Identification and characterization of circRNA-miRNA network regulating flavonoid biosynthesis during hormone treatment in Ginkgo biloba

Circular RNAs (circRNAs) are critical regulators of plant growth, development, and stress adaptation; however, their roles in the biosynthesis of flavonoids, a class of pharmaceutically valuable secondary metabolites, remain poorly understood. Low flavonoid content in Ginkgo biloba leaves severely restricts its medicinal and industrial applications. Exogenous hormones effectively promote flavonoid accumulation, but the underlying circRNA mediated competitive endogenous RNA (ceRNA) regulatory mechanisms remain unclear. In this study, we treated G. biloba leaves with 24-epibrassinolide (EBR) and ethephon (ETH) and identified their optimal functional concentrations: 0.8 mg/L EBR increased quercetin and kaempferol accumulation, while 200 mg/L ETH elevated isorhamnetin content. Whole transcriptome sequencing identified 45 and 47 differentially expressed circRNAs (DECs), 135 and 289 differentially expressed miRNAs (DEMs), and 2232 and 2719 differentially expressed mRNAs (DEGs) under EBR and ETH, respectively. GO and KEGG analyses indicated that circRNAs host genes were consistently enriched in protein homeostasis pathways, indicating a core role of circRNAs in hormone induced stress responses. At the mRNA level, EBR primarily affected phenylpropanoid metabolism, whereas ETH modulated photosynthesis and stress responses. At the miRNA level, ETH uniquely enriched autophagy and DNA repair pathways, consistent with its robust global stress response signature. EBR significantly upregulated the flavonol synthesis genes GbFLS5, GbFLS6, and GbF3’H4 , along with GbMYB8 . Conversely, ETH induced the methylated flavonoid synthesis gene GbOMT and GbbHLH144. By integrating two independent methods, we further constructed putative ceRNA networks and screened key regulatory pairs involved in flavonoid biosynthesis, such as chr8_part1:429472903|429562118-novel_miR_202/448- GbbHLH6 . Given that the functions of these ceRNA modules are preliminarily predicted, further experimental validation is required. This study reveals the divergent circRNA-miRNA-mRNA regulatory mechanisms underlying EBR and ETH induced flavonoid accumulation, providing promising targets for molecular breeding and industrial utilization of high flavonoid yielding G. biloba resources.