Test of the linear relation between T_H and T_m was carried out for aqueous M(II)Cl_2 solutions (M(II)=Mg, Ca, Sr, and Ba). The linearity between T_H and T_m is excellent except the MgCl_2 solution. The linear relation does not hold for aqueous MgCl_2 solution in a stringent sense although it is a good approximation for describing the relation between T_H and T_m.

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Ability of plants to regulate the hydraulic conductivity inside the plant is crucial for sound growth and survival under adverse environment. In this manuscript, we demonstrated that low-root-temperature induces a decline in root hydraulic conductivity (Lp_r), which causes severe reduction in leaf water potential, stomatal conductance and hence transpiration in rice seedlings. In order to examine hydraulic regulation of rice plants at molecular levels, we identified 33 aquaporins in the genome sequences and characterized them by organ specific localization, water transport activity and stress responsibility of gene expression. We suggest that the low-root-temperature dependent decline in Lp_r, which is observed for several hours, is caused by the inactivation of aquaporins rather than by decrease in their abundances. We also showed that expressions of several root aquaporins are up-regulated during the subsequent acclimation process under low-root-temperature.

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Bryophytes such as mosses and liverworts generally possess a high degree of dehydration stress tolerance allowing them to survive long-term desiccation and freezing without causing significant damages. Abscisic acid (ABA), known to be involved in regulation of seed maturation and stomata closure in higher plants, has been postulated to play a key role in development of the dehydration stress tolerance of bryophytes. In the moss Physcomitrella patens, the presence of conserved cellular mechanisms of ABA-induced signaling and gene expression for the stress tolerance have been suggested. In contrast, very little is known about the mechanisms underlying ABA responses in liverworts. Our physiological analyses using the liverwort Marchantia polymorpha showed the role of ABA in dormancy and sugar metabolism. Recent molecular approaches have revealed the presence of conserved mechanisms for ABA signaling leading to gene expression and stress tolerance in liverworts.

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Virtually every cell responds to mechanical stimuli, by which cells can regulate their growth, shape, motility and differentiation. Mechanosensors are the major player in this cell function and mechanosensitive (MS) channels are the only established molecular class of mechanosensors to date. Among them, bacterial MscL is the best studied one owing to its resolved 3D structure. MscL is activated (opened) simply by tension increase in the plasma membrane, contributing to the cell volume regulation against hyopotonic challenge. To explore the biophysical mechanisms of MscL activation, we performed molecular dynamics (MD) simulations of structural changes of the MscL protein in response to tension increase. We found that MscL senses the tension increase mainly with Phe78 at the lipid-water interface, which induces a subtle change in the channel gate composed of hydrophobic amino acids that stabilizes the closed state of MscL. MscL opening is triggered by this subtle change and is accelerated by the penetration of water molecules into the gate region.

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Alkaliphiles, which are capable of growth in an extremely alkaline environment, serve as a model system to understand the physiological mechanisms allowing microbial growth under extreme environmental conditions. The internal pH of alkaliphiles is kept 2 units lower than the external pH 10 or higher. In alkaliphilic Bacillus, Na^+-dependent pH homeostasis involves the Na^+/H^+ antiport activities as well as Na^+ re-entry routes. A predominant role was evident for the NaChBac (voltage-dependent) Na channel of alkaliphilic B. pseudofirmus OF4 at sub-optimal [Na^+]. NaChBac mutants exhibit a significant loss of pH homeostasis capacity. Alkaliphilic Bacillus halodurans C-125 produces negatively charged acidic polymers that are associated with the peptidoglycan as secondary cell-wall polymers. Mutant strains which introduced mutations in genes encoding acidic polymers biosynthesis result in a growth defect according by elevated cytoplasmic pH. Alkaliphilic Bacillus clausii KSM-K16 was recently identified as the first bacterium in which a single stator-rotor of bacterial flagellar motor uses both protons and sodium ions for ion-coupling, depending upon the environmental pH.

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An African chironomid, Polypedilum vanderplanki is known as the only insect with a capability of an extreme desiccation tolerance, namely anhydrobiosis. In the 1950s and 1960s, Hinton strenuously studied anhydrobiosis in this insect from a physiological standpoint; however, nobody has afterward investigated the phenomenon. In 2000, research on mechanisms of the anhydrobiosis was resumed due to succeed in establishment of a rearing system of P. vanderplanki. This review is focused on the latest findings on physiological and molecular mechanisms underpinning induction of anhydrobiosis in P. vanderplanki.

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Mechanical force plays an important role in the interactions of cells with the extracellular matrix (ECM) in many tissues. The cell-ECM mechanical interactions affect cellular behaviors such as adhesion, proliferation, differentiation, and survival by activating intracellular signal transduction pathways. However, the molecular mechanisms on mechanical interactions between cells and ECMs remain incomplete owing to the difficulty to find out a molecule or event cues the signal of force transduction. To answer this question, it is necessary to measure the force exerted to the all adherent point and amount of activated molecules recruited to all that point at same time. For this purpose, we developed a new 3D micro- hydrogel pillars substrate based on a two- photon initiate polymerization technique. We found that cell can migrate on top of the substrate and cellular forces can be measured by deflection of each pillar during cellular migration.

