Journal of Experimental Botany, Vol. 52, No. 364, pp. 2105-2114,
November 1, 2001
© 2001 Oxford University Press
Original Papers |
Desiccation tolerance of protoplasts isolated from pea embryos
Department of Biology, University of South Dakota, 414 E. Clark Street, Vermillion, SD 57069, USA
Received 22 March 2001; Accepted 23 June 2001
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
To facilitate studies of desiccation tolerance at the cellular level, a technique to isolate protoplasts from desiccation-tolerant pea (Pisum sativum L. cv. Alaska) embryos has been developed. Using FDA (fluorescein diacetate) as a probe, viability of the protoplasts was investigated before and after drying to determine whether the protoplasts could survive desiccation in a manner similar to the tissue from which they were isolated. Protoplasts were isolated from 12 h imbibed pea axes, suspended in several different sugar solutions, then dried to water contents less than 0.2 g H2O g-1 DW. Protoplasts only survived drying if the rate was rapid (<2 h), while slow drying (24 h) was lethal. Maximal survival (75%) was obtained after drying protoplasts with a mixture of sucrose and raffinose, while pure sucrose and trehalose were somewhat less effective protectants. Low survival was obtained after drying protoplasts with monosaccharides and pure raffinose. Protoplasts isolated from germinated seedlings did not survive dehydration below 0.2 g H2O g-1 DW. Transmission electron microscopy revealed that dried desiccation-tolerant protoplasts appeared shrunken, with folded membranes, while dried protoplasts from sensitive tissue had disrupted membranes. While isolated protoplasts maintained some of the desiccation tolerance of orthodox seeds, their inability to survive complete drying and their sensitivity to drying rate is similar to the behaviour of recalcitrant embryos.
Key words: Desiccation tolerance, protoplast isolation, Pisum sativum L., pea, embryo.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Desiccation tolerance of plant embryos is a complex phenomenon, with many factors contributing to a cell's ability to survive the progressive loss of water. Although the ability of orthodox seeds to survive desiccation is important to agriculture, complete understanding of the mechanisms that permit these cells to tolerate extremely low water contents has been elusive. Studies of the phenomenon of desiccation tolerance have generally been carried out at two levels: in vivo studies of the response of whole tissues to desiccation and rehydration (Senaratna and McKersie, 1983
a, b; Lalonde and Bewley, 1985; Koster and Leopold, 1988; Berjak et al., 1990; Blackman et al., 1995; Leprince et al., 1995; Reisdorph and Koster, 1999; Walters et al., 2001), and in vitro studies of model systems, including isolated sugars, lipids, proteins, membranes, and organelles (Santarius, 1973; Caffrey et al., 1988; Crowe and Crowe, 1988a, b; Koster et al., 1994, 2000; Sun et al., 1996). Studies of seed desiccation tolerance at the level of individual cells or protoplasts have been lacking, yet could provide valuable information to bridge the gap between model systems and whole tissues.
Protoplasts from a variety of systems have been used successfully to study mechanisms of freezing damage, believed to result primarily from cellular dehydration during extracellular ice formation (Steponkus and Webb, 1992). Protoplasts isolated from leaves of winter rye, oat and Arabidopsis displayed freezing tolerances similar to those of the intact tissues, and the behaviour of their plasma membranes has been shown to be crucial to survival of freeze-induced dehydration (Webb et al., 1994; Uemura et al., 1995). In contrast, protoplasts isolated from Jerusalem artichoke tubers were significantly more tolerant of freezing than were the tubers themselves, and it was suggested that attachments between the cell wall and the plasma membrane might cause lethal damage to the membrane during freezing of the intact tissue (Murai and Yoshida, 1998). Although the tolerance of the protoplasts did not always coincide with that of the intact tissues, membrane damage was thought to be the lethal event in all cases. In desiccation-sensitive embryos, membranes are also considered to be a primary site of dehydration damage (Senaratna and McKersie, 1983a, b; Leopold, 1986), and preventing damage to membranes is thought to be important for desiccation tolerance (Leopold, 1986). Seed tissues damaged by desiccation have increased rates of electrolyte leakage (Senaratna and McKersie, 1983a, b; Koster and Leopold, 1988; Blackman et al., 1995; Leprince et al., 1995), suggesting that plasma membranes have become more permeable as a result of the dehydration stress.
Despite the interest in membranes as a site of damage to sensitive tissues, little direct information exists on the behaviour of the plasma membranes of seed embryo cells during desiccation and rehydration. Use of desiccation-tolerant and desiccation-sensitive protoplasts might facilitate the direct observation of cellular events, particularly those associated with the behaviour of the plasma membrane, that occur during dehydration and rehydration. The goal of this study was to determine whether protoplasts isolated from seed embryonic axes tolerate desiccation in a manner similar to the intact embryos, and if so, to use these protoplasts to develop a system for the study of desiccation tolerance at this cellular level. To achieve this goal, protoplasts were isolated from pea (Pisum sativum L. cv. Alaska) embryonic axes at points of germination both before and after the loss of desiccation tolerance, they were subjected to several drying protocols, and their survival was assessed using the fluorescent probe fluorescein diacetate (FDA).
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant materials and protoplast isolation
Pea seeds (Pisum sativum L. cv. Alaska) were purchased from Gurney's Seed and Nursery Co. (Yankton, SD, USA) and were stored at 4 °C until use. For most experiments, seeds were imbibed in wet germination paper rolls (Anchor Paper Co., St Paul, MN, USA) in the dark at 25 °C for 12 h (a time when the axes are still desiccation-tolerant; Reisdorph and Koster, 1999
), and the axes were then detached for use in protoplast isolation. Some seeds were allowed to germinate for 1 week in the dark at 28 °C to obtain etiolated seedlings that were no longer desiccation-tolerant. To isolate embryonic axes from seeds nearer the onset of germination, dry seeds were cracked with a mortar and pestle, and both intact and broken axes were collected and soaked in water for 4 h.
