Intracellular glasses and seed survival in the dry state

So-called orthodox seeds can resist complete desiccation and survive the dry state for extended periods of time. During drying, the cellular viscosity increases dramatically and in the dry state, the cytoplasm transforms into a glassy state. The formation of intracellular glasses is indispensable to survive the dry state. Indeed, the storage stability of seeds is related to the packing density and molecular mobility of the intracellular glass, suggesting that the physico-chemical properties of intracellular glasses provide stability for long-term survival. Whereas seeds contain large amounts of soluble non-reducing sugars, which are known to be good glass formers, detailed in vivo measurements using techniques such as FTIR and EPR spectroscopy reveal that these intracellular glasses have properties that are quite different from those of simple sugar glasses. Intracellular glasses exhibit slow molecular mobility and a high molecular packing, resembling glasses made of mixtures of sugars with proteins, 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 low molecular mobility and a high stability, which serves as an ecological advantage, keeping the seeds stable under adverse conditions of temperature or water content that bring the tissues out of the glassy state.     

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.     

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.     

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.     

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.     

Unraveling protein stabilization mechanisms: Vitrification and water replacement in a glass transition temperature controlled system

The aim of this study was to elucidate the role of the two main mechanisms used to explain the stabilization of proteins by sugar glasses during drying and subsequent storage: the vitrification and the water replacement theory. Although in literature protein stability is often attributed to either vitrification or water replacement, both mechanisms could play a role and they should be considered simultaneously. A model protein, alkaline phosphatase, was incorporated in either inulin or trehalose by spray drying. To study the storage stability at different glass transition temperatures, a buffer which acts as a plasticizer, ammediol, was incorporated in the sugar glasses. At low glass transition temperatures (< 50 °C), the enzymatic activity of the protein strongly decreased during storage at 60 °C. Protein stability increased when the glass transition temperature was raised considerably above the storage temperature. This increased stability could be attributed to vitrification. A further increase of the glass transition temperature did not further improve stability. In conclusion, vitrification plays a dominant role in stabilization at glass transition temperatures up to 10 to 20 °C above storage temperature, depending on whether trehalose or inulin is used. On the other hand, the water replacement mechanism predominately determines stability at higher glass transition temperatures.     

Glass formation and desiccation tolerance in seeds

The formation of intracellular glass may help protect embryos from damage due to desiccation. Soluble sugars similar to those found in desiccation tolerant embryos were studied with differential scanning calorimetry. Those sugars from desiccation tolerant embryos can form glasses at ambient temperatures, whereas those from embryos that do not tolerate desiccation only form glasses at subzero temperatures. It is concluded that tolerant embryo cells probably contain sugar glasses at storage temperatures and water contents, but intolerant embryo cells probably do not.     

Thermodynamic aspects of vitrification

Vitrification is a process in which a liquid begins to behave as a solid during cooling without any substantial change in molecular arrangement or thermodynamic state variables. The physical phenomenon of vitrification is relevant to both cryopreservation by freezing, in which cells survive in glass between ice crystals, and cryopreservation by vitrification in which a whole sample is vitrified. The change from liquid to solid behavior is called the glass transition. It is coincident with liquid viscosity reaching 10¹³ Poise during cooling, which corresponds to a shear stress relaxation time of several minutes. The glass transition can be understood on a molecular level as a loss of rotational and translational degrees of freedom over a particular measurement timescale, leaving only bond vibration within a fixed molecular structure. Reduced freedom of molecular movement results in decreased heat capacity and thermal expansivity in glass relative to the liquid state. In cryoprotectant solutions, the change from liquid to solid properties happens over a ∼10 °C temperature interval centered on a glass transition temperature, typically near −120 °C (±10 °C) for solutions used for vitrification. Loss of freedom to quickly rearrange molecular position causes liquids to depart from thermodynamic equilibrium as they turn into a glass during vitrification. Residual molecular mobility below the glass transition temperature allows glass to very slowly contract, release heat, and decrease entropy during relaxation toward equilibrium. Although diffusion is practically non-existent below the glass transition temperature, small local movements of molecules related to relaxation have consequences for cryobiology. In particular, ice nucleation in supercooled vitrification solutions occurs at remarkable speed until at least 15 °C below the glass transition temperature.     

