Mapping zygote-to-adult developmental cell phylogeny in Arabidopsis thaliana reveals a 3-cell rule of branching

Similar to the evolutionary history of all species on Earth, the developmental lineage history of all cells in a multicellular organism is stored in a phylogenetic tree. Mapping the zygote-to-adult developmental cell phylogeny of a complex organism is of tremendous value but technically challenging. We here adapted SMALT, a powerful cell lineage tracing system developed in our laboratory, to Arabidopsis thaliana to record the zygote-to-adult developmental lineages of two plant individuals. SMALT performed efficiently in A. thaliana, with an average of ~50 barcoding mutations accumulated on the 1-kb barcode sequence of each cell in three-month-old plants. Using the barcoding mutations we reconstructed, with high statistical confidence and at single-cell resolution, the phylogenetic tree for thousands of cells sampled from various shoot branches of each plant. The cell phylogenies show cells of every shoot branch are derived from exactly three founder cells, each belonging to one of three early-determined lineages. The 3-cell pattern holds for primary, secondary and tertiary branches, and even for single leaves or flowers/siliques. Integrating SMALT with single-cell RNA sequencing revealed the three founder cells responsible for establishing three germ layers of each branch/organ, which in turn updates our understandings of plant germ layers to single-cell resolution. We further showed that the 3-cell rule represents the optimal strategy for an indeterminate plant to manage its stem cell pool, with a general analytical framework proposed to unify the distinct strategies used in plant and animal organogenesis.