Supplementary Materials and methods
Localization of GFP-AtPGR fusion proteins in transgenic plants
For the subcellular localization analysis of the AtPGR (At5g19930) protein in transgenic
Arabidopsis, the AtPGR cDNA fragment was amplified using the following primers: 5’GAATTCATGGAAACGTCGCCGCAATT-3’ (EcoRI site is shown in italics) and 5’AAGCTTTCAGAAAATGTACACAGA-3’ (HindIII site is shown in italics). The PCR
products were inserted into the pEGAD vector at the EcoRI and HindIII cloning sites. The
nucleotide sequence of the new construct was confirmed by DNA sequencing. For the
intracellular localization of GFP-AtPGR fusion proteins in transgenic plants, root samples
were mounted on microscope slides and observed using an Olympus FluoView1000 confocal
microscope (Olympus, Tokyo, Japan). Confocal images were obtained and processed using
FV10-ASW 1.7A computer software (Olympus).
Analysis of GUS activity
In order to generate the AtPGR promoter-driven GUS construct, the 1.5-kb upstream genomic
fragment from the AtPGR translation start codon was PCR amplified and digested by PstI and
BamHI. The fragment was cloned into the vector pCAMBIA1391, resulting in a
transcriptional fusion of the AtPGR promoter with the GUS coding region. The following
primers
were
utilized
for
the
CATCTGCAGCCATCATACTCTCGCAGGA-3’
promoter:
AtPGR
and
5’5’-
CATGGATCCGAGAATCTGTTGACGACGAAG-3’. The nucleotide sequence of the new
construct was confirmed by DNA sequencing. The construct was introduced into
Agrobacterium tumefaciens strain GV3101, which was utilized for the transformation of
Arabidopsis plants using a previously described vacuum infiltration technique (Bechtold and
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Pelletier 1998). Homozygous lines (T3 generation) from 10 independent transformants were
obtained, and five lines were selected for GUS staining. Hygromycin resistance of the T2
generation from these five selected lines was segregated as a single locus. Histochemical
staining for GUS activity in transgenic plants was conducted as described previously
(Jefferson et al. 1987). Whole seedling or various tissues were immersed in 1 mM 5-bromo4-chloro-3-indolyl--glucuronic acid solution in 100 mM sodium phosphate, pH 7.0, 10 mM
EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide and 0.1% Triton X100, and then incubated for 4 h at 37 oC. Chlorophyll was cleared from the plant tissues by
immersion in 70% ethanol.
Extraction of RNA and RT-PCR
Total RNA was extracted from the frozen samples using the Plant RNeasy extraction kit
(Qiagen, Valencia, CA, USA). In order to remove any residual genomic DNA in the
preparation, the RNA was treated with RNAse-free DNAse I in accordance with the
manufacturer’s instructions. The concentration of RNA was spectrophotmetrically quantified
and 5 g of total RNA was separated on a 1.2% formaldehyde agarose gel to verify the
concentration and monitor the extraction integrity. RT-PCR was employed to measure the
levels of AtPGR expression in RNAi transgenic plants, using 500 ng of total RNA together
with the following primers: AtPGR: forward (5’-ACTGGAAGAAATGGAAACGTCGCC-3’)
and reverse (5’-AGGCAGCTAAGAGTCCTGCCTT-3’); Actin8 (At1g49240): forward (5’TGCCTATCTACGAGGGTTTC-3’) and reverse (5’- GTCCGTCGGGTAATTCATAG-3’).
After 25 PCR amplification cycles, 20 µL of each RT-PCR product was loaded onto a 1.2%
(w/v) agarose gel to visualize the amplified DNA.
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Quantitative real-time PCR (qPCR)
Total RNA was extracted from the variously-treated 10-day-old Arabidopsis seedlings using
an RNeasy Plant Mini kit (Qiagen). qPCR was carried out using the SensiMix One-Step kit
(Quantance, London, UK) and a Rotor-Gene 6000 quantitative PCR apparatus (Corbett
Research, Mortlake, NSW, Australia).
Arabidopsis Actin8 was used as the internal control.
Results were analyzed using RG6000 1.7 software (Corbett Research). Quantitative analysis
was carried out using the Delta Delta CT method (Livak and Schmittgen 2001). Each sample
was run in three independent experiments. The reaction primers utilized were: AtPGR,
upstream
5’-ACTGGAAGAAATGGAAACGTCGCC-3’
AGGCAGCTAAGAGTCCTGCCTT-3’;
CAB1
and
(At1g29930),
downstream
5’-
upstream
5’-
GGACTTGCTTTACCCCGG-3’ and downstream 5’-GAAGGACATAAAACGAATCC-3’;
Actin8,
upstream
5’-TGCCTATCTACGAGGGTTTC-3’
and
downstream
5’-
GTCCGTCGGGTAATTCATAG-3’.
Stress tests
For the Glc, Frc and Man stress tests, seeds were sown on MS medium supplemented with
5% Glc, 6% Frc and 0.1% Man, respectively, grown in a growth chamber, and assessed for
percentage of cotyledon greening after 10 days. Experiments were conducted in triplicate for
each line (50 seeds each). To test osmotic stress, seeds were sown on MS medium
supplemented with 350 mM mannitol. Growth and phenotypic assessment was as described
for the sugars. For 2-DG stress test, seeds were sown on MS medium supplemented with 0,
0.01, 0.025, 0.05 and 0.1 mM 2-DG. Growth and phenotypic assessment was as described for
the sugars. For 3-OMG stress test, seeds were sown on MS medium supplemented with 0, 20,
50, and 150 mM 3-OMG. Growth and phenotypic assessment was as described for the sugars.
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Supplementary References
Bechtold N, Pelletier G (1998) In planta Agrobacterium-mediated transformation of adult
Arabidopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82:259-266
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: b-glucuronidase as a sensitive
and versatile gene fusion marker in higher plants. EMBO J 6: 3901-3907
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time
quantitative PCR and the 2-CT method. Methods 25: 402-408
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