Cytoplasmic Vitrification and Survival of Anhydrobiotic Organisms

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Comp. Biochem. Physiol. Vol. 117A, No. 3, pp. 327–333, 1997 ISSN 0300-9629/97/$17.00 Copyright  1997 Elsevier Science Inc. 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 physical mechanism of stress tolerance in anhydrobiotic organisms. He argued that cytoplasmic vitrification could offer Some microscopic animals, microbes and plant tissues many advantages for anhydrobiotic organisms to survive ex- evolved special mechanisms that enable them to tolerate treme desiccation. In the vitreous state, deteriorative reac- extreme desiccation and to survive for an extended period tions that threaten the survival of organisms would be sup- in the desiccated state. They are in a unique living state pressed because of the extremely high viscosity, complete known as anhydrobiosis. Such anhydrobiotic organisms in- dehydration is avoided due to the lower water vapor pres- clude bacteria spores, fungal spores, yeast cells, nematodes, sure of the vitreous state and solute crystallization is pre- rotifers, tardigrades, cysts, pollen, plant seeds and resurrec- vented. The vitreous state can also resist change of intracel- tion plants (9,18). More than 98% of their body water may lular pH and ionic strength during dehydration (6). be removed, and yet upon rehydration they are able to re- The vitreous state is confirmed in several dry biological cover their full metabolic capacity immediately. systems, including fungal spores, Artemia cysts and plant In the past decade, studies found that anhydrobiotic or- seeds [(4,5,18,27); Sun and Leopold, unpublished]. How- ganisms share some common features associated with their ever, the role of cytoplasmic vitrification in desiccation tol- desiccation tolerance (9). The dry organisms usually con- erance has not been critically examined. We scrutinize new tain high concentrations of soluble carbohydrates that stabi- evidence and discuss how vitrification, crystallization and lize membranes and macromolecules upon desiccation phase separation contribute to the survival or death of dry (7,9,18). These carbohydrates interact with membranes and biological systems. macromolecules and can replace water molecules during dehydration (7,9,10). By doing so, carbohydrates are able to protect anhydrobiotic organisms from desiccation damage. DESICCATION TOLERANCE Burke (6) suggested cytoplasmic vitrification as a bio- AND CYTOPLASMIC VITRIFICATION

Address reprint requests to: W. Q. Sun; School of Biological Sciences, Fac- Survival to extreme desiccation requires anhydrobiotic or- ulty of Science, National University of Singapore, Kent Ridge Crescent, ganisms to withstand enormous stresses that few organisms Singapore 119260, Republic of Singapore. Tel. 65-772-7932; Fax 65-779- 5671; E-mail: [email protected]. can tolerate (12,20). Water is the most important compo- Received 7 September 1995; accepted 18 January 1996. nent in living systems. It confers a structural order on mem- 328 W. Q. Sun and A. C. Leopold branes and proteins in cells and is involved in every life tion study showed no evidence of cytoplasmic crystalliza- process. As water is removed from the cells, a series of tion in either desiccation-tolerant and desiccation-intoler- events may occur: the increase of solute concentration, ant systems during dehydration (27). With phospholipid change of intracellular pH and ionic strength, the accelera- membrane model systems, Crowe et al. (8) recently made tion of destructive reactions, denaturation of proteins and similar observations during freeze drying. Their data indi- disruption of membranes. These events could disrupt all cated the importance of direct interaction between carbo- synthesis and metabolism and destroy the structural organi- hydrates and membranes for the preservation of membranes zation of cells and macromolecules. in addition to vitrification. However, as water is removed from the cells of anhy- During dehydration, the desiccation-intolerant tissues drobiotic organisms, the cytoplasm are expected to become are damaged at hydrations far above the water content vitrified (i.e., enter into a vitreous state), because these or- for cytoplasmic vitrification. Because the phase curve of ganisms accumulate high contents of soluble carbohydrates cytoplasmic vitrification is the same for both desiccation- that are known to be good vitrifying agents (6,11,14,17,30). intolerant and desiccation-tolerant tissues (Fig. 1), the The liquid-to-glass transition during cell dehydration can desiccation-tolerant tissues are not expected to become result in the immobilization of cellular structures and bio- vitrified at the similar hydration levels that begin to damage chemical components and therefore preserve their biologi- the desiccation-intolerant tissues. This fact is a strong argu- cal structures and capacities by minimizing stress damages. ment against the proposition that the cytoplasmic vitrifica- The vitreous state has been confirmed in fungal spores, tion is involved in the desiccation tolerance. It appears to Artemia cysts and plant seeds with various techniques, in- us that cytoplasmic vitrification cannot provide sufficient cluding differential scanning calorimetry, electron spin res- protection for anhydrobiotic organisms to survive cell dehy- onance and thermal stimulated current (4,5,30; Sun and dration. If cytoplasmic