Solute Accumulation and Compartmentation during the Cold Acclimation of Puma Rye

During cold acclimation of Puma rye (Secale cereale L. cv Puma), the intracellular osmotic potential nearly doubles. During this period, the accumulation of glycinebetaine, proline, and soluble sugars was monitored. The amount of glycinebetaine increased from 290 to 1300 micrograms per gram fresh weight during the 4-week acclimation period. Proline content did not change during the first 3 weeks of acclimation but then increased from 27 to 580 micrograms per gram fresh weight during the next 3 weeks. The total soluble sugar content more than doubled by the second week of cold acclimation, increasing from 11 to 26 milligrams per gram fresh weight. Most of this increase can be attributed to the accumulation of sucrose and raffinose, whose levels increased from 2.4 and 0 to 11 and 5 milligrams per gram fresh weight, respectively. The content of monosaccharides, predominantly glucose, remained at a constant 10 milligrams per gram fresh weight throughout the acclimation period. A comparison of the sugar content of protoplasts versus vacuoles isolated from cold-acclimated leaves revealed that the extravacuolar volume contained monosaccharides, sucrose, and raffinose. Thus, the increased amounts of sucrose and raffinose that occur during cold acclimation are present in compartments external to the vacuole and may contribute to cryoprotection.     

Factors affecting the survival of frozen-thawed mouse spermatozoa

Mouse epididymal spermatozoa were frozen in solutions containing various compounds with different molecular weights, and the factors affecting the postthawing survival were examined. Monosaccharides (glucose, galactose) had almost no protective effect regardless of the concentration and the temperature of exposure. On the other hand, disaccharides (sucrose, trehalose) and trisaccharides (raffinose, melezitose) resulted in higher survival rates, especially at a concentration of around 0.35 mol/kg H(2)O (0.381-0.412 Osm/kg). Macromolecules, such as PVP10, Ficoll 70, bovine serum albumin, and skim milk had almost no effect, but compounds with a molecular weight of about 800, such as metrizamide and Nycodenz, had some protective effect. When a raffinose solution was supplemented with 10% metrizamide, resulting in an osmolality of approximately 0.400 Osm/kg, a high survival rate was obtained. Solutions at about 0.400 Osm/kg containing trehalose alone, trehalose + metrizamide, raffinose alone, and raffinose + metrizamide, were all effective for sperm freezing; frozen-thawed sperm could fertilize oocytes, and the resultant embryos could develop to live young after transfer. For freezing mouse spermatozoa, aqueous solutions at approximately 0.400 Osm/kg containing a disaccharide or a trisaccharide seem to be effective.     

Addition of oligosaccharide decreases the freezing lesions on human red blood cell membrane in the presence of dextran and glucose

Although incubation with glucose before freezing can increase the recovery of human red blood cells frozen with polymer, this method can also result in membrane lesions. This study will evaluate whether addition of oligosaccharide (trehalose, sucrose, maltose, or raffinose) can improve the quality of red blood cell membrane after freezing in the presence of glucose and dextran. Following incubation with glucose or the combinations of glucose and oligosaccharides for 3 h in a 37 °C water bath, red blood cells were frozen in liquid nitrogen for 24 h using 40% dextran (W/V) as the extracellular protective solution. The postthaw quality was assessed by percent hemolysis, osmotic fragility, mean corpuscle volume (MCV), distribution of phosphatidylserine, the postthaw 4 °C stability, and the integrity of membrane. The results indicated the loading efficiency of glucose or oligosaccharide was dependent on their concentrations. Moreover, addition of trehalose or sucrose could efficiently decrease osmotic fragility of red blood cells caused by incubation with glucose before freezing. The percentage of damaged cell following incubation with glucose was 38.04 ± 21.68% and significantly more than that of the unfrozen cells (0.95 ± 0.28%, P < 0.01). However, with the increase of the concentrations of trehalose, the percentages of damaged cells were decreased steadily. When the concentration of trehalose was 400 mM, the percentage of damaged cells was 1.97 ± 0.73% and similar to that of the unfrozen cells (P > 0.05). Moreover, similar to trehalose, raffinose can also efficiently prevent the osmotic injury caused by incubation with glucose. The microscopy results also indicated addition of trehalose could efficiently decrease the formation of ghosts caused by incubation with glucose. In addition, the gradient hemolysis study showed addition of oligosaccharide could significantly decrease the osmotic fragility of red blood cells caused by incubation with glucose. After freezing and thawing, when both glucose and trehalose, sucrose, or maltose were on the both sides of membrane, with increase of the concentrations of sugar, the percent hemolysis of frozen red blood cells was firstly decreased and then increased. When the total concentration of sugars was 400 mM, the percent hemolysis was significantly less than that of cells frozen in the presence of dextran and in the absence of glucose and various oligosaccharides (P < 0.01). However, when both glucose and trehalose were only on the outer side of membrane, with increase of the concentrations of sugars, the percent hemolysis was increased steadily. Furthermore, addition of oligosaccharides can efficiently decrease the osmotic fragility and exposure of phosphatidylserine of red blood cells frozen with glucose and dextran. In addition, trehalose or raffinose can also efficiently mitigate the malignant effect of glucose on the postthaw 4 °C stability of red blood cells frozen in the presence of dextran. Finally, addition of trehalose can efficiently protect the integrity of red blood cell membrane following freezing with dextran and glucose. In conclusion, addition of oligosaccharide can efficiently reduce lesions of freezing on red blood cell membrane in the presence of glucose and dextran.     

