Easy Come, Easy Go: Capillary Forces Enable Rapid
Refilling of Embolized Primary Xylem Vessels1[OPEN]
Vivien Rolland 2*, Dana M. Bergstrom, Thomas Lenné, Gary Bryant, Hua Chen, Joe Wolfe,
N. Michele Holbrook, Daniel E. Stanton 3, and Marilyn C. Ball
Plant Science Division, Research School of Biology (V.R., T.L., D.E.S., M.C.B.), and Centre for Advanced
Microscopy (H.C.), Australian National University, Acton, Australian Capital Territory 2601, Australia;
Australian Antarctic Division, Department of Environment, Kingston, Tasmania 7050, Australia (D.M.B.);
Center for Molecular and Nanoscale Physics, School of Applied Sciences, RMIT University, Melbourne,
Victoria 3001, Australia (G.B.); School of Physics, University of New South Wales, Sydney, New South
Wales 2052, Australia (J.W.); and Department of Organismic and Evolutionary Biology, Harvard University,
Cambridge, Massachusetts 02138 (N.M.H.)
ORCID IDs: 0000-0002-9232-1287 (V.R.); 0000-0001-8484-8954 (D.M.B.); 0000-0001-5483-7592 (G.B.); 0000-0001-6573-517X (H.C.);
0000-0002-6713-9328 (D.E.S.); 0000-0001-9170-940X (M.C.B.).
Protoxylem plays an important role in the hydraulic function of vascular systems of both herbaceous and woody plants, but
relatively little is known about the processes underlying the maintenance of protoxylem function in long-lived tissues. In this study,
embolism repair was investigated in relation to xylem structure in two cushion plant species, Azorella macquariensis and Colobanthus
muscoides, in which vascular water transport depends on protoxylem. Their protoxylem vessels consisted of a primary wall with
helical thickenings that effectively formed a pit channel, with the primary wall being the pit channel membrane. Stem protoxylem
was organized such that the pit channel membranes connected vessels with paratracheal parenchyma or other protoxylem vessels
and were not exposed directly to air spaces. Embolism was experimentally induced in excised vascular tissue and detached shoots
by exposing them briefly to air. When water was resupplied, embolized vessels refilled within tens of seconds (excised tissue) to a
few minutes (detached shoots) with water sourced from either adjacent parenchyma or water-filled vessels. Refilling occurred in
two phases: (1) water refilled xylem pit channels, simplifying bubble shape to a rod with two menisci; and (2) the bubble contracted
as the resorption front advanced, dissolving air along the way. Physical properties of the protoxylem vessels (namely pit channel
membrane porosity, hydrophilic walls, vessel dimensions, and helical thickenings) promoted rapid refilling of embolized conduits
independent of root pressure. These results have implications for the maintenance of vascular function in both herbaceous and
woody species, because protoxylem plays a major role in the hydraulic systems of leaves, elongating stems, and roots.
1
This work was supported by the Australian Antarctic Science
Program (grant nos. 3095 and 4192 to M.C.B., D.M.B., and G.B.)
and the Australian Research Council (Discovery Project grant no.
DP110105380 to M.C.B., G.B., and N.M.H.).
2
Present address: Australian Research Council Centre of Excellence for Translational Photosynthesis, Plant Science Division, Research School of Biology, Australian National University, Acton,
Australian Capital Territory 2601, Australia.
3
Present address: Department of Ecology, Evolution, and Behavior, University of Minnesota-Twin Cities, 100 Ecology Building, 1987
Upper Buford Circle, Saint Paul, MN 55108.
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Vivien Rolland ([email protected]).
V.R., D.M.B., T.L., G.B., N.M.H., and M.C.B. designed the research;
V.R., D.M.B., T.L., H.C., and M.C.B. performed the research; V.R.,
G.B., J.W., D.E.S., and M.C.B. analyzed the data; V.R., D.M.B., G.B.,
J.W., N.M.H., and M.C.B. wrote the article.
[OPEN]
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1636
There is a pressing need to understand how plants
manage the maintenance of water transport from roots
through leaves under changing environmental conditions (Allen et al., 2010; Choat et al., 2012). The problem arises because water is transported through the
xylem under tension (i.e. under negative absolute
pressure). As tension increases, conduits become increasingly vulnerable to cavitation, which causes the
conduits to lose their ability to transport water. Conduits can become embolized during normal diurnal
function as a result of tensions induced by transpiration and in response to environmental conditions such
as drought or freezing stress (Zimmermann and Tyree,
2002). Vulnerability to cavitation and embolism formation suggests that plants have mechanisms to regain lost
hydraulic capacity, either through the formation of new
conduits or by refilling embolized ones.
The vulnerability of conduits to embolisms and the
capacity for repair are related to the structural diversity
of xylem tissue (Zwieniecki and Holbrook, 2009; Lens
et al., 2011; Cai et al., 2014). In vascular plants, the
classification of xylem tissues depends on the meristem
that produced them (Evert and Eichhorn, 2006).
