Image analysis
Image sequences were analysed using IMAGEJ (NIH), see
http://www.opensourceov.org/ for details. Briefly, the pixel
values of each image were subtracted from the next in the sequence to
quantify any differences between images. Pixel differences indicate a
change in light transmission or reflection as water-filled vessels fill
with air during cavitation events (Brodribb et al., 2016). Due to
the frequency of image capture, cavitation events can be separated from
other “noise” due to movement, leaf shrinkage, or drying of hydrogel
by filtering differences that are not fast changes confined to the
xylem. Noise was removed using pixel thresholding leaving only pixels
associated with cavitation events. The embolism area per image was
calculated as the summed pixels and expressed as cumulative embolism, a
percentage of the total cavitated area in pixels per sequence.
Cumulative embolism was plotted against water potential to create
vulnerability curves (Bourbia et al., 2020; T. J. Brodribb, Carriqui,
Delzon, & Lucani, 2017) and derive the water potential at which 50% of
total xylem were cavitated (P50).