Plant Physiol. (1998) 118: 975-986
Drying Increases Intracellular Partitioning of Amphiphilic
Substances into the Lipid Phase1
Impact on Membrane Permeability and Significance for Desiccation
Tolerance
Elena A. Golovina,
Folkert A. Hoekstra*, and
Marcus A. Hemminga
Timiryazev Institute of Plant Physiology, Botanicheskaja 35, Moscow, 127276, Russia (E.A.G.); and Departments of Plant Physiology
(E.A.G., F.A.H.) and Molecular Physics (M.A.H.), Wageningen
Agricultural University, Wageningen, The Netherlands
 |
ABSTRACT |
Previously we proposed that
endogenous amphiphilic substances may partition from the aqueous
cytoplasm into the lipid phase during dehydration of
desiccation-tolerant organ(ism)s and vice versa during rehydration.
Their perturbing presence in membranes could thus explain the transient
leakage from imbibing organisms. To study the mechanism of this
phenomenon, amphiphilic nitroxide spin probes were introduced into the
pollen of a model organism, Typha latifolia, and their
partitioning behavior during dehydration and rehydration was analyzed
by electron paramagnetic resonance spectroscopy. In hydrated pollen the
spin probes mainly occurred in the aqueous phase; during dehydration,
however, the amphiphilic spin probes partitioned into the lipid phase
and had disappeared from the aqueous phase below 0.4 g water
g
1 dry weight. During rehydration the probes reappeared
in the aqueous phase above 0.4 g water g1 dry
weight. The partitioning back into the cytoplasm coincided with the
decrease of the initially high plasma membrane permeability. A charged
polar spin probe was trapped in the cytoplasm during drying. Liposome
experiments showed that partitioning of an amphiphilic spin probe into
the bilayer during dehydration caused transient leakage during
rehydration. This was also observed with endogenous amphipaths that
were extracted from pollen, implying similar partitioning behavior. In
view of the fluidizing effect on membranes and the antioxidant
properties of many endogenous amphipaths, we suggest that partitioning
with drying may be pivotal to desiccation tolerance, despite the risk
of imbibitional leakage.
| |
INTRODUCTION |
All dry biological systems release various substances of
relatively low Mr when placed in water.
This is a clear indication of the increased membrane permeability
associated with cellular dehydration. In the case of
desiccation-tolerant (anhydrobiotic) organisms, the leakage generally
declines with the progression of rehydration, whereas
desiccation-sensitive organisms continue to leak. But even in
desiccation-tolerant organisms, excessive leakage can ensue,
particularly when imbibition occurs at low temperatures and/or when the
organism is very dry.
Several hypotheses have attempted to explain the mechanism of leakage
during water uptake (Simon, 1974
; Crowe et al., 1989; Hoekstra et al.,
1992a). The prevailing hypothesis is based on the observation that
liposomal membranes become transiently leaky during the phase change
from the gel phase to the liquid-crystalline phase (Hammoudah et al.,
1981). Defects at the boundary between these phases are held
responsible for the increased permeability. In dehydrating and
rehydrating organisms membranes can undergo phase changes and thus
become transiently leaky (Crowe et al., 1989; Hoekstra et al., 1992a,
1992b). For example, during imbibition of dry pollen the transition of
membrane lipids from the gel phase into the liquid-crystalline phase
coincides with an instantaneous loss of endogenous solutes, an event
that the pollen does not survive.
When pollen membranes are manipulated to assume the liquid-crystalline
state before the uptake of liquid water, thus avoiding the phase
transition during imbibition, solute leakage is considerably less and
viability is preserved. However, even with their membranes in the
liquid-crystalline phase, dry anhydrobiotes still leak to a certain
extent upon imbibition (Hoekstra et al., 1992a; Tetteroo et al., 1996).
This leakage is transient, but nevertheless may constitute 50% of the
total amount of endogenous solutes present, usually without affecting
viability but with some loss of vigor. The aim of the present work was
to elucidate the mechanism of this transient imbibitional leakage that
is not associated with a change from the gel phase to the
liquid-crystalline phase.
Increased permeability may result from the presence of compounds
associated with the membrane surface or located more deeply in the core
of the membrane (Maher and Singer, 1984). The production of large
amounts of disaccharides during desiccation of anhydrobiotes is well
known (Crowe et al., 1984). Hydrogen bonding of these sugars to the
polar headgroups of the membrane phospholipids is considered important
in the tolerance of desiccation. The spacing of the individual
phospholipid molecules that results from this hydrogen bonding prevents
the dehydration-induced increase of Tm in
liposomes, thereby keeping the membranes in the liquid-crystalline phase (Crowe et al., 1987). It has been argued that this mechanism also
works in vivo and plays a key role in desiccation tolerance and
prevention of imbibitional leakage (Crowe et al., 1992). However, the
content of sugars in dry organisms may not be sufficient to fully
protect the membranes, even if all sugars were hydrogen bonded to the
phospholipids (Hoekstra et al., 1997).
Flavonols were considered in the search for compounds other than sugars
that may depress the dehydration-induced increase of
Tm in membranes (Hoekstra et al., 1997).
Quercetin, for example, a flavonol with strong antioxidant activity
(Terao et al., 1994) that can be found in pollen as 0.2% to 1% of the
dry matter (Ylstra et al., 1992; Vogt and Taylor, 1995), decreases the
Tm of dry palmitoyl-oleoyl PC liposomes by
30°C (Hoekstra et al., 1997). This occurs at the low molar ratio of
quercetin to phospholipid of 1:10. Quercetin is inserted in the more
apolar part of the liposomal membrane and increases the disorder of the
acyl chains there. The amphipathic behavior of flavonols was suggested
to lead to partitioning from the cytoplasm into membranes during dehydration and to prevent gel-phase formation by membrane fluidization (Hoekstra et al., 1997). The increased disorder could cause some transient leakage during rehydration, which could explain the observed
initial leakage from imbibing anhydrobiotes. Although partitioning of
amphiphilic substances into membranes and the associated physiological
consequences have been thoroughly studied (e.g. Rowe et al., 1998), to
our knowledge, this phenomenon has never been considered in relation to
the desiccation of anhydrobiotic organisms.
