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Seasonal Changes in the Freezing Behavior of Xylem Ray Parenchyma Cells in Four Boreal Hardwood Species
Cryobiology 38, 81– 88 (1999) Article ID cryo.1998.2149, available online at http://www.idealibrary.com on
Seasonal Changes in the Freezing Behavior of Xylem Ray Parenchyma Cells in Four Boreal Hardwood Species Katsushi Kuroda,* Jun Ohtani,* Masatoshi Kubota,† and Seizo Fujikawa† ,1 *Faculty of Agriculture and †Institute of Low Temperature Science, Hokkaido University, Sapporo, 060-0819 Japan The freezing behavior of xylem ray parenchyma cells in several boreal hardwood species, namely, Betula platyphylla, Populus canadensis, P. sieboldii, and Salix sachalinensis, was examined by differential thermal analysis (DTA), cryo-scanning electron microscopy (Cryo-SEM), and freeze-fracture replica electron microscopy. Although DTA profiles of samples harvested in summer and in winter suggested that the xylem ray parenchyma cells in all four species responded to freezing stress by extracellular freezing, Cryo-SEM showed clearly that the xylem ray parenchyma cells in all these species responded to freezing stress by shallow supercooling in summer and by extracellular freezing in winter. It is suggested that DTA failed to reveal the true freezing behavior of xylem ray parenchyma cells because of an overlap of temperature ranges between the high-temperature exotherm and the low-temperature exotherm and/or because of the limited extent of the LTE. The seasonal changes in freezing behavior of xylem ray parenchyma cells in all these boreal species, which are results of seasonal cold acclimation, support the hypothesis that a gradual shift of freezing behavior in xylem ray parenchyma cells from shallow supercooling in hardwood species that grow in tropical zones to extracellular freezing in hardwood species that grow in cold areas might be a result of the evolutionary adaptation of hardwood species to cold climates. © 1999 Academic Press Key Words: xylem ray parenchyma cells; extracellular freezing; supercooling; cold acclimation; cryoscanning electron microscope (Cryo-SEM).
via the gradual increase in supercooling ability that is seen in hardwood species in temperate zones (10), to extracellular freezing in hardwood species in colder regions (2). In red osier dogwood (Cornus sericea), one of most freezing-tolerant woody species examined to date, the freezing behavior of the xylem ray parenchyma cells shifts seasonally from shallow supercooling in summer to extracellular freezing in winter (4). These features of red osier dogwood support the hypothesis that the freezing behavior of xylem ray parenchyma cells in hardwood species might be strongly dependent upon temperatures in the growth environment, which are related both to geography and to season. However, to date, red osier dogwood is the only tree examined that exhibits seasonal changes in freezing behavior. In this study, we examined the seasonal freezing behavior of the xylem ray parenchyma cells in several hardwood species that belong to the genera Batula, Populus, and Salix, all of which have been reported to exhibit extracellular freezing around xylem ray parenchyma cells
Xylem ray parenchyma cells in woody species respond to freezing of the xylem water either by deep supercooling or by extracellular freezing (1, 2, 8). Many woody species growing in temperate zones respond to freezing of the apoplastic water in xylem by deep supercooling in the xylem ray parenchyma cells, whereas woody species belonging to the genera Betula, Populus, and Salix, which grow in colder regions where minimum winter temperatures fall below 240°C, respond to freezing of apoplastic water in xylem by extracellular freezing in the xylem ray parenchyma cells (7, 13). The freezing behavior of xylem ray parenchyma cells in hardwood species tends to change with the temperatures in the areas where they grow. Such behavior changes gradually from shallow supercooling in hardwood species that grow in tropical and subtropical zones (11),
Received September 24, 1998; accepted December 15, 1998. 1 To whom correspondence should be addressed. Fax: 81-011-706-7142. E-mail: [email protected]. 81
0011-2240/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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upon freezing (13). Our goal was to obtain further evidence of the dependence on growth temperatures of the freezing behavior of xylem ray parenchyma cells in hardwood species.
with a transmission electron microscope (1200 EX; JEOL Co. Ltd.). RESULTS
DTA of Xylem MATERIALS AND METHODS
Materials For materials, twigs (all of more than 5 years of age) were collected from Betula platyphylla, Populus canadensis, P. sieboldii, and Salix sachalinensis (all the trees were more than 20 years old and were growing on the campus of Hokkaido University in Sapporo). Small blocks, including the second or third annual rings, were cut out from the fresh xylem.
