Integrated mass spectrometry imaging and single-cell transcriptome atlas strategies provide novel insights into taxoid biosynthesis and transport in Taxus mairei stems.

Taxol, which is an widely used important chemotherapeutic agent, which was originally isolated from Taxus stem barks. However, the precise distribution of taxoids and the transcriptional regulation of taxoid biosynthesis across Taxus stems are largely unknown. Here, we used MALDI-IMS analysis to visualize taxoid distribution across Taxus mairei stems and single-cell RNA sequencing to generate expression profiles. The first single cell T. mairei stem atlas was created and provided a spatial distribution pattern of Taxus stem cells. Cells were reordered using a main developmental pseudotime trajectory which provided temporal distribution patterns in Taxus stem cells. Most known taxol biosynthesis-related genes were primarily expressed in epidermal, endodermal, and xylem parenchyma cells, which caused uneven taxoid distribution across T. mairei stems. We developed a single-cell perspective to screen novel transcription factors (TFs) during regulated taxol biosynthesis. Several novel TFs, such as endodermal cell-specific MYB47 and xylem parenchyma cell-specific NAC2 and bHLH68 were implicated as potential regulators of taxol biosynthesis. Furthermore, an ATP-binding cassette family transporter gene, ABCG2, was proposed as a potential taxoid transporter candidate. We generated the first Taxus stem metabolic single-cell atlas and identified molecular mechanisms underpinning the cell-specific transcriptional regulation of the taxol biosynthesis pathway.