Aequorin-Based Ca2+ Imaging in Plants

Seedlings were grown on half-strength MS medium for five days. Reconstitution of aequorin was performed in vivo by spraying seedlings with 10 µM coelenterazine and followed by incubation at 21°C in the dark for 12–16h. For surfactant treatment, 0.01% or 0.1% of silwet L-77 (Sigma) was added to the coelenterazine solution. For Ca²⁺ inhibitor treatments, rice roots were treated with different concentrations of GdCl₃, LaCl₃, neomycin and thapsigargin, respectively for 30min before 0.25M NaCl and 1mM H₂O₂ treatment. Treatments and aequorin luminescence imaging were performed at room temperature using a ChemiPro HT system as described previously (Jiang et al., 2013 (link)). The recording was started about 5 s prior to treatment and luminescence images were acquired for 3min. For the analysis of time courses of increase in [Ca²⁺]_i, each exposure time was 30 s and the images were taken continuously for several minutes. To avoid the interference of chloroplast auto-fluorescence signal in the aequorin luminescence imaging, all the treatments were performed in the complete darkness. To record the chloroplast auto-fluorescence signal, seedlings were first exposed to strong light for 1min. After that, the light was turned off and the chloroplast auto-fluorescence was recorded. WinView/32 and Meta Morph 7.7 were used to analyse recorded luminescence images.