Treatment of A. thaliana leaves with calcium channel blocker and calcium chelators abolishes ROS production after leaves wounding. Droplets containing different concentrations of verapamil (10 μM, 100 μM, 1 mM, 10 mM), EGTA (1 mM, 10 mM, 50 mM, 100 mM) and oxalate (100 μM, 1 mM, 10 mM, 50 mM) were applied on leaves for 3 hours. Leaves were then either wounded and stained with DCF-DA and visualized at a fluorescence microscope with two sets of filters (ROS in green and chlorophyll autofluorescence in red, upper panels), or were tested for cell viability using trypan blue (lower panels). Densitometric analysis of the ROS signal is displayed on the right of each image series. Asterisks represent significant differences using Student's t test relative to water-treated control; *P < 0.05, **P < 0.01.
Beneloujaephajri et al. BMC Plant Biology 2013 13:160 doi:10.1186/1471-2229-13-160