TY - JOUR
AU - Koster, Karen L.
AB - Abstract Desiccation tolerance of protoplasts isolated from germinating pea (Pisum sativum L. cv. ‘Alaska’) embryonic axes depends, in part, on the osmotic strength and composition of the suspending medium. To determine the reason for this dependence and whether treatment with different solutions results in different types of damage, protoplast recovery and survival were assessed after dehydration to a range of water contents. Protoplasts were derived from germinating axes that had intermediate desiccation tolerance. Protoplasts were isolated and resuspended in buffers containing sucrose/raffinose (85:15, w/w) or sorbitol, which were isotonic or hypertonic to the cells of the embryonic axis, then were flash-dried to a range of water contents. Protoplasts were rehydrated and stained with fluorescein diacetate (FDA) to assess survival and to estimate two types of membrane injury: lysis and the loss of semipermeability. In all treatments, protoplast survival dropped sharply during the initial phase of dehydration due to lysis. Protoplast survival was greater in hypertonic sucrose/raffinose buffer than in isotonic sucrose/raffinose buffer, or in the latter made hypertonic by the addition of sorbitol. When sorbitol was substituted for sucrose/raffinose in either the isolation or desiccation buffer, or both, protoplast survival at intermediate and low hydrations decreased due to a loss of membrane semipermeability. The results indicate that additional sucrose/raffinose is beneficial for the desiccation tolerance of protoplasts, the benefit is not due to a simple osmotic effect, and the benefit is greatest at water contents less than 0.5 g g−1 DW, where the presence of the sugars appears to protect membrane semipermeability. Desiccation tolerance, membrane injury, pea seeds Pisum sativum, protoplasts, sucrose Abbreviations Abbreviations DW dry weight FDA fluorescein diacetate HAI hours after the onset of imbibition MES 2-[N-morpholino] ethanesulphonic acid s.e.m. standard error of the mean Introduction Desiccation tolerance is fundamental to agricultural productivity because it enables the storage of orthodox seeds from one growing season to the next, as well as facilitating the long-term storage of genetic resources. Numerous factors contribute to seed desiccation tolerance, including the accumulation of sugars that stabilize membranes and other cellular structures during drying (Koster and Leopold, 1988; Bryant et al., 2001). Membranes are particularly vulnerable to the physical stresses that accompany dehydration and rehydration and are, therefore, considered a primary site of damage in desiccation sensitive cells (Steponkus, 1984; Crowe et al., 1992; Bryant et al., 2001). Protoplasts have been used to study membrane behaviour during freezing and freeze-induced dehydration (Gordon-Kamm and Steponkus, 1984; Uemura and Steponkus, 1989, 2003), and these studies have been extended to describe the desiccation tolerance of protoplasts isolated from pea embryonic axes (Xiao and Koster, 2001; Koster et al., 2003). As embryos lose desiccation-tolerance during germination, protoplasts derived from the embryos also lose tolerance. Protoplasts isolated from desiccation-tolerant axes at 12 HAI (hours after the onset of imbibition) generally survived desiccation, while protoplasts isolated near and after the completion of germination were significantly less tolerant (Koster et al., 2003). Desiccation tolerance of embryo protoplasts also depended on the sugar composition and osmotic strength of the suspending solution. Tolerance was maximal when protoplasts were isolated and dried in a mixture of sucrose and raffinose (Xiao and Koster, 2001), similar to the sugars found in many orthodox embryos (Amuti and Pollard, 1977; Koster and Leopold, 1988). A monosaccharide, glucose, and the sugar alcohols sorbitol and mannitol were less effective protectants (Xiao and Koster, 2001). The osmotic strength of the sugar solutions had its greatest effect on survival when protoplasts were isolated from axes at the completion of germination, i.e. at 24 HAI. For these protoplasts with intermediate tolerance, isolation and dehydration using hypertonic solutions led to improved survival compared with protoplasts isolated and dried with isotonic solutions (Koster et al., 2003). These results led to the speculation that the desiccation tolerance of the protoplasts may have been enhanced by sucrose uptake from the suspending solution, as sucrose has been correlated with desiccation tolerance (Hoekstra and van Roekel, 1988; Koster and Leopold, 1988; Buitink et al., 2003). Another possibility is that isolation of protoplasts in a hypertonic solution resulted in the preferential isolation of protoplasts with elevated intracellular osmotic strength, possibly derived from cells that were not as far advanced in the germination process and that were, thus, intrinsically more desiccation tolerant than cells possessing lower osmotic strength (Koster et al., 2003). The current study addresses the effects of solution composition and osmotic strength on membrane damage during dehydration of protoplasts derived from pea embryonic axes at 24 HAI. Freeze-induced dehydration is known to cause different types of membrane damage to leaf mesophyll cells; the type of damage depends on the extent of dehydration and the freezing tolerance of the tissue (Steponkus, 