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

Abstract

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.

Complexities, similarities, and differences in circadian regulation in the green lineage

“…Tokitaka Oyama presented an investigation of a noncell‐autonomous circadian rhythm and its characteristics. Using single‐cell level imaging with a duckweed species ( Lemna minor ), his group succeeded in monitoring dual‐color bioluminescence reporters (Arabidopsis CIRCADIAN CLOCK‐ASSOCIATED1::luciferase + ( AtCCA1::LUC+ ) under the circadian clock and 35S::modified click‐beetle red‐color luciferase ( CaMV35S::PtRLUC )) (Muranaka & Oyama, 2016; Watanabe et al ., 2023). Both AtCCA1::LUC+ and CaMV35S::PtRLUC had bioluminescence rhythms with periods of c .…”

In my own time: A non–cell-autonomous circadian regulation in plant cells

“…The combination of new biosensors with micrografting can be a strong tool for the identification of new molecules regulating non–cell-autonomous rhythms. This study by Watanabe et al 2023 opens a window to an exciting niche of research in biological rhythms, which may help to solve the question of what makes the non-autonomous clock go on its own time.…”

“…In this issue of Plant Physiology , Watanabe et al 2023 demonstrated that the rhythmic behavior of the luminescent reporter AtCCA1::LUC + depends on the autonomous cell clock, whereas a second reporter, CaMV35S::PtRLUC , does not. Rather, CaMV35S::PtRLUC depends on the communication with the neighboring cells.…”

“…C , F) To disrupt the non–cell-autonomous clock, duckweed cells were incubated in a plasmolysis-inducing solution. Adapted from Watanabe et al, 2023 . Figure created by J.M.U in Affinity designer (Version 2.0.4).…”

“… Watanabe et al 2023 report the disruption of the autonomous cell signaling by overexpressing the members of the evening complex. It would be very interesting to resolve whether overexpression of morning-expressed clock genes would have the same effect on cell-to-cell communication, excluding that this is a dependent function of the evening complex.…”