Identification of novel regulators required for early development of vein pattern in the cotyledons by single-cell RNA-sequencing

The leaf veins of higher plants contain a highly specialized vascular system comprised of xylem and phloem cells that transport water, organic compounds and mineral nutrients. The development of the vascular system is controlled by phytohormones that interact with complex transcriptional regulatory networks. Before the emergence of true leaves, the cotyledons of young seedlings perform photosynthesis that provides energy for the sustainable growth and survival of seedlings. However, the mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood, in part due to the complex cellular composition of this tissue. To better understand the development of leaf veins, we analyzed 14 117 single cells from 3-day-old cotyledons using single-cell RNA sequencing. Based on gene expression patterns, we identified 10 clusters of cells and traced their developmental trajectories. We discovered multiple new marker genes and developmental features of leaf veins. The transcription factor networks of some cell types indicated potential roles of CYCLING DOF FACTOR 5 (CDF5) and REPRESSOR OF GA (RGA) in the early development and function of the leaf veins in cotyledons. These new findings lay a foundation for understanding the early developmental dynamics of cotyledon veins. The mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood. In this study, we comprehensively characterized the early differentiation and development of leaf veins in 3-day-old cotyledons based on single-cell transcriptome analysis. We identified the cell types and novel marker genes of leaf veins and characterized the novel regulators of leaf vein.