vitrification does not adequately pro- Leopold, unpublished). The thermal stimulated current and tect anhydrobiotic organisms during cell dehydration, what x-ray diffraction techniques permitted us to study whether possible benefits does it have for them? cytoplasmic vitrification plays any protective role during cell dehydration. We compared cytoplasmic vitrification in THE VITREOUS STATE AND desiccation-tolerant and desiccation-intolerant systems and SURVIVAL IN THE DESICCATED STATE failed to detect any difference of cytoplasmic vitrification that could be associated with the difference in desiccation It has been hypothesized that the vitreous state is associated tolerance of two biological systems (Fig. 1). X-ray diffrac- 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 in- versely 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 transla- tion 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 hypothe- sis may take more than 10 years to be completed, if experi- ments are to be conducted under physiological conditions. Recently, we used a mathematical approach to investi- gate 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 FIG. 1. Phase diagrams of glass transition of desiccation- equation, we have calculated the maximum temperature tolerant (soybean axis) and desiccation-intolerant (red oak (Tmax) for long-term storage of corn and soybeans over a cotyledon) tissues. Transition from glass to liquid state oc- range of water contents (Fig. 2). The temperature for long- curs upon crossing glass transition curve by increasing either term storage drops dramatically as water contents are ele- temperature at constant water content or water content when temperature is kept constant. Glass transition temper- vated; Tmax (curves) for long-term seed storage is in good atures (Tg) of red oak cotyledons were measured with the agreement with the glass transition temperature (Tg) (data thermal stimulated depolarization current (27). 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 (ϳ0.02 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.

and Leopold, unpublished data). In the vitreous state, free radical release is significantly inhibited. However, as hydra- FIG. 2. The vitreous state and the survival of plant seeds in tion level of the tissue increases and glass transition temper- the desiccated state. (A) Soybean and (B) corn. The phase ature decreases, the trapped free radicals are released. This diagram of seed glass transition (Tg, data points) is superim- result suggests that the accelerated deterioration of dry bio- posed onto the maximum temperature curve for a mean survival period over 50 years (T , curves). T shows good logical systems, when not in the vitreous state, is probably max g associated with the free radical release that causes damages. agreement with Tmax, indicating that the vitreous state has to be maintained for long-term seed survival. Glass transition The vitreous state also retards the nonenzymatic Maillard temperatures (data points) of seed axes and embryos are reactions involved in the deterioration of dry organisms adopted from Williams and Leopold (30) and Bruni and Leo- (29). In food products, lipid oxidation and Maillard reac- pold (4,5). Maximum temperatures for long-term storage are calculated by solving the seed viability equation (28). tions are commonly prevented in the vitreous state (16,24). Dashed line shows the extrapolated region from experimen- These observations provide evidence that the vitreous tal data. 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 the maintenance of the vitreous state is required for long- viscosity. term survival of the dry organisms. When seeds are not in the vitreous state, seed deterioration would be expected to CYTOPLASMIC CRYSTALLIZATION be accelerated (26). AND PHASE SEPARATION Free radical–induced reactions occur in many dry biological systems such as lyophilized virus and bacteria, mem- As we have already shown, the loss of the vitreous state branes, dry blood products and animal tissues. The loss of would accelerate the deterioration of dry biological systems. viability of dry cells and the deterioration of other dry prod- The vitreous state is thermodynamically unstable, and its ucts are correlated with the production and release (i.e., physical stability depends on the extremely high viscosity disappearance) of free radicals during storage (13). Figure 3 of such a system. When the vitreous state is lost (by either shows the release of organic free radicals in soybean embry- increasing temperature or increasing water content that de- onic axes in relation to the loss of the glassy state (Sun presses Tg), the viscosity is rapidly lowered and the solution 330 W. Q. Sun and A. C. Leopold 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. Crys- tallization and phase separation are important threats to processing and preservation of biological materials such as enzymes, genetic materials, membranes, cells and pharma- ceuticals. 