DTA of xylem, with a cooling rate of 0.1°C/ min, produced only one distinct high-temperature exotherm (HTE), which was produced by the freezing of apoplastic water in xylem. There was no low-temperature exotherm (LTE) of the type produced by the intracellular freezing of xylem ray parenchyma cells upon breakdown of supercooling. These results were similar for all species examined in winter and in summer (Fig. 1). Furthermore, neither slower cooling (0.01°C/
Freezing For differential thermal analysis (DTA), the small blocks were frozen at different cooling rates indicated in text from 3 to 250°C, without ice seeding, basically as described previously (3, 4, 6, 11). For electron microscopy, small blocks of sample in specimen holders were also frozen at different cooling rates indicated in text from 23°C to desired temperatures, after ice seeding at the starting temperature. After equilibration at each final temperature, samples were cryofixed with freon 22. Control samples were cryofixed from room temperature (3, 4, 6, 11). DTA DTA was performed by a method similar to that described in previous reports (3, 4, 6, 11). Electron Microscopy Cryo-scanning electron microscopy (CryoSEM) and electron microscopy of freeze-fracture replicas were performed as described in previous reports (3, 4, 6, 11). In brief, a cryofixed sample was transferred to a cold stage in the specimen preparation chamber of CryoSEM (JSM 840A, equipped with CRU-40; JEOL Co. Ltd.), equilibrated at 2105°C, fractured, etched, and evaporated with platinum– carbon. After observation with Cryo-SEM, the evaporated film (freeze replica) was extracted from the sample. Freeze replicas were observed
FIG. 1. DTA profiles for B. platyphylla harvested in summer (a) and in winter (b), for P. canadensis in summer (c) and in winter (d), for P. sieboldii in summer (e) and in winter (f), and for S. sachalinensis in summer (g) and in winter (h).
SEASONAL CHANGES IN FREEZING BEHAVIOR OF XYLEM CELLS
83
FIG. 2. (a) Cryo-SEM and (b) freeze-fracture replica micrographs showing xylem ray parenchyma cells in a control sample that was cryofixed from room temperature without slow cooling. Xylem from B. platyphylla harvested in summer.
min) nor faster cooling (2.5°C/min) generated any evidence of an LTE (data not shown). These results indicate that the xylem ray parenchyma cells in these woody species might respond to the freezing of apoplastic water in xylem by extracellular freezing. Assessment of the Freezing Behavior of Xylem Ray Parenchyma Cells by Cryo-SEM Observations by Cryo-SEM clearly revealed differences in freezing behavior of xylem ray parenchyma cells that had been cryofixed from desired temperatures after cooling. Cryofixation of samples from room temperature, without cooling (control samples), exhibited circular or ellipsoidal profiles of cross-fractured xylem ray parenchyma cells with crystals of small intracellular ice (less than 50 nm in diameter, determined from freeze-fracture replicas) in the cytoplasm (Figs. 2a and 2b). These ice crystals in control samples were undoubtedly produced by the cryofixation of liquid water. After cryofixation, supercooled cells resembled the control samples, with respect to both the profiles of cells and the size of intracellular ice crystals (Fig. 3). Cryofixation of samples, in which intracellular freezing had occurred during slow cooling upon the breakdown of supercooling, re-
vealed significantly larger (more than 1 mm in diameter) intracellular ice crystals (Fig. 4). Cryofixation of samples that had undergone extracellular freezing during slow cooling revealed more or less shrunken and deformed cells without any evidence of intracellular ice (Fig. 5a). Observations of freeze-fracture replicas confirmed that these extracellularly frozen cells contained no intracellular ice after slow freezing, at least below 220°C (Fig. 5b). In extracellularly frozen cells, the endoplasmic reticulum (ER) was reorganized into multiplex lamellae, which were evidence of dehydration (5). Desiccated cells were also observed as cells with an empty lumen or as cells with collapsed cytoplasm within cell walls that retained the original, ellipsoidal profile of the cell wall (Fig. 6). Freezing Behavior of Xylem Ray Parenchyma Cells, as Observed by Cryo-SEM The freezing behavior of xylem ray parenchyma cells observed by Cryo-SEM was different from that predicted by DTA, which had suggested the occurrence of extracellular freezing in samples harvested both in winter and in summer. Table 1 summarizes the results of Cryo-SEM after slow freezing (5°C/day) for the xylem ray parenchyma cells of all species ex-
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SEASONAL CHANGES IN FREEZING BEHAVIOR OF XYLEM CELLS
amined. There were clear differences between winter and summer in the freezing behavior of these xylem ray parenchyma cells. In winter, xylem ray parenchyma cells in all the species examined exhibited extracellular freezing. By contrast, in summer, supercooling was apparent. In summer, even very slow cooling at 2.5°C/day failed to induce extracellular freezing (not shown). In samples harvested both in winter and in summer, there were small numbers of desiccated cells after such slow cooling. The desiccated cells were essentially produced by freezing below 210°C and they showed a tendency to increase by freezing to lower temperatures. Although it is not clear why desiccation occurred, a possibility of cavitation of cellular water during slow freezing has been suggested (12). In winter, although all the xylem ray parenchyma cells (apart from a few desiccated cells) in all species examined underwent extracellular freezing during slow cooling to lower temperatures, the critical cooling rates that induced extracellular freezing differed among species. Table 2 shows results of analysis by Cryo-SEM of freezing behavior of xylem ray parenchyma cells that were frozen at 0.1°C/min to 250°C. At this comparatively high rate of cooling, which corresponded to that during DTA, all xylem ray parenchyma cells with the exception of desiccated cells of B. platyphylla underwent extracellular freezing. In other species, however, small numbers of intracellularly frozen cells were produced. In summer, although all xylem ray paren-
