Low temperature and high light dependent dynamic photoprotective strategies in Arabidopsis thaliana.

Arabidopsis thaliana has been recognized as a chilling tolerant species based on analysis of resistance to low temperature stress, however, the mechanisms involved in this tolerance are not yet clarified. The low temperature-induced effects are exacerbated when plants are exposed to low temperatures in the presence of high light irradiance but the experimental data on the impact of light intensity during cold stress and its influence during recovery from stress are rather limited. The main objective of this study was to re-examine the photosynthetic responses of A. thaliana plants to short term (6 days) low temperature stress (12/10°C) under optimal (150 µmol m-2 s-1 ) and high light (500 µmol m-2 s-1 ) intensity and the subsequent recovery from the stress. Simultaneous measurements of the in vivo and in vitro functional performance of both photosystem II (PSII) and photosystem I (PSI), as well as, net photosynthesis, low temperature (77 K) chlorophyll fluorescence and immunoblot analysis of the relative abundance of PSII and PSI reaction center proteins were used to evaluate the role of light in the development of possible protective mechanisms during low temperature stress and the consequent recovery from exposure to low temperature and different light intensities. The results presented clearly suggest that Arabidopsis plants can employ a number of highly dynamic photoprotective strategies depending on the light intensity. These strategies include one based on LHCII quenching and two other quenching mechanisms localized within the PSII and PSI reaction centers, which are all expressed to different extent depending on the severity of the photoinhibitory treatments under low temperature stress conditions.

Differential temperature effects on dissipation of excess light energy and energy partitioning in lut2 mutant of Arabidopsis thaliana under photoinhibitory conditions.

The high-light-induced alterations in photosynthetic performance of photosystem II (PSII) and photosystem I (PSI) as well as effectiveness of dissipation of excessive absorbed light during illumination for different periods of time at room (22 °C) and low (8-10 °C) temperature of leaves of Arabidopsis thaliana, wt and lut2, were followed with the aim of unraveling the role of lutein in the process of photoinhibition. Photosynthetic parameters of PSII and PSI were determined on whole leaves by PAM fluorometer and oxygen evolving activity-by a Clark-type electrode. In thylakoid membranes, isolated from non-illuminated and illuminated for 4.5 h leaves of wt and lut2 the photochemical activity of PSII and PSI and energy interaction between the main pigment-protein complexes was determined. Results indicate that in non-illuminated leaves of lut2 the maximum rate of oxygen evolution and energy utilization in PSII is lower, excitation pressure of PSII is higher and cyclic electron transport around PSI is faster than in wt leaves. Under high-light illumination, lut2 leaves are more sensitive in respect to PSII performance and the extent of increase of excitation pressure of PSII, ΦNO, and cyclic electron transport around PSI are higher than in wt leaves, especially when illumination is performed at low temperature. Significant part of the excessive light energy is dissipated via mechanism, not dependent on ∆pH and to functioning of xanthophyll cycle in LHCII, operating more intensively in lut2 leaves.

Effects of flavonol glycosides on liposome stability during freezing and drying.

Flavonoids are a large and diverse group of plant secondary metabolites that are mainly present as glycosides. They are often accumulated in response to abiotic stresses such as UV radiation, drought, cold and freezing. The most extensively studied function of flavonoids is their antioxidant activity although their importance as antioxidants in plants has been questioned. We therefore aim to study effects of flavonols on cellular stress tolerance that are independent of their antioxidant function. Here we investigate the effects of the glycosylated flavonols kaempferol-3-O-glucoside, kaempferol-7-O-glucoside, quercetin-3-O-glucoside and quercetin-3-O-rhamnoside on liposome stability after freezing and drying. Insertion of flavonols in lipid bilayers destabilized egg phosphatidylcholine (EPC) liposomes and to a lesser extent vesicles made from equal proportions of EPC and egg phosphatidylethanolamine (EPE) during a freeze-thaw cycle, while liposomes containing the unsaturated non-bilayer lipid 18:2 PE were either unaffected or slightly stabilized. In general, the kaempferol derivatives were more destabilizing for liposomes during freezing than the quercetin derivatives. Fourier-transform infrared spectroscopy revealed that all flavonols were localized in the interfacial region of the lipid bilayers, forming H-bonds with the lipid phosphate and carbonyl groups. The phase transition temperature of dry 16:0/18:1 PC (POPC) and POPC/EPE liposomes was decreased by 75°C and 55°C, respectively. Changes in the vibration bands attributed to the phenolic ring structures of the flavonols in the presence of liposomes provided further evidence of interactions of these molecules in particular with the interfacial region of the bilayers.

