Introduction
Water is transported under negative pressure through a plant in a
complex network of xylem cells (Dixon and Joly 1895). Being under
negative pressure means the water column is at constant risk of
interruption by large bubbles (Tyree and Sperry 1989). When tension
increases, such as when soil water is limiting or when evaporative
demand exceeds hydraulic conductivity, there is an increasing likelihood
of embolism formation, which blocks the internal flow of water (Urli et
al. 2013; Brodribb and Cochard 2009). The primary source of gas leading
to embolism formation is believed to be air entrance through the
nanoscale pores of pit membranes from neighbouring gas-filled conduits
(Choat et al. 2015b; Tyree and Sperry 1989; Guan et al. 2021; Kaack et
al. 2021). If water stress is not relieved, more xylem conduits will
experience embolism, leading to progressive declines in hydraulic
conductivity, and eventual failure of the hydraulic system, with
consequences for photosynthetic performance and potential dieback of
organs (Urli et al. 2013; Brodribb and Cochard 2009; Adams et al. 2017;
Cardoso et al. 2020a).
A suite of adaptations have evolved to reduce the likelihood of embolism
spread when hydrated xylem is exposed to negative pressure, with species
native to seasonally dry environments having xylem that is highly
resistant to embolism formation (Choat et al. 2012). These adaptations
range from gross anatomical differences in xylem conduits, like reduced
conduit diameter, length, interconnectivity, or increased cell-wall
thickness (Hacke et al. 2001; Blackman et al. 2010; Jacobsen et al.
2019; Scoffoni et al. 2017b; Schumann et al. 2019), through to
micro-anatomical variation such as increased pit membrane thickness (Li
et al. 2016; Kaack et al. 2019), or in conifers, an increased torus
overlap (Bouche et al. 2014). Considerable focus has been placed on
establishing relationships between xylem anatomy and emergent traits
that surmise embolism resistance, such as the water potential at which
50% of the xylem is embolised (P50 ) (Choat et
al. 2018; Brodribb et al. 2020b). Less emphasis has been placed on
understanding if individual xylem conduits have specific thresholds at
which embolism will form (Jacobsen et al. 2019).
Assuming that most declines in xylem hydraulic conductance during
drought are due to embolism then the gradual decline in hydraulic
conductance as a drought progresses (Sergent et al. 2020), suggests that
there might be a range of water potentials at which embolism events will
occur across a population of xylem conduits. This range of water
potential is typically quantified by the slope of vulnerability curves,
with a narrow range and a steep slope characterising vulnerable species,
and a wider range and flatter slope more embolism resistant species
(Kaack et al. 2021). Methods that are capable of visualizing individual
embolism events support the idea that individual embolism events will
occur over a wide range of water potentials in many species in both stem
and leaf xylem (Jacobsen et al. 2019; Venturas et al. 2016; Johnson et
al. 2020; Knipfer et al. 2015). In stems the first embolism events are
often observed near the primary xylem (Choat et al. 2015a), or in some
cases the largest volume vessels (Jacobsen et al. 2019; Johnson et al.
2020; Knipfer et al. 2015), suggesting these conduits embolise first. In
leaves, the first embolism events are almost always observed in the
midrib and proceed, as leaf water potential declines, through the
increasingly higher orders of veins (Skelton et al. 2017; Brodribb et
al. 2016a; Scoffoni et al. 2017b).
The apparently wide range of water potentials at which embolism
formation will occur could also be explained by a temporal aspect of
embolism spread, because propagation is largely known to occur from an
embolised to a non-embolised conduit, suggesting that pre-existing
embolism or the availability of gas affects the actual spreading process
(Guan et al. 2021; Wason et al. 2021). Once initial embolism has formed
in xylem conduits, extrinsic factors beyond the anatomy of a single
conduit, such as the proximity of water-filled conduits to embolised
ones, cut-open conduits, and hydraulic segmentation can influence the
likelihood of embolism formation (Choat et al. 2010; Knipfer et al.
2015; Choat et al. 2015b; Torres-Ruiz et al. 2016; Lamarque et al. 2018;
Guan et al. 2021). Gas-filled conduits may act as a source of embolism
propagation if a plant experiences a subsequent drought, thereby
rendering neighbouring water-filled conduits more vulnerable. Pit
membranes in the bordered pits between xylem conduits are believed to
prevent this spread of air between conduits (Choat et al. 2008). In
conifers with torus and margo pit membranes, aspiration of the torus
prevents the spread of air from embolized tracheids into neighbouring
sap-filled tracheids (Liese and Bauch 1967; Hacke et al. 2004;
Pittermann et al. 2005). In angiosperms, the multiple pore constrictions
in pit membranes with a given thickness fulfil a similar function, with
the most narrow pore constriction determining mass flow of gas (Kaack et
al. 2021).
The pattern of embolism events in leaves suggest that the proximity to
air-filled neighbours influences the vulnerability of individual
conduits (Guan et al. 2021). While often initiated in the midrib,
embolism can display an unpredictable pattern of progression across the
network of leaf veins, with large areas, comprised of many hundreds of
xylem conduits, often observed embolizing simultaneously (Brodribb et
al. 2016b). In detached leaves in which all of the xylem conduits in a
petiole are embolized, the seeding of embolism throughout the leaf
venation network is accelerated, occurring at higher water potentials
than in an intact leaf on dehydration (Guan et al. 2021).
The relative importance of individual conduit traits versus the
hydration status of surrounding conduits on determining the embolism
resistance of an individual xylem conduit remains largely unknown. There
is evidence suggesting gas-filled conduits make water filled neighbours
more vulnerable (Knipfer et al. 2015; Choat et al. 2015b; Torres-Ruiz et
al. 2016), yet pit membranes greatly reduce the spread of gas into
water-filled conduits from embolized neighbours (Choat et al. 2008). To
what extent does the presence of pre-existing embolism influence the
vulnerability of the remaining xylem? Here we designed an experiment in
which to test four hypotheses related to this question. The first
hypothesis is that individual conduits have a specific water potential
threshold at which an embolism will occur, and that pit membranes reduce
the likelihood of embolism spread between gas and water-filled conduits
under a narrow range of pressure differences. Challenges in testing this
hypothesis are the lack of accurate water potential measurements at the
individual conduit level (Bouda et al. 2019), and the uncoupling of
pressure-driven embolism propagation from temporal effects. The second
hypothesis is that pre-existing embolism changes the apparent embolism
resistance of remaining individual water-filled conduits. If each
conduit has a high fidelity to an individual threshold at which embolism
will occur, then the most vulnerable conduits will consistently
experience embolism earliest in drought, such that if drought is abated
and refilling does not occur, then the relative embolism resistance of
the remaining xylem may appear to be higher than the conduits with a low
embolism resistance. Additionally, we hypothesize that on rehydration no
refilling of embolized conduits will occur and that leaf xylem behaves
in a similar way to stem xylem.
To test these hypotheses, we selected five angiosperm species with xylem
consisting of vessels and one vessel-less angiosperm species, all with
uniform pit membranes, and three conifers with tracheid-based xylem
separated by torus-margo pit membranes. Embolism was observed using the
optical method, which is non-destructive, and permits a clear
visualization of embolism events, through a cycle of dehydration to a
variable degree of embolism, rehydration, and then subsequent
dehydration to complete desiccation.