Constructs used in the transient assay and for generating transgenic Arabidopsis plants
All the constructs generated in this study were made using standard recombinant DNA techniques and verified by DNA sequencing. The following primers were used in generating the PCR products. The position of each primer in the GST6 promoter is indicated, and the restriction site used for each primer is shown in parenthesis.
Primer 1: 5′-GGGCTGCAGTTGACATGTATATAATCACC-3′, from –783 to –764 (5′PstI site);
Primer 2: 5′-GTTATGTCATTGATGACGACCG-3′, from –432 to
–413 (5′PstI site);
Primer 3: 5′-GGGGTCGACGGAATTGGATGAAGAAG-3′, from
–9 to +8 (5′SalI site);
Primer 4: 5′-GGGGAGATCTCCCTTGTTCCTTTCTCCCCC-3′, from –58 to –38 (5′BglII site);
Primer 5: 5′-GTTATGTCATTGATGACGACCGTGCA-3′, from –432 to –413 (5′PstI site);
Primer 6: 5′-GGGCTGCAGCAGTCAATAAATCCGTGATTTC-3′,
from –211 to –190 (5′PstI site);
Primer 7: 5′-GGGCTGCAGGACGTGCCTGTGGGTAGTGGG-3′,
from –127 to –107 (5′PstI site);
Primer 8: 5′-GGGCTGCAGGAAACAAAATTCCCCTCAC-3′, from
–229 to –211 (5′PstI site);
Primer 9: 5′-GGGCTGCAGTTAGATGGATAACAATGCAAT-3′,
from –183 to –163 (5′PstI site);
Primer 10: 5′-GGGCTGCAGCAATGCAATAAATAAAGGGG-3′,
from –171 to –152 (5′PstI site);
Primer 11: 5′-GGGCTGCAGGGGGACCCAATAAAAAAAAAGG-3′, from –155 to –134 (5′PstI site);
Primer 12: 5′-GGGCTGCAGCCCACTACCCACAGGCACGTC-3′,
from –106 to –126 (5′PstI site);
Primer 13: 5′-GGGCTGCAGTTTATTTATTGCATTGTTATCC-3′,
from –156 to –177 (5′PstI site).
The GST6 promoter fragments from –783 to +8 and –432 to +8 were isolated by PCR with GST6 specific primers (Primer 1 or 2 in combination with Primer 3) and the GST6 genomic clone (Chen et al. 1996) as the PCR template. The PCR fragments were digested with PstI and SalI, gel purified and subcloned into pDR101C (Riggs & Chrispeels 1987), which contains the luciferase coding sequence and nos terminator, to give MPSO-GST6–783 and MPSO-GST6–432. Site-directed mutagenesis was performed using Promega’s ‘Altered Sites in vitro Mutagenesis System’ to generate the 2 bp mutation of the ocs element within the 791 and 440 bp GST6 promoter fragments, following the manufacturer’s instructions. The mutant promoter fragments were subcloned into the PstI/SalI sites of pDR101C to give MPSO-GST6–783/M1 and MPSO-GST6–432/M1. The GST6 minimal promoter was constructed using Primers 3 and 4. The PCR fragment was digested with BglII and SalI, gel purified and subcloned into the BglII/SalI sites of pDR101C to give MPSO-GST6–58. For the ocs element one-copy construct, two complementary oligonucleotides (Primer 5 and a complementary one) were synthesized with PstI restriction sites flanking the GST6 ocs element. The oligonucleotides were annealed and subcloned into the PstI site of pBluescript SK + 0. A clone that contained the 5′ end of the GST6 ocs element closest to the EcoRI site in the polylinker was isolated and digested with PstI and BamHI. The PstI/BamHI fragment of the GST6 ocs element was subcloned into MPSO-GST6–58, which had been digested with PstI and BglII to give MPSO-OCS–58. For the deletion constructs, Primers 6 and 7 were used in combination with Primer 3 to generate 219 and 134 bp PCR fragments, which were then digested with PstI and SalI and subcloned into pDR101C to give MPSO-GST6–211 and MPSO-GST6–126. MPSO-GST6–291 was prepared by digesting the GST6 440 bp promoter fragment with RsaI to generate a PstI/RsaI fragment from –432 to –292 and an RsaI/SalI fragment from –291 to +8, respectively. The RsaI/SalI fragment was subcloned into the EcoRV/SalI sites of pBluescript SK +. The resulting plasmid was digested with PstI and SalI and the PstI/SalI fragment was subcloned into pDR101C to give MPSO-GST6–291. For the deletion constructs that contain the region between –211 and –106, Primers 6, 9, 10 and 11 were used in combination with Primer 12 to generate the 106, 78, 65 and 50 bp PCR fragments, respectively, which were then digested with PstI and subcloned into MSPO-GST6–58 at the PstI site. Meanwhile, Primer 6 was used in combination with Primer 13 to generate a 56 bp PCR fragment, which was also digested with PstI and subcloned into MSPO-GST6–58 at the PstI site. To generate the internal deletion constructs from –211 to –127 or from –155 to –127 within the GST6 791 bp fragment, Primer 8 or Primer 13 was used in combination with Primer 1 to amplify a 573 or 628 bp PCR fragment. The PCR fragments were digested with PstI, gel purified, and subcloned into the PstI site of MPSO-GST6–126 to give MPSO-GST6–783del-1 or MPSO-GST6–783del-2.
