Plants were grown at 20ºC for seven days and subject to a symmetrical cycle of acclimation, cold, freeze-thaw, and deacclimation. Chilling began by decreasing the temperature overnight from 20ºC to 4ºC at a rate of 1.3ºC•h-1 and maintaining temperatures of 4 ºC in the day and 2ºC at night for 5 days. Freeze-thaw cycling lasted 12 days with day temperatures of 4ºC and night temperatures gradually decreasing from -2ºC the first night to -4ºC for three nights and -10ºC for four nights, then recovering to -4ºC for three nights and -2ºC for one night. This treatment was designed to allow daily freeze-thaw cycling and protein synthesis. Chilling conditions (4ºC day, 2ºC night) were resumed for five days, followed by deacclimation with increasing temperature to 20ºC overnight and maintaining for three days. Sampling was done at four different times, each at the 11th hour of light to avoid circadian effects: 1) before chilling treatment, 2) five days after initiation of chilling treatment, 3) eight days into freeze-thaw treatment and 4) three days into de-acclimation.
Barley cv. Morex plants were grown in steam-sterilized soil in a growth chamber (Model GC-15, EGC Chagrin Falls, OH) with 12 h photopheriod (148 µmol m-2 s-1 average photosynthetic active radiation) at 23ºC, 12 h dark at 20ºC and 70% relative humidity.
Total RNA was isolated using TRIzol reagent (Invitrogen) from frozen crown samples taken from above soil tissue excluding most fully extended lamina. The protocol is available on the Arabidopsis Information Resource website at http://www.arabidopsis.org/info/2010_projects/comp_proj/AFGC/RevisedAFGC/site2RnaL.htm. RNA was purified using Qiagen RNeasy spin columns with DNase treatment. Quality was assessed by running 25-250 ng on a RNA Lab-On-A-Chip (Caliper Technologies) using an Agilent Bioanalyzer 2100 (Agilent Technologies).
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.
MAS files from the GCOS software were used for determining the ‘present’ gene list. Probe set intensity values above the level set by the gcRMA normalization processor were imported into GeneSpring GX 7.3 software (Silicon Genetics, CA, USA), which was used for the rest of the analysis.