Deep small-RNA sequencing uncovers a diverse spectrum of microRNAs putatively regulating biosynthesis and variation of specialized metabolites in Thymus daenensis

Abstract

Thymus daenensis Celak., is an ideal model for delving into the intricate regulatory mechanisms involved in polyphenol biosynthesis and thymol-rich essential oil (EO) production. While, there has been an effort to explore metabolic pathways in thyme species at mRNA level, but mechanisms responsible for post-transcriptional control, in particular microRNAs (miRNAs), are understood only poorly. Here, the sequencing of approximately 75 million small-RNA reads resulted in identification of 618 conserved miRNAs, 50 multi-member families, and 28 novel miRNAs. A comparative analysis of the small-RNAome profile was conducted between two distinct genotypes: Zagheh-11, characterized by low EO content but rich in carvacrol and triterpenic acids, and Malayer-21, which exhibited higher levels of thymol, EO, and rosmarinate. The divergence in their terpenome appears to stem from a genotype-specific control over MEP and MVA pathways, primarily influenced by 10 differentially expressed miRNAs. The expression patterns of key miRNAs and their putative targets ([miR396c, DXS], [miR477a, ispD], [miR135a, ispE], [miR9b-5p, MVD and MVK], [miR181a-3p, ACAT]) were validated using qRT-PCR. Furthermore, we uncovered an extensive network consisting of 139 miRNAs that target 47 enzyme genes in the biosynthetic pathways of secondary metabolites. The majority of the 13 928 identified target unigenes were enriched in pathways of secondary metabolites, carbon and amino acid metabolism. We found miR166 and miR159 as the most abundantly expressed miRNAs in T. daenensis. We examined the conservation and phylogenetic status of the miR159, miR164, and miR396 families, highlighting their profound impacts on production of specialized metabolites. The putative regulatory behavior of tda-miRNAs herein presents a promising foundation for strategically altering metabolic pathways, enabling the controlled production of targeted drug compounds.