Tomato (Lycopersicon esculentum Mill.) plants, which normally do not accumulate glycinebetaine (GB), are susceptible to chilling stress. Exposure to temperatures below 10℃ causes various symptoms of injury, and greatly decreases fruit set (i.e., the number of fruits per plants) in most cultivars. Conventional breeding efforts have failed to produce cultivars with satisfactory cold tolerance. Therefore, we have transformed tomato (cv. Moneymaker) with the cod A. gene of Arthrobacter globiformis, which encodes choline oxidase to catalyze the conversion of choline to GB. These transgenic plants express the cod A gene and synthesize choline oxidase, while accumulating up to 0.23μmol of GB per gram fresh weight in their leaves. During various phases of growth and development, from seed germination to fruit production, the GB-accumulating plants are more tolerant of chilling stress than their wild-type counterparts. Moreover, at the reproductive stage, our transgenic tomato plants yield, on average, 30% more fruit after an episode of chilling stress. We demonstrate here that introducing the biosynthetic pathway of GB into tomato via metabolic engineering is an effective strategy for improving fruit production under cold-stress conditions.

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Ultrafast pump-probe (PP) spectroscopy was applied to the measurements of microscopic viscosity of the aqueous solutions of saccharides. A dye molecule, malachite green (MG), the electronic excited state lifetime of which depends on solvent viscosity, was used as a probe molecule. The viscosity of the solution measured by a viscometer increased nonlinearly with saccharide concentration and this increase was the largest for the trehalose solution. The decay of the PP signal of MG was fitted with a multi-exponential function and it showed that the molecular dynamics occurring in the ultrafast time region, <200 fs, was not affected by the viscosity. The dynamics slower than 1 ps showed clear viscosity dependence and at high concentration of disaccharide, the picosecond decay became bi-exponential. This indicates that heterogeneity of the solvent has increased, i.e. the excited state lifetime of MG molecules trapped in the vicinity of the aggregated sugar molecules becomes longer because they sense higher microscopic viscosity. The time constants for the picosecond decay were plotted against the viscosity. Although trehalose showed the largest nonlinear increase of the viscosity with the concentration, the viscosity dependence of the picosecond time constants was the weakest among the measured saccharides. This result indicates that aggregated hydrogen-bonding network of trehalose efficiently interrupts macroscopic translational motion of the solution, although its effect on microscopic molecular motion, such as the rotation of the phenyl group, is small.

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Freezing temperature and incidence of freezing injury of rough rice grains with 11 moisture content levels in the range of 15.1% to 26.5% were determined in this study. Differential scanning calorimetry (DSC) was used to measure the freezing temperature of rice. Rice grains with a moisture content of less than 20.8% on average did not freeze even at a temperature of -55℃. Rice grains with a moisture content of 22.1% on average froze at about -35℃. Incidence of freezing injury of rice grains was determined by the germination rate because grains that suffered freezing injury did not germinate. Rice grains with a moisture content of less than 17.8% on average germinated after being stored at -80℃ for 11 days. Thus, no grain with a moisture content of less than 17.8% froze even at a temperature of -80℃. Rice for long-term storage is always dried until the moisture content becomes around 16%. Thus, there is no need for concern about freezing and freezing injury of rice stored in farm silos under natural conditions in winter in Hokkaido.

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For aqueous solutions of various mono-, di-, and oligosaccharides thermodynamic properties have been investigated at low temperatures using differential scanning calorimetry. The amount of unfrozen water, Uw, increases linearly with the glass transition temperatures of anhydrous carbohydrates. Uw also shows a linear relationship with partial molar isentropic compressibility K^0_<S,2>. More unfrozen water is induced in the presence of a given carbohydrate, with more negative value of K^0_<S,2>, acting as a structure breaker to the three dimensional hydrogen-bond network of water. Relative amount of unfrozen water for disaccharides studied is associated with the combination of constitutive monosaccharides and attendant molecular structure features such as the position and type of the glycosidic linkage between the constituent units. These results suggest that for a series of the above thermodynamic parameters there is a common systematic dependency on molecular structures of carbohydrates.

