RESEARCH New Phytol. (2000), 148, 239–255
Xylem conduits of a resurrection plant
contain a unique lipid lining and refill
following a distinct pattern after
desiccation
H. - J. W A G N E R", H. S C H N E I D E R", S. M I M I E T Z", N. W I S T U B A",
M. R O K I T T A#, G. K R O H N E$, A. H A A S E#    U. Z I M M E R M A N N"*
" Lehrstuhl fuW r Biotechnologie, Biozentrum, UniversitaW t WuW rzburg, Germany
# Lehrstuhl fuW r Experimentelle Physik V (Biophysik), UniversitaW t WuW rzburg, Germany
$ Abteilung fuW r Elektronenmikroskopie, Biozentrum, UniversitaW t WuW rzburg, Germany
Received 14 March 2000 ; accepted 9 June 2000

The axial and radial refilling with water of cut dry branches (up to 80 cm tall) of the resurrection plant
Myrothamnus flabellifolia was studied in both acro- and basipetal directions by using "H-NMR imaging. NMR
measurements showed that the conducting elements were not filled simultaneously. Axial water ascent occurred
initially only in a cluster of a very few conducting elements. Refilling of the other conducting elements and of the
living cells was mainly achieved by radial extraction of water from these initial conducting elements. With time,
xylem elements in a few further regions were apparently refilled axially. Radial water spread through the
tissue occurred almost linearly with time, but much faster in the acropetal than in the basipetal direction.
Application of hydrostatic pressure (up to 16 kPa) produced similar temporal and spatial radial refilling patterns,
except that more conducting elements were refilled axially during the first phase of water rise. The addition of
raffinose to the water considerably reduced axial and radial spreading rates. The polarity of water climbing was
supported by measurements of the water rise in dry branches using the ‘ light refraction ’ (and, sometimes, the ‘ leaf
recurving’) method. Basipetal refilling of the xylem conduit exhibited biphasic kinetics ; the final rise height did
not exceed 20–30 cm. Three-cm-long branch pieces also showed a directionality of water climbing, ruling out the
possibility that changes in the conducting area from the base to the apex of the branches were responsible for this
effect. The polarity of water ascent was independent of gravity and also did not change when the ambient
temperature was raised to c. 40mC. At external pressures of 50–100 kPa the polarity disappeared, with basipetal
and acropetal refill times of the xylem conduit of tall branches becoming comparable. Refilling of branches dried
horizontally (with a clinostat) or inverted (in the direction of gravity) showed a pronounced reduction of the
acropetal water rise to or below basipetal water climbing level (which was unaffected by this treatment). Unlike
water, benzene and acetone climbing showed no polarity. In the case of benzene, the rise kinetics (including the
final heights) were comparable with those measured acropetally for water, whereas with acetone the rise height was
less. Transmission electron microscopy of dry branches demonstrated that the inner surfaces of the conducting
tracheids and vessels were lined with a continuous osmiophilic (lipid) layer, as postulated by the kinetic analysis
and light microscopy studies. The thickness of the layer varied between 20 and 80 nm. The parenchymal and
intervessel pits as well as numerous tracheid corners contained opaque inclusions, presumably also consisting of
lipids. Electron microscopy of rehydrated plants showed that the lipid layer was either thinned or had
disintegrated and that numerous vesicle-like structures and lipid bodies were formed (together with various
intermediate structural elements). These, many other data and the physical–chemical literature imply that several
(radial) driving forces (such as capillary condensation, Marangoni forces, capillary, osmotic and turgor pressure
forces) operate when a few conducting elements become axially refilled with water. These forces apparently lead
to an avalanche-like radial refilling of most of the conducting elements and living cells, and thus to the removal
of the ‘ internal cuticle ’ and of the hydrophobic inclusions in the pits. The polarity of water movement presumably
results from high resistances in the basipetal direction, which are created by local gradients in the thickness of the
*Author for correspondence (tel j49 931 888 4508 ; fax j49 931 888 4509 ; e-mail zimmerma!biozentrum.uni-wuerzburg.de).
240
RESEARCH H.-J. Wagner et al.
lipid film as a result of draining under gravity in response to drought. There are striking similarities in morphology
and function between the xylem-lining lipid film and the lung surfactant film lining the pulmonary air spaces of
mammals.
Key words : Myrothamnus flabellifolia, xylem refilling, polarity of refilling, lipid lining, NMR imaging,
resurrection plants.

We have previously studied the acropetal water rise
kinetics in dry woody branches of Myrothamnus
flabellifolia under various laboratory and field conditions (Schneider et al., 2000). Water rise in the
xylem conduit was observed by light refraction
changes and the recurving of the folded leaves that
occurred at the advancing water front. The axial
water advance in M. flabellifolia occurred in a very
few conducting elements at a much higher rate than
in many others. However, the spatial and temporal
resolution of the methods was too limited to allow
study of the axial and radial refilling of the other
conducting elements and tissue cells.
Analysis of experimental data and light microscopy (Schneider et al., 1999) support the view that
the refilling of the dry xylem of M. flabellifolia is
apparently hampered by a hydrophobic (lipid) film
on the inner wall of the conducting elements. This
film must be removed or thinned before wetting can
occur and water can rise to the apex. The data
strongly suggest that : hydrostatic (root) pressure can
disintegrate the lipid film ; and both radial and axial
water spread are necessary for water to rise under its
own capillary pressure (as in cut branches). Disintegration of the lipid film apparently required a
feature characteristic of interfacial (Marangoni)
streaming. Streaming at an aqueous–organic interface is induced by surface tension gradients that can
be created by temperature gradients or composition
gradients in the lipid film. If gradients exist in the
surface properties, there will be polarity in water rise
(i.e. basipetal and acropetal refilling rates will differ).
However, such effects should not occur when water
is replaced by purely hydrophobic, organic liquids.
Water rise kinetics derived from visual and light
microscopy studies alone (Schneider et al., 1999) are
unlikely to be definitive because their analysis
involves many approximations. Other lines of evidence are required to consolidate the theoretical
considerations and the conclusions drawn from such
studies.
This study focuses on wetting dynamics and the
polarity of the refilling processes of the xylem of cut
dry branches of M. flabellifolia. Both acro- and
basipetal rise of water and organic solvents were
studied using electron microscopy and NMR
imaging as well as the ‘ light refraction ’ method.
Unlike the ‘ light refraction ’ and ‘ leaf recurving ’
methods (Schneider et al., 2000), NMR imaging
allows noninvasive determination of the water
refilling pattern at the level of a very few conducting
elements of ‘ air-dry ’ branches. Thus, the advancing
water front and the radial water spread can be
determined very accurately for hydrostatic-,
osmotic- and\or capillary-pressure-driven water rise
both in acro- and basipetal directions.
In combination with electron microscopy of dry
and refilled vessels and tracheids of M. flabellifolia,
NMR imaging of the transient axial and radial
refilling pattern gives a detailed insight into the
mechanisms of wetting and water ascent on the level
of the xylem conducting element, as well as on that
of the tissue.
