The role of macromolecular stability in desiccation tolerance

The work presented in this thesis concerns a study on the molecular interactions that play a role in the macromolecular stability of desiccation-tolerant higher plant organs. Fourier transform infrared microspectroscopy was used as the main experimental technique to assess macromolecular structures within their native environment.Protein secondary structure and membrane phase behavior of Typha latifolia pollen were studied in the course of accelerated aging. The overall protein secondary structure of fresh pollen highly resembled that of aged pollen, which indicates that endogenous proteins in these pollen are very stable, at least with respect to their conformation. In contrast, large changes in membrane phase behavior were detected between fresh and aged pollen. Membranes isolated from fresh pollen occurred mainly in the liquid crystalline phase at room temperature, whereas the membranes of aged pollen were at least partly in the gel phase (Chapter 2).The in situ heat stability of the proteins in this pollen was studied as a function of the water content of the pollen. Temperature-induced denaturation of proteins was accompanied by the formation of intermolecular extended-sheet structures. Below 0.16 g H 2 O g -1dry weight (DW), the temperature at which the proteins began to denature increased rapidly and the extent of protein structural rearrangements due to heating decreased (Chapter 3).Inspection of the overall protein secondary structure of thin slices of embryo axes of onion, white cabbage and radish seeds did not show signs of protein aggregation and denaturation after long-term dry storage. It was concluded 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 at least several decades of open storage (Chapter 4).Adaptations in overall protein secondary structure in association with the acquisition of desiccation tolerance were studied using isolated immature maize embryos. Isolated immature maize ( Zea mays ) embryos acquire tolerance to rapid drying between 22 and 25 days after pollination (DAP) and to slow drying from 18-DAP onwards. In fresh, viable 20- and 25-DAP embryo axes, the overall protein secondary structure was identical, and this was maintained after flash drying. On rapid drying, 20-DAP axes showed signs of protein breakdown and lost viability. Rapidly dried 25-DAP embryos germinated and had a protein profile similar to the fresh control. On slow drying, the-helical contribution in both the 20- and 25-DAP embryo axes increased when compared with that in the fresh controls, and survival of desiccation was high. The protein profile in dry mature axes resembled that after slow drying of the immature axes. Rapid drying resulted in an almost complete loss of membrane integrity in 20-DAP embryo axes and much less so in 25-DAP axes. After slow drying, membrane integrity was retained in both the 20- and 25-DAP axes. It was concluded that slow drying of excised immature embryos leads to an increased proportion of-helical protein structures in their axes, which coincides with additional tolerance of desiccation stress (Chapter 5).A novel FTIR method was used to study glasses of pure carbohydrates and glasses in the cytoplasm of desiccation-tolerant plant organs. The method is based on a temperature study of the position of the OH-stretching vibration band (OH). The glass transition temperatures ( Tg s) of several dry carbohydrate glasses determined by this FTIR method resembled those of produced by other methods. FTIR analysis gives additional information on the molecular properties of glassy structures. The shift ofOH with temperature - the wavenumber-temperature coefficient (WTC) - is indicative of the average strength of hydrogen bonding in glasses. The WTC was found to be higher in sugar glasses having higher Tg . This suggests that carbohydrate glasses are more loosely packed when they have higher Tg . For Typha latifolia pollen and dried Craterostigma plantagineum leaves similarOH vs temperature plots were obtained as for pure carbohydrate glasses, indicating that a glass transition was observed. The data suggested that the carbohydrates that are present in the cytoplasm of these plant organs are the primary components contributing to the glassy state (Chapter 6).In order to find a relation between desiccation tolerance and physical stability, the heat stability of proteins and the properties of the glassy matrix in several dry maturation-defective mutant seeds of Arabidopsis thaliana were studied. Proteins in dried wild-type seeds did not denature up to 150°C. In dried desiccation-sensitive