response of xylem ray parenchyma cells of (cornus sericea l.)

Plant Physiol. (1994) 104: 737-746

Response of Xylem Ray Parenchyma Cells of Red Osier Dogwood (Cornus sericea L.) to Freezing Stress'

Microscopic Evidence of Protoplasm Contraction

Zoran Ri&* and Edward N. Ashworth

Center for Plant Environmental Stress Physiology, Department of Horticulture, Purdue University, West Lafayette, Indiana 47907

Freezing behavior of wood tissue of red osier dogwood (Cornus sericea 1.) cannot be explained by current concepts of freezing resistance. Previous studies indicated that water in wood tissue presumably froze extracellularly. However, it was observed that xylem ray parenchyma cells within these tissues could survive temperatures as low as -80°C and the walls of these cells did not collapse during freezing (S.R. Malone and E.N. Ashworth [1991] Plant Physiol 95: 871-881). This observation was unexpected and is inconsistent with the current hypothesis of cell response during freezing. Hence, the objective of our study was to further examine the mechanism of freezing resistance of wood tissue of red osier dogwood. We studied freezing stress response of xylem ray parenchyma cells of red osier dogwood using freeze substitution and transmission electron microscopy. Wood samples were collected in winter, spring, and summer of 1992. Specimens were cooled from 0°C to -60°C at 5"C/h. Freezing stress did not affect the structural organization of wood tissue. However, the xylem ray parenchyma cells showed two unique responses to a freezing stress: protoplasm contraction and protoplasm fragmentation. Protoplasm contraction was evident at all freezing temperatures and in tissues collected at different times of the year. Cells with fragmented protoplasm, however, were noticed only in tissues collected in spring and summer. Protoplasm contraction in winter tissue occurred without apparent damage to the protoplasm. In contrast, protoplasm contraction in spring and summer tissues was accompanied by substantial damage. No evidence of intracellular ice formation was observed in parenchyma cells exposed to freezing stress. Differences in protoplasm contraction and appearance of cells with fragmented protoplasm likely indicated seasonal changes in cold hardiness of the wood tissue of red osier dogwood. We speculate that the appearance of fragmented protoplasm may indicate that cells are being injured by an alternative mechanism in spring and summer.

Current concepts of freezing resistance recognize two mechanisms by which woody plant tissues survive freezing temperatures: freezing avoidance and freezing tolerance (Burke et al., 1976; George et al., 1982; Wisniewski and

This work was supported by the Cooperative States Research Service, U.S. Department of Agriculture, under agreement No. 90- 37264-5702. This is Purdue University Agricultura1 Experiment Sta- tion Article No. 13776.

* Corresponding author; fax 1-605-677-6557. 737

Ashworth, 1986). Tissues that survive low temperatures by freezing avoidance display deep supercooling characteristics. During a freezing stress, tissue water, presumably within xylem ray parenchyma cells, remains liquid at low temperatures (supercools) by remaining isolated from extracellular ice (Burke, 1979). As a result, the parenchyma cells of supercooling tissues do not dehydrate and they retain their original shape and volume during freezing (Burke et al., 1976; Ash- worth et al., 1983, 1993; Malone and Ashworth, 1991). Tissues displaying freezing avoidance are moderately hardy and cannot survive temperatures below -4OOC (George et al., 1974; Burke et al., 1976; Becwar et al., 1981).

Tissues that resist low temperatures by freezing tolerance lose their cellular water to extracellular ice (George et al., 1982). During freezing, ice forms outside the living cells at temperatures slightly below O°C and grows in extracellular spaces. Since the plasma membrane and cell wall provide an effective barrier to ice (Steponkus, 1984), a difference in water potential between extracellular ice and the intracellular water is established. The formation of a water potential gradient results in the movement of intracellular water through the plasma membrane and cell wall to extracellular ice. As tissue temperature declines, cells become progressively dehydrated. The reduction in cell water content is accompanied by a reduction in cell volume and collapse of the cell wall (Ashworth et al., 1988; Pearce, 1988; Malone and Ash- worth, 1991; Pearce and Ashworth, 1992). This type of freezing behavior is also called extracellular freezing (Burke et al., 1976; Burke and Stushnoff, 1979). Differential thermal analysis of tissues exhibiting extracellular freezing records only one exotherm, the HTE (Malone and Ashworth, 1991). The HTE is linked to freezing of water in xylem vessels and extracellular spaces, and this freezing event is not injurious (Quamme et al., 1972; Burke et al., 1976). Some tissues exhibiting extracellular freezing are cold hardy and can survive temperatures below -4OoC, in some cases as low as -196OC (Sakai, 1960; Guy et al., 1986).

