SummaryThe plant circadian oscillation system is based on the circadian clock of individual cells and coordinates the circadian behavior of the plant body. To observe the cellular circadian behavior of both the oscillator and its output in plants, we developed the dual-color bioluminescence monitoring system that automatically measured the luminescence of two luciferase reporters simultaneously at a single-cell level. We selected a yellow-green-emitting firefly luciferase (LUC+) and a red-emitting luciferase (PtRLUC) that is a mutant form of Brazilian click beetle ELUC. We used AtCCA1::LUC+ and CaMV35S::PtRLUC to observe the cellular behavior of the oscillator and output, respectively. These bioluminescent reporters were introduced into the cells of a duckweed, Lemna minor, by particle bombardment. Time series of the bioluminescence of individual cells in a frond were obtained using a dual-color bioluminescence monitoring system with a green-pass- and red-pass filter. Luminescence intensities from the LUC+ and PtRLUC of each cell were calculated from the filtered luminescence intensities. We succeeded in reconstructing the bioluminescence behaviors of AtCCA1::LUC+ and CaMV35S::PtRLUC in the same cells. Under prolonged constant light conditions, AtCCA1::LUC+ showed a robust circadian rhythm in individual cells in an asynchronous state in the frond, as previously reported in studies using other plants. In contrast, CaMV35S::PtRLUC stochastically showed circadian rhythms in a synchronous state. Thus, we clearly demonstrated the uncoupling between the oscillator and output in individual cells. This dual-color bioluminescence monitoring system is a powerful tool to analyze various stochastic phenomena accompanying large cell-to-cell variation in gene expression.Significance statementWe succeeded in establishing the world’s first dual-color bioluminescence monitoring system at a single-cell level that enables simultaneous measurement of the luminescence activities of two reporter genes in plants. This system is a strong tool to analyze stochastic phenomena, and we clearly demonstrated the uncoupling of rhythmic behavior between two bioluminescent reporters in individual cells that stochastically occurred in the same plant.

The plant circadian oscillation system is based on the circadian clock of individual cells. Circadian behavior of cells has been observed by monitoring the circadian reporter activity such as bioluminescence of AtCCA1::LUC+. To deeply analyze different circadian behaviors in individual cells, we developed the dual-color bioluminescence monitoring system that automatically measured the luminescence of two luciferase reporters simultaneously at a single-cell level. We selected a yellow-green-emitting firefly luciferase (LUC+) and a red-emitting luciferase (PtRLUC) that is a mutant form of Brazilian click beetle ELUC. We used AtCCA1::LUC+ and CaMV35S::PtRLUC. CaMV35S::LUC+ was previously reported as a circadian reporter with a low amplitude rhythm. These bioluminescent reporters were introduced into the cells of a duckweed, Lemna minor, by particle bombardment. Time series of the bioluminescence of individual cells in a frond were obtained using a dual-color bioluminescence monitoring system with a green-pass- and red-pass filter. Luminescence intensities from the LUC+ and PtRLUC of each cell were calculated from the filtered luminescence intensities. We succeeded in reconstructing the bioluminescence behaviors of AtCCA1::LUC+ and CaMV35S::PtRLUC in the same cells. Under prolonged constant light conditions, AtCCA1::LUC+ showed a robust circadian rhythm in individual cells in an asynchronous state in the frond, as previously reported. In contrast, CaMV35S::PtRLUC stochastically showed circadian rhythms in a synchronous state. These results strongly suggested the uncoupling of cellular behavior between these circadian reporters. This dual-color bioluminescence monitoring system is a powerful tool to analyze various stochastic phenomena accompanying large cell-to-cell variation in gene expression.

The circadian clock is responsible for the temporal regulation of various physiological processes in plants. Individual cells contain a circadian oscillator consisting of a clock gene circuit that coordinates physiological rhythms within the plant body in an orderly manner. The coordination of time information has been studied from the perspective of cell–cell local coupling and long-distance communication between tissues based on the view that the behavior of circadian oscillators represents physiological rhythms. Here, we report the cellular circadian rhythm of bioluminescence reporters that are not governed by the clock gene circuit in expressing cells. We detected cellular bioluminescence rhythms with different free-running periods in the same cells using a dual-color bioluminescence monitoring system in duckweed (Lemna minor) transfected with Arabidopsis CIRCADIAN CLOCK ASSOCIATED 1::luciferace + (AtCCA1::LUC+) and Cauliflower mosaic virus 35S::modified click-beetle red-color luciferase (CaMV35S::PtRLUC) reporters. Co-transfection experiments with the two reporters and a clock gene-overexpressing effector revealed that the AtCCA1::LUC + rhythm, but not the CaMV35S::PtRLUC rhythm, was altered in cells with a dysfunctional clock gene circuit. This indicated that the AtCCA1::LUC + rhythm is a direct output of the cellular circadian oscillator, whereas the CaMV35S::PtRLUC rhythm is not. After plasmolysis, the CaMV35S::PtRLUC rhythm disappeared, whereas the AtCCA1::LUC + rhythm persisted. This suggests that the CaMV35S::PtRLUC bioluminescence has a symplast/apoplast-mediated circadian rhythm generated at the organismal level. The CaMV35S::PtRLUC-type bioluminescence rhythm was also observed when other bioluminescence reporters were expressed. These results reveal that the plant circadian system consists of both cell-autonomous and non-cell-autonomous rhythms that are unaffected by cellular oscillators.

SummaryThe circadian clock is responsible for the temporal regulation of various physiological phenomena in plants. Individual cells contain the cellular circadian clock consisting of the clock gene circuit. Physiological rhythms are coordinated in the plant body in an orderly manner. Multiple circadian rhythms with different free running periods (FRPs) can coexist in the same plant. In addition to different circadian properties between tissues/organs, uncoupled circadian rhythms in the same cell have been reported. The relationship between the cellular circadian clock and the various circadian outputs with different FRPs is currently unknown. Using a dual-color bioluminescence monitoring system in Lemna minor transfected with the AtCCA1::LUC+ and CaMV35S::PtRLUC reporters, cellular bioluminescence rhythms with different FRPs were detected in the same cells. Co-transfection experiments with the two reporters and a clock gene overexpressing effector revealed that the circadian properties of the AtCCA1::LUC+ rhythm, but not those of the CaMV35S::PtRLUC rhythm, were altered in the cells with a dysfunctional clock gene circuit. This indicates that the AtCCA1::LUC+ rhythm is a direct output of the cellular circadian clock while the CaMV35S::PtRLUC rhythm is not. After plasmolysis, the CaMV35S::PtRLUC rhythm disappeared while the AtCCA1::LUC+ rhythm persisted. This suggested that CaMV35S::PtRLUC is a symplast/apoplast mediated circadian rhythm generated at an organismal level. This idea is consistent with the observation that the phases of CaMV35S::PtRLUC cellular rhythms remain highly synchronized while those of AtCCA1::LUC+ rhythms gradually get desynchronized. The plant circadian system consists of both cell-autonomous rhythms and non-cell-autonomous rhythms that are unaffected by the cellular clock.