Discussion
Experiment 1 and 2 show that spreading of drought-induced embolism and
thus embolism resistance can be strongly affected by the proximity of
the xylem area studied to pre-existing embolism such as cut xylem
tissue. The observation that embolism initiation (PEP12)
occurs over a > 1MPa range of xylem water potential for
four out of six species studied challenges the assumption that embolism
spreads once a certain threshold difference has been reached. While
cut-open conduits may facilitate embolism spreading, hydraulic
segmentation may limit this potential artefact. As predicted, embolism
spreading in minor veins was also affected by the proximity to cut
conduits, but showed a rather limited, local distribution without
affecting the entire leaf. Also, we found no difference between the
optical and pneumatic method for five species, despite considerable
variation in the P12 values of both methods. The
consequences and broader significance of these findings present some old
questions and assumptions in a new light, and provides a novel gas
diffusion hypothesis as a possible mechanism for embolism spreading.
Embolism spreading depends
on pre-existing embolism
Since embolism spreading happens largely from one embolised conduit to a
neighbouring one, as predicted by the air-seeding hypothesis
(Zimmermann, 1983; Sperry and Tyree, 1988), the observation that
embolism formation may depend on proximity to an existing gas source
such as cut-open conduits or pre-existing embolised conduit is not
surprising. Embolism formation appears to be unlikely if a conduit is
not connected to a pre-existing embolism. Novel, de novo embolism
formation has been observed in very few conduits that are not connected
to embolised ones based on microCT (Brodersen et al. , 2013, Choatet al. , 2015, 2016), and embolism formation in seemingly isolated
conduits could occasionally be observed in our experiments.
However, the rather
two-dimensional view associated with the optical method, its limited
resolution to accurately detect narrow vessel ends (Oskolski and Jansen,
2009), and its shortcoming to detect pre-existing embolism, did not
allow us to confirm that these conduits were completely disconnected
from neighbouring gas sources.
If availability of a pre-existing gas source or embolised conduit is
important, then where does the gas come from to induce embolism in
intact xylem? It is possible that there is almost always an embolised
conduit available, perhaps in primary xylem or in older xylem from an
older growth ring. This would be an obvious gas source if functional,
sap-filled conduits show any direct connection with these embolised
conduits via bordered pits. However, these pre-existing gas sources may
be limited due to poor connectivity or compartmentalisation of the
hydraulic network (Kitin et al. , 2004; Morris et al. ,
2016). Since vessels and tracheids do not share pits with non-conductive
fibres (Sano et al. , 2011), it is unlikely that air entry from
these cells or intercellular spaces will contribute to embolism
formation in conduits.
Hydraulic segmentation
reduces embolism spreading in xylem tissue
While the proximity of a studied xylem area to cut conduits seems to be
important, the speed of embolism spreading over a certain distance also
depends on the vessel dimensions. If each individual vessel would
embolise separately (Johnson et al. , 2020), wide and long vessels
would show a faster propagation of embolism over a given distance than
narrow, short vessels. Spreading of embolism would especially be reduced
in xylem with a high degree of hydraulic constrictions, making xylem
patches at the distal side of embolised conduits seemingly more
resistant to embolism. The four species that showed a reduced embolism
resistance in detached leaves as compared to leaves attached to
branches, have open vessels running directly from the base of the
petiole into the midrib (Table 1, Figure 2). Since the maximum vessel
length in petioles of L. tulipifera and B. pendula were
shorter than the petiole length (Table 1), both species showed
relatively small differences in embolism resistance between detached
leaves and leaves attached to a stem segment (Fig. 1c, e).
Wide and long vessels in the midrib and secondary veins were found to
embolise before the high 3rd to 5thvein orders. This pattern confirms various studies based on the optical
method and microCT observations (Brodribb et al. , 2016a, b;
Scoffoni et al. , 2017; Klepsch et al. , 2018). However, the
observation of local spreading of embolism in minor veins near cut
vessels in experiment 2 supports the hypothesis that embolism can spread
from pre-existing gas sources, and in minor veins prior to embolism
formation in the large vessels of major veins (Fig. 3). This observation
suggests that proximity to a gas source is the main driver for embolism
spreading, and not the conduit diameter per se. Nevertheless, the
limited and short dimensions of minor veins (Chatelet et al. ,
2006; Lechthaler et al. , 2019; Hua et al. , 2020) are a
plausible explanation for why artificially induced embolism near cuts in
minor veins propagate locally, rather than spread across the lamina.
Short conduits in minor vein orders may have been selected for in order
to maximise leaf hydraulic function over the life span of a leaf. Minor
orders of leaf veins are believed to be prone to xylem embolism
formation over the life of a leaf due to localized damage caused by
insects, pathogens or mechanical damage to the lamina, and because they
sustain the most negative pressures (Brodribb et al. , 2010).
Considerable restriction to the extent of embolism spread through minor
vein orders would ensure a limited impact on the hydraulic capacity of a
leaf that sustains localised damage to the lamina.
It is also possible that wide and long vessels are more likely connected
to a pre-existing embolism than narrow, short conduits, and that large
vessels show a higher amount of intervessel pit membrane area than
narrow, short vessels. In other words, the reason why large and wide
vessels are likely to embolise first, may reflect a difference in the
rate of air entry, which is caused by their connectivity to a gas
source, and not any inherent difference in embolism resistance per se.
We are not aware of an alternative mechanism that would explain why wide
conduits are more vulnerable to embolism than narrow ones. Pit membrane
thickness, which is strongly associated with embolism resistance (Liet al. , 2016; Kaack et al. , 2019), was found not to be
related to conduit diameter (Kotowska et al. , 2020; Wu et
al. , 2020).
Vulnerability segmentation
may reflect hydraulic segmentation, but not intrinsic differences in
embolism resistance per se
The observation that embolism formation in leaves of an individual tree
may occur under highly variable (> 1 MPa) xylem water
potentials, suggests that embolism resistance may represent a relative
trait that does not capture the absolute, intrinsic embolism resistance
of its xylem. It is likely that hydraulic segmentation includes highly
reduced conduit dimensions, especially with respect to conduit length
and width, with a high number of interconduit end walls over a short
stretch of xylem tissue. Conduit end walls have been suggested to hold
up embolism spreading at least temporarily, with pit membranes
functioning as safety valves and preventing further spreading of
embolism due to their tiny pores (Zhang et al. , 2017, 2020; Kaacket al. , 2019; Johnson et al. , 2020). Moreover, narrow and
short tracheids or fibriform vessels may be more confined than long and
wide vessels, with a small interconduit pit membrane area for air entry
in narrow tracheids.
Quantifying embolism resistance across the entire xylem pathway could be
complicated by measuring artefacts or the proximity to pre-existing
embolism, such as cut xylem. In our study, comparison of xylem embolism
resistance of leaves attached to long stem segments with embolism
resistance of stem xylem based on previous papers (Klepsch et
al. , 2018; Zhang et al. , 2018), indicates that leaf xylem of all
six species was between 0.5 and > 1 MPa more embolism
resistant than stem xylem. Our result for B. pendula was
consistent with Klepsch et al. (2018), with leaf xylem being more
resistant than stem xylem. Most angiosperms species, however, showed
that stem xylem was either more embolism resistant, or equally resistant
than leaf xylem (Zhu et al. , 2016; Skelton et al. , 2018;
Losso et al. , 2019).
Therefore, caution is needed to
directly compare absolute values of embolism resistance between organs,
since measured values of embolism resistance could be relative
estimations only, especially if destructive methods are used and
cut-open xylem accelerates embolism spreading.