Transcriptome-Wide Identification of miRNA Targets under Nitrogen Deficiency in Populus tomentosa Using Degradome Sequencing

miRNAs are endogenous non-coding small RNAs with important regulatory roles in stress responses. Nitrogen (N) is an indispensable macronutrient required for plant growth and development. Previous studies have identified a variety of known and novel miRNAs responsive to low N stress in plants, including Populus. However, miRNAs involved in the cleavage of target genes and the corresponding regulatory networks in response to N stress in Populus remain largely unknown. Consequently, degradome sequencing was employed for global detection and validation of N-responsive miRNAs and their targets. A total of 60 unique miRNAs (39 conserved, 13 non-conserved, and eight novel) were experimentally identified to target 64 mRNA transcripts and 21 precursors. Among them, we further verified the cleavage of 11 N-responsive miRNAs identified previously and provided empirical evidence for the cleavage mode of these miRNAs on their target mRNAs. Furthermore, five miRNA stars (miRNA*s) were shown to have cleavage function. The specificity and diversity of cleavage sites on the targets and miRNA precursors in P. tomentosa were further detected. Identification and annotation of miRNA-mediated cleavage of target genes in Populus can increase our understanding of miRNA-mediated molecular mechanisms of woody plants adapted to low N environments.