Plant Physiol. (1998) 118: 1253-1264
The Responses of Cytochrome Redox State and Energy Metabolism to
Dehydration Support a Role for Cytoplasmic Viscosity in Desiccation
Tolerance1
Olivier Leprince* and
Folkert A. Hoekstra
Department of Biomolecular Sciences, Laboratory of Plant
Physiology, Wageningen Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
 |
ABSTRACT |
To characterize the depression of
metabolism in anhydrobiotes, the redox state of cytochromes and energy
metabolism were studied during dehydration of soaked cowpea
(Vigna unguiculata) cotyledons and pollens of
Typha latifolia and Impatiens
glandulifera. Between water contents (WC) of 1.0 and 0.6 g
H2O/g dry weight (g/g), viscosity as measured by electron
spin resonance spectroscopy increased from 0.15 to 0.27 poise. This
initial water loss was accompanied by a 50% decrease in respiration
rates, whereas the adenylate energy charge remained constant at 0.8, and cytochrome c oxidase (COX) remained fully oxidized.
From WC of 0.6 to 0.2 g/g, viscosity increased exponentially. The
adenylate energy charge declined to 0.4 in seeds and 0.2 in pollen,
whereas COX became progressively reduced. At WC of less than 0.2 g/g,
COX remained fully reduced, whereas respiration ceased. When dried
under N2, COX remained 63% reduced in cotyledons until WC
was 0.7 g/g and was fully reduced at 0.2 g/g. During drying under pure
O2, the pattern of COX reduction was similar to that of
air-dried tissues, although the maximum reduction was 70% in dried
tissues. Thus, at WC of less than 0.6 g/g, the reduction of COX
probably originates from a decreased O2 availability as a
result of the increased viscosity and impeded diffusion. We suggest
that viscosity is a valuable parameter to characterize the relation
between desiccation and decrease in metabolism. The implications for
desiccation tolerance are discussed.
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INTRODUCTION |
Tolerance of desiccation requires tissues to cope with physical,
biochemical, and structural stresses associated with the loss of water.
Two mechanisms of protection against the deleterious effects of water
loss have received major attention: (a) the synthesis of the so-called
late-embryogenesis-abundant proteins (Close, 1996
; Kermode, 1997) and
(b) the accumulation of di- and oligosaccharides (Leprince et al.,
1990; Horbowicz and Obendorf, 1994; Vertucci and Farrant, 1995). In
model systems these sugars protect the structural integrity of
dehydrated membranes and proteins (Crowe et al., 1992; Hoekstra et al.,
1997). They also participate in the formation of a glassy state (Burke,
1986; Green and Angell, 1989; Leopold et al., 1994). In glasses,
diffusion and chemical reactions are severely slowed because the
viscosity is assumed to be 1012-fold higher than
in a liquid. However, there are numerous examples of pollen and seeds
in which there is no apparent correlation between the expression of
desiccation tolerance and the nature and concentration of protectants
(Hoekstra et al., 1994, 1997; Vertucci and Farrant, 1995). To our
knowledge there is no convincing evidence yet that the in vitro
protective mechanisms of sugars also function in situ (Hoekstra et al.,
1997). Glasses are formed in both dried, desiccation-sensitive and
desiccation-tolerant seeds and pollen and appear to have identical
properties (Sun et al., 1994; Buitink et al., 1996).
An alternative hypothesis for desiccation tolerance
proposes that anhydrobiotes are capable of evading damage from
oxidative and peroxidative reactions during drying (Leprince et al.,
1994; Vertucci and Farrant, 1995). Indeed, loss of viability in
dehydrating embryonic tissues and pollen has been associated with
various symptoms of free radical-induced injury (Senaratna et al.,
1987; Hendry et al., 1992; Van Bilsen and Hoekstra, 1993; Leprince et al., 1994). Such damage probably results from the formation of ROS
during the water loss and subsequent dry storage. Under normal physiological conditions, tight metabolic coupling in mitochondria, together with antioxidant defense mechanisms, maintains the
concentrations of ROS at nonharmful levels (Halliwell and Gutteridge,
1984; Cadenas, 1989; Skulachev, 1996). During dehydration of
desiccation-tolerant tissues, a tight control of ROS production and
metabolism is expected. However, this control may be lost in
desiccation-sensitive tissues during dehydration, resulting in an
overproduction of ROS (Leprince et al., 1994), as occurs in other
oxidative stress conditions (McKersie, 1991; Foyer et al., 1994;
Inzé and Van Montagu, 1995). We speculated that a coordinated
down-regulation of metabolism must occur during drying to achieve
desiccation tolerance (Leprince et al., 1994, 1995). Before
investigating such a hypothesis, we should characterize the relations
between the loss of water and the mechanisms that control the
generation of ROS in tissues that retain physiological and cellular
integrity during drying. Surprisingly, the significance of the changes
in the physical properties of water during drying for metabolism has
drawn little attention. This is in marked contrast to the wealth of
studies of the transition in metabolism during imbibition before
germination (Bewley and Black, 1994) and the relationship between the
hydration layers of cellular and molecular structures and oxidative
processes (for review, see Leopold and Vertucci, 1989).
Under physiological conditions, the generation of ROS greatly depends
on the availability of molecular O2 and on the
redox states of the electron-transfer components (Cadenas, 1989;
Skulachev, 1996). In mitochondria, such components must be reduced to
build up a membrane potential and produce ATP. However, this reduction must be regulated, since it also favors the leakage of electrons from
the mitochondrial electron-transport chain to molecular
O2 and the generation of ROS (Halliwell and
Gutteridge, 1984; Cadenas, 1989; Skulachev, 1996). A parallel between
desiccation sensitivity and oxidative stress can be assumed because
they are both associated with high metabolic rates and are
characterized by similar symptoms of injury (Leprince et al., 1994,
1995; Vertucci and Farrant, 1995). Based on this assumption, we
assessed noninvasively the redox states of respiratory chain components
and energy metabolism during drying. These redox states can be
estimated in situ by spectrophotometry at low temperature, as
demonstrated by the pioneering work of Wilson and Bonner (1971) on
intact peanut cotyledons during imbibition.
