Folkert A. Hoekstra

Comparative Analysis of the Heat Stable Proteome of Radicles of Medicago truncatula Seeds during Germination Identifies Late Embryogenesis Abundant Proteins Associated with Desiccation Tolerance

A proteomic analysis was performed on the heat stable protein fraction of imbibed radicles of Medicago truncatula seeds to investigate whether proteins can be identified that are specifically linked to desiccation tolerance (DT). Radicles were compared before and after emergence (2.8 mm long) in association with the loss of DT, and after reinduction of DT by an osmotic treatment. To separate proteins induced by the osmotic treatment from those linked with DT, the comparison was extended to 5 mm long emerged radicles for which DT could no longer be reinduced, albeit that drought tolerance was increased. The abundance of 15 polypeptides was linked with DT, out of which 11 were identified as late embryogenesis abundant proteins from different groups: MtEm6 (group 1), one isoform of DHN3 (dehydrins), MtPM25 (group 5), and three members of group 3 (MP2, an isoform of PM18, and all the isoforms of SBP65). In silico analysis revealed that their expression is likely seed specific, except for DHN3. Other isoforms of DNH3 and PM18 as well as three isoforms of the dehydrin Budcar5 were associated with drought tolerance. Changes in the abundance of MtEm6 and MtPM25 in imbibed cotyledons during the loss of DT and in developing embryos during the acquisition of DT confirmed the link of these two proteins with DT. Fourier transform infrared spectroscopy revealed that the recombinant MtPM25 and MtEm6 exhibited a certain degree of order in the hydrated state, but that they became more structured by adopting α helices and β sheets during drying. A model is presented in which DT-linked late embryogenesis abundant proteins might exert different protective functions at high and low hydration levels.

IS VITRIFICATION INVOLVED IN DEPRESSION OF THE PHASE TRANSITION TEMPERATURE IN DRY PHOSPHOLIPIDS

Recent literature has suggested that the depression of the phase transition temperature (Tm) in dry phospholipids by sugars may be ascribed to vitrification of the stabilizing solute, rather than by the direct interaction between sugar and phospholipid we have proposed. Koster et al. ((1994) Biochim. Biophys. Acta 1193, 143-150) claim that the only necessity is that the glass transition (Tg) for the sugar exceed Tm for the lipid. Evidence is presented in the present paper that this is not sufficient. Based on the vitrification hypothesis of Koster et al., the predicted order of effectiveness in depressing Tm in dry dipalmitoylphosphatidylcholine (DPPC) is dextran > or = hydroxyethyl starch > stachyose > raffinose > trehalose > sucrose > glucose. In fact, the opposite order was seen. The effect of raffinose, sucrose, or trehalose on Tm in dry DPPC depends on the thermal history of the sample, as we have reported previously. When DPPC dried with trehalose is heated for the first time, Tm is about 55 degrees C, but on the second and subsequent heating scans Tm falls to about 25 degrees C. Koster et al. suggest that this effect is due to heating the sample above Tg rather than to melting the hydrocarbon chains. We present evidence here that all that is required is for the chains to be melted. Further, we show that retention of residual water by DPPC dried with trehalose depends on the drying temperature, but is independent of drying temperature with glucose, a finding that is consistent with direct interaction. We conclude that vitrification is not in itself sufficient to depress Tm in dry phospholipids.

A Fourier transform infrared microspectroscopy study of sugar glasses: application to anhydrobiotic higher plant cells

