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\begin{document}

\title{No gas source, no problem: proximity to pre-existing embolism and
segmentation affect embolism spreading in angiosperm xylem by gas
diffusion}



\author[1]{Xinyi Guan}%
\author[1]{Luciano Pereira}%
\author[2]{Scott McAdam}%
\author[3]{Kun-Fang Cao}%
\author[4]{Steven Jansen}%
\affil[1]{Institute of Systematic Botany and Ecology, Ulm University,}%
\affil[2]{Purdue University}%
\affil[3]{State Key Laboratory for Conservation and Utilisation of Subtropical Agro-Bioresources, Guangxi University}%
\affil[4]{Institute of Systematic Botany and Ecology, Ulm University}%


\vspace{-1em}



  
  \date{June 15, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Embolism spreading in dehydrating angiosperm xylem is driven by gas
movement between embolised and sap-filled conduits. Here, we examine how
proximity to pre-existing embolism and hydraulic segmentation affect
embolism propagation. Based on the optical method, we compared xylem
embolism resistance between detached leaves and leaves attached to
branches, and between intact leaves and leaves with cut minor veins for
six species. Moreover, we directly compared the optical and pneumatic
method on detached leaves. Embolism resistance of detached leaves was
significantly lower than leaves attached to stems, except for two
species with all vessels ending in their petioles. Cutting of minor
veins showed limited embolism spreading in minor veins near the cuts
prior to major veins. Moreover, there was strong agreement in embolism
resistance between the optical and pneumatic method, with minor
differences occurring during early stages of embolism formation. We
conclude that embolism resistance may represent a relative trait,
depending on the proximity and connectivity to pre-existing embolism as
a gas source. Since embolism formation may not rely on a certain
pressure difference threshold between functional and embolised conduits,
we suggest that embolism is facilitated by pressure-driven gas
diffusion, while hydraulic segmentation can prevent embolism propagation
by reducing gas diffusion.%
\end{abstract}%



\sloppy


\textbf{No gas source, no problem: proximity to pre-existing embolism
and segmentation affect embolism spreading in angiosperm xylem by gas
diffusion}

Xinyi Guan\textsuperscript{1}, Luciano Pereira\textsuperscript{1,2,3},
Scott A. M. McAdam\textsuperscript{4}, Kunfang Cao\textsuperscript{5,6},
Steven Jansen\textsuperscript{1}

1. Institute of Systematic Botany and Ecology, Ulm University,
Albert-Einstein-Allee 11, 89081, Ulm, Germany

2. Laboratory of Plant Physiology ``Coaracy M. Franco'', Center R\&D in
Ecophysiology and Biophysics, Agronomic Institute (IAC), Campinas,
Brazil

3. Laboratory of Crop Physiology, Department of Plant Biology, Institute
of Biology, P.O. Box 6109, University of Campinas (UNICAMP), Campinas
13083-970, Brazil

4. Purdue Center for Plant Biology, Department of Botany and Plant
Pathology, Purdue University, 915 W. State St. West Lafayette, IN,
47907, USA.

5. Plant Ecophysiology and Evolution Group, State Key Laboratory for
Conservation and Utilisation of Subtropical Agro-Bioresources, Guangxi
University, Nanning, Guangxi, 530004, China.

6. Guangxi Key Laboratory of Forest Ecology and Conservation, College of
Forestry, Guangxi University, Nanning, Guangxi, 530004, China.

* Corresponding author:\emph{xinyi.guan@uni-ulm.de}

Funding information: The German Research Foundation (DFG, project Nr.
410768178)

\textbf{Abstract}

Embolism spreading in dehydrating angiosperm xylem is driven by gas
movement between embolised and sap-filled conduits. Here, we examine how
proximity to pre-existing embolism and hydraulic segmentation affect
embolism propagation. Based on the optical method, we compared xylem
embolism resistance between detached leaves and leaves attached to
branches, and between intact leaves and leaves with cut minor veins for
six species. Moreover, we directly compared the optical and pneumatic
method on detached leaves. Embolism resistance of detached leaves was
significantly lower than leaves attached to stems, except for two
species with all vessels ending in their petioles. Cutting of minor
veins showed limited embolism spreading in minor veins near the cuts
prior to major veins. Moreover, there was strong agreement in embolism
resistance between the optical and pneumatic method, with minor
differences occurring during early stages of embolism formation. We
conclude that embolism resistance may represent a relative trait,
depending on the proximity and connectivity to pre-existing embolism as
a gas source. Since embolism formation may not rely on a certain
pressure difference threshold between functional and embolised conduits,
we suggest that embolism is facilitated by pressure-driven gas
diffusion, while hydraulic segmentation can prevent embolism propagation
by reducing gas diffusion.

\textbf{Key-words}

Bordered pits, embolism, vessel network, angiosperm xylem, drought
stress, optical method, pneumatic method, gas diffusion

\textbf{Acknowledgements}

We thank Dr. H. Jochen Schenk (California State University, Fullerton)
and Lucian Kaack (Ulm University) for valuable comments on an early
version of this paper, Dr. Melvin Tyree (Zhejiang Normal University) for
useful discussions about pneumatic experiments, two anonymous reviewers
for helpful suggestions, and various colleagues from the botanical
garden of Ulm University for assistance with sampling. XG, KC, and SJ
acknowledge financial support by the German Research Foundation (DFG,
project Nr. 410768178). SAMM acknowledges the Humboldt Foundation for a
postdoctoral fellowship at Ulm University. The authors declare that
there is no conflict of interest.

\textbf{No gas source, no problem: proximity to pre-existing embolism
and segmentation affect embolism spreading in angiosperm xylem by gas
diffusion}

\textbf{Running title:} Pre-exist embolism affects embolism spreading

Xinyi Guan\textsuperscript{1}, Luciano Pereira\textsuperscript{1,2,3},
Scott A. M. McAdam\textsuperscript{4}, Kunfang Cao\textsuperscript{5,6},
Steven Jansen\textsuperscript{1}

1. Institute of Systematic Botany and Ecology, Ulm University,
Albert-Einstein-Allee 11, 89081, Ulm, Germany

2. Laboratory of Plant Physiology ``Coaracy M. Franco'', Center R\&D in
Ecophysiology and Biophysics, Agronomic Institute (IAC), Campinas,
Brazil

3. Laboratory of Crop Physiology, Department of Plant Biology, Institute
of Biology, P.O. Box 6109, University of Campinas (UNICAMP), Campinas
13083-970, Brazil

4. Purdue Center for Plant Biology, Department of Botany and Plant
Pathology, Purdue University, 915 W. State St. West Lafayette, IN,
47907, USA.

5. Plant Ecophysiology and Evolution Group, State Key Laboratory for
Conservation and Utilisation of Subtropical Agro-Bioresources, Guangxi
University, Nanning, Guangxi, 530004, China.

6. Guangxi Key Laboratory of Forest Ecology and Conservation, College of
Forestry, Guangxi University, Nanning, Guangxi, 530004, China.

* Corresponding author: xinyi.guan@uni-ulm.de

\textbf{Abstract}

Embolism spreading in dehydrating angiosperm xylem is driven by gas
movement between embolised and sap-filled conduits. Here, we examine how
proximity to pre-existing embolism and hydraulic segmentation affect
embolism propagation. Based on the optical method, we compared xylem
embolism resistance between detached leaves and leaves attached to
branches, and between intact leaves and leaves with cut minor veins for
six species. Moreover, we directly compared the optical and pneumatic
method on detached leaves. Embolism resistance of detached leaves was
significantly lower than leaves attached to stems, except for two
species with all vessels ending in their petioles. Cutting of minor
veins showed limited embolism spreading in minor veins near the cuts
prior to major veins. Moreover, there was strong agreement in embolism
resistance between the optical and pneumatic method, with minor
differences occurring during early stages of embolism formation. We
conclude that embolism resistance may represent a relative trait,
depending on the proximity and connectivity to pre-existing embolism as
a gas source. Since embolism formation may not rely on a certain
pressure difference threshold between functional and embolised conduits,
we suggest that embolism is facilitated by pressure-driven gas
diffusion, while hydraulic segmentation can prevent embolism propagation
by reducing gas diffusion.

