Osmotic Stress Causes Mechanical Freeze-Thaw Damage to Thylakoids In Vitro and In Vivo

Solute influx into the thylakoid lumen during freezing in vitro causes a volume increase. After freezing at -20°C for 3 h in sucrose solutions at initial concentrations above 0.1 osmolal thylakoids showed a threefold increase in volume after thawing compared to unfrozen controls. Electron microscopy showed that thylakoids frozen in situ swell during thawing. Depending on the freezing conditions, extensive osmotic stress may lead to mechanical membrane rupture with the concomitant release of the lumenal protein plastocyanin. Membrane rupture can also be shown in vitro under non-freezing conditions, although to a lesser degree, it is proportional to the permeability of the solutes used to suspend the membranes. Thylakoids frozen and thawed in vitro are less extensible than unfrozen controls, leading to rupture under less severe osmotic stress and thereby increasing damage.