The effect of arbutin on membrane integrity during drying is mediated by stabilization of the lamellar phase in the presence of nonbilayer-forming lipids.

Arbutin (4-hydroxyphenyl-beta-glucopyranoside) is a solute accumulated to high concentrations in drought and frost resistant plants. Arbutin can inhibit membrane lysis, both free radical-mediated and enzymatic in nature, and it has been suggested that arbutin might contribute to membrane stabilization in these plants. However, we found that arbutin destabilized phosphatidylcholine vesicles during drying and rehydration, which appears to be inconsistent with the proposed protective function of arbutin for membranes. We also found, however, that arbutin stabilizes membranes containing nonbilayer-forming lipids during freezing. We now report that, in liposomes containing the nonbilayer-forming lipids monogalactosyldiacylglycerol (MGDG) or phosphatidylethanolamine (PE), arbutin served a protective function during drying, as measured by retention of carboxyfluorescein (CF) and extent of vesicle fusion. In hydrated samples containing these lipids, arbutin stabilized the lamellar liquid crystalline phase. Therefore, the interaction between arbutin and lipid membranes and the resulting effects on membrane stability depend, in a complex manner, on the lipid composition of the membrane.

Antifreeze proteins differentially affect model membranes during freezing.

Over the past decade antifreeze proteins from polar fish have been shown either to stabilize or disrupt membrane structure during low temperature and freezing stress. However, there has been no systematic study on how membrane composition affects the interaction of antifreeze proteins with membranes under stress conditions. Therefore, it is not possible at present to predict which antifreeze proteins will protect, and which will damage a particular membrane during chilling or freezing. Here, we analyze the effects of freezing on spinach thylakoid membranes and on model membranes of varying lipid composition in the presence of antifreeze protein type I (AFP I) and specific fractions of antifreeze glycoproteins (AFGP). We find that the addition of galactolipids to phospholipid model membranes changes the effect each protein has on the membrane during freezing. However, the greatest differences observed in this study are between the different types of antifreeze proteins. We find that AFP type I and the largest molecular weight fractions of AFGP induce concentration dependent leakage from, and are fusogenic to the liposomes. This is the first report that an antifreeze protein induces membrane fusion. In contrast, the smallest fraction of AFGP offers a limited degree of protection during freezing and does not induce membrane fusion at concentrations up to 10 mg/ml.

Cabbage cryoprotectin is a member of the nonspecific plant lipid transfer protein gene family.

We have recently purified a protein (cryoprotectin) from the leaves of cold-acclimated cabbage (Brassica oleracea) to electrophoretic homogeneity, which protects thylakoids isolated from the leaves of nonacclimated spinach (Spinacia oleracea) from freeze-thaw damage. Sequencing of cryoprotectin showed the presence of at least three isoforms of WAX9 proteins, which belong to the class of nonspecific lipid transfer proteins. Antibodies raised against two synthetic peptides derived from the WAX9 proteins recognized a band of approximately 10 kD in western blots of crude cryoprotectin preparations. This protein and the cryoprotective activity could be precipitated from solution by the antiserum. We show further that cryoprotectin is structurally and functionally different from WAX9 isolated from the surface wax of cabbage leaves. WAX9 has lipid transfer activity for phosphatidylcholine, but no cryoprotective activity. Cryoprotectin, on the other hand, has cryoprotective, but no lipid transfer activity. The cryoprotective activity of cryoprotectin was strictly dependent on Ca(2+) and Mn(2+) and could be inhibited by chelating agents, whereas the lipid transfer activity of WAX9 was higher in the presence of ethylenediaminetetraacetate than in the presence of Ca(2+) and Mn(2+).

Non-disaccharide-based mechanisms of protection during drying.

Few tissues or organisms can survive the removal of nearly all their intra and extracellular water. These few have developed specialized adaptations to protect their cellular components from the damage caused by desiccation and rehydration. One mechanism, common to almost all such organisms, is the accumulation of disaccharides within cells and tissues at the onset of dehydration. This adaptation has been extensively studied and will not be considered in this review. It has become increasingly clear that true desiccation tolerance is likely to involve several mechanisms working in concert; thus, we will highlight several other important and complimentary adaptations found especially in the dehydration-resistant tissues of higher plants. These include the scavenging of reactive oxygen species, the down-regulation of metabolism, and the accumulation of certain amphiphilic solutes, proteins, and polysaccharides.

