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