Freezing of isolated thylakoid membranes in complex media. VI. The effect of pH.

Thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were used as a model biomembrane system for evaluating the significance of the hydrogen ion activity for cryoprotection. After freeze-thaw treatment in a buffered complex medium adjusted to various pH, light-induced photosynthetic membrane reactions were determined at optimum proton concentration. When thylakoids were suspended at hydrogen ion activities above and below the physiologically important pH range, irreversible inhibition of membrane functions was significantly less distinct after freezing at -15 degrees C than after storage for the same time at 0 degree C. It is suggested that thylakoid preservation at subfreezing temperatures could be due to temperature- and concentration-induced changes of the proton activity in the unfrozen part of the system and retardation of the temperature-dependent aging processes of the isolated membranes. In addition, the increase in the concentration of cryoprotective compounds during freezing could stabilize chloroplast membranes against the deleterious effect of unfavorable high and low proton concentrations. Thylakoid injury brought about by lowering the pH was primarily due to dissociation of the chloroplast coupling factor (CF1), which increased the proton permeability of the membranes and caused inhibition of photophosphorylation. In media adjusted to more alkaline pH, inactivation of the water oxidation system was an initial result of membrane damage. Then, noncyclic photophosphorylation was limited by photosystem II-mediated electron flow. Photosystem I-driven electron transport was substantially more stable over a wide pH range.