AN ABSCISIC-ACID ANALOG INHIBITS ABSCISIC ACID-INDUCED FREEZING TOLERANCE AND PROTEIN ACCUMULATION, BUT NOT ABSCISIC ACID-INDUCED SUCROSE UPTAKE IN A BROMEGRASS (BROMUS-INERMIS LEYSS) CELL-CULTURE

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a brom egrass (Bromus inermis Leyss) cell culture and induces the accumulatio n of several heat-stable proteins. Two stereoisomers of an ABA analog, 2'3' dihydroacelylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing toleranc e in these cells. Freezing tolerance was unchanged in the presence of (-) DHA (LT(50) - 9 degrees C), and no increase in heat-stable protein accumulation was detected; however; the (+) enantiomer increased the freezing tolerance (LT(50) - 13 degrees C) and induced the accumulatio n of these polypeptides. All three forms of ABA increased freezing tol erance in the brome-grass cells, although (-) ABA was less effective t han either(+) or (+/-) ABA when added at equal concentrations. Cells p retreated with 20 or 50 mu M (+/-) DHA displayed lower levels of freez ing tolerance following the addition of 2.5, 7.5 or 25 mu M (+/-) ABA. Full freezing tolerance could be restored by increasing the concentra tion of (+/-) ABA to > 25 mu M. Pretreatment of cells with (-) DHA (20 or 50 mu M) had no effect on freezing tolerance when 25 mu M (+) ABA was added. The induction of freezing tolerance by 25 mu M (-) ABA was completely inhibited by the presence of 20 mu M (-) DHA. The accumulat ion of ABA-responsive heat-stable proteins was inhibited by pretreatme nt with 20 mu M (-) DHA in cells treated with 2.5 or 7.5 mu M (+/-) AB A, and in cells treated with 25 mu M (-) ABA. The accumulation of thes e polypeptides was restored when (+/-) or (+) ABA was added at a conce ntration of 25 mu M. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen wit h the other ABA-responsive proteins. The effects of the various isomer s of ABA and DHA on cell osmolarity and sucrose uptake was also invest igated, In both cases, (+/-) and (+) ABA had pronounced effects on the parameters measured, whereas (-) ABA treated cells gave substantially different results. In both sucrose uptake and cell osmolarity, DHA ha d no significant effect on the results obtained following, (+/-) or () ABA treatment. Maximum freezing tolerance was only observed in cells when both heat-stable protein accumulation and sucrose uptake were ob served.