The intrinsically disordered protein LEA7 from Arabidopsis thaliana protects the isolated enzyme lactate dehydrogenase and enzymes in a soluble leaf proteome during freezing and drying.

The accumulation of Late Embryogenesis Abundant (LEA) proteins in plants is associated with tolerance against stresses such as freezing and desiccation. Two main functions have been attributed to LEA proteins: membrane stabilization and enzyme protection. We have hypothesized previously that LEA7 from Arabidopsis thaliana may stabilize membranes because it interacts with liposomes in the dry state. Here we show that LEA7, contrary to this expectation, did not stabilize liposomes during drying and rehydration. Instead, it partially preserved the activity of the enzyme lactate dehydrogenase (LDH) during drying and freezing. Fourier-transform infrared (FTIR) spectroscopy showed no evidence of aggregation of LDH in the dry or rehydrated state under conditions that lead to complete loss of activity. To approximate the complex influence of intracellular conditions on the protective effects of a LEA protein in a convenient in-vitro assay, we measured the activity of two Arabidopsis enzymes (glucose-6-P dehydrogenase and ADP-glucose pyrophosphorylase) in total soluble leaf protein extract (Arabidopsis soluble proteome, ASP) after drying and rehydration or freezing and thawing. LEA7 partially preserved the activity of both enzymes under these conditions, suggesting its role as an enzyme protectant in vivo. Further FTIR analyses indicated the partial reversibility of protein aggregation in the dry ASP during rehydration. Similarly, aggregation in the dry ASP was strongly reduced by LEA7. In addition, mixtures of LEA7 with sucrose or verbascose reduced aggregation more than the single additives, presumably through the effects of the protein on the H-bonding network of the sugar glasses.

Response of Tomato Plants, Ailsa Craig and Carotenoid Mutant tangerine, to Simultaneous Treatment by Low Light and Low Temperature.

Tomato (Solanum lycopersicum L.) plants, wild type Ailsa Craig, and carotenoid mutant tangerine that accumulates prolycopene instead of all-trans-lycopene were exposed to a combined treatment by low light and low temperature for 5 days. The ability of plants to recover from the stress after development for 3 days at control conditions was followed as well. The suffered oxidative stress was evaluated by the extent of pigment content, lipid peroxidation, membrane stability, and H2O2 generation. The level of MDA content under combined treatment in tangerine implies that the mutant demonstrates lower sensitivity to stress in comparison with Ailsa Craig. The oxidative protective strategy of plants was estimated by following the antioxidant and antiradical activity of phenolic metabolites, including anthocyanins, as well as the activities of antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT). Presented results revealed that the oxidative stress was much stronger expressed after exposure of both types of plants to low light combined with low temperature compared to that after treatment with only low light. The most significant antioxidant protection was provided by phenolic substances, including anthocyanins. The lower sensitivity of tangerine plants to low light can be attributed to the higher activity of the antioxidant enzyme CAT.

Effect of Low Light on Photosynthetic Performance of Tomato Plants-Ailsa Craig and Carotenoid Mutant Tangerine.

The effects of a five-day treatment with low light intensity on tomato plants-Ailsa Craig and tangerine mutant-at normal and low temperatures and after recovery for three days under control conditions were investigated. The tangerine tomato, which has orange fruits, yellowish young leaves, and pale blossoms, accumulates prolycopene rather than all-trans lycopene. We investigated the impact of low light at normal and low temperatures on the functioning and effectiveness of photosynthetic apparatuses of both plants. The photochemical activities of Photosystem I (PSI) and Photosystem II (PSII) were assessed, and the alterations in PSII antenna size were characterized by evaluating the abundance of PSII-associated proteins Lhcb1, Lhcb2, CP43, and CP47. Alterations in energy distribution and interaction of both photosystems were analyzed using 77K fluorescence. In Aisla Craig plants, an increase in thylakoid membrane fluidity was detected during treatment with low light at a low temperature, while for the tangerine mutant, no significant change was observed. The PSII activity of thylakoids from mutant tangerine was more strongly inhibited by treatment with low light at a low temperature while low light barely affected PSII in Aisla Craig. The obtained data indicated that the observed differences in the responses of photosynthetic apparatuses of Ailsa Craig and tangerine when exposed to low light intensity and suboptimal temperature were mainly related to the differences in sensitivity and antenna complexes of PSII.

