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Deep supercooling of shoot and bud tissues of Picea abies
Forest Ecology and Management, 20 (1987) 97-103
97
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Deep Supercooling of Shoot and Bud Tissues of P i c e a abies PAWEL PUKACKI
Polish Academy of Sciences, Institute of Dendrology, PL-62-035 Kdrnik (Poland) (Accepted 7 August 1986)
ABSTRACT Pukacki, P., 1987. Deep supercooling of shoot and bud tissues ofPicea abies. For. Ecol. Manage., 20: 97-103. Using differential thermal analysis ( D T A ) it was shown that actively growing or developing buds and shoots of Norway spruce die as a result of a single freezing of tissue water, which takes place at temperatures between - 8 and - 16 ° C. O n the other hand, in winter-hardened organs, tissue water freezes in two successive stages, each of which preceded by supercooling of the tissue water. At first,there is freezing of supercooled water in the extracellular spaces and then there occurs the crystallization by deep supercooling of the remaining tissue water; in winter-resting buds this occurs in the range from - 17 to - 27 ° C. Dead or dehardened buds do not demonstrate the tendency towards deep supercooling. The role of water content and of barriers restrictingwater movement from cytoplasm to the extracellular ice are discussed.
INTRODUCTION
Tissue of actively growing shoots and buds of trees from the temperate climatic zones die when frozen to about - 5 to - 1 0 ° C (Larcher, 1975). Other investigations have shown that death is caused by the formation of ice, which destroys the cell structure (e.g. Levitt, 1980). On the other hand, in winter, the tissue water during cooling at first becomes supercooled. This state is unstable, and under some conditions a spontaneoustransition of the deeply supercooled tissue water into a stable phase occurs. The temperature of this change is described as the homogenous ice-nucleation temperature ( George et al., 1974). It has been experimentally established that the temperature crystallization of small pure water droplets 10/lm in diameter occurs at a temperature of nucleation of -38.1°C (Burke and Stushnoff, 1979). For typical homogenous plant solutions, the nucleation temperature (Tn), according to Rasmussen and MacKenzie (1972), is related to the melting temperature (Tm) as follows: 0378-1127/87/$03.50
© 1987 Elsevier Science Publishers B.V.
98 Tn = - 3 8 . 1 ° C + (1.8+_0.4) Tm In many species of herbaceous and woody plants Tm varies from 0.69 ° to 2.94 ° C (Burke and Stushnoff, 1979). The temperature of the phase change can be registered using methods such as differential thermal analysis (DTA), differential scanning calorimetry (DSC), or pulsed nuclear magnetic resonance spectroscopy (NMR; Burke et al., 1976; Burke and Stushnoff, 1979). Temperature curves in a differential thermal analysis reveal the occurrence of ice crystallization. Graham and Mullin (1976) and Sakai (1979) suggest that, in tissues of woody plants, there exist barriers restricting the spread of ice. It is uncertain whether this p h e n o m e n o n exists primarily because of morphological features of tissues (George et al., 1974) or because of energy bartiers restricting the movement of water from frozen tissues (Bervaes et al., 1977 ). Most probably, the rate of crystallization, and the degree of supercooling of tissue water, will depend on several factors such as physico-chemical properties of water, ice adhesions, phasic changes in the lipids of cell membranes, structure of tissues, and their hydration and stage of preparation for low-temperature stress. In the results presented below, seasonal differences are demonstrated in the supercooling of tissue water in relation to the freezing tolerance of buds and of Norway spruce. MATERIALSAND METHODS
Plant material. One-year-old shoots were collected from 19-year-old trees of Picea abies (L.) Karst. growing on an experimental area of the Institute of Dendrology in KSrnik (Giertych, 1970). The shoots were stored in polythene bags at 1 °C for 2-3 days before experiments. DTA analysis. Differential thermal analysis was used to determine the crystallization of supercooled tissue water as described by Quamme et al. (1972). Shoot and bud tissue samples were taken from the current seasonal growth. A copper-constantan thermocouple was placed in contact with the sample, and the tissue wrapped in parafilm. The differential temperature was recorded between a sample and a dried reference which were both frozen in an aluminium block at 0.3°C/min. The presented DTA analyses are the means of six samples. Sample moisture content was determined after each analysis, and is expressed as water percent of fresh weight. Hardiness determination. Tests of frost-hardiness were conducted using 10-cm lengths of current-season shoots. Samples were frozen to temperatures between - 5 and - 3 5 ° C, at - 5 °C intervals. The cooling rate was 3.5 ° C/h, and each
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Fig. 1 (left). Freezing curves determined by DTA for actively growing shoot and bud of Norway spruce. Fig. 2 (right). Typical profile of DTA for winter- hardy Norway spruce buds. Living bud (A), and after freeze injury (B).