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Protoxylem Properties Enable Rapid Embolism Repair
Primary xylem is produced by apical meristems and
includes both protoxylem and metaxylem conduits,
which are distinguished by their wall structure and the
timing of their development. Protoxylem matures during organ elongation, which results in loss of function
due to stretching in some tissues and species, while in
many others, functionality is maintained throughout
the life of the organ. In contrast, metaxylem matures in
elongated tissue. In herbaceous plants, primary xylem is
the major hydraulic system of the roots, stems, and
leaves. In woody plants, the primary xylem remains the
main hydraulic system of the leaves, while the radial
growth of stems occurs through the activity of a vascular cambium, which produces secondary xylem with
only metaxylem conduits. As a woody plant grows, the
secondary xylem (and hence the metaxylem) thus becomes of increasing importance to stem hydraulic
function. However, protoxylem remains an integral
component of the plant hydraulic system due to its
function in leaves and elongating stems and roots.
As discussed in a recent review (Brodersen and McElrone, 2013), refilling of embolized vessels has been shown
to depend on the generation of positive pressure by roots
in many monocots, herbaceous plants, and a few woody
species. However, many species lack root pressure; thus,
attention has focused on so-called novel refilling, which
involves adjacent living cells in the repair of embolized
metaxylem or secondary xylem in stems of mature plants.
Novel refilling has been studied with a variety of methods
to visualize temporal variation in the presence and subsequent absence of embolized vessels, including cryoscanning electron microscopy (Cryo-SEM; Canny, 1997;
McCully et al., 2014), double staining (Zwieniecki and
Holbrook, 1998; Zwieniecki et al., 2000), NMR imaging
(Holbrook et al., 2001; Zwieniecki et al., 2013), and highresolution x-ray computed tomography (Lee and Kim,
2008; Brodersen et al., 2010; Kim and Lee, 2010; Lee et al.,
2013; Suuronen et al., 2013). These observations, in combination with other measurements, led to a working hypothesis of an osmotically driven repair mechanism in
which sugars pumped into embolized vessels by adjacent
paratracheal parenchyma provide the osmotic pressure
difference that refills the vessel (Nardini et al., 2011).
Little is known about embolism and its repair in
protoxylem, which has structural features that make it
potentially more vulnerable to embolism than metaxylem in the same plant or tissue (Choat et al., 2005). These
include a greater exposed area of the primary cell wall
with annular or helical thickenings instead of secondary
walls. This could enhance stretching of the primary wall
when large pressure differences develop between functional and embolized vessels, thereby decreasing the
pressure required for air seeding of bubbles (Choat et al.,
2004). Choat et al. (2005) suggested that greater vulnerability of protoxylem to embolism might underpin the
roles of petioles, leaves, and small stems in the hydraulic
segmentation hypothesis of Zimmermann (1983), in
which sacrifice of the most easily replaceable tissues
protects the function of the main structure of a plant
during water stress. If ease of protoxylem embolism
were to contribute to the function of hydraulic fuses
during mild water stress, then ease of refilling would be
required to rapidly reset the system.
This study focuses on embolism repair in two distantly
related, vascular species, Azorella macquariensis (Apiaceae)
and Colobanthus muscoides (Caryophyllaceae), that depend
exclusively on protoxylem for vascular water transport.
Both species form cushions, with the former being an endemic, keystone species in the alpine zone of subantarctic
Macquarie Island and the latter being a regional endemic
that plays a major role in rocky coastal areas often within
the supralittoral zone (Selkirk et al., 1990; Orchard, 1993).
Both species are of ecological interest, because the subantarctic region is under increasing threat from climate change
(Adams, 2009). Specifically, the climate on Macquarie Island is progressively changing from one that is perpetually
wet and misty to one with increased exposure to periodic
drying (Bergstrom et al., 2015). Dieback of alpine vegetation was first observed in 2008, and by 2010, extensive and
unprecedented decline of A. macquariensis led to its listing
as critically endangered (Bricher et al., 2013).
In this study, protoxylem structure was studied in
relation to embolism repair. Refilling of gas-filled vessels
was compared between excised tissue and that in intact,
detached shoots. The results showed that the physical
properties of the protoxylem facilitated refilling by capillary forces and that rapid refilling in detached shoots
supplied with water occurred without root pressure.
RESULTS
Ultrastructure and Organization of Protoxylem in
C. muscoides and A. macquariensis
The ultrastructure of protoxylem elements was
similar in C. muscoides and A. macquariensis (Figs. 1 and
2). In both species, helical secondary wall thickenings
occurred in each protoxylem element (Figs. 1, A and C,
and 2F). These helices intermittently fused within an
element (Fig. 2B), and the thickenings of adjacent elements tended to align with one another (Fig. 2C).
Perpendicular walls marked the junction of two protoxylem elements, whereas the end walls of protoxylem vessels were inclined (Figs. 1, B and D, and 2D).
Longitudinal sections of frozen hydrated protoxylem
elements revealed that helical thickenings effectively
formed a channel-shaped pit, with the primary wall
being the pit channel membrane (Fig. 2, A and E). Pit
channel geometry was highly variable within individual protoxylem elements (Fig. 2, A9 and A99). Finally, water was sublimed from hydrated vessels to
reveal the ultrastructure of the primary wall forming
the pit channel membranes. They were organized as a
loose mesh with relatively large pores (Fig. 2C9).
