When the first leaf was fully emerged the plants were treated for 6 d at 3 C, 12-h light (150 mmol m22 s21)/1 C, 12-h dark. Control plants were harvested after 8 d at 20 C. All samples were collected in the middle of the light period. This experiment was conducted three times to yield three independent biological replicates. To analyze the involvement of the photooxidative stress, seeds of wild type and mutants were imbibed for 3 h in water containing 0 (control) or 50 mM norfluorazon. The hydrated seeds were grown in a growth-controlled environment chamber as above except that light intensity was 10 mmol m22 s21 for the first 8 d and 200 mmol m22 s21 for the next 6 d. During the growth, treated plants were watered two times with 50 mM norfluorazon.For analysis of Cbf expression, treated samples were collected after 4, 8, and 24 h of cold treatment in the light.
Wild type and mutants were germinated in peat pots and grown in a controlled-environment chamber for 8 d at 20 C with 12-h photoperiod (300 mmol m22 s21).
For RNA isolation, two methods were applied: RNA from embryo and endosperm collected during seed development was isolated by Trizol (Gibco BRL Life Technologies, Rockville, MD) and passed through an RNeasy spin column (Qiagen, Hilden, Germany) for further clean up as recommended by Affymetrix, Inc. (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetric, Inc., Santa Clara, CA). For embryo and endosperm collected after imbibition, PURESCRIPT® RNA isolation kits (Gentra Systems, Inc., Minneapolis, MN) and RNeasy spin column (Qiagen, Hilden, Germany) were used. Quality of RNA was assessed on an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).
The BioArray High-Yield RNA Transcript Labeling Kit (Enzo Diagnostics) was then used to synthesize biotin-labeled cRNA from template cDNA by in vitro transcription.
10 µg labeled, fragmented cRNA was hybridized at 45°C with rotation for 16 hours in an Affymetrix GeneChip Hybridization Oven 320 on Affymetrix Barley1 Genechip arrays. The arrays were washed and stained using streptavidin phycoerythrin on an Affymetrix Fluidics Station 400.
The arrays were scanned on a Hewlett-Packard GeneArray scanner.