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Ultrastructural study of deep supercooling of xylem ray parenchyma cells from Styrax obassia
Micron, Vol. 25, No. 3, pp. 241-252, 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0968~.328/94 $7.00+ 0.00
Pergamon
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Ultrastructural Study of Deep Supercooling of Xylem Ray Parenchyma Cells from Styrax obassia SEIZO FUJIKAWA,*~: KATSUSHI K U R O D A t and KAZUMI FUKAZAWAt *Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan t Section of Wood Biology, Faculty of Agriculture, Hokkaido University, Sapporo, Japan (Received 30 November 1993; Revised 1 February 1994)
Abstract--Some of the water in the xylem of a woody species (Styrax obassia Sieb. et Zucc.), collected during the summer, appeared to exhibit deep supercooling and the breakdown of supercooling at around -20°C, as determined by differential thermal analysis (DTA). Ultrastructural observations of the low-temperature behaviour of the xylem, using both cryo-scanning electron microscopy (cryo-SEM) and freeze-fracture replica techniques, provided direct evidence that xylem ray parenchyma cells can be deeply supercooled to around - 2 0 ° C and that the breakdown of supercooling produces intracellular freezing in the ray parenchyma cells. Ultrastuctural observations also confirmed that the supercooling of ray parenchyma cells was basically unaffected by the presence or absence of bulk water in the neighbouring tracheary elements, and that individual ray parenchyma cells acted as isolated droplets of water. Furthermore, the present ultrastructural study provides clear evidence that intracellular freezing upon the breakdown of deep supercooling results in severe damage to plasma membranes. Nonetheless, the characteristics of supercooling reappeared in the ray parenchyma cells upon recooling.
Key words: Styrax obassia, xylem ray parenchyma cells, deep supercooling, intracellular freezing, cryo-scanning electron microscopy (cryo-SEM), freeze-fracture replica.
INTRODUCTION Living xylem ray parenchyma cells of several woody species are believed to avoid freezing by deep supercooling in response to low temperatures (George and Burke, 1977). The water in xylem ray parenchyma cells in several woody species in winter can be supercooled to -40°C, which is known as the homogeneous nucleation temperature of pure water. A comprehensive list of woody species that exhibit deep supercooling in the xylem has been reported (George et al., 1982). The development of deep supercooling in xylem ray parenchyma cells of woody species has been suggested from the results of studies that mainly involved differential thermal analysis (DTA), in addition to studies using differential scanning calorimetry (DSC) and nuclear magnetic reasonance (NMR) (George and Burke, 1977). Other relevant data have been obtained by relating specific results to the range of temperatures over which injury to cells is observed (Burke et al., 1976). Electron microscopy using a variety of cryo-techniques has the potential to provide direct evidence about the lowtemperature behaviour of ray parenchyma cells, whether they are in a supercooling, intracellular freezing or extracellular freezing state, at desired low temperatures. In previous ultrastructural studies, however, no clear evidence of the low-temperature behaviour of the xylem ray parenchyma cells of woody species was obtained. Using an ultrathin-sectioning method with samples
Correspondence to Dr S. Fujikawa, Institute of Low Temperature Science, Hokkaido University, Sapporo, 060 Japan. Fax: 81-11-7165698. 241
prepared by a modified freeze-substitution technique (MacKenzie et al., 1975), Wisniewski and Ashworth (1985) observed the ultrastructure of xylem ray parenchyma cells, during freezing, in which the formation of intracellular ice crystals had been predicted. However, the presence of intracellular ice crystals in the ray parenchyma cells was not demonstrated. This failure may have originated from the use of a modified freeze-substitution technique in which freeze-substitution and fixation were performed under partially melting conditions with respect to ice. Ashworth et al. (1988) and Malone and Ashworth (1991) observed the low-temperature behaviour of xylem parenchyma cells from several woody species by scanning electron microscopy (SEM) using samples prepared by a freeze-substitution technique. However, observations of freeze-substituted samples by SEM have not provided clear ultrastructural evidence of the freezing behaviour of ray parenchyma cells. These earlier studies (Ashworth et al., 1988; Malone and Ashworth, 1991) showed only the absence of any detectable collapse of cell walls in the ray parenchyma cells upon freezing. From the prediction that, if extracellular freezing were to take place in the ray parenchyma cells during freezing, collapse of cell walls would occur as a result of extensive cellular dehydration, it was concluded that the absence of any collapse of the walls of ray parenchyma cells reflected the occurrence of deep supercooling and/or intracellular freezing. Using the same electron microscopic technique, however, Malone and Ashworth (1991) were unable to observe the collapse of the cell walls in ray parenchyma cells upon freezing of some woody species that had been predicted by DTA to exibit extracellular freezing. In their study, cryo-SEM was
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also used to examine the low-temperature behaviour of the xylem ray parenchyma cells, but they failed to obtain ultrastructural information beyond that obtained by the freeze-substitution technique. The present study was undertaken to obtain direct ultrastructural information about the low-temperature behaviour of the xylem ray parenchyma cells in woody species. In this study, xylem of Styrax obassia, collected during the summer, was chosen as a material that exibited characteristics of deep supercooling upon examination by DTA. The low-temperature behaviour in xylem ray parenchyma cells of Styrax obassia was examined by cryo-SEM and by freeze-fracture replica techniques and the results are compared with the profiles obtained by DTA.
MATERIALS AND METHODS
Materials Internodes (about 7 mm in diameter) of two-year-old twigs were collected from Styrax obassia Sieb. et Zucc., from July to September, 1993. The source of the twigs was a tree growing on the campus of Hokkaido University, Sapporo.
DTA (differential thermal analysis) Samples of fresh debarked twig internodes were cut into lengths of about 3 cm and utilized as fresh xylem for characterization of the low-temperature behaviour by DTA. In some cases, xylem soaked in distilled water for 30 min was used instead of fresh xylem. Oven-dried xylem, heated at 120°C overnight, was used as a reference. The samples were split longitudinally into two parts near the center. The junction of a 36-gauge copper constantan thermocouple was placed in contact with the split xylem of one of the cut halves, sealed with the other cut half and the entire sample was wrapped in aluminum foil. A pair of one fresh and one oven-dried sample was placed in a glass bottle; the bottle was closed and placed in a freezer (MDF-192; Sanyo Co. Ltd, Tokyo) and kept at 3°C. The temperature of the freezer was controlled by a programmable digital temperature controller (ES-100P; Tajiri Co. Ltd, Sapporo). The temperature of freezing events was determined from the difference between the output from the fresh (or water-soaked) sample and that from the oven-dried reference sample with a hybrid recorder (HR1300; Yokogawa Co. Ltd, Tokyo).
Preparation of samples Jbr electron microscopy Fresh xylem was cut into small blocks (about 3 x 3 x 4 mm a in the radial, longitudinal and tangential directions, respectively). A sample of xylem was inserted into a small hole in a specimen holder for electron microscopy. A small portion of xylem was exposed beyond the hole, revealing the tangential plane of the current year upon fracturing. Spaces between the fresh xylem and hole in the
holder were filled with starch paste. The spaces in xylem that had been soaked in distilled water were filled with distilled water• These samples were placed in petri dishes, which were closed and transferred to a freezer kept at 3°C.
Coolin9 or warmin9 of samples The cooling or warming of samples both for electron microscopy and DTA was performed in the same way. The samples were first equilibrated at 3°C for 30 min and then they were cooled to the desired temperature at the desired rate. Some samples were repeatedly frozen and thawed. Samples for both DTA and electron microscopy with different dimensions were shown to experience exactly the same cooling and warming events over the ranges of rates of change in temperature (0.2-0•01 °C/rain) used in this study• The samples for electron microscopy were cryofixed by abrupt immersion in melting freon 22 ( - 1 5 0 ° C ) soon after the desired temperature had been attained, and they were stored in liquid nitrogen prior to examination. As control, samples were cryofixed from room temperature.
