We have observed year to year variation in seasonal changes in freezing tolerance of wheat and herbage crops in Sapporo. From induction in early autumn to wintering time in December, which is cold acclimation period, air temperature is the main factor for determining development of freezing tolerance. In detail, perennial grasses and legume induce hardening when daily minimum air temperature fall below 10℃ from late September to early October. Since then cold hardiness develops almost linearly in proportion to decrease Jn air temperatures till the field is covered with snow. The hardening process consists of at least two stages with a turning point at around 0℃. In the first stage where positive temperature prevails, plant hardiness correlates with reduction of water. Water content increases hardly in the second stage at freezing temperatures, but plant hardiness continues to develop. Plant size influences on freezing tolerance less than on snow tolerance, but it needs more than three weeks of prehardening growth at optimum growth temperature to exhibit its ability in full. After attainment of the maximum hardiness in late December, freezing tolerance diminishes slowly under snow cover. The degree of decreasing hardiness is related with amount of depletion of total sugar content during under-snow period. The snow tolerant variety of wheat could keep metabolism in low level under snow due to be less sensitive to comparatively high temperature around 0℃, whereas the freezing tolerant wheat variety responds much to such temperatures resulting in exhibiting high level of hardiness by using much energy.

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Cold acclimation processes involve dramatic alterations in protein synthesis and gene expression in a variety of plant species. Most soluble proteins are coagulated by heating at 100℃ for 10 minutes, but some highly hydrophilic cold-regulated proteins (CORs) remain soluble in aqueous solution. In our previous studies, we analyzed heat-stable proteins and their mRNAs during cold acclimation of spinach. Heat-stable COR with a molecular mass of 85 kDa (COR85) was the most evident in levels of protein synthesis and mRNA accumulation among all heat-stable CORs. Increased levels of COR 85 correlated with the development of freezing tolerance during cold acclimation. Interestingly, COR 85 accumulated specifically in the leaves and stems and not roots of the cold-acclimated plants. Consistent with this observation, freezing tolerance was induced in leaves and stems, but not in roots. COR 85 was a member of Group II LEA protein family and inducible by drought, salt stress, and abscisic acid. We obtained evidence that COR 85 is involved in cryoprotection of a freezing-sensitive enzyme. Among the nine proteins with a variety of hydrophilicity, COR 85 was the most effective in protecting against freezing denaturation of lactate dehydrogenase in vitro. On the other hand, some soluble proteins were coimmunoprecipitated with COR 85, suggesting that they may interact with COR 85 in vivo. These data allow us propose the hypothesis that COR 85 may be associated with some stress-sensitive enzymes.

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Seventeen cDNA clones corresponding to hardening-induced Chlorella (hiC) genes were isolated by differential screening of a cDNA library from 6 h-hardened cells of Chlorella vulgaris C-27, a frost-hardy strain. Northern analysis showed that all hiC transcripts are specific to low temperature and their induction patterns are diverse. Genomic Southern and dot analyses of 2 strains of Chlorella (C. vulgaris C-27, frost-hardy; C. ellipsoidea C-102, chilling-sensitive) revealed that 10 out of 17 hiC genes are not detected in C-102 and the other 7 hiC genes were not expressed in C-102 except only one gene. The expression of these 16 genes is probably necessary for the acquisition of freezing tolerance. Homology searches of the sequences of the hiC clones resulted in the following data: hid, chaperonin-60β polypeptide; hiC2, desiccation-related protein; hiC3, thioredoxin peroxidase; hiC4, ribosomal protein L 2; hiC6, group III LEA (Late Embryogenesis Abundant) protein; hiC7, peptidyl-prolyl cis-trans isomerase; hiC8, ribosomal protein S-27; hiCIO, zeste protein; hiC12, group III LEA protein (different from hiC6). On the other hand, 31 soluble proteins, which were induced or increased during hardening, were isolated by 2D-HPLC and SDS-PAGE. The N-terminal sequences of 7 out of 31 proteins were determined. The results of homology search were as follows : 14-kDa and 10-kDa proteins (identical with HIC6 and HIC12 proteins, respectively), group III LEA proteins; 22-kDa protein, thioredoxin peroxidase ; 18-kDa and 16.5-kDa proteins, photosystem II oxygen-evolving enhancer protein 2 ; the two other, no homology. The results obtained showed that the LEA protein (HIC 6) is highly involved in the development of frost hardiness. Introduction of the lea gene enhanced freezing tolerance in yeast.

