Appendix S3: Experimental procedures
Materials
Wild-type and T-DNA insertion lines of Arabidopsis were obtained from the
European Arabidopsis Stock Centre (Scholl et al., 2000), except for the u19-2
homozygous line (WiscDsLox430F05) which was kindly provided by P.
Goldsbrough, Purdue University.
DNA isolation and PCR reactions
DNA was isolated from WT and T-DNA insertion lines using CTAB method
(Murray and Thompson 1980), or a buffer containing SDS (200 mM Tris-HCl pH
7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS), followed by precipitation in 2propanol and 70% ethanol.
Amplification conditions were: 4 min at 94°C,
followed by 30 to 35 cycles of 20 sec at 94°C, 30 sec at 60°C, 1 minute at 72°C,
and an extension of 5 min at 72°C after the final cycle. Amplification was carried
out in a 50 μl volume consisting of final concentration of 1X PCR buffer (supplied
with the Promega Taq polymerase, Catalog # M8305), 2 mm MgCl2, 0.2 mM
deoxynucleotide (dNTP) mixture (Bioline), 0.2 μM each sense and antisense
primer, 2.5 units Taq polymerase (Promega), and 50-100 ng template DNA.
Reaction products were separated by electrophoresis on a 1% agarose gel in 1X
TAE buffer (Sambrook et al., 1989). Fermentas GeneRuler 1 kb DNA ladder
(SM0311) was used as size marker in agarose gels. Primers used in PCR
assays are given in supplementary table 1.
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Measurement of gene expression
Total RNA was prepared from 100 mg two week-old seedling tissue using
RNeasy plant RNA minipreps (Qiagen, Dorking, UK) or from whole plants using
Tri reagent (Sigma). For gel blot analysis, 10 µg per sample was electrophoresed
through 1.0% agarose (Life Technologies, Paisley, UK) formaldehyde gels
(Sambrook et al., 1989). RNA was transferred to nylon membranes (Roche,
Mannheim, Germany) by capillary action. Blots were pre hybridised and
hybridised in 50% formamide as described previously (Knight et al., 1999) at
42C. Blots were washed twice in each of the following successively: 2X SSC
(1X SSC is 0.15 M sodium chloride, 0.15 M sodium citrate, pH7), 0.1% SDS
followed by 1X SSC, 0.1% SDS and finally 0.1X SSC, 0.1 % SDS at 42C.
Probes were prepared by PCR or digestion of ESTs and labelled with 32P-CTP by
the random priming method using DNA “Ready-to-go” labelling beads (GE
Healthcare, Little Chalfont, UK). For semi-quantitative RT-PCR assays cDNA
synthesis was performed on 5 g total RNA using a cDNA synthesis kit
(Fermentas, St. Leon-Rot, Germany, catalog number K1632) with 5 μM genespecific antisense or sense primer and 5 μM anchored oligo dT primer or genespecific sense or antisense primers and 10 units of MMLV reverse transcriptase.
cDNA was diluted 10-20 fold with water and 2-5 μl was used in semi-quantitative
PCR assays. PCR conditions were the same as for genomic DNA PCR but the
cycle numbers varied. For realtime-PCR assays, cDNA was prepared from 1 µg
RNA using random hexamer primers and Superscript II reverse transcriptase
(Invitrogen) according to the manufacturer’s protocol. cDNA was treated with
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RNase H and RNase A after reverse transcription. Quantitative real-time PCR
was carried out using a SYBR green mastermix (Applied Biosystems) and an ABI
Prism 7000 Sequence Detection System (Applied Biosystems). TPC1 and
UBQ10
(At4g05320)
expression
levels
were
determined
by
absolute
quantification against a standard curve derived from a cDNA dilution series.
TPC1 expression levels were normalized using UBQ10. Realtime-PCR
conditions for all primer combinations were: 95°C for 10 min; 40 cycles of (95°C
for 15 sec, 60°C for 1 min), followed by a melting curve protocol to determine the
specificity of primers. Primers used in real-time PCR are given in supplementary
table 1.
Proteomic analysis of glutathione transferases
For each line, approx. 4 g of root culture tissue (DeRidder et al., 2002) was
harvested and glutathione transferases were purified using glutathione affinity
chromatography as described (DeRidder et al., 2002). The purified samples were
analysed by 2D-PAGE as described (DeRidder et al., 2002) except that the entire
sample was used and 12% acrylamide gels were used for 2nd dimension
separation. Gels were stained with Coomassie Brilliant Blue and proteins in the
stained spots were identified by peptide mass fingerprinting (Loutre et al., 2003).
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References for the Appendix S3
DeRidder, B.P., Dixon, D.P., Beussman, D.J., Edwards, R., Goldsbrough,
P.B. (2002) Induction of glutathione S-transferases in Arabidopsis by
herbicide safeners. Plant Physiol. 130, 1497-505.
Knight, H., Veale, E., Warren, G.J. and Knight, M.R. (1999) The sfr6 mutation
in Arabidopsis suppresses low-temperature induction of genes dependent on
the CRT/DRE sequence motif. Plant Cell, 11, 875–886.
Loutre, C., Dixon, D.P., Brazier, M., Slater, M., Cole, D.J., Edwards, R. (2003).
Isolation of a glucosyltransferase from Arabidopsis thaliana active in the
metabolism of the persistent pollutant 3,4-dichloroaniline. Plant J. 34, 485-93.
Murray, M.G. and Thompson, W.F. (1980) Rapid isolation of high molecular
weight plant DNA. Nucleic Acids Res. 8, 4321-4325.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A
Laboratory Manual. New York: Cold Spring Harbour.
Scholl, R.L., May, S.T., Ware, D.H. (2000) Seed and molecular resources for
Arabidopsis. Plant Physiol. 124, 1477-1480.
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