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Although organ transplantation is established as a therapeutic procedure for various terminal organ dysfunctions, there are many unsolved problems such as shortage of donors and primary graft failure. A more reliable organ preservation solution could resolve these problems. Therefore, we have developed new ET-Kyoto solution characterized by extracellular type ion composition, non-reducing disaccharide of trehalose, and vascular endothelial protective agents (dibutyryl cAMP, nitroglycerin, N-acetyl cysteine). We confirmed the superior efficiency of the solution in canine lung transplantation and have applied it to clinical lung transplantation at Kyoto University Hospital since April 2002.

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Here we report on the mechanism of antioxidant function of trehalose for unsaturated fatty acids (UFAs). The spin-lattice relaxation times (T_1) of ^1H and ^<13>C NMR spectra are measured to investigate the interaction between trehalose and UFAs. It is found that for the ^1H and ^<13>C signals assigned to the olefin double bonds in UFA, the T_1 values decrease with increasing concentration of trehalose and the change reaches a plateau at an integer molar ratio of trehalose/UFA. Such T_1 changes are found only for mixed systems of trehalose and UFAs possessing cis double bond (s). Similar experiments are performed for other sugar/UFA mixtures, but no apparent T_1 changes were observed for the UFA protons. In addition, it is suggested from the ^<13>C-T_1 measurements that the OH-6'(OH-6) and OH-3 (OH-3') hydroxyl groups of trehalose are responsible for the interaction with UFAs. Computer modeling study indicates that trehalose forms a stable complex with an olefin double bond through OH…π and CH…O types of hydrogen bonding. Furthermore, a significant increase in the activation energy is found for hydrogen abstraction reaction from the activated methylene group of UFA. Therefore, these results indicate that one trehalose molecule stoichiometrically interacts with one cis-olefin double bond of UFA and hence it has a significant depression effect on the oxidation of UFA.

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Effect of sodium tetraborate (borax, Na_2B_4O_7) on the thermal properties and protein secondary structure of a freeze-dried pharmaceutical formulation model was studied. Addition of sodium tetraborate raised frozen solution T_g' (glass transition temperature of maximally freeze-concentrated solutes) and freeze-dried solid T_g (glass transition temperature) of bovine serum albumin and disaccharide (sucrose, trehalose) combinations, indicating borate-saccharide complex formation. Sodium tetraborate reduced structure-stabilizing effect of sucrose and trehalose against lyophilization-induced structural perturbation to some extent. The borate-saccharide complex should improve protein storage stability in dispense of decreased structure-stabilization during the freeze-drying process.

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The objective of this project was to develop new on-farm rice storage techniques at a temperature below ice point, known as super-low-temperature storage, using fresh chilly air in winter. Two on-farm storage experiments were conducted in two country elevators in Hokkaido from 1996 to 2000. When rough rice was aerated from the bottom to the top of the silo in winter, the temperature of whole rice grains in the silo fell below ice point. At the end of the storage period in summer, the temperature of rice grains in the center of the silo was still below ice point, whereas the temperature of rice grains near the silo wall increased to about 20℃. However, there was no deterioration in the quality of rice grains near the silo wall. The quality of rice stored in the silo was preserved at a level similar to that of freshly harvested rice. The combination of rice storage at a temperature below ice point and utilization of fresh chilly air in winter enables the quality of rice to be preserved at a high level without the requirement of any cooling unit and electricity. The use of new techniques for super-low-temperature storage in cold regions of Japan has been increasing in recent years. In Hokkaido, 23 country elevators have been constructed since 1996. The total storage capacity of rough rice in Hokkaido has been increasing in recent years and reached 104,000 t in 2002.

