Plant Physiol. (1998) 118: 531-541
Influence of Water Content and Temperature on Molecular Mobility
and Intracellular Glasses in Seeds and Pollen1
Julia Buitink*,
Mireille M.A.E. Claessens,
Marcus A. Hemminga, and
Folkert A. Hoekstra
Wageningen Agricultural University, Laboratory of Plant Physiology,
Arboretumlaan 4, 6703 BD Wageningen, The Netherlands (J.B., F.A.H.,
M.M.A.E.C.); and Laboratory of Molecular Physics, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands (J.B., M.M.A.E.C., M.A.H.)
 |
ABSTRACT |
Although the occurrence of
intracellular glasses in seeds and pollen has been established,
physical properties such as rotational correlation times and viscosity
have not been studied extensively. Using electron paramagnetic
resonance spectroscopy, we examined changes in the molecular mobility
of the hydrophilic nitroxide spin probe 3-carboxy-proxyl during melting
of intracellular glasses in axes of pea (Pisum sativum
L.) seeds and cattail (Typha latifolia L.) pollen. The
rotational correlation time of the spin probe in intracellular glasses
of both organisms was approximately 10
3 s. Using the
distance between the outer extrema of the electron paramagnetic
resonance spectrum (2Azz) as a measure of molecular mobility, we found a sharp increase in mobility at a definite temperature during heating. This temperature increased with decreasing water content of the samples. Differential scanning calorimetry data on
these samples indicated that this sharp increase corresponded to
melting of the glassy matrix. Molecular mobility was found to be
inversely correlated with storage stability. With decreasing water
content, the molecular mobility reached a minimum, and increased again
at very low water content. Minimum mobility and maximum storage
stability occurred at a similar water content. This correlation suggests that storage stability might be at least partially controlled by molecular mobility. At low temperatures, when storage longevity cannot be determined on a realistic time scale, 2Azz
measurements can provide an estimate of the optimum storage conditions.
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INTRODUCTION |
Deterioration of seeds and pollen during storage involves many
physical and chemical changes, such as disrupted intracellular integrity, decreased activities of enzymes, lipid peroxidation and
deesterification, and Maillard reactions (Priestley, 1986
; Wilson and
McDonald, 1986; Wettlaufer and Leopold, 1991; Van Bilsen and Hoekstra,
1993; Van Bilsen et al., 1994). Since the formation of glasses in
dehydrating biological tissues has been established, this physical
phenomenon has been put forward as a prominent factor in the control of
deterioration rates during storage (Burke, 1986; Williams and Leopold,
1989; Leopold et al., 1994; Leprince and Walters-Vertucci, 1995;
Buitink et al., 1996). A glass is a thermodynamically unstable
solid-state with an extremely high viscosity (Franks et al., 1991). Its
formation is promoted by low water content of tissues and by low
temperatures. Both factors are also known to extend the longevity of
seeds and pollen (Roberts, 1972; Roberts and Ellis, 1989; Vertucci and
Roos, 1993; Buitink et al., 1998), and improved storage stability was
observed when glasses were present (Sun and Leopold, 1994; Sun, 1997;
Buitink et al., 1998). It is assumed that the high viscosity of
intracellular glasses decreases molecular mobility and impedes
diffusion within the cytoplasm, thus slowing down deleterious reactions
and changes in structure and chemical composition during aging (Sun and
Leopold, 1993; Sun, 1997). However, the molecular mobility and
viscosity in biological glasses has received little
attention.
Molecular mobility has been studied using EPR spectroscopy by labeling
polymers and food materials with a suitable, stable spin probe (Steffen
et al., 1992; Blackburn et al., 1996; Dzuba, 1996; Hemminga and Van den
Dries, 1998). From the EPR spectra of the spin probe,
R can be assessed (Kumler and Boyer, 1976; Kovarskii et al., 1978; Spielberg and Gelerinter, 1982; Ohta and Kuwata, 1985; Roozen and Hemminga, 1990; Roozen et al., 1991; Dzuba et
al., 1993). Whereas R values of 1012 to
109 s can be calculated from conventional EPR spectra
(Knowles et al., 1976), ST-EPR spectroscopy further expands this
range to very slow (106 - 103 s;
Hemminga, 1983) and ultra slow (103 - 102 s; Van den Dries et al., 1998) molecular motions.
ST-EPR spectroscopy has been successfully applied to determine
R values of spin probes in sugar glasses (Roozen et al.,
1991; Van den Dries et al., 1998) and organic liquids at low
temperatures (Ito, 1983). In glassy Suc-water and malto-oligosaccharide
mixtures, R decreases by several orders of magnitude
upon approaching Tg (Roozen et al., 1991).
