Freezing adaptation and the injurious mechanisms of biology are diverse. Upon freezing of extracellular water in biological system, chilling sensitive plant cells are subjected to intracellular freezing due to lack of barrier property against penetration of extracellular ice through both cell walls and chilling-injured plasma membranes. Similar property of cell walls and plasma membranes which allow for extracellular ice penetration exists in central tissues of dormant bud in cold-hardy boreal trees. Thus, in such dormant buds, extraorgan freezing occurs to avoid formation of extracellular ice in adjacent with the central tissue cells. In the most biological cells, on the other hand, plasma membranes as well as cell walls become barrier against penetration of extracellular ice producing extracellular freezing. In extracellular freezing, there are two types of mechanisms to produce injury. One type is injury caused by direct effects of freezing-induced concentrated solutions. Such type of injury, furthermore, may divide into two cases, injury which occurs even during very fast non-equilibrium freezing (at cooling rates faster than minute level) and injury which occurs by very long time (more than day level) at equilibrium freezing. The former case is injury during cryopreservation in freezing sensitive cells and latter case is injury during long time exposure to freezing under high subzero temperatures even in cold-hardy plants. Exposure of cells in such concentrated solutions produced diverse kinds of ultrastructural changes in plasma membranes which closely related on occurrence of injury. Second type of injury by extracellular freezing is produced by physical effect due to cellular shrinkage by dehydration as well as due to cellular deformation by growth of extracellular ice crystals. Such extracellular freezing-induced physical effects produced membrane fusion in plasma membranes as a serious cause of injury. The most useful protective mechanism to prevent membrane fusions by such physical effects of extracellular freezing is accumulation of compartible solutes including cryoprotectants in case of cryopreservation. Upon formation of extracellular ice, some of cells in trees that develop thick and rigid cell walls are not affected by the presence of extracellular ice and keep liquid state of intracellular water by supercooling to very low temperatures for long time periods without dehydration. Such cells keeping supercooling accumulate supercooling-promoting diverse kinds of polyphenols.

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Neutron imaging is a useful method for studying the water distribution of intact plants, due to the strong interaction of neutrons with hydrogen. This interaction provides high image contrast, even in the presence of small quantities of water. Moreover, the combination of neutron imaging with low-contrast tracer D_2O enables direct visualization of water flow and calculation of water flow rates at the tissue level in plants, at high resolution. This article introduces these two visualization methods, and reports our most recent experimental results using these methods.

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When a near-infrared femtosecond laser is focused in water, several kinds of nonlinear phenomena, such as shockwave emission and cavitation bubble generation, are induced at the laser focal point. We have succeeded to induce crystallization by leading these phenomena in a supersaturated solution of protein. The mechanism of the crystallization would be strongly related with conformation change of water molecule binding with protein. Here, I introduce nonlinear phenomena when the laser is focused on water and representative example of the laser-induced crystallization.

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"Hydrophobicity" plays crucially important roles in a variety of phenomena in aqueous environments. It is widely believed that the hydrophobicity originates from the inability of nonpolar solutes to participate in hydrogen bonds of water. If this is true, the hydrophobicity should be strengthened when the hydrogen bonding is enhanced, for example, by lowering the temperature. However, there are experimental observations manifesting that the hydrophobicity is weakened at low temperatures. Here we clarify the true physical origin of the hydrophobicity by investigating cold denaturation of a protein as an important example. The major conclusions are as follows^ The hydrophobicity is caused primarily by the entropic excluded-volume effect; and its characteristics are ascribed to the interplay of the exceptionally small molecular size and the strongly attractive interaction of water, and not necessarily to its hydrogen-bonding properties.

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Freezing and desiccation injury in living organism (mechanical stress, fusion, aggregation and degeneration) is believed to be associated fundamentally with the behavior of water molecules in the organism living under such an extreme condition. This common understanding highlights the importance of water molecule, namely bound water, which interacts with biomolecules and/or cryo/lyo-protective agents. In this review, first the roles of bound water in biosystems are discussed, followed by introducing the several quantifying method of bound water. Thereafter, the recent author's results from the dielectric spectroscopy of bound water in gelatin-gel are discussed, in which the relationship between relaxation time of bound water and freezing / desiccation is suggested.

