Long-term monitoring of bioluminescence circadian rhythms of cells in a transgenic Arabidopsis mesophyll protoplast culture.

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 clock-gene 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.