As a result of drying, the cytoplasm of desiccation-tolerant organisms, such as seed and pollen, enters into a highly viscous, solid-like, semi-equilibrium state: the glassy state. The work in this dissertation is focussed on the function and characteristics of intracellular glasses in these organisms.
It was established that intracellular glasses are formed in both desiccation-tolerant and -intolerant pollen (chapter 1). However, desiccation-intolerant pollen loses its viability during drying before intracellular glasses are formed. This indicates that desiccation tolerance is not related with the formation of glasses during drying. Storage of cattail ( Typha latifolia ) pollen under different water contents and temperatures revealed the existence of an optimum water content for survival at a constant relative humidity (11-15%) (chapter 2). The water content corresponding to this relative humidity shifted to higher values with lower storage temperatures, and was found to be associated with the Brunauer, Emmet, and Teller monolayer value. Drying of the pollen below these water contents had detrimental effects on longevity. 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. There was a change in ageing kinetics of cattail pollen associated with the melting of the intracellular glass. Above the glass transition temperature (T g ) the activation energy of the ageing rates increased two to three times. This suggests that the presence of glasses in the dry state improves storage stability by decreasing viscosity and, thus, ageing rate. It was concluded that T g curves might be useful for predictions of storage longevity above optimum water contents. However, they cannot be used solely to predict the precise conditions of optimum storage. Subsequently, we sought for a more direct measurement to assess the viscosity of the cytoplasm of tissues.
For this purpose, we used electron paramagnetic resonance (EPR) spectroscopy to study the molecular mobility of the hydrophilic nitroxide spin probe 3-carboxy-proxyl (CP) that was incorporated into embryonic axes of pea seeds and cattail pollen. Using the distance between the outer extrema of the EPR spectrum (2 A zz ) as a measure of molecular mobility, a sharp increase in mobility was observed at a definite temperature during heating (chapter 3). This temperature increased with decreasing water content of the samples, and was found to be associated with the melting of the glassy state as measured by differential scanning calorimetry (DSC). Molecular mobility was found to be inversely correlated with storage stability: the higher the molecular mobility, the shorter the longevity: with decreasing water content, the molecular mobility reached a minimum, in concert with ageing rates. At very low water contents, both molecular mobility and ageing rates increased again. Minimum mobility and maximum storage stability occurred at similar water contents, suggesting that storage stability might be partially controlled by molecular mobility. To understand the nature of the changes in 2 A zz in spectra of CP in the tissues, echo detected (ED) EPR spectroscopy was employed (chapter 4). The shape of the ED EPR spectrum revealed the presence of librational motion of the spin probe, a motion typically present in glassy materials. The change in 2 A zz appeared to be the result of librational motion of the spin probe.
With the use of saturation transfer (ST) EPR spectroscopy, a more quantitative measure of molecular mobility was acquired: the rotational correlation time (τ R ), which corresponds to the time it takes for the spin probe to rotate a radian around its axis (chapter 5). At room temperature,τ R of CP in pea embryonic axes increased during drying from 10 -11 s in de hydrated state to 10 -4 s in the dry state. At T g ,τ R was constant at10 -4 s for all water contents studied. The temperature dependence ofτ R at all water contents studied followed Arrhenius behaviour with a break at T g . The temperature effect onτ R above T g was much smaller in pea axes as found previously for sugar and polymer glasses. Thus, the melting of the intracellular glass by raising the temperature caused only a moderate increase in molecular mobility in the cytoplasm as compared to a huge increase in amorphous sugars.
The application of saturation transfer EPR spectroscopy to biological tissues enabled a quantitative comparison between storage stability and molecular mobility in different tissues (section III). The temperature and moisture dependence of ageing rates of seeds and pollen was found to correlate with the rotational motion of CP in the cytoplasm (chapter 6-8). An increase in the temperature resulted in a faster rotational motion in the cytoplasm of cattail pollen, analogous to faster ageing rates (chapter 6). Decreasing the water content of the pollen resulted in a decrease in rotational motion until a minimum was reached, after which rotational motion slightly increased again. The water 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 of the pollen.
We also investigated the relationship between the longevity of lettuce seeds and the molecular mobility in the cytoplasm of their radicles (chapter 7). Longevity of lettuce seeds was predicted using the viability equation of Ellis and Roberts. Increasing the temperature resulted in faster rotational motion and shorter longevity. There was a linear relationship between the logarithms of rotational motion and estimated longevity for temperatures above 5°C, which is the same temperature range for which experimental data were used to obtain the viability constants of the viability equation. Below 5°C, there was a deviation from linearity, which might stem from inaccurate predictions by the viability equation at low temperatures.
Chapter 8 further demonstrates that there is a linear relationship between the logarithms of rotational motion in the cytoplasm of seed and pollen of several plant species and their ageing rates or longevities. This linearity suggests that cytoplasmic mobility might be an important controlling factor of ageing rates. The linear relationship between the two parameters could be used to predict lifespan of germplasm at low temperatures (at which experimental determination of longevity is practically impossible) by simply measuring theτ R values at these low temperatures (chapter 7 and 8). Based on the predictions using the linear regression between ageing rate and rotational motion of CP in pea embryonic axes, an optimum water content of storage was found. This optimum water content shifted to higher values with lowering the storage temperature, as was found previously for cattail pollen based on experimental data (chapter 2). It was predicted that the longevity of seeds at high (0.12 to 0.16 g/g) water content is much higher than previously suggested on the basis of the viability equation. The predictions show that drying germplasm too far leads to decreased longevity compared to storage of germplasm at higher water contents, suggesting that current storage protocols might have to be re-examined.
Desiccation-tolerant organisms contain large amounts of soluble sugars. This, and the fact that sugars are excellent glass-formers has led to the suggestion that sugars play an important role in intracellular glass formation. The presence and amounts of oligosaccharides have been found to correlate with longevity. Furthermore, oligosaccharide glasses are known to increase the T g and viscosity of model sucrose glasses. This suggests that oligosaccharides might enhance the stability of intracellular glasses (chapter 9 and 10). Osmo-priming, i.e. pre-imbibition of seeds in an osmotic solution, can result in a decrease in oligosaccharide content and longevity. Priming pea seeds decreased the total oligosaccharide content in the embryonic axes (chapter 9). Despite the change in oligosaccharide:sucrose ratio, no differences in T g values were detected in the dry axes before and after priming as determined by DSC. Also no difference was found between the rotational mobility of CP in dry untreated axes and that of dry primed axes. Chapter 10 demonstrates that osmo-priming of impatiens and bell pepper seeds resulted in considerable decreases in longevity and oligosaccharide contents, while sucrose contents increased. Again, no differences in the T g curves were found between control and primed impatiens seeds. Similarly, there was no difference in rotational motion of CP in the cytoplasm between control and primed impatiens seeds and between control and primed bell pepper embryonic axes. It was concluded that oligosaccharides in seeds do not appear to 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.
To understand the nature and composition of biological glasses we investigated 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. Two distinct changes in the temperature dependence of molecular mobility were detected 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, at which the molecular mobility abruptly increased over several orders of magnitude, was found to correspond with a critical temperature (T c ) where the dynamics of the system changed from solid-like to liquid-like. The temperature interval between T g and T c increased with increasing molecular weight of the sugars. The interval between T g and T c in biological tissues was over 50°C, implying that the stability remained high even at temperatures far above T g . A comparably high T c -T g interval was found for the molecular mobility of poly-L-lysine, suggesting 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 .