Electron microscopy Cryofixed samples were processed for observation in a cryo-SEM (JSM 840A; J E O L Co. Ltd, Tokyo) by the method described previously (Fujikawa et al., 1988; Fujikawa, 1991). In brief, a cryofixed sample was transferred to a cold stage kept at - 1 0 5 ° C in the specimen-preparation chamber of the cryo-SEM, allowed to equilibrate for 10 min, and then fractured. The fracture plane was etched for 10 sec, replicated by evaporation of platinum-carbon for 10 sec and reinforced by evaporation of carbon for 20 sec. The samples were transferred to the cold stage of the SEM, held at - 164°C, and the secondary emission image was observed at an accerelating voltage of 5 kV. After observation and photographic recording by cryoSEM, the samples were removed from the system• Xylem was dissolved by immersion in 100% HzSO 4 overnight and subsequent immersion in commercial bleach for 1 hr, and then replicas were mounted on grids. Freeze-replicas were also produced independently of cryo-SEM, using a freeze-etching apparatus (JFD 7000; J E O L Co. Ltd) by exactly the same procedures as described above for the preparation of samples for cryo-SEM. The freeze-replicas were examined at 100 kV with a transmission electron microscope (1200 EX; JEOL Co. Ltd).
RESULTS
DTA profiles Cooling of fresh xylem from 3 to - 5 0 ° C at a rate of 0.1 °C/rain produced a high temperature exotherm (HTE) at around - 5 ° C and then a clear low-temperature exotherm (LTE) between - 1 4 ° C (initial temperature) and - 2 6 ° C (terminal temperature) with an approximate
Supercooling of Xylem
midpoint at - 2 0 ° C (Fig. la). This DTA profile obtained by cooling the fresh xylem was unchanged when samples had been soaked in water for 30 min were similarly analyzed (Fig. lb). Repeating the freezing and thawing between 3 and - 5 0 ° C brought about a slight shift in the LTE to higher temperatures with a gradual reduction in the height of the peak of the LTE, with increased cycles of treatment (Fig. lc). Slower cooling at a rate of 0.01°C/min from 3 to - 5 0 ° C produced profiles similar to those obtained upon cooling at a rate of 0.1°C/min, although the temperature range of the LTE became slightly narrower, between - 1 6 and -23°C, with a distinct reduction in the height of the peak of the exotherm (Fig. ld).
Ultrastructure of ray parenchyma cells in controls Fresh xylem was cryofixed from room temperature to provide details of the ultrastructure of control ray parenchyma cells without exposure to low-temperature stress. The cross-fracture views of control ray parenchyma cells varied from a typical ellipse (Fig. 2a) to a
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more rectangular ellipse (Fig. 3b). Cryo-SEM barely revealed any intracellular ice crystals, as a result of cryofixation, because of very small size of the crystals (less than 50 nm in diameter; Fig. 2b). Observations of freezefracture replicas clearly revealed, however, the presence of very small, intracellular ice crystals in the ray parenchyma cells (Fig. 2c). The ultrastructure of the cytoplasm was well preserved as a result of the formation of these very small ice crystals. The lumen of vessels in the fresh xylem was not generally filled with water (Fig. 3a). Only a small amount of water (visible as ice crystals) was observed in parts of vessels, in particular in the pit regions (Fig. 3b).
Ultrastructure of ray parenchyma cells at low temperatures Cooling offresh xylem at 0.1°C/min Fresh xylem was cooled at a rate of 0.1°C/min from 3 to
-50°C, and samples were cryofixed at desired temperatures during the cooling. Xylem cryofixed at - 1 0 ° C (a temperature above the LTE) revealed ultrastructure that was identical to that of the ray parenchyma cells in controls, and very small, intracellular ice crystals were again visible (Fig. 4). The xylem cryofixed at - 1 7 , - 2 0 and - 2 3 ° C (temperatures within the LTE) had ray parenchyma cells with large intraceUular ice crystals (more than 0.5/tm in diameter), which were easily visible by cryo-SEM (Fig. 5a and the cell on the left in Fig. 5b), in addition to ray parenchyma cells with very small ice crystals, similar to those in controls (the cell on the right as indicated by an arrow in Fig. 5b). In this temperature range (temperatures within the LTE), both types of ray parenchyma cells, namely, those with and those without large intracellular ice crystals, were randomly distributed in ray tissue (Figs 5b,c). The percentage of ray parenchyma cells with large intracellular ice crytstals increased gradually with the reduction in temperature (Table 1). Samples cryofixed at - 3 0 ° C (a temperature below the LTE) yielded ray parenchyma cells with large intracellular ice crystals exclusively (Fig. 6).
Effects of soaking in water
0
t
I
,
I
-10 -20 TEMPERATURE (C)
,
I -30
Fig. 1. DTA profiles during cooling of xylem. The numbers in each profile are the temperatures of the initiation (left) and termination (right) of the LTE. All the profiles are means from 3 preparations. (a) Cooling of fresh xylem from 3 to - 5 0 ° C at a rate of 0.1°C/min. (The profiles below - 3 5 ° C are omitted in all DTA profiles since no further changes were observed.) (b) Cooling of water-soaked xylem from 3 to - 5 0 ° C at a rate of 0.1°C/rain. (c) Repeating cooling of fresh xylem; (c-I) first cooling from 3 to - 5 0 ° C at a rate of 0.1°C/rain; (c-2) second cooling in the same way as c-l, after the c-1 sample had been held at - 5 0 ° C for 2 hr, warmed from - 5 0 to 3°C at a rate of 0.2°C/min, and held at 3°C for 2 hr; and (c-3) third cooling in the same way as c-l, after the same thawing process as c-2, (d) Cooling of water-soaked xylem from 3 to - 5 0 ° C at a rate of 0.01°C/min.
Xylem soaked with water for 30 min was cryofixed from room temperature. As a result of this treatment, the lumen of all vessels were filled with water (visible as ice crystals, Fig. 7). In the presence of water in the lumen of neighbouring vessels, however, low-temperature behaviour in the ray parenchyma cells (at cooling rates of both 0.1 and 0.01 °C/min), as observed by cryo-electron microscopy, was similar to that of fresh xylem (Table 1), reflecting the similar DTA profiles.
Effects of repeated freezing and thawing Xylem that had been frozen and thawed between 3 and - 50°C revealed major destruction of the cytoplasm when cryofixed at 3°C (Fig. 8a).The cytoplasm of ray parenchyma cells contained numerous small vesicles, instead of
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Supercooling of Xylem
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Fig. 3. Cryo-SEMphotographsof fresh xylemthat was cryofixedfromroom temperature.(a) A fracturedvesselwithoutwater in the lumen. (b) Pits in a vesselwith water (as ice crystals,arrows) and adjacent ray parenchymacells. the comparatively large cytoplasmic organelles in the ray parenchyma cells visible before freezing and thawing (compare Fig. 8a with Fig. 2b). Freeze-replicas of these cells also showed destruction of the plasma membranes. The plasma membranes showed evidence of partial vesiculation (arrows in Figs 8b,c), in addition to the formation of circular areas free of intramembrane particles (IMPs) (asterisks in Figs 8b,c). Although frozen and thawed xylem showed major destruction of the plasma membranes of ray parenchyma cells, the low-temperature behaviour of the cells during recooling, as observed by cryo-electron microscopy (Fig. 8c), was similar with that of samples before freezing and thawing (Table 1), as reflected by the DTA profiles. While cooling to temperatures above the LTE produced only small intracellular ice crystals (Fig. 8c), cooling to temperatures below the LTE produced only large intra-
cellular ice crystals in the ray parenchyma cells (not shown).