Cryo-SEM and fresh sections revealed that the protoxylem was in contact with neighboring cells (Figs. 2
and 3). In both species, the vascular tissue was organized as a ring around the pith (Fig. 3, A–D), and
xylem vessels were in contact with other xylem vessels
and/or parenchyma cells but not with intercellular gas
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Rolland et al.
Figure 1. Confocal micrographs of protoxylem vessels excised from
C. muscoides (A and B) and A. macquariensis (C and D). A and C, Rupture
of protoxylem elements revealed that highly fluorescent secondary wall
thickenings were helical (arrowheads). B and D, Protoxylem elements were
joined together by perpendicular junctions (arrowhead), and vessels were
connected at junctions of inclined end walls (dashed boxes). Bars = 20 mm.
spaces, at least in well-hydrated tissues. In A. macquariensis, large spaces (up to 100 mm in width; Fig. 3,
E–G) filled with gas or water occurred in the stem
cortex, well distanced from the xylem.
Embolisms Resorb Quickly in Protoxylem Excised from
C. muscoides
Dehydration of excised protoxylem tissue by exposure
to ambient air for a few minutes or less was sufficient to
create embolisms that spread in a matter of seconds
through several protoxylem elements (Fig. 4, A and B). The
pit channels of embolized vessels were filled with gas (Fig.
4B9). However, upon the placement of a drop of water on
the protoxylem tissue, the pit channels filled with water
within seconds (Fig. 4C9) and embolisms disappeared in
tens of seconds, regardless of whether they were contained
within a single vessel or spanned the junction of two
vessels (Fig. 4, C–N; Supplemental Movies S1 and S2).
The kinetics of embolism repair were measured in
excised xylem for 14 individual bubbles with a temporal resolution of 0.5 s (Fig. 5). Because junctions affect
gas diffusion, meniscus shape, and water flow, which
could affect rates of bubble resorption, bubbles were
classified as free (n = 9; Fig. 5A) or trapped (n = 3; Fig.
5B), depending on whether they occurred away from a
junction or at a junction, respectively. Additionally, a
category named twin bubbles (n = 1; Fig. 5C) was created to regroup a free bubble and a trapped bubble
found close together in the same protoxylem element.
These twin bubbles likely resulted from the splitting of
a larger bubble into two parts.
Free bubbles resorbed at a similar rate (Fig. 5D). The
rate of decrease in length (slope of the graph) varied
between 0.65 and 1.95 mm s21. These values were calculated for bubble lengths greater than 15 mm to ensure
that bubbles were rod shaped and in contact with the
walls. In contrast, the trapped bubbles showed varied
behavior, with some junctions accelerating resorption
while others impeded it (Fig. 5E). The rate of decrease in
length to a minimum of 15 mm varied between 0.6 and
6.4 mm s21. The twin bubbles showed an interesting
behavior (Fig. 5F). The initial resorption rate was similar
in both bubble types, but after about 20 s, the free
bubble started to resorb faster until it was fully resorbed. Then, the remaining bubble started resorbing
faster, possibly because resorption of the free bubble
allowed more rapid dispersion of dissolved gas.
Time-dependent changes in volume and surface
area were calculated for all free bubbles. The rate of
volume change (Fig. 5G) increased with increasing
bubble radius (Fig. 5I). In contrast, the rates of change
in bubble surface area during dissolution were similar
regardless of width (Fig. 5H).
Protoxylem Embolisms Resorb Quickly in Detached
Shoots in C. muscoides
Experiments were then conducted to determine
whether patterns of embolism repair in detached shoots
were similar to those observed in excised xylem tissue
(Fig. 6). The embolism shown in Figure 6 originally
spanned several junctions and filled at least three separate protoxylem vessels, including two elements
within one vessel (Fig. 6, A and B). Immediately after
cavitation formation, the pit channels between secondary wall thickenings of xylem elements were filled with
gas (Fig. 6, B and B9). The refilling of individual elements followed a consistent pattern. First, the pit
channels filled with water within a few minutes, simplifying the shape of the bubbles to essentially a rod
with two menisci. Once the pit channels were filled,
then the bubbles decreased in length and all but one
were dissolved in less than 8 min (Fig. 6, B–M and F9).
The meniscus of the remaining bubble moved away
from the field of view shortly after recording ended.
