Research
The impact of lipid distribution, composition and mobility
on xylem water refilling of the resurrection plant
Myrothamnus flabellifolia
Blackwell Publishing Ltd.
H. Schneider1, B. Manz2, M. Westhoff 1, S. Mimietz1, M. Szimtenings3, T. Neuberger3, C. Faber3, G. Krohne4,
A. Haase3, F. Volke2 and U. Zimmermann1
Lehrstuhl für Biotechnologie, Biozentrum der Universität, Am Hubland, D−97074 Würzburg, Germany; 2Fraunhofer Institut für Biomedizinische Technik,
1
Ensheimer Strasse 48, D−66386 St Ingbert, Germany; 3Lehrstuhl für Experimentelle Physik V (Biophysik) der Universität, Am Hubland, D−97074 Würzburg,
Germany; 4Abteilung für Elektronenmikroskopie, Biozentrum der Universität, Am Hubland, D−97074 Würzburg, Germany
Summary
Author for correspondence:
U. Zimmermann
Tel: +49 931 8884508
Fax: +49 931 8884509
Email: [email protected]
Received: 7 January 2003
Accepted: 22 April 2003
doi: 10.1046/j.1469-8137.2003.00814.x
• Lipids play a crucial role in the maintenance of the structural and functional integrity of the water-conducting elements and cells of the resurrection plant Myrothamnus flabellifolia during complete dehydration.
• Lipid composition, mobility and distribution within the internodal and nodal xylem
regions (including short shoots and leaves) were investigated in the presence and
absence of water by using various nuclear magnetic resonance (NMR) spectroscopy
and imaging techniques differing greatly in the level of spatial resolution and acquisition of lipid parameters.
• Significant findings include: a discontinuity in the branch xylem between an inner
zone where no water moves and an outer zone where the water moves; the blocking
of water movement in the inner zone by lipids that are not dispersed by water, and
the facilitation of water advance in the xylem elements and pits of the outer zone
by water-dispersed lipids; the relative impermeability of leaf trace xylem to the rehydrating water and, hence, the relative hydraulic isolation of the leaves.
• These results elucidated part of the strategy used by the resurrection plant to cope
with extreme drought and to minimize transpirational water loss upon hydration.
Key words: Myrothamnus flabellifolia, xylem refilling, lipid lining, phospholipid, leaf
trace, NMR imaging and spectroscopy, diffusion, transmission electron microscopy.
© New Phytologist (2003) 159: 487–505
Introduction
Water availability is one of the factors severely limiting the
productivity of agricultural crops in arid and semi-arid areas.
Irrigation is the current strategy to overcome water shortage.
However, the scale of problems of soil salinization associated
with irrigation schemes is considerable and continuing to
grow (Läuchli & Lüttge, 2002). Genetic engineering of
drought-tolerant crop plants therefore provides a promising
alternative approach for the management of areas where
usable fresh water is limited. Substantial progress in this field
can be expected if the genes and the physiological processes
contributing to drought tolerance are identified in plants
© New Phytologist (2003) 159: 487–505 www.newphytologist.com
growing naturally on dry stands. These plants have evolved
various, very efficient mechanisms to cope with drought.
Thus, their investigation at the molecular and membrane
level through whole-plant responses up to the ecosystem level
may pave the way for the development of drought-tolerant
crops.
Among the great diversity of drought-tolerant plants
occurring in arid regions, the poikilohydric resurrection plant
Myrothamnus flabellifolia is one of the most interesting candidates (Canny, 2000). This plant is a woody, multi-stemmed
shrub that grows up to 1–2 m high in shallow soils on rocky
outcrops in southern Africa. Upon drought, the leaves close
like tiny inverted sunshades and become small brown rolls
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that furl very tightly to the branches. After rain, the leaves
unfold and display their green adaxial surfaces within about
1 d, even after extremely long periods of drought. While the
molecular biology, biochemistry and physiology of drought
tolerance of M. flabellifolia have been studied in detail
(Gündel, 1968; Hoffmann, 1968; Vieweg & Ziegler, 1969;
Bewley & Krochko, 1982; Suau et al., 1991; Goldsworthy,
1992; Wilson & Drennan, 1992; Bianchi et al., 1993; Drennan
et al., 1993; Sherwin & Farrant, 1996; Hartung et al.,
1998; Farrant et al., 1999; Scott, 2000), the mechanistic
aspects of the refilling process of the dry xylem elements have
been addressed by very few groups (Child, 1960; Gaff, 1977;
Goldsworthy, 1992; Sherwin et al., 1998).
Recently, 1H nuclear magnetic resonance (NMR) imaging,
as well as light and electron microscopy studies on branches
of M. flabellifolia have shown that an assembly of driving
forces including root pressure and capillary forces as well
as xylem surface tension and osmotic pressure gradients is
involved in axial water ascent and radial refilling of the tissue
cells of the resurrection plant (Schneider et al., 1999, 2000b;
Wagner et al., 2000; Zimmermann et al., 2001). For the
interactive operation of the various forces during water lifting,
conditioning of the xylem elements by lipids seems to be of
extremely high relevance. Controlled phase separation of
lipids and water and lipid disposal within the xylem elements
during dehydration are apparently of equal importance for the
following rehydration/dehydration cycle.
Refinement of our current knowledge of xylem refilling
and consolidation of the inferences drawn previously call for
a spatial analysis of the lipid composition, distribution and
mobility throughout the entire branch and the corresponding
local interplay of lipids with water upon wetting. At present,
data are available only for the internodes (Schneider et al.,
1999, 2000b; Wagner et al., 2000; Zimmermann et al., 2001).
By contrast, our knowledge about the nodal regions of the branch,
including leaf traces, short shoots and leaves is poor. Understanding of the strategies adopted by M. flabellifolia to allocate
water most efficiently also deserves – among other things – a
detailed examination of the breakdown and disposal of lipids
in conducting and nonconducting xylem elements.
As demonstrated here, substantial information about the
putative role of xylem lipids in withstanding drying and facilitating subsequent rehydration could be obtained by light and
electron microscopy and in particular by the numerous complementary variants offered by NMR spectroscopy and imaging (Kuchenbrod et al., 1995, 1996, 1998; Volke & Pampel,
1995; Bentrup, 1996; Manz & Callaghan, 1997; Manz et al.,
1997, 1999; Meininger et al., 1997; Volke et al., 1997; Pampel
et al., 1998; Rokitta et al., 1999; Seymour et al., 1999;
Wagner et al., 2000; Wistuba et al., 2000; Peuke et al., 2001;
Zimmermann et al., 2001). In contrast to the microscopy
techniques, the noninvasive NMR methods allowed measurements on lipid composition, mobility and distribution on the
same branch both in the presence and absence of water.
The experimental evidence given here corroborates the
hypothesis that axial water rise and water spreading to the
leaves and other tissue parts is under the control of lipids arising from their uneven distribution, mobility and composition. The results also support our previous view (Schneider
et al., 2000b) that a combination of several methods is
required for investigations of such a specialised plant system
in order to avoid misinterpretations.
Materials and Methods
Plant material
Unless stated otherwise, all experiments were carried out on
cut branches of M. flabellifolia Welw. 40–80 cm long and
2.5–5 mm basal diameter. After collection from dry plants
grown at the farm Glücksburg located at Outjo, Namibia, the
branches were transported to the University of Würzburg,
Germany, and stored there at an ambient relative humidity
of c. 30–50% before use. Under these conditions water uptake
is negligible, thus the branches are termed ‘air-dry’ in the
following.
Nuclear magnetic resonance spectroscopy and imaging
experiments
Three NMR magnets were used because of the different
requirements for measuring the chemical properties, the
spatial distribution and the movement of lipid and water
molecules in M. flabellifolia.
Experimental setups For magic angle spinning (MAS) spectroscopy, three-dimensional (3-D) imaging, long-term imaging
of vessel refilling and diffusion measurements a Bruker DMX
400 NMR spectrometer (Bruker, Rheinstetten, Germany)
with a vertical 9.4 T magnet and a proton resonance frequency
of 400 MHz was used. Single-slice imaging, acquisition of
T1 maps, chemical shift imaging and spectroscopic refilling
studies were conducted by using a horizontal 300-MHz
Bruker BIOSPEC 70/20 spectrometer with a magnetic field
strength of 7.0 T (Wagner et al., 2000). High-resolution
imaging was performed by using a vertical 750-MHz Bruker
AVANCE 750 WB spectrometer with a magnetic field
strength of 17.6 T.
The MAS spectroscopy and high-resolution imaging were
conducted on branch pieces whereas all other experiments
were performed on entire cut branches. The samples were
placed inside the NMR probe head containing the radiofrequency (r.f.) emitter/receiver coil, tuned to the Larmor frequency of protons (i.e. 300 MHz, 400 MHz and 750 MHz)
and inserted into the magnet. The two-channel 1H/broadband probe head of the DMX 400 is commercially available
(Bruker) and allowed not only 1H, but also 13C NMR measurements. The probe heads of the BIOSPEC and AVANCE
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750 WB were used exclusively for proton NMR experiments.