Response of Pea Plants (Pisum sativum cv. Ran 1) to NaCl Treatment in Regard to Membrane Stability and Photosynthetic Activity.

Salinity is one of the most extreme abiotic stress factors that negatively affect the development and productivity of plants. The salt-induced injuries depend on the salt tolerance of the plant species, salt concentration, time of exposure and developmental stage. Here, we report on the response of pea plants (Pisum sativum L. cv Ran 1) to exposure to increasing salt concentrations (100, 150 and 200 mM NaCl) for a short time period (5 days) and the ability of the plants to recover after the removal of salt. The water content, membrane integrity, lipid peroxidation, pigment content and net photosynthetic rate were determined for the pea leaves of the control, treated and recovered plants. Salt-induced alterations in the primary photosynthetic reactions and energy transfer between the main pigment-protein complexes in isolated thylakoid membranes were evaluated. The pea plants were able to recover from the treatment with 100 mM NaCl, while at higher concentrations, concentration-dependent water loss, the disturbance of the membrane integrity, lipid peroxidation and an increase in the pigment content were detected. The net photosynthetic rate, electron transport through the reaction centers of PSII and PSII, activity of PSIIα centers and energy transfer between the pigment-protein complexes were negatively affected and were not restored after the removal of NaCl.

Antioxidative Defense, Suppressed Nitric Oxide Accumulation, and Synthesis of Protective Proteins in Roots and Leaves Contribute to the Desiccation Tolerance of the Resurrection Plant Haberlea rhodopensis.

The desiccation tolerance of plants relies on defense mechanisms that enable the protection of macromolecules, biological structures, and metabolism. Although the defense of leaf tissues exposed to solar irradiation is challenging, mechanisms that protect the viability of the roots, yet largely unexplored, are equally important for survival. Although the photosynthetic apparatus in leaves contributes to the generation of oxidative stress under drought stress, we hypothesized that oxidative stress and thus antioxidative defense is also predominant in the roots. Thus, we aimed for a comparative analysis of the protective mechanisms in leaves and roots during the desiccation of Haberlea rhodopensis. Consequently, a high content of non-enzymatic antioxidants and high activity of antioxidant enzymes together with the activation of specific isoenzymes were found in both leaves and roots during the final stages of desiccation of H. rhodopensis. Among others, catalase and glutathione reductase activity showed a similar tendency of changes in roots and leaves, whereas, unlike that in the leaves, superoxide dismutase activity was enhanced under severe but not under medium desiccation in roots. Nitric oxide accumulation in the root tips was found to be sensitive to water restriction but suppressed under severe desiccation. In addition to the antioxidative defense, desiccation induced an enhanced abundance of dehydrins, ELIPs, and sHSP 17.7 in leaves, but this was significantly better in roots. In contrast to leaf cells, starch remained in the cells of the central cylinder of desiccated roots. Taken together, protective compounds and antioxidative defense mechanisms are equally important in protecting the roots to survive desiccation. Since drought-induced damage to the root system fundamentally affects the survival of plants, a better understanding of root desiccation tolerance mechanisms is essential to compensate for the challenges of prolonged dry periods.

Different Responses to Water Deficit of Two Common Winter Wheat Varieties: Physiological and Biochemical Characteristics.

Since water scarcity is one of the main risks for the future of agriculture, studying the ability of different wheat genotypes to tolerate a water deficit is fundamental. This study examined the responses of two hybrid wheat varieties (Gizda and Fermer) with different drought resistance to moderate (3 days) and severe (7 days) drought stress, as well as their post-stress recovery to understand their underlying defense strategies and adaptive mechanisms in more detail. To this end, the dehydration-induced alterations in the electrolyte leakage, photosynthetic pigment content, membrane fluidity, energy interaction between pigment-protein complexes, primary photosynthetic reactions, photosynthetic and stress-induced proteins, and antioxidant responses were analyzed in order to unravel the different physiological and biochemical strategies of both wheat varieties. The results demonstrated that Gizda plants are more tolerant to severe dehydration compared to Fermer, as evidenced by the lower decrease in leaf water and pigment content, lower inhibition of photosystem II (PSII) photochemistry and dissipation of thermal energy, as well as lower dehydrins' content. Some of defense mechanisms by which Gizda variety can tolerate drought stress involve the maintenance of decreased chlorophyll content in leaves, increased fluidity of the thylakoid membranes causing structural alterations in the photosynthetic apparatus, as well as dehydration-induced accumulation of early light-induced proteins (ELIPs), an increased capacity for PSI cyclic electron transport and enhanced antioxidant enzyme activity (SOD and APX), thus alleviating oxidative damage. Furthermore, the leaf content of total phenols, flavonoids, and lipid-soluble antioxidant metabolites was higher in Gizda than in Fermer.