lec1-1 , lec1-3 and abi3-5 seeds, protein denaturation occurs at temperatures below 100°C. In desiccation-tolerant abi3-7 and abi3-1 seeds, protein denaturation commenced above 120 and 135°C, respectively. The maximal rate of change ofH with temperature was much higher in abi3-5 , lec1-1 and lec1-3 mutant seeds than in wild-type, abi3-1 , and abi3-7 seeds. This was interpreted as a higher molecular packing density in dried desiccation-tolerant than in dried desiccation-sensitive seeds, which is associated with a higher, respectively lower protein denaturation temperature. The generally lower physical stability of the desiccation-sensitive mutant seeds coincides with a lack of biochemical adaptations that normally occur in the later stages of seed development (Chapter 7).The relation between physical stability and desiccation tolerance was also studied in slowly dried (desiccation-tolerant) and rapidly dried (desiccation-sensitive) carrot somatic embryos. Although protein denaturation temperatures were similar in the embryos after slow or rapid drying, the extent of protein denaturation was higher in the rapidly dried embryos. Slowly dried embryos are in a glassy state at room temperature, whereas no clearly defined glass transition temperature was observed in the rapidly dried embryos. Moreover, the molecular packing density of the cytoplasmic glassy matrix was higher in the slowly dried embryos. While sucrose is the major soluble carbohydrate after rapid drying, on slow drying, the trisaccharide umbelliferose accumulates at the expense of sucrose. Dry umbelliferose and sucrose glasses have almost similar Tg s. Both umbelliferose and sucrose depressed the transition temperature of dry liposomal membranes equally well; prevented leakage from dry liposomes after rehydration, and preserved the secondary structure of dried proteins. The similar protecting properties in model systems and the apparent interchangeability of both sugars in viable dry somatic embryos suggest no special role for umbelliferose in the improved physical stability of the slowly dried somatic embryos. It was suggested that LEA proteins, which are synthesized during slow drying together with the sugars, are responsible for the increased stability of the slowly dried embryos (Chapter 8).The dehydration-sensitive polypeptide, poly-L-lysine was used as a model to study dehydration-induced conformational transitions of this polypeptide as influenced by drying rate and carbohydrates. In solution poly-L-lysine adopts a random coil conformation. Upon slow drying of small droplets of the polypeptide solution over a period of several hours, the polypeptide adopts an extended-sheet conformation. Upon fast air-drying within 2-3 minutes, the aqueous polypeptide structure is preserved. Slow air-drying in the presence of sugars also preserves the aqueous conformation and results in the formation of a glassy state having a higher Tg than that of sugar alone. The importance of direct sugar - polypeptide interaction in stabilization during slow air-drying was studied by drying the polypeptide in the presence of glucose, sucrose or dextran. Compared to dextran (and sucrose to a lesser extent), glucose gives superior protection, while having the lowest Tg and the best interacting properties. It was suggested that during slow drying, a protectant with sufficient interaction is required for preservation of the aqueous protein structure (Chapter 9).The structure of a D-7 LEA (late embryogenesis abundant)-like protein protein isolated from Typha latifolia pollen was studied using FTIR. In solution, the protein adopts a random coil conformation. Fast air-drying (5 minutes) leads to the formation of-helical structure, whereas slow drying (few hours) leads to both-helical and intermolecular extended-sheet structures. When dried in the presence of sucrose, the protein adopts predominantly-helical conformation, irrespective of drying rate. Drying of a mixture of LEA protein and sucrose results in the formation of a glassy state having higher Tg and a higher average strength of hydrogen bonding than a pure sucrose glass. It was suggested that LEA proteins might be involved in the formation of a tight molecular network in the dehydrating cytoplasm of anhydrobiotic organisms, which may contribute to desiccation tolerance (Chapter 10).Taken together, in situ FTIR studies can give additional information on the molecular organization in desiccation-tolerant cells. The added value of this approach is that molecular structures and inter-molecular interactions can be studied in intact biological systems (Chapter 11).</p

