JXB Advance Access originally published online on April 28, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Journal of Experimental Botany, Vol. 54, No. 387, pp. 1607-1614,
June 1, 2003
© 2003 Oxford University Press
Changing desiccation tolerance of pea embryo protoplasts during germination
Received 7 October 2002; Accepted 14 March 2003
Department of Biology, The University of South Dakota, 414 E. Clark Street, Vermillion, SD 57069, USA
1 To whom correspondence should be addressed. Fax: +1 605 677 6557. E-mail: kkoster{at}usd.edu
2 Present address: Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA.
3 Present address: Department of Biology, Northern State University, Aberdeen, SD 57401, USA.
4 This value was originally called the CMC, the critical moisture content, by Reisdorph and Koster (1999). However, the term ‘critical water content’ is often used to describe the water content at which damage first becomes apparent (the ‘threshold water content’ of Reisdorph and Koster). WC50 is a less ambiguous expression for quantifying the 50% damage point.
5 For convenience, sampling times are designated as hours from the onset of imbibition. Germination was completed by 24 h of imbibition, and radicles at 36 h of imbibition were in a post-germinative stage.
6 Arcsin transformation converts 50% to 45%; therefore, 45% survival was used in the transformed data for calculation of critical moisture contents.
7 WC50s were determined on a dry matter basis (g g–1 DW) rather than by equilibration to defined water potentials because the slower drying rates involved in equilibrium drying led to death of the entire population of protoplasts (Xiao and Koster, 2001). Water potentials corresponding to these water contents were extrapolated as described in Table 1.
Abbreviations: ANCOVA, analysis of covariance; DW, dry weight; FDA, fluorescein diacetate; MES, 2-(N-morpholino)ethanesulphonic acid; WC50, water content at which 50% of a population is killed.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Protoplasts were isolated from pea (Pisum sativum L. cv. Alaska) embryonic axes during and after germination to determine whether the loss of desiccation tolerance in the embryos also occurs in the protoplasts. At all times studied, protoplast survival decreased as water content decreased; however, the sensitivity to dehydration was less when the protoplasts were isolated from embryos that were still desiccation-tolerant (12 h and 18 h of imbibition) than when protoplasts were derived from axes that were sensitive (24 h and 36 h of imbibition). The water content at which 50% of the population was killed (WC50) increased throughout germination and early seedling growth for both the intact tissue and the protoplasts derived from them. Prior to radicle emergence, protoplasts were less desiccation-tolerant than the intact axes; however, protoplasts isolated from radicles shortly after emergence had lower WC50s than the intact radicles. A comparison of protoplast survival after isolation and dehydration in either 500 mM sucrose/raffinose or 700 mM sucrose revealed no difference in tolerance except at 24 h of imbibition, when protoplasts treated in the more concentrated solution had improved tolerance of dehydration. Although intact epicotyls are generally more desiccation-tolerant than radicles, protoplasts isolated separately from epicotyls and radicles did not differ in tolerance. Collectively, these data suggest that protoplasts gradually lose desiccation tolerance during germination, as do the orthodox embryos from which they were derived. However, even prior to radicle emergence, protoplasts display a sensitivity to progressive dehydration that is similar to that shown by recalcitrant and ageing embryos.
Key words: Desiccation tolerance, embryo, germination, pea, Pisum sativum L., protoplast isolation.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Desiccation tolerance of orthodox seeds depends upon modifications of cellular biochemistry and ultrastructure that allow the embryo to withstand the physical and metabolic stresses that accompany dehydration. For example, the presence of di- and oligosaccharides, late embryogenesis abundant (LEA) proteins, and antioxidants have been correlated with desiccation tolerance in embryos (Senaratna et al., 1985; Koster and Leopold, 1988; Leprince et al., 1990; Blackman et al., 1992; Vertucci and Farrant, 1995; Pammenter and Berjak, 1999), and it is thought that these compounds can stabilize cellular structures during drying. While these and other factors correlate with desiccation tolerance, the physical and chemical mechanisms by which they enable cells to survive are still uncertain. Experiments done on model membranes and proteins suggest that some of the compounds associated with tolerance can indeed stabilize dried systems in vitro (Crowe et al., 1984; Koster et al., 2000); however, the mere presence of the protectant is not sufficient to confer tolerance (see review by Pammenter and Berjak, 1999). For example, cell anatomy also plays a role in the desiccation tolerance of the tissue. Cells in desiccation-tolerant embryos are generally smaller in size, with accumulated dry matter filling much of the volume, and internal membranes are less abundant (Farrant and Walters, 1998).
