Function of floral fragrance-related microRNAs and their targets in Hedychium coronarium

Background Hedychium coronarium is highly valued for its intense fragrance, which may be influenced by the expression of microRNAs (miRNAs). miRNAs are a class of small RNAs that play conserved and pivotal regulatory roles throughout plant growth and development, modulating various aspects of plant metabolism. However, the specific functions of miRNAs in the growth and development of H. coronarium remain largely unexplored. Results Therefore, to identify miRNAs in H. coronarium and evaluate their relationship with the synthesis of floral fragrance compounds, we analyzed the volatile compounds and the miRNA patterns at three developmental stages (F1, F5, F9). Our results showed that the volatile emissions of major floral compounds (eucalyptol, ocimene, and linalool) increased with the flower development. Using small RNA sequencing, 171 conserved miRNAs from 24 miRNA families and 32 novel miRNAs were identified. Degradome sequencing revealed 102 mRNA degradation sites corresponding to 90 target genes from 30 miRNA families. The qRT-PCR results indicated that the expression levels of hco-miR393a and hco-miR167n were consistent with the release pattern of floral fragrance compounds, whereas the expression levels of HcTIR1 and HcARF8 were inversely correlated with hco-miR393a and hco-miR167n expression. Tobacco co-transformation demonstrated that HcTIR1 and HcARF8 are respective targets of hco-miR393a and hco-miR167n. Additionally, treatments with exogenous IAA and the auxin inhibitor PCIB affected both the release of floral fragrance compounds in H. coronarium and the expression of hco-miR393a and hco-miR167n. STTM and VIGS experiments indicated that hco-miR167n and hco-miR393a positively regulate the metabolism of floral fragrance compounds, while HcARF8 and HcTIR1 act as negative regulators. Dual-luciferase and yeast one-hybrid assays demonstrated that HcARF8 binds to the promoter of the terpene synthase gene HcTPS8, thereby regulating the synthesis of fragrance compounds. Conclusions This is the first report to identify miRNAs in H. coronarium and elucidate their expression profiles in petal tissues across different developmental stages. These findings provide new insights into the molecular regulation of floral fragrance compound synthesis and underscore the role of miRNAs in the Zingiberaceae family of plants.