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We have investigated the effect of guanidine hydrochloride (GdnHCl) on the hydration structure of 1-butyl-3-methylimidazolium chloride ([bmim][Cl]) used as a protein model compound. It was found that the addition of GdnHCl induces the change in the hydration structure of [bmim][Cl]. Our results indicate that the interaction between water molecules and GdnHCl is an important factor for the protein denaturation.

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We have investigated the low temperature freezing-induced changes in secondary structure of α/β- and β-proteins by the use of FT-IR spectroscopy. From the IR spectral analyses, we found that the low-temperature ice crystallization induces the increases of the β-sheet structure in α/β-proteins and the random coil structure in β-proteins. Our results indicate that the changes in secondary structure in aqueous proteins solutions at 77K depend on the contents of the β-sheet and the α-helix structures which are originally contained in the proteins.

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We attempted cryopreserved cultured rice cells by desiccation method. As routinely subcultured rice cell did not survive desiccation, the cells were precultured with 0.4M sucrose for 5 days. Precultured cell were desiccated under controlled relative humidity (RH) and cryopreserved in liquid nitrogen. High survival rate was obtained in the desiccated slowly under 70% RH for 18 h, however the cells desiccated rapidly under 8% RH did not survived both desiccation and cryopreservation. A culture medium for regrowth appeared to be important to enhance the survival of preserved cells. We removed NH_4NO_3 from and added glutamine to MS medium for the higher survival rate when cultured cryopreserved cells. These results suggest that the preculture treatment, slow desiccation under controlled relative humidity and suitable regrowth culture medium were required for the successful preservation.

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Triops cancriformis, which belongs to an aquatic crustacean family, primarily inhabits paddy fields in Japan. Triops's eggs can enter cryptobiosis (i.e., a metabolic dormancy), and become tolerant to dryness, low and high temperatures, and vacuums, and this ability allows for adaptability under the "half dry" conditions in paddy fields. In this study, to elucidate the mechanism underlying cryptobiosis induction and termination, we analysed trehalose, a cryptobiotic compatible solute, in T. cancriformis egg. Newly laied eggs contained very little trehalose and followed by accumulation of trehalose up to 1.2% of per dry mass with its dehydration proceeds. After rehydration most of eggs did not hatch and trehalose remained. This is unique because most of cryptobiotic organisms resume immediately after rehydration.

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Structured water and gas hydrate have some potential effect on the preservation of vegetables similar to cryopreservation. However, no practical method is proposed to observe the degree of water structured or how rigidly a gas hydrate is formed. Therefore, we examined the usage of fluorescence imaging in order to estimate the degree of water structured and to detect where gas hydrate is formed in a sample. Uranine solution was pressurized under a given Xe gas pressure and time-course fluorescence images were obtained by fluorescence microscope. It was found that the averaged intensity of fluorescence image was decreasing as time passed, i.e., the degree of water structured was strengthened. This phenomenon was inferred to be caused by quenching due to the concentration of fluorescent dye. By using this method, the change in the degree of water structured and positions where gas hydrates formed were visually distinguished.

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Most of the leaf tissues of cold hardy bamboos employ deep supercooling as the mechanisms of cold hardiness. Their mechanisms of deep supercooling and freezing injury still remain unknown. Here, we analyzed precise freezing behaviors in leaf tissues of Sasa kurilensis. Differential thermal analyses of both leaf blades and leaf sheath revealed that a large low temperature exotherm (LTE) initiated around -20℃. To reveal freezing behaviors during the LTE, we observed their freezing process using cryomicroscopy. Leaf blades have a ladder-like vein network. Mesophyll cells are compartmentalized in a tissue unit surrounded by lateral and longitudinal veins. The large LTE released from the leaf blade is most likely a combination of multiple freezing events: all the mesophyll cells in a tissue unit froze together in two sequential incidents (indicated by darkening of the unit) and tissue units randomly froze one after another in the first half of the LTE. In the latter half of the LTE, cells in the hypodermis and other tissues froze independently one after another (some of which were accompanied by bubble formation). Both types of freezing events were most likely to be intracellular freezing except for bubble formation (cavitation). LTE corresponded well with injuries of the tissues. The large LTE released from the leaf sheath arose from the random (intracellular) freezing of cell clusters. The tissue was not separated by lateral veins but the cluster of cells froze together (cluster size was different in a freezing incident from another). LTE corresponded well with injuries of the leaf sheath. In both leaf blades and leaf sheath, acoustic emission (AE) was observed during LTE. AE in leaf blades was well coincided with the bubble formation (cavitation) in bulliform cells and/or intracellular freezing in hypodermis. While that in the leaf sheath was more likely coincided with intracellular freezing of the component cells.