Recent studies have shown that freezing behavior of some wood tissues cannot be explained by current concepts of

Abbreviations: HTE, high-temperature exotherrn; LTSEM, low- temperature scanning electron microscopy; TEM, transmission electron microscopy.

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freezing resistance. Using differential thermal analysis and LTSEM, Malone and Ashworth (1991) studied freezing behavior of wood tissue of red osier dogwood (Cornus sericea L.). They observed that the freezing curve of the wood tissue showed only a HTE, suggesting that tissue water froze extracellularly. Contrary to predictions, cell walls of xylem ray parenchyma cells did not collapse during freezing, yet could survive temperatures as low as -8OOC. This observation was unlike what has been observed in herbaceous plants (Pearce, 1988; Pearce and Ashworth, 1992) and bark tissues (Ash- worth et al., 1988; Malone and Ashworth, 1991), in which, in response to a freezing stress, both cell wall collapse and reduction in cell volume were observed.

An alternative explanation regarding the freezing behavior of xylem ray parenchyma cells of red osier dogwood is required. The LTSEM experiments of Malone and Ashworth (1991) that demonstrated that the cell walls of xylem ray parenchyma cells do not collapse during freezing raise an important question. If the cell walls of these cells do not contract, how can cells dehydrate and lose water to extracellular ice? One possibility is that during a freezing stress, the protoplasm contracts within the rigid cell wall. The objective of this study was to test this hypothesis by examining the status of the plasma membrane and cytoplasm in relation to the cell wall. Therefore, we employed freeze substitution and TEM to study the response of xylem ray parenchyma cells of freeze-tolerant red osier dogwood (Malone and Ashworth, 1991) to a freezing stress.

The freeze-substitution technique we used was modified in our laboratory to fulfill the requirements of our experi- mental design and overcome difficulties associated with wood preparation for TEM (Ristic and Ashworth, 1993a). This technique utilizes two general steps. The first step involves the rapid freezing of tissues to immobilize cell contents; the second step involves replacement of frozen water with a freeze-substitution fluid at low temperatures (Harvey, 1982; Howard and O’Donnell, 1987). Successful preservation of tissue morphology and cell ultrastructure depends upon both steps.

Woody tissues are difficult to prepare for TEM using freeze substitution. The major obstacles are the inability to cool specimens rapidly and avoid intracellular ice formation and the inability to infiltrate tissues with epoxy resins (Ristic and Ashworth, 1993a). Efforts to maximize the cooling rate have utilized propane jet freezers (Moor et al., 1976), high-pressure cryofixation (Kaeser et al., 1989), and a variety of coolants including liquid propane, ethane, Freon, and subcooled nitrogen (Elder et al., 1982; Harvey, 1982; Ryan et al., 1987). The most favorable results regarding successful preservation of tissue morphology and cell ultrastructure have been obtained using liquid propane and high-pressure freezers (Harvey, 1982; Kaeser et al., 1989). Unfortunately, these conditions were not readily adaptable to the necessary design of our freezing experiment. To avoid melting of tissues prior to quench cooling, the manipulation of specimens during transfer from the freeze stress to the quench coolant had to be minimized. This precluded loading specimens into a high- pressure freezer. We compromised and used rapid manual transfer of the specimen directly into the coolant. In addition, since we needed to conduct the quench freezing immediately

adjacent to electrical cooling equipment used for laboratory freezing experiments, we did not wish to use the efficient, but extremely flammable, liquid propane as a quench cooler. Hence, Freon 12 was substituted for safety reasons. Difficul- ties associated with specimen infiltration with epoxy resin have been resolved by developing a new infiltration method for preparation of freeze-substituted wood tissues for TEM (Ristic and Ashworth, 1993a).

Despite these limitations, we expected our freeze-substitution method to be successful. Our primary objective in this study was to determine the status of the plasma membrane and cytoplasm in freeze-stressed xylem ray parenchyma cells of red osier dogwood during freezing. Therefore, even if small ice crystals caused by quench freezing were formed in the xylem parenchyma cells, we would not expect them to severely clistort the interna1 cell structure and would be able to evaluate the status of the protoplasm in relation to the cell wall.

