Mapping the zygote-to-adult developmental cell phylogeny in Arabidopsis thaliana reveals a three-cell rule of...

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 developed e-SMALT, a powerful cell lineage tracing system integrated with single-cell RNA sequencing, in Arabidopsis thaliana to record the zygote-to-adult developmental lineages of two plant individuals. The system performed efficiently in A. thaliana, with an average of ~50 barcoding mutations accumulated on the 1-kb barcode sequence of each cell in 3-month-old plants. Using the barcoding mutations, we reconstructed the phylogenetic tree for thousands of cells sampled from various shoot branches of each plant, with high statistical confidence and at single-cell resolution. The cell phylogenies show that cells of every shoot branch are derived from exactly three founder cells, each belonging to one of three early-determined lineages. The three-cell pattern holds for primary, secondary and tertiary branches, and even for single flowers/siliques. Incorporating single-cell RNA sequencing data revealed the three founder cells responsible for establishing the three germ layers of each branch/organ, which in turn updates our understanding of plant germ layers to single-cell resolution. We further showed that the three-cell rule reflects an adaptive strategy for an indeterminate plant to manage its stem cell pool, suggesting an analytical framework to unify the distinct strategies between plants and animals in organogenesis.