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<Transcriptome profiling by DNA microar … <Transcriptome profiling by DNA microarray>
Total RNA samples were extracted using an RNeasy Plant Mini Kit (Qiagen, http://www.qiagen.com/). Transcriptome profiling using Affymetrix ATH1 GeneChips (24K) was performed according to the manufacturer’s instructions. The R package from Bioconductor (http://www.bioconductor.org/) was used for the normalization (MAS5) of the CELL files. After filtering using Student’s t-test
(P < 0.05) between the wild type and other plants, all genes were then filtered with a fold change of ≥2.0 in the expression level between the wild type and each mutant. Only 907 genes in the other lines were considered. The fold change value was calculated as log2 (Dataset S1). Gene Ontology analysis (TAIR10; http://www.arabidopsis.org/) was performed to annotate 245 biotic and abiotic stress-related genes. The log2-transformed value was used for HCA using MeV 4.8. The data discussed here were also deposited in NCBI’s Gene Expression Omnibus (Edgar et al., 2002) and are accessible through the GEO Series accession number GSE51215.
<Real-time PCR>
Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) and cDNA was synthesized using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, http://www.invitrogen. com/) according to the manufacturer's instructions. The primers for MYB12 and PAP1 were used as described previously
(Stracke et al., 2007; Gonzalez et al., 2008). The PCR programme for amplification was as follows: 95°C for 20 sec, 95°C for 3 sec, and 60°C for 30 sec (40 cycles).
<Assay of radical scavenging activity>
Radical scavenging activity was measured using DPPH as described previously (Nakajima et al., 2004; Tohge et al., 2005b).
<Oxidative stress treatment using methyl viologen>
Three-week-old plants grown on GM plates were transferred to GM plates containing 10 lM methyl viologen (Wako Pure Chemical Industries) and were incubated for 21 days. After the stress treatment, frozen aerial parts were extracted with 20 ll MeOH per milligram fresh weight (FW). The UV absorption of the remaining chlorophylls (663 nm) was measured.
<Drought stress treatment>
This experiment was conducted using Arasystem (Beta-Tech BVBA, http://www.arasystem.com/). Seeds of each line were sown in soil [Pro-Mix BX (Premier Tech Horticulture Inc., http://www.pthorticulture.com/): vermiculite = 2:1, supplemented with fertilizer] and were grown in the plant growth room. After germination, three plants per pot were used for the drought stress
experiments. The plants were last watered on day 20 after germination.
Three-week-old plants were exposed to drought stress by stopping watering. Trays with pots were turned 90° every day. The plants were rewatered after 16 days. The number of surviving plants was counted 3 days after watering.
<3,3′-Diaminobenzidine staining>
Three-week-old plants were incubated in 2-ml tubes with 2 ml of water for 3 days in the growth chamber to assess drought stress. The detached leaves of the incubated plants were used for DAB staining (Thordal-Christensen et al., 1997).
<Water loss experiment>
Two-week-old plants were transferred from agar plates to soil conditions
(five plants per pot). After a 1-week conditioning period, the plants were subjected to drought stress by withholding water.The weight of the pots was calculated at each time point. Another experiment was performed using detached leaves. The weight of 10 leaves from 3-week-old plants grown on GM medium was calculated at each time point. medium was calculated at each time point.
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