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

Hawthorn ( Crataegus spp.) is a valuable genus of medicinal and edible plants in the Rosaceae family that are rich in bioactive compounds. Despite the availability of chromosome‑level genomes and bulk transcriptomes for Crataegus species, the leaf cellular composition, developmental trajectories, and cell type-specific expression of biosynthetic pathways remain unexplored at single‑cell resolution. Here, we applied complementary protoplast‑based single‑cell RNA sequencing (scRNA‑seq) and nucleus‑based snRNA‑seq to construct the first single‑cell atlas of hawthorn leaves. We optimized isolation protocols for two Crataegus species ( C. pinnatifida var. major N. E. Br. and C. scabrifolia (Franch.) Rehder), generated four single‑cell transcriptomic libraries across platforms, and profiled 32,292 high‑quality cells to explore the developmental landscape of hawthorn leaves. The cells were clustered into sixteen groups that we annotated into nine major leaf cell types (mesophyll, pavement, guard, xylem, metaxylem, phloem parenchyma, companion, sieve element, and meristematic cells), revealing extensive cellular heterogeneity and candidate marker genes. Pseudotime reconstruction revealed branched developmental trajectories, and cell‑type‑resolved profiling revealed the cell‑specific expression of flavonoid biosynthetic genes. This single‑cell atlas lays a foundation for mechanistic investigations of tissue‑specific metabolite biosynthesis and offers a cellular framework to guide future functional studies and trait improvement breeding in hawthorn.