Glass formation in plant anhydrobiotes: survival in the dry state

Anhydrobiotes can resist complete dehydration and survive the dry state for extended periods of time. During drying, cytoplasmic viscosity increases dramatically and in the dry state, the cytoplasm transforms into a glassy state. Plant anhydrobiotes possess large amounts of soluble non-reducing sugars and their state diagrams resemble those of simple sugar mixtures. However, more detailed in vivo measurements using techniques such as Fourier transform infrared spectroscopy and electron paramagnetic resonance spectroscopy reveal that these intracellular glasses are complex systems with properties quite different from those of simple sugar glasses. Intracellular glasses exhibit a high molecular packing and slow molecular mobility, resembling glasses made of mixtures of proteins and sugars, which potentially interact with additional cytoplasmic components such as salts, organic acids, and amino acids. Above the glass transition temperature, the cytoplasm of biological systems still exhibits a high stability and low molecular mobility, which could serve as an ecological advantage. All desiccation-tolerant organisms form glasses upon drying, but desiccation-sensitive organisms generally lose their viability during drying at water contents at which the glassy state has not yet been formed, suggesting that other factors are necessary for desiccation tolerance. Nevertheless, the formation of intracellular glasses is indispensable to survive the dry state. Storage stability of seeds and pollens is related to the molecular mobility and packing density of the intracellular glass, suggesting that the characteristic properties of intracellular glasses provide stability for long-term survival.     

High critical temperature above T(g) may contribute to the stability of biological systems

In this study, we characterized the molecular mobility around T(g) in sugars, poly-L-lysine and dry desiccation-tolerant biological systems, using ST-EPR, (1)H-NMR, and FTIR spectroscopy, to understand the nature and composition of biological glasses. Two distinct changes in the temperature dependence of the rotational correlation time (tau(R)) of the spin probe 3-carboxy-proxyl or the second moment (M(2)) were measured in sugars and poly-L-lysine. With heating, the first change was associated with the melting of the glassy state (T(g)). The second change (T(c)), at which tau(R) abruptly decreased over several orders of magnitude, was found to correspond with the so-called cross-over temperature, where the dynamics changed from solid-like to liquid-like. The temperature interval between T(g) and T(c) increased in the order of sucrose < trehalose < raffinose 50 degrees C, implying that the stability above T(g) improved in the same order. These differences in temperature-dependent mobilities above T(g) suggest that proteins rather than sugars play an important role in the intracellular glass formation. The exceptionally high T(c) of intracellular glasses is expected to provide excellent long-term stability to dry organisms, maintaining a slow molecular motion in the cytoplasm even at temperatures far above T(g).     

Is there a role for oligosaccharides in seed longevity? An assessment of intracellular glass stability

We examined whether oligosaccharides extend seed longevity by increasing the intracellular glass stability. For that purpose, we used a spin probe technique to measure the molecular mobility and glass transition temperature of the cytoplasm of impatiens (Impatiens walleriana) and bell pepper (Capsicum annuum) seeds that were osmo-primed to change oligosaccharide content and longevity. Using saturation transfer electron paramagnetic resonance spectroscopy, we found that the rotational correlation time of the polar spin probe 3-carboxy-proxyl in the cytoplasm decreased, together with longevity, as a function of increasing seed water content, suggesting that longevity may indeed be regulated by cytoplasmic mobility. Osmo-priming of the seeds resulted in considerable decreases in longevity and oligosaccharide content, while the sucrose content increased. No difference in the glass transition temperature was found between control and primed impatiens seeds at the same temperature and water content. Similarly, there was no difference in the rotational motion of the spin probe in the cytoplasm between control and primed impatiens and bell pepper seeds. We therefore conclude that oligosaccharides in seeds do not affect the stability of the intracellular glassy state, and that the reduced longevity after priming is not the result of increased molecular mobility in the cytoplasm.     

