De novo Transcriptome Sequencing of Cold-treated Kentucky Bluegrass (Poa pratensis) and Analysis of the Genes Involved in Cold Tolerance

Kentucky bluegrass (Poa pratensis) is strongly resistant to cold stress. Although the molecular mechanism of the plant response to cold stress has been widely documented in model plants, little is known about the cold tolerance of Kentucky bluegrass at the genomic level. Here, we compared the transcriptomes of Kentucky bluegrass under cold treatment (-5°C) and a control treatment (at 20°C) by RNA-seq and de novo assembly. Totally 75,934 unigenes were generated, among which 53,762 were successfully annotated in public databases. Upon comparing the transcriptomes of the control and cold-treated plants, 3,896 unigenes were identified as differentially expressed. Among these genes, 2,410 were down-regulated and 1,486 were up-regulated in the cold-treated plants. A few previously reported cold-induced proteins, antioxidant enzymes, and osmoregulation proteins were identified, and their expression levels were estimated. Moreover, ten differentially expressed genes were selected for qRT-PCR verification. Their expression patterns were consistent with the results of the RNA-seq. Additionally, the transcription factor families, i.e., ethylene response factors, heat stress transcription factors, NAC proteins, WRKY domaincontaining proteins, and auxin response factors, were identified as differentially expressed genes. The identification of these involved genes will facilitate studies on the transcriptional regulation of cold tolerance in plants. Keywords: Kentucky bluegrass; Cold tolerance; Transcriptome; Differentially expressed genes; Transcriptional regulation Introduction Cold stress, such as chilling and freezing temperatures, is a major environmental factor that influences plant distribution, growth and development [1]. A few plants cope with cold stress by acquiring cold tolerance, a process termed cold acclimation. During this process, various biochemical and physiological changes occur and make the plant more tolerant under chilling or freezing conditions [2]. Cold acclimation involves the signal transduction of various genes responding to cold stress, transcriptional regulation and posttranslational regulation of transcription factors [1]. The induction of cold responsive (COR) genes have been shown to result in cold acclimation for alfalfa [3]. Many transcription factors involved in cold tolerance have been identified [4]. C-repeat binding factors (CBFs), also known as dehydration-responsive element-binding (DREB) proteins, are a group of transcription factors that are well-studied in plant cold stress. CBFs can bind to the promoters of COR genes and activate their expression [1]. The ectopic expression of CBF1 activates the expression of COR and induces cold tolerance in Arabidopsis [5]. Regulators upstream of CBF have also been identified, including ICE1, MYB15, CAMTA and HOS1 [1,6-8]. Although considerable studies have documented the molecular mechanism of plant cold tolerance, deep studies are necessary to highlight the transcriptional regulation of cold tolerance. Kentucky bluegrass (Poa pratensis) is a perennial, cool-season species of grass native to Europe and is widely used for making lawns in parks, gardens, golf courses and sports fields [9]. This plant is strongly resistant to cold stress and can survive at temperatures as low as -38°C. This adaptability suggests that Kentucky bluegrass