Cooling at 0.01°C/rain Water-soaked xylem was cooled at a rate of 0.0 l°C/min from 3 to -50°C. The low-temperature behaviour of the ray parenchyma cells, as observed by cryo-electron microscopy, was similar to that of samples cooled at a rate of 0.1°C/min (Table 1), as reflected also by the DTA profiles. Furthermore, although the cooling of xylem from 3 to - 5 0 ° C at a rate of 0.01°C/min with water soaking was the most extreme condition employed in this study and might be expected to facilitate dehydration of ray parenchyma cells during cooling, no detectable collapse of ray parenchyma cells occurred (Figs 9a,b). Extracellular spaces between ray cells (arrows in Fig. 9a) did not
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Fig. 4. A cryo-SEMphotograph of ray parenchymacells in fresh xylem,cooled to - 10°C (temperatureabove the LTE) at a rate of 0.1°C/min, showing the absence of large intracellular ice crystals. contain ice crystals, suggesting the absence of any dehydration of ray parenchyma cells.
DISCUSSION In this study, cryo-SEM and freeze-fracture replica techniques were used to obtain ultrastructural information about the low-temperature behaviour of xylem ray parenchyma cells in Styrax obassia, in order to assess the validity of results obtained by DTA. In the analysis of the deep supercooling of woody species by DTA, the initial large peak, which is referred to as the HTE, is believed to represent the freezing of the bulk water contained within tracheary elements, whereas the peak observed at lower temperatures in some woody species, referred to as the LTE, is believed to represent the freezing of cellular water contained within the xylem parenchyma cells (Burke et al., 1976). By contrast, woody species that do not produce an LTE during cooling are believed to experience extracellular freezing around the xylem ray parenchyma cells (Burke et al., 1976). The DTA profiles upon cooling of the xylem of Styrax obassia collected in summer included a clear LTE around - 2 0 ° C (the midpoint). Many studies have shown changes in temperatures of the LTE dependent upon seasons. In summer, the midpoint temperatures of the LTE are around - 2 0 ° C in the xylem of apple (Malus pumila) (Quamme et al., 1972), flowering dogwood (Cornus florida) and peach (Prunus persica) (Wisniewski and Ashworth, 1986), and in the xylem of many species of very hardy deciduous trees grown in Japan (Kaku and Iwaya, 1978). In winter, the LTE of these woody species shifts to around - 4 0 ° C . The present ultrastructural observations by cryoelectron microscopy revealed two distinct types of intracellular ice crystal in the ray parenchyma cells of the xylem of Styrax obassia during cooling: very small
intracellular ice crystals ( < 50 nm), which were barely detected by observation with cryo-SEM; and very large intracellular ice crystals (>0.5 lam), which were easily detected by cryo-SEM. Since very small intracellular ice crystals were also produced in cryofixed control samples without exposure to cooling, they can reasonably be deduced to be intracellular ice crystals that are produced as a result of cryofixation. The freeze-fracture replica observations showed that the cryofixation method employed in this study converted water in the ray parenchyma cells into very small ice crystals which produced no detectable damage to cytoplasmic structures. By contrast, the large intracellular ice crystals can be deduced to be intracellular ice crystals produced during cooling as a result of the breakdown of deep supercooling. The lower the rate of cooling, the larger were the ice crystals that formed (Fujikawa, 1988). Our ultrastructural evidence, based on the above assumptions, of the low-temperature behaviour of ray parenchyma cells in fresh xylem of Styrax obassia cooled at 0.1°C/min, corresponds closely to the DTA profiles, supporting previous predictions. It is considered, from the ultrastructural results, that samples cooled to temperatures above the LTE experience supercooling in the majority of ray parenchyma cells because only very small intracellular ice crystals are produced in the ray parenchyma cells. Because large intracellular ice crystals were produced at lower temperatures, the temperatures within and below the LTE, the formation of small intracellular ice crystals produced at higher temperatures, the temperatures above the LTE, can refer to the result of cryofixation, but not the result during slow cooling. When temperatures for initiation of freezing were higher, larger ice crystals must be formed than in the case of lower initiation temperatures (Fujikawa, 1988). The samples cooled to temperatures within the LTE showed evidence of intracellular freezing by forming large intracellular ice crystals as a result of the breakdown of deep supercooling
Supercooling of Xylem
Fig. 5. Cryo-SEM photographs of ray parenchyma cells in fresh xylem, cooled to the temperatures (within the LTE) indicated below at a rate of 0.1 °C/min. (a) A photograph of a sample cooled to - 2 0 ° C , showing a cell with large intracellular ice crystals. Some of the eutectic material in the cytoplasm is indicated by arrows. (b) A sample cooled to - 23°C showing two neighbouring ray cells with (leftside cell) and without (right-side cell, indicated by an arrow) large intracellular ice crystals. (c) A sample cooled to - 17°C showing ray tissue in which ray parenchyma cells with and without (indicated by arrows) large intracellular ice crystals are mixed together. The star indicates a ray parenchyma cell fractured at the end wall.
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Table 1. Percentage* of ray parenchyma cells with large (> 0.5 Mmdiameter) intracellular ice crystals Temperature ("C) Sample
20
Fresh xylem cooled at 0.1°C/min Water-soaked xylem coated at 0.1°C/min Fresh xylem recooled at 0.1°C/mint Water-soaked xylem cooled at 0.01°C/min
0 0 (0)$ 0
-10
-17
-20
-23
-30
2 0 1 0
38
69 61 67 68
88 ---
100 100 100 100
---
*Each percentage was obtained from observations of 100 ray parenchyma cells, from at least 2 samples. tFresh xylem, which were cooled from 3 to - 50°C at a rate of 0.1°C/min. held at - 50°C for 2 hr, warmed to 3°C at a rate of 0.2°C/min, and held at 3°C for 2 hr, was recooled at a rate of 0.1°C/min to the indicated temperatures. SThis value was obtained at 3°C.
Fig. 6. A cryo-SEM photograph showing the ultrastructure of ray parenchyma cells in fresh xylem, cooled to -30°C (temperature below the LTE) at a rate of 0.1°C/min, showing that all ray cells contained large intracellular ice crystals. *See legend to Fig. 5c.
