Chloroplast membrane damage during freezing: the lipid phase

In order to study the effect of freeze damage to chloroplast membranes microviscosity of spinach thylakoids was probed by stearic acid spin labels. Changes in ESR parameters have been determined either as a function of temperature or during freezing at ?15 °C as a function of time. An empirical parameter $\text{h}{}_{\mathrm{+}}\text{h}{}_0$ (ratio of height of a low field line component h₊ over height of the central line h₀) proved to be very sensitive to minute changes in membrane structure.In cryoprotected chloroplast membranes Arrhenius plot breaks indicative of phase changes are observed at +15 and ?10 °C. Breaks in the Arrhenius plots were not observed in vesicles prepared from chloroplast lipids by sonication. Instead, a melting zone was indicated below ?30 °C.Freeze damage of thylakoids during storage at ?15 °C is reflected in an increase of $\text{h}{}_{\mathrm{+}}\text{h}{}_0$ and a decrease in central line width W₀. At +20 °C, differences between the ESR parameters of active as compared to freeze-damaged membranes could be detected, if the osmolarity of the suspending medium exceeded 200 mosm. The observed changes in line shapes are interpreted as an increase in mobility and/or orientation of the lipids following the swelling of thylakoids. They do not indicate a disorganization of the lipid phase. Sedimentation experiments indicated that the freeze-damaged swollen membranes still exhibited osmotic responses. It is suggested that freezing which is known to dissociate proteins from the membranes altered the charge distribution of the membranes leading first to membrane swelling and finally, by the opening of hydrophilic channels, to membrane collapse.