The present paper is aimed at gaining insight into changes in the
molecular mobility that accompany glass formation in anhydrobiotes. We
used EPR and ST-EPR spectroscopy to characterize the molecular motion
of CP, the polar nitroxide spin probe that we incorporated into axes of
pea (Pisum sativum L.) seeds and cattail (Typha
latifolia L.) pollen. We show that the distance between
the outer extrema in powder spectra produced by conventional EPR
spectroscopy can be used to detect changes in molecular motion, and
that ST-EPR spectroscopy allows an estimation of
R. We discuss the possible relationship
between glasses, molecular mobility, and storage stability in these
organisms.
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MATERIALS AND METHODS |
Plant Material and Treatments
Mature male inflorescences of cattail (Typha latifolia
L.) were collected from field populations near Wageningen, The
Netherlands, in 1996, and allowed to shed their pollen in the
laboratory. Pollen (94% germination) was cleaned by sieving through a
fine copper mesh, dried in dry air to 0.05 to 0.08 g water
g1 dry weight, and stored at 
20°C until
use. Pea (Pisum sativum cv Karina) seeds (99% germination)
were obtained from Nunhems Zaden (Haelen, The Netherlands) and stored
at 5°C until use.
The polar nitroxide spin probe CP (Sigma) was used for spin labeling of
pea seeds and cattail pollen. Pollen (3 g) was prehydrated in water
vapor for 16 h at 5°C to about 0.7 g water
g1 dry weight, and then mixed at 25°C with 6 mL of liquid germination medium containing 2.5 mM CP. The
germination medium 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. After a few
minutes, an additional 20 mL of the germination medium was added, and
the pollen was recovered by filtration. The pollen was then mixed with
20 mL of a solution of 1 mM CP and 120 mM of
the broadening agent potassium ferricyanide. Ferricyanide broadens
spin-probe signals in the solution surrounding the cells to
invisibility (Golovina and Tikhonov, 1994; Golovina et al., 1997).
After 5 more min, the pollen was recovered by filtration, spread out in
a large Petri dish, and rapidly dried in a flow of dry air (3% RH) in
a drying box to a water content of less than 0.05 g water
g1 dry weight.
The pollen retained high viability after labeling and drying. To
determine the R at high water content
(1.5-0.5 g water g1 dry weight), pollen
samples were taken regularly during drying for EPR measurements. After
drying, the pollen was stored over various saturated salt solutions or
P2O5 (Winston and Bates,
1960) at 25°C for at least 3 d to obtain the various water
contents.
Pea seeds were rolled in germination paper and soaked in tap water
overnight at 15°C, and then axes were isolated. Subsequently, the
axes were incubated in a solution containing 1 mM CP and
200 mM ferricyanide at room temperature. After 45 min, the
axes were rinsed with distilled water and dried at 35% RH for 3 d. To determine the R at high water content
(1.5-0.5 g water g1 dry weight), pea axes were
regularly sampled during drying for EPR measurements. After drying, the
axes were stored over the saturated salt solutions or
P2O5 at 25°C to obtain
the various water contents.
With every sample taken for EPR measurements, a sample treated
similarly was taken for determination of water content. Water contents
were analyzed by weighing the samples before and after heating at
96°C for 36 to 48 h (Buitink et al., 1996) and calculating the
water loss on a dry-weight basis.
EPR and ST-EPR Measurements
EPR spectra were recorded with an X-band EPR spectrometer (model
300E, Bruker Analytik, Rheinstetten, Germany). Microwave power was kept
low (200 µW or 2 mW) to avoid saturation. Modulation amplitude was
0.4 G for pea axes and 1 G for cattail pollen.
Samples with various water contents were loaded into a 3-mm-diameter
EPR capillary. For each measurement, the capillary was filled for a
length of 5 cm with pollen or with two isolated pea axes. To prevent
water loss during the measurements, the capillaries were sealed at both
sides. Temperature was controlled using a controller with liquid
nitrogen vapor as the coolant. Samples were rapidly cooled to 150°C
and allowed to equilibrate for 30 min, after which scans were recorded
at 10°C increments with equilibration for 5 min after each increment.
