Circadian Rhythms in Arabidopsis Protoplasts

Seeds were sown in 1/2 MS-solid medium and stratified in darkness at 4 °C for 3 days, and germinated seedlings were entrained for 3 weeks under ND conditions at 22–23 °C. To examine circadian oscillations in protoplasts, mesophyll protoplasts were isolated from the lower epidermal layer of 3-week-old Arabidopsis seedlings. Whole seedlings were soaked in 10 mL of an enzyme solution (400 mM mannitol, 20 mM KCl, 20 mM MES-KOH [pH 5.7], 10 mM CaCl₂, 1% Cellulase R10, 0.5% Macerozyme R10, and 0.1% bovine serum albumin) for 16 h in the dark. The isolated protoplasts were filtered through sterile 100 mm stainless mesh and resuspended in W5 solution. The pCCA1:LUC and p35S:ELF3 plasmids were prepared by PEG purification. Then, the recombinant constructs were transiently introduced into Arabidopsis protoplasts via PEG-mediated transformation [23 (link)].
Luminescence rhythms were monitored using the Tristar2 LB 942 Multimode Microplate Reader (Berthold Technologies, Wildbad, Germany). The circadian period was estimated using the Fast Fourier Transform-Nonlinear Least Squares (FFT-NLLS) suite of programs available in the Biodare2 software.