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Thermal unfolding of ribonuclease A (RNase), lysozyme, and α-chymotripsinogen A was analyzed in various aqueous solutions. The equilibrium between [unfolding]/[folding] ratio was correlated well to the water activity (aw) of the solution, which was described well by the modified Wyman-Tanford equation. This shows the important role of aw in protein stability. From this analysis, the change in hydration number (Δi) upon protein unfolding was obtained. Thus obtained Δi was much smaller than that based on the change in the accessible surface area in protein unfolding. By the integration of the reciprocal Wyman-Tanford plot, the free energy difference (ΔΔG) for protein stability in a solution and in pure water was calculated. TheΔΔG showed linear relationships with the concentration of solute, in most cases. This provides a theoretical basis for the empirical linear extrapolation model (LEM).

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Trehalose is known to act as a protectant against various environmental stresses, including drying, freezing, oxidation, ethanol stress, osmotic stress and so on. In this review, we discuss the origin of such a function of trehalose based on its hydration property. First, we summarize the solution properties of trehalose obtained from a variety of physicochemical measurements. Second, several results of recent computer simulations are described to provide atomistic level picture of the hydration property of this sugar. It is noted that trehalose has a highly anisotropic hydration shell, and thus it has amphiphilic nature, which allows complex formation not only with hydrophilic molecules but also with hydrophobic ones. Third, we describe our recent study showing that trehalose can form a stable intermolecular complex with benzene in aqueous solution. On the basis of this result, we propose a new mechanism for the inhibition effect of trehalose on aggregation of β-amyloid. Finally, it is stressed that the origin of these unique functions of trehalose is ascribed to the structural peculiarity of this sugar, that is, the presence of rigid α,α-1,1 glycosidic bond.

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Freezing tolerance is one of the most important characteristics for plants living at subzero temperatures in winter. When ice crystals form and grow in the extracellular spaces they may bring cells, especially cell surface, to the dehydration and mechanical stresses. Many physiological studies have supported the hypothesis that the enhancement of freezing tolerance in plants during cold acclimation is closely associated with an increase in the cryostability of plasma membrane. In Arabidopsis, many of the plasma membrane proteins including dehydrins, lipocalin and synaptotagmin increase during cold acclimation. Although there was little information about the freeze-induced mechanical stress tolerance, we have revealed that the extracellular calcium increases the tolerance to freeze-induced mechanical stress, the mechanism of which is involved in the membrane resealing via plant synaptotagmin, SYT1. Recently, besides Arabidopsis, we confirmed the calcium-dependent freezing tolerance in three monocot and one dicot. In addition, freezing tolerance tests of three monocots in the presence of anti-SYT1 antibody demonstrated that the calcium-dependent freezing tolerance results in the membrane repair. Finally, our results support the idea that the calcium-dependent membrane repair is a common mechanism of freeze-induced mechanical stress tolerances for many angiosperms.

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Dimethyl sulfoxide (DMSO) is one of the useful cryoprotective agents for a variety of cells and tissues allowing prolonged storage at subzero temperatures. Since most of the optimum DMSO concentrations for the different cells and tissues cryopreservations are similar, we predict the reason for the optimum DMSO concentration by measuring the thermodynamic properties of culture medium including DMSO and by observing the toxicity of DMSO on the neonatal rat cardiac myocytes. The melting point measurements suggest that the larger DMSO concentration medium has lower melting points, which would prevent the cell damage by ice reformation during the thawing process. However, such medium has higher toxic effect on the cryopreserved cells. These two contrary factors can predict the optimum concentration of DMSO in the medium for the cryopreservation of cells including the cardiac myocytes.

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We have investigated the additive effect of 1-butyl-3-methylimidazolium acetate ([bmim][acetate]), which is one of the imidazolium-based ionic liquid (IL), on the stability of secondary structure of lysozyme in water by the use of FT-IR spectroscopy. From the IR spectral analyses, we found that the population of the α-helical structure of lysozyme decreases up to 6 M. However, the population of the α-helical structure at 10 M is higher than that at 6 M. This result means that the degree of protein unfolding becomes smaller above 6 M. Our result shows that [bmim][acetate] has a possibility for a new preservation agent.

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The purpose of this study was to examine use of freeze-drying microscopy (FDM) analysis to obtain maximum allowable product temperature during primary drying segment of pharmaceutical lyophilization process. FDM analysis of frozen solutions containing non-crystalline lyoprotectants (trehalose, sucrose, PVP 29,000) showed beginning of physical collapse at temperatures (T_c) several degrees higher than their glass transition temperature of maximally freeze-concentrated solutes (T_g') obtained by thermal analysis. The T_c and T_g' depended on the solute concentration, scanning rate, and cell pressure during the analysis. Loss of the cake structure upon lyophilization of the solutions at the primary drying shelf temperatures above their T_cs indicated relevance of the FDM analysis for the process development.