  
Plant material
Cut branches of Myrothamnus flabellifolia Welw.
(40–80 cm in length and 2n5–5 mm in diameter)
collected from dry intact plants grown at
Glu$ cksburg, a farm at Outjo, Namibia, were used
throughout the experiments. For basipetal rise
kinetics, the apex of branches was removed.
Hydraulic conductivity measurements
The hydraulic conductivity of the xylem of dry
branches was determined by measurement of the
rate of acro- or basipetal water flow through 10-cmlong leafless branch pieces by means of a pressure
chamber equipped with a manometer. Pressures up
to 100 kPa were applied.
Xylem refilling measurements
Acro- and basipetal refilling of the conducting
elements of the xylem of M. flabellifolia with water
and organic solvents were studied in the laboratory
and in the field under various conditions by the ‘ dye
rise ’, the ‘ light refraction ’ and, when possible, the
‘ leaf recurving ’ methods (Schneider et al., 2000).
The NMR imaging experiments were carried out
exclusively in the laboratory.
The principle and conditions of the determination
of T -weighted "H-NMR spin echo images have
"
been described by Kuchenbrod et al. (1995). Briefly,
in the NMR experiments a Bruker BIOSPEC 70\20
spectrometer equipped with a laboratory-made climate chamber was used. The magnetic field strength
RESEARCH Refilling mechanisms of Myrothamnus flabellifolia II
was 7 T. The horizontal bore had an effective
diameter of 12 cm. A Helmholtz-type radio frequency coil (diam. 2 cm), which could be tuned onto
the proton resonance frequency of 300 MHz, was
used as a transmitter and receiver coil.
The plant–radio frequency coil assembly, in an
open perspex tube, was introduced horizontally into
the bore. The projecting cut end of the horizontal
branch was placed in tap water while the other end of
the perspex tube was connected via Teflon tubing to
the climate chamber. We used a two-dimensional
cross-sectional NMR spin echo imaging sequence.
Slice selection was performed using a frequencyselective gaussian 90m pulse of 500 µs duration. The
slice gradient was 59 mT\m ; the 180m pulse was not
selective. The sequences were used to produce an
image of 128i128 pixels (zero-filled to 256i256
picture elements). The spin echo time (TE) was
adjusted to 8 ms and the repetition time (TR) to
0n5 s. The spatial resolution was 45i45 µm#, the slice
thickness was 2 mm and the field of view (FOV) was
5n5i5n5 mm#. The signal intensity of the T "
weighted "H-NMR spin echo images did not change
upon variation of the repetition time demonstrating
that the images reflected purely spin-densityweighted images and thus changes in local water
concentration within branch cross-sections.
During the experiments, temperature and rh
within the bore were 20mC and 30–40%, respectively.
Screening experiments using a light source installed
outside the magnet showed that light intensity
(c. 700 µmol m−# s−") had no effect on the water rise
kinetics and pattern. Therefore, most of the experiments were performed in the dark.
Electron microscopy
For TEM studies on green branches, the outer bark
of c. 2-cm-long rehydrated-branch pieces was removed, then, to minimize fixation artefacts, the
pieces were fixed by two different procedures : twostep fixation, a 24-h treatment with a cacodylatebuffered Karnovsky solution containing 2% formaldehyde and 2n5% glutaraldehyde, followed by washing in cacodylate buffer and subsequent post-fixation
in 2% osmium tetroxide (OsO ) (Plattner & Zing%
sheim, 1987) ; one-step fixation overnight in a
cacodylate-buffered solution containing 0n1% glutaraldehyde and 2% OsO . After dehydration in
%
ethanol, the fixed material was embedded in Epon
resin. Transverse sections were cut with an ultramicrotome (MT-7000, Ultra, RMC, Tucson, AZ,
USA) and stained, unless stated otherwise, with
uranyl acetate and lead citrate. Electron micrographs
were taken with a transmission electron microscope
(EM 900, Zeiss, Oberkochen, Germany).
For dry-branch fixation, 2-mm-thick branch crosssections were sealed in glass flasks with osmium
241
vapour (Perner, 1965) for a few days. The material
was then embedded in Epon resin and treated as
already described.

Because of technical restrictions, horizontally
oriented branches were mostly used in NMR studies
of xylem refilling, so that water ascent was studied in
the absence of gravity. Because of the fixed position
of the magnet during each refilling experiment, each
NMR experiment yielded one data point for the rise
time and the corresponding height of water (i.e. the
time at which a "H-signal could be recorded in the
NMR cross-section was determined).
NMR measurements supported the finding of
Schneider et al. (2000), that water applied externally
did not penetrate through the bark into the xylem
(i.e. that water rise was only possible through the
xylem conduit when a cut end was in water). To
estimate the time dependence of acro- and basipetal
water rise, NMR images were made 4n5–40 cm above
the cut end of leafy (or leafless) branches of similar
morphology. The times and rise heights are listed in
Table 1. Because there were no differences in rise
time and height between leafy and leafless branches,
data were pooled. These parameters were in the
range for acropetal water rise measured by the ‘ light
refraction ’ and ‘ leaf recurving ’ methods in horizontal branches (Schneider et al., 2000). A few
experiments with upright branches (using a vertical
11n7-T NMR magnet) also yielded good agreement
with the kinetic data of Schneider et al. (2000)
(Table 1). Thus, NMR imaging supported the
accuracy of the ‘ kinetic ’ techniques.
Table 1 shows that basipetal water rise was
considerably delayed compared with acropetal, giving evidence for a strong polarity of water movement
in the xylem conduit of M. flabellifolia.
Nonuniform axial and radial xylem refilling
Consistent with the findings of Schneider et al.
(2000), the NMR measurements showed that the
axially advancing water front was not uniform
throughout the entire xylem conduit. Figs 1 and 2
show typical sequences of "H-NMR images for acroand basipetal refilling, acquired c. 6 cm from the cut
base and apex. In both cases, a "H-signal was
initially recorded only in a very small region
(commonly 4–5 pixels ; resolution 45i45 µm#)
indicating that (acro- and basipetal) refilling apparently occurred only in one or in a very few adjacent
conducting elements. Because of the limited geometric resolution of NMR imaging it was not possible
to match unequivocally these initial signal intensity
‘ spots ’ to vessels and\or tracheids as seen in the
corresponding microscopic cross-sections (Fig. 1f).
242
RESEARCH H.-J. Wagner et al.
Table 1. Rise heights and times of acro- and basipetal water movement in horizontal and upright branches
extracted from NMR images as shown in Figs 1 and 2
Acropetal-horizontal
Basipetal-horizontal
Acropetal-upright
Height (cm)
Height (cm)
Time (min)
Height (cm)
Time (min)
13p4
5.5
260
10
40
54p13
6
245
11
42
120p10
6
363
13
60
245
375
6.2
–
386
–
–
–
–
–
6.0p0.4
(n l 11)
12.3p1.0
(n l 3)
23.2p10
(n l 3)
35
39.5
Time (min)
For mean valuespSD, n, the number of individual experiments. See text for further details.