An additional factor involved in desiccation tolerance is the dynamic behaviour of cell structures during dehydration. In particular, the behaviour of plasma membranes is important as cells shrink and re-expand during a cycle of desiccation and rehydration (Gordon-Kamm and Steponkus, 1984). For mesophyll cells exposed to freeze-induced dehydration, this is one factor that contributes to freezing tolerance, and studies using leaf protoplasts have defined a progression of damages that accrue as the temperature drops and freeze-induced dehydration becomes more extensive (Uemura et al., 1995). During cold acclimation, cells develop the means to prevent the types of damage that occur in non-acclimated tissues, but they remain vulnerable to lesions that occur at lower temperatures.
Although these and other studies (Murai and Yoshida, 1998) using isolated protoplasts have revealed much about the mechanisms of freezing damage and tolerance, protoplasts have only recently been used in studies of desiccation tolerance (Xiao and Koster, 2001). Protoplasts isolated from desiccation-tolerant pea (Pisum sativum L.) embryos were dried and rehydrated with approximately 75% survival, while protoplasts isolated from 1-week-old seedlings did not survive similar desiccation (Xiao and Koster, 2001). Thus, the tolerance of the protoplasts in some respects resembled that of the tissue from which they were obtained. However, tolerance differed in that it depended strongly on the drying rate; slow drying of the protoplasts led to their complete destruction, while the intact embryonic axes survived slow drying. Examination of the ultrastructure of the dried protoplasts using transmission electron microscopy revealed that the plasma membranes of desiccation-tolerant protoplasts were intact and folded around the cell contents, while the membranes of sensitive protoplasts from seedlings had ruptured during dehydration (Xiao and Koster, 2001). Therefore, the behaviour of the plasma membrane during drying was deemed crucial to the survival of the protoplasts from desiccation-tolerant embryos.
As orthodox pea seeds germinate, the tissues of the embryo become increasingly sensitive to dehydration (Reisdorph and Koster, 1999). During this transition period, the composition of the cells changes, both in chemistry and ultrastructure. Di- and oligosaccharides are replaced by monosaccharides (Koster and Leopold, 1988), while dehydrins and other desiccation-related proteins disappear (Baker et al., 1995). Stored dry matter is broken down and replaced by water-filled vacuoles, cell membranes proliferate, and the cells expand as germination progresses (Ramsay, 2002). The loss of desiccation tolerance for pea embryos during germination has been quantified as the progressive increase in the WC504, the water content at which 50% of the embryos were killed during dehydration (Reisdorph and Koster, 1999). It is thought that the gradual loss of tolerance reflects the sequential loss of protective mechanisms during germination (Vertucci and Farrant, 1995; Reisdorph and Koster, 1999; Sun, 1999). To investigate these protective mechanisms, however, it is necessary to define the various mechanisms of damage to cells that differ in their desiccation tolerance. The use of protoplasts that exhibit a range of tolerances will facilitate such investigations, particularly studies of membrane behaviour during dehydration. The goal of this study was to examine the desiccation tolerance of pea embryo protoplasts further and to determine whether protoplasts isolated from germinating embryos become progressively more sensitive to dehydration, as do the intact embryos.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Plant materials and protoplast isolation
Pea seeds (Pisum sativum L. cv. Alaska) purchased from Gurney’s Seed and Nursery Co. (Yankton, SD, USA) were stored at 4 °C until use. Seeds were hydrated in wet germination paper rolls (Anchor Paper Co., St Paul, MN, USA) in the dark at 28 °C for periods ranging from 12 h to 36 h. Under these conditions, radicles begin to elongate between 18 h and 24 h, and most have emerged from the seed coat by 24 h. The embryos become increasingly sensitive to dehydration after 18 h and are killed by desiccation at 36 h of imbibition5 (Reisdorph and Koster, 1999). For each lot of seeds used, the time-course for germination and the loss of tolerance were confirmed to be the same as reported by Reisdorph and Koster (1999).
