2.4 In situ detection of reactive oxygen species
ROS cellular localization was determined by confocal microscopy in an Eclipse TE–2000–E2 Confocal Laser Scanning Microscope (Nikon), essentially as described by Mayta et al. (2018). Discs (110 mm2 in diameter) were collected during the light period from leaves belonging to the fourth node of 5 different plants per line, vacuum-infiltrated in the dark with 50 µM DCFDA in 10 mM Tris-HCl pH 7.5 and 0.1% (v/v) Tween-20, incubated in the dark for 1 h in the same solution, washed briefly and mounted in water. Imaging was performed by scanning 5 optical slices (with an interval of ∼1 µm) of the palisade parenchyma immediately below the epidermis. Fluorescence intensities were estimated using Fiji software (Schindelin et al.2012), using the z-projections of the different stacks.