Root tips, which represent the initial stage of taproot development, serve as an ideal model for investigating plant growth and secondary metabolism. However, studies of root tips in Panax species have been limited, restricting our understanding of cell fate transitions during early root development and the cellular heterogeneity associated with ginsenosides biosynthesis. To address this gap, we conducted single-cell RNA sequencing (scRNA-seq) and spatial metabolomics analyses on the root tips of three Panax species: Panax notoginseng, Panax ginseng, and Panax quinquefolium. Our research reconstructed the developmental trajectory of the early endodermis and revealed epidermis-specific expression patterns of key enzyme genes involved in ginsenosides biosynthesis. We identified several novel transcription factors (TFs): IAA29 (which positively regulates endodermis suberization) and MYB2/MYB78 (positive regulators of ginsenosides biosynthesis), validated by dual-LUC reporter and electrophoretic mobility shift assay (EMSA). Conserved and divergent ligand-receptor interaction patterns across the three Panax species were discovered, with the FAD gene family exhibiting tissue- and species-specific expression. Cell-specific genes expression was confirmed by RNA in situ hybridization. Mass spectrometry imaging (MSI) mapped ginsenosides spatial distribution, while LC–MS/MS verified species-specific biosynthesis. This study presents a single-cell transcriptional landscape of early differentiation and cell type-specific ginsenosides accumulation in the Panax genus.

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