Protoplasts were isolated using the procedure described by Xiao and Koster (2001) with some modifications. At 18 h of imbibition, all axes were used; however, at 24 h and 36 h, axes that had not yet emerged from the seed coat were discarded, to improve the uniformity of the population. Embryonic axes were chopped into small pieces (1–2 mm3), rinsed using deionized water, and transferred to an osmotically adjusted digestion solution containing 2% (w/v) Onozuka Cellulase RS and 0.1% (w/v) Pectolyase Y-23 (Karlan, Santa Rosa, CA) in 5 mM CaCl2 and 20 mM MES (2-[N-morpholino]ethanesulphonic acid) (pH 5.5). After 3–5 h, the digested tissue was filtered using a nylon mesh with 62 µm diameter pores, and the liberated protoplasts were sedimented at 250 g for 10 min at room temperature. The protoplast pellet was resuspended using 1–3 ml of an osmotically adjusted buffer solution containing 5 mM CaCl2 and 20 mM MES (pH 5.5).
The osmotic strength and composition of the digestion and resuspension buffers were compared in two sets of experiments. In one set, designated OS 700, the osmotic strength of the digestion solution was adjusted with 700 mM sorbitol, and 700 mM sucrose was used for resuspension. In a second set (OS 500), a 500 mM mixture of sucrose and raffinose (85:15, w/w) that is representative of the sugar composition of desiccation-tolerant maize embryos (Koster and Leopold, 1988; Koster, 1991) was used throughout.
Pea epicotyls and radicles lose desiccation tolerance at different rates during germination, with differences in tolerance becoming measurable at 18 h, as radicles begin to elongate (Reisdorph and Koster, 1999); therefore, one set of experiments compared the desiccation tolerance of protoplasts isolated from these tissues during germination and radicle elongation. Axes from seeds at 18, 24, and 36 h of imbibition were separated into epicotyls and radicles before protoplast isolation and resuspension in the OS 700 solutions. Viability and desiccation tolerance of the protoplasts were assessed as described below.
Protoplast viability, drying, and water contents
Viability of isolated protoplasts was assessed using FDA (fluorescein diacetate, 0.005%, w/v) before and after drying treatments (Widholm, 1972). The accumulation of FDA relies on the integrity of the plasma membrane, and thus is comparable to measures of leakage from cells. Protoplasts were counted on a haemocytometer grid using a fluorescence microscope (Leica, Laborlux S) and were considered viable if they fluoresced brightly, were round, and exhibited normal morphology (Fig. 1).
|
To evaluate the desiccation tolerance of the protoplasts, preweighed 100 µl aliquots of the resuspended protoplasts were dehydrated in a chamber containing silica gel with a stream of dry air flowing over the samples at approximately 10 l min–1 (Xiao and Koster, 2001). Samples were removed periodically over 6 h to obtain protoplasts that had a range of water contents. The partially dehydrated samples were weighed to determine how much water had been lost and were then rehydrated to their original volume with deionized water containing FDA (0.005%, w/v). After 15 min, the rehydrated protoplasts were counted to assess viability, as described above.
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 4–8 separate grids, and each experiment was then replicated at least twice. Because the range of water contents was achieved by a non-equilibrium drying protocol, exact replication of water contents could not be achieved from one experiment to the next; therefore, each point on a graph represents the mean and standard error determined from a single experimental trial.
