miR172-Mediated Repression of APETALA2-like Genes Regulates Floral Meristem Activity During Double-Flower Formation in Camellia japonica

The miRNA172–APETALA2 (AP2) regulatory module is a conserved mechanism governing floral development in plants. Disruption of the miR172 target sites in AP2 genes has been shown to be key to the domestication of double flowers in ornamental species. Camellia japonica, a woody ornamental plant with diverse floral forms, serves as an important model for studying double-flower formation. In this study, we characterized two AP2-like transcription factors, CjAP2-1 and CjAP2-2, which possess evolutionarily conserved miR172-binding sites and exhibit broad expression across floral tissues. To investigate the role of the miR172–AP2 module in C. japonica, we identified four members of the miR172 family and demonstrated that miR172 is directly involved in the cleavage of CjAP2-1 and CjAP2-2 transcripts. Through bulked amplicon sequencing of cultivars with diverse floral forms, we uncovered natural variations at the miR172-binding sites of CjAP2-1 and CjAP2-2, which can potentially disrupt miR172-mediated mRNA cleavage. We showed that two dinucleotide mutations (CjAP2-1-mut5 and CjAP2-1-mut9) significantly reduced the miR172-mediated repression of CjAP2-1 transcripts. Functional analysis in Arabidopsis revealed that overexpression of the CjAP2-1-mut5 variant caused significant floral abnormalities, including ectopic formation of reproductive organs, loss of floral determinacy, and fusion of floral organs. Further analysis of downstream genes indicated that key regulators of floral homeotic and meristem activity were markedly altered in the transgenic plants. Our findings demonstrate that perturbations in the miR172–AP2 regulatory relationship underlie the formation of double flowers in C. japonica by altering floral meristem determinacy and organ identity.