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In order to demonstrate the validity for aqueous solutions, "Three-Phase Freezing Model" was applied to coffee solutions. The one-dimensional freezing processes of 10, 20, and 40% solutions have been numerically simulated based on the thermo-physical properties as well as the phase diagram obtained by DSC-method, while the freezing experiments were carried out in the similar conditions to those of the simulation. The results showed that the experimental and simulated freezing curves were in good agreement, and that the phase diagram and eutectic temperature of a solution were important for accurate prediction. Then, it is concluded that the freezing model was proved to be applicable in the concentration range of 0-40%.

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Effects of sodium/potassium phosphate buffers on residual activities of lactate dehydrogenase (LDH) were investigated in view of its preservation in the ambient temperature storage. Two preliminary refrigeration methods were also examined. From the results, there is no significant difference between sodium and potassium phosphate buffers and the preliminary refrigeration in -85℃ freezer is better than in liquid nitrogen on residual activities.

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The optimum low temperature and frozen conditions are presented based on the artificial neural network analysis between measured viscoelastic properties of cooked rice and sensory evaluations for ordinary adults as well as the aged. For maintaining the "preference" felt by adults, the temperature conditions for one-day and long-term storage were demonstrated to be above 15℃ and below -25℃, respectively. Ice crystals within a kernel of cooked rice were distributed at both inter- and inner-cellular spaces, showing relatively large crystals in the central portion during frozen storage at -5℃. Then the characteristic preferences of adults and the aged for cooked rice were revealed by artificial neural network analysis based on the results of the sensory evaluation. These results demonstrated that the models were useful for predicting "preference" for each group based on the measured parameters such as viscoelastic properties and scores of sensory evaluation for cooked rice.

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Residual biological activity of lactate dehydrogenase (LDH) freeze-dried in present of four kinds of disaccharides (i.e. trehalose (TRE), sucrose (SUC), maltose (MAL) and lactose (LAC)) was examined, and the stabilizing effect of dissacharides was compared and discussed based on water replacement and glass transition hypothesis. Every disaccharide protected LDH from stress induced by freeze-drying, although the protecting ability was almost same degree among them. However, the more residual moisture content of sample raised the LDH residual activity. This result will be explained by steric hindrance of water replacement by dissacharide. Although LDH lost all of the activity within one month during storage at the temperature above T_g, when the sample was in glassy state at the same temperature, the activity was enough kept. This will result from the immobilizing effect by glass transition. On the other hand, when the sample was stored below the T_g, TRE and SUC enough protected LDH during at least three months. However, MAL and LAC destabilized LDH by the browning reaction to LDH. It is thought that TRE has non-reactivity to protein and high T_g will work to protein as the most effective lyoprotectant among four disaccharides through the current research.

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Homogeneous nucleation temperatures (T_H's) of emulsified aqueous solutions of polyethylene glycols (average molecular weight=200, 300, 400 and 600) were measured as a function of pressure. It is shown that emulsified polyethylene glycol solutions of R=50 (R: moles of water/moles of solute) are all glass-forming at high pressures.

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Germination of seeds is a complex physiological process triggered by imbibition of water. Although germination does not occur even though the seeds on a mother plant are in a humid surrounding, wheat (Triticum aestivum L.) caryopses are characterized by relatively weak embryo dormancy and are susceptible to preharvest sprouting, vivipary. We focus on dynamic state of water in wheat seeds for the purpose of screening characteristics in wheat three cultivars having different susceptibility in preharvest sprouting. Although Shirogane-Komugi is known as one of the susceptible preharvest sprouting cultivars, there was no relationship of ABA sensitivity to vivipary since the mature seeds easily loose their dormancy. Spin-spin NMR relaxation times (T_2) in wheat indicated that cellular water in the dry seeds mainly consisted of tightly bound water. Although there are no influence of ABA on T_2 in non-sprouting seeds, ABA strongly reduced T_2 in endosperms for sprouting seeds. These results indicate that ABA prohibited both water uptake and water mobility in sprouting seeds while mobility of water bound with starch appears to remain unchanged in non-sprouting seed. The water in the endosperms of the ABA-treated seeds have more restricted mobility than in those treated by DW. Especially, the rate of long T_2 values reduced by ABA was the highest in Shirogane-Komugi and the tendency was consisted with their vivipary of three cultivars tested. Therefore, it is suggested that the T_2 reflected a primary event of sprouting for wheat grains. In conclusion, the water compartments determined by the T_2 in endosperms of sprouting seeds clearly reflected an ABA sensitivity and thus the parameters would be useful for vivipary screening in wheat cultivars.

