Molecular Cloning of Abscisic Acid-Responsive mRNAs Expressed during the Induction of Freezing Tolerance in Bromegrass (Bromus inermis Leyss) Suspension Culture

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Abstract
Abscisic acid (ABA) increases the freezing tolerance of bromegrass (Bromus inermis Leyss) cell-suspension cultures at 23[deg] C and elicits many metabolic changes similar to those observed during cold acclimation. Induction and maintenance of freezing tolerance by ABA is accompanied by the expression of novel polypeptides and translatable RNAs. The objective of this study was to isolate and characterize ABA-responsive cDNAs associated with ABA-induced freezing tolerance in bromegrass cell cultures. Among the 16 ABA-responsive cDNA clones isolated, 9 were expressed only with ABA treatment, 7 showed increased transcript level, and 1 was transiently expressed. Cold responsiveness was determined in three clones with increased transcript levels and in the transiently expressed clone. Deacclimation of ABA-hardened cells was a relatively slow process, because all of the novel transcripts persisted for at least 7 d after cells were cultured in ABA-free medium. Preliminary sequencing of cDNAs has identified several clones that share high sequence homology with genes associated with sugar metabolism, osmotic stress, and protease activity. Clone pBGA61 was fully sequenced and tentatively identified as an NADPH- dependent aldose reductase. The predicted amino acid sequence of the coding region shared 92% similarity with that predicted for barley aldose reductase cDNA. It is proposed that expression of genes related to sugar metabolism and osmotic stress may be required for ABA-induced hardening.