Many plants living in temperate regions acquire freezing tolerance after exposure to non-freezing low temperatures over a certain period. This phenomenon is known as cold acclimation. To search novel genes involved in cold acclimation, mutants of Arabidopsis thaliana that have an increased freezing tolerance without cold acclimation were isolated using the T-DNA activation-tagging method. Calli derived from hypocotyls of about 33,000 seedlings were co-cultivated with Agrobacterium tumefaciens harboring pPCVICEn4HPT that contains enhancers originated from the 35S promoter of cauliflower mosaic virus, to generate the activation-tagging callus lines. The T-DNA-inserted calli were selected for their ability to grow in the callus-inducing medium (CIM) containing hygromycin for two weeks and to remain alive after a subsequent freezing treatment at -14℃ for 3 h. Consequently, only 47 transformant lines were found to survive. In 34 of these lines, loci of insertions of T-DNA were determined by thermal asymmetric interlaced-PCR (TAIL-PCR): single T-DNA insertions were seen in 28 lines, while double insertions in 6 lines. Furthermore, we harvested seeds from regenerated plants of these tagging lines and re-evaluated freezing tolerance of the calli from the seedlings. The calli from 6 mutant lines were more freezing-tolerant than that from the wild-type plants. In two of these mutant lines examined with semi-quantitative reverse transcription-PCR (RT-PCR), a gene located near the insertion of enhancer elements was activated. A basic local alignment search tool (BLAST) search showed that the gene activated in the one line encodes a protein with an Armadillo-like helical motif and that activated in the other line encodes a protein belonging to a fibrillin family

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Water deficit is a main constraint on crop production in the semi-arid areas. Cowpea (Vigna unguiculata (L.) WALE) is one of the most drought resistant crops. Gene expression of aquaporins, water channel proteins, is induced by water stress indicating the necessity of facilitated water transport to the shoot. In order to identify the drought resistant characteristics in cowpea plants, transpiration rate, water content of leaves and roots in relation to aquaporin were studied. Following transpiration rate of cowpea leaves markedly decreased with the decrease in soil water until 7 days after treatment (DAT), it proceeded with minor change during 45 DAT while there was no change in water content of leaves and roots throughout the drought stress. Aquaporin-related proteins with a relative molecular mass of 30 kDa were detected in microsomal membrane fractions extracted from leaf and root tissues of both control and drought-stressed cowpea plants by immunoblot with an anti-aquaporin specific antibody. As a result, there were no differences in drought stress as well as organs. Interestingly, phosphorylation of serine residue at 30 kDa as a same size with the aquaporin-related protein, was detected in microsomal polypeptides extracted from roots of drought- stressed cowpea, but not in those of leaves. These results indicate that further studies on phosphorylation of aquaporins will reveal the water status characterizing the marked drought resistance in cowpea.

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Trehalose is a non-reducing disaccharide of glucose that has functions as a protectant in the stabilization of biological structures and enhances the tolerance of organisms to abiotic stress. In this study, we focused on the tomato (Solanum lycopersicum L.) trehalose-6phosphate synthase (TPS) gene and analyzed the gene expression and the protective function for environmental stresses. By semi-quantitative RT-PCR analysis of tomato leaves treated with four different stresses such as 50 μM ABA, drought, 250 mM NaCl, chilling (10℃), it was found that an expression of tomato TPS gene was most strongly induced by chilling stress. Although ^1H-NMR spin-spin relaxation time T_2 and water content of tomato plants treated with chilling stress were higher than those of control, those treated with chilling stress and 100 mM trehalose maintained the same level of control. By 2,3,5- trip he nyltetrazolium chloride (TTC) reductive method, it was observed that trehalose reduced chilling injury. These results suggested that TPS may be involved in chilling tolerance of tomato plants.

