Long-term monitoring of bioluminescence circadian rhythms of cells in a transgenic <i>Arabidopsis</i> mesophyll protoplast culture

Abstract

The circadian system of plants is based on the cell-autonomously oscillating circadian clock. In the plant body, these cellular clocks are associated with each other, but their basic and intrinsic properties are still largely unknown. Here we report a method that enables long-term monitoring of bioluminescence circadian rhythms of a protoplast culture in a complete synthetic medium. From the leaves of Arabidopsis transgenic plants carrying the luciferase gene under a clockgene promoter, mesophyll protoplasts were isolated and their bioluminescence was automatically measured every 20 min for more than one week. Decreasing luminescence intensities were observed in protoplasts when they were cultured in a Murashige and Skoog-based medium and also in W5 solution. This decrease was dramatically improved by adding the phytohormones auxin and cytokinin to the MS-based medium; robust circadian rhythms were successfully monitored. Interestingly, the period lengths of bioluminescence circadian rhythms of protoplasts under constant conditions were larger than those of detached leaves, suggesting that the period lengths of mesophyll cells in leaves were modulated from their intrinsic properties by the influence of other tissues/cells. The entrainability of protoplasts to light/dark signals was clearly demonstrated by using this monitoring system. By analyzing the circadian behavior of isolated protoplasts, the basic circadian system of plant cells may be better understood.

Monitoring single-cell bioluminescence of <i>Arabidopsis</i> leaves to quantitatively evaluate the efficiency of a transiently introduced CRISPR/Cas9 system targeting the circadian clock gene <i>ELF3</i>

“…Protoplasts would be suitable for investigations of cell-autonomous gene function by using the transiently introduced CRISPR/Cas9 system. Recently, we succeeded in stably monitoring the long-term bioluminescence circadian rhythms of Arabidopsis protoplasts (Nakamura and Oyama 2018). By monitoring the bioluminescence rhythms of individual protoplasts for a long term, it would be possible to experimentally reevaluate the mutation induction efficiencies of protoplasts.…”

Adaptive diversification in the cellular circadian behavior of Arabidopsis leaf- and root-derived cells

“…1d and 3c). Previous studies have shown that the circadian rhythm damped in a diluted culture of protoplasts derived from shoot apex and leaves 12,27 . We assumed that intercellular communication might play a role in the maintenance of coherent rhythms among cells.…”

“…As previously reported 27–30 , we prepared protoplasts from the leaves and roots of an Arabidopsis CCA1::LUC transgenic plant. Protoplasts of leaves are mainly mesophyll cells 27 and those of roots are presumedly a heterogeneous population with different cell types 31,32 . Cells derived from the protoplasts of leaves and roots are hereafter referred to as leaf-derived cells and root-derived cells, respectively.…”

“…Even under such asynchronous conditions, it has been suggested that intercellular communication influences the circadian behavior of cells 12,13,20–24 . The use of isolated cells (protoplasts) and suspension-culture cells, which prevents the influence of tissue/organ systems 25–27 , has been adopted for cell-based studies on the circadian rhythms of plant. Cell density affects the circadian behavior of cells, which implies that intercellular communication between isolated shoot apex cells influences the circadian rhythm 12 .…”

The Plant Circadian Oscillator

“…Studies in protoplasts support the presence of an autonomous clock in each plant cell [128,129,130]. To what extent are these cell-autonomous clocks coupled?…”