1984; Uemura et al., 1995). One type of injury, lysis, results when hydraulic pressure disrupts the plasma membrane, while a second type of injury, the loss of semipermeability [Referred to as the ‘loss of osmotic responsiveness (LOR)’ by Steponkus (1984) and colleagues.], results when membranes fuse and form non-bilayer phases at low water contents (Webb and Steponkus, 1993; Uemura et al., 1995). One of this study's goals was to determine whether similar types of membrane damage occur during the dehydration of seeds that are becoming sensitive to desiccation as they germinate, and thus, whether changes in membrane behaviour contribute to changes in desiccation tolerance. To estimate the incidence of these two types of membrane injury during progressive dehydration, both the number of intact protoplasts and their ability to retain fluorescein before and after drying in the presence of different osmotica were counted. The results indicate that the type and extent of membrane damage to embryo protoplasts is influenced by sugar composition and osmotic strength of the extracellular solution. Materials and methods Plant material Seeds of Pisum sativum L. cv. ‘Alaska’ (Main Street Seed and Supply Co., Bay City, MI, USA) were stored at 4 °C until use. Imbibition of the seeds took place within wetted rolls of germination paper (Anchor Paper Co., St Paul, MN, USA) in the dark at 28 °C for 24 h prior to protoplast isolation. At 24 HAI, the radicles had begun to protrude through the seed coat, signalling the completion of germination and the loss of desiccation tolerance. Only 33±8% of radicles survived when seeds were desiccated below 0.2 g g−1 DW at this time (data not shown). Solute potential and water potential measurements In order to determine the correct osmotic strengths for use in isotonic and hypertonic treatments of protoplasts, the solute potential of the radicles was estimated. Radicle sections were viewed through a microscope after 15 min incubation in sorbitol solutions having a range of osmotic strengths; the solute potential of the solution that produced incipient plasmolysis in at least 50% of the cells was taken as the average solute potential of the radicle cells. The water potential of germinating radicles was determined by placing five 2–3 mm long sections into a C-51 or C-52 vapour pressure osmometer chamber; these were allowed to equilibrate for 30 min prior to reading the thermocouple output using an HR-33 microvoltmeter (Wescor Inc., Logan, UT, USA). Protoplast isolation and desiccation Protoplasts were isolated as previously described (Koster et al., 2003) with minor modifications. At 24 HAI, 50 axes that had penetrated the seed coat (5–7 mm total radicle length) were detached, and the tip and epicotyls were discarded to ensure that protoplasts would be derived primarily from radicle cortex cells. Radicles were cut into 1–2 mm3 fragments, rinsed with deionized water, and transferred to 5 ml of an isolation solution containing 2% (w/v) Onozuka Cellulase RS, 0.1% (w/v) Pectolyase Y-23 (both from Karlan Research Products Corp., Santa Rosa, CA, USA), 0.2% (w/v) α-amylase, 5 mM CaCl2, 20 mM 2-[N-morpholino]ethanesulphonic acid (MES, pH 5.5) and the appropriate osmoticum, described below. After 4.5 h, the digested tissue was filtered through nylon mesh (62 μm pore diameter) and rinsed four times with 10 ml of an osmotically adjusted ‘desiccation’ solution. The filtrate was centrifuged at 250 g for 10 min at 22 °C. The pelleted protoplasts were resuspended in 1 ml of the desiccation solution. Osmotic treatments Isotonic and hypertonic sucrose/raffinose solutions (85:15, w/w) (Koster et al., 2000, 2003) were used to test the effect of osmotic strength on the incidence of different membrane injuries during dehydration. The isotonic solution (−1.25 MPa) contained 0.4 M sucrose/raffinose, and the hypertonic solution (−1.95 MPa) contained 0.6 M sucrose/raffinose. Each solution also contained 5 mM CaCl2 and 20 mM MES (pH 5.5). The effects of sugar composition on membrane injury were tested using hypertonic solutions and replacing the sucrose/raffinose mix with sorbitol (−1.98 MPa) during protoplast isolation and/or desiccation. To test whether improved tolerance after isolation in hypertonic sucrose/raffinose resulted from elevated sucrose in the medium, protoplasts were isolated and desiccated in a solution containing 0.4 M sucrose/raffinose, supplemented with sorbitol to achieve a solute potential of −1.95 MPa. Water potentials of all solutions were measured on a vapour pressure osmometer as described above. Protoplast viability, drying, and water contents Membrane integrity was used as an index of protoplast viability and was assessed using fluorescein diacetate (FDA) before and after drying, as previously described by Koster et al. (2003). Protoplasts were counted on a haemocytometer grid using a fluorescence microscope (Laborlux S, Leica, Bannockburn, IL, USA), with at least four grids counted at each sampling time. Only round protoplasts with a normal morphology were counted. Protoplasts that appeared intact, but did not retain the FDA, were also counted to measure non-viable protoplasts that had not lysed (referred to as ‘leaky’ protoplasts below). Protoplast diameters were calculated using haemocytometer gridlines as a scale and measuring images collected by a digital camera and displayed on a video monitor at ×200 