Conventional EPR spectroscopy can detect changes in R of
spin probes ranging from 1012 to 109 s,
which corresponds to the lifetime of the probe in a given orientation. In this motional range, the EPR spectrum of nitroxides consists of three lines (Fig. 1, top
spectrum), and R can be calculated according to the
method of Knowles et al. (1976):
![&tgr;<SUB>R</SUB> = 6.5 × 10<SUP>−10</SUP>&Dgr;B<SUB><UP>0</UP></SUB><UP>[(</UP>h<SUB><UP>0</UP></SUB><UP> /</UP>h<SUB><UP>−1</UP></SUB><UP>)</UP><SUP><UP>½</UP></SUP><UP> − 1]</UP>](/content/vol118/issue2/fulltext/531/img001.gif)
|
(1)
|
where h1 and
h0 are the heights of the high-field and
central lines in the EPR spectra, respectively, and
B0 is the line width of the central line
in Gauss. The rotational motion of the spin probe was assumed to be
isotropic. The R of partially hydrated (above
approximately 0.25 g water g1 dry weight)
pollen and pea axes was determined using Equation 1.
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| Figure 1.
EPR spectra of CP in cattail pollen at various
water contents (g/g, g water g1 dry weight). Spectra were
recorded at room temperature. The h+1,
h0, and h1 peaks are
shown in the top spectrum. The distance between the two outer extrema,
2Azz, is indicated in the bottom spectrum.
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|
The characteristic R for spin probes in
organic glasses near and above Tg is
approximately 105 s or higher (Dzuba et al.,
1984; Roozen et al., 1991; Van den Dries et al., 1998). These
R values cannot be determined using Equation 1, because the shapes of the lines in the spectra change due to the
appearance of a powder spectrum (Fig. 1, below 0.25 g water
g1 dry weight). In these powder spectra the distance
between the two outer extrema (2Azz) is
temperature dependent for a number of glass-forming substances at and
above their Tg (Kumler and Boyer, 1976;
Kovarskii et al., 1978; Spielberg and Gelerinter, 1982; Ohta and
Kuwata, 1985; Dzuba et al., 1993) (Fig. 1). We made use of this
parameter to obtain a qualitative measurement of the molecular mobility
of CP.
From the R one can derive the viscosity of the
matrix in which the spin probe is rotating, according to the modified
Stokes-Einstein equation (Roozen et al., 1991):
|
(2)
|
where 
is the solvent viscosity, kb
is Boltzmann's constant, V is the volume of the rotating
molecule, T is the absolute temperature,
0 is the zero viscosity
R, and k is a dimensionless slip
parameter.
ST-EPR spectroscopy was used in the motional region for
R > 107 s. This
method is based on the diffusion and recovery of saturation between
different portions of the powder spectrum in competition with field
modulation (Hemminga, 1983). For ST-EPR spectroscopic measurements, the
second harmonic quadrature absorption signal was detected under the
following conditions: a field-modulation amplitude of 5 G, microwave
power of 100 mW, and field-modulation frequency of 50 kHz (Hemminga et
al., 1984). The phase was set with the self-null method (Thomas et al.,
1976).
In ST-EPR spectroscopy, R values are usually
obtained empirically using reference material with known viscosity.
Here, spectra of CP in anhydrous glycerol were used to construct a
calibration curve according to the method of Van den Dries et al.
(1998). Because the viscosity for anhydrous glycerol is known over a
broad temperature range, R of CP in glycerol
can be obtained from Equation 2. Spectra of CP in anhydrous glycerol
were recorded every 3°C, and the values of the line-shape parameters
L"/L
and C/C (explained in Fig. 5) were calculated for each
temperature (data not shown). From the curves representing the
line-shape parameters of CP in glycerol against
R, the R values of CP
in the axes and pollen were obtained by interpolation of the
corresponding line-shape parameters.
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| Figure 5.
ST-EPR spectra of CP in pea axes at 0.08 g
water g1 dry weight recorded at various temperatures.
Scans were recorded at 100 mW, a modulation frequency of 50 kHz, and a
modulation amplitude of 5 G.
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Differential Scanning Calorimetry
Pollen and pea axes with different water contents were
hermetically sealed in aluminum pans for differential scanning
calorimetry. Second-order transitions of the samples were determined
using a differential scanning calorimeter (Pyris 1, Perkin-Elmer)
calibrated for temperature with indium (156.6°C) and methylene
chloride (95°C) standards and for energy with indium (28.54 J
g1). Baselines were determined using an empty
pan, and all thermograms were baseline corrected. Scans were taken from
100°C to 120°C at a rate of 10°C min1.