MATERIALS AND METHODS

Plant Material

Twigs (diameter 3-5 mm) of red osier dogwood (Comus sericea L.) from the current season’s growth were collected from plantings adjacent to the Purdue University campus in February, April, and August of 1992. To minimize dehydration during handling, samples were placed on moistened filter paper in a Petri dish and immediately transported to the laboratory. Samples were transported to the laboratory in a box containing crushed ice.

Laboratory Freezing Stress

Controlled laboratory freezing of specimens was similar to that described by Malone and Ashworth (1991). Twigs were cut with a razor blade into 0.5- to 1-mm-thick cross-sections on moistened filter paper and placed on a depressed flat surface of an aluminum block that had previously been placed in the -8OOC freezer and cooled to O°C using a programmable temperature controller (Omega Engineering, Inc., Stamford, CT). The aluminum surface was lightly coated with vegetable oil to keep specimens from sticking. To minimize dehydration, inverted Petri dishes and an aluminum cover were placed over the specimens. Specimens were al- lowed to equilibrate at O°C for 30 min, cooled to -6OOC at 5OC/h, and collected at O, -5, -10, -20, -30, -40, -50, and -6OOC. Ice crystals were placed near the samples to initiate ice nucleation. Tools used to handle the samples were pre- cooled to appropriate temperatures to avoid warming the samples. During freezing, plate temperature was constantly monitored with an attached thermocouple. When appropriate temperatures were reached, specimens were rapidly transferred using prechilled forceps into melted Freon 12 and quench frozen.

Freeze Substitution

Specimens were prepared for TEM using the method of Ristic and Ashworth (1993a). Briefly, samples that had been quench frozen in melted Freon 12 were quickly transferred

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to liquid nitrogen. Samples were subsequently transferred from liquid nitrogen to vials containing freeze-substitution fluid (2% osmium tetroxide in 100% methanol [w/v]) that had previously been cooled to -8OoC, Substitution was car- ried out at -8OOC for 14 d. Substituted specimens were gradually warmed to room temperature over 48 h. To facili- tate embedding, specimens were rehydrated in a graded series of methanol and treated with 25 m sodium phosphate buffer (pH 6.9) at room temperature for 48 h. Following treatment with phosphate buffer, tissues were dehydrated in a graded series of acetone (30, 50, 70, 80, 90, 95, 100%). Acidified 2,2-dimethoxypropane was added to 100% acetone (Muller and Jacks, 1975). Specimens were infiltrated with Spurr resin (Spurr, 1969) at room temperature. Polymeriza- tion was camed out at 6OoC for 24 h. Thin sections (80-99 nm) were cut on a Reichert Ultracut E ultramicrotome using a diamond knife. Sections were stained with 4% uranyl acetate in 70% ethanol (w/v) for 20 min and in 0.2% aqueous lead citrate (w/v) for 2 min and viewed with a Philips 400 transmission electron microscope at 80 kV.

Chemical Fixation

Samples (about 1 mm3) were fixed with 4% glutaraldehyde and 4% paraformaldehyde in 25 m~ sodium phosphate buffer (pH 6.9) for 6 h at room temperature. Specimens were then rinsed and held overnight in phosphate buffer. Postfix- ation with 2% osmium tetroxide (in the above buffer) was for 6 h at room temperature. Following postfixation, specimens were dehydrated in a graded series of acetone (30, 50, 70, 80, 90, 100%) and gradually infiltrated (2 h each at 20, 40, 60, 80, and 100% embedding media in acetone) with Spurr embedding medium (Spurr, 1969). Specimens were held ovemight in pure Spurr resin prior to polymerization at 6OoC for 24 h. Specimens were sectioned, stained, and viewed as described above.

RESULTS

Assessment of the Freeze-Substitution Method

Control wood tissue of red osier dogwood prepared by freeze substitution was similar to that prepared by chemical fixation. A11 cells appeared regular in shape, and no structural disorganization of the tissue was observed. Vessel members, fibers, and xylem ray parenchyma cells (Fig. 1, a and b) did not exhibit any signs of distortion and/or collapse. In addition, no evidence of cell-wall separation within the middle lamella was noticed.

