Protoplasts were isolated from 5-6 week-old plants by incubating rosette leaves for 12-14 hours in N&M medium containing 1% cellulase “onozuka” R-10 and 0.25% macerozyme R10 (Yakult Honsha Co., Ltd., Japan). Protoplasts were separated by centrifugation, washed once with CPW medium and precipitated by centrifugation at low speed for 5 min.
Growth protocol
Seeds of Arabidopsis thaliana Landsberg erecta (Ler) ecotype and kyp-2 mutant were sown and incubated at 4oC for 4 days and then grown under long-day conditions (14 h light) at 21oC
Extracted molecule
total RNA
Extraction protocol
Total RNAs were prepared from leaves and protoplasts using RNeasy Plant Mini Kit according to the manufacturer’s protocol (Qiagen)
Label
biotin
Label protocol
The probe Preparation, cDNA synthesis, cRNA reactions and hybridizationaccording to Affymetrix GeneChip® Expression Analysis Technical Manual was carried out in the Weizmann Institute microarray unit.
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on ATH1 Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using Affymetrix 3000G
Description
no additional information
Data processing
Data analysis was performed using Partek®. Additional quality assessment and visualizations were done using SpotFire DecisionSite (Tibco). Microarray CEL files were read into Partek and preprocessed by Robust Multiarray Averaging (RMA, Irizarry et al 2003) using default parameters, followed by quantile normalization and probe summarization using median polish. Expression signals were generated and displayed on log 2 scale. Signals distribution plots, principal component analysis and hierarchical clustering of the normalized samples were examined, in order to assess the quality of the microarrays. Statistical hypothesis testing for identification of differentially expressed genes was done using 2-Way ANOVA in Partek, where the fixed effects were the “tissue” (leaf or protoplast) and “source plant” (Ler or kyp). “Sources of variation analysis” in Partek showed a much higher average F-ratio of the tissue effect compared to the average F-ratio of the source plant effect (466 vs. 20.4, respectively). P-values were calculated for each effect as well as for the interaction between them, and further adjusted for multiple tesing by step-up FDR according to Benjamini and Hochberg (1995). Fold change values are represented on linear scale, where positive and negative values indicate up- and down-regulation, respectively. Differentially expressed genes were defined as those having absolute expression signal > 5 in at least one of the arrays, FDR adjusted p-value < 0.05 in any of the effects or interaction, and fold change value either below -1.3 or above 1.3. Among the 1,499 differentially expressed genes, 1,484 genes showed significant tissue effect, 109 genes showed significant source plant effect and 37 genes showed significant interaction. 98 genes had both tissue and source plant significant effect. Hierarchical clustering applied to the differentially expressed genes using the Pearson correlation coefficient as a measure of similarity (data not shown), yielded two main clusters according to “tissue”. Together with the sources of variation analysis and the numbers of differentially expressed genes in each contrast, these data suggests a much more robust tissue effect compared to the source plant effect.
