Genome-wide identification of microRNAs associated with osmotic stress and elucidation of the role of miR319 in Medicago ruthenica seedlings

Abstract

Drought is a major environmental stress that affects plant growth, development, and productivity. Medicago ruthenica, a leguminous forage, has garnered attention owing to its resistance to abiotic stress. The purpose of the current study was to explore genes conferring drought resistance to M. ruthenica. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression in plants and are associated with developmental plasticity and abiotic/biotic stress responses. Here, high-throughput small RNA, mRNA, and degradome sequencing analyses were performed to analyze miRNAs and their potential target genes in the leaves of M. ruthenica seedlings under osmotic stress conditions. In total, 591 miRNAs were identified. A comparison of the expression levels showed that 15 miRNAs (14 upregulated and 1 downregulated) were significantly differentially expressed following PEG6000 treatment compared with those in the control (0 h). Most miRNAs are highly conserved between M. ruthenica and Medicago truncatula. Using TargetFinder, 11 target genes were predicted; the expression of these target genes negatively correlated with that of five miRNAs related to osmotic stress response. miR319 downregulated the expression of teosinte branched/cycloidea/proliferating cell factor 4 (TCP4), which encodes plant-specific transcription factors, more significantly in the leaves than in the roots. These results were confirmed using quantitative real-time polymerase chain reaction, northern blotting, RLM 5ʹRACE, and a Nicotiana benthamiana transient expression system. The miR319-TCP4 module may act as a homeostasis factor in M. ruthenica roots following drought injury, and it is conserved among plant species.