magnification. The diameters of at least 58 protoplasts (live or leaky) were measured after resuspension in isotonic or hypertonic sucrose/raffinose, or hypertonic sorbitol. Protoplast suspensions were dehydrated under a stream of dry air flowing at 10 l min−1, as described by Koster et al. (2003). Samples were removed from the drying chamber at 30 min intervals over a period of 4.5 h, weighed to determine the amount of water lost, and rehydrated to their original volume using deionized water containing FDA (0.005%, w/v). The total numbers of intact fluorescent (live) and non-fluorescent (leaky) protoplasts were counted on a haemocytometer slide as before, and the percentage of protoplasts remaining in each category (live versus leaky) was calculated relative to the initial numbers. Relative changes in the numbers of living and leaky protoplasts can be used to infer the relative importance of lysis and loss of membrane semipermeability as types of damage affecting the population. During dehydration and rehydration, live protoplasts may have one of three fates. First, if their plasma membranes remain undamaged, protoplasts will retain fluorescein and be counted as ‘live’. Second, if lysis occurs, a decrease in the total number of protoplasts will be counted. Finally, if the plasma membrane loses semipermeability, then the protoplast will remain intact but not retain fluorescein; these protoplasts are classified as ‘leaky’. Leaky protoplasts were detected in all populations prior to desiccation and it was unknown whether they lysed or remained intact during desiccation. Because of this confounding variable, the absolute values of the incidence of lysis and loss of membrane semipermeability could not be determined. However, relative changes in the numbers of living and leaky protoplasts can be used to infer the relative importance of the two types of damage. Water contents of the dehydrated protoplast suspensions were calculated on a dry mass basis (g g−1 DW) by comparing the air-dried mass of the sample, prior to its rehydration for protoplast counting, to the mean oven-dried mass of a set of 4–5 replicate samples. The oven-dried mass was obtained by drying the replicate samples at 70 °C in vacuo with P2O5 for at least 16 h (Koster et al., 2003). Statistical analysis Changes in water content of the suspensions during desiccation were analysed by one-way ANOVA, with drying time as the fixed variable and water content as the dependent variable. Means of % survival and % leaky protoplasts within treatments were arc sin-square root transformed to correct for inequality of error variance (Snedecor and Cochran, 1980), and were compared by one-way ANOVA, using water content as the fixed variable and % survival (number of live protoplasts normalized to the initial total number of live protoplasts) or % leaky protoplasts (number of leaky protoplasts normalized to the initial total number of leaky protoplasts) as the dependent variable. In order to compare survival and leakiness across several treatments, arc sin-square root transformed means were compared via ANCOVA, with treatment as a fixed factor and water content as a covariate. Statistical analysis was performed using SPSS for Windows 11.5.0. Results Solute potential and water potential of embryos To determine the appropriate water potentials for protoplast isolation solutions, the water and solute potentials of germinating pea radicles were measured. Both quantities increased rapidly from 12 to 24 HAI; water potential remained constant after this time, although further slight increases in solute potential occurred through 48 HAI (Fig. 1). Based on these results, it was determined that the 0.4 M sucrose/raffinose solution (Ψ= −1.25 MPa) was isotonic to radicle cells at 24 HAI, and the 0.6 M sucrose/raffinose solution (Ψ= −1.95 MPa) was hypertonic. Fig. 1 View largeDownload slide Water potential and solute potential of germinating pea radicles. Water potential and solute potential differed significantly (F=21.25; P < 0.001), time (the covariate) had a highly significant effect (F=94.87; P <0.001), and there was a significant interaction between them (F=7.83; P <0.01). Each point represents the mean of n ≥5 radicles (solute potential) or n ≥5 groups of five radicles (water potential), error bars represent s.e.m. Fig. 1 View largeDownload slide Water potential and solute potential of germinating pea radicles. Water potential and solute potential differed significantly (F=21.25; P < 0.001), time (the covariate) had a highly significant effect (F=94.87; P <0.001), and there was a significant interaction between them (F=7.83; P <0.01). Each point represents the mean of n ≥5 radicles (solute potential) or n ≥5 groups of five radicles (water potential), error bars represent s.e.m. Protoplast size Live protoplasts isolated and resuspended in isotonic sucrose/raffinose were larger than those in hypertonic sorbitol solution, while protoplasts in the hypertonic sucrose/raffinose were intermediate in size (Table 1). Leaky protoplasts in the isotonic solution were the same size as live protoplasts in the same solution; however, leaky protoplasts in the hypertonic solutions tended to be larger than live protoplasts in the same solutions. Table 1 Protoplast sizes after isolation and resuspension in experimental osmotica Osmoticum  Live protoplasts   Leaky protoplasts     Radius (μm)  Calculated volume (μm3)  Radius (μm)  Calculated volume (μm3)  Isotonic sucrose/raffinose  6.77 (0.27) a  1300  6.39 (0.23) a  1090  Hypertonic sucrose/raffinose  6.21 (0.58) ab  1000  7.14 (0.25) b  1520  Hypertonic sorbitol  5.78 (0.20) b  806  6.36 (0.19) a  1070  Osmoticum  Live protoplasts   Leaky protoplasts     Radius (μm)  Calculated volume (μm3)  Radius (μm)  Calculated volume (μm3)  Isotonic sucrose/raffinose  6.77 (0.27) a  1300  6.39 (0.23) a  1090  Hypertonic sucrose/raffinose  6.21 (0.58) ab  1000  7.14 (0.25) b  1520  Hypertonic sorbitol  5.78 (0.20) b  806  6.36 (0.19) a  1070  For protoplast radii, n=40 haemocytometer grids counted, numbers in parentheses are standard errors of the mean (s.e.m). There was no significant difference between the radii of live and leaky protoplasts from the isotonic sucrose/raffinose treatment, as determined by an independent Student's t test, but leaky protoplasts were significantly larger than live protoplasts in both hypertonic sucrose/raffinose (P <0.01) and hypertonic sorbitol treatments (P <0.05). Osmoticum had a significant effect on radii of live protoplasts (F=5.07; P <0.01) and leaky protoplasts (F=3.86; P <0.05); values sharing the same letter within each column are not significantly different according to ANOVA analysis. Mean protoplast volumes were calculated from the means of the measured radii using the formula V=4/3πr3. View Large Water content during drying Water contents as a function of drying time for isotonic and hypertonic sucrose/raffinose solutions and for the hypertonic sorbitol solution are shown in Fig. 2. During the first 2 h, water content dropped rapidly in all three treatments, while only small decreases occurred during the final 2.5 h (Fig. 2, inset). Differences in drying rates during the first 0.5 h, when much of the damage to protoplasts occurred (described below, Figs 3, 4) were compared by ANCOVA. Drying rates of the isotonic sucrose/raffinose samples (3.48 g g−1 DW h−1) and the hypertonic sorbitol samples (3.56 g g−1 DW h−1) did not differ; during this interval, water contents of the hypertonic sorbitol samples were slightly, but significantly, greater than those of the isotonic sucrose/raffinose samples (F=12.81, P <0.01). Hypertonic sucrose/raffinose samples had lower water contents and dried more slowly (2.04 g g−1 DW h−1) during this interval than either the hypertonic sorbitol (F=43.37, P <0.001) or the isotonic sucrose/raffinose samples (F=23.21, P <0.001). After 4–4.5 h of drying, hypertonic sucrose/raffinose samples had water contents that were slightly, but significantly, greater (0.22 g g−1 DW) than those of isotonic sucrose/raffinose (0.17 g g−1 DW; F=21.66, P <0.001) or hypertonic sorbitol solutions (0.15 g g−1 DW; F=27.45, P <0.001). Water contents of the latter two treatments did not differ after 4–4.5 h of drying. Fig. 2 View largeDownload slide Water content of protoplast samples suspended in isotonic sucrose/raffinose, hypertonic sucrose/raffinose, or hypertonic sorbitol solutions during drying. Lines are drawn as an aid to the eye. The inset graph more clearly shows the water contents between 2–4.5 h of drying. Each point represents the mean of n ≥4 samples, error bars represent s.e.m. Fig. 2 View largeDownload slide Water content of protoplast samples suspended in isotonic sucrose/raffinose, hypertonic sucrose/raffinose, or hypertonic sorbitol solutions during drying. Lines are drawn as an aid to the eye. The inset graph more clearly shows the water contents between 2–4.5 h of drying. Each point represents the mean of n ≥4 samples, error bars represent s.e.m. Fig. 3 View largeDownload slide Survival and leakiness of protoplasts after isolation and desiccation in solutions containing: (A) isotonic sucrose/raffinose, (B) hypertonic sucrose/raffinose, and (C) hypertonic sucrose/raffinose/sorbitol. Both survival (open bars) and leakiness (filled bars) were normalized to the initial number of viable or leaky protoplasts, respectively. Water content variation within each group was 7.7% of the mean, on average. Bars marked by the same letter in the same font were not significantly different, according to Fischer's LSD test (P >0.05). Error bars represent s.e.m. For isotonic sucrose/raffinose (A), water content had a significant effect on survival (F=12.68; P <0.001) and leakiness (F=12.68; P <0.001); n ≥12 haemocytometer grids counted; n for WC=0.22 was 36, and n for WC=0.15 was 24. For hypertonic sucrose/raffinose (B), water content had a significant effect on survival (F=5.83; P <0.001) and no effect on leakiness; n ≥12 haemocytometer grids counted, n for WC=0.25 was 48. For hypertonic sucrose/raffinose/sorbitol (C), water content had a significant effect on survival (F=21.84; P <0.001) and leakiness (F=4.48; P <0.01); n ≥8 haemocytometer grids counted, n for WC=0.21 was 16. Fig. 3 View largeDownload slide Survival and leakiness of protoplasts after isolation and desiccation in solutions containing: (A) isotonic sucrose/raffinose, (B) hypertonic sucrose/raffinose, and (C) hypertonic sucrose/raffinose/sorbitol. Both survival (open bars) and leakiness (filled bars) were normalized to the initial number of viable or leaky protoplasts, respectively. Water content variation within each group was 7.7% of the mean, on average. Bars marked by the same letter in the same font were not significantly different, according to Fischer's LSD test (P >0.05). Error bars