The Tg values were determined by the onset
and midpoint of the temperature range over which the change in specific
heat occurred. All analyses were performed with Perkin-Elmer software.
| |
RESULTS |
Molecular Motion in Pollen and Seeds
Figure 1 shows representative EPR spectra of CP in cattail pollen
at different water contents recorded at room temperature. When the
water content was decreased from 0.53 to 0.25 g water g1 dry weight (top two spectra), the relative
amplitudes of the outer spectral lines decreased and the width of the
central peak increased. Using Equation 1, the
R of CP in hydrated pollen and pea axes
(approximately 1.2 g water g1 dry weight)
was calculated to be approximately 1011 s (data
not shown). In the partially dehydrated state (0.53 g water
g1 dry weight), R of
CP in cattail pollen was calculated as 1.6 × 1010 s (Fig. 1, top spectrum). At 0.25 g
water g1 dry weight (Fig. 1, second spectrum
from the top), R of CP in pollen was 7.9 × 1010 s. The R of CP
in pea axes at 0.5 g water g1 dry weight
was 3.6 × 1010 s (data not shown). At a water content
below 0.25 g water g1 dry weight, a powder
spectrum (characterized by the two broad peaks at the extremes)
overlapped the mobile spectrum (three sharp lines separated by a
distance of 15 G). The bottom spectrum shown in Figure 1 has the
characteristic shape of a powder spectrum, indicative of slow molecular
mobility of the spin probe with R >108 s.
With the appearance of this powder spectrum below 0.25 g water g1 dry weight, the R
of CP cannot be calculated using Equation 1, because the line shapes
are distorted. The distance between the two broad peaks at the extremes
is referred to as 2Azz (Fig. 1, bottom spectrum).
Similar spectra were obtained for pea axes in relation to water
content (data not shown).
Figure 2 shows EPR spectra of CP in dry
pea axes and cattail pollen (both having 0.07 g water
g1 dry weight) at a range of temperatures. At
least two overlapping spectra contributed to the total spectrum
observed: a powder spectrum at all temperatures and a mobile spectrum
above 20°C, the contribution of which increased with increasing
temperature. Note that the contribution of the mobile component to the
total spectrum is considerably larger for pollen at 70°C than for pea
axes at 90°C.
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| Figure 2.
EPR spectra of CP in pea axes and cattail pollen
recorded at various temperatures. Both contained approximately
0.07 g water g1 dry weight.
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At 150°C, we assumed that the motion of the probe was completely
immobilized and therefore we took the corresponding
2Azz values as the maximum values. At this low
temperature, the value of 2Azz gives information
about the polarity of the spin probe's environment in the tissue
(Knowles et al., 1976). In pea axes the maximum
2Azz decreased with decreasing water content from 74 to 70 G, whereas in cattail pollen, it changed from 72.5 to 71.5 G (Fig. 3).
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| Figure 3.
Change in the distance between the outer extrema
of the EPR spectra (2Azz) of CP in pea axes ( ) and
cattail pollen ( ) at 150°C as a function of water content. dw,
Dry weight.
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|
Because we observed a powder spectrum of CP in pea axes and cattail
pollen at low water contents (<0.2 g water g1
dry weight), R cannot be directly calculated
from the EPR spectra using Equation 1. However, the change in
2Azz with temperature can be used as an estimate
of molecular motion (Van et al., 1974; Dzuba, 1996). A decrease in
2Azz is indicative of an increase in molecular
mobility. Figure 4 shows these changes in
2Azz with temperature. When the temperature of
pea axes and cattail pollen increased, the 2Azz
slowly decreased, then abruptly decreased above a definite temperature.
With increasing water contents, this abrupt decrease in
2Azz, denoting an abrupt increase in molecular mobility, commenced at lower temperatures. At a water content of 0.002 g water g1 dry weight in pea axes the decrease
in 2Azz was less clear.
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| Figure 4.
Comparison of the distance between the outer
extrema of the EPR spectra (2Azz) of CP in pea axes and
cattail pollen at different water contents against temperature.
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We used ST-EPR to obtain an indication of the magnitude of change in
R (reflecting the lifetime of the probe in a
given orientation) that corresponds to the change in
2Azz with temperature. This technique is based on
recording spectra under saturation conditions, which yields line shapes
that are sensitive to R >107 s (Hyde and
Dalton, 1979). The ST-EPR spectra can be well characterized by
independent line-shape parameters such as the line-height ratios L"/L
and C/C (Fig. 5). These ratios are
dependent on the R of the spin probe. To
determine the R of CP corresponding to a
certain line-height ratio, anhydrous glycerol was used as a reference
solvent (this solvent was previously used as a reference in sugar
glasses in Roozen et al. [1991] and Van den Dries et al. [1998]).
Although the composition of the cytoplasm in seeds and pollen is not
comparable to glycerol, at present the use of glycerol will give the
best approximation of the relationship between the line-height ratios
and R. Extrapolation of the
R of CP in seeds and pollen to the
corresponding viscosity is not valid, because the Stokes-Einstein law
fails at temperatures below approximately 1.3 Tg
(Liu and Oppenheim, 1996).
