Experimental design
We applied three different experiments to investigate the potential effect of cut-open xylem on embolism spreading.
Experiment 1: Comparison of detached leaves with leaves attached to a short and long stem segment
Embolism resistance of leaf xylem was measured using the optical method. For each of the six species studied, we considered three different types of samples: (1) a detached leaf with a cut petiole, (2) a single leaf and petiole connected to a short (0.5 cm) stem segment, regardless of the vessel length in stem xylem, and (3) a leaf attached to a long branch, which was at least two times the maximum vessel length as measured for stem xylem. Depending on the species, the branch length of the latter samples had a length of ca. 80 to 160 cm. Four to five replicates were tested for each sample type. The dehydration time for each leaf or branch usually took 10 to 48 hours. L. tulipiferaand B. pendula were the slowest dehydrating species. Also, the dehydration speed of detached leaves was much faster than leaves attached to a long branch for F. sylvatica , P. avium , andC. betulus (Table S1).
Experiment 2: Comparison of intact leaves vs leaves with cut-open minor veins
To compare the potential impact of cut-open narrow vessels or tracheids in leaf xylem on embolism spreading, the optical method was applied on two adjacent leaves attached to a long branch, which was more than twice the maximum vessel length in stem xylem. This approach was applied to all six species. Two to three leaf pairs were tested for each species. Two mature, healthy and adjacent leaves were selected and placed under the stereomicroscope. In one leaf, we cut a few minor veins (3rd or 4th vein order) with a razor blade. Four to six cuts were made and the length of each cut was about 1 to 2 mm. The other leaf selected was kept intact. Cuts on leaves were made at the beginning of the dehydration procedure, and images were taken every 10 seconds to obtain a high temporal resolution of embolism formation within the first 10 minutes after making the cuts. Moreover, a transparent tape was applied to both leaf areas observed to avoid any potential difference in dehydration between the cut and intact leaf. After the first ten minutes of scanning the leaf, images were taken every five minutes.
Experiment 3: Comparison of the optical method with the Pneumatron
Both the optical method and the Pneumatron were applied to the same detached leaves to estimate embolism resistance of xylem tissue. The Pneumatron was connected to the cut leaf petiole, while the optical method was applied to the upper part of the leaf blades, as far away from the cut petiole as possible. In this way, we obtained the highest hydraulic distance between both methods, with the optical method focussing on the intact vessels in the upper leaf veins, and the Pneumatron measuring gas diffusion between the cut-open and first series of intact conduits of the petiole, and probably the lower part of the leaf blade. For each species, four replicates were tested.