Importance of Realistic Freeze-Thaw Protocols

The relative effect of a freeze-thaw cycle on photosynthesis, respiration, and ion leakage of potato leaf tissue was examined in two potato species, Solanum acaule Bitt. and Solanum commersonii Dun. Photosynthesis was found to be much more sensitive to freezing stress than was respiration, and demonstrated more than a 60% inhibition before any impairment of respiratory function was observed. Photosynthesis showed a slight to moderate inhibition when only 5 to 10% of the total electrolytes had leaked from the tissue (reversible injury). This was in contrast to respiration which showed no impairment until temperatures at which about 50% ion leakage (irreversible injury) had occurred. The influence of freeze-thaw protocol was further examined in S. acaule and S. commersonii, in order to explore discrepancies in the literature as to the relative sensitivities of photosynthesis and respiration. As bath cooling rates increased from 1°C/hour to about 3 or 6°C/hour, there was a dramatic increase in the level of damage to all measured cellular functions. The initiation of ice formation in deeply supercooled tissue caused even greater damage. As the cooling rates used in stress treatments increased, the differential sensitivity between photosynthesis and respiration nearly disappeared. Examination of agriculturally relevant, climatological data from an 11 year period confirmed that air cooling rates in the freezing range do not exceed 2°C/hour. It was demonstrated, in the studies presented here, that simply increasing the actual cooling rate from 1.0 to 2.90C/hour, in frozen tissue from paired leaflet halves, meant the difference between cell survival and cell death. While the effects of freezing on a number of essential cellular and subcellular processes have been extensively studied (1 1, 13, 14), there has been little effort to relate systematically the sequential development of injury to several of these processes during the imposition of realistic freezing and thawing stress on an intact tissue system. To the contrary, many of the investigations in this area rely solely on results obtained with isolated systems, such as chloroplasts or thylakoid membranes, which can lead to conclusions that are often non consistent with those derived from intact tissue (8, 9). Al'This work was supported by the College of Agriculture and Life Sciences, University of Wisconsin, Madison. 2Present address: Department of Agronomy, University of Kentucky, Lexington, KY 40546. though one can gain valuable information from such studies, regarding some aspects of freeze-induced impairment of photosynthetic functions, one can clearly not gain an understanding of the interactions among the various cellular compartments within an intact tissue during freeze-thaw stress. For example, Singh et al. (27) have demonstrated that fully functional plant mitochondria can be isolated from cells which have been lethally damaged by freezing stress. Prolonged exposure ofmitochondria to the altered cellular environment, however, was found to result in the loss of function. Little work has been done on the effect ofthe reversible or transitory changes in the cellular environment on organellar function. The general preoccupation with dramatic events during experimental freezing tests, such as the rather sudden loss of cellular functions within a very narrow temperature range, has led most investigators to overlook very early events in the initial stages of freezing injury. The results of Palta et al. (7, 18, 19, 23) have shown that freeze-induced functional alterations of the cellular membrane transport proteins are gradual and reversible. Alterations in membrane transport properties would clearly result in an altered cellular environment. In spite ofthis altered environment, no changes in the ultrastructure of the chloroplasts nor the mitochondria are observed at incipient (reversible) injury, while swelling and ultrastructural disorganization are observed following a lethal (irreversible injury) freeze-thaw stress (17, 21, 22). The protocols frequently used to study freezing tolerance often produce stresses which have little relevance to freezing stress in the natural environment and tend to obscure subtle changes occurring during incipient injury. Levitt (1 1) noted (but provided no data) that air cooling rates in nature are usually less than 1 to 2°C/h and that ice nucleation generally occurs before tissue temperature reaches -2°C. In spite of this common observation, some recent studies investigating the mechanism of freezing injury have used cooling rates of 6 to 20°C/h (10, 25) and 60°C/h (3, 24). In some studies there is no indication that the nucleation temperature was controlled (9, 10, 25). Many studies investigating the mechanism of freezing injury to the photosynthesis apparatus using isolated membrane preparations have utilized cooling rates of 20 to 1 50C/h (4, 5, 26). The studies on freezing stress presented here were designed to investigate the alterations in cell membrane, chloroplast, and mitochondrial function following a realistic, freeze-thaw