Elena A. Golovina

Membrane Stabilization in the Dry State

Abstract We discuss current ideas of how membranes in desiccation-tolerant plant organ(ism)s are protected from the deleterious effect of complete water removal. Results of studies with model membranes showed that sugars play a major role in preventing fusion, phase transitions and most likely also phase separations. The sugars ability to form a stable glass and to interact directly with the phosphate of the phospholipid polar headgroup is the requirement for the protection of dry liposomes. Disaccharides alone fulfil these requirements. Dry membranes of desiccation tolerant plants in situ often have elevated phase transition temperatures (Tm) that are readily restored upon rehydration. Elevated Tm may point to insufficient interaction of sucrose with the polar headgroups. Attempts to observe this interaction in situ by analyzing the asymmetric phosphate stretching band failed. Thus, we suggest factors other than sugars in the suppression of Tm in intact cells and provide suggestions concerning potential roles of amphipathic compounds in this regard.

Membrane fluidity adjustments in ethanol-stressed Oenococcus oeni cells

ABSTRACT The effect of ethanol on the cytoplasmic membrane of Oenococcus oeni cells and the role of membrane changes in the acquired tolerance to ethanol were investigated. Membrane tolerance to ethanol was defined as the resistance to ethanol-induced leakage of preloaded carboxyfluorescein (cF) from cells. To probe the fluidity of the cytoplasmic membrane, intact cells were labeled with doxyl-stearic acids and analyzed by electron spin resonance spectroscopy. Although the effect of ethanol was noticeable across the width of the membrane, we focused on fluidity changes at the lipid-water interface. Fluidity increased with increasing concentrations of ethanol. Cells responded to growth in the presence of 8% (vol/vol) ethanol by decreasing fluidity. Upon exposure to a range of ethanol concentrations, these adapted cells had reduced fluidity and cF leakage compared with cells grown in the absence of ethanol. Analysis of the membrane composition revealed an increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells grown in the presence of 8% (vol/vol) ethanol. Preexposure for 2 h to 12% (vol/vol) ethanol also reduced membrane fluidity and cF leakage. This short-term adaptation was not prevented in the presence of chloramphenicol, suggesting that de novo protein synthesis was not involved. We found a strong correlation between fluidity and cF leakage for all treatments and alcohol concentrations tested. We propose that the protective effect of growth in the presence of ethanol is, to a large extent, based on modification of the physicochemical state of the membrane, i.e., cells adjust their membrane permeability by decreasing fluidity at the lipid-water interface.

An Electron Paramagnetic Resonance Spin-Probe Study of Membrane-Permeability Changes with Seed Aging

We developed an electron paramagnetic resonance spin-probe technique to study changes in the barrier properties of plasma membranes in wheat (Triticum aestivum L.) seeds during aging under dry storage. The estimation of these barrier properties was based on the differential permeability of membranes for the stable free radical 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy and the broadening agent ferricyanide. The line-height ratio between the water and lipid components in the electron paramagnetic resonance spectra of 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (R value) allowed for the quantitative assessment of the plasma membrane permeability in small samples, enabling separate studies of the axis, scutellum, aleurone layer, and starchy endosperm tissue. High R values corresponded to low permeability and vice versa. Starchy endosperm cells had completely permeable plasma membranes even in mature, viable seeds. The loss of germinability with aging coincided with a considerably increased plasma membrane permeability of the embryo axis cells, but not of the scutellum and aleurone layer cells. The threshold R value for the individual axes associated with viability loss was established at 5 to 6, with the total ranging from 0 to more than 12. We suggest that the R value of an individual axis is the result of contributions from all individual cells, each of them characterized by a different permeability. The loss of viability, therefore, corresponds to the accumulation of cells having permeability above a critical level.

Long-Term Stability of Protein Secondary Structure in Dry Seeds

Abstract Changes in protein secondary structure during seed aging were studied by in situ Fourier transform infrared microspectroscopy. Seeds of onion, white cabbage and radish, harvested in 1969, were stored at 15–20°C and 30% relative humidity. Fresh control seeds were harvested in 1994 and stored under the same conditions. In 1995, the germination capacity of seeds was >90% for the 1994 harvest and zero for the 1969 harvest. Inspection of the amide-I bands in Fourier self-deconvolved infrared spectra of thin slices of embryo axes of the various seeds did not reveal major changes in relative peak height and band position of the different protein secondary structures with aging. No protein aggregation and denaturation were found after long-term storage. Heating of dry viable radish seeds up to 145°C did not cause appreciable protein denaturation. In contrast, boiling hydrated radish seeds for 5 min led to decoiling of the α-helical structure and the appearance of a band at 1627 cm−1. This band is characteristic of intermolecular extended β-sheet structures. We conclude that, despite the loss of viability and the long postmortem storage period, secondary structure of proteins in desiccation tolerant dry seed is very stable and conserved during several decades of open storage.