Storage behavior of Typha latifolia pollen at low water contents: Interpretation on the basis of water activity and glass concepts

Germplasm must be stored under optimal conditions to maximize longevity and efficiently maintain genetic resources. In order to identify optimal storage conditions, we investigated the effects of temperature (−5 to 45°C) and water content (<0.17 g H₂O g⁻¹ dry weight) on longevity of Typha latifolia L. pollen. Longevity was highest at water contents corresponding to storage relative humidity (RH) of 11‐15% which corresponded to the shoulder of water sorption isotherms. Also coinciding with this shoulder were abrupt changes in heat capacity of water present in the pollen. Consistent with changes in isotherms with temperature and the concept of critical RH for storage, optimum water contents increased with decreasing temperature. An attempt was made to explain the aging behavior according to the glass concept. The water content‐temperature combinations of optimal storage were found to be below the glass transition curve, indicating that optimum storage conditions are achieved when intracellular glasses are present. We also found a change in activation energy of aging in Arrhenius plots around T_g, demonstrating a change in aging kinetics when the glassy state is lost. We concluL that T_g curves cannot be used solely to predict precise conditions of optimum storage, but might be useful for predictions of storage longevity above optimum water contents. The data imply that too much drying reduces longevity and should be avoided, particularly when cryogenic storage is considered.     

Calorimetric studies of the state of water in seed tissues

To understand the physical state of water in hydrating biological tissues, thermodynamic properties of water in cotyledons of pea and soybean with moisture contents ranging from 0.01 g H₂O/g dw to 1.0 g H₂O/g dw were studied using differential scanning calorimetry. The heat capacity of the tissues increased abruptly at moisture contents above 0.08 and 0.12 g H₂O/g dw for soybean and pea cotyledons, respectively. Melting transitions of water were observed at moisture contents >0.23 and 0.26 g H₂O/g dw for soybean and pea. However, freezing of water was not observed unless moisture contents exceeded 0.33-0.35 g H₂O/g dw. In both seed tissues, the temperatures of the freezing and melting varied with moisture content and showed hysterisis. The energy of the transition also varied with moisture content and was similar to the heats of fusion and crystallization of pure water only at moisture contents >0.54 and 0.58 gH₂O/g dw for soybean and pea seeds, respectively. The thermal properties of water change distinctly as seed moisture content changes: at least five states or water can be identified.     

Plant vitrification solution 2 lowers water content and alters freezing behavior in shoot tips during cryoprotection

Plant shoot tips do not survive exposure to liquid nitrogen temperatures without cryoprotective treatments. Some cryoprotectant solutions, such as plant vitrification solution 2 (PVS2), dehydrate cells and decrease lethal ice formation, but the extent of dehydration and the effect on water freezing properties are not known. We examined the effect of a PVS2 cryoprotection protocol on the water content and phase behavior of mint and garlic shoot tips using differential scanning calorimetry. The temperature and enthalpy of water melting transitions in unprotected and recovering shoot tips were comparable to dilute aqueous solutions. Exposure to PVS2 changed the behavior of water in shoot tips: enthalpy of melting transitions decreased to about 40 J g H2O(-1) (compared to 333 J g H2O(-1) for pure H2O), amount of unfrozen water increased to approximately 0.7 g H2O g dry mass(-1) (compared to approximately 0.4 g H2Og dry mass(-1) for unprotected shoot tips), and a glass transition (T(g)) at -115 degrees C was apparent. Evaporative drying at room temperature was slower in PVS2-treated shoot tips compared to shoot tips receiving no cryoprotection treatments. We quantified the extent that ethylene glycol and dimethyl sulfoxide components permeate into shoot tips and replace some of the water. Since T(g) in PVS2-treated shoot tips occurs at -115 degrees C, mechanisms other than glass formation prevent freezing at temperatures between 0 and -115 degrees C. Protection is likely a result of controlled dehydration or altered thermal properties of intracellular water. A comparison of thermodynamic measurements for cryoprotection solutions in diverse plant systems will identify efficacy among cryopreservation protocols.     

Binding study of riboflavin-binding protein with riboflavin and its analogues by differential scanning calorimetry

Thermal unfolding parameters of hens' egg-white riboflavin-binding-protein (RBP) were measured by differential scanning calorimetry. Thermal denaturation scans of apoRBP and RBP complexes with riboflavin and its analogues (FMN, N10 DL-glyceryl isoalloxazine, and N10 -hydroxypentyl isoalloxazine) have been measured. It was found that ligand binding causes increase of RBP thermal stability, as manifested by a change of denaturation temperature from 60.8°C for apoRBP to 72.8°C for RBP—Rf complex. For RBP—FMN complex, the denaturation temperature of 73.0°C was even higher than for the RBP—Rf complex. The other two flavin analogues showed transition temperatures in between 66.9°C and 68.8°C, respectively. Analysis of excess heat capacity data showed that the best fit was the sum of two independent thermal transitions. One of the transitions, which contributed 70% to the total heat effect, has transition temperature in the broad range of 60.5–73.2°C; the other transition temperature is in the narrower range of 65.4–71.1°C. The observed transitions can be related to RBP domains.     