To isolate protoplasts, the embryonic axes were chopped into small pieces (1–2 mm3) using a razor blade. The minced axes were rinsed using deionized water and were transferred to a digestion solution containing 2% (w/v) Onozuka Cellulase RS and 0.1% (w/v) Pectolyase Y-23 (Karlan Research Products Corp., Santa Rosa, CA, USA) in 5 mM CaCl2 and 20 mM MES (2-[N-morpholino]ethane sulphonic acid) (pH 5.5), with either 500 mM sugars (for 4 h and 12 h imbibed axes) or 200 mM sugars (for 1 week germinated seedlings) as osmotica. The sugar concentrations used were those that generated the maximal recovery of protoplasts at each germination time. Most isolations were carried out in a mixture of sucrose and raffinose (85:15, w/w), representative of the sugar composition of desiccation-tolerant maize embryos and possessing known hydration and glass-forming properties (Koster and Leopold, 1988; Koster, 1991). Axis tissue was allowed to digest for 4 h, then the liberated protoplasts were partially purified by filtering the enzyme solution through a nylon mesh having 62 µm diameter pores (for 4 h and 12 h imbibed axes) or 125 µm diameter pores (for 1 week germinated seedlings). The filtered solution was centrifuged at 250 g for 10 min. The pelleted protoplasts were resuspended in a buffered solution containing 5 mM CaCl2 and 20 mM MES (pH 5.5) with either 500 mM (for 4 h and 12 h imbibed axes) or 200 mM sugars (for 1 week germinated seedlings) as osmotica.
Viability of the liberated protoplasts was tested using the probe fluorescein diacetate (FDA) at a final concentration of 0.005% (w/v) (Widholm, 1972). Protoplasts were counted on a haemocytometer slide using a fluorescence microscope (Leica GmbH, Wetzlar, Germany). Protoplasts that fluoresced brightly and exhibited normal morphology were considered to be viable.
Assessing the desiccation tolerance of isolated protoplasts
The general treatment contained three parts: drying of the protoplasts, determination of the water content of the dried protoplast samples, then rehydration and measurement of protoplast survival. Initial attempts were made to dry the protoplasts to defined low water potentials by allowing them to equilibrate in sealed chambers with saturated salt solutions (Rockland, 1960). This drying protocol required about 24 h for sample equilibration and resulted in complete loss of viability for the protoplasts; therefore, faster drying rates were used in subsequent experiments. To dry the protoplasts rapidly, 100 µl of the resuspended protoplasts was loaded into each of two sets of three small preweighed plastic dishes. The filled dishes were weighed then transferred to a chamber containing silica gel desiccant. Dry air that had passed through a tube containing silica gel was blown through the chamber for 4 h at a flow rate of approximately 10 l min-1. After this air-drying treatment, one set of the three small dishes was weighed to determine how much water had been lost, then the protoplasts were immediately rehydrated to their original volume with deionized water containing FDA (0.005%, w/v). After 15 min, the rehydrated protoplasts were viewed on a haemocytometer slide using the fluorescence microscope. The number of fluorescent protoplasts was counted to assess viability.
Survival percentages were obtained by dividing the number of surviving protoplasts by the number of fresh protoplasts in the initial suspension. For each experimental trial, the mean number of protoplasts per volume of rehydrated protoplasts was compared to the mean number of protoplasts per volume of the freshly prepared protoplasts. Each of these means was based on counts from five separate slides, and each experiment was then replicated five times.
Water contents of the air-dried samples were determined in each experiment using the remaining set of three dishes of dried protoplast suspensions. These dishes were weighed, then transferred to an oven for drying at 70 °C in vacuo with P2O5 for at least 24 h. The oven dry weight was obtained and was used to calculate the water content in g H2O g-1 dry weight (DW) of the air-dried samples using the following formula:
 |
Experimental treatments of protoplasts
Effect of imbibition time on protoplast desiccation tolerance:
To determine whether protoplasts would reflect the desiccation tolerance of the intact tissue from which they were obtained, protoplasts were isolated from both desiccation-tolerant pea embryonic axes (4 h and 12 h imbibed) and from desiccation-sensitive seedlings (1 week germinated), and their survival was tested after dehydration as described above.
Effect of sugars as osmotica on protoplast desiccation tolerance:
Because sugars have been shown to differ in their ability to protect membranes during drying (Crowe et al., 1984a, b, 1986; Crowe and Crowe, 1988a, b; Caffrey et al., 1988; Sun et al., 1994, 1996), the effect of different sugars on protoplast desiccation tolerance was also tested. Protoplasts were isolated from 12 h imbibed pea seed axes as described above, except that 500 mM sorbitol was used as an osmoticum during the isolation. Aliquots of the isolated protoplasts were then resuspended in the following sugars: sucrose, trehalose, raffinose, sorbitol, glucose, mannitol, and the sucrose/raffinose mix, each at a concentration of 500 mM. Next, 100 µl of protoplast suspension with each of the different sugars as osmotica was loaded into plastic dishes and dried under a stream of dried air as described above. After drying, the protoplasts were rehydrated and stained with FDA (0.005%, w/v) to assess survival.