DISCUSSION
The results of this study revealed patterns in embolism repair that were consistent between excised
xylem and detached shoots. In excised tissue, water
entered embolized protoxylem vessels and the bubbles
were fully dissolved within 100 s (Fig. 5). These results
demonstrated that the physical properties of the vessels themselves generated sufficient capillary forces to
enable refilling in the absence of externally applied
pressure. Although less rapid, refilling also occurred in
stems of detached shoots, with the longer time most
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Protoxylem Properties Enable Rapid Embolism Repair
Figure 2. Cryo-SEM images of protoxylem elements in intact and fully hydrated stems of
C. muscoides (A–D) and A. macquariensis (E and
F). A and E, Longitudinal sections through icefilled protoxylem, showing that secondary wall
thickenings (filled arrowheads) and pit channels
(empty arrowheads) connected a vessel with adjacent parenchyma (A) or another ice-filled vessel
(E). A9 and A99, Higher magnification of the white
dashed boxes in A highlighting that pit channels
from a single protoxylem element had different
sizes (brackets) and shapes (dashed lines). B and
C, Longitudinal sections in which ice was sublimed by prolonged etching, showing that helical
secondary wall thickenings intermittently fused
with each other (arrowheads in B) and aligned
with thickenings of adjacent vessels (arrowheads
in C). C9, Higher magnification of the white
dashed box in C, showing the loose mesh-like
structure (arrowhead) of the protoxylem wall. D,
Longitudinal section through the junction of two
protoxylem vessels. F, Transverse section through
protoxylem elements and their helical secondary
wall thickenings (arrowheads). Bars = 10 mm (A–
F) and 2 mm (A9, A99, and C9).
likely due to the slower movement of water into
embolized protoxylem through pit channels connecting vessels with adjacent cells. Nevertheless, the total
time required to resorb visible bubbles was less than 8
min (Fig. 6). These measurements were made in detached shoots, and hence refilling occurred in the absence of positive root pressure in well-watered plants.
As well as a surface tension (i.e. a force per unit of
length [newtons m–1] in the plane of the interface), g can
also be expressed as a specific surface free energy (J m–2 =
newtons m–1). For a pure water-air interface, g is about
0.07 newtons m–1 or 0.07 J m–2. The Young-Laplace relation gives the pressure difference DP across a curved
surface with a surface tension g. If the surface is locally
spherical with radius r, the pressure difference is:
Expansion and Disappearance of a Bubble
The expansion and disappearance of a bubble in a
xylem vessel involve surface tension, capillarity, and
pressure across a meniscus. The surface between two
pure fluids (such as water and air) has a surface tension g.
DP ¼ 2g=r
where the pressure is higher on the concave side of the
interface. In the examples of interest here, liquid water
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Rolland et al.
Figure 3. Bright-field (A–D) and Cryo-SEM (E–G) images showing the
general architecture of C. muscoides (A and B) and A. macquariensis
(C–G) stems. A and C, Transverse sections of fresh shoots of C. muscoides (A) and A. macquariensis (C) stained with Toluidine Blue to
highlight the distribution of the protoxylem (blue). B and D, Higher
magnification of the white dashed boxes in A and C, respectively,
showing that in both species the protoxylem (white arrowheads) was
organized in a ring around the pith. The stem cortex was densely
packed with parenchyma cells in C. muscoides but loosely packed in
A. macquariensis (gaps highlighted with black arrowheads). E, Transverse section of a cryopreserved, intact, and fully hydrated A. macquariensis stem, showing densely packed protoxylem conduits
(general area highlighted with the dashed circle) and large spaces filled
by water (filled arrowhead) or air (empty arrowhead) between parenchyma cells distant from the protoxylem. The outer stem boundary is
highlighted by a black dashed line. F and G, Higher magnification of
water-filled (F) and air-filled (G) intercellular spaces among parenchyma. Bars = 100 mm (A, C, and E) and 50 mm (B, D, F, and G).
is always observed to be on the convex side, so the
bubble has a higher pressure than the water. To put a
scale on the equation above, for a pressure difference
of 1 atm, the radius of a clean, spherical air-water
meniscus would be 1.4 mm; smaller radii give higher
pressure differences and vice versa.
The saturated vapor pressure of water at ordinary
temperatures is only approximately 1 kPa or 0.01 atm.
So the pressure exerted by water vapor in the bubble
can usually be ignored in comparison with atmospheric pressure PA or with the pressures and tensions
commonly encountered in plant water relations.
Hence, if a bubble has a significant internal pressure, it
must contain other gases, usually air; conversely, if it
contains no air, its internal pressure is negligible.
Now consider a small bubble, in water with an absolute pressure P, whose water-vapor interface is
spherical with radius r (the bubble need not be a
complete sphere; part of its boundary may be vaporvessel wall interface). If there is only water vapor in
the bubble, then its internal pressure is negligible, so P
in the water, which is on the convex side of the meniscus, is P = –2g/r. (Because of the attraction between
water molecules, P may be less than 0, a phenomenon
that is necessary for transpiration in tall trees.) Rearranging the preceding equation gives a critical radius
for the bubble: rc = – 2g/P. So, with sufficiently small
radius r , rc, such a bubble spontaneously closes, even
against an absolute tension P , 0 in the water. However, once r . rc, a bubble in bulk water expands.
Next, consider the expansion of a bubble of water
vapor in a hypothetical vessel with r . rc, whose walls
are completely rigid, have a contact angle of zero with
water, and are filled with water that contains no dissolved gases and has a constant (negative) pressure P.
This bubble will expand until it deforms into the spaces
of the wall and will continue doing so until all the
menisci in the walls have the critical radius rc = – 2g/P.
