In the anhydrobiosis state of desiccation-tolerant organisms such as sleeping chironomid (Polypedilum vanderplanki), metabolic reactions are completely suspended but the increase of their entropy is suppressed well unlike in the death (substrate) state. Probably there exists “entropy barrier” to be able to suppress entropy increase in the body of anhydrobiotic organisms. The entropy barrier is thought to be constructed from desiccation protectants such as trehalose and late embryogenesis abundant (LEA) proteins. In the past three decades, we have investigated the structure and function of these protectants from both experimental and theoretical viewpoints. Here, we summarize the main results of those studies. In the first part, we summarize the solution and solid-state properties of trehalose and the underlying mechanism of torehalose-induced desiccation tolerance in P. vanderplanki. In the second part, we summarize the results for the structure and function of LEA peptides, which are model compounds whose amino acid sequences consist of two or four tandem repeats of the 11-mer motif found commonly in native group 3 LEA proteins.

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During the fermentation of dough and the production of baker’s yeast, yeast cells are exposed to numerous and multiple environmental stresses including freezing, air-drying, and high-sucrose, so-called baking-associated stresses. In addition, such stress conditions could induce oxidative stress in yeast cells with an increase in reactive oxygen species level due to the denaturation of proteins including antioxidant enzymes and the severe damage to mitochondrial membrane or respiratory chain. To avoid lethal damage, baker’s yeast cells need to acquire a variety of stress-tolerant mechanisms. For example, proline and arginine are important amino acids involved in the stress tolerance of baker’s yeast. In fact, the engineering of proline and arginine metabolism is a promising approach for the development of stress-tolerant baker’s yeast. We believe that not only baker’s yeast but also other important industrial yeasts with higher tolerance to various stresses could contribute to yeast-based industry for the effective production of bread doughs and alcoholic beverages or the breakthrough of bioethanol production.

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Anhydrobiosis (“life without water”) represents an extreme example of tolerance adaptation to water loss, where an organism can survive in an ametabolic state until water returns. The sleeping chironomid Polypedilum vanderplanki inhabiting in semiarid region of Africa is the only insect known to be capable of anhydrobiosis. In the dehydrated larvae, massive accumulated trehalose is thought to replace the water in its tissues. Simultaneously, highly hydrophilic proteins called the late embryogenesis abundant (LEA) proteins are expressed in huge quantities and act as a molecular shield to defend other proteins and cell membranes against aggregation and denaturation. Trehalose together with LEA proteins forms a glassy matrix, which protects the biological molecules and maintains the structural integrity of larvae in the anhydrobiotic state. Meanwhile, reparation factors are likely to be involved in the anhydrobiosis. Recently the draft genome analysis of this insect has been accomplished. We determine that the genome of the sleeping chironomid specifically contains clusters of multi-copy genes encoding desiccation protectants and reparation factors. Here, we summarized recent topics on the molecular mechanisms underlying the anhydrobiosis in P. vanderplanki.

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The viscosity of potassium chloride-glucose-water ternary system with varying concentrations was investigated in this study. The density of the ternary system was measured with a Gay-Lussac-type pycnometer. The flow time of the ternary system was measured with an Ubbelohde viscometer. The viscosity of the solutions was calculated from the density and the flow time. All measurements were conducted at 25.0 ± 0.01°C. The viscosity curve is not simple. It decreases at lower KCl concentration then increase gradually with increasing KCl concentration. The tendency is emphasized with increasing glucose concentration. An increase or decrease in the dependence of viscosity on KCl concentration is seen in the binary KCl-water system as a function of temperature.

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Flavonol glycosides, which are a kind of flavonoid, are generally known as antioxidative substances and accumulated in various plant species. Recent studies showed that some flavonol glycosides possess anti-ice nucleation activities (or supercooling-facilitating activities), which results in the depression of freezing temperatures of the solutions in the presence of ice nucleating substances such as Erwinia ananas and silver iodide. In this study, supercooling-facilitating activities in the crude extracts from leaves, which may include supercooling-facilitating polyphenols, were compared among 65 plants (22 woody plants, 43 herbaceous plants) by droplet freezing assay using E. ananas as an ice nucleator. Relatively high supercooling-facilitating activities (>2˚C) were detected in the crude extracts from 14 plants. Since it is expected that the application of crude extracts with relatively high supercooling-facilitating activities may protect plant tissues from frost damages by ice-nucleating bacteria (Pseudomonas, Erwinia, etc.) on the surface of the tissues at relatively high subzero temperatures (about -2 ~ -3˚C), further characterization were examined using crude extract of Sasa senanensis with relatively high supercooling-facilitating activity. When partial purification was examined, the HPLC recovered fraction which may include flavone glycosides, which are also a kind of flavonoid, showed supercooing-facilitating activity.