To verify our hypothesis we used an EPR spin-probe technique using a
number of mostly amphipathic spin probes with different polarities. The
parameters of the EPR spectra of these spin probes depend on the
polarity of the environment (Marsh, 1981), which allows the location in
hydrophilic and hydrophobic environments of the cell to be
distinguished. Thus, we studied partitioning of the spin-probe
molecules into the lipid phase of the anhydrobiotic pollen of
Typha latifolia, with drying and the release of these molecules into the cytoplasm with rehydration to explain imbibitional leakage. The disturbing effect of amphipaths, both synthetic and extracted from pollen, on membranes during drying and rehydration was
also verified in a model membrane system.
| |
MATERIALS AND METHODS |
Plant Material and Treatments
Mature male inflorescences of Typha latifolia L. were
collected from field populations near Wageningen, The Netherlands, in 1995, and allowed to shed their pollen in the laboratory. Pollen was
cleaned by sieving through a fine copper mesh, dried in dry air to 0.05 to 0.08 g water g1 DW, and stored in
closed plastic vials (20 mL) at 
20°C until use. Before all
experiments, the dry pollen was washed three times in hexane to remove
apolar hydrocarbons from the pollen wall, and the adhering hexane was
removed by evaporation. This treatment is necessary to obtain
homogeneous hydration and does not impair viability (Iwanami and
Nakamura, 1972).
Elevated moisture content was obtained by exposing the pollen at 12°C
to a stream of water vapor-saturated air produced at 24°C. In this
humid atmosphere the moisture content of the pollen reached 0.7 g
water g1 DW within a few hours, and
1.5 g water g1 DW within 20 h. In the
course of this vapor hydration, samples of approximately 100 mg were
taken for studies of plasma membrane permeability.
The germination medium used for imbibition consisted of 1.6 mM H3BO3, 1.3 mM
Ca(NO3)2·4H2O,
0.8 mM
MgSO4·7H2O, 1.0 mM KNO3, and 0.2 M Suc in
2 mM sodium-phosphate-citrate buffer, pH 5.9.
For every sample taken for EPR, two samples (50-80 mg) were taken for
determination of moisture content. Water content was analyzed by
weighing the samples before and after heating for 4 h at 105°C
and calculating the water loss.
EPR Measurements
EPR spectra were obtained at room temperature with an X-band EPR
spectrometer (model 300E, Bruker Analytik, Rheinstetten, Germany).
Microwave power was 2 mW, and the modulation amplitude was 1 G. Four
spectra were accumulated to improve the signal-to-noise ratio.
Determination of Plasma Membrane Permeability
Plasma membrane permeability of the pollen was determined by an
EPR technique using the water-soluble nitroxide radical TEMPONE (1 mM) as the spin probe and ferricyanide (120 mM)
as the broadening agent (Golovina and Tikhonov, 1994; Golovina et al.,
1997). TEMPONE easily penetrates through the plasma membrane into cells
because of its amphipathic nature and relatively small size. In
contrast, the charged ferricyanide ions cannot penetrate through intact membranes. Inside cells TEMPONE partitions between hydrophilic (cytoplasmic water) and hydrophobic (membranes and oil bodies) compartments.
The EPR spectrum of TEMPONE in pollen (Fig.
1) is the result of the superposition of
two spectra, slightly different in shape, arising from TEMPONE
molecules in the polar cytoplasm and in the apolar membranes and/or
lipid bodies. The superposition of these two spectra can be resolved in
the high-field spectral region only. The TEMPONE signal from outside
the grains is fully broadened by ferricyanide. Because ferricyanide
cannot penetrate through an intact plasma membrane, the signal from
TEMPONE inside the aqueous cytoplasm is not broadened. If there are
defects in the plasma membrane, some ferricyanide ions will be able to
penetrate into the cell and to some extent broaden the signal arising
from TEMPONE located in the cytoplasm. This has no influence on the lipid signal, because the lipid environment is inaccessible to ferricyanide ions, allowing the use of the apolar peak as the internal
standard for the amount of pollen examined. Thus, the line-height ratio
of the lipid peak to the water peak (L/W) depends only on the number of
ferricyanide ions that have penetrated the cell through the plasma
membrane and can be used to characterize plasma membrane permeability
(Fig. 1).
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| Figure 1.
EPR spectra of TEMPONE in T. latifolia pollen. Pollen of approximately 0.37 g water
g1 DW was incubated in a solution of 1 mM
TEMPONE and 120 mM ferricyanide directly (a) and indirectly
(b) after a previous 5 min of rehydration in germination medium. The
TEMPONE at the outside of the pollen was fully broadened by
ferricyanide. The permeability of the plasma membrane to ferricyanide
can be calculated as the ratio between the line heights of the lipid
fraction (left) and the aqueous cytoplasmic fraction (right) in the
high-field region of the spectrum (L/W).
|
|
The following protocols were carried out to discriminate between the
plasma membrane permeability at the onset of imbibition and after
completion of rehydration (5 min; F.A. Hoekstra and E.A.
Golovina, unpublished data). One sample was directly incubated in a 10-mL tube containing 5 mL of a 1 mM TEMPONE/120
mM ferricyanide (K3Fe[CN]6) solution at
22°C (the direct method). After 5 min of gentle shaking the sample
was recovered by filtration (plasma/serum filters, Greiner, Alphen a/d
Rijn, The Netherlands), and then loaded into a capillary (2 mm
diameter, filled for 1 cm). A very small amount of the solution was
injected on top of the sample to prevent drying, and the EPR spectra of
TEMPONE were recorded. A second sample was incubated in a 10-mL tube
containing 5 mL of liquid germination medium for 5 min at 22°C with
gentle shaking, after which the medium was filtered off (the indirect
method). Subsequently, the pollen was incubated in 5 mL of the
TEMPONE/ferricyanide solution. After 5 min the pollen was recovered by
filtration, loaded in the capillary as described above, and EPR spectra
were recorded.
Determination of Partitioning
To study the in situ partitioning of amphiphilic substances during
drying and rehydration, an EPR spin-probe technique was also used. For
this purpose TEMPONE was used as the model amphiphilic compound.
Because of differences in the isotropic hyperfine splitting constants
of EPR spectra in hydrophilic and hydrophobic environments, the
location of TEMPONE in these environments could be easily determined.