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Plants respond to conditions of drought and cold stress with various physiological and developmental changes to tolerate these stresses. Many genes are induced by drought and cold stress, and they seem to function in the protection of cells from dehydration in plants. Abscisic acid (ABA) plays important roles in the tolerance of plants to drought and cold stress. Many genes that respond to drought and/or cold stress are also induced by exogenous application of ABA. It appears that dehydration triggers the production of ABA, which, in turn, induces various genes. Czs-acting and tram-acting factors involved in ABA-induced gene expression have been extensively analyzed. Several genes that are induced by drought and cold stress, are not responsive to exogenous ABA treatments. These findings suggest the existence of ABA-independent as well as ABA-dependent signal-transduction cascades between the initial stress signal and the expression of specific genes. Analyses of the promoters of drought- and cold-inducible genes have revealed a novel as-acting element (DRE) that function in the ABA-independent gene expression by dehydration or low temperature. Many genes for factors involved in the signal transduction cascades, such as protein kinases and transcription factors, are regulated by these stress signals. Molecular analyses of these genes should provide a better understanding of the signal transduction cascades under drought and cold stress.

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Mechanisms of freezing resistance in plant cells show, seemingly, to be very diverse processes. However, when we consider about adaptation processes of plants, in long-time scale, to cold areas, there may be a chain or a commonness in the wide variety of adaptation mechanisms of plants to freezing temperatures. In this brief review, the continuity or the commonness of the adaptation mechanisms in plaint cells to freezing temperatures was indicated.

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The maintenance of fruits and vegetables at low temperature have been proved to be effective in keeping their quality, but it depends on the degree of chilling sensitivity of harvested products which varies due to species, cultivars, maturity, plant part and physiological condition. In the present study, physiological responses of horticultural products to low temperature and the associated metabolic changes were discussed in relation to their shelf life and chilling injury, indicating some phenomena of metabolic changes and dysfunction induced by low temperature such as changes in phenolics metabolism and the resultant discoloration in water convolvulus (Ipomoea aquatica Forsk.) and egg plant fruit, alternation of respiratory chain and mitochodrial function in cucumber fruit and potatoes, and the complexity of responses to low temperature and the induced metabolic events in potatoes.

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In the northern part of Japan, cool-weather damage in rice production is very severe in climate damages. And it has occurred about 25 times during the past century (once every 4 years on an average) in Hokkaido. Rice plants has 2 types of cool-weather damage; delayed growth type and destructive sterile one. Between them, it is very important for the latter to solve physiological mechanism to cool stress and to establish the countermeasures for conquering cool-weather damage. Destructive sterility to cool stress mainly suffer the damage at booting stage and flowering one during pollen developmental process. The most sensitive stage, the trough in fertility curve to cool stress at booting stage is the early microspore stage from tetrad to early microspore phase. And the next sensitive stage is just before pollen germination at flowering stage. At booting stage, the decrease of pollen number per anther to the stress is closely related to the sterility. Pollen activity to the stress, however, is not clear to the tolerance. At flowering stage, the cool tolerance is influenced after decision of pollen number in an anther. So, one of the important reaction to coolness is the abnormal digestion of starch to sucrose just before the germination of the pollens. Besides pollen differentiation and development mechanism, it is necessary for more strong cool tolerance to solve the mechanism of fertilization on the stigma, and more stable and higher in-vitro germination because of very short longevity of rice pollens.