in some ray p a r e n c h y m a cells. The n u m b e r of ray p a r e n c h y m a cells in which intracellular freezing occurred increased gradually u p o n reduction of cooling temperatures within the temperature range of the LTE, and at temperatures below the L T E all ray p a r e n c h y m a cells experienced intracellular freezing. C o r r e s p o n d e n c e between the results of D T A and our ultrastructural analysis of the low-temperature behaviour of ray p a r e n c h y m a cells was obtained under all the conditions employed in this study. Ultrastructural studies indicated clearly that, while there was very little water in fresh xylem, the majority of lumens of vessels adjacent to the ray p a r e n c h y m a cells were completely filled with water after soaking in water of xylem for 30 min. However, the presence or absence of water in the lumen of adjacent vessels did not bring a b o u t changes in either the D T A profiles or the results of ultrastructural analysis of the low-temperature behaviour of ray p a r e n c h y m a cells in the xylem of Styrax obassia. It has been shown that soaking the xylem of flowering d o g w o o d (Cornusflorida) and peach (Prunus persica) with buffered water for more than one day produces a shift or disappearance of the
L T E (Wisniewski and Davis, 1989; Wisniewski et al., 1991). However, these studies did not ascribe the change in the L T E to the direct effects of water in the xylem. In these studies, the changes in the L T E caused by prolonged soaking in water were deemed to be due to degradation of some cell wall structures, in particular in the pit wall regions of ray p a r e n c h y m a cells, by activation of endogenous cellulases during prolonged soaking in water (Wisniewski and Davis, 1989). Similar changes in the L T E were produced by the treatment of xylem with cellulase (Wisniewski et al., 1991) and were inhibited by the addition of cyclohexamide to xylem during soaking in water (Wisniewski and Davis, 1989). Slower cooling (0.01°C/min) of the xylem of Styrax obassia reduced the height of the peak of the L T E (albeit not quantitatively), but the temperature range for the L T E was barely changed as c o m p a r e d to that observed u p o n cooling at 0.1'~C/min. Despite the reduction of the height of the peak of the L T E by slower cooling, ultrastructural observations revealed the b r e a k d o w n of supercooling at temperatures below the L T E in all ray p a r e n c h y m a cells. It has been shown that the temperature
Supercoolingof Xylem
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Fig.7. Acryo-SEMphotographofwater-soakedxylem,cooledto - 20°Cat a rate of0.1°C/min,showingicecrystalsin the lumenofa vessel(upper)and adjacentray parenchymacells(below).Water-soakedxylem,cryofixedfromroomtemperature,alsoshowssimilar appearance withthis figurein the lumenof a vessel.*Seelegendto Fig. 5c. at which the LTE occurs is essentially unaffected by the cooling rate in the xylem of Haralson apple (Pyrus malus) (Quamme et al., 1973). However, studies using xylem from apple trees (Malus pumila) collected in autumn and early winter (the seasons of natural cold acclimation) showed a clear shift in the midpoint temperature of the LTE from - 20 to - 30°C after holding the xylem at 1.3 to - 5°C for 20-150 min during cooling at a rate of l°C/min (Hong and Sucoff, 1982). This change is thought to be a result of artificial cold acclimation of the xylem during the holding period (Hong and Sucoff, 1982). Gusta et al. (1983) showed that some water in the xylem ray parenchyma cells of woody species, such as red ash (Fraxinus pennsylvanica) and American elm (Ulmus americana), is gradually lost during prolonged cooling. The gradual dehydration results in a downward shift in temperatures of the LTE because the concentration of cell solutes due to dehydration depresses the homogeneous nucleation temperature of water (Rasmussen and MacKenzie, 1972). Consequently, differences in cooling rates may significantly affect the temperatures of the LTE in these woody species (Gusta et al., 1983). In the xylem of Styrax obassia it is suggested, however, that, even at the lowest cooling rates employed in this study (0.01°C/min), dehydration of the ray parenchyma cells did not occur, given the absence of ice crystals in the intercellular spaces between ray parenchyma cells and the minimal changes in temperatures of the LTE as compared with more rapid cooling. Freezing of xylem to temperatures below the LTE
results in cell death, probably as a result of the formation of intracellular ice crystals upon the breakdown of supercooling in the ray parenchyma cells (Burke et al., 1976). In this ultrastructural study, it appeared that, after freezing and thawing, the plasma membranes in ray parenchyma cells showed numerous vesiculations and IMP-free areas, probably due to intracellular freezing during the first cooling cycle. Such ultrastructural changes in the plasma membrane have been related to the occurrence of significant damage to cells by freezing (Fujikawa, 1988). Despite the occurrence of severe damage to the plasma membranes by freezing and thawing, recooling of such xylem again gave a clear LTE. Similar results of DTA after freezing and thawing have been reported (Quamme et al., 1973; Gusta et al., 1983), strongly supporting the role of the cell wall in the deep supercooling of the ray parenchyma cells (Quamme et al., 1973). Hong et al. (1980) indicated that the relationship between the LTE and cell injury is roughly quantitative, and they suggested that the total xylem parenchyma does not freeze as a unit but rather as small groups of cells. Making a careful examination of the LTE by calorimetric analysis, George and Burke (1977) showed that the LTE is composed of many isolated freezing events, suggesting that each individual ray may be a freezing unit in shagbark hickory. Hong and Sucoff (1980) suggested that individual cells or small groups of cells constituted the freezing unit. The present ultrastructural study of the xylem of Styrax obassia supports the suggestion of Hong
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Fig. 8. Ultrastructure of ray parenchyma cells after freezing and thawing (the fresh xylem was cooled from 3 to - 50'C at a rate of 0.1 °C/min, kept at - 5 0 ° C for 2 hr and thawed from - 5 0 to Y'C at a rate of 0.2°C/min). (a) A cryo-SEM photograph of frozen and thawed xylem cryofixed at 3°C showing destruction of the cytoplasm. (b) A freeze-replica photograph of frozen and thawed xylem cryofixed at 3°C, showing the protoplasmic fracture face (PFF) of a broken plasma membrane with IMP-free areas (asterisks) and vesiculation (arrows). Small intracellular ice crystals are visible in the cytoplasm (CY). (c) A freeze-replica photograph of frozen and thawed xylem recooled to - 10°C at a rate of 0.1 °C/min, showing the P F F of the plasma m e m b r a n e with similar structures to those in (b). CW, cell wall.
Supercooling of Xylem
251
Fig. 9. Cryo-SEM photographs showing the ultrastructure of ray parenchyma cells in xylem cooled to - 50 °C at a rate of 0.01°C/min, showing (a) the absence of ice in the intercellular spaces (arrows), and (b) formation of large intracellular ice crystals. Some eutectic material in the cytoplasm is indicated by arrows. *See legend to Fig. 5c.
and Scoff (1980), showing r a n d o m mixing of both supercooled and intracellularly frozen ray p a r e n c h y m a cells in ray tissue at temperatures within the LTE. In conclusion, the present study indicates that cryo-electron microscopy, using both c r y o - S E M and freeze-fracture replica techniques, can provide direct information about the low-temperature behaviour of ray p a r e n c h y m a cells. The ultrastructural results correspond to the results obtained by D T A with respect to the lowtemperature behaviour in xylems of Styrax obassia which showed clear L T E during cooling. Further extensive ultrastructural studies are continuing in order to assess
low-temperature behaviour of xylem ray p a r e n c h y m a cells in w o o d y species which show different types of D T A profile during cooling.
REFERENCES Ashworth, E. N., Echlin, P., Pearce, R. S. and Hayes, T. L., 1988. Ice formation and tissue response in apple twigs. Plant Cell Environ., 11, 703-710. Burke, M. J., Gusta, L. V., Quamme, H. A., Weiser, C. J. and Li, P. H., 1976. Freezing and injury in plants. A. Rev. Plant Physiol., 27, 507 528.
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Fujikawa, S., 1988. Artificial biological membrane ultrastructural changes caused by freezing. Electron Microsc. Rev., 1, 113-140. Fujikawa, S., 1991. Freeze-fracture techniques. In: Electron Microscopy in Biology, A Practical Approach, Harris, J. R. (ed.). IRL Press, Oxford, pp. 173-201. Fujikawa, S., Suzuki, T., Ishikawa, T., Sakurai, S. and Hasegawa, Y., 1988. Continuous observation of frozen biological materials with cryo-scanning electron microscope and freeze-replica by a new cryosystem. J. Electron Microsc., 37, 315-322. George, M. F. and Burke, M. J., 1977. Cold hardiness and deep supercooling in xylem of shagbark hickory. Plant Physiol., 59, 319-325. George, M. F., Becwar, M. R. and Burke, M. J., 1982. Freezing avoidance by deep supercooling of tissue water in winter-hardy plants. Cryobiology, 19, 628~39. Gusta, L. V., Tyler, N. J. and Chen, T. H.- H., 1983. Deep supercooling in woody taxa growing north of the - 4 0 ° C isotherm. Plant Physiol., 72, 122-128. Hong, S. G. and Sucoff, E., 1980. Unit of freezing of deep supercooling water in woody xylem. Plant Physiol., 66, 40-45. Hong, S. G. and Sucoff, E., 1982. Rapid increase in deep supercooling of xylem parenchyma. Plant Physiol., 69, 697-700. Hong, S. G., Sucoff, E. and Lee-Stadelmann, O. Y., 1980. Effects of freezing deep supercooled water on the viability of ray cells. Bot. Gaz., 141, 464468. Kaku, S. and Iwaya, M., 1978. Low temperature exotherms in xylem of evergreen and deciduous broad-leaved trees in Japan with reference to freezing resistance and distribution range. In: Plant Cold Hardiness and Freezin O Stress, Li, P. H. and Sakai, A. (eds.). Academic Press, New York, pp. 227-239.
