Is vitrification involved in depression of the phase transition temperature in dry phospholipids?

AbstractRecent 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, 14–150) 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 ≥ 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°C, but on the second and subsequent heating scans Tm falls to about 25°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

Membrane chemical stability and seed longevity

Here, we investigate the relationships between the chemical stability of the membrane surface and seed longevity. Dry embryos of long-lived tomato and short-lived onion seeds were labeled with 5-doxyl-stearic acid (5-DS). Temperature-induced loss of the electron spin resonance signal caused by chemical conversion of 5-DS to nonparamagnetic species was used to characterize the membrane surface chemical stability. No difference was found between temperature plots of 5-DS signal intensity in dry onion and tomato below 345 K. Above this temperature, the 5-DS signal remained unchanged in tomato embryos and irreversibly disappeared in onion seeds. The role of the physical state and chemical status of the membrane environment in the chemical stability of membrane surfaces was estimated for model systems containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) dried alone or in the presence of trehalose or glucose. Fourier transform infrared spectroscopy was used to follow temperature-induced structural changes in dry POPC. Spin-label technique was used to relate the chemical stability of 5-DS with the dynamic properties of the bilayer and 5-DS motion behavior. In all the models, the decrease in 5-DS signal intensity was always observed above Tm for the membrane surface. The 5-DS signal was irreversibly lost at high temperature when dry POPC was embedded in a glucose matrix. The loss of 5-DS signal was moderate when POPC was dried alone or in the presence of trehalose. Comparison of model and in vivo data shows that the differences in longevity between onion and tomato seeds are caused by differences in the chemical status of the membrane surface rather than the degree of its immobilization

Imbibitional Chilling Injury in Pollen: Involvement of the Respiratory Chain

Chilling injury is sustained by dry pollen of Typha latifolia L. upon hydration in germination medium at 0°C. This injury is evidenced as poor germination, low vigor, and depressed respiration. Isolated mitochondria showed multiple sites of impaired electron transport. Besides losses of cytochrome (Cyt) c and NAD(+), the activities of membrane-bound enzyme complexes such as Cyt oxidase, NADH-duroquinone oxidoreductase, succinate-duroquinone oxidoreductase, and malate-duroquinone oxidoreductase were severely affected. Similarly, as in isolated mitochondria, in situ tests of mitochondrial activity showed that Cyt c was partially lost from its site of action. Re-addition of the lost Cyt c to the grains restored the N,N,N′,N′-tetramethyl p-phenylenediamine dihydrochloride plus ascorbate-mediated electron transport from Cyt c to O(2), but did not significantly accelerate the overall O(2) uptake. Electron flow to duroquinone in the injured grains was low, indicating that lesions at the substrate side of ubiquinone determine the rate of O(2) consumption. Leakage of NAD(+), and also of adenylate phosphates and Krebs cycle substrates out of the injured grains, was considerable. Increasing the initial moisture content of the grains strongly enhanced their resistance to cold hydration. Below 17% moisture content (fresh weight basis), the decrease in vigor closely matched the loss of NAD(+) and adenosine phosphates. Vitality was irreversibly lost by cold hydration below 10 to 12% initial moisture content. Injury to dry pollen was prevented by imbibition at 27°C. Decrease of vigor and increased leakage, however, started below 20°C, and complete loss of vitality occurred below 10°C. These results are interpreted as evidence that loss of membrane integrity is the primary cause of imbibitional chilling injury

The Responses of Cytochrome Redox State and Energy Metabolism to Dehydration Support a Role for Cytoplasmic Viscosity in Desiccation Tolerance

To characterize the depression of metabolism in anhydrobiotes, the redox state of cytochromes and energy metabolism were studied during dehydration of soaked cowpea (Vigna unguiculata) cotyledons and pollens of Typha latifolia and Impatiens glandulifera. Between water contents (WC) of 1.0 and 0.6 g H(2)O/g dry weight (g/g), viscosity as measured by electron spin resonance spectroscopy increased from 0.15 to 0.27 poise. This initial water loss was accompanied by a 50% decrease in respiration rates, whereas the adenylate energy charge remained constant at 0.8, and cytochrome c oxidase (COX) remained fully oxidized. From WC of 0.6 to 0.2 g/g, viscosity increased exponentially. The adenylate energy charge declined to 0.4 in seeds and 0.2 in pollen, whereas COX became progressively reduced. At WC of less than 0.2 g/g, COX remained fully reduced, whereas respiration ceased. When dried under N(2), COX remained 63% reduced in cotyledons until WC was 0.7 g/g and was fully reduced at 0.2 g/g. During drying under pure O(2), the pattern of COX reduction was similar to that of air-dried tissues, although the maximum reduction was 70% in dried tissues. Thus, at WC of less than 0.6 g/g, the reduction of COX probably originates from a decreased O(2) availability as a result of the increased viscosity and impeded diffusion. We suggest that viscosity is a valuable parameter to characterize the relation between desiccation and decrease in metabolism. The implications for desiccation tolerance are discussed