Freeze-Thaw Injury to Isolated Spinach Protoplasts and Its Simulation at Above Freezing Temperatures

Abstract

Possibilities to account for the mechanism of freeze-thaw injury to isolated protoplasts of Spinacia oleracea L. cv. Winter Bloomsdale were investigated. A freeze-thaw cycle to-3.9 C resulted in 80% lysis of the protoplasts. At-3.9 C, protoplasts are exposed to the equivalent of a 2.1 osmolal solution. Isolated protoplasts behave as ideal osmometers in the range of concentrations tested (0.35 to 2.75 osmolal), arguing against a minimum critical volume as a mechanism of injury. Average protoplast volume after a freeze-thaw cycle was not greatly different than the volume before freezing, arguing against an irreversible influx of solutes while frozen. A wide variety of sugars and sugar alcohols, none of which was freely permeant, were capable of protecting against injury which occurred when protoplasts were frozen in salt solutions. The extent of injury was also dependent upon the type of monovalent ions present, with Li = Na > K = Rb = Cs and Cl-Br > I, in order of decreasing protoplast survival. Osmotic conditions encountered during a freeze-thaw cycle were established at room temperature by exposing protoplasts to high salt concentrations and then diluting the osmoticum. Injury occurred only after dilution of the osmoticum and was correlated with the expansion of the plasma membrane. Injury observed in frozen-thawed protoplasts was correlated with the increase in surface area the plasma membrane should have undergone during thawing, supporting the contention that contraction of the plasma membrane during freezing and its expansion during thawing are two interacting lesions which cause protoplast lysis during a freeze-thaw cycle. Research concerned with freezing injuryin plants and work on microorganisms and animal tissues have essentially been two distinct fields of study and little effort has been made to ".... incorporate the results into more effective concepts of freezing injury" (12). Direct comparisons between the two systems have not and should not have been made since the freezing processes of the two systems are quite disparate. With the possible exception of algae, the environment of plant cells frozen in vivo is considerably different from that of single mammalian cells frozen in vitro. Although the latter are suspended in isotonic aqueous solutions, the former are osmotically stable in H20 due, in large part, to pressure developed by constraints on the cell membrane by a relatively rigid cell wall. Ice, which surrounds single cells when suspensions are frozen (16), forms only in isolated regions of some plant tissues (6), presumably due to the sparsity of extracellular water available for nucleation and crystallization and/or the location offavorable nucleation sites (7). These disparities between in vivo and in vitro studies preclude a comparison between the two systems. 'Department of Agronomy Series Paper 1214. 2Present address:

Dynamics of membrane exchange of the plasma membrane and the lysis of isolated protoplasts during rapid expansions in area

“…Acclimated plants were transferred to 13~176 (11.5-hr photoperiod) for one week and then to 2~ (10-hr photoperiod) for four weeks, after which they were fully acclimated (LTs0 -25 to -30~ Protoplasts were enzymically isolated from excised leaves in a solution of 1.5% (wt/vol) Cellulysin (Calbiochem, LaJolla. Calif.) 0.5% macerase (Calbiochem), and 0.3% potassium dextran sulfate as described previously (Wiest & Steponkus, 1978) with the following modifications. The leaves were brushed with carborundum (329 grit) prior to digestion.…”

Microtubules in Mesophyll Cells of Nonacclimated and Cold-Acclimated Spinach

“…The concentration of the solutions was gradually increased to simulate the freezing rate (2°C/h). Osmotic concentration, based on the vapor pressure of ice at each test temperature, simulated the freezeinduced dehydration at various temperatures (19). Three concentrations of osmotica were examined: 1.…”

Mechanical study of the deformation and rupture of the plasma membranes of protoplasts during osmotic expansions

“…The area increase at which 50% of a population lyse is called the Tolerable Surface Area Increment (TSAI), and it is largely independent of the time course of the osmotic treatment (Wiest & Steponkus, 1978). Plant protoplasts isolated from leaf mesophyll cells are spherical over a range of osmotic pressures, and their plasma membranes appear smooth and continuous in both light and electron micrographs.…”

“…Those isolated from cold-acclimated plants can be expanded in area by typically 60% without lysis. In either case, some individuals will expand rather more than the mean value without lysis (Wiest & Steponkus, 1978).…”