As a result of the water loss during drying, diffusion of
O2 to mitochondria may be impeded. This may have
a beneficial effect, as there is less of a chance to form ROS
(Skulachev, 1996). However, it also may induce anoxia during drying. It
is interesting that survival of invertebrates in the dry state has been
associated with tolerance to anoxia (Hofmann and Hand, 1994; Hand and
Hardewig, 1996). In these animal systems, anoxia induces a coordinated
suppression of the catabolic and anabolic biosynthetic pathways of
energy metabolism (Hand and Hardewig, 1996). These observations
prompted us to examine further the interaction between
O2 diffusion as a function of WC and redox states
of Cyts. Because of the physiological significance of glasses in
seeds and pollen (Leopold et al., 1994; Leprince and Walters-Vertucci,
1995), we further assessed the relation between loss of water and
increase in viscosity during drying. For this purpose, ESR spectroscopy
was used to study the rotational motion of nitroxide spin-probes that
were inserted into the cytoplasm. From the rotational diffusion
coefficient, the viscosity of the cytoplasmic surrounding was
calculated during drying (Freed and Fraenkel, 1963; Hemminga et al.,
1993). From the relation between viscosity and the
O2 diffusion coefficient, the diffusion rates of
O2 to mitochondria during drying were estimated.
| |
MATERIALS AND METHODS |
Plant Material, Hydration, and Drying Treatments
Seeds of cowpea (Vigna unguiculata L. Walp.),
germination > 90%, were purchased locally and allowed to soak
overnight at 15°C in wet paper towels. Before drying or incubation
with chemicals, the cotyledons were excised and blotted dry on filter
paper. The cotyledons were placed in a sealed chamber flushed with dry
air, N2, or 100% O2 for
intervals up to 24 h at 22°C or at 4°C in a cold room (RH
about 3%). Pollen grains of Typha latifolia L. and Impatiens glandulifera Royle were harvested in Wageningen,
The Netherlands. Fresh, mature I. glandulifera pollen was
used immediately (>92% germination), whereas mature T. latifolia pollen was first dried and stored at 
20°C (>95%
germination). To obtain hydrated T. latifolia pollen, the
material was rehydrated overnight at 4°C in 100% RH. Rehydrated
pollen was then uniformly spread on a Petri dish and dried back at
20°C and 55% to 65% RH while samples were taken for analysis. WC
were assessed gravimetrically by comparing the sample weights before
and after drying for 38 h at 96°C and are expressed on a dry-
weight basis.
In Situ Determination of Redox States of Cyts
Pollen (300 mg) was loaded directly into a spectroscope cuvette
with a low-temperature attachment. To avoid changes in WC that may
occur between sampling and loading, all manipulations were carried out
in a glove box at 3%, 55%, or 100% RH, depending on the initial WC
of the sample. After loading, the cuvette was sealed with tape and
transferred into a Dewar flask filled with liquid
N2.
Eight to 16 cowpea cotyledons were frozen in liquid
N2 and ground into a homogeneous powder using a
mortar and pestle. Frozen powder was then loaded into the spectroscope
cuvette that was precooled in liquid N2. To
reduce condensation of water at the surface of the frozen material,
grinding and loading were performed in a glove box at 3% RH. For
drying experiments under anoxia and hyperoxia, the glove box was also
purged with pure N2 and O2, respectively. At the time the cuvette was filled, an additional aliquot
of pollen or powdered frozen cotyledons was always secured in a small
tube for WC determination.
In vivo spectra of Cyts were recorded in the visible region at liquid
N2 temperature using a 1- or 3-mm-pathlength
cuvette on an Aminco DW2a or DW2000 spectrophotometer (SLM Instruments, Urbana, IL) in double-beam mode, interfaced to a personal computer. A
slit width of 3 mm and a scan rate of 30 nm/min were used. Instrumental noise was reduced by averaging 5 to 10 successive scans. Spectra of
powdered cotyledons and pollen were routinely recorded against insoluble PVP (Polyclar AT) powder as a reference, which is assumed to
have light-scattering properties similar to the biological material
(Chance, 1954). The effects of various reducing conditions on the
spectra were determined to ascertain that the light-scattering effects
of our material did not produce artifacts and to determine the maximum
absorbance values for reduced Cyts as a calibration for 100%
reduction. Chemically reduced spectra were obtained from hydrated
cotyledons following incubation for 4 h with 1 mM KCN or 1 h in 5% (w/v) sodium dithionite prior to drying. Difference spectra were obtained either mathematically, using the spectra obtained
in the conditions described above, or by scanning the reduced samples
against an aerated hydrated sample in the reference cuvette. The
proportion of reduced Cyts was determined as the difference between
absorbances at the peaks corresponding to Cyts b + c and a aa3 and those of the baseline. The
baseline was taken as the tangent line connecting points on the
absorption scan from about 540 to 565 nm (Cyts b + c) and
590 to 608 nm (Cyt a aa3). Actual concentrations of Cyts could
not be calculated because of: (a) overlapping absorption peaks of Cyts
from microsomal and mitochondrial origin (Fig. 1); (b) the unknown
geometrical structure of the powdered material, which makes it
impossible to determine accurately the light pathlength; and (c) the
dependence of molar extinction coefficient of reduced Cyts on the
osmotic potential or water activity of the solvent (Kornblatt and Hoa, 1990).
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| Figure 1.
Absorbance spectra at various WC of powdered
cowpea cotyledons (A, Scan 1-6, WC is 1.22, 0.90, 0.52, 0.39, 0.29, and
0.15 g/g, respectively) and intact I. glandulifera
pollen (B, WC is 1.2 and 0.09 g/g in scan 1 and 2, respectively).
