A guiding role of the Arabidopsis circadian clock in cell differentiation revealed by time-series single-cell RNA sequencing.

Circadian rhythms and progression of cell differentiation are closely coupled in multicellular organisms. However, whether establishment of circadian rhythms regulates cell differentiation or vice versa has not been elucidated due to technical limitations. Here, we exploit high cell fate plasticity of plant cells to perform single-cell RNA sequencing during the entire process of cell differentiation. By analyzing reconstructed actual time series of the differentiation processes at single-cell resolution using a method we developed (PeakMatch), we find that the expression profile of clock genes is changed prior to cell differentiation, including induction of the clock gene LUX ARRYTHMO (LUX). ChIP sequencing analysis reveals that LUX induction in early differentiating cells directly targets genes involved in cell-cycle progression to regulate cell differentiation. Taken together, these results not only reveal a guiding role of the plant circadian clock in cell differentiation but also provide an approach for time-series analysis at single-cell resolution.

Time-Series Single-Cell RNA-Seq Data Reveal Auxin Fluctuation during Endocycle.

Appropriate cell cycle regulation is crucial for achieving coordinated development and cell differentiation in multicellular organisms. In Arabidopsis, endoreduplication is often observed in terminally differentiated cells and several reports have shown its molecular mechanisms. Auxin is a key factor for the mode transition from mitotic cell cycle to endocycle; however, it remains unclear if and how auxin maintains the endocycle mode. In this study, we reanalyzed root single-cell transcriptome data and reconstructed cell cycle trajectories of the mitotic cell cycle and endocycle. With progression of the endocycle, genes involved in auxin synthesis, influx and efflux were induced at the specific cell phase, suggesting that auxin concentration fluctuated dynamically. Such induction of auxin-related genes was not observed in the mitotic cell cycle, suggesting that the auxin fluctuation plays some roles in maintaining the endocycle stage. In addition, the expression level of CYCB1;1, which is required for cell division in the M phase, coincided with the expected amount of auxin and cell division. Our analysis also provided a set of genes expressed in specific phases of the cell cycle. Taking these findings together, reconstruction of single-cell transcriptome data enables us to identify properties of the cell cycle more accurately.

Importance of epidermal clocks for regulation of hypocotyl elongation through PIF4 and IAA29.

Circadian clocks adjust an organism's environmentally relevant physiological responses.. In plants, a decentralized circadian clock system has recently been proposed. Epidermal clock function is crucial for cell elongation; thus, epidermis-specific overexpression of CCA1 caused smaller cotyledons and longer hypocotyls under 27°C, concomitant with elevated night time levels of PIF4 mRNA. However, which tissue's clock regulates PIF4 expression is still an open question. Here we tested spatial expression patterns of PIF4 and its downstream target IAA29 with or without epidermal clock perturbation. Using an epidermal-specific expression system, we revealed that epidermal clock perturbation increases PIF4 expression in both epidermis and mesophyll. However, IAA29 expression is mainly regulated in the epidermis, implying the potential importance of epidermis for regulation of cell elongation through PIF4 and IAA29. We conclude that the circadian clock in epidermis regulates cell elongation mainly in epidermis, and there is also another inter-tissue signaling pathway from epidermis to mesophyll.

Decentralized circadian clocks process thermal and photoperiodic cues in specific tissues.

The circadian clock increases organisms' fitness by regulating physiological responses(1). In mammals, the circadian clock in the suprachiasmatic nucleus (SCN) governs daily behavioural rhythms(2). Similarly, in Arabidopsis, tissue-specific circadian clock functions have emerged, and the importance of the vasculature clock for photoperiodic flowering has been demonstrated(3-5). However, it remains unclear if the vasculature clock regulates the majority of physiological responses, like the SCN in mammals, and if other environmental signals are also processed by the vasculature clock. Here, we studied the involvement of tissue-specific circadian clock regulation of flowering and cell elongation under different photoperiods and temperatures. We found that the circadian clock in vascular phloem companion cells is essential for photoperiodic flowering regulation; by contrast, the epidermis has a crucial impact on ambient temperature-dependent cell elongation. Thus, there are clear assignments of roles among circadian clocks in each tissue. Our results reveal that, unlike the more centralized circadian clock in mammals, the plant circadian clock is decentralized, where each tissue specifically processes individual environmental cues and regulates individual physiological responses. Our new conceptual framework will be a starting point for deciphering circadian clock functions in each tissue, which will lead to a better understanding of how circadian clock processing of environmental signals may be affected by ongoing climate change(6).