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 (Zhuet al. , 2013; Martin‐StPaul 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 et al. , 2018; Kaack 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 et al. , 2008;
Tixier et al. , 2014; Wason et al. , 2018; Avila et
al. , submitted). Although embolism spreading from previously embolised
conduits has been well presented in many textbooks and papers
(Zimmermann, 1983; Crombie et al. , 1985; Choat et al. ,
2016; Lamarque et al. , 2018), various basic questions about this
process remain unclear (Kaack 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 (Kitinet al. 2004; Sano et al. , 2011; Hochberg 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 (Ψ50, 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 (Pereiraet al. , 2016, 2020a; Zhang et al. , 2018; Oliveira 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 et al. , 2013; Hochberg 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 et al. , 2016a, b, 2017; Choat et al. ,
2016; Lamarque 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; Levionnoiset 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 et al. , 2006; Loepfe 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 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 et al. , 2016; Klepsch et al. ,
2018; Skelton et al. , 2018; Levionnois 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 Vitis vinifera and Laurus nobilis , for instance,
were suggested to underestimate embolism resistance (Choat et
al. , 2010, Torres-Ruiz et al. , 2015; Lamarque et al. ,
2018). Removal of leaves in seedlings of the ring-porous speciesQuercus robur was found to result in artificial embolism
formation in stem xylem based on microCT (Choat 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 Quercusand Populus (Bréda et al. , 1993; Tyree et al. ,
1992; Skelton 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 et al. , 2013; Torres-Ruiz 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 et al. ,
2011; Levionnois 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 et al. , 1984; André et
al. , 1999, André, 2005, Wolfe 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 et al. , 2019; Johnsonet 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 et al. ,
2017; Klepsch 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 et al. , 2016, Zhang et al. , 2018). Direct
comparison of the pneumatic and optical method to detached leaves ofEucalyptus camaldulensis suggested no significant difference for
this species (Pereira 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.