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-like 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. By dissecting time-resolved transcriptional dynamics, we discovered that dedifferentiation is accompanied by the activation of stem-cell and meristematic marker genes and associated with prevalent chromatin remodeling. Despite discovery of a shared regulatory program underlying early dedifferentiation, temporal modeling of diverse somatic cell types uncovered extensive bias in dedifferentiation potential, including epithem-derived dedifferentiated cells, variable dedifferentiation rates, and diversity in early reprogramming outcomes as a major source of in vitro heterogeneity. 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.