Bromus inermis Leyss cell cultures treated with 75 micromolar abscisic acid (ABA) at both 23 and 3°C developed more freezing resistance than cells cultured at 3°C. Protein synthesis in cells induced to become freezing tolerant by ABA and low temperature was monitored by I'4Cileucine incorporation. Protein synthesis continued at 3°C, but net cell growth was stopped. Most of the major proteins detected at 23°C were synthesized at 3WC. However, some proteins were synthesized only at low temperatures, whereas others were inhibited. ABA showed similar effects on protein synthesis at both 23 and 3°C. Comparative electrophoretic analysis of I'4Cileucine labeled protein detected the synthesis of 19, 21 and 47 kilodalton proteins in less than 8 hours after exposure to exogenous ABA. Proteins in the 20 kilodalton range were also synthesized at 3°C. In addition, a 31 kilodalton protein band showed increased expression in freezing resistant ABA treated cultures after 36 hours growth at both 3 and 23°C. Quantitative analysis of I'4Clleucine labeled polypeptides in two-dimensional gels confirmed the increased expression of the 31 kilodalton protein. Two-dimensional analysis also resolved a 72 kilodalton protein enriched in ABA treated cultures and identified three proteins (24.5, 47, and 48 kilodaltons) induced by low temperature growth.Freezing resistance is induced in some plants (20,27) and plant suspension cultures (6) by exposure to low temperatures.Endogenous levels of ABA have been shown to increase during high and low temperature growth conditions (8). Following application of exogenous ABA, improved cold resistance has been observed in alfalfa seedlings (22), potatoes (5), and suspension cultures of some plant species (4,19) and callus cultures (14). A novel aspect of these studies is that ABA can substitute for the cold requirement in inducing freezing resistance (4, 5).Cycloheximide inhibits cold hardening in winter wheat (26), winter rape (12), and potato stem cultures (5), which suggests protein synthesis is required for the induction of freezing resistance. However, little is understood about the effects of low temperature or ABA on protein synthesis or gene expression. Electrophoretic analysis ofproteins synthesized during cold hardening of winter wheat, rye (7, 23), rape (13,17), and mulberry trees (28) has shown increased or decreased synthesis of certain proteins in hardier varieties. The relationship of most of these proteins to freezing resistance in plants is probably equivocal, since other complicating processes such as vernalization, dormancy, adjustment to low temperature growth and development occur concomitantly.We have used bromegrass suspension cultures as a model system to study cold acclimation and ABA induced freezing tolerance. Our approach has been to compare protein changes occurring during ABA induced hardening responses at 23°C with those during low temperature hardening. The correlation of specific protein changes occurring during ABA induced freezing tolerance at both low (3°C) an...