Identification of circRNAs in industrial hemp (Cannabis sativa L.) and prediction of regulatory networks underlying female differentiation

Abstract

Introduction Circular RNAs (circRNAs) are important regulators of plant development and stress responses, but their roles in 6-benzylaminopurine (6-BA)-induced female flower differentiation in industrial hemp remain unclear. Methods Industrial hemp plants were treated with 60 mg·L-1 6-BA, and female flower samples were collected at different developmental stages. Genome-wide high-throughput sequencing was used to identify circRNAs. Selected circRNAs and miRNAs were validated by qRT-PCR. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed, and putative circRNA–miRNA–mRNA networks and the protein-coding potential of circRNAs were computationally evaluated. Results A total of 264 circRNAs were identified. Multiple circRNAs exhibited stage-specific responses to 6-BA. Cs_circ_67024327 showed the strongest induction at the early female flower stage, whereas Cs_circ_44508988 and Cs_circ_21994222 exhibited distinct responses from the early to middle developmental stages. CsSPT5, a flowering-time candidate gene located near the SDR/PAR boundary, was downregulated at the shoot apical meristem stage but upregulated at the early and middle female flower stages. No statistically significant GO terms or KEGG pathways were detected after multiple-testing correction. Computational analyses suggested that several circRNAs may function as miRNA sponges, and some circRNAs contained conserved domains indicating potential protein-coding capacity. Discussion These findings provide preliminary evidence that 6-BA-responsive circRNAs may participate in female flower differentiation in industrial hemp through predicted ceRNA interactions or mechanisms related to their coding potential. These putative regulatory mechanisms require further experimental validation.