Spectra were recorded at 77 K using PVP in the reference cuvette. C
(cotyledons) and D (pollen) show the effect of drying on the redox
state of Cyts in more detail using difference spectra. In C, scan 1 (hydrated control) was subtracted from each individual scan (2-6). In
D, scan 1 was subtracted from scan 2. Peaks and the trough (D) are
indicated by upward and downward arrows, respectively.
|
|
Respiration Measurements
O2 uptake and CO2
release were measured in cotyledons of cowpea and T. latifolia pollen using a gas chromatograph with two columns in
series, essentially as described by Hoekstra and Bruinsma (1980). The
first column (Porapack R, 80-100 mesh, Chrompack International BV,
Middelburg, The Netherlands) was operated at 65°C, and the second one
(Mol Sieve 5A, 50-90 mesh) was operated at 25°C as a loop after the
catharometric detector. The carrier gas was He. For calibration, pure
CO2 and air were used, assuming a gas composition at atmospheric pressure of 20.95% O2, 0.93% Ar,
and 78.09% N2. Three to five cotyledons that
were dried to different extents were sealed in 7-mL sterile vessels and
incubated at 25°C. Rates of gas exchange were calculated by linear
regression analysis of three measurements taken every 20 min
to 4 h, depending on respiration rate. After respiration measurement,
the sample WC was approximately 5% less than before
measurement.
Adenylate Extraction and AEC Measurements
At intervals during drying, samples of 250 mg of cotyledons and 50 mg of pollen were homogenized in 10% TCA and then the TCA was removed
by diethyl ether (Al-Ani et al., 1985). ATP content was determined
using the luciferin-luciferase system (catalog no. 1699695, Boehringer
Mannheim). Analyses of ADP and AMP were performed after enzymatic
conversion to ATP (Swedes et al., 1975).
Assessment of Viscosity and O2 Diffusion Coefficient
Using ESR of Nitroxide Spin-Probes
Fully hydrated cotyledons of cowpea were cut into
3-mm3 pieces and incubated for 15 min at room
temperature in an aerated solution of 2 mM spin-probe
(4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy or CP, Sigma).
Subsequently, potassium ferricyanide was added to the solution to a
final concentration of 150 mM and incubation was prolonged
for 30 min. Pieces of cotyledons were briefly washed in distilled water
and allowed to dry at ambient conditions (22°C, 50%-70% RH) before
the ESR measurements. For T. latifolia pollen, loading of
the spin-probes, incubation, and washing procedures were performed in
liquid germination medium according to the method of Buitink et al.
(1998). Ferricyanide anions broaden the triplet ESR signal of
spin-probes via spin-spin interactions (Golovina et al., 1997). Because
ferricyanide cannot pass through intact membranes, the contribution of
the extracellular spin-probe to the total ESR signal of our material is
invisible. ESR spectra were recorded at 5°C and 25°C on an ESR
X-band spectrometer (model ESP 300, Bruker Analytik, Reinstetten,
Germany). Modulation amplitude was 1 G and microwave power was 6 mW.
Other parameters were set as necessary to obtain the optimal
signal-to-noise ratio.
To assess the cytoplasmic viscosity, 
R values
for anisotropic tumbling in liquid solution were derived from ESR
spectra according to the method of Freed and Fraenkel (1963):
![&tgr;<SUB>R</SUB>=6.5× 10<SUP>−6</SUP> B<SUB>+1</SUB> ([h<SUB>+1</SUB>/h<SUB>−1</SUB>]<SUP>1/2</SUP>−1)](/content/vol118/issue4/fulltext/1253/img001.gif)
|
(1)
|
where h+1 and
h
1 are the amplitudes of the low- and
high-field lines of the spectra, respectively (Fig. 5, scan 1),
B+1 is the line width of the low-field line
(in tesla). Viscosity values were then determined using the following
modified Stokes-Einstein relation (Keith and Snipes, 1974; Hemminga et al., 1993):
|
(2)
|
where 
is the viscosity of the solvent, V is the
volume of the rotating molecule, kb is the
Boltzmann's constant, and T is the absolute temperature.
0 is the zero viscosity rotational correlation
time, which is negligible (J. Buitink and M.A. Hemminga, personal
communication). k is a dimensionless interaction parameter taking into account the fact that nonspherical molecules displace the
solvent molecules as they rotate (Hemminga et al., 1993). O2-diffusion coefficients were calculated from
the experimental correlation between viscosity of aqueous solutions and
temperature for a binary mixture (Reid et al., 1987):
![D<SUB>O<SUB>2</SUB></SUB> = 7.4 × 10<SUP>−8</SUP>(T[2.26 M<SUB>H<SUB>2</SUB>O</SUB>]<SUP>1/2</SUP>/V<SUP>0.6</SUP><SUB>O<SUB>2</SUB></SUB>)](/content/vol118/issue4/fulltext/1253/img003.gif)
|
(3)
|
where T is the absolute temperature,
MH2 is the molecular weight of
water, and VO2 is the molar volume
of O2.
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| Figure 5.
ESR spectra of CP introduced into hydrated (WC is
1.2 g/g) cowpea cotyledons (scan 1) and dried to various extents (scans
2-5). In scan 1, the spectral parameters used to calculate
R are shown. Water contents (expressed on a dry-weight
basis) are indicated next to each spectrum. The asterisk indicates
spectral features characteristic of a rigid-solid-state-like
spectrum.
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|
Determination of Tg
Three milligrams of cotyledons dried to different extents were
hermetically sealed into aluminum differential scanning calorimetry pans. Glass transitions were determined using Pyris-1 differential scanning calorimetry (Perkin-Elmer) using the settings described by
Leprince and Walters-Vertucci (1995). The Tg was determined by the
onset of the temperature range at which changes in specific heat,
characteristic of a glass transition, occurred (Leprince and
Walters-Vertucci, 1995).
Experimental Design and Statistical Treatment
All drying experiments were performed twice or three times and the
resulting data were pooled in the graphics. Furthermore, the value for
each measurement represents an average of 3 to 16 cotyledon pairs or
12 × 106 pollen grains. As an aid to the
eye, the data were fitted using the software Tablecurve 2D (Jandel
Software, San Rafael, CA), with third-order polynomial
regression or asymmetric transition functions (i.e. y = a/(1 + x/b)c;
y = a + b/(1 + (x/c)d); y = a + b/x + clnx/x2)
giving adjusted r2 varying between 0.908 and 0.988.
| |
RESULTS |
In Situ Spectrophotometric Determination of Redox States of Cyts
The effects of decreasing WC on the absorbance spectra of powdered
cowpea cotyledons and intact I. glandulifera pollen are shown in Figure 1, A and B, respectively.
