Abstract
The miR156 family, crucial for phase transition and stress responses in plants, remains functionally uncharacterized in the ecologically and commercially important gymnosperm Larix kaempferi. This study systematically investigated L. kaempferi miR156 through phylogenetic analysis, structural prediction, expression profiling during somatic embryogenesis, and heterologous functional validation in Arabidopsis. Four MIR156 family members (LkMIR156s) were identified in Larix kaempferi, each with a characteristic stem-loop structure and highly conserved mature sequences. Computational predictions indicated that these LkMIR156s target four LkSPL family genes (LkSPL1, LkSPL2, LkSPL3, and LkSPL9). qRT-PCR analysis showed that mature LkmiR156s expression remained relatively low during early embryonic development but was significantly upregulated at the cotyledonary stage (21-42 days). Precursor transcript levels peaked earlier (around 28 days) than those of the mature LkmiR156, which remained highly expressed throughout cotyledonary embryo development. This sustained high expression coincided with cotyledon morphogenesis and embryonic dormancy. Functional validation via heterologous overexpression of LkMIR156b1 in Arabidopsis resulted in increased rosette leaf numbers (42.86% ± 6.19%) and individual leaf area (54.90% ± 6.86%), phenotypically consistent with the established role of miR156 in growth regulation. This study reveals the temporal expression dynamics of LkmiR156s during L. kaempferi somatic embryogenesis and its coordinated expression patterns with cotyledon development and embryonic dormancy. The functional conservation of the miR156-SPL module was confirmed in a model plant, providing key molecular insights into the developmental regulatory network of conifers. These findings offer potential strategies for optimizing somatic embryogenesis techniques in conifer species.