Proc. Nati. Acad. Sci. USA
Vol. 86, pp. 520-523, January 1989
Biophysics
Membrane phase transitions are responsible for imbibitional
damage in dry pollen
JOHN H. CROWE*t, FOLKERT A. HOEKSTRA**, AND Lois M. CROWE*
*Department of Zoology, University of California, Davis, CA 95616; and tPlant Physiology Department, Agricultural University, Wageningen,
The Netherlands
Communicated by John D. Baldeschwieler, September 26, 1988
ABSTRACT
We have found that the most probable cause
of the leakage seen when dry cells or organisms such as seeds,
pollen, or yeast cells are plunged into water is a gel to liquid
crystalline phase transition in membrane phospholipids accom-
panying rehydration. By using Fourier transform infrared
spectroscopy we have recorded infrared spectra ofCH2 stretch-
ing vibrations in dry and partially hydrated intact pollen grains
of Typha latfolia. The vibrational frequency changes abruptly
as phospholipids pass through the gel to liquid crystalline phase
transition. Below the apparent transition, viable pollen shows
low germination and high leakage when placed in water, but
above the transition germination increases and leakage de-
creases. The apparent transition temperature falls with in-
creasing water content, much as in pure phospholipids. By
using this phenomenon, it was possible to construct a hydra-
tion-dependent phase diagram for the intact pollen. This phase
diagram has immediate applications since it has high predictive
value for the viability of the pollen when it is placed in water.
When dry seeds (1, 2), pollen (3), yeast cells (4), and anhy-
drobiotic microscopic animals (5) are rapidly rehydrated they
leak their soluble cellular contents into the surrounding
medium, as a result of which they are often killed. This
imbibitional leakage has long been known to be a major
source of damage to the dry organisms, particularly if the
imbibition is accomplished at low temperatures (6). Eco-
nomic losses result if, for example, dry soybean seeds are
imbibed in the field during cold weather. Consequently,
numerous workers have investigated the physical basis for
the leakage. Simon (1, 7) suggested that it is due to formation
ofnonbilayer phases, particularly the inverse hexagonal (HI,)
phase in the plasma membrane of the dry cells. According to
this hypothesis, the presence of HI, phase lipids would be
incompatible with maintenance ofa low-permeability bilayer.
When the cell is rehydrated, the HI, lipids would then enter
lamellar phase, and leakage would be abated. This appealing
idea was quickly embraced by the scientific community, and
many workers began to search for HI, phase lipids in plasma
membranes of dry cells. However, there is good evidence
that HI, lipids are not found in membranes of dry but viable
organisms (8-10), and we have sought an alternative expla-
nation for the leakage.
When pure phospholipids are dehydrated their transition
temperature rises, in some lipids by 70'C or more (11, 12).
Consequently, a fully hydrated phospholipid that is in liquid
crystalline phase at room temperature might very well un-
dergo a transition to gel phase when it is dried at the same
temperature, followed by a transition back to liquid crystal-
line phase when it is rehydrated (see ref. 13 for recent
discussion). It is also well known that leakage occurs when
hydrated phospholipid vesicles are heated through their
thermotropic gel to liquid crystalline phase transition (14) or
when dry gel phase vesicles are rehydrated and pass through
the gel to liquid crystalline transition (15). Thus, it seems
possible a priori that the leakage from dry organisms during
rehydration might be due to a gel to liquid crystalline lipid
phase transition. According to this idea, membrane phos-
pholipids in the dry organism would be in gel phase. When the
dry organism is placed in water, the gel phase lipids would
undergo a transition to liquid crystalline phase, during which
leakage would occur. We now show that imbibitional leakage
from dry intact pollen grains is almost certainly due to such
transitions.
MATERIALS AND METHODS
Sample Preparation. Pollen of cattail (Typha latifolia) was
chosen as a model system to use in these studies primarily
because the pollen is available in large quantities. The pollen
was collected from the field and stored at -20'C until use.
The only preparation given to the pollen samples before the
experiments were conducted was to remove a hydrocarbon
layer on the outside of the cell wall and to adjust the water
content. In a recently published study (16), a crystalline
phase hydrocarbon in the same pollen studied here was
detected by x-ray diffraction. This crystalline phase was
attributed to a hydrocarbon on the surface of the pollen, the
presence of which not surprisingly makes it impossible to
obtain infrared spectra of cellular portions of the intact
pollen, as described below. Thus, this extracellular hydro-
carbon was removed by washing the dry pollen in hexane
briefly, after which treatment the pollen remain fully viable.