freezing temperature was held for 24 h. After thawing at 1 ° C for 24 h the shoots were placed in basins with water at 20°C and 100% relative humidity for 14 days. The extent of oxidative browning of shoots and bud tissues was used to rate injury. The 'injury temperature' was the highest temperature at which more than 50% injury occurred relative to the unfrozen controls. In one variant of the experiment, the 10-cm shoots were dehardened using long days, temperature of 20°C, and 100% relative humidity, for 7 days (Pukacki, 1981 ). RESULTS
Differential thermal analysis (DTA) of actively growing shoots and buds revealed one exotherm showing ice formation at about - 10 °C (Fig. 1 ). In the autumn, at the time when spruces become hardened, water crystallization occurs at lower temperatures. Fig. 2A shows the three distinct exothermic points at consecutive stages of tissue water supercooling. The first exotherm is usually observed between - 8 and - 1 6 ° C . In hardened tissues, this crystallization takes place outside the cells and does not cause any freezing injury. The second exotherm, at about - 1 8 to - 2 0 ° C , is much smaller and is also non-lethal; This second exotherm also occurs in buds previously injured by freezing (Fig. 2B ). The second exotherm is caused by freezing of water that is exuded from the cells to join the extracellular ice that was formed during the first phase of crystallization ( Quamme et al., 1972 ). The migration of water to extracellular nuclei of ice is permitted by a reduction in the resistance of cell membranes to water transfer, and the existence of water vapour pressure gradients between the supercooled water and the ice crystals outside. A further lowering of tissue temperatures leads to the occurrence of deep supercooling of tissue water, and finally its crystallization at a homogenous nucleation temperature, giving 'low
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Fig. 3 (left). DTA freezing profiles of Norway spruce bud and shoot. Winter-hardy tissue (A), and after dehardening (B). Fig. 4 (right). DTA freezing profiles of overwintering buds of four Polish Norway spruce provenances: Kowary 98; Istebna 99; Nowy Targ 103; and Wetlina 104. Solid circles on the DTA profiles indicate the temperature causing visual injury in separate viability experiments on buds collected from the same plants.
temperature exotherms' (LTE) at about - 2 7 °C (Fig. 2A). Spruce buds which have been killed by freezing do not undergo deep supercooling (Fig. 2B ). Winter buds that were dehardened in long days at 20 °C did not produce LTEs and their two high-temperature exotherms occurred at higher temperatures than in hardened buds (Fig. 3 ). Norway spruce from Polish provenances produced LTEs at different temperatures, all preceded by high-temperature exotherms (Fig. 4). The temperatures causing visual injury in separate tests were about 5 °C lower than the LTE temperature, possibly because of the large temperature steps used in the freezing tests. Provenances Istebna 99 and Kowary 98 were most frost-hardy; LTE's occurred in these populations at about - 24 ° C and visual injury occurred at - 3 0 ° C . The tendency to deep supercooling varied seasonally as shown by the presence of LTE's in shoots and buds sampled on 11 occasions during the year (Fig. 5). LTE's occurred during the period of winter rest, but not during the period of vegetative growth. In the period from autumn to late spring, the supercooling LTE changed from - 1 7 to - 2 7 °C in buds. In shoots of the two studied spruce populations it was not possible to register LTE below a temperature of - 21 ° C (Fig. 5). The temperature of first exotherm formation was significantly correlated with the water content of the tissues (Fig. 6; r--0.75 for buds and r = 0 . 7 8 for shoots). Thffs, freezing in the extracellular spaces occurred at a lower temperature when the tissues were most dehydrated. The tissue water content had no demonstrable effect on the temperature crystallization of deeply supercooled water.