As the viscosity for glycerol is known over a broad temperature range,
the corresponding R could be calculated
according to Equation 2. Subsequently, the two line-height ratios were
obtained from ST-EPR scans of anhydrous glycerol for a range of
temperatures and plotted against the corresponding
R. Those calibration curves were used to
obtain R from line-height ratios calculated
from ST-EPR scans of CP in pea axes (Fig. 5). The
R values for pea axes (0.08 g water
g1 dry weight) calculated according to both
line-height ratios and plotted against the temperature are shown in
Figure 6. R
values of CP in pea axes were in the range of
102 to 106 s. The
R values of CP in the pollen were in the same
range (data not shown). From Figure 6 it can be seen that there is a
difference in the R derived from the L"/L
ratio and the C/C ratio. Furthermore, it can be observed that the
R obtained from the C/C ratio shows an
increase in mobility with increasing temperature comparable to the
2Azz curves (Fig. 4).
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| Figure 6.
R of CP in pea axes at 0.08 g
water g1 dry weight as a function of temperature. The
R values were obtained by comparing the L"/L ratio and
the C/C ratio with those of CP in anhydrous glycerol.
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One can speculate on what causes the difference in
R obtained from both line-height ratios. The
R obtained from the L"/L ratio probably
reflects overall isotropic rotational motion, whereas the
R derived from C/C also reflects some
anisotropic motion. According to Dzuba (1996), the change in
2Azz can be described by a librational model.
Unlike rotational motion, in which the spin probe rotates entirely
randomly, librational motion assumes that the spin probe rotates within
a cone given by a small angle (
). Therefore, it could be that the
anisotropic motion seen in the R obtained from
the C/C ratio arises from librational motion. Whether the change in
2Azz with temperature is due to librational motion is currently under investigation. As ST-EPR is a laborious technique, more investigations are needed to make full use of this
technique as a method to determine molecular mobility. Meanwhile, we
will consider the measurements of 2Azz as an
estimate of molecular motion.
Molecular Motion and Intracellular Glasses
As shown in Figure 4, a sharp increase in the molecular motion of
CP was noticeable when the temperature of the sample was increased.
Because the temperature corresponding to this sharp increase depended
on water content, an attempt was made to explain this behavior
according to the glass theory. From the plot of 2Azz against temperature we derived two
characteristic temperature points (see inset in Fig.
7 for details): at the intercept
(midpoint Tg) and at the point of deviation
from a straight line (onset Tg). Figure 7
shows plots of the temperature at which the breaks occurred compared
with the water content in pea axes. For cattail pollen a similar type
of plot was obtained (data not shown). At low water contents (<0.002 g
water g1 dry weight) the characteristic
temperature points were difficult to determine exactly, as the legs
below and above the glass transition did not show a sharp drop in
2Azz. The curves in Figure 7 are remarkably
similar to state diagrams of intracellular glasses in seeds (Leopold et
al., 1994; Leprince and Walters-Vertucci, 1995) and pollen (Buitink
et al., 1996).
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| Figure 7.
Effects of water content on the onset and midpoint
of abrupt changes in the distance between the outer extrema of EPR
spectra of CP in pea axes. The inset shows how the onset and midpoint
temperatures were determined. Onset Tg was
taken as the point of deviation from a straight line, and midpoint
Tg as the intercept of the two lines (the
line drawn through the data points is meant as an aid to
visualization). dw, Dry weight.
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To ascertain whether the sharp changes in molecular motion were due to
the melting of intracellular glasses, the onset
Tg was measured by differential scanning
calorimetry and compared with the onset Tg
as determined by EPR (Fig. 8). Both
curves closely matched one another, the EPR data being slightly lower
than the differential scanning calorimetry data. The
Tg measured by differential scanning
calorimetry has been found to occur above the
Tg measured at a molecular level
(Kalichevsky et al., 1992). The midpoint of
Tg from EPR measurements was situated
slightly above the midpoint of the Tg
measured by differential scanning calorimetry (data not shown). The
Tg for dry pollen (62°C) was lower than
that for dry pea axes (92°C), which may be related to the high level
of oligosaccharides in the latter (Amuti and Pollard, 1977;
Saleki-Gerhardt and Zografi, 1994). The constant value of
Tg measured by differential scanning
calorimetry when the last 2% of water was removed may indicate that
the first small amount of water does not contribute to plasticization
of the glass. It is possible that this water is not present in the
glass but, rather, is located in some other part of the tissue (e.g.
cell walls).
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| Figure 8.
The relationship between
Tg and water content in pea axes and cattail
pollen. The open symbols show the Tg
determined as the onset of the second-order-like transitions measured
by differential scanning calorimetry during heating with a scanning
rate of 10°C min1. The closed symbols show the onset of
the sudden decrease in 2Azz (derived from Fig. 4). dw, Dry
weight.