Plant fructans stabilize phosphatidylcholine liposomes during freeze-drying.

Fructans have been implicated as protective agents in the drought and freezing tolerance of many plant species. A direct proof of their ability to stabilize biological structures under stress conditions, however, is still lacking. Here we show that inulins (linear fructose polymers) isolated from chicory roots and dahlia tubers stabilize egg phosphatidylcholine large unilamellar vesicles during freeze-drying, while another polysaccharide, hydroxyethyl starch, was completely ineffective. Liposome stability was assessed after rehydration by measuring retention of the soluble fluorescent dye carboxyfluorescein and bilayer fusion. Inulin was an especially effective stabilizer in combination with glucose. Analysis by HPLC showed that the commercial inulin preparations used in our study contained no low molecular mass sugars that could be responsible for the observed stabilizing effect of the fructans. Fourier transform infrared spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature of dry egg PtdCho by more than 20 degrees C in the presence of inulin. A direct interaction of inulin with the phospholipid in the dry state was also indicated by dramatic differences in the phosphate asymmetric stretch region of the infrared spectrum between samples with and without the polysaccharide.

Lipid composition determines the effects of arbutin on the stability of membranes.

Arbutin (hydroquinone-beta-D-glucopyranoside) is an abundant solute in the leaves of many freezing- or desiccation-tolerant plants. Its physiological role in plants, however, is not known. Here we show that arbutin protects isolated spinach (Spinacia oleracea L.) thylakoid membranes from freeze-thaw damage. During freezing of liposomes, the presence of only 20 mM arbutin led to complete leakage of a soluble marker from egg PC (EPC) liposomes. When the nonbilayer-forming chloroplast lipid monogalactosyldiacylglycerol (MGDG) was included in the membranes, this leakage was prevented. Inclusion of more than 15% MGDG into the membranes led to a strong destabilization of liposomes during freezing. Under these conditions arbutin became a cryoprotectant, as only 5 mM arbutin reduced leakage from 75% to 20%. The nonbilayer lipid egg phosphatidylethanolamine (EPE) had an effect similar to that of MGDG, but was much less effective, even at concentrations up to 80% in EPC membranes. Arbutin-induced leakage during freezing was accompanied by massive bilayer fusion in EPC and EPC/EPE membranes. Twenty percent MGDG in EPC bilayers completely inhibited the fusogenic effect of arbutin. The membrane surface probes merocyanine 540 and 2-(6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosph ocholi ne (NBD-C(6)-HPC) revealed that arbutin reduced the ability of both probes to partition into the membranes. Steady-state anisotropy measurements with probes that localize at different positions in the membranes showed that headgroup mobility was increased in the presence of arbutin, whereas the mobility of the fatty acyl chains close to the glycerol backbone was reduced. This reduction, however, was not seen in membranes containing 20% MGDG. The effect of arbutin on lipid order was limited to the interfacial region of the membranes and was not evident in the hydrophobic core region. From these data we were able to derive a physical model of the perturbing or nonperturbing interactions of arbutin with lipid bilayers.

Release of two peripheral proteins from chloroplast thylakoid membranes in the presence of a Hofmeister series of chaotropic anions.

The dissociation of two peripheral spinach (Spinacia oleracea L.) thylakoid membrane proteins, coupling factor CF1 and ferredoxin-NADP+-oxidoreductase, in the presence of chaotropic sodium salts has been investigated, using monospecific antibodies against the proteins as probes. Release of both proteins followed the Hofmeister series of anions (Cl- < NO3- < Br- < I- < SCN-). In mixtures, the different salts had an additive effect. In addition, there were also qualitative differences in the action of the anions, such that NaI and NaSCN led to a different concentration and time dependence of the dissociation of the peripheral proteins from thylakoids. An analysis of the temperature dependence of protein release showed that the more chaotropic ions reduced the activation energy required for the dissociation of the proteins from their binding sites on the membrane. The addition of sugars (glucose, sucrose, or trehalose) reduced the amount of protein released from the membranes in the presence of NaI or NaSCN.