Whereas the probe head of the AVANCE 750 WB was commercially available (Bruker) the BIOSPEC probe head was
constructed by the Department of Biophysics of the University of Würzburg, Germany (Wagner et al., 2000).
Spectroscopy For MAS spectroscopy, small pieces cut from
an air-dry branch of M. flabellifolia were inserted into a
commercial ZrO2 MAS rotor of 4 mm outer diameter, with a
spherical inset in order to obtain highest spectral resolution
(Pampel et al., 1998). For the MAS spectra of hydrated
samples deionized water was added to the wood pieces. The
data were acquired at a spinning speed of 15 kHz and a
repetition time of 3 s. For the 1H spectra 64 transients were
averaged and for the 13C spectra 2048 transients were
averaged.
For spectroscopic refilling studies, slice-selective proton
spectra without spatial resolution were acquired every minute.
A slice thickness of 2 mm was selected. Since the signal load
and therefore the sensitivity of the coil changed during refilling, a benzene-filled tube was used as a reference.
Imaging Single-slice proton distribution images of air-dry
as well as of rehydrated branch material were made by using
a T1-weighted two-dimensional cross-sectional spin echo
imaging sequence as described by Wagner et al. (2000): the
slice thickness was between 0.5 mm and 3 mm; the echo time
and the repetition time were adjusted to 10 ms and 0.2–1 s,
respectively; the spatial resolution was 40 × 40 µm2 or
55 × 55 µm2, depending on the field of view.
Three-dimensional lipid distribution images of air-dry
branches were acquired by using a standard 3-D spin echo
pulse sequence. In the case of rehydrated branches, proton signals arising from lipid were suppressed by application of an
inversion pulse before the spin echo experiment, thus yielding
images that displayed almost exclusively the distribution of
free water. For both types of experiment the echo time and
repetition time were adjusted to 4.6 ms and 0.9 s, respectively.
The field of view was 10 mm along the radial and 20 mm
along the axial direction, resulting in an isotropic voxel resolution of 78 µm. Three-dimensional reconstructions were
made for air-dry branches both from very weak signal intensities (for imaging of the surface of the branch) and from signal intensities which were by a factor of two to three higher
than the background signal (for imaging of proton-rich
branch regions).
Quantitative T1 images (so-called T1 maps) were obtained
by using a saturation recovery technique introduced by Freeman and Hill (1971). To this end, eight images of 0.5 mm or
1 mm thickness with repetition times between 40 ms and 3 s
were acquired. The T1 map was then calculated on a pixelby-pixel basis, with an error tolerance of 27%. For this type
of experiment, a spatial resolution of 25 × 50 µm2 or
55 × 55 µm2 was achieved, depending on the field of view.
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Long-term NMR imaging of water rise was done by acquiring one multislice data set every 15 min over a duration of
20 h. Each data set consisted of 16 individual cross-sectional
images with an in-plane voxel resolution of 117 µm, a slice
thickness of 500 µm and separating space between neighbouring slices of 1 mm. The field of view was 15 × 15 mm2.
High-resolution proton imaging was conducted on about
2-cm-long pieces cut from air-dry branches and trimmed to a
diameter of c. 2 mm and 450 µm. The pieces were positioned
in glass tubes with inner diameters of 2.2 mm and 530 µm,
respectively. The tubes were introduced into solenoid microcoils with an inner diameter of 2.5 mm (Bruker) and 700 µm,
respectively (constructed by the Department of Biophysics of
the University of Würzburg, Germany). High-resolution proton distribution images were obtained by using a standard
3-D spin echo pulse sequence, with an echo time of 3 ms, a repetition time of 1 s and 10 averages. In the case of the 2.5-mm
coil, the voxel resolution was 14 × 14 × 100 µm3; for the
700 µm coil a voxel resolution of 6 × 6 × 100 µm3 was
achieved. Each three-dimensional data set of a branch region
of 6 mm length was acquired within 24–66 h.
Spectroscopic imaging 1H NMR chemical shift imaging
(CSI) experiments were used for separation and spatial
attribution of lipid and water signals to structural features.
The experiments were conducted as described by Meininger
et al. (1997), except that water suppression was not
performed. A slice thickness of 5 mm was chosen in order to
obtain sufficient signal intensity. Selection of a spatial
resolution of 39 × 39 µm2, one averaging, a matrix size of
128 × 128 pixels, an echo time of 6.5 ms and a repetition
time of 500 ms resulted in an experiment duration of
116 min for each image.
Diffusion experiments Diffusion experiments were performed
on 10-cm-long branch pieces placed into glass tubes of 5 mm
diameter, which were closed in order to exclude evaporation.
Diffusion coefficients were measured using a pulsed-gradient
stimulated-echo (PGSTE) pulse sequence. The gradient pulse
duration δ and separation ∆ were 4 ms and 50 ms, respectively,
and the gradient amplitude, g, was increased from zero to
1 T m−1 in 32 increments. Axial and radial diffusion coefficients
were measured separately by changing accordingly the
direction of the applied gradient pulse. The resulting
attenuating diffusion curves were fitted by exponential curves.
From the curves the self-diffusion coefficients were extracted
as described elsewhere (Callaghan, 1991; see Appendix). The
intrinsic error of the method is c. 10%.
In addition, a two-dimensional map of the water selfdiffusion coefficient along the axial direction was acquired with
a slice thickness of 3 mm and an in-plane voxel resolution
of 55 µm. The gradient pulse duration and separation were
2 ms and 50 ms, respectively, and the gradient amplitude was
incremented in 16 steps from zero to 0.45 T m−1. With this
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experimental arrangement, self-diffusion coefficients lower
than 10−11 m2 s−1 could not be resolved.
Light and electron microscopy
Light microscopic lipid distribution images were obtained by
preparing freehand transverse sections of air-dry branch
material using lipid-free razor blades. The sections were
subsequently stained by using 0.2% w : v Nile red dissolved
in a 1 : 1 mixture of isopropanol glycerol. Stained sections
were mounted in glycerol and examined at an excitation
wavelength of 450–490 nm by means of an epifluorescence
microscope equipped with a photography device (Axiophot;
Zeiss, Oberkochen, Germany).
For transmission electron microscopy (TEM), 1- to 2-mmthick air-dry branch pieces were fixed with osmium tetroxide
vapour as described by Wagner et al. (2000). Fixed specimens
were embedded in Epon resin. After polymerization, the specimens were cut transversally with an ultra-microtome (MT7000, Ultra; RMC, Tucson, AZ, USA). Image contrast was
enhanced by post-staining of the ultrathin sections with uranyl acetate and lead citrate. Microphotographs were taken
with a transmission electron microscope (EM 900 and EM
10, respectively; Zeiss).
For electron microscopy studies of lipid–water interactions
during the refilling process, the basal cut end of air-dry
branches was placed in a gold sol (average particle size 5 nm;
BB International, Cardiff, UK). Because of the colloidal features of the sol, water climbing was greatly reduced (Schneider
et al., 2000b). After 45 min a refilling height of c. 4 cm was
reached. 2-mm pieces were cut from the branch region of the
actual water front and embedded in LR-White. The specimens were then treated as described above, except that short
post-staining times were chosen in order to obtain a weak contrast. In a second set of experiments small pieces of air-dry
branches were subjected to aqueous fixation following a twostep Karnovsky protocol. Briefly, this consisted of 24-h glutaraldehyde/formaldehyde fixation followed by a treatment with
OsO4 as described by Wagner et al. (2000). Fixed specimens
were embedded in LR-White and then treated as described
above, with conventional post-staining times in order to
obtain sufficient contrast.
Results
Lipid spectra and diffusion in air-dry branch wood
Spatially nonresolved NMR spectroscopy provided detailed
information about the chemical composition of the branch
wood of M. flabellifolia. Typical 1H NMR spectra of small
air-dry branch pieces measured by using the conventional
nonspinning mode as well as MAS are shown in Fig. 1. It
is obvious that the resolution of the conventional spectrum
is much less than that of the MAS spectrum, and more
Fig. 1 1H and 13C NMR spectra of branch wood pieces of Myrothamnus flabellifolia (a–c and d, respectively). For comparison, measurements
were performed successively on the same sample in the air-dry (a, b and d) and rehydrated state (c) using the 9.4 T magnet. Spectrum a was
recorded by using the conventional nonspinning mode, whereas spectra b –d represent high-resolution magic angle spinning (MAS) recordings.
Some characteristic peaks are marked and can be assigned as follows: H1 = 0.9 ppm, terminal CH3; H2 = 1.3 ppm, CH2 of aliphatic chain;
H3 = 1.6 ppm, CH2 β to carboxyl; H4 = 2.1 ppm, CH2 α to double bond; H5 = 5.3 ppm, CH=CH group (Volke & Pampel, 1995; Lunati et al.,
2001); C1 = 13.9 ppm, terminal CH3; C2 = 22.8 ppm, CH2–CH3; C3 = 33.5 ppm, CH2=CO; C4 = 54 ppm, γ CH3 of phospholipid;
C5 = 60 ppm, αCH3 of phospholipid; C6 = 129.4 ppm, CH2 (Volke et al., 1997). The asterisk in spectrum b marks the broad resonance peak
corresponding to bound water.
