background ecotype: Columbia genotype/variation: wild type tissue: Shoots time under flooding (h): 0.5 tissue: Shoots time under flooding (h): 0
Treatment protocol
For flooding treatment, 14-day-old plants were placed between two foamed plastic strips as a floating platform. To minimize the effects of mechanical stress, the platforms with plants were put on filter papers that were wetted with half-strength MS for 1 h as pre-treatment. The platforms were then transferred to liquid half-strength MS 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. For submergence treatment, the plates with 14-day-old plants were submerged into half-strength MS that was bubbled with 3% oxygen balanced with nitrogen for the time indicated.
Growth protocol
Experiments were carried out on Arabidopsis thaliana ecotype Columbia-0. The ethylene-signaling mutant, ein2-5 was obtained from the Arabidopsis Biological Resource Center, Ohio State University, USA. Seeds were surface-sterilized for 20 min in 0.5% of sodium hypochloride 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 Murashige and Skoog (MS) medium (Duchefa Biochemie BV, Haarlem, Netherlands) containing 0.5% sucrose, and kept at 4 oC in the dark for 3 days. Plates were then transferred to a growth chamber, placed vertically, and grown at 22 C under a long photoperiod with 16 h of light (~81 μmol s-1 m-2) and 8 h of darkness for 5 days. The 5-day-old seedlings were transplanted onto fresh plates, and the plates were placed vertically to prevent roots from growing into medium. The transplanted seedlings were grown in the growth chamber until they were 14 days 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
Shoots of Arabidopsis (Columbia) without flooding treatment
For flooding treatment, 14-day-old plants were placed between two foamed plastic strips as a floating platform. To minimize the effects of mechanical stress, the platforms with plants were put on filter papers that were wetted with half-strength MS for 1 h as pre-treatment. The platforms were then transferred to liquid half-strength MS 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. For submergence treatment, the plates with 14-day-old plants were submerged into half-strength MS that was bubbled with 3% oxygen balanced with nitrogen for the time indicated.
Growth protocol
Experiments were carried out on Arabidopsis thaliana ecotype Columbia-0. The ethylene-signaling mutant, ein2-5 was obtained from the Arabidopsis Biological Resource Center, Ohio State University, USA. Seeds were surface-sterilized for 20 min in 0.5% of sodium hypochloride 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 Murashige and Skoog (MS) medium (Duchefa Biochemie BV, Haarlem, Netherlands) containing 0.5% sucrose, and kept at 4 oC in the dark for 3 days. Plates were then transferred to a growth chamber, placed vertically, and grown at 22 C under a long photoperiod with 16 h of light (~81 μmol s-1 m-2) and 8 h of darkness for 5 days. The 5-day-old seedlings were transplanted onto fresh plates, and the plates were placed vertically to prevent roots from growing into medium. The transplanted seedlings were grown in the growth chamber until they were 14 days 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
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 with Axon GenePix 4000B and GenePix 6.0 (Axon Instruments, Inc., Union City, CA, USA), respectively.
Description
Biological replicate 3 of 4.
Data processing
The exported GenePix Result (GPR) files that were imported into GeneSpring 7.3 (Silicon Genetics, Redwood, CA, USA) via LOWESS Normalization.
