Materials and methods
The five vessel-bearing angiosperm species used in this study wereIlex verticillata (L.) A.Gray [Aquifoliaceae],
Rhododendron hirsutum L. [Ericaceae], Ficus religiosa L.
[Moraceae], Tilia cordata Mill. [Malvaceae] andLindera benzoin L. [Lauraceae], the vessel-less angiosperm
was Drimys winteri J.R. Forst & G.Forst. [Winteraceae], and
three conifer species were Agathis robusta (C.Moore ex. F.Muell)
Bailey [Araucariaceae], Tsuga canadensis (L.) Carrière
[Pinaceae] and Torreya californica Torr. [Taxaceae].
Plants of Ti. cordata , and Ts. canadensis were grown
outside in the grounds of the Botanical Gardens of Ulm University, Ulm
(Germany) (48° 25’ N, 9° 57’ E), F. religiosa , A. robusta ,
and D. winteri in the glasshouses of the Botanical Gardens of Ulm
University, I. verticillata , L. benzoin and To.
californica were grown in the glasshouses of Purdue University, West
Lafayette (Indiana, USA) (40° 25’ N, 86° 54’ W, elevation: 187 m).R. hirsutum was collected in the Allgäu Alps near Oberstdorf
(Germany) (47° 25’ N, 10° 17’ E, elevation: 2600 m). Care was taken to
ensure no pre-existing embolism was present in the stems prior to
experiments, with all plants grown under well-watered conditions for the
duration of the growing season prior to harvesting. All stems were
collected between May and June 2019 prior to dawn and were more hydrated
than -0.4 MPa. Furthermore, air-filled xylem conduits appear as a blank
area in the xylem when image processing is performed using the optical
method. In all experiments no blank areas were detected in the observed
areas of xylem at the completion of the experiment, providing support
for no substantial, pre-existing embolism being present in any sample
measured prior to the experiment.
Terminal branches ranging from 0.3 to 2.0 m long (all exceeding the
length of the longest vessel determined by air injection for the five
angiosperm species) were collected prior to dawn, cut under water, and
bagged for approximately 1 h, so that all experiments started with a
stem water potential (Ψstem) after equilibration of at
least -0. 4 MPa. Stems were measured in all species, except L.
benzoin in which experiments were conducted on leaves. Stems and leaves
were fixed under a stereo microscope (SZMT2, optika, Italy) or in a
Raspberry Pi clamp (opensourceOV.org). Vulnerability curves for stems
were conducted on a region of the stem in which the bark was gently
removed by hand, without touching the xylem beneath, and an adhesive gel
(Tensive) immediately applied to the surface of the stem xylem to avoid
dehydration and provide greater optics. A glass cover slip was placed on
top of the adhesive gel to aid imaging. A stem psychrometer (ICT
International, version 4.4) was then attached to the stem beyond the
length of the longest vessel from the area imaged and
Ψstem measurements were collected every 10 min. Branches
were allowed to naturally dry while images were captured every 3 min.
All branches were rehydrated across a range of dehydrated
Ψstem to build a data set spanning a range of variation
in the percentage of embolism at the point of rehydration across the
species selected. At the point of rehydration, the end of the branch was
excised under water (removing c. 1 cm from the cut end) until
Ψstem had fully recovered. Rehydration with water
potentials approaching 0 MPa generally occurred in less than 60 mins in
all species. Branches were allowed to remain at a relaxed, high water
potential for a variable amount of time ranging from 30 mins to 5 h. The
cut end of the stem was removed from water once rehydrated, and the
branch was then allowed to bench dry a second time until all conduits
had visibly embolized. Stem or leaf images were analysed using ImageJ
(version 1.52h, NIH, USA) to quantify the accumulation of embolized
xylem area through time. Optical vulnerability curves were constructed
as described by Brodribb et al. (2016b).
Based on the dehydration-rehydration-dehydration curves, we were able to
determine the area of xylem that was embolised on the second cycle of
dehydration at a more hydrated Ψ than when rehydration occurred
(E pr) as follows:
\begin{equation}
\ E_{\text{pr}}=\left(\frac{E_{r2}-E_{r1}\ }{100-E_{r1}\ }\right)\ X\ 100\nonumber \\
\end{equation}Where \(E_{r1}\) is the percentage of accumulated embolized xylem area
at the moment of rehydration and \(E_{r2}\) was the percentage of
accumulated embolized xylem area that occurred prior to when plants
reached the same Ψ at which rehydration occurred.
Based on the dehydration-rehydration-dehydration curves, we also
determined the P 50 (calculated by considering all
events of embolism obtained using the
dehydration-rehydration-dehydration curves), and an apparentP 50 (P 50r) that was
obtained based only on the embolism events that occurred on the second
cycle of dehydration. From these values we were able to calculate the
percentage change in P 50 due to pre-existing
embolism using the follow equation:\(\text{Percent\ change\ in\ apparent\ }P_{50\ }=\left(\frac{P_{50}-P_{50r}\ }{P_{50}\ }\right)\ X\ 100\)
We conducted the experiment at least once in all species, three times inI. verticillata and R. hirsutum and twice in L.
benzoin and F. religiosa . For To. californica , three
rehydration curves were performed at the same \(E_{r1}\) and the results
are presented as a mean of these three experiments (Supplementary Figure
S1).