Image acquisition and analysis
A Zeiss 700 laser scanning confocal microscope was used for all analyses. Fluorescence signals of CoroNa Green (excitation 488 nm, 5% power, 493 nm emission filter) and CoroNa Green and SNARF-1 co-stain (excitation 488 nm, 15% power with 555 nm emission filter, 555 nm, 20% power with 566 nm emission filter) were collected with 20x objectives in CoroNa Green incubation buffer. Fluorescent signals of suberin (excitation 488 nm, 5% power, 493 nm emission filter) were collected with 20x object in 50% (V/V) glycerol in deionized water.
Quantification was performed using ImageJ software (Schneider et al., 2012). Briefly, the microscopy images were separated into groups based on xylem development. For each CoroNa Green sample imaged, a 3D maximum projection was assembled from z-stack using the Zen Black (Zeiss) software and exported as a single image file to Image J, where Na+ positive vacuoles were counted and recorded. The same export settings were used for all images of an experiment. For Fluorol Yellow 088 fluorescence quantification, lines were drawn through cell layer perpendicular to the radial cell walls of export 3D maximum projection images using the segmented line tool in ImageJ, and the signal intensity values along the line calculated by ImageJ. The values corresponding to the radial cell walls were used to obtain the average fluorescence value for the entire endodermis / exodermis cell layer in each section. For suberized cell count, the total number of cells and the total number of suberized cells in endodermis / exodermis were counted in ImageJ. The images used in figures were exported with Zen Black as described above and assembled using Illustrator