Electric field-induced fusion of isolated vacuoles and protoplasts of different developmental and metabolic provenience

Using an electric field pulse technique, we induced fusion between vacuoles and protoplasts of Kalanchoë daigremontiana , between protoplasts from etiolated and green leaf mesophyll, and between mesophyll protoplasts from plants of different physiological properties ( Avena sativa : C₃ mechanism of photosynthesis, Kalanchoë daigremontiana : crassulacean acid metabolism). Close membrane contact amongst protoplasts or between protoplasts and vacuoles (as required for fusion) was achieved by the application of an alternating, non-uniform electric field to the suspension. Due to the dielectrophoresis effect the cells attach to each other along the field lines. The fusion process is initiated by the injection of an electric field pulse of high intensity and short duration (μs range). The field intensity has to be sufficiently high to induce reversible breakdown in the area of close membrane contact. After the application of the field pulse, the fusion process is initiated and completed within seconds to a few minutes, depending on the material investigated.
Fusion occurs between protoplasts and vacuoles as well as between protoplasts of different species. Both tonoplast and plasma membranes completely intermingled, indicating that in contrast to suggestions in the literature these membranes are compatible. Furthermore the cytoplasms of etiolated and green protoplasts obviously do not mix after fusion is completed, as etioplasts and chloroplasts kept separated from each other. In all experiments the volume of the fusion product equalled the sum of the compartments that underwent fusion. The wide spectrum of possible applications resulting from these fusion experiments in relation to metabolic problems is discussed.     

High frequency fusion of plant protoplasts by electric fields

Mesophyll cell protoplasts of Vicia faba were collected by dielectrophoresis in a highly inhomogeneous alternating electric field (sine wave, 5 to 10 V peak-to-peak value, 500 kHz, electrode distance 200 m). Under these conditions, the cells formed aggregates of two or three on the electrodes or bridges consisting of 4 to 6 protoplasts between the electrodes. This pearl chain arrangement of the cells was only stable for the duration of the applied field. By the additional application of a high single field pulse (square wave, 15 V, 50 s), it was possible to induce cell fusion within the aggregates or bridges. This electrically stimulated fusion of cells proceeded at room temperature and under physiological pH-conditions, without the use of chemical reagents, and gave a high yield. Smaller fused aggregates formed spheres within a few minutes. During the dielectrophoretically induced adhesion of the protoplasts to one another, the field strength must be chosen such that dielectric breakdown of the membrane is avoided, but at the same time, the strength of the subsequently applied single field pulse must be high enough to induce dielectric breakdown at the sites of contact between the protoplast membranes. From these results, one can conclude that in addition to close contact between membranes, the prerequisite for electrically stimulated cell fusion is dielectric breakdown which leads to changes in the membrane conductance, permeability, and probably fluidity.Presented at II Congress FESPP, Santiago de Compostela, Spain, 27.7.–1.8.1980, and Gordon Research Conference of Bioelectrochemistry, Tilton, New Hampshire, USA, 4.8.–8.8.1980     

Tissue specific variation of C-glycosylflavone patterns in oat leaves as influenced by the environment

A comparison is made between the flavone patterns accumulating in epidermal tissues and in the mesophyll of oat primary leaves grown in a phytotron and under field conditions. In developing leaves cultivated under standard conditions, varying patterns of two vitexin-derived O-rhamnosides and of one isovitexin O-arabinoside are produced in the basal region as the result of basal meristem activity. These patterns are tissue specific and differ quantitatively in the epidermis and the mesophyll. During the course of subsequent growth and differentiation, this pattern is constant as the compounds are moved upwards due to basipetal leaf growth. In comparison, the flavone patterns generated in the basal section of leaves grown in the field do not vary significantly. There is the additional accumulation of isoorientin O-arabinoside. Again flavone patterns are tissue specific, but in contrast to standard growth they are modified characteristically in those leaf tissues which are already morphologically differentiated. It is possible that the isovitexin moiety of the O-arabinoside is oxidized to the corresponding isoorientin derivative in the mesophyll. Moreover, field-grown leaves show a two-fold increase in flavone content in each leaf epidermis and a six-fold increase in the mesophyll when compared to the corresponding tissues of phytotron-grown leaves.     

Soybean leaf photosynthesis in relation to maturity classification and stage of growth

Leaf photosynthetic rates were measured on field-grown soybeans during the 1980 season. Comparisons were made between different cultivars and isolines representative of maturity groups I–IV. Mature, fully expanded leaves at different nodes on the plant were measured in high light to determine which had the highest potential photosynthetic rates at any one time. Successive leaves during the growing season had maximum rates which increased from about 22 mol CO₂ m^-2 s^-1 on 25 June to a peak of 30–44 mol CO₂ m^-2 s^-1 in early August.The persistency and eventual decline in the maximum rate was associated with the maturity group and related dates of flowering, pod fill and onset of senescence. Early maturing cultivars (groups I and II) had higher peak rates (38–44 mol CO₂ m^-2 s^-1) than later maturing cultivars (30–35 mol CO₂ m^-2 s^-1, groups III and IV). However, the photosynthetic rates of early maturing cultivars declined rapidly after attaining their peak, whereas the leaves of later maturing cultivars maintained their photosynthetic activity for much longer.     

