effect of freezing on thylakoid membranes in the presence of organic acids

The effect of salts of organic acids on washed and non-washed chloroplast membranes during freezing was investigated. Thylakoids were isolated from spinach leaves (Spinacia oleracea L.) and, prior to freezing, salts of various organic acids or inorganic salts or both were added. Freezing occurred for 3 to 4 hours at -25 C. After thawing membrane integrity was investigated by measuring the activity of cyclic photophosphorylation. At very low NaCl levels (1 to 3 mM, washed thylakoids) salts of organic acids either could not prevent membrane inactivation in the course of freezing (succinate) or were effective only at relatively high concentrations (0.1 M or more of acetate, pyruvate, malate, tartrate, citrate). If NaCl was present at higher concentrations (e.g., 0.1 M) some organic acids, e.g. succinate, malate, tartrate, and citrate, were able to protect frost-sensitive thylakoids at surprisingly low concentrations (10 to 20 mM). Other inorganic salts such as KCl, MgCl2, NaNO3 could also induce protection by organic acids which otherwise were ineffective or poorly effective. For effective protection, a more or less constant ratio between inorganic salt and organic acid or between two or more organic acids had to be maintained. Departure to either side from the optimal ratio led to progressive inactivation. The unspecificity of the protective effect of organic acids suggests that these compounds protect colligatively. There are also indications that, in addition, more specific interaction with the membranes contributes to protection. At temperatures above the freezing point, the presence of salts of organic acids decreased the rate of membrane inactivation by high electrolyte concentrations.