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A trial was carried out to determine the effects of different fertility regimes on the cold tolerance of tall fescue. Tall fescue was grown on soil amended with thermophile sewage sludge compost and chemical fertilizer; soil without amendment was also prepared as the control. After establishing, grass was used for NMR determination. Temperature was lowered in every 5℃-step from 25 to -30℃. T_1s were measured using saturation recovery method. T_2s were measured using CPMG method and solid echo method. There was no change in Arrhenius plot of T_1 in fescue leaves responded to lowering temperature from 25 to 0℃; then a slight increase was observed from -5 to -10℃ for almost treatments. On the contrary, T_2 of fescue leaves gradually increased along with decreasing temperature from 25 to -15℃. Subsequently, a sharp decline in T_2 was observed at temperature ranging from -20 to -30℃ for all treatments. The increase up to 60-70% of T_2 short fraction ratio probably indicates that tissue water began freezing at these temperatures. Results from NMR determination suggested that T_1 was affected by water content (r=0.87); T_1 value of the control was lowest corresponded to the lowest amount of water in the leaves. The supercooling ability was highest for fescue grown on compost-fertilizer blend. Freezing temperature of -18.8℃ in this treatment was approximately 2 to 4℃ lower than the others. It means that fescue grown on compost-fertilizer tolerated cold stress better than fescue grown on other amendments.

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Osmotic dehydration was examined as a preventive method of tissue softening of agricultural products by the freezing injury. Carrot and potato tissues were dehydrated in 50% (w/w) sucrose solution at 10℃ for 24 hours. Injury of cell plasma membrane by freezing-thawing or osmotic dehydration was analyzed by the electrical measurement. Rheological properties of cell tissues were determined by the vibrating reed method. Cell plasma membrane of carrot tissue was relatively resistant to osmotic dehydration. Therefore, viscoelasticity of rehydrated carrot tissue pretreated by the osmotic dehydration before freezing-thawing showed 2-3 times higher that of control without osmotic dehydration, suggesting the effect the osmotic dehydration to prevent tissue softening after freezing-thawing. On the contrary, cell plasma membrane of potato tissue received a serious damage by the osmotic dehydration so that pretreatment by the osmotic dehydration was not effective for the prevention of freezing injury in this case. This result suggested some difference in the cell membrane structure between potato and carrot.

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Osmotic dehydration was examined as a preventive method of tissue softening of agricultural products by the freezing injury. Carrot and potato tissues were dehydrated in 50% (w/w) sucrose solution at 10℃ for 24 hours. Injury of cell plasma membrane by freezing-thawing or osmotic dehydration was analyzed by the electrical measurement. Rheological properties of cell tissues were determined by the vibrating reed method. Cell plasma membrane of carrot tissue was relatively resistant to osmotic dehydration. Therefore, viscoelasticity of rehydrated carrot tissue pretreated by the osmotic dehydration before freezing-thawing showed 2-3 times higher that of control without osmotic dehydration, suggesting the effect the osmotic dehydration to prevent tissue softening after freezing-thawing. On the contrary, cell plasma membrane of potato tissue received a serious damage by the osmotic dehydration so that pretreatment by the osmotic dehydration was not effective for the prevention of freezing injury in this case. This result suggested some difference in the cell membrane structure between potato and carrot.

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Change in the cellular structure during hydration and dehydration is a crucial factor for the successful cryopreservation. In this study, the swelling process of polymer gel beads, used as models of biological cells, was followed in the aqueous sugar solutions by the measurement of nuclear magnetic relaxation times. It was found that the swelling was retarded in the presence of sucrose molecules, and the preferential water absorption by the gel beads was indicated.