The peak at 599 to 603 nm that is attributable to the heme
aa3 complex of COX (Chance, 1954; Lance and Bonner, 1968; Ikuma, 1972) increased 2- and 10-fold upon drying of cowpea cotyledons and I. glandulifera pollen,
respectively. The identity of this complex was confirmed by its
sensitivity to cyanide (see next paragraph; Fig. 2A) and Ar (data not
shown). To avoid possible light-scattering artifacts when assessing
concentrations of Cyts, we studied the effects of drying on COX using
difference spectra. Difference spectra were obtained by subtracting the
absorbance spectrum of a hydrated sample from the spectra of samples
having various WC (Fig. 1, C and D). They are referred to as air-dried minus hydrated difference spectra. For both cowpea cotyledons and
I. glandulifera pollen, air-dried minus hydrated difference spectra showed an increase in absorbance at 599 to 603 nm during drying, indicating an increase in the proportion of reduced COX. Results similar to those in Figure 1, C and D, were obtained when the
dried material was directly scanned against the hydrated material in
the reference cuvette instead of scanning against PVP followed by
spectra subtraction.
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| Figure 2.
A, The effect of chemical reduction on Cyt redox
state of hydrated, powdered cotyledons. Difference spectra were
obtained by subtracting the spectra of nontreated samples from spectra
of samples obtained after chemical reduction with 1 mM KCN
(scan 2) and 5% sodium dithionite (scan 3). The dried minus hydrated
difference spectrum (scan 1) is shown as a control. B, The effect of
drying plus chemical reduction on Cyt redox state of powdered
cotyledons. Incubation with KCN or sodium dithionite were performed
before drying. Difference spectra were obtained after subtraction of an
untreated, dried sample from a dried KCN-treated sample (scan 1) and
from a dried sodium-dithionite-treated sample (scan 2) of similar WC.
Peaks and troughs are indicated by upward and downward arrows,
respectively.
|
|
In addition to the presence of Cyt
aa3, cowpea cotyledons
contained additional Cyt components. The presence of these components was confirmed by the presence of peaks or troughs in the various difference spectra shown in Figure 2A.
Scan 1 exhibits a peak at 520 nm of unknown origin and one trough
between 558 and 560 nm that indicates the presence of b-type
Cyts. In scans 2 and 3 (Fig. 2A) a peak is observed at 549 nm that is
attributable to a c-type Cyt. In light of previous studies
of microsomal membranes isolated from legume seedlings (Hendry et al.,
1981; Lüthje et al., 1997), it is most likely that b-
and c-type Cyts observed in cowpea cotyledons are from
plasma membrane oxidases. In I. glandulifera pollen, the
difference spectra (Fig. 1D) exhibits a peak at 549 nm and a trough at
558 nm, which may correspond to mitochondrial Cyt c and
b, respectively. In T. latifolia pollen, we found
features similar to I. glandulifera pollen, although the
maximum absorbance of Cyt c and COX was 10-fold lower (data not shown).
To further ascertain that the changes in absorbance observed in the
air-dried minus hydrated difference spectra were the result of actual
changes in redox states of the Cyts, we examined chemically reduced
spectra that were obtained after incubating hydrated cotyledons with
KCN or sodium dithionite. Spectra of untreated, hydrated cotyledons
were then subtracted from these chemically reduced spectra to obtain
reduced minus air-oxidized difference spectra (Fig. 2A, scans 2 and 3).
The extent of COX reduction (599-603 nm peak) with the chemicals in
the hydrated state (scans 2 and 3) was identical to that of the
untreated, dried minus hydrated difference spectrum (control, scan 1).
However, compared with this control spectrum, the COX peaks in the
chemically reduced difference spectra were at lower wavelengths. To
assess whether the water loss or the extent of reduction could shift
the peak to a higher wavelength, difference spectra were obtained by
subtracting a spectrum of hydrated cotyledons from spectra of
cotyledons that were dried in the presence of either KCN or sodium
dithionite.
In the presence of KCN, the spectrum (data not shown) was remarkably
similar to the air-dried minus hydrated difference spectrum (Fig. 2,
scan 1). Indeed, no peak was discernible in the difference spectrum
between cotyledons dried in the presence and absence of KCN (Fig. 2B,
scan 1). The difference spectrum between cotyledons dried in the
presence and absence of sodium dithionite showed a peak at 599 nm and a
trough at 607 nm (Fig. 2B, scan 2), originating from the fact that the
COX peak in the presence of sodium dithionite is not sensitive to water
loss. The difference between the effects of KCN and sodium dithionite
may stem from differences in the mode of reduction of COX (i.e. via
blocking electron transport to O2 or direct
reduction, respectively). From these observations we conclude
that the absorbance peak in the 599-603 nm region is
associated with reduced COX. Furthermore, the desiccation-induced increase of this peak (Fig. 1) indicates that COX becomes increasingly reduced in both cotyledons and pollen during drying.
Additional information about the redox state of b-type Cyts
in cotyledons can be derived by comparing the absorbance spectra in
Figure 1A and the difference spectra in Figure 2. The absorbance spectra of hydrated material (Fig. 1A, scan 1) showed a double peak at
approximately 558 nm. When spectra from hydrated cotyledons that were
reduced with sodium dithionite were subtracted from spectra of
untreated, hydrated cotyledons, no peak was observed at 558 nm in the
difference spectra (Fig. 2A, scan 3). It follows that sodium dithionite
had no effect on the redox states of Cyt b. Thus, we
conclude that microsomal Cyts were mostly reduced in the hydrated,
untreated material. After drying, their redox state changed, as
demonstrated by the trough at 558 nm in the dried minus hydrated
spectrum (Fig. 2A, control, scan 1). When spectra from dried cotyledons
that were reduced by sodium dithionite prior to drying were subtracted
from spectra of untreated, dried material, the peak reappeared at 558 nm in the difference spectra (Fig. 2B, scan 2), indicating that sodium
dithionite kept the Cyt b reduced during drying. From these
observations we conclude that the microsomal Cyts become more oxidized
during drying of untreated material. Such oxidation can be monitored by
comparing scans 1 through 6 in Figure 1, A and C. The absence of peaks
in the difference spectrum of the dried material in the presence and
absence of KCN suggests that cyanide does not lead to reduction of
microsomal Cyts.
