The aro-miR166b/c-ArHDZ22 module regulates reproductive development in Anoectochilus roxburghii

Anoectochilus roxburghii (Wall.) Lindl., a rare and precious traditional Chinese medicinal plant, faces the challenge of seed developmental failure due to incomplete endosperm development. The miR166-HD-Zip Ⅲ module has been reported to be involved in the regulation of reproductive development. However, the function of miR166-HD-Zip Ⅲ module in A. roxburghii remains unclear. In this study, 38 ArHD-Zip TFs were identified from the full-length transcriptome of A. roxburghii. Among them, ArHDZ22 was found to promote flowering and seed development through phylogenetic analysis, sequence analysis and overexpression functional verification experiments. Subsequently, aro-miR166b/c that may regulate ArHDZ22 were screened by Small RNA sequencing, K-means analysis, differential expression analysis and qRT-PCR. The Dual-luciferase assay and tobacco transient transformation demonstrated that aro-miR166b/c could degrade ArHDZ22 directly. In addition, the overexpression experiments in Arabidopsis thaliana showed that aro-miR166b/c was able to regulate reproductive development by targeting ArHDZ22. Overall, this study revealed the regulatory role of the aro-miR166b/c-ArHDZ22 module in reproductive development, further elucidating the molecular mechanism of the reproductive development of A. roxburghii, and also provides a solid theoretical basis for its efficient breeding and germplasm innovation of A. roxburghii.