With time, the signal region always increased radially
from the initial conducting ‘ spot ’ while the maximum signal intensity of individual pixels remained
unchanged, indicating that the size of the waterfilled region expanded continuously. The spreading
was frequently accompanied by "H-signals appearing in a separate, second or (considerably delayed)
third or fourth region of the cross-section (Figs 1b,c,
2a,b). This indicated conducting elements unrelated
to the first becoming axially refilled with water. The
size of these water-filled areas also increased over
time, forming differently shaped "H-signal patterns.
As partly shown in Figs 1 and 2, there were
temporary crescent, cross, triangular or semicircular
shapes before a final ring-shaped pattern of uniform
signal intensity developed. The time dependence of
the increase in total signal intensity extracted from
the images is presented in Figs 1e and 2d. It is
evident that the increase in total signal intensity
occurred almost linearly with time but that the
slopes depended on the direction of water movement.
For acropetal water rise, the slope was c. 2% min−",
basipetally it was much lower, c. 0n2% min−" (values
referred to the final water-filled area of the crosssection). There were similar results from images
further from the cut end (up to c. 40 cm under
acropetal conditions). Hydrated branches which
were dehydrated and again refilled showed a similar,
but delayed, time-dependent pattern of the "Hsignals. Interestingly, the initial "H-signals were
recorded in regions that were not identical with those
of the first hydration cycle. Thus, these ‘ prominent ’
conducting elements reflect statistical variations in
the drying process, not structural characteristics of
the xylem conduit.
Comparison of NMR images with microscopic
cross-sections (Fig. 1f) showed that the ring-shaped
rehydrated area comprised the xylem conducting
area, the living tissue cells, the phloem and the
phelloderm. The refilling of the phloem, which can
be clearly seen from the NMR images (Figs 1d, 2b),
occurred after c. 30–40 min under acropetal con-
ditions. The water-conducting ring was apparently
near a core of nonconducting elements, because the
extremely weak background signal intensity (detectable throughout the entire cross-section before
application of water ; Figs 1a,b, 2a) did not change in
this region during refilling. This background signal
intensity might reflect very small amounts of water
(remaining from the drying process) or arise from
the protons of lipids within the xylem (see discussion
of TEM studies in the ‘ Xylem anatomy and ultrastructure ’ section). Interestingly, when NMR images
of dry branches with high signal yield were acquired
(Fig. 1a) an additional structural region of extremely
low signal intensity became visible, separating the
conducting and nonconducting xylem portions.
Estimation of the rehydrated area of branches of
different diameters from the "H-NMR images or by
eye showed that the rehydrated area occupied
64n3p12n6% (n l 78) of the total cross-sectional
area of the branches. The absolute conducting area
decreased slightly towards the apex.
Polarity of water rise
As observed by Child (1960) basipetal water or dye
movement was extremely slow when the apex was
not removed. Only the leaves immersed in water and
just above the water level re-expanded. When the
branch apex was cut, basipetal xylem refilling with
water or dye (e.g. ink, fluorescein) occurred, but still
very slowly in agreement with NMR imaging (Fig.
3). After c. 6 h, there was a significant increase in
the refilling rate, but it remained much less than
under acropetal conditions. Final heights of only
20–30 cm were reached after c. 27 h (Fig. 3).
Consistent with this, water uptake was greatly
reduced and the onset of transpiration occurred after
8–11 h (i.e. considerably delayed compared with
acropetal refilling (Schneider et al., 2000)). Basipetal
water rise generally showed more scatter than
acropetal. The biphasic climbing pattern (not predicted by Eqn 9 ; Schneider et al., 2000) apparently
RESEARCH Refilling mechanisms of Myrothamnus flabellifolia II
(a)
(b)
(c)
(d)
(e)
(f)
243
Fig. 1. Typical transverse "H-NMR images of acropetal water rise in cut (dry) 70-cm-long, horizontally
oriented leafy branches of Myrothamnus flabellifolia. (a) High-resolution image of a dry branch (obtained by 32
averages) indicating some background signal intensity and a region of extremely low signal intensity separating
the conducting and nonconducting xylem areas. Note that the cortex and phloem exhibit no signal intensity
(arrow). For measurement of the water refilling kinetics, images of lower resolution (no averages) were taken
on a regular basis 6n5 cm from the basal cut end. Images (b–d) were acquired 12, 30 and 70 min after water
application. (e) Total "H-signal intensity in the cross-section with time, after subtraction of the constant
background signal of the dry branch. For comparison, a typical light microscopic cross-section of a rehydrated
branch is shown (f). The arrows in (a), (d) and (f) mark the phloem.
occurred in all branches, independent of the length
of branch piece and orientation (upright, inverted or
horizontal). As in the case of acropetal water
movement (Schneider et al., 2000) and in agreement
with the NMR measurements (Table 1), removal of
the leaves did not change significantly the rate of
water ascent or the final rise height (Fig. 3).
Interestingly, when a ramified branch was refilled in
244
RESEARCH H.-J. Wagner et al.
(a)
(b)
(c)
(d)
Fig. 2. Typical transverse "H-NMR images of basipetal water rise in a cut, horizontally oriented branch of
Myrothamnus flabellifolia. Images were taken on a regular basis 6n2 cm from the apical cut end, those shown
here (a) 386 min (b) 600 min and (c) 990 min after water application. (d) Increase in the total "H-signal intensity
in the cross-section with time (after subtraction of the constant background signal of the dry branch). The
arrow in (b) marks the phloem.
the basipetal direction, water rise occurred instantaneously acropetally in the side branch when the
water front reached the branching point.
The polarity of refilling was also clear in branches
of uniform morphology loaded with ink from both
cut ends simultaneously, and it was also found that
acropetal water rise was much faster than water
ascent in the basipetal direction (data not shown).
Even when 3-cm-long branch pieces (taken at regular
intervals from 60–70-cm-tall branches) were used,
the polarity in water movement remained, indicating
that this phenomenon was an intrinsic property of
the entire branch.
Green branches dehydrated at 20–24mC and 45mC
in the upright position showed slightly reduced
acropetal water rise kinetics upon refilling (in
agreement with NMR imaging), but the polarity for
water movement did not disappear. However,
branches dried when inverted or horizontal (by
using a clinostat-like set-up ; Block et al., 1986)
usually showed a dramatic reduction of acropetal
refilling, with basipetal water rise remaining
unaffected. The acropetal rise heights measured after
24 h varied considerably (usually between 3 cm and
25 cm). Basipetal water rise could only be significantly reduced (to c. 5 cm) when the branches were
heated in an oven for 5 d at 100mC.
The asymmetry of water movement remained
with ambient temperature elevated to c. 40mC.