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Group-3 late embryogenesis abundant (G3LEA) proteins are expressed in anhydrobiotic organisms experiencing desiccation stress and their major segments consist of repeated and conserved 11-mer amino acid units. However, little information has been obtained with regard to the properties of these repeating units. Here, we synthesized three model peptides, each of which consists of two repeating units, and investigated their structures in solution and dry states by FT-IR spectroscopy. Furthermore, we examined how the structures of these peptides are influenced by addition of trehalose because this sugar is also produced massively in the dried cells of anhydrobiotic organisms. Consequently, it was shown that the secondary structure of these peptides transform from random coils to a-helical coiled coils in the response to dry stress, even in the presence of trehalose. In conclusion, the 11-mer repeating units act as core segments which could drive the structuralization of G3LEAs in the dry state.

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Effects of the dehydration rate and the co-existing salts were investigated on the conformational structure of a LEA protein from P. vanderplanki, PvLEA2, which is composed of 180 amino acid residues, with a molecular weight of 20.6 kDa. The aqueous solution of the desalted pure LEA protein at 1mg/ml was vaporized at a constant rate in an environment with a given relative humidity (RH) kept constant. The resulting LEA protein with the residual water content being as low as 3 wt% had the content of a-helical structure at ca. 60% relative to the whole, except for the case of drying at RH = 98% which yielded ca 50% content of a-helical structure of the dehydrated LEA protein. The conformational structure of the dried LEA protein depended on the kinds of co-existing salts, being predominantly a-helical and 6-sheet structures in the presence of NaCl and CaCl_2, respectively. These findings suggest that it is controversial to apply the ion-sequestration mechanism to the relationship among the dehydrated LEA protein and all kinds of ionic species.

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According to our recent NMR study, trehalose forms an intermolecular complex with benzene in aqueous solution, To elucidate the main driving force of such complex formation, we calculated the potential of mean force (PMF) as a function of the distance (R) between the center of mass of trehalose and that of benzene, PMF exhibited the single minimum at a value of R≈5Å, where the benzene molecule was located around the C1(C1') and C2(C2') methine groups and the exocyclic methylene groups of the trehalose. This structure was in good agreement with that predicted from the ^1H-^1H NOESY measurements. On the other hand, the MD simulation of the aqueous solution of trehalose indicated that dehydration pockets are present around C1(C1') and C2(C2') atoms and the exocyclic methylene groups. The positions of such pockets were shown to coincide with the regions where the benzene is located with a high probability. In conclusion, benzene binds to trehalose in a fashion that dehydration penalty, accompanied by intermolecular complex formation, is minimized.

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Physical properties and protein-stabilizing effects of pharmaceutical excipient combinations in freeze-drying were studied. Frozen solutions containing basic amino acids (e.g., L-arginine) and organic acids (e.g., citric acid) showed thermal transition of maximally freeze-concentrated solute (T_g') that were significantly higher than those of the single-solute frozen solutions. Freeze-drying of solutions containing some basic amino acids and dicarboxylic acids resulted in glass-state amorphous solids that protected secondary structure of bovine serum albumin (BSA) and activity of lactate dehydrogenase (LDH) from lyophilization-induced changes. Thermal analysis and infrared spectroscopy suggested contribution of electrostatic interactions and hydrogen-bonding to decrease mobility of molecules in the glass-state amorphous solids.

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In cryobiology, the freezing of tissues needs further detailed clarification. This study investigated microscopically the freezing behavior and subsequent post-thaw histological change in a complex tissue, namely, aortic media, which consisted mainly of intricately intertwined elastic fibers, collagen fibers, and smooth muscle cells. The freezing behavior was observed using two methods: optical microscopy of a section of a frozen sample and field emission scanning electron microscopy of a frozen sample. Comparison of the relation between the freezing behavior and the post-thaw histological changes observed in this study for aortic media and that previously reported for muscle tissues indicates that the presumption of freezing behavior based on the observed histological changes depends on the type of tissue.

