The optical method
The optical method following Brodribb et al. (2016a, b) was applied to quantify the amount of leaf xylem embolism. A healthy, mature and undamaged leaf from each branch was fixed under a stereo microscope (Axio Zoom.V16, Zeiss, Jena, Germany) or in optical clamps (for more details, see http://www.opensourceov.org/). The surface area of the leaf, which was scanned for both the stereo microscope and clamps, was about 1 cm2. In general, images were taken every 5 min, and the water potential was simultaneously monitored with a stem psychrometer at 10 min intervals (see below). Then, images were processed using the Fiji version of ImageJ (Schindelin et al. , 2012) and the “OpenSourceOV ImageJ Toolbox” was used to analyse the images. Image stacks were made to determine changes in the brightness of leaf veins, which was due to embolism formation. The Percentage of Embolised Pixels (PEP) was quantified over time at decreasing xylem water potentials, with PEP50 representing the xylem water potential corresponding to 50% of total embolised pixels (Brodribb et al. , 2016b).