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Fig. 5 (left). Seasonal changes in DTA freezing profiles of shoots and buds of two Norway spruce provenances: Kowary 98; and Zwierzyniec 121. Fig. 6 (right). Relationship between the high temperature exotherms of Norway spruce tissues and the water content. DISCUSSION
Buds and shoots of overwintering spruce produce barriers that restrict rapid ice formation (Figs. 2, 3, 4 ). Tissue water supercools and freezes in stages, resulting in DTA profiles with two or three clear exotherms. After dehardening there is an increase in the temperature at which successive exotherms occur (Figs. 3, 5 ). In winter-hardy buds the low-temperature exotherm occurred at - 2 7 ° C , whereas after their dehardening the lowest exotherm occurred at - 16.5 ° C. It may presumed that, in dehardened tissues, barriers to ice formation are eliminated so that the tissues freeze at higher temperatures. The extracellular and probably the intertissue crystallization ( Sakai, 1982 ) depends to a large extent on the facility for water transport out of the cytoplasm into the intertissue spaces. Olien and Smith (1977), when analysing the process of freezing plant tissues, assigned a considerable role to the adhesive energy which occurs between extracellular ice, cell-wall polymers and the plasmolemma. It is believed that, at temperatures close to 0 ° C, the amount of water asso-
102
ciated with structural proteins increases as well as the amount of hydrogen bonding (Weiser, 1970; Pukacki, 1979). This physico-chemical state exists in hardened tissues (Tumanov and Krasavcev, 1959). High temperatures such as the 20 ° C used in the dehardening experiment also influence the ultrastructure of cells. Bervaes et al. (1977) have shown that hardened pine needles and tissues of apple tree bark have higher energy barriers to water movement from the cytoplasmic colloids to the ice crystals than do nonhardened tissues. The existence of these barriers delays cytoplasmic crystallization. After dehardening the energy potential of these barriers is substantially lowered, and as a result ice forms within the cells at a higher temperature. Thus the degree of tissue injury increases, together with a lowering of the negative activation energy. Dead buds did not produce low-temperature exotherms (Fig. 2). The cell membranes did not prevent the migration of water and ions from the cytoplasm to the intercellular spaces and the easy movement of ice crystals into cells. However, extracellular freezing (the first exotherm) occurred at lower temperatures than in living shoots (Fig. 2B ). This was probably caused by an increase in the concentration of the intercellular solution following one-way movement of ions through the damaged cell membranes (Dexter et al., 1932; Ashworth et al., 1983; Pukacki, 1984). The tendency to deep supercool changed during the season (Fig. 5). The content of water of the tissues had little effect on this phenomenon, in contrast to results reported by Kaku et al. (1982) and Quamme (1983), possibly because there was little variation in the water content of tissues in this study. Undoubtedly, water content had a significant role in the supercooling of extracellular water, i.e. on the temperature of the high-temperature exotherms (Fig. 6). The results in the present paper suggest that the freezing injuries of overwintering sensitive organs in spruce depend on the specific freezing. Freezing avoidance by deep supercooling appears to be a common survival mechanism in these tissues. However, the details of biophysical and/or biochemical mechanisms which allow deep supercooling are not known. ACKNOWLEDGEMENTS
This study was supported by research fund MR II/16, coordinated by the Institute of Dendrology, Kdrnik.
REFERENCES Ashworth, E.N., Rowse, D.J. and Billmyer, L.A., 1983. The freezing of water in woody tissues of apricot and peach and the relationship to freezing injury. J. Am. Soc. Sci., 198: 299-303. Bervaes, J.C.A.M., Ketchie, D.O. and Kuiper, P.J.C., 1977. Kinetics of freezing damage in apple bark and pine needles. Physiol. Plant., 40: 35-38.