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Many important crops are indigenous to subtropical climate and are, in general, highly sensitive to(low or nonfreezing temperatures ranged between 0 and 12℃. In northern climates, plants encounter occasionally unusual chill weather in the early growing season, causing various types of injury. To improve the productivity of crops in the northern areas, it is important to understand the mechanisms of injury and adaptation to chilling temperatures. The phenomena of chilling injury occurring in the field exhibit complicated aspects due to the composite environmental factors including temperature, light and water relation, and also due to some biological factors including developmental stages and tissue differentiations. All of these factors are mutually interrelated with respect to the mode of injury and diferences in the tolerance to cold. In this article, I will discuss about the possible relationship between those physical and biological factors and the mode of injury. Explanation will be given to the mechanism of cold-adaptation of plants on bases of fluidity of membrane lipids, primary membrane transports and cell homeostasis, water ralation and suppression or elimination of the formation of oxygen radicals.

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The survivability after cryopreservation of plant tissues can be increased by pretreatments such as cold acclimation, air drying and preculture on a medium supplemented with abscisic acid or a high concentration of sugar. It is not clear why these pretreatments increase freezing-survivability of the tissues. In this report, in order to clarify the mechanism involved, the effect of preculture (on a medium supplemented with sugar) on freezing-survivability, sugar and water content, the cell structure of asparagus (Asparagus officinalis L.) shoot tips was investigated. Furthermore, the effect of cold acclimation on freezing tolerance and sugar components was examined in in vitro cultured shoots of blueberried honeysuckle (Lonicera caerulea L. var. emphyllocaryx NAKAI).

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In 1990, Fabre and Dereuddre reported successful cryopreservation of shoot tips of Solanum species by new protocol called encapsulation-dehydration. This procedure involves three important treatments : 1. alginate encapsulation of cultured materials, that can protect materials from stresses; 2. pretreatment with concentrated sucrose medium, that promotes dehydration tolerance; 3. dehydration in the air, which increases vitrification tendency. This method has been applied to the cryopreservation of shoot tips and somatic embryos of other species.

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The development of a simple and reliable method for cryopreservation would allow much more widespread use of cryopreserved cultured plant cells and meristems. Potentially valuable cryogenic procedures would be vitrification and encapsulation/dehydration techniques. These new procedures increased the number of species to be cryopreserved over the last few years. In the vitrification method, cells and meristems are sufficiently dehydrated by a highly concentrated vitrification solution (PVS2) at 0 or 25℃. Thus, the procedure eliminates the need for controlled slow freezing and permits cells and meristems to be cryopreserved by direct transfer into liquid nitrogen. Successfully vitrified and warmed meristems directly developed shoots without inter-mediary callus formation. The encapsulation/dehydration technique is easy to handle and alleviates dehydration process. However, the problems are the lower rate of survival, the later recovery growth and the longer dehydration process compared with the vitrification procedure. To solve these problems, a novel cryogenic procedure, encapsulation/vitrification method, was presented. In this method, meristems were trapped into alginate-coated beads containing a mixture of 0. 4M sucrose and 2M glycerol. These encapsulated meristems (30-40) were dehydrated in a 100ml beaker containing 50 ml PVS2 for about 100 min at 0℃. This procedure is easy to handle and produces high levels of survival. Thus, the method promises for cryopreserving meristems, hairy roots, meristems and somatic embryos.

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The establishment of method for cryopreservation of human red cells in 1960s' made an important contribution to the development of modern biology and medicine. In this lecture, the current status of cryopregervation of human blood cells, such as red cells, platelets, granulocytes, monocytes, lymphocytes and hematopoietic stem cells are reviewed. The challenge to find a successful method to cryopreserve granulocytes and platelets, which still remain as difficult cells to be cryopreserved, should make ways for new therapies.

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