MacKenzie, A. P., Kuster, T. A. and Luyet, B. J., 1975. Freeze-fixation at high subzero temperatures. Cryobiology, 12, 427~139. Malone, S. R. and Ashworth, E. N., 1991. Freezing stress response in woody tissues observed using low-temperature scanning electron microscopy and freeze substitution techniques. Plant Physiol., 95, 871 88l. Quamme, H., Stushnoff, C. and Weiser, C. J., 1972. The relationship of exotherms to cold injury in apple stem tissue. J. Am. Soc. hortic. Sci., 97, 608-613. Quamme, H., Weiser, C. J. and Stushnoff, C., 1973. The mechanism of freezing injury in xylem of winter apple twigs. Plant Physiol., 51, 273577. Rasmussen, D. H. and MacKenzie, A. P., 1972. Effect of solute on icesolution interfacial free energy; calculation from measured homogeneous nucleation temperature. In: Water Structure at the Water Polymer Interface, Jellnek, H. H. G. (ed.). Plenum, New York, pp. 12(~145. Wisniewski, M. and Ashworth, E. N., 1985. Changes in the ultrastructure of xylem parenchyma cells of peach (Prunus persica) and red oak (Quercus rubra) in response to a freezing stress. Am. J. Bot., 72, 1364-1376. Wisniewski, M. and Ashworth, E. N., 1986. Seasonal variation in deep supercooling and dehydrative resistance. HortScience, 21, 503 505. Wisniewski, M. and Davis, G., 1989. Evidence for the involvement of a specific cell wall layer in regulation on deep supercooling of xylem parenchyma. Plant Physiol., 91, 151-156. Wisniewski, M., Davis, G. and Schaffer, K., 1991. Mediation of deep supercooling of peach and dogwood by enzymatic modifications in cell-wall structure. Planta, 184, 254-260.
Pergamon
0968-4328(94)E0005-A
I
Ultrastructural Study of Deep Supercooling of Xylem Ray Parenchyma Cells from Styrax obassia SEIZO FUJIKAWA,*~: KATSUSHI K U R O D A t and KAZUMI FUKAZAWAt *Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan t Section of Wood Biology, Faculty of Agriculture, Hokkaido University, Sapporo, Japan (Received 30 November 1993; Revised 1 February 1994)
Abstract--Some of the water in the xylem of a woody species (Styrax obassia Sieb. et Zucc.), collected during the summer, appeared to exhibit deep supercooling and the breakdown of supercooling at around -20°C, as determined by differential thermal analysis (DTA). Ultrastructural observations of the low-temperature behaviour of the xylem, using both cryo-scanning electron microscopy (cryo-SEM) and freeze-fracture replica techniques, provided direct evidence that xylem ray parenchyma cells can be deeply supercooled to around - 2 0 ° C and that the breakdown of supercooling produces intracellular freezing in the ray parenchyma cells. Ultrastuctural observations also confirmed that the supercooling of ray parenchyma cells was basically unaffected by the presence or absence of bulk water in the neighbouring tracheary elements, and that individual ray parenchyma cells acted as isolated droplets of water. Furthermore, the present ultrastructural study provides clear evidence that intracellular freezing upon the breakdown of deep supercooling results in severe damage to plasma membranes. Nonetheless, the characteristics of supercooling reappeared in the ray parenchyma cells upon recooling.
Key words: Styrax obassia, xylem ray parenchyma cells, deep supercooling, intracellular freezing, cryo-scanning electron microscopy (cryo-SEM), freeze-fracture replica.
INTRODUCTION Living xylem ray parenchyma cells of several woody species are believed to avoid freezing by deep supercooling in response to low temperatures (George and Burke, 1977). The water in xylem ray parenchyma cells in several woody species in winter can be supercooled to -40°C, which is known as the homogeneous nucleation temperature of pure water. A comprehensive list of woody species that exhibit deep supercooling in the xylem has been reported (George et al., 1982). The development of deep supercooling in xylem ray parenchyma cells of woody species has been suggested from the results of studies that mainly involved differential thermal analysis (DTA), in addition to studies using differential scanning calorimetry (DSC) and nuclear magnetic reasonance (NMR) (George and Burke, 1977). Other relevant data have been obtained by relating specific results to the range of temperatures over which injury to cells is observed (Burke et al., 1976). Electron microscopy using a variety of cryo-techniques has the potential to provide direct evidence about the lowtemperature behaviour of ray parenchyma cells, whether they are in a supercooling, intracellular freezing or extracellular freezing state, at desired low temperatures. In previous ultrastructural studies, however, no clear evidence of the low-temperature behaviour of the xylem ray parenchyma cells of woody species was obtained. Using an ultrathin-sectioning method with samples
Correspondence to Dr S. Fujikawa, Institute of Low Temperature Science, Hokkaido University, Sapporo, 060 Japan. Fax: 81-11-7165698. 241
prepared by a modified freeze-substitution technique (MacKenzie et al., 1975), Wisniewski and Ashworth (1985) observed the ultrastructure of xylem ray parenchyma cells, during freezing, in which the formation of intracellular ice crystals had been predicted. However, the presence of intracellular ice crystals in the ray parenchyma cells was not demonstrated. This failure may have originated from the use of a modified freeze-substitution technique in which freeze-substitution and fixation were performed under partially melting conditions with respect to ice. Ashworth et al. (1988) and Malone and Ashworth (1991) observed the low-temperature behaviour of xylem parenchyma cells from several woody species by scanning electron microscopy (SEM) using samples prepared by a freeze-substitution technique. However, observations of freeze-substituted samples by SEM have not provided clear ultrastructural evidence of the freezing behaviour of ray parenchyma cells. These earlier studies (Ashworth et al., 1988; Malone and Ashworth, 1991) showed only the absence of any detectable collapse of cell walls in the ray parenchyma cells upon freezing. From the prediction that, if extracellular freezing were to take place in the ray parenchyma cells during freezing, collapse of cell walls would occur as a result of extensive cellular dehydration, it was concluded that the absence of any collapse of the walls of ray parenchyma cells reflected the occurrence of deep supercooling and/or intracellular freezing. Using the same electron microscopic technique, however, Malone and Ashworth (1991) were unable to observe the collapse of the cell walls in ray parenchyma cells upon freezing of some woody species that had been predicted by DTA to exibit extracellular freezing. In their study, cryo-SEM was
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also used to examine the low-temperature behaviour of the xylem ray parenchyma cells, but they failed to obtain ultrastructural information beyond that obtained by the freeze-substitution technique. The present study was undertaken to obtain direct ultrastructural information about the low-temperature behaviour of the xylem ray parenchyma cells in woody species. In this study, xylem of Styrax obassia, collected during the summer, was chosen as a material that exibited characteristics of deep supercooling upon examination by DTA. The low-temperature behaviour in xylem ray parenchyma cells of Styrax obassia was examined by cryo-SEM and by freeze-fracture replica techniques and the results are compared with the profiles obtained by DTA.
MATERIALS AND METHODS
Materials Internodes (about 7 mm in diameter) of two-year-old twigs were collected from Styrax obassia Sieb. et Zucc., from July to September, 1993. The source of the twigs was a tree growing on the campus of Hokkaido University, Sapporo.
