Comp. Biochem. Physiol. Vol. 117A, No. 3, pp. 327–333, 1997
Copyright  1997 Elsevier Science Inc.
ISSN 0300-9629/97/$17.00
PII S0300-9629(96)00271-X
Cytoplasmic Vitrification and
Survival of Anhydrobiotic Organisms
Wendell Q. Sun* and A. Carl Leopold†
*School of Biological Sciences, Faculty of Science, National University of
Singapore, Kent Ridge Crescent, Singapore 119260, Republic of Singapore, and
†Boyce Thompson Institute for Plant Research at Cornell University, Ithaca, NY 14853, U.S.A.
ABSTRACT. We examine the relationship between cytoplasmic vitrification and survival of anhydrobiotic
organisms under extreme desiccation condition. The ability of anhydrobiotic organisms to survive desiccation is
associated with the accumulation of carbohydrates. Spores, yeasts and microscopic animals accumulate trehalose,
whereas pollen, plant seeds and resurrection plants contain sucrose and oligosaccharides such as raffinose and
stachyose. During dehydration, these carbohydrates and other components help the organisms enter into the
vitreous state (cytoplasmic vitrification). The immobilization by vitrification may minimize stress damages on
the cellular structures and protect their biological capabilities during dehydration and rehydration; however,
cytoplasmic vitrification alone is found to be insufficient for anhydrobiotic organisms to survive extreme dehydration. The survival of dry organisms in the desiccated state requires the maintenance of the vitreous state. When
the vitreous state is lost, free radical oxidation, phase separation and cytoplasmic crystallization would occur
and impose real threat to the survival of dry organisms. comp biochem physiol 117A;3:327–333, 1997.  1997
Elsevier Science Inc.
KEY WORDS. Anhydrobiosis, carbohydrate crystallization, dehydration, desiccation tolerance, dry organism,
free radical, glass transition, phase separation, seed longevity
INTRODUCTION
Some microscopic animals, microbes and plant tissues
evolved special mechanisms that enable them to tolerate
extreme desiccation and to survive for an extended period
in the desiccated state. They are in a unique living state
known as anhydrobiosis. Such anhydrobiotic organisms include bacteria spores, fungal spores, yeast cells, nematodes,
rotifers, tardigrades, cysts, pollen, plant seeds and resurrection plants (9,18). More than 98% of their body water may
be removed, and yet upon rehydration they are able to recover their full metabolic capacity immediately.
In the past decade, studies found that anhydrobiotic organisms share some common features associated with their
desiccation tolerance (9). The dry organisms usually contain high concentrations of soluble carbohydrates that stabilize membranes and macromolecules upon desiccation
(7,9,18). These carbohydrates interact with membranes and
macromolecules and can replace water molecules during dehydration (7,9,10). By doing so, carbohydrates are able to
protect anhydrobiotic organisms from desiccation damage.
Burke (6) suggested cytoplasmic vitrification as a bioAddress reprint requests to: W. Q. Sun; School of Biological Sciences, Faculty of Science, National University of Singapore, Kent Ridge Crescent,
Singapore 119260, Republic of Singapore. Tel. 65-772-7932; Fax 65-7795671; E-mail: [email protected].
Received 7 September 1995; accepted 18 January 1996.
physical mechanism of stress tolerance in anhydrobiotic organisms. He argued that cytoplasmic vitrification could offer
many advantages for anhydrobiotic organisms to survive extreme desiccation. In the vitreous state, deteriorative reactions that threaten the survival of organisms would be suppressed because of the extremely high viscosity, complete
dehydration is avoided due to the lower water vapor pressure of the vitreous state and solute crystallization is prevented. The vitreous state can also resist change of intracellular pH and ionic strength during dehydration (6).
The vitreous state is confirmed in several dry biological
systems, including fungal spores, Artemia cysts and plant
seeds [(4,5,18,27); Sun and Leopold, unpublished]. However, the role of cytoplasmic vitrification in desiccation tolerance has not been critically examined. We scrutinize new
evidence and discuss how vitrification, crystallization and
phase separation contribute to the survival or death of dry
biological systems.
