Selective osmotic effect on diffusion of plasma membrane lipids in maize protoplasts.

Abstract

Osmotic levels in the range typically used during plant protoplast isolation and incubation were investigated with regard to effects on the lateral diffusion of lipid probes in the plasma membrane. The lateral diffusion coefficient of a fluorescent sterol probe in the plasma membrane of maize (Zea mays L.) root protoplasts in a medium containing 0.45 M mannitol was 4 times faster than when the medium contained 0.9 M mannitol. The lateral diffusion coefficient of a fluorescent phospholipid probe, however, did not change over this range of mannitol concentrations. Similar diffusion characteristics were observed when the medium contained trehalose instead of mannitol. Slower lateral diffusion of the sterol probe at higher osmolality was also observed when KCI/CaCl2-based osmotic media were used with protoplasts isolated by a mechanical, rather than by an enzymic, method. Extraction and quantitation of total lipids from protoplasts showed that both the phospholipid and sterol contents per protoplast decreased with increasing osmolality, while the sterol/phospholipid ratio increased. These results demonstrate that osmotic stress induces selective changes in both the composition and biophysical properties of plant membranes.Evidence for the existence of lipid domains in plant and other biological membranes has come from a variety of experiments (1). One source of such evidence has been studies of lipid dynamics involving long-range motions-i.e., motions over distances that are large compared to molecular dimensions. Use of fluorescence techniques to measure such lipid motions in the plasma membrane of plant protoplasts was first reported by Metcalf et al. (2). In that work, fluorescence redistribution after photobleaching was used to measure the lateral diffusion of various fluorescent lipid probes in protoplasts from suspension-cultured soybean cells. Two diffusion coefficients, one in the 2-5 x 1010 cm2/sec range and another in the 1-6 x 10-9 cm2/sec range, were simultaneously observed under certain conditions of temperature and probe concentration. The mobile fraction, summed for the two diffusing species, was in the 0.56-0.76 range-i.e., a significant portion of the lipid probe moved so slowly as to appear immobile on the time scale of the experiment. These heterogeneous diffusion parameters were interpreted as evidence for the existence of lipid domains in the plasma membrane of soybean protoplasts (1).Fluorescence photobleaching recovery experiments carried out in our laboratory (3, 4) have yielded results that are comparable to those reported by Metcalf et al. (2). Fluorescent lipid probes (5) were used that enabled the consistent resolution of two diffusion coefficients over a range of conditions. The lateral diffusion coefficient of a fluorescent sterol probe in the plasma membrane of root cortical protoplasts from two different maize lines was 3-7 x 10-10 cm2/sec for temperatures in the 3.50C-370C range (3). Over the same temperature range, the diffusion coefficient of a fluorescent phospholipid prob...

Photon emission from melanoma cells during brief stimulation by patterned magnetic fields: is the source coupled to rotational diffusion within the membrane?

“…Circumferential movements, as manifested by the lateral diffusion of lipids within the plasma cell membrane, have been inferred by a variety of methods (Lenaz 1987;Furtula et al 1990). The diffusion coefficients (D) of selected lipids, such as the mobile lipophilic quinones involved with electron transfer, range from 10 -6 cm 2 /s (Lenaz 1987) to 3 × 10 -9 cm 2 /s (Furtula et al 1990). The equivalent diffusion length (4Dt) 1/2 , where t is time, in one second would be between 2 × 10 -5 m/s to 5.5 × 10 -7 m/s.…”

Effects of low temperature on lateral diffusion in plasma membranes on maize (Zea mays L.) root cortex protoplasts: relevance to chilling sensitivity

“…characteristics (Metcalf. Wang & Schindler 1986;Caldwell & Whitman 1987;Dugas et al 1989;Walko & Nothnagel 1989;Furtula et al 1990;). For the lipid probes LY-DC,2:()PE and LY-Sito, there was a slight tendency for the mobile' fraction to increase with increasing temperature in the range from 2 to 21 °C (Figs 3b & 4b).…”

“…Cortical tissue was allowed to digest for 2 h before being filtered through a nylon cloth having 63 fiva diameter pores to separate protoplasts from cell wall debris. Protoplast isolation continued according to the procedure of Gronwald & Leonard (1982), except that mannitol concentrations were 450 mol m~^ (Furtula, Khan & Nothnagel 1990).…”

“…Observation of lateral diffusion of the fluorescent probes in the plasma membranes of maize protoplasts was made using FPR. The instrumentation and methods used have been previously described (Dugas et al 1989;Furtula et al 1990;Nothnagel et al 1990). Curve fitting based on the theoretical models of Axelrod et al (1976) was used to obtain the lateral diffusion coefficient, D, and the mobile fraction, m, of the fiuorescent molecules.…”

Lipid content in higher plants under osmotic stress

“…It is evident an increase of PG, PC and PI in stressed sample and, conversely to leaves, a decrease of BPG and PE. Selective changes in lipid content and in biophysical properties of plant membranes have been reported for maize protoplast under osmotic shock [14]. In particular, a loss of phospholipids, mainly those containing unsaturated fatty acyl chains, and a concomitant increase in neutral lipids such as triacylglycerols has been documented.…”