Water contents of the dehydrated protoplast suspensions were estimated using a set of three replicate samples that were weighed (‘air-dried weight’) and replaced in the drying chamber each time samples were removed for the determination of survival. At the end of the entire air-drying period, the three samples were dried at 70 °C in vacuo with P2O5 for at least 16 h to obtain their oven-dried weight. Water contents for each sampling time were then calculated (g g–1 DW) using the following formula:
[(air-dried weight – oven-dried weight)/oven-dried weight]
Statistical analysis
Protoplast survival percentages were calculated by comparing mean numbers of viable protoplasts before and after dehydration. To correct for possible distribution abnormalities associated with data that are expressed as percentages, the percentage survival values were arcsin transformed to provide data that were nearly normally distributed (Zar, 1996). For each imbibition time, transformed protoplast survival data as a function of water content were fit using linear regression, and the resulting equations were used to calculate WC50, the water content at which 50%6 of the protoplasts were killed by dehydration. ANCOVA was used to analyse differences in the survival of protoplasts isolated from different tissues (radicle versus epicotyl), using different osmotic strengths (OS 500 versus OS 700), and from different imbibition times.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Protoplasts isolated from pea embryonic axes prior to the completion of germination are rounded and lack a large central vacuole (Fig. 1A). Instead, the contents of the protoplasts largely comprise accumulated dry matter, including protein bodies and starch (Xiao and Koster, 2001), giving them a dense appearance. The density of the protoplasts makes it difficult to separate them from cellular debris during isolation. Nonetheless, viable protoplasts can be distinguished and counted when treated using the fluorescent stain FDA and viewed under UV light (Fig. 1B).
The desiccation tolerance of protoplasts isolated at different times during and shortly after germination is shown in Figs 2 (OS 700) and 3 (OS 500). For all times tested, protoplast survival decreased with decreasing water content (P <0.0001). Linear regression gave reasonably good fits to the arcsin transformed data from most times and was, therefore, used to facilitate comparison among the data sets (not shown).
|
|
Figure 2 shows the survival of protoplasts isolated, resuspended and dried in buffer containing 700 mM sugars (OS 700). Comparisons of the survival of protoplasts isolated separately from epicotyl versus radicle tissues in OS 700 solutions found no significant difference between protoplasts from these two tissues at 18 h, 24 h, or 36 h of imbibition (P >0.05) (Fig. 2). As a result of this finding, survival data from radicle and epicotyl protoplasts were pooled for ANCOVA analysis among imbibition times for the protoplasts in OS 700 treatments. The regression lines shown in Fig. 2 were derived from the pooled, arcsin transformed data and were back-transformed for ease of viewing. These regression lines were used to compare the desiccation tolerance of the protoplasts from different imbibition times using ANCOVA (Fig. 2). According to this analysis, protoplasts isolated from embryonic axes at 18 h and 24 h of imbibition had similar desiccation tolerance; neither the slopes nor the intercepts of lines from the arcsin-transformed data were significantly different (P >0.05) (Fig. 2A, B). The survival of protoplasts from axes at 36 h (Fig. 2C) was, however, significantly different from the others (P <0.0001) and suggests that protoplasts at 36 h were less desiccation-tolerant than those isolated at earlier imbibition times in the OS 700 solutions.
Survival data for protoplasts isolated from radicles at 12, 18, 24, and 36 h of imbibition and dried in 500 mM sucrose/raffinose solution (OS 500) are presented in Fig. 3. Although epicotyl and radicle protoplasts in the OS 700 treatments did not differ in their survival of dehydration (Fig. 2), only radicle tissues were used in the OS 500 experiments. Comparison of survival lines among the imbibition times revealed that protoplasts from axes at 12 h and 18 h did not differ significantly from one another in their desiccation tolerance (P >0.05) (Fig. 3A, B), but they were significantly more tolerant than protoplasts from axes at 24 h and 36 h of imbibition (P <0.05) (Fig. 3C, D). Protoplasts isolated from axes at 24 h and 36 h were similar to one another in their tolerance (P >0.05) (Fig. 3C, D). This is in contrast to the analysis of the protoplasts isolated and dried in the 700 mM sugar solutions, for which protoplasts from axes at 24 h and 36 h of imbibition differed in tolerance (Fig. 2B, C).