The loading of the spin probe into the pollen was as follows: Pollen (3 g) was prehydrated in water vapor to about 0.8 g water g1 DW and then mixed at 25°C with
approximately 6 mL of the liquid germination medium. After a few
minutes another 20 mL of germination medium was added and the pollen
was recovered by filtration. The restricted addition of liquid kept
leakage of endogenous solutes to a minimum (F.A. Hoekstra and E.A.
Golovina, unpublished results). The pollen was then mixed with
20 mL of a solution containing 1 mM spin probe and 120 mM ferricyanide (necessary for quenching of the EPR signal
from TEMPONE in the adhering extracellular solution). After 5 min the
pollen was recovered by filtration, spread out in a large Petri dish,
and allowed to slowly dry on the laboratory bench at room temperature.
At intervals, samples were loaded into capillaries (2 mm diameter) for
EPR analysis. The nitroxide spin probes TEMPO and TEMP-amine were
used in addition to TEMPONE.
For repartition experiments during rehydration of dry pollen in humid
air, the same procedures of spin-probe loading were followed as
outlined above, except that the 120 mM ferricyanide was
replaced by 10 mM. Because ferricyanide caused some
membrane deterioration during the long interval between loading and
analysis, the concentration had to be less than 120 mM. In
fact, the lack of extracellular EPR signal in the vapor-rehydration
experiments made the use of ferricyanide as a quencher superfluous.
However, some ferricyanide (10 mM) was necessary as an
external electron acceptor to prevent reduction of TEMPONE to a
non-free-radical form (Kaplan et al., 1973; Miller, 1978). The filtered
pollen sample loaded with the spin probe was rapidly dried overnight in
a flow of dry air (3% RH) in a dry box, and retained good
viability during the procedure. The dried pollen was rehydrated in
vapor-saturated air at 12°C, and at intervals samples were loaded
into the capillaries under humid conditions.
Partitioning of the preloaded spin probes during drying and vapor
rehydration of the pollen was estimated from the line-height ratios in
the high-field region of the spectrum in two different ways: with the
amplitude of the lipid peak as a percentage of the sum of the
amplitudes of the lipid and the aqueous peaks (L/[L + W]), and as the
partition parameter P (W/L) (Fig.
2).
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| Figure 2.
EPR spectra of preloaded TEMPONE in T. latifolia pollen at different levels of dehydration and
rehydration. Vapor-prehydrated pollen was incubated for 5 min in
germination medium that was replaced by TEMPONE/ferricyanide. After 5 more min, the pollen was filtered and EPR spectra were recorded in the
hydrated state (2.70 g water g1 DW) (a); after 2 h
of exposure to laboratory air (0.80 g water g1 DW) (b);
after 5 h of exposure to laboratory air (0.05 g water
g1 DW) (c); and after forced rehydration of dried pollen
in humid air (0.85 g water g1 DW) (d). The arrow in c
points to the anisotropic spectrum, representing slow-moving TEMPONE
molecules trapped in a solid environment. The dotted line represents
the position of the lipid peak of the spectra.
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Extraction of Amphipathic Compounds from Pollen
Hexane-washed pollen (2.5 g) was suspended in 10 mL of deionized
water and rapidly frozen. The frozen material was pressed through the
orifice of a homogenizer (AB-Biox, Järfälla, Sweden) at
20°C at 20,000 p.s.i. The crushed pollen was mixed with 100 mL of
chloroform:methanol (1:1, v/v), followed by 5 min of mild ultrasonic
treatment. After low-speed centrifugation (1000g) the supernatant was mixed with 15 mL of a 0.9% NaCl solution. After phase
separation the chloroform layer at the bottom of the glass tube was
collected and dried by passage over a column of anhydrous Na2SO4. After volume
reduction, the material was loaded on a BioSil A (Bio-Rad) column (18 cm in length, 16 mm in diameter) that was prewashed with 110 mL of
chloroform. After elution of neutral lipids with 150 mL of chloroform,
the amphipathic substances were recovered with 100 mL of acetone. After
removal of the acetone the material was suspended in 0.5 mL of water
for use in the liposome experiments.
Alternatively, amphipathic substances were recovered from an aqueous
pollen extract. After homogenization as described above, the homogenate
was thawed and subsequently centrifuged for 10 min at
30,000g, and the supernatant was recentrifuged at
150,000g for 30 min. The supernatant was led over three
coupled Sep-Pak C18 cartridges (no. 51910, Waters), and the cartridges were washed with 10 mL of water.
Amphipathic substances were eluted from the cartridges by 10 mL each of
increasing concentrations of methanol in water (v/v): 20%, 50%, 80%,
and 100%. After vacuum evaporation of the methanol and freeze drying
of the aqueous extract, the solid material was thoroughly mixed with
250 µL of water for use in the liposome experiments.
Liposome Studies
EggPC (100 mg) in chloroform, purchased from Fluka, was used
without further purification. The chloroform was evaporated in a stream
of nitrogen and, after the sample was dried under vacuum for at least
2 h, 1 mL of water containing 500 mg of Suc (Pfanstiehl, Inc.,
Waukegan, IL) was added (Suc:eggPC = 5:1, w/w). After five freeze-thaw cycles and vortexing, liposomes were produced by extrusion through one 100-nm pore-size polycarbonate filter (Nuclepore Corp., Pleasanton, CA) 35 times using the LiposoFast extruder (Avestin, Inc.,
Ottawa, Canada) according to the method of MacDonald et al. (1991).
These liposomes were used for EPR studies.
To produce liposomes with encapsulated CF, the same procedure was
followed, but the dried eggPC (20 mg) was mixed with 2 mL of 100 mM CF (purified according to the method of Klausner et al.
