Stomatal Aperture Imaging and Analysis

After staining, imaging of stomata was performed with a Nikon Eclipse 90i microscope equipped with a CCD camera using a TRITC filter Ex 540/25 DM 565 BA 605/55 (Nikon). Image analysis was performed using ImageJ software (https://imagej.nih.gov/ij/). For better visualization of the guard cells the option “sharpen” in ImageJ was used. The width and the length of the stomatal aperture were measured as shown on Fig 1A, and the stomatal aperture index (SAI) was calculated by division of the aperture width through the length. The SAI of at least 30 stomata per leaf was calculated, and three leaves per each treatment / time point were used for statistical analysis. Data were analyzed using Student’s t-test. The confocal images were captured under Leica SP8 microscope at following settings: excitation at 488 nm, emission 505–545 nm (green fluorescence); excitation at 561 nm, emission at 600–640 nm (red fluorescence), 20x objective, using Leica Application Suite (LAS) software.