Effect of time and water content on protoplast desiccation tolerance:
There were small, but statistically significant, differences among the sample water contents after air-drying the protoplast suspensions in the experiments using different sugars; therefore, further experiments were conducted to compare survival after drying the samples to similar water contents. Protoplasts were isolated from 12 h imbibed pea seed axes, then 100 µl of protoplast suspension in the sucrose/raffinose mix (500 mM) was dehydrated over silica gel under a stream of dried air at 10 l min-1 for 4 h, where the samples reached a water content of about 0.18 g H2O g-1 DW. After drying, the samples were transferred to chambers containing either CaSO4 (Drierite, WH Hammond Drierite Co., Xenia, OH, USA) or saturated NaCl solution, where they were incubated for 24 h and 48 h. At room temperature, CaSO4 dries gases to a dew point of -73.3 °C (Fisher Scientific, Pittsburgh, PA, USA), corresponding to a relative humidity (RH) of about 48%, while the RH above a saturated NaCl solution is about 75% (Rockland, 1960). The water content of the samples incubated over CaSO4 for 24 h dropped to around 0.12 g H2O g-1 DW, while the water content of the samples incubated over the saturated NaCl solution for 24 h increased to around 0.28 g H2O g-1 DW. After 24 h and 48 h of incubation, the samples were rehydrated and stained with FDA to assess survival.
Statistical analyses:
The residualized data from the above experiments met normal distribution and equal variance criteria, so Student's t-test and ANOVA were used to determine whether observed differences were significant.
Preparation of protoplasts for electron microscopy
Fresh protoplasts suspended in sucrose/raffinose solution were fixed for 3 h at 22 °C in 2% (w/v) gluteraldehyde and 2% (w/v) paraformaldehyde in 0.1 M phosphate buffer, pH 6.8, that was osmotically adjusted with sucrose/raffinose to be isotonic to the protoplast suspension medium. Fixed protoplasts were centrifuged at 250 g, rinsed twice in osmotically adjusted phosphate buffer (pH 6.8), and were then post-fixed with 2% (w/v) OsO4 for 2 h. Protoplasts were rinsed twice then dehydrated through a graded series of acetone before infiltration in Spurr epoxy resin and polymerization at 60 °C for 24 h.
Protoplasts that had been dried under a stream of air were fixed using a freeze-substitution protocol based on that of Ristic and Ashworth (Ristic and Ashworth, 1993). Dishes containing dried protoplast suspensions were quench-frozen in liquid nitrogen, then were transferred into a solution of 2% (w/v) OsO4 in methanol precooled to -80 °C. After incubating for one week at -80 °C, the samples were gradually warmed to 4 °C over 24 h, and fixed protoplasts were recovered by centrifugation at 250 g for 10 min. The pelleted protoplasts were rinsed once with phosphate buffer (pH 6.8) then were dehydrated and embedded as described above.
Sections were cut using a Reichert Ultracut UCT ultramicrotome (Leica Inc., Dearfield, IL) and were collected on carbon-coated grids. The sections were stained first with 4% (w/v) uranyl acetate in 70% (v/v) ethanol for 20 min, then with 0.2% (w/v) aqueous lead citrate for 2 min. The stained sections were viewed using a JEOL-1210 transmission electron microscope at 80 kV.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Drying rate and water contents of protoplast suspensions
The rate of water loss from protoplast suspensions dried under a stream of air at 10 l min-1 is shown in Fig. 1
. Using this drying protocol, most of the water in the sample evaporated within 1.5 h (Fig. 1, inset), followed by a slower removal of additional water. This biphasic rate curve is characteristic of diffusive processes. Drying to known equilibrium water potentials was not feasible in these experiments because slow drying rates (about 24 h) caused the destruction of all protoplasts; however, it is possible to compare the final water contents achieved using the rapid air-drying procedure to water contents obtained after equilibration of sugar solutions at a range of water potentials (Koster, 1991). This analysis shows that the water contents attained by the protoplast suspensions after air-drying (0.18–0.10 g H2O g-1 DW) were similar to those obtained by equilibration to water potentials between -60 and -100 MPa (about 65% and 50% RH).
|
Effect of imbibition time on protoplast desiccation tolerance
Protoplasts isolated from 4 h and 12 h imbibed axes and from 1 week germinated axes were tested to determine whether their desiccation tolerance would decline during germination in a manner similar to that of the embryos from which they were isolated. Figure 2 shows that protoplasts isolated from 4 h and 12 h imbibed pea axes retained significant tolerance of dehydration to water contents less than 0.2 g H2O g-1 DW. The protoplasts isolated from 1 week germinated pea axes were not desiccation-tolerant. Greater desiccation tolerance was observed in the protoplasts isolated from 12 h imbibed axes (76%) than in those from 4 h imbibed axes (62%) (P<0.05). The water contents attained after drying were not statistically different among the samples (P>0.05).
|
Effect of sugars as osmotica on protoplast desiccation tolerance
As shown in Fig. 3, the survival of protoplasts dried with the sucrose/raffinose mix, pure sucrose, or trehalose in the suspension buffer was higher than that of protoplasts dried in glucose, sorbitol, mannitol, or raffinose. The highest survival was obtained from protoplasts dried in the sucrose/raffinose mixture, which represents the sugar composition of desiccation-tolerant maize embryos (Koster and Leopold, 1988). The lowest survival was obtained in the mannitol solution. The mannitol and raffinose solutions sometimes crystallized during drying, becoming opaque and white, and this resulted in complete destruction of the protoplasts in those samples. Crystallization of these sugars also led to highly variable mean water contents for the mannitol and raffinose samples because crystalline samples had almost no water remaining (Fig. 3). When the results obtained for mannitol and raffinose were excluded from the analysis, the water contents of samples containing the sucrose/raffinose mix, pure sucrose, and trehalose were significantly higher after air-drying than were the water contents of samples dried in glucose and sorbitol. The higher post-drying water contents may have contributed to the higher survival of protoplasts in these samples. To test this, the following set of experiments was performed where the water contents of all the samples were more comparable.
