In general, Cold-responsive genes could be classified into two groups: 1) functional proteins, which directly protect plants against environmental stresses, and 2) regulatory proteins, which regulate the expression of downstream target genes in the stress response. The first group mainly comprises enzymes involved in the biosynthesis of various osmoprotectants, such as late embryo genes is abundant proteins, antifreeze proteins, chaperones, and detoxification enzymes. The second group mainly includes transcription factors and protein kinases. The best-characterized transcription factors involved in the plant cold response are the class of AP2/ERF, one kind of subfamily was known as CBF/DREB, which regulate cold-responsive gene expression by binding to DRE/ CRT cis-elements in the promoter region of cold-responsive genes. Changes in the expression of cold-responsive contribute to the differences in plant cold tolerance. For example, Solanum commersonii and S. tuberosum, which are closely SB203580 p38 MAPK inhibitor related species that differ in their cold acclimation abilities, exhibit considerable differences in the expression levels of cold-responsive genes. Chen et al. found that the activities of some detoxification enzymes, such as catalase, superoxide dismutase, peroxidase and esterase are increased in response to cold stress, whereas the plant’s metabolic activity is decreased. Some cold-induced genes have been cloned from Eucalyptus plants. For example, four CBF paralogs were previously isolated from E. gunnii, and qRT-PCR analysis demonstrated that they exhibited complementary expression profiles in a range of natural standard and cold conditions. Navarro et al.found overexpression of EguCBF1a or EguCBF1b in the cold-sensitive E. urophylla?E. grandis hybrid could enhance its freezing tolerance. Given the importance of cold-responsive genes in plant cold tolerance, studying the cold response at the transcription level may be a key step in identifying specific tolerance mechanisms. Next generation sequencing provides a high throughput approach for analyzing genes involved a particular process at transcription level. Compared to the traditional sequencing techniques, NGS is more robust and demonstrates greater resolution and inter-lab portability compared to several microarray platforms. NGS could detect millions of transcripts and is beneficial to explore new genes and their expression profiling independent of a reference genome. For example, cDNA libraries for E. gunnii have been constructed to identify genes involved in cell protection, LEA/dehydrin accumulation, and cryoprotection. Despite its obvious potential, these next generation sequencing methods have not been applied for E. dunnii yet. The goal of this study was to construct a comprehensive transcriptome to investigate the molecular mechanism of cold tolerance in E. dunnii. Based on the expression profiles of these transcripts, we hypothesize that the Orn pathway may play a less important role than the Glu pathway during cold acclimation or that it may represent an alternative pathway for cold acclimation in E. dunnii. Free proline accumulation is affected not only by the proline biosynthesis pathway but also by the proline degradation pathway.