Xylem ray parenchyma cells from control tissues were adequately preserved by freeze substitution. No signs of protoplasm contraction were noticed. Subcellular structures were visible, although evidence of small intracellular ice crystals, caused by quench freezing, was observed (Fig. lb; ice crystals can be recognized as white grains throughout the cell). The ice crystals, however, did not severely disrupt cell ultrastructure. Cell organelles, such as vacuoles, mitochondria, and chloroplasts, were well defined (Fig. lb).

Winter Tissue

Freezing stress did not disrupt the structural organization of wood tissue of red osier dogwood. Vessel members, fibers, and xylem ray parenchyma cells were regularly shaped, and no distortion and/or cell-wall separation within the middle lamella were noticed, as would be expected if individual cells had collapsed.

However, exposure to freezing stress resulted in protoplasm contraction of the xylem ray parenchyma cells. Signs of contraction were noticed at -5OC (Fig. lc) and were subsequently observed at a11 other freezing temperatures (Figs. 1, d-f, and 2, a-c). Protoplasm contracted along the radial axes of the parenchyma cells.

Generally, xylem ray parenchyma cells with contracted protoplasm did not appear damaged by a freezing stress. Cytoplasm was dense and darkly stained, and interna1 structures such as vacuoles, chloroplasts, and mitochondria were visible (Figs. l e and 2b). Interestingly, the intemal membranes of contracted protoplasm were negatively stained (Figs. l e and 2b). Furthermore, numerous light-gray stained droplets were observed in many chloroplasts, and these droplets appeared not to be surrounded by membranes. Plasma membrane of the contracted protoplasm was tightly ap- pressed to the cytoplasm and no exocytotic and/or endocy- totic vesicle formation was noticed at any freezing temperature. Occasionally, however, signs of damage to the plasma membrane were observed. Plasma membranes appeared broken, and pieces of broken membrane were attached to the cell wall (Fig. l f ) . The xylem ray parenchyma cells appeared to be free of intracellular ice crystals. Signs of intracellular ice formation were not observed in either contracted protoplasm or the space between contracted protoplasm and the cell wall (Figs. 1, c-f, and 2, a-c).

Spring and Summer Tissues

Spring and summer wood tissues of red osier dogwood showed similar responses to a freezing stress. The gross structural organization of the tissues was not affected by freezing. However, the xylem ray parenchyma cells showed two distinct responses: protoplasm contraction and protoplasm fragmentation.

Evidence of protoplasm contraction was noticed at -5OC, and cells with contracted protoplasm were subsequently observed at a11 other freezing temperatures (Figs. 3, b-e,’and 4, b and c). The cytoplasm of contracted protoplasm appeared dense and darkly stained. Furthermore, it seemed that protoplasm suffered substantial damage during contraction. Nu- merous small pieces of protoplasm remained attached to the cell wall (Fig. 3, c-e). Many protoplasmic projections from both contracted protoplasm and pieces of broken protoplasm were also noticed (Fig. 3e). In addition, plasma membrane of contracted protoplasm appeared damaged. The membrane was either not visible (Fig. 4b) or, if visible, it was frequently swollen and broken (Fig. 4c).

In many cells, the protoplasm appeared fragmented rather then contracted. Xylem ray parenchyma cells displayed fragmented protoplasm at -5OC and other freezing temperatures (Figs. 3f and 4a). Pieces of fragmented protoplasm were dense

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Figure 1. The effect of freezing stress on xylem ray parenchyma cells of wood tissue of C. ser/cea collected in winter of1992. Tissue was subjected to laboratory freezing stress at a 5°C/h cooling rate, harvested at appropriate temperatures,and prepared for TEM using chemical fixation (a) and the freeze-substitution technique (b-f). Evidence of protoplasmcontraction was noticed at —5°C (c-e) and other freezing temperatures (f). a, Xylem ray parenchyma cell in control tissue,cw, Cell wall; asterisk, vacuole; arrow, mitochondria; bar = 1 MM. b, Xylem ray parenchyma cell in control tissue. Asterisk,Vacuole; black arrow, chloroplast; arrow, mitochondria; bar = 1 P.M. c, Tissue freeze stressed at —5°C. cw, Cell wall;arrow, contracted protoplasm; star, empty space between cell wall and contracted protoplasm; bar = 2 MM. d, Tissuefreeze stressed at —5°C. Arrow, Contracted protoplasm; star, empty space between cell wall and contracted protoplasm;bar = 1 MM. e, Tissue freeze stressed at —5°C. Asterisk, Vacuole; arrow, chloroplast; bar = 1 MM. f, Tissue freeze stressedat -30°C. Arrowhead, Broken plasma membrane attached to the cell wall; bar = 1 MM.