85
chyma cells (apart from a few desiccated cells) in all species examined underwent intracellular freezing upon the breakdown of supercooling during slow cooling (5°C/day), the temperature for intracellular freezing differed among species (Table 1). While intracellular freezing in S. sachalinensis occurred between 210 and 215°C, intracellular freezing in P. sieboldii occurred, in a majority of cells, below 230°C (Table 1). Intracellular freezing in B. platyphylla and P. canadensis occurred at temperatures between 210 and 230°C (Table 1). We examined the effects of cooling rates on supercooling ability in the xylem ray parenchyma cells of S. sachalinensis and P. sieboldii in summer. Samples were cooled rapidly at 2.5°C/min (to indicated temperatures and held overnight) and freezing behavior was examined by Cryo-SEM (Table 3). The results showed that intracellular freezing occurred at higher temperatures by rapid cooling (Table 3) than by slow cooling (Table 1), suggesting the partial dehydration of cells and the consequent depression of supercooling temperatures due to the dehydration-induced concentration of cellular solutes by slow cooling (9). DISCUSSION
Hardwood species in the genera Betula, Populus, and Salix are distributed in the coldest areas of the North hemisphere, where minimum air temperatures in winter can fall below 240°C (13). Previous DTA studies showed that, upon cooling, the xylem of these species produces only an HTE and not an LTE in all seasons. In
FIG. 3. Cryo-SEM micrograph showing xylem ray parenchyma cells after supercooling. Xylem ray parenchyma cells of B. platypylla that had been harvested in summer, frozen to 215°C at a rate of 5°C/day and cryofixed. FIG. 4. Cryo-SEM micrograph showing xylem ray parenchyma cells after intracellular freezing upon breakdown of supercooling. Xylem from B. platyphylla that had been harvested in summer, frozen to 220°C at a rate of 5°C/day, and cryofixed. FIG. 5. (a) Cryo-SEM and (b) freeze-fracture replica micrographs showing xylem ray parenchyma cells after extracellular freezing. (a) Xylem ray parenchyma cells of B. platyphylla had been harvested in winter, frozen to 250°C at a rate of 0.1°C/min, and cryofixed. (b) Xylem ray parenchyma cells of B. platyphylla that had been harvested in winter, frozen to 230°C at a rate of 0.1°C/min, and cryofixed. Arrows show multiplex lamellae of the ER beneath the plasma membrane (P). FIG. 6. Cryo-SEM micrograph showing xylem ray parenchyma cells after desiccation. Xylem from B. platyphylla had been harvested in summer, frozen to 225°C at a rate of 5°C/day, and cryofixed.
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KURODA ET AL. TABLE 1 Cryo-SEM Results Showing Freezing Behavior of Xylem Ray Parenchyma Cells in Hardwood Species at a Cooling of 5°C/day
Temperature (°C): B. platyphylla winter
summer
P. gerlica winter
summer
P. sieboldii winter
summer
S. sachalinensis winter
summer
25
210
215
220
225
230
235
240
245
250
SC IF EF D SC IF EF D
— — — — 100 0 0 0
— 0 100 a 0 99 1 0 0
— — — — 75 25 0 0
— — — — 15 85 0 0
— — — — 4 96 0 0
0 0 100 0 0 98 0 2
— — — — — — — —
— — — — — — — —
— — — — — — — —
0 0 100 0 — — — —
SC IF EF D SC IF EF D
— — — — — — — —
— — — — 97 3 0 0
— — — — — — — —
— — — — 8 81 0 11
— — — — — — — —
— — — — 2 86 0 12
— — — — — — — —
— — — — — — — —
— — — — — — — —
0 0 80 20 — — — —
SC IF EF D SC IF EF D
— — — — — — — —
— — — — 100 0 0 0
— — — — — — — —
— — — — 90 10 0 0
— — — — — — — —
— — — — 60 32 0 8
— — — — — — — —
— — — — — — — —
— — — — — — — —
0 0 89 11 0 92 0 8
SC IF EF D SC IF EF D
— — — — — — — —
— — — — 100 0 0 0
— — — — 0 100 0 0
— — — — — — — —
— — — — — — — —
— — — — — — — —
— — — — — — — —
— — — — — — — —
— — — — — — — —
0 0 87 13 — — — —
Note. The number indicates percentage of supercooled cells (SC), intracellularly frozen cells (IF), extracellulary frozen cells (EF), and desiccated cells (D). a Cells on way of extracellular freezing, showing slight shrinkage of cell walls and formation of small intracellular ice as a result of cryofixation.
DTA, the absence of an LTE is explained in terms of extracellular freezing in xylem ray parenchyma cells. The presence of an LTE is explained in terms of supercooling (2, 8). DTA in the present study revealed the absence of an LTE in all samples in summer and in winter,
leading to the interpretation that the xylem ray parenchyma cells in these species respond to freezing of the apoplastic water in xylem by extracellular freezing both in summer and in winter. In the present and in previous studies (4, 11),
87
SEASONAL CHANGES IN FREEZING BEHAVIOR OF XYLEM CELLS TABLE 2 Cryo-SEM Results Showing Freezing Behavior of Xylem Ray Parenchyma Cells of Hardwood Species Harvested in Winter at a Cooling of 0.1°C/min to 250°C 250
Temperature (°C): B. Platyphylla SC IF EF D P. gerlica SC IF EF D P. sieboldii SC IF EF D S. sachalinensis SC IF EF D
0 0 96 4 0 19 81 0 0 28 72 0 0 22 67 11
Note. The number indicates percentage of supercooled cells (SC), intracellularly frozen cells (IF), extracellularly frozen cells (EF), and desiccated cells (D).
