ABSCISIC ACID-INDUCED HEAT TOLERANCE IN BROMUS-INERMIS LEYSS CELL-SUSPENSION CULTURES - HEAT-STABLE, ABSCISIC ACID-RESPONSIVE POLYPEPTIDES IN COMBINATION WITH SUCROSE CONFER ENHANCED THERMOSTABILITY

Increased heat tolerance is most often associated with the synthesis o f heat-shock proteins following pre-exposure to a nonlethal heat treat ment. In this study, a bromegrass (Bromus inermis Leyss cv Manchar) ce ll suspension cultured in a medium containing 75 mu M abscisic acid (A BA) without prior heat treatment had a 87% survival rate, as determine d by regrowth analysis, following exposure to 42.5 degrees C for 120 m in. In contrast, less than 1% of the control cells survived this heat treatment. The heat tolerance provided by treatment with 75 mu M ABA w as first evidenced after 4 d of culture and reached a maximum toleranc e after 11 d of culture. Preincubation with sucrose partially increase d the heat tolerance of control cells and rendered ABA-treated cells t olerant to 45 degrees C for 120 min (a completely lethal heat treatmen t for control cells). Comparative two-dimensional polyacrylamide gel e lectrophoresis of cellular protein isolated from heat-tolerant cells i dentified 43 ABA-responsive proteins of which 26 were heat stable (did not coagulate and remained soluble after 30 min at 90 degrees C). Eig ht heat-stable, ABA-responsive proteins ranging from 23 to 45 kD had s imilar N-terminal sequences. The ABA-responsive (43-20 kD), but none o f the control heat-stable, proteins cross-reacted to varying degrees w ith a polyclonal antibody directed against a conserved, lysine-rich de hydrin sequence. A group of 20- to 30-kD heat-stable, ABA-responsive p roteins cross-reacted with both the anti-dehydrin antibody and an anti body directed against a cold-responsive winter wheat protein (Wcs 120) . In ABA-treated cells, there was a positive correlation between heat- and pH-induced coagulation of a cell-free homogenate and the heat tol erance of these cells. At 50 degrees C, control homogenates coagulated after 8 min, whereas cellular fractions from ABA-treated cells showed only marginal coagulation after 15 min. In protection assays, additio n of heat-stable, ABA-responsive polypeptides to control fractions red uced the heat-induced coagulation of cell-free homogenates. Sucrose (8 %) alone and control, heat-stable fractions enhanced the thermostabili ty of control fractions, but the most protection was conferred by ABA- responsive, heat-stable proteins in combination with sucrose. These da ta suggest that stress-tolerance mechanisms may develop as a result of cooperative interactions between stress proteins and cell osmolytes, e.g. sucrose. Hypotheses are discussed implicating the role of these p roteins and osmolytes in preventing coagulation and denaturation of ce llular proteins and membranes.