The protective effect of sugars on chloroplast membranes during temperature and water stress and its relationship to frost,desiccation and heat resistance

Summary Freezing, desiccation and high-temperature stress may under certain conditions result in inactivation of electron transport (DCIP reduction) and cyclic photophosphorylation of isolated chloroplast membranes of spinach (Spinacia oleracea L.). When sugars are present during temperature and water stress, the thylakoids may be partially or completely protected. This membrane stabilization depends on the concentration of sugars and their molecular size. The trisaccharide raffinose is, on a molar basis, more effective than the disaccharide sucrose and the latter more than the monosaccharide glucose. An uncoupling effect and a stimulation of electron transport can be observed during freezing, desiccation and heat treatment, e.g. electron transport reactions are less sensitive to temperature and water stress than is photophosphorylation. As sugars are known to accumulate in winter, unspecific membrane stabilization by sugars may help to explain the often reported parallel development of frost, drought and heat resistance in many plants during winter.Dedicated to Professor Otto Stocker, Darmstadt, on the occasion of his 85th birthday.     

Cryoprotection of spinach chloroplast membranes by dextrans

The cryoprotective properties of dextrans have been investigated in freezing experiments with isolated spinach thylakoids (Spinacia oleracea L.). The activity of cyclic photophosphorylation was used as an assay for membrane integrity.Dextrans of average molecular weights between 10,000 and 70,000 daltons proved to be fairly nontoxic to chloroplast membranes. On a molar basis, cryoprotective action increased with increasing molecular weight; on a unit weight basis, the cryoprotective effectiveness of different dextrans was comparable. In the presence of low dextran concentrations which are not sufficient for complete membrane preservation, the effectiveness of the polymers could be considerably increased by the addition of electrolytes. This is in contrast to cryoprotection exerted by sugars. At a given dextran concentration, membrane activity is a function of the electrolyte concentration and follows an optimum curve. If membrane-toxic action of the electrolytes and salt crystallization during freezing which complicate the situation, are not taken into consideration, the increase in membrane protection during freezing by salts was dependent on the concentration of the salts and was not much influenced by the nature of the cations and anions. At 0 °C, dextrans delayed the inactivation of thylakoids suspended in NaCl solutions.From the results it is concluded that cryoprotection produced by dextrans is caused in part by specific membrane stabilization.     

Effects of freezing on the release and inactivation of chloroplast coupling factor 1

The three high-molecular-weight subunits of chloroplast coupling factor (CF₁) are the primary proteins released from pyrophosphate-washed thylakoids exposed to freezing. Identical subunit profiles are found in the supernatant proteins of thylakoids exposed to different intensities of freezing stress by the inclusion of sugars with varying degrees of cryoprotective efficiency. Isolated CF₁ is inactivated by freezing in the presence of NaCl, glucose, and sucrose but raffinose can protect against loss of enzymatic activity during freezing. The low specific activity of the supernatant proteins released from the thylakoid and the inability to recover the Ca²⁺-dependent ATPase activity lost from the membrane suggest that inactivation accompanies release of CF₁ during freezing.     