Heat shock tolerance and antioxidant activity in moth bean seedlings treated with tetcyclacis

Moth bean (Vigna aconitifolia (Jacq.) Marechal cv. Jaadia) seeds were germinated in the presence of 0, 18, or 36 M solutions of the gibberellin biosynthesis inhibitor, tetcyclacis. After 72 h, seedlings were exposed to 22 or 48°C for 90 min. The 48°C temperature dramatically increased total electrolyte and sugar leakage from the seedlings, particularly in the controls. Tetcyclacis reduced electrolyte and sugar leakage at 48°C by 15–35% compared to the 48°C controls. High temperature increased malondialdehyde concentration in control seedlings but not in treated seedlings indicating that tetcyclacis inhibited high temperature-induced lipid peroxidation. Relative to the control, tetcyclacis tended to increase the total activities of catalase and peroxidase in the seedlings. In contrast, tetcyclacis tended to decrease ascorbic acid oxidase activity, particularly at 48°C. These results suggest that tetcyclacis conferred at least some heat shock tolerance to moth bean seedlings. This increased tolerance was correlated with increased activities of some antioxidant systems.     

Viscous solutions, networks and the glass transition in high sugar galactomannan and kappa-carrageenan mixtures

Small-deformation rotational oscillation was used to examine the effect of small additions of galactomannan and kappa-carrageenan on the vitrification of glucose syrup at a total level of solids of 83%. The method of reduced variables allowed construction of composite curves covering the glass transition and glassy state (from 10(5) to 10(9.5) Pa) over a wide frequency range (up to 15 orders of magnitude). The combined WLF/free volume framework was employed to determine the rheological glass transition temperature (T(g)), fractional free volume and thermal expansion coefficient of the samples. It was found that the WLF-predicted glass transition temperature matched the cross over of experimental modulus traces in the passage from the glass transition (GG') to the glassy state (GG"). This coincides with the mechanistic transformation from free volume effects to the Arrhenius-type phenomena, thus ascribing physical significance to the rheological T(g). The T(g) value of 83% glucose syrup at a scan rate of 2 degrees C min(-1) was -25.3 degrees C. Replacing, for example, 1% glucose syrup with guar gum shifted the T(g) of the mixture to -19.7 degrees C. Network formation via the K(+)-supported junction zones of the kappa-carrageenan chains further increased the T(g) to about -1 degrees C. It appears that the low rates of relaxation processes and diffusion mobility in the presence of a polysaccharide network accelerate the collapse of the free volume thus inducing vitrification of the high sugar/polysaccharide mixture at high temperatures.     

Thermal stability of bovine-brain myelin membrane

The thermal behaviour of bovine-brain myelin membrane has been studied by high-sensitivity differential scanning calorimetry, Fourier-transform infrared spectroscopy and thermal gel analysis. Spectroscopic results indicate that protein transitions take place between 60°C and 90°C, while thermal gel analysis has provided the thermal denaturation profiles of myelin proteolipid, DM-20 protein and the Wolfgram Fraction. An irreversible calorimetric transition centred at 80.3 ± 0.2°C with a specific enthalpy of 4.7 ± 0.6 J/g of total protein has been assigned to the thermal denaturation of myelin proteolipid and DM-20 protein. The effects of the myelin storage conditions, scan rate, ionic strength and pH on this calorimetric transition have also been investigated. The thermal transition of the proteolipid practically disappears after treatment of the myelin with different amounts of chloroform-methanol 2:1 (v/v), a treatment which is generally used in proteolipid purification. On the other hand, the addition of several detergents to myelin only causes minor modifications to this transition, which then occurs at about 70°C, with a specific enthalpy of between 2.5 and 3.6 J/g of total protein. These results appear to show that detergents preserve the native conformation of the proteolipid far more than do organic solvents. Hence the use of detergents would seem to be the appropriate method for proteolipid purification.Abbreviations DSC Differential scanning calorimetry - TGA Thermal gel analysis - FTIR Fourier-transform infrared spectroscopy - PLP Proteolipid protein - MBP Myelin basic protein - DM-20 Protein DM-20 - WF Wolfgram fraction - BSA Bovine serum albumine - SDS Sodium dodecyl sulfate - ANSA 4-amino-3-hydroxynaphthalene-1-sulphonic acid - OG -d-glucopyranoside - PAGE Polyacrylamide gel electrophoresis - Chaps 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate - CNS Central nervous system Correspondence to: P. L. Mateo     

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.     

