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.