In this study, analysis of freezing-induced ion leakage revealed that freezing tolerance of the perennial ryegrass cv. Caddyshack was continuously increased from 1 d to 14 d of cold acclimation. About 3,000 quality ESTs were generated from two cDNA libraries that were constructed from cold-acclimated (CA) and nonacclimated (NA) perennial ryegrass cv. Caddyshack plant materials. Comparative analysis of EST indicated that over 60 EST groups were either increased or decreased three times or more in the CA library than in the NA library. Reverse transcriptase PCR (RT-PCR) analysis confirmed that the expression patterns for 19 out of the 23 selected genes were consistent with the EST abundance analysis. A cross-species microarray study was performed by using perennial ryegrass RNA to hybridize with Affymetrix barley gene chips and the results revealed that selection of proper background correction and normalization methods is critical for the success of cross-species microarray studies. By using a specific three-step normalization method, i.e. IdealMM + scaling + tukey.biweight, a total of 2,870 (12.6%) genes out of approximately 2,2000 genes on the barley gene chip were identified as differentially regulated by either 1 d or 7 d of cold acclimation. Quantitative reverse transcriptase PCR (qRT-PCR) analysis confirmed that the expression patterns of fourteen selected genes are consistent with the signal intensity patterns observed in the microarray study.

Gene ontology (GO) analysis of differentially regulated genes identified through either comparative EST analysis or microarray study indicates that diverse biological processes, cellular components or molecular functions responded differentially to cold acclimation. Our results suggest that many cold-regulated (COR), dehydration-responsive (Dhn), and ice recrystallization inhibition (IRI) genes were greatly up-regulated by cold acclimation. Up-regulation of the biological processes of embryonic development, gene expression regulation, translation, and down-regulation of the biological process of photosynthesis are important in increasing freezing tolerance of perennial ryegrass. Meanwhile, differential regulation of the biological processes of cell communication, metabolites and energy precursor generation and response to extracellular stimulus, are also important for plant to adapt to cold temperatures. The molecular function of structural molecule activity, enzyme regulator activity, RNA binding, and receptor activity were up-regulated, and oxygen binding and receptor binding were down-regulated during cold acclimation.

Two genes encoding IRI proteins, LpIRI-a and LpIRI-b, were identified in the perennial ryegrass cv. CaddyShack. Amino acid alignments among the IRI proteins revealed that many conserved repetitive IRI-domain motifs (NxVxxG/NxVxG) are present in both LpIRI-a and LpIRI-b proteins. qRT-PCR analysis revealed that both LpIRI-a and LpIRI-b genes were up-regulated by dozens of fold after 1 h of cold acclimation, and the transcripts kept accumulating up to thousands of fold by 7 d of cold acclimation. qRT-PCR analysis also revealed that both genes were not responsive to ABA, NaCl, and drought treatment in perennial ryegrass. Overexpression of the LpIRI-a and LpIRI-b in Arabidopsis showed that both genes can increase the survival rates and decrease freezing-induced ion leakage of the transgenic plants after they were subjected to freezing temperatures. Expression of LpIRI-a and LpIRI-b proteins in E. coli suggested that the expressed exogenous proteins enhanced freezing tolerance of the host E. coli cells.