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.