Identification of Conserved and Novel Cold-Responsive MicroRNAs in Trifoliate Orange (Poncirus trifoliata (L.) Raf.) Using High-Throughput Sequencing

Pre-exposure to a period of low but non-freezing temperature, a phenomenon known as cold acclimation, can enhance freezing tolerance. However, the genetic regulatory network controlling cold acclimation of this plant remains unclear. MicroRNAs (miRNAs) have been shown to play an important role in abiotic stress responses. To have a deeper understanding on the cold acclimation, the miRNAs before and after low temperature treatment in trifoliate orange (Poncirus trifoliata (L.) Raf.) were characterized using deep sequencing. A total of 107 conserved miRNAs, belonging to 44 miRNA families, and 5 potential novel miRNAs were identified. Bioinformatics analysis indicated that 36 conserved and 5 novel miRNAs were either up- or downregulated by cold, respectively; ten of these cold-responsive miRNAs were confirmed by quantitative RT-PCR assay. Time-course expression analysis further validated cold responsiveness of the five selected conserved miRNAs. The predicted target genes of cold-responsive miRNAs encode a variety of proteins implicated in abiotic stress responses, including the well-characterized transcription factors. In addition, expression patterns of two target genes were inversely related to the relevant miRNAs. The present study gains insight into molecular mechanisms underlying the cold acclimation-mediated freezing tolerance at posttranscriptional level and unravels cold-responsive miRNAs of significant value for stress-oriented genetic engineering.