103 Burke, M.J., Stushnoff, C., 1979. Frost hardiness:A discussionof possible molecular causes of injurywith particularreferenceto deep supercoolingof water. In: H. Mussell and R.C. Staples (Editors),Stress Physiology in Crop Plants.Wiley, New York, pp. 197-225. Burke, M.J., Gusta, L.V., Quarnme, H.A., Weiser, C.J. and Li, P.H., 1976. Freezing and injury in plants.Annu. Rev. Plant Physiol.,27: 507-528. Dexter, S.T.,Tottingham, W.E. and Graber, L.F., 1932. Investigationsof hardiness of plants by measurement of electricalconductivity.Plant Physiol.,7: 63-78. George, M.F., Burke, M.J., Pellett,H.M. and Johanson, A.G., 1974. Low temperature exotherms and woody plant distribution.HortScience, 9: 519-522. Gierty~h,. M., 1970. A provenance experiment on Norway spruce Picea abies (L.) Karst. established in 1969. (Polish ed.) Arbor. Kdrnickie, 15: 263-276. Graham, P.R. and Mullin, R., 1976. The determination of lethal freezing temperatures in buds and stems of deciduous azalea by a freezing curve method. J. Am. Soc. Hortic. Sci., 101: 3-7. Kaku, S., Iwaya, M. and Jeon, K.B., 1982. Supercooling ability and cold hardiness of rhododendron flower buds with reference to winter water relations. In: P.H. Li and A. Sakai (Editors), Plant Cold Hardiness and Freezing Stress: Mechanisms and Crop Implications. Academic Press, London, vol. II, pp. 357-366. Larcher, W., 1975. Physiological Plant Ecology. Springer, Berlin, 252 pp. Levitt, J., 1980. Responses of Plants to Environmental Stresses. Vol. II, Chilling, Freezing, and High Temperature Stresses (2nd edition) Academic Press, New York, 607 pp. Olien, C.R. and Smith, M.N., 1977. Ice adhesions in relation to freeze stress. Plant Physiol., 60: 499-503. Pukacki, P., 1979. Dependence of electrical impedance of Magnolia shoots on temperature. Arbor. Kdrnickie, 24: 187-192. Pukacki, P., 1981. Freezing tolerance of Polish Norway spruce provenances. Arbor. K6rnickie, 26: 151-161. Pukacki, P., 1984. The occurrence of deep supercooling in the genus Picea. In: T. Holubowicz (Editor), Second symp. winter hardiness in woody perennials. Acta Hortic., 168: 101-107. Quamme, H.A., 1983. Relationship of air temperature to water content and supercooling of overwintering peach flower buds. J. Am. Soc. Hortic. Sci., 108: 697-701. Quamme, H.A., Stushnoff, C. and Weieer, C.J., 1972. The relationship of exotherms to cold injury in apple tissues. J. Am. Soc. Hort. Sci., 97: 608-613. Rasmussen, D.H. and Mackenzie, A.P., 1972. Effect of solute on ice-solution interfacial free energy; calculation from measured homogeneous nucleation temperatures. In: H.H.G. Jellinek (Editor), Water Structure at the Water-Polymer Interface. Plenum Press, New York, pp. 126-145. Sakai, A., 1979. Freezing avoidance of primordial shoots of very hardy conifer buds. Low Temp. Sci. Ser. B, 37: 1-9. Sakai, A., 1982. Freezing tolerance of shoots and flower primordia of coniferous buds by extraorgan freezing. Plant Cell Physiol., 23: 1219-1227. Tumanov, I.I. and Krasavcev, O.A., 1959. Zakalivanie severnych drevesnych rastenij atricatelnymi temperaturami (Hardening of northern woody plants by negative temperative treatment). Fiziol. Rast., 6: 654-667. Weiser, C.J., 1970. Cold resistance and injury in woody plants. Science, 169: 1269-1278.
