A 24‐nt miR9560 modulates the transporter gene <i>BrpHMA2</i> expression in <i>Brassica parachinensis</i>

MicroRNAs (miRNAs) control gene expression in plant through transcript cleavage and translation inhibition. Recently, 24-nt miRNAs have been shown to direct DNA methylation at target sites, regulating the neighboring gene expression. Our study focused on miR9560, a 24-nt miRNA induced by cadmium (Cd) stress in Brassica rapa ssp. parachinensis (B. parachinensis). Phylogenetic analysis revealed miR9560 predominantly emerged in the Rosanae superorder and was conserved in Brassicaceae, with potential target sites adjacent to transporter family genes HMAs. RNA gel blotting showed that mature miR9560 was only detected in various Brassica crops roots after Cd stress. In B. parachinensis, miR9560's putative target site is upstream of BrpHMA2, an afflux-type Cd transporter. In a transient expression system of B. parachinensis protoplasts, the expression of miR9560 increased the DNA methylation upstream of BrpHMA2, reducing the transcription of BrpHMA2. This regulation was also observed in Arabidopsis wild-type protoplasts but not in the mutants dcl234 and ago4 with impairments in the RNA-dependent DNA methylation (RdDM) pathway. We deduced that miR9560 modulates BrpHMA2 expression via the RdDM pathway, potentially regulating Cd uptake and movement in B. parachinensis. Furthermore, this regulatory mechanism may extend to other Brassica plants. This study enhances our comprehension of 24-nt miRNAs role in regulating Cd accumulation within Brassica plants.