Synchrony of plant cellular circadian clocks with heterogeneous properties under light/dark cycles

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The rapid assessment of gene function is crucial in biological research. The CRISPR/Cas9 system is widely used as a tool for targeted gene editing in many organisms including plants. Previously, we established a transient gene expression system for investigating cellular circadian rhythms in duckweed. In this system, circadian reporters and clock gene effectors-such as overexpressors, RNA interference (RNAi), and CRISPR/Cas9-were introduced into duckweed cells using a particle bombardment method. In the present study, we applied the CRISPR/Cas9 system at a single cell level to Arabidopsis thaliana, a model organism in plant biology. To evaluate the mutation induction efficiency of the system, we monitored single-cell bioluminescence after application of the CRISPR/Cas9 system targeting the ELF3 gene, which is essential for robust circadian rhythmicity. We evaluated the mutation induction efficiency by determining the proportion of cells with impaired circadian rhythms. Three single guide RNAs (sgRNAs) were designed, and the proportion of arrhythmic cells following their use ranged from 32 to 91%. A comparison of the mutation induction efficiencies of diploid and tetraploid Arabidopsis suggested that endoreduplication had a slight effect on efficiency. Taken together, our results demonstrate that the transiently introduced CRISPR/Cas9 system is useful for rapidly assessing the physiological function of target genes in Arabidopsis cells.

mentioning

confidence: 99%

Monitoring single-cell bioluminescence of Arabidopsis leaves to quantitatively evaluate the efficiency of a transiently introduced CRISPR/Cas9 system targeting the circadian clock gene ELF3

Kanesaka

Okada

Ito

et al. 2019

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“…Thus, analysis of physiological behavior at the single-cell level in the whole plant is important for understanding the physiology of these cyclic processes (Libault et al 2017;Muranaka and Oyama 2018). Circadian rhythms at the single-cell level have been analyzed in intact plants such as the duckweed Lemna gibba following transfection with a circadian bioluminescence reporter using the particle bombardment method (Muranaka and Oyama 2016;Muranaka et al 2013;Okada et al 2017). These analyses clearly demonstrate heterogeneous circadian behaviors in individual cells in the same frond (leaf-like structure of duckweed).…”

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confidence: 99%

Use of a duckweed species, Wolffiella hyalina, for whole-plant observation of physiological behavior at the single-cell level

Isoda

Oyama

2018

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We developed a new model system to analyze physiological behavior at the single-cell level in whole plants. Wolffiella hyalina is a species of rootless duckweed, which has a thin and very small structure and can grow rapidly on the surface of culture medium. Epidermal and mesophyll cells were transfected with a reporter gene using particle bombardment and were observed at the single-cell level in the whole living plant. An EM-CCD camera system with a macro zoom microscope was used to capture time-lapse images of bioluminescence, and we successfully detected circadian rhythms in individual cells that expressed a luciferase gene under the control of a circadian promoter. We also detected individual S-phase cells in meristematic tissues of intact W. hyalina plants by using a 5-ethynyl-2′-deoxyuridine (EdU)labeling assay. Our observations indicated that low-molecular-weight compounds could access the inside of the plant body. Thus, W. hyalina showed the experimental characteristics suitable for single-cell analyses that could be combined with whole-plant observations and/or pharmacological analyses/chemical biology.

“…By measuring rhythmicity in individual leaf cells in transgenic Arabidopsis plants with fluorescence-tagged CCA1, cell-specific differences in clock properties were demonstrated (Yakir et al 2011). Furthermore, by developing a single-cell bioluminescence imaging system for monitoring cellular gene expression in duckweed, the circadian behavior of individual cells in an intact plant was quantitatively analyzed Oyama 2016, 2018;Muranaka et al 2013;Okada et al 2017). Individual cellular circadian clocks oscillate with their own frequencies and respond independently to external light/dark signals.…”

mentioning

confidence: 99%

Long-term monitoring of bioluminescence circadian rhythms of cells in a transgenic Arabidopsis mesophyll protoplast culture

Nakamura

Oyama

2018

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The circadian system of plants is based on the cell-autonomously oscillating circadian clock. In the plant body, these cellular clocks are associated with each other, but their basic and intrinsic properties are still largely unknown. Here we report a method that enables long-term monitoring of bioluminescence circadian rhythms of a protoplast culture in a complete synthetic medium. From the leaves of Arabidopsis transgenic plants carrying the luciferase gene under a clockgene promoter, mesophyll protoplasts were isolated and their bioluminescence was automatically measured every 20 min for more than one week. Decreasing luminescence intensities were observed in protoplasts when they were cultured in a Murashige and Skoog-based medium and also in W5 solution. This decrease was dramatically improved by adding the phytohormones auxin and cytokinin to the MS-based medium; robust circadian rhythms were successfully monitored. Interestingly, the period lengths of bioluminescence circadian rhythms of protoplasts under constant conditions were larger than those of detached leaves, suggesting that the period lengths of mesophyll cells in leaves were modulated from their intrinsic properties by the influence of other tissues/cells. The entrainability of protoplasts to light/dark signals was clearly demonstrated by using this monitoring system. By analyzing the circadian behavior of isolated protoplasts, the basic circadian system of plant cells may be better understood.