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.
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Abbreviations
- ABA:
-
abscisic acid
- DHA:
-
2′,3′ dihydroacetylenicabscisyl alcohols
- DMSO:
-
dimethyl sulfoxide
- LT50 :
-
temperature at which 50% of cells are killed
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The authors would like to acknowledge the technical assistance of Angela Bollman, Bruce Ewan and Angela Shaw. This work was supported by grants from the Natural Science and Engineering Research Council of Canada to L.V.G. and N.H.L., and a grant from the University of Saskatchewan to R.W.W.
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Wilen, R.W., Fu, P., Robertson, A.J. et al. 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. Planta 200, 138–143 (1996). https://doi.org/10.1007/BF00196661
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DOI: https://doi.org/10.1007/BF00196661