DTA failed to detect the freezing exotherm of xylem ray parenchyma cells, by intracellular freezing upon the breakdown of supercooling,
as an LTE in the present boreal hardwood species in summer (4) and in tropical and subtropical hardwood species (11). The failure of DTA to detect an LTE in these species can be explained by an overlap of the temperature range between the HTE and the LTE and/or the limited extent of an LTE that is due to a small number of ray parenchyma cells within the xylem. We showed that Cryo-SEM in combination with observations of freeze-fracture replicas is the most reliable method for correct assessment of the freezing behavior of xylem ray parenchyma cells (3, 4, 6). Present results also revealed clear differences in the freezing behavior of xylem ray parenchyma cells, with supercooling, intracellular freezing upon the breakdown of supercooling, extracellular freezing, and desiccation all being observed. Cryo-SEM showed that with freezing of water in apoplastic spaces, the xylem ray parenchyma cells in all hardwood species examined were supercooled in summer, but the freezing behavior changed to extracellular freezing in winter. Similar seasonal changes in the freezing behavior of xylem ray parenchyma cells with respect to supercooling and extracellular freezing have also been observed by Cryo-SEM in red osier dogwood (4), which is also distributed in the coldest geographic areas (13). The cooling rates that resulted in extracellular freezing
TABLE 3 Cryo-SEM Results Showing Freezing Behavior of Xylem Ray Parenchyma Cells of Hardwood Species Harvested in Summer at a Cooling of 2.5°C/min Temperature (°C): B. platyphylla SC IF EF D P. sieboldii SC IF EF D
25
210
215
220
225
230
100 0 0 0
63 37 0 0
20 80 0 0
0 100 0 0
0 100 0 0
— — — —
100 0 0 0
90 5 0 5
78 20 0 2
40 55 0 5
— — — —
0 95 0 5
Note. The number indicates percentage of supercooled cells (SC), intracellularly frozen cells (IF), extracellularly frozen cells (EF), and desiccated cells (D).
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of xylem ray parenchyma cells varied among species. However, the cooling rates that produced extracellular freezing of the xylem ray parenchyma cells of all species examined were sufficiently high to bring about extracellular freezing in a natural winter. Cooling rates below 0.1°C/min, which resulted in extracellular freezing in all species examined, are apparently far higher cooling rates than those that occur in nature. The present and previous results indicate one common property of the xylem ray parenchyma cells in all hardwood species examined, which are distributed in the coldest areas of the Northern hemisphere (7, 13) and which have been reported in previous studies to adapt to freezing by extracellular freezing (7, 13). In all these species, the freezing behavior changes from shallow supercooling in summer to extracellular freezing in winter. It has been indicated that the freezing behavior of xylem ray parenchyma cells in hardwood species exhibits a tendency of change depending upon geographic changes in relation with winter temperatures (3, 4, 6, 11). The freezing behavior of xylem ray parenchyma cells changes from shallow supercooling in tropical and subtropical woody species (11), via deep supercooling in temperate woody species (3), to extracellular freezing in cool temperate woody species (4), probably as a result of evolutionarily developed acclimation to cold (3, 4, 6, 11). Seasonal changes in freezing behavior of xylem ray parenchyma cells, with respect to supercooling and extracellular freezing, in hardwood species in cold areas might, thus, be a reflection of evolutionary adaptation. ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Education, Science and Culture of Japan; from the Japan Society for the Promotion of Science; and from the Cooperative Research Funds of the Institute of Low Temperature Science of Hokkaido University.
