A secondary-metabolic compound, 3-O-feruloylquinic acid, was isolated from winter wheat (Triticum aestivum L. cv Chihokukomugi) and identified by NMR, MS and UV spectral analyses. HPLC analysis showed that the accumulation of the compound in winter wheat was triggered by exposure to low temperature. In addition, ESR spectral analysis showed that the compound exhibited antioxidative activity.

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Shoot apices of blue honeysuckle (Lonicera caerulea L. var. emphyllocalyx Nakai) in vitrcrcultures were successfully cryopreserved utilizing two methods of encapsulation-dehydration and vitrification. Encapsulation-dehydration, however, seems preferable to vitrification, since the highest survival rate of apices (93.3±3.3%) in encapsulation-dehydration was higher than that (50.0±5.8%) in vitrification. The highest survival was achieved when moisture content in beads was reduced to 23% by air-drying with silica gel for 4 hours in encapsulation-dehydration. In both cases, cold acclimation of cultures at 5℃ for 2 weeks was essential. However, in the present study it was shown that immersion of alginate-coated apices for 16 hours in loading solution supplemented with 0.5M glycerol and 0.1mM salicylic acid resulted in a high survival rate (93.3±6.7%) that can eliminate the cold acclimation process.

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Carrot, broccoli and potato tissue were osmotically dehydrated by immersion in 50%(w/w) sucrose solution and their rheological properties were measured before and after freezing-thawing-rehydration. Although the dynamic elasticity and viscosity became smaller after freezing-thawing as compared to the fresh sample, the dehydrated samples, except potato, showed much higher retention of rheological properties than the untreated sample. In measurement of impedance, the radius of the Cole-Cole arc, an index of the intactness of cell plasma membrane, showed good correlations with the rheological properties for carrot and broccoli. The osmotically dehydrated sample showed much less drip than the untreated sample after freezing-thawing-rehydration. These results indicate that osmotic dehydrofreezing protects the cell structure, in particular the cell plasma membrane, against freezing injury to give reduced softening after freezing-thawing.

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It is well known that an application of low temperature for a certain period induces freezing tolerance in plants including Arabidopsis plant. Although many studies have been conducted to elucidate molecular mechanisms of the acquisition of freezing tolerance, it is still difficult in some cases to determine how Arabidopsis responds to these treatments at the cellular level. To study such responses, we initiated characterization of kinetics of cold acclimation process with Arabidopsis T87 suspension cultured cells. In this study, we describe the changes of freezing tolerance, sugar content and gene expression during cold acclimation. These results may provide new information about molecular mechanism of cold acclimation in plants at the cellular level.

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Dynamic states of water in seeds of rice (Oryza sativa L., cv. Hinohikari) plants grown at low/high temperatures during seed maturation were examined by NMR relaxation times (T_1 and T_2). Dry weight of grain grown at 20℃ gradually increased while both 25 and 30℃ enhanced growth rate at early- to mid-stage. T_<1s> in the seeds treated by three different temperatures related to their water content until 36 DAF (days after flowering). T_2 values of grain treated both at 25 and 30℃ sharply dropped to less than 1ms after 22 DAF, while those treated at 20℃ remained more or less unchanged until 29 DAF. Therefore, the low temperature maintained free water in grain one week longer than the high temperature treatment. The 20℃-treatment produced thin rice kernels which were lower than 1.8mm in thickness, while 30℃-treatment enhanced the kernel size over 2.0mm in thickness. The present results indicated that the ^1H-NMR spectroscopy was useful for early diagnosis of temperature stresses on rice kernel development.

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Seeds of almost all plants growing in areas with marked seasonal temperature variations require a period of cold treatment prior to germination. Wild rice, Zizania palustris L. and Z. aquatica L., are annual aquatic plants having deep dormant seeds. Since water in seeds plays an important role not only in physiological but also in chemical processes, dynamic states of water in wild rice seeds were determined during stratification. Wild rice seeds required at least 12 weeks of low temperature (3℃) treatment to break dormancy. The significant prolongation in T_1 was observed in the seeds chilled for 16 weeks whereas water content did not change. Zizania seeds indicating typical recalcitrant seed, have not tightly bound water observed in orthodox seeds such as wheat and rice plants. The prolongation of T_1 in germinating seeds mainly depended upon increase in T_1 of long fraction derived from free water. The prolongation of T_1 of the long fraction in the chilled seeds of wild rice suggested that the dormancy break entirely occurred at 16 weeks. These tendencies were more manifested in T_1 compared to T_2. Changes in molecular dynamics of water indicated that dormancy could be easily monitored by T_1. Therefore, the water compartments in endosperms of germinating seeds would reflect potentiality breaking of dormancy in seeds of wild rice as an aquatic plant.

