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 analysis was used to visualize various secondary metabolites in leaf sections of Taxus mairei,confirming the tissue-specific accumulation of different active metabolites.Single-cell sequencing was used to produce expression profiles of 8846 cells,with a median of 2352 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 T.mairei leaves.Our data were used to create the first Taxus leaf metabolic single-cell atlas and to reveal spatial and temporal expression patterns of several secondary metabolic pathways.According to the cell-type annotation,most taxol biosynthesis genes are expressed mainly in leaf mesophyll cells;phenolic acid and flavonoid biosynthesis genes are highly expressed in leaf epidermal cells(including the stomatal complex and guard cells);and terpenoid and steroid biosynthesis genes are expressed specifically in leaf mesophyll cells.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 establishes the transcriptional landscape of major cell types in T.mairei leaves at a single-cell resolution and provides valuable resources for studying the basic principles of cell-type-specific regulation of secondary metabolism.