For the transgenic studies, the MPSO-GST6–783 and MPSO-GST6–783/M1 constructs were digested with PstI and SacI to release the promoter–luciferase fusion fragments, which were subcloned into pUC19. The resulting plasmids were digested with HindIII and SacI to release the fragments containing the GST6–783–luciferase fusion or the GST6–783/M1–luciferase fusion plus 10 nucleotides of flanking polylinker sequence from pUC19. These fragments were used to replace the 35S–GUS fusion fragment in the pBI121 vector at the HindIII and SacI sites to give pBI-GST6–783–Luc and pBI-GST6–783/M1–Luc. The mobilization of the GST6 one-copy ocs element (fused to the GST6–58 minimal promoter and luciferase reporter gene MPSO-OCS-58) to pBI121 was done in a similar way, and the corresponding construct was called pBI-OCS–Luc.
Transformation of Arabidopsis thaliana
All pBI constructs were transformed into Agrobacterium tumefaciens strain A2260 and then into Arabidopsis thaliana (ecotype Columbia) using the in planta transformation method (Bechtold et al. 1993). The transformed lines were selected for resistance to 50 mg l–1 kanamycin sulfate. T2 seeds from independent transgenic lines grown in Murashige and Skoog (MS) medium containing 50 mg l–1 kanamycin were germinated and used for the subsequent treatments and luciferase assays.
Plant material, growth conditions, treatment with SA or H2O2 and luciferase assay
Transgenic Arabidopsis thaliana was grown on MS medium at 22°C with a day length of 16 h. Eight-day-old seedlings were transferred to MS liquid medium with or without 400 μm SA or 1 mm H2O2 for the period indicated in the figures. Whole-cell extracts were then prepared from roots or the aerial parts of the transgenic plants in 300 μl of luciferase grinding buffer (0.2 m potassium phosphate pH 7.2, 1 mm DTT, 0.5 mm phenylmethanesulfonyl fluoride). For the luciferase assay, 100 μl of the cell extract was added to 100 μl luciferase assay buffer containing 50 mm HEPES pH 7.6, 20 mm MgCl2, 0.5 mg ml–1 BSA and 10 mm ATP. A luminometer (MGM instruments, Hamden, CT, USA) was then used to count the emitted photons for 20 sec. Duplicate luminescence values for each sample were averaged and background luminescence from the reaction buffer was subtracted. To standardize luciferase activity for each sample, the amount of total protein in each cell extract was determined using the Bradford assay (Bio-Rad laboratories, Hercules, CA, USA) according to manufacturer’s instructions, and the relative luciferase activity for each sample was determined and expressed as U per 20 sec mg–1 protein.
Carrot protoplast isolation, electroporation and luciferase assay
Carrot suspension cells (RCWC) were generously provided by Dr Danniel Gallie from the University of California, Riverside, CA, USA. Protoplasts were isolated from 4-day-subcultured suspension cells which had been depleted of 2,4-D for 2 days, using the procedure of Gallie & Young (1994) and Ulmasov et al. (1994) with the following modification. The carrot suspension cells were sedimented at 60 g for 5 min in a Sorval SH-3000 swinging bucket rotor, resuspended in 25 ml of protoplast isolation enzyme solution (Ulmasov et al. 1994) and shaken gently at room temperature for 4–5 h in the dark. Light microscopy was used to monitor the completion of the protoplast preparation. Then protoplasts were pelleted at 40 g for 5 min, gently washed once with W5 solution (Ulmasov et al. 1994) and once with electroporation buffer (Gallie & Young 1994), and resuspended in electroporation buffer. Protoplasts were counted using a hemocytometer under the light microscope, resuspended to a final concentration of 1 × 106 ml–1, and left on ice for at least 30 min before electroporation.
For electroporation, 5 μg of each construct was mixed with 400 μl of carrot protoplasts. The DNA and protoplast mixture was transferred into an ice-cold cuvette (Bio-Rad Laboratories) and electroporation was carried out at 500 μF and 450 V with an electroporator (Bio-Rad Laboratories). After electroporation, protoplasts were transferred immediately into protoplast growth medium (Gallie & Young 1994) with or without 2,4-D or SA, and incubated in the dark for 16–20 h. The protoplasts were then collected by centrifugation at 40 g for 3 min and lysed in 300 μl luciferase grinding buffer. The lysates were used for the luciferase assay as described before except that the emitted photons were counted for 30 sec. The amount of total protein was determined and the relative luciferase activity for each sample was expressed as U per 30 sec mg–1 protein. For each construct, two duplicate electroporations were conducted with the same protoplast preparation, and at least three independent protoplast preparations were used to calculate the average activity and standard deviation for each construct.