To study the response of the Cyt redox states to water loss, the
absorbance of mitochondrial Cyts in cotyledons and pollen was plotted
as a function of WC obtained after various times of drying (Fig.
3). In hydrated pollen of I. glandulifera (Fig. 3A) and T. latifolia (Fig. 3B) and
in hydrated cotyledons (Fig. 3C), the A599
was low. Because the a a3 complex is mostly oxidized in
steady-state physiological conditions (Chance, 1954; Kariman et al.,
1983; Le
tan et al., 1993), we assumed that the absorbance values
in the hydrated state correspond to near 100% oxidized levels. During
drying, the A599-603 did not increase
substantially until a WC of 0.6 g/g was reached. Thereafter, it
increased markedly until 0.2 to 0.3 g/g. Below these WC values the
absorbance reached a constant value that was equivalent to those
obtained after reduction with sodium dithionite or KCN. In cotyledons,
KCN kept COX reduced regardless of WC (Fig. 3C). The
A599-603 values obtained in dried
cotyledons and pollen were similar to those measured in dry, quiescent
material (data not shown), indicating that the increase in absorbance
was not due to synthesis of Cyts during drying. The progressive red
shift in the maximum absorbance from 599 to 603 nm during drying is
particularly noteworthy (Fig. 3, D and E). Treating cotyledons with KCN
prior to drying did not interfere with the red shift in cowpea
cotyledons (Fig. 3E). Such shifts have been previously observed in the
Soret maximum of isolated beef heart COX exposed to decreasing water
activity (Kornblatt and Hoa, 1990). However, the reasons why the
chromophores are perturbed in our material during drying remain to be
elucidated. In I. glandulifera pollen, the effects of
reducing WC on absorbance of Cyt c was also recorded (Fig.
3A). The plot of the proportion of reduced mitochondrial Cyt
c versus WC was similar to that of COX, indicating that the
redox changes are synchronous during drying.
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| Figure 3.
A to C, The effects of drying on the absorbance of
the  -band of Cyt c ( ) and COX ( ) in I. glandulifera pollen (A), T. latifolia pollen
(B), and cowpea cotyledons (C). For cowpea cotyledons the effect of 1 mM KCN is also shown ( ). D to E, The effect of drying on
the absorbance maximum of the -peak of COX for T. latifolia pollen (D) and cowpea cotyledons (E) in the absence
() and presence () of 1 mM KCN. DW, Dry weight.
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Effects of O2 Supply on COX Reduction during Drying
At least two factors may contribute to the increased reduction of
mitochondrial Cyts: (a) the lack of O2, as suggested by studies of mammalian (Kariman et al., 1983), fungal (Letan et al., 1993), and plant tissues (Lance and Bonner, 1968), and (b) viscosity that changes the solvent properties around COX (Escamilla et
al., 1989; Kornblatt and Hoa, 1990). However, the interpretation of the
COX redox behavior in our material during drying is further complicated
because O2 availability is also related to
viscosity. To test the effects of O2
availability, hydrated cotyledons of cowpea were dried for different
intervals in a sealed chamber under a stream of 100%
N2 or 100% O2. Under
anoxia, only a 60% reduction in COX was observed in the hydrated state
(Fig. 4A). Thus, traces of
O2 may have penetrated the tissues and partially oxidized the COX before having been fixed in liquid
N2. Beginning at a WC of 0.7 g/g, the extent of
reduction increased to 100% at 0.3 g/g. Thus, O2
availability could be considered to be a factor controlling the onset
of COX reduction. Drying cotyledons in the presence of 100%
O2 did not affect the pattern of reduction of COX
(Fig. 4A). It was estimated that approximately 70% ± 5.8% reduction
was achieved in the dry state, based on the average (±SE)
of the data points corresponding to the four lowest WC values. The
composition of the atmosphere during drying did not influence the shift
in the position of the absorbance maximum (Fig. 4B). However, the
resulting 70% COX reduction, despite the high concentration of
O2 present during drying, suggests that there is
a barrier limiting O2 availability to COX.
Therefore, we investigated the relation between viscosity and the COX
redox state during drying.
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| Figure 4.
The effects of O2 concentration on the
reduction (A) and absorbance maximum (B) of COX during drying of cowpea
cotyledons. Treatments were 100% N2 ( ), air (), and
100% O2 ( ). The curves represent fits
(r2 = 0.915) to the control (air)
experiments. DW, Dry weight.
|
|
Changes in Cytoplasmic Viscosity and the O2-Diffusion
Coefficient during Drying
To estimate cytoplasmic viscosity, we studied the effects of
drying on the rotational motion of CP that was introduced into the
cytoplasm of cowpea cotyledon cells (Fig.
5) and T. latifolia pollen.
With decreasing WC, both the amplitudes of and the distance between the
left (low-field) and right (high-field) peaks decreased. At WC of less
than 0.32 g/g (Fig. 5, scan 5), the high-field peak became
progressively distorted and two additional components appeared at the
extremes of the spectra. These components are characteristic of a
solid-state-like spectrum in which the spin-probe is almost immobilized. The theory that predicts R is
only valid for liquid-state spectra that are representative of an
(an)isotropic tumbling of the spin-probe (for theoretical details, see
Hemminga et al., 1993). Thus, spectra of CP introduced in samples with
WC of less than 0.25 g/g cannot be used to calculate
R. Therefore, the corresponding viscosities
are unknown. Before drying, the cytoplasmic viscosities of both
T. latifolia and cowpea cotyledons were approximately 0.15 poise (Fig. 6A), similar to values
previously reported (Keith and Snipes, 1974). No significant changes
were observed until the cotyledons and the pollen reached a WC of 0.7 and 0.9 g/g, respectively. Below these values the viscosity increased
exponentially with further drying. A viscosity of 3 poise in pollen and
10 poise in cotyledons was reached at approximately 0.3 g/g (Fig. 6A). At this WC, the Tg is approximately 70°C (data not shown). As Tg
progressively approaches a value close to room temperature with further
drying, viscosities are expected to increase considerably. Similar
results were obtained when 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy was used instead of CP.
