A non-cell-autonomous circadian rhythm of bioluminescence reporter activities in individual duckweed cells.

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 noncell-autonomous rhythms that are unaffected by cellular oscillators.

Prolonged Light Exposure Induces Circadian Impairment in Aquaporin-4-Knockout Mice.

Astrocytes are densely present in the suprachiasmatic nucleus (SCN), which is the master circadian oscillator in mammals, and are presumed to play a key role in circadian oscillation. However, specific astrocytic molecules that regulate the circadian clock are not yet well understood. In our study, we found that the water channel aquaporin-4 (AQP4) was abundantly expressed in SCN astrocytes, and we further examined its circadian role using AQP4-knockout mice. There was no prominent difference in circadian behavioral rhythms between Aqp4-/- and Aqp4+/+ mice subjected to light-dark cycles and constant dark conditions. However, exposure to constant light induced a greater decrease in the Aqp4-/- mice rhythmicity. Although the damped rhythm in long-term constant light recovered after transfer to constant dark conditions in both genotypes, the period until the reappearance of original rhythmicity was severely prolonged in Aqp4-/- mice. In conclusion, AQP4 absence exacerbates the prolonged light-induced impairment of circadian oscillations and delays their recovery to normal rhythmicity.

Interspecific divergence of circadian properties in duckweed plants.

The circadian clock system is widely conserved in plants; however, divergence in circadian rhythm properties is poorly understood. We conducted a comparative analysis of the circadian properties of closely related duckweed species. Using a particle bombardment method, a circadian bioluminescent reporter was introduced into duckweed plants. We measured bioluminescence circadian rhythms of eight species of the genus Lemna and seven species of the genus Wolffiella at various temperatures (20, 25, and 30°C) and light conditions (constant light or constant dark). Wolffiella species inhabit relatively warm areas and lack some tissues/organs found in Lemna species. Lemna species tended to show robust bioluminescence circadian rhythms under all conditions, while Wolffiella species showed lower rhythm stability, especially at higher temperatures. For Lemna, two species (L. valdiviana and L. minuta) forming a clade showed relatively lower circadian stability. For Wolffiella, two species (W. hyalina and W. repanda) forming a clade showed extremely long period lengths. These analyses reveal that the circadian properties of species primarily reflect their phylogenetic positions. The relationships between geographical and morphological factors and circadian properties are also suggested.

Detection of Uncoupled Circadian Rhythms in Individual Cells of Lemna minor using a Dual-Color Bioluminescence Monitoring System.

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. By 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.

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

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.