\textbf{Key-words}

Bordered pits, embolism, vessel network, angiosperm xylem, drought
stress, optical method, pneumatic method, gas diffusion

\textbf{Author contributions}

XG performed the experiments, with help from LP and SAMM. SJ and XG
conceived the original ideas. SJ with KC supervised the project. XG
wrote the manuscript with support from SJ, and revisions from all
authors.

\textbf{Introduction}

Xylem sap in plants is frequently transported under negative pressure
(Dixon and Joly, 1895; Jansen and Schenk 2015). Under conditions of low
soil water content and/or high transpiration rates, the tensile force of
xylem sap may increase considerably, which could lead to interruption of
water transport in tracheary elements by large gas bubbles (embolism).
Understanding the frequency and mechanism behind embolism formation in
plant species is important because the amount of embolised conduits may
affect the transport of xylem sap, and therefore photosynthesis
(Zhu\emph{et al.} , 2013; Martin-StPaul \emph{et al.} , 2017). There is
strong and convincing evidence that drought-induced embolism formation
occurs via bordered pits in cell walls of adjacent conduits (Zimmermann,
1983; Sperry \& Tyree, 1988; Jansen \emph{et al.} , 2018; Kaack \emph{et
al.} , 2019). It has frequently been assumed that once the pressure
difference between sap-filled conduits (under negative pressure) and
embolised ones (under atmospheric pressure) exceeds a certain threshold,
embolism spreads from an embolised conduit to a neighbouring one via the
mesoporous pit membranes of bordered pits (Choat \emph{et al.} , 2008;
Tixier \emph{et al.} , 2014; Wason \emph{et al.} , 2018; Avila \emph{et
al.} , submitted). Although embolism spreading from previously embolised
conduits has been well presented in many textbooks and papers
(Zimmermann, 1983; Crombie \emph{et al.} , 1985; Choat \emph{et al.} ,
2016; Lamarque \emph{et al.} , 2018), various basic questions about this
process remain unclear (Kaack \emph{et al.} , 2019). Since gas movement
across pit membranes may involve two different processes, namely mass
flow and diffusion, we prefer the general term embolism spreading or
propagation instead of air-seeding, which includes mass flow of gas
across a pit membrane only.

An important question is whether spreading of embolism in xylem tissue
is facilitated by the presence of pre-existing embolised conduits,
because this would raise questions about the absolute value of embolism
resistance, and whether or not the mechanism behind embolism spreading
depends on a certain pressure difference. Pre-existing embolism could be
embolised conduits from a previous growth ring or protoxylem
(Kitin\emph{et al.} 2004; Sano \emph{et al.} , 2011; Hochberg \emph{et
al.} , 2016). Embolised conduits and local spreading of embolism could
also occur when herbivores or xylem feeding insects damage conduits, or
when a plant organ experiences other types of damage or die-back.
Artificial embolism spreading may occur when xylem tissue has been cut
open to take embolism resistance measurements, because when a
transpiring plant is cut in air, the air-water meniscus is quickly
pulled back into the conduit lumina until it stops at an interconduit
pit membrane (Zimmermann, 1983). A widely used approach to evaluate
embolism resistance is to measure the xylem water potential that
corresponds to 50\% loss of hydraulic conductance (\selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{50},
MPa), while the xylem water potential corresponding to 50\% of the total
amount of gas that can be extracted from a dehydrated xylem tissue has
been suggested as an alternative, direct approach of embolism
quantification (Pereira\emph{et al.} , 2016, 2020a; Zhang \emph{et al.}
, 2018; Oliveira \emph{et al.} , 2019). Both experimental approaches
rely on cut plant organs either due to the requirements to measure
hydraulic conductivity, or gas diffusion kinetics of dehydrating
samples. Moreover, dehydration of a cut branch or leaf can proceed much
faster than dehydration of an intact plant (Cochard \emph{et al.} ,
2013; Hochberg \emph{et al.} , 2017). Other methods, however, such as
microCT observations and the optical method can be used to quantify
embolism in a non-destructive way in intact plants (Brodribb \emph{et
al.} , 2016a, b, 2017; Choat \emph{et al.} , 2016; Lamarque \emph{et
al.} , 2018).

The amount of embolism propagation could be limited by hydraulic
segmentation, which represents a hydraulic constriction or bottleneck of
the conduit network (Zimmermann, 1983; Tyree \& Ewer, 1991;
Levionnois\emph{et al.} , 2020). In a broad sense, hydraulic
segmentation has also been described as compartmentalisation,
connectivity, sectoriality, or modularity, and may include narrow
conduit dimensions and/or poorly interconnected conduits, which increase
the resistance of the hydraulic pathway (Ellmore \emph{et al.} , 2006;
Loepfe \emph{et al.} , 2007; Espino \& Schenk, 2009). It has frequently
been suggested that these constrictions of the hydraulic pathway may
cause a difference in embolism resistance, which is defined as
vulnerability segmentation (Tyree and Ewers, 1991; Levionnois \emph{et
al.} , 2020). Leaves, for instance, are said to be less embolism
resistant than stem xylem based on the vulnerability segmentation
hypothesis, although results across a broad range of species are mixed
and could partly be explained by the different methods used (Zhu
\emph{et al.} , 2016; Klepsch \emph{et al.} , 2018; Skelton \emph{et
al.} , 2018; Levionnois \emph{et al.} , 2020). Also, it has not been
tested yet whether vulnerability segmentation is affected by
pre-existing embolism.

In a few studies, considerable differences in embolism resistance have
been reported between intact plants and xylem tissue. Cut-open stem
xylem of \emph{Vitis vinifera} and \emph{Laurus nobilis} , for instance,
were suggested to underestimate embolism resistance (Choat \emph{et al.}
, 2010, Torres-Ruiz \emph{et al.} , 2015; Lamarque \emph{et al.} ,
2018). Removal of leaves in seedlings of the ring-porous
species\emph{Quercus robur} was found to result in artificial embolism
formation in stem xylem based on microCT (Choat \emph{et al.} , 2016).
In a few species, however, the bench dehydration method, which is a
widely applied method for hydraulic estimations of embolism resistance,
was found to show no difference in embolism resistance between cut,
dehydrating branches and dehydration of intact plants of
\emph{Quercus}and \emph{Populus} (Br\selectlanguage{ngerman}éda \emph{et al.} , 1993; Tyree
\emph{et al.} , 1992; Skelton \emph{et al.} , 2018). While more species
need to be studied to understand a possible artefact associated with
embolism spreading from cut-open xylem, two explanations could be
suggested for this observed discrepancy, i.e. why cut-open xylem may
reduce embolism resistance and facilitate embolism propagation. First,
it is possible that the cutting of conduits with sap under negative
pressure introduces a cutting artefact, with embolism formation due to a
sudden pressure drop (Wheeler \emph{et al.} , 2013; Torres-Ruiz \emph{et
al.} , 2015). Second, embolism spreading could be prevented by hydraulic
segmentation, which may occur at growth rings, nodes, and the transition
between organs, such as leaf petioles or side branches (Sano \emph{et
al.} , 2011; Levionnois \emph{et al.} , 2020). Indeed, vessels are known
not to run completely randomly, but may end near nodes, side branches,
stem-petiole transitions, and between the vascular bundles of the
petiole and major veins (Salleo \emph{et al.} , 1984; André \emph{et
al.} , 1999, André, 2005, Wolfe \emph{et al.} , 2016).

