Nicotiana benthamiana plants were grown in a glasshouse at 22°C and using natural light with daylight extension to 16 h. Plants were grown until they had six leaves and the youngest leaves over 1 cm long were infiltrated with Agrobacterium and maintained in the glasshouse for the duration of the experiment.
Agrobacterium tumefaciens, GV3101(MP90)  were cultured on Lennox agar (Invitrogen) supplemented with 50 μg.ml-1 kanamycin (Sigma) and incubated at 28°C. A 10 μl loop of confluent bacterium were re-suspended in 10 ml of infiltration media (10 mM MgCl2, 0.5 μM acetosyringone), to an OD600 of 0.2, and incubated at room temperature without shaking for 2 h before infiltration. Infiltrations were performed according to the methods of Voinnet et al. (2003) . Approximately 300 μl of this Agrobacterium mixture was infiltrated into a young leaf of N. benthamiana and transient expression was assayed from three to 14 days after inoculation.
Each of the promoter-LUC fusions in pGreenII 0800-LUC were used in transient transformation by mixing 100 μl of Agrobacterium transformed with the reporter cassette and 900 μL of a second Agrobacterium strain transformed with a cassette that contained the TF gene fused to the 35S promoter in either a pART27-derived or pGreenII 62-SK binary vectors described below.
RPH-138: GTGAGAGGTCCTAAGCTTATGTCCGGTTAT; RPH-139: CTTGACTGGCGAGAATTCCCACGATCTCTTT; RPH-140: ATTGACAAGGATGGATCCCTACATTCTGGA; RPH-141: CACGATCTCTTTTTCCGTCATCGTCT; RPH-146: TGGCCTTTATGAGGGAATTCCTGATTTTTC; RPH-179: TGGCGGTTTTGGTACCCCGGGTCAAC; RPH-180: CCATCACCATGGTAGTATACACCAAC; RPH-198: CACACAGTTGGGAGGAGTTGCTGTCCC; RPH-199: CTTGCGAACTTCTTCGACGGTCACCAT; RPH-212: AATTGGTACCGATATCGAGCTC; RPH-213: AGCTGAGCTCGATATCGGTACC; RPH-332: ACTCCTCGACTGTCACCATGGTTGCTTG; RPH-333: GCGGAAGGGTACCGAATTCATAGCAACTGG
Construction of pGreenII-62-SK
The cauliflower mosaic virus (CaMV) 35S expression cassette (Kpn I-Bgl II) of pJIT-62  was inserted into the multiple cloning site (MCS) (Kpn I-Bam HI) of pGreenII-0000 . Flanking sequences from the LB to the 35S-promoter and from the CaMV-terminator to the RB were deleted by digestion, (Stu I-Kpn I and Spe I-Hpa I respectively), T4 pol and re-ligation to produce pGreenII-62-1. The pUC9 MCS was converted to the pBluescript MCS by ligating two oligonucleotides (RPH-212 and RPH-213) to the Eco RI-Hind III cut vector, generating Kpn I and Sac I sites, then inserting a Kpn I-Sac I fragment from pBluescript SKII+ (Stratagene).
Construction of the plasmids containing genes for enzymes and TFs
The HortResearch apple EST libraries were constructed in either the Lambda ZAP Express or the Lambda ZAP II vector systems (Stratagene), resulting in cDNAs cloned unidirectionally as Eco RI-Xho I fragments in pBK-CMV or pBluescript SK-, respectively.
Genes encoding various enzymes and transcription factors were cloned into one of two plant expression vectors derived from the pART7/27 binary vector system . Both vectors carry the same transcriptional regulatory signals for plant gene expression, namely the CaMV35S promoter and octopine synthase terminator. The T-DNA border elements, chimeric kanamycin selectable marker and vector backbone are identical in both vectors. The two derivatives differ in that one is suitable for conventional restriction enzyme cloning of cDNAs whilst the second facilitates Gateway recombination cloning of the cDNAs.
In order to improve the compatibility of restriction sites in the multiple cloning site of pART7 with those of the pBK-CMV or pBluescript SK- EST library vectors, the Xho I-Xba I region of pART7 was replaced with the Sal I-Xba I multiple cloning site region of pBK-CMV, generating pSAK7. cDNAs from the EST libraries were cloned into pSAK7 as either Eco R1-Xho I or Bam HI-Xho I fragments, placing them under the transcriptional control of the CaMV 35S promoter. The 35S-cDNA-ocs 3' cassette was then cloned as a Not 1 fragment into pART27, to generate the plant gene expression construct.
