genetic background: Columbia genotype: wt tissue: seedling age: 9d time (h): 3 treatment: submerged
Treatment protocol
Plates with plants on the surface of the medium were placed into half-strength MS liquid medium that was bubbled with 3 % oxygen balanced with nitrogen for the indicated times. The liquid MS medium was pretreated with 3% oxygen for one hour before use.
Growth protocol
Experiments were carried out on Arabidopsis thaliana ecotype Columbia-0. Two T-DNA insertion mutant lines of WRKY22, wrky22-ko1 (SALK_047120) and wrky22-ko2 (SALK_098205), were obtained from the Arabidopsis Biological Resource Center, Ohio State University, USA. All seeds were surface-sterilized with 0.5% of sodium hypochloride for 20 minutes and washed at least five times with sterilized water. Seeds were sown on plates with 0.55% of phytagel (Sigma-Aldrich, St. Louis, MO, USA) in half-strength MS medium (Duchefa Biochemie BV, Haarlem, Netherlands) containing 0.5% sucrose at pH 5.7, and kept at 4oC in the dark for 3 days to achieve uniform germination. The plates were then transferred to a growth chamber and placed vertically, and plants were grown at 22ºC under a 16-hour light (81 μmol s-1 m-2)/8-hour dark cycle until 9-day-old.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from shoots or roots using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacture's description using a 1:2 ratio of sample:TRIZOL reagent. Total RNA was subjected to DNase treatment using the TURBO RNA free kit (Ambion) according to the manufacture's instructions. Genomic DNA contamination and RNA integrity were tested on an Agilent 2100 Bioanalyzer.
Label
Cy5
Label protocol
10 µg of total RNA were primed with 1.25 µl of 100 µM Oligo dTV DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 500 µM each dATP, dCTP, dGTP, with 100 µM dTTP, 400 µM aminoallyl-dUTP. The aminoallyl-labeled cDNA was hydrolyzed to obtain single stranded labeled DNA with addition of 10 µl of 0.5 M EDTA and 10 µl of 1 N NaOH at 65℃ for 15 min followed by neutralization with 25 µl of 1 M HEPES, pH 7.4. The labed DNA was then purified with Microcon-30 concentrator by washing with water for three times. NHS-ester Cy dyes were used to couple with the aminoally-cDNA at room temperature for 1 h. Unreacted NHS-ester Cy dyes were quenched with addition of 4.5 µl of 4 M hydroxylamine and removed by PCR clean up kit (QIAGEN). For details, see http://ipmb.sinica.edu.tw/microarray/protocol.htm.
Channel 2
Source name
Arabidopsis (Columbia) without Submergence Treatment
treatment: control genotype: wt genetic background: Columbia tissue: seedling age: 9d
Treatment protocol
Plates with plants on the surface of the medium were placed into half-strength MS liquid medium that was bubbled with 3 % oxygen balanced with nitrogen for the indicated times. The liquid MS medium was pretreated with 3% oxygen for one hour before use.
Growth protocol
Experiments were carried out on Arabidopsis thaliana ecotype Columbia-0. Two T-DNA insertion mutant lines of WRKY22, wrky22-ko1 (SALK_047120) and wrky22-ko2 (SALK_098205), were obtained from the Arabidopsis Biological Resource Center, Ohio State University, USA. All seeds were surface-sterilized with 0.5% of sodium hypochloride for 20 minutes and washed at least five times with sterilized water. Seeds were sown on plates with 0.55% of phytagel (Sigma-Aldrich, St. Louis, MO, USA) in half-strength MS medium (Duchefa Biochemie BV, Haarlem, Netherlands) containing 0.5% sucrose at pH 5.7, and kept at 4oC in the dark for 3 days to achieve uniform germination. The plates were then transferred to a growth chamber and placed vertically, and plants were grown at 22ºC under a 16-hour light (81 μmol s-1 m-2)/8-hour dark cycle until 9-day-old.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from shoots or roots using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacture's description using a 1:2 ratio of sample:TRIZOL reagent. Total RNA was subjected to DNase treatment using the TURBO RNA free kit (Ambion) according to the manufacture's instructions. Genomic DNA contamination and RNA integrity were tested on an Agilent 2100 Bioanalyzer.
Label
Cy3
Label protocol
10 µg of total RNA were primed with 1.25 µl of 100 µM Oligo dTV DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 500 µM each dATP, dCTP, dGTP, with 100 µM dTTP, 400 µM aminoallyl-dUTP. The aminoallyl-labeled cDNA was hydrolyzed to obtain single stranded labeled DNA with addition of 10 µl of 0.5 M EDTA and 10 µl of 1 N NaOH at 65℃ for 15 min followed by neutralization with 25 µl of 1 M HEPES, pH 7.4. The labed DNA was then purified with Microcon-30 concentrator by washing with water for three times. NHS-ester Cy dyes were used to couple with the aminoally-cDNA at room temperature for 1 h. Unreacted NHS-ester Cy dyes were quenched with addition of 4.5 µl of 4 M hydroxylamine and removed by PCR clean up kit (QIAGEN). For details, see http://ipmb.sinica.edu.tw/microarray/protocol.htm.
Hybridization protocol
Arrays in this study were carried out with the Agilent Arabidopsis Gene Expression Microarray, V4 version 4× 44k array (Agilent, Santa Clara, CA, USA), based on the manufacturer's two-color microarray protocols. Hybridyzation experiments were performed at the DNA Microarray Core facility, Institute of Plant and Microbial Biology, Academia Sinica, as described on http://ipmb.sinica.edu.tw/microarray/protocol.htm.
Scan protocol
Array signals were detected and analyzed using the Agilent DNA Microarray Scanner G2565CA and Agilent Feature Extraction 10.7.1.1 software, respectively.
Description
Biological replicate 3 of 4.
Data processing
The acquired results were then imported into GeneSpring 11.5 (Agilent Technologies, Santa Clara, CA, USA) using LOWESS Normalization.
