Loss of Adenosine Triphosphate Synthesis Caused by Freezing and Its Relationship to Frost Hardiness Problems

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Abstract
Chloroplasts and mitochondria, isolated from leaves of spinach and wheat in aqueous buffer containing NaC1, are no longer able to form ATP from ADP and inorganic phosphate after being frozen in water. Since freezing stimulates transport of electrons from water to ferricyanide in chloroplasts, loss of ATP synthesis is considered to be an uncoupling effect In mitochondria, oxygen uptake is inhibited by freezing. By feeding intact leaves of spinach and barley with labelled phosphate, carbon dioxide and sucrose it was shown that freezing affects ATP synthesis not only in isolated cell organelles but also in the intact cell. Loss of ATP synthesis by isolated chloroplasts and mitochondria can be completely or partially prevented by the addition of small amounts of sugars such as glucose, sucrose or raffinose prior to freezing. The protective action of sugars is abolished by addition of somewhat more than equimolar concentrations of a number of inorganic salts. On this basis a model system has been constructed containing chloroplasts, sucrose and NaCl in balanced concentrations; this system in some ways reflects the response of intact cells to freezing. Since cysteine and glutathione, in contrast to sugars, fail to protect the chloroplast system against frost inactivation, and since no SH-group oxidation in chloroplasts due to freezing could be detected, the sulfhydryl-disulfide theory of frost hardiness is rejected. Instead, a working hypothesis is proposed, which explains the action of frost on the cell by hydrogen-bond rupture in lipoprotein systems.