MicroRNA repertoire and responses of Hypnum cupressiforme to a heavy metal-enriched urban environment

MicroRNAs (miRNAs) are important post-transcriptional regulators of plant development and environmental responses, but their diversity and functions remain poorly characterised in bryophytes. Here, we characterised the miRNA repertoire of the moss Hypnum cupressiforme and examined selected miRNAs following prolonged exposure to a heavy metal-enriched urban environment. Small RNA sequencing of H. cupressiforme from the Strandzha Mountain, Bulgaria, identified 18 mature miRNA sequences using a conservative homology-based approach, including conserved and bryophyte-associated families. Four abundant miRNAs - hcu-miR319a, hcu-miR533, hcu-miR536a and hcu-miR538a - yielded candidate targets in the Physcomitrium patens transcriptome. Their expression was assessed by RT-qPCR in moss collected from the Strandzha Mountain and after approximately five months at a moss-wall installation in Kuklen, Plovdiv Region, Bulgaria, where the accumulation of potentially toxic elements has previ­ously been documented. The expression levels of hcu-miR319a, hcu-miR533 and hcu-miR538a were significantly reduced, whereas hcu-miR536a showed a non-significant downward trend. The predicted targets of the three responsive miRNAs suggest potential links with developmental regulation, starch metabolism and protein turnover. Together with previously documented physiological responses at the same moss-wall installation, these findings indicate that miRNA regulation may contribute to the response of H. cupressiforme to prolonged urban environmental exposure. The identified miRNAs provide candidates for further investigation of small-RNA-mediated regulation and their potential use as molecular indicators in moss-based environmental biomonitoring.