Structural transitions in the intrinsically disordered plant dehydration stress protein LEA7 upon drying are modulated by the presence of membranes.

Dehydration stress-related late embryogenesis abundant (LEA) proteins have been found in plants, invertebrates and bacteria. Most LEA proteins are unstructured in solution, but some fold into amphipathic α-helices during drying. The Pfam LEA_4 (Group 3) protein LEA7 from the higher plant Arabidopsis thaliana was predicted to be 87% α-helical, while CD spectroscopy showed it to be largely unstructured in solution and only 35% α-helical in the dry state. However, the dry protein contained 15% ß-sheets. FTIR spectroscopy revealed the ß-sheets to be largely due to aggregation. ß-Sheet content was reduced and α-helix content increased when LEA7 was dried in the presence of liposomes with secondary structure apparently influenced by lipid composition. Secondary structure was also affected by the presence of membranes in the fully hydrated state. A temperature-induced increase in the flexibility of the dry protein was also only observed in the presence of membranes. Functional interactions of LEA7 with membranes in the dry state were indicated by its influence on the thermotropic phase transitions of the lipids and interactions with the lipid headgroup phosphates.

Influence of drying on the secondary structure of intrinsically disordered and globular proteins.

Circular dichroism (CD) spectroscopy of five Arabidopsis late embryogenesis abundant (LEA) proteins constituting the plant specific families LEA_5 and LEA_6 showed that they are intrinsically disordered in solution and partially fold during drying. Structural predictions were comparable to these results for hydrated LEA_6, but not for LEA_5 proteins. FTIR spectroscopy showed that verbascose, but not sucrose, strongly affected the structure of the dry proteins. The four investigated globular proteins were only mildly affected by drying in the absence, but strongly in the presence of sugars. These data highlight the larger structural flexibility of disordered compared to globular proteins and the impact of sugars on the structure of both disordered and globular proteins during drying.

The Role of Alternative Electron Pathways for Effectiveness of Photosynthetic Performance of Arabidopsis thaliana, Wt and Lut2, under Low Temperature and High Light Intensity.

A recent investigation has suggested that the enhanced capacity for PSI-dependent cyclic electron flow (CEF) and PSI-dependent energy quenching that is related to chloroplast structural changes may explain the lower susceptibility of lut2 to combined stresses-a low temperature and a high light intensity. The possible involvement of alternative electron transport pathways, proton gradient regulator 5 (PGR5)-dependent CEF and plastid terminal oxidase (PTOX)-mediated electron transfer to oxygen in the response of Arabidopsis plants-wild type (wt) and lut2-to treatment with these two stressors was assessed by using specific electron transport inhibitors. Re-reduction kinetics of P700+ indicated that the capacity for CEF was higher in lut2 when this was compared to wt. Exposure of wt plants to the stress conditions caused increased CEF and was accompanied by a substantial raise in PGR5 and PTOX quantities. In contrast, both PGR5 and PTOX levels decreased under the same stress conditions in lut2, and inhibiting PGR5-dependent pathway by AntA did not exhibit any significant effects on CEF during the stress treatment and recovery period. Electron microscopy observations demonstrated that under control conditions the degree of grana stacking was much lower in lut2, and it almost disappeared under the combined stresses, compared to wt. The role of differential responses of alternative electron transport pathways in the acclimation to the stress conditions that are studied is discussed.

Reactivation of the Photosynthetic Apparatus of Resurrection Plant Haberlea rhodopensis during the Early Phase of Recovery from Drought- and Freezing-Induced Desiccation.