|
Effect of time and water content on protoplast desiccation tolerance
To lower their water content further, the dried protoplasts in the sucrose/raffinose mix were transferred to a chamber containing anhydrous CaSO4 (about 48% RH), where they were incubated for 24 h and 48 h. Figure 4A shows that the water content of the samples decreased to around 0.12 g H2O g-1 DW after 24 h and decreased another 0.02 g H2O g-1 DW during an additional 24 h incubation. These water contents are consistent with those achieved after equilibration of a sucrose/raffinose solution to a water potential of -100 MPa (Koster, 1991). The survival of protoplasts in the sucrose/raffinose mix also dropped after 24 h incubation over CaSO4 (Fig. 4A), but was still greater than the survival achieved by drying rapidly to the same water content in glucose or sorbitol (compare Figs 3
and 4A), confirming that the sucrose/raffinose mix conferred greater tolerance of desiccation to the protoplasts than did the monosaccharides.
|
Additional samples of protoplasts air-dried in the sucrose/raffinose mix were placed over a saturated NaCl solution, which generates a relative humidity of about 75% at room temperature (Rockland, 1960
). Incubation of samples at this higher humidity led to sample water contents around 0.28 g H2O g-1 DW (Fig. 4B), a value consistent with the water content of a sucrose/raffinose solution equilibrated at a water potential of -40 MPa (Koster, 1991). The survival of protoplasts decreased to 20% after 24 h. After 48 h at the higher water content, protoplast survival dropped further, to 8% (Fig. 4B). Thus, incubation at the intermediate water content (0.28 g H2O g-1 DW) was more damaging to the protoplasts than was incubation at the lower water content (0.10 to 0.12 g H2O g-1 DW).
Appearance of protoplasts
Protoplasts isolated from 12 h imbibed embryonic axes were small, averaging 10 µm in diameter, round, and lacked a large central vacuole (Fig. 5A). The cytosol appeared dense. The most conspicuous features were the nucleus and numbers of protein and lipid bodies. The contents of the protein bodies appeared to be diffuse, as degradation of the stored proteins was occurring. Lipid bodies were found in clusters around the periphery of the cell, though some were also observed deeper in the cytosol. In many protoplasts, large starch grains were also apparent. Sheets of membrane, presumed to be regions of endoplasmic reticulum, were observed in many protoplasts.
|
By contrast, protoplasts isolated from 1 week germinated seedlings were larger, averaging 20–25 µm in diameter, and had a conspicuous large central vacuole (Fig. 6A
). The cytosol formed a thin layer around the periphery of the protoplast. The nucleus and other organelles, including mitochondria and structures that appeared to be the remnants of protein bodies, were located in this cytosolic region.
|
Air-dried protoplasts from 12 h imbibed tissue appeared shrunken, with the plasma membrane folded around the compacted cellular structures (Fig. 5B
). Protein bodies in the dehydrated protoplasts (Fig. 5B) were smaller than in the fresh protoplasts (Fig. 5A), and clusters of lipid bodies were visible throughout the cytosol and around the periphery of the shrunken protoplasts (Fig. 5B). The nuclear envelope was folded and the contents of the nucleus appeared dense. All membranes appeared to be intact in the dehydrated protoplasts from desiccation-tolerant embryos.
Dehydrated protoplasts from 1 week germinated seedlings were difficult to detect using transmission electron microscopy because almost all protoplasts were destroyed during the dehydration treatment, so that mostly debris remained. The few dried protoplasts that were observed had disorganized contents with ruptured plasma membranes (Fig. 6B).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Desiccation tolerance of seed embryos has been studied at both organismal and subcellular levels. The goal of this research was to try to bridge this gap by developing a system for studying seed desiccation tolerance at the level of isolated protoplasts. Protoplasts isolated from desiccation-tolerant pea embryonic axes were dried to water contents less than 0.20 g H2O g-1 DW, then rehydrated, and had high levels of survival as measured by FDA fluorescence (Fig. 2
). Protoplasts that were isolated from desiccation-sensitive axes did not survive dehydration (Fig. 2), confirming that the ability to survive the drying treatment depended on properties intrinsic to the protoplasts, and not merely on the protocols used. This is an important characteristic of the protoplast system. In simpler model systems using membrane vesicles isolated from a desiccation-sensitive organism, drying in the presence of disaccharides can lead to the preservation of the membranes’ functional integrity (Crowe et al., 1984b). Thus, for a relatively simple system, such as isolated membranes, the drying protocol can confer desiccation tolerance, while for a more complex system like the protoplasts used here, the drying protocol alone did not lead to survival of protoplasts isolated from desiccation-sensitive tissue.
