immediately frozen in liquid N2. All of the harvests were performed in the 2nd or in the 16th hr of the light period or after different periods of darkness (2 and 5 min and 10 hr). Frozen tissues were powdered, lyophilized using Edwards High Vacuum LTD, model EF2 and stored over CaCl2 at -27 C for 1 or 2 weeks.Simultaneously, the fresh and dry weights oftissues, comparable to those taken for nucleotides analysis, were determined.Extraction and Determination of Nucleotides. Adenine nucleotides were extracted by putting 25 mg of dried tissue into 10 ml of boiling deionized H20 for 3 min. The extract was then rapidly cooled in an ice bath and centrifuged at 0 C to remove insoluble residue. The supernatant fraction was used directly for an assay. In order to determine the ATP, ADP, and AMP contents, 0.2 ml of the extract was incubated in HEPES buffer with P-enolpyruvate, pyruvate kinase, and adenylate kinase according to Pradet (15). After incubation samples were immediately assayed by the luciferine-luciferase method, according to Saint John (16). A Beckman liquid scintillation system (model LS-100) was used to measure light produced in the reaction.Enzymes and Chemicals. All enzymes and chemicals were products of Sigma Chemical Company. RESULTSFrost tolerance of leaves increased slightly during plant growth at 5 C for 8 days (Fig. 1) and the marked increase of Tk2 to -14 C was noted after lowering the temperature to 0 C. However, the cold treatment did not increase the frost tolerance of roots (Fig. 1, upper curves): their Tk. varied from -4 C to -5.5 C and did not change during the whole hardening period.The content of ATP in the control, non-cold-treated tissue did not change significantly in the course of experiment (Fig. 2). Fluctuations observed in roots were probably due to the changeable proportion of young and older roots in the sample. Some ATP increase noted in roots at the end of experiment was probably due to the new root formation which usually occurs when plants are cultured in a water medium.When plants were subjected to cold treatment significant changes in ATP content occurred in the tissues studied (Fig. 2). In roots, ATP content was maintained at the level of the control during the first 4 days of cold treatment and then, after 8 and 14 days it decreased to about 50% of the initial value.In leaves, a 2-fold increase of ATP content was noted during the first 4 days of the cold treatment which persisted as the cold treatment continued. Decreasing the temperature from 5 to 0 C had no further effect on ATP content. Since the cold treatment brought about marked changes of both frost tolerance and ATP content in leaves only, further experiments were performed on that tissue.In order to check whether the higher ATP content in leaves of frost-tolerant plants was due only to photosynthetic phosphoryl-

The effects of chilling and freezing temperature on membrane permeability and ATP content were studied in the leaves of cucumber (Cucumis sativus L.) and winter rape (Brassica napus L. var. oleifera L.) leaves, grown at different temperatures. In the winter rape leaves, the endogenous ATP content was modified by application of dinitrophenol (DNP) solutions of different concentrations. The low temperature‐induced changes in membrane permeability (as monitored by the conductivity method) were found to be associated with ATP decrease, both in the chilling‐sensitive and chilling‐resistant (subjected to freezing) plants. In tissues showing reversible injuries, changes in ATP content preceded those in membrane permeability and the adenylate energy charge was affected slightly. In tissues showing irreversible membrane damage, the ATP content was always below 0.4 μmol (g dry weight)−1 and the adenylate energy charge was near 0.5. DNP treatment increased freezing sensitivity of winter rape leaves. In the cold‐hardened winter rape leaves, however, freezing and thawing did not significantly affect ATP content or the energy charge, although the specimen showed a rather large increase in membrane permeability. In these leaves ATP content recovered about 20 h after a freezing and thawing treatment. It is proposed that a decrease in ATP supply might be the primary reason for the membrane leakiness at low temperature, both in chilling‐sensitive and chilling‐resistant (subjected to freezing) plants. The conclusion is, however, not true for the cold‐acclimated, frostadapted cells.

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