Effects of Freezing on Spinach Leaf Mitochondria and Thylakoids in Situ and in Vitro

The sensitivity of spinach (Spinacia oleracea L.) leaf mitochondria and chloroplast membranes to subzero temperature stress was compared after freezing of the membrane systems in situ and in vitro. Respiratory and photosynthetic activities were measured polarographically.When leaves were frozen under controlled conditions for 2 hours to various minimum temperatures and mitochondria and chloroplasts isolated after thawing, the membrane systems showed a nearly simultaneous inactivation of respiratory and photosynthetic activities between -5 to -7 C. At that temperature range in both membrane systems phosphorylation became only slightly more affected than electron transport, i.e. after freezing in situ conspicuous uncoupling of phosphorylation from electron transport was not observed.In contrast, mitochondria and thylakoids isolated from the same preparation of intact leaves under comparable conditions using NaCl as osmoticum exhibited differences in sensitivity towards freezing for 2 to 4 hours at -25 C in vitro. In the absence of cryoprotectants, photophosphorylation of isolated thylakoids became completely uncoupled from electron transport which was increased several-fold compared with the unfrozen controls. Inactivation of respiratory functions of isolated mitochondria followed the same pattern as observed after freezing in situ. In the presence of sucrose for protection of thylakoids significantly higher concentrations of the cryoprotectant were necessary than for preservation of mitochondria. Thus, under the conditions used in this study chloroplast membranes proved to be more sensitive to freezing in vitro than mitochondria.