Max-Planck-Institut für Molekulare Pflanzenphysiologie, D-14424 Potsdam, Germany
Received on July 17, 2001; accepted on August 31, 2001.
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Abstract |
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Key words: desiccation/fructans/glucans/liposomes/oligosaccharides
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Introduction |
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Because membranes are the primary targets of both freezing and desiccation injury in cells (Steponkus, 1984; Crowe et al., 1992
; Oliver et al., 2001
), a role of fructans in cellular stress tolerance should involve the stabilization of membranes under stress conditions. Recent reports indicate that a high molecular mass (degree of polymerization [DP] > 25,000) bacterial levan is able to directly interact with membranes (Demel et al., 1998
; Vereyken et al., 2001
). In addition, a cyclic bacterial fructan (cycloinulohexaose) protected liposomes during freezing and freeze-drying (Ozaki and Hayashi, 1996
), and inulin preparations from chicory roots and dahlia tubers stabilized liposomes during freeze-drying (Hincha et al., 2000
).
This latter finding is particularly relevant because the chicory and dahlia inulin preparations of polysaccharides of a DP between 10 and 30 were protective (Hincha et al., 2000), whereas the other investigated polysaccharides, hydroxyethyl starch and dextran, were completely ineffective in stabilizing membranes during freeze-drying (Crowe et al., 1994
; Hincha et al., 2000
). The available evidence suggests that this ineffectiveness is due to the inability of hydroxyethyl starch and dextran to interact with the membrane lipids in the dry state and depress the gel to liquid-crystalline lipid phase transition temperature (Tm) of the dry membranes (Crowe et al., 1996
, 1997; Hincha et al., 2000
; Tsvetkova et al., 1998
). This has been attributed to the large size of the polymers, which would sterically prevent them from interacting with membrane lipids.
It has, however, been shown that even oligosaccharides of different chain length vary significantly in their ability to stabilize membranes. Malto-oligosaccharides up to DP 3 (maltotriose) are good membrane protectants during freezing and drying, and homologous saccharides of DP 4 and above are much less effective (Miyajima et al., 1986; Suzuki et al., 1996
; Nagase et al., 1997
). On the other hand, because the chicory inulins showed clear evidence of hydrogen bonding to phosphatidylcholine headgroups in dry membranes (Hincha et al., 2000
), the question arose of whether fructans might have fundamentally different properties than glucans that would enable them to interact much more effectively with membranes.
To answer this question, we have compared fructans and glucans of equal chain lengths for their ability to stabilize phosphatidylcholine liposomes during air-drying. We found that with increasing DP fructans reduced leakage of a soluble marker better than glucans, but that glucans afforded better protection against membrane fusion. Using Fourier transform infrared (FTIR) spectroscopy we were able to show that the ability of glucans to hydrogen bond to the head groups of dry lipids decreased dramatically with increasing DP, whereas chain length hardly affected the ability of fructans to interact.
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Results |
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Discussion |
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These differences were closely related to the differences observed in the ability of the sugars to form hydrogen bonds with the phosphate in EPC headgroups (Figure 9). While both disaccharides induced a downfield shift in the phosphate peak by at least 15 wave numbers, the difference between control samples containing only EPC and those containing glucans became progressively less with increasing DP, until the peaks almost coincided. The difference between the peak of control samples and of samples containing the DP 5 fructan, however, was still 13 wave numbers. In addition, all spectra from samples containing fructans showed a clear shoulder, centered around 1220 cm1, indicating very strong hydrogen bonding between the fructans and at least a subpopulation of the EPC head groups (Crowe et al., 1996).
It is interesting to note that the inability of polymeric glucans to directly interact with membrane lipids has been previously attributed to the large size of these molecules (Crowe et al., 1996, 1997; Tsvetkova et al., 1998
; Hincha et al., 2000
). We have presented evidence here that size-related effects of steric hindrance can not fully account for the observed effects of oligo- or polymeric sugars, as we have directly compared glucans and inulins of the same DP. Likewise, it has recently been shown (Vereyken et al., 2001
) that a bacterial fructan of DP > 25,000 was able to interact with fully hydrated lipid model membranes, but a smaller dextran was not. These findings indicate that much more specific (although currently not fully understood) properties of the different sugars than just size determine their ability to interact with membranes.
