limb, petal tube, stigma/style, ovary and sepal tissues collected at 08:00 h (1 h after the onset of light) and at 20:00 h (1 h after the onset of darkness). To determine transcript levels during floral development, total RNA was isolated from whole flowers collected at 20:00 h (1 h after the onset of darkness) at various developmental stages including small bud (1 cm), medium bud (3–4 cm), tube expansion (just prior to anthesis), anthesis (complete limb expansion just prior to pollen release) and post-anthesis (2 days after pollen release). For rhythmic and ethylene-regulated expression, tissue was collected as previously described (Underwood et al., 2005). For post-pollination transcript analysis, MD and line 44568 flowers were pollinated 1 day prior to anthesis or set aside as a non-pollinated control. Beginning at 10:00 h, both pollinated and non-pollinated MD and 44568 flowers were collected, and the petal limb removed as spatial analysis revealed the petal limb as the target tissue primary containing PhCFAT transcript. This process was repeated at 12 h intervals up to 60 h after pollination. For PhCFAT RNAi transcript analysis, whole flowers (at anthesis) from both MD and PhCFAT RNAi lines were collected at 20:00 h (1 h after the onset of darkness).
In all cases, total RNA was extracted from collected tissues as previously described (Underwood et al., 2005). Quantitative RT-PCR utilizing Taqman One-Step RT-PCR reagents (Applied Biosystems, Foster City, CA, USA), the following primers and probe (PhCFAT forward primer, 5′-GCAAGTGTTGGACAGCTCAAGCAA-3′; PhCFAT reverse primer, TCTTGTTAGGGCTAGGCATTGGCA; PhCFAT probe, FAM – TGATGAAGCAGCCATCGTTGTCTCCT-3BHQ1), a series of in vitro-transcribed PhCFAT standards, and 1 µl of 100 ng µl−1 RNA were then used to quantify mean PhCFAT levels as previously described (Underwood et al., 2005).
To determine the transcript levels of other floral volatile-related genes (PhBPBT, PhBSMT1, PhPAAS, and PhIGS1) in PhCFAT RNAi lines and MD whole flowers, total RNA (see above) was analyzed utilizing real-time RT-PCR (Applied Biosystems, Foster City, CA, USA) or One-Step RT-PCR (Qiagen Inc., Valencia, CA, USA). For PhBPBT transcript analysis, real-time RT-PCR analysis utilizing the following primers and probe (PhBPBT reverse primer, 5′-GAAATAAGAAAGGTGAGAATGGGATT-3′; PhBPBT forward primer, 5′-AGCTCCTTGACGAATTTTTCCA-3′; PhBPBT probe, 5′-/56FAM/TGGTCCCTATATGTTTGCCTGGCTTTGC/3BHQ_1/ -3′), and a dilution series of in vitro-transcribed PhBPBT standards, was used to determine mean PhBPBT transcript levels in PhCFAT RNAi and MD petunia. For PhBSMT1 transcript analysis, real-time RT-PCR (as described by Underwood et al., 2005) was utilized to determine mean PhBSMT1 transcript levels in PhCFAT RNAi and MD petunia.
To determine PhPAAS and PhIGS1 transcript levels, PhCFAT RNAi and MD total RNA was analyzed using One-Step RT-PCR. Utilizing the following primers (PhPAAS forward primer, 5′-TCCTTGTAGTTCTAGTACTGCTGGAA-3′; PhPAAS reverse primer, 5′-TCAACAGCAGTTGTTGAAGTAGTTC-3′; PhIGS1 forward primer, 5′-GCCTATGTCATGCCATTGAA-3′; PhIGS1 reverse primer, 5′-TCTTTAATTGTGTAGGCTGC-3′; ubiquitin forward primer, 5′-AACATACAGAAGGAGTCAACAC-3′; ubiquitin reverse primer, 5′-AGAAGTCACCACCACGAAG-3′), 100 ng of whole-flower total RNA from two separate MD and PhCFAT RNAi plants of lines 6, 15 and 16 was analyzed. The RT-PCR reaction was then amplified utilizing the following program: 50°C for 30 min; 94°C for 15 min; 30 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 1 min; final incubation at 72°C for 10 min. Aliquots (15 μl) of each product were then run (20 min at 100 V) on a 1% agarose gel, and photographed using a Polaroid Fotodyne camera (Polaroid Corporation, Pasadena, CA, USA). Transcript levels of a poly-ubiquitin petunia homolog were analyzed to provide an RNA loading control.
For the rhythmic emission experiment, volatiles were collected and analyzed as previously described (Underwood et al., 2005). For comparative analysis of volatiles emitted from PhCFAT RNAi and MD, seven flowers each were collected from five plants of each line (PhCFAT RNAi lines 6, 15 and 16, and MD) at 20:00 h (1 h after the onset of darkness) for two consecutive nights that followed sunny days. In all cases, volatiles were then collected for 1 h and analyzed as previously described (Schmelz et al., 2001). The eluted volatiles were quantified using flame-ionization gas chromatography (model 5890, series II; Hewlett-Packard, Palo Alto, CA, USA), and the resulting data were converted to (µg g−1 fresh weight) h−1. To determine a percentage difference for each compound, measured values of volatile emitted by flowers from PhCFAT RNAi lines were then divided by the average value of the corresponding volatile emitted by MD flowers over the same sample period. The percentage difference from MD (mean ± SE) was then calculated for each transgenic line.
