Detection of uncoupled circadian rhythms in individual cells of Lemna minor using a dual-color bioluminescence monitoring system

Abstract

The 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 worlds 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.