Lateral and Rotational Mobilities of Lipids in Specific Cellular Membranes of Eucalyptus gunnii Cultivars Exhibiting Different Freezing Tolerance

Abstract

Two cell lines of Eucalyptus gunnii have been shown to keep their differential frost tolerance at the cellular level after longterm culture. They have been used to investigate the fluidity of specific cell membranes in relation with frost tolerance. Protoplasts and isolated vacuoles were obtained from both cell lines. In addition, purified plasma membrane and tonoplast (the vacuolar membrane) were separated from a crude microsomal fraction through free-flow electrophoresis. The lateral and rotational mobilities of lipids in these different membranes were studied by two biophysical techniques: fluorescence recovery after photobleaching (FRAP) and fluorescence polarization. After labeling the vacuoles isolated from the frost-sensitive cells with 1-oleoyl-2-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminocaproyl phosphatidylcholine, a single mobile component was observed with a diffusion coefficient of 2.4 x 10-9 cm2 s-i and a mobile fraction close to 100% at a temperature of 23°C. When using isolated vacuoles from the frost tolerant line, a higher lateral diffusion of tonoplast lipids was found with a diffusion coefficient of 3.2 x 10'9 cm2 s-1, still with a mobile fraction close to 100%. No convincing data were obtained when performing fluorescence recovery after photobleaching experiments on protoplasts. Fluorescence polarization experiments confirmed the differential behavior of the two cell lines for tonoplast and also for plasma membrane. In addition, they showed that intrinsically tonoplast exhibited a higher fluidity than plasma membrane. Our results provide the first information on the fluidity of tonoplast and on the compared properties of two important plant membranestonoplast and plasma membrane-through the use of two complementary biophysical approaches. In addition, they suggest there is a correlation between membrane fluidity and cold tolerance. The potential interest of plant vacuole as a natural model system in membrane studies is emphasized.

Introduction of Specific Carbohydrates into Eucalyptus gunnii Cells Increases their Freezing Tolerance

“…This is reported to result in an increase in membrane fluidity (Cadenhead and Demchak, 1969) and could prevent membrane injury during dehydration . Similar effects could explain our previous results on Eucalyptus membranes (Leborgne et al, 1992).…”

“…Previous work (Leborgne et al, 1992) on the comparison of membrane fluidity between the two Eucalyptus cell lines 832 and 867 showed that the resistant cell line 832 has a higher fluidity. The fluidity can be influenced not only by lipid composition but also by solutes which bind to the lipid head groups.…”

“…Previous data have shown that some soluble carbohydrates accumulate during acclimation and are more abundant in the resistant clones (Leborgne, unpublished work). Furthermore, results from our laboratory (Teulibres et al, 1989;Leborgne et al, 1992) have shown that Eucalyptus cell suspension cultures are convenient and appropriate biological material for studying the mechanisms of cold resistance. Using such simplified systems, and also protoplasts derived from these cell suspensions, we estimated the sugar content in different culture conditions.…”

Proteolipidic Composition of Exosomes Changes during Reticulocyte Maturation

“…The DPH probe reports on the conformational and rotational mobilities of lipid acyl chains and enabled the discrimination between the different possible states of the lipid phase by comparing the measurements with those obtained for model membranes (29). As references, we chose suspensions of lipid vesicles of various compositions as follows: POPC for the disordered liquid (l d ), POPC/chol (6:4), DPPC/chol (6:4), and POPC/SM/chol (4: (Fig.…”

“…Steady-state Fluorescence Polarization Experiments-The experiments were carried out with a homemade automatic system (29). Labeled samples were placed in a closed housing system, thermostatically controlled by Peltier elements.…”