The difference between protoplasts from the OS 500 and OS 700 treatments is also seen when the WC50s are compared (Table 1). Protoplasts from radicles at 12 h and 18 h of imbibition had WC50 
0.25 g g–1 DW, indicating that more than 50% of the protoplasts survive desiccation to this low water content, regardless of the osmotic strength of the suspending buffer. Protoplasts from radicles at 36 h of imbibition were desiccation sensitive, with WC50s of about 1.1 to 1.2 g g–1 DW, again regardless of the sugar concentration in the buffer. However, protoplasts from axes at 24 h of imbibition were significantly more tolerant of desiccation (P <0.001) when they were in the OS 700 treatment (WC50=0.35 g g–1 DW) than they were in the OS 500 treatment (WC50=1.08 g g–1 DW).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
During germination, pea seeds become more sensitive to desiccation, as indicated by increasing WC50 values for growth and leakage (Reisdorph and Koster, 1999). The protoplasts studied here also displayed increasing sensitivity to desiccation as germination progressed and radicles elongated. Protoplasts from desiccation-tolerant axes at 12 h and 18 h of imbibition had relatively high survival rates, even when dried to water contents less than 0.2 g g–1 DW (Figs 2, 3). However, protoplasts from axes that were losing tolerance (24 h) were less tolerant of drying, as indicated by their increasing WC50s (Table 1). Few protoplasts isolated from sensitive axes at 36 h survived drying to water contents less than 0.2 g g–1 DW (Figs 2, 3). Thus, in this aspect, the desiccation tolerance of the protoplasts is similar to that of the tissue from which it was isolated, as previously reported by Xiao and Koster (2001).
Although the desiccation tolerance of axis-derived protoplasts resembles that of the embryos in some aspects, the previous report suggested that there were important differences between the tolerance of protoplasts and that of the tissues from which they were isolated (Xiao and Koster, 2001). The most obvious distinction noted was a dependence on the drying rate, such that protoplasts only survived water contents less than 0.2 g g–1 DW if dried quickly (<2 h), while intact axes readily survived slower drying rates (
24 h). This dependence on drying rate is linked to a dependence on water content (Figs 2, 3). Survival of protoplasts from all imbibition times decreased as their water content dropped. However, this effect was less severe for protoplasts from axes at 12 h and 18 h of imbibition than it was for protoplasts from later imbibition times, which were damaged at higher water contents, i.e. earlier in the drying process (Figs 2, 3). By contrast, survival of intact pea axes did not depend on their water content until 18 h of germination, when some axes were beginning to lose desiccation tolerance (Reisdorph and Koster, 1999). In the intact tissues, the hydration dependence for damage was only observed below certain threshold water contents, suggesting that drying to higher water contents did not significantly damage the tissues. The threshold water contents for damage increased as germination progressed and desiccation tolerance was lost (Reisdorph and Koster, 1999). Thus, the extent of dehydration influences the survival of protoplasts and of intact tissues that are losing desiccation tolerance. Embryos of desiccation sensitive recalcitrant species also display a non-linear dependence of survival on water content. Recalcitrant embryos often survive with little damage until they are dried below a critical water content (or water potential), below which survival declines precipitously (Dussert et al., 1999; Liang and Sun, 2000; Sun and Liang, 2001; Walters et al., 2001). These critical water contents are characteristic for different species and cultivars and are used to compare the relative desiccation tolerance of these embryos.
A further distinction between desiccation tolerance of protoplasts and the tissue from which they were isolated is suggested by the data shown in Fig. 2. Although within intact axes, the radicle tissue loses desiccation tolerance prior to the epicotyl (Koster and Leopold, 1988; Reisdorph and Koster, 1999), protoplasts isolated separately from these two tissues displayed no significant differences in their survival after dehydration (Fig. 2). For example, at 24 h of imbibition, most intact radicles are quite desiccation-sensitive and are killed by water contents less than 1.3 g g–1 DW, while greater than 80% of epicotyls survive water contents of 0.2 g g–1 DW (Reisdorph and Koster, 1999). By contrast, most protoplasts isolated at 24 h in the OS 700 buffer survived desiccation to about 0.35 g g–1 DW, regardless of whether they were derived from radicle or epicotyl tissue.
A broader comparison of the WC50s for intact tissues and protoplasts (Table 1) also shows differences in their desiccation tolerance. Early in germination (18 h), both tissues and protoplasts have WC50s between 0 and 0.25 g g–1 DW (Table 1) (Reisdorph and Koster, 1999). Although protoplasts isolated at these early stages of germination have low calculated WC50 values, their survival at water contents less than 0.2 g g–1 DW was less than that of the intact tissue. At 24 h of imbibition, the WC50s began to increase for protoplasts and for intact radicles, while epicotyls remained mostly tolerant. At 36 h, intact radicles were quite sensitive to drying, with a WC50 of 2.6 g g–1 DW (approximately –0.8 MPa)7 but their protoplasts were somewhat more tolerant, with WC50s of 1.09 and 1.19 g g–1 DW (approximately –6 MPa) for the OS 500 and OS 700 treatments, respectively. The values for protoplasts isolated from radicles at 36 h are more equivalent to the WC50, estimated on a water potential basis, for the relatively tolerant epicotyls at 36 h of imbibition (Table 1).
