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Rice ERF OsEATB restricts GA biosynthesis - Plant Physiology

Rice ERF OsEATB restricts GA biosynthesis - Plant Physiology

seedlings calculated are

seedlings calculated are the ones with no visible change of phenotype. These experiments were repeated twice and similar results were obtained each time. Microarray analysis Control and transgenic seedlings were grown to the 4-leaf-stage in half-strength Murashige and Skoog medium under a 14/10 h light/dark cycle at 28°C. RNA samples were extracted using an RNeasy mini kit (Cat#74106, Qiagen, GmBH, Germany) following the manufacturer’s instructions. To determine integration of RNA, the RNA integrity number (RIN) was determined using an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, USA). Microarray analyses were carried out using a competitive hybridization method using the Affymetrix microarray system. All procedures were carried out according to the manufacturer's protocols. To obtain biotin-labeled cRNA, 1 μg total RNA was amplified, labeled, and purified using the GeneChip 3’IVT Express kit (Cat#901229, Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. Array hybridization and washing was performed using the GeneChip® Hybridization, Wash and Stain kit (Cat#900720, Affymetrix, Santa Clara, CA, US) in the 645 Hybridization Oven (Cat#00-0331-220V, Affymetrix, Santa Clara, CA, US) and the Fluidics Station 450 (Cat#00-0079, Affymetrix, Santa Clara, CA, US) according to the manufacturer’s instructions. Slides were scanned by a GeneChip® Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US) and analyzed with Command Console Software 3.1 (Affymetrix, Santa Clara, CA, US) with default settings. Raw data were normalized by the MAS 5.0 algorithm with Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US). The normalized log2 signal intensity data was generated by the MAS 5.0 algorithm. All microarray data have been deposited in a public database. The dataset was normalized by the LOWESS dye normalization method. Spots with low fluorescence intensity (i.e. =1 or

with 15 ml 80% (v/v) methanol at 4°C for 12 h. Before grinding of plant tissues, the following labeled GAs were added as internal standards: [2H 2 ] GA1 (1.00ng/g), [2H 2 ] GA4 (2.00 ng/g), [2H 2 ] GA12 (2.00 ng/g), [2H 2 ] GA24 (6.00 ng/g), and [2H 2 ] GA53 (4.00ng/g). After centrifugation (10,000 g, 4°C, 20 min), the supernatant was collected and passed through a C-18 SPE-cartridge (12 mL,1.5 g) preconditioned with 8 ml water, 8 ml methanol, and 8 ml 80%(v/v) methanol. The eluate was pooled and evaporated under a nitrogen gas stream and redissolved in 3 ml water. The solution was acidified with 360 μl 0.1 mol/L hydrochloric acid and repeatedly extracted with ethyl ether (10 × 0.5 ml). The ether phases were combined, dried under nitrogen gas, and redissolved in 112 μl acetonitrile. Then, 180 μl Et3N (20 μmol/mL) and 108 μl 3-bromoacetonyltrimethylammonium bromide (20 μmol/mL) were added and the reaction solution was vortexed for 10 min. The mixture was evaporated to dryness under a stream of nitrogen gas, and then the residue was dissolved in 30 μl water. The resulting sample solution was injected by 25 kV × 1 min and separated by 100-cm amino groups, coated capillary electrophoresis coupled with electrospray ionization quadrupole-time of flight mass spectrometry for analysis (Chen et al., 2011). Data Availability All microarray data from this work are available from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) (www.ncbi.nlm.nih.gov/geo) under the series entry GSE28229. Accession Numbers Sequence data from this article can be found in the GenBank/EMBL database under the following accession numbers: OsEATB,EU622934; OsCPS2,AK072928; GA20ox2,AY114310; OsCPS1, AK100333; OsKS1, AK119442; OsKO2, AK071743; OsKAO, AK120757; XET,AF443603; UROD,AF119232; GOX,AF022740; SLR1,AB262980; OsERFf3,AB036883; CPD,AK111418; OsERF15,ABH04236; OsBIERF1,AAV98700; OsBIERF2,AAV98701; OsBIERF3,CAC39058; 25

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