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Figure 2. The effect of freezing stress on xylem ray parenchymacells of wood tissue of C. ser/cea collected in winter of 1992. Tissuewas subjected to laboratory freezing stress at a 5°C/h cooling rate,harvested at appropriate temperatures, and prepared for TEM usingthe freeze-substitution technique. The protoplasm of xylem rayparenchyma cells contracted in response to a freezing stress, a,Tissue freeze stressed at -40°C. fb, Fiber; bar = 2 MM. b, Tissuefreeze stressed at -40°C. Star, Empty space between cell wall andcontracted protoplasm; arrowheads, negatively stained cell mem-branes; bar = 0.5 MM. c, Tissue freeze stressed at -50°C. Bar =2 MM.

and darkly stained and, in many cells, were tightly appressedto the cell wall (Fig. 5). Plasma membrane and internalstructures of the fragmented protoplasm were disrupted andnot recognizable (Fig. 4a). Criteria for distinguishing cellswith fragmented protoplasm from those with contracted pro-toplasm were based on the size and general morphology ofthe protoplasm. In cells with contracted protoplasm, a largeportion of the protoplasm was contracted (Figs. 3, b-e, and4, b and c). In contrast, in cells with fragmented protoplasmseveral separate pieces of protoplasm were noticed, and therewas no apparent connection between them (Figs. 3f and 5).Furthermore, in cells with fragmented protoplasm, manystarch grains were located outside the protoplasm, indicatingdamage to the chloroplasts (Fig. 3f). By contrast, starch grainswere never observed outside the protoplasm in cells withcontracted protoplasm.

As in the winter tissue, no signs of intracellular ice forma-tion were noticed in the spring and summer tissues. In bothcells with contracted protoplasm and cells with fragmentedprotoplasm, evidence of intracellular ice crystals was notobserved in any tissues exposed to a freeze-stress treatment.

DISCUSSION

The freeze-substitution technique is based on the premisethat quench freezing preserves tissue and cell structure in-stantly, and, therefore, allows examination of tissue and cellmorphology in their native states. The advantages of thistechnique have been well documented (Dempsey et al., 1975;Heuser et al., 1976; Browning and Gunning, 1977; Chandler,1984; Reger et al., 1984; Lancelle et al., 1985, 1986; Schneideret al., 1985; Torri-Tarelli et al., 1985; Hippe and Hermanns,1986; Risric and Ashworth, 1993a). This technique has beenused to examine ulrrastructural changes in the leaf (Harveyand Pihakaski, 1989, 1990) and morphological changes ofthe wood tissues (Ashworth et al., 1988; Malone and Ash-worth, 1991) arising from freezing stress. It has also beendemonstrated that this technique can be used to satisfactorilypreserve cell ultrastructure and morphology of wood tissue(Risric and Ashworth, 1993a).

Using freeze substitution and TEM we observed proto-plasm contraction in xylem ray parenchyma cells of red osierdogwood after exposure to a laboratory freezing stress. Thiswas seen at all freezing temperatures and in tissues collectedat different times of the year.

What is the mechanism that could explain protoplasmcontraction in the xylem ray parenchyma cells of red osierdogwood? There are two possibilities. One possibility is thatduring freezing, cell walls of xylem ray parenchyma cellsremained rigid and the protoplasm contracted. The alterna-tive would be that both the cell wall and the protoplasmcontracted but the cell wall regained its original shape duringspecimen preparation for TEM. We favor the former possi-bility and do not believe that the cell wall contracted duringfreezing for several reasons.

