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Methods S1. Supplemental materials and methods
DNA constructs and plant transformation
All primers used to amplify the STRS genes for various constructs are listed in Table S1. All
PCR amplification for cloning was performed using high fidelity Prime STAR HS DNA
Polymerase (Takara Bio Inc.). For overexpression of the STRS genes, STRS1 (XbaI and SmaI)
and STRS2 (BamH1 and Sma1) cDNAs were cloned into the pBluescript II KS (+) vector. These
constructs were used to amplify XbaI-start codon-FLAG tag-STRS1 (no start codon)-Sma1 and a
BamH1-start codon-c-Myc tag-STRS2 (no start codon)-Sma1 fragments which were inserted
downstream of the CaMV35S promoter in a modified pBluescript II KS (+) vector from which
the SpeI-BamH1-SmaI sites had been removed from the MCS. The resulting pro35S:STRS-OX
constructs were digested with PstI and SalI and then ligated into the pGreenII 0229
Agrobacterium binary vector.
For organ-specific STRS expression, the upstream intergenic regions of STRS1 (211 bp)
and STRS2 (241 bp) were amplified from genomic DNA and ligated into the BamHI/EcoRI sites
of pKS Bluescript to form pKS::proSTRS constructs. The UIDA (GUS) reporter gene (including a
NOS terminator) was subcloned from pCAMBIA (www.cambia.org) into the EcoRI/HindIII sites
of pKS::proSTRS1 to form pKS::proSTRS1:GUS. The proSTRS1:GUS fragment was then
subcloned
into
the
SpeI/HindIII
sites
of
the
pGreenII
0229
vector
to
form
pGreen::proSTRS1:GUS. To create the proSTRS2:GUS fusion, a SpeI-EcoRI STRS2 fragment
from pKS::proSTRS2 was used to replace the STRS1 promoter to form pGreen::proSTRS2:GUS.
As a negative control, a “promoterless” GUS construct was prepared by removing the STRS2
promoter fragment from pGreen::proSTRS2:GUS with SpeI/EcoRI, filling in the 5´-overhangs
and self-ligation of the remaining plasmid. For the “long promoter” (lp) pro:STRS:GUS fusions,
either an 856 bp (proSTRS1lp) or a 1104 bp (proSTRS1lp) SpeI/EcoRI fragment of upstream
DNA was used to replace the short STRS2 promoter fragment in pGreen::proSTRS2:GUS to form
pGreen::proSTRS1lp:GUS
and
pGreen::proSTRS2lp:GUS,
respectively.
For
STRS
promoter:genomic STRS-GUS fusion constructs, SalI/Pst1 proSTRS1lp:gSTRS1 and SalI/KpnI
proSTRS2lp:gSTRS2 fragments were amplified from genomic DNA and ligated in frame with the
GUS gene contained in the pRITA cloning vector. Each proSTRSlp:gSTRS-GUS fragment was
subcloned into pGreenII 0229 using the same respective restriction sites.
For the proSTRS2:gSTRS2-GFP construct, EGFP was amplified from the Gateway GFP
destination
vector
pK7WGF2
and
cloned
into
the
KpnI
and
ApaI
sites
of
pRITA::proSTRSlp:gSTRS-GUS upstream of the GUS gene and in frame with the genomic STRS2
gene. To prevent read-through into the GUS gene, a stop codon was introduced with the ApaI
reverse primer.
For pro35S:GFP-STRS fusion constructs, STRS cDNAs were amplified and cloned into
pENTR/D-TOPO vector using the pENTR directional TOPO Cloning Kit (Invitrogen). The STRS
cDNAs were then subcloned into the binary Gateway EGFP vector pK7WGF2 using Gateway
technology (Karimi et al., 2002).
For ATPase and RNA-unwinding activities, STRS1 (SalI and NotI) and STRS2 (SalI and
XhoI) cDNAs were cloned in frame into the pET28a 6×His-tag E.coli expression vector
(Novogen). GFP-STRS1 (NdeI and SalI) and GFP-STRS2 (SalI and XhoI) DNA was amplified
from the respective Gateway GFP-STRS vectors and subcloned into pET28a.
The vectors for plant transformation were transferred into Agrobacterium tumefaciens
strain GV3101 and the strains used to transform either wild-type Arabidopsis or the strs mutants
via the floral dip method (Clough and Bent, 1998). Transformants were selected by spraying
with 300 μl l-1 BASTA (Glufosinate ammonium, Bayer CropScience). All transformants
employed for analysis were T3 generation plants harboring a single homozygous copy of the
transgene.
Quantitative real-time PCR
Isolation of total RNA, preparation of cDNA and primer design were performed essentially
according to Kant et al. (2006). All primer sequences are shown in Table S1. qPCR was
performed with an ABI PRISM 7500 Sequence Detection System (SDS) (Applied Biosystems).
Each reaction contained 5 μl PerfeCTa® SYBR® Green Fast Mix® (Quanta Biosciences), 40 ng
cDNA and 100-500 nM of gene-specific primer in a final volume of 10 μl. PCR amplifications
were performed using the following conditions: 95 °C for 30 s, 40 cycles of 95 °C for 5 s
(denaturation) and 60 °C for 35 s (annealing/extension). Data were analyzed using the SDS 1.3.1
software (Applied Biosystems). To check the specificity of annealing of the primers, dissociation
kinetics was performed at the end of each PCR run. All reactions were performed in triplicates.
The relative quantification values for each target gene were calculated by the 2-ΔΔCT method
(Livak and Schmittgen, 2001) using UBQ10 as an internal reference gene for comparing data
from different PCR runs or cDNA samples. To ensure the validity of the 2-ΔΔCT method, twofold
serial dilutions of cDNA from unstressed Arabidopsis thaliana were used to create standard
curves, and the amplification efficiencies of the target and reference genes were shown to be
approximately equal.