Interactions of proline, serine, and leucine with isolated spinach thylakoids: solute loading during freezing is not related to membrane fluidity.

We have investigated the physical mechanisms through which amino acids influence freeze-thaw damage to isolated spinach (Spinacia oleracea L.) thylakoid membranes. The cryoprotective amino acid proline reduced osmotic membrane rupture during thawing by reducing solute loading of thylakoids during freezing for 1 h to -5 degreesC. Evidence for binding of proline to the thylakoid surface was obtained by determining the partitioning of the hydrophobicity-sensitive dye merocyanine 540 between the bulk solution and the membranes in the presence of different concentrations of proline. This binding led to a decrease in lipid fluidity at the membrane surface, as measured with fluorescence depolarization spectroscopy using the probe trimethylammonium-diphenylhexatriene (TMA-DPH). The hydrophobic core of the membranes, as probed with 1,6-diphenyl-1,3,5,-hexatriene, was not influenced. Surprisingly, cryotoxic concentrations of both serine and leucine resulted in the same amount of reduction of thylakoid lipid fluidity, without reducing solute loading during freezing. In the case of these cryotoxic amino acids, unknown additional interactions with the membranes must result in membrane destabilization during a freeze-thaw cycle.

Interactions of arbutin with dry and hydrated bilayers.

The glycosylated hydroquinone arbutin (4-hydroxyphenyl-beta-D-glucopyranoside) is abundant in certain resurrection plants, which can survive almost complete dehydration for prolonged periods. Little is known about the role of arbutin in vivo, but it is thought to contribute toward survival of the plants in the dry state. We have investigated the interactions of arbutin with model membranes under conditions of high and low hydration, as well as the possible participation of arbutin in carbohydrate glasses formed at low water contents. Retention of a trapped soluble marker inside large unilamellar vesicles and fusion of vesicles was monitored by fluorescence spectroscopy. Effects of arbutin on glass-transition temperatures and hydrated membrane phase-transition temperatures were measured by differential scanning calorimetry. The possible insertion of arbutin into membrane bilayers was estimated by following arbutin auto-fluorescence. Evidence is presented that arbutin does not change the glass-transition temperature of a sucrose/trehalose glass, but that arbutin does interact with hydrated membranes by insertion of the phenol moiety into the lipid bilayer. This interaction causes increased membrane leakage during air-drying by a mechanism other than vesicle-vesicle fusion. Implications of these effects on the dehydrated plant cells, as well as possible methods of obviating the damage, are discussed.

The effects of chloroplast lipids on the stability of liposomes during freezing and drying.

Chloroplast thylakoids contain four classes of lipids, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol (cpPG). We have investigated the effects of these lipids on the stability of large unilamellar vesicles made from egg phosphatidylcholine (EPC), by substitution of different fractions of EPC in the membranes by the various chloroplast lipids. Damage to liposomes after freezing to - 18 degrees C was measured as carboxyfluorescein leakage or fusion between vesicles. The presence of all chloroplast lipids increased leakage. However, the maximum amount of leakage and the concentration dependence were dramatically different between the different lipids. Only SQDG induced vesicle fusion, while the non-bilayer lipid MGDG did not. The presence of MGDG in the membranes led to more leakage than the presence of another non-bilayer lipid, egg phosphatidylethanolamine (EPE). In EPE-containing liposomes, leakage was strongly associated with fusion. Combinations of different chloroplast lipids had an additive effect on leakage induced by freezing. Most of the leakage from galactolipid-containing vesicles occurred during the first 15 min of freezing at - 18 degrees C. After a 3 h incubation period, most leakage occurred between 0 degrees C and - 10 degrees C. Lowering the temperature to - 22 degrees C had only a small additional effect. Incubation of liposomes at - 10 degrees C in the presence of 2.5 M NaCl without ice crystallization, approximately the same concentration obtained by freezing to - 10 degrees C, resulted in very little leakage. Air drying of liposomes to low water contents resulted in massive leakage, both from pure EPC vesicles and from vesicles containing galactolipids. The latter vesicles showed more leakage at any given water content than EPC vesicles.