[1981]), and 0.25 M Suc in 1 mM Tes buffer,
pH 7.5. The suspension was then layered onto a Sephadex G-50 column
(50-80 mesh). Elution was with 1 mM Tes buffer, pH 7.5, to
separate the vesicles from excess Suc and CF. In the opened lid of an
Eppendorf tube, 10 µL of the liposome suspension was mixed with
another 20 µL of water and Suc and/or amphipaths as desired. Drying
of the 30-µL droplets in the opened lids was carried out by exposure
to a stream of dry air (RH < 3%) in a dry box for at
least 3 h at 25°C in the dark (0.02 g water
g1 DW). One milliliter of 1 mM Tes
buffer, pH 7.5, was then added and after closure of the lids the lipid
mixture was suspended by vortexing. Leakage of CF from the vesicles was
assessed with a fluorometer (excitation wavelength = 490 nm and
emission wavelength = 515 nm; Aminco, Silver Spring, MD) according
to methods described previously (Hoekstra and Van Roekel, 1988).
| |
RESULTS AND DISCUSSION |
Decline of Membrane Permeability with Rehydration
Figure 1 (spectrum a) shows an EPR spectrum of TEMPONE in pollen
(initial moisture content of 0.37 g water
g1 DW) that was allowed to imbibe in the
presence of TEMPONE/ferricyanide (1:120 mM) solution. This
rehydration is referred to as direct incubation. Spectrum b shows such
a spectrum after first allowing the pollen to imbibe in germination
medium for 5 min, followed by the addition of TEMPONE/ferricyanide. We
refer to this treatment as indirect incubation. From the spectra it
becomes clear that the aqueous contribution to the spectrum (W) has
increased with the previous 5 min of rehydration in germination medium.
Apparently, more ferricyanide ions could enter pollen to broaden the
water signal if ferricyanide was present at the onset of imbibition rather than being added after 5 min. Because the spin-probe method provides information on membrane permeability (L/W) at the moment of
addition of the spin probe, the difference between the spectra shows
that plasma membranes are more permeable at the onset of imbibition
than after rehydration is complete. After 5 min the permeability did
not continue to decrease with time of rehydration (data not shown).
Full rehydration in medium apparently led to minimum permeability
values. Thus, it is expected that with increasing initial moisture
content of the pollen, the permeability of plasma membranes at the
onset of imbibition decreases and finally approaches this minimum
value (if plasma membranes are not irreversibly disrupted).
Plasma Membrane Permeability at the Onset of Imbibition as
Influenced by Initial Moisture Content
Plasma membrane permeability at the onset of imbibition decreased
more or less linearly with increasing initial moisture content of the
pollen and reached a minimum value at approximately 1.2 g water
g1 DW (direct rehydration in
TEMPONE/ferricyanide solution; Fig. 3).
When the TEMPONE/ferricyanide solution was added after completion of
rehydration (Fig. 3, indirect), low permeability was observed for
moisture contents above 0.06 water g1 DW. The
elevated permeability at the onset of imbibition (Fig. 3, direct) is
reduced to the same low value upon completion of rehydration,
indicating that the structural defects of the dried plasma membrane
have been restored upon rehydration. In contrast, the high permeability
at initial moisture contents below 0.06 g water
g1 DW is of a more drastic nature, because
membranes were still permeable after 5 min of preincubation in
germination medium (indirect curve). The membrane phospholipids of
pollen with moisture content below 0.06 g water
g1 DW were in the gel phase (Crowe et al.,
1989; Hoekstra et al., 1992a). As a consequence of imbibition, a
transition to the liquid-crystalline phase occurred, which is supposed
to cause the complete loss of endogenous solutes and viability.
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| Figure 3.
Permeability of plasma membranes of T. latifolia pollen of different initial moisture contents upon
direct hydration in TEMPONE/ferricyanide (1 mM:120
mM) solution (direct incubation) or after a previous
hydration in liquid germination medium for 5 min (indirect incubation),
followed by substitution for TEMPONE/ferricyanide solution. Imbibition
temperature was 22°C. Plasma membrane permeability was expressed as
L/W, in arbitrary units.
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The region of initial moisture contents just above 0.06 g water
g1 DW is characterized by a rather high initial
permeability of the plasma membranes (Fig. 3, direct). Although
membranes are already in the liquid-crystalline phase before imbibition
(Crowe et al., 1989; Hoekstra et al., 1992a) and therefore no phase
change is involved, approximately 40% to 50% of the endogenous
solutes may leak out during this rehydration period. This may reduce
vigor but generally does not impair germination capacity (Hoekstra et al., 1992a; F.A. Hoekstra and E.A. Golovina, unpublished
results). Furthermore, high initial moisture contents of approximately
1.1 to 1.2 g water g1 DW result in less
leakage and improved pollen vigor.
Because the high initial leakage during rehydration is not associated
with a membrane phase transition, another mechanism for this phenomenon
was sought. High membrane permeability can be caused by fluidizing
membrane perturbants (Maher and Singer, 1984; Casal et al., 1987). We
previously raised the possibility that endogenous amphipaths may
partition from the aqueous phase into the lipid phase during
dehydration and vice versa during rehydration (Hoekstra et al., 1997).
Accordingly, the imbibitional leakage may stem from the perturbing
effect of such compounds. To study this possibility in more detail we
conducted experiments with the spin probe TEMPONE as a model
amphipathic compound loaded into pollen to follow its partitioning with
drying and rehydration.
Partitioning of Amphipaths during Dehydration and Rehydration
EPR spectra of TEMPONE in hydrated pollen show a considerable
contribution from the aqueous cytoplasm (Fig. 2, spectrum a), characterized by a hyperfine splitting constant of about 17 G (distance
between peaks measured in magnetic field units). The small peak
directly to the left of the water line in the high-field region of the
spectrum (Fig. 2, spectrum a) is typical of TEMPONE in a lipidic
environment (hyperfine splitting of 14.0-14.5 G; for review, see
Marsh, 1981). The distribution of TEMPONE over the aqueous and lipidic
environments depends on the partitioning coefficient of this spin probe
and the volume of both environments available. Lipid is present in
T. latifolia pollen as approximately 13.3% of the dry
matter and consists of 8.0% neutral lipids and 5.3% polar lipids
(Hoekstra et al., 1991). Drying of the pollen to 0.8 g water
g1 DW caused a considerable decrease in the
polar peak and an increase in the lipid peak (Fig. 2, spectrum b).
Further drying to 0.05 g water g1 DW
resulted in the complete disappearance of the water component from the
spectrum (Fig. 2, spectrum c). The lipid character of the remaining
lines follows from the value of the hyperfine splitting constant
(14.0-14.5 G). Also, a slight sign of an anisotropic spectrum can be
observed (minute bulge at the left side of the spectrum), which
indicates that some slow-moving TEMPONE molecules are trapped in a
solid environment. The considerable increase in the line height of the
lipid component with drying shows that partitioning of TEMPONE into the
lipid phase has occurred. This phenomenon was described before in
lyophilized yeast cells, but to our knowledge has not been discussed in
terms of the physiological consequences (Keith and Snipes, 1974).
