Mass spectrometry imaging and single-cell transcriptional profiling reveal the tissue-specific regulation of bioactive ingredient biosynthesis in Taxus leaves

Taxus leaves provide the raw industrial materials for taxol, a natural antineoplastic drug widely used in the treatment of various cancers. However, the precise distribution, biosynthesis, and transcriptional regulation of taxoids and other active components in Taxus leaves remain unknown. Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-IMS) analysis was used to visualize various secondary metabolites in T. mairei leaf sections, confirming the tissue-specific accumulation of different active metabolites. Single-cell sequencing was applied to produce expression profiles of 8846 cells, with a median of 2,352 genes per cell. Based on a series of cluster-specific markers, cells were grouped into 15 clusters, suggesting a high degree of cell heterogeneity in the T. mairei leaves. Our data created the first Taxus leaf metabolic single-cell atlas and reveal spatial and temporal expression patterns of several secondary metabolic pathways. According to the cell type annotation, most of the taxol biosynthesis genes are expressed mainly in leaf mesophyll cells, phenolic acid and flavonoid biosynthesis genes are expressed highly in leaf epidermis cells (including stomatal complex and guard cells), and terpenoid and steroid biosynthesis genes are expressed specifically in leaf mesophyll cells. Furthermore, a number of novel and cell specific transcription factors involved in secondary metabolite biosynthesis were identified, including MYB17, WRKY12, WRKY31, ERF13, GT_2, and bHLH46. Our research established the transcriptional landscape of the major cell types in T. mairei leaves at single-cell resolution and provided valuable resources for studying the basic principles of cell type specific regulation of secondary metabolism.