Using LTSEM, Malone and Ashworth (1991) examined theresponse of xylem ray parenchyma cells of red osier dogwoodto a freezing stress. This technique does not involve chemicalfixation and permits observation of tissue in the frozen state.Malone and Ashworth (1991) observed that xylem ray paren-

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Figure 3. The effect of freezing stress on xylem ray parenchyma cells of wood tissue of C. ser/cea collected in spring of1992. Tissue was subjected to laboratory freezing stress at a 5°C/h cooling rate, harvested at appropriate temperatures,and prepared for TEM using the freeze-substitution technique. Xylem ray parenchyma cells showed two distinct responses:protoplasm contraction (b-e) and fragmentation of protoplasm (0. a, Xylem ray parenchyma cell in control tissue. Bar =2 /iM. b, Tissue freeze stressed at —10°C. White arrow, Cell wall; black arrow, contracted protoplasm; curved blackarrow, space between cell wall and contracted protoplasm; bar = 2 ^M. c, Tissue freeze stressed at — 10°C. vs, Vesselmember; arrow, cell wall; black arrow, contracted protoplasm; arrowheads, pieces of broken protoplasm attached to thecell wall; bar = 2 MM. d, Tissue freeze stressed at —30°C. Arrow, Contracted protoplasm; arrowhead, pieces of brokenprotoplasm attached to the cell wall; bar = 2 JIM. e, Tissue freeze stressed at —30°C. Arrow, Cytoplasmic projection;arrowhead, pieces of broken protoplasm attached to the cell wall; asterisk, vacuole; bar = 1 I*M. f, Tissue freeze stressedat — 40°C. fb, Fiber; white arrow, cell wall; black arrow, piece of fragmented protoplasm; arrowhead, starch grain; star,cell lumen; bar = 2 MM-

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Figure 4. The effect of freezing stress on xylemray parenchyma cells of wood tissue of C. seri-cea collected in spring of 1992. Tissue wassubjected to laboratory freezing stress at a 5°C/h cooling rate, harvested at appropriate tem-peratures, and prepared for TEM using thefreeze-substitution technique. Xylem ray pa-renchyma cells showed two distinct responses:fragmentation of protoplasm (a) and proto-plasm contraction (b and c). a, High magnifi-cation of the xylem ray parenchyma cell pre-sented in Figure 3f. cw, Cell wall; arrow, pieceof fragmented protoplasm; asterisk, starchgrain; star, cell lumen; bar = 1 JIM. b, Tissuefreeze stressed at — 40°C. cw, Cell wall; n,nucleus; white arrow, contracted protoplasm;black arrow, pieces of broken protoplasm at-tached to the cell wall; asterisk, starch grain;bar = 1 MM. c, Tissue freeze stressed at — 50 °C.cw, Cell wall; arrow, plasma membrane; arrow-head, damage on the plasma membrane; aster-isk, starch grain; bar = 0.2 HM.

chyma cell walls did not collapse in response to either labo-ratory freezing stress or freezing stress in the field. Moreover,they also noticed that xylem ray parenchyma cells had areduced volume of protoplasm. This reduction in protoplasmvolume is consistent with protoplasm contraction observedin our present study.

Ashworth et al. (1993) studied the response of xylem rayparenchyma cells of red osier dogwood to a dehydrativestress using LTSEM. They exposed the wood tissue of redosier dogwood to a dehydrative stress by incubating speci-mens at a range of constant RHs that correspond to the vaporpressure over ice at -20, -40, -60, and -80°C. The woodtissue was dehydrated below the fiber saturation moisturepoint (30%). However, no collapse of xylem ray parenchyma

cell walls was noticed in response to dehydration. Similarly,no evidence of cell-wall separation was found in specimensof red osier dogwood exposed to numerous freeze-thawcycles in the field (Ashworth et al., 1993).

The lignified secondary cell walls of the wood are rigidand do not have sufficient elasticity to permit significantcontraction (U.S. Forest Products Laboratory, 1974; Ash-worth et al., 1993). When compressed, wood from mosthardwood nonsupercooling species can reversibly changedimensions less than 1 % in the direction perpendicular to thelongitudinal axis and approximately one-twentieth as muchin the direction parallel to the longitudinal axis at a maximumapplied external force (U.S. Forest Products Laboratory,1974). Furthermore, wood is dimensionally stable at moisture

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Figure 5. The effect of freezing stress on xylem ray parenchymacells of wood tissue of C. ser/cea collected in spring of 1992. Tissuewas subjected to laboratory freezing stress at a 5°C/h cooling rate,harvested at -60°C, and prepared for TEM using the freeze-substi-tution technique. Note fragmented protoplasm in the xylem rayparenchyma cell, vs, Vessel member; fb, fiber; arrow, cell lumen;arrowhead, piece of fragmented protoplasm; bar = 2 /IM.

