Identification of Radish (Raphanus sativus L.) miRNAs and Their Target Genes to Explore miRNA-Mediated Regulatory Networks in Lead (Pb) Stress Responses by High-Throughput Sequencing and Degradome Analysis

Increasing evidence has revealed that microRNA (miRNA)-mediated gene regulation plays a significant role in response to heavy metal stresses. However, there is little information available about the expression patterns or roles of miRNAs under lead toxicity stress in plants. The radish is an important root vegetable crop with a fleshy taproot as the edible part. It was of vital importance to investigate the response mechanisms and explore the regulatory network at the molecular level under the heavy metal stresses in radish. In the present study, using high-throughput sequencing and degradome analysis, a genome-wide identification of radish miRNA and their targets under the exposure of Pb stress was conducted. A total of 74 known and 173 potential novel miRNAs were successfully identified from two radish root libraries of one untreated control (CK) and one Pb-stressed (Pb500). Of these, 25 known and nine novel miRNAs were significantly differentially expressed and identified as Pb-responsive miRNAs. Degradome analysis revealed that 1,979 miRNA-mRNA target transcripts could potentially be cleaved. Gene Ontology (GO) analysis revealed that these target transcripts were predominately involved in the regulation of transcription, defense responses, and binding related terms. The identified target genes for Pb-responsive miRNAs were mainly involved in stress-related signal sensing and transduction, specific metal uptake and homeostasis mechanisms. Additionally, the expression patterns of 20 Pb-responsive miRNAs and six target genes were validated by quantitative real-time PCR (qRT-PCR). These results provide fundamental insights into the miRNA-mediated regulatory networks and molecular mechanisms underlying plant responsiveness to Pb stresses.