DESICCATION TOLERANCE
AND CYTOPLASMIC VITRIFICATION
Survival to extreme desiccation requires anhydrobiotic organisms to withstand enormous stresses that few organisms
can tolerate (12,20). Water is the most important component in living systems. It confers a structural order on mem-
328
branes and proteins in cells and is involved in every life
process. As water is removed from the cells, a series of
events may occur: the increase of solute concentration,
change of intracellular pH and ionic strength, the acceleration of destructive reactions, denaturation of proteins and
disruption of membranes. These events could disrupt all
synthesis and metabolism and destroy the structural organization of cells and macromolecules.
However, as water is removed from the cells of anhydrobiotic organisms, the cytoplasm are expected to become
vitrified (i.e., enter into a vitreous state), because these organisms accumulate high contents of soluble carbohydrates
that are known to be good vitrifying agents (6,11,14,17,30).
The liquid-to-glass transition during cell dehydration can
result in the immobilization of cellular structures and biochemical components and therefore preserve their biological structures and capacities by minimizing stress damages.
The vitreous state has been confirmed in fungal spores,
Artemia cysts and plant seeds with various techniques, including differential scanning calorimetry, electron spin resonance and thermal stimulated current (4,5,30; Sun and
Leopold, unpublished). The thermal stimulated current and
x-ray diffraction techniques permitted us to study whether
cytoplasmic vitrification plays any protective role during
cell dehydration. We compared cytoplasmic vitrification in
desiccation-tolerant and desiccation-intolerant systems and
failed to detect any difference of cytoplasmic vitrification
that could be associated with the difference in desiccation
tolerance of two biological systems (Fig. 1). X-ray diffrac-
FIG. 1. Phase diagrams of glass transition of desiccation-
tolerant (soybean axis) and desiccation-intolerant (red oak
cotyledon) tissues. Transition from glass to liquid state occurs upon crossing glass transition curve by increasing either
temperature at constant water content or water content
when temperature is kept constant. Glass transition temperatures (Tg ) of red oak cotyledons were measured with the
thermal stimulated depolarization current (27).
W. Q. Sun and A. C. Leopold
tion study showed no evidence of cytoplasmic crystallization in either desiccation-tolerant and desiccation-intolerant systems during dehydration (27). With phospholipid
membrane model systems, Crowe et al. (8) recently made
similar observations during freeze drying. Their data indicated the importance of direct interaction between carbohydrates and membranes for the preservation of membranes
in addition to vitrification.
During dehydration, the desiccation-intolerant tissues
are damaged at hydrations far above the water content
for cytoplasmic vitrification. Because the phase curve of
cytoplasmic vitrification is the same for both desiccationintolerant and desiccation-tolerant tissues (Fig. 1), the
desiccation-tolerant tissues are not expected to become
vitrified at the similar hydration levels that begin to damage
the desiccation-intolerant tissues. This fact is a strong argument against the proposition that the cytoplasmic vitrification is involved in the desiccation tolerance. It appears to
us that cytoplasmic vitrification cannot provide sufficient
protection for anhydrobiotic organisms to survive cell dehydration. If cytoplasmic vitrification does not adequately protect anhydrobiotic organisms during cell dehydration, what
possible benefits does it have for them?
THE VITREOUS STATE AND
SURVIVAL IN THE DESICCATED STATE
It has been hypothesized that the vitreous state is associated
with the survival of anhydrobiotic organisms in dry state
(6,11,30). From the theoretical consideration, the vitreous
state would serve as a biophysical barrier to the deteriorative
processes of dry biological systems due to its extremely high
viscosity. Most physical and chemical processes in cells are
diffusion limited. The rate of deleterious reactions is inversely correlated with the cytoplasmic viscosity. In the vitreous state, the cytoplasm is so viscous that diffusional
movements are almost arrested, and therefore the deterioration should be greatly inhibited. For example, the translation through one molecular distance is around 300,000 years
in the vitreous state (20). However, experimental evidence
is still lacking, partly because any study testing this hypothesis may take more than 10 years to be completed, if experiments are to be conducted under physiological conditions.