A theory of the electric field-induced phase transition of phospholipid bilayers

Improving the statistical mechanical model of Jacobs et al. (Jacobs, R.E., Hudson, B. and Andersen, H.C. (1975) Proc. Natl. Acad. Sci. U.S. 72, 3993–3997) we have constructed a model which describes not only the temperature but also the external field dependence of the membrane structure of phospholipid bilayers. In addition to the interactions between head groups, between hydrocarbon chains, and the internal conformational energy of the chains (which were considered in Jacobs' model), our model includes the energy of deformation and the field energy as well.By the aid of this model we can explain the phenomenon of dielectric breakdown, the non-linearity of current-voltage characteristics, and the mechanism of membrane elasticity.The free energy of the membrane, the average number of the gauche conformations in the hydrocarbon interior and at the membrane surface, gauche distribution along the chain, the membrane thickness, area and volume are calculated at different temperatures and voltages. The calculation also gives the temperature dependence of Young's modulus and that of the linear thermal expansion coefficient.     

A pulsed NMR study of lipids,bound water and sodium ions in macroscopically-oriented lecithin/water lyotropic liquid crystal model membrane systems

The temperature and orientation dependence of pulsed NMR ‘free induction decay’ signals have been studied in detail for lipid bilayers macroscopically-oriented between glass slides. Results for the lipid molecules (¹H, ³¹P), bound water (²H₂O) and ions dissolved in the aqueous phase (²³Na) are presented. Bilayers of egg-lecithin, dimyristoyl lecithin and potassium oleate have been investigated. In the liquid crystal phase all the signals, including those from bound water and ions exhibit a |3 cos²? ? 1| dependence on orientation of the bilayer normal to the magnetic field. In the case of DML samples, some orientation dependence of both ¹H and ²H signals persists in the gel phase, indicating that the lipid molecules retain a degree of reorientational freedom about their long axes in this phase. At the gel-liquid crystal transition the ²H quadrupole spittings undergo a discontinuous change. Results are interpreted in terms of a model in which water molecules are bound to individual lipid head groups and reorient with them, while sodium ions are located in the aqueous channel between bilayers.     

Planarian activity differences when maintained in water pre-treated with magnetic fields: a nonlinear effect

There have been multiple claims that exposing water to a static magnetic field affects its properties which influence living systems. To test this hypothesis, planarian subsequent to dissection were maintained in spring water that had been previously exposed for only one day to one of three (16, 160, or 1,600 G) intensity static magnetic fields or to a reference condition. Although there was no significant difference in regeneration rates over the subsequent seven-day period, there was a statistically significant nonlinear effect for planarian mobility and diffusion rates. Both mobility rates and diffusion velocity of a liquid within the water that had been exposed to the 16 G field was about twice that for water exposed to the other intensities. These results imply that nonlinear biophysical effects may emerge under specific conditions of intensity ranges for particular volumes of water.     

Variations of soil structure and microbial population in a compost amended soil

Changes in soil structure and in microbial population were recorded in a long term field experiment over the growing season of maize (June–November). Determinations were made on samples from plots which had received, for two years, the following treatments: mineral fertilizers, farmyard manure and three rates of compost. Seasonal variations were observed for the stability of soil aggregates, total porosity, pore size distribution, mycorrhizal infection and aerobic cellulolytic microorganisms. The stability of the soil aggregates changed in a similar way to that found for both mycorrhizal infection and the number of aerobic cellulolytic microorganisms. Physical characteristics were not affected in any instance by the organic dressings and microbiological populations were generally influenced only by the higher doses of compost.     

Neurochemical effects of a 20 kHz magnetic field on the central nervous system in prenatally exposed mice

C57/B1 mice were exposed during pregnancy (gestation days 0–19) to a 20 kHz magnetic field (MF). The asymmetric sawtooth-wave form magnetic field in the exposed racks had a flux density of 15 μT (peak to peak). After 19 days, the exposure was terminated, and the mice were housed individually under normal laboratory conditions. On postnatal day (PD) 1, PD21, and PD308, various neurochemical markers in the brains of the offspring were investigated and the brains weighed. No significant difference was found in the whole brain weight at PD1 or PD21 between exposed offspring and control animals. However, on PD308, a significant decrease in weight of the whole brain was detected in exposed animals. No significant differences were found in the weight of cortex, hippocampus, septum, or cerebellum on any of the sampling occasions, nor were any significant differences detected in protein-, DNA-level, nerve growth factor (NGF), acetylcholine esterase- (AChE), or 2′,3′-cyclic nucle-otide 3′-phosphodiesterase- (CNP; marker for oligodendrocytes) activities on PD21 in cerebellum. Cortex showed a more complex pattern of response to MF: MF treatment resulted in a decrease in DNA level and increases in the activities of CNP, AChE, and NGF protein. On PD308, the amount of DNA was significantly reduced in MF-treated cerebellum and CNP activity was still enhanced in MF-treated cortex compared to controls. Most of the effects of MF treatment during the embryonic period were similar to those induced by ionizing radiation but much weaker. However, the duration of the exposure required to elucidate the response of different markers to MF seems to be greater and effects appear later during development compared to responses to ionizing radiation. © 1995 Wiley-Liss, Inc.