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comparable with that obtained by 1H NMR-CSI experiments
described previously (Zimmermann et al., 2001). The
spectrum obtained by using the conventional mode showed a
single broad resonance peak in the range between 0 ppm
and 3 ppm By contrast, the 1H MAS spectrum exhibited
five prominent sharp peaks. A sharp peak is also seen at
5.3 ppm The peaks at 1.3 ppm (indicated by H2) and
2.1 ppm (H4) are characteristic for (–CH 2 –) n and for
–CH2CH=CHCH2–, respectively, whereas the peak at
5.3 ppm (H5) is typical for –CH=CH– and/or CH of
glycerol (Volke & Pampel, 1995; Lunati et al., 2001). The cooccurrence of these peaks can be taken as evidence for lipids
in the wood (Volke & Pampel, 1995; Pampel et al., 1998;
Lunati et al., 2001). In the range of the occurrence of water
resonances (i.e. between 4 ppm and 5 ppm), a broad peak of
extremely low intensity is seen which assumed a maximum
value at about 4.2 ppm (Fig. 1, asterisk in curve b). The shape
of the peak indicates that the sample contained very small
amounts of strongly bound water (about 3%).
Additional information about the lipid composition was
obtained by 13C MAS spectroscopy. A typical spectrum of airdry branch pieces is depicted in Fig. 1 (curve d). The broad
background signal seen over the entire frequency range is
caused by immobilized cellulose, cellulose-like materials and
NMR probe material (F. Volke & B. Manz, unpubl. results).
The peaks of high intensity at 33.5 ppm (C3), 60 ppm (C5)
and 54 ppm (C4) are characteristic for –CH2C=O, αCH2
and γCH2 groups of phospholipids (Volke et al., 1997), even
though the phospholipid βCH3 peak expected at 64 ppm
could not be resolved. Other parts of the peak pattern are typical for 13C NMR MAS spectra of phospholipids consisting of
a glycerol backbone, saturated and unsaturated fatty acids
(e.g. see the spectra of pure palmitoyl-oleoyl-phosphatidyl
choline in Volke et al., 1997).
Thus, we can conclude from MAS spectroscopy that branches of M. flabellifolia contain large amounts of (phospho-)
lipids. Measurements of the self-diffusion coefficient of protons by using a 1H NMR PGSTE experiment corroborated
this conclusion. In contrast to hydrated branches (see further
below), proton diffusion measured on air-dry branches can
be assigned exclusively to the diffusion of lipids (Volke et al.,
1994a). A typical diffusion curve of a 10-cm branch piece
is shown in Fig. 2 (curve a). From the linear curve, a value
of 1.6 × 10−12 m2 s−1 was calculated. This value agrees well
with the self-diffusion coefficient of pure phospholipid
molecules (Lindblom & Wennerström, 1977; Lindblom
et al., 1977; Volke et al., 1994a; Galle & Volke, 1995).
Lipid spectra and diffusion in hydrated branch wood
In the case of fully hydrated branch material, the 1H NMR
signals are dominated by the abundant water and not by
lipids. 1H MAS spectroscopy studies on hydrated branch
material (Fig. 1, curve c) revealed a pattern of resonance peaks
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Fig. 2 Typical (half-logarithmic) Stejskal–Tanner plot of the lateral
(curves a and b) and axial (curve c) proton self-diffusion measured on
a 10-cm long branch piece of Myrothamnus flabellifolia in the air-dry
(curve a) and hydrated state (curves b and c). Curves were measured
by using the 9.4 T NMR magnet and applying a pulsed-gradient
stimulated-echo (PGSTE) pulse sequence. Curve a could be fitted by
one exponential (corresponding to one-component Brownian
motion), whereas curve b and c could only be fitted by four
exponential curves, indicating the involvement of more diffusion
components. From the curves, the self-diffusion coefficients were
extracted as described by Callaghan (1991). For further details, see
text and Appendix.
which was identical to that of air-dry branch wood (Fig. 1,
curve b), except that instead of the single resonance peak at c.
4.2 ppm a double peak of high intensity appeared in this
frequency range. The peaks at 4.7 ppm and 4.5 ppm can be
attributed to free and bound water, respectively (Zhou et al.,
1999). The high intensity of the 4.5 ppm peak can be traced
back to enhanced binding of water to the lipids whereas the
shift of the bound water resonance from c. 4.2 ppm (curve b)
to 4.5 ppm indicates an increased mobility of the respective
water molecules (Lindblom & Wennerström, 1977;
Lindblom et al., 1977; Volke et al., 1994a,b; Galle & Volke,
1995).
By analogy to air-dry branches, 1H NMR PGSTE experiments can also yield information about lipid diffusion in
hydrated branches. By appropriate selection of the NMR
parameters, axial and radial diffusion of protons attached to
lipids can be separated from that of protons attached to free
(or bound) water (see the M aterials and Methods section).
Figure 3 shows a two-dimensional distribution map of the
self-diffusion coefficients of protons measured along the axial
direction in the internodal region of a fully hydrated Myrothamnus branch. In the central xylem area no diffusive signal
attenuation was recorded, indicating that the self-diffusion
coefficients of the protons must be below 10−11 m2 s−1 (see the
Materials and Methods section). Considering the value of
the self-diffusion coefficient of lipids measured on air-dry
branches (see earlier and Fig. 2, curve a), it can be concluded
that this central area does not conduct water. By contrast, the
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Fig. 3 Two-dimensional distribution map of the self-diffusion
coefficients of the rehydrated 10-cm branch piece of Fig. 2
determined in axial direction by using a spatially resolved
pulsed-gradient stimulated-echo (PGSTE) experiment (in-plane
resolution 55 µm). The central xylem area exhibits no diffusion signals
indicating that the axial self-diffusion coefficient of protons in this
region must be below 10−11 m2 s−1. By contrast, the average axial
self-diffusion coefficient of the surrounding xylem region (cx) is
calculated to be about 10−9 m2 s−1, demonstrating that only this
area is involved in water conductance.
surrounding xylem area is obviously water-conducting. The
average value of the self-diffusion coefficient was determined
to be 1 × 10−9 m2 s−1, thus being comparable to that of bulk
water (2.3 × 10−9 m2 s−1; Mills, 1973). This indicates that
this diffusion coefficient must be assigned to the waterconducting xylem elements.
By using the same NMR approach, but without spatial resolution, it was possible to determine the average self-diffusion
coefficients of lipids, bound and free water in axial and radial
direction. Typical diffusion curves in radial and axial direction
are given in Fig. 2 (curves b and c, respectively). The diffusion
curves could be consistently fitted by four exponential components, independent of the direction. The fit of the first, very
fast exponential component yielded a self-diffusion coefficient
of 1.25 × 10−9 m2 s−1. This value is practically identical to that
of the free xylem water displayed in the diffusion map of Fig. 3.
Analysis of the second component yielded a self-diffusion
coefficient of 2.7 × 10−10 m2 s−1. Such a value is expected when
water is interacting with the polar regions of the lipids (Volke
et al., 1994a). The self-diffusion coefficient of 4.5 × 10−11
m2 s−1 calculated from the fit of the third component is typical for water molecules that are bound to pure lipids. This can
be concluded from experiments in which the diffusion coefficients of lipids were measured in terms of the concentration
of water molecules per lipid molecule (Volke et al., 1994a).
Comparison with curve a (measured on air-dry branches)
shows that the fourth, very slow component arose from lipid
diffusion (self-diffusion coefficient 1.6 × 10−12 m2 s−1).
Fig. 4 Light microscopy image of a cross-section of an air-dry
Myrothamnus branch prepared from the nodal region c. 10 cm away
from the basal cut end and stained with an anhydrous solution of the
lipophilic dye Nile red. Excitation at 450 –490 nm results in a yellow
fluorescence when the dye is bound to lipids; p, pith; lta, lipid-rich
tracheid assemblies; lt, leaf trace. Bar, 200 µm.
Spatial distribution of lipids in the nodes and internodes
of air-dry branches
Assignment of the (phospho-) lipids to specific structures and
compartments within the branch wood of M. flabellifolia was
obtained by fluorescence light microscopy and 1H NMR
imaging experiments. A typical fluorescence light microscopy
image of a cross-section through the node of an air-dry branch
taken at c. 5 cm height and stained with the lipophilic
fluorescent dye Nile red is shown in Fig. 4. The staining
suggested that abundant lipids were present in the decussately
arranged leaf traces and in the cells delineating the rhomboidshaped pith. Some lipid deposits were also seen within the
pith. Throughout the rays, lipids seemed to be evenly
distributed. In some images of the node (see Fig. 4) ring
fragments consisting of tracheids with lipid-filled lumena
were seen. Assignment of their location to the conducting or
nonconducting area was not possible. Staining of internodal
regions taken between 2 cm and 20 cm height gave no clearcut indication of similar lipid-rich ring fragments. Investigation of short shoots and leaves also revealed the presence
of lipids (data not shown).