represent s.e.m. For isotonic sucrose/raffinose (A), water content had a significant effect on survival (F=12.68; P <0.001) and leakiness (F=12.68; P <0.001); n ≥12 haemocytometer grids counted; n for WC=0.22 was 36, and n for WC=0.15 was 24. For hypertonic sucrose/raffinose (B), water content had a significant effect on survival (F=5.83; P <0.001) and no effect on leakiness; n ≥12 haemocytometer grids counted, n for WC=0.25 was 48. For hypertonic sucrose/raffinose/sorbitol (C), water content had a significant effect on survival (F=21.84; P <0.001) and leakiness (F=4.48; P <0.01); n ≥8 haemocytometer grids counted, n for WC=0.21 was 16. Fig. 4 View largeDownload slide Survival and leakiness of protoplasts after isolation and desiccation in hypertonic solutions. (A) Isolation in sorbitol and desiccation in sucrose/raffinose solution; (B) isolation in sucrose/raffinose and desiccation in sorbitol solution; and (C) isolation and desiccation in hypertonic sorbitol solution. Both survival (open bars) and leakiness (filled bars) were normalized to the initial number of viable or leaky protoplasts, respectively. Water content variation within each group was 7.7% of the mean, on average. Bars marked by the same letter in the same font were not significantly different, according to Fischer's LSD test (P >0.05). Error bars represent s.e.m. For (A), water content had a significant effect on survival (F=15.23; P <0.001) and no effect on leakiness; n ≥8 haemocytometer grids counted, n for WC=0.22 was 32. For (B), water content had a significant effect on survival (F=40.22; P <0.001) and leakiness (F=40.22; P <0.001); n ≥8 haemocytometer grids counted, n for WC=0.18 was 32. For (C), water content had a significant effect on survival (F=37.15; P <0.001) and leakiness (F=37.15; P <0.001); n ≥8 haemocytometer grids counted, n for WC=0.16 was 32. Fig. 4 View largeDownload slide Survival and leakiness of protoplasts after isolation and desiccation in hypertonic solutions. (A) Isolation in sorbitol and desiccation in sucrose/raffinose solution; (B) isolation in sucrose/raffinose and desiccation in sorbitol solution; and (C) isolation and desiccation in hypertonic sorbitol solution. Both survival (open bars) and leakiness (filled bars) were normalized to the initial number of viable or leaky protoplasts, respectively. Water content variation within each group was 7.7% of the mean, on average. Bars marked by the same letter in the same font were not significantly different, according to Fischer's LSD test (P >0.05). Error bars represent s.e.m. For (A), water content had a significant effect on survival (F=15.23; P <0.001) and no effect on leakiness; n ≥8 haemocytometer grids counted, n for WC=0.22 was 32. For (B), water content had a significant effect on survival (F=40.22; P <0.001) and leakiness (F=40.22; P <0.001); n ≥8 haemocytometer grids counted, n for WC=0.18 was 32. For (C), water content had a significant effect on survival (F=37.15; P <0.001) and leakiness (F=37.15; P <0.001); n ≥8 haemocytometer grids counted, n for WC=0.16 was 32. Because the water content of the protoplast samples did not change greatly after 2.5 h of drying, samples obtained after 2.5 h were grouped for analysis of damage according to water content values shown on the x-axes (Figs 3, 4). Thus, n values for the drier samples were much greater than for wetter samples (n values indicated in figure legends, Figs 3, 4). Recovery and survival after rehydration, isotonic versus hypertonic sucrose/raffinose Survival of protoplasts isolated and desiccated in isotonic sucrose/raffinose solution dropped to 53% after a moderate dehydration from 5.47 to 3.61 g g−1 DW (Fig. 3A, open bars). Survival did not diminish during further drying to 1.24 g g−1 DW, but decreased to 37% at lower water contents. The initial drop in survival was accompanied by a decrease in the number of leaky protoplasts (Fig. 3A, filled bars), indicating that more protoplasts were damaged by lysis than by loss of semipermeability as a result of moderate dehydration. At lower water contents, the numbers of leaky protoplasts did not change significantly. Protoplasts isolated in hypertonic sucrose/raffinose solution also had an initial drop in viability to 60% as the water content fell from 3.55 to 2.44 g g−1 DW during the first 30 min of drying (Fig. 3B, open bars). There was no further decrease in survival at lower water contents. Leaky protoplasts did not change in number during dehydration, suggesting that the initial loss of protoplast viability was accompanied by the lysis of an equal proportion of protoplasts. Survival of protoplasts in the hypertonic sucrose/raffinose solution was greater (>50%, Fig. 3B) at water contents <1.0 g g−1 DW than it was in the isotonic sucrose/raffinose solution (Fig. 3A). The improved desiccation tolerance of protoplasts isolated and dried in hypertonic sucrose/raffinose may have resulted from the preferential isolation of protoplasts with lower solute potentials, which might have been closer to the embryonic state (Fig. 1). Alternatively, protoplasts isolated in hypertonic sucrose/raffinose may have taken up more sucrose from the concentrated external solution during isolation, resulting in elevated internal sucrose levels and increased desiccation tolerance, as suggested previously (Koster et al., 2003). To discriminate between these possibilities, protoplasts were isolated and