Vanderauwera et al. 10.1073/pnas.0706668104.

Supporting Information

Files in this Data Supplement:

SI Figure 4
SI Table 2
SI Table 3
SI Table 4
SI Table 5
SI Table 6
SI Table 7
SI Table 8
SI Table 9
SI Table 10
SI Table 11
SI Table 12
SI Table 13
SI Methods




SI Figure 4

Fig. 4. Schematic overview of the differentially expressed transcripts identified in the genome-wide transcriptome analysis. For each source of variability affecting mRNA levels: HL stress time course (T), genotype (G), and the combined effect of HL stress and genotype (G&T), the number of differentially expressed is presented.





SI Methods

Microarray Analysis and Data Processing.

RNA was isolated from pools of 30 seedlings with TRIzol Reagent (Invitrogen, Carlsbad, CA). Total RNA concentrations were determined with an ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE), and quality was examined with the RNA 6000 Nano Assay Kit and 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Reverse transcription, RNA labeling (15 mg of total RNA), hybridization to Genechip Arabidopsis ATH1 Genome Arrays (Affymetrix, Santa Clara, CA), and scanning were performed according to the manufacturer's instructions (Affymetrix; www.affymetrix.com/) at the VIB Microarray Facility (Leuven, Belgium). Raw data (CEL files) were processed with the statistical algorithm Robust Multiarray Average (gcRMA) (51-53) with the affy package of R/Bioconductor (54). The gcRMA-processed data were subjected to a two-factor ANOVA with the MultiExperiment Viewer of the Microarray Software Suite TM4 from The Institute for Genome Research (55). A multiple testing correction was performed on the p-values of the F-statistics to adjust the false discovery rate by using QVALUE (http://genomine.org/qvalue/) (22). Genes with P values <0.001 and q values <0.05 were retained for further analysis. Before clustering, these significant profiles were processed: expression values were obtained by averaging the inverse log-transformed gcRMA-processed values of the two independent replicates, and only genes with an average absolute expression value (over the eight time points) >10 were retained. Within the data sets of "genotype-significant" and "time-significant" genes, many genes were also significant for the other main effect "time" or "genotype," respectively. Genes significant for both main effects were omitted from the main effect data sets "time-significant" and "genotype-significant" and combined with the "interaction-significant" genes into the "time and genotype-significant" data set. Resulting data sets were log transformed, median centered across each gene, and subjected to hierarchical average linkage clustering (with a Euclidian distance metric) with Cluster/Treeview (56). Array element annotations were compiled by The Arabidopsis Information Resource (www.Arabidopsis.org/) (57) and according to the affy25K_array_elements-2004-06-01 version.

Quantitative RT-PCR.

Expression characteristics of eight genes were validated on RNA samples used for microarray hybridizations and on RNA from a biological repeat experiment. First-strand cDNA was prepared from 2.5 mg of total RNA, Superscript II RNaseH- Reverse Transcriptase (Invitrogen) and a oligo(dT)15 primer. Five microliters of a 1:12 diluted first-strand cDNA was used as a template in the subsequent PCR, which was performed on the iCycler iQ (BioRad, Hercules, CA) with 200 nM primers and Platinum Supermix-UGD (2´) (Invitrogen) supplemented with fluorescein in a final volume of 25 ml per reaction, according to manufacturer's instructions. All PCRs were performed at least in triplicate. Universal ProbeLibrary probes #138, #88, #143, #25, #110, #63, and #46 (Roche Diagnostics, Brussels, Belgium) were used for At1g05680 (UGT74E2), At5g66400 (RAB18), At5g52300 (LTI65), At5g52310 (RD29A), At4g34000 (ABF3), At2g4660 (AtHB7), and At5g62470 (AtMYB96), respectively. Actin-related protein 7 (At3g60830) was used as housekeeping gene with Universal ProbeLibrary probe #147. For At2g04050, gene expression levels were quantified with the Platinum SYBR green Supermix-UGD (2´) (Invitrogen) in combination with gene-specific primer pairs 5'-TTCATCCCACTACTCGGTTTC-3' and 5'-AAACAAAAGCCCAGCAAACAGG-3' for At2g04050, and 5'-ACTCTTCCTGATGGACAGGTG-3' and 5'-CTCAACGATTCCATGCTCCT-3' for actin-related protein 7 (At3g60830).

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53. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed, TP (2003) Biostatistics 4:249-264.

54. Gautier L, Cope L, Bolstad BM, Irizarry RA (2004) Bioinformatics 20:307-315.

55. Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Currier T, Thiagarajan M, et al. (2003) BioTechniques 34:374-378.

56. Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Proc. Natl. Acad. Sci. USA 95:14863-14868 [Err. Proc. Natl. Acad. Sci. USA 96:10943].

57. Rhee SY, Beavis W, Berardini TZ, Chen G, Dixon D, Doyle A, Garcia-Hernandez M, Huala E, Lander G, Montoya M, et al. (2003) Nucleic Acids Res 31:224-228.