Mechanism of Hyperosmotic and Extracellular Freezing Injury

The vome of isolated protoplasts of rye (Secale cereale L. cv Puma) in a suspending solution at constant concentration is shown to be neglgbly changed by tensions in the plasma membrane which approach that tension necessary to lyse them. This aflows a detailed investigation of the plasma membrane stress-strain relation by micropipette aspiration. Over periods less than a second, the membrane behaves as an elastic two-dimensional fluid with an area modulus ofelasticity of230 milnewtons per meter. Over longer periods, the stress-strain relation approaches a surface energy law-the resting tension is independent of area and has a value of the order 100 micronewtons per meter. Over longer periods the untensioned area, which is defhied as the area that would be occupied by the molecules in the membrane at any given time if the tension were zero, increases with time under large imposed tensions and decreases under sufficiently small tension. It is proposed that these long term responses are the result of exchange of material between the plane of the membrane and a reservoir of membrane material. The irreversibility of large contractions in area is demonstrated directly, and the behavior of protoplasts during osmoticaLy induced cycles of contraction and expansion is explained in terms of the membrane stress-strain relation. When the volume of isolated protoplasts is reduced by transferring them to a medium with greater osmotic pressure, the area of their plasma membranes is also reduced and they regain their spherical shape. Thus, osmotic expansions and contractions of spherical protoplasts both produce changes in the area of the plasma membrane. Steponkus and co-workers (22, 23, 25) have shown that one may ascribe to a population of protoplasts an absolute surface area increment, greater expansions than which cause the plasma membrane to lyse. This increment is independent of the extent of contraction. One form of freeze-thaw injury suffered by protoplasts from nonacclimated tissue is the result of incompletely reversible contractions of the plasma membrane during freezing of the suspending medium (22, 23, 25). These studies suggest that, during large deformation, the mechanical properties of the plasma membrane of isolated protoplasts are qualitatively different from l This material is, in part, based on work supported by the National Science Foundation under Grant PCM-8021688 and the United States Department of Energy under Contract DE-AC02-81R10917. Department of Agronomy Series Paper 1426. 2 CufTent address: School of Physics, The University of New South Wales, P.O. Box 1, Kensington, N. S. W. 2033, Australia. those of other membranes hitherto studied, and that particular mechanical properties may, in part, confer on a given protoplast its propensity to lyse during a given freeze-thaw cycle, or other cycle of osmotic contraction and expansion. Previous experimental and theoretical studies of the mechanical properties of biological membranes have been almost exclusively of sea urchin eggs (4, 15) and RBC3 (16, 19, 24) and have usually considered only small or zero changes in area. The mechanical properties of RBC have been considered in several sophisticated analyses (3, 5) and the classical treatment of viscoelasticity has been extended to describe the SSR of RBC (6). Tensions have been measured in the membranes of protoplasts from giant algal cells (12) but, as in Reference 15, changes in area were not reported. Previously (28), we have reported briefly the measurement of the SSR of the plasma membrane of isolated rye protoplasts, using the elastimeter of Mitchison and Swann (15). In this paper we report: (a) an analysis of the use of the elastimeter which demonstrates that, in this system, changes in area may be determined more accurately than in other systems hitherto studied; (b) measurements ofthe SSR ofthe protoplast plasma membrane including its resting tension, its elastic modulus, the time dependence of area changes caused by imposed constant tensions, and the change with time ofthe tension necessary to maintain a given deformation in area; and (c) a direct demonstration of the critical role of membrane contraction in lysis caused by osmotically induced contraction and expansion. MATERIALS AND METHODS Seedlings of rye (Secale cereale L. cv Puma) were grown as previously described (25) for 2 weeks at 20/15°C (day/night, 13h photoperiod). Under these conditions, the seedlings did not cold acclimate and 50%o of the crowns survived a freeze-thaw cycle to -2°C. Protoplasts were enzymically isolated in 0.53 Osm sorbitol solutions (in which their average size is inferred to be approximately equal to that in vivo) using a method previously described (25), except that the leaves were brushed with carborundum powder instead of being finely chopped prior to digestion. Protoplasts ranged in radius from 7 to 25 ,um in the 0.53 Osm suspending media. Except as noted, those with radii of 20 ± I ,um were routinely chosen for the experiments reported here. The elastimeter of Mitchison and Swann (15) comprises a micropipette which abuts the cell and a manometer with which a negative pressure is applied (Fig. 1). Pipettes were made by pulling 1-mm diameter glass tubing on a commercial electrode puller. The tips were then snapped off and only those with a neat planar 3Abbreviations: RBC, red blood cells; N, Newton; Osm, osmolal; SSR, stress-strain relation; TSAI, tolerable surface area increment.