97
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Deep Supercooling of Shoot and Bud Tissues of P i c e a abies PAWEL PUKACKI
Polish Academy of Sciences, Institute of Dendrology, PL-62-035 Kdrnik (Poland) (Accepted 7 August 1986)
ABSTRACT Pukacki, P., 1987. Deep supercooling of shoot and bud tissues ofPicea abies. For. Ecol. Manage., 20: 97-103. Using differential thermal analysis ( D T A ) it was shown that actively growing or developing buds and shoots of Norway spruce die as a result of a single freezing of tissue water, which takes place at temperatures between - 8 and - 16 ° C. O n the other hand, in winter-hardened organs, tissue water freezes in two successive stages, each of which preceded by supercooling of the tissue water. At first,there is freezing of supercooled water in the extracellular spaces and then there occurs the crystallization by deep supercooling of the remaining tissue water; in winter-resting buds this occurs in the range from - 17 to - 27 ° C. Dead or dehardened buds do not demonstrate the tendency towards deep supercooling. The role of water content and of barriers restrictingwater movement from cytoplasm to the extracellular ice are discussed.
INTRODUCTION
Tissue of actively growing shoots and buds of trees from the temperate climatic zones die when frozen to about - 5 to - 1 0 ° C (Larcher, 1975). Other investigations have shown that death is caused by the formation of ice, which destroys the cell structure (e.g. Levitt, 1980). On the other hand, in winter, the tissue water during cooling at first becomes supercooled. This state is unstable, and under some conditions a spontaneoustransition of the deeply supercooled tissue water into a stable phase occurs. The temperature of this change is described as the homogenous ice-nucleation temperature ( George et al., 1974). It has been experimentally established that the temperature crystallization of small pure water droplets 10/lm in diameter occurs at a temperature of nucleation of -38.1°C (Burke and Stushnoff, 1979). For typical homogenous plant solutions, the nucleation temperature (Tn), according to Rasmussen and MacKenzie (1972), is related to the melting temperature (Tm) as follows: 0378-1127/87/$03.50
© 1987 Elsevier Science Publishers B.V.
98 Tn = - 3 8 . 1 ° C + (1.8+_0.4) Tm In many species of herbaceous and woody plants Tm varies from 0.69 ° to 2.94 ° C (Burke and Stushnoff, 1979). The temperature of the phase change can be registered using methods such as differential thermal analysis (DTA), differential scanning calorimetry (DSC), or pulsed nuclear magnetic resonance spectroscopy (NMR; Burke et al., 1976; Burke and Stushnoff, 1979). Temperature curves in a differential thermal analysis reveal the occurrence of ice crystallization. Graham and Mullin (1976) and Sakai (1979) suggest that, in tissues of woody plants, there exist barriers restricting the spread of ice. It is uncertain whether this p h e n o m e n o n exists primarily because of morphological features of tissues (George et al., 1974) or because of energy bartiers restricting the movement of water from frozen tissues (Bervaes et al., 1977 ). Most probably, the rate of crystallization, and the degree of supercooling of tissue water, will depend on several factors such as physico-chemical properties of water, ice adhesions, phasic changes in the lipids of cell membranes, structure of tissues, and their hydration and stage of preparation for low-temperature stress. In the results presented below, seasonal differences are demonstrated in the supercooling of tissue water in relation to the freezing tolerance of buds and of Norway spruce. MATERIALSAND METHODS
Plant material. One-year-old shoots were collected from 19-year-old trees of Picea abies (L.) Karst. growing on an experimental area of the Institute of Dendrology in KSrnik (Giertych, 1970). The shoots were stored in polythene bags at 1 °C for 2-3 days before experiments. DTA analysis. Differential thermal analysis was used to determine the crystallization of supercooled tissue water as described by Quamme et al. (1972). Shoot and bud tissue samples were taken from the current seasonal growth. A copper-constantan thermocouple was placed in contact with the sample, and the tissue wrapped in parafilm. The differential temperature was recorded between a sample and a dried reference which were both frozen in an aluminium block at 0.3°C/min. The presented DTA analyses are the means of six samples. Sample moisture content was determined after each analysis, and is expressed as water percent of fresh weight. Hardiness determination. Tests of frost-hardiness were conducted using 10-cm lengths of current-season shoots. Samples were frozen to temperatures between - 5 and - 3 5 ° C, at - 5 °C intervals. The cooling rate was 3.5 ° C/h, and each
99
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60
1
I
180 120 TIME, rain.