Sugar compartmentation in frost-hardy and partially dehardened cabbage leaf cells

In frost-hardy and partially dehardened leaves of Brassica oleracea L. var. sabellica L. the distribution of cryoprotective sugars and of chloride between chloroplasts and the nonchloroplast part of leaf cells was investigated using the nonaqueous isolation technique as a means of cell fractionation. In chloroplasts of frost-hardy leaves high concentrations of sucrose and raffinose and comparatively low concentrations of chloride have been found. The ratios between sugars and chloride were so as to ascertain complete protection of the frost-sensitive thylakoid membranes during freezing. During dehardening, sugars decreased especially in the chloroplasts. There was a conversion of sucrose and raffinose into monosaccharides. This led to a large increase in the concentration of glucose and fructose in the nonchloroplast parts of the cells. There is evidence that the sugar concentration in the vacuole increased at the expense of sugars located in chloroplasts and cytoplasm. The quantity of sugars that remained in the chloroplasts did not appear to be sufficient for complete membrane protection at very low freezing temperatures.     

Comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation

Mouse sperm has proven to be more difficult to cryopreserve than sperm of other mammalian species. Published reports show that only three cryoprotectant agents (CPAs), alone or combined, have been studied: glycerol and dimethyl sulfoxide (DMSO), as permeating agents, and raffinose, as a nonpermeating agent. To date, the most consistent results for mouse sperm cryopreservation have been achieved by use of raffinose/skim milk as cryoprotectant with rapid cooling at 20 degrees C per minute. In this study, we compared the cryoprotection provided by permeating (glycerol, formamide, propanediol, DMSO, adonitol) or nonpermeating (lactose, raffinose, sucrose, trehalose, d-mannitol) compounds for freezing mouse sperm. Different solutions were made using 3% skim milk solution as the buffer or extender in which all different cryoprotectant agents were dissolved at a concentration of 0.3 M, with a final osmolality of approx. 400 mOsm. Sperm samples from CB6F1 (hybrid) and C57BL/6J (inbred) mice collected directly into each CPA were frozen/thawed under identical conditions. After thawing and CPA elimination (centrifugation) raffinose (59%), trehalose (61%), and sucrose (61%) sustained the best motility (P = < 0.1) of the nonpermeating agents, whereas the best of the permeating agents was DMSO (42%). Membrane integrity was analyzed and showed that the simple exposure (prefreeze) to sugars was less harmful than the exposure to glycols. Coincidentally, sperm frozen in trehalose (41%), raffinose (40.5%), and sucrose (37.5%) were the samples less injured among all different postthawed CPA tested. The in vitro fertilization results demonstrated that hybrid mouse spermatozoa frozen with sugars (lactose 80%, raffinose 80%, trehalose 79% of two-cell embryos production) were more fertile than those frozen with glycols (glycerol 11%).     

Effect of sugar type on the survival of frozen-thawed rhesus monkey (Macaca mulatta) sperm

Sperm-freezing extenders supplemented with sugar or a combination of different sugars are widely used for the cryopreservation of nonhuman primate spermatozoa. Understanding which sugar or combination of sugars offers the highest level of cryoprotection would be beneficial for the development of sperm-freezing extenders. In the present study we aimed to investigate the effect of glucose, lactose, and raffinose separately or in combination on the cryosurvival of rhesus monkey spermatozoa. Toward that end, we prepared eight extenders by adding various types of sugars to a basic medium (BM): G-BM (0.3 M glucose), L-BM (0.3 M lactose), R-BM (0.3 M raffinose), LG-BM (0.15 M lactose+0.15 M glucose), RG-BM (0.15 M raffinose+0.15 M glucose), LR-BM (0.15 M lactose+0.15 M raffinose), and LRG-BM (0.1 M lactose+0.1 M raffinsoe+0.1 M glucose). A saline control (0.157 M sodium chloride) was also used. The results showed no significant difference in post-thaw motility when spermatozoa were frozen with G-BM, L-BM, LG-BM, RG-BM, and LRG-BM, but the post-thaw motility was significantly lower when it was frozen with R-BM, LR-BM, and the saline control. The highest plasma membrane integrity was achieved when spermatozoa were frozen with G-BM, L-BM, LG-BM, RG-BM, and LRG-BM, and the highest acrosome integrity was achieved with G-BM, L-BM, LG-BM, RG-BM, LRG-BM, and the saline control. The results indicate that the various sugars offered different protective effects. For the cryopreservation of rhesus monkey spermatozoa, glucose (monosaccharide) and lactose (disaccharide) were shown to be more suitable than raffinose (trisaccharide) for preserving spermatozoal motility, plasma membrane, and acrosome. Specifically, raffinose was detrimental to sperm acrosome integrity.     