Why might protoplasts isolated from elongated radicles at 36 h be more tolerant of desiccation than the tissues from which they were isolated? One possibility is that the treatment of the radicle protoplasts—suspension in concentrated sucrose or sucrose/raffinose solutions and rapid drying—helped them to survive desiccation to lower water contents than they would have survived within the tissue. In a prior study, protoplasts dried in sucrose or sucrose/raffinose solutions had a greater survival of desiccation than protoplasts dried in monosaccharide solutions (Xiao and Koster, 2001). The presence of di- and oligosaccharides in desiccation-tolerant cells has been documented (Crowe et al., 1984; Hoekstra and Van Roekel, 1988; Koster and Leopold, 1988), although the mechanism or mechanisms by which these sugars may protect cellular structures is still debated (Bryant et al., 2001). Possible effects of sugars on the desiccation tolerance of embryo protoplasts are discussed more fully by Xiao and Koster (2001).
The differences between the buffers used in these experiments did not significantly affect the desiccation tolerance of the protoplasts, except at 24 h of imbibition, when protoplasts isolated and dried in the OS 700 buffers were significantly more tolerant than those in the OS 500 buffers (Table 1). At 24 h, the osmotic potential of most pea radicle cells is about –1.3 MPa (data not shown), which is about isotonic to the solutions used in the OS 500 treatment, but hypotonic to the OS 700 solutions. One possibility to explain the differential survival at this time is that somewhat different populations of protoplasts were isolated in each solution. At 24 h of imbibition, the radicles are elongating, so cell size and content are becoming more variable. It is conceivable that use of the 700 mM sugars would lead to the preferential isolation of protoplasts with higher internal osmotic strengths, and that this population of protoplasts would lyse when isolated in 500 mM sugars due to the osmotic influx of water. The protoplasts with the more concentrated internal solutes may be derived from cells that were closer to the embryonic state than were the protoplasts isolated in the 500 mM sugars. In this scenario, the improved survival of the OS 700 protoplasts at low water contents reflects their developmental state—they were derived from cells that had not yet lost tolerance.
A second possibility is that suspension of the OS 700 protoplasts in a hypertonic sucrose solution created a gradient for the transport of sucrose into the protoplasts. At 24 h of imbibition, the endogenous intracellular sucrose content has decreased and has largely been replaced by monosaccharides (Koster and Leopold, 1988). Thus, suspending the protoplasts in either a sucrose or a sucrose/raffinose solution at slightly acidic pH, as in these experiments, might facilitate the loading of the protoplasts with the di- and oligosaccharides that are typically associated with desiccation tolerance. This is especially likely given the abundance of proton–sucrose symports in plants and, more specifically, in seeds (Bush, 1994; Patrick and Offler, 2001). In this scenario, the stronger gradient created by the 700 mM sugars, compared with the 500 mM sugars, would allow more sucrose to move into the protoplasts during isolation and suspension, thus raising the intracellular sugar content. This might then confer increased dehydration resistance to the protoplasts. Similar incubation in sucrose solutions has resulted in elevated sugar contents and improved freezing tolerance in eucalyptus suspension cells (Leborgne et al., 1995; Travert et al., 1997). A related study found that increasing the sucrose levels in Arabidopsis leaves by incubating intact seedlings in sucrose solutions significantly improved the freezing tolerance of mesophyll protoplasts derived from the seedlings (Uemura and Steponkus, 1998). In the latter study, sucrose was thought to act as both a metabolic substrate and a compatible solute to protect internal structures during freeze-induced dehydration. The extent to which sugars move across the plasma membranes of embryo-derived protoplasts during isolation, suspension, and drying is not known and merits further investigation.
