Cytoplasmic Vitrification and Survival of Anhydrobiotic Organisms

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Cytoplasmic Vitrification and Survival of Anhydrobiotic Organisms 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 Vitri®cation and Survival of Anhydrobiotic Organisms Wendell Q. Sun* and A. Carl Leopold² *School of Biological Sciences, Faculty of Science, National University of Singapore, Kent Ridge Crescent, Singapore 119260, Republic of Singapore, and ²Boyce Thompson Institute for Plant Research at Cornell University, Ithaca, NY 14853, U.S.A. ABSTRACT. We examine the relationship between cytoplasmic vitri®cation 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 raf®nose and stachyose. During dehydration, these carbohydrates and other components help the organisms enter into the vitreous state (cytoplasmic vitri®cation). The immobilization by vitri®cation may minimize stress damages on the cellular structures and protect their biological capabilities during dehydration and rehydration; however, cytoplasmic vitri®cation alone is found to be insuf®cient for anhydrobiotic organisms to survive extreme dehydra- tion. 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 or- ganisms. He argued that cytoplasmic vitri®cation 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 con®rmed 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 vitri®cation 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 vitri®cation, 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 de- hydration (7,9,10). By doing so, carbohydrates are able to protect anhydrobiotic organisms from desiccation damage. DESICCATION TOLERANCE Burke (6) suggested cytoplasmic vitri®cation 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 vitri®cation. 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 vitri®ed (i.e., enter into a vitreous state), because these or- for cytoplasmic vitri®cation. Because the phase curve of ganisms accumulate high contents of soluble carbohydrates cytoplasmic vitri®cation 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- vitri®ed 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 vitri®ca- The vitreous state has been con®rmed 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 vitri®cation cannot provide suf®cient 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 vitri®cation 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 bene®ts does it have for them? cytoplasmic vitri®cation plays any protective role during cell dehydration. We compared cytoplasmic vitri®cation in THE VITREOUS STATE AND desiccation-tolerant and desiccation-intolerant systems and SURVIVAL IN THE DESICCATED STATE failed to detect any difference of cytoplasmic vitri®cation 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 vit- reous state, the cytoplasm is so viscous that diffusional movements are almost arrested, and therefore the deteriora- tion 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
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