In this paper, we aim to test to what extent cut-open angiosperm xylem
has an effect on embolism spreading in leaves across a diverse selection
of six temperate species. In the first and the second experiment we
investigate if embolism resistance of leaf xylem was affected by the
proximity to cut-open conduits. We hypothesise that leaf xylem would be
more vulnerable to embolism for detached leaves with a cut petiole
compared to leaves attached to stem segments. However, not only the
proximity to cut-open vessels, but also hydraulic segmentation at the
stem-leaf, or the petiole-leaf blade transition could affect embolism
spreading, and may prevent a potential artefact in measurements of
embolism resistance near cut xylem tissue. We included species with both
deciduous and marcescent leaves (i.e. species that retain dead leaves on
the plant), and diffuse porous and ring-porous wood, because hydraulic
segmentation can be associated with leaf phenology and vessel
dimensions. If pit membranes in bordered pits of vessels and tracheids
would function as safety valves that avoid the spreading of embolism
from embolised to functional conduits, it is possible that embolism
spreading is reduced by the number of interconduit endwalls and/or the
connectivity between conduits (Kaack \emph{et al.} , 2019;
Johnson\emph{et al.} , 2020). Species that show hydraulic segmentation,
may have safety valves composed of many tracheids and/or narrow,
fibriform vessels.

Since drought-induced embolism is frequently reported to initiate in
large vessels, while narrow and short vessels or tracheids embolise
typically later at lower xylem water potentials (Scoffoni \emph{et al.}
, 2017; Klepsch \emph{et al.} , 2018), we tested if embolism spreading
in minor veins with narrow and short conduits would also be affected by
the proximity to a cut-open vein. In this second experiment, we expect
that narrow and short vessels near cut minor veins would embolise before
embolism occurs in the large vessels of major veins, which would make
narrow vessels seemingly more vulnerable than wide ones. We also predict
that embolism spreading in minor veins near artificially induced cuts
has a rather limited, local distribution due to the short dimensions of
minor veins.

Finally, we aimed to test whether or not embolism spreading differs
between intact vessels that are close to cut-open vessels in a petiole,
and intact vessels in leaf veins that are located further away from
cut-open xylem. For this reason, the Pneumatron and the optical
vulnerability method were applied to the same detached leaf. If the
pneumatic method would be subject to a potential artefact due to gas
extraction from intact vessels that are neighbouring embolised, cut
conduits, this method could systematically underestimate embolism
resistance compared to the optical method. The pneumatic method, which
estimates the changing gas volume in intact vessels during dehydration,
showed a good agreement with hydraulic methods applied to stem segments
(Pereira \emph{et al.} , 2016, Zhang \emph{et al.} , 2018). Direct
comparison of the pneumatic and optical method to detached leaves
of\emph{Eucalyptus camaldulensis} suggested no significant difference
for this species (Pereira \emph{et al.} , 2020a), although a larger
number of species should be tested to confirm this finding.

The three complementary sets of experiments will help us to address the
question of whether embolism spreading in angiosperm xylem relies on a
certain pressure difference threshold between embolised and functional
conduits.

\textbf{Materials and methods}

\textbf{Study site and plant material}

The six angiosperm species studied included \emph{Betula pendula}
,\emph{Carpinus betulus} , \emph{Fagus sylvatica} , \emph{Liriodendron
tulipifera} , \emph{Prunus avium} , and \emph{Quercus petraea} . Samples
were collected at Ulm University, Germany (48°25'20.3'' N, 9°57'20.2''
E). Except for \emph{L. tulipifera} , all species are common, native
angiosperm trees to Ulm. Various individuals of \emph{L. tulipifera}were
grown at the Botanical garden of Ulm University. Most species studied
were diffuse porous, except for \emph{Q. petraea} , which was
ring-porous. Also, \emph{C. betulus} and \emph{Q. petraea} were
considered as species with marcescent leaves during winter, with partial
marcescent leaves in \emph{F. sylvatica} . The remaining three species
are deciduous. We selected four to five mature trees for each species.
Healthy, mature and sun-exposed branches were sampled between June and
September 2019 for experiment 1 and 3, and in May 2020 for experiment 2.

\textbf{Vessel length measurements}

Maximum vessel length of stems and leaf petioles was determined by
applying the air injection method (Greenidge, 1952). After connecting
the basipetal part of a stem or petiole to a syringe, a ca. 150 kPa
pressure was applied, while the acropetal part was kept under water.
Successive cuts at the proximal stem/petiole end were made under water
with a razor blade at intervals of 10 mm for stems and 2 mm for leaves
until the first continuous stream of air bubbles could be seen emerging
from the cut end. The corresponding length was then measured and
recorded as the maximum vessel length of a stem or leaf petiole
(MVL\textsubscript{stem} and MVL\textsubscript{petiole}, respectively).
At least six stems or leaves were taken for these measurements (Figure
S1).

We measured the maximum vessel length at the stem-leaf transition for
leaves attached to 0.5 cm long stem segments. This approach was applied
to six samples per species based on the air injection method. The
syringe was connected to the short stem sample, and the leaf was
shortened until bubbles could be seen emerging from the cut end.

The vessel length distribution of leaf xylem was measured with a
Pneumatron device (Pereira \emph{et al.} , 2020b). Instead of injecting
air (Cohen \emph{et al.} , 2003; Wang \emph{et al.} , 2014; Pan \emph{et
al.} , 2015), the amount of gas that could be sucked up via cut-open
conduits allowed us to measure the air conductivity of open vessels
while shortening leaf petioles. We then plotted the air conductivity of
the cut-open vessels against the petiole length. The average vessel
length was obtained by fitting the vessel length equation from
Sperry\emph{et al.} (2005) to our data.

We defined a segmentation index as the maximum vessel length at the
petiole end divided by the petiole length. This index indicated to what
extent the longest vessels from the petiole end run into the leaf blade.
Values \textless{} 1 indicated that vessels ended before the leaf blade
started, while values \textgreater{} 1 suggested that at least some
vessels starting at the petiole end run directly into the midrib of the
leaf blade.

\textbf{Xylem embolism resistance of leaves}

\textbf{Sample preparation}

All samples were collected before 9:00 in the morning. After cutting
samples from a tree in air, large branches were immediately put in a
water-filled bucket and a ca. 20 cm stem segment was cut from the stem
base under water to avoid air entry as much as possible. The length of
the remaining branch samples was between 80 and 160 cm, which was much
longer than the maximum vessel length (MVL\textsubscript{stem}). Samples
were covered up with a dark plastic bag, transferred to the lab within
10 min, and then rehydrated for more than two hours.

\textbf{The optical method}

The optical method following Brodribb \emph{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 cm\textsuperscript{2}. 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 \emph{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 PEP\textsubscript{50} representing the xylem
water potential corresponding to 50\% of total embolised pixels
(Brodribb \emph{et al.} , 2016b).

\textbf{Pneumatic measurements}

An automatic Pneumatron apparatus was applied to measure gas diffusion
kinetics of desiccating leaves (Pereira \emph{et al.} , 2020a,
Jansen\emph{et al.} , In press). The principle of this apparatus was
similar to the manual approach of Pereira \emph{et al.} (2016) and Zhang
\emph{et al.} (2018), but with a much higher accuracy and temporal
resolution. When samples for pneumatic measurements were prepared, the
cut-open conduits embolised intentionally, which happened quickly by
shaving the sample ends with a fresh razor blade in air.

