Systematic Identification and Functional Study of Vitamin B6-Related PDX2 Genes in the Ginkgo biloba Genome

Vitamin B6 is an essential coenzyme involved in various metabolic processes critical for plant growth and development. However, its biosynthesis and regulatory mechanisms remain poorly understood in the ancient gymnosperm Ginkgo biloba. In this study, we identified two members of the PDX2 gene family (Gb_34755 and Gb_34990) through genome-wide analysis and characterized their molecular and functional properties. Bioinformatic analysis revealed distinct physicochemical traits and subcellular localizations, with Gb_34755 predicted in the cytoplasm and Gb_34990 in both chloroplasts and cytoplasm. Both proteins contain the glutaminase-related PLN02832 domain, indicating involvement in VB6 biosynthesis. Chromosomal mapping placed the genes in transcriptionally active regions on chromosomes 6 and 9. Phylogenetic analysis showed close evolutionary relationships between Ginkgo PDX2 genes and those in ferns and gymnosperms, distinct from angiosperms. Promoter analysis revealed differential enrichment of cis-elements: Gb_34990 harbored low-temperature and salicylic acid-responsive elements, while Gb_34755 showed motifs related to development. Gene expression profiling indicated significant upregulation (p < 0.05) of both genes during the late developmental stages of Ginkgo kernels, coinciding with peak VB6 content. Functional validation via transient overexpression in Nicotiana benthamiana confirmed a positive regulatory role, with VB6 levels increasing from 3.38 μg/g to 12.17 μg/g (p < 0.05). This study provides the first comprehensive functional analysis of the PDX2 gene family in Ginkgo and confirms their critical role in VB6 biosynthesis. These findings enhance our understanding of vitamin metabolism in gymnosperms and present promising targets for metabolic engineering in plants.