Where conventional cloning was more problematic, due to the lack of suitable restriction sites, a Gateway-adapted version of the pART7/27 plant transformation system was utilised. This Gateway-adapted version was produced by cloning the CaMV35S promoter, multiple cloning site and octopine synthase transcriptional terminator cassette of pART7 as a Not I fragment into pART27. Subsequently, the 1711 bp Gateway RfA cassette (Invitrogen Corp.) was cloned into the Sma I site of the multiple cloning site to generate pHEX2 (35S-att R1-CmR-ccd B-att R2-ocs 3'). cDNAs from the EST libraries were cloned into pHEX2 using Gateway recombination technology and all Gateway reactions were performed as recommended by the manufacturer (Invitrogen Corp.). cDNAs were amplified using universal primers designed to the multiple cloning site regions of the pBK-CMV or pBluescript SK-. The primers used for pBluescript SK- clones were 5-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCCCGGGCTGCAGGAATTC-3' and 5'- GGGGACCACTTTGTACAAGAAAGCTGGGTCCGGGCCCCCCCTCGAG-3' and the primers used for pBK-CMV clones were 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGACACTAGTGGATCCAAAGAATTC-3' and 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTGCCGCTCTAGAAGTACTCTCGAG-3'
Amplification with these primers results in PCR products with att B ends which were recombined with the att P sites of the Gateway pDONR201 vector, creating pENTRY vectors. All pENTRY vector clones were sequence verified to ensure the fidelity of the cDNA sequence. Gateway att P × att R reactions were then carried out with the pENTRY vector and the pHEX2 Destination vector, to generate the plant gene expression construct.
Enzyme expression plasmids. The apple cDNA clone of MdC4H1 [GenBank: DQ075002] was inserted into pGreenII 62-SK as an Eco RI-Xho I fragment. The genes for L-galactose dehydrogenase [GenBank: AY176585] and L-galactose-1-P phosphatase [GenBank: AY787585] from kiwifruit were cloned as previously described  and transformed into Agrobacterium using standard methods.
Construction of pGreenII 0800-LUC
The Renilla reporter gene pRL-null (Promega, Madison, WI) was modified to remove the mammalian 5'UTR intron by digestion with Nhe I-Spe I, T4 Pol, followed by re-ligation. This gene was inserted into the expression cassette p35S-2 , and the flanking sequences deleted as above. This cassette was inserted into the Hpa I site of pGreenII 0000, to produce pGreen 0800-1. A 35S-LUC-expression cassette was inserted into pGreenII 0800 to produce pGreenII 0800-35S-LUC. The flanking sequence between the CaMV terminator and the RB was deleted (Spe I-Stu I, T4 pol, ligation). Finally the pBluescript MCS replaced the 35S promoter to produce pGreenII 0800-LUC. These vectors are available on request.
Promoter cloning into pGreenII 0800-LUC
A 0.97 kb region of the Arabidopsis CHS promoter (At5g13930) was amplified by PCR from the Arabidopsis ecotype Columbia using the primers RPH-179 and RPH-180, then digested with Kpn I and Nco I and cloned into the MCS of pGreenII 0800-LUC. The 1.04 kb pea CHS-1a promoter [GenBank: X80007] was subcloned into pGreenII 0800-LUC as an Eco RI-Nco I fragment . A 0.92 kb Petunia CHS-A promoter [GenBank: X14591] was amplified by PCR using primers RPH-332 and RPH-333 from a V26 genomic DNA, digested with Kpn I and Nco I and cloned into pGreenII 0800-LUC. The 1.3 kb Apple CHS1 promoter [GenBank: DQ022678] was isolated from Malus domestica Royal Gala, using the Genome Walker kit (Clonetech) with gene specific primers RPH-198 and RPH-199, then cloned into pGEM T-easy (Promega) and subcloned as a Sal I-Nco I fragment into pGreenII 0800-LUC. pwo-Polymerase (Roche) was used for all PCR amplifications and cloned genes were sequenced to confirm no sequence modifications were incorporated.
