Influence of Water Content and Temperature on Molecular Mobility and Intracellular Glasses in Seeds and Pollen

Abstract

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.

Molecular mobility in the cytoplasm: An approach to describe and predict lifespan of dry germplasm

“…It is assumed that the high viscosity of intracellular glasses decreases molecular mobility and impedes diffusion within the cytoplasm, thus slowing deleterious reactions and changes in structure and chemical composition during aging (12,(18)(19)(20)(21). Molecular mobility is increasingly considered a key factor influencing storage stability, because it is thought to control the rate of detrimental aging reactions responsible for reducing shelf life (18)(19)(20)(21)(22)(23)(24).…”

“…For random rumbling, R roughly corresponds to the average time for a molecule to progress one radian (25). The time scale of the motion detected by this technique ranges from 10 Ϫ7 to 10 Ϫ3 s, encompassing an extremely wide range of temperatures and mcs (24,26,27). We have previously assessed the rotational motion of the polar spin probe 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl (CP) incorporated in the cytoplasm of seeds and pollen as a function of mc and temperature (24,27).…”

“…The time scale of the motion detected by this technique ranges from 10 Ϫ7 to 10 Ϫ3 s, encompassing an extremely wide range of temperatures and mcs (24,26,27). We have previously assessed the rotational motion of the polar spin probe 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl (CP) incorporated in the cytoplasm of seeds and pollen as a function of mc and temperature (24,27). It was shown that the temperature dependence of the rotational motion of the spin probe in seeds changed around the glass transition temperature (T g ), with the activation energy of rotational motion changing by a factor of three when tissues were stored below or above T g (27).…”

“…Germination experiments were performed to ensure that incubation in the spin probe͞ ferricyanide solution and subsequent drying back did not affect the percentage of germination. Labeling of Typha pollen was performed as described (24). Briefly, pollen was prehydrated in water vapor for 16 h at 5°C to an mc of 0.7 g͞g and then mixed at 25°C with liquid germination medium containing 2.5 mM CP, after which the pollen was recovered by filtration.…”

Dry storage of sperm: applications in primates and domestic animals

“…Trehalose, a disaccharide found in high concentrations in many desiccation-tolerant animals and plants (Crowe et al 1984Gadd et al 1987;Westh and Ramlov 1991;Bianchi et al 1993;Drennan et al 1993;Crowe and Crowe 2000), has been the lyoprotectant of choice for many cellular dehydration studies Wolkers et al 2001;Acker et al 2002) owing to its ability to replace the hydrogenbonded water molecules and depress the phase transition in dehydrated samples (Crowe and Crowe 1988;Harrigan et al 1990;Leslie et al 1994;Tsvetkova et al 1998).Trehalose also has a high glass transition temperature (T g ; Koster et al 1994Koster et al , 2000Sun et al 1996;Crowe et al 1997;Buitink et al 1998) owing to the stability of the glycosidic bond (Kacurakova and Mathlouthi 1996;Schebor et al 1999). The high glass transition state for trehalose allows living cells to be placed into a static glassy state at ambient temperature following the removal of cellular water.…”