DTA (differential thermal analysis) Samples of fresh debarked twig internodes were cut into lengths of about 3 cm and utilized as fresh xylem for characterization of the low-temperature behaviour by DTA. In some cases, xylem soaked in distilled water for 30 min was used instead of fresh xylem. Oven-dried xylem, heated at 120°C overnight, was used as a reference. The samples were split longitudinally into two parts near the center. The junction of a 36-gauge copper constantan thermocouple was placed in contact with the split xylem of one of the cut halves, sealed with the other cut half and the entire sample was wrapped in aluminum foil. A pair of one fresh and one oven-dried sample was placed in a glass bottle; the bottle was closed and placed in a freezer (MDF-192; Sanyo Co. Ltd, Tokyo) and kept at 3°C. The temperature of the freezer was controlled by a programmable digital temperature controller (ES-100P; Tajiri Co. Ltd, Sapporo). The temperature of freezing events was determined from the difference between the output from the fresh (or water-soaked) sample and that from the oven-dried reference sample with a hybrid recorder (HR1300; Yokogawa Co. Ltd, Tokyo).
Preparation of samples Jbr electron microscopy Fresh xylem was cut into small blocks (about 3 x 3 x 4 mm a in the radial, longitudinal and tangential directions, respectively). A sample of xylem was inserted into a small hole in a specimen holder for electron microscopy. A small portion of xylem was exposed beyond the hole, revealing the tangential plane of the current year upon fracturing. Spaces between the fresh xylem and hole in the
holder were filled with starch paste. The spaces in xylem that had been soaked in distilled water were filled with distilled water• These samples were placed in petri dishes, which were closed and transferred to a freezer kept at 3°C.
Coolin9 or warmin9 of samples The cooling or warming of samples both for electron microscopy and DTA was performed in the same way. The samples were first equilibrated at 3°C for 30 min and then they were cooled to the desired temperature at the desired rate. Some samples were repeatedly frozen and thawed. Samples for both DTA and electron microscopy with different dimensions were shown to experience exactly the same cooling and warming events over the ranges of rates of change in temperature (0.2-0•01 °C/rain) used in this study• The samples for electron microscopy were cryofixed by abrupt immersion in melting freon 22 ( - 1 5 0 ° C ) soon after the desired temperature had been attained, and they were stored in liquid nitrogen prior to examination. As control, samples were cryofixed from room temperature.
Electron microscopy Cryofixed samples were processed for observation in a cryo-SEM (JSM 840A; J E O L Co. Ltd, Tokyo) by the method described previously (Fujikawa et al., 1988; Fujikawa, 1991). In brief, a cryofixed sample was transferred to a cold stage kept at - 1 0 5 ° C in the specimen-preparation chamber of the cryo-SEM, allowed to equilibrate for 10 min, and then fractured. The fracture plane was etched for 10 sec, replicated by evaporation of platinum-carbon for 10 sec and reinforced by evaporation of carbon for 20 sec. The samples were transferred to the cold stage of the SEM, held at - 164°C, and the secondary emission image was observed at an accerelating voltage of 5 kV. After observation and photographic recording by cryoSEM, the samples were removed from the system• Xylem was dissolved by immersion in 100% HzSO 4 overnight and subsequent immersion in commercial bleach for 1 hr, and then replicas were mounted on grids. Freeze-replicas were also produced independently of cryo-SEM, using a freeze-etching apparatus (JFD 7000; J E O L Co. Ltd) by exactly the same procedures as described above for the preparation of samples for cryo-SEM. The freeze-replicas were examined at 100 kV with a transmission electron microscope (1200 EX; JEOL Co. Ltd).
RESULTS
DTA profiles Cooling of fresh xylem from 3 to - 5 0 ° C at a rate of 0.1 °C/rain produced a high temperature exotherm (HTE) at around - 5 ° C and then a clear low-temperature exotherm (LTE) between - 1 4 ° C (initial temperature) and - 2 6 ° C (terminal temperature) with an approximate
Supercooling of Xylem
midpoint at - 2 0 ° C (Fig. la). This DTA profile obtained by cooling the fresh xylem was unchanged when samples had been soaked in water for 30 min were similarly analyzed (Fig. lb). Repeating the freezing and thawing between 3 and - 5 0 ° C brought about a slight shift in the LTE to higher temperatures with a gradual reduction in the height of the peak of the LTE, with increased cycles of treatment (Fig. lc). Slower cooling at a rate of 0.01°C/min from 3 to - 5 0 ° C produced profiles similar to those obtained upon cooling at a rate of 0.1°C/min, although the temperature range of the LTE became slightly narrower, between - 1 6 and -23°C, with a distinct reduction in the height of the peak of the exotherm (Fig. ld).
Ultrastructure of ray parenchyma cells in controls Fresh xylem was cryofixed from room temperature to provide details of the ultrastructure of control ray parenchyma cells without exposure to low-temperature stress. The cross-fracture views of control ray parenchyma cells varied from a typical ellipse (Fig. 2a) to a
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,
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o
-14~~-26
c-1 ~
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c-2 ~
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-13.s ~ - 2 3
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243
more rectangular ellipse (Fig. 3b). Cryo-SEM barely revealed any intracellular ice crystals, as a result of cryofixation, because of very small size of the crystals (less than 50 nm in diameter; Fig. 2b). Observations of freezefracture replicas clearly revealed, however, the presence of very small, intracellular ice crystals in the ray parenchyma cells (Fig. 2c). The ultrastructure of the cytoplasm was well preserved as a result of the formation of these very small ice crystals. The lumen of vessels in the fresh xylem was not generally filled with water (Fig. 3a). Only a small amount of water (visible as ice crystals) was observed in parts of vessels, in particular in the pit regions (Fig. 3b).
Ultrastructure of ray parenchyma cells at low temperatures Cooling offresh xylem at 0.1°C/min Fresh xylem was cooled at a rate of 0.1°C/min from 3 to
-50°C, and samples were cryofixed at desired temperatures during the cooling. Xylem cryofixed at - 1 0 ° C (a temperature above the LTE) revealed ultrastructure that was identical to that of the ray parenchyma cells in controls, and very small, intracellular ice crystals were again visible (Fig. 4). The xylem cryofixed at - 1 7 , - 2 0 and - 2 3 ° C (temperatures within the LTE) had ray parenchyma cells with large intraceUular ice crystals (more than 0.5/tm in diameter), which were easily visible by cryo-SEM (Fig. 5a and the cell on the left in Fig. 5b), in addition to ray parenchyma cells with very small ice crystals, similar to those in controls (the cell on the right as indicated by an arrow in Fig. 5b). In this temperature range (temperatures within the LTE), both types of ray parenchyma cells, namely, those with and those without large intracellular ice crystals, were randomly distributed in ray tissue (Figs 5b,c). The percentage of ray parenchyma cells with large intracellular ice crytstals increased gradually with the reduction in temperature (Table 1). Samples cryofixed at - 3 0 ° C (a temperature below the LTE) yielded ray parenchyma cells with large intracellular ice crystals exclusively (Fig. 6).
Effects of soaking in water
0
t
I
,
I
-10 -20 TEMPERATURE (C)
,
I -30
Fig. 1. DTA profiles during cooling of xylem. The numbers in each profile are the temperatures of the initiation (left) and termination (right) of the LTE. All the profiles are means from 3 preparations. (a) Cooling of fresh xylem from 3 to - 5 0 ° C at a rate of 0.1°C/min. (The profiles below - 3 5 ° C are omitted in all DTA profiles since no further changes were observed.) (b) Cooling of water-soaked xylem from 3 to - 5 0 ° C at a rate of 0.1°C/rain. (c) Repeating cooling of fresh xylem; (c-I) first cooling from 3 to - 5 0 ° C at a rate of 0.1°C/rain; (c-2) second cooling in the same way as c-l, after the c-1 sample had been held at - 5 0 ° C for 2 hr, warmed from - 5 0 to 3°C at a rate of 0.2°C/min, and held at 3°C for 2 hr; and (c-3) third cooling in the same way as c-l, after the same thawing process as c-2, (d) Cooling of water-soaked xylem from 3 to - 5 0 ° C at a rate of 0.01°C/min.