Theoretical and experimental studies on freezing point depression and vapor pressure deficit as methods to measure osmotic pressure of aqueous polyethylene glycol and bovine serum albumin solutions

For survival in adverse environments where there is drought, high salt concentration or low temperature, some plants seem to be able to synthesize biochemical compounds, including proteins, in response to changes in water activity or osmotic pressure. Measurement of the water activity or osmotic pressure of simple aqueous solutions has been based on freezing point depression or vapor pressure deficit. Measurement of the osmotic pressure of plants under water stress has been mainly based on vapor pressure deficit. However, differences have been noted for osmotic pressure values of aqueous polyethylene glycol (PEG) solutions measured by freezing point depression and vapor pressure deficit. For this paper, the physicochemical basis of freezing point depression and vapor pressure deficit were first examined theoretically and then, the osmotic pressure of aqueous ethylene glycol and of PEG solutions were measured by both freezing point depression and vapor pressure deficit in comparison with other aqueous solutions such as NaCl, KCl, CaCl(2), glucose, sucrose, raffinose, and bovine serum albumin (BSA) solutions. The results showed that: (1) freezing point depression and vapor pressure deficit share theoretically the same physicochemical basis; (2) theoretically, they are proportional to the molal concentration of the aqueous solutions to be measured; (3) in practice, the osmotic pressure levels of aqueous NaCl, KCl, CaCl(2), glucose, sucrose, and raffinose solutions increase in proportion to their molal concentrations and there is little inconsistency between those measured by freezing point depression and vapor pressure deficit; (4) the osmotic pressure levels of aqueous ethylene glycol and PEG solutions measured by freezing point depression differed from the values measured by vapor pressure deficit; (5) the osmotic pressure of aqueous BSA solution measured by freezing point depression differed slightly from that measured by vapor pressure deficit.     

Carbohydrate Changes during Maturation of Cucumber Fruit : Implications for Sugar Metabolism and Transport

Changes in the carbohydrate profiles in the mesocarp, endocarp, and seeds of maturing cucumber (Cucumis sativus, L.) fruit were analyzed. Fruit maturity was measured by a decrease in endocarp pH, which was found to correlate with a loss in peel chlorophyll and an increase in citric acid content. Concentrations of glucose and fructose (8.6-10.3 milligrams per gram fresh weight, respectively) were found to be higher than the concentration of sucrose (0.3 milligrams per gram fresh weight) in both mesocarp and endocarp tissue. Neither raffinose nor stachyose were found in these tissues. The levels of glucose and fructose in seeds decreased during development, but sucrose, raffinose, and stachyose accumulated during the late stages of maturation. Both raffinose and stachyose were found in the seeds of six lines of Cucumis sativus L. This accumulation of raffinose saccharides coincided with an increase in galactinol synthase activity in the seeds. Funiculi from maturing fruit were found to be high in sucrose concentration (4.8 milligrams per gram fresh weight) but devoid of both raffinose and stachyose. The results indicated that sucrose is the transport sugar from the peduncle to seed, and that raffinose saccharide accumulation in the seed is the result of in situ biosynthesis and not from direct vascular transport of these oligosaccharides into the seeds.     

Evidence for a role of raffinose in stabilizing photosystem II during freeze–thaw cycles

A role of non-reducing sugars like sucrose and raffinose in the protection of plant cells against damage during freezing has been proposed for many species, but reports on physiological effects are conflicting. Non-aqueous fractionation of mesophyll cell compartments in Arabidopsis thaliana was used to show that sucrose and raffinose accumulate in plastids during low temperatures, pointing to a physiological role in protecting the photosynthetic apparatus. Comparing a previously described raffinose synthase (RS) mutant of A. thaliana with its corresponding wild type, accession Col-0, revealed that a lack of raffinose has no effect on electrolyte leakage from leaf cells after freeze–thaw cycles, supporting that raffinose is not essential for protecting the plasma membrane. However, in situ chlorophyll fluorescence showed that maximum quantum yield of PS II photochemistry (F _v/F _m) and other fluorescence parameters of cold acclimated leaves subjected to freeze–thaw cycles were significantly lower in the raffinose synthase mutant than in the corresponding wild type, indicating that raffinose is involved in stabilizing PS II of cold acclimated leaf cells against damage during freezing.     