Haberlea rhodopensis is a unique desiccation-tolerant angiosperm that also survives winter frost. As, upon freezing temperatures, H. rhodopensis desiccates, the taxon is proposed to survive low temperature stress using its desiccation tolerance mechanisms. To reveal the validity of this hypothesis, we analyzed the structural alterations and organization of photosynthetic apparatus during the first hours of recovery after drought- and freezing-induced desiccation. The dynamics of the ultrastructure remodeling in the mesophyll cells and the restoration of the thylakoid membranes shared similarities independent of the reason for desiccation. Among the most obvious changes in thylakoid complexes, the proportion of the PSI-LHCII complex strongly increased around 70% relative water content (RWC), whereas the proportion of Lhc monomers decreased from the beginning of rehydration. We identified enhanced levels of cyt b6f complex proteins that contributed to the enhanced electron flow. The high abundance of proteins related to excitation energy dissipation, PsbS, Lhcb5, Lhcb6 and ELIPs, together with the increased content of dehydrins contributed to the preservation of cellular integrity. ELIP expression was maintained at high levels up to 9 h into recovery. Although the recovery processes from drought- and freezing-induced desiccation were found to be similar in progress and time scale, slight variations indicate that they are not identical.

Interaction of two intrinsically disordered plant stress proteins (COR15A and COR15B) with lipid membranes in the dry state.

COR15A and COR15B form a tandem repeat of highly homologous genes in Arabidopsis thaliana. Both genes are highly cold induced and the encoded proteins belong to the Pfam LEA_4 group (group 3) of the late embryogenesis abundant (LEA) proteins. Both proteins were predicted to be intrinsically disordered in solution. Only COR15A has previously been characterized and it was shown to be localized in the soluble stroma fraction of chloroplasts. Ectopic expression of COR15A in Arabidopsis resulted in increased freezing tolerance of both chloroplasts after freezing and thawing of intact leaves and of isolated protoplasts frozen and thawed in vitro. In the present study we have generated recombinant mature COR15A and COR15B for a comparative study of their structure and possible function as membrane protectants. CD spectroscopy showed that both proteins are predominantly unstructured in solution and mainly alpha-helical after drying. Both proteins showed similar effects on the thermotropic phase behavior of dry liposomes. A decrease in the gel to liquid-crystalline phase transition temperature depended on both the unsaturation of the fatty acyl chains and lipid headgroup structure. FTIR spectroscopy indicated no strong interactions between the proteins and the lipid phosphate and carbonyl groups, but significant interactions with the galactose headgroup of the chloroplast lipid monogalactosyldiacylglycerol. These findings were rationalized by modeling the secondary structure of COR15A and COR15B. Helical wheel projection indicated the presence of amphipathic alpha-helices in both proteins. The helices lacked a clear separation of positive and negative charges on the hydrophilic face, but contained several hydroxylated amino acids.

Stabilization of Dry Sucrose Glasses by Four LEA_4 Proteins from Arabidopsis thaliana.

Cells of many organisms and organs can withstand an (almost) total water loss (anhydrobiosis). Sugars play an essential role in desiccation tolerance due to their glass formation ability during dehydration. In addition, intrinsically disordered LEA proteins contribute to cellular survival under such conditions. One possible mechanism of LEA protein function is the stabilization of sugar glasses. However, little is known about the underlying mechanisms. Here we used FTIR spectroscopy to investigate sucrose (Suc) glass stability dried from water or from two buffer components in the presence of four recombinant LEA and globular reference proteins. Buffer ions influenced the strength of the Suc glass in the order Suc < Suc/Tris < Suc/NaP. LEA proteins strengthened the sugar H-bonded network and the molecular structure in the glassy state. The position of νOH peak and the wavenumber-temperature coefficient (WTCg) provided similar information about the H-bonded network. Protein aggregation of LEA proteins was reduced in the desiccation-induced Suc glassy state. Detailed knowledge about the role of LEA proteins in the stabilization of dry sugar glasses yields information about their role in anhydrobiosis. This may open the possibility to use such proteins in biotechnical applications requiring dry storage of biologicals such as proteins, cells or tissues.

Effect of membrane fluidity on photosynthetic oxygen production reactions.

The effect of changes of membrane fluidity on the oxygen evolving capability of isolated thylakoids was investigated. Alteration of the lipid phase fluidity was achieved by incorporation of the plant sterol stigmasterol. Incorporation of stigmasterol in the lipid bilayer of thylakoid membranes results in rigidization of the hydrophobic phase of thylakoid membranes and decreases the degree of packing of the lipid head groups. These changes of lipid order are accompanied by a reduction of oxygen evolution, measured with 1,4-benzoquinone as an electron acceptor, and by a more pronounced inhibition of PSI-mediated electron transport. By analysis of the parameters of oxygen flash yields and oxygen burst under continuous illumination it was shown that after treatment with stigmasterol: 1.) the number of active oxygen-evolving centres decreased; 2.) the remaining active oxygen-evolving centres were not affected in respect to the oscillation pattern; 3.) the contribution of the slow oxygen-evolving centres in oxygen burst yield was increased. The effect of stigmasterol was compared with the well-studied effect of cholesterol. Results were discussed in terms of determining the role of lipid order for the organization and functioning of the photosynthetic machinery.