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Upon dehydration, the sleeping chironomid (Polypedilum vanderplanki) larvae decrease water content and simultaneously increase trehalose content, to eventually enter into a state of a metabolically suspended animation, i.e., anhydrobiosis, after 48 h of the treatment. Intriguingly, the desiccated larvae can resume the activity once rehydrating. In addition to trehalose content elevation, expression of genes encoding stress-related proteins, such as HSP and LEA proteins, are thought to be deeply involved in desiccation tolerance. However, fine molecular mechanisms underlying anhydrobiosis have remained to be elucidated. Exhaustive gene expression analysis using microarray technique showed that a large number of genes annotated as related to protein degradation mechanisms, including ubiquitin-proteasome system, ER-associated protein degradation and lysosomal hydrolases was upregulated in the larvae upon dehydration. Based on the result, we expected that protein degradation might play an important role to invoke desiccation tolerance.

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Structural stability of lecithin liposomes during freeze-thawing was studied by investigating their freezing behavior and effects of ice nucleators. DSC cooling traces of the liposome suspension showed exotherms due to the freezing of extra- and intraliposomal solutions. The exotherm due to freezing of the intraliposomal solution hardly changed even if freezing was repeated after thawing. Liposomes became larger when frozen in the liquid nitrogen followed by thawing, and the extent of the exotherm due to freezing of the intraliposomal solution increased as a result. In the presence of Pseudomonas syringae, freezing temperature of the extraliposomal solution shifted to a higher subzero temperature and amount of freeze-dehydration from the liposome increased, which caused the decrease in the exothermic heat due to the intraliposomal freezing. Judging from the leakage of encapsulated ATP, it was indicated that the vesicular structure of lecithin liposomes was hardly destroyed by the intraliposomal freezing, nor was it destroyed by the substantial freeze-dehydration.

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Cowpea (Vigna unguiculata (L.)Walp.) is one of the most important legume in semi-arid region. In this study, we aimed to investigate the physiological response in three cowpea lines producing with low yield caused by drought stress at flowering stage. Prolonged drought stress decreased seed yield and pod number in the three lines tested. A drought susceptible line, IT95K-238-3, showed a sharp decline in the number of pods as well as in the number of flowers under drought stress. The number of flower bud formation decreased and that of flower bud abscission increased during drought stress. These effects were severe in the drought susceptible line. Leaf stomatal conductance and relative water content of peduncle apex markedly decreased in the most susceptible line exposed to drought stress. Based on these results, it was suggested that both stomatal closure in leaves and the decline of water content in peduncle apex induced by drought stress restricted flower bud formation and enhanced flower bud abscission. As a result, these factors caused a reduction of the number of flowers and seed yield.

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Pre-harvest sprouting (PHS) in wheat causes devastating damage to the quality of wheat flour owing to the degradation of seed starch during germination. In this study, we investigated whether nuclear magnetic resonance (NMR) relaxation times (T_1, T_2) could be used to characterize PHS in wheat (Triticum aestivum L.) cultivars, 'Shirogane-Komugi' (sprouting susceptible), 'Norin61' (sprouting resistant) and 'Chikugoizumu'(intermediate type). The PHS rate of 'Shirogane-Komugi' markedly increased to about 65% at 28 DAP while PHS rate of 'Norin61' was almost zero at the stage. The water contents in the wheat seeds of the three cultivars were not significantly different during ripening stages. T_2 values of long fraction in 'Shirogane-Komugi' were about 130 ms and those of 'Chikugoizumi' indicated around 75 ms at 28 DAP. On the other hand, those of 'Norin61' decreased and was about 60 ms at 28 DAP. T_2 values of long fraction of all of the three cultivars markedly decreased to less than 1 ms at 42 DAP. These results suggested that free water in 'Shirogane-Komugi', 'Chikugoizumi' and 'Norin61' seeds was maintained until 28, 21 and 14 DAP, respectively. The PHS sensitive cultivar, 'Shirogane-Komugi' kept free water 14 days longer than the resistant one. Therefore, NMR relaxation time, especially, T_2 is effective for early screening of pre-harvest sprouting characteristics.