Vapor rehydration of dry pollen previously loaded with
TEMPONE/ferricyanide (1 mM:10 mM) caused the
lipid component to decrease and the water component to reappear (Fig.
2, spectrum d). We interpret this to mean that TEMPONE partitioning
into the lipid phase is reversible, depending on the volume of water
available.
To follow the partitioning of the spin label from a water to a lipid
environment with drying, we plotted the amplitude of the lipid peak as
a percentage of the amplitudes of both the lipid and the aqueous peaks
(Fig. 4A). The product of the amplitude (hi) and the square of the width
(
sHi2) of a peak is used to
approximate the amount of spin probe in each environment
(Ai = hisHi2).
However, if the peak widths do not change, peak amplitudes can be used
instead (Shimshick and McConnell, 1973). Because we did not notice
pronounced changes in peak widths with drying, we used amplitude
characteristics in Figure 4 to describe partitioning. The amounts of
TEMPONE in the lipid component increased slightly with drying of the
fully hydrated sample, to increase more rapidly at moisture contents
below 1.2 g water g1 DW. TEMPONE was out
of the solution below approximately 0.4 g water
g1 DW. The rehydration curve follows the same
pattern but with some hysteresis. The starting point of repartitioning
was the same (0.4 g water g1 DW), but the probe
moved into the water earlier with respect to moisture content. We
speculate that this was the result of different relative amounts of
sorbed and bulk water between samples with the same moisture content
when dried or rehydrated, possibly caused by the hysteresis of water
sorption by polymers (Slade and Levine, 1991).
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| Figure 4.
The effect of dehydration and rehydration on the
percentage of TEMPONE (TN) in the lipid phase of T. latifolia pollen (L × 100/[L + W]) (A), and the
partition parameter P (W/L) as calculated from
amplitudes of the EPR spectra (B). TEMPONE was preloaded as described
in ``Materials and Methods''. Dehydration of fully hydrated pollen
was performed on the laboratory bench at room temperature; rehydration
of dried pollen was in vapor-saturated air at 12°C.
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The same data presented as the ratio between the water and lipid
components (the partition parameter P) are shown in Figure 4B. This presentation emphasizes the partitioning at high moisture contents, which was less clear in Figure 4A. This parameter is sensitive to slight changes in the amplitude of the lipid component. The scatter of the P values at elevated water contents may
be attributable to the calculation of this parameter using the
considerable line heights of the water contribution to the spectra
divided by the very small line heights of the lipid component. During rehydration, P was 0 until it began to increase rapidly at
moisture contents above 0.4 g water g1 DW.
Apparently, partitioning of TEMPONE from the lipidic phase into the
aqueous phase had not begun until the water content had reached
0.4 g water g1 DW. Because of the
dehydration curve shown in Figure 4B, we attempted to describe the
driving force for the partitioning of TEMPONE from the aqueous phase to
the lipid phase.
Supposing that the changes in the size of the water compartment (the
cytoplasm and vacuole) are responsible for the partitioning into the
lipid compartment, the size of which remained the same, we considered
the following: the amount of spin probe in water (Aw) or lipid
(Al) compartments can be calculated as the
product of concentration (Cw,
Cl) and volume
(Vw, Vl)
|
(1)
|
The ratio between the amounts of spin probe in both compartments
is:
|
(2)
|
If we express the partition coefficient as:
|
(3)
|
then:
|
(4)
|
Supposing that the partition coefficient k and the size
of the lipid compartment Vl do not change
with drying, then:
|
(5)
|
where B = k/Vl, which is constant.
According to Equation 5, the partition parameter P, defined
as Aw/Al, has a
linear dependence on moisture content in the range where bulk water is
present. As mentioned above, amplitudes in the high-field region of the spectrum can be used instead of the amounts of spin probe in
each environment when the widths do not change (Shimshick and
McConnell, 1973). Figure 4B shows the relationship between the
partition parameter P and moisture content expressed on a DW
basis for dehydrating pollen. Indeed, the dependence is approximately
linear over the high range above approximately 1 g water
g1 DW, but deviates from this linearity below
this moisture content. The comparatively low values of P may
be attributed to the slightly increased viscosity below 1 g water
g1 DW and the steep increase in viscosity below
0.6 g water g1 DW (Leprince and Hoekstra,
1998). From the data obtained it is not completely clear whether the
deviation of P from the expected straight line is caused by
water-line broadening as a result of increased viscosity, or whether
the increased viscosity of the cytoplasm itself promotes partitioning
into the more liquid lipid phase. However, the very slight increase in
line width of the water peak between 1.0 and 0.6 g water
g1 DW (data not shown) favors the latter
explanation.
Effect of Polarity of Molecules on Dehydration-Induced Partitioning
Insight into the partitioning behavior of molecules with different
polarity during dehydration was obtained using TEMPamine and TEMPO as
model molecules. The EPR spectrum of the more polar, charged TEMPamine
in fully hydrated pollen has the typical isotropic shape of a nitroxide
spin probe in water solution, with a distance between peaks of 17 G
(Fig. 5, spectrum a). There was no sign of TEMPamine in the lipid environment, but a clear indication of an
anisotropic spectrum (low-field side) was apparent, which can be
explained by the binding of some charged TEMPamine molecules to the
surface of immobile structures such as proteins. After dehydration this
spectrum disappeared completely, and a new complex spectrum
appeared instead (Fig. 5, spectrum b). This spectrum of
TEMPamine in dehydrated pollen is characterized by signal contributions from two different environments. The two outer extremes of the spectrum
represent the high- and low-field peaks of an anisotropic spectrum
characterized by restricted mobility of the spin probe. The two small,
narrow lines with a hyperfine splitting of about 14.5 G can be
attributed to TEMPamine molecules localized in the lipid environment.
The central component of the spectrum was not resolved.
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| Figure 5.
EPR spectra of preloaded TEMPamine in T. latifolia pollen. Prehydrated pollen was incubated for 5 min in
germination medium that was replaced by a TEMPamine/ferricyanide (1 mM:120 mM) solution. After 5 more min, the
pollen was filtered and EPR spectra were recorded in the hydrated state
in the presence of minute amounts of the solution (a) and after 5 h of exposure of the pollen to air (moisture content = 0.14 g
water g1 DW) (b).