A Calorimetric Study of the Glass Transition Behaviors in Axes of Bean Seeds with Relevance to Storage Stability

Although the presence of intracellular aqueous glasses has been established in seeds, their physiological role in storage stability is still conjectural. Therefore, we examined, using differential scanning calorimetry, the thermal behavior of glass transitions in axes of bean (Phaseolus vulgaris L.) with water contents (WC) between 0 and 1 g H2O/g dry weight (g/g) and temperatures between -120 and +120[deg]C. Three types of thermal behaviors associated with the glass transition were observed. The appearance, the glass -> liquid transition temperature, and the amount of energy released during these transitions were dependent on the tissue WC. No glass transitions were observed at WC lower than 0.03 and higher than 0.45 g/g. A brief exposure to 100[deg]C altered the glass properties of tissues with WC between 0.03 and 0.08 g/g but did not affect the thermal behavior of glasses with higher WC, demonstrating that thermal history is important to the intracellular glass behavior at lower WC. Correspondence of data from bean to models predicting the effects of glass components on the glass -> liquid transition temperature suggests that the intracellular glasses are composed of a highly complex sugar matrix, in which sugar and water molecules interact together and influence the glass properties. Our data provide evidence that additional glass properties must be characterized to understand the implications of a glassy state in storage stability of seeds.     

Characterization of molecular mobility in seed tissues: an electron paramagnetic resonance spin probe study.

The relationship between molecular mobility (tauR) of the polar spin probe 3-carboxy-proxyl and water content and temperature was established in pea axes by electron paramagnetic resonance (EPR) and saturation transfer EPR. At room temperature, tauR increased during drying from 10(-11) s at 2.0 g water/g dry weight to 10(-4) s in the dry state. At water contents below 0.07 g water/g dry weight, tauR remained constant upon further drying. At the glass transition temperature, tauR was constant at approximately 10(-4) s for all water contents studied. Above Tg, isomobility lines were found that were approximately parallel to the Tg curve. The temperature dependence of tauR at all water contents studied followed Arrhenius behavior, with a break at Tg. Above Tg the activation energy for rotational motion was approximately 25 kJ/mol compared to 10 kJ/mol below Tg. The temperature dependence of tauR could also be described by the WLF equation, using constants deviating considerably from the universal constants. The temperature effect on tauR above Tg was much smaller in pea axes, as found previously for sugar and polymer glasses. Thus, although glasses are present in seeds, the melting of the glass by raising the temperature will cause only a moderate increase in molecular mobility in the cytoplasm as compared to a huge increase in amorphous sugars.     

Influence of Water Content and Temperature on Molecular Mobility and Intracellular Glasses in Seeds and Pollen

Although the occurrence of intracellular glasses in seeds and pollen has been established, physical properties such as rotational correlation times and viscosity have not been studied extensively. Using electron paramagnetic resonance spectroscopy, we examined changes in the molecular mobility of the hydrophilic nitroxide spin probe 3-carboxy-proxyl during melting of intracellular glasses in axes of pea (Pisum sativum L.) seeds and cattail (Typha latifolia L.) pollen. The rotational correlation time of the spin probe in intracellular glasses of both organisms was approximately 10⁻³ s. Using the distance between the outer extrema of the electron paramagnetic resonance spectrum (2A_zz) as a measure of molecular mobility, we found a sharp increase in mobility at a definite temperature during heating. This temperature increased with decreasing water content of the samples. Differential scanning calorimetry data on these samples indicated that this sharp increase corresponded to melting of the glassy matrix. Molecular mobility was found to be inversely correlated with storage stability. With decreasing water content, the molecular mobility reached a minimum, and increased again at very low water content. Minimum mobility and maximum storage stability occurred at a similar water content. This correlation suggests that storage stability might be at least partially controlled by molecular mobility. At low temperatures, when storage longevity cannot be determined on a realistic time scale, 2A_zz measurements can provide an estimate of the optimum storage conditions.     