Water contents of the samples were adjusted by equilibrating
them over known relative humidities. At equilibrium the
samples were transferred to the sample chamber in a cold
room at 40C to minimize changes in water content during the
transfer. To obtain dry samples, the pollen grains were
evacuated on a VirTis lyophilizer for at least 24 hr at 0.01
mmHg, after which they were removed from the lyophilizer
under vacuum and transferred to a glove box flushed with dry
nitrogen. The samples were removed from the vacuum flask
in the glove box and loaded in the sample chamber.
Infrared Spectroscopy. Infrared spectroscopy was done
with a Perkin-Elmer 1750 Fourier transform infrared spec-
trometer assisted by a Perkin-Elmer 7500 data station.
Temperature control was by means of a Peltier device, and
sample temperature was recorded with a thermocouple
against the window. The samples were loaded in a monolayer
between two BaF2 windows for acquisition of the spectra.
Ten spectra were recorded at each temperature and stored
without further data processing. Frequencies of the CH2
bands were computed on the data station to the nearest 0.1
wavenumber by the center of gravity algorithm of Cameron
et al. (17).
Abbreviations: Tm, midpoint of phase transition; Gm, midpoint of
germination curve.
tTo whom reprint requests should be addressed.
520
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Proc. Natl. Acad. Sci. USA 86 (1989)
521
Measurement of Germination and Leakage. Germination of
the pollen and leakage ofNAD' were measured as described
(3).
RESULTS AND DISCUSSION
Use of Infrared Spectroscopy to Measure Phase Transitions.
We have found that Fourier transform infrared spectroscopy
is particularly useful for measuring phospholipid phase tran-
sitions in intact, dry pollen. Spectra were obtained between
3000 and 2800 wavenumbers, the region where CH2 vibra-
tions in hydrocarbon chains are strongest. With increasing
temperature the vibrational frequency (or wavenumber) of
these bands increases abruptly in pure phospholipids (18) and
in intact cells (19) as they pass through the gel to liquid
crystalline transition temperature. Surprisingly, it was pos-
sible to detect CH2 bands clearly in the spectra from intact
pollen (Fig. 1). When the pollen was heated in the spectrom-
eter (14, 15) and scans were taken at different temperatures,
a clear increase in vibrational frequency of the CH2 groups
was seen (Fig. 1). This elevation in frequency as a function
of temperature can be recorded to produce the results shown
in Fig. 2, and the transition temperature can be estimated
from the midpoint of this frequency change (Tm in Fig. 2).
Infrared spectroscopy is far more useful for measuring
phase transitions in a complex mixture of lipids, proteins,
etc., such as that found in a biological membrane or in the
pollen studied here than is the more widely used differential
scanning calorimetry for reasons that we will document in
detail elsewhere (20). Briefly, when differential scanning
calorimetry scans are done on a native biological membrane
small melting endotherms are seen during warming that
probably represent melting of small domains of gel phase
lipid. These small endotherms are often of such low enthalpy
that
it is difficult to distinguish them from noise in the
baseline. When the same sample is studied with infrared
spectroscopy, by contrast, successive melting of small gel
phase domains results in a smooth, progressive increase in
the average vibrational frequency. The result is the kind of
curve seen in Fig. 2. The apparent Tm can be extracted from
the midpoint of the curve by probit analysis, as described
elsewhere (20).
Hydration-Dependent Phase Transitions in Intact Pollen.
Spectra similar to those shown in Fig. 1 were taken from
Liquid crystalline
~Gel
(D0
C:
.0
2950
2850
Wavenumber, cm-'
FIG. 1.
Representative infrared spectra for intact pollen grains of
T. latifolia containing about 0.7 g of water per g of dry weight. As
temperature is increased the vibrational frequency (wavenumber) for
the CH2 bands increases (14, 15).
_'
cuo
v9 M2Y/
io/
*
E
g Dry Weight A:
/
0.00
2852
A
U)
/
~~~~~~0.14!