Although large numbers of protoplasts isolated from desiccation-tolerant embryos did survive drying to water contents less than 0.2 g H2O g-1 DW (Fig. 2), their desiccation tolerance was not the same as that of the tissue from which they were isolated. First, protoplasts isolated from both 4 h and 12 h imbibed axes did not exhibit as high a survival percentage as intact embryos, which have 100% survival of desiccation to water contents less than 0.1 g H2O g-1 DW (Reisdorph and Koster, 1999). Second, the protoplasts did not survive slow drying over a 24 h period, while embryos imbibed for 12 h readily survive this slow drying (Reisdorph and Koster, 1999). Protoplasts only survived dehydration if the drying rate was rapid (Fig. 1), which is reminiscent of the behaviour of many recalcitrant seeds that can survive to water contents less than 0.4 g H2O g-1 DW if dried rapidly (Berjak et al., 1990; Pammenter and Berjak, 1999; Walters et al., 2001). These authors suggest that recalcitrant seeds survive dehydration to low water contents only if flash-dried because rapid drying minimizes the amount of time that the embryo has intermediate moisture contents, at which unregulated metabolism can damage the cells (Leprince et al., 1999; Pammenter and Berjak, 1999; Walters et al., 2001). The protoplasts’ enhanced sensitivity to drying rate compared to the intact embryo may, therefore, reflect differences in the metabolic activity of the two systems. Leprince and co-workers have shown that the metabolic status of tissues is a component of desiccation tolerance and have hypothesized that metabolism must be down-regulated before cells can achieve complete tolerance (Leprince et al., 1995, 1999, 2000). Removal of the cell wall during protoplast isolation is known to alter some metabolic processes, including lipid metabolism (Browse et al., 1988), and may have altered the ability of the embryo protoplasts to slow their metabolism during dehydration.
The damaging effect of intermediate water contents on protoplasts was apparent in samples that were incubated at 75% relative humidity after first being air-dried (Fig. 4B). While protoplast survival diminished in these samples and in samples incubated over CaSO4 (RH 
48%) (Fig. 4A), the decrease was significantly greater in the samples held at the higher humidity, suggesting that deleterious reactions causing the loss of viability were accelerated in the wetter samples. The damaging effects of unregulated metabolism that occur at intermediate water contents can be reduced by further drying because this reduces metabolic activity. Walters et al. showed that lowering the water content resulted in a greater reduction of metabolism in desiccation-tolerant embryos than in desiccation-sensitive embryos (Walters et al., 2001). The results of this study are consistent in that protoplasts survived longer when dried to lower water contents (Fig. 4). Differences in longevity at 75% RH between protoplasts (<2 d) and intact pea axes (>100 d; Walters et al., 2001) also suggest that the protoplasts' metabolic activity is greater than that of the embryos.
Other than the drying rate, the factor that had the most significant impact on protoplast survival was the sugar composition of the suspension buffer in which the protoplasts were dried (Fig. 3). Survival was greatest in the sucrose/raffinose mixture, followed by pure sucrose and trehalose, while monosaccharides and the trisaccharide raffinose were not protective (Fig. 3). The presence of di- and oligosaccharides has been associated with desiccation tolerance in vivo in many species (Crowe et al., 1984a; Hoekstra and Van Roekel, 1988; Koster and Leopold, 1988), and these sugars have also been shown to preserve the structure and function of isolated membranes during desiccation in vitro (Crowe et al., 1984b; Caffrey et al., 1988; Koster et al., 1994, 2000; Sun et al., 1996). Thus, the survival of protoplasts dried in the sucrose/raffinose mixture, pure sucrose, and trehalose is consistent with previous findings.
The mechanism by which the sucrose/raffinose mixture and the pure disaccharides stabilized protoplasts during drying in these experiments is unclear. Several mechanisms, not mutually exclusive, have been proposed to explain the protective effects of sugars during desiccation, and these are considered in the context of the protoplast system. One suggestion is that sugars, especially disaccharides, form hydrogen bonds to replace those formed by water with membrane lipids and proteins (Crowe and Crowe, 1988b). Hydrogen bonding between sugars and phospholipids has been reported for samples with water contents 
0.01 g H2O g-1 DW (Crowe and Crowe, 1988b); however, evidence for direct hydrogen bonding between sugars and phospholipids at the somewhat higher water contents (around 0.18 g H2O g-1 DW) experienced by the dried protoplast samples has not been presented. Thus, it is not known whether this mechanism can explain stabilization by sugars when some water molecules are present.
Another mechanism by which sugars might prevent damage to dehydrated systems is by acting as osmotic spacers that limit the close approach of membranes and hydrophilic macromolecules during drying and, thus, minimize physical stresses that can cause damage (Wolfe and Bryant, 1999; Bryant et al., 2001). This mechanism can explain stabilization by sugars at all water contents less than about 0.2 g H2O g-1 DW. The osmotic effects of the intracellular sugars are presumably important in stabilizing cellular structures in these experiments; however, because they are non-specific, they cannot account for the differential survival achieved using different sugars (Fig. 3).
Vitrification has been invoked as a means by which sugars can slow diffusive processes and, thereby, stabilize dried cells (Burke, 1986; Bruni and Leopold, 1991; Koster, 1991; Buitink et al., 2000). The temperature at which solutions vitrify depends upon both the water content and the molecular weight of the solute; for the sugars used in these experiments, vitrification at room temperature only occurs at water contents less than about 0.1 g H2O g-1 DW (Koster, 1991; Sun et al., 1996; Koster et al., 2000). At the water contents attained by the air-dried protoplast samples, the sugar solutions were likely in a ‘rubbery’ state rather than a glass; however, molecular mobility is still limited by the high viscosity of the solution under these conditions (Buitink et al., 2000). This factor may have contributed to the differential survival of the protoplasts because the air-dried solutions of di- and oligosaccharides were closer to the glassy state, and thus would have restricted molecular mobility more than the monosaccharide solutions at the lowest water contents achieved.
