Transcriptome Changes in Response to Cold Acclimation in Perennial Ryegrass as Revealed by a Cross‐Species Microarray Analysis

Freezing tolerance in perennial ryegrass (Lolium perenne L.) can be dramatically increased by a period of cold acclimation. To understand the mechanisms of cold acclimation and freezing tolerance in L. perenne, a cross‐species microarray study was conducted by using total RNA from cold‐acclimated and nonacclimated L. perenne to hybridize with Affymetrix Barley1 GeneChips from barley (Hordeum vulgare L.). Our results indicate that selection of proper background correction and normalization method is critical for the success of a barley–perennial ryegrass cross‐species microarray study. By using a specific three‐step normalization method, a total of 2870 (12.6%) genes out of approximately 22,000 genes on the barley gene chip were identified as differentially regulated by either 1 or 7 d of cold acclimation. Quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR) analysis confirmed that the expression patterns of 14 selected genes are consistent with the signal intensity patterns observed in the microarray study, supporting the validity of using a cross‐species microarray in studying gene expression in species for which genomic resources remain scarce. Gene ontology analysis of differentially regulated genes indicates that different biological processes, cellular components, and molecular functions responded differently to cold acclimation in observable temporal patterns, suggesting that perennial ryegrass gradually acquires freezing tolerance by systematically regulating gene expression in an orderly manner.