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Comparing the spectra of TEMPamine in hydrated and dehydrated pollen
(Fig. 5, spectra a and b), we conclude that only a small part of the
TEMPamine molecules partitioned into the lipid phase during
dehydration. The majority of the TEMPamine molecules were trapped in
the dried cytoplasm, their charge apparently preventing their
partitioning into the lipid phase during dehydration. The small amount
of spin-probe molecules in the lipid phase may be explained by the
presence of the neutral form of TEMPamine molecules, possibly due to
changes in ion strength and pH of the cytoplasm with drying.
A considerable number of TEMPO molecules were present in the lipid
phase of fully hydrated pollen grains (Fig.
6, spectrum a), as resolved in the
high-field part of the spectrum. The more apolar nature of TEMPO
compared with TEMPONE explains why the aqueous contribution to the
spectrum is rather low, even for hydrated pollen. The spectrum of TEMPO
in dried pollen is characterized by three narrow lines with a hyperfine
splitting of 14.5 G, which is typical of lipid surroundings (Fig. 6,
spectrum b). A very slight indication of an anisotropic spectrum (left
side) is visible. It is clear that upon drying almost all TEMPO
molecules partitioned out of the aqueous environment into the lipid
environment, similarly to TEMPONE.
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| Figure 6.
EPR spectra of preloaded TEMPO in T. latifolia pollen. Prehydrated pollen was incubated for 5 min in
germination medium that was replaced by a TEMPO/ferricyanide (1 mM:120 mM) solution. After 5 more min, the
pollen was filtered and EPR spectra were recorded in the hydrated state
in the presence of minute amounts of the solution (a) and after 5 h of exposure of the pollen to air (moisture content = 0.16 g
water g1 DW) (b). The arrow in b points to the
anisotropic spectrum, representing slow-moving TEMPONE molecules
trapped in a solid environment.
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Figure 7 shows the increase in the
percentage of TEMPO in the lipid phase during dehydration. At 0.8 g water g1 DW, all spin-probe molecules had
disappeared from the aqueous surroundings and moved to the lipid
surroundings. The slight amount of immobilized TEMPO was not accounted
for in the calculation of this percentage.
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| Figure 7.
The effect of dehydration on the percentage of
TEMPO (TO) in the lipid phase of T. latifolia pollen, as
calculated from the amplitudes of the EPR spectra. The TEMPO was
preloaded as described in ``Materials and Methods''.
Dehydration of the fully hydrated pollen was performed on the
laboratory bench at room temperature.
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Partitioning of Amphipaths into EggPC Liposomal Membranes upon
Dehydration
From the spectra of TEMPONE in dry pollen (Fig. 2, spectrum c) one
cannot determine whether the site of partitioning is the membranes,
lipid bodies, or both. Because only partitioning into membranes can
influence permeability, we investigated whether the spin probe is able
to partition into membranes with drying. A mixture of eggPC liposomes,
Suc, and TEMPONE plus ferricyanide was allowed to dehydrate. Suc (mass
ratio of Suc:eggPC = 5:1) was added to prevent liposome fusion and
phase transitions with drying (Crowe et al., 1996), which may mimic the
situation in the cytoplasm (pollen contains 23% Suc on a DW basis,
which gives a 4:1 mass ratio of Suc:phospholipid [Hoekstra et al.,
1992c]). The ferricyanide was used to quench the EPR signal from
outside the liposomes.
Figure 8A shows EPR spectra of TEMPONE in
the liposome suspension before and after drying. In the spectrum of the
hydrated liposome sample, the aqueous peak was less pronounced than
that in the spectrum of hydrated pollen (Fig. 2, spectrum a). This may
be explained by the approximately six-times-higher amount of lipid per
trapped volume in liposomes than in pollen grains. Drying of the
liposome sample resulted in the disappearance of the water line from
the EPR spectrum of TEMPONE. In this dry sample (0.02 g water
g1 DW) only one component with a hyperfine
splitting of approximately 14.5 G could be observed, which is typical
of a hydrocarbon environment. The shape of the lipid spectrum is
attributable to a decreased rotational motion of the spin-probe
molecules and indicative of a viscous environment (Marsh, 1981).
Compared with the much more fluid character of the environment of the
spin probe in dry pollen (Fig. 2, spectrum c), this may indicate that
in the latter case a considerable number of TEMPONE molecules had
partitioned into lipid bodies. Another possibility could be that
membranes are more fluid in dry pollen (mainly 18:2 as the unsaturated
fatty acid) than in the dry, lyoprotected eggPC (mainly 18:1)
liposomes.
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| Figure 8.
A, EPR spectra of TEMPONE (1 mM) in
suspensions of eggPC liposomes (100 mg/mL) containing 120 mM ferricyanide before and after dehydration for 3 h
in a stream of dry air (3% RH) at 24°C. The liposomes were produced
in the presence of Suc (mass ratio of Suc:eggPC = 5:1). B, EPR
spectrum of TEMPONE in Suc glass.
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These changes in EPR spectra unambiguously demonstrate that
partitioning of amphipathic molecules such as TEMPONE from the aqueous
environment into membranes with drying is possible in principle. There
was no indication that TEMPONE was trapped in the Suc glass. The EPR
spectrum of TEMPONE in a pure Suc glass (i.e. without liposomes) (Fig.
8B) clearly indicates the solid environment of the TEMPONE molecules
typically seen with trapping of the probe in a glass (Marsh, 1981;
Dzuba et al., 1993).
TEMPONE-Induced Leakage from Desiccated EggPC Liposomes
If amphipathic substances in general are responsible for the
transient leakage from dry anhydrobiotes during rehydration, then it
should be possible to simulate this in liposomes as a model system. It
was shown in Figure 8 that TEMPONE can partition into eggPC liposomes
upon dehydration. Figure 9 shows the
effect of such a partitioning of TEMPONE as a model amphipathic
substance on the leakage of CF from eggPC liposomes. When 30 µL of a
lyoprotected liposome suspension without TEMPONE was dried for 3 h
in a stream of dry air, CF retention was close to 90% upon
rehydration. However, when dried in the presence of TEMPONE (at a molar
ratio of TEMPONE:eggPC of 1 and higher), CF leakage was considerable.