Recently, we used a mathematical approach to investigate the possible role of vitreous state in the survival of
seeds during dry storage (28). The Tg as a function of water
content has been recently reported for soybean and corn
embryos. With the equations derived from the seed viability
equation, we have calculated the maximum temperature
(Tmax ) for long-term storage of corn and soybeans over a
range of water contents (Fig. 2). The temperature for longterm storage drops dramatically as water contents are elevated; Tmax (curves) for long-term seed storage is in good
agreement with the glass transition temperature (Tg ) (data
points) in both species. The data have clearly shown that
Vitrification of Anhydrobiotic Organisms
329
FIG. 3. Effect of glass transition on the release of organic free
radicals in soybean seed axes. Free radical content is measured with electron spin resonance method, and maximum
free radical level is registered at low water content (,0.02
g/g dw). As hydration level of the tissue increases and glass
transition temperature decreases, the trapped free radicals
become unstable and are released. Free radical release increases significantly after the vitreous state is melted. The
data of free radical contents are extracted from Priestly
et al. (21) and reinterpreted by the present authors.
FIG. 2. The vitreous state and the survival of plant seeds in
the desiccated state. (A) Soybean and (B) corn. The phase
diagram of seed glass transition (Tg , data points) is superimposed onto the maximum temperature curve for a mean survival period over 50 years (Tmax , curves). Tg shows good
agreement with Tmax , indicating that the vitreous state has
to be maintained for long-term seed survival. Glass transition
temperatures (data points) of seed axes and embryos are
adopted from Williams and Leopold (30) and Bruni and Leopold (4,5). Maximum temperatures for long-term storage are
calculated by solving the seed viability equation (28).
Dashed line shows the extrapolated region from experimental data.
the maintenance of the vitreous state is required for longterm survival of the dry organisms. When seeds are not in
the vitreous state, seed deterioration would be expected to
be accelerated (26).
Free radical–induced reactions occur in many dry biological systems such as lyophilized virus and bacteria, membranes, dry blood products and animal tissues. The loss of
viability of dry cells and the deterioration of other dry products are correlated with the production and release (i.e.,
disappearance) of free radicals during storage (13). Figure 3
shows the release of organic free radicals in soybean embryonic axes in relation to the loss of the glassy state (Sun
and Leopold, unpublished data). In the vitreous state, free
radical release is significantly inhibited. However, as hydration level of the tissue increases and glass transition temperature decreases, the trapped free radicals are released. This
result suggests that the accelerated deterioration of dry biological systems, when not in the vitreous state, is probably
associated with the free radical release that causes damages.
The vitreous state also retards the nonenzymatic Maillard
reactions involved in the deterioration of dry organisms
(29). In food products, lipid oxidation and Maillard reactions are commonly prevented in the vitreous state (16,24).
These observations provide evidence that the vitreous
state contributes to the stability of anhydrobiotic organisms
in dry state. The vitreous state may provide a kinetic stability (i.e., real-time stability) due to its extremely high bulk
viscosity.
CYTOPLASMIC CRYSTALLIZATION
AND PHASE SEPARATION
As we have already shown, the loss of the vitreous state
would accelerate the deterioration of dry biological systems.
The vitreous state is thermodynamically unstable, and its
physical stability depends on the extremely high viscosity
of such a system. When the vitreous state is lost (by either
increasing temperature or increasing water content that depresses Tg ), the viscosity is rapidly lowered and the solution
330
becomes unstable, being vulnerable to crystallization and
phase separation. It is of particular interest to study what
effects cytoplasmic crystallization and phase separation may
have on anhydrobiotic organisms and what mechanism the
organisms adopt to increase their chance of survival. Crystallization and phase separation are important threats to
processing and preservation of biological materials such as
enzymes, genetic materials, membranes, cells and pharmaceuticals.
Carbohydrates may protect the dry organisms, only if
they are available on hydrophilic sites of cellular membranes and macromolecules and do not become crystallized
(18). It is conceivable that cytoplasmic crystallization
would lead to the death of the dry organism due to the destruction of structural organization in the cells. We tried
with an x-ray diffraction study to detect the presence of
cytoplasmic crystallization in dry seeds but failed to show
any evidence (27,28). However, Fig. 4 shows an interesting
observation that suggests a possible link between cytoplasmic crystallization and the death of dry organisms (Sun
and Leopold, unpublished data). The data reveal that seed
longevity of 16 species (orthodox seeds) is correlated to the
carbohydrate compositions, each with different crystallization rates. Seed species with carbohydrate composition having high crystallization rate tends to lose seed viability more
rapidly during dry storage.