In addition to the importance of the suspending sugar solution on protoplast desiccation tolerance, the rapid drying rate (1.5–2.5 g g–1 DW h–1 during the first 2 h) probably contributed to the improved survival of radicle protoplasts compared with the intact radicles at later stages of germination (Table 1). Flash drying, as this non-equilibrium drying technique is sometimes termed, is known to improve the survival of desiccation-sensitive embryos (Pammenter and Berjak, 1999) and is thought to help the cells move quickly through intermediate water contents at which damage from uncontrolled metabolism is most severe (Leprince et al., 1999; Walters et al., 2001). Xiao and Koster (2001) speculated that the sensitivity of embryo protoplasts to the drying rate results from an inability to down-regulate metabolism as they dry. The co-ordinated slowing of metabolic processes during dehydration is thought to be crucial for complete desiccation tolerance (Leprince et al., 1995, 1999, 2000), and it is possible that, unlike intact tolerant embryos, protoplasts cannot slow their metabolism as drying occurs and, as such, may exhibit some of the characteristics of recalcitrant embryos.
|
Conclusions |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
These experiments demonstrate that the desiccation tolerance of protoplasts isolated from pea embryonic axes during the course of germination and radicle elongation decreases together with that of the tissue from which they were isolated. Furthermore, at all stages tested, protoplast survival depended strongly on the water content, such that viability was gradually lost as drying progressed. This hydration dependence for damage is similar to that observed in embryos that are losing desiccation tolerance during germination (Reisdorph and Koster, 1999). It is also reminiscent of the desiccation sensitivity of some recalcitrant species, although the hydration dependence of the intact embryos is usually only observed below certain critical water potentials (Walters, 1999; Sun and Liang, 2001).
Despite the similarity of protoplast desiccation tolerance to that of the intact tissue, there are also important differences, in particular, the strong dependence of protoplast survival on a rapid drying rate. The drying rate dependence suggests that metabolic damage contributes to protoplast desiccation sensitivity. In this manner, the protoplasts are similar to recalcitrant species and to tolerant species undergoing ageing (Walters et al., 2001). It further suggests that the process of isolating protoplasts alters their ability to regulate their metabolism, particularly during dehydration, and may imply that interactions between the plasma membrane and the cell wall are needed for full desiccation tolerance. Future studies of protoplast ultrastructure, membrane behaviour, and metabolism during dehydration will reveal additional details about mechanisms of desiccation damage and tolerance in seed embryos.
|
Acknowledgements |
|---|
We thank Dr Karen Olmstead for guidance on statistical analysis and Dr Matsuo Uemura for helpful discussions and comments on the manuscript. Portions of this research were supported by the National Science Foundation under Grant OSR-9452894 and by the South Dakota Future Fund. JLR was the recipient of a Grant-in-Aid from Sigma Xi and a graduate student research grant from NSF-EPSCoR at The University of South Dakota.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Baker EH, Bradford KJ, Bryant JA, Rost TL. 1995. A comparison of desiccation-related proteins (dehydrin and QP47) in peas (Pisum sativum). Seed Science Research 5, 185–193.
Blackman SA, Obendorf RL, Leopold AC. 1992. Maturation proteins and sugars in desiccation tolerance of developing soybean seeds. Plant Physiology 100, 225–230.
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]
Bush DR. 1994. Proton-coupled sugar and amino acid transporters in plants. Annual Review of Plant Physiology and Plant Molecular Biology 44, 513–542.[Web of Science]
Crowe JH, Crowe LM, Chapman D. 1984. Preservation of membranes in anhydrobiotic organisms: the role of trehalose. Science 223, 701–703.
Dussert S, Chabrillange N, Engelmann F, Hamon S. 1999. Quantitative estimation of seed desiccation sensitivity using a quantal response model: application to nine species of the genus Coffea L. Seed Science Research 9, 135–144.
Farrant JM, Walters C. 1998. Ultrastructural and biophysical changes in developing embryos of Aesculus hippocastanum in relation to the acquisition of tolerance to drying. Physiologia Plantarum 104, 513–524.[CrossRef]
Gordon-Kamm WJ, Steponkus PL. 1984. The influence of cold acclimation on the behavior of the plasma membrane following osmotic contraction of isolated protoplasts. Protoplasma 123, 161–173.[CrossRef][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.