Glassy State and Seed Storage Stability: The WLF Kinetics of Seed Viability Loss at T>Tgand the Plasticization Effect of Water on Storage Stability

The relationship between the glassy state in seeds and storage stability was examined, using the glass transition curve and a seed viability database from previous experiments. Storage data for seeds at various water contents were studied by Williams–Landel–Ferry (WLF) kinetics, whereas the glass transition curves of seeds with different storage stability were analysed by the Gordon–Taylor equation in terms of the plasticization effect of water on seed storage stability. It was found that the critical temperatures (T_c) for long-term storage of three orthodox seeds were near or below their glass transition temperatures (T_g), indicating the requirement for the presence of the glassy state for long-term seed storage. The rate of seed viability loss was a function of T-T_gat T>T_g, which fitted the WLF equation well, suggesting that storage stability was associated with the glass transition, and that the effect of water content on seed storage was correlated with the plasticization effect of water on intracellular glasses. A preliminary examination suggested a possible link between the glass transition curve and seed storage stability. According to the determined WLF constants, intracellular glasses in seeds fell into the second class of amorphous systems as defined by Slade and Levine (Critical Reviews in Food Science and Nutrition30: 115–360, 1991). These results support the interpretation that the glassy state plays an important role in storage stability and should be a major consideration in optimizing storage conditions.     

Carbohydrate polymers in amorphous states: an integrated thermodynamic and nanostructural investigation

The effect of water on the structure and physical properties of amorphous polysaccharide matrices is investigated by combining a thermodynamic approach including pressure- and temperature-dependent dilatometry with a nanoscale analysis of the size of intermolecular voids using positron annihilation lifetime spectroscopy. Amorphous polysaccharides are of interest because of a number of unusual properties which are likely to be related to the extensive hydrogen bonding between the carbohydrate chains. Uptake of water by the carbohydrate matrices leads to a strong increase in the size of the holes between the polymer chains in both the glassy and rubbery states while at the same time leading to an increase in matrix free volume. Thermodynamic clustering theory indicates that, in low-moisture carbohydrate matrices, water molecules are closely associated with the carbohydrate chains. Based on these observations, we propose a novel model of plasticization of carbohydrate polymers by water in which the water dynamically disrupts chains the hydrogen bonding between the carbohydrates, leading to an expansion of the matrix originating at the nanolevel and increasing the number of degrees of freedom of the carbohydrate chains. Consequently, even in the glassy state, the uptake of water leads to increased rates of matrix relaxation and mobility of small permeants. In contrast, low-molecular weight sugars plasticize the carbohydrate matrix without appreciably changing the structure and density of the rubbery state, and their role as plasticizer is most likely related to a reduction of the number of molecular entanglements. The improved molecular packing in glassy matrices containing low molecular weight sugars leads to a higher matrix density, explaining, despite the lower glass transition temperature, the reduced mobility of small permeants in such matrices.     

The effects of moisture and temperature on the ageing kinetics of pollen: interpretation based on cytoplasmic mobility

This study shows that characterization of the molecular mobility in the cytoplasm of pollen provides a new understanding of the effects of moisture and temperature on ageing rates. Using EPR spectroscopy, we determined the rotational motion of the polar spin probe, 3-carboxy-proxyl, in the cytoplasm of Typha latifolia pollen, under different temperature and moisture content conditions. Increasing the temperature resulted in faster rotational motion, analogous to faster ageing rates. With decreasing moisture content, rotational motion first decreased until a minimum was reached, after which rotational motion slightly increased again. The moisture content at which this minimal rotational motion was observed increased with decreasing temperature, comparable to the pattern of ageing rate. A significant linear relationship was found between ageing rates and rotational motion in the cytoplasm, suggesting that these parameters are causally linked. Upon melting of the intracellular glass, a twofold increase in activation energy of rotational motion and ageing rate was observed. In contrast, melting of the sucrose glass resulted in an increase in rotational motion of five orders of magnitude. The difference in rotational motion upon melting glasses of pollen or sucrose suggests that other molecules beside sugars play a role in intracellular glass formation in pollen.     