These differences in the ability of the different sugars to interact with membrane lipids and depress Tm were also reflected in their ability to stabilize liposomes during drying. The differences in CF leakage (Figures 2, 4, 6), however, were not as clear-cut as those observed by FTIR spectroscopy, because CF leakage is influenced not only by lipid phase transitions but also by vesicle fusion. In the concentration range used, the effects on fusion were much stronger than the effects on leakage (Figure 2). This is in agreement with previous findings (Crowe et al., 1985), that much higher concentrations of the disaccharide trehalose were necessary to completely prevent leakage than were necessary to prevent fusion.
For the glucans, we found an increased ability to prevent fusion with increasing DP (Figures 4 and 5). In parallel, the ability to prevent leakage decreased. As a decrease in fusion should normally translate into a decrease in leakage, this could only mean that leakage due to lipid phase transitions increased very strongly with DP and thus surpassed the decrease in leakage associated with increased protection against fusion. This is in good agreement with the FTIR data, as discussed above. For the fructans, the reverse argument is applicable, as their ability to prevent leakage increased with increasing DP, although their ability to prevent fusion decreased.
The reduced ability of the inulins to prevent fusion with increasing DP is a surprising observation. In general, protection against fusion by sugars has been related to their ability to form glasses (vitrify) during drying (Crowe et al., 1998). Because the propensity of oligomeric substances to vitrify has in many cases been found to increase with increasing DP (Levine and Slade, 1988
; Slade and Levine, 1991
), we would have expected increased protection against fusion from both fructans and glucans with increasing chain length. However, only the glucans, at least up to DP 5, fulfilled this expectation and for the glucans an increase in glass transition temperature (Tg) with increasing DP has indeed been reported before (Orford et al., 1990
; Slade and Levine, 1991
). For the inulins, no conclusive data are available from the literature, as only commercial mixtures have been investigated and no information about composition and purity has been supplied (Schaller-Povolny et al., 2000
; Hinrichs et al., 2001
). With these preparations, however, the expected increase in Tg with increasing average DP was observed. It is therefore not clear at this point why the pure inulins (Figure 1) used in our experiments showed reduced protection against fusion with increasing DP.
Another unexpected finding was that the chicory inulin that had provided protection against leakage during freeze-drying (Hincha et al., 2000) was completely ineffective during air-drying (Figures 2, 3, 6). The most likely reason is that the chicory inulin precipitated from solution during the slow air-drying process and thus was not available for membrane stabilization or glass formation. Visual inspection of the dried samples showed a powdery appearance, whereas all other sugars gave a clear, transparent film. We assume that in the freeze-drying process the chicory inulin was immobilized in the vicinity of the membranes during freezing in liquid nitrogen and therefore precipitation was prevented and protection became possible.
The fructan concentrations we have used in our fusion and leakage experiments may be expected to be present in plant cells, at least for example after cold acclimation (Livingston and Henson, 1998). Oligomeric glucans, on the other hand, have only been found in total concentrations between 1 and 2 mg ml1 in plant cells (van de Wal et al., 1998
). Glucans could interfere with starch synthesis and probably other metabolic functions in plants and are therefore not expected to be accumulated to higher concentrations. In general, it may be expected that oligomeric or polymeric sugars that are not directly involved either in photosynthetic carbon fixation or glycolysis could be accumulated in plant cells to higher concentrations under stress than other sugars, without negative effects on primary metabolism and growth. We therefore conclude from our results that fructans may have unique properties that would make them ideal solutes to stabilize plant cells under stress. We are currently testing this hypothesis using transgenic plants that accumulate fructans in their leaves.
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Materials and methods |
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Sugar analysis
Malto- and fructo-oligosaccharides were analyzed by HPLC using a CarboPac PA-100 anion exchange column on the Dionex DX-300 gradient chromatography system (Dionex, Sunnyvale, CA) coupled with pulsed amperometric detection by a gold electrode. The column was equilibrated in 0.15 M NaOH and was eluted with a linear gradient of 1 M NaAc in 0.15 M NaOH as described in detail in previous publications (Hellwege et al., 1997, 1998).
Preparation of liposomes
EPC was dried from chloroform under a stream of N2 and stored under vacuum overnight to remove traces of solvent. Liposomes were prepared from hydrated lipids using a hand-held extruder with two layers of polycarbonate membranes with 100 nm pores (MacDonald et al., 1991; Avestin, Ottawa, Canada).