If the spaces in the wall are sufficiently large (r . rc), the
bubble will penetrate through them, expanding into
any nearby volumes of water under tension.
For two reasons, however, the bubble may not reach
this state. First, real walls are not infinitely rigid. When
a negative pressure is applied to its contents, a real
vessel shrinks, and when that pressure becomes less
negative, it expands. As the bubble expands, the water
pressure can become less negative, and from the
equation above, this increases rc. If P becomes less
negative until rc becomes larger than the radius of the
vessel, the bubble could stabilize at a length smaller
than that of the vessel. Second, the surrounding water
will usually contain dissolved air (i.e. air can diffuse
into the bubble, raising the pressure from near zero).
In the long term, the air in the bubble can approach
equilibrium with the air in the surrounding water,
which in turn approaches equilibrium with atmospheric
air, with pressure PA. In that hypothetical equilibrium,
the water surrounding the bubble has a pressure PA
– 2g/r, where r is the radius of all the air-water interfaces of the bubble. At this stage, r might equal the
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Protoxylem Properties Enable Rapid Embolism Repair
Figure 4. Bright-field images showing rapid embolism repair in protoxylem excised from C. muscoides. A, Protoxylem element
with embolism induced by dehydration in air. B, Protoxylem with an embolism spanning the end walls of two linked vessels
(black arrowhead). C to N, Time series showing that the addition of water triggered rapid resorption of embolisms both in the
absence (C–H) and the presence (I–N) of a junction (black arrowheads). B9 and C9, Higher magnification of the dashed boxes in
B and C, respectively, showing that upon embolism, pit channels were initially filled with air (empty arrowheads) but filled with
water (filled arrowheads) within a few seconds. Bars = 20 mm (A–N) and 5 mm (B9 and C9).
radius of the vessel, if it is only partially filled, or that of
spaces in the wall, depending on how far the bubble has
expanded. In either case, the absolute water pressure PA
– 2g/r is less than PA. If water at atmospheric pressure is
subsequently available (as provided in our experiments),
then that water will flow into the vessel walls and then
the vessel itself. If water continues flowing down its
pressure gradient until the water in the vessel also has
pressure PA, then the air in the bubble is at pressure PA +
2g/r. At this stage, if the air dissolved in the water in the
walls and/or vessel is in equilibrium with that outside
(at PA), then the air in the bubble (which is at pressure
higher than atmospheric by 2g/r) will dissolve into the
surrounding water. This is a mechanism that allows
refilling in the absence of osmotic effects.
Protoxylem Refilling Is a Two-Step Process
In our experiments, refilling of embolized protoxylem occurred in two phases: (1) water refilled the xylem pit channels, simplifying bubble shape to a rod
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Figure 5. Kinetics of embolism repair for different categories of bubbles in excised protoxylem of C. muscoides. A to C, Diagrams of the different categories of bubbles analyzed in this study. Free bubbles (A) are free-floating, trapped bubbles (B) span a
junction or have an end trapped in a junction, and twin bubbles (C) are two bubbles close together in the same protoxylem
element. D to F, Graphs showing time-dependent changes in the lengths of individual free bubbles (D), trapped bubbles (E), and
twin bubbles (F). The dashed line in D highlights the minimum bubble length of 15 mm that was used in calculations of bubble
volumes shown in I. The arrowhead in F highlights the point at which the free bubble (purple line) is fully resorbed and at which
the trapped bubble (pink line) starts to resorb faster. G and H, Graphs showing the time-dependent changes in volume (G) and
surface area (H) of individual free bubbles. I, Graph showing the correlation between the rate of decrease in volume and the
radius (rm) of individual free bubbles. Lines show linear fits with rm (solid line, P = 0.0004, adjusted r2 = 0.87) and rm2 (dashed
line, P = 0.0015, adjusted r2 = 0.81).
with two menisci; and (2) the bubble contracted as the
resorption front advanced, dissolving air along the
way. In the first phase of refilling, water could have
moved radially through the pit channel membranes
from adjoining cells into the embolized vessel, consistent with previous observations on foliar metaxylem
in sunflower (Helianthus annuus; Canny, 1997) and
stem secondary xylem in grapevine (Vitis vinifera;
Brodersen et al., 2010). Alternatively, or in addition,
water could have moved axially through the end wall
of an adjoining water-filled vessel into the pit channel
of the embolized vessel. Several physical properties of
the protoxylem contributed to refilling of excised,
embolized vessels. A large proportion of the primary
wall was exposed, forming the membrane of pit
channels bounded by secondary, helical thickenings.