desiccated in a hypertonic solution that contained the same concentrations of sucrose and raffinose as the isotonic sucrose/raffinose solution, but that was amended with sorbitol to achieve the same solute potential as the hypertonic sucrose/raffinose solution. Protoplast survival decreased to 66% during the initial drying from 4.42 to 3.19 g g−1 DW (Fig. 3C, open bars), with a further steady decline to 23% at 0.21 g g−1 DW. The drop in the percentage of leaky protoplasts indicates that more protoplasts were damaged by lysis than by loss of semipermeability during dehydration. Comparison of the three treatments using ANCOVA with water content as the covariate showed that the increased amount of sucrose/raffinose in the hypertonic treatment (Fig. 3B) significantly improved desiccation tolerance (F=10.88, P <0.001); there was no difference between the two treatments having the same amount of sucrose/raffinose (Fig. 3A, C; P=0.291). Increased sucrose/raffinose also eliminated changes in the percentage of leaky protoplasts (Fig. 3B), suggesting that it reduced the incidence of lysis relative to the other treatments. Recovery and survival after rehydration, hypertonic treatments in which sucrose/raffinose was replaced by sorbitol To determine if sucrose/raffinose has the most beneficial effect during protoplast isolation or desiccation, sucrose/raffinose was replaced with sorbitol in either the isolation or desiccation solution, or both. When sorbitol replaced sucrose/raffinose in the isolation solution, survival dropped to 54% during the initial drying from 3.77 to 2.53 g g−1 DW, and there was a second decrease to 35% when water contents dropped below 1.03 g g−1 DW (Fig. 4A, open bars). The percentage of leaky protoplasts increased at lower hydrations, suggesting that the loss of semipermeability was a major form of injury to these protoplasts (Fig. 4A, filled bars). A similar trend in survival was noted when sucrose/raffinose was present during isolation, but was replaced by sorbitol in the desiccation solution (Fig. 4B). Protoplast survival decreased to 58% during the initial dehydration from 6.22 to 4.37 g g−1 DW, then remained constant until protoplasts were dried to the lowest water contents. After desiccation to 0.18 g g−1 DW, only 13% of protoplasts survived (Fig. 4B, open bars). There was an initial slight but significant reduction in the percentage of leaky protoplasts, indicating that more protoplasts were damaged by lysis than by loss of semipermeability during the initial phase of drying. When sucrose/raffinose was absent from both the isolation and desiccation solutions (Fig. 4C), survival decreased to 47% during the initial dehydration from 5.88 to 3.93 g g−1 DW, and there was a further decline to 7% as water contents fell to 0.16 g g−1 DW. The percentage of leaky protoplasts did not significantly change until protoplasts were dried to 0.31 g g−1 DW, where there was a large increase (Fig. 4C, filled bars), indicating that more protoplasts were damaged by loss of semipermeability than by lysis at these lowest hydrations. When results from all hypertonic treatments were analysed, protoplast survival differed significantly among treatments (P <0.001); improved survival in treatments with sucrose/raffinose indicates that desiccation tolerance was enhanced by sucrose/raffinose availability. The presence of sucrose/raffinose in the desiccation buffer particularly enhanced survival of drying to the lowest hydration levels (Figs 3B, 4A). Discussion In this study, the effects of solution composition and osmotic strength on the desiccation tolerance of protoplasts isolated from germinating pea embryonic axes were examined. Pea embryos at 24 HAI are completing germination and are classified as having intermediate tolerance (Reisdorph and Koster, 1999). During this phase, the sucrose concentration in the embryo is decreasing, while the concentration of monosaccharides is increasing (Koster and Leopold, 1988). The presence of sucrose is considered an important factor contributing to desiccation tolerance in seeds (Koster and Leopold, 1988; Black et al., 1996; Buitink et al., 2003) and other plant tissues (Bianchi et al., 1991; Ghasempour et al., 1998); thus it is possible that supplemental sucrose could enhance desiccation tolerance in tissues with intermediate tolerance. Results shown in Fig. 3 confirm this. Survival was improved when radicle protoplasts were isolated and dried in hypertonic compared with isotonic sucrose/raffinose solutions (Fig. 3A, B). However, when hypertonic solutions were used having the same amount of sucrose and raffinose as isotonic sucrose/raffinose solutions, made hypertonic by added sorbitol, protoplast desiccation tolerance did not differ from that of protoplasts in the isotonic sucrose/raffinose solutions (Fig. 3A, C). Thus, the improved desiccation tolerance conferred by the hypertonic sucrose/raffinose solution resulted from the increase in extracellular sucrose and/or raffinose, and possibly, sugar uptake into the protoplasts. The improvement in desiccation tolerance resulting from sucrose and raffinose was further confirmed by data shown in Fig. 4. In these experiments, sorbitol replaced sucrose/raffinose as the osmoticum in either the isolation solution, the desiccation solution, or both. In all cases, the use of sorbitol as an osmoticum led to a decrease in protoplast desiccation tolerance, particularly