More insight into the lipid distribution was obtained by 1H
NMR images which allowed the noninvasive acquisition of
sequential cross-sectional images of nodal and internodal
regions, even – if necessary – throughout the entire intact
branch. In the case of air-dry branches proton distribution
images reflect the spatial lipid distribution (see earlier and also
Zimmermann et al., 2001). Thus, the signal intensity is a
measure of lipid abundance. Four typical cross-sectional spin
echo images selected from a 3-D data set are presented in
Fig. 5a. The data set was acquired over a length of 2 cm of an
intact branch (at a height of c. 5 cm, including nodal and
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Fig. 5 Four typical two-dimensional 1H NMR lipid distribution images (a) and the corresponding three-dimensional reconstruction
(b) measured on a dry intact Myrothamnus branch at a height of 5 cm. The entire set of the data was acquired (by using the 9.4 T magnet)
over a length of 2 cm, including nodal and internodal regions (in-plane resolution 78 µm). Some furled leaves and a short shoot are marked
by asterisks and an arrow, respectively. The images reveal lipid accumulation in the pith periphery (pp), in the leaf traces (lt) and in lipid ‘pieces’
(lp). These lipid pieces form a hollow, fragmented cylinder as indicated by the three-dimensional reconstruction (pieces exhibiting reflected
images of edge curvature are marked by asterisks). They are apparently identical to the lipid-rich tracheid assemblies (lta) seen in Fig. 4 and
are located in the nonconducting area which is separated from the conducting one by a ‘black ring’ (br). For further details, see text. Bar in
(a), 2 mm.
internodal regions), by using the 9.4 T NMR magnet. Consistent with Fig. 4, the sequential scans of 78 µm thickness
and 78 × 78 µm2 in-plane resolution revealed a weak background lipid signal throughout the entire branch both in
radial and longitudinal direction (Fig. 5a) and throughout the
leaves (asterisks in Fig. 5a). The short shoots apparently
contained more lipids, as indicated by the relatively high
proton signal intensity (arrow in Fig. 5a). Extremely high
lipid amounts were found in the leaf traces and also in the
rhomboidal pith region. However, unlike the light microscopy results (Fig. 4) NMR imaging showed that the lipids
were apparently restricted to the periphery of the pith. (Note
that high lipid concentrations were occasionally found in the
outermost tissue region of branches collected at sites of
extremely high ambient temperature (up to 45°C) and
irradiation (up to 3000 µmol m−2 s−1). In addition, the relative humidity could assume values as low as 10%.)
An interesting finding was that the homogeneous lipid
background signal distribution was interrupted by a faint ring
of extremely low signal intensity (Figs 5a, 6, 11b). This ring
was found in internodes (Zimmermann et al., 2001) as well as
in nodes, with varying thickness from c. 50 µm to 750 µm.
The position of the ring-shaped structure was exactly at the
boundary between the central nonconducting and the surrounding conducting xylem area (see Figs 3, 6, and 11). The
contrast of this structure which is termed ‘black ring’ could be
enhanced by increasing the slice thickness to 1–3 mm (see, for
example, Figure 11b). The ‘black ring’ could be resolved
(more or less clearly) in all images taken from intact branches
at 7.0 or 9.4 T up to a height of c. 40 cm. In some branches,
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Fig. 6 1H NMR chemical shift images of a Myrothamnus branch
taken at c. 10 cm height. The measurements were performed by
using the 7.0 T magnet with an in-plane resolution of 39 µm. (a) The
spatial distribution of the major lipid resonance at 1.5 ppm in the
air-dry state; (b) the spatial distribution of the resonance of free water
at 4.8 ppm in the re-hydrated state; lp, lipid piece; br, black ring. Bars,
1 mm.
additional ‘black ring’-like structures could be seen towards
the centre of the nonconducting area.
The position of the ‘black ring’ allowed the assignment of
the ‘lipid ring fragments’ seen in the light microscopy images
(Fig. 4) to the nonconducting area. These structures could be
resolved very clearly by 1H NMR spin echo imaging as well
as by 1H NMR chemical shift imaging of the major lipid resonance at c. 1.5 ppm (Figs 5a and 6a). Thus, it was possible
to demonstrate that their shape could assume even smaller
fractions of a circle than observed by light microscopy. In
some slices, particularly of internodal regions (taken at heights
of up to c. 40 cm), lipid-rich structures were not found, in
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agreement with the staining experiments. Inspection of
Fig. 5a shows that the position of the lipid-rich structures
could vary considerably when successive images of the same
branch were analysed. However, projection of the lipid-rich
structural elements of the various two-dimensional images
into the same plane suggested that they formed a closed ‘lipid
ring’. Studies on different branches demonstrated that the
location of this ‘lipid ring’ – when present – varied between
the pith and the ‘black ring’. In Fig. 5a the structures almost
coincided with the ‘black ring’, whereas in the case of Fig. 6a
a ‘lipid ring’ element was located half-way between the pith
and the ‘black ring’. The NMR imaging experiments at 7.0 T
and 9.4 T also revealed that ‘lipid ring’ structures were present
only in seven out of 27 branches and that occasionally up to
three ‘lipid ring’ elements arranged serially in the nonconducting area were found.
Figure 5b is a 3-D reconstruction of the regions of high signal intensity of the branch in Fig. 5a. The lipids delineating
the pith and the two-dimensional ‘lipid ring’ elements seen in
Fig. 5a apparently form hollow cylinders oriented parallel to
the branch surface. However, unlike the pith lipid continuum, the cylinder surface corresponding to the ‘lipid ring’ was
fragmented. The size of the pieces increased towards the nodal
region, there forming a more continuous layer. Interestingly,
consecutive longitudinally arranged pieces occasionally exhibited reflected images of edge curvature (see asterisks in
Fig. 5b), indicating that they had formed a unity before
growth-induced rupture. Light microscopy suggested that the
pieces of the ‘fragmented lipid cylinder’ corresponded to
lipid-rich tracheid assemblies.
Correlation of low lipid signal intensities with ray lipid
content and grouping
High-resolution 1H NMR imaging at 17.6 T allowed
assignment of the ‘black ring’ and the weak lipid background
signal to lipid structures seen in stained branch sections by
light microscopy (Fig. 4). Because of the required microcoil
dimensions, only small branch pieces of about 2 cm length
(taken at different heights between 10 cm and 30 cm) could
be investigated. Two typical images recorded close to and
within the node by using a 2.5-mm coil (resolution
14 × 14 µm2) are depicted in Fig. 7a and 7b, respectively. The
presence of high concentrations of lipids at the pith periphery
and in the fragments of the cylindrical structure of Fig. 5b can
be taken as further evidence that these areas represent typical
sites of lipid accumulation in the branches of M. flabellifolia.
The spatial resolution was even high enough to display the
uniseriate rays separately because of their high lipid content.
Figure 7b shows that ray grouping was quite dense and
disordered in the leaf traces, in contrast to the other areas
where the rays were uniformly oriented in radial direction.
Between the rays (i.e. in the xylem elements), no lipid signals
were recorded. This indicated that the lumena of the xylem
elements were not filled completely with lipids, as were the
lumena of the tracheid assemblies forming the ‘fragmented
lipid cylinder’. It is also clear that the weak lipid background
signals seen at 7.0 T and 9.4 T must be assigned mainly to the
rays.
This was further confirmed by using a 700-µm coil which
increased the spatial resolution to 6 × 6 µm2. As seen in
Fig. 7c, the image resolution was high enough to resolve single
(or at least two) lipid-rich parenchyma cells. The abundance
of lipids in the rays became obvious when the dimensions of
the cells were calculated from the lipid images in Fig. 7c. The
average width was with one or two pixels (i.e. 6 µm or 12 µm)
in good agreement with the corresponding light microscopy
value (6 ± 1 µm; n = 30). The average length was 22 ± 9 µm
(n = 26), which was similar to the microscopy value of
23 ± 7 µm (n = 30).
Interestingly, in contrast to low-resolution 1H NMR imaging, the ‘black ring’ was not always clearly distinguishable in
high-resolution 1H NMR images (Fig. 7a,b), although the
imaged areas covered both conducting and nonconducting
xylem areas (as judged by eye and by 1H NMR imaging at
7.0 T). Comparison of a high-resolution 1H NMR image of
a branch segment showing a ‘black ring’ (Fig. 7d) with the
corresponding low-resolution image (data not shown) demonstrated that this structure arose from ray parenchyma cells
exhibiting a lower average lipid content than the neighbouring cells. Furthermore, the ray grouping varied considerably at
the boundary between conducting and nonconducting xylem
areas, a consequence of the insertion of new rays towards the
conducting area (see inset in Fig. 7d). Close to this boundary
the spaces between the rays were, on average, relatively large
compared with the density of ray grouping in the adjacent
nonconducting and conducting regions. Together with the
finding that the NMR signals arise mainly from the ray cell
lipids, these anatomical features induce differences in 1H
(lipid) signals per voxel at lower resolution. As a result, highintensity signals arise in areas where the ray cells are lipid-rich
and densely grouped, but not in areas where the cells contain
less lipid and also large interstices exist. This so-called ‘partialvolume effect’ creates a ‘black ring’ especially at low image
resolutions.