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Protoxylem Properties Enable Rapid Embolism Repair
Figure 6. Bright-field images showing rapid embolism resorption in detached shoots in C. muscoides. A and B, A large embolism spanned four protoxylem elements highlighted in blue, yellow, pink, and green. The blue and yellow elements belonged
to the same vessel and were connected by a perpendicular junction (black arrowheads), while inclined end walls (black dashed
boxes) marked junctions with separate vessels highlighted in green and pink. B9, Higher magnification of the area enclosed by
the white dashed box in B, showing that gas filled the vessel lumen and pit channels at 0 s. C to M, Time series of images
depicting embolism repair. C, The green element was the first to refill with water. D, Short lag phase during which pit channels
became refilled with water along much of the length of the pink and yellow elements. E, Water entry and bubble resorption split
the embolism in the pink element into two bubbles (white arrowhead). F and G, The two bubbles in the pink element shrank
(filled white arrowheads) and were completely dissolved (empty white arrowheads) within 27 s of their formation. F9, Higher
magnification of the white dashed box in F, showing that the pit channels refilled with water (white arrowheads) prior to passage
of the bubble resorption front through the blue element. The junction between yellow and blue elements is highlighted with a
black arrowhead. H, Water entered the yellow element from pit channels on a side wall (white arrowhead). I, The bubble split
into two (white arrowhead). J and K, On the left, the resorption front advanced to the junction with the blue element within 17 s
(black arrowheads), while the free bubble on the right shrank (filled white arrowhead) and was fully resorbed (empty white
arrowhead) within 39 s. L and M, After a lag phase, the resorption front (white arrowhead) moved through the junction (black
arrowhead) into the blue element and continued to advance to the left. Bars = 20 mm.
The primary wall structure, as revealed by the sublimation of ice from frozen vessels, consisted of an open
weave of microfibrils, creating numerous passages for
lateral exchange of water between adjacent vessels or
cells (Fig. 2C9). These walls were also thin, and so the
path length for water movement across pit channel
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Rolland et al.
when examined on the macroscopic scale. Because the
interfacial energy scales with the microscopic area, a
textured hydrophilic surface can exhibit a smaller
macroscopic contact angle than a flat surface of the
same material, as shown schematically in Figure 7.
Kohonen and Helland (2009) analyzed the effects of
wall structures (wall sculpturing, in their terminology)
on wettability. They presented models for different
structures on the internal surfaces of xylem vessels and
showed that these structures can improve wetting
compared with a flat surface. In this case, they argued,
the gaps between the surface structures act somewhat
like small capillaries (or wicks), enhancing water uptake. This microscopic structure reduces the contact
angle at the macroscopic level, which in turn increases
the force that draws water across the surface, somewhat like the way in which the attraction of water to
the fibers draws a fluid up a wick.
The model most relevant to our study assumes
that helical thickenings can be modeled by assuming
that the surface is covered with cylinders of width
w separated by a center-to-center distance d. For
this model, Kohonen and Helland (2009) showed
Figure 7. Schematic highlighting how hemiwicking enhances vessel
refilling by capillary forces. In all three sketches, the microscopic
contact angle has the same large value. A, The case of a vessel without
helical thickenings (smooth wall), for which the macroscopic and
microscopic contact angles are equal. B, A low-magnification view
of a vessel with helical thickenings. The high-magnification view of
the same vessel shows how the large value of the microscopic
contact angles on the helical thickenings (empty black arrowhead)
leads to a macroscopic contact angle of zero in the low-magnification
view (filled black arrowhead). The zero macroscopic contact angle
gives a lower radius of curvature and, therefore, increases the
pressure differential across the meniscus, facilitating refilling by
capillary forces.
membranes was short, again enhancing rates of water
entry into embolized vessels. Finally, the rapid influx of
water into the pit channels of embolized vessels indicated that the pit channel membranes were highly
permeable to water.
Protoxylem Helical Thickenings Promote Rapid
Embolism Repair
The two-phase pattern of refilling implied that the
protoxylem wall structure plays a significant role in
rewetting and bubble dissolution. The contact angle is
that between the solid surface and the liquid-gas interface, and it is determined by the interfacial energies
of the solid-gas, solid-liquid, and liquid-gas interfaces.
A textured surface, when examined on a scale smaller
than that of the texture, has a larger surface area than
Figure 8. Schematic of the experimental setup to create embolisms in
detached shoots of C. muscoides and observe their repair.
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Protoxylem Properties Enable Rapid Embolism Repair
that hemiwicking (i.e. enhanced wetting) occurs if the
following condition holds:
cosuE .
d 2 wuE
d þ wðp 2 uE Þ
where uE (in radians) is the intrinsic or microscopic
contact angle, which is also that for a microscopically
smooth surface. In the case of C. muscoides protoxylem,
d is approximately 3 mm and w is approximately 1.5
mm (values estimated from Fig. 4). Taking values of uE
as those (42°–55°) reported for the contact angle that
water makes with smooth xylem lumen in a range of
woody species (Zwieniecki and Holbrook, 2000; Kohonen and Helland, 2009), the inequality above is satisfied for any contact angle smaller than 81° (i.e. even for
surfaces that are only slightly hydrophilic). For a vessel
with radius R and macroscopic contact angle u, the
pressure difference across the meniscus is (2g/R)cosu.
Hemiwicking that reduces the macroscopic contact angle to zero, therefore, increases the pressure difference
by the factor 1/cosu. For the range of uE cited above
(42°–55°), the pressure across the meniscus would be
increased by 35% to 74%. Therefore, the presence of
helical thickenings in the protoxylem contributes considerably to the elimination of embolisms (Fig. 7).
