Supporting Information
Stress experiments
All stress experiments were performed with Arabidopsis ecotype Landsberg
erecta (Ler). Seeds were surface-sterilized with 70% ethanol 4 times 1 min and
washed with sterile water 5 times 1 min. Sterilized seeds were plated on MS
medium with vitamins (Duchefa Biochemie B.V., Haarlem, Netherlands) and
0.8% agar. Plated seeds were kept at 4°C for 3 days in the dark and then grown
under continuous light of 12 µmol photons m-2s-1 for 5days at 22°C in E-66HO/2
growth chamber (Percival Scientific, Inc., Perry, Iowa, USA) equipped with
compact fluorescent lamps F55BX/840 (GE Lighting AG, Zurich, Switzerland).
Plates with “non pre-treated” seedlings were left at the same conditions for 1
more day, while plates with “pre-treated” seedlings were transferred to 120
µmol photons m-2s-1 light for 24h at 22°C in the same growth chamber.
Subsequently, light was switched off for 1h and temperature in the growth
chamber was decreased to 12°C. At the end of this dark treatment, Fv/Fm of
control seedlings was measured (0 time point in and Figure 8B). Light was then
switched on and 300 µmolm-2s-1 light intensity and 12°C stress treatment was
applied for up to 144h. Fv/Fm was measured at certain time points as shown in
Figure 8B. For each measurement, separate plates were used.
Array hybridization and evaluation.
Experimental procedures are described according to MIAME (Minimum
Information About a Microarray Experiment) standards for plant genomics
(Brazma et al., 2001; Zimmermann et al., 2006). Affymetrix Arabidopsis ATH1
GeneChips® were used for the experiment (Affymetrix, Santa Clara, CA).
The entire experiment was performed four times, providing independent
biological replicates. Each of the four experiments was carried out as follows: the
two lines, Ler wild-type and soldat10, were grown on eight MS agar plates with
vitamins (Duchefa) for 6 days under 12 μmolm-2s-1 continuous light at 22°C in
the same growth chamber equipped with the same lamps as used for the stress
experiments. On each of the eight plates, approximately 50 Ler wild-type and 50
soldat10 seeds were put one by one. Seedlings from the eight separate plates
were pooled to a container with liquid nitrogen and total RNA was isolated
according to (Melzer et al., 1990). Total RNA from 2 independent experiments
was pooled to create biological replicates: the biological replicate I consisted of
equal amounts of total RNA from experiments 1 and 2 and the biological
replicate II consisted of equal amounts of total RNA from experiments 3 and 4.
The quality of RNA of the biological replicates was determined with a NanoDrop
ND 1000 (NanoDrop Technologies, Delaware, USA) and a Bioanalyzer 2100
(Agilent, Waldbronn, Germany). Total RNA samples (2 µg) were reversetranscribed into double-stranded cDNA, in vitro transcribed in presence of
biotin-labeled nucleotides using a IVT Labeling Kit (Affymetrix Inc., P/N 900449,
Santa Clara, CA), purified and quantified using BioRobot Gene Exp – cRNA Target
Prep (Qiagen AG, Switzerland). The labeled cRNA quality was determined using
Bioanalyzer 2100.
Biotin-labeled cRNA samples (15 µg) were fragmented randomly to 35–200 bp at
94°C in Fragmentation Buffer (Affymetrix Inc., P/N 900371, Santa Clara, CA) and
were mixed in 300 µl of Hybridization Mix (Affymetrix Inc., P/N 900720, Santa
Clara, CA) containing a Hybridization Controls and Control Oligonucleotide B2
(Affymetrix Inc., P/N 900454, Santa Clara, CA), before hybridization to
GeneChip® Arabidopsis ATH1 Genome arrays for 16 h at 45°C. Arrays were then
washed using an Affymetrix Fluidics Station 450 FS450_0004 protocol. An
Affymetrix GeneChip Scanner 3000 (Affymetrix Inc., Santa Clara, CA) was used to
measure the fluorescent intensity emitted by the labeled target.
Raw data processing was performed using the Affymetrix GCOS 1.4 software
(Affymetrix Inc., Santa Clara, CA). After hybridization and scanning, probe cell
intensities were calculated and summarized for the respective probe sets by
means of the MAS5 algorithm (Hubbell et al., 2002). To compare the expression
values of the genes from chip to chip, global scaling was performed, which
resulted in the normalization of the trimmed mean of each chip to a target
intensity (TGT value) of 500 as detailed in the statistical algorithms description
document of Affymetrix (2002). Quality control measures were considered
before performing the statistical analysis. These included adequate scaling
factors (between1 and 3 for all samples) and appropriate numbers of present
calls calculated by application of a signe-drank call algorithm (Liu et al., 2002).
The efficiency of the labeling reaction and the hybridization performance was
controlled with the following parameters: Present calls and optimal 3' /5'
hybridization ratios (around 1) for the housekeeping genes (GAPDH and ACO7),
for the poly A spike in controls and the prokaryotic control (BIOB, BIOC, CREX,
BIODN).
Microarray data analysis.
Microarray data were analyzed with GeneSpring GX 7.3.1 software (Silicon
Genetics) as described by (Laloi et al., 2007). Only those transcripts that were
called present or marginal in at least both duplicates of the same line were taken
into account. A cutoff value of 2-fold change was adopted to identify genes that
were differentially expressed in soldat10 compared with Ler wild type. For each
gene, the average expression value in the two biological replicates of soldat10
was compared to the average expression value in the two biological replicates of
Ler wild type.
Descriptions of each gene are from TAIR. Localization of some proteins encoded
by genes up-regulated in soldat10 was predicted with Wolf PSORT program
which can be found under proteomics tools at www.expasy.ch .
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