The closed symbols in Fig. 3 represent data from
measurements of basipetal, antigravitational water
rise in inverted branches both in the laboratory and
(some) in the field. Despite the large scatter of the
data it is evident that temperature accelerated initial
water rise, associated with the disappearance of the
biphasic climbing profile. However, the final water
rise heights were comparable with those measured at
20–24mC, at least up to 23 h (Fig. 3).
Basipetal versus acropetal xylem refilling under
hydrostatic and osmotic pressures
Basipetal water ascent increased and the polarity of
water movement disappeared upon pressure ap-
RESEARCH Refilling mechanisms of Myrothamnus flabellifolia II
245
Fig. 3. Influence of leaves and ambient temperature on the basipetal water rise kinetics in the xylem of cut dry
branches of Myrothamnus flabellifolia against gravity (after removal of the apex) using the ‘ light refraction ’
method of Schneider et al. (2000). Leafy branches (open squares) ; leafless branches enclosed in silicone rubber
and\or parafilm (open circles) ; laboratory ambient temperature 20–24mC. Leafy branches at c. 40mC in the
laboratory (closed triangles) and in the field (closed circles). Data show mean values (pSD) of at least three
independent experiments. For clarity only half of the bars of the SD are shown.
Fig. 4. Effect of external-pressure application on the basipetal refilling kinetics of leafy and leafless branches
of Myrothamnus flabellifolia against gravity at ambient temperature 20–24mC and pressure 100 kPa. Filled
symbols, acropetal rise in leafy branches, ‘ leaf recurving ’ method ; open symbols, basipetal rise in leafy
branches, ‘ leaf recurving ’ method ; crossed circles, basipetal rise in leafless branches sealed with silicone
rubber, ‘ light refraction ’ method of Schreider et al. (2000) ; data points of three different branches. Note that
the basipetal rise kinetics of the leafy branches varied considerably when branches were not protected against
radial water loss through lesions.
plication to the water reservoir. Typical rise kinetics
measured with the ‘ leaf recurving ’ and ‘ light
refraction ’ methods under laboratory conditions are
shown in Fig. 4. Pressures of c. 100 kPa were needed
to refill the empty conducting elements of 50–70-cm-
tall branches basipetally against gravity. Complete
refilling occurred in
1 h. For basipetal refilling
only, it was necessary to seal lesions along the
branches (e.g. where leaves had been removed),
otherwise water escaped from these sites, reducing
246
RESEARCH H.-J. Wagner et al.
Table 2. Changes in the axial hydraulic conductivity
of 10-cm-long leafless branch pieces with pressure (Pext),
applied for 20 min in both acro- and basipetal directions
(laboratory conditions ; temperature : 20–24mC)
Pext
(kPa)
25
50
100
LP (10−' m s−" kPa−")
Acropetal-upright
Basipetal-inverted
1.7p1.0
(n l 5)
3.4p1.2
(n l 5)
3.5p0.8
(n l 6)
0.4p0.3
(n l 6)
2.8p0.3
(n l 5)
3.6p1.2
(n l 6)
Values are meanspSD ; n, number of individual experiments.
final heights (Fig. 4). Best results were obtained
when all leaves were removed and the branches were
completely covered with silicone rubber. Apparently, the axial hydraulic resistance for basipetal
water rise was much larger than the hydraulic
resistance in radial direction. Measurements of the
hydraulic conductivity in the acro- and basipetal
directions gave further support for this assumption.
When pressures of 50–100 kPa were applied, the
polarity in the hydraulic conductivity observed at
lower pressures disappeared while, simultaneously,
the hydraulic conductivities in both directions
increased (Table 2). NMR imaging showed (Fig. 5)
that pressure application did not change the pattern
of radial water spread for acro- and basipetal refilling
(Figs 1, 2). However, the total signal intensity was
biphasic with time for pressure-driven acropetal
water rise (Fig. 5d) even though the complete radial
refilling time remained almost constant compared
with acropetal refilling under capillary pressure (Fig.
1e). The biphasic refilling process in Fig. 5d could be
approximated by two straight lines. The slope of the
fast refilling phase was 4n7% min−" and that of the
second, slow one 0n8% min−". The arithmetic mean
value of the two slopes corresponded quite well with
the slope of c. 2% min−" extracted from the curve in
Fig. 1e. Therefore, the data suggested that the first
phase reflected axial water movement in the conducting elements and the second the ‘ true ’ radial
water spreading rate through the apoplastic space
and tissue cells. This interpretation is apparently
also in agreement with the larger ‘ rate-limiting
radii ’ of the ‘ conducting elements ’ that were
extracted from fitting of the corresponding water rise
kinetics under pressure (Schneider et al., 2000).
However, when the branches were immersed in
50 mM raffinose solution, both axial (acropetal) water
ascent and radial spreading were considerably retarded (Fig. 6). Application of c. 20 kPa in the presence
of raffinose usually resulted in a re-establishment of
the axial and radial flow rates (data not shown).
Acropetal and basipetal rise of organic solvents
Unlike that of water, benzene rise showed no
polarity. Measurements of benzene rise in vertical,
leafy, dry branches (using the ‘ light refraction ’
method) yielded similar results for both rate and
final height in acropetal and basipetal directions
(Fig. 7). Benzene rose as fast as or faster than water
in the acropetal direction (Fig. 7). In horizontal
branches (i.e. in the absence of gravity) benzene rise
rate and final height were slightly greater than those
of water, and symmetric in the two directions. Leaf
recurving was never observed during benzene
refilling, indicating that benzene apparently did not
change the properties of the conducting elements of
the branches. This was also demonstrated by
experiments in which water ascent was studied in
branches pre-treated with benzene. After complete
evaporation of the benzene from the treated
branches, water rise showed the same time profile
and polarity (together with leaf recovery) as in the
controls (data not shown). Consistent with this,
staining with fluorescein diacetate (Romeis, 1989)
and microscopic inspections of the transverse
sections of benzene-treated branches gave no evidence of cell damage. By contrast, immersion in
benzene of transverse sections of untreated branches
resulted in severe damage to the tissue cells. This
indicated that the benzene in the xylem conduit did
not come into direct contact with the cell
constituents.
Acro- and basipetal ascents for pure acetone were
also symmetric, even though the final rise heights
were c. 30 cm (i.e. lower than for benzene ; Fig. 7).
Acetone is more hydrophilic than benzene and is
completely miscible with water. When the acetone
was gradually replaced by water it was found that the
polarity between acro- and basipetal liquid rise
appeared (Fig. 8). At a ratio of c. 40 : 60 for
acetone : water, the polarity in liquid movement was
clearly developed. It is interesting to note that pure
acetone always induced recurving of the leaves but
only occasionally did they unfold. After evaporation
of acetone from treated branches there were reduced
final rise heights for acropetal water refilling (12–18
cm after 48 h) and leaf unfolding was generally
suppressed. This was evidence that acetone had
interfered with the cells (those involved in axial
water ascent ; Schneider et al., 2000).