The second phase of refilling began once the pit
channels were filled with water and the bubble shape
was simplified to a rod with two curved menisci. Then,
bubble volume declined with the advance of the resorption front driven by pressure generated by surface
tension. This effect was also enhanced by helical thickenings, as the diameters of the rod-shaped bubbles were
smaller than those of the vessels containing them,
thereby allowing surface tension to overcome greater
pressure differences. Using the Young-Laplace equation, pressure differences across bubble menisci were
estimated to range between 26 and 77 kPa for large- and
small-diameter protoxylem vessels, respectively. Such
pressures are high enough to force rapid redissolution
of air bubbles within the protoxylem, consistent with
theoretical calculations showing that compression of an
embolus to as little as 2% above atmospheric pressure
(i.e. 2 kPa) should cause resorption within 1,000 s or less
even in much larger metaxylem vessels (Pickard, 1989).
The two phases of refilling raise questions about the
relative importance of vessel walls and bubble geometry to rates of bubble resorption. If reabsorption of gas
were limited by the interfacial area of the two hemispherical bubble ends (area, 4pr2), then one would
expect the bubble volume decrease rate to be proportional to r2 and the length decrease rate to be independent of bubble width. If resorption at the ends
were negligible and if reabsorption into the vessel
walls (area, 2prL) were to limit the resorption of gas,
then the absolute rate of bubble volume loss would be
proportional to bubble radius for bubbles of the same
length and the length decrease rate would be proportional to 1/r. The data shown in Figure 5I do not
distinguish between these two simplified models (i.e.
volume decrease rate fr and fr2). Qualitatively,
however, these results indicate that the rates were
dominated by a combination of small bubble size and
high positive pressure exerted on the bubbles.
The Protoxylem as a Two-Way Switch: Easily Embolized
and Easily Refilled
These results have far-reaching implications for the
function of both herbaceous and woody species. Many
herbaceous species depend on protoxylem for water
transport, and a capacity for rapid repair could enhance
the recovery of vascular function upon rewetting after
mild desiccation. In contrast, mature stems of long-lived
woody species depend mainly on secondary xylem for
water transport. However, recent studies using highresolution x-ray computer tomography have found that
embolism occurred first in protoxylem surrounding the
pith and propagated outward through radial files of
xylem conduits (Brodersen et al., 2013). These observations suggest that protoxylem is an Achilles’ heel
rendering the vascular system vulnerable to loss of
function during drought stress (Rockwell et al., 2014).
However, protoxylem is also of major importance for
water transport in young stems, petioles, and leaves, all
of which would be subject to lower water potentials
than the main stem of woody plants during rapid
transpiration. Choat et al. (2005) pointed out that the
greater vulnerability of protoxylem to embolism in
these tissues could enable protoxylem to function as
fuses, protecting the function of the main stem by effectively turning water off at the tap if water potentials
of more distal organs declined to perilously low levels.
Here, we show that protoxylem vessels are also able to
rapidly repair embolisms once water becomes available.
This would enhance their hypothesized function as
fuses but might also provide a readily restorable pathway for water transport that could provide a source of
water for the repair of embolized conduits in the secondary xylem. Thus, while protoxylem may be an
Achilles’ heel for stem function under extreme conditions, they may also promote the recovery of function
upon rewatering. In this sense, their easy come, easy go
behavior may offer more benefits than detriments to the
integrated function of plants in complex, highly variable
environments.
MATERIALS AND METHODS
Plant Material
Samples from two species of cushion plants, Azorella macquariensis (Apiaceae) and Colobanthus muscoides (Caryophyllaceae), were collected in April
2012 on Macquarie Island, a subantarctic Australian territory with a cool
and very humid oceanic climate. Living cushions were transferred in refrigerated conditions to a temperature-controlled facility on the Australian
National University campus, where specimens were maintained in sealed
transparent containers at 4°C to 6°C, with 16 h of daily low-intensity illumination (200 mmol photosynthetically active radiation m22 s21), similar to the
cool and misty conditions characteristic of the growing season on Macquarie
Island.
Plant Physiol. Vol. 168, 2015
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Rolland et al.
Anatomical Characterization of Xylem Using
Confocal Microscopy
Xylem of C. muscoides and A. macquariensis was excised from fresh shoots and
mounted in water. Samples were excited with a wavelength of 405 nm, and light
emitted between 411 and 490 nm was collected using the LSM 780 confocal laserscanning microscope (Carl Zeiss), a 403 water immersion objective (numerical
aperture [NA] = 1.1), and the Zen 2011 software package (Carl Zeiss).
regression was then fit between the calculated rates of decrease in bubble
volume and the corresponding radius (rm) and radius squared (rm2).
The meniscus pressure differential (Δp) for each bubble was calculated
using the Laplace-Young equation:
Dp ¼ ð2gÞ=rm
where water surface tension (g) was assumed to be 72 millinewtons m21 at
room temperature. This assumes no surfactants, which is the condition least
favorable for cavitation.