Pneumatic measurements were taken every 15 min. For this a 40 kPa of
absolute pressure was created with a vacuum pump, which extracted gas
from a petiole end via a solenoid valve. The amount of gas extracted in
a discharge tube with known volume could be measured with a pressure
sensor. The vacuum pump reached 40 kPa (i.e., the initial pressure
P\textsubscript{i}) within less than a second. Pressure data were
recorded in a SD card at a time interval of 500ms. The final pressure
(P\textsubscript{f}) was taken after 30s. According to the ideal gas
law, the moles of air extracted from vessels (\selectlanguage{greek}Δ\selectlanguage{english}n) could be calculated
following the equation below:

\selectlanguage{greek}Δ\selectlanguage{english}n = n\textsubscript{f}-n\textsubscript{i} =\(\frac{(Pf-Pi)\times V}{\text{RT}}\) (Eqn 1)

where n\textsubscript{i} and n\textsubscript{f} represented the moles of
air molecules at the initial and final pressure, V was the fixed volume
of the discharge tube (1.1 mL), R was the gas constant (8.134 kPa L
mol\textsuperscript{-1} K\textsuperscript{-1}), and T was the room
temperature in the lab (around 25\selectlanguage{ngerman}°C). Since a small V is needed to
increase the measurement precision when a tiny amount of air is sucked
from plant tissue, which is the case for detached leaves
(Pereira\emph{et al.} , 2020a, Jansen \emph{et al.} , In press), V was
estimated as the maximum gas volume that could be extracted when leaves
were fully dehydrated (AD\textsubscript{max}, see below) divided by
510.2 (Pereira\emph{et al.} , 2020a). The volume of air discharged (AD,
\selectlanguage{greek}µ\selectlanguage{english}L) from vessels could then be calculated based on the ideal gas law,
with P\textsubscript{atm} being the atmospheric pressure: AD =
10\textsuperscript{6}\selectlanguage{ngerman}×\selectlanguage{greek}Δ\selectlanguage{english}n R T/ P\textsubscript{atm} (Eqn 2),

Finally, the Percentage of Air Discharged (PAD, \%) was calculated:

PAD =
100\selectlanguage{ngerman}×(AD-AD\textsubscript{min})/(AD\textsubscript{max}-AD\textsubscript{min})
(Eqn 3)

where AD\textsubscript{min} was the minimum volume of air discharged
when the leaf was well hydrated, and AD\textsubscript{max} was the
maximum volume of air discharged when the leaf was strongly dehydrated.

Vulnerability curves were generated by plotting PAD or PEP against the
corresponding leaf water potential (\selectlanguage{greek}Ψ\selectlanguage{english}), with a fitting by the following
equation (Pammenter and Vander Willigen, 1998):

PAD or PEP =100/ (1+exp (\emph{S} /25) (\selectlanguage{greek}Ψ-\selectlanguage{english}P\textsubscript{50})) (Eqn 4)

\emph{S} represented the slope of the fitted curve, and
P\textsubscript{50} represented the water potential at 50\% of air
discharged, or 50\% of the total embolised pixels of the leaf area
scanned. Values of P\textsubscript{12} (water potential at 12\% of air
discharged or embolised pixels) and P\textsubscript{88} (water potential
at 88\% of air discharged or embolised pixels) were calculated following
the equations by Domec and Gartner (2001):

P\textsubscript{12} = 2/(\emph{S} /25) +P\textsubscript{50} (Eqn 5)

P\textsubscript{88} = -2/(\emph{S} /25) +P\textsubscript{50} (Eqn 6)

\textbf{Water potential measurements}

Psychrometers (PSY1, ICT International, Armidale, NSW, Australia) were
applied to obtain water potential values from the leaves that were
simultaneously measured with the optical and pneumatic method. Sand
paper with a grit size of 400 was used to carefully remove a small area
(10 mm\textsuperscript{2}) of leaf cuticle under a stereomicroscope,
while paying special attention not to abrade the vascular bundles and
introduce air-entry into the xylem. Vaseline was used to seal the
psychrometer chamber to the abraded surface of the leaf. Water potential
measurements were taken every 10 min, and the measurements were stopped
when leaves were completely desiccated, or the water potential showed no
further decrease over a long period. The duration of measurements was
between one and two days.

At the beginning of each dehydration experiment, the leaf water
potential dropped rapidly within one hour. After that, the water
potential decreased slowly and steadily. To test the accuracy of the
xylem water potential values taken with psychrometers, we applied two
different approaches. Firstly, for leaves that were attached to long
branches, we measured not only leaf xylem water potential, but also stem
xylem water potential with another stem psychrometer (Fig. S2). This
comparison provided a reasonably good match between both organs,
although differences became more pronounced at high levels of
dehydration for some species, with xylem water potential decreasing more
quickly for leaves than stems. An exception was \emph{F. sylvatica} ,
which had more negative xylem water potentials for leaves than stems
when water potentials in leaves were less negative than -5 MPa.
Secondly, water potential values of detached leaves that were measured
with the Pneumatron and optical method were compared to a second set of
leaves. Therefore, two leaves with a similar size were excised from the
same branch at the same time. One leaf was attached to a psychrometer,
while the other one was left to dry under similar conditions (same light
intensity, temperature, and humidity). The water potential of the second
leaf was measured with a pressure chamber (PMS Instrument Company,
Albany, OR, USA) at an interval of 20 to 60 min to validate the accuracy
of the stem psychrometer measurements during the first hours of
dehydration. These tests revealed an overall good agreement between both
methods for three species tested (\emph{C. betulus} , \emph{F.
sylvatica} , and \emph{Q. petraea} ; Fig. S3).

\textbf{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. \emph{L. tulipifera}and
\emph{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 \emph{F. sylvatica} , \emph{P. avium} ,
and\emph{C. 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
(3\textsuperscript{rd} or 4\textsuperscript{th} 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.

\textbf{Data analysis}

Vulnerability curves were plotted and fitted using SigmaPlot 14 (Systat
Software Inc., Erkrath, Germany). After testing data for normal
distribution and homogeneity of variance, a one-way ANOVA was applied to
test for significant difference between xylem embolism resistance of a
detached leaf, and a leaf attached to a short or long branch. An
independent t-test was applied to determine whether the optical and
pneumatic method differed. Statistics were performed in SPSS 22 (IBM,
Armonk, New York, USA), and all graphs were drawn in SigmaPlot.

\textbf{Results}

\textbf{Petiole vessel length measurements}

\emph{L. tulipifera} and \emph{Q. petraea} had the longest petiole
vessels of the six species studied, with an average length of 8.19 \selectlanguage{ngerman}±
1.47 and 8.02 ± 1.33 cm (mean ± standard deviation), respectively.
Despite having the longest petioles and the largest leaf surface area,
vessels of \emph{L. tulipifera} were always shorter than the petiole
length, indicating that vessels at the base of the petiole ended well
before the base of the lamina. Cut-open vessels in the petiole of
\emph{B. pendula} also ended before the base of the lamina. Petiole
vessels of \emph{Q. petraea} , however, were found to run from the
petiole base up to the middle of the midrib. A similar observation was
found for cut-open vessels in \emph{F. sylvatica} and \emph{P. avium} ,
which reached to half the length of the midrib. \emph{C. betulus} had
petiole xylem with vessels that were only slightly longer than the
petiole (Table 1).

\emph{Q. petraea} had the longest average vessel length in petiole
xylem, with a value of 5.1 ± 0.9 cm, which was followed by \emph{L.
tulipifera} (3.52 ± 1.17 cm). The mean vessel length in petioles
of\emph{B. pendula} , \emph{F. sylvatica} , and \emph{C. betulus} was
shorter than 1 cm. Since the petiole of \emph{P. avium} had a pronounced
notch, we were unable to obtain accurate vessel length measurements for
this species with the Pneumatron, even when using glue or parafilm to
avoid any leakage (Table 1).

\textbf{Experiment 1: Does embolism spreading depend on the proximity to
cut conduits?}

The shape of the vulnerability curves obtained was consistently
sigmoidal for the three sample types, i.e. detached leaves, leaves
attached to a short stem segment, and leaves attached to a long stem
segment (Figure 1). Embolism expansion in leaf veins started typically
in major veins and proceeded to minor veins (Figure S4). We did not see
different patterns in the progression of embolism formation among
detached leaves and leaves attached to a short or long stem segment.