Rather than being protective, both pure raffinose and mannitol had damaging effects on protoplasts during drying (Fig. 3). These sugars frequently crystallized during drying, and when this occurred, all the protoplasts in those samples were completely destroyed. Crystallization of the sugars could damage the protoplasts in at least two ways. First, growing crystals could physically penetrate the protoplast plasma membranes and disrupt the protoplasts. In addition, crystallization has been shown to hinder the ability of sugars to affect membrane lipid phase behaviour and, thus, might render the sugars non-protective during desiccation (Caffrey et al., 1988; Koster et al., 1996).
The monosaccharides glucose and sorbitol were also unable to stabilize pea protoplasts during dehydration (Fig. 3). The accumulation of monosaccharides is associated with the loss of desiccation tolerance during seed germination (Koster and Leopold, 1988), and monosaccharides generally fail to protect membrane vesicles and liposomes from desiccation damage in vitro (Crowe et al., 1986). Thus, their inability to protect embryo-derived protoplasts during dehydration is consistent with data from prior studies using both whole tissues and isolated membranes. Glucose is a reducing sugar and, thus, can initiate non-enzymatic glycosylation of free amines (Baynes et al., 1989), particularly at intermediate water contents (Kaanane and Labuza, 1989). The non-enzymatic glycosylation of proteins has been suggested as a cause of seed deterioration during storage at 75% RH (Wettlaufer and Leopold, 1991; Sun and Leopold, 1995) and may have contributed to the inability of glucose to preserve protoplast integrity during dehydration. By contrast, the sugar-alcohol sorbitol lacks a carbonyl group and is not a participant in non-enzymatic glycosylation of amines (Baynes et al., 1989). Thus, the inability of sorbitol to protect these embryo-derived protoplasts during dehydration cannot be ascribed to non-enzymatic glycosylation by this sugar.
It is not known whether significant exchange between the intracellular sugars and the sugars of the suspension buffer occurred during isolation or drying of the protoplasts. At 12 h of imbibition, pea embryos contain large amounts of sucrose and stachyose (Koster and Leopold, 1988), so it was presumed that isolation in a buffer containing sucrose and raffinose would result in minimal loss of sucrose to the isolation buffer via diffusion. During isolation in sorbitol for the experiments on the effects of different sugars, it is possible that sucrose diffused out of the protoplasts and sorbitol may have diffused in. In this set of experiments, the sugars in the resuspension buffer (the sucrose/raffinose mix, pure sucrose, trehalose, raffinose, glucose, sorbitol, or mannitol) may also have been exchanged into the protoplasts during the air-drying treatment. This possibility does not significantly affect the conclusion that the sucrose/raffinose mixture and the disaccharides were more protective of protoplasts during drying. It simply widens the scope of where (intracellular versus extracellular) the sugars were exerting their protective effects.
Conservation of plasma membrane surface area is critical to survival of freeze-induced dehydration by protoplasts (Steponkus and Webb, 1992). Protoplasts from desiccation-tolerant pea embryos appeared to conserve the surface area of the plasma membrane during osmotic contraction by extensive folding around the cell components (Fig. 5B). This contrasts with freeze-tolerant mesophyll protoplasts that form tethered extrusions from the surface (Gordon-Kamm and Steponkus, 1984). Folding of the plasma membrane and cell walls has been observed in dry embryos (Webb and Arnott, 1982) and desiccation-tolerant ‘resurrection plants’ (Platt et al., 1994). This folding may represent the mechanism by which plasma membrane surface area is conserved by desiccation-tolerant plant cells. Folding of the cell wall with the plasma membrane is not always associated with enhanced tolerance to dehydration, however. The greater tolerance of protoplasts compared to intact tissues of Jerusalem artichoke to freeze-induced dehydration was attributed to a loosening of the mechanical tension exerted by the cell wall on the plasma membrane during dehydration (Murai and Yoshida, 1998).
By contrast to the intact appearance of most dried protoplasts from desiccation-tolerant embryos, dried protoplasts from desiccation-sensitive seedlings were completely disrupted (Fig. 6B), suggesting that damage occurred during the dehydration process and was not dependent upon the behaviour of the plasma membrane during rehydration. Further studies are in progress to understand better the behaviour of the membranes of both tolerant and sensitive protoplasts during dehydration and rehydration.
From these experiments, it is concluded that protoplasts isolated from desiccation-tolerant pea embryos retained some of the tolerance of the intact tissue; however, significant differences existed between the tolerance of the protoplasts and that of the intact embryos. Protoplasts were similar to intact embryos in that the presence of sucrose and raffinose correlated with maximal desiccation tolerance (Fig. 3). Plasma membrane behaviour was also similar, with folding of the plasma membrane during dehydration occurring in both protoplasts (Fig. 5B) and intact embryos (Webb and Arnott, 1982). Rapid deterioration of protoplasts stored at 0.28 g H2O g-1 DW (Fig. 4B) was similar to the deterioration of seeds that occurs during storage at intermediate water contents and probably reflects metabolic dysfunction (Leprince et al., 2000; Walters et al., 2001). The metabolic activity of the protoplasts may have caused the most pronounced difference between the protoplasts and the intact embryo, namely, the inability of the protoplasts to survive slow drying over 24 h. The protoplasts’ enhanced sensitivity to drying rate is similar to that of recalcitrant embryos (Berjak et al., 1990; Pammenter and Berjak, 1999; Walters et al., 2001). This response may reflect an inability of the protoplasts to shut down their metabolism during dehydration, as has been suggested for recalcitrant seeds (Leprince et al., 1999; Pammenter and Berjak, 1999; Walters et al., 2001).