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The change in molecular mobility during melting of intracellular
glasses can be measured as a function of temperature or water content,
the relationship being reflected by the state diagram (Fig. 8). To
determine the change in molecular mobility as a function of water
content instead of temperature, it is necessary to correct for the
polarity change of the environment in which CP is present for each
water content (Fig. 3). Therefore, using curves similar to those shown
in Figure 4, the mobility at a certain water content was expressed as
the difference between the maximum 2Azz (at
150°C, where the spin probe is assumed to be immobilized) and the
2Azz measured at the desired temperature. We
refer to this parameter as Azz. Thus, an
increase in Azz represents a relative increase in
molecular mobility compared with the completely immobilized situation at 150°C (i.e. the more the value departs from zero, the higher the molecular mobility). Figures
9 and
10 show
the dependence of the molecular mobility (Azz)
on water content in pea axes and pollen, respectively. Between
approximately 0.2 and 0.1 g water g1
dry weight, the mobility decreased with decreasing water content at all
temperatures analyzed. When the tissues reached approximately 0.1 to
0.05 g water g1 dry weight, the
Azz reached a constant value, indicating that the mobility had reached a minimum. When water contents were decreased further, below approximately 0.05 g water
g1 dry weight, mobility increased again for
pollen (Fig. 10). For pea axes, the mobility slightly increased again
or reached a constant level at very low water content (Fig. 9). The
water content corresponding to the minimum mobility (lowest
Azz) shifted to higher values with decreasing
temperatures.
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| Figure 9.
Change in Azz, calculated as the
difference between the maximum 2Azz (150°C) and
2Azz at the indicated temperatures, with water content for
spectra of CP in pea axes. Curves were fitted with a third-order
polynomial. dw, Dry weight.
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| Figure 10.
Change in Azz, calculated as the
difference between the maximum 2Azz (at 150°C) and
2Azz at the indicated temperatures, with water content
spectra of CP in cattail pollen. Curves were fitted with a third-order
polynomial. dw, Dry weight.
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| Figure 11.
Correlation between water content (wc) of optimum
storage longevity for pea seeds and cattail pollen and the water
content of minimum mobility (lowest value of Azz). Water
contents for optimum storage longevity for cattail were determined by
Buitink et al. (1998). The water content of optimum storage longevity
was found to increase with decreasing temperatures of storage (45°C,
35°C, 25°C, 15°C, and 5°C). Water contents of optimum storage
longevity for pea seeds stored in the light were derived from Vertucci
et al. (1994). With decreasing storage temperature (45°C, 35°C,
25°C, 15°C, 5°C, and 5°C), the water contents of optimum
storage increased. The water content at which minimum mobility was
observed was determined from the minima of the third-order polynomial
equations derived from curves similar to Figures 9 and 10 at
temperatures comparable to the storage temperatures (45°C, 35°C,
25°C, 15°C, 5°C, and 5°C). dw, Dry weight.
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| |
DISCUSSION |
Behavior of Nitroxide Spin Probes in Dehydrating
Organisms
To characterize molecular motion using EPR, a spin probe must be
introduced into the material. Depending on the polarity of the spin
probe, it will partition into the apolar oil phase, polar aqueous
cytoplasm, or both. Several amphipathic spin probes, such as TEMPO and
4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy, completely partition into
the lipid phase during drying of cattail pollen (Hoekstra and Golovina,
1998). We found that the more polar
4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy partially partitioned into
the lipid phase of cattail pollen and pea axes during drying (data not
shown). Because we were interested in the molecular mobility in the
cytoplasm rather than in the lipid phase, we avoided the use of these
spin labels and instead used the polar nitroxide spin probe CP.
There are several reasons why we believe that CP was not present in the
lipid phase in the dry organisms. Upon melting of the oil, which
occurred in cattail pollen at approximately 20°C and in pea axes at
40°C and 20°C, as determined by differential scanning
calorimetry (data not shown), we never observed an abrupt appearance of a mobile EPR spectrum. Furthermore, during drying of
pollen at 25°C, when the oil is liquid (Fig. 1), we did not observe the typical lipid signal with a hyperfine splitting constant of
14 G (Knowles et al., 1976). The resolution of the spectrum was high
enough to distinguish a possible oil contribution from the aqueous
contribution. When samples of elevated water content were heated above
80°C, two peaks were observed in the high-field part of the spectrum,
one attributable to the water signal and the other to the lipid signal
(data not shown).