The distribution of solutes and water in dry cells is typically heterogeneous and in a nonuniform manner between
each of the different domains (20). We previously suggested
that the gradual loss of the vitreous state during seed aging
might be caused by phase separation (i.e., de-mixing) of cytoplasmic glass domains (26). Under conditions of accelerated aging, low cytoplasmic viscosity would facilitate separation of cytoplasmic matters and therefore help form various
glass domains. Phase separation might prevent carbohydrates from protecting cellular membranes and macromolecules by making them unavailable. Phase separation of the
cytoplasmic glasses were observed recently in maize embryos
of several lines (Fig. 5) (2). Two major glass domains, one
at 20°C and another at 45°C, were detected. An increase
in the proportion of high temperature domains appears to
be correlated with seed longevity. The preliminary data suggest an association of phase separation with seed longevity.
It has been noted that oligosaccharides, especially raffinose, seem to have a special contribution to the survival of
plant seeds. Raffinose content has been correlated with the
vigor of aged seeds (1). The phase separation of cytoplasmic
glass is correlated with the raffinose level. As raffinose content increases, cytoplasmic glass seems more likely to stay
at the high temperature domain (Fig. 5). Beneficial effects
of raffinose can also be seen from Fig. 4, where it prevented
sucrose crystallization. Smythe (25) reported that raffinose
and stachyose were the two most effective sugars that inhibit sucrose crystallization. They inhibited sucrose crystallization because of their similarity to sucrose in the fructose-
W. Q. Sun and A. C. Leopold
FIG. 4. (A) Crystallization tendency of carbohydrate mix-
tures and (B) seed longevity of 16 species (orthodox) with
different carbohydrate compositions. The rate of crystallization of sucrose/oligosaccharide mixtures, measured with
light refraction, increases when the mixture is dominated
with a single carbohydrate. Seeds with very low or high oligosaccharide content are found to lose their viability more
rapidly, suggesting a possible role of crystallization in seed
death at the desiccated state. B is drawn with the data from
Horbowics and Obendorf (15) and Lin and Huang (19).
glucose moieties and their lack of fit onto the surface of the
growing crystal was due to the additional galactose moiety.
Plant seeds may adopt the mechanism to improve their survival in the desiccated state by using oligosaccharides such
as raffinose and stachyose to prevent phase separation and
cytoplasmic crystallization.
We know little how anhydrobiotic organisms that accumulate trehalose prevent phase separation and crystallization, because like sucrose, trehalose may crystallize easily.
Glasses of pure trehalose, when stored slightly above room
temperature, become crystallized after a few days, even under very dry conditions (22). However, studies with sugar
model systems suggested that protein may play a significant
role in preventing crystallization of trehalose glasses. It has
been observed that the presence of a small amount of pro-
Vitrification of Anhydrobiotic Organisms
331
FIG. 5. Phase separation of
the cytoplasmic glass in corn
embryos. Two major glass domains are observed with the
method of thermally stimulated depolarization current
(TSDC). Mean viability period ( P50 ) is from accelerated
aging study at 30°C and 75%
RH. The data are adopted
from Bernal-Lugo and Leopold (2) and reinterpreted by
us. Phase separation appears
to be related to carbohydrate
composition and is associated
with seed longevity.
tein inhibited crystallization of trehalose glasses (22). A recent study on the crystallization behavior of bovine somatotropin (bSt)/sucrose glasses has found that the protein
inhibited crystallization by increasing crystallization temperature of the sugar glasses (23).
