Single-cell transcriptomics of tobacco roots and leaves

Nicotine, a key alkaloid in tobacco, is synthesized in the roots, transported to the leaves via the xylem, and stored in vacuoles. While previous studies on nicotine biosynthesis and transport have largely relied on genomics and bulk RNA analyses, single-cell transcriptomics offers unprecedented resolution to explore these processes within heterogeneous cell populations. Here, we present high-resolution single-cell transcriptome atlases for the roots of Nicotiana sylvestris and N. tabacum, as well as for the leaves of N. tabacum treated with or without methyl jasmonate (MeJA). In the root single-cell landscapes, we confirmed that nicotine biosynthesis predominantly occurs in cortical cells. Trajectory analysis of xylem cell differentiation revealed a conserved developmental trajectory between N. sylvestris and N. tabacum, despite species-specific gene contributions. In leaves, integration of high-definition weighted gene co-expression network analysis (hdWGCNA) with differentially expressed gene (DEG) data from untreated and MeJA-treated plants identified key nicotine transport gene VAMP714. Furthermore, by comparing genes upregulated in the tobacco cortex relative to tomato and those commonly expressed in the cortex of both N. sylvestris and N. tabacum, we identified potential hub genes involved in nicotine biosynthesis, such as QPT2, GSTU19, and ATX1. Together, our findings provide novel insights into the cellular and molecular mechanisms underlying nicotine biosynthesis and transport, establishing a foundation for future single-cell studies in tobacco and other alkaloid-producing plants.