REFERENCES 1. Ashworth, E. N. Responses of bark and wood cells to freezing. Adv. Low Temp. Biol. 3, 65–106 (1996). 2. Burke, M. J., Gusta, L. V., Quamme, H. A., Weiser, C. J., and Li, P. H. Freezing and injury in plants. Annu. Rev. Plant Physiol. 27, 507–528 (1976). 3. Fujikawa, S., Kuroda, K., and Fukazawa, K. Ultrastructural study of deep supercooling of xylem ray parenchyma cells from Styrax obassia. Micron 25, 241–252 (1994). 4. Fujikawa, S., Kuroda, K., and Ohtani, J. Seasonal changes in the low-temperature behaviour of xylem ray parenchyma cells in red osier dogwood (Cornus sericea L.) with respect to extracellular freezing and supercooling. Micron 27, 181–191 (1996). 5. Fujikawa, S., and Takabe, K. Formation of multiplex lamellae by equilibrium slow freezing of cortical parenchyma cells of mulberry and its possible relationship to freezing tolerance. Protoplasma 190, 189 –203 (1996). 6. Fujikawa, S., Kuroda, K., and Ohtani, J. Seasonal changes in dehydration tolerance of xylem ray parenchyma cells of Stylax obassia twigs that survive freezing temperatures by deep supercooling. Protoplasma 197, 34 – 44 (1997). 7. George, M. F., Burke, M. J., Pellett, H. M., and Johnson, A. G. Low temperature exotherms and woody plant distribution. HortScience 9, 519 –522 (1974). 8. George, M. F., Becwar, M. R., and Burke, M. J. Freezing avoidance by deep undercooling of tissue water in winter-hardy plants. Cryobiology 19, 628 – 639 (1982). 9. Gusta, L. V., Tyler, N. J., and Chen, T. H.-H. Deep undercooling in woody taxa growing north of the 240°C isotherm. Plant Physiol. 72, 122–128 (1983). 10. Kaku, S., and Iwaya, M. Low temperature exotherms in xylems of evergreen and deciduous broad-leaved trees in Japan with reference to freezing resistance and distribution range. In “Plant Cold Hardiness and Freezing Stress” (P. H. Li and A. Sakai, Eds.), pp. 227–239. Academic Press, New York, 1978. 11. Kuroda, K., Ohtani, J., and Fujikawa, S. Supercooling of xylem ray parenchyma cells in tropical and subtropical hardwood species. Trees 12, 97–106 (1997). 12. Rajashekar, C. B., and Lafta, A. Cell-wall changes and cell tension in response to cold acclimation and exogeneous abscisic acid in leaves and cell culture. Plant Physiol. 111, 605– 612 (1996). 13. Sakai, A., and Larcher, W. “Frost Survival of Plants, Responses and Adaptation to Freezing Stress.” Springer-Verlag, Berlin/Heidelberg/New York, 1987.
Seasonal Changes in the Freezing Behavior of Xylem Ray Parenchyma Cells in Four Boreal Hardwood Species Katsushi Kuroda,* Jun Ohtani,* Masatoshi Kubota,† and Seizo Fujikawa† ,1 *Faculty of Agriculture and †Institute of Low Temperature Science, Hokkaido University, Sapporo, 060-0819 Japan The freezing behavior of xylem ray parenchyma cells in several boreal hardwood species, namely, Betula platyphylla, Populus canadensis, P. sieboldii, and Salix sachalinensis, was examined by differential thermal analysis (DTA), cryo-scanning electron microscopy (Cryo-SEM), and freeze-fracture replica electron microscopy. Although DTA profiles of samples harvested in summer and in winter suggested that the xylem ray parenchyma cells in all four species responded to freezing stress by extracellular freezing, Cryo-SEM showed clearly that the xylem ray parenchyma cells in all these species responded to freezing stress by shallow supercooling in summer and by extracellular freezing in winter. It is suggested that DTA failed to reveal the true freezing behavior of xylem ray parenchyma cells because of an overlap of temperature ranges between the high-temperature exotherm and the low-temperature exotherm and/or because of the limited extent of the LTE. The seasonal changes in freezing behavior of xylem ray parenchyma cells in all these boreal species, which are results of seasonal cold acclimation, support the hypothesis that a gradual shift of freezing behavior in xylem ray parenchyma cells from shallow supercooling in hardwood species that grow in tropical zones to extracellular freezing in hardwood species that grow in cold areas might be a result of the evolutionary adaptation of hardwood species to cold climates. © 1999 Academic Press Key Words: xylem ray parenchyma cells; extracellular freezing; supercooling; cold acclimation; cryoscanning electron microscope (Cryo-SEM).
via the gradual increase in supercooling ability that is seen in hardwood species in temperate zones (10), to extracellular freezing in hardwood species in colder regions (2). In red osier dogwood (Cornus sericea), one of most freezing-tolerant woody species examined to date, the freezing behavior of the xylem ray parenchyma cells shifts seasonally from shallow supercooling in summer to extracellular freezing in winter (4). These features of red osier dogwood support the hypothesis that the freezing behavior of xylem ray parenchyma cells in hardwood species might be strongly dependent upon temperatures in the growth environment, which are related both to geography and to season. However, to date, red osier dogwood is the only tree examined that exhibits seasonal changes in freezing behavior. In this study, we examined the seasonal freezing behavior of the xylem ray parenchyma cells in several hardwood species that belong to the genera Batula, Populus, and Salix, all of which have been reported to exhibit extracellular freezing around xylem ray parenchyma cells
Xylem ray parenchyma cells in woody species respond to freezing of the xylem water either by deep supercooling or by extracellular freezing (1, 2, 8). Many woody species growing in temperate zones respond to freezing of the apoplastic water in xylem by deep supercooling in the xylem ray parenchyma cells, whereas woody species belonging to the genera Betula, Populus, and Salix, which grow in colder regions where minimum winter temperatures fall below 240°C, respond to freezing of apoplastic water in xylem by extracellular freezing in the xylem ray parenchyma cells (7, 13). The freezing behavior of xylem ray parenchyma cells in hardwood species tends to change with the temperatures in the areas where they grow. Such behavior changes gradually from shallow supercooling in hardwood species that grow in tropical and subtropical zones (11),
Received September 24, 1998; accepted December 15, 1998. 1 To whom correspondence should be addressed. Fax: 81-011-706-7142. E-mail: [email protected]. 81
0011-2240/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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upon freezing (13). Our goal was to obtain further evidence of the dependence on growth temperatures of the freezing behavior of xylem ray parenchyma cells in hardwood species.
with a transmission electron microscope (1200 EX; JEOL Co. Ltd.). RESULTS
DTA of Xylem MATERIALS AND METHODS
Materials For materials, twigs (all of more than 5 years of age) were collected from Betula platyphylla, Populus canadensis, P. sieboldii, and Salix sachalinensis (all the trees were more than 20 years old and were growing on the campus of Hokkaido University in Sapporo). Small blocks, including the second or third annual rings, were cut out from the fresh xylem.