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Water in seeds plays an important role not only in physiological but also in chemical processes. Especially, germination for soybean seed was inhibited by rapid water uptake and the role of water mobilization is very important for soybean germination. One of the best and most precise methods of analyzing water conditions in biological tissues is nuclear magnetic resonance (NMR). Most seeds treated by imbibition through the hilum germinated, whereas seeds treated through the seed coat and soaking hardly germinated. Water content of radicle treated by imbibition through the hilum remarkably increased, and prolongation of NMR relaxation time (T_2) indicated appearance of free water. On the other hand, water content and T_2 in cotyledons did not indicate significant difference in all treatments. From these results, it was suggested that T_2 of the radicle and cotyledon of soybean seeds strongly depended on characteristics of water status and thus reflected the difference of metabolic activity during seed germination.

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Change in the deep supercooling (DSC) ability of xylem tissues of beech (Fagus crenata L.) twigs was detected by a differential thermal analysis (DTA). DSC abilities of xylem tissue shown as peaks of low temperature exotherms in DTA were about -30℃ in summer and -40℃ in winter. DSC ability of beech twigs in winter was lowered to about -30℃ by heat treatment at 60℃ for 10 min but was not lowered by heat treatment at 50℃ for 10 min. In order to find protein factors related to DSC ability, changes in the protein composition of xylem tissues caused by seasonal cold acclimation and heat treatments were analyzed by two-dimensional gel electrophoresis. About 350 protein spots were detected in the crude soluble fraction of xylem tissues in winter. Seventy-five protein spots were induced during seasonal cold acclimation. Fifty-six of the cold acclimation-induced proteins remained after heat treatment of xylem tissues at 50℃ for 10 min, and 12 of the 56 proteins were decreased by heat treatment of xylem tissues at 60℃ for 10 min.

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Xylem parenchyma cells of most boreal trees including conifers adapt to subfreezing temperature by deep supercooling. Present study showed that crude extracts from xylem of several conifers had effect to promote supercooling of small water droplets with AgI as an ice-nucleating substance. Fractionation of the crude extracts from xylem of Larix kaempferi indicated that any substances except for soluble sugars were responsible for the promotion of supercooling, possibly by inhibiting or reducing ice nucleation. We also showed that the extracts from xylem of L. kaempferi had effect to promote supercooling, not only of small water droplets (2μl) but also of bulk water (1ml).

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Acid precipitates in snow may be a stress factor that affects the growth of wintering plants. The objective of this study was to determine the influence of acid-snow stress on leaves of wintering plants by in vitro experiments. Three different experiments, equilibrium freezing, prolonged freezing and repeated freeze-thawing, were carried out in order to simulate freeze-thawing of acid snow during winter. The results showed that acidification in the process of freeze-thawing caused enhancement of freezing injury of wintering plants.

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Cultured rice cells and moss protonemata were preserved in liquid nitrogen by desiccation method. Although rice cells cultured in MS medium containing 3% sucrose did not survive desiccation, the cells cultured in the medium containing higher concentrations (0.4M or 0.6M) of sucrose survived desiccation and preservation in liquid nitrogen. Viability of the cells after preservation in liquid nitrogen was enhanced when cultured in the medium without ammonium ion. This result accorded with the result of rice cells preserved in liquid nitrogen by pre-freezing method. Moss protonemata without pre-culture with the higher concentration of sucrose survived desiccation and preservation in liquid nitrogen. However, for the higher survival rate after preservation, it was needed that the speed of desiccation was decreased.

