miR828 positively regulates proanthocyanidin accumulation in sainfoin (Onobrychis viciifolia)

miR828, a highly conserved noncoding RNA, participates in the regulation of metabolic pathways associated with proanthocyanidins (PAs) in plants. This study aimed to identify miR828 in sainfoin (Onobrychis viciifolia), clone the OvmiR828c gene, predict its potential target genes, and investigate its function through heterologous overexpression in Arabidopsis thaliana. The findings indicated that the stem-loop sequence of OvmiR828c is 91 base pairs in length and can form a stable stem-loop secondary structure. The mature miR828 sequence is located at the 5' end of the precursor, while the upstream regulatory region contains multiple cis-acting elements, including MYB-binding sites and elements related to circadian rhythm. The heterologous overexpression of OvmiR828c in A. thaliana transgenic lines results in a significant increase in seed coat pigmentation and PA accumulation. Through functional annotation and expression pattern analysis of eight predicted target genes, MYB transcription factors, methyltransferase family genes, cysteine synthases, and kinesin family proteins were preliminarily identified. These findings contribute to the understanding of the regulatory network formed by miR828 and its target genes in PA biosynthesis, laying a foundation for subsequent in-depth exploration of this metabolic pathway.