Spatial distribution of lipids in air-dry branches at the
ultrastructural level
The characteristic lipid distribution pattern in the internodal
and nodal regions as revealed by 1H NMR imaging called for
a detailed reinvestigation of the ultrastructural features of the
nonconducting and conducting areas by TEM. (It should be
noted that no information about the lipid composition and
arrangements in the ray cells could be obtained for technical
reasons.) As shown by Wagner et al. (2000), osmiophilic
substances seen by TEM in branches of M. flabellifolia can be
assigned to lipids. This could also be demonstrated in this
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Fig. 7 High-resolution 1H NMR lipid distribution images of 2-cm long pieces of air-dry Myrothamnus branches. Data in (a), (b) and (d) were
acquired with a 17.6 T magnet by using a 2.5-mm microcoil (in-plane resolution 14 µm), data in (c) were acquired using a 700-µm microcoil
(in-plane resolution 6 µm). High-resolution imaging required trimming of the branch pieces to the dimensions of the microcoil: (a) and (b) show
cross-sections of the nodal region of the branch after removal of the bark and of the outermost part of the conducting xylem area, (c) shows a
small part of the conducting xylem area and (d) shows a section around the boundary between the nonconducting and conducting areas. The
evaluation of the structural arrangement of the rays (straight lines) together with the ‘black ring’ (dotted lines) is depicted in the inset of (d);
pp, pith periphery; lp, lipid piece; lt, leaf trace; rc, ray cell; br, black ring. Bars in (a), (b) and (d), 500 µm; bar in (c), 200 µm.
study by comparison of the light microscopy and TEM
results. Figure 8a shows that the lumena of the tracheid
assemblies forming the ‘fragmented lipid cylinder’ in the
nonconducting area (Fig. 5b) were completely filled with a
highly osmiophilic substance that corresponded to the lipids
stained with Nile red (see earlier). The extraordinarily dark
staining with osmium tetroxide suggested a high degree of
double bonds associated with unsaturated fatty acids as
predicted by MAS (see earlier and Plattner & Zingsheim,
1987). The inner walls of the tracheids were also covered with
a weakly stained lipid lining of c. 100 nm thickness.
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Occasionally, it was found that the tracheid edges and pits
contained opaque bulk lipid inclusions (Fig. 8a). The faint
staining of the xylem lining and pit and edge inclusions
suggested that these lipids were less unsaturated than the
lipids in the lumen. The TEM images of the other part of the
nonconducting area exhibiting a weak background signal in
the 1H NMR images (Fig. 5a) showed that the lumena of the
tracheids and vessels were empty (Fig. 8b). However, it is
evident that lipid linings were continuously covering the inner
xylem walls. These linings were apparently double- (Fig. 8b)
or multiple-layered (Fig. 8c). A very thin (about 20 nm) layer
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Fig. 8 Electron micrographs of ultrathin cross-sections of air-dry Myrothamnus branches through the nonconducting (a–c) and conducting areas
(e), as well as through the boundary region of both areas (d) and through a leaf trace (f). The specimens were either fixed with osmium tetroxide
vapour (a, b, d–f) or unfixed (c). Ultrathin sections were stained with uranyl acetate and lead citrate. (a) Lipid-rich tracheid assembly (= lipid
piece in Fig. 5); (b) typical part of the nonconducting area with double-layered xylem wall lining as well as lipid pit and edge inclusions; (c) higher
magnification of a multiple-layered xylem lining; (d) intervessel pits at the periphery of the nonconducting area (right) facing the conducting
area (left); (e) intervessel pit of the conducting area with thin lipid lining and lipid edge inclusions; (f) vessel and tracheids with completely lipidfilled lumena, composite wall linings and/or lipid pit and edge inclusions. Staining of the structural elements with osmium tetroxide obviously
ranged from opaque to black, suggesting lipophilic substances of different chemical composition. Note that electron-dense material is found
within the thin lipid lining, even in the absence of OsO4 staining; cx, conducting xylem; nx, nonconducting xylem; pi, pit inclusion; ei, tracheid
edge inclusion; arrows indicate thin, darkly stained lipid layers. Bars in (a), (b), (d) and (f), 1 µm; bars in (c) and (d), 500 nm.
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Fig. 9 Transmission electron micrographs documenting lipid–water interactions during refilling of conducting xylem elements. (a) A branch after
45 min exposure of the cut end to a gold sol. Numerous gold particles that have obviously penetrated the xylem lipid lining and the pit lipid
inclusions are seen in the inset at higher magnification. (b) Intermediate states of disintegration of the xylem lipid lining and removal of lipids
from a pit in the conducting xylem under formation of granula and blebs, respectively. (c) The nonconducting xylem of the branch sample used
in (b), with disintegrated lipid pit and edge inclusions, but with intact thick composite lipid wall lining. The intermediate states were preserved
by fixation of an air-dry branch piece in an aqueous Karnovsky solution for 24 h (including OsO4 treatment); ll, lipid lining; pi, pit inclusion; ei,
edge inclusion. Bars, 1 µm.
could always be resolved attached to the xylem wall, which
was characterized by high staining contrast (corresponding
to unsaturated lipids). Interestingly, the contrast remained
pronounced when unfixed, post-stained specimens were
investigated (Fig. 8c). This finding indicates (Nagl, 1981)
that the thin layer contains elements of atomic numbers
higher than those of carbon and oxygen (e.g. phosphorous as
suggested by MAS; see earlier). This layer was overlain by a
second layer of c. 60–80 nm. Frequently, particularly towards
the branch centre, lipid linings could be revealed consisting of
several alternating thin and thick layers with a total thickness
of more than 300 nm (Fig. 8c). Interestingly, the inner walls
(including the middle lamella) of the mostly lipid-filled pits
connecting the xylem elements were always covered with a
similar double- or multiple-layered lipid lining, thus forming
a continuum with the lipid lining of the xylem elements
(Fig. 8b,c). By contrast, the numerous pits connecting the
nonconducting xylem elements with the ray cells never
exhibited middle lamellas covered with a thick, composite
wall lining (data not shown). A notable exception was also the
pits at the periphery of the nonconducting area facing the
conducting area. The middle lamellas of these pits were
covered with only a thin lipid lining (Fig. 8d).
In the conducting area, continuous xylem lipid linings
were also observed, but their thickness was only about 20 nm
(Fig. 8d,e). These linings consisted of a single, darkly stained
lipid layer which also extended into the intervessel pits
(Fig. 8e). The lipid filling of the intervessel pits and of the
numerous pit connections between the ray cells and the xylem
elements of the conducting xylem region appeared very variable from branch to branch. Frequently, pits were found
which were either empty or only partly filled with lipids.
When present, lipids were found predominantly in the pit
edges (Fig. 8e; see also Fig. 9a).
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Disintegration of xylem lipid deposits upon hydration
The variation in the thickness of the xylem lipid lining and the
associated differences in the lipid filling of the pits as well as
the obvious variations in the chemical composition of the
xylem lipids raise the question about the functional relevance
of these compounds in xylem refilling. The TEM experiments
gave some insight into the early sequence of interactions
between lipids and water upon wetting.
In the first set of experiments a gold sol was used for visualization of the water front. Consistent with previous dye
climbing and NMR imaging results (Schneider et al., 2000b;
Wagner et al., 2000), gold particles were found only in a few
clusters of conducting xylem elements. A typical crosssectional TEM image of this region taken after 45-min exposure
of the cut end of the branch to a gold sol is provided in Fig. 9a.
It is evident that the lipid linings of the inner walls of the
xylem are decorated with numerous tiny gold particles (black
spots), partly infiltrating the lipid layer (see inset in Fig. 9a).
Local disintegration of the lipid linings has apparently not yet
occurred. Interestingly, a high number of gold particles are
distributed in the lipid remnants of the pit. These findings
suggest that water can easily penetrate the lipids of a few
xylem elements predestined for refilling.
Experiments in which cut pieces of air-dry branches were
kept in aqueous fixation solution for 24 h suggested that
water penetration into the lipids is followed by a massive disintegration of the organized lipid structures. Several stages of
these processes on the nanometre scale can be resolved as indicated in Fig. 9b. In the lower xylem element, partial lipid
removal from the walls had obviously already taken place. The
pit of this element is also empty. By contrast, in the adjacent
upper xylem element disintegration of the lipid lining has
proceeded, but is not completed. Highly osmiophilic granular
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remnants can be seen that are still attached to the wall. (Note
that such granular remnants can still be found in fully
hydrated branches (Wagner et al., 2000) suggesting that such
lipid–water interactions are maintained.) Removal of lipid
from the pit has obviously just started, as suggested by the
emission of a lipid bleb into the xylem lumen.
Inspection of the nonconducting area of the specimen in
Fig. 9b revealed that the lipid inclusions of the pits and tracheid
edges were dissolved upon contact with the aqueous fixation
solution, but not the thick composite lipid lining (Fig. 9c).