The rubber‐to‐glass transition in high sugar agarose systems

The small and large deformation properties of agarose in the presence of high levels of sugar were investigated. Mixtures can be described as lightly cross‐linked rubbers, which undergo vitrification upon cooling. The combined Williams–Landel–Ferry (WLF)/free volume framework was used to derive the glass transition temperature, the fractional free volume, and the thermal expansion coefficient of the glass. Sucrose‐rich cosolute crystallizes, but addition of the polymer encourages intermolecular interactions, which transform the mixture into a high viscosity glass. The mechanical properties of glucose syrup, a noncrystalline sugar, follow WLF behavior in the glass transition region and revert to an Arrhenius‐type prediction in the glassy state. Measurements on sugar samples and agarose–sugar mixtures were resolved into a basic function of temperature alone and a basic function of frequency (time) alone. The former traces the energetic cost of vitrification, which increases sharply with decreasing temperature. The latter, at long time scales, is governed by the infinite molecular weight of the agarose network. In the region of short times, the effect of free volume is active regardless of the sample composition. © 1999 John Wiley & Sons, Inc. Biopoly 49: 267–275, 1999     

Mechanisms associated with cellular desiccation tolerance in the animal extremophile artemia

Using differential scanning calorimetry, we demonstrated the presence of biological glasses and measured the transition temperatures in dry encysted embryos (cysts) of the brine shrimp, Artemia franciscana. Cysts from the following three geographic locations were studied: San Francisco Bay (SFB); the Great Salt Lake, Utah (GSL); and the Mekong Delta, Vietnam (VN; these cysts were produced from previous sequential inoculations of SFB cysts into growth ponds). Values for the glass transition temperature, T(g), were highest in VN cysts. This study indicates that the composition and properties of these biological glasses can be altered by natural selection and thermal adaptation. To our knowledge, T(g) values for all three kinds of cysts were significantly higher than those for any other desiccation-tolerant animal system. To gain insight into the significance of T(g), we examined the thermal stability of these dry cysts at 80 °C. GSL cysts were the least tolerant, by far, with VN cysts being extremely tolerant and SFB cysts not far behind. Those results correlated with the thermal transition values. Also measured were alcohol-soluble carbohydrates, ~90% of which is the disaccharide trehalose, a known component of biological glasses. Amounts in the GSL cysts were significantly less than those in the other two kinds of cysts. Several stress proteins were measured in the three groups of cysts, with all of them being in lesser amounts in GSL cysts compared with the SFB and VN cysts. We interpret the data in terms of mechanisms involved with desiccation tolerance and thermal conditions at the sites of cyst collection.     

Misfolding and aggregation of vacuolar glycoproteins in plant cells

Phaseolin and lectin-related polypeptides, the abundant oligomeric glycoproteins of bean seeds, are synthesized on the endoplasmic reticulum (ER) and then transported to the storage vacuole via the Golgi apparatus. Glycosylation and folding are among the major modifications these proteins undergo in the ER. Although a recurrent role of N-glycosylation is on protein folding, in previous studies on common bean (Phaseolus vulgaris) seeds we demonstrated that the oligosaccharide side-chains are not required for folding, intracellular transport and activity of storage glycoproteins. We show here that in lima bean (Phaseolus lunatus), incubation of the developing cotyledon with tunicamycin to prevent glycosylation has a dramatic effect on the intracellular transport of the storage glycoproteins. When lacking their glycans, phaseolin and lectin-related polypeptides misfold and are retained in the ER as mixed aggregates to which the chaperone BiP irreversibly associates. The lumen of the ER becomes enlarged to accommodate the aggregated polypeptides. Intracellular transport of legumin, a naturally unglycosylated storage protein, is mostly unaffected by the inhibitor, indicating that the observed phenomenon specifically occurs on glycoproteins. Furthermore, recombinant lima bean phaseolin synthesized in tobacco protoplasts is also correctly folded and matured in the presence of tunicamycin. To our knowledge, this is the first report that describes in detail the block of intracellular transport of vacuolar glycoproteins in plant cells due to aggregation following glycosylation inhibition.