Trehalose Increases Freeze-Thaw Damage in Liposomes Containing Chloroplast Glycolipids

Chloroplast thylakoids contain three classes of glycolipids, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG). We have investigated the stability of large unilamellar vesicles made from egg phosphatidylcholine (EPC) and different chloroplast glycolipids during freezing to -18 degreesC, as a function of the presence of three sugars: glucose, sucrose, or trehalose. Contrary to the situation in thylakoids, where cryoprotection increases from glucose < sucrose < trehalose, liposomes containing 50% DGDG showed the opposite behavior. In fact, carboxyfluorescein leakage increased over the control values (freezing in the absence of sugar) in the presence of trehalose. This effect was not seen in vesicles made from pure EPC, or a mixture of EPC and MGDG, or EPC and SQDG. Liposomes made from mixtures of all three glycolipids, however, showed even more leakage in the presence of trehalose than liposomes containing only DGDG and EPC. Copyright 1998 Academic Press.     

Enrichment in Specific Soluble Sugars of Two Eucalyptus Cell-Suspension Cultures by Various Treatments Enhances Their Frost Tolerance via a Noncolligative Mechanism

A cell-suspension culture obtained from the hybrid Eucalyptus gunnii/Eucalyptus globulus was hardened by exposure to lower temperatures, whereas in the same conditions cells from a hybrid with a more frost-sensitive genotype, Eucalyptus cypellocarpa/Eucalyptus globulus, were not able to acclimate. During the cold exposure the resistant cells accumulated soluble sugars, in particular fructose and sucrose, with a limited increase in cell osmolality. In contrast, the cell suspension that was unable to acclimate did not accumulate soluble sugars in response to the same cold treatment. To an extent similar to that induced after a cold acclimation, frost-hardiness of the cells increased after a 14-h incubation with specific soluble sugars such as sucrose, raffinose, fructose, and mannitol. Such hardening was also observed for long-term cultures in mannitol-enriched medium. This cryoprotective effect of sugars without exposure to lower temperatures was observed in both the resistant and the sensitive genotypes. Mannitol was one of the most efficient carbohydrates for the cryoprotection of eucalyptus. The best hardiness (a 2.7-fold increase in relative freezing tolerance) was obtained for the resistant cells by the cumulative effect of cold-induced acclimation and mannitol treatment. This positive effect of certain sugars on eucalyptus freezing tolerance was not colligative, since it was independent of osmolality and total sugar content.     

Cryoprotective leaf proteins.

Leaves of frost-resistant plants contain a number of soluble proteins which are capable of protecting isolated biomembranes against inactivation during freezing. Such proteins have not been found in non-hardy summer material. The pattern of protective proteins was not uniform in hardy material of different origin and appeared to change with the season. Cryoprotective proteins were isolated by preparative gel electrophoresis. Molecular weights of different proteins as determined by their electrophoretic mobility in sodium dodecyl sulfate gels were between 10000 and 20000. Circular dichroism measurements failed to indicate helical structures. The amino acid composition of 2 active proteins revealed a high content of polar amino acids. The proteins were heat-stable. They were, on a molar basis, more than 1000 times as effective in protecting thylakoid membranes against freezing damage as low-molecular-weight cryoprotectants such as sucrose, glycerol or dimethylsulfoxide. Very low concentrations of the proteins increased cryoprotection provided by sucrose. Of a number of oligopeptides of known composition, only a few were cryoprotective. Their activity was very small as compared with that of the active proteins. The concentration of the cryoprotective proteins in hardy leaves appeared to be high enough for a significant contribution of the proteins to the frost tolerance of resistant plants.     