Lipid-rich areas and their functional role in rehydration
of the leaves
1H
Cross-sectional
NMR imaging of water rise in the internodes of M. flabellifolia confirmed the results obtained by the
gold sol TEM experiments, and by previous NMR studies
(Schneider et al., 2000a; Wagner et al., 2000; Zimmermann
et al., 2001), that axial water ascent occurred initially only in
a cluster of very few conducting elements. The present investigations revealed that these ‘starter elements’ were frequently
located nearby the ‘black ring’ structure mentioned earlier
(data not shown). Refilling of the other conducting elements
and of the living cells was mainly achieved by radial water
spreading from these initial conducting elements. Crosssectional NMR spin echo images through the internode as
well as chemical shift images of the resonance of free water at
4.8 ppm gave further evidence that the ‘black ring’, but not
the ‘fragmented lipid cylinder’ delineates the conducting area
from the central nonconducting one (see earlier and Fig. 6b).
Analysis of the refilling kinetics suggested that refilling of
the conducting elements and cells was completed after 40–
60 min, as also found recently (Wagner et al., 2000). This is,
however, not the case, as shown by 1H NMR spectroscopy.
The sensitivity of NMR spectroscopy is significantly greater
than that of NMR imaging, but this is at the expense of spatial
resolution. Figure 10 shows a typical time dependence of the
signal intensity of the lipid resonance (curve a) and the resonance
of free water (curve b) measured at 1.5 ppm and 4.8 ppm,
respectively. Consistent with the MAS measurements (see
Fig. 1), only a lipid signal, but no signal of free water could be
recorded before rehydration (see the first 20 min in Fig. 10).
After the appearance of the water front at the selected slice
(marked with a double-headed arrow at t = 40 min in
Fig. 10), the signal intensity at 4.8 ppm increased almost
linearly with time. After about 40 min (i.e. after c. 80 min in
Fig. 10) the signal intensity increase continued further, but at
a considerably lower rate. By contrast, the intensity of the
lipid signal remained nearly unaltered, indicating that significant lipid shifts between the selected slice and other parts of
the branch did not occur during the refilling process.
The refilling data obtained here by NMR spectroscopy
(Fig. 10) are consistent with the corresponding NMR imaging results of Wagner et al. (2000), provided that only the first
Fig. 10 Typical time dependence of the 1H NMR signal intensity of
the lipid resonance at 1.5 ppm (curve a) and the water resonance at
4.8 ppm (curve b) of a virtual slice of a branch internode during
refilling. Measurements were performed by using a 7.0-T magnet.
The data were extracted from a series of 1H NMR spectra which had
been acquired every minute. Application of tap water to the cut
branch end and appearance of the water front at the selected slice are
indicated by an upwardly directed arrow and a double-headed arrow,
respectively. It is evident that the signal intensity of the water
resonance increased biphasically during refilling, whereas the signal
intensity of the lipid resonance remained unaltered.
part of the biphasic curve is considered. The slope of the timedependent signal intensity curve determined spectroscopically
(1.5% min−1) agrees quite well with that deduced from the
previous NMR imaging measurements (2% min−1; Wagner
et al., 2000). The second, slow refilling phase seen only by NMR
spectroscopy (0.4% min−1) emphasizes the view that refilling
of the branch tissue apparently takes much more time than
60 min. Water is obviously extracted from the branch tissue
continuously during rehydration, presumably as a result of the
refilling of the leaves and the associated onset of transpiration
(Schneider et al., 2000b). This raises the question of how water
is extracted from the conducting elements into the leaf traces.
The leaf traces are characterized by an extraordinarily dense
arrangement of ray cells and by a high number of tracheids
(Figs 4 and 7b). Vessels are seen only sporadically. Large
amounts of lipids apparently separate the tracheids and vessels
of the leaf traces from the conducting elements of the main
branch, as suggested by the NMR images (Figs 5a and 7b)
and, in particular, by TEM of air-dry branches. The TEM
studies revealed that not only the ray cells, but also the
tracheids and vessels contained abundant lipid. Occasionally,
‘pearl chain’ alignments of completely lipid-filled tracheids
and /or vessels were seen which were similar to those forming
the ‘fragmented lipid cylinder’ (Fig. 8f ). The majority of the
tracheids and vessels showed composite lipid linings of the
walls consisting of a thin and thick layer (Fig. 8f ), which
extended into the intervessel pits, as found for the xylem elements in the nonconducting area (see earlier). The pits, and
frequently also the tracheid edges, were completely filled with
lipids (Fig. 8f ). The obvious similarities of these structural
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Fig. 11 Cross-sectional single-slice 1H NMR spin echo images (a, b) and corresponding T1 map (c) of the node region of a branch before
(b) and after 24-h refilling (a, c). The data sets were acquired by using a 7.0-T magnet (in-plane resolution 55 µm). Surprisingly, for the
re-hydrated branch (a) the signal intensity in the leaf traces (lt) is as low as in the central nonconducting area and apparently equal to that of
the air-dry branch (b). The short T1 relaxation times (c) can be taken as additional evidence that the high lipid accumulation in the leaf traces
seen in Fig. 5 represents a high resistance for water movement to the leaves. Note also that the conducting area in the T 1-weighted spin echo
image (a) consists of concentric rings characterized by high, but variable signal intensities, reflecting presumably seasonal variations in the
diameter of the xylem elements. Consistent with this, highest signal intensity is found at the periphery of the branch comprising immature xylem,
phloem and cortex; pp, pith periphery; lp, lipid piece; lt, leaf trace; br, black ring; cx, water-conducting xylem area. Bars, 1 mm. Note that in
(a) the signal intensity of the ‘lipid piece’ was enhanced by computer processing.
Fig. 12 Cross-sectional (a) and longitudinal (b) images from a three-dimensional 1H NMR data set displaying the distribution of free water
within an intact Myrothamnus branch that had been watered for 3 d. The data set had been acquired at a height of c. 20 cm by using a 9.4-T
magnet, with an in-plane resolution of 78 µm. Lipid signals were suppressed by application of an inversion pulse, thus yielding images that
displayed almost exclusively water signals. Note that the leaf traces contain no significant amounts of water, as also suggested by Fig. 11. The
main branch is apparently connected hydraulically with the adjacent short shoots only via a water-filled xylem (and/or cell) continuum of very
small cross-section at the periphery of the leaf traces (arrow in a); lt, leaf trace; ss, short shoot; l, leaf. Bars, 1 mm.
elements with the features of the central nonconducting area
(Fig. 8b) can be taken as evidence that water movement into
the leaves must be quite restricted. This assumption was supported by single-slice NMR images made in the plane of the
nodes of rehydrated branches. As depicted in Fig. 11a, images
taken at the sites of the oppositely arranged leaf traces did not
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reveal any increase in signal intensity upon 24-h rehydration
compared with images of the same branch in the air-dry state
(Fig. 11b). Water signals in these areas were also not observed
after long-term rehydration, as revealed by cross-sectional
water distribution images from 3-D experiments made on a
branch that had been watered for 3 d (Fig. 12a). (Note that
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the lipid signals were suppressed as described in the Materials
and Methods section when performing these experiments.)
The T1 maps of rehydrated branches yielded similar results. A
typical T1 map of the branch of Fig. 11a,b is given in Fig. 11c.
For the conducting xylem area the average T1 values were
850–1160 ms, whereas in the central nonconducting area
(including the pith and the ‘fragmented lipid cylinder’) as well
as in the leaf traces the T1 values were 400–500 ms (i.e. significantly lower). This shows that the water content in the leaf
traces of M. flabellifolia was apparently kept extremely low by
the lipids in the tracheids, at the expense of water movement
through a few peripheral conducting xylem elements and /or
the numerous cells (arrow in Fig. 12a) to the short shoots and
then to the leaves. These organs exhibited water-related signal
intensities, as demonstrated by the cross-sectional and longitudinal water distribution images displayed in Fig. 12a,b.
Discussion
The results obtained by TEM, as well as by various NMR
spectroscopy and imaging techniques differing greatly in the
level of spatial resolution and acquisition of lipid parameters,
have provided an integrated view about the important role of
lipids in xylem and cell conditioning for dehydration and
rehydration of the branches of M. flabellifolia. This species has
obviously developed very economic strategies based on lipid
distribution, composition and mobility, in order to use water
efficiently and to minimize transpirational water loss.
The xylem of the internodes and nodes of the branch occupies at least 80% of the cross-sectional area. About half of the
xylem can be assigned to the conducting pathway (Wagner
et al., 2000). This region is separated from the nonconducting
region by a concentric ring-shaped area of variable thickness
(c. 50–750 µm) and low 1H signal intensity as identified in
dry branches by NMR imaging. This ‘black ring’ arose from
an ‘NMR anomaly’ induced by pronounced differences in the
lipid content and density of the rays between this area and the
adjacent conducting/nonconducting regions.
Both the conducting and nonconducting xylem regions
contained a similar distribution of lipids within the ray cells,
which yielded a uniformly low 1H NMR background signal.