A
_o
-3o~,
I
240
T
B l
0 40 -213 -30 -40 REFERENCE TEMPERATURE,°C
Fig. 1 (left). Freezing curves determined by DTA for actively growing shoot and bud of Norway spruce. Fig. 2 (right). Typical profile of DTA for winter- hardy Norway spruce buds. Living bud (A), and after freeze injury (B).
freezing temperature was held for 24 h. After thawing at 1 ° C for 24 h the shoots were placed in basins with water at 20°C and 100% relative humidity for 14 days. The extent of oxidative browning of shoots and bud tissues was used to rate injury. The 'injury temperature' was the highest temperature at which more than 50% injury occurred relative to the unfrozen controls. In one variant of the experiment, the 10-cm shoots were dehardened using long days, temperature of 20°C, and 100% relative humidity, for 7 days (Pukacki, 1981 ). RESULTS
Differential thermal analysis (DTA) of actively growing shoots and buds revealed one exotherm showing ice formation at about - 10 °C (Fig. 1 ). In the autumn, at the time when spruces become hardened, water crystallization occurs at lower temperatures. Fig. 2A shows the three distinct exothermic points at consecutive stages of tissue water supercooling. The first exotherm is usually observed between - 8 and - 1 6 ° C . In hardened tissues, this crystallization takes place outside the cells and does not cause any freezing injury. The second exotherm, at about - 1 8 to - 2 0 ° C , is much smaller and is also non-lethal; This second exotherm also occurs in buds previously injured by freezing (Fig. 2B ). The second exotherm is caused by freezing of water that is exuded from the cells to join the extracellular ice that was formed during the first phase of crystallization ( Quamme et al., 1972 ). The migration of water to extracellular nuclei of ice is permitted by a reduction in the resistance of cell membranes to water transfer, and the existence of water vapour pressure gradients between the supercooled water and the ice crystals outside. A further lowering of tissue temperatures leads to the occurrence of deep supercooling of tissue water, and finally its crystallization at a homogenous nucleation temperature, giving 'low
100
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190 °
270 °
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160 ' 0 T I M E , min.
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Fig. 3 (left). DTA freezing profiles of Norway spruce bud and shoot. Winter-hardy tissue (A), and after dehardening (B). Fig. 4 (right). DTA freezing profiles of overwintering buds of four Polish Norway spruce provenances: Kowary 98; Istebna 99; Nowy Targ 103; and Wetlina 104. Solid circles on the DTA profiles indicate the temperature causing visual injury in separate viability experiments on buds collected from the same plants.
temperature exotherms' (LTE) at about - 2 7 °C (Fig. 2A). Spruce buds which have been killed by freezing do not undergo deep supercooling (Fig. 2B ). Winter buds that were dehardened in long days at 20 °C did not produce LTEs and their two high-temperature exotherms occurred at higher temperatures than in hardened buds (Fig. 3 ). Norway spruce from Polish provenances produced LTEs at different temperatures, all preceded by high-temperature exotherms (Fig. 4). The temperatures causing visual injury in separate tests were about 5 °C lower than the LTE temperature, possibly because of the large temperature steps used in the freezing tests. Provenances Istebna 99 and Kowary 98 were most frost-hardy; LTE's occurred in these populations at about - 24 ° C and visual injury occurred at - 3 0 ° C . The tendency to deep supercooling varied seasonally as shown by the presence of LTE's in shoots and buds sampled on 11 occasions during the year (Fig. 5). LTE's occurred during the period of winter rest, but not during the period of vegetative growth. In the period from autumn to late spring, the supercooling LTE changed from - 1 7 to - 2 7 °C in buds. In shoots of the two studied spruce populations it was not possible to register LTE below a temperature of - 21 ° C (Fig. 5). The temperature of first exotherm formation was significantly correlated with the water content of the tissues (Fig. 6; r--0.75 for buds and r = 0 . 7 8 for shoots). Thffs, freezing in the extracellular spaces occurred at a lower temperature when the tissues were most dehydrated. The tissue water content had no demonstrable effect on the temperature crystallization of deeply supercooled water.