During drying the hyperfine splitting constant of CP decreased from
16.5 to 15.5 G (Fig. 1). A decrease in hyperfine splitting constant is
associated with a decrease in the polarity of the spin probe
environment (Knowles et al., 1976). During drying the apparent polarity
of the cytoplasmic environment decreased, possibly because of the
decrease of the dielectric constant of the environment associated
with the loss of water. This is also substantiated by the observation
of the decrease in 2Azz with water loss at 150°C (Fig. 3). It is interesting to note that cattail pollen and
pea axes differ in the extent of their 2Azz
decrease. Therefore, it seems evident that changes in water content
are not solely responsible for this difference and that some
intrinsic factors (e.g. salts or proteins) also contribute to the
decrease in 2Azz during drying.
Molecular Mobility in Biological Glasses
Although the presence of intracellular glasses in seeds and pollen
has been established, little is known about their viscosity and
molecular mobility upon melting. We investigated the molecular motion
of a spin probe in the cytoplasm of dry tissues to establish the
relationship between glasses, molecular mobility, and storage stability.
During drying of cattail pollen and pea axes, the
R of CP increases from
1011 s in the hydrated state to
102 s in the dry state. These values are
consistent with values obtained in organic and inorganic glasses (Ito,
1983; Dzuba et al., 1984; Roozen et al., 1991), but contradict a
previous study on molecular mobility in dry soybean axes, in which the
R of TEMPO was found to be approximately
1010 s (Bruni and Leopold, 1990). Referring to
the tendency of this spin probe to partition into oil during drying, we
attribute this contradiction to the fact that these authors might have
observed TEMPO in oilbodies. They nevertheless observed an abrupt
change in molecular mobility around Tg. It is
interesting that TEMPO in the lipid phase seems to be able to
"sense" the glass transition of the cytoplasmic surroundings.
Molecular mobility was assessed in relation to water content and
temperature. The change in distance between the two outer extrema of
the powder spectrum (2Azz) with increasing
temperature revealed a sharp increase in molecular mobility at a
certain temperature that depended on the sample water content. This
sharp increase was closely associated with the
Tg as measured by differential scanning
calorimetry. When R was monitored during
melting of the glass, there was a decrease of four orders of magnitude,
from 102 to 106 s.
We found a close correlation between changes in
R, determined by the C/C ratio of ST-EPR
spectra and changes in 2Azz derived from
conventional EPR spectra during melting of intracellular glasses
(compare Figs. 4 and 6). This correlation indicates that a change in
2Azz represents a change in molecular mobility. One can
speculate on the type of molecular mobility that gives rise to the
change in 2Azz. Echo-detected EPR spectroscopy of
nitroxide spin probes dissolved in organic glasses has revealed that
the nitroxides undergo librational motions (Dzuba, 1996). This type of
motion is described by a model that assumes that the spin probe rotates
within a cone given by a small angle, (Dzuba et al., 1992; Dzuba,
1996). Since we measured a R for CP in
intracellular glasses of 102 to
104 s, it seems unlikely that the change in
2Azz was due to a change in overall rotational
motion; most likely it represents a change in librational motion, as
has been established in wheat embryos (Dzuba et al., 1996). This is
further supported by our ST-EPR study, in which some indication for
anisotropic motion arising from libration comes from the different
values of R deduced from the line-height ratios
L"/L and C/C (Fig. 6). For the interpretation of our results,
however, a motional model that describes the change of
2Azz is not needed.
Storage Stability in Relation to Molecular Mobility and
Intracellular Glasses
It has been shown that upon formation of glasses, the storage
stability of seeds and pollen improves (Sun and Leopold, 1994; Sun,
1997; Buitink et al., 1998). The impact of intracellular glasses on the
storage behavior of seeds and pollen has been ascribed to the high
viscosity in the glass. Indeed, glasses are known to slow down
detrimental reactions such as the rate of browning reactions (Karmas et
al., 1992) and to increase the stability of enzymes (Chang et al.,
1996). Although the presence of glasses has been associated with
increased storage stability of seeds and pollen, there is not much
known about the relationship between molecular mobility and storage
stability. Our data on molecular motion in pea axes and cattail pollen
in relation to water content and temperature enable a comparison with
storage behavior, which is also known to depend on water content and
temperature (Vertucci et al., 1994; Buitink et al., 1998).
We found a close relationship between the molecular mobility of CP and
storage behavior in both pollen and seed axes. With decreasing water
content molecular mobility, expressed as a change in the outer
extrema of the EPR spectra (Azz), decreased,
whereas storage stability increased. Figure 11 clearly demonstrates
that molecular mobility and storage stability are linked; the water content for optimum storage at various temperatures corresponds closely
to the water content at which minimum mobility is observed (data from
Vertucci et al., 1994; Buitink et al., 1998). Although the curves do
not converge exactly with the 1:1 line, the slight deviation might be
explained from errors in the determination of water content or the lack
of an exact determination of the water content for optimum storage
stability. Furthermore, the water content of minimum mobility was
determined from the minima of the third-order polynomial equations.
