Integrating scRNA-seq and snRNA-seq with spatial transcriptomics to unlock the xylem puzzle.

BACKGROUND: Xylem development is a dynamic, continuous process fundamental to secondary growth in woody plants and to biomass accumulation on earth. While single-cell RNA sequencing (scRNA-seq) enables reconstruction of early xylem differentiation trajectories, its reliance on protoplast isolation excludes late-stage cells with thickened secondary cell walls, leaving key phases such as secondary cell wall deposition and programmed cell death poorly characterized. RESULTS: We perform single-nucleus RNA sequencing (snRNA-seq) of the stem-developing xylem of Populus and integrate previous scRNA-seq datasets to reconstruct a comprehensive developmental landscape of xylem formation. Anatomical validation confirms that scRNA-seq profiles predominantly represent early-stage stem-developing xylem, while snRNA-seq enriches for deeper, secondary cell wall depositing layers. Integrated analysis reveals a spatially and transcriptionally defined secondary cell wall zone, supported by both lignin autofluorescence and its correlation with laser capture microdissection-derived transcriptomes. Differential expression and gene ontology analyses uncover enrichment for lignin biosynthesis and programmed cell death associated genes, suggesting that secondary cell wall formation and programmed cell death initiation are transcriptionally coordinated. Unsupervised clustering and machine learning by support vector machine classification further reveal greater transcriptomic heterogeneity among early-stage xylem cells compared to late-stage cells. CONCLUSIONS: Our findings demonstrate the high compatibility and complementarity of scRNA-seq and snRNA-seq platforms. This integrated approach not only overcomes key limitations of individual technologies but also provides a practical and scalable framework for resolving complex developmental trajectories in plants. It successfully reconstructs the full continuum of xylem development, from cambial progenitors through secondary cell wall formation and to programmed cell death.