A comparative study of the electrochemical properties of vitamin B-6 related compounds at physiological pH

A comparative study of vitamin B6 group and related compounds in buffered solutions using electrochemical techniques has been performed at neutral pH. Irreversible bi- or tetra-electronic processes are observed for these substances, and the electron transfer coefficient (αn) calculated. It was concluded that either the first or second electron transfer were the rate determining step of the electrode process. The diffusion coefficient of these substances was calculated and the values given follow an inverse tendency to the molecular size. For aldehydes the values obtained were corrected of the hydration reaction. It is important to remark that catalytic waves were reported for the first time for these compounds. Using a model involving the nitrogen of the basic structure the kinetic constants were calculated for most of them

Heat stability of proteins in desiccation tolerant cattail pollen (Typha latifolia): A Fourier transform infrared spectroscopic study.

Secondary structure and aggregation behavior of proteins, as determined in situ in Typha latifolia pollen, were studied by means of Fourier transform infrared microspectroscopy. The amide-I band, arising from the peptide backbone, was recorded over a temperature range from -50 to 120°C at different hydration levels of the pollen. Dehydration increased the denaturation temperature of the proteins and decreased the extent of protein structural rearrangements due to heating. Below 0.16 g H2O/g dry weight (DW), the temperature at which the proteins began to denature increased rapidly. In fully hydrated pollen, denaturation commenced above 60°C, whereas in very dry pollen (0.01 g H2O/g DW) it did at approximately 116°C. Temperature-induced aggregation of proteins was accompanied by the appearance of an infrared band in the region between 1625 and 1630 cm-1 and a weak band around 1692 cm-1. These bands are characteristic of intermolecular extended β-sheet structures. The α-helical band position (band around 1657 cm-1) did not shift substantially over a temperature range from -40 to 120°C at all the water contents tested, indicating that α-helical structures are particularly heat stable. We show here that the proteins in dry desiccation-tolerant pollen are particularly heat stable

In situ FTIR assessment of desiccation-tolerant tissues

This essay shows how Fourier transform infrared (FTIR) microspectroscopy can be applied to study thermodynamic parameters and conformation of endogenous biomolecules in desiccation-tolerant biological tissues. Desiccation tolerance is the remarkable ability of some organisms to survive complete dehydration. Seed and pollen of higher plants are well known examples of desiccation-tolerant tissues. FTIR studies on the overall protein secondary structure indicate that during the acquisition of desiccation tolerance, plant embryos exhibit proportional increases in alpha-helical structures and that beta-sheet structures dominate upon drying of desiccation sensitive-embryos. During ageing of pollen and seeds, the overall protein secondary structure remains stable, whereas drastic changes in the thermotropic response of membranes occur, which coincide with a complete loss of viability. Properties of the cytoplasmic glassy matrix in desiccation-tolerant plant organs can be studied by monitoring the position of the OH-stretching vibration band of endogenous carbohydrates and proteins as a function of temperature. By applying these FTIR techniques to maturation-defective mutant seeds of Arabidopsis thaliana we were able to establish a correlation between macromolecular stability and desiccation tolerance. Taken together, in situ FTIR studies can give unique information on conformation and stability of endogenous biomolecules in desiccation-tolerant tissues

FT-IR spectroscopy of the major coat protein of M13 and Pf1 in the phage and reconstituted into phospholipid systems

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Induction of desiccation tolerance in plant somatic embryos : how exclusive is the protective role of sugars?

Plant somatic embryos usually lack desiccation tolerance. They may acquire such a tolerance upon preculture in the presence of abscisic acid (ABA), followed by slow drying, but not fast drying. ABA causes torpedo-shaped somatic embryos to lose their chlorophyll, suspend growth, exhibit low rates of respiration, and maintain elevated sucrose contents. The subsequent slow drying leads to a partial conversion of sucrose into oligosaccharides and the expression of dehydrin transcripts. Slow-dried, desiccation-tolerant somatic embryos have stable membranes, retain their native protein secondary structure, and have a densely packed cytoplasmic glassy matrix. Fast-dried, desiccation-sensitive somatic embryos experience some loss of phospholipids and an increase in free fatty acids. Their proteins show signs of denaturation and aggregation, and the glassy matrix has reduced hydrogen bonding. The reduced conversion of sucrose into oligosaccharides appears not to underlie dehydration injury. Proteins in slow-dried somatic embryos, not pretreated with ABA, also show signs of denaturation, which might be attributed to low sugar contents. We conclude that by reducing cellular metabolism, ABA maintains high sugar contents. These sugars contribute to the stability of membranes, proteins, and the cytoplasmic glassy matrix, whereas slow drying permits a further fine tuning of this stability. Partitioning of endogenous amphiphiles from the cytoplasm into membranes during drying may cause membrane perturbance, although it might confer protection to membranes in the case of amphiphilic antioxidants. The perturbance appears to be effectively controlled in desiccation-tolerant systems but not in sensitive systems, for which we suggest dehydrins are responsible. In this context, the low desiccation tolerance in the presence of ample sugars is discussed

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 (T(g)), as determined by other methods. The temperature at the intersection point decreased with increasing water content, which further validates that, indeed, T(g) was observed. T(g) values that were determined for dry glucose, sucrose, maltose, trehalose and raffinose glasses were 27, 57, 91, 108 and 108°C, respectively. The shift of vOH with temperature, the wavenumber-temperature coefficient (WTC), was higher in sugar glasses having higher T(g). This suggests that glasses are more loosely packed when they have higher T(g). 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 T(g) in dry pollen was ca. 45°C and in dry plant leaves ca. 65°C, with WTC values comparable to those observed in the carbohydrates. The T(g) 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