Both acropetal and basipetal rise of organic liquids
followed Eqn 9 of Schneider et al. (2000) (with
appropriate values for the viscosity and density of
the liquids). However, the data could only be fitted
(Fig. 7) by assuming radial extraction of the organic
liquids from the xylem conduit. The best fits for the
benzene rise kinetics were obtained when the
radial extraction rate (Vl) was assumed to be c.
0n6–1i10−"' m# s−" (Table 3). For pure acetone, Vl
was slightly smaller. In the light of the nontoxic
RESEARCH Refilling mechanisms of Myrothamnus flabellifolia II
(a)
(b)
(c)
(d)
Fig. 5. Typical series of transverse "H-NMR images of acropetal water rise in a leafy (horizontally oriented)
branch of Myrothamnus flabellifolia under a pressure of c. 13 kPa (corresponding to the root pressure of intact
plants ; Schneider et al., 2000). Images were taken regularly 13 cm from the basal cut end, those shown here
(a) 2 min, (b) 10 min and (c) 50 min after water application. (d) Total "H-signal intensity in the cross-section
with time (after subtraction of the constant background signal of the dry branch). Note that in contrast with
Fig. 1, the refilling kinetics is biphasic, with a fast axial phase (a) and a slow phase reflecting the ‘ true ’ radial
water spreading rate (r).
(a)
(b)
Fig. 6. Typical transverse "H-NMR images of acropetal water rise in a leafy (horizontally oriented) branch of
Myrothamnus flabellifolia in the presence of 50 mM raffinose. Images were taken regularly 5n5 cm from the basal
cut end, those shown here (a) 63 min and (b) 130 min after application of the sugar solution, when c. 50% of
the conducting area was refilled.
247
248
RESEARCH H.-J. Wagner et al.
Fig. 7. Acro- and basipetal ascent of benzene and acetone in comparison with the corresponding water rise
kinetics of Myrothamnus flabellifolia. Measurements were taken under laboratory conditions at an ambient
temperature of 20–24mC by the ‘ light refraction ’ method (except for acropetal water loading, when the ‘ leaf
recurving ’ method was used). Circles, benzene ; triangles, acetone ; squares, water ; open symbols, acropetal
rise ; filled symbols, basipetal rise. Data show mean values (p SD) of at least three independent experiments.
Solid lines, best curve fits obtained for liquid ascent using Eqn 9 of Schneider et al. (2000).
Fig. 8. Acro- and basipetal rise heights of acetone\water mixtures measured after 2 h, under laboratory
conditions in Myrothamnus flabellifolia at an ambient temperature of 20–24mC by the ‘ light refraction ’ method
of Schneider et al. (2000). Open columns, acropetal rise ; closed columns, basipetal rise. Data show mean values
(pSD) of four independent experiments.
effect of benzene on the living cells, the finding of a
radial extraction rate for benzene was somewhat
surprising, but might have been due to the high
solubility of this liquid in the lipids of the radial
apoplastic and cellular pathways (see the ‘ Xylem
anatomy and ultrastructure ’ section).
The contact angles for benzene and acetone were
c. 50–60m and 84m, respectively, depending on the
assumptions for surface tension (Table 3). The
contact angle values of benzene were smaller than
those of water (Schneider et al., 2000) as expected for
xylem walls with hydrophobic rather than hydrophilic features.
Xylem anatomy and ultrastructure
Microscopic investigations showed numerous
tracheids and mostly solitary vessels. The per-
RESEARCH Refilling mechanisms of Myrothamnus flabellifolia II
249
Table 3. Values of the radial water extraction rate, Vl, the contact angle, Θ, and the surface tension, σ, obtained
from fitting the rise kinetics of acetone and benzene in Fig. 7 by using Eqn 9 of Schneider et al. (2000) (ratelimiting radius, r l 1 µm ; ambient temperature, 21mC)
Vl
(10−"' m# s−")
σ cos Θ
Viscosity
(10−$ kg m−" s−")
Density
(kg m−$)
σ
(10−# N m−")
Θ (m)
Fig.
Acetone
0.4
0.0023
0.316
788
0.6–1.0
0.0139
0.625
879
85
84
83
62
56
46
7
Benzene
3.0
2.5
2.0
3.0
2.5
2.0
centages of the contributions of the tracheids and
vessels to the conducting area were estimated to be
67n3p5n5% and 19n7p4n7%, respectively (n l 5).
The tracheids were rhomboidal in cross-section and
thick-walled, whereas the vessels were roughly
circular (or, more accurately, polyhedral) and thinwalled.
The radial and tangential diameters of the
tracheids were in the order of 3–6 µm and 10–16 µm,
respectively. At the branch base, c. 60% of the
vessels had a diameter of 20–29 µm and c. 33% had
a diameter of 30–39 µm. Only c. 7% of the vessels
exhibited diameters of 40–49 µm. These data agree
with those of Carlquist (1976), who reported a mean
value of 36 µm for the diameter of the vessels, but
stand in contrast to the value of 14n4 µm published
by Sherwin et al. (1998).
The mean vessel and tracheid lengths (determined
from macerated branch pieces ; Sherwin et al.,
1998) were 479p41 µm (nl 9) and 557p67 µm (n l
16), respectively. These values also agree well with
those of Carlquist (1976). The perforation plates
were of a modified scalariform (reticulate) type ; the
intervessel and parenchymal pit sizes were c. 1–2 µm.
Ray parenchyma cells were abundant (area percentage of the conducting xylem : 13n1p2n5% ; n l 5)
and exclusively uniseriate, with tangential and radial
diameters of 16n8p4n8 µm and 46n5p10n5 µm,
respectively (n l 12). In the light of the results
presented in Schneider et al. (2000) these cells were
presumably involved intimately in radial water
spreading.
TEM gave further evidence for the hydrophobic
features of the inner walls of the conducting
elements. Fig. 9a–c demonstrates that the inner
surfaces of the dry-fixed (empty) tracheids and
vessels were lined with a continuous, osmiophilic
layer of quite variable thickness (20–80 nm). This
finding is consistent with previous experiments
(Schneider et al., 1999) suggesting that the inner
walls of dry conducting elements are covered by lipid
layers stainable with Sudan III. There were some
hints that the large variation in thickness depended,
at least partly, on evaporation of lipids. Extremely
thick, multi-layered lipid linings were found in the
7
inner, nonconducting area of the xylem (from which
no "H-NMR signal could be recorded ; Figs 1, 2, 5,
6). The layers in both nonconducting and conducting
regions frequently appeared continuous even within
the intervessel pits (Fig. 9a). Sometimes, relatively
large, irregularly shaped and osmiophilic aggregations were found attached to the inner walls
of a tracheid (Fig. 9c). Omission of post-staining of
the dry-fixed sections with uranyl acetate and lead
citrate yielded similar results, indicating that the
osmiophilic xylem lining and inclusions consist
mainly of lipids, not of proteins or other electrondense compounds.
TEM images showed opaque inclusions in parenchymal and intervessel pits and in numerous corners
of the tracheids (Fig. 9a,b). The osmiophilic nature
of the inclusions indicated lipid composition. There
was some evidence that the number of lipidcontaining pits decreased gradually towards the apex
and that the pits of the very apex did not contain any
osmiophilic material.
