Mitochondrial area quantification

Roots of 6-d-old seedlings were stained with MitoTracker green FM (200 nM; Molecular Probes) for 45 min. Mitochondrial images were obtained using confocal microscopy (Eclipse C1 Plus Confocal microscope using EZ-C1 3.80 imaging software and Ti-Control) at different times (0, 1, 3, and 6 h after HS). Automated quantification of mitochondrial area was performed using the Mito-Morphology macro for ImageJ software (version 1.39; National Institutes of Health) as described previously (Dagda et al., 2009). In brief, the green channel of cells stained with MitoTracker green FM was converted to grayscale and inverted (to show mitochondria-specific fluorescence as black pixels), and the threshold was set to optimally resolve individual mitochondria. The macro traces mitochondrial outlines using the command “analyze particles.”