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, 828–832.
Leborgne N, Teulieres C, Travert S, Rols MP, Teissie J, Boudet AM. 1995. Introduction of specific carbohydrates into Eucalyptus gunnii cells increases their freezing tolerance. European Journal of Biochemistry 229, 710–717.[Web of Science][Medline]
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, Deltour R, Thorpe PC, Atherton NM, Hendry GAF. 1990. The role of free radicals and radical processing systems in loss of desiccation tolerance in germinating maize (Zea mays L.). New Phytologist 116, 573–580.[CrossRef][Web of Science]
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.[CrossRef]
Liang Y, Sun WQ. 2000. Desiccation tolerance of recalcitrant Theobroma cacao embryonic axes: the optimal drying rate and its physiological basis. Journal of Experimental Botany 51, 1911–1919.
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 and 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.
Patrick JW, Offler CE. 2001. Compartmentation of transport and transfer events in developing seeds. Journal of Experimental Botany 52, 551–564.
Ramsay JL. 2002. Ultrastructure of pea embryonic axes (Pisum sativum L. cv. Alaska) and the associated loss of desiccation tolerance during germination. PhD dissertation, The University of South Dakota.
Reisdorph N, Koster KL. 1999. Progressive loss of desiccation tolerance in germinating pea (Pisum sativum) seeds. Physiologia Plantarum 105, 266–271.
Senaratna T, McKersie BD, Stinson RH. 1985. Antioxidant levels in germinating soybean seed axes in relation to free radical and dehydration tolerance. Plant Physiology 78, 168–171.
Sun WQ. 1999. Desiccation sensitivity of recalcitrant seeds and germinated orthodox seeds: can germinated orthodox seeds serve as a model system for studies of recalcitrance? In: Marzalina M, Khoo KC, Jayanthi N, Tsan FY, Krishnapillay B, eds. Recalcitrant seeds. Proceedings of IUFRO Seed Symposium 1998. Kuala Lumpur: Forest Research Institute Malaysia, 29–42.
Sun WQ, Liang Y. 2001. Discrete levels of desiccation sensitivity in various seeds as determined by the equilibrium dehydration method. Seed Science Research 11, 317–323.[Web of Science]
Travert S, Valerio L, Fouraste I, Boudet AM, Teulieres C. 1997. Enrichment in soluble sugars of two eucalyptus cell-suspension cultures by various treatments enhances their frost tolerance via a non-colligative mechanism. Plant Physiology 114, 1433–1442.[Abstract]
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]
Uemura M, Steponkus PL. 1998. Effect of artificial manipulation of intracellular sugar content on the incidence of freeze-induced membrane lesions of protoplasts isolated from Arabidopsis thaliana leaves. Plant Physiology 117, S-109.
Vertucci CW, Farrant JM. 1995. Acquisition and loss of desiccation tolerance. In: Kigel J, Galili G, eds. Seed development and germination. Marcel Dekker, New York, 237–271.
Walters C. 1999. Levels of recalcitrance in seeds. In: Marzalina M, Khoo KC, Jayanthi N, Tsan FY, Krishnapillay B, eds. Recalcitrant seeds. Proceedings of IUFRO Seed Symposium 1998. Kuala Lumpur: Forest Research Institute Malaysia, 1–13.
Walters C, Pammenter NW, Berjak P, Crane J. 2001. Desiccation damage, accelerated ageing and respiration in desiccation-tolerant and -sensitive seeds. Seed Science Research 11, 135–148.[Web of Science]
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]
Xiao L, Koster KL. 2001.Desiccation tolerance of protoplasts isolated from pea embryos. Journal of Experimental Botany 52, 2105–2114.
Zar JH. 1996. Biostatistical analysis, 3rd edn. New Jersey: Prentice-Hall.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  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]
|
|
|
|
|
|
J. M. R. Faria, J. Buitink, A. A. M. van Lammeren, and H. W. M. Hilhorst Changes in DNA and microtubules during loss and re-establishment of desiccation tolerance in germinating Medicago truncatula seeds J. Exp. Bot., August 1, 2005; 56(418): 2119 - 2130. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||