Xylem soaked with water for 30 min was cryofixed from room temperature. As a result of this treatment, the lumen of all vessels were filled with water (visible as ice crystals, Fig. 7). In the presence of water in the lumen of neighbouring vessels, however, low-temperature behaviour in the ray parenchyma cells (at cooling rates of both 0.1 and 0.01 °C/min), as observed by cryo-electron microscopy, was similar to that of fresh xylem (Table 1), reflecting the similar DTA profiles.
Effects of repeated freezing and thawing Xylem that had been frozen and thawed between 3 and - 50°C revealed major destruction of the cytoplasm when cryofixed at 3°C (Fig. 8a).The cytoplasm of ray parenchyma cells contained numerous small vesicles, instead of
J~
f
Supercooling of Xylem
245
Fig. 3. Cryo-SEMphotographsof fresh xylemthat was cryofixedfromroom temperature.(a) A fracturedvesselwithoutwater in the lumen. (b) Pits in a vesselwith water (as ice crystals,arrows) and adjacent ray parenchymacells. the comparatively large cytoplasmic organelles in the ray parenchyma cells visible before freezing and thawing (compare Fig. 8a with Fig. 2b). Freeze-replicas of these cells also showed destruction of the plasma membranes. The plasma membranes showed evidence of partial vesiculation (arrows in Figs 8b,c), in addition to the formation of circular areas free of intramembrane particles (IMPs) (asterisks in Figs 8b,c). Although frozen and thawed xylem showed major destruction of the plasma membranes of ray parenchyma cells, the low-temperature behaviour of the cells during recooling, as observed by cryo-electron microscopy (Fig. 8c), was similar with that of samples before freezing and thawing (Table 1), as reflected by the DTA profiles. While cooling to temperatures above the LTE produced only small intracellular ice crystals (Fig. 8c), cooling to temperatures below the LTE produced only large intra-
cellular ice crystals in the ray parenchyma cells (not shown).
Cooling at 0.01°C/rain Water-soaked xylem was cooled at a rate of 0.0 l°C/min from 3 to -50°C. The low-temperature behaviour of the ray parenchyma cells, as observed by cryo-electron microscopy, was similar to that of samples cooled at a rate of 0.1°C/min (Table 1), as reflected also by the DTA profiles. Furthermore, although the cooling of xylem from 3 to - 5 0 ° C at a rate of 0.01°C/min with water soaking was the most extreme condition employed in this study and might be expected to facilitate dehydration of ray parenchyma cells during cooling, no detectable collapse of ray parenchyma cells occurred (Figs 9a,b). Extracellular spaces between ray cells (arrows in Fig. 9a) did not
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Fig. 4. A cryo-SEMphotograph of ray parenchymacells in fresh xylem,cooled to - 10°C (temperatureabove the LTE) at a rate of 0.1°C/min, showing the absence of large intracellular ice crystals. contain ice crystals, suggesting the absence of any dehydration of ray parenchyma cells.
DISCUSSION In this study, cryo-SEM and freeze-fracture replica techniques were used to obtain ultrastructural information about the low-temperature behaviour of xylem ray parenchyma cells in Styrax obassia, in order to assess the validity of results obtained by DTA. In the analysis of the deep supercooling of woody species by DTA, the initial large peak, which is referred to as the HTE, is believed to represent the freezing of the bulk water contained within tracheary elements, whereas the peak observed at lower temperatures in some woody species, referred to as the LTE, is believed to represent the freezing of cellular water contained within the xylem parenchyma cells (Burke et al., 1976). By contrast, woody species that do not produce an LTE during cooling are believed to experience extracellular freezing around the xylem ray parenchyma cells (Burke et al., 1976). The DTA profiles upon cooling of the xylem of Styrax obassia collected in summer included a clear LTE around - 2 0 ° C (the midpoint). Many studies have shown changes in temperatures of the LTE dependent upon seasons. In summer, the midpoint temperatures of the LTE are around - 2 0 ° C in the xylem of apple (Malus pumila) (Quamme et al., 1972), flowering dogwood (Cornus florida) and peach (Prunus persica) (Wisniewski and Ashworth, 1986), and in the xylem of many species of very hardy deciduous trees grown in Japan (Kaku and Iwaya, 1978). In winter, the LTE of these woody species shifts to around - 4 0 ° C . The present ultrastructural observations by cryoelectron microscopy revealed two distinct types of intracellular ice crystal in the ray parenchyma cells of the xylem of Styrax obassia during cooling: very small
intracellular ice crystals ( < 50 nm), which were barely detected by observation with cryo-SEM; and very large intracellular ice crystals (>0.5 lam), which were easily detected by cryo-SEM. Since very small intracellular ice crystals were also produced in cryofixed control samples without exposure to cooling, they can reasonably be deduced to be intracellular ice crystals that are produced as a result of cryofixation. The freeze-fracture replica observations showed that the cryofixation method employed in this study converted water in the ray parenchyma cells into very small ice crystals which produced no detectable damage to cytoplasmic structures. By contrast, the large intracellular ice crystals can be deduced to be intracellular ice crystals produced during cooling as a result of the breakdown of deep supercooling. The lower the rate of cooling, the larger were the ice crystals that formed (Fujikawa, 1988). Our ultrastructural evidence, based on the above assumptions, of the low-temperature behaviour of ray parenchyma cells in fresh xylem of Styrax obassia cooled at 0.1°C/min, corresponds closely to the DTA profiles, supporting previous predictions. It is considered, from the ultrastructural results, that samples cooled to temperatures above the LTE experience supercooling in the majority of ray parenchyma cells because only very small intracellular ice crystals are produced in the ray parenchyma cells. Because large intracellular ice crystals were produced at lower temperatures, the temperatures within and below the LTE, the formation of small intracellular ice crystals produced at higher temperatures, the temperatures above the LTE, can refer to the result of cryofixation, but not the result during slow cooling. When temperatures for initiation of freezing were higher, larger ice crystals must be formed than in the case of lower initiation temperatures (Fujikawa, 1988). The samples cooled to temperatures within the LTE showed evidence of intracellular freezing by forming large intracellular ice crystals as a result of the breakdown of deep supercooling
Supercooling of Xylem
Fig. 5. Cryo-SEM photographs of ray parenchyma cells in fresh xylem, cooled to the temperatures (within the LTE) indicated below at a rate of 0.1 °C/min. (a) A photograph of a sample cooled to - 2 0 ° C , showing a cell with large intracellular ice crystals. Some of the eutectic material in the cytoplasm is indicated by arrows. (b) A sample cooled to - 23°C showing two neighbouring ray cells with (leftside cell) and without (right-side cell, indicated by an arrow) large intracellular ice crystals. (c) A sample cooled to - 17°C showing ray tissue in which ray parenchyma cells with and without (indicated by arrows) large intracellular ice crystals are mixed together. The star indicates a ray parenchyma cell fractured at the end wall.
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Table 1. Percentage* of ray parenchyma cells with large (> 0.5 Mmdiameter) intracellular ice crystals Temperature ("C) Sample
20
Fresh xylem cooled at 0.1°C/min Water-soaked xylem coated at 0.1°C/min Fresh xylem recooled at 0.1°C/mint Water-soaked xylem cooled at 0.01°C/min
0 0 (0)$ 0
-10
-17
-20
-23
-30
2 0 1 0
38
69 61 67 68
88 ---
100 100 100 100
---
*Each percentage was obtained from observations of 100 ray parenchyma cells, from at least 2 samples. tFresh xylem, which were cooled from 3 to - 50°C at a rate of 0.1°C/min. held at - 50°C for 2 hr, warmed to 3°C at a rate of 0.2°C/min, and held at 3°C for 2 hr, was recooled at a rate of 0.1°C/min to the indicated temperatures. SThis value was obtained at 3°C.