Fig. 9d–f shows electron micrographs of sections
of water-filled branches made at 2–4 cm from the
base 24 h after acropetal water ascent against gravity.
A very thin, continuous osmiophilic film (20 nm)
still covered the inner surface of the waterconducting tracheids and vessels. Frequently, fluffy
and\or tiny granular aggregations were found on the
thin lipid layer (Fig. 9e). The lumen of the vessels
and tracheids contained abundant lipid bodies
and\or vesicle-like structures (Fig. 9f), especially
near the intervessel and parenchymal pits. Additionally, numerous osmiophilic ‘ threads ’ (Fig. 9e)
were often found, especially near the thin lipid film.
Most interestingly from the water spreading perspective, the pits were lipid-free or only sometimes
contained small vesicles or granular remnants. These
findings were independent of the fixation procedure
used (see the Materials and Methods section).
TEM could not elucidate the effects of benzene
treatment on the thickness and\or integrity of the
lipid lining because, owing to its chemical nature,
any benzene remaining on the xylem walls despite
evaporation would stain with OsO . However,
%
electron microscopy revealed (consistent with the
RESEARCH H.-J. Wagner et al.
250
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 9. Transmission electron micrographs of dry and rehydrated branch cross-sections of Myrothamnus
flabellifolia. (a–c) Dry-fixed branches. V, vessel ; T, tracheid ; arrows, lipid linings of variable thickness ;
asterisks, lipid inclusions in tracheid corner and intervessel pits ; a, lipophilic aggregations attached to a tracheid
wall. (d–f) Rehydrated branches. P, parenchyma cell ; t, lipophilic threads (also marked by frame) ; g, granular
deposits of variable size ; v, vesicle-like structures. Pits are empty and lipid linings (arrows) appear disintegrated
and\or partly removed. Bar, 1 µm.
light-microscopic results) that benzene rising in dry
branches did not damage the ray parenchyma cells,
since ultrastructural features such as membranebounded lipid bodies appeared unaltered between
normal and benzene-pre-treated, rehydrated branch
material (data not shown).

The mathematical treatment of the water rise kinetics
in dry branches of M. flabellifolia given in Schneider
et al. (2000) was limited because the inner surface of
the xylem apparently has time-varying wettability
properties upon contact with water.
The uncertainties included in the theoretical
analysis of water rise under its own capillary force as
well as under hydrostatic and osmotic pressure are
obviously greatly reduced by the observations
reported here.
Evidence for a lipid film on the inner surface of the
xylem walls
The electron micrographs give clear indications of
an osmiophilic (lipid) film of variable thickness
covering the inner walls of the conducting and
nonconducting tracheids and the vessels of dry
branches. Interestingly, the intervessel and parenchymal pits of dry branches were also completely
filled with lipids. The lipid deposits on the inner
xylem walls (together with the lipid-filled pits) might
provide an efficient barrier, preventing complete
water loss from the tissue cells during dehydration.
This is in accordance with Gaff (1977) who showed
that ‘ air-dry ’ branches still contain c. 7% water.
Electron microscopy also showed that part of the
film was removed, thinned or disintegrated on water
penetration, resulting, through intermediate stages,
in the formation of vesicle-like structures and lipid
bodies. Simultaneously, the lipid inclusions of the
pits were displaced by water to a large extent.
Ultrastructural investigations do not allow systematic studies of the lipid film and pit topography
of the inner xylem walls over the entire length of the
branches (i.e. on a micrometer or smaller scale).
Thus, there remain some questions as to the
continuum of the lipid film, particularly towards the
apex. However, there are four other kinds of
information which favour the view of a continuous
rather than an interrupted lipid film over the entire
inner surface of the xylem conduit.
RESEARCH Refilling mechanisms of Myrothamnus flabellifolia II
The high contact angles (or equivalently the very
low surface tensions) from water rise kinetics argue
for a far-ranging continuous lipid film rather than for
a frequently interrupted one, or numerous empty
pits.
Extremely hydrophobic liquids such as benzene
spread rapidly axially in both directions, presumably
because of the relatively low contact angle (Table 3).
Even though the radial extraction rate (on the
conducting-element level) was comparable with that
of water, deterioration of tissue cells was not
observed, thus indicating that the solubility of the
benzene in the lipid films was quite high. The local
rearrangements of the film and the inclusions in the
pits were apparently reversible, as shown by water
refilling of benzene-pre-treated branches.
The concentration- and molecular-size-dependent
reduction of axial and radial water movement by
sugars is also convincing evidence for a continuous
solute-reflecting lipid film (Schneider et al., 2000).
The fourth type of experiment which favours the
concept of a lipid film continuum is the nonuniform
refilling pattern of the conducting elements with
water.
Radial refilling pattern and driving forces
Summarizing the evidence. Water, under its own
capillary pressure, initially rises acropetally in a few
neighbouring conducting elements in the xylem of
M. flabellifolia (Figs 1, 2, 5, 6). Most of the empty
elements and the tissue cells are apparently refilled
by radial water movement although axial rise might
occur in a few additional elements. The basipetal
pattern is qualitatively similar but strongly retarded.
NMR images of dry branches of European trees
(e.g. birch) show a completely different pattern,
where water ascends simultaneously in the large
vessels (data not shown). The different refilling
patterns are especially striking because birch and
Myrothamnus both exhibit a diffuse-porous xylem
with comparable large-vessel diameter.
In M. flabellifolia the number of active conducting
elements could only be increased significantly by
pressurization, which did not change the pattern and
average velocity of radial refilling (Fig. 5 ; Schneider
et al., 2000).
The mechanism of xylem refilling with water
under its own capillary pressure can be pictured as
follows (Fig. 10).
At the very end of the dehydration process a few
xylem elements might be covered by a lipid film that
is extremely thin and\or imperfect (e.g. due to
trapped air bubbles). NMR imaging on branches
subjected to several rehydration\dehydration cycles
suggests that this occurs randomly. Surface heterogeneity leads to a film which will be weakened by
penetration of water (Adamson & Gast, 1997 ;
Schneider et al., 2000). This will certainly facilitate
251
axial water rise in some elements. Apart from the
(relatively poor) wettability, the capillary driving
force is initially effective at its maximum value, as
the surface tension is close to that of pure water.
Then the surface tension drops because of lipid
removal from the walls and the pits. Axial water
movement is retarded and the water-filled conducting element(s) can function as a source for radial
water movement to nearby vessels, tracheids and
parenchyma cells, thereby replacing the lipid inclusions within the intervessel and parenchymal pits
by water. The questions are : what are the driving
forces for this process ; and in which conducting
element (vessel or tracheid ?) does axial water ascent
start ?
Kinetic analysis is compatible with vessels and\or
tracheids being initially involved and NMR resolution is insufficient to distinguish between these
alternatives. However, the dominant role of the
vessels can be inferred from the NMR images when
the radial driving forces are considered.