The Lack of Lutein Accelerates the Extent of Light-induced Bleaching of Photosynthetic Pigments in Thylakoid Membranes of Arabidopsis thaliana.

The high light-induced bleaching of photosynthetic pigments and the degradation of proteins of light-harvesting complexes of PSI and PSII were investigated in isolated thylakoid membranes of Arabidopsis thaliana, wt and lutein-deficient mutant lut2, with the aim of unraveling the role of lutein for the degree of bleaching and degradation. By the means of absorption spectroscopy and western blot analysis, we show that the lack of lutein leads to a higher extent of pigment photobleaching and protein degradation in mutant thylakoid membranes in comparison with wt. The highest extent of bleaching is suffered by chlorophyll a and carotenoids, while chlorophyll b is bleached in lut2 thylakoids during long periods at high illumination. The high light-induced degradation of Lhca1, Lhcb2 proteins and PsbS was followed and it is shown that Lhca1 is more damaged than Lhcb2. The degradation of analyzed proteins is more pronounced in lut2 mutant thylakoid membranes. The lack of lutein influences the high light-induced alterations in organization of pigment-protein complexes as revealed by 77 K fluorescence.

Tomato plants acclimate better to elevated temperature and high light than to treatment with each factor separately.

The influence of two factors - high temperature and high light intensity, acting separately or simultaneously on the pigment composition, fluorescent characteristics, membrane integrity and synthesis of protective substances was investigated in tomato plants (Solanum lycopersicum cv. M 82). Moderate elevated temperatures (38/29 °C) were applied under optimum or high light intensity for 2 and 6 days and after that the plants are allowed to recover for 5 days at optimum conditions. Parameters of chlorophyll fluorescence were used to evaluate the alterations of photosystem I and photosystem II activity and malondialdehyde content was determined as a measure of stress-induced peroxidation of membrane lipids. The response of treated plants to high light and elevated temperature was estimated by analyzing the accumulation of anthocyanins. Both stress factors exhibit different impact on studied parameters - high light intensity influences considerably quantum yield of photosystem II and photochemical quenching that is compensated to some extent when applied at elevated temperature. High temperature reduces strongly non-photochemical quenching. Data obtained show that after two days under particular conditions, the plants tend to acclimate, but this is achieved after longer treatment - 6 days. During the recovery period the activity of photosystem I and the quantum yield of photosystem II recover almost completely, while the values of non-photochemical quenching although slightly higher, did not reach the levels at the beginning of treatment.

Differential destabilization of membranes by tryptophan and phenylalanine during freezing: the roles of lipid composition and membrane fusion.

The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.

Specific interactions of tryptophan with phosphatidylcholine and digalactosyldiacylglycerol in pure and mixed bilayers in the dry and hydrated state.

Amphiphilic solutes play an important role in the desiccation tolerance of plant cells, because they can reversibly partition into cellular membranes during dehydration. Their effects on membrane stability depend on their chemical structure, but also on the lipid composition of the host membrane. We have shown recently that tryptophan destabilizes liposomes during freezing. The degree of destabilization depends on the presence of glycolipids in the membranes, but not on the phase preference (bilayer or non-bilayer) of the lipids in mixtures with the bilayer lipid phosphatidylcholine. Here, we have investigated the influence of tryptophan on the phase behavior and intermolecular interactions in dry and hydrated bilayers made from the phospholipid egg phosphatidylcholine and the plant chloroplast glycolipid digalactosyldiacylglycerol, or from a mixture (1:1) of these lipids, using Fourier-transform infrared spectroscopy. To distinguish effects of the hydrophobic ring structure of tryptophan from those of the amino acid moiety, we also performed experiments with the hydrophilic amino acid glycine. Our data show that there are specific interactions between tryptophan and either phospholipid or glycolipid in the dry state, as well as H-bonding interactions between the lipids and both solutes. In the rehydrated state, the H-bonding interactions between amino acids and lipids are mostly replaced by interactions between water and lipids, while the hydrophobic interactions between lipids and tryptophan mostly persist.