Cell-specific dedifferentiation in Arabidopsis thaliana

Single somatic plant cells are capable of dedifferentiation and reversion to totipotency. However, understanding of dedifferentiation remains incomplete, limiting efforts to reprogram plant cell identities with engineered properties. Here, we describe the transcriptional dynamics of 125,091 Arabidopsis thaliana whole seedling protoplasts along a time course of dedifferentiation and varied chemical perturbations using single-cell RNA sequencing. We observed widespread cell-type-specific transcriptional reprogramming concomitant with cell wall digestion, including a guard cell-specific somatic embryogenic program initiated by ectopic LEC2 expression. Contrasting cell states between chemical perturbations across real time revealed that phytohormones antagonize cell aging, promote retention of transcriptional diversity, and selectively suppress wound responses to prioritize acquisition of totipotency. By dissecting time-resolved transcriptional dynamics, we discovered that dedifferentiation is non-uniform, accompanied by the emergence of heterogeneous stem cell-like identities, and associated with prevalent chromatin remodeling at the onset of dedifferentiation. Modeling dedifferentiation trajectories of diverse somatic cell types uncovered extensive bias in stem potential, including the identification of epithem-derived stem-like cells, variable dedifferentiation rates, and diversity in early reprogramming outcomes as a major source of dedifferentiation heterogeneity, despite discovery of a shared regulatory program underlying early dedifferentiation. Taken together, these data showcase how initial somatic cell identity, phytohormone signaling, chromatin remodeling, and reprogramming heterogeneity combine to coordinate the early transcriptional events defining cellular dedifferentiation.