at lower water contents. As previously hypothesized, the improved desiccation tolerance in sucrose/raffinose solutions may have resulted from sucrose uptake by the protoplasts. Pisum sativum seeds possess both active and passive sucrose transporters (Tegeder et al., 1999), although their abundance and localization in germinating embryos is unknown to us. Protoplasts in our experiments, suspended in sucrose solutions with concentrations well into the range of linear uptake (Lichtner and Spanswick, 1981), should have taken up sucrose by passive diffusion. Moreover, it is likely that protoplasts in hypertonic sucrose/raffinose solutions took up more sucrose than those in isotonic sucrose/raffinose because of the enhanced sucrose gradient in the former. Increased intracellular sucrose levels could, thus, account for the improved survival of protoplasts in the hypertonic sucrose/raffinose treatment. A report of a raffinose transporter in pea embryos has not been found, so the role of raffinose remains unknown in this system. The intermediate size of live protoplasts isolated in hypertonic sucrose/raffinose solution further suggests that these protoplasts took up sucrose from the external solution. Live protoplasts isolated in hypertonic sorbitol had approximately 62% of the volume of protoplasts isolated in isotonic sucrose/raffinose (Table 1). This value corresponds to the ratio of the solution water potentials (−1.25 versus −1.98 MPa) and suggests that the protoplasts behaved as perfect osmometers during isolation. The intermediate size of the protoplasts isolated in hypertonic sucrose/raffinose solutions may have resulted from uptake of sucrose by the protoplasts, resulting in the osmotic uptake of water. The leaky protoplasts in each treatment displayed less osmotic responsiveness, although those in the hypertonic sucrose/raffinose were larger than leaky protoplasts in the other solutions (Table 1). The ability of sucrose and some other sugars to stabilize dried membranes previously has been noted (Crowe et al., 1992; Koster et al., 2000); however, desiccation tolerance requires not only that membranes be stabilized when dry, but also that they remain intact during the processes of dehydration and rehydration. Types of membrane injury resulting from dehydration vary with the extent of water loss. Membrane injury resulting from freeze-induced dehydration has been described by Steponkus and colleagues for mesophyll cells of several species (Steponkus and Lynch, 1989; Webb et al., 1994; Uemura et al., 1995). In those cells, lysis occurred primarily during thawing and the consequent rehydration and expansion of non-cold-acclimated protoplasts that had been mildly dehydrated by freezing to temperatures between −2 °C and −6 °C. The loss of membrane semipermeability occurred below −6 °C, when dehydration was more extensive and membranes were brought close together as cells contracted. The close approach of membranes can cause phase changes and fusions that result in the inability of the membrane to function as a permeability barrier (Wolfe and Bryant, 1999). In this study of embryo protoplasts, two forms of membrane injury, lysis and the loss of semipermeability, can also be distinguished. In these experiments, lysis accounts for the initial drop in protoplast survival noted in each treatment (Figs 3, 4). During the first 30 min of drying, protoplast samples sustained the greatest rate of water loss (Fig. 2), although the water contents remained relatively high (>2.5 g g−1 DW). The rapid efflux of water during the initial stages of drying may have caused membrane rupture, as observed by Xiao and Koster (2001) in protoplasts isolated from 1-week-old pea seedlings. The extent of lysis during the early stages of drying was more pronounced in the isotonic sucrose/raffinose solution (Fig. 3A) than in the hypertonic sucrose/raffinose solution (Fig. 3B), which had a slower drying rate (Fig. 2). Protoplasts isolated from earlier stages of germination (12–18 HAI), when the embryos were more tolerant of desiccation, did not display this initial drop in survival (Koster et al., 2003). Thus, it is possible that, during germination, there is a decrease in membrane hydraulic conductivity, which would result in increased resistance to water efflux. The initial drop in protoplast survival in response to mild dehydration was observed in all treatments; however, protoplast survival at lower water contents varied considerably among the treatments. In the isotonic sucrose/raffinose treatment, there was a small drop in survival at water contents less than 1 g g−1 DW (Fig. 3A), while there was no further decrease in survival in the hypertonic sucrose/raffinose treatment (Fig. 3B). Protoplasts that were dried in sorbitol had the poorest survival of drying to water contents <0.5 g g−1 DW (Fig. 4B, C). This drop in survival at low hydrations was accompanied by increased numbers of leaky protoplasts, suggesting that loss of semipermeability was an important type of membrane injury. The loss of semipermeability was not as important a form of injury in protoplasts dried in the presence of sucrose/raffinose, supporting the concept that sucrose plays an important role in membrane protection at low hydrations (Koster et al., 2000; Bryant et al., 2001). The loss of membrane semipermeability during protoplast desiccation is thought to result from the damaging effect of