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| Figure 6.
A, The effects of drying on cytoplasmic viscosity
in cowpea cotyledons () and T. latifolia pollen
(). B, The effects of drying on calculated O2-diffusion
coefficients in cowpea cotyledons dried at 22°C () and 5°C ()
and in T. latifolia pollen () dried at room
temperature. DW, Dry weight.
|
|
The O2-diffusion coefficient is inversely
proportional to the viscosity of the medium (Nobel, 1983; Reid et al.,
1987). It must be pointed out that the experimental correlation used to calculate O2-diffusion coefficients is valid only
for binary systems and becomes less accurate with increasing
viscosities (Reid et al., 1987). Therefore, in this study, the relation
between O2-diffusion coefficients versus WC is
regarded as qualitative (Fig. 6B). At room temperature the
O2-diffusion coefficients for both T. latifolia and cowpea cotyledons prior to drying were approximately
9.5 × 109 m2 s1. During drying of both materials, their
respective O2-diffusion coefficients decreased
exponentially below 0.8 g/g. The O2-diffusion coefficient measured at 5°C in hydrated cotyledons of cowpea was significantly lower than that at 25°C. However, this difference rapidly disappeared during the loss of water. These observations suggest that O2 diffusion through the tissues is
progressively impeded upon the loss of water and increase in viscosity.
Relation among Viscosity, Temperature, and Redox State of COX
As mentioned earlier in this paper, an increased viscosity may
directly or indirectly contribute to increased COX reduction. On the
one hand, an increase in viscosity reduces the O2
availability and the environment around COX may experience anoxia. On
the other hand, an increase in viscosity could impede the
conformational changes of COX that are necessary for the intramolecular
electron transfer to O2 and proton pumping
(Einarsdottir et al., 1995; Wittung and Malmström, 1996). When
the extent of COX reduction in cowpea cotyledons and T. latifolia pollen were plotted against viscosity, a similar
relation appeared for both materials despite their differences in
cellular composition and structure (Fig. 7A). The viscosity corresponding to the
onset of COX reduction was approximately 0.4 poise for both pollen and
cotyledons. Since temperature also affects viscosity, measurements of
COX reduction were carried out in cowpea cotyledons during drying at
22°C and 5°C. When dried at 5°C, the onset of reduction shifted
toward higher viscosity values (Fig. 7A). At 1.2 poise, the extent of COX reduction was 18% ± 2% (n = 2) and 56% ± 7%
(n = 3) at 5°C and 22°C, respectively. In the
driest state at 5°C, 63% ± 7% of maximal reduction was achieved,
as estimated by the average (±SE) of the data points
corresponding to the three lowest WC values. This indicates that
viscosity cannot be the sole factor involved in the reduction of COX.
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| Figure 7.
The response of COX reduction to viscosity and
temperature during drying of cowpea cotyledons (, 22°C; ,
5°C) and in T. latifolia pollen (, room
temperature). Reduction levels are plotted as a function of viscosity
(A) or as a function of sample WC (B). Viscosity values were taken from
the equation fitting the viscosity versus WC plot of Figure 6A. The
relation between viscosity and COX reduction in T. latifolia pollen is also shown. The curves represent fits
(r2 = 0.915) to the control (22°C)
experiments. DW, Dry weight.
|
|
In vitro studies have demonstrated that the transfer of electrons from
the Cyt a to the Cyt a3 CuB center of COX requires water movements
in and out of the enzyme (Kornblatt and Hoa, 1990; Kornblatt and
Kornblatt, 1992). Such water movements can be inhibited by decreasing
water activity or osmotic potential, which results in the reduction of
the Cyt a (Escamilla et al., 1989; Kornblatt and Hoa, 1990).
To test whether a decreased water activity in the cotyledons caused the
reduction of Cyt a, we compared the A599-603 from material dried at 22°C and
5°C (Fig. 7B). From water sorption isotherms (data not shown), it can
be derived that, for the same water activity, the WC of tissues
equilibrated at 5°C is higher than that of tissues equilibrated at
22°C. The plot of the levels of reduced COX versus WC shows that the
WC corresponding to the onset of reduction of COX is lower in tissues dried at 5°C than at 22°C (Fig. 7B). If water activity were
responsible for the increase in reduction of COX, the WC corresponding
to the onset of reduction would have shifted to higher WC, which is in
contrast to what is observed in Figure 7B. Instead, it is likely that
the reduced temperature during drying and the consequent reduction in
metabolism maintained a concentration of dissolved O2 high enough to maintain a significant
proportion of COX in the oxidized state during drying.
Response of Respiration and Energy Metabolism to Drying in Cowpea
Cotyledons
To examine the relationship between energy metabolism and changes
in viscosity, O2 uptake and
CO2 release rates were measured in the gas phase
and plotted as a function of WC and viscosity during drying (Fig.
8, A and B). In addition, we determined
the AEC as an estimate of the metabolic activity (Pradet and Raymond, 1983). The plots of gas-exchange rates versus WC show that respiration declined progressively as water was lost (Fig. 8A). At WC of less than
0.27 g/g, gas exchange was not detectable. The decline in respiration
is more pronounced when plotted as a function of viscosity rather than
WC (Fig. 8B). At viscosities greater than 0.27 poise, gas-exchange
rates decreased exponentially.
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| Figure 8.
The effect of drying on several metabolic
parameters in cowpea cotyledons. A and B, CO2 release ()
and O2 uptake (). C and D, Respiratory quotient values
(CO2 output/O2 input). E and F, AEC values
during drying () and in dry tissues before imbibition (). Data
were plotted as a function of WC (A, C, and E) and viscosity (B, D, and
F). Viscosity values were taken from the equation fitting the viscosity
versus WC plot of Figure 6A. DW, Dry weight.
|
|
When the samples were dried, respiratory quotient values
(CO2 release/O2
consumption) decreased from 0.9 to 0.6. It is unlikely that such a low
value is a sign of breakdown of storage lipid. Since the amount of oil
present in cowpea cotyledons is less than 2% of the dry weight (data
not shown), storage lipid is not a likely candidate for respirable
substrate during drying. Thus, the low respiratory quotient at the
onset of drying may be due to the generation of ROS. At WC between 0.8 and 0.27 g/g, the respiratory quotient increased to 1.2 (Fig. 8C). It
is noteworthy that a WC of 0.8 g/g corresponds to the WC at which
O2 diffusion starts to decrease exponentially.