There was considerable variation in the PEP\textsubscript{12},
PEP\textsubscript{50} and PEP\textsubscript{88} values among the three
types of samples for several species. Comparison of detached leaves with
leaves attached to short stem segments showed a significant difference
(\emph{P} \textless{} 0.05) in PEP\textsubscript{50} for \emph{C.
betulus} , \emph{F. sylvatica} , \emph{P. avium} and \emph{Q.
petraea}(Figure 1, Table S2). Detached leaves of these species showed a
ca. 1.5 MPa less negative PEP\textsubscript{50} value compared to
PEP\textsubscript{50} values of leaves on short stem segments. A minor
difference in PEP\textsubscript{50} with no significant difference was
obtained for \emph{L. tulipifera} and \emph{B. pendula} (Figure 1, Table
S2).

A positive, exponential correlation (\emph{R²} = 0.52,
\emph{P}\textless{} 0.05) was found for the shift in embolism resistance
between detached leaves and leaves attached to a short stem segment, and
the segmentation index of leaf xylem (Figure 2). Leaves with a
segmentation index \textgreater{} 1 were strongly affected by the
cut-open vessels at the petiole end, resulting in a shift in
PEP\textsubscript{12}, PEP\textsubscript{50} and PEP\textsubscript{88}
of 1MPa or more between detached leaves and leaves attached to stem
segments. \emph{L. tulipifera} and \emph{B. pendula} , which had all
vessels ending within their petiole, were clearly less affected by the
proximity to cut xylem conduits compared to the four other species with
vessels running from the petiole end into the midrib.

A significant difference in PEP\textsubscript{12} and
PEP\textsubscript{50} was also found between leaves attached to a short
branch, and those on a long branch for \emph{F. sylvatica} . No
significant difference in xylem embolism resistance was found between
leaves on a short stem segment and leaves on a long stem piece for the
other species studied, except for PEP\textsubscript{88} values
of\emph{B. pendula} and \emph{P. avium} . Any similarity or
dissimilarity in PEP\textsubscript{50} between the three types of
samples was mostly reflected in PEP\textsubscript{12} and
PEP\textsubscript{88} (Figure 1, Table S2).

\textbf{Experiment 2: Does the proximity to cut conduits also affect
embolism spreading in minor leaf veins?}

The percentage of cumulative embolised xylem area in leaves with
cut-open minor veins and intact leaves was plotted against time for the
six species studied (Figure S5). For leaves with several artificial cuts
in the 3\textsuperscript{rd} or 4\textsuperscript{th} order veins, no
immediate embolism formation was detected after the cutting, except for
the vessels that were cut open. In all six species, however, we observed
embolism events in minor veins adjacent to the cuts during the first
hours of desiccation (Figure 3, Figure S4). These embolism events in
minor veins, with relatively narrow and short vessels, occurred prior to
embolism formation in the wide and long vessels of major veins. Since
the amount of embolised pixels near the cuts had a local effect only and
was relatively small compared to the total amount of embolised pixels
that could be detected in the entire leaf area, there was no large
overall difference in embolism spreading between intact and cut leaves.
Therefore, the embolism spreading patterns did not differ when the
percentage of embolised pixels of the entire scanned leaf area was
plotted against time. After artificially induced embolism events
occurred near the cuts, embolism spreading followed in the major veins,
and then spread to the minor veins in both intact and cut leaves (Figure
3).

\textbf{Experiment 3: Is the Pneumatron underestimating embolism
resistance compared to the optical method?}

The optical method and Pneumatron were simultaneously applied to the
same detached leaves for all six species. We were unable to obtain
Pneumatron measurements for \emph{B. pendula} , because the gas volume
that could be extracted from the leaves was too small to meet the
sensitivity requirements of the tube volume, even if we reduced the
volume of the discharge tube to the minimum volume of 1.1 mL.

Vulnerability curves based on the two methods were obtained for five
species (Figure 4). The PEP\textsubscript{12}, PEP\textsubscript{50} and
PEP\textsubscript{88} values of detached leaves obtained with the
optical method in experiment 1 (Fig.1, Table S2) did not differ
significantly from those obtained in experiment 3 (Table S3). A
difference in embolism spreading from cut conduits to intact ones versus
spreading between intact conduits would especially be reflected in
PAD\textsubscript{12} and PEP\textsubscript{12} values. Indeed, average
PAD\textsubscript{12} values based on the Pneumatron were lower than the
average PEP\textsubscript{12} values based on the optical method for
four species, except for \emph{P. avium} . The difference between
PAD\textsubscript{12} and PEP\textsubscript{12} was more than 0.6 MPa
for \emph{Q. petraea} , \emph{F. sylvatica} , and \emph{C. betulus} ,
although these differences were not statistically significant. Also,
there was considerable variation among the samples tested (Fig. 5a),
with several samples showing a slightly more negative
PAD\textsubscript{12} value than PEP\textsubscript{12} for \emph{P.
avium} and \emph{L. tulipifera} .

Despite minor differences between PEP\textsubscript{12} and
PAD\textsubscript{12}, the vulnerability curves based on the optical and
pneumatic method showed strikingly similar patterns for the five species
tested, with a strong correlation between PAD\textsubscript{50} and
PEP\textsubscript{50}, and between PAD\textsubscript{88} and
PEP\textsubscript{88} (Fig. 5b, c). When intraspecific differences in
embolism resistance were found within a species, both methods matched
each other very well (Figure 4, 5, Figure S6). Although curves based on
the optical method had a relatively steeper slope compared to the
pneumatic curves for \emph{F. sylvatica} , \emph{C. betulus} and
\emph{Q. petraea} , this difference was not significant. No significant
difference was found between PAD\textsubscript{50} and
PEP\textsubscript{50}, and between PAD\textsubscript{88} and
PEP\textsubscript{88}. Nevertheless,\emph{P. avium} showed a 0.67 MPa
difference (\emph{P} = 0.287) between PEP\textsubscript{50} and
PAD\textsubscript{50}, and a 0.61MPa difference (\emph{P =} 0.454)
between PEP\textsubscript{88} and PAD\textsubscript{88} (Table S3).

\textbf{Discussion}

Experiment 1 and 2 show that spreading of drought-induced embolism and
thus embolism resistance can be strongly affected by the proximity of
the xylem area studied to pre-existing embolism such as cut xylem
tissue. The observation that embolism initiation (PEP\textsubscript{12})
occurs over a \textgreater{} 1MPa range of xylem water potential for
four out of six species studied challenges the assumption that embolism
spreads once a certain threshold difference has been reached. While
cut-open conduits may facilitate embolism spreading, hydraulic
segmentation may limit this potential artefact. As predicted, embolism
spreading in minor veins was also affected by the proximity to cut
conduits, but showed a rather limited, local distribution without
affecting the entire leaf. Also, we found no difference between the
optical and pneumatic method for five species, despite considerable
variation in the P\textsubscript{12} values of both methods. The
consequences and broader significance of these findings present some old
questions and assumptions in a new light, and provides a novel gas
diffusion hypothesis as a possible mechanism for embolism spreading.

\textbf{Embolism spreading depends on pre-existing embolism}

Since embolism spreading happens largely from one embolised conduit to a
neighbouring one, as predicted by the air-seeding hypothesis
(Zimmermann, 1983; Sperry and Tyree, 1988), the observation that
embolism formation may depend on proximity to an existing gas source
such as cut-open conduits or pre-existing embolised conduit is not
surprising. Embolism formation appears to be unlikely if a conduit is
not connected to a pre-existing embolism. Novel, de novo embolism
formation has been observed in very few conduits that are not connected
to embolised ones based on microCT (Brodersen \emph{et al.} , 2013,
Choat\emph{et al.} , 2015, 2016), and embolism formation in seemingly
isolated conduits could occasionally be observed in our experiments.
However, the rather two-dimensional view associated with the optical
method, its limited resolution to accurately detect narrow vessel ends
(Oskolski and Jansen, 2009), and its shortcoming to detect pre-existing
embolism, did not allow us to confirm that these conduits were
completely disconnected from neighbouring gas sources.