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Spider silk was indicated to have an ice nucleation activity. In this study, ice nucleation activity of the spider silk was confirmed by the observation using a CCD video camera and an environmental scanning electron microscope (ESEM). Moreover, "equilibrium freezing" of water in polymer gels was induced by the addition of the spider silk used as an ice nucleator, which was shown by the measurement of differential scanning calorimetry. Ice nucleation activity of the spider silk, here indicated, may find an application in the field of biotechnology.

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Larvae of Polypedilum vandezplanki can enter the state of cryptobiosis in which organisms are almost completely dehydrated and the metabolic activity is undetectable. One of the most characteristic phenomena is remarkable accumulation of trehalose in the cryptobiotic larva. This study aimed to clarify an importance of vitrified trehalose in cryptobiosis. Slowly-dehydrated larva (Slow sample) accumulated much trehalose, successfully entered the state of cryptobiosis, and showed high temperature tolerance, whereas quickly-dehydrated one (Quick sample) with a small amount of trehalose failed to do. Differential scanning calorimetric (DSC) analysis demonstrated that glass transition took place only in Slow sample, suggesting that a large amount of trehalose in the larval body is actually vitrified. When Slow samples were exposed to high relative humidity such as 93 or 98% for 5 days, the glassy state was lost and the recovery rate was lowered, although the trehalose content did not change so much. On the other hand, those exposed to 38% or 60% r.h. maintained the glassy state and the ability for recovery. However, their glass-transition temperatures declined, and consequently they became sensitive to high temperature. The results in the present study suggest vitrification of trehalose in the larval body is essential for induction and maintenance of cryptobiosis in P. vanderplanki.

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The effect of trehalose on the aggregation of β-amyloid (Aβ) was investigated using quartz crystal microbalance (QCM) and circular dichroism spectroscopy (CD). Here we prepared three types of host Aβ-guest Aβ systems differing in a combination of their secondary structures: namely, β-sheet-β-sheet (system (1)), β-sheet-random coil (system(2)) and random coil-random coil (system(3)). The host Aβ was fixed on the electrode of QCM, and the guest Aβ was dissolved in a buffer solution. The host-guest interaction was monitored through a frequency shift (ΔF) of the quartz vibration: a larger ΔF value means the occurrence of a larger degree of host-guest aggregation. When disaccharide (trehalose, neotrehalose or maltose) was added in the above system, the time dependent profile of ΔF was significantly affected. In systems (1) and (2), any of these disaccharides depressed significantly the host-guest aggregation: maltose and trehalose exhibited the strongest effect in systems (1) and (2), respectively. Interestingly, in system(3), trehalose rather promoted the aggregation compared with the control (without disaccharide), while both maltose and neotrehalose depressed the aggregation as much as in the cases of systems (1) and (2). The results of systems (2) and (3) imply that trehalose more strongly interacts with Aβ in a random coil than that in p-sheets. In fact, CD measurements indicated that trehalose retarded the transformation of Aβ from a random coil to β-sheet. Taken together, these results open up the possibility that trehalose modifies the aggregation process of Aβ through its preferential interaction with the random coil state of Aβ.

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Trehalose is a disaccharide that attracts much attention as a stress protectant. In this study, we investigated the interaction between trehalose and benzene compounds. The solubility of benzene compounds increased by the addition of trehalose. Trehalose increased the fluorescence intensity of benzene and p-cresol. The spin-lattice relaxation times (T_1) of ^1H and ^<13>C NMR spectra were measured to investigate the interaction between trehalose and benzene. The T_1 values for the ^1H signals assigned to benzene proton, decrease with increasing concentration of trehalose and the changes reaches plateaus at integer ratios of trehalose to benzene. On the other hand, from the ^<13>C-T_1 measurements for trehalose, the T_1 values of the C-2(C-2') and C-6'(C-6) are found to remarkably change by addition of benzene. ^1H-^1H NOESY measurement for a mixture of trehalose and benzene provide direct evidence for the interaction between the C6'(C-6) and C2(C2') sites of the sugar and the double bonds of benzene. Computer modeling study indicates that trehalose forms a stable complex with a benzene double bond though OH…π and CH…O types of hydrogen bonding. Therefore, these results indicate that one trehalose molecule stoichiometrically interacts with benzene compounds.