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A method of preserving actively-growing cultured plant cells through exposure to liquid drying (L-drying) was examined. In vitro grown cells from a moss species (Pogonatum inflexum) were exposed to L-drying after washing and resuspending in liquid culture medium with or without trehalose. L-drying itself decreased survival rapidly with little further effect from temperature of storage, although survival was markedly improved by addition of trehalose into suspension medium. Cells suspended in medium containing trehalose survived L-drying at rates comparable to controls. Dropping the trehalose suspended cells on a piece of aluminum foil before L-drying was important for their subsequent survival through these procedures because the cells dropped on filter paper did not survived L-drying. Cells prepared by L-drying were successfully regrown after 4 weeks storage at 5°C. Thus, L-drying method, widely used for preservation of microorganisms, appears to be a technique which could be used for the preservation of actively-growing cultured plant cell.

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Many plants which experience subzero temperatures in winter increase freezing tolerance by exposure to non-freezing temperature, a phenomenon known as cold-acclimation. Recently, using a confocal fluorescent microscopy with cryostage, we found the extracellular freezing-induced membrane dynamics (cryodynamics) of the endoplasmic reticulum (ER) in cells of Welsh onion which can survive the winter below -40°C. For example, the extracellular freezing quickly led to stop the streaming of ER networks and then the networks collapse into vesicles. Furthermore, in cold-acclimated cells, the networks and streaming of ER are recovered within one hour after thawing. In this study, we focused on the generality of this cryodynamics in various plants which were collected in and near Iwate University in Morioka. In herbaceous plants sampled in winter, the streaming of ER networks was observed at nonfreezing temperatures, but in woody plants sampled from winter to summer the ER streaming was not observed. The ER streaming in herbaceous plant cells was quickly stopped by extracellular freezing but not by supercooling, while the freeze-induced ER vesiculation was not observed. In woody plants, the ER cryodynamics like that observed in herbaceous plants was not observed. Taken together, our results indicate that the extracellular freezing generally may result in the stop of ER streaming if ER streaming occurs in cells in the nonfreezing condition.

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Temperatures higher or lower than physiological temperature cause thermal stress, inducing cell damage and death. These thermal effects are utilized in various medical treatments. One cellular response to stress is morphological change. For example, vesicles and blebs often appear on the surface of the cell membrane. Therefore, gaining a better understanding of the time-series of morphological changes in cells is important, especially in relation to cell death. This study investigated the dynamics of morphological changes, including mobility of adherent cultured cells, under low-temperature stress near physiological temperature, using time-lapse three-dimensional imaging with digital holographic microscopy (DHM), which shows the thickness distribution of cells. The dynamics were roughly classified into two categories: (a) “normal behavior” and (b) “cell damage and death–related behavior.” Based on DHM data, the volume, projected area, average and maximum thickness, and position of the cells were analyzed to investigate deformation of cells, cell growth and division, formation of blebs, collapse of cell structure, and mobility (velocity) of cells.

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Abscisic acid (ABA) controls plants’ tolerance to environmental stress such as freezing and desiccation. We previously reported that AR7, the ABA-insensitive mutant of Physcomitrella patens has a defect in the ARK gene encoding the group B3 MAP kinase kinase kinase, with a single amino acid change from serine to phenylalanine in the non-kinase domain having a putative regulatory function. By ultraviolet mutagenesis, we obtained over one hundred ABA-insensitive mutants, from which two ark nonsense mutants were identified. These mutants showed inhibited protonemal growth with elongated gametophore in the absence of ABA, in contrast to AR7 showing protonemal growth similar to wild type. These results indicate that ARK is involved in regulation of plant growth in addition to stress responses.

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It is known that trehalose has the ability to protect biotissue against dry and low temperature stresses. We observed by high speed atomic force microscopy that lipid bilayers on mica bearing some defects are restored with addition of trehalose solution (5 wt%). Moreover, the orientation and structure of lipid bilayer changed in presence of trehalose with high concentration (10 wt%).

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For the investigation of cryoprotective effect of antifreeze glycoprotein (AFGP) on the nematode Caenorhabditis elegans and the Antarctic nematode Panagrolaimus davidi, we observed the freezing behavior and examined survival rate after freeze-thawing of the nematodes. When the Antarctic nematode P. davidi was cooled slowly to -75°C in the presence of 0.5% AFGP dissolved in M9 buffer, percentage of the unfrozen nematodes increased and most of them survived after thawing. In the case of nematode C. elegans that was cooled slowly, the nematodes in AFGP dissolved in M9 buffer could not survive though most of the nematodes in 0.5% AFGP dissolved in 5% DMSO could survive after freeze-thawing. The cryoprotective effect of AFGP was observed even when P. davidi was quenched by dipping it into LN2. When the nematodes were dipped in 0.5% AFGP dissolved in 5% DMSO and quenched with a cryo-plate, 79% of them could survive after thawing. Thus, it is clear that AFGP has cryoprotective effect on the nematodes.

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