Introduction
Xylem sap in plants is frequently transported under negative pressure
(Dixon and Jolly, 1896; 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 efficiency of water, and therefore photosynthesis
(Zhu et 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 is based on two processes, namely mass flow and
diffusion, we prefer the general term embolism spreading 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, which
may occur in conduits from a previous growth ring or protoxylem (Kitinet al. 2004; Sano et al. , 2011; Hochberg et al. ,
2016). If this would be correct, the mechanism behind embolism spreading
may not dependent on xylem pressure only (Avila et al. ,
submitted). Embolised conduits could also occur when a herbivore or
xylem feeding insect damages conduits, or when a plant organ experiences
die-back, which may result in local embolism spreading. 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 the 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 approach (Oliveira et al. , 2019, Pereira et
al. , 2016, 2019). Both experimental approaches rely on cut plant organs
either due to the requirements to measure hydraulic conductivity, or the
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; Choat et al. ,
2016; Lamarque et al. , 2018).
The amount of embolism propagation could be limited by hydraulic
segmentation of the conduit network, which represents a hydraulic
constriction or bottleneck (Zimmermann, 1983; Tyree et al. , 1991;
Levionnois 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 et al. , 2006;
Loepfe et al. , 2007; Espino & Schenk, 2009).
In a few studies, considerable differences in embolism resistance have
been reported between intact plants and xylem tissue in cut organs ofVitis vinifera and Laurus nobilis , with cut-open xylem
potentially underestimating stem embolism resistance (Choat et
al. , 2010, Torres-Ruiz et al. , 2015; Lamarque et al. ,
2018). 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 ofQuercus and Populus (Breda et al. , 1993; Tyreeet 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, three explanations could be
suggested for the observed discrepancy. First, it is possible that the
cutting of conduits with sap under negative pressure introduces a
cutting artefact, although artificially embolised conduits near stem
ends can be removed before hydraulic measurements are made (Wheeleret al. , 2013; Torres-Ruiz et al. , 2015). A second
explanation is that embolism spreading could be avoided by hydraulic
segmentation, which may occur at the transition between organs, growth
rings, and nodes (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). Thirdly, embolism may also occur in conduits that are not
connected to embolised conduits, although the frequency of such
de novo process is unclear
(Brodersen et al. , 2013; Choat et al. , 2015, 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 experiment we aimed to
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 transition could affect embolism
spreading, and may prevent a potential artefact in measurements of
embolism resistance near cut xylem tissue. We therefore 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). Xylem tissue that shows hydraulic segmentation
could include many tracheids and/or narrow, fibriform vessels (Rančićet al. , 2010). Species that show little or no hydraulic
segmentation, may not have these safety valves. Removal of leaves in
seedlings of the ring-porous species Quercus robur , for instance,
was found to result in potential embolism formation in the stem based on
microCT observations (Choat et al. , 2016).
Whether or not embolism spreading depends directly on vessel dimensions
was tested in a second experiment by cutting minor leaf veins.
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. , 2016;
Klepsch et al. , 2018). These observations may give the impression
that wide conduits are more vulnerable to embolism, although any
functional explanation for such differential embolism resistance remains
unclear. Indeed, pit membrane thickness, which is a major determinant of
vulnerability to embolism (Li et al. , 2016; Kaack et al. ,
2019), is not related to conduit dimensions (Klepsch et al. ,
2018; Wu et al. , 2020; Kotowska et al. , 2020). If the
proximity of a gas source would determine embolism spreading, 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.
Finally, we applied a methodological comparison of embolism resistance
in leaf xylem between the optical method and the pneumatic method. If
the pneumatic method would be subject to a potential artefact due to gas
extraction from intact vessels that are neighbouring or connected to
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).
While direct comparison of the pneumatic and optical method to detached
leaves of Eucalyptus camaldulensis suggested no significant
difference for this species (Pereira et al. , 2019), a larger
number of species should be tested to generalise this finding.
The three complementary sets of experiments will contribute to a better
understanding of the driving forces behind embolism spreading in xylem
tissue.