Effect of Cold Acclimation on the Incidence of Two Forms of Freezing Injury in Protoplasts Isolated from Rye Leaves

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Abstract
The freezing tolerance and incidence of two forms of freezing injury (expansion-inducing lysis and loss of osmotic responsiveness) were determined for protoplasts isolated from rye leaves (Secale cereale L. cv Puma) at various times during cold acclimation. During the first 4 weeks of the cold acclimation period, the LT50 (i.e. the minimum temperature at which 50% of the protoplasts survived) decreased from -5.degree. C to -25.degree. C. In protoplasts isolated from nonacclimated leaves (NA protoplasts), expansion-induced lysis (EIL) was the predominant form of injury at the LT50. However, after only 1 week of cold acclimation, the incidence of EIL was reduced to less than 10% at any subzero temperature; and loss of osmotic responsiveness was the predominant form of injury, regardless of the freezing temperature. Fusion of either NA protoplasts or protoplasts isolated from leaves of seedlings cold acclimated for 1 week (1-week ACC protoplasts) with liposomes of dilinoleoylphosphatidylcholine also decreased the incidence of EIL to less than 10%. Fusion of protoplasts with dilinoleoylphosphatidylcholine diminished the incidence of loss of osmotic responsiveness, but only in NA protoplasts or 1-week ACC protoplasts than were frozen to temperatures over the range of -5 to -10.degree. C. These results suggest that the cold acclimation process, which results in a quantitative increase in freezing resistance, involves several different qualitative changes in the cryobehavior of the plasma membrane.