In the nonconducting area the intervessel pits were mostly
completely clogged by lipid inclusions. The majority of the
lumena of the xylem elements were free of lipids. However,
extremely high lipid concentrations were found at the pith
periphery and, interestingly, in some tracheid assemblies.
Three-dimensional 1H NMR imaging reconstructions
showed that these tracheid assemblies were not randomly
arranged. They formed a fragmented hollow cylinder made
up of ‘lipid pieces’ – at least throughout the 2-cm long branch
regions used for imaging. The physiological role of this peculiar structure is not clear at present. It seems likely that it
represents a relict of a continuous cylindrical assembly of lipidrich tracheids that served as a protection against complete
water loss during extreme drought, particularly in the juvenile
plant since ‘fragmented lipid rings’ are found only in the central xylem area. The finding of occasionally up to three of
these cylindrical structures can also be taken as support for
this assumption, because extremely dry periods are not unusual in the southern part of Africa. Disruption and associated
fragmentation of these ‘protecting sheaths’ apparently
occurred during internodal elongation. This explains straightforwardly the finding that the average size of the fragments
was much larger in the nodes than in the internodes, and that
the lipid fragments occasionally exhibited reflected images of
edge curvature. Lipid filling of these tracheid assemblies is
apparently achieved by the outermost cell layers of the branch
located in the region of the cambium and /or cortex. This is
suggested by NMR images of some dry branches collected
from sites of extremely high ambient temperature and irradiation as well as of low relative humidity (Schneider et al.,
1999). These occasionally showed unusually high lipid signal
intensities in the outermost tissue region.
Although this certainly deserves further investigation, it
seems clear that the lipid-rich tracheid /vessel assemblies in the
leaf traces are obviously used for the restriction of water supply to the leaves. The NMR investigations suggested that only
a few – if any – xylem elements were water-conducting. The
majority of the xylem elements were unable to conduct water,
a result which contradicts the current view about the function
of leaf traces (Esau, 1977). Nonwettability was obviously
achieved by thick composite lipid wall linings (which
extended into the pits) and by additional filling of the pits
with bulk lipids. The reduced conducting area explains the
recent observation (Schneider et al., 2000b) that upon water
supply to the cut end of the air-dry branches, leaf recurving
always occurs with a delay of a few minutes after arrival of the
water front at a certain height. Together with the morphological peculiarity of leaf furrow-located stomata (Schneider
et al., 1999), reduction of the conducting area in the leaf
traces seems to be the strategy of M. flabellifolia to minimize
water loss by evaporation. The refilling studies by means of 1H
NMR spectroscopy support this view. The main refilling
phase was finished after about 40 min, in agreement with previous NMR imaging data (Wagner et al., 2000). However,
owing to the higher sensitivity of NMR spectroscopy a subsequent, very slow refilling phase could be resolved, which obviously reflected compensation of water evaporation from tissue
cells and /or cell wall material.
The resurrection plant M. flabellifolia apparently uses a
similar strategy to Bulnesia sarmientoi to counteract excessive
transpirational water loss under extreme environmental conditions. This tree, which grows up to 14-m tall, rooting in
highly saline water at fairly high ambient temperatures (up to
40°C) facilitates axial water ascent at the expense of water supply to the leaves by clogging of a large part of the xylem of
twigs at intermediate heights with mucopolysaccharides and
proteins (Zimmermann et al., 2002). These NaCl-binding
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compounds are apparently optimal for the generation of
hydraulic resistances when the plant is faced with salinity, but
not with drought.
For M. flabellifolia the central problem is the reactivation of
the xylem in the conducting area for water ascent after
drought. As shown here, the xylem lipid linings as well as the
bulk lipid inclusions of the pits and tracheid edges disintegrate upon rehydration. Lipid blebs and granular remnants
are formed, which are presumably the precursors of the innumerable lipid bodies found in the lumena of water-filled
xylem elements (Schneider et al., 1999; Wagner et al., 2000).
An additional source for the formation of the lipid bodies
upon wetting may be lipid secretion into the xylem by the ray
cells, which are found in extraordinarily high abundance in
M. flabellifolia (Carlquist, 1976). Because of the numerous
pits between the ray cells and the xylem elements (see earlier)
the ray cells can be regarded as contact cells. Such cells are
known to release metabolites (such as lipids, sugars, proteins,
etc.) into the adjacent xylem elements (Sauter et al., 1973;
Esau, 1977). The finding that the ray cells of the ‘black ring’
area contain less lipid than those in the adjacent nonconducting and conducting areas could be taken as an indication for
the release of lipids into the neighbouring xylem elements.
This assumption would straightforwardly explain the transformation of conducting xylem elements in the ‘black ring’
area into nonconducting ones during seasonal growth.
The crucial point is the penetration of the lipid lining by
water upon wetting required for axial water ascent and radial
spreading, respectively. Mandatory for this process is apparently the thickness as well as the composition of the lipid lining of the xylem elements and pits. In the nonconducting
area, the inner walls of the xylem elements and pits are covered
by a very thick (sometimes more than 300 nm) lipid lining
consisting of alternating thin (about 20 nm) and thick (about
60–80 nm) layers of high and low TEM contrast, respectively.
Wetting and subsequent breakdown of these composite lipid
linings upon contact with aqueous fixation solutions was
never observed. This suggests that the thick, composite lipid
linings contain a large amount of neutral, nonwettable lipids,
which are presumably concentrated in the 60–80-nm-thick
layers. This conclusion can be drawn from the observation
that in the conducting area the inner walls of the xylem elements and pits are exclusively covered by a single 20-nm-thick
layer which is water-wettable. The high TEM contrast of this
layer, even in the absence of osmium tetroxide staining, indicates the presence of phospholipids, as mentioned earlier. The
13C MAS spectroscopy on air-dry branch wood also provided
strong evidence for significant amounts of phospholipids. The
value of the self-diffusion coefficient of 1.6 × 10−12 m2 s−1
deduced from the 1H NMR PGSTE experiments on dry
branches is also typical for these compounds (Lindblom &
Wennerström, 1977; Lindblom et al., 1977; Volke et al.,
1994a; Galle & Volke, 1995). A major component of the
phospholipids seen in the 13C MAS spectra seemed to be
© New Phytologist (2003) 159: 487–505 www.newphytologist.com
phosphatidyl choline (Fig. 1). The 13C MAS spectrum of
Fig. 1 shows many other signals which must be attributed to
lipids or phospholipids (e.g. phosphatidylethanolamines).
However, they could not be identified unambiguously
because of signal overlapping. The high resolution of the MAS
spectra was achieved at the expense of spatial resolution. The
possibility can therefore not completely be excluded that the
signals recorded in the 13C MAS spectra and in the 1H NMR
PGSTE experiments resulted from the rays or, more likely,
from both compartments.
Phospholipids are known to bind significant amounts of
water (Crowe & Crowe, 1984; Cornell et al., 1974; Gawrisch
et al., 1978, 1992, 1995; Arnold et al., 1981; Volke et al.,
1982). The self-diffusion coefficients deduced from 1H NMR
PGSTE experiments performed on hydrated branches in axial
and radial direction are consistent with this finding. As mentioned earlier, self-diffusion coefficients of 4.5 × 10−11 m2 s−1
and 2.7 × 10−10 m2 s−1 are typical for water bound to lipids or
interacting with the polar regions of phospholipids (Volke
et al., 1994a; Lindblom & Wennerström, 1977; Lindblom
et al., 1977). Furthermore, the rapid gold sol penetration of
the lipids of the conducting area can only be explained if
phospholipids or, more generally speaking, amphiphilic lipids
are involved (taking into account that the pit lipids exhibited
only a low TEM contrast compared with the xylem linings).
