Simulation of in situ freezing damage of the photosynthetic apparatus by freezing in vitro of thylakoids suspended in complex media.

Chloroplast thylakoid membranes were isolated from leaves of unhardened and cold-acclimated spinach (Spinacia oleracea L.). For freezethaw treatment, the membranes were suspended in complex media composed to simulate the solute concentrations in the chloroplast stroma in the unhardened and hardened states of the leaves. In particular, high concentrations of amino acids were applied for simulating the hardened state. After frost treatment, photosynthetic activities and chlorophyll fluorescence parameters of the thylakoids were tested to determine the degree of freezing damage. The results revealed a pattern of freezing injury similar to that observed upon frost treatment of thylakoids in situ. A major manifestation of damage was the inhibition of photosynthetic electron transport. Uncoupling of photophosphorylation, which is the dominating effect of freezing of thylakoids suspended in binary solutions (e.g., containing one sugar and one inorganic salt), was also visible but less pronounced in the complex media. Thylakoids obtained from cold-acclimated leaves did not exhibit an increased frost tolerance in vitro, as compared with thylakoids from unhardened plants. The results, furthermore, indicated a strong protective effect of free amino acids at the concentrations and composition found in chloroplasts of hardened leaves. The presence of inorganic salts in the complex media slightly stabilized rather than damaged the membranes during freezing. It is concluded that inactivation of thylakoids in situ may be understood as the destabilizing action of the combined solutes surrounding the thylakoids, occurring when solute concentration is raised due to freezing of water.