[beta]-1,3-Glucanase Is Cryoprotective in Vitro and Is Accumulated in Leaves during Cold Acclimation.

We have used isolated spinach (Spinacea oleracea L.) thylakoid membranes to investigate the possible cryoprotective properties of class I [beta]-1,3-glucanase (1,3-[beta]-D-glucan 3-glucanohydrolase; EC 3.2.1.39) and chitinase. Class I [beta]-1,3-glucanase that was purified from tobacco (Nicotiana tabacum L.) protected thylakoids against freeze-thaw injury in our in vitro assays, whereas class I chitinase from tobacco had no effect under the same conditions. The [beta]-1,3-glucanase acted by reducing the influx of solutes into the membrane vesicles during freezing and thereby reduced osmotic stress and vesicle rupture during thawing. Western blots probed with antibodies directed against tobacco class I [beta]-1,3-glucanase showed that in spinach and cabbage (Brassica oleracea L.) leaves an isoform of 41 kD was accumulated during frost hardening under natural conditions.

The lytic activity of the bee venom peptide melittin is strongly reduced by the presence of negatively charged phospholipids or chloroplast galactolipids in the membranes of phosphatidylcholine large unilamellar vesicles.

We have investigated the dependence of the lytic activity of the bee venom peptide melittin on the lipid composition of its target membrane. The lysis of large unilamellar liposomes, measured as loss of the fluorescent dye carboxyfluorescein, in the presence of melittin was strongly reduced when the negatively charged lipids phosphatidylglycerol (PG) or phosphatidylserine (PS), or the plant chloroplast lipids monogalactosyldiacylglycerol (MGDG) or digalactosyldiacylglycerol (DGDG) were incorporated into egg phosphatidylcholine (EPC) membranes. This reduction was evident at concentrations below 10 wt% of the additional lipids. It was not due to reduced binding of melittin to the vesicles. It was also not related to a reduced insertion depth of the peptide into the bilayer, as shown by quenching of the intrinsic tryptophan fluorescence of the peptide by the aqueous quencher sodium nitrate. Fourier transform infrared spectroscopy (FTIR) revealed specific interactions of the peptide with the headgroups of the inhibitory lipids. The phosphate peak in PG was shifted by two wavenumbers after the addition of melittin. There was no shift in EPC or PS. Instead, in PS the COO- peak was strongly distorted in the presence of melittin. These data indicate ionic interactions between the basic peptide and the negative charges on the membrane surface. The galactolipids are uncharged. Here the evidence points to hydrogen bonding between melittin and OH-groups of the sugar headgroups. Liposomes containing DGDG were the only case where we found evidence for changes in fatty acyl chain motion due to the presence of melittin, from the CH2-scissoring peaks.

Purification and Characterization of a Cryoprotective Protein (Cryoprotectin) from the Leaves of Cold-Acclimated Cabbage.

We have purified a protein (cryoprotectin) from the leaves of cold-acclimated cabbage (Brassica oleracea L.) that protects thylakoids from nonacclimated spinach (Spinacia oleracea L.) against freeze-thaw damage. The procedure involves precipitations by heat, ammonium sulfate, and the glycosaminoglycan heparin and column chromatography on Polyamide 6 and a C18 reverse-phase matrix. After reverse-phase chromatography we obtained a single band of an apparent molecular mass of 7 kD when fractions that showed cryoprotective activity were analyzed by sodium dodecyl sulfate gel electrophoresis and silver staining. Gel-filtration experiments confirmed that the active protein is a monomer of 7 kD native molecular mass. This 7-kD protein could be purified only from cold-acclimated cabbage, but not from plants grown under nonacclimating conditions. Using peroxidase-labeled lectins, we show that cryoprotectin is a glycoprotein and that the saccharide moiety contains [alpha]1-3-linked fucose.

