No circadian clock-gene circuit in the generation of cellular bioluminescence rhythm of CaMV35S::PtRLUC in duckweed

The 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. ### Competing Interest Statement The authors have declared no competing interest.