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

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 roles of miRNAs in the growth and development of H. coronarium remain largely uncharacterized.

To identify miRNAs in H. coronarium and assess their potential role in the synthesis of floral fragrance compounds, we analyzed the volatile compounds and miRNA expression patterns at three developmental stages (F1, F5, F6). Our findings revealed that the volatile emissions of major floral compounds, including eucalyptol, ocimene, and linalool, increased as the flowers progressed through development. Small RNA sequencing identified 171 conserved miRNAs from 24 miRNA families, along with 32 novel miRNAs. Degradome sequencing uncovered 102 mRNA degradation sites corresponding to 90 target genes from 30 miRNA families. Quantitative RT-PCR (qRT-PCR) analysis showed that the expression of hco-miR393a and hco-miR167n mirrored the release pattern of floral fragrance compounds, while the expression of HcTIR1 and HcARF8 inversely correlated with those of hco-miR393a and hco-miR167n. Co-transformation experiments in tobacco confirmed that HcTIR1 and HcARF8 are direct targets of hco-miR393a and hco-miR167n, respectively. Additionally, treatments with exogenous IAA and the auxin inhibitor PCIB modulated both the release of floral volatiles and the expression of hco-miR393a and hco-miR167n. STTM and VIGS experiments further indicated that hco-miR167n and hco-miR393a positively regulate floral fragrance metabolism, while HcARF8 and HcTIR1 act as negative regulators. Finally, dual-luciferase and yeast one-hybrid assays demonstrated that HcARF8 binds to the promoter of the terpene synthase gene HcTPS8, thereby regulating the biosynthesis of floral fragrance compounds.

This study represents the first comprehensive identification of miRNAs in H. coronarium and the characterization of their expression profiles in petal tissues at various developmental stages. These findings offer novel insights into the molecular mechanisms governing the synthesis of floral fragrance compounds and highlight the critical role of miRNAs in the regulation of metabolic processes within the Zingiberaceae family.