Leakage experiments
For leakage experiments, an appropriate amount of lipid was hydrated in 0.25 ml of 100 mM CF, 10 mM TES, 0.1 mM ethylenediamine tetra-acetic acid (pH 7.4). After extrusion, the vesicles were passed through a NAP-5 column (Sephadex G-25; Pharmacia) equilibrated in TES-EDTA-NaCl (TEN) buffer (10 mM TES, 0.1 mM ethylenediamine tetra-acetic acid [pH 7.4], 50 mM NaCl), to remove the CF not entrapped by the vesicles. The eluted samples were then diluted with TEN to a lipid concentration of approximately 10 mg ml1. Liposomes (40 µl) were mixed with an equal volume of concentrated solutions of fructans and glucans in TEN and 20-µl aliquots were filled into the wells of 60-well microplates. The plates were dried in desiccators at 28°C and 0% relative humidity for 24 h in the dark.
Damage to the liposomes was determined after rehydration with 20 µl TEN buffer. For leakage measurements, 10 µl of sample were diluted in the wells of black 96-well plates in 0.3 ml TEN. Measurements were made with a Fluoroskan Ascent (Labsystems, Helsinki, Finland) fluorescence microplate reader at an excitation wavelength of 444 nm and an emission wavelength of 555 nm. Fluorescence of CF is strongly quenched at the high concentration inside the vesicles and is increased when CF is released into the medium. The total CF content of the vesicles (100% leakage value) was determined after lysis of the membranes with 5 µl 1% Triton X-100. The values were corrected for the quenching of CF fluorescence by Triton X-100. The figures show the means ± SD from three parallel samples. Where no error bars are visible, they were smaller than the symbols.
Fusion experiments
For liposome fusion experiments, two liposome samples were prepared in TEN. One contained 1 mol% each of NBD-PE and Rh-PE in EPC, the other contained only EPC. After extrusion, liposomes were combined at a ratio of 1:9 (labeled:unlabeled), resulting in a lipid concentration of 10 mg ml1. Liposomes (40 µl) were mixed with an equal volume of concentrated solutions of solutes in TEN and 20-µl aliquots were filled into the inside of the caps of 1.5-ml microcentrifuge tubes. Samples were dried as described above and were rehydrated by filling 1 ml TEN buffer into a tube and then quickly closing and inverting the tube. Membrane fusion was measured by resonance energy transfer (Struck et al., 1981) with a Kontron SFM 25 fluorometer (Bio-Tek Instruments, Neufahrn, Germany) as described (Hincha et al., 1998
; Oliver et al., 1998b
). The figures show the means ± SD from three parallel samples. Where no error bars are visible, they were smaller than the symbols.
FTIR spectroscopy
Spectra were obtained from samples containing sugar and EPC liposomes at a weight ratio of 1:2, corresponding to the highest sugar concentration used in the leakage and fusion experiments. Liposomes were extruded in the presence of the sugars, so that the sugars were present on both sides of the membranes. Fifty microliters of sample were spread on a CaF2 window and dried under the same conditions as described above. The window with the dried sample was fixed in a cuvette holder connected to a temperature control unit (Specac Eurotherm, Worthington, UK). The cuvette holder was placed in a vacuum chamber with windows which was placed in the infrared beam. Temperature was controlled by a liquid N2 reservoir and an electrical heater. Temperature was measured with a thermocouple attached to the cuvette holder next to the sample. The sample was first heated to 50°C for at least 15 min under vacuum to remove residual moisture the lipid had taken up during sample handling. The effectiveness of this procedure was verified by the absence of a water band in the FTIR spectra at 1650 cm1. The sample was then cooled to 30°C and after a 5-min equilibration the temperature was increased at a constant rate of 1°C min1. Spectra were recorded with a Perkin-Elmer GX 2000 Fourier-transform infrared spectrometer, assisted by a computer equipped with the Spectrum 2000 software. The peak frequencies of the CH2 symmetric stretch band around 2850 cm1 were estimated by eye after normalization of absorbance and baseline flattening, using the interactive abex and flat routines, respectively (Tsvetkova et al., 1998). Tm was estimated by eye as the midpoints of the lipid melting curves (Crowe et al., 1997
). The peaks from the phosphate asymmetric stretch vibrations of different samples were compared after normalization of absorbance (abex) and baseline flattening in the 13001200 cm1 region. Because the peaks from samples containing inulins showed a strong broadening and the appearance of a shoulder when compared to controls and samples containing glucans, all phosphate peaks were further treated with the deconvolution routine included in the Spectrum software to enhance these spectral differences.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 Present address: PlantTec Biotechnologie GmbH; Hermannswerder 14, D-14473 Potsdam, Germany
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References |
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