Glassy State and Seed Storage Stability: A Viability Equation Analysis

Dry seeds exist generally in a glassy (or vitrified) state.The high viscosity of the glassy state would be expected tohave a retarding effect on deteriorative reactions in the cytoplasm.Thus the glassy state may be considered to be a biophysicalbarrier for seed deterioration. The present study aims to testthe hypothesis that seed storage stability is associated withthe glassy state. With the equations derived from the seed viabilityequation, we have calculated the maximum temperature (Tmax)for long-term storage of corn, pea and soybeans. The Tmax forlong-term seed storage is found to be in a good agreement withthe glass transition temperature (Tg) in each instance, suggestingthat seed deterioration would be accelerated when seeds arenot in the glassy state. Experiments with soybeans given acceleratedageing show that the loss of glassy state is followed by a rapiddecrease in seed viability. These observations provide indirectevidence that the glassy state may play a significant role inseed storage stability.Copyright 1994, 1999 Academic Press Glassy state, seed longevity, storage stability, viability analysis     

Mechanisms of seed ageing under different storage conditions for Vigna radiata (L.) Wilczek: lipid peroxidation,sugar hydrolysis,Maillard reactions and their relationship to glass state transition

Two primary biochemical reactions in seed ageing (lipid peroxidation and non-enzymatic protein glycosylation with reducing sugars) have been studied under different seed water contents and storage temperatures, and the role of the glassy state in retarding biochemical deterioration examined. The viability loss of Vigna radiata seeds during storage is associated with Maillard reactions; however, the contribution of primary biochemical reactions varies under different storage conditions. Biochemical deterioration and viability loss are greatly retarded in seeds stored below a high critical temperature (approximately 40 degrees C above glass transition temperature). This high critical temperature corresponds to the cross-over temperature (T(c)) of glass transition where molecular dynamics changes from a solid-like system to a normal liquid system. The data show that seed ageing slows down significantly, even before seed tissue enters into the glassy state.     

Temperature dependency of molecular mobility in preserved seeds

Although cryogenic storage is presumed to provide nearly infinite longevity to cells, the actual timescale for changes in viability has not been addressed theoretically or empirically. Molecular mobility within preserved biological materials provides a first approximation of the rate of deteriorative reactions that ultimately affect shelf-life. Here, temperature effects on molecular mobility in partially dried seeds are calculated from heat capacities, measured using differential scanning calorimetry, and models for relaxation of glasses based on configurational entropy. Based on these analyses, glassy behavior in seeds containing 0.07 g H(2)O/g dm followed strict Vogel-Tamman-Fulcher (VTF) behavior at temperatures above and just below the glass transition temperature (Tg) at 28 degrees C. Temperature dependency of relaxation times followed Arrhenius kinetics as temperatures decreased well below Tg. The transition from VTF to Arrhenius kinetics occurred between approximately 5 and -10 degrees C. Overall, relaxation times calculated for seeds containing 0.07 g H(2)O/g dm decreased by approximately eight orders of magnitude when seeds were cooled from 60 to -60 degrees C, comparable to the magnitude of change in aging kinetics reported for seeds and pollen stored at a similar temperature range. The Kauzmann temperature (T(K)), often considered the point at which molecular mobility of glasses is practically nil, was calculated as -42 degrees C. Calculated relaxation times, temperature coefficients lower than expected from VTF kinetics, and T(K) that is 70 degrees C below Tg suggest there is molecular mobility, albeit limited, at cryogenic temperatures.     

Pools of water in anhydrobiotic organisms: A thermally stimulated depolarization current study

The ability to survive the removal of water in anhydrous biosystems is especially remarkable as a departure from the manifold structural and functional dependences on the presence of H₂O molecules. Identifiable pools of water present in dry soybean axes were investigated by means of the thermally stimulated depolarization current method. Samples were examined in the temperature range 100-340 K and over water contents (h, in gram H₂O per gram sample dry weight) ranging from h = 0.05 to 0.30 g/g. Three water-dependent relaxation mechanisms were detected; one attributed to dipolar reorientation of H₂O molecules hydrogen-bonded to other water molecules, one to reorientation of CH₂OH groups, and one to a glass transition in sugar-water domains. These glassy domains can protect intracellular components against destruction in the dehydrated state. Interestingly, protecting glassy domains were not found in dehydration intolerant seeds, supporting the hypothesis that the ability to withstand dehydration is associated with intracellular glass formation. A model for the state of cell water at interfaces is proposed.     

