Antibodies against individual thylakoid membrane proteins as molecular probes to study chemical and mechanical freezing damage in vitro

The release of proteins and the loss of biochemical activities under mechanical and chemical stresses during freezing of isolated thylakoid membranes were investigated, using polyacrylamide gel electrophoresis, single radial immunodiffusion and the measurement of cyclic photophosphorylation. Antibodies against purified proteins derived from the stromal (coupling factor CF1, ferredoxin-NADP⁺ reductase) and the lumenal side (plastocyanin) of the membrane vesicles were used as probes. Low initial solute concentrations were employed to generate mechanical stress. Chemical stresses were manipulated by varying the molar ratios of cryotoxic to cryoprotective solutes at high initial solute concentrations. Constant low amounts of ferredoxin-NADP⁺ reductase were lost from the membranes during freezing, irrespective of the composition of the suspending media. Damage at high initial osmolalities was accompanied by the release of CF1, which was influenced by the ratio of potentially cryotoxic to cryoprotective solutes, as demanded by the colligative theory of membrane cryopreservation. CF1 release and loss of cyclic photophosphorylation were linearly correlated at different ratios of salt to sucrose. However, the correlation data revealed that CF1 release could account for only part of the observed cryoinjury. Plastocyanin release was predominant at low initial osmolalities and was not influenced by the chemical composition of the suspending media. This indicates mechanical damage by membrane rupture. Under these circumstances loss of plastocyanin and loss of cyclic photophosphorylation were linearly correlated. Loss of photophosphorylation could be prevented by the addition of up to 1.2 mg plastocyanin/ml prior to freezing. It could also be ameliorated to a large extent by raising the phenazine methosulfate concentration in the test assay from 30 to 230 μM. This indicates that the membranes are able to reseal after rupture, maintaining a proton gradient upon illumination and that it is the loss of plastocyanin from their lumen that inhibits cyclic photophosphorylation.