Lch-miR156b-directed repression of LcSPL11 regulates flowering in Liriodendron chinense

MicroRNAs (miRNAs) are small regulatory RNAs that control gene expression through sequence-specific interactions with target transcripts and play pivotal roles in plant developmental phase transitions. Among them, the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) module constitutes a conserved regulatory pathway controlling flowering time in diverse species, yet its molecular function in woody perennials with extended juvenile phases remains poorly understood. Here, seven miR156 family members were identified in Liriodendron chinense by small RNA sequencing, and the Lch-miR156b-LcSPL11 regulatory module was functionally characterized. 5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE) and dual-luciferase assays demonstrated that Lch-miR156b directly cleaves and represses LcSPL11 transcripts. Expression profiling further associated the Lch-miR156b-LcSPL11 module with flowering-time variation in L. chinense. In heterologous assays, ectopic expression of Lch-miR156b in Arabidopsis thaliana delayed flowering, whereas overexpression of LcSPL11, including a miRNA-resistant variant (rLcSPL11), accelerated floral transition. In addition, LcSOC1 and LcFUL showed expression patterns similar to that of LcSPL11, their promoters contained conserved GTAC cis-elements, and yeast one-hybrid assays confirmed direct binding of LcSPL11 to both promoters. Together, these findings reveal a conserved miRNA-mediated regulatory cascade in which Lch-miR156b modulates flowering through LcSPL11 and its downstream targets LcSOC1 and LcFUL, providing mechanistic insight into flowering control in a woody basal angiosperm.