If availability of a pre-existing gas source or embolised conduit is
important, then where does the gas come from to induce embolism in
intact xylem? It is possible that there is almost always an embolised
conduit available, perhaps in primary xylem or in older xylem from an
older growth ring. This would be an obvious gas source if functional,
sap-filled conduits show any direct connection with these embolised
conduits via bordered pits. However, these pre-existing gas sources may
be limited due to poor connectivity or compartmentalisation of the
hydraulic network (Kitin \emph{et al.} , 2004; Morris \emph{et al.} ,
2016). Since vessels and tracheids do not share pits with non-conductive
fibres (Sano \emph{et al.} , 2011), it is unlikely that air entry from
these cells or intercellular spaces will contribute to embolism
formation in conduits.

\textbf{Hydraulic segmentation reduces embolism spreading in xylem
tissue}

While the proximity of a studied xylem area to cut conduits seems to be
important, the speed of embolism spreading over a certain distance also
depends on the vessel dimensions. If each individual vessel would
embolise separately (Johnson \emph{et al.} , 2020), wide and long
vessels would show a faster propagation of embolism over a given
distance than narrow, short vessels. Spreading of embolism would
especially be reduced in xylem with a high degree of hydraulic
constrictions, making xylem patches at the distal side of embolised
conduits seemingly more resistant to embolism. The four species that
showed a reduced embolism resistance in detached leaves as compared to
leaves attached to branches, have open vessels running directly from the
base of the petiole into the midrib (Table 1, Figure 2). Since the
maximum vessel length in petioles of \emph{L. tulipifera} and \emph{B.
pendula} were shorter than the petiole length (Table 1), both species
showed relatively small differences in embolism resistance between
detached leaves and leaves attached to a stem segment (Fig. 1c, e).

Wide and long vessels in the midrib and secondary veins were found to
embolise before the high 3\textsuperscript{rd} to
5\textsuperscript{th}vein orders. This pattern confirms various studies
based on the optical method and microCT observations (Brodribb \emph{et
al.} , 2016a, b; Scoffoni \emph{et al.} , 2017; Klepsch \emph{et al.} ,
2018). However, the observation of local spreading of embolism in minor
veins near cut vessels in experiment 2 supports the hypothesis that
embolism can spread from pre-existing gas sources, and in minor veins
prior to embolism formation in the large vessels of major veins (Fig.
3). This observation suggests that proximity to a gas source is the main
driver for embolism spreading, and not the conduit diameter per se.
Nevertheless, the limited and short dimensions of minor veins (Chatelet
\emph{et al.} , 2006; Lechthaler \emph{et al.} , 2019; Hua \emph{et al.}
, 2020) are a plausible explanation for why artificially induced
embolism near cuts in minor veins propagate locally, rather than spread
across the lamina. Short conduits in minor vein orders may have been
selected for in order to maximise leaf hydraulic function over the life
span of a leaf. Minor orders of leaf veins are believed to be prone to
xylem embolism formation over the life of a leaf due to localized damage
caused by insects, pathogens or mechanical damage to the lamina, and
because they sustain the most negative pressures (Brodribb \emph{et al.}
, 2010). Considerable restriction to the extent of embolism spread
through minor vein orders would ensure a limited impact on the hydraulic
capacity of a leaf that sustains localised damage to the lamina.

It is also possible that wide and long vessels are more likely connected
to a pre-existing embolism than narrow, short conduits, and that large
vessels show a higher amount of intervessel pit membrane area than
narrow, short vessels. In other words, the reason why large and wide
vessels are likely to embolise first, may reflect a difference in the
rate of air entry, which is caused by their connectivity to a gas
source, and not any inherent difference in embolism resistance per se.
We are not aware of an alternative mechanism that would explain why wide
conduits are more vulnerable to embolism than narrow ones. Pit membrane
thickness, which is strongly associated with embolism resistance
(Li\emph{et al.} , 2016; Kaack \emph{et al.} , 2019), was found not to
be related to conduit diameter (Kotowska \emph{et al.} , 2020; Wu
\emph{et al.} , 2020).

\textbf{Vulnerability segmentation may reflect hydraulic segmentation,
but not intrinsic differences in embolism resistance per se}

The observation that embolism formation in leaves of an individual tree
may occur under highly variable (\textgreater{} 1 MPa) xylem water
potentials, suggests that embolism resistance may represent a relative
trait that does not capture the absolute, intrinsic embolism resistance
of its xylem. It is likely that hydraulic segmentation includes highly
reduced conduit dimensions, especially with respect to conduit length
and width, with a high number of interconduit end walls over a short
stretch of xylem tissue. Conduit end walls have been suggested to hold
up embolism spreading at least temporarily, with pit membranes
functioning as safety valves and preventing further spreading of
embolism due to their tiny pores (Zhang \emph{et al.} , 2017, 2020;
Kaack\emph{et al.} , 2019; Johnson \emph{et al.} , 2020). Moreover,
narrow and short tracheids or fibriform vessels may be more confined
than long and wide vessels, with a small interconduit pit membrane area
for air entry in narrow tracheids.

Quantifying embolism resistance across the entire xylem pathway could be
complicated by measuring artefacts or the proximity to pre-existing
embolism, such as cut xylem. In our study, comparison of xylem embolism
resistance of leaves attached to long stem segments with embolism
resistance of stem xylem based on previous papers (Klepsch \emph{et al.}
, 2018; Zhang \emph{et al.} , 2018), indicates that leaf xylem of all
six species was between 0.5 and \textgreater{} 1 MPa more embolism
resistant than stem xylem. Our result for \emph{B. pendula} was
consistent with Klepsch \emph{et al.} (2018), with leaf xylem being more
resistant than stem xylem. Most angiosperms species, however, showed
that stem xylem was either more embolism resistant, or equally resistant
than leaf xylem (Zhu \emph{et al.} , 2016; Skelton \emph{et al.} , 2018;
Losso \emph{et al.} , 2019). Therefore, caution is needed to directly
compare absolute values of embolism resistance between organs, since
measured values of embolism resistance could be relative estimations
only, especially if destructive methods are used and cut-open xylem
accelerates embolism spreading.

\textbf{Embolism spreading may not rely on a fixed pressure difference
threshold, but on pressure-driven diffusion}

The observation of embolism spreading under highly variable xylem water
potentials in experiment 1 and 2 indicates that embolism formation may
not always rely on a certain threshold of the pressure difference
between a functional and embolised conduit, as frequently assumed based
on the air-seeding hypothesis, and quantitatively estimated by the
Young-Laplace equation (Sperry and Tyree, 1988; Choat \emph{et al.} ,
2008). Even if some of our xylem water potential measurements may not be
fully accurate due to a pressure gradient heterogeneity (Bouda \emph{et
al.} , 2019), the \textgreater{} 1 MPa difference in
PEP\textsubscript{50} values between detached leaves and leaves
connected to stems provides solid evidence that embolism formation in
xylem tissue from the same organ of a species may occur under different
xylem water potentials. This finding is not entirely new, and in line
with earlier differences in embolism resistance between intact plants
and cut plants (Choat \emph{et al.} , 2010; Torres-Ruiz \emph{et al.} ,
2015; Lamarque \emph{et al.} , 2018). For instance, a ca. 4 MPa
difference in P\textsubscript{50} was found for \emph{Laurus
nobilis}based on microCT observation of cut branches and intact
seedlings (Nardini \emph{et al.} , 2017; Lamarque \emph{et al.} , 2018).
Similar to our results, Skelton \emph{et al.} (2018) compared the
vulnerability curves of cut branches and intact plants based on the
optical method for\emph{Quercus wislizenii} , and found a 1.5 MPa
difference in P\textsubscript{50} between leaves attached to a long, cut
branch, and leaves from an intact plant. The finding that cut plant
material can be more vulnerable to embolism spreading than intact plants
raises concerns about embolism resistance measurements of plant material
samples with pre-existing embolism, the possible induction of embolism
due to cutting, and the application of the bench dehydration method on
cut plant material (Sperry and Tyree, 1988).