Effective cryoprotection of thylakoid membranes by ATP

Freezing of isolated spinach thylakoids in the presence of NaCl uncoupled photophosphorylation from electron flow and increased the permeability of the membranes to protons. Addition of ATP prior to freezing diminished membrane inactivation. On a molar basis, ATP was at least 100 times more effective in protecting thylakoids from freezing damage than low-molecularweight carbohydrates such as sucrose and glucose. The cryoprotective effectiveness of ATP was increased by Mg²⁺. In the absence of carbohydrates, preservation of thylakoids during freezing in 100 mM NaCl was saturated at about 1–2 mM ATP, but under these conditions membranes were not fully protected. However, in the presence of small amounts of sugars which did not significantly prevent thylakoid inactivation during freezing, ATP concentrations considerably lower than 0.5 mM caused nearly complete membrane protection. Neither ADP nor AMP could substitute for ATP. These findings indicate that cryoprotection by ATP cannot be explained by a colligative mechanism. It is suggested that ATP acts on the chloroplast coupling factor, either by modifying its conformation or by preventing its release from the membranes. The results are discussed in regard to freezing injury and resistance in vivo.Abbreviations CF₁ chloroplast coupling factor - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PMS phenazine methosulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Cryopreservation of spermatozoa of the terrestrial Puerto Rican frog, Eleutherodactylus coqui

World-wide reports of amphibian population declines have led to increased interest in the reproductive biology of anurans. As a model system, here we present evidence for the effective cryoprotection of sperm from the Puerto Rican frog, Eleutherodactylus coqui, using mixtures of fetal bovine serum (FBS) and dimethylsulfoxide (Me(2)SO), glycerol or sucrose extenders. Using a fluorescent dye exclusion assay, we found that 53.9 and 50.4% of all sperm with intact membranes prior to freezing maintained membrane integrity after rapid freezing and thawing when protected with either a FBS/glycerol or FBS/sucrose solution, respectively. The methods reported here may be useful for similar work with many of the more than 700 other species in this genus.     

Freezing of isolated thylakoid membranes in complex media. VIII. Differential cryoprotection by sucrose, proline and glycerol

The cryoprotective efficiency of sucrose, proline and glycerol for chloroplast membranes isolated from spinach leaves ( Spinacia oleracea L. cv. Monatol) was determined after freeze-thaw treatment in media containing the predominant inorganic electrolytes of the chloroplast stroma. In most cases, the protective capacity of equimolar concentrations of the cryoprotectants followed the order sucrose > proline > glycerol. The lower the freezing temperature the less cryoprotectant was necessary for comparable preservation of the capacity of photosynthetic electron transport. Likewise, the cryoprotective efficiency of sucrose for cyclic photophosphorylation and light-induced proton gradient increased with decreasing freezing temperature. In contrast, while proline effectively stabilized these membrane reactions at mild and moderate freezing temperatures, it was much less efficient at more severe freezing stress. Cryoprotection of photophosphorylation and proton gradient formation at given initial concentrations of glycerol was largely independent of the freezing temperature. While dissociation of the peripheral part of chloroplast coupling factor (CF₁) during freeze-thaw treatment cannot be prevented in the presence of lower initial concentrations of proline and glycerol and. at mild freezing temperatures, of sucrose, the latter may stabilize this protein complex at least under more severe freezing conditions. The differences in the cryoprotective efficiency of the solutes are discussed relative to their non-ideal activity-concentration profiles, solution properties and penetration behaviour across the thylakoid membrane.     

Lipid-sugar interactions : relevance to anhydrous biology

The ability of seeds and other anhydrous plant forms to survive the withdrawal of water must involve a mechanism for protecting the integrity of cellular membranes. Evidence from animal systems implicates sugars as protective components, and we have tested the changes in mesomorphic phase state of phospholipid model membranes upon hydration and dehydration in the presence of sucrose and/or sucrose plus raffinose. X-ray diffraction studies of dry dimyristoylphosphatidylcholine (DMPC) indicate that the presence of sucrose lowers the chain order/disorder transition temperature to that of hydrated lipid; likewise, the lamellar repeat spacings showed the dry DMPC/sucrose mixture to be similar to that of the hydrated lipid. These results support the proposed potential of sugars to substitute for water in biomembranes. If sucrose is to serve as a protectant during desiccation of seeds, its tendency to crystallize would lessen its effectiveness. Raffinose is known to serve as an inhibitor of sucrose crystallization, and is abundant in seeds. The addition of raffinose to make DMPC/sucrose/raffinose mixtures (1/1/0.3 mass ratio) prevented sucrose crystallization, suggesting this as a possible in vivo role for raffinose.