Comprehensive analysis of AHL genes in Malus domestica reveals the critical role of MdAHL6 in flowering induction.

AT-hook motif nuclear localized (AHL) genes are crucial in various biological processes, yet the AHL gene family in apples has remained largely unexplored. In this study, we isolated 36 MdAHL genes from the apple genome and grouped them into two distinct clades. We characterized the gene structure, conserved motifs, protein biochemical properties, and promoter regions of the MdAHL genes. Transcriptional analysis revealed that MdAHL genes are preferentially and predominantly expressed in flowers and leaves. Notably, during the floral induction phase, the MdAHL6 gene exhibited remarkably high transcriptional activity. Overexpression of MdAHL6 resulted in shortened hypocotyls and delayed flowering by regulating hypocotyl- and floral-related genes. Y1H, EMSA, GUS activity, and molecular docking assays revealed that MdAHL6 directly binds to AT-rich regions, inhibiting the expression of FLOWERING LOCUS T (MdFT). Furthermore, Y2H, pull-down, and BiFC assays demonstrated a physical interaction between MdAHL6 and the class II knotted-like transcription factor MdKNOX19, which significantly enhances the inhibitory effect of MdAHL6 on MdFT expression. This comprehensive initial analysis unveils the critical role of the MdKNOX19-MdAHL6-MdFT module in flowering induction and lays a theoretical foundation for future functional exploration.