Ageing increases the sensitivity of neem (Azadirachta indica) seeds to imbibitional stress

Imbibitional stress was imposed on neem (Azadirachta indica) seeds by letting them soak for 1 h in water at unfavourable, low temperatures before further incubation at 30degreesC. Sensitivity to low imbibition temperatures increased with a decrease in seed moisture content (MC). To investigate a possible involvement of seed age in the extent of imbibitional damage, initially high-quality seed lots that differed in storage history (10 weeks versus 10 months) were examined at 4 and 7% (fresh weight basis). After 10 months of storage, the 7% MC seeds had become sensitive to imbibitional stress. Further drying (1 week) to 4% MC affected aged seeds more than non-aged seeds. Barrier properties of cellular membranes in axes excised after 1 d of rehydration were estimated using a spin-probe technique. The proportion of cells with intact membranes increased with increasing imbibition temperature. For each temperature tested, there were more cells with leaky membranes after 10 months than after 10 weeks of dry storage. Localization of embryo cells displaying loss of turgor and abnormal cellular structure was accomplished using cryo-planing, followed by cryo-scanning electron microscopy. Inspection of the cryo-planed surfaces confirmed that imbibitional damage was temperature dependent, occurring at the periphery. Ageing increased the number of imbibitionally damaged, peripheral cell layers. Germination was estimated to fail when less than 70% of axis cells were alive. We conclude that ageing increases the sensitivity to imbibitional stress. Both the fast ageing and the sensitivity to imbibitional stress might explain the apparent controversies about neem seed desiccation tolerance and storage behaviour.

Spin-labeling study of membranes in wheat embryo axes. 1. Partitioning of doxyl stearates into the lipid domains

The interaction of lipid soluble spin labels with wheat embryo axes has been investigated to obtain insight into the structural organization of lipid domains in embryo cell membranes, using conventional electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopy. Stearic acid spin labels (n-SASL) and their methylated derivatives (n-MeSASL), labelled at different positions of their doxyl group (n=5, 12 and 16), were used to probe the ordering and molecular mobility in different regions of the lipid moiety of axis cell membranes. The ordering and local polarity in relation to the position of the doxyl group along the hydrocarbon chain of SASL, determined over the temperature range from -50 to +20 degrees C, are typical for biological and model lipid membranes, but essentially differ from those in seed oil droplets. Positional profiles for ST-EPR spectra show that the flexibility profile along the lipid hydrocarbon chain does exist even at low temperatures, when most of the membrane lipids are in solid state (gel phase). The ordering of the SASL nitroxide radical in the membrane surface region is essentially higher than that in the depth of the membrane. The doxyl groups of MeSASLs are less ordered (even at low temperatures) than those of the corresponding SASLs, indicating that the MeSASLs are located in the bulk of membrane lipids rather than in the protein boundary lipids. The analysis of the profiles of EPR and ST-EPR spectral parameters allows us to conclude that the vast majority of SASL and MeSASL molecules accumulated in embryo axes is located in the cell membranes rather than in the interior of the oil bodies. The preferential partitioning of the doxyl stearates into membranes demonstrates the potential of the EPR spin-labelling technique for the in situ study of membrane behavior in seeds of different hydration levels.

Desiccation Tolerance and Long Term Structural Stability

Suspended life in completely desiccated organisms requires the protection of proteins and phospholipids in the cellular membranes. Desiccation tolerant organisms usually contain large amounts of sugars. Di-, tri- and tetra—saccharides in particular, play a major role in preventing fusion, phase transitions and most likely phase separations in dry membranes. The ability of the sugars to form a glass and to directly interact with the polar headgroups of the phospholipids are the hypothesized mechanisms involved in this protection. Sugars also protect labile proteins from major structural rearrangements during dehydration.