Fourier transform infrared microspectroscopy (FTIR) was used to study glasses of pure carbohydrates and in the cytoplasm of desiccation tolerant plant organs. The position of the OH stretching vibration band (vOH) shifted with temperature. Two linear regression lines were observed in vOH against temperature plots. The temperature at the point of intersection between these two lines coincided with the glass transition temperature (Tg), as determined by other methods. The temperature at the intersection point decreased with increasing water content, which further validates that, indeed, Tg was observed. Tg values that were determined for dry glucose, sucrose, maltose, trehalose and raffinose glasses were 27, 57, 91, 108 and 108 degrees C, respectively. The shift of vOH with temperature, the wavenumber-temperature coefficient (WTC), was higher in sugar glasses having higher Tg. This suggests that glasses are more loosely packed when they have higher Tg. For Typha latifolia pollen and dried Craterostigma plantagineum leaves we obtained similar vOH vs. temperature plots as for carbohydrate glasses, indicating that a glass transition was observed. The Tg in dry pollen was ca. 45 degrees C and in dry plant leaves ca. 65 degrees C, with WTC values comparable to those observed in the carbohydrates. The Tg values in these tissues decreased with increasing water contents. Our data suggest that the carbohydrates that are present in the cytoplasm are primary factors contributing to the glassy state. We conclude that FTIR provides new insights in the structure of glasses in carbohydrates and in biological tissues.

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.

DEHYDRATION-INDUCED CONFORMATIONAL CHANGES OF POLY-L-LYSINE AS INFLUENCED BY DRYING RATE AND CARBOHYDRATES

The conformation of hydrated and air-dried poly-L-lysine in thin films was studied using Fourier transform IR spectroscopy in the amide-I region. Hydrated poly-L-lysine has a random coil conformation. Upon slow drying of small droplets of the polypeptide solution over a period of several hours, an extended beta-sheet conformation is adopted. This conformational transition can be prevented by fast air-drying within 2-3 min. Slow air-drying in the presence of sucrose also preserves the aqueous conformation and results in the formation of a glassy state. Comparison of shifts of the OH band with temperature indicates that sucrose/poly-L-lysine mixtures form a molecularly more densely packed glassy matrix, having a higher glass transition temperature (Tg), than sucrose alone. Whether direct interaction of sugar and polypeptide or glass formation is involved in the stabilization during slow air-drying was studied by drying in the presence of glucose or dextran. Compared with dextran (and sucrose to a lesser extent), glucose gives superior protection. Dried glucose has the lowest Tg and the best interacting properties. We conclude that either immobilization by fast air-drying or sufficient interaction with a protectant through hydrogen bonding (slow drying) plays the leading role in the preservation of the aqueous protein structure.

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.

Calorimetric Properties of Dehydrating Pollen (Analysis of a Desiccation-Tolerant and an Intolerant Species)

The physical state of water in the desiccation-tolerant pollen of Typha latifolia L. and the desiccation-sensitive pollen of Zea mays L. was studied using differential scanning calorimetry in an attempt to further unravel the complex mechanisms of desiccation tolerance. Melting transitions of water were not observed at water content (wc) values less than 0.21 (T. latifolia) and 0.26 (Z. mays) g H2O/g dry weight. At moisture levels at which melting transitions were not observable, water properties could be characterized by changes in heat capacity. Three hydration regions could be distinguished with the defining wc values changing as a function of temperature. Shifts in baseline power resembling second-order transitions were observed in both species and were interpreted as glass-to-liquid transitions, the glass-transition temperatures being dependent on wc. Irrespective of the extent of desiccation tolerance, both pollens exhibited similar state diagrams. The viability of maize pollen at room temperature decreased gradually with the removal of the unfrozen water fraction. In maize, viability was completely lost before grains were sufficiently dried to enter into a glassy state. Apparently, the glassy state per se cannot provide desiccation tolerance. From the existing data, we conclude that, although no major differences in the physical behavior of water could be distinguished between desiccation-tolerant and -intolerant pollens, the physiological response to the loss of water varies between the two pollen types.

