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.
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
To estimate leaf vulnerability to embolism, both the optical method and
the Pneumatron were applied to the same detached leaves. 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.