28 29 2 The circadian clock regulates various physiological responses. To achieve this, both 30 animals and plants have distinct circadian clocks in each tissue that are optimized for 31 that tissue's respective functions. However, if and how the tissue-specific circadian 32 clocks are involved in specification of cell types remains unclear. Here, by implementing 33 a single-cell transcriptome with a new analytics pipeline, we have reconstructed an 34 actual time-series of the cell differentiation process at single-cell resolution, and 35 discovered that the Arabidopsis circadian clock is involved in the process of cell 36 differentiation through transcription factor BRI1-EMS SUPPRESSOR 1 (BES1) 37 signaling. In this pathway, direct repression of LATE ELONGATED HYPOCOTYL 38 (LHY) expression by BES1 triggers reconstruction of the circadian clock in stem cells. 39 The reconstructed circadian clock regulates cell differentiation through fine-tuning of 40 key factors for epigenetic modification, cell-fate determination, and the cell cycle. Thus, 41 the establishment of circadian systems precedes cell differentiation and specifies cell 42 types. 43 44The circadian clock is involved in various physiological responses to regulate a large set of 45 genes in both animals and plants 1 . Circadian clocks in each tissue regulate different responses 46 consistent with tissue-specific sets of circadian-regulated genes 2,3 . The tissue-specific clocks 47 increased number of free-ending vascular bundles 6 , indicating abnormal vascular cell 55 differentiation in the clock mutant. 56To investigate the involvement of the plant circadian clock in cell differentiation, we 57 performed detailed observations of clock mutants, cca1; lhy; timing of CAB expression 1 58(cca1 lhy toc1) as well as a knockdown of BROTHER OF LUX ARRHYTHMO (also known as 59 NOX) by artificial microRNA in a lux arrhythmo/phytoclock1 mutant background (lux nox). 60We confirmed that the clock mutants affect development of vascular bundles, guard cells, and 61 root cells ( Supplementary Fig. 1a-c), suggesting that the plant circadian clock is generally 62 involved in the process of cell differentiation. We then utilized the vascular cell differentiation 63 induction system, referred to as VISUAL 7 , for further investigation of the molecular 64 mechanisms driving the clock-mediated cell differentiation. In this system, vascular cells 65 (including both xylem and phloem cells) are induced from mesophyll cells through the stem 66 cells and vascular stem cells (Fig. 1a). A loss-of-function mutation in BRI1-EMS 67 SUPPRESSOR 1 (BES1) showed severe defects in vascular cell differentiation, as shown 68 previously 8 . We found that clock mutants showed significant defects in vascular cell 69 differentiation (Fig. 1b). Perturbation of endogenous circadian rhythms by random light/dark 70 conditions also inhibited vascular cell differentiation ( Fig. 1c), suggesting the requirement of 71 a functional circadian clock for cell differentiation. 72
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.
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