Internal volatile extraction
To determine PhCFAT RNAi internal volatile levels, three flowers each were collected from five plants of each line (PhCFAT RNAi lines 6, 15 and 16, and MD) at 20:00 h (1 h after the onset of darkness) following each of two sunny days. In each case, the corollas (petal limb and tube) were removed, immediately frozen, and ground using mortar and pestle. Internal volatiles were then extracted as previously described (Schmelz et al., 2004) with two deviations (no HCl added to sample, and 170°C instead of 200°C during the vapor-phase extraction). Samples were supplemented with nonyl acetate as an internal standard and analyzed by flame-ionization gas chromatography with the resulting data converted to µg g−1 fresh weight. For coniferyl aldehyde and homovanillic acid, tissue was ground and extracted with dichloromethane overnight. The extract was analyzed by GC–MS and compared with authentic standards. PhCFAT RNAi sample replicates were then divided by average MD values for corresponding compounds obtained from the same day to determine a percentage difference for each sample. Mean percentage differences from MD (±SE) were then calculated for each transgenic line.
Expression of PhCFAT in Escherichia coli and purification of recombinant protein
The coding region of petunia PhCFAT was amplified by PCR using forward and reverse primers (5′-CACATATGGGAAACACAGACTTTCATG-3′ and 5′-CATGGATCCTCAATAAGTAGCAGTAAGGTCC-3′, respectively), and subcloned into the NdeI–BamHI site of the expression vector pET-28a containing an N-terminal hexa-histidine tag (Novagen, Madison, WI, USA). Sequencing revealed that no errors were introduced during PCR amplifications. Expression was performed in E. coli Rosetta cells grown in LB medium with 50 µg ml−1 kanamycin and 37 µg ml−1 chloramphenicol at 18°C. Induction, harvesting, and protein purification by affinity chromatography on nickel–nitriloacetic acid agarose (Qiagen Inc.) were performed as described previously (Negre et al., 2002). Eluted fractions with the highest PhCFAT activity were desalted on Econo-Pac® 10DG columns (Bio-Rad Laboratories, Hercules, CA, USA) into 50 mm Tris–HCl buffer (pH 7.5) with 2% glycerol, and examined by SDS–PAGE gel electrophoresis followed by staining of the gel with Coomassie brilliant blue. The purity of the isolated protein (99%) was taken into account for Kcat determination. Total protein concentration was determined by the Bradford method (Bradford, 1976) using Bio-Rad protein reagent and BSA as a standard.
Enzyme activity was measured by determining how much of the 14C-labelled acetyl group of acetyl CoA was transferred to the side chain of coniferyl alcohol. The standard reaction mixture (50 µl) contained purified PhCFAT protein (18 μg) and 140 μm acetyl CoA (containing 0.08 µCi; Amersham Biosciences UK Ltd, Buckinghamshire, UK) in assay buffer (50 mm citric acid, pH 6.0, 1 mm DTT or 50 mm Tris-HCl, pH 7.5, 1 mm DTT). After incubation for 15 min at room temperature, the product was extracted with 100 µl hexane, and 50 µl of the organic phase was counted in a liquid scintillation counter (model LS6800; Beckman, Fullerton, CA, USA). The raw data (cpm, counts per minute) were converted to nanokatals (nkat, nanomoles of product produced per second) based on the specific activity of the substrate and the efficiency of counting. Controls included assays using boiled protein with and without the alcohol substrate. For pH optimum determination, assays were performed in 50 mm Tris/Na phosphate/Na citrate buffer at pH values ranging from 3.5 to 8.5. Once the pH optimum had been determined, other buffers were used to maximize coniferyl alcohol acyltransferase (CFAT) activity with coniferyl alcohol as a substrate. At pH 6.0, CFAT activity was significantly higher in 50 mm citrate buffer than in 50 mm Tris/Na phosphate/Na citrate buffer at pH 6.0, while at pH 7.5, CFAT activity was significantly higher in 50 mm Tris–HCl than in 50 mm Tris/Na phosphate/Na citrate buffer at pH 7.5. Assays performed at overlapping pH values between two buffers (50 mm citrate and 50 mm Tris–HCl) gave very similar activities, and ratios between activities at pH 6.0 and 7.5 were consistent with those in 50 mm Tris/Na phosphate/Na citrate buffer at corresponding pH values. Thus, 50 mm citrate, pH 6.0, and 50 mm Tris–HCl, pH 7.5, buffers were used in the biochemical analysis of CFAT protein.
Product verification was performed using TLC. For TLC analysis, the standard reaction was scaled to 1 ml using non-radiolabeled acetyl CoA (Sigma, St Louis, MO, USA). The reaction product was extracted in 1 ml hexane, concentrated to approximately 10 µl, spotted onto a pre-coated silica gel TLC plate (PE SIL G/UV; Whatman, Maidstone, UK), and co-chromatographed with authentic standards using ethyl acetate:hexane (7:3 v/v) as a solvent.
For kinetic analysis, an appropriate enzyme concentration was chosen so that the reaction velocity was proportional to the enzyme concentration and linear with respect to incubation time for at least 15 min. Kinetic data were evaluated by hyperbolic regression analysis. PhBPBT and PhPAAS activities in the presence of 2 mm coniferyl aldehyde and homovanillic acid were determined as described previously (Boatright et al., 2004; Kaminaga et al., 2006).
Coupled in vitro reaction
The coupled reaction with PhCFAT and PhIGS1 was carried out in a 100 mm MES-KOH buffer (pH 6.5) containing 0.5 mm coniferyl alcohol, 0.3 mm acetyl CoA, 0.5 mm NADPH and 2 μg of each purified enzyme in a total volume of 150 μl. After incubation at room temperature for 20 min, the reaction solution was extracted with 1 ml of hexane, and the hexane solution was concentrated with liquid N2. A fraction (2 μl) was injected into the GC-MS apparatus for analysis. Control reactions (substituting PhBPBT for PhCFAT, or omitting PhCFAT from the PhIGS1 coupled reaction) were carried out and analyzed in an identical fashion.