Abscisic Acid-Induced Chilling Tolerance in Maize Suspension-Cultured Cells

Abstract

The induction of chilling tolerance by abscisic acid (ABA) in maize (Zea mays L. cv Black Mexican Sweet) suspension cultured cells was examined. Cell viability during exposure to chilling was estimated by triphenyl tetrazolium chloride reduction immediately after chilling and a filter paper growth assay. Both methods yielded comparable results. Chilling tolerance was induced by transferring 5-day-old cultures (late log phase) to a fresh medium containing ABA (10 to 100 micromolar). The greatest chilling tolerance was achieved with ABA at 100 micromolar. Growth of cells was inhibited at this concentration. After a 7-day exposure to 40C in the dark, the survival of ABA-treated cells (100 micromolar ABA, 280C for 24 h in the dark) was sevenfold greater than untreated cells. Effective induction of chilling tolerance was first observed when cells were held at 280C for 6 hours after adding ABA. No tolerance was induced if the culture was chilled at the inception of ABA treatment. Induction of chilling tolerance was inhibited by cycloheximide. These results indicate that ABA is capable of inducing chilling tolerance when ABA-treated cells are incubated at a warm temperature before exposure to chilling, and this induction requires de novo synthesis of proteins.

Relationship between Proline and Abscisic Acid in the Induction of Chilling Tolerance in Maize Suspension-Cultured Cells

“…The CH, at 1 PM, inhibits the ABA-induced chilling tolerance by 60% in maize suspension-cultured cells with a similar degree of inhibition in protein synthesis ( Fig. 5A; Xin and Li, 1992). In contrast, Pro-induced chilling tolerance was not inhibited by CH at 1 p~ (Fig.…”

“…3). We have reported that ABA-induced chilling tolerance can be inhibited by CH (Xin and Li, 1992). To determine further the relationship of Pro-induced chilling tolerance to ABA, we examined the effect of CH on the Proinduced chilling tolerance.…”

“…We have shown that 1 p~ CH does not injure the cells but is sufficient to inhibit the ABA-induced chilling tolerance in maize suspension-cultured cells (Xin and Li, 1992). To compare the effects of CH on the protein synthesis and chilling tolerance in ABA-and Pro-treated cells, 2 g of cells (fresh weight) were transferred to culture medium containing 100 PM ABA, 100 m~ Pro, or neither with CH at concentrations of 0, 0