Sucrose influx and mechanical damage by osmotic stress to thylakoid membranes during an in vitro freeze-thaw cycle

Abstract

The effects of a freeze-thaw cycle on thylakoid volume were studied by micro-haematocrit centrifugation. At initial osmolalities prohibiting mechanical freezing damage thylakoids showed a volume increase after freezing, indicating solute uptake. At initial osmolalities below 0.1 osmolal thylakoids lost volume, apparently because of membrane rupture. Rupture occurred at a volume smaller than the maximum volume of unfrozen controls. Resealing of the membranes after freeze-induced rupture was demonstrated by the ability of thylakoids frozen at low initial osmolalities to act as osmometers in response to changes in the external solute concentration. From experiments conducted at 0°C the permeability coefficient for sucrose across thylakoid membranes was calculated as P = (1.3 ± 0.25) · 10 −10 m/s , independent of the applied concentration gradient. The permeability coefficient at −20°C was estimated from samples frozen in the presence of 500 mM sucrose, which yielded complete cryoprotection. A value of P = 0.18 · 10 −10 m/s was calculated and from this a Q 10 of 2.67 could be derived for the diffusion of sucrose across thylakoid membranes. From the comparison of P values at 0°C and 5°C a Q 10 of 2.89 was calculated for diffusion under non-freezing conditions. It is suggested that mechanical freezing damage to thylakoid membranes is brought about by the diffusion of solutes into the intrathylakoid space and by loss of membrane material from the vesicles under strongly hypertonic conditions. During thawing osmotic water influx leads to thylakoid swelling and membrane rupture.