Protoplasts isolated from pea embryonic axes provide a new system for the study of desiccation tolerance at the cellular level. Differences in response to dehydration between protoplasts and intact axes may provide an insight into the role of the cell wall in desiccation tolerance, while microscopic examination of the protoplasts may reveal details about the response of the plasma membrane to desiccation that could not be obtained from intact embryos or isolated membranes.
|
Acknowledgments |
|---|
We gratefully acknowledge the technical support of Lezlee Zeigler, Erica Schipper and Nichole Reisdorph. We also thank Dr Zoran Ristic and Lesley Barton for their assistance with electron microscopy, and we are indebted to Dr Christina Walters for her helpful comments on the manuscript. This material is based upon work supported by the National Science Foundation under Grant OSR-9452894 and by the South Dakota Future Fund.
|
Notes |
|---|
1 Present address: Structural Biology Department, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
2 To whom correspondence should be addressed. Fax: +1 605 677 6557. E-mail: kkoster{at}usd.edu
|
Abbreviations |
|---|
ANOVA, analysis of variance; DW, dry weight; FDA, fluorescein diacetate; MES, 2-(N-morpholino)ethane sulphonic acid; RH, relative humidity.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Baynes JW, Watkins NG, Fisher CI, Hull CJ, Patrick JS, Ahmed MU, Dunn JA, Thorpe SR. 1989. The Amadori product on protein: structure and reactions. In: Baynes JW, Monnier VM, eds. The Maillard reaction in aging, diabetes, and nutrition. New York: Alan R Liss, Inc., 43–68.
Berjak P, Farrant JM, Mycock DJ, Pammenter NW. 1990. Recalcitrant (homoiohydrous) seeds: the enigma of their desiccation-sensitivity. Seed Science and Technology18, 297–310.[Web of Science]
Blackman SA, Obendorf RL, Leopold AC. 1995. Desiccation tolerance in developing soybean seeds: the role of stress proteins. Physiologia Plantarum 93, 630–638.
Browse J, Somerville CR, Slack CR. 1988. Changes in lipid-composition during protoplast isolation. Plant Science 56, 15–20.[Web of Science]
Bruni F, Leopold AC. 1991. Glass transitions in soybean seed. Relevance to anhydrous biology. Plant Physiology 96, 660–663.
Bryant G, Koster KL, Wolfe J. 2001. Membrane behaviour in seeds and other systems at low water content: the various effects of solutes. Seed Science Research 11, 17–25.[Web of Science]
Buitink J, van den Vries IJ, Hoekstra FA, Alberda M, Hemminga MA. 2000. High critical temperature above Tg may contribute to the stability of biological systems. Biophysical Journal 79, 1119–1128.[Web of Science][Medline]
Burke MJ. 1986. The glassy state and survival of anhydrous biological systems. In: Leopold AC, ed. Membranes, metabolism, and dry organisms. Ithaca: Comstock Publishing Associates, 358–363.
Caffrey M, Fonseca V, Leopold AC. 1988. Lipid–sugar interactions. Relevance to anhydrous biology. Plant Physiology 86, 754–758.
Crowe JH, Crowe LM. 1988a. Factors affecting the stability of dry liposomes. Biochimica et Biophysica Acta 939, 327–334.[Medline]
Crowe JH, Crowe LM, Chapman D. 1984a. Preservation of membranes in anhydrobiotic organisms: the role of trehalose. Science 223, 701–703.
Crowe LM, Crowe JH. 1988b. Trehalose and dry dipalmitoylphosphatidylcholine revisited. Biochimica et Biophysica Acta 946, 193–201.[Medline]
Crowe LM, Mouradian R, Crowe JH, Jackson SA, Womersley C. 1984b. Effects of carbohydrates on membrane stability at low water activities. Biochimica et Biophysica Acta 769, 141–150.[Medline]
Crowe LM, Womersley C, Crowe JH, Appel L, Rudolph A. 1986. Prevention of fusion and leakage in freeze-dried liposomes by carbohydrates. Biochimica et Biophysica Acta 861, 131–140.
Gordon-Kamm WJ, Steponkus PL. 1984. The behaviour of the plasma membrane following osmotic contraction of isolated protoplasts: implications in freezing injury. Protoplasma 123, 83–94.[Web of Science]
Hoekstra FA, Van Roekel T. 1988. Desiccation tolerance of Papaver dubium L. pollen during its development in the anther. Possible role of phospholipid composition and sucrose content. Plant Physiology 88, 626–632.
Kaanane A, Labuza TP. 1989. The Maillard reaction in foods. In: Baynes JW, Monnier VM, eds. The Maillard reaction in aging, diabetes, and nutrition. New York: Alan R Liss, Inc., 301–327.
Koster KL. 1991. Glass formation and desiccation tolerance in seeds. Plant Physiology 96, 302–304.
Koster KL, Lei YP, Anderson M, Martin S, Bryant G. 2000. Effects of vitrified and non-vitrified sugars on phosphatidylcholine fluid-to-gel phase transitions. Biophysical Journal 78, 1932–1946.[Web of Science][Medline]
Koster KL, Leopold AC. 1988. Sugars and desiccation tolerance in seeds. Plant Physiology 88, 829–832.
Koster KL, Sommervold CL, Lei YP. 1996. The effect of storage temperature on interactions between dehydrated sugars and phosphatidylcholine. Journal of Thermal Analysis 47, 1581–1596.
Koster KL, Webb MS, Bryant G, Lynch DV. 1994. Interactions between soluble sugars and POPC (1-palmitoyl-2-oleoylphosphatidylcholine) during dehydration: vitrification of sugars alters the phase behaviour of the phospholipid. Biochimica et Biophysica Acta 1193, 143–150.[Medline]
Lalonde L, Bewley JD. 1985. Desiccation of imbibed and germinating pea axes causes a partial reversal of germination events. Canadian Journal of Botany 63, 2248–2253.
