2
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 whi ch 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 g roup 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 (Conti g149_at) and 26S proteasome regulatory particle triple-A ATPase subunit 3 (HVSMEk0005D04r2_s_at) were chosen from microarray dat a for internal controls. Genes for which we performed r eal-time RT-PCR are indicated by asterisks. PCR was performed by a GeneAmp PCR system 9700 (Applie d Biosystems) with Ex taq DNA polymerase (Takara). The primers used in this study are listed in Supplemental Ta ble 5. The PCR products were separated on agarose gel, stained with ethidium bromide and visualized by FMBIO II (Hitachi Software, Tokyo, Japan).

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Page 1: Supplemental Figure 2

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).

Page 2: Supplemental Figure 2

Amy6-4

Amy32b

EPB1

Cat-B

Hypothetical protein

Aleurone RNase

Dhn1,7,8

LEA

HVA22

Cont GA3 -GlcY ABA CH JA -GlcY

A

B

group

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

+ GA3

*

*

*

*

*

*

*

*

**

**

*

*


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