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The phytohormone abscisic acid (ABA) plays an important role in freezing tolerance in plants. Treatment with ABA increased freezing tolerance of protonema cells of the moss Physcomitrella patens. ABA triggered morphological changes in cellular organelles, such as reduction in sizes of chloroplasts and vacuoles, and physiological changes such as accumulation of soluble sugars, especially that of sucrose, and boiling-stable proteins. We used protein synthesis inhibitors, protein serine/threonine kinase inhibitors and protein serine/threonine phosphatase inhibitors to examine cellular events associated with ABA-induced freezing tolerance. The results indicated that a protein synthesis inhibitor cycloheximide dramatically decreased freezing tolerance of the ABA-treated cells and accumulation of boiling-stable proteins. A phosphatase inhibitor okadaic acid also decreased ABA-induced freezing tolerance in P. patens. These inhibitors, however, did not have any effect on ABA-induced accumulation of sucrose. These results suggest critical roles of de novo synthesis of nuclear encoded protein and phosphatase-mediated signal transduction in ABA-induced cellular processes leading to freezing tolerance, and that sucrose only plays a limited role in these processes.

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Most of xylem parenchyma cells of boreal hard wood species respond to subfreezing temperature by deep supercooling. Because xylem tissue cells that adapt by deep supercooling have the least resistance to subfreezing temperature among woody tissues, the limit of supercooling ability of xylem parenchyma cells is an essential factor for the survival of woody plants in cold areas. However, the deep supercooling mechanism of xylem parenchyma cell has not yet been clarified. In this study, accumulation of soluble sugars during seasonal cold acclimation in birch (Betula platyphylla var. japonica Kara) xylem tissue has been examined to discuss the influences of the intracellular sugars to the supercooling ability of xylem parenchyma cells. The supercooling ability of xylem parenchyma cells of birch seasonally changed. Fructose, glucose, sucrose, raffinose and stachyose were mainly accumulated in birch xylem parenchyma cells. Seasonal change in the amount of sucrose, raffinose and stachyose was positively correlated with the supercooling ability in xylem parenchyma cells. Although the proportion of each sugar composition was different between xylem parenchyma cells that undergo deep supercooling and bark parenchyma cells that undergo extracellular freezing, the major components of soluble sugars which accumulated in xylem cells were same with those of bark cells. However, the concentration of soluble sugars in xylem parenchyma cells might be higher than that of bark cells or cambial cells. It seems that the intracellular soluble sugars participate, at least in part, to the increase of supercooling ability of xylem parenchyma cells via increasing intracellular osmotic concentration. However, such a freezing temperature depression alone cannot explain high supercooling ability of xylem parenchyma cells, suggesting presence of other factors which may promote supercooling.

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Xylem ray parenchyma cells (XRPCs) of Larix kaempferi respond to subfreezing temperatures by deep supercooling in winter. We carried out differential display to isolate genes expressed in association with increase in the magnitude of deep supercooling ability in XRPCs of L. kaempferi, and identified ten genes preferentially expressed during winter seasons. These genes encoded proteins with sequence similarity to NBS/LRR protein (WXL1), organic anion transporter (WXL2), H-pyrophosphatase (WXL3), aminoalcoholphosphotransferase (WXL4), flavonol 3-O-glucosyltransferase (WXL5), histone H2A (WXL6), LRR-containing F-box protein (WXL7), reverse transcriprase (WXL8), a protein similar to Arabidopsis protein T20 L15, 20 (WXL9) and an unknown protein (WXL10). These genes were differentially expressed during fall and winter seasons. Expression of transcripts of WXL4 and 9 increased from October, while that of WXL1, 2, 3, 5, 6, 7, 8 and 10 dramatically increased in November. Transcripts of WXL3 and 9 were similarly expressed in xylem and cortex tissues, while those of WXL1, 2, 4, 5, 6, 7, 8 and 10 were preferentially expressed in xylem tissue.