contents above the fiber saturation point (U.S. Forest Prod-ucts Laboratory, 1974). The fiber saturation point, whichaverages about 30% of the maximum wood moisture content(U.S. Forest Products Laboratory, 1974), is defined as themoisture content at which cell walls are completely saturatedbut no water exists in cell cavities and extracellular spaces.Wood dimensions change very little even after completeremoval of cellular water. It shrinks most in the tangentialdirection, about one-half as much radially, and only slightlyalong the longitudinal axis. Typical values of tangential andradial shrinkage as wood is completely dried range for mosthardwood species from 6 to 12 and 3 to 7%, respectively(U.S. Forest Products Laboratory, 1974). The combined ef-fects of tangential and radial shrinkage can distort the shapeof wood pieces because of the difference in shrinkage andthe curvature of annual rings (U.S. Forest Products Labora-tory, 1974). The behavior of cells within the wood tissuewould likely be similar.

Based on physical and mechanical properties of the wood,it is therefore not surprising that the cell walls of xylem rayparenchyma cells of red osier dogwood did not collapse inresponse to either a freezing (Malone and Ashworth, 1991)or a dehydrative stress (Ashworth et al., 1993). Furthermore,we observed no evidence of structural disorganization and/or distortion of the wood tissue after exposure to freezing, as

would be expected if the wood tissue changed its dimensions.We also noticed no evidence of cell-wall layers separating infreeze-stressed samples. Due to the tight cell-to-cell connec-tions in tissue regions where xylem ray parenchyma cells arelocated, it would be difficult to imagine that cell-wall collapsecould have occurred without severe tearing and/or disruptionof xylem ray parenchyma cell walls as well as walls ofadjacent wood cells.

The contraction of the protoplasm indicated that during afreezing stress xylem ray parenchyma cells were dehydrated.Presumably, cellular water from parenchyma cells was lostto extracellular ice. Dehydration of xylem ray parenchymacells indicated by protoplasm contraction is consistent withNMR measurements of red osier dogwood stems duringexposure to a freezing stress (Burke et al., 1974; Harrison etal., 1978). Burke et al. (1974) observed a decline in the liquidwater content of red osier dogwood stems during freezing.

Although protoplasm contraction seems a likely responseof xylem ray parenchyma cells to freezing, an importantquestion remains unanswered. What is present between thecell wall and contracted protoplasm? Two possibilities seemlikely. Either ice was present in the space between the cellwall and contracted protoplasm, or air occupied that space.

Formation of ice crystals between the cell wall and proto-plasm has occasionally been reported in plant cells exposedto freezing. Asahina (1978), for example, noticed that icecrystals were formed between the cell wall and plasma mem-brane of Tradescantia staminal hair cells under slight super-cooling conditions. Therefore, it is possible that ice crystalswere also present between the cell wall and contracted pro-toplasm of xylem ray parenchyma cells of red osier dogwood.If the ice crystals were present between the cell wall andcontracted protoplasm of parenchyma cells of red osier dog-wood, then the question arises, Where did the ice come from?Ice must either have spread from extracellular ice and grownthrough the cell wall or been initiated in the thin film ofwater between the plasma membrane and the cell wall. Inmost plants, the cell wall is an effective barrier to the spreadof ice (Steponkus, 1984). Whether this is true for red osierdogwood is not known.

If ice had initiated between the cell wall and plasmamembrane, it is surprising that evidence of intracellular iceformation was never observed. In many cells contractedprotoplasm was damaged. This was particularly apparent inthe spring and summer tissues. Yet there was no evidencethat ice had spread into the cytoplasm of damaged cells. Itcould be argued that intracellular ice was not observed inxylem ray parenchyma cells because of protoplasm dehydra-tion. The protoplasm dehydrates only after extracellular icehas formed and a Water potential gradient between extracel-lular ice and the intracellular water has been established.Water would have to be withdrawn from parenchyma cellsbefore growing ice crystals reach the protoplasm, and thisseems unlikely.

The alternative that air was present between the cell walland contracted protoplasm seems attractive. Water couldhave been removed from xylem ray parenchyma cells to theempty lumen of neighboring fibers, either as liquid or vapor.However, at present we have no evidence supporting thispossibility.

