Genome-Wide Identification of Ginkgo biloba SPL Gene Family and Expression Analysis in Flavonoid Biosynthesis and Water Stress

SQUAMOSA promoter-binding protein-like (SPL) transcription factors specific to plants are vital for regulating growth, development, secondary metabolite biosynthesis, and responses to both biotic and abiotic stresses. Despite their importance, no systematic investigations or identifications of the SPL gene family in Ginkgo biloba have been conducted. In this study, we identified 13 SPL genes within the Ginkgo biloba reference genome, spanning seven chromosomes, and categorized these genes into six groups based on their phylogenetic relationships with Arabidopsis thaliana SPL gene families. Our analysis of gene structure, conserved domains, motifs, and miR156 target predictions indicates that GbSPLs are highly conserved across evolutionary timelines. Furthermore, synteny analysis highlighted that dispersed duplication events have expanded the SPL gene family in Ginkgo biloba. Examination of the cis-regulatory elements revealed that many GbSPL genes possess motifs associated with light, hormones, and stress, implying their involvement in flavonoid biosynthesis and adaptation to environmental conditions. RNA-Seq and qRT-PCR expression profiles of GbSPL genes across various tissues and low- and high-flavonoid leaves and during both short-term and long-term water stress illustrated their roles in flavonoid biosynthesis and responses to water stress. Subcellular localization experiments showed that GbSPL2 and GbSPL11 proteins are situated within the nucleus. Our research offers the first systematic characterization of the SPL gene family in Ginkgo biloba, establishing a valuable foundation for understanding their evolutionary background and functional roles in flavonoid biosynthesis and water stress response.