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Freezing resistance of xylem ray parenchyma cells of birch (Betula platyphylla L.) by deep supercooling increases during seasonal cold acclimation. When apoplast substances were partially eluted by an acidic solution from winter xylem tissue, freezing resistance by deep supercooling of the tissue was decreased. Several major proteins were detected in the acid-extractable fraction of xylem tissue, and the amounts of those proteins were increased in winter. Immunoblot analysis showed that some of the major proteins are similar. N-terminal amino acid sequencing revealed that they are similar to the pathogenesis-related proteins.

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When tubers of Helianthus tuberosus L. (Jerusalem artichoke) were frozen at -10℃ by equilibrium freezing, the tubers were injured lethally after thawing. In plasma membrane fraction prepared from sublethally frozen tubers, the levels of some plasma membrane proteins, named frost-susceptible proteins (FSPs), decreased as compared with that from unfrozen tubers. Response of FSP120, one of the major FSPs with a molecular mass of 120 kDa, to freezing treatment were characterized using the antibody to FSP120 in this study. The total amount of FSP120 in the crude extract scarcely declined after sublethal freezing treatment of tubers although the levels of FSP120 in the crude microsomes decreased after freezing treatment. These results suggest that FSP120 might be released from plasma membranes during freezing treatment of the tubers of Jerusalem artichoke.

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The size of micro-capillaries within cell walls of xylem parenchyma cells of woody plants has been estimated by the plasmolysis method using hypertonic solutions consisted of various size of molecules ranging from 0.8 nm to 5.2 nm. All xylem parenchyma cells examined, including xylem ray parenchyma cells exhibiting typical deep supercooling to -40℃, produced distinct plasmolysis by treatment with these all hypertonic solutions, indicating that size of cell wall micro-capillaries in xylem cells exceeded more than 5.2 nm. Water located in cell wall micro-capillaries in size larger than 5.2 nm cannot supercool at least below -20℃. Thus, it is suggested that the cause of deep supercooling of xylem parenchyma cells in woody plants cannot be explained solely by structural property of cell walls in relation with the size of micro-capillaries.

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To determine how enhancement of a scavenging system of reactive oxygen species is involved in the mitigation or delay of the occurrence of chilling injury, transgenic rice plants that constitutively overexpress wheat catalase were investigated. After chilling treatment (5℃) for up to 8 days, the transgenic rice exhibited less injury than did the wild type. Furthermore, the transgenic rice recovered better from chilling stress than the wild type when they were returned to non-chilling temperatures after the chilling treatment for 2 to 4 days. The advantages of the transgenic rice in chilling tolerance were consistent with the results that, under the chilling process or the rewarming process after the chilling treatment, there were less amount of hydrogen peroxide, less extent of plasma membrane-associated injury, and less amount of lipid peroxides in the transgenic rice than in the wild type. These suggest that the introduction of catalase into rice plants results in increased chilling tolerance as a consequence of increased membrane stability due to effective removal of reactive oxygen species by high activity of catalase transformed.

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Complex impedance was measured to analyze ice crystal morphology for frozen gelatin solutions from 5Hz to 100kHz at -20℃. Samples with different freezing rate were prepared by program freezer over a temperature range of -120 to -20℃. The maximum vales of dielectric loss (ε") were different from freezing rate at 1250Hz. A Micro-Slicer Image Processing System (MSIPS) has been applied to observe the ice crystal structure formed in frozen gelatin solutions. The equivalent diameters of ice crystals were measured quantitatively, based on images obtained by multi-slicing of a frozen sample with the thickness of 5 μm. The equivalent diameter of ice crystals was in the range of 31.7 μm up to 54.3 μm, and then at the freezing rate of 37.0 to 219.5℃ h^<-1>. The electric conductivity of whole sample was in the range of 1.24×10^<-6> up to 3.63×10^<-6>. The study therefore suggests that measuring the dielectric properties of frozen liquid food materials may be associated with ice crystal morphology and may be of considerable use of understanding the internal structure of frozen materials.

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