If embolism spreading for given species may not rely on a certain
pressure threshold, which mechanism does then trigger embolism? We
believe that this is a highly important question in our understanding of
water transport, even though this question cannot be fully answered
based on the available evidence. We speculate that gas diffusion across
interconduit pit membranes plays a role in determining the amount of gas
dissolved in xylem sap, which may affect embolism nucleation. Although
mass flow is theoretically 10\textsuperscript{5} times faster than
diffusion, gas diffusion in xylem is overall much faster and more common
than mass flow. The main reason seems to be that gas diffusion takes
place continuously over very large areas, while mass flow requires gas
movement through multi-layered, tiny pore constrictions of mesoporous
pit membranes (Kaack \emph{et al.} , 2020). However, the continuous
nature of gas diffusion and the high amounts of gas
(CO\textsubscript{2}and O\textsubscript{2}) surrounding conduits (Spicer
and Holbrook, 2005; Teskey \emph{et al.} , 2008) do not mean that gas
concentration in xylem sap is always under equilibrium with gas in
embolised conduits, as has frequently been assumed (Hammel 1967; Yang
and Tyree, 1992; Wheeler\emph{et al.} , 2013; Schenk \emph{et al.} ,
2016). Gas solubility of xylem sap is affected by pressure and
temperature (Mercury \emph{et al.} , 2003; Schenk \emph{et al.} , 2016;
Lidon \emph{et al.} , 2018). Due to a nearly constant atmospheric
pressure in a cut-open vessel, but a considerably variable liquid water
pressure in sap-filled conduits, the driving force for gas diffusion is
the unbalanced, anisobaric situation between embolised conduits and
functional conduits. We ignore here the effect of temperature on gas
solubility of xylem sap as all experiments were conducted in an
air-conditioned lab under similar temperature (Schenk \emph{et al.} ,
2016). Moreover, oversolubility of gas may occur in nanoporous cell
walls and mesoporous pit membranes, as is known for nanoconfined
environments in general (Pera-Titus \emph{et al.} , 2009; Ho\emph{et
al.} , 2015; Coasne \& Farrusseng, 2019), which could contribute to
concentration gradients of gas dissolved in xylem sap.

A recently embolised intact vessel is not immediately filled with gas
under atmospheric pressure, but eventually achieves equilibrium of its
gas pressure with the atmosphere, depending on how fast gas is attracted
from surrounding gas sources via diffusion. While cut-open vessels are
immediately filled with air and reach atmospheric pressure immediately,
intact vessels that embolise are initially filled with water vapour (ca.
2.4 kPa). It has been modelled that gas diffusion takes from 20 min to
several hours to obtain atmospheric pressure (ca. 101.3 kPa) in
embolised, intact vessels, which may depend on the distance to the
nearest gas phase, and the interconduit pit membrane area for gas
diffusion (Yang \& Tyree, 1992; Wang \emph{et al.} , 2015a, b). Although
gas diffusion happens across conduit cell walls and pit membranes, the
micropores (\textless{} 2 nm) in hydrated walls are much smaller than
the 5 to 50 nm dimensions of pit membrane pores (Donaldson \emph{et al.}
, 2019; Kaack \emph{et al.} , 2019). Therefore, it is reasonable to
assume that gas diffusion across hydrated, 200 to 600 nm thick pit
membranes is much faster than across the much thicker layers of
secondary cell walls (Yang \emph{et al.} , submitted). Indeed, axial gas
diffusion in wood is found to be about one to two orders of magnitude
larger than radial diffusion (Sorz \& Hietz, 2006). Moreover, end-wall
resistivity of conduits has been suggested to be proportional to lumen
resistivity (Sperry \emph{et al.} , 2005; Hacke \emph{et al.} , 2006),
and conductance of gas increases to the 4\textsuperscript{th} power with
conduit diameter or pore diameter according to Hagen-Poiseuille's
equation.

Further research is clearly needed to investigate how gas diffusion may
contribute to the very high gas solubility of xylem sap (Schenk \emph{et
al.} , 2016), whether oversolubility occurs due to nanoconfined spaces
such as pit membranes and cell walls (Pera-Titus \emph{et al.} , 2009;
Ho\emph{et al.} , 2015; Coasne \& Farrusseng, 2019), how gas-xylem sap
interfaces are affected by the dynamic surface tension of xylem sap
lipids (Yang \emph{et al.} , 2020), and how surfactant-coated
nanobubbles may contribute to the gas concentration of xylem sap and
embolism formation (Schenk \emph{et al.} , 2015, 2017; Jansen \emph{et
al.} , 2018; Park \emph{et al.} , 2019).

\textbf{The optical method and Pneumatron show close similarity in
measuring embolism resistance}

We found a strong agreement between the optical method and the
Pneumatron, with no significant difference between either
P\textsubscript{12}, P\textsubscript{50} or P\textsubscript{88} values
obtained from the optical method and the Pneumatron in five deciduous
species (Figure 4, Table S2). This finding is in line with the results
from Pereira \emph{et al.} (2020a) for \emph{Eucalyptus camaldulensis} ,
and supports the hypothesis that the pneumatic method measures gas
extraction from embolism events in intact vessels (Jansen \emph{et al.}
, In press; Yang \emph{et al.} , submitted). The fast and
straightforward approach of taking pneumatic methods on small samples
such as individual leaves makes the Pneumatron device also suitable for
field observations and embolism resistance measurements at the
intraspecific and intratree level.

While a high coefficient of determination
(\emph{R\textsuperscript{2}}=0.91) indicated good agreement between
PEP\textsubscript{50} and PAD\textsubscript{50} values (Fig. 5b), the
vulnerability curves with the pneumatic method were less steep than with
the optical method for four species studied, except for \emph{L.
tulipifera} . A somewhat weaker, but still significant correlation was
found between the PEP\textsubscript{12} and PAD\textsubscript{12} values
based on both methods (Fig. 5a). It is possible that the presence of gas
in cut-open vessels affects the process of embolism spreading as
discussed above, and this may explain why the percentage of gas
discharged by the Pneumatron was slightly higher than the percentage of
the cumulative embolised pixels during early stages of dehydration in
four out of five species studied (Figure 3, Figure 5A). A similar
finding was reported in Pereira \emph{et al.} (2020a). However, the
opposite was found for\emph{P. avium} , which may have slower gas
diffusion due to its thick pit membranes as compared to the other
species (Kaack \emph{et al.} , 2020). If intact vessels become
embolised, but gas diffusion across pit membranes is slow, atmospheric
pressure will not be quickly reached in a recently embolised vessel
(Wang \emph{et al.} , 2015a, b).

The xylem area selected to apply the optical method was deliberately
chosen in the upper part of the leaf blade, where vessels in the leaf
veins are separated from cut vessels at the petiole end by at least one
and most likely various intervessel walls, as shown based on the maximum
vessel length in petioles. Since the Pneumatron extracts gas from the
petiole end, the tight similarity in embolism resistance between both
methods suggests that the gas extracted with the Pneumatron comes from
intact, embolised conduits. It is possible that there could be some
overlap between vessels from which gas is extracted with the Pneumatron
and those that are visualised with the optical method (Fig. 6). Yet, it
remains unclear over how many end walls the Pneumatron is able to
extract gas. We speculate that this number of end walls depends at least
partly on the pit membrane thickness, the complete or partial hydration
of the pit membrane, and whether or not porous medium characteristics of
interconduit pit membranes change during dehydration, since these would
determine gas diffusion considerably (Crombie \emph{et al.} , 1985;
Kaack\emph{et al.} , 2019; Zhang \emph{et al.} , 2020).