The solute permeability of thylakoid membranes is reduced by low concentrations of trehalose as a co-solute.

The different efficiencies of sucrose and trehalose in protecting isolated spinach (Spinacia oleracea L.) thylakoids against freeze-thaw damage is quantitatively related to their ability to reduce the solute loading of the vesicles during freezing. In the present paper we show that this effect is based on a reduction of the solute permeability of the membranes. Permeability was measured with 14C-labeled glucose at temperatures between 0 and 10 degrees C. Glucose permeability was reduced by both sucrose and trehalose, with trehalose effective at much lower concentrations than sucrose. An analysis of the temperature dependence of glucose permeability in the presence and absence of trehalose revealed that a 50% reduction in permeability resulted from a 10% increase in activation energy and a 30% decrease in activation entropy. Using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene (DPH), we found that the reduced permeability of the membranes in the presence of trehalose was unaccompanied by a reduction in lipid fluidity. This also excluded the possibility of a solute-induced liquid crystalline to gel phase transition. A reduced partitioning of the hydrophobicity-sensitive dye merocyanine 540 into thylakoids and into membranes containing 50% digalactosyldiacylglycerol in the presence of trehalose as compared to sucrose and glucose showed that the lipid headgroup region of these membranes became less accessible for solutes. No significant difference in merocyanine partitioning in the presence of trehalose as compared to sucrose or glucose was apparent when monogalactosyldiacylglycerol dispersions or phosphatidylcholine vesicles were investigated.

Galactose-Specific Lectins Protect Isolated Thylakoids against Freeze-Thaw Damage.

We have measured freeze-thaw damage to isolated spinach (Spinacia oleracea L.) chloroplast thylakoid membranes in the presence of different galactose-specific seed lectins to determine whether the binding of proteins to the membrane surface can lead to cryoprotection. Of the seven lectins investigated, five were protective to different degrees and two showed no measurable effect. Protection was afforded by a reduction of the solute permeability of the membranes. This reduced the solute influx during freezing and thereby osmotic rupture of the thylakoid vesicles during thawing. Using model membranes and fluorescently labeled lectins, we could show that the proteins bound exclusively to the digalactosyl lipids in the membranes. Binding was a prerequisite for the protective effect, because the presence of up to 5 mM galactose in the samples completely inhibited both binding of the lectins to thylakoid and model membranes and cryoprotection. The degree of binding was, in contrast, not related to the cryoprotective efficiency of different lectins; cryoprotection was a function of the hydrophobicity of the proteins.

Cryotoxicity of antifreeze proteins and glycoproteins to spinach thylakoid membranes--comparison with cryotoxic sugar acids.

We have used thylakoids from spinach (Spinacia oleracea L.) chloroplasts to test the effects of antifreeze proteins (AFP) from the starry flounder (Platichthys stellatus; AFP-SF) and from the antarctic eel pout (Austrolycichthys brachycephalus; AFP-AB), and antifreeze glycoproteins (AFGP) from the antarctic fish Dissostichus mawsoni on biological membranes during freezing. Freeze-thaw damage, measured as the release of the lumenal protein plastocyanin from the thylakoid vesicles, was strongly increased in the presence of all proteins tested. Measurements of the time dependence of plastocyanin release in a simplified artificial chloroplast stroma medium showed that all the fish proteins increased damage during the initial rapid phase while only AFGP increased plastocyanin release during the linearly time dependent slow phase. A slow plastocyanin release is also seen in the absence of freezing. It is increased by the presence of AFGP and AFP-AB, but not by AFP-SF. In order to distinguish between the contribution of the polypeptide and the carbohydrate part of AFGP on freeze-thaw damage we investigated the effects of galactose and N-acetylgalactosamine. While galactose was protective, N-acetylgalactosamine increased the rate of plastocyanin release in an artificial stroma medium at -20 degrees C. It had no effect on the rapid phase of damage and was also ineffective at 0 degree C. The same was found for several other sugar derivatives (N-acetylglucosamine, gluconic acid, glucuronic acid, galacturonic acid). From these data we conclude that the increased plastocyanin release during the rapid phase of freeze-thaw damage is a function of the polypeptide part of AFGP. The increased rate of plastocyanin loss at longer incubation times both at 0 degree C and at -20 degrees C may be mediated by the N-acetylgalactosamine moiety of the AFGP, but is strongly amplified by the polypeptide.