hydration forces that become large when hydrophilic surfaces, such as membranes, are brought into close apposition (Wolfe and Bryant, 1999; Bryant et al., 2001). The presence of soluble sugars between membranes helps limit the close approach of their surfaces during dehydration and, thus, helps stabilize the membranes during desiccation (Koster et al., 2000; Bryant et al., 2001). Given the likelihood that sucrose was taken up by protoplasts and thus could exert protective effects on the membranes at low hydrations, the results presented here (Fig. 3B versus Fig. 4) are in accord with this concept. Uemura and Steponkus (2003) reported similar protection against freeze-induced membrane injury after incubation of Arabidopsis thaliana leaves in sucrose. Incubation of leaves in 10–35 mM sucrose reduced expansion-induced lysis, and incubation in 30–400 mM sucrose reduced the loss of semipermeability in protoplasts isolated from the leaves. Incubation in sorbitol had no effect on lysis, but preserved some membrane semipermeability when protoplasts were frozen to −5 °C (Uemura and Steponkus, 2003). In related studies using the Arabidopsis mutant sfr4 that is deficient in sucrose synthesis during cold acclimation, Uemura et al. (2003) found extensive loss of membrane semipermeability in frozen–thawed protoplasts from cold-acclimated leaves. However, incubation of the leaves in sucrose solution led to a decrease in this type of damage. In both these studies, the authors conclude that exposure to elevated sucrose helps prevent the loss of semipermeability during freezing, possibly due to the sugar's ability to limit the close apposition of membranes during freeze-induced dehydration (Uemura and Steponkus, 2003; Uemura et al., 2003). In addition to its role as an osmotic and volumetric spacer between cellular membranes, sucrose is also a primary metabolic substrate, and thus may be metabolized by the protoplasts. Uemura and Steponkus (2003) speculated that some of the protective effects of sucrose against freeze-induced membrane damage resulted from the cells' use of sucrose to provide carbon and energy for the modification of membrane lipids. This is more likely to have occurred extensively during the several days of sucrose incubation used by those authors than in the few h of incubation during protoplast isolation and drying in our study. Nevertheless, the fate of any internalized sucrose should be investigated to assess the contribution of sucrose metabolism to the protection measured in these and other studies. In this study, it was found that the additional sucrose in the hypertonic compared to the isotonic sucrose/raffinose treatment resulted in a reduction of lysis (Fig. 3A, B), an effect that was not achieved by the addition of sorbitol to the isotonic sucrose/raffinose solution (Fig. 3C). As noted above, the decreased lysis may have resulted from the slower rate of water loss from protoplasts in the hypertonic sucrose/raffinose solution (Fig. 2). When sorbitol replaced sucrose/raffinose in the isolation solution, loss of semipermeability increased relative to lysis (Fig. 4A). When sucrose/raffinose was present in the isolation solution but not in the desiccation solution, damage from loss of semipermeability was lessened (Fig. 4B). Since protoplasts were more likely to have taken up sucrose during the isolation phase, protoplasts isolated in sucrose/raffinose may have contained more sucrose than those in treatments in which sucrose was absent from the isolation phase; the treatments in which sucrose/raffinose was present in the isolation solution suffered the least from loss of semipermeability. From these experiments, it is concluded that desiccation tolerance of pea embryo protoplasts is enhanced by isolation and drying in a hypertonic sucrose/raffinose solution. Protection cannot be replicated by the use of sorbitol, thus it is not a simple osmotic effect. Protection of protoplast membranes during drying may result from sucrose uptake into the protoplasts, especially during isolation. Protection against the loss of membrane semipermeability is greater when sucrose is in the isolation solution, rather than the desiccation solution; supplemental sucrose in either solution reduces the incidence of lysis. These results suggest that sucrose uptake by protoplasts can decrease the incidence of membrane injury during drying. Elevated levels of intracellular sucrose can hinder the close approach of membranes and other cellular structures during dehydration and thus could minimize damaging hydration forces at low water contents. Future studies may determine whether the observed membrane protection results entirely from increased levels of intracellular sucrose or also from products of sucrose metabolism. We thank Joni Wipf for technical assistance, Dr Karen Gaines and Dr Jim Novak for statistical advice, and Dr Matsuo Uemura for helpful comments on the manuscript. This project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2003-35100-13364 to KLK. 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TI - Sugar effects on membrane damage during desiccation of pea embryo protoplasts
JF - Journal of Experimental Botany
DO - 10.1093/jxb/erj208
DA - 2006-06-23
UR - https://www.deepdyve.com/lp/oxford-university-press/sugar-effects-on-membrane-damage-during-desiccation-of-pea-embryo-8NN0dsM6Yo
SP - 2303
EP - 2311
VL - 57
IS - 10
DP - DeepDyve
ER -