DTA of xylem, with a cooling rate of 0.1°C/ min, produced only one distinct high-temperature exotherm (HTE), which was produced by the freezing of apoplastic water in xylem. There was no low-temperature exotherm (LTE) of the type produced by the intracellular freezing of xylem ray parenchyma cells upon breakdown of supercooling. These results were similar for all species examined in winter and in summer (Fig. 1). Furthermore, neither slower cooling (0.01°C/
Freezing For differential thermal analysis (DTA), the small blocks were frozen at different cooling rates indicated in text from 3 to 250°C, without ice seeding, basically as described previously (3, 4, 6, 11). For electron microscopy, small blocks of sample in specimen holders were also frozen at different cooling rates indicated in text from 23°C to desired temperatures, after ice seeding at the starting temperature. After equilibration at each final temperature, samples were cryofixed with freon 22. Control samples were cryofixed from room temperature (3, 4, 6, 11). DTA DTA was performed by a method similar to that described in previous reports (3, 4, 6, 11). Electron Microscopy Cryo-scanning electron microscopy (CryoSEM) and electron microscopy of freeze-fracture replicas were performed as described in previous reports (3, 4, 6, 11). In brief, a cryofixed sample was transferred to a cold stage in the specimen preparation chamber of CryoSEM (JSM 840A, equipped with CRU-40; JEOL Co. Ltd.), equilibrated at 2105°C, fractured, etched, and evaporated with platinum– carbon. After observation with Cryo-SEM, the evaporated film (freeze replica) was extracted from the sample. Freeze replicas were observed
FIG. 1. DTA profiles for B. platyphylla harvested in summer (a) and in winter (b), for P. canadensis in summer (c) and in winter (d), for P. sieboldii in summer (e) and in winter (f), and for S. sachalinensis in summer (g) and in winter (h).
SEASONAL CHANGES IN FREEZING BEHAVIOR OF XYLEM CELLS
83
FIG. 2. (a) Cryo-SEM and (b) freeze-fracture replica micrographs showing xylem ray parenchyma cells in a control sample that was cryofixed from room temperature without slow cooling. Xylem from B. platyphylla harvested in summer.
min) nor faster cooling (2.5°C/min) generated any evidence of an LTE (data not shown). These results indicate that the xylem ray parenchyma cells in these woody species might respond to the freezing of apoplastic water in xylem by extracellular freezing. Assessment of the Freezing Behavior of Xylem Ray Parenchyma Cells by Cryo-SEM Observations by Cryo-SEM clearly revealed differences in freezing behavior of xylem ray parenchyma cells that had been cryofixed from desired temperatures after cooling. Cryofixation of samples from room temperature, without cooling (control samples), exhibited circular or ellipsoidal profiles of cross-fractured xylem ray parenchyma cells with crystals of small intracellular ice (less than 50 nm in diameter, determined from freeze-fracture replicas) in the cytoplasm (Figs. 2a and 2b). These ice crystals in control samples were undoubtedly produced by the cryofixation of liquid water. After cryofixation, supercooled cells resembled the control samples, with respect to both the profiles of cells and the size of intracellular ice crystals (Fig. 3). Cryofixation of samples, in which intracellular freezing had occurred during slow cooling upon the breakdown of supercooling, re-
vealed significantly larger (more than 1 mm in diameter) intracellular ice crystals (Fig. 4). Cryofixation of samples that had undergone extracellular freezing during slow cooling revealed more or less shrunken and deformed cells without any evidence of intracellular ice (Fig. 5a). Observations of freeze-fracture replicas confirmed that these extracellularly frozen cells contained no intracellular ice after slow freezing, at least below 220°C (Fig. 5b). In extracellularly frozen cells, the endoplasmic reticulum (ER) was reorganized into multiplex lamellae, which were evidence of dehydration (5). Desiccated cells were also observed as cells with an empty lumen or as cells with collapsed cytoplasm within cell walls that retained the original, ellipsoidal profile of the cell wall (Fig. 6). Freezing Behavior of Xylem Ray Parenchyma Cells, as Observed by Cryo-SEM The freezing behavior of xylem ray parenchyma cells observed by Cryo-SEM was different from that predicted by DTA, which had suggested the occurrence of extracellular freezing in samples harvested both in winter and in summer. Table 1 summarizes the results of Cryo-SEM after slow freezing (5°C/day) for the xylem ray parenchyma cells of all species ex-
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KURODA ET AL.