:3
0~~/
-2851
1
0.091
U,~~
~~~Tmea~e
/
I
>
~~~Tm
2850
28491
~
A
A
-20
-10
0
10
20
30
40
Temperature, 00
FIG. 2.
Vibrational frequencies for CH2 symmetric stretch as a
function of temperature in samples of pollen grains with differe.it
water contents. Water contents (expressed as g of water per g of dry
weight) are shown in the numbers by each curve. The midpoint of the
transition (Tm) is indicated for two of the curves (15).
pollen at different water contents, and the resulting vibra-
tional frequencies were measured, yielding the curves in Fig.
2. According to these data, the apparent Tm changes with
water content. The four representative plots shown in Fig. 2
suggest that Tm rises from about -60C in the hydrated poller
to about 320C when the pollen is dried. In pollen with
intermediate water contents, intermediate Tm values were
obtained (Fig. 2).
Assignment of Phase Transitions to Phospholipids. The fol-
lowing studies were conducted to establish that the data seen
in Figs. 1 and 2 represent phase transitions in membrane
phospholipids. The procedures used in these studies and the
detailed results are presented elsewhere (20). Briefly, the
major hydrocarbons in these pollen grains are triglycerides,
phospholipids, and a hydrocarbon on the surface that can be
removed by washing the pollen grains briefly in hexane. This
treatment does not alter viability, so all studies reported here
were conducted with pollen from which this extracellular
hydrocarbon had been removed. To distinguish between
transitions in the remaining triglycerides and phospholipids,
we isolated both ofthese lipid fractions and measured their Tm
values with differential scanning calorimetry and Fourier
transform infrared spectroscopy. The triglycerides undergo
melting at 10-15TC. This transition does not change with water
content, indicating that the hydration-dependent transitions
seen in Fig. 2 cannot be due to melting of the triglycerides. By
contrast, the fully hydrated phospholipids have a Tm of -60C,
whereas that of the dry phospholipids is in excess of 30'C. We
conclude that the hydration-dependent transit:rns recorded in
Fig. 2 can be assigned to the phospholipids.
Physiological Significance of the Phase Transition. Our
hypothesis that membrane phase transitions are responsible
for imbibitional damage in dry pollen suggests that the
transition temperature should closely predict rates of leakage
from the pollen during rehydration and the subsequent
germination success. Physiological data on leakage from and
germination of the pollen as a function of imbibitional
temperature (17) indeed agree well with the measurement of
Tm. Pollen grains with various water contents (produced by
equilibrating the pollen to known relative humiditie" were
imbibed in germination medium at several temperatures.
Leakage of NAD+ was recorded immediately, and germina-
tion was measured subsequently. Some representative re-
sults, shown in Fig. 3, demonstrate that germination
ini-
creases with temperature coincidentally with the increase in
vibrational frequency of the CH2 bands. Furthermore, leak-
age declines over the same temperature range as germination
and vibrational frequency increase (Fig. 3). It is po, 3ible to
extract from the germination/temperature plots the midpoint
Biophysics: Crowe et al.

Proc. Natl. Acad. Sci. USA 86 (1989)
c0
Cu
C
.)0
so
0)
w
0)
sDl
0
10
20
30
Temperature, 0C
E0
0.0
E
ID
30
' 2849
40
FIG. 3.
Vibrational frequencies for CH2 symmetric stretch as a function of temperature in a pollen sample containing about 0.1 g of water
per g ofdry weight. Also shown are data for germination ofand leakage ofNAD' from pollen adjusted to about 0.1 g of water per g of dry weight
by e ,posing them to moist air before they were imbibed in germination medium at the indicated temperatures. Germination and leakage data
are expressed as % of maximal values recorded. The temperature at which germination reaches half-maximal value (Gm) is also indicated.
of the germination curve, a parameter we have called Gm in
Fig. 3. It is apparent from the data shown in Fig. 3 that Gm
is approximately equal to Tm.
Phase Diagram for Intact Pollen. Data similar to those
shown in Fig. 3 were produced for pollen with a variety of
water contents, and Tm and Gm were extracted from plots of
germination or vibrational frequency ofthe CH2 bands. From
these data we have constructed the hydration-dependent
phase diagram for the intact pollen shown in Fig. 4. The phase
diagram shows unquestionably that Tm rises steeply when
water content is decreased below about 0.25 g of H20 per g
of dry weight, a figure that agrees well with values for
hydration dependence for Tm in pure phospholipids (8).
