Freezing Injury and Uncoupling of Phosphorylation from Electron Transport in Chloroplasts

Abstract

Suimmary. Freezing of chloroplast membranes uncouples 'photophosphorylation fron electron transport and inactivates the light-dependent and thiol-requiring ATPase, con-formational changgs and the light-dependent proton uptake. AllI of these energy requiring activities can be protected against inactivation by addition of sucrose prior to freezing. The direct relation to 'photophosphorylation is demonstrated iby the quantitatively similar response of photophosphorylation and the other activities to sucrose protection. Salts interfere w'ith tihe protection afforded by sucrose. In contrast to the light-dependent ATPase, the ATPase activities which are unmasked by digestion with trypsin show no significant response to freezing. Similarly, the chloro-plast coupling 'factor, Which is released !from the membranes by ethylenediamine tetraacetic acid treatment, survives freezing. The membranes, which are depleted of the factor, are damaged by ,freezing. The results suggest that uncoupling of phosphorylation from electron transport is caused by interference of freezing with a 'structure involved in the formation of a non-phosphorylated higgh energy state of chloroplasts. They are best explained on the basis of Mitchell's theory of phosphorylation. Since freezing alters the qpermeability properties of chloroplast membranes-frozen membrane vesicles no longer function as osmometers-it may be assumed that freezing uncouples phosphorylation from electron transport by preventing the formation of a pH igradient across the vesicle membranes owin'g to proton leakage through the membranes. From the results, the basic injury icaused by freezing appears to consist in the alteration of permeability 'properties of biological membranes (lue to the dehydration which accompanies 'freezing.