Sub-cellular localization of GFP/RFP fusion proteins
For transient expression in protoplasts, leaves from 2.5 week-old Arabidopsis WT plants and
various gene silencing mutants were used for isolation and transformation of protoplasts as
described (http://molbio.mgh.harvard.edu/sheenweb/protocols_reg.html). Fluorescence signals
were examined 12-16 hours after transformation. Protoplasts were subsequently stained with 4,
6- diamidino-2-phenylindole (DAPI). For transient transformation of hydroponically-grown
roots (Figure S5), pro35S:GFP-STRS seeds were germinated on 0.5 X MS plates (0.75% agar)
and 7 day-old seedlings were transferred to a hydroponics system according to Gibeaut et al.
(1997). Transformation was performed as described (Levy et al., 2005). Three days after
transformation, the transformed roots were placed in a cover slip chamber (Nalge Nunc
International) followed by flushing the roots with liquid MS medium or MS supplemented with
200 mM NaCl. For GFP-STRS localization in stable transgenic plants, pro35S:GFP-STRS1 and
pro35S:GFP-STRS2 seedlings were germinated and grown on 200 μm mesh (Saati Tech)
overlaid on MS medium in plates. The mesh with the seedlings was then transferred either to
fresh MS medium or MS supplemented with 200 mM NaCl. For kinetics of stress-mediated
STRS relocalization, 10 day-old seedlings grown upon mesh on MS medium were transferred to
a cover slip chamber (Nalge Nunc International) and flushed with liquid MS or MS
supplemented with 200 mM NaCl, 500 mM mannitol or 100 M ABA. For heat stress,
MS/mesh-grown seedlings were exposed to 45 ºC. Three independent experiments were
performed for each stress treatment. Confocal imaging was performed with an inverted Zeiss
LSM 510 laser scanning microscope (Carl Zeiss) with a ×40 or ×63 oil-immersion objective lens.
For imaging GFP/RFP alone or together with DAPI, the single-track and multiple-track facilities
of the confocal microscope were used, respectively. For imaging GFP and RFP, 488 nm and 561
nm excitation wavelengths were used, respectively. For the DAPI stain, 405 nm excitation was
used. Fluorescence was detected using a 505-550 nm and 630-680 nm band-pass filter for GFP
and RFP, respectively. A 420-480 nm band-pass filter was used for the DAPI stain, and a 650 nm
long-pass filter was used for chlorophyll autofluorescence. Post-acquisition image processing
was performed by the LSM 5 Image Browser software (Carl Zeiss). The data were exported as 8bit TIFF files and processed by Ulead Photo Impact Version 3.0 software (Corel Corporation).
Expression and purification of the recombinant STRS proteins
His-tagged STRS constructs were transformed into E.coli competent cells BL-21 (DE3)
(Novagen). Small-scale expression experiments were carried out to select the optimal conditions
for solubility of the expressed proteins. Cells were grown at 37 °C in Luria Broth (LB) to an
OD600 of 0.4. Isopropyl -D-thiogalactoside (IPTG) was added to a final concentration of 0.2
mM and growth continued for 4 hours at 37 °C. Recombinant proteins were purified through
affinity chromatography. Cells were lysed for 45 min on ice adding 2.5 volumes per gram of
pellet of Buffer A (0.1 M NaPO4, 0.01 M Tris-HCl pH 8, 0.01%, 5 mM NP-40 Imidazole) in the
presence of lysozyme (1 mg ml-1), protease inhibitors (1:10,000 dilution, Sigma) and 1 mM
PMSF. The lysate was sonicated 5 times for 10 s and the suspension was centrifuged at 30,000 x
g for 1.5 h at 4 °C. The supernatant was applied to an affinity FPLC-NiNTA-column (His Trap
HP, GE Healthcare Life Sciences), equilibrated with Buffer B (50 mM Tris-HCl pH 8, 250 mM
NaCl, 25 mM Imidazole, 20% glycerol). Proteins bound to the column were eluted with a linear
gradient (0.025-0.5 M Imidazole) in Buffer B. The presence of the recombinant protein in the
collected fractions was checked by staining (Coomassie Blue) and western blotting with anti6xHis anti-His tag polyclonal antibodies (Cell Signaling TECHNOLOGY®).
References
Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacteriummediated transformation of Arabidopsis thaliana. Plant J., 16, 735-743.
Gibeaut, D.M., Hulett, J., Cramer, G.R. and Seemann, J.R. (1997) Maximal biomass of
Arabidopsis thaliana using a simple, low maintenance hydroponic method and favorable
environmental conditions. Plant Physiol., 115, 317-319.
Kant, S., Kant, P,. Raveh, E. and Barak, S. (2006) Evidence that differential gene expression
between the halophyte, Thellungiella halophila, and Arabidopsis thaliana is responsible for
higher levels of the compatible osmolyte proline and tight control of Na+ uptake in T.
halophila. Plant Cell Environ., 29, 1220-1234.
Karimi, M., Inze, D. and Depicker, A. (2002) GATEWAY vectors for Agrobacteriummediated plant transformation. Trends Plant Sci., 7, 193-195.
Levy, M., Rachmilevitch, S. and Abel, S. (2005) Transient Agrobacterium-mediated gene
expression in the Arabidopsis hydroponics root system for subcellular localization studies.
Plant Mol. Biol. Rep., 23, 179-184.
Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using realtime quantitative PCR and the 2-ΔΔCT method. Methods, 25, 402-408.