maize inbred line: B73 tissue: lamina sixth leaf treatment: nitrate supply reduced to 0.15mM KNO3 plant age: day 30 after germination
Treatment protocol
Control and low temperature treated plants were supplied with full-strength Hoagland solution (15mM KNO3, 5mM CaCl2, 2mM MgSO4, 2mg/L Fe, 0.5mM KH2PO4, 50µM H3BO4, 10µM MnCl2, 1µM ZnSO4, 0.3µM CuSO4, 0.5µM Na2MoO4). For low N treatment, the nitrate content was reduced to 0.15mM KNO3, for low P treatment the phosphate content of the nutrient solution was 0.1mM KH2PO4. For low temperature treatment, conditions in one chamber were switched to night temperatures of 4°C at day seven after germination while all other settings were kept constant.
Growth protocol
For all experiments, B73 seeds were incubated on wet filter paper for three days, and uniform seedlings were transferred into pots of 1.5L volume containing nutrient-poor peat soil (Basissubstrat 2, Klasmann & Deilmann, Germany). Fertilization started at day seven of the experiment with modified Hoagland solutions. The low temperature experiment was performed in two Snijders climate chambers (IMAGO F3000, Snijders Scientific) at 14h days with 28°C and 80% humidity, followed by 10h of night at 20°C and 50% humidity. Light intensity on soil level was ca. 600µmol m-2 s-1, and ca. 800µmol m-2 s-1 at the position of the highest leaves. All seedlings were allowed to establish in the pots under control conditions. The nutrient deficiency experiments (N, P) were performed in a CLF climate chamber (PlantMaster PGR 3045, CLF Plant Climatics GmbH, Germany) with a diurnal rhythm of 14h light (ca. 200 µmol m-2 s-1 at level of the soil surface and ca. 650 µmol m-2 s-1 just under the light source which was adjusted according to plant height to give maximal possible irradiance) at 28°C and 80% humidity, and 10h night at 20°C and 50% humidity.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from four biological replicates per treatment after the method described by Logemann et al. (1987) using 100mg of frozen leaf material. The isolated RNA was purified with the RNeasy purification kit according to the manufacturer's instructions (Qiagen, Germany). The quality was checked with the Agilent 2100 Bioanalyzer using the RNA 6000 Nano kit.
Label
Cy3
Label protocol
Cy3-labeled cRNA were prepared according to the Agilent protocol from 500ng total RNA (One-Color Microarray-based gene expression analysis v.5.5).
Hybridization protocol
After fragmentation, an aliquot of 1.65 µg of Cy3-labeled cRNA were hybridisied for 17h at 65°C on Mais_array (Agilent-025271). Arrays were washed according to the protocol (One-Color Microarray-based gene expression analysis v.5.5).
Scan protocol
Slides were scanned immediately after washing on the Agilent G2565B Microarray Scanner.
Data processing
The scanned images were analyzed with Feature Extraction Software version 11.7.1 (Agilent) using default parameters (protocol GE-v5_95_Feb07). The processed signals were log2 transformed and normalised to the 75th percentile followed by median base line correction using the Agilent GeneSpring GX 11.0 software. Normalisation was done across all samples.
