Transcriptomic analysis of Arabidopsis wild-type (Col-0), phyb-9, jaz10 and jaz10phyB-9 plants to methyl jasmonate (MeJA)

We used Affymetrix 1.0 ST arrays to compare the transcriptome of 4 weeks‐day‐old plants sprayed with 250uM of MeJA or mock (control). For the experiments we used wild-type (Col-0), phyB-9 and jaz10 single mutants and jaz10phyb double mutant. Samples were harvested after 3hours of induction with MeJA or mock. We performed a first analysis with the transcriptomes of wild‐type (Col-0) to identify those group of genes that are up‐ and down‐regulated by MeJA treatment (2 fold-changes). We founded 719 genes whose expression was statistically significantly different between the two treatments. To gain knowledge into the regulation of phyB over these set of 719 genes, we contrasted their expression levels against phyB transcriptome. By cluster analysis, we identified five major groups of genes, where in two of them the transcript levels increased in presence of MeJA (clusters 1 and 2) and three whose expression decrease after MeJA treatment (Cluster 3, 4 and 5). We used a factorial experimental design comprising four genotypes (Col-0, phyb-9, jaz10 and jaz10phyb-9) and two treatments (MeJA or mock). 4 weeks‐day‐old plants were sprayed with MeJA (250μM) or mock solution and harvested 3hs later. Two replicates per treatment were performed for col and tree replicates for phyb-9. RNA was extracted using The Spectrum Plant Total RNA Kit (Sygma), prepared, labelled and hybridized to the arrays in accordance with the manufacturer’s instructions for the GeneChip Arabidopsis Gene 1.0 ST Array (Affymetrix). Raw microarray data were processed and analyzed using Affymetrix Expression Console® software. Genes with ‘absent’ calls and a signal of <50 units in all replicate experiments were filtered out. Significantly differentially expressed genes were identified by performing profile analysis using Significance Analysis of Microarrays with a p-value <0.05. A test filter was performed to work only with those genes for which the ratio of expression showed at least a 2‐fold change between “col mock” vs “col MeJA”.