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The consequences of freezing stress and subsequent dehydration were apparent in the xylem ray parenchyma cells of red osier dogwood. The appearance of light-gray droplets located in the chloroplasts was evident after freezing. The droplets did not appear to be surrounded by membranes, and it is possible that they represent accumulation of osmiophilic material derived from the chloroplast during freezing. Fur- thermore, cellular membranes of freeze-stressed parenchyma cells appeared negatively stained. Similar observation of negatively stained cell membranes was reported in freeze- stressed leaf tissues of nonacclimated and cold-acclimated Secale cereale L. (Harvey and Pihakaski, 1989, 1990) prepared for TEM by either freeze substitution or chemical fixation. We do not know what caused cellular membranes to appear negatively stained. It is possible that during dehydration, cytoplasm became dense and peripheral regions of the membrane lipid bilayer stained darker than the membrane itself. Altematively, properties of cell membranes may change during freezing, which could alter the membrane response to fixation. We believe that the former possibility is less likely. Ultrastructural consequences of cellular dehydration have been studied in many species (Nir et al., 1969, 1970; Alieva et al., 1971; Fellows and Boyer, 1976; Vieira da Silva, 1976; Hallam and Luff, 1980; Ristic and Cass, 1991, 1992), but no observations of negatively stained membranes were reported in these studies. Therefore, it seems likely that the appearance of negatively stained membranes is the result of both low temperatures and cellular dehydration influencing membrane properties.

Striking differences in protoplasm contraction were noticed among wood tissues of red osier dogwood collected at different times of the year. Protoplasm contraction in the winter tissue appeared to occur without significant damage to the protoplasm. In contrast, protoplasm contraction in the spring and summer tissues was accompanied by substantial damage to the protoplasm. This damage was manifested by broken protoplasm whose pieces remained attached to the cell wall after the protoplasm contracted. The differences in protoplasm contraction suggested that parenchyma cells from the spring and summer tissues differed from those from the winter tissue in terms of the strength of the connection between the plasma membrane and the cell wall. It is tempt- ing to speculate that connections between the plasma membrane and the cell wall are due to associated adhesion mole- cules, and that these components change during cold acclimation and deacclimation.

Cells with fragmented protoplasm were apparent in freeze- stressed spring and summer tissues of red osier dogwood. Similar cells were also observed in the wood tissue of flow- ering dogwood (Cornus florida L.) after exposure to freezing stress (Ristic and Ashworth, 1993b). Intracellular ice formation has been proposed to be the source of freezing injury, but only in wood tissues that exhibit the deep supercooling characteristics (Burke et al., 1976; George and Burke, 1977; George et al., 1982; Ashworth et al., 1983). It is known that red osier dogwood does not exhibit deep supercooling characteristics (Hanison et al., 1978; Malone and Ashworth, 199 1). Moreover, intracellular ice was not observed in freeze- stressed xylem ray parenchyma cells. Thus, it seems likely

that another source of freezing injury must exist in the wood tissue of red osier dogwood.

Based on Malone and Ashworth (1991), LTSEM, and our TEM observations, we conclude that the xylem ray parenchyma cells of red osier dogwood display a unique response to freezing stress. During laboratory freezing, cell walls of parenchyma cells remain rigid (Malone and Ashworth, 1991) and the protoplasm contracts. In the winter tissue, protoplasm contraction occurs without significant damage to the protoplasm. In contrast, in the spring and summer tissues protoplasm contraction is accompanied by substantial damage to the protoplasm. In addition, the parenchyma cells from the spring and summer tissues show a unique type of injury. This injury is manifested by fragmented protoplasm with indistin- guishable plasma membrane and cell ultrastructures. Differ- ences in protoplasm contraction and appearance of cells with fragmented protoplasm likely reflect seasonal changes in cold hardiness of the wood tissue of red osier dogwood.

ACKNOWLEDCMENTS

The authors are grateful to Drs. Mike Hasegawa and Avtar Handa of the Purdue University Department of Horticulture for critically reading the manuscript, and Dr. John Senft of the Purdue University Department of Forestry and Natural Resources for helpful discus- sions. Thanks are also due to Dr. Charles Bracker and Mrs. Deb Sherman of the Purdue University Electron Microscopy Center for their help and cooperation and Mrs. Vicki Stirm for her excellent technical assistance.

Received July 12, 1993; accepted October 28, 1993. Copyright Clearance Center: 0032-0889/94/104/0737/10.

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