CONTRIBUTION OF CARBOHYDRATES
TO CYTOPLASMIC VITRIFICATION
The accumulation of soluble carbohydrates in cells is associated with the desiccation-tolerant state in anhydrobiotic organisms. Upon dehydration, animals and microorganisms
synthesize trehalose (see 9), whereas desiccation-tolerant
plant tissues accumulate sucrose and oligosaccharides such
as raffinose and stachyose (see 18). In many plant seeds,
anhydrobiotic animals and microorganisms, soluble carbohydrates constitute between 15 and 25% of their total dry
mass. It is believed that soluble carbohydrates are responsible for the cytoplasmic vitrification in anhydrobiotic organisms (6,14,17,30). Because cells contain much more than
soluble carbohydrates, two questions arise as to the role of
carbohydrates in cytoplasmic vitrification. Can carbohydrates alone form the cytoplasmic glass needed for the cells
to avoid desiccation stress? Are other cytoplasmic components such as proteins involved in cytoplasmic vitrification?
Figure 6 shows the phase diagrams of glass transitions for
corn embryos and the carbohydrate mixes similar to those
found in vivo (Sun and Leopold, unpublished data). The
phase diagram for embryo tissues is distinctively different
from that of carbohydrate mixes, indicating that the presence of carbohydrates alone does not fully account for the
vitreous state in anhydrobiotic organisms. The study on the
dependence of glass formation on hydration and tempera-
ture between carbohydrate mixes and embryo tissues suggests the involvement of some factor other than sugars. It
should be a highly hydrophilic polymer-like component in
the cytoplasmic vitrification of embryo tissues. This polymerlike component may have a strong effect on the property and
organization of water in the cells, so that it can significantly
increase Tg of the cytoplasmic glass at low water content
FIG. 6. Glass transition phase diagrams for corn embryos and
the representative carbohydrate mix (a mass ratio of 4 : 1 for
sucrose and raffinose). Two phase diagrams do not match
each other, showing that carbohydrates alone are not sufficient to form the vitreous state in seeds. Glass transition of
the carbohydrate mixture is measured with differential scanning calorimetry [s, data of this study; n, data from (17)].
The Tg data of corn embryos are adopted from Williams and
Leopold (30) and Bruni and Leopold (4,5).
W. Q. Sun and A. C. Leopold
332
(,0.15 g/g dry weight), possibly by inhibiting the plasticizing effect of water. On the other hand, its presence may
lower Tg at high water content by resisting freeze-induced
dehydration (Fig. 6). A good candidate of the polymer-like
component is protein in the biological tissues. We suspect
that this polymer-like component is comprised of desiccation stress proteins (i.e., dehydrin and water stress protein)
that are commonly present in anhydrobiotic organisms. In
soybean seeds, desiccation tolerance is quantitatively correlated with the level of desiccation stress proteins (3). It
should be noted that not all proteins could play the role of
the polymer-like component for cytoplasmic vitrification.
Sarciaux et al. (23) showed that bovine somatotropin at a
concentration as high as 20% did not significantly influence
Tg of sucrose glasses.
In conclusion, there is no doubt that cytoplasmic vitrification plays an important role in the survival of anhydrobiotic organisms during desiccation and subsequent dry
storage. The existing data, although limited, show that cytoplasmic vitrification is associated with desiccation tolerance
and the long-term storage stability of dry biological systems.
Much evidence presented in this article is from our studies
on plant seeds. However, it is believed that anhydrobiotic
animals may use the same biophysical mechanisms to escape
stresses and damages, because on the cellular and molecular
scales the physical principles of desiccation damage should
be the same regardless of whether the organism is an animal,
a microbe or a plant.
It is acknowledged that at least two biophysical mechanisms may be involved in protecting anhydrobiotic organisms under desiccation stress. One relates to the protective
effect of hydrophilic polyols (carbohydrates and other compatible solutes) by the direct interaction with membranes
and macromolecules or by water replacement. The other
concerns the role of carbohydrates in cytoplasmic vitrification (liquid-to-glass transition) that may preserve the biological structures and their capacities by immobilization
during cell dehydration. These two mechanisms are not mutually exclusive. Sugar molecules in the vitreous state may
be available for substituting water at the hydrophilic sites of
macromolecules and membrane systems and therefore offer
them an environment that is similar to a dilute aqueous
solution.
Currently, our understanding of biological glasses is based
principally on the studies of the kinetic effect of the glass
transition. To understand how the vitreous state affects the
property and stability of dry biological systems, we should
extend our studies into areas such as structures, dynamics
and molecular interactions of the biological glasses.
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