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The DSC dehydration scans of raffinose pentahydrate (R5W) reveal three symmetrical endotherms of which only two depend on the heating rate. Enthalpies corresponding to each of the two lower endotherms corresponded to 20% of the total enthalpy change. They were therefore assigned to R4W and R3W, respectively. XRD-DSC measurement shows the thermodynamic and structural changes occurring during a complete dehydration of R5W. On the removal of the first water molecule, to yield a R4W stoichiometry small peaks appear at ca. 8°(2θ), which do not appear in the R5W diffractogram. In order to obtain some additional information about possible existence of intermediate hydrates, a simulation was performed on the effects of removing each one of the five water molecules in turn from their different locations in R5W. From these results the R4W crystal structure was estimated.

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Uncoupled OD stretching Raman spectra were measured for aqueous solutions of magnesium salts, urea, thiourea, DMF, and DMSO. From the wavenumber changes of the uncoupled OD stretching Raman spectrum (ν A) with solute concentration, it is shown that urea is classified as a structure maker. There are three types of the ν A wavenumber variations in aqueous electrolyte solutions with solute concentration. There is little ν A wavenumber variation in aqueous DMSO solution with solute concentration.

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Two types of spectroscopic measurements were carried out to determine the effect of microscopic viscosity on molecular dynamics in saccharide aqueous solutions. Femtosecond pump-probe (PP) spectroscopy was carried out to monitor diffusional phenyl ring rotation of malachite green (MG) molecule which occurred in a time scale of picoseconds. Fluorescence correlation spectroscopy (FCS) was also carried out to monitor the translational diffusion of rhodamine 6G (R6G) molecules in the vicinity of a focusing point of an objective lens, which occurred in a time scale of sub-milliseconds. The PP signal exhibited multi-exponential decay, indicating a microscopic inhomogeneity of the viscosity. On the other hand, time correlation function obtained by FCS measurement exhibited single exponential decay, indicating that the inhomogeneity of the saccharide solution was averaged out and has disappeared in the sub-millisecond time scale.

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The elastic and thermal properties of lower aminoalcohols with different chemical structures were measured by Brillouin scattering and Modulated Differential Scanning Calorimetry (MDSC). The glass transition of pure substances of propylene glycol (PG), 1-amino-2-propanol (12AP) and 2-amino-1-propanol (21AP) were investigated. It is found that the glass transition temperatures and their glass-forming tendency and behavior of elastic properties are affected by the structure of molecules. The substitution effect of hydroxyl to amino moiety was resulted in stronger intermolecular interactions, higher glass transition temperature and more rigid in network structure in AP. It is found that the tendency of vitrification in AP series was much lower than PG since they are easily felled into crystallization. The structural relaxation time was derived by using the simple viscoelastic theory and it obeys the Arrhenius law. The results indicated similar tendency, therefore the relaxation process which coupled to acoustic wave is deduced to mainly their similar backbone structure.

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The enthalpy relaxation rates of freeze-dried bovine serum albumin (BSA) with or without a small amount of water were investigated in a temperature range of 50-300K by using adiabatic calorimetry. While not found in anhydrous BSA, spontaneous enthalpy relaxation was observed due to a glass transition from 120K to 180K in BSA with 1.85% moisture. A typical glass transition temperature in the wide enthalpy relaxation range was estimated as 155K based on an empirical interpretation. Since globular proteins with a small amount of water are generally immobilized at room temperature, this low-temperature glass transition is interpreted as attributed to the rearrangement of water molecules within BSA.

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Effect of solute sizes on their miscibility in the freeze-concentrated phase among ice crystals was studied using poly(vinylpyrrolidone) (PVP), Ficoll, sucrose, and dextran as models. Thermal analysis of the frozen solutions containing various concentration ratios of higher molecular weight polymers showed two transitions (glass transition temperatures of maximally freeze-concentrated solutes: T_g's) near the individual polymer T_g's at wide concentration ratios, indicating separation into different phases. Smaller polymer combinations showed a single transition of the solute-mixture concentrated phase that moved between the intrinsic T_g's depending on the concentration ratios. The polymer size and concentration ratios, as well as the structure and co-solute compositions, are important factor that determine their miscibility in frozen solutions.

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