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By means of fluorescence correlation spectroscopy (FCS), a decay component faster than the translational diffusion of the fluorescent probe molecule was observed in highly concentrated aqueous solutions of saccharides. We concluded that the fast decay is due to the increased lifetime of the excited triplet (T_1) state of the probe molecule. The lifetime of T_1 state was too short to be observed by FCS in aquous solution, because of the quenching by oxygen molecules (O_2). With high concentration of saccharides, the quenching is reduced because the encounter of the O_2 molecule with the probe molecule is interrupted. Time-resolved transient absorption spectroscopy was carried out and it was confirmed that T_1 state lifetime lengthened in highly concentrated viscous saccharide solutions. It was concluded that either the concentration or translational diffusion rate of oxygen molecule are reduced.

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Germination phenomena in wheat seeds treated by temperatures, plant hormones and ascorbic acid (AsA) were investigated. At 28 days after pollination (DAP), none of the seeds treated by ABA or AsA germinated while the seeds treated by distilled water alone and GA_3 germinated, and there was no temperature dependence. At 35 DAP, germination of the seeds treated at 10℃ was promoted significantly while that treated by AsA was markedly suppressed. In mature seeds, germination treated by GA_3 was promoted at higher temperatures while AsA remarkably suppressed seed germination and shoot elongation in all temperatures tested. In ^1H-NMR analysis, the spin-spin relaxation times (T_2) of germinating seeds treated by ABA and AsA showed about 34% and 19% of the control. These results indicated that AsA significantly suppressed both germination and seedling growth of wheat without temperature dependence.

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For the establishment of successful cryopreservation of Caenorhabditis elegans, freezing survival of the nematode was investigated in the presence of cryoprotectant. Dimethylsulfoxide (DMSO) was found, as a result, to be more effective in the cryoprotective activity than glycerol. Highest activity of DMSO was obtained at the concentration of ca. 5 v/v % in the M9 buffer. In the presence of DMSO, survival rate of the nematode decreased with the life stage from L1 to L4. It also depended on the dipping time in the DMSO solution before freezing: it increased with the time until about 1 hour but decreased with further dipping time. Cooling rate, lowest temperature of cooling and initiation temperature of freezing also affected freezing survival. Slow cooling prevented complete freezing in a larger proportion of nematodes than fast cooling. When accompanied by the treatment of ice seeding, slow cooling caused higher survival rate.

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Protoplasts were isolated from cultured cells of Pogonatum inflexum by treatment of enzyme solution containing 2%(w/v) Driselase, 0.6 M mannitol and 5 mM CaCl_2. Isolated protoplasts were re-suspended in different liquid medium and desiccated in a chamber maintaining 50% humidity. Protoplasts survived the desiccation and a subsequent cryopreservation in liquid nitrogen (desiccation-cryopreservation). Composition of suspension medium influenced the survival rate of desiccated protoplasts. Desiccated and desiccated-cryopre served protoplasts were also cultured and clarified to retain the cell division potential.

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Recently, the long-term preservation of various microalgae has become possible. However, it remains still unsuccessful with Spirulina sp. which forms a peculiar spiral trichome. In this study, we conducted freeze-thawing treatment of Spirulina sp. aiming at its long-term preservation. The spiral trichome of Spirulina sp. was found to be destroyed into short trichomes by the treatment and the cells were damaged one after another from both ends of trichome eventually. We found, however, that the spiral trichome was less destructed by removing water around the trichome before freezing. Spirulina sp. was extremely susceptible to drying. Finally, viability above 50% was obtained with Spirulina sp. in the stationary phase by partial drying followed by 2-step freezing.

