Measurement of cytosolic calcium concentration

The radish mesophyll protoplasts were isolated according to the methods of Yoo et al. (2007) and Hagimori and Nagaoka (1992) with minor modifications. 0.5 mm leaf strips were cut from the middle part of the leaves and transferred to the enzyme solution [0.5 M MES (pH 7.5) containing 1.5% (w/v) cellulase R10, 0.4% macerozyme R10, 0.4 mM mannitol, 20 mM KCl, 10 mM CaCl₂, and 0.1% BSA] immediately. After vacuum infiltrated in the dark for 30 min at 25°C, the leaf strips were transferred to an incubator to continue the digestion in the dark for 4 h at room temperature. Then the enzyme solution was washed twice with W5 solution. The protoplasts were collected after centrifugation and re-suspended in MMG [4 mM 4-morpholineethanesulfonic acid (MES, pH 5.7) containing 0.4 M mannitol and 15 mM MgCl₂] solution. Protoplasts were kept at room temperature.

Cytosolic calcium concentration was measured by the calcium fluorescent probe Fluo-3/AM (Molecular Probes) based on the method of Yan et al. (2015) and Zhang et al. (1998). Briefly, 1 mM Fluo-3/AM in anhydrous DMSO (5 μL) was added to radish mesophyll protoplasts. After incubation at 4°C in the dark for 2 h, the protoplasts were washed three times with isotonic solution to wash away the residual dye. Then the protoplasts were incubated with incubation solution [containing 20 mM Fluo-3/AM, 0.5 M mannitol, 4 mM MES (pH 5.7), and 20 mM KCl] at 25°C for 1 h. The fluorescence of Ca²⁺ was measured after various reagents (as described in Figure 2) were added. Images of 25 protoplasts for each treatment in three independent experiments were observed using a PE (Ultra View VOX) laser scanning confocal microscope (LSCM) with the 488 nm excitation wavelength. The emission fluorescence was filtered by a 515 nm filter to eliminate the autofluorescence of chlorophyll. The fluorescence of protoplasts on acquired images were analyzed by image J software (NIH). Data were calculated as means ± SE of pixel intensities.