Exploring miRNA-target modules regulating flower opening and senescence in Osmanthus fragrans through integrated transcriptome, miRNAome, and degradome analysis

Osmanthus fragrans, one of China's ten traditional flowers, is extensively utilized in the ornamental, food, and cosmetics industries. The processes of flower opening and senescence are crucial to its life cycle, but the optimal commercial harvest period lasts only 2-3 days, presenting a significant bottleneck for industrial development. MicroRNAs (miRNAs) are key noncoding small RNA (sRNA) that regulate plant growth, development, and stress responses. However, the regulatory network between miRNAs and mRNAs, particularly in the context of flower opening and senescence in O. fragrans, remains poorly understood. In this study, we identified 135 miRNAs through sRNA sequencing, comprising 46 known and 89 novel miRNAs, with 73 exhibiting differential expression. Furthermore, 529 target genes for 112 miRNAs were identified using degradome sequencing, with 57 targets classified into categories 0-2. Among these, 26 were transcription factors, including 12 SBPs, 5 NACs, 3 ARFs, 2 AP2s, and 2 GRFs. A regulatory network was constructed for all these target pairs. Through the network and weighted gene co-expression network analysis (WGCNA) of differentially expressed genes (DEGs), we identified key miRNA-mRNA modules regulating flower opening (ofr-miR156j-OfSPL2/SPL9 and ofr-miR396a5p-OfGRF1) and flower senescence (ofr-miR164b-5p-OfNAC079 and ofr-miR394b-5p-OfFBXO6). These modules were further validated through bioinformatics analysis and qRT-PCR assays. This study represents the first systematic identification of miRNAs involved in O. fragrans flower opening and senescence, establishing a comprehensive regulatory network that provides a foundation for future research into miRNA-mediated regulation of these processes.