Above the line, the phospholipids will be in liquid crystalline
phase; below the line, they will be in gel phase. Also shown
in Fig. 4 are the values for Gm, which agree with those for Tm;
as Tm falls with increasing water content, Gm falls coinci-
dentally. Above this line, the pollen grains do not leak when
they are placed in water, and they show maximal germina-
tion. Below the line, they leak extensively and show minimal
germination.
Mechanism of Imbibitional Leakage: A Hypothesis. Based
fin the data presented in Figs. 1-4, we suggest the following
hypothesis concerning the cause of imbibitional leakage,
summarized in Fig. 5. When pollen, and probably other
organisms are dehydrated, Tm for membrane phospholipids
30 9'
O
Tm
*Gm
(20
\
0
Liquid Crystalline,
E
10 t
0
Germinative, Not Leaky
E
1
Gel.
0 -
Leaky,
Not Germinative
-iG ~O
0
0.2
0.4
0.6
0.8
2.5
H20, g per g of dry weight
FIG. 4.
Hydration-dependent phase diagram for pollen obtained
by extracting values for Tm from the data shown in Fig. 2 and similar
data not shown. For comparison, Gm values extracted from germi-
nation data shown in Fig. 3 and similar data for pollen imbibed at
other water contents are also shown.
increases. As a result, when the cells are rehydrated, the
phospholipids undergo a transition from gel to liquid crys-
talline phase. Consequently the cells leak their contents to
the medium, and they are killed. By contrast, if the dry cells
are heated to above the gel to liquid crystalline phase
transition temperature before they are placed in water, they
do not undergo a phase transition during rehydration and they
do not leak (Fig. 5). Similarly, if the cells are partially
rehydrated by exposing them to water vapor before they are
placed in bulk water, Tm may be reduced so that the partially
hydrated phospholipids are in liquid crystalline phase. In
either case, during rehydration phospholipids in membranes
of the dry cells pass from liquid crystalline phase to liquid
crystalline phase when they are rehydrated, and they do not
leak (Fig. 5). If one is to predict the conditions under which
this situation can be obtained, it is essential to have available
a detailed phase diagram of the kind shown in Fig. 4.
Utility of the Phase Diagram. It is now feasible to produce
phase diagrams similar to that shown in Fig. 4 for all cells that
are capable of surviving dehydration, including plant
propagules of commercial importance. By using such phase
diagrams it will be possible to predict the combination of
temperature and water content at the time the cells are placed
in bulk water that will result in minimal leakage and maximal
germination. This procedure should be particularly useful
since a full phase diagram can be constructed in a matter of
a few hours with Fourier transform infrared spectroscopy,
whereas the comparable germination studies under the vari-
ety of conditions that would be required may take weeks or
even months to complete. We conclude that the results
presented here not only explain an old puzzle-imbibitional
damage in dry organisms-but also may have considerable
practical importance.
This research was supported by Grants DMB 85-18194 from the
National Science Foundation and R/A-62 from National Sea Grant.
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Biophysics: Crowe et al.

Proc. Natl. Acad. Sci. USA 86 (1989)
523
Biophysics: Crowe et al.
00
0
0)
0
0
00
Hydrated Bilayer
(Liquid Crystalline)
nI
B.ow
Below
w
Dry Bilayer
(Gel)
0
0
0
0
Rehydration
Phase Transition.
00
NoLeakage
l
u
o
0
m
Rehydration.
,:
(
No Phase Transition,
|
No Leakage
0
)
3
0.
00
0000
00
Above Tm
(Liquid Crystalline)
FIG. 5.
Diagrammatic representation of the proposed mechanism for imbibitional leakage. As a hydrated bilayer (water molecules are
represented by small, open circles) in liquid crystalline phase is dehydrated, it may enter gel phase, depending on the temperature. If this dry
bilayer is not heated to above its transition temperature (Tm) before it is returned to water (upper pathway), it will undergo a phase transition
during rehydration during which it would be expected to leak. On the other hand, if it is heated to above Tm or partially hydrated by exposure
to water vapor (lower pathway), the bilayer can pass through the phase transition in the absence of bulk water. Under these conditions it will
not undergo a phase transition when it is placed in water and it will not leak.
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-
ll
Al
r..