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Anhydrobiotes, which have a high degree of desiccation tolerance, form a glassy state during extreme desiccation. Vitrification has previously been found in cultured plant cells treated with sucrose-enriched medium in a dry state. We have found that desiccation tolerance of Marchantia polymorpha cells is increased by preculture in a medium containing 0.5 M sucrose. However, it was difficult to induce the same high degree of desiccation tolerance in Arabidopsis thaliana cells by the same method. Using cultured M. polymorpha and A. thaliana cells, we examined the exact relationship of desiccation tolerance to glass formation. Thermal analysis by differential scanning calorimetry (DSC) showed that the glass transition temperature (Tg) in M. polymorpha precultured cells was higher than that in A. thaliana precultured cells at the same water content. A. thaliana precultured cells were vitrified easily but did not survive in this severe condition. The results suggested that precultured plant cells are easily vitrified at ambient temperatures after desiccation. For the development of desiccation tolerance in cultured plant cells, it is important to protect the cells against damage caused by dehydration before vitrification.

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Developments of cryopreservation techniques on primary rat cortical cells were carried out to observe the effect of cooling rates. Primary rat cortical cells were dispersed in the cryopreserving medium including 5 vol% dimethylsulfoxide, and then they were frozen by various cooling rates ranged from -3K/min to -150K/min. The survival ratio was estimated qualitatively by using the phase-contrast microscope. The feasibility of the neural activity was verified by measuring its spontaneous action potential with a multi-electrode array system. Based on these characterizations, the lower cooling rate of the dispersed sample was more feasible for the cryopreservation of the primary rat cortical cells.

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We have investigated the coordination behavior of the acetate ion in aqueous rare earth chloride (LnCl_3; Ln^<3+>=La^<3+>-Lu^<3+>) solutions in the glassy state by a Raman spectroscopy. The series behavior of the symmetric Raman Ln-OH_2 stretching band (v_w) for the [Ln(H_2O)_n]^<3+> complex and the Raman C-C stretching band (vcc) for the acetate ions is discussed in conjunction with the coordination number change in the middle of the series. It is concluded that the Ln^<3+>-acetate complex changes from a polymeric chain structure to a bidentate ligand.

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We have investigated the coordination behavior of the rare earth (Ln^<3+>) ion in N-methylaceatmide (NMA) solution at 77 K by a Raman spectroscopy. Series behavior of the symmetric Raman Ln-Cl stretching band (v_<Ln-Cl>) and the Raman amide III band (v_<AIII>) of the NMA is discussed in conjunction with the coordination number change in the middle of the series. It is concluded that the elimination of NMA molecule induces the coordination number change of the Ln^<3+> ion.

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The terahertz frequency band bridges the molecular motions of dielectric relaxations and oscillatory, intermolecular motions. The terahertz time domain spectroscopy (THz-TDS) provides the solutions to observe optical properties in the terahertz frequency region. To understand the terahertz dynamics of glass formers, preliminary we demonstrate the aqueous solutions of well known glass formers, ethylene glycol, and propylene glycol. The frequency dependence of complex permittivity in the terahertz region is determined by dual thickness geometry. The dielectric constant of aqueous solutions shows clear concentration dependences. The combination of Cole-Davidson and Debye equations is applied to determine relaxation time and strength of main relaxation and the relaxation at terahertz region. The fast relaxation process can be attributed to the fast motions of OH moiety of water and glass forming materials.

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We studied the cluster structure of ethylene glycol aqueous solutions by Brillouin and Raman scattering. We measured the ultrasonic sound velocity of the sample by Brillouin scattering. From the concentration dependence of the sound velocity, we discussed the properties of cluster structure related to glass transitions. The number of H_2O molecule neighboring a EG becomes a little higher with increasing temperature and the association between EG molecules and H_2O molecules weakened with increasing temperature. We showed that Brillouin scattering is a powerful tool to investigate the dynamics of cluster structure in liquid. The OD stretching bond was measured by Raman scattering to discuss the hydrogen bonds among molecules.

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Homogeneous ice nucleation (T_H) and melting (T_m) temperatures were measured for emulsified aqueous solutions of NaCl, methanol, and (CH_3)_4NCl as a function of solute concentration. There is a linear relation between T_H and T_m for aqueous NaCl and (CH_3)_4NCl solutions, but not for aqueous methanol solution. Qualitative interpretation is given for the non-linear relation between T_H and T_m for aqueous methanol solution.

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