Genome-Wide Identification of GRF and GIF Genes in Larix kaempferi and Their Potential Association with Somatic Embryogenesis, Hormone Responses, and miR396-Mediated Regulation

This study systematically identified and characterized the Growth-regulating factors ( GRFs ) and GRF-interacting factors ( GIFs ) gene families in Larix kaempferi , revealing five LkaGRF and three LkaGIF genes. All LkaGRF proteins contain conserved QLQ and WRC domains, while LkaGIF proteins possess the SSXT domain, confirming their structural conservation across land plants. Phylogenetic analysis placed most LkaGRFs in a gymnosperm-specific clade, indicating ancient lineage diversification, whereas LkaGIFs showed both conserved and gymnosperm-specific branches. Predicted miR396 target sites were detected in all LkaGRF transcripts, and 5′ RLM-RACE further supported miR396-directed cleavage of LkaGRF3 and LkaGRF5. Promoter analysis revealed abundant hormone- and stress-responsive cis-elements. Expression profiling demonstrated that both gene families are significantly upregulated at day 28 during somatic embryogenesis, corresponding to the critical stage of cotyledon differentiation. Hormonal treatments showed that most LkaGRFs are rapidly downregulated by IAA, ABA, GA, and SA, LkaGRF1 showed a generally sustained repressed expression pattern under all five hormone treatments, whereas LkaGRF3 and LkaGRF5 were continuously and significantly downregulated by IAA, ABA, GA, and SA. These results support the potential involvement of the miR396-GRF/GIF module in somatic embryogenesis and hormone-responsive developmental processes in L. kaempferi , and provide candidate regulators for future functional studies in conifer embryogenic systems.