This leakage occurred during rehydration and not during drying, because
no green fluorescence was visible in the dried liposomes. The
lyoprotective Suc matrix (Suc:eggPC = 5:1) was always present in
these experiments to prevent complete leakage (Crowe et al., 1987).
Virtually no encapsulated CF was lost from the TEMPONE-treated
liposomes when they were kept hydrated for the 3-h period. These data
clearly indicate that partitioning of the amphipathic compound into
membranes during drying can cause leakage from the liposomes during
rehydration.
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| Figure 9.
Retention of CF by eggPC liposomes that were dried
for 3 h in a stream of dry air (3% RH) at 24°C in the presence
of Suc (mass ratio of Suc:eggPC = 5:1) and varying amounts of
TEMPONE. The CF retention of the nondried controls kept for 3 h at
24°C is also indicated.
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Pollen Amphipath-Induced Leakage from Desiccated EggPC Liposomes
The extent to which endogenous amphipaths from anhydrobiotic
organisms are also able to cause leakage is shown in Table
I. Endogenous amphipaths from T. latifolia pollen were obtained by two different methods: from an
organic solvent extract and from an aqueous extract. Aliquots of these
extracts were added to eggPC liposomes. Both extracts resulted in
leakage of CF during rehydration of the dried liposomes, although there
was also some leakage observed without drying. The change of color from
yellow-orange to green during drying of the droplets containing the
amphipath-treated liposomes indicates that some CF leakage had occurred
before the liposomes were dry. These data indicate how effective
endogenous amphipaths can be at increasing plasma membrane permeability
during dehydration, leading to considerable leakage during
rehydration.
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Table I.
Retention of CF by eggPC liposomes as influenced by
Suc and extracts of amphipaths from T. latifolia pollen
Liposomes were air dried and rehydrated or kept hydrated. The extracts
of amphipaths were obtained by organic solvent extraction or by aqueous
extraction, as described in "Materials and Methods," and suspended
in water. The aqueous extract used was the fraction that was eluted
from reversed-phase (C18) cartridges by 50% methanol:water
(v/v).
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Is the TEMPONE-Induced Increase of Liposome Permeability Transient?
Increased permeability of the pollen plasma membranes during
rehydration was shown to be transient (Fig. 3). Therefore, we also
expected the high permeability of TEMPONE-treated liposomes at
rehydration to be transient after repartitioning of the amphipathic molecules back into the aqueous phase. Figure
10 shows the effects of direct and
indirect application of TEMPONE/ferricyanide to the liposomes on the
shape of EPR spectra, and indicates that this expectation is correct.
In a control experiment (Fig. 10A), the permeability of eggPC liposomes
without previous TEMPONE treatment was tested. Compared with the
TEMPONE spectrum of the fresh liposome sample, that of the dried sample
had a slightly reduced water peak on direct imbibition in
TEMPONE/ferricyanide solution, which was restored when rehydration was
first in water, and then (after a few minutes) by the addition of a
TEMPONE/ferricyanide solution. This slightly reduced water peak
resulting from the penetration of some ferricyanide into the liposomes
upon rehydration corresponds to the approximately 10% leakage of CF
from the control liposomes in Figure 9 (molar ratio of
TEMPONE:eggPC = 0).
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| Figure 10.
EPR spectra of TEMPONE in suspensions of eggPC
liposomes containing Suc (mass ratio of Suc:eggPC = 5:1). The
suspensions (10-µL droplets) were dried in the absence (A) or
presence (B) of TEMPONE (molar ratio of TEMPONE:eggPC = 1:1). Dry
samples without previous TEMPONE (A) were rehydrated in
TEMPONE/ferricyanide (1 mM:120 mM; direct) or
first in water, followed after a few minutes by TEMPONE/ferricyanide
(indirect; final concentration 1 mM:120 mM).
Samples with TEMPONE administered before drying (B) were diluted in 300 mM ferricyanide (the nondried and the direct treatment) or
first in water for a few minutes followed by 600 mM
ferricyanide. The final molar ratio of TEMPONE/ferricyanide was in
either case approximately 1:120.
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When liposomes were dried in the presence of TEMPONE (molar ratio of
TEMPONE:eggPC = 1:1), the aqueous contribution to the TEMPONE
spectrum was low when the ferricyanide was added directly, and high
(comparable with the nondried control) when it was added a few minutes
after rehydration (Fig. 10B). We interpret this to mean that
permeability of the liposomes for ferricyanide was high in the direct
treatment but low in the indirect treatment after partitioning of the
majority of TEMPONE molecules back into the aqueous phase. Apparently,
after rehydration the TEMPONE-treated liposomes were still intact.
Relevance of Partitioning to the Transient Leakage from
Anhydrobiotes during Rehydration
We have made an attempt here to explain the mechanism of the high
plasma membrane permeability of anhydrobiotes during rehydration using
in situ EPR of stable nitroxide spin probes in pollen as a model
organism. The permeability of rehydrating pollen strongly decreases
with increasing initial moisture content (Fig. 3), and coincides with
repartitioning of the added amphipathic spin probes from the lipid
phase into the aqueous cytoplasmic phase during rehydration (Fig. 4).
The correlation between plasma membrane permeability and partitioning
expressed as percentage of spin probe in the lipid phase is shown in
Figure 11.
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| Figure 11.
Correlation between plasma membrane permeability
at the onset of rehydration of T. latifolia pollen
having different initial moisture content (data from Fig. 3, direct)
and the associated percentage of amphiphilic TEMPONE partitioned in the
lipid phase of the pollen. The percentage TEMPONE in the lipid phase
was calculated from the equation fitting the rehydration curve of
Figure 4A.
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Liposome experiments were conducted to extend the evidence of the
relationship between partitioning and permeability from a correlative
level to a more causal level. It was shown that when used as a model
amphipath, TEMPONE causes considerable leakage (Fig. 9) from
rehydrating eggPC liposomes in which it had partitioned during drying
(Fig. 8). However, the leakage occurred only at a high molar ratio of
TEMPONE:eggPC (1:1 and higher). Nondried liposomes did not leak. We
have demonstrated here that the leakage was caused solely by the
presence of TEMPONE during drying. After full rehydration the liposomal
membranes closed up again (Fig. 10) in a manner similar to that which
occurred in fully rehydrated pollen, as concluded from the reduced
permeability of the plasma membranes (Fig. 3). It is tempting to assume
that the partitioning behavior of the spin probes reflects similar
behavior of endogenously occurring amphipaths in the pollen, which is
responsible for imbibitional leakage.