Construction of hp-cassette
A short hairpin was built by asymmetric amplification of a region of the LUC gene: primers RPH-138 and RPH-146 were used to amplify a 454 bp fragment introducing a Hin dIII and Eco RI site near the end of the PCR product. The second PCR product corresponding to the antisense region of the hairpin was a 283 bp PCR product of amplification using primers RPH-140 and RPH-139. The first primer, RPH-140, was from the same region as RPH-138 but introduced a Bam HI site near the end of the PCR product. RPH-139 was 142 bp closer to RPH-138 and RPH-140 than RPH146, and also introduced an Eco RI site near the end of the amplified product. The PCR products were digested with appropriate restriction enzymes and these two digested PCR products were used in a 3-way ligation to Hin dIII-Bam HI cut pNOS-7  to produce the NOS-hpLUC cassette. This NOS-hpLUC was modified to replace the loop region with an intron by amplifying the NOS-hpLUC cassette with primer RPH-141, common to both the sense and antisense region, with pwo-polymerase. An intron sequence from a kiwifruit terpene synthase genes [GenBank: DQ026298] was also amplified with primers RPH-099 and RPH-100. The intron amplification product was treated with polynucleotide kinase (NEB) and used in a ligation with the non-phosphorylated amplification product of the NOS-hpLUC cassette. The resulting cassettes either contained an intron in +ve (RPH-099 to RPH-100 relative to the NOS promoter) or -ve (RPH-100 to RPH-099 relative to NOS promoter) configuration. Accordingly the +ve intron configuration had a splicable GT to AG intron arrangement, the -ve intron configuration did not. The nos-hpLUC cassette and intron containing derivatives were inserted into a 35S-LUC, 35S-REN cassette; pGreenII 0800 35S-LUC, as an Eco RV fragment. In this way the reporter cassettes were generated with the intron containing NOS-hpLUC cassette in both orientations. pGreenII 0800-3.6 had the NOS promoter directing transcription in the same orientation as the 35S-REN and towards the T-DNA Right border (forward). pGreenII 0800-3.1 had the NOS promoter directing transcription of the hpLUC in the same orientation as the 35S-LUC and towards the T-DNA left border (reverse) (Fig. 6A).
Construction of pSoup-P19
The pSoup helper plasmid  was converted to a T-DNA carrying version by inserting a Bgl II fragment that contained the T-DNA from pGreen0000 into the unique Bam HI site of pSoup creating pSoup0000. A 35S-P19-CaMV fusion was isolated from pBIN-61-P19  as a Sac I-Bgl II partial and inserted into the pSoup0000 multiple cloning site to produce pSoup-P19.
0.5 mM cinnamic acid, pH 7.0 (trans-cinnamic acid 99+%, Aldrich, Milwaukee) was infiltrated into the tobacco leaves after Agrobacterium infiltration and allowed to incubate in the leaf for up to 2 hours before extraction.
The infiltrated leaves were removed from the N. benthamiana plant and frozen in liquid nitrogen. The material was crushed in a pestle and mortar before extraction with 10 ml of ethyl acetate for 18 hours. Post extraction, the plant material was removed by filtering and the organic solvent evaporated. The residue was re-dissolved in 1 mL methanol and used for HPLC analysis.
Twenty μl of the methanolic extracts were run on a HP1100 HPLC system on a Vydac® RP 300Å C18 HPLC column (250 × 4.6) (Phenomenex, CA, USA). The mobile phases were A: 1% phosphoric acid and B: 100% acetonitrile (BDH). The separation of the product and the substrate was performed using gradient elution  at room temperature with a flow rate of 1 ml/min. The substrate and product were monitored using UV at 275 and 310 nm. The peak areas were quantified with cinnamic and p-coumaric acid (trans 98%, Aldrich, Milwaukee) standards.
Galactose dehydrogenase and Galactose-1-P phosphatase assays
Tobacco leaves were harvested approximately 70 hours after infiltration with Agrobacterium containing the plasmid encoding the respective genes under control of the 35S promoter. Tissue was frozen in liquid nitrogen, protein was extracted as described by  and assayed as described by [18, 17].
Firefly Luciferase and Renillia luciferase were assayed using the dual luciferase assay reagents (Promega, Madison, USA). After inoculation and a transient incubation of 2–4 days, 2 cm leaf discs were harvested and ground in 500 μl of Passive Lysis Buffer. Five μl of a 1/100 dilution of this crude extract was assayed in 40 μl of Luciferase Assay Buffer, and the chemiluminescence measured. 40 μl of Stop and Glow™ buffer was then added and a second chemiluminescence measurement made. Absolute RLU were measured in a Turner 20/20 luminometer, with a 5 second delay and 15-second measurement. Data was collected as ratio or, for multiple data points (e.g. several leaves of different ages were infiltrated), the regression-gradient and regression-standard-error were used as a measure of relative promoter strength. Ratios are without units as both the light measurement and protein concentrations are identical. Background controls were run with only the promoter-LUC, 35S-REN reporter plasmid (no TF). In some cases, positive controls were run using a TF with known activity.