The response of the respiratory quotient to drying showed a different
pattern when plotted as a function of viscosity (Fig. 8D). With a
2-fold increase in viscosity, respiratory quotient values first
decreased to 0.6 and then increased to 1.2 at a viscosity of 0.8 poise
(Fig. 8D). They did not change with a further increase in viscosity.
This suggests that during drying the tissues did not ferment, despite
the reduction of the O2-diffusion coefficient. An
increased breakdown of organic acids (e.g. citrate, malate) is a likely
possibility for the increase in respiratory quotient values.
During the loss of water from the hydrated state to 0.5 g/g WC, the AEC
remained constant at approximately 0.8 (Fig. 8E). At WC of less than
0.5 g/g, the AEC decreased to 0.4. Similar observations have been made
for other plant systems in which the energy metabolism was limited by a
total or partial deprivation of O2 (Pradet and
Raymond, 1983; Raymond et al., 1985). In both dry seeds and pollen
before imbibition, AEC was approximately 0.2 (Fig. 8E), as previously
observed in seeds (Raymond et al., 1985). When plotted against
viscosity, the AEC declined almost linearly (Fig. 8F).
| |
DISCUSSION |
Desiccation Induces the Reduction of Mitochondrial Cyts in
Anhydrobiotes
The relations between redox status and metabolic functions of Cyts
have been extensively characterized in seeds by spectrophotometry of
isolated mitochondria (Lance and Bonner, 1968; Wilson and Bonner, 1971;
Ikuma, 1972; Sowa et al., 1993) and isolated microsomal membranes
(Hendry et al., 1981; Lüthje et al., 1997). Extraction of
mitochondria from dry tissues often leads to loss of structural and
functional integrity (Hoekstra and van Roekel, 1983) or, at best, gives
a low yield of intact material (Attucci et al., 1991). Moreover, the
necessary use of aqueous solvents during isolation from drying tissues
implies that the changes in redox states of the Cyts observed in vitro
may not reflect the actual in vivo redox state before the extraction.
Therefore, we recorded spectra of intact or powdered material during
drying. Our noninvasive analysis of the -band of Cyts c
and aaa3 in pollen and
aaa3 in cowpea cotyledons
shows clearly that drying at a WC of less than 0.6 g/g induces a steady
increase in the extent of reduction of mitochondrial Cyts. Furthermore,
these Cyts remained reduced at values less than 0.2 g/g. The glassy
state is formed at room temperature when the WC is less than 0.12 g/g
in cowpea cotyledons (data not shown) and 0.08 g/g in T. latifolia pollen (Buitink et al., 1996, 1998). Thus, both Cyt
c and COX are fully reduced before a glassy state is formed
during drying. In intact organisms, the only conditions known to induce
total reduction in the terminal portion of the Cyts are anoxia (Lance
and Bonner, 1968; Kariman et al., 1983; Letan et al., 1993) and
treatment with inhibitors that block the intramolecular transfer of
electrons to the O2-binding site of the COX
(Lance and Bonner, 1968). Such conditions also reduced COX in our
material before drying (Figs. 1 and 2). Furthermore, our results
confirm earlier suggestions that in dry pollen and seeds mitochondria
are intact and the electron-transport chain is functional (Wilson and
Bonner, 1971; Hoekstra and Van Roekel, 1983; Leopold and Vertucci,
1989; Attuci et al., 1991).
Impeded O2 Diffusion Is a Likely Factor Responsible for
the Desiccation-Induced Reduction of Mitochondrial Cyts
Two observations point to a critical role for the
O2 availability in the reduction of COX when the
tissues are dried at a WC less than 0.6 g/g: (a) during drying of
cowpea cotyledons and T. latifolia pollen, the exponential
increase in viscosity and the corresponding decrease in the
O2-diffusion coefficient (Fig. 6, A and B)
coincided with the onset of the COX reduction; (b) the AEC decreased to
values similar to those found in germinating seeds under anoxia (Fig.
8E; Pradet and Raymond, 1983; Al-Ani et al., 1985). Thus, we suggest
that the diffusion of O2 becomes strongly impeded
by the increase in solvent viscosity, which may restrict the
O2 supply to the mitochondria and reduce COX.
However, the observation that increasing [O2]
during drying did not have an effect on the onset of COX reduction
(Fig. 4A) challenges this suggestion. Two arguments are put forward to
address this question. First, the decrease in diffusion rates during
drying of our material could be so large that increasing the
O2 gradient from outside to inside of the tissues
will not significantly increase the O2 flux to
the mitochondria and O2 availability to the COX.
Second, previous studies (Al-Ani et al., 1985; Raymond et al., 1985)
have shown that the respiratory metabolism in hydrated seeds is
sustained at O2 partial pressures as low as 0.2 kPa. Furthermore, at 21 kPa, COX is saturated with substrates, judging
from the low Km for
O2 (Kariman et al., 1983; Letan et al.,
1993). These observations show that at 21 kPa and greater there is
sufficient O2 to keep COX fully oxidized.
Therefore, drying hydrated seeds or pollen at an
O2 partial pressure greater than 21 kPa is not
expected to influence COX activity.
One may argue that the in vivo reduction of Cyts may be attributed to
mechanisms other than O2 deprivation. For
instance, it has been shown that the reduction of
O2 by COX is inhibited in conditions that prevent
the solvent entry and expulsion between Cyt a and Cyt
a3 during the COX catalytic cycle
(Escamilla et al., 1989; Kornblatt and Kornblatt, 1992). Such water
movements can be inhibited by decreasing the water activity of the
medium (Kornblatt and Hoa, 1990). We showed that manipulating the water activity of cowpea cotyledons during drying did not affect the onset of
COX reduction accordingly (Fig. 7). Therefore, the dependence of COX
reduction on impeded water movements within our material is unlikely.