A candidate force is capillary condensation
together with Marangoni- and capillary-tensiondriven water flow. A curved surface not only changes
the pressure of the liquid below the surface but also
reduces the equilibrium vapour pressure (p) from
that of bulk water (p ) (Carman, 1953) according to
!
the thermodynamic relationship :
ln
0pp!1 l MP
RT
Eqn 1
ρ
(M, ρ, R and T have their usual meaning (i.e.
molecular weight, density, gas constant and absolute
temperature) and P is the capillary pressure.)
Replacement of P by 2 σ cos Θ\r (see preceding
paper) leads to the Kelvin equation :
ln
0pp!1 l 2 cosrRTM
σ
ρ
Θ
Eqn 2
(r, radius of the lumen of the conducting element.)
Redistribution of liquids between interconnected
capillaries of different sizes can occur through
distillation and\or flow of liquid. According to Eqn
2 large capillaries have higher vapour pressure than
small ones. Thus, distillation occurs from the large
to the small capillary. Capillary condensation will
always take place when the contact angle is
90m. This condition is fulfilled for the xylem of
M. flabellifolia (Schneider et al., 2000). A water film
is formed on the wall of the empty capillary (because
the capillary pressure of a plane surface is zero)
followed by water movement along the surface down
to the bottom of the vertical capillary due to gravity
(see Fig. 10). The result is liquid condensation and
refilling of the lumen of the small capillary. The
capillary suction pressure of the condensed liquid
will induce an additional liquid flow from large to
small capillary because the capillary pressure (P) is
252
RESEARCH H.-J. Wagner et al.
Fig. 10. Schematic diagram of the mechanism of radial
refilling on the level of a large and a small conducting
element, a vessel and a tracheid (left-hand side) ; and on
the xylem\tissue level (right-hand side). l, lipid lining ; ld,
disintegrated lipid lining ; lp, lipid pit inclusion ; cf,
capillary force ; Vl, radial extraction rate ; vrad, velocity of
radial spreading in the tissue (l slope of the slow phase of
the biphasic signal intensity time curve in Fig. 5d) ; P,
turgor pressure.
inversely proportional to the radius. The large vessel
drains out when no further water is available.
Otherwise, its displaced water is immediately compensated by corresponding water uptake from the
water bath. Radial redistribution of water will be
completed when the height of the meniscus is
uniform at all points, and is accompanied by a
corresponding uniformity of P and p\p .
!
The mechanism of capillary condensation strongly
suggests that one or a few vessels are filled first and
that refilling of the tracheids of the dry branches of
M. flabellifolia (in the absence of root or external
pressure) occurs mostly radially. However, there is
one important point to note. Radial refilling also
obviously occurred in the absence of gravity because
most of the NMR imaging studies were performed
on horizontal branches. Thus, before capillary
pressure can become as effective as capillary condensation there must be further forces which
facilitate the disintegration of the lipid film and the
refilling of the lumen of the empty small tracheids
from the cut end. One possibility is that liquid
condensation in the empty tracheids is promoted by
the corners, leading to an increase in capillary
pressure and thus in enhanced spreading rates (Finn,
1989). The other possibility is that osmotic or,
particularly, interfacial forces are involved. Interfacial forces should be generated because once water
has been condensed on the lipid film surface tension
is reduced. Thus, a very steep surface tension
gradient will be established between the lipid\water
mixture and the pure water at the cut end, resulting
in Marangoni convection and rapid removal or
disintegration of the lipid film (Fig. 10 ; Schneider et
al., 2000).
Thus we can conclude that several (radial) driving
forces come into operation when a few conducting
elements become refilled with water (Fig. 10).
Capillary condensation, Marangoni forces and capillary forces together with the generation of turgor
pressure apparently lead to an avalanche-like increase of the refilling rate of the dry conducting
elements and cells in the neighbourhood of the
originally water-filled elements. Thus, it becomes
clear why the rate of refilling on the tissue level is
much greater than the water extraction rates on the
vessel level (Schneider et al., 2000).
The slope of the slow phase of the NMR signal
intensity increase (Fig. 6d) presumably reflects the
‘ true ’ velocity of radial spreading, vrad, through the
tissue. Taking the dimensions of the waterconducting area of the branch cross-section into
account, vrad is roughly calculated to be 1n3i10−( m
s−". Assume the hydraulic conductivity of the radial
pathway is c. 7i10−* m s−" kPa−" (Zimmermann
& Steudle, 1975), Eqn 7 of Schneider et al. (2000)
indicates a pressure of c. 19 kPa which is comparable
with the value of the root pressure measured in intact
plants (Schneider et al., 2000). Furthermore, the
NMR measurements support the hypothesis of a
constant radial driving force made in the kinetic
analysis as the "H-signal intensity in the NMR
cross-sections increased almost linearly with time
(except at the very end of the radial refilling process,
where equilibrium is reached). Most importantly,
this was independent of the height and orientation of
the branches.
Polarity of xylem refilling
The flow in the xylem of hydrated plants, including
M. flabellifolia (Schneider et al., 1999), is generally
acropetal, from roots to leaves. This is due to the
chemical potential gradient of water within the
conduit generated by transpirational water loss and
not usually to rectifying properties of the waterconducting elements (Malone, 1993). Thus, refilling
of empty conducting elements with water under its
own capillary pressure should not show any polarity
in water ascent. The modified capillary equations of
Lucas (1918), Washburn (1921) and Rideal (1922)
also predict identical filling rates both in acro- and
basipetal directions (Eqns 8–10, Schneider et al.,
2000). Consistent with the theory, refilling experiments on dry branches of European trees, such as
birch, beech and willow, have not yielded any
evidence for differences between acro- and basipetal
water (and benzene) ascent (data not shown).
By contrast, a distinctly different phenomenon
was observed for basipetal refilling of dry branches
of M. flabellifolia, in which basipetal ascent of water
was extremely slow and much lower final heights
RESEARCH Refilling mechanisms of Myrothamnus flabellifolia II
were reached than in the case of acropetal rise.
Basipetal ascent also showed a biphasic pattern and
not the ‘ Nt-dependence ’ of acropetal rise (modified
by the assumption of radial water extraction).
The initial water rise was extremely retarded and
variable from branch to branch, suggesting that
basipetal penetration of water into the conducting
elements was initially hampered. Once water had
climbed up to c. 10 cm, the water rise kinetics showed
qualitatively (but not quantitatively) similar features
to acropetal rise kinetics. Increasing temperature
apparently facilitated initial water penetration because the first phase of the biphasic water rise pattern
disappeared, but polarity remained. This finding
might be related to the decrease in viscosity.
Application of pressures of about 100 kPa accelerated
basipetal water rise considerably and the polarity
disappeared. However, complete refilling of the
xylem of the branches required prevention of radial
water loss through lesions. This and the hydraulic
conductivity measurements demonstrated clearly
that the radial hydraulic resistance under basipetal
conditions was much lower than the axial resistance,
in contrast with acropetal water rise. This was a
surprising result because NMR imaging showed that
radial water spread was greatly retarded compared
with the findings for acropetal water rise.
