Supplemental Figure 2 Validation of microarray analysis and examination of the effects of defense-related signaling on GA signaling by semi-quantitative RT-PCR. RT-PCR was performed using cDNA from 1.2 g total RNA isolated from protoplasts which were the same RNA samples as used in Fig. 3. Genes were categorized into three groups. Group A is GA-inducible genes, group B is ABA-inducible genes and group C is -GlcY-inducible genes. Amplified product of dehydrin genes was thought to be a mixture of Dhn1, Dhn7 and Dhn8. PolyA-binding protein(Contig546_at), GAPDH (Contig149_at) and 26S proteasome regulatory particle triple-A ATPase subunit3 (HVSMEk0005D04r2_s_at) were chosen from microarray data for internal controls. Genes for which we performed real-time RT-PCR are indicated by asterisks. PCR was performed by a GeneAmp PCR system 9700 (Applied Biosystems) with Ex taq DNA polymerase (Takara). The primers used in this study are listed in Supplemental Table 5. The PCR products were separated on agarose gel, stained with ethidium bromide and visualized by FMBIO II (Hitachi Software, Tokyo, Japan).
+ GA3 Cont GA3-GlcY ABA CH JA -GlcY group Amy6-4 Amy32b EPB1 Cat-B Hypothetical protein Aleurone RNase Dhn1,7,8 LEA HVA22 * * A * * B * Blue copper-binding protein FAD-linked oxidoreductase Ferritin Oxalate oxidase Pirin Permatin PR5 AP2 transcription factor Chitinase class I Chitinase III Peroxidase HvBWMK1 * * * * C * * * * * polyA-binding protein GAPDH 26S proteasome regulatory particle triple-A ATPase subunit3