Leopold AC (ed.). 1986. Membranes, metabolism and dry organisms. Ithaca: Comstock Publishing Associates.
Leprince O, Buitink J, Hoekstra FA. 1999. Axes and cotyledons of recalcitrant seeds of Castanea sativa Mill. exhibit contrasting responses of respiration to drying in relation to desiccation sensitivity. Journal of Experimental Botany 50, 1515–1524.
Leprince O, Harren FJM, Buitink J, Alberda M, Hoekstra FA. 2000. Metabolic dysfunction and unabated respiration precede the loss of membrane integrity during dehydration of germinating radicles. Plant Physiology 122, 597–608.
Leprince O, Vertucci CW, Hendry GAF, Atherton NM. 1995. The expression of desiccation-induced damage in orthodox seeds is a function of oxygen and temperature. Physiologia Plantarum 94, 233–240.
Murai M, Yoshida S. 1998. Evidence for the cell wall involvement in temporal changes in freezing tolerance of Jerusalem artichoke (Helianthus tuberosus L.) tubers during cold acclimation. Plant Cell Physiology 39, 97–105.
Pammenter NW, Berjak P. 1999. A review of recalcitrant seed physiology in relation to desiccation-tolerance mechanisms. Seed Science Research 9, 13–37.
Platt KA, Oliver MJ, Thomson WW. 1994. Membranes and organelles of dehydrated Selaginella and Tortula retain their normal configuration and structural integrity. Protoplasma 175, 57–65.
Reisdorph NA, Koster KL. 1999. Progressive loss of desiccation tolerance in germinating pea (Pisum sativum) seeds. Physiologia Plantarum 105, 266–271.
Ristic Z, Ashworth EN. 1993. New infiltration method permits use of freeze-substitution for preparation of wood tissues for transmission electron microscopy. Journal of Microscopy 171, 137–142.
Rockland LB. 1960. Saturated salt solutions for the static control of relative humidity between 5 °C and 40 °C. Analytical Chemistry 32, 1375–1376.
Santarius KA. 1973. The protective effects of sugars on chloroplast membranes during temperature and water stress and its relationship to frost, desiccation and heat resistance. Planta 113, 105–114.[Web of Science]
Senaratna T, McKersie BD. 1983a. Dehydration injury in germinating soybean (Glycine max L. Merr.) seeds. Plant Physiology 72, 620–624.
Senaratna T, McKersie BD. 1983b. Characterization of solute efflux from dehydration injured soybean (Glycine max L. Merr.) seeds. Plant Physiology 72, 911–914.
Steponkus PL, Webb MS. 1992. Freeze-induced dehydration and membrane destabilization in plants. In: Somero GN, Osmond CB, Bolis CL, eds. Water and life: comparative analysis of water relationships at the organismic, cellular and molecular level. Berlin: Springer-Verlag, 338–362.
Sun WQ, Irving TC, Leopold AC. 1994. The role of sugar, vitrification and membrane phase transition in seed desiccation tolerance. Physiologia Plantarum 90, 621–628.
Sun WQ, Leopold AC. 1995. The Maillard reaction and oxidative stress during aging of soybean seeds. Physiologia Plantarum 94, 94–104.
Sun WQ, Leopold AC, Crowe LM, Crowe JH. 1996. Stability of dry liposomes in sugar glasses. Biophysical Journal 70, 1769–1776.[Web of Science][Medline]
Uemura M, Joseph RA, Steponkus PL. 1995. Cold acclimation of Arabidopsis thaliana. Effect on plasma membrane lipid composition and freeze-induced lesions. Plant Physiology 109, 15–30.[Abstract]
Walters C, Pammenter NW, Berjak P, Crane J. 2001. Desiccation damage, accelerated aging and respiration in desiccation tolerant and sensitive seeds. Seed Science Research (in press).
Webb MA, Arnott HJ. 1982. Cell wall conformation in dry seeds in relation to the preservation of structural integrity during desiccation. American Journal of Botany 69, 1657–1668.[Web of Science]
Webb MS, Uemura M, Steponkus PL. 1994. A comparison of freezing injury in oat and rye: two cereals at the extremes of freezing tolerance. Plant Physiology 104, 467–478.[Abstract]
Wettlaufer SH, Leopold AC. 1991. Relevance of Amadori and Maillard products to seed deterioration. Plant Physiology 97, 165–169.
Widholm JM. 1972. The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technology 47, 189–194.[Web of Science][Medline]
Wolfe J, Bryant G. 1999. Freezing, drying, and/or vitrification of membrane-solute-water systems. Cryobiology 39, 103–129.[Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Altamirano-Hernandez, M. G. Lopez, J. A. Acosta-Gallegos, R. Farias-Rodriguez, and J. J. Pena-Cabriales Influence of Soluble Sugars on Seed Quality in Nodulated Common Bean (Phaseolus vulgaris L.): The Case of Trehalose Crop Sci., May 31, 2007; 47(3): 1193 - 1205. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Halperin and K. L. Koster Sugar effects on membrane damage during desiccation of pea embryo protoplasts J. Exp. Bot., July 1, 2006; 57(10): 2303 - 2311. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Koster, N. Reisdorph, and J. L. Ramsay Changing desiccation tolerance of pea embryo protoplasts during germination J. Exp. Bot., June 1, 2003; 54(387): 1607 - 1614. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||