Indeed, endogenous amphipaths extracted from the pollen also caused
leakage at the appropriate dilution, much more during drying and
rehydration than when the liposomes were kept hydrated (Table I). An
important question that arises is whether sufficient endogenous
amphipaths are available to generate the imbibitional leakage in situ.
At a phospholipid content of approximately 6% on a DW basis in the
pollen (Hoekstra et al., 1992c), the amphipaths would have to be
present at a few percent of the DW (depending on their
Mr). This is entirely feasible, considering the
large quantities of amphipaths such as flavonoids that are present in pollens and seeds (Gorobets et al., 1982; Wiermann and Vieth, 1983;
Ceska and Styles, 1984; Liao et al., 1989; Zerback et al., 1989; Rizk
et al., 1992; Ylstra et al., 1992; Vogt and Taylor, 1995).
Amphipathic substances may fluidize membranes, disturb the packing
order of membrane components, and increase permeability (Maher and
Singer, 1984; Casal et al., 1987). There is circumstantial evidence
that such fluidization occurs when anhydrobiotic organisms are
desiccated. Previously, we used Fourier-transformed IR data to describe
the phase behavior of membranes in dehydrating seeds (Hoekstra et al.,
1993) and leaves of the resurrection plant Craterostigma plantagineum (Hoekstra et al., 1997), and these data may support the fluidization hypothesis. The peak position of the
CH2 symmetric stretching vibration in the gel
phase at low temperature was approximately 1 wave number higher in the
dry specimens than in the hydrated controls (Table
II). Consequently, the change in wave
number with the phase transition was less extensive in organisms when
they were dry than when they were hydrated. This means that the
vibrational freedom of the CH in the acyl chains is higher in the dry
state than in the hydrated state in the gel phase. We interpret this as
fluidization of the dry bilayer in situ caused by partitioning. In
contrast, there was no difference between hydrated and lyophilized isolated membranes with respect to band position (wave number) in the
gel phase, nor between hydrated and lyophilized eggPC liposomes (Table
II). During the isolation of membranes the amphipathic substances are
likely to have partitioned to the isolation medium.
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|
Table II.
Band position of the CH2 symmetric
stretching vibration in Fourier-transformed IR spectra of hydrated and
dried specimens at 30°C (below the onset of the phase transition)
Specimens used were leaves and isolated membranes of the
desiccation-tolerant resurrection plant C. plantagineum
(Hoekstra et al. 1997) and defatted, desiccation-tolerant alfalfa
somatic embryos (Hoekstra et al., 1993). As a comparison, IR data are
given for eggPC liposomes (F.A. Hoekstra, unpublished results).
Dehydration of isolated membranes and liposomes was by freeze drying;
dehydration of leaves and somatic embryos was by air drying.
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Consequences of Partitioning of Endogenous Amphipaths for
Desiccation Tolerance and Storage Stability
The transient leakage of solutes during rehydration may not be the
only consequence of amphipath partitioning into membranes with
dehydration. Very early during dehydration the decrease in the volume
of the aqueous compartment leads to partitioning into the lipid
environment (Fig. 4B). This may provide an early signaling of the
imminent drought/desiccation stress by membrane fluidization and the
associated changes in ion pump activity (for review, see Shinitzky,
1984). We believe that similar signaling may apply to osmotic and
freezing stress. During dehydration TEMPO and TEMPONE completed
partitioning into the lipid phase at relatively high moisture contents
(0.8 and 0.4 g water g1 DW, respectively;
Figs. 4 and 7). Under these conditions there is still freezable water
(Buitink et al., 1996), albeit of high viscosity (Leprince and
Hoekstra, 1998).
If endogenous amphipaths have a partitioning behavior similar to
TEMPONE/TEMPO, which is likely considering the mechanism discussed
above, this could have important consequences for the storage of
anhydrobiotes at slightly elevated moisture contents (e.g. between 0.2 and 0.7 g water g1 DW). Under these
conditions cells would have increased membrane permeability. This may
be one of the reasons for the strongly reduced longevity of seeds in
this moisture-content range (Priestley, 1986). It is reasonable to
suppose that not only the plasmalemma but also the internal membranes
have increased permeability, because amphipathic compounds may
partition into any membrane type. Lipid bodies could then have a
protective role in that they can harbor a considerable amount of the
more apolar amphiphilic compounds.
The beneficial effect of endogenous amphipaths in anhydrobiotes may
include the ability to depress the transition temperature of dry
membranes and, thus, to prevent the formation of a gel phase (Hoekstra
et al., 1997) and to act as antioxidants inside of membranes (Wang and
Zheng, 1992; Terao et al., 1994; Saija et al., 1995). A partitioning
behavior as outlined above would then be most effective to give
automatic antioxidant protection to membranes in dehydrating organisms
and, thus, to extend storage longevity. That these membrane-fluidizing
compounds also increase permeability may be an inevitable disadvantage.
Because of this dual effect of amphipath partitioning, we suggest that
there is tight control of this phenomenon in desiccation-tolerant
organisms.
| |
FOOTNOTES |
1
This work was supported by stipends from the
Wageningen Agricultural University and the Netherlands Organization for
Scientific Research to E.A.G.
*
Corresponding author; e-mail
folkert.hoekstra{at}algem.pf.wau.nl; fax 31-317-484740.
Received June 1, 1998;
accepted August 11, 1998.
| |
ABBREVIATIONS |
Abbreviations:
CF, 5(6)carboxyfluorescein.
DW, dry weight.
EPR, electron paramagnetic resonance.
PC, phosphatidylcholine.
TEMPamine, 4-amino-2,2,6,6-tetramethyl-1-piperidinyloxy.
TEMPO, 2,2,6,6-tetramethyl-1-piperidinyloxy.
TEMPONE, 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy.
Tm, gel phase to liquid-crystalline phase
transition temperature.
| |
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Top
Abstract
Introduction