Control of electron transfer by protein dynamics in photosynthetic reaction centers

Abstract

Trehalose and glycerol are low molecular mass sugars/polyols that have found widespread use in the protection of native protein states, in both short- and long-term storage of biological materials, and as a means of understanding protein dynamics. These myriad uses are often attributed to their ability to form an amorphous glassy matrix. In glycerol, the glass is formed only at cryogenic temperatures, while in trehalose, the glass is formed at room temperature, but only upon dehydration of the sample. While much work has been carried out to elucidate a mechanistic view of how each of these matrices interact with proteins to provide stability, rarely have the effects of these two independent systems been directly compared to each other. This review aims to compile decades of research on how different glassy matrices affect two types of photosynthetic proteins: (i) the Type II bacterial reaction center from Rhodobacter sphaeroides and (ii) the Type I Photosystem I reaction center from cyanobacteria. By comparing aggregate data on electron transfer, protein structure, and protein dynamics, it appears that the effects of these two distinct matrices are remarkably similar. Both seem to cause a “tightening” of the solvation shell when in a glassy state, resulting in severely restricted conformational mobility of the protein and associated water molecules. Thus, trehalose appears to be able to mimic, at room temperature, nearly all of the effects on protein dynamics observed in low temperature glycerol glasses.     

The role of sugar, vitrification and membrane phase transition in seed desiccation tolerance

In a search for the mechanism of desiccation tolerance, a comparison was made between orthodox (desiccation-tolerant) soybean ( Glycine max [L.] Merrill) and recalcitrant (desiccation-intolerant) red oak ( Quercus rubra L.) seeds. During the maturation of soybean seeds, desiccation tolerance of seed axes is correlated with increases in sucrose, raffinose and stachyose. In cotyledons of mature oak seeds, sucrose levels are equal to those in mature soybeans, but oligosaccharides are absent. By using the thermally stimulated current method, we observed the glassy state in dry soybean seeds during maturation. Oak cotyledons showed the same phase diagram for the glass transition as did mature soybeans. By using X-ray diffraction, we found the maturation of soybeans to be associated with an increased ability of membranes to retain the liquid crystalline phase upon drying, whereas the mature oak cotyledonary tissue existed in the gel phase under similar dry conditions. These findings lead to the conclusion that the glassy state is not sufficient for desiccation tolerance, whereas the ability of membranes to retain the liquid crystalline phase does correlate with desiccation tolerance. An important role for soluble sugars in desiccation tolerance is confirmed, as well as their relevance to membrane phase changes. However, the presence of soluble sugars does not adequately explain the nature of desiccation tolerance in these seeds.     

Investigating variables and mechanisms that influence protein integrity in low water content amorphous carbohydrate matrices

Biopharmaceutical proteins are often formulated and freeze dried in agents that protect them from deleterious reactions that can compromise activity and authenticity. Although such approaches are widely used, a detailed understanding of the molecular mechanisms of protein stabilization in low water content amorphous glasses is lacking. Further, whilst deterioration chemistries are well described in dilute solution, relatively little is known about the extent and mechanisms by which protein integrity is compromised in the glassy state. Here we have investigated the relationship between protein modification and rate thereof, with variation of pH, carbohydrate excipient, temperature and the glass transition temperature using a model protein, lysozyme. Mass spectrometry analysis and peptide mapping confirm that protein modifications do occur in the glassy state in a time‐, temperature‐, and carbohydrate excipient‐dependent manner. There were clear trends between the buffer pH and the primary modification detected (glycation). Most importantly, there were differences in the apparent reactivities of the lysine residues in the glass compared with those previously determined in solution, and therefore, the well‐characterized solution reactivity of this reaction cannot be used to predict likely sites of modification in the glassy state. These findings have implications for (i) the selection and combinations of formulation components, particularly with regard to glycation in the glassy state, and (ii) the design of procedures and methodologies for the improvement of protein stability in the glassy state. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Molecular mobility and dynamic site heterogeneity in amorphous lactose and lactitol from erythrosin B phosphorescence