Anatomical Characterization of Xylem Using Cryo-SEM
Single living shoots (i.e. current season growth of stem plus leaves) of
C. muscoides and A. macquariensis were frozen in a fully hydrated state by immersion in LN2. Frozen stems were cut to a convenient size in LN2, mounted
on an aluminum stub using room-temperature Tissue Tek (Sakura Finetechnical), and immediately placed back in LN2. Frozen samples were placed in a
cryomicrotome (Leica EM UC7; Leica Microsystems) at 290°C, planed to the
desired depth using glass knives at 2100°C. Samples were then transferred to
the cryostage (CT1500 LN2 cryo stage; Oxford Instruments) of a Cryo-SEM
device (4300SE/N; Hitachi High Technologies), where they were etched at
290°C for as long as desired, coated with gold, and imaged using the Hitachi
software package (Hitachi High Technologies).
Anatomical Characterization of Fresh Shoots Using BrightField Microscopy
Transverse sections of fresh shoots of both C. muscoides and A. macquariensis
were cut with a sledge microtome (G.S.L.1, S. Lucchinetti; Schenkung Dapples) and stained with a toluidine blue solution (0.05% [w/v] in water).
Samples were observed using a DM6000B microscope (Leica Microsystems)
with a 203 multiimmersion objective (NA = 0.7), and images were taken with
a SPOT Flex camera (SPOT Imaging Solutions).
In Vitro Generation and Resorption of Embolisms
Fresh shoots of C. muscoides were dissected with forceps under water as follows:
(1) most of the tissue surrounding the xylem was removed to expose the xylem
tissue; (2) the sheet containing the xylem was slit in half along its length, and the pith
was gently removed; and (3) strands of xylem vessels were teased apart in
groups small enough to allow good imaging and large enough to preserve their
connections, structure, and integrity. Xylem vessels were then exposed to air
at room temperature. Dehydration was visually monitored using a dissecting
microscope, and within minutes, bubbles formed within intact vessels. At this
point, excised xylem was covered with a drop of water and a coverslip and placed
on the stage of the DM6000B microscope (Leica Microsystems) with 633 and
1003 oil immersion objectives (NA = 1.4). Images were taken every 0.5 s with a
SPOT Flex camera (SPOT Imaging Solutions). Movies with two frames per
second were generated from these image series using ImageJ 1.48n (National
Institutes of Health).
Calculation of Bubble and Xylem Properties
Measurements of bubble dimensions were made using ImageJ 1.47n (National Institutes of Health). Bubble length (L) was measured for each bubble as
the distance between the apices of the menisci. Meniscus radius (rm) was
calculated for each bubble from the mean of the radii of both menisci across
several time points when the bubble had a rod shape. When a bubble became
a sphere, its radius (rs) was determined at each time point as half of L. Bubble
volume (V) and surface area (S) were calculated for the rod- and sphereshaped bubbles using:
Rod :
Sphere :
V ¼ ðL–2rm Þpr2m þ ð4=3Þpr3m
V ¼ ð4=3Þpr3s
S ¼ 2ðL–2rm Þprm þ 4 pr2m
S ¼ 4 pr2s
Bubble width was calculated from the mean of three measurements per bubble
(one near each meniscus and one in the middle of the bubble) at several time points.
Linear regressions were conducted in R (version 2.15.3) to assess the effects
of bubble dimensions on rates of bubble size reduction. Rates of change in
bubble volume and surface area were determined by linear regression. Because
nearly resorbed bubbles change shape as well as size, a conservative cutoff
of 15 mm in length was applied to keep bubble shape comparable. A linear
Generation and Resorption of Embolisms in
Detached Shoots
Single fresh shoots of C. muscoides were removed from a cushion, covered
with water, and placed on the stage of a dissecting microscope. Using forceps,
most of the tissue surrounding the xylem was removed over an approximately
1-cm window to expose xylem vessels to air, leaving the rest of the plant
untouched (Fig. 8A). The exposed xylem was then allowed to dehydrate at
room temperature for several minutes under a gentle air flow generated with a
small plastic pipette while the rest of the shoot was kept wet. Dehydration was
visually monitored using a dissecting microscope, and embolisms could be
detected within minutes. Once bubbles appeared, further dehydration was
blocked by the addition of a drop of paraffin oil (Gold Cross) and a coverslip
over the detached portion of the shoot (Fig. 8B). Samples were observed using
the DM6000B microscope (Leica Microsystems) with a 203 multi-immersion
objective (NA = 0.7), and images were taken with a SPOT Flex camera (SPOT
Imaging Solutions).
Supplemental Data
The following supplemental materials are available.
Supplemental Movie S1. Movie of embolism repair in excised C. muscoides
protoxylem (free bubble).
Supplemental Movie S2. Movie of embolism repair in excised C. muscoides
protoxylem (trapped bubble).
ACKNOWLEDGMENTS
We thank the Australian Antarctic Division for logistic support enabling
the collection of plant material from Macquarie Island and three anonymous
reviewers for insightful comments on an earlier version of the article.
Received March 4, 2015; accepted June 18, 2015; published June 19, 2015.
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