Small RNA sequencing identifies cucumber miRNA roles in waterlogging-triggered adventitious root primordia formation

The formation of adventitious roots (ARs) is a key morphological adaptation of cucumber (Cucumis sativus L.) to waterlogging stress. MicroRNAs (miRNAs) constitute a group of non-coding small RNAs (sRNA) that play crucial roles in regulating diverse biological processes, including waterlogging acclimation. However, which specific miRNAs and how they are involved in waterlogging-triggered de novo AR primordia formation are not fully known. Here, Illumina sRNA sequencing was applied to sequence six sRNA libraries generated from the waterlogging-tolerant cucumber Zaoer-N after 48 h of waterlogging and the control. A total of 358 cucumber miRNAs, 312 known and 46 novel, were obtained. Among them, 23 were differentially expressed, with 10 and 13 being up- and downregulated, respectively. A qPCR expression study confirmed that the identified differentially expressed miRNAs were credible. A total of 657 putative miRNA target genes were predicted for the 23 miRNAs using an in silico approach. A gene ontology enrichment analysis revealed that target genes functioning in cell redox homeostasis, cytoskeleton, photosynthesis and cell growth were over-represented. In total, 58 of the 657 target genes showed inverse expression patterns compared with their respective miRNAs through a combined analysis of sRNA- and RNA-sequencing-based transcriptome datasets using the same experimental design. The target gene annotation included a peroxidase, a GDSL esterases/lipase and two heavy metal-associated isoprenylated plant proteins. Our results provide an important framework for understanding the unique miRNA patterns seen in responses to waterlogging and the miRNA-mediated formation of de novo AR primordia in cucumber.