Single-nucleus RNA-seq resolves spatiotemporal developmental trajectories in the tomato shoot apex

Single cell transcriptomics is revolutionizing our understanding of development and response to environmental cues1-3. Recent advances in single cell RNA sequencing (scRNA-seq) technology have enabled profiling gene expression pattern of heterogenous tissues and organs at single cellular level and have been widely applied in human and animal research4,5. Nevertheless, the existence of cell walls significantly encumbered its application in plant research. Protoplasts have been applied for scRNA-seq analysis, but mostly restricted to tissues amenable for wall digestion, such as root tips6-10. However, many cell types are resistant to protoplasting, and protoplasting may yield ectopic gene expression and bias proportions of cell types. Here we demonstrate a method with minimal artifacts for high-throughput single-nucleus RNA sequencing (snRNA-Seq) that we use to profile tomato shoot apex cells. The obtained high-resolution expression atlas identifies numerous distinct cell types covering major shoot tissues and developmental stages, delineates developmental trajectories of mesophyll cells, vasculature cells, epidermal cells, and trichome cells. In addition, we identify key developmental regulators and reveal their hierarchy. Collectively, this study demonstrates the power of snRNA-seq to plant research and provides an unprecedented spatiotemporal gene expression atlas of heterogeneous shoot cells.