SEASONAL CHANGES IN FREEZING BEHAVIOR OF XYLEM CELLS
amined. There were clear differences between winter and summer in the freezing behavior of these xylem ray parenchyma cells. In winter, xylem ray parenchyma cells in all the species examined exhibited extracellular freezing. By contrast, in summer, supercooling was apparent. In summer, even very slow cooling at 2.5°C/day failed to induce extracellular freezing (not shown). In samples harvested both in winter and in summer, there were small numbers of desiccated cells after such slow cooling. The desiccated cells were essentially produced by freezing below 210°C and they showed a tendency to increase by freezing to lower temperatures. Although it is not clear why desiccation occurred, a possibility of cavitation of cellular water during slow freezing has been suggested (12). In winter, although all the xylem ray parenchyma cells (apart from a few desiccated cells) in all species examined underwent extracellular freezing during slow cooling to lower temperatures, the critical cooling rates that induced extracellular freezing differed among species. Table 2 shows results of analysis by Cryo-SEM of freezing behavior of xylem ray parenchyma cells that were frozen at 0.1°C/min to 250°C. At this comparatively high rate of cooling, which corresponded to that during DTA, all xylem ray parenchyma cells with the exception of desiccated cells of B. platyphylla underwent extracellular freezing. In other species, however, small numbers of intracellularly frozen cells were produced. In summer, although all xylem ray paren-
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chyma cells (apart from a few desiccated cells) in all species examined underwent intracellular freezing upon the breakdown of supercooling during slow cooling (5°C/day), the temperature for intracellular freezing differed among species (Table 1). While intracellular freezing in S. sachalinensis occurred between 210 and 215°C, intracellular freezing in P. sieboldii occurred, in a majority of cells, below 230°C (Table 1). Intracellular freezing in B. platyphylla and P. canadensis occurred at temperatures between 210 and 230°C (Table 1). We examined the effects of cooling rates on supercooling ability in the xylem ray parenchyma cells of S. sachalinensis and P. sieboldii in summer. Samples were cooled rapidly at 2.5°C/min (to indicated temperatures and held overnight) and freezing behavior was examined by Cryo-SEM (Table 3). The results showed that intracellular freezing occurred at higher temperatures by rapid cooling (Table 3) than by slow cooling (Table 1), suggesting the partial dehydration of cells and the consequent depression of supercooling temperatures due to the dehydration-induced concentration of cellular solutes by slow cooling (9). DISCUSSION
Hardwood species in the genera Betula, Populus, and Salix are distributed in the coldest areas of the North hemisphere, where minimum air temperatures in winter can fall below 240°C (13). Previous DTA studies showed that, upon cooling, the xylem of these species produces only an HTE and not an LTE in all seasons. In
FIG. 3. Cryo-SEM micrograph showing xylem ray parenchyma cells after supercooling. Xylem ray parenchyma cells of B. platypylla that had been harvested in summer, frozen to 215°C at a rate of 5°C/day and cryofixed. FIG. 4. Cryo-SEM micrograph showing xylem ray parenchyma cells after intracellular freezing upon breakdown of supercooling. Xylem from B. platyphylla that had been harvested in summer, frozen to 220°C at a rate of 5°C/day, and cryofixed. FIG. 5. (a) Cryo-SEM and (b) freeze-fracture replica micrographs showing xylem ray parenchyma cells after extracellular freezing. (a) Xylem ray parenchyma cells of B. platyphylla had been harvested in winter, frozen to 250°C at a rate of 0.1°C/min, and cryofixed. (b) Xylem ray parenchyma cells of B. platyphylla that had been harvested in winter, frozen to 230°C at a rate of 0.1°C/min, and cryofixed. Arrows show multiplex lamellae of the ER beneath the plasma membrane (P). FIG. 6. Cryo-SEM micrograph showing xylem ray parenchyma cells after desiccation. Xylem from B. platyphylla had been harvested in summer, frozen to 225°C at a rate of 5°C/day, and cryofixed.
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KURODA ET AL. TABLE 1 Cryo-SEM Results Showing Freezing Behavior of Xylem Ray Parenchyma Cells in Hardwood Species at a Cooling of 5°C/day
Temperature (°C): B. platyphylla winter
summer
P. gerlica winter
summer
P. sieboldii winter
summer
S. sachalinensis winter
summer
25
210
215
220
225
230
235
240
245
250
SC IF EF D SC IF EF D
— — — — 100 0 0 0
— 0 100 a 0 99 1 0 0
— — — — 75 25 0 0
— — — — 15 85 0 0
— — — — 4 96 0 0
0 0 100 0 0 98 0 2
— — — — — — — —
— — — — — — — —
— — — — — — — —
0 0 100 0 — — — —
SC IF EF D SC IF EF D
— — — — — — — —
— — — — 97 3 0 0
— — — — — — — —
— — — — 8 81 0 11
— — — — — — — —
— — — — 2 86 0 12
— — — — — — — —
— — — — — — — —
— — — — — — — —
0 0 80 20 — — — —
SC IF EF D SC IF EF D
— — — — — — — —
— — — — 100 0 0 0
— — — — — — — —
— — — — 90 10 0 0
— — — — — — — —
— — — — 60 32 0 8
— — — — — — — —
— — — — — — — —
— — — — — — — —
0 0 89 11 0 92 0 8
SC IF EF D SC IF EF D
— — — — — — — —
— — — — 100 0 0 0
— — — — 0 100 0 0
— — — — — — — —
— — — — — — — —
— — — — — — — —
— — — — — — — —
— — — — — — — —
— — — — — — — —
0 0 87 13 — — — —
Note. The number indicates percentage of supercooled cells (SC), intracellularly frozen cells (IF), extracellulary frozen cells (EF), and desiccated cells (D). a Cells on way of extracellular freezing, showing slight shrinkage of cell walls and formation of small intracellular ice as a result of cryofixation.