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Fig. 5 (left). Seasonal changes in DTA freezing profiles of shoots and buds of two Norway spruce provenances: Kowary 98; and Zwierzyniec 121. Fig. 6 (right). Relationship between the high temperature exotherms of Norway spruce tissues and the water content. DISCUSSION
Buds and shoots of overwintering spruce produce barriers that restrict rapid ice formation (Figs. 2, 3, 4 ). Tissue water supercools and freezes in stages, resulting in DTA profiles with two or three clear exotherms. After dehardening there is an increase in the temperature at which successive exotherms occur (Figs. 3, 5 ). In winter-hardy buds the low-temperature exotherm occurred at - 2 7 ° C , whereas after their dehardening the lowest exotherm occurred at - 16.5 ° C. It may presumed that, in dehardened tissues, barriers to ice formation are eliminated so that the tissues freeze at higher temperatures. The extracellular and probably the intertissue crystallization ( Sakai, 1982 ) depends to a large extent on the facility for water transport out of the cytoplasm into the intertissue spaces. Olien and Smith (1977), when analysing the process of freezing plant tissues, assigned a considerable role to the adhesive energy which occurs between extracellular ice, cell-wall polymers and the plasmolemma. It is believed that, at temperatures close to 0 ° C, the amount of water asso-
102
ciated with structural proteins increases as well as the amount of hydrogen bonding (Weiser, 1970; Pukacki, 1979). This physico-chemical state exists in hardened tissues (Tumanov and Krasavcev, 1959). High temperatures such as the 20 ° C used in the dehardening experiment also influence the ultrastructure of cells. Bervaes et al. (1977) have shown that hardened pine needles and tissues of apple tree bark have higher energy barriers to water movement from the cytoplasmic colloids to the ice crystals than do nonhardened tissues. The existence of these barriers delays cytoplasmic crystallization. After dehardening the energy potential of these barriers is substantially lowered, and as a result ice forms within the cells at a higher temperature. Thus the degree of tissue injury increases, together with a lowering of the negative activation energy. Dead buds did not produce low-temperature exotherms (Fig. 2). The cell membranes did not prevent the migration of water and ions from the cytoplasm to the intercellular spaces and the easy movement of ice crystals into cells. However, extracellular freezing (the first exotherm) occurred at lower temperatures than in living shoots (Fig. 2B ). This was probably caused by an increase in the concentration of the intercellular solution following one-way movement of ions through the damaged cell membranes (Dexter et al., 1932; Ashworth et al., 1983; Pukacki, 1984). The tendency to deep supercool changed during the season (Fig. 5). The content of water of the tissues had little effect on this phenomenon, in contrast to results reported by Kaku et al. (1982) and Quamme (1983), possibly because there was little variation in the water content of tissues in this study. Undoubtedly, water content had a significant role in the supercooling of extracellular water, i.e. on the temperature of the high-temperature exotherms (Fig. 6). The results in the present paper suggest that the freezing injuries of overwintering sensitive organs in spruce depend on the specific freezing. Freezing avoidance by deep supercooling appears to be a common survival mechanism in these tissues. However, the details of biophysical and/or biochemical mechanisms which allow deep supercooling are not known. ACKNOWLEDGEMENTS
This study was supported by research fund MR II/16, coordinated by the Institute of Dendrology, Kdrnik.
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