Nevertheless, in the dry state, the low water activity may well prevent
COX from being reoxidized by O2, which should still diffuse, albeit slowly, through the glassy cytoplasm. Indeed, we
observed that COX in dried cowpea cotyledons remained reduced for at
least 6 months of open storage.
Another mechanism that may reduce COX during drying involves the
presence of electron donors in the mitochondrial matrix. As water is
removed from the cell, the concentrations of various reducing
substances (e.g. ascorbate) is increased. Consequently, the likelihood
that these substances may act as electron donors to Cyt c
and COX increases during drying. However, two sets of data do not
support this hypothesis. First, we found that the extent of reduction
of the ubiquinone-10 pool in cowpea cotyledons was 50% to 67% in both
hydrated and dried conditions (O. Leprince and A.M. Wagner,
unpublished results). This finding indicates that the electron
transport between the ubiquinone pool and COX is maintained throughout
drying. Furthermore, in hydrated seeds of several species, the electron
transport in mitochondria occurs at a WC greater than 0.3 g/g (Leopold
and Vertucci, 1989). Second, we have demonstrated that the microsomal
Cyts in cowpea cotyledons are progressively oxidized during drying.
This behavior is in marked contrast to that of mitochondrial Cyts,
which are being reduced during drying (Figs. 1-3). If the
concentration of potential electron donors to the Cyts increased during
drying in all cellular compartments, the Cyts of both microsomal and
mitochondrial origin would be reduced.
A Functional Role for Cytoplasmic Viscosity in Depressing
Metabolism and Altering Cyt Redox States during Drying
Our study showed that viscosity can be regarded as a new, valuable
parameter to characterize the effects of drying on metabolism. Characteristically, the loss of water in anhydrobiotes is associated with a decrease in respiration rates until a WC of about 0.3 g/g, below
which no gas exchange can be detected (Fig. 8A; Leopold and Vertucci,
1989; Vertucci and Farrant, 1995). The precise causes that trigger the
metabolic arrest during drying are not known. Electron
microscopy studies have suggested that the progressive shutdown
of metabolism is associated with a general dedifferentiation of
organelles (Leprince et al., 1990; for review, see Vertucci and
Farrant, 1995). In cowpea we observed a biphasic pattern in the
relation between viscosity and respiration. At the onset of drying the
initial increase in viscosity to 0.27 poise was associated with a
decrease in gas-exchange rates to 50% of their initial values before
drying. In contrast, the plot of respiration rates versus WC (Fig. 8A)
shows a linear decline in respiration during drying. However, the
initial increase in viscosity did not impede the electron transport
within the mitochondrial chains, nor did it dramatically affect the AEC
values. Thus, despite the loss of water, the system is still able to
control the supply and demand of energy. We suggest that changes in
medium viscosity and diffusion processes during drying may play a role
in the control of energy supply and demand. Indeed, several studies of
mitochondria showed that both the rates of electron transport and the
mobility of the redox components within the lipid bilayer are more
sensitive to changes in the medium bulk viscosity than in membrane
microviscosity (Fato et al., 1993; Chazotte, 1994; Esmann et al.,
1994).
The suggestions that there is a control of energy supply and demand
during drying and that the reduction of COX during drying likely
results from a desiccation-induced decrease in O2
availability are further supported by studies of metabolism depression
in animal anhydrobiotes. Indeed, survival in the quiescent state of
lower organisms is associated with tolerance to acute anoxia at high WC
(Crowe et al., 1992; Hand and Hardewig, 1996). In Artemia
cysts, the depression of metabolism (e.g. protein synthesis) is
controlled by O2 limitation and acidic pH
(Hofmann and Hand, 1994). In these animal systems, anoxia induces a
coordinated suppression of both catabolism and anabolism (Hand and
Hardewig, 1996). Whether the O2 availability
directly controls the depression of metabolism in drying seeds and
pollen remains to be clarified.
Implications for Desiccation Tolerance
Results of this study suggest that a regulated increase in
viscosity may be regarded as a mechanism that controls the depression of metabolism during drying. An increase in viscosity may induce anoxia
in dehydrating tissues before the cytoplasm enters a glassy state. It
remains to be determined whether this strategy is beneficial or
detrimental for desiccation tolerance. Anoxic conditions during dehydration would strongly diminish the danger of overproducing ROS.
The formation of ROS is a random process that is strictly dependent on
molecular collisions with O2 (Cadenas, 1989;
Skulachev, 1996). Thus, entering into anoxia can be seen as a mechanism
to escape from oxidative damage as suggested by Skulachev (1996). A
rapid increase in viscosity during the onset of drying can be seen as a
mechanism to slow metabolism and decrease O2
concentration, thereby decreasing the chances of generating ROS. It
follows that the onset of drying is the critical phase for ROS
formation, when the electron transport is still active and
O2 is present at high concentrations. However, it
can also be argued that a rapid increase in viscosity during early
drying is detrimental because the induction of anoxia may increase the
concentrations of metabolic end products, such as acetaldehyde and
protons, to toxic levels. To clarify this ambivalence, the changes in
viscosity during drying must be compared in desiccation-tolerant and
-intolerant organisms. Furthermore, techniques that enable us to
measure noninvasively [O2] and ROS during
drying need to be developed.
| |
FOOTNOTES |
1
This work was supported by a grant from the
Wageningen Agricultural University and the Laboratory of Plant
Physiology to O.L.
*
Corresponding author; e-mail
olivier.leprince{at}guest.pf.wau.nl; fax 31-317-484740.
Received April 6, 1998;
accepted August 20, 1998.
| |
ABBREVIATIONS |
Abbreviations:
AEC, adenylate energy charge.
COX, Cyt
c oxidase.
CP, 3-carboxy-proxyl.
ESR, electron spin
resonance.
g/g, g H2O/g dry weight.
ROS, reactive oxygen
species.
Tg, glass transition temperature.
R, rotational
correlation time.
WC, water content(s).
| |
ACKNOWLEDGMENTS |
The authors wish to thank J. Buitink for critically reading the
manuscript and Dr. A.M. Wagner (Free University of Amsterdam, The
Netherlands) for the measurements of ubiquinone pools.
| |
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Abstract
Introduction