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Rattail fescue (Vulpia myuros (L.) C.C. Gmel.), a winter annual grass weed, originated in the Mediterranean region, and widely distributed in temperate region across the world. Lodging characteristics of the rattail fescue are utilized as a crop cultivated in orchard garden for the purpose of various weed control, protecting against soil erosion and maintaining ground temperature. Although mechanism of lodging resistance in crops has been studied, the causative factor of lodging in rattail fescue remains unknown. Lodging of the plant culm was enhanced by 25℃ and followed by 20, 30, 15℃ treatments. NMR spin-lattice relaxation times (T_1 and T_2 ) of fresh leaves and culms in Arrhenius plots decreased when temperature elevated from 0 to 50℃. However, there was no distinctive feature in water status of the tissues between 15 and 30℃. Furthermore, electrolyte leakage, contents of water, chlorophyll and nitrogen of the plant leaves and other organs did not significantly change when the plants began to lodge. In contrast, node angle markedly decreased due to remarkable cell elongation of convex side in individual nodes, especially in the basal node at the lodging stage. These results indicate that lodging characteristics of rattail fescue coincided with initiation of epinasty of the basal node.

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Supercooling-facilitating (anti-ice nucleation) substances, which are several flavonol glycosides and hydrolyzable tannin, are found in xylem parenchyma cells in trees to keep a liquid state of intracellular water to -40℃ in winter. It is gradually discovered that other associated compounds also have supercooling-facilitating activity in the solution containing ice-nucleating bacteria, Erwinia ananas. These supercooling-facilitating substances are expected to be used in real world application. However, for their applications, judging the supercooling activity not only by standard droplet freezing assay in solutions containing different kinds of ice nucleators but also by more practical methods using a large volume of solutions under shaking may be required. In this study, we measured the supercooling-facilitating activity of several kinds of flavonol glycosides and tannins by droplet freezing assay in solutions with various ice nucleators and by shaking assay in bulk solutions. It was revealed that supercooling activity of substances tested in this study varied according to measurement condition, such as types of ice nucleator, volume of solution, concentration of substances and the presence or absence of shaking.

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Arabidopsis thaliana increases its freezing tolerance upon exposure to chilling temperature (e.g., cold acclimation, CA). CA results in changes of lipid and protein compositions in the plasma membrane. We recently found that one of the dynamin-related proteins (DRP1E) that are associated with endocytosis pathway accumulated in the plasma membrane during CA. Thus, we hypothesize that DRP1E involves in CA-induced modification of the plasma membrane. To address this question, we characterized a T-DNA insertion mutant of DRP1E (drp1e) in the CA process. CA increased freezing tolerance in both wild type and drp1e plants but the extent was much less in drp1e mutant. Furthermore, the effect of DRP1E knockout on the extent of cold acclimation was more apparent in younger leaves than in older (and mature) leaves and considerable differences in cold-responsive proteins were found in the plasma membrane of younger leaves between the wild type and drp1e plants. These results suggest that DRP1E have critical roles in freezing tolerance in young leaves.

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Many plants living under subzero temperatures in winter increase freezing tolerance by exposure to non-freezing temperature, which is known as cold acclimation. In cold-acclimated cells, unique cryobehaviors of the plasma membrane and endoplasmic reticulum (ER) have been reported but their physiological meaning or mechanism is largely unknown. Allium fistulosumis a cold-hardy Welshonion which survives winter of-40℃ in Saskatchewan, Canada, andintact cells inthe single epidermal layer, which is easily peeled from leaf sheath, were observed. The cryobehavior of ER in these epidermal cells that were stained with ER-selective fluorescent dye (ER-Tracker) was observed using a confocal fluorescent microscope with cryostage. According to our observations, cold acclimation increased ER volume per cell and extracellular freezing induced ER vesiculation through the breakdown of the ER network. Freeze-induced ER vesicles in cold-acclimated cells were larger and more abundant than those in non-acclimated cells. ER vesiculation may be associated with extracellular calcium because freeze-induced ER vesicles tended to be more abundant in the presence of calcium than in the absence of calcium. Furthermore, ER vesiculation also occurred in Arabidopsis root cells, suggesting a possibility that ER vesiculationis conserved in monocotyledonous and dicotyledonous plants. After thawing, the ER network was recovered only in cold-acclimated cells, suggesting that the dynamics of ER during freeze/thaw cycles are associated with freezing tolerance.

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