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Freezing process of plants is an important issue but only few analyses have been made and mechanisms involved remain unsolved. This is because freezing proceeds too fast and there have been no good methods to analyze the process. To address this problem, we utilized digital infra-red thermography and observed freezing events in wintering rabbit eye blueberry stems to characterize the initiation and spread of freezing. Referential images (raw images minus reference image) clearly visualized where the first freezing events occurred and how they spread in the stems. Differential images (differences between each neighboring images) more precisely visually detected where the first freezing occurred, in which direction and how fast the freezing front proceeded, how many times a portion of the stem froze and whether the heat release was from the surface or inside. Freezing curves for each spot on the stem extracted from the thermography images also allowed us to analyze freezing behaviors of the tissues. Differential images visualized the first freezing event (ice nucleation), which tended to be sharp and narrow temperature rise (probably from the surface) and slow in propagation. This was followed by the second freezing event, which tended to be vague and broad temperature rise (probably from inside) and fast in propagation. Freezing curve of the ice nucleation site clearly detected these two freezing events, the first small heat release and the second large heat release. Differential thermal analysis (DTA) revealed that excised bark had exotherm at much higher temperature than xylem and pith tissues. From these data, the following procedure of freezing in blueberry stems was hypothesized: 1) freezing initiates in the bark, 2) this slowly spreads in both directions or in one direction, 3) in some places this is transmitted to the xylem, 4) freezing in the xylem rapidly proceeds to cover the whole stem. A high ice nucleation activity in the bark seems to initiate spontaneous freezing of the stem.

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The plant growth regulator abscisic acid (ABA) is involved in triggering responses to various environmental stresses such as freezing and desiccation in higher plants but little is known about its role in lower plants, especially in liverworts. In the present study, effect of ABA on sugar accumulation and stress tolerance was examined using the liverwort Marchantia polymorpha. It was found that isolated gemmae of M. polymorpha cultured in a medium containing ABA accumulated higher amount of soluble sugars than did gemmae cultured without ABA. The sugar accumulation was further enhanced by addition of sucrose in the medium. The gemmae responded to ABA better than thalli in respect of sugar accumulation. Freezing tests showed that survival rate after freezing of 10μM ABA-treated samples increased as the sucrose concentration in the medium increased, but the survival was very little in non-treated samples. Desiccation tests showed the highest survival rate in samples treated with 5% sucrose and 10μM ABA. These results indicate that ABA plays a role in accumulation of sucrose in gemmae and enhances stress tolerance in liverworts.

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Cryopreservation of the nematode Caenorhabditis elegans in the adult stage is of importance as the nematode is a powerful model organism. In this study, we applied the protocol previously established for cryopreservation of the L4 nematode to the adult one. Survival rate of 84% was obtained as a result. When ice nucleation active bacteria, Pseudomonas syringae, was used for the simplification of ice seeding procedure instead of the use of a pre-cooled needle, survival rate of 82% was obtained after thawing. Moreover, a simple method of cryopreservation using a deep freezer and polystyrene foam was developed for a practical purpose. According to the observation under a cryomicroscope, eggs in the nematode body were found to freeze at ca. -20℃ when cooled at the rate of 0.2℃/min after ice seeding at -3℃ although the nematode body remained unfrozen down to -100℃.

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A DSC study was conducted to analyze the exotherm due to freezing of the suspension of Caenorhabditis elegans for the establishment of its cryopreservation. In the presence of Pseudomonas syringae used as an ice nucleator, temperature of freezing initiation of the nematode suspension was increased to ca. -4℃ from that obtained in the absence of the bacteria, ca. -23℃. Then, the freezing exotherm was deconvoluted into two parts, i.e., a peak part due to freezing of outside nematodes accompanied by that of inside nematodes inoculated from outside ice, and the following part due to freezing of water dehydrated from nematodes overlapped with spontaneous freezing of water inside nematodes. Results of the analysis well corresponded with those obtained with the cryomicroscopic observation.

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Cold acclimation (CA) results in alterations of plasma membrane (PM) lipid and protein compositions in plants, which is critical to increase their freezing tolerance. Recent studies indicate that PM is considered not homogeneous and contains sterol/sphingolipid-enriched microdomains. In plant cells, however, the function of plasma membrane microdomains is not yet fully understood. The present study aims to investigate lipid and protein compositions of microdomains isolated from non-acclimated (NA) and cold-acclimated (CA) leaves of oat and rye that differ considerably in freezing tolerance. We found that there are considerable differences in lipid and protein compositions in total PM and DRM (detergent-resistant plasma membrane) between the two species as well as before and after CA. From these results, we will discuss potential roles of plasma membrane microdomains in CA and freezing tolerance in plants.

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