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 <em>Typha latifolia</em> 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 <em>in situ</em> 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<img src="/wda/abstracts/2440_b.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>-sheet structures. Below 0.16 g H ₂ O g <sup>-1</SUP>dry 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 ( <em>Zea mays</em> ) 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<img src="/wda/abstracts/2440_a.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="alpha" border="0"/>-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<img src="/wda/abstracts/2440_a.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="alpha" border="0"/>-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 (<img src="/wda/abstracts/2440_v.gif" ALIGN="AbsMiddle" width="10" height="11" ALT="v" border="0"/>OH). The glass transition temperatures ( <em>T</em>_g 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 of<img src="/wda/abstracts/2440_v.gif" ALIGN="AbsMiddle" width="10" height="11" ALT="v" border="0"/>OH 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 <em>T</em>_g . This suggests that carbohydrate glasses are more loosely packed when they have higher <em>T</em>_g . For <em>Typha latifolia</em> pollen and dried <em>Craterostigma plantagineum</em> leaves similar<img src="/wda/abstracts/2440_v.gif" ALIGN="AbsMiddle" width="10" height="11" ALT="v" border="0"/>OH <em>vs</em> 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 <em>Arabidopsis thaliana</em> were studied. Proteins in dried wild-type seeds did not denature up to 150°C. In dried desiccation-sensitive <em>lec1-1</em> , <em>lec1-3</em> and <em>abi3-5</em> seeds, protein denaturation occurs at temperatures below 100°C. In desiccation-tolerant <em>abi3-7</em> and <em>abi3-1</em> seeds, protein denaturation commenced above 120 and 135°C, respectively. The maximal rate of change of<img src="/wda/abstracts/2440_v.gif" ALIGN="AbsMiddle" width="10" height="11" ALT="v" border="0"/>H with temperature was much higher in <em>abi3-5</em> , <em>lec1-1</em> and <em>lec1-3</em> mutant seeds than in wild-type, <em>abi3-1</em> , and <em>abi3-7</em> 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 <em>T</em>_g 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<img src="/wda/abstracts/2440_b.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>-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 <em>T</em>_g 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 <em>T</em>_g 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 <em>Typha latifolia</em> pollen was studied using FTIR. In solution, the protein adopts a random coil conformation. Fast air-drying (5 minutes) leads to the formation of<img src="/wda/abstracts/2440_a.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="alpha" border="0"/>-helical structure, whereas slow drying (few hours) leads to both<img src="/wda/abstracts/2440_a.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="alpha" border="0"/>-helical and intermolecular extended<img src="/wda/abstracts/2440_b.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>-sheet structures. When dried in the presence of sucrose, the protein adopts predominantly<img src="/wda/abstracts/2440_a.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="alpha" border="0"/>-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 <em>T</em>_g 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, <em>in situ</em> 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).