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Actual time-series of single-cell transcriptomics reveals circadian clocks as key regulators of cell differentiation in Arabidopsis


 The basis of toti/pluripotency is elaborate regulation of cell-cycle progression and cell-fate determination. Circadian clocks are involved in this process, but the underlying mechanisms have not been studied due to technical limitations. In particular, there is a lack of research on the universality of cell differentiation mechanisms in multicellular organisms using plants with high cell-fate plasticity. Here, exploiting in vivo single-cell RNA sequencing and a new actual time reconstitution method, PeakMacth, we analyzed actual time-series of cell reprogramming and differentiation processes at single-cell resolution, and found that the circadian clock modulates cell differentiation via BES1-mediated GSK3 signaling, which has a β-catenin-like function in Arabidopsis. In this pathway, the clock gene LUX in meristematic stem cells directly targets the CYCD and RETINOBLASTOMA-RELATED (RBR) genes, which are commonly involved in cell-cycle progression and cell-fate determination in plants and animals. In addition, the rhythmicity of the circadian clock was associated with cell state, and the establishment of the circadian rhythm preceded cell differentiation. Thus, our study not only reveals the involvement of the circadian clock in the differentiation of plant stem cells but also demonstrates functionally analogous features in the regulatory system of cell differentiation across species.