Proteins from frost-hardy leaves protect thylakoids against mechanical freeze-thaw damage in vitro.

We have isolated protein fractions from cold-acclimated, frost-hardy cabbage (Brassica oleracea L.) and spinach (Spinacia oleracea L.) leaves which protect isolated thylakoids from non-hardy spinach against mechanical membrane rupture during an in-vitro freeze-thaw cycle. No protective activity was found in similar preparations from non-hardy leaves. The proteins protected the membranes from damage by reducing their solute permeability during freezing and by increasing their expandability during thawing. The proteins act by increasing the resistance of the membranes against the osmotic stress to which they are exposed during a freeze-thaw cycle. In the absence of cryoprotectants this stress results in membrane rupture.

Membrane rupture is the common cause of damage to chloroplast membranes in leaves injured by freezing or excessive wilting.

The effects of freezing and desiccation of spinach leaves (Spinacia oleracea L. cv Yates) on the thylakoid membranes were assessed using antibodies specific for thylakoid membrane proteins. The peripheral part of the chloroplast coupling factor ATPase (CF1) was used as a molecular marker for chemical membrane damage by chaotropic solutes. Plastocyanin, a soluble protein localized inside the closed thylakoid membrane system, was a marker for damage by mechanical membrane rupture. After freezing and wilting of leaves which resulted in damage, very little CF1 was detached from the membranes, whereas almost all plastocyanin was released from the thylakoids. It is suggested that in vivo dehydration both by freezing and desiccation results in membrane rupture rather than in the dissociation of peripheral thylakoid membrane proteins.

Polypeptide pattern and enzymic character of vacuoles isolated from barley mesophyll protoplasts.

Intact chloroplasts and vacuoles were isolated from mesophyll protoplasts of barley. The chloroplasts occupied about 15% of the cellular volume and contained 75% of the protein, whereas the vacuoles occupied about 80% of the volume and contained less than 4% of total cellular protein. Contamination of the vacuolar fraction by foreign protein is included in these values. Chlorophyll was absent from the vacuolar fraction, but less than 1% of several extra-vacuolar marker proteins were still present. The vacuoles contained hydrolytic enzymes. Several of them (α-mannosidase, α-galactosidase, N-acetylglucosaminidase) were soluble, whereas part of the activity of others semimented with the tonoplasts during centrifugation. Attached proteins could be released from the membranes during freezing in the presence of NaCl. One-dimensional gel electrophoretic separation of soluble vacuolar proteins under non-denaturing conditions yielded more than 10 protein bands. A comparative analysis was performed of thylakoids and vacuoles which were subfractionated into tonoplasts and soluble vacuolar constituents. Sodium dodecyl sulfate gel electrophoresis separated about 15 polypeptides of the soluble fraction which reacted with silver reagent. The tonoplast fraction yielded about 20 bands. A similar number of bands was observed when vacuoles incubated with the (14)C-labelled SH-reagent N-ethylmaleimide were analysed for radioactive polypeptides. Silverstaining of the polypeptides and their SH-content did not correlate. Several polypeptides of the vacuolar fraction had molecular weights very similar to the molecular weights of known chloroplast proteins. However, with the exception of the two subunits of ribulose-1,5-bisphosphate carboxylase, contamination of the vacuolar fraction by chloroplast proteins could be ruled out as a possible cause of the close correspondence. The lipophilic carboxylic-group reagent N,N'-dicyclohexylcarbodiimide ([(14)C]DCCD) reacted with several polypeptides of thylakoids and tonoplasts. However, the labelling patterns were different. The most heavily labelled polypeptide of thylakoids was the 8-kDa polypeptide of the basal part of the coupling factor CF0. Tonoplast polypeptides heavily labelled with [(14)C]DCCD had molecular weights of 24, 28, and 56 kDa. The vacuolar 8-kDa polypeptide remained unlabelled.