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

Abstract

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a bromegrass (Bromus inermis Leyss) cell culture and induces the accumulation of several heat-stable proteins. Two stereoisomers of an ABA analog, 2′3′ dihydroacetylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing tolerance in these cells. Freezing tolerance was unchanged in the presence of (−) DHA (LT50 -9°C), and no increase in heat-stable protein accumulation was detected; however, the (+) enantiomer increased the freezing tolerance (LT50 -13°C) and induced the accumulation of these polypeptides. All three forms of ABA increased freezing tolerance in the bromegrass cells, although (−) ABA was less effective than either (+) or (±) ABA when added at equal concentrations. Cells pretreated with 20 or 50 μM (−) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 μM (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 μM. Pretreatment of cells with (−) DHA (20 or 50 μM) had no effect on freezing tolerance when 25 μM (+) ABA was added. The induction of freezing tolerance by 25 μM (−) ABA was completely inhibited by the presence of 20 μM (−) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 μM (−) DHA in cells treated with 2.5 or 7.5μM (+) ABA, and in cells treated with 25 μM (−) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 μM. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen with the other ABA-responsive proteins. The effects of the various isomers of ABA and DHA on cell osmolarity and sucrose uptake was also investigated. 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 had 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 observed.