ABSTRACT: BACKGROUND:Cope's gray treefrog, Dryophytes chrysoscelis, withstands the physiological challenges of corporeal freezing, partly by accumulating cryoprotective compounds of hepatic origin, including glycerol, urea, and glucose. We hypothesized that expression of genes related to cryoprotectant mobilization and stress tolerance would be differentially regulated in response to cold. Using high-throughput RNA sequencing (RNA-Seq), a hepatic transcriptome was generated for D. chrysoscelis, and gene expression was compared among frogs that were warm-acclimated, cold-acclimated, and frozen. RESULTS:A total of 159,556 transcripts were generated; 39% showed homology with known transcripts, and 34% of all transcripts were annotated. Gene-level analyses identified 34,936 genes, 85% of which were annotated. Cold acclimation induced differential expression both of genes and non-coding transcripts; freezing induced few additional changes. Transcript-level analysis followed by gene-level aggregation revealed 3582 differentially expressed genes, whereas analysis at the gene level revealed 1324 differentially regulated genes. Approximately 3.6% of differentially expressed sequences were non-coding and of no identifiable homology. Expression of several genes associated with cryoprotectant accumulation was altered during cold acclimation. Of note, glycerol kinase expression decreased with cold exposure, possibly promoting accumulation of glycerol, whereas glucose export was transcriptionally promoted by upregulation of glucose-6-phosphatase and downregulation of genes of various glycolytic enzymes. Several genes related to heat shock protein response, DNA repair, and the ubiquitin proteasome pathway were upregulated in cold and frozen frogs, whereas genes involved in responses to oxidative stress and anoxia, both potential sources of cellular damage during freezing, were downregulated or unchanged. CONCLUSION:Our study is the first to report transcriptomic responses to low temperature exposure in a freeze-tolerant vertebrate. The hepatic transcriptome of Dryophytes chrysoscelis is responsive to cold and freezing. Transcriptomic regulation of genes related to particular pathways, such as glycerol biosynthesis, were not all regulated in parallel. The physiological demands associated with cold and freezing, as well as the transcriptomic responses observed in this study, are shared with several organisms that face similar ecophysiological challenges, suggesting common regulatory mechanisms. The role of transcriptional regulation relative to other cellular processes, and of non-coding transcripts as elements of those responses, deserve further study.