\textbf{Acknowledgements}

We thank Dr. H. Jochen Schenk (California State University, Fullerton)
and Lucian Kaack (Ulm University) for valuable comments on an early
version of this paper, Dr. Melvin Tyree (Zhejiang Normal University) for
useful discussions about pneumatic experiments, two anonymous reviewers
for helpful suggestions, and various colleagues from the botanical
garden of Ulm University for assistance with sampling. XG, KC, and SJ
acknowledge financial support by the German Research Foundation (DFG,
project nr. 410768178). SAMM acknowledges the Humboldt Foundation for a
postdoctoral fellowship at Ulm University. The authors declare that
there is no conflict of interest.

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\textbf{Tables}

\textbf{Table 1.} Overview of the petiole length and vessel length
dimensions for six temperate tree species studied. Maximum vessel length
measurements were based on air injection, while the average length of
vessels starting at the basipetal end of the petiole was estimated with
a Pneumatron. Values indicate mean +- standard deviation (n = 5 or 6).\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
& Petiole length (cm) & Max. vessel length at the petiole end (cm) &
Average vessel length at the base of the petiole (cm) & Average vessel
length at the base of the petiole (cm) & Max. vessel length in the stem
(cm) & Max. vessel length in the stem (cm) & Max. vessel length for
0.5cm stem + petiole + leaf (cm)\tabularnewline
\midrule
\endhead
\emph{B. pendula} & 2.03\selectlanguage{ngerman}±0.48 & 1.07\selectlanguage{ngerman}±0.26 & 0.83\selectlanguage{ngerman}±0.09 & 0.83\selectlanguage{ngerman}±0.09 &
13.64\selectlanguage{ngerman}±0.80 & 13.64\selectlanguage{ngerman}±0.80 & 0\selectlanguage{ngerman}±0\tabularnewline
\emph{C. betulus} & 1.37\selectlanguage{ngerman}±0.13 & 1.44\selectlanguage{ngerman}±0.30 & 0.96\selectlanguage{ngerman}±0.12 & 0.96\selectlanguage{ngerman}±0.12 &
12.50\selectlanguage{ngerman}±4.99 & 12.50\selectlanguage{ngerman}±4.99 & 0.37\selectlanguage{ngerman}±0.10\tabularnewline
\emph{F. sylvatica} & 1.23\selectlanguage{ngerman}±0.06 & 3.41\selectlanguage{ngerman}±0.27 & 0.66\selectlanguage{ngerman}±0.43 & 13.58\selectlanguage{ngerman}±3.87 &
13.58\selectlanguage{ngerman}±3.87 & 0\selectlanguage{ngerman}±0 & 0\selectlanguage{ngerman}±0\tabularnewline
\emph{L. tulipifera} & 8.68\selectlanguage{ngerman}±1.39 & 8.19\selectlanguage{ngerman}±1.47 & 3.52\selectlanguage{ngerman}±1.17 & 10.25\selectlanguage{ngerman}±0.58 &
10.25\selectlanguage{ngerman}±0.58 & 7.27\selectlanguage{ngerman}±1.76 & 7.27\selectlanguage{ngerman}±1.76\tabularnewline
\emph{P. avium} & 3.53\selectlanguage{ngerman}±1.00 & 5.47\selectlanguage{ngerman}±0.83 & / & 22.3\selectlanguage{ngerman}±1.97 & 22.3\selectlanguage{ngerman}±1.97 &
2.29\selectlanguage{ngerman}±1.18 & 2.29\selectlanguage{ngerman}±1.18\tabularnewline
\emph{Q. petraea} & 1.17\selectlanguage{ngerman}±0.21 & 8.02\selectlanguage{ngerman}±1.33 & 5.1\selectlanguage{ngerman}±0.90 & 80.38\selectlanguage{ngerman}±11.5 &
80.38\selectlanguage{ngerman}±11.5 & 5.78\selectlanguage{ngerman}±1.47 & 5.78\selectlanguage{ngerman}±1.47\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{Figure legends}

\textbf{Figure 1} . Leaf xylem vulnerability curves of six deciduous
species using the optical method applied to different samples: a
detached leaf (red), leaf attached to a short stem segment (blue), and
to a long branch (green). For each sample type, four to five replicates
were measured (pale colours). Bright lines are regression lines for each
type of sample, and grey lines indicate 95\% confidential intervals.
PEP\textsubscript{50} values (MPa) of the three types of samples were
presented with mean ± standard deviation (n = 4 or 5), with different
small letters showing significant difference between each sample type
(\emph{P} \textless{}0.05). Note that the curve fitting may give the
impression that embolism occurs at positive xylem pressures, while the
raw data make clear that embolism spreading never occurs at a xylem
water potential that is less negative than -0.1 MPa.

\textbf{Figure 2} . Relationship between the segmentation index and the
difference in PEP\textsubscript{12} (red), PEP\textsubscript{50}(yellow)
and PEP\textsubscript{88} (green) values between detached leaves and
leaves attached to a short stem segment. A segmentation index was
defined as the maximum vessel length divided by the petiole length, with
vessels running into the leaf blade when the value is \textgreater{} 1,
and the longest vessels ending before the leaf blade when the value is
\textless{}1.

\textbf{Figure 3} . Maps of embolism events between an intact leaf (A-F)
and a leaf with cuts in minor veins (G-L) of \emph{Quercus petraea} .
The cut and intact leaf pair were attached to the same long branch, with
images taken for both leaves after a certain desiccation time (shown on
the left). White solid lines represent artificial cuts of the
3\textsuperscript{rd} or 4\textsuperscript{th} veins, and white arrows
point at embolism in minor veins near the cuts, which happened much
earlier than embolism in major veins.

\textbf{Figure 4} . Xylem vulnerability curves of leaves of five
deciduous species based on the optical and pneumatic method. Both
methods were applied to the same detached leaf. Pale and bright red
lines represent raw data and regression lines of the optical method,
respectively. Blue lines represent raw data (pale blue) and regression
lines (bright blue) based on the pneumatic method. For each species,
four leaves were tested, and P\textsubscript{50} values (MPa) of both
methods were presented.

\textbf{Figure 5} . Correlation between paired values of
P\textsubscript{12} (a), P\textsubscript{50} (b), and
P\textsubscript{88} (c) as measured with the optical and pneumatic
method. Each dot represents a single leaf, solid black lines indicate
the regression line, blue dashed lines indicate the 95\% confidence
intervals, and black dashed lines indicate the 1:1 line.

\textbf{Figure 6} . Illustration of the optical method and pneumatic
measurements on detached leaves and possible embolism spreading during
dehydration after 5 and 10 hours. The Pneumatron was connected at the
basipetal end of the petiole, while the leaf area scanned with the
optical method (pale yellow square) is separated from the cut-open
vessels (white) by at least several end walls. (a) The initial status
when leaves were fresh, and all intact vessels were water-filled (blue).
(b, c) Embolism spreads to various intact vessels (green), from which
gas is extracted with the Pneumatron, and which are observed with the
optical method when these are within the scanned leaf area. It is
assumed here that the Pneumatron extracts gas across two end walls.
Arrows indicate gas diffusion through intervessel walls towards the
Pneumatron. Embolism formation in vessels only captured with the optical
method are coloured yellow. Adapted from Jansen \emph{et al.} (In
press).

\selectlanguage{english}
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