Fig. 6. A cryo-SEM photograph showing the ultrastructure of ray parenchyma cells in fresh xylem, cooled to -30°C (temperature below the LTE) at a rate of 0.1°C/min, showing that all ray cells contained large intracellular ice crystals. *See legend to Fig. 5c.
in some ray p a r e n c h y m a cells. The n u m b e r of ray p a r e n c h y m a cells in which intracellular freezing occurred increased gradually u p o n reduction of cooling temperatures within the temperature range of the LTE, and at temperatures below the L T E all ray p a r e n c h y m a cells experienced intracellular freezing. C o r r e s p o n d e n c e between the results of D T A and our ultrastructural analysis of the low-temperature behaviour of ray p a r e n c h y m a cells was obtained under all the conditions employed in this study. Ultrastructural studies indicated clearly that, while there was very little water in fresh xylem, the majority of lumens of vessels adjacent to the ray p a r e n c h y m a cells were completely filled with water after soaking in water of xylem for 30 min. However, the presence or absence of water in the lumen of adjacent vessels did not bring a b o u t changes in either the D T A profiles or the results of ultrastructural analysis of the low-temperature behaviour of ray p a r e n c h y m a cells in the xylem of Styrax obassia. It has been shown that soaking the xylem of flowering d o g w o o d (Cornusflorida) and peach (Prunus persica) with buffered water for more than one day produces a shift or disappearance of the
L T E (Wisniewski and Davis, 1989; Wisniewski et al., 1991). However, these studies did not ascribe the change in the L T E to the direct effects of water in the xylem. In these studies, the changes in the L T E caused by prolonged soaking in water were deemed to be due to degradation of some cell wall structures, in particular in the pit wall regions of ray p a r e n c h y m a cells, by activation of endogenous cellulases during prolonged soaking in water (Wisniewski and Davis, 1989). Similar changes in the L T E were produced by the treatment of xylem with cellulase (Wisniewski et al., 1991) and were inhibited by the addition of cyclohexamide to xylem during soaking in water (Wisniewski and Davis, 1989). Slower cooling (0.01°C/min) of the xylem of Styrax obassia reduced the height of the peak of the L T E (albeit not quantitatively), but the temperature range for the L T E was barely changed as c o m p a r e d to that observed u p o n cooling at 0.1'~C/min. Despite the reduction of the height of the peak of the L T E by slower cooling, ultrastructural observations revealed the b r e a k d o w n of supercooling at temperatures below the L T E in all ray p a r e n c h y m a cells. It has been shown that the temperature
Supercoolingof Xylem
249
Fig.7. Acryo-SEMphotographofwater-soakedxylem,cooledto - 20°Cat a rate of0.1°C/min,showingicecrystalsin the lumenofa vessel(upper)and adjacentray parenchymacells(below).Water-soakedxylem,cryofixedfromroomtemperature,alsoshowssimilar appearance withthis figurein the lumenof a vessel.*Seelegendto Fig. 5c. at which the LTE occurs is essentially unaffected by the cooling rate in the xylem of Haralson apple (Pyrus malus) (Quamme et al., 1973). However, studies using xylem from apple trees (Malus pumila) collected in autumn and early winter (the seasons of natural cold acclimation) showed a clear shift in the midpoint temperature of the LTE from - 20 to - 30°C after holding the xylem at 1.3 to - 5°C for 20-150 min during cooling at a rate of l°C/min (Hong and Sucoff, 1982). This change is thought to be a result of artificial cold acclimation of the xylem during the holding period (Hong and Sucoff, 1982). Gusta et al. (1983) showed that some water in the xylem ray parenchyma cells of woody species, such as red ash (Fraxinus pennsylvanica) and American elm (Ulmus americana), is gradually lost during prolonged cooling. The gradual dehydration results in a downward shift in temperatures of the LTE because the concentration of cell solutes due to dehydration depresses the homogeneous nucleation temperature of water (Rasmussen and MacKenzie, 1972). Consequently, differences in cooling rates may significantly affect the temperatures of the LTE in these woody species (Gusta et al., 1983). In the xylem of Styrax obassia it is suggested, however, that, even at the lowest cooling rates employed in this study (0.01°C/min), dehydration of the ray parenchyma cells did not occur, given the absence of ice crystals in the intercellular spaces between ray parenchyma cells and the minimal changes in temperatures of the LTE as compared with more rapid cooling. Freezing of xylem to temperatures below the LTE
results in cell death, probably as a result of the formation of intracellular ice crystals upon the breakdown of supercooling in the ray parenchyma cells (Burke et al., 1976). In this ultrastructural study, it appeared that, after freezing and thawing, the plasma membranes in ray parenchyma cells showed numerous vesiculations and IMP-free areas, probably due to intracellular freezing during the first cooling cycle. Such ultrastructural changes in the plasma membrane have been related to the occurrence of significant damage to cells by freezing (Fujikawa, 1988). Despite the occurrence of severe damage to the plasma membranes by freezing and thawing, recooling of such xylem again gave a clear LTE. Similar results of DTA after freezing and thawing have been reported (Quamme et al., 1973; Gusta et al., 1983), strongly supporting the role of the cell wall in the deep supercooling of the ray parenchyma cells (Quamme et al., 1973). Hong et al. (1980) indicated that the relationship between the LTE and cell injury is roughly quantitative, and they suggested that the total xylem parenchyma does not freeze as a unit but rather as small groups of cells. Making a careful examination of the LTE by calorimetric analysis, George and Burke (1977) showed that the LTE is composed of many isolated freezing events, suggesting that each individual ray may be a freezing unit in shagbark hickory. Hong and Sucoff (1980) suggested that individual cells or small groups of cells constituted the freezing unit. The present ultrastructural study of the xylem of Styrax obassia supports the suggestion of Hong
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Fig. 8. Ultrastructure of ray parenchyma cells after freezing and thawing (the fresh xylem was cooled from 3 to - 50'C at a rate of 0.1 °C/min, kept at - 5 0 ° C for 2 hr and thawed from - 5 0 to Y'C at a rate of 0.2°C/min). (a) A cryo-SEM photograph of frozen and thawed xylem cryofixed at 3°C showing destruction of the cytoplasm. (b) A freeze-replica photograph of frozen and thawed xylem cryofixed at 3°C, showing the protoplasmic fracture face (PFF) of a broken plasma membrane with IMP-free areas (asterisks) and vesiculation (arrows). Small intracellular ice crystals are visible in the cytoplasm (CY). (c) A freeze-replica photograph of frozen and thawed xylem recooled to - 10°C at a rate of 0.1 °C/min, showing the P F F of the plasma m e m b r a n e with similar structures to those in (b). CW, cell wall.
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Fig. 9. Cryo-SEM photographs showing the ultrastructure of ray parenchyma cells in xylem cooled to - 50 °C at a rate of 0.01°C/min, showing (a) the absence of ice in the intercellular spaces (arrows), and (b) formation of large intracellular ice crystals. Some eutectic material in the cytoplasm is indicated by arrows. *See legend to Fig. 5c.
and Scoff (1980), showing r a n d o m mixing of both supercooled and intracellularly frozen ray p a r e n c h y m a cells in ray tissue at temperatures within the LTE. In conclusion, the present study indicates that cryo-electron microscopy, using both c r y o - S E M and freeze-fracture replica techniques, can provide direct information about the low-temperature behaviour of ray p a r e n c h y m a cells. The ultrastructural results correspond to the results obtained by D T A with respect to the lowtemperature behaviour in xylems of Styrax obassia which showed clear L T E during cooling. Further extensive ultrastructural studies are continuing in order to assess
low-temperature behaviour of xylem ray p a r e n c h y m a cells in w o o d y species which show different types of D T A profile during cooling.
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