In the absence of considerable embolism individual conduits have specific thresholds for embolism resistance
Our data suggest that in the absence of considerable embolism in neighbouring xylem conduits, individual conduits have a relatively unique, and highly conserved threshold Ψ at which embolism will occur. This threshold, however, is not sustained when there is a high proportion of embolized conduits in the xylem (more than 60%), an observation which supports the gas availability hypothesis proposed by Guan et al. (2021). We suggest that early during a drought, pit membranes provide a sufficient barrier to prevent embolism spread from gas-filled to neighbouring water-filled conduits, yet once more than 60% of conduits are gas-filled, diffusion of gas into water-filled conduits can be facilitated and occurs at relatively minor pressure differences across conduits.
Observations of variation in embolism resistance across xylem conduits have been reported by microCT imaging (Knipfer et al. 2015; Choat et al. 2015b; Torres-Ruiz et al. 2016; Jacobsen et al. 2019). A number of key anatomical traits vary across populations of xylem conduits that are associated with embolism resistance, including (t/b )3 and vessel diameter, although we have no mechanistic explanation of how these traits affect embolism spreading (Hacke et al. 2001; Blackman et al. 2010; Jacobsen et al. 2019; Scoffoni et al. 2017b). In addition to anatomical differences that might set this variation in inter-conduit embolism resistance, variation across age class of xylem might also account for some of the spread in embolism resistance thresholds between conduits, with older xylem in Vitisbeing more vulnerable to embolism formation during drought (Brodersen et al. 2013), or more resistant within a single growing season (Sorek et al. 2021).
In angiosperms, pore constrictions in multi-layered pit membranes and/or a relatively high degree of isolation within the hydraulic conduit network provides added protection from the spreading of pre-existing embolism into water-filled conduits during drought (Johnson et al. 2020; Schenk et al. 2008; Avila et al. 2021). The narrow size of pore constrictions (< 50 nm) and the highly variable pore size dimensions do not allow mass flow of gas from an embolised to a water-filled conduit under negative pressure (Yang et al. 2020; Kaack et al. 2019; Kaack et al. 2021). Surfactants coating the cellulose of pit membranes are believed to promote gas entry from a neighbouring embolised vessel into a water-filled conduit, but at the same time mitigate embolism spreading (Schenk et al. 2021). In addition to providing a physical barrier for the spreading of gas between an embolized and water-filled conduit, pit membranes may provide a short-term buffer to the further spreading of embolism, particularly when there are small numbers of embolized conduits. Upon embolism formation the water vapour in a conduit is at a negative pressure, and modelling suggests that it may take between 20 min and 10 h for this negative gas pressure to equilibrate with atmospheric pressure, during which time the embolized conduit can draw gas slowly across cell walls and rapidly across pit membranes from neighbouring gas filled, but also water-filled conduits, potentially buffering water-filled conduits from embolism (Wang et al 2015). Our experiments provide evidence that pit membranes can act as safety valves to delay the spread of embolism between neighbouring gas- and water-filled conduits.
Upon rehydration we did not observe conduit refilling (refilling of conduits have been observed using the optical method in the hydroids of a moss (Brodribb et al. 2020a)). Refilling of xylem on excised stem rehydration has been reported to occur (Trifilò et al. 2014), although the validity of these observations has been challenged (Lamarque et al. 2018). We did however observe a small degree of embolism formation persisting in a few species after rehydration (Figure 1B). This lag time in embolism cessation may be due to a slower, heterogenous reduction in the pressure difference between conduits in the area of xylem that we were observing with the optical method (Bouda et al. 2019), a phenomenon exacerbated by the presence of widespread embolism and associated decline in hydraulic conductivity. Furthermore, there may be a temporal component to embolism resistance, such that the longer a conduit experiences a negative Ψ the more likely the chance of changes in water vapour and gas concentration in embolised conduits, or gas dissolved in xylem sap, which may trigger embolism spread (Guan et al. 2021, Kaack et al. 2021).
While embolism formation on rehydration was observed in all species, there appeared to be a tendency for more embolism formation after rehydration in conifer species (Supplementary Figure S1). To test whether pit membrane anatomy or conduit anatomy might explain why in some conifer species there was an exacerbated embolism spreading after rehydration we included the angiosperm species D. winteri in our sampling, a vessel-less species with homogeneous pit membranes, as opposed to torus-margo pit membranes (Zhang et al. 2020). Unlike some conifers, the formation of embolism after rehydration in D. winteri was limited (Supplementary Figure S1C), suggesting that even though this species has only tracheids, an altered conformation caused by embolism is able to create a highly gas impermeable structure that protects the neighbouring conduits from air invasion (Zhang et al. 2020).
Other factors might also provide an explanation for the greater absence of embolism occurrence in angiosperm xylem after rehydration and on a second cycle of dehydration until reaching the Ψ at which the branch was rehydrated, including the increased separation of conduits imbedded in a matrix of non-conductive xylem tissue, such as fibres and parenchyma that might offer a physical barrier for rapid air propagation through the xylem (Johnson et al. 2020; Avila et al. 2021), particularly compared to the tracheid based xylem of conifers, which is largely homogeneous and comprised of closely packed tracheids. The close proximity of tracheids with tracheid tips slightly bent and overlapping multiple neighbouring tracheids may facilitate a more rapid spread of embolism on a second dehydration cycle in conifers (Torres-Ruiz et al. 2016; Choat et al. 2015b), while the 3D reconstruction of vessel networks deserves more attention for angiosperms (Wason et al. 2021).