Single-cell RNA-sequencing profiles reveal the developmental landscape of hawthorn leaves.

Hawthorn (Crataegus spp.) is an economically and medicinally important genus in Rosaceae. Its leaves accumulate valuable secondary metabolites, including flavonoids, and have long been used in traditional Chinese medicine. Despite available chromosome-level genome assemblies and bulk transcriptome data, the cellular composition of hawthorn leaves, their developmental trajectories, and the cell-type-specific distribution of biosynthetic gene expression remain uncharacterized. Here we report the first single-cell transcriptomic atlas of hawthorn leaves, generated by integrating protoplast-based scRNA-seq and nucleus-based snRNA-seq from two species (C. pinnatifida var. major N. E. Br. and C. scabrifolia (Franch.) Rehder). From four libraries, we profiled 32,292 high-quality cells, resolved sixteen transcriptional clusters, and annotated nine canonical cell types: mesophyll, pavement, guard, xylem, metaxylem, phloem parenchyma, companion, sieve element, and meristematic cells. Cell-type-resolved interspecific comparison identified 642 differentially expressed genes between species, with divergent enrichment of photosynthesis and volatile biosynthesis processes in C. pinnatifida var. major and hypoxia-response processes in C. scabrifolia. Pseudotime analysis reconstructed branched developmental trajectories converging on mesophyll dominance at late stages, consistent with classical dicotyledonous leaf organogenesis. Cell-type-resolved profiling demonstrated spatial partitioning of flavonoid biosynthetic genes across multiple cell populations, with upstream enzymes concentrated in epidermal and phloem-associated cells and downstream enzymes in mesophyll and xylem cells. This atlas provides a cellular framework for mechanistic study of tissue‑specific secondary metabolite and for future functional and breeding applications in hawthorn.