The interactions of phospholipids (or related amphiphilic
lipids) with water at the molecular level and the breakdown of
phospholipid layers are well understood (Arnold et al., 1979;
Crowe & Crowe, 1984; McIntosh, 1996; Huang & Epand,
1997; Gutberlet et al., 1998). Some of the phospholipids,
especially phosphatidyl choline and phosphatidylserine, form
crystalline lamellar structures in the dry state. Upon water
binding to the polar heads the lamellar phase turns over into
the nonlamellar inverted hexagonal II (HII) phase if the
dynamic molecular shape of the lipids assumes a noncylindrical geometry. Whereas phosphatidylethanolamines readily
form HII phases upon contact with water, other types of
phospholipids (e.g. phosphatidyl cholines), even when fully
hydrated, require additional factors like changes in temperature, fatty acid composition or electrostatic interactions to
force them into the HII phase (Huang & Epand, 1997;
Gutberlet et al., 1998). The tubes or micelles formed by the
HII structure are water-conducting, thus facilitating penetration of water into the lipid layer. The HII structure is unstable:
with increasing water penetration multilamellar liposomes
or – in the presence of some fatty acids – lipid bodies are
formed (partly by fusion). The phase transitions of the phospholipids are accompanied by characteristic changes in 31P NMR
spectra (Cullis et al., 1983; Crowe & Crowe, 1984; Smith &
Ekiel, 1984) which could also recently be resolved by preliminary experiments on branches of M. flabellifolia (F. Volke &
B. Manz, unpubl. data).
The kinetics (as well as the temperature) of the phase
transitions of pure phospholipids depend on the lipid
501
502 Research
composition and charge distribution within the lipid molecules,
but may also change when other compounds, such as trehalose
and sucrose, are present (Crowe & Crowe, 1984; Crowe et al.,
1984a; Smith & Ekiel, 1984; Hoekstra et al., 1997; Wolfe &
Bryant, 1999; Koster et al., 2000; Oliver et al., 2002). These
carbohydrates are well-known to protect membranes against
dehydration and were also found in high amounts in cells of
M. flabellifolia and other desiccation-tolerant organisms
(Crowe & Crowe, 1984, 2000; Crowe et al., 1984a,b; Gadd
et al., 1987; Bianchi et al., 1993; Drennan et al., 1993; Oliver
et al., 2002). Preliminary studies on M. flabellifolia have
shown that the xylem sap of hydrated plants also contains trehalose, sucrose and other lipid-interacting sugars in variable
amounts. Taking the relatively slow dehydration process into
consideration one can assume that spatial accumulation and
depletion, respectively, results in an uneven distribution of
these sugars throughout the xylem elements of the conducting
area. The associated differences in lipid conditioning could
easily explain why axial water ascent in dry branches is
favoured initially by only a few elements (Schneider et al.,
2000a,b; Wagner et al., 2000; Zimmermann et al., 2001).
The involvement of carbohydrates in xylem lipid conditioning certainly deserves further investigation. The focus of the
future research must also include elucidation of the mechanisms protecting M. flabellifolia plants in the field against
repetitive drought. Under laboratory conditions the branches
of the resurrection plant lose their ability to refill entirely after
the first cycles of rehydration and dehydration (Wagner et al.,
2000). Therefore, analysis of sugars and other solutes which
are secreted into the xylem sap during hydration and during
the first phase of dehydration is of equally high importance.
This knowledge and the findings reported here and in previous publications will facilitate the identification of the
involved genes – an important step on the long way towards
genetically engineered, drought-tolerant crops. Knowledge of
protecting carbohydrates and their interaction with phospholipids will also certainly lead to novel strategies in cryopreservation of mammalian and plant cells. Generation of
artificial anhydrobiosis in mammalian and plant cells is of
great biomedical and biotechnological interest because the
compromising effects of freezing and thawing on cells can be
minimised or completely eliminated (Beattie et al., 1997;
Wolfe & Bryant, 1999; Crowe & Crowe, 2000). Technologies
for the injection of balanced mixtures of lipids, disaccharides
and other related compounds into cells without adverse sideeffects are available (Zimmermann & Neil, 1996). Thus, generation of artificial anhydrobiosis seems to be a goal that can
soon be realized.
Acknowledgements
The authors are very grateful to C. Gehrig, D. Bunsen and
K. Schwuchow for skilful technical assistance as well as to
J. Schiller and H.-J. Wagner for helpful discussions. We thank
D. Haddad and A. Purea for construction of the 700-µm
microcoil used for the high-resolution 1H NMR imaging
studies. The permission of the Namibian government as
well as of J. Hofmann, the owner of the farm Glücksburg
near Outjo, Namibia, for collection of samples of M.
flabellifolia is greatly appreciated. This work was supported by
grants of the BMBF (16SV1329) and of the Deutsche
Forschungsgemeinschaft (Schwerpunkt ‘Apoplast’, Zi 99/9–
3, and HA 1232/13–1) to U.Z. and A.H.
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Appendix
Nuclear magnetic resonance spectroscopy and imaging
– some background information
A detailed description of NMR techniques can be found in a
variety of textbooks (Abragam, 1961; Fukushima & Roeder,
1981; Shaw, 1984; Ernst et al., 1987; Paudler, 1987; Slichter,
1990). The underlying principle of magnetic resonance is that
many nuclei carry a spin and therefore a magnetic moment.
If a sample with nuclear spins is placed inside a strong, external
magnetic field, B0, the interaction of the magnetic moment with
B0 causes the nuclear spins to align and thus create a small
magnetization vector within the sample. This magnetization
can be manipulated by the application of radio frequency (r.f.)
pulses of a given power and duration. After the application
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Wistuba N, Reich R, Wagner H-J, Zhu JJ, Schneider H, Bentrup F-W,
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Zhou Z, Sayer BG, Hughes DW, Stark RE, Epand RM. 1999. Studies of
phospholipid hydration by high-resolution magic-angle spinning nuclear
magnetic resonance. Biophysical Journal 76: 387–399.
Zimmermann U, Neil GA. 1996. Electromanipulation of cells. Boca Raton,
FL, USA: CRC Press.
Zimmermann U, Wagner H-J, Heidecker M, Mimietz S, Schneider H,
Szimtenings M, Haase A, Mitlöhner R, Kruck W, Hoffmann R,
König W. 2002. Implications of mucilage on pressure bomb
measurements and water lifting in trees rooting in high-salinity water.
Trees 16: 100–111.
Zimmermann U, Wagner H-J, Szimtenings M, Schneider H, Haase A.
2001. Restoration of the hydraulic system in a resurrection plant: fitting
the theory with the facts. New Phytologist 151: 314–317.
‘gyromagnetic ratio’ of the nucleus under investigation. For a
hydrogen nucleus (proton), γ has a value of 2.68 × 108 Hz T−1
(Abragam, 1961; Fukushima & Roeder, 1981; Ernst et al., 1987).
Depending on the selection of the NMR parameters,
the chemical properties, the spatial distribution, molecular
mobility and the movement of (lipid and water) molecules
can be determined.
In solid or liquid samples, the spins are located in atoms or
molecules and are therefore surrounded by electron clouds
which interact with the spin angular momentum. This gives
rise to small variations of the local magnetic field and therefore of the Larmor frequency. This effect, known as ‘chemical
shift’ (given in units of Hz or, more commonly, of ppm), is
characteristic for a specific chemical environment and is used
as a fingerprint to identify molecules (Ernst et al., 1987;
Meininger et al., 1997).
The intrinsic magnetic moment of each spin dipole interacts also with neighbouring spin dipoles. In the case of liquid
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Research
samples, where molecular tumbling motion is more rapid
than dipolar interaction, the effect of the latter is averaged to
zero. In the case of solid samples, where the internuclear vectors have fixed orientations, this dipolar interaction leads to a
severe broadening of the resonance peaks. One approach to
overcome broad resonance peaks in solids and/or systems with
spatially restricted motions is called magic angle spinning (MAS;
Andrew et al., 1959). The basic principle of this technique is
spinning of the sample about an axis inclined by 54.7° (the
so-called magic angle) in relation to the polarizing field. If the
rotation speed is fast enough (typically several kHz), static,
through-space dipolar couplings are drastically reduced for
nonrigid solids, and thus high-resolution NMR spectra can be
obtained (Volke & Pampel, 1995; Pampel et al., 1998).
Nuclear magnetic resonance imaging is based on the fact
that the Larmor frequency is proportional to the polarizing
magnetic field. If in addition to the external magnetic field,
B0, a uniform magnetic field gradient is applied, this frequency experiences a spatial signature, thus allowing the
acquisition of two- and three-dimensional spin distribution
data sets (Mansfield & Morris, 1982; Callaghan, 1991;
Kuchenbrod et al., 1995; Manz & Callaghan, 1997). Threedimensional images can be reconstructed from these data sets
by using the appropriate software.
Displacement of molecules can be measured by using the
pulsed-gradient stimulated-echo (PGSTE) NMR method
(Stejskal & Tanner, 1965). As in the case of imaging, this
method uses magnetic field gradients, but in the so-called difference mode. A matched pair of gradient pulses of duration
δ and amplitude g, separated by a delay ∆, is applied along
with a sequence of r.f. pulses. Increasing values of δ, g, or ∆
lead to a phase shift as well as an attenuation of the NMR
signal. The phase shift arises from correlated molecular displacements (e.g. flow) over the well-defined interval ∆. For
noncorrelated, Brownian motion the amplitude E of the echo
signal is given by E = exp(–γ 2g 2δ 2D∆), where D is the molecular self-diffusion coefficient. By measuring the NMR signal
with increasing gradient strength g, flow velocities (Kuchenbrod
et al., 1998) as well as self-diffusion coefficients can be
determined (Stejskal & Tanner, 1965; Kuchenbrod et al.,
1995; Manz & Callaghan, 1997). In the latter case, a plot of
ln E vs γ 2g 2δ 2∆ (Stejskal–Tanner plot) yields a straight line
with slope –D for each component of Brownian motion. Spatially resolved maps of flow velocities and self-diffusion coefficients can be obtained by combining the PGSTE method
with imaging techniques (Callaghan, 1991; Callaghan & Xia,
1991; Callaghan et al., 1994; Kuchenbrod et al., 1995, 1996,
1998; Manz et al., 1997, 1999; Seymour et al., 1999).
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