Differences in the susceptibility of plant membrane lipids to peroxidation

Peroxidation of three membrane lipid preparations from plants was initiated using Fe-EDTA and ascorbate and quantified as the production of aldehydes and loss of esterified fatty acids. Using liposomes prepared from commercial soybean asolecithin, the degree of peroxidation was shown to be dependent on: the free radical dose, which was varied by the ascorbate concentration; the presence of tocopherol in the liposome; the configuration, of the liposome, multilamellar or unilamellar; and time after initiation. There were dramatic interactions among these factors which led to the conclusion that in comparing the susceptibility of different membrane preparations it is essential to examine the kinetics of the peroxidation reactions. The composition of the liposome was a major determinant of the degree of peroxidation and of the type of degradative reactions initiated by the oxygen free radicals. A fresh polar lipid extract from Typha pollen had very similar fatty acid composition to the soybean asolecithin, but was more resistant to peroxidation as shown by less aldehyde production and increased retention of unsaturated fatty acids after treatment. Similarly, microsomal membranes from the crowns of non-acclimated and cold acclimated winter wheat (Triticum aestivum L.) seedlings had a much higher linolenic acid content than soybean asolecithin but was much more resistant to peroxidation. In the winter wheat microsomes, the loss of esterified fatty acids was not selective for the unsaturated fatty acids; consequently, even with 40% degradation, the degree of unsaturation in the membrane did not decrease. These different reaction mechanisms which occur in plant membranes may explain why measurements of fatty acid unsaturation fail to detect peroxidative reactions during processes such as senescence, aging and environmental stress.

Protein synthesis of binucleate and trinucleate pollen and its relationship to tube emergence and growth

Under humid conditions, both bi- and trinucleate pollen species incorporate, on the average, very low amounts of leucine, e.g., 0.4 pmol min-1mg pollen-1. During germination in vitro, however, the two types of pollens greatly differ in their capacity for protein synthesis.Binucleate pollen species such as Typha, which are characterized by slow respiration in humid air and prolonged lag periods during germination in vitro, contain large amounts of monoribosomes at dehiscence. Polyribosomes are formed soon after the pollen is wetted in the germination medium, and a considerable incorporation of leucine is initiated after 10–15 min. More rapidly respiring, binucleate pollen showing a short lag period, such as Tradescantia, may already contain many polysomes at dehiscence and incorporate leucine within 2 min of germination. However, rapidly respiring, trinucleate Compositae pollen contains very limited amounts of ribosomal material and never attains any substantial level of incorporation. Cycloheximide completely inhibits both protein synthesis and tube emergence and growth in the slowly respiring, binucleate pollen species. The more rapidly respiring types are less dependent on protein synthesis, while germination of the phylogenetically advanced, trinucleate Compositae pollen proceeds completely independently. It is concluded that the level of phylogenetic advancement of the male gametophyte is characterized by its overall state of metabolic development at dehiscence rather than by the number of its generative cells.

Mitochondrial development and activity of binucleate and trinucleate pollen during germination in vitro

Bi-and trinucleate pollen generally differ in the extent of their mitochondrial development at anther dehiscence and in the rate of their attainment of maximum-phosphorylative capacity during germination in vitro, as judged from experiments with representatives of both groups.The typically trinucleate pollen of Aster tripolium L. immediately respired at a high rate, maintaining a high energy charge. Mitochondria attained maximum electron-transducing capacity within 2 min of incubation, while tube growth started within 3 min. In contrast, the binucleate pollen of Typha latifolia L. only gradually reached a relatively low rate of respiration, concomitant with a temporary decrease in energy charge, upon immersion in the germination medium. Development of the mitochondrial, electrontransducing system occurred in about 75 min, after which the first pollen tubes emerged. Starting from a poor differentiation, mitochondria became increasingly normal in appearance as germination proceeded.The binucleate pollen of Nicotiana alata Link et Otto and Tradescantia paludosa Anders. et Woods. showed intermediate characteristics: Nicotiana resembled Typha but mitochondria developed at a higher rate; Tradescantia germinated more rapidly and resembled the trinucleate pollen of Aster.Inhibitors of mitochondrial or cytoplasmic protein synthesis failed to affect the development of the mitochondrial, respiratory capacities during pollen germination. It is concluded that the duration of the lag period is determined by the level and rate of mitochondrial development and not by the division of the generative cell.