We have used phosphorescence from erythrosin B to characterize the molecular mobility and dynamic heterogeneity in dry films of amorphous lactose and lactitol from -25 to 120 degrees C. The phosphorescence emission spectra red-shifted and broadened with temperature in both sugars, indicating that both the rate of dipolar relaxation and the extent of inhomogeneous broadening increased dramatically at higher temperature. Phosphorescence intensity decays were well fit using a stretched exponential decay model; the rate constant for non-radiative quenching due to collisions with the matrix was calculated from the lifetimes. Arrhenius plots of this rate were non-linear, increasing very gradually at low and dramatically at high temperatures in both sugars. The rate of quenching was significantly lower in a 1:1 (wt/wt) mixture of lactose/lactitol in both the glass and the melt, providing strong evidence that specific interactions within the mixture lowered the matrix mobility. The lifetimes varied systematically with emission wavelength in both matrixes; analysis of the temperature dependence indicated that the activation energy for non-radiative quenching of the triplet state varied somewhat with emission wavelength. Time-resolved emission spectra collected as a function of delay time following pulsed excitation exhibited significant shifts to higher energy as a function of time. These data support a photophysical model in which erythrosin B molecules are distributed among matrix sites that vary such that blue-emitting sites with slower rates of matrix dipolar relaxation also have slower rates of molecular collisions. The amorphous matrixes of lactose and lactitol in both the glass and the melt state are thus characterized by dynamic site heterogeneity in which different sites vary in terms of their overall molecular mobility.     

The glassy state in corn embryos

The possibility is examined whether seeds may survive the desiccated state in part by vitrification, or the formation of a glassy state. Embryos excised from viable corn (Zea mays L.) seeds at low moisture contents show a series of low temperature first- and second-order phase transitions in the differential scanning calorimeter. These embryos produce normal seedlings if moistened. The thermal events can be duplicated almost entirely in both extracted lipids and purified commercial corn oil. They are therefore associated primarily with these bulk lipids, since membrane phospholipids are present in too small an amount to produce a detectable signal. When the bulk lipids have been extracted, a glass transition appears in the remaining material. At low water contents, it occurs above +40°C and systematically falls to below −60°C as the water content of the embryo rises to 20%. These data are consistent with our hypothesis that the desiccated state in seeds is a glassy state, and that imbibition of water reduces the glass transition temperature below ambient, allowing biochemical activity to resume.     

The preservation of liposomes by raffinose family oligosaccharides during drying is mediated by effects on fusion and lipid phase transitions

Raffinose family oligosaccharides (RFO) have been implicated as protective agents in the cellular dehydration tolerance, especially of many plant seeds. However, their efficacy in stabilizing membranes during dehydration has never been systematically investigated. We have analyzed the effects of sucrose, raffinose, stachyose, and verbascose on liposome stability during air-drying. With increasing degree of polymerization (DP), the RFO were progressively better able to stabilize liposomes against leakage of aqueous content and against membrane fusion after rehydration. Indeed, there was a very tight linear correlation between fusion and leakage for all RFO. These data indicate that increased protection of liposomes against leakage with increasing DP is due to better protection against fusion. This is in accord with the higher glass transition temperature of the longer chain oligosaccharides. Further evidence for the influence of glass transitions on membrane stability in the dry state was provided by experiments testing the temperature dependence of membrane fusion. During incubation at temperatures up to 95 degrees C for 2 h, fusion increased less with temperature in the presence of higher DP sugars. This indicates that RFO with a higher glass transition temperature are better able to protect dry membranes at elevated temperatures. In addition, Fourier-transform infrared (FTIR) spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature of dry liposomes in the presence of all investigated sugars. However, the RFO became slightly less effective with increasing chain length, again pointing to a decisive role for preventing fusion. A direct interaction of the RFO with the lipids was indicated by a strong effect of the sugars on the phosphate asymmetric stretch region of the infrared spectrum.