Especially at lower temperatures, the exact water content of minimum
mobility is difficult to assess; there seems to be a plateau of minimum
mobility present. To present the data clearly, we calculated a single
value of minimum mobility, which should be considered with caution.
At low water content, molecular mobility seems to increase again. A
similar observation has been made by Seitz et al. (1981), who observed
that in Artemia cysts at lower hydration levels, the water
self-diffusion coefficients increased slightly. Clegg et al. (1982)
suggested from NMR studies on Artemia cysts that the
increased mobility of water at a low water content might be due to a
displacement of the water from polar-binding sites in the cell by
sugars. Another explanation is that at these low water contents the
spin probe partitions into a more mobile environment. However, it
should be noted that this environment cannot be the lipid phase. CP in
these phases will rotate faster than 108 s at
room temperature. Therefore, the spectrum of CP in the oil phase will
show the characteristic three sharp lines of a mobile spectrum and does
not contribute to the 2Azz of the powder
spectrum.
Optimum Storage Conditions Predicted by Molecular Mobility
Recent studies report a water-content limit below which seed
longevity did not increase further (Ellis et al., 1989, 1990), and
which had an adverse effect on seed viability and seed vigor (Vertucci
and Roos, 1993; Vertucci et al., 1994; Buitink et al., 1998). It is
thought that the removal of the last remaining water molecules may
destabilize biological structures (Sun, 1997; Buitink et al., 1998) or
enhance the lifetime of free radicals due to the loss of water as a
quencher (Karel, 1975). However, we observed an increase in molecular
mobility for cattail pollen (which also occurred to a lesser extent in
pea axes) when it was dried to very low water contents. This increase
in molecular mobility also might be responsible for the decreased
storage stability observed at these low water contents.
In some cases, state diagrams can be used to predict the optimal
storage conditions (Sun, 1997). For pea axes the optimum water contents
of storage at a certain temperature were found to coincide with
Tg (Fig. 8; Vertucci et al., 1994).
However, other seed species show a divergence of the optimal
storage stability from the Tg curve (Sun, 1997).
This is also true for cattail pollen (Buitink et al., 1998), in
which we found that the optimum storage conditions coincided
with the water content at which molecular mobility was minimum
(Fig. 11). This minimum mobility occurred below
Tg. Therefore, we propose that measurements
of molecular mobility rather than state diagrams be used to predict
optimum storage conditions.
Based on the relationship between the minimum molecular mobility and
the water content of optimal storage, we made an attempt to predict the
optimum storage conditions at subzero temperatures. This was found to
be 20°C and 60°C for pea axes (Fig. 9, E and F) and 20°C
and 40°C for cattail pollen (Fig. 10, E and F). In practice it will
not be possible to analyze storage behavior at these low temperatures
on a realistic time scale. At 20°C the minimum molecular motion of
pea can be estimated at approximately 0.10 g water
g1 dry weight, and at 60°C between 0.14 and
0.2 g water g1 dry weight. A similarly
elevated optimum water content for low-temperature storage was
predicted by Vertucci and Roos (1993) on the basis of thermodynamic
considerations. At 20°C the minimal molecular motion of pollen was
approximated at 0.1 g water g1 dry weight,
and at 40°C between 0.15 and 0.2 g water
g1 dry weight. With lower temperatures, an
increase in the water content at which minimum mobility was observed
became evident. This implies that too much drying increases mobility
and reduces longevity and should be avoided, particularly when
cryogenic storage is considered. Where determinations of optimal
storage conditions of seeds and pollen by germination assays take too
long to perform, measurements of 2Azz might be considered
instead.
| |
FOOTNOTES |
1
This research was financially supported by the
Netherlands Technology Foundation (STW) and was coordinated by the Life
Sciences Foundation.
*
Corresponding author; e-mail julia.buitink{at}algem.pf.wau.nl; fax
31-317-484740.
Received March 24, 1998;
accepted June 21, 1998.
| |
ABBREVIATIONS |
Abbreviations:
CP, 3-carboxy-proxyl.
EPR, electron paramagnetic
resonance.
ST-EPR, saturation-transfer EPR.
R, rotational correlation time.
TEMPO, 2,2,6,6-tetramethyl-1-piperidinyloxy.
Tg, glass-to-liquid transition temperature.
| |
ACKNOWLEDGMENTS |
The authors thank Drs. Olivier Leprince and Elena Golovina for
critically reading the manuscript and Mark Alberda for excellent technical assistance.
| |
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Abstract
Introduction