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 de- struction 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 hav- ing high crystallization rate tends to lose seed viability more rapidly during dry storage. The distribution of solutes and water in dry cells is typi- cally 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 cy- FIG. 4. (A) Crystallization tendency of carbohydrate mix- toplasmic glass domains (26). Under conditions of acceler- tures and (B) seed longevity of 16 species (orthodox) with ated aging, low cytoplasmic viscosity would facilitate separa- different carbohydrate compositions. The rate of crystallization of sucrose/oligosaccharide mixtures, measured with tion of cytoplasmic matters and therefore help form various light refraction, increases when the mixture is dominated glass domains. Phase separation might prevent carbohy- with a single carbohydrate. Seeds with very low or high oli- drates from protecting cellular membranes and macromole- gosaccharide content are found to lose their viability more cules by making them unavailable. Phase separation of the rapidly, suggesting a possible role of crystallization in seed cytoplasmic glasses were observed recently in maize embryos death at the desiccated state. B is drawn with the data from Horbowics and Obendorf (15) and Lin and Huang (19). 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 glucose moieties and their lack of fit onto the surface of the be correlated with seed longevity. The preliminary data sug- growing crystal was due to the additional galactose moiety. gest an association of phase separation with seed longevity. Plant seeds may adopt the mechanism to improve their sur- It has been noted that oligosaccharides, especially raffi- vival in the desiccated state by using oligosaccharides such nose, seem to have a special contribution to the survival of as raffinose and stachyose to prevent phase separation and plant seeds. Raffinose content has been correlated with the cytoplasmic crystallization. vigor of aged seeds (1). The phase separation of cytoplasmic We know little how anhydrobiotic organisms that accu- glass is correlated with the raffinose level. As raffinose con- mulate trehalose prevent phase separation and crystalliza- tent increases, cytoplasmic glass seems more likely to stay tion, because like sucrose, trehalose may crystallize easily. at the high temperature domain (Fig. 5). Beneficial effects Glasses of pure trehalose, when stored slightly above room of raffinose can also be seen from Fig. 4, where it prevented temperature, become crystallized after a few days, even un- sucrose crystallization. Smythe (25) reported that raffinose der very dry conditions (22). However, studies with sugar and stachyose were the two most effective sugars that in- model systems suggested that protein may play a significant hibit sucrose crystallization. They inhibited sucrose crystal- role in preventing crystallization of trehalose glasses. It has lization because of their similarity to sucrose in the fructose- 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 Leo- pold (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 re- ture between carbohydrate mixes and embryo tissues sug- cent study on the crystallization behavior of bovine somato- gests the involvement of some factor other than sugars. It tropin (bSt)/sucrose glasses has found that the protein should be a highly hydrophilic polymer-like component in inhibited crystallization by increasing crystallization tem- thecytoplasmic vitrification ofembryo tissues. Thispolymer- perature of the sugar glasses (23). like component may have a strong effect on the property and organization of water in the cells, so that it can significantly increase T of the cytoplasmic glass at low water content CONTRIBUTION OF CARBOHYDRATES g 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 responsi- ble 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 FIG. 6. Glass transition phase diagrams for corn embryos and corn embryos and the carbohydrate mixes similar to those the representative carbohydrate mix (a mass ratio of 4:1 for found in vivo (Sun and Leopold, unpublished data). The sucrose and raffinose). Two phase diagrams do not match phase diagram for embryo tissues is distinctively different each other, showing that carbohydrates alone are not suffi- from that of carbohydrate mixes, indicating that the pres- cient to form the vitreous state in seeds. Glass transition of the carbohydrate mixture is measured with differential scan- ence of carbohydrates alone does not fully account for the ning calorimetry [᭺, data of this study; ᭝, data from (17)]. vitreous state in anhydrobiotic organisms. The study on the The Tg data of corn embryos are adopted from Williams and dependence of glass formation on hydration and tempera- Leopold (30) and Bruni and Leopold (4,5). 332 W. Q. Sun and A. C. Leopold

(Ͻ0.15 g/g dry weight), possibly by inhibiting the plasticiz- raffinose content and seed glassy state. Seed Sci. Res. 5:75– ing effect of water. On the other hand, its presence may 80;1995. 3. Blackman, S.B.; Obendorf, R.L.; Leopold, A.C. Desiccation lower Tg at high water content by resisting freeze-induced tolerance in developing soybean seeds: the role of stress pro- dehydration (Fig. 6). A good candidate of the polymer-like teins. Physiol. Plant. 93:630–638;1995. component is protein in the biological tissues. We suspect 4. Bruni, F.; Leopold, A.C. Cytoplasmic glass formation in maize that this polymer-like component is comprised of desicca- embryos. Seed Sci. Res. 2:251–253;1992. tion stress proteins (i.e., dehydrin and water stress protein) 5. Bruni, F.; Leopold, A.C. Pools of water in anhydrobiotic organisms: a thermally stimulated depolarization current study. that are commonly present in anhydrobiotic organisms. In Biophys. 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