DTA, the absence of an LTE is explained in terms of extracellular freezing in xylem ray parenchyma cells. The presence of an LTE is explained in terms of supercooling (2, 8). DTA in the present study revealed the absence of an LTE in all samples in summer and in winter,
leading to the interpretation that the xylem ray parenchyma cells in these species respond to freezing of the apoplastic water in xylem by extracellular freezing both in summer and in winter. In the present and in previous studies (4, 11),
87
SEASONAL CHANGES IN FREEZING BEHAVIOR OF XYLEM CELLS TABLE 2 Cryo-SEM Results Showing Freezing Behavior of Xylem Ray Parenchyma Cells of Hardwood Species Harvested in Winter at a Cooling of 0.1°C/min to 250°C 250
Temperature (°C): B. Platyphylla SC IF EF D P. gerlica SC IF EF D P. sieboldii SC IF EF D S. sachalinensis SC IF EF D
0 0 96 4 0 19 81 0 0 28 72 0 0 22 67 11
Note. The number indicates percentage of supercooled cells (SC), intracellularly frozen cells (IF), extracellularly frozen cells (EF), and desiccated cells (D).
DTA failed to detect the freezing exotherm of xylem ray parenchyma cells, by intracellular freezing upon the breakdown of supercooling,
as an LTE in the present boreal hardwood species in summer (4) and in tropical and subtropical hardwood species (11). The failure of DTA to detect an LTE in these species can be explained by an overlap of the temperature range between the HTE and the LTE and/or the limited extent of an LTE that is due to a small number of ray parenchyma cells within the xylem. We showed that Cryo-SEM in combination with observations of freeze-fracture replicas is the most reliable method for correct assessment of the freezing behavior of xylem ray parenchyma cells (3, 4, 6). Present results also revealed clear differences in the freezing behavior of xylem ray parenchyma cells, with supercooling, intracellular freezing upon the breakdown of supercooling, extracellular freezing, and desiccation all being observed. Cryo-SEM showed that with freezing of water in apoplastic spaces, the xylem ray parenchyma cells in all hardwood species examined were supercooled in summer, but the freezing behavior changed to extracellular freezing in winter. Similar seasonal changes in the freezing behavior of xylem ray parenchyma cells with respect to supercooling and extracellular freezing have also been observed by Cryo-SEM in red osier dogwood (4), which is also distributed in the coldest geographic areas (13). The cooling rates that resulted in extracellular freezing
TABLE 3 Cryo-SEM Results Showing Freezing Behavior of Xylem Ray Parenchyma Cells of Hardwood Species Harvested in Summer at a Cooling of 2.5°C/min Temperature (°C): B. platyphylla SC IF EF D P. sieboldii SC IF EF D
25
210
215
220
225
230
100 0 0 0
63 37 0 0
20 80 0 0
0 100 0 0
0 100 0 0
— — — —
100 0 0 0
90 5 0 5
78 20 0 2
40 55 0 5
— — — —
0 95 0 5
Note. The number indicates percentage of supercooled cells (SC), intracellularly frozen cells (IF), extracellularly frozen cells (EF), and desiccated cells (D).
88
KURODA ET AL.
of xylem ray parenchyma cells varied among species. However, the cooling rates that produced extracellular freezing of the xylem ray parenchyma cells of all species examined were sufficiently high to bring about extracellular freezing in a natural winter. Cooling rates below 0.1°C/min, which resulted in extracellular freezing in all species examined, are apparently far higher cooling rates than those that occur in nature. The present and previous results indicate one common property of the xylem ray parenchyma cells in all hardwood species examined, which are distributed in the coldest areas of the Northern hemisphere (7, 13) and which have been reported in previous studies to adapt to freezing by extracellular freezing (7, 13). In all these species, the freezing behavior changes from shallow supercooling in summer to extracellular freezing in winter. It has been indicated that the freezing behavior of xylem ray parenchyma cells in hardwood species exhibits a tendency of change depending upon geographic changes in relation with winter temperatures (3, 4, 6, 11). The freezing behavior of xylem ray parenchyma cells changes from shallow supercooling in tropical and subtropical woody species (11), via deep supercooling in temperate woody species (3), to extracellular freezing in cool temperate woody species (4), probably as a result of evolutionarily developed acclimation to cold (3, 4, 6, 11). Seasonal changes in freezing behavior of xylem ray parenchyma cells, with respect to supercooling and extracellular freezing, in hardwood species in cold areas might, thus, be a reflection of evolutionary adaptation. ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Education, Science and Culture of Japan; from the Japan Society for the Promotion of Science; and from the Cooperative Research Funds of the Institute of Low Temperature Science of Hokkaido University.
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