Polarity in capillary-pressure-driven water movement can have several causes. A directionality in
water rise is expected if the conducting area decreases
continuously from the base to the apex. Estimations
of the total conducting area from NMR images and
from microscopic examinations of cross-sections of
M. flabellifolia have indeed shown that this is the
case. However, the decrease in conducting area (and
diameter of the conducting elements) was too small
to account for the observed effects and there was
pronounced polarity even in only 3-cm-long branch
pieces. Changes in the conducting area should also
lead to polarity with benzene and acetone.
We are driven to the conclusion that polarity for
water rise in the dry xylem of M. flabellifolia must be
related to the special features of the conducting
elements. From a theoretical standpoint (Schneider
et al., 2000), gradients in surface roughness of the
walls of the conducting elements could cause marked
differences between acropetal and basipetal water
rise (axially and radially). However, electron microscopy gave no indication of ultrastructural
elements which could straightforwardly explain the
directionality. Therefore, the most likely explanation
for the polarity is that gradients in wettability exist
which are closely coupled with the disintegration of
the lipids from the xylem walls and from the pits. If
lipid disintegration is facilitated in the basipetal
direction from the very beginning of penetration,
water rise would be reduced immediately by the
dramatic drop in surface tension. Thus, radial water
movement both on the level of the conducting
253
elements and on the tissue level would be greatly
retarded compared with acropetal rise, as found by
the NMR experiments.
An axial gradient in wettability can arise from a
gradient in lipid composition and\or thickness.
Experiments with a plane soap film stretched across
a rectangular frame suggest (Adamson & Gast, 1997)
that a gradient in thickness is more likely than a
gradient in composition (which would imply material
separation during the drying process). When a new
film is raised vertically in a closed system (to avoid
evaporation) it is initially relatively thick. Drainage
begins immediately, causing the film thickness to
decrease from bottom to top.
Such a process is likely during dehydration of the
branches in their natural environment. It is reasonably certain that drying of the xylem conduit occurs
from apex to base. Accordingly, lipid spreading and
formation of the film on the inner xylem walls will
occur in the direction of gravity. Drainage of the film
combined with thinning is initiated immediately
above the continuously receding water front,
resulting in ‘ saw tooth-like ’ profiles of the condensed
(multilamellar) lipid film on small (at least 3 cm)
scales. The formation of such structures implies a
liquid–solid transition which depends on the gel-toliquid transition temperature. We can speculate that
this might be facilitated by the local temperature
gradient at the air–water interface created by the
cooling of the receding water due to evaporation.
A gradient in thickness is difficult to determine by
electron microscopy. However, the electron micrographs have shown that the thickness of the lipid film
varied between 20 and 80 nm. This overall variability
in thickness seems to substantiate the earlier considerations. Additional evidence in support of this
explanation is given by the experiments in which the
drying process was performed in the absence, or in
the direction, of gravity. Subsequent acropetal rise
heights were greatly reduced and were sometimes
even less than basipetal ones, most probably because
of inappropriate lipid deposition on the walls and in
the pits. This clearly indicates that the directionality
of water movement in the dry branches of
M. flabellifolia reflects the ‘ finger prints ’ of the
formation of the lipid layers during drying.
What is the chemical composition of the lipid layer ?
The composition of the lipid layer on the inner
xylem walls of M. flabellifolia is not known. It is
difficult to extract the xylem lipids separately from
those of the cells which might be different (cellular
lipids are presumably, at least partly, volatile (Da
Cunha & de Lurdes Rodrigues Roque, 1974)).
However, dipalmitoyl phosphatidylcholine (DPPC)
or analogous compounds are promising candidates.
DPPC is insoluble in benzene and preliminary
experiments have shown (G. Binder, H. Schneider,
254
RESEARCH H.-J. Wagner et al.
U. Zimmermann, unpublished) that palmitic acid is
a major component of extracted lipids. DPPC is a
powerful surfactant, reducing the surface tension of
water to nearly zero (Johansson et al., 1994). The
transition temperature of DPPC (41mC for the fully
hydrated lipid) is decreased significantly by
phosphatidylglycerol or trehalose (i.e. by compounds
found in M. flabellifolia ; Bangham et al., 1979 ;
Crowe et al., 1984a,b ; Drennan et al., 1993 ; Crowe
& Crowe, 2000). Furthermore, there are striking
similarities in morphology and function between the
xylem lining and the surfactant film lining the
pulmonary air spaces of mammals, which contains
80% DPPC (Morley et al., 1978 ; Bangham et al.,
1979 ; Shelley et al., 1982 ; Oosterlaken-Dijksterhuis
et al., 1991 ; Schu$ rch et al., 1994, 1995 ; von Nahmen
et al., 1997). The main function of the surfactant
system is reduction of surface tension at the alveolar
air–liquid interface at the end of expiration to
minimize the capillary pressure created by decreased
alveolar radius (Johansson et al., 1994). Thus, the
alveolar air–liquid interfaces have features comparable with those of the xylem of M. flabellifolia
during refilling.

From these considerations we can conclude that the
lipid deposits on the inner xylem walls of M.
flabellifolia provide an efficient barrier against complete water loss during drought, as postulated
recently (Schneider et al., 1999). Water contact leads
to the generation of several forces ridding the plant
of the ‘ internal impregnation ’, even when root
pressure is not available. Filling takes longer without
root pressure but the mechanisms of axial and radial
refilling of conducting elements and cell rehydration
remain the same.
The xylem of other woody plants and trees also
seems impregnated with hydrophobic compounds
(data not shown ; Scott, 1966 ; Laschimke &
Laschimke, 1998). The key differences between M.
flabellifolia and other species are the lipid inclusions
in the pits and the apparently thicker lipid lining of
the walls that is disintegrated and partly removed
during the rehydration process, finally forming lipid
bodies and vesicle-like structures. There is preliminary evidence that these lipid spheres might
play a very important role, apart from transpirationinduced water flow, in the water ascent of hydrated
plants by generation of Marangoni streaming at their
interfaces. This, the nature of the lipids and the
possible involvement of hydrophobic proteins in
lipid film formation await further exploration.
              
The authors are grateful to C. Gehrig for expert preparation of the ultramicrotome sections, to H. Zimmermann
and R. Hagedorn, Humboldt-Universita$ t zu Berlin, and to
R. Reich for critical discussion, to S. Shirley for helpful
comments on revision of the manuscript and to K. Lindner
for screening TEM studies. The plant material was
collected with kind permission of J. Hofmann, the owner
of the farm Glu$ cksburg near Outjo, Namibia. This work
was supported by grants of the German Space Agency
(DLR 50WB 9643) and of the Deutsche Forschungsgemeinschaft (Schwerpunkt ‘ Apoplast ’, Zi 99\9–1 and
Graduiertenkolleg GRK 64\2) to U. Z. and A. H.
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