Abstract
Anthocyanins, biosynthesized through the flavonoid pathway, critically determine floral coloration and ornamental value in plants. While floral development has been extensively studied in Gerbera hybrida, the microRNA-mediated regulation of anthocyanin biosynthesis remains unclear. In this study, we identified and characterized the precursor of gerbera microRNA156 (GhmiR156), which exhibits a typical stem-loop secondary structure. The mature GhmiR156 sequence shows 93.65% similarity with miR156 from other plants. Through target prediction analysis, we identified five potential target genes of GhmiR156, all encoding SQUAMOSA Promoter-Binding Protein-Like (SPL) transcription factors. Among these, the gene c35442.graph_c0, which shares the highest similarity with AtSPL2 in Arabidopsis, was designated as GhSPL2. Expression analysis revealed an inverse correlation between GhmiR156 and GhSPL2 across different tissues and developmental stages of ray florets. This regulatory relationship was further validated by RLM-5'RACE, which showed that GhmiR156 directly targets and cleaves GhSPL2 mRNA, subsequently supported by dual-luciferase reporter assays and Western blotting analysis. Subcellular localization analysis indicated that GhSPL2 is a nuclear-localized protein, consistent with AtSPL2. Functional analyses revealed that overexpression of GhSPL2 suppressed anthocyanin accumulation by downregulating key biosynthetic genes GhPAL, GhF3H and GhUFGT. Conversely, overexpression of GhmiR156 represses GhSPL2 expression, thereby alleviating its inhibitory effect on anthocyanin accumulation in ray florets, and exhibits an increase in anthocyanin content. Collectively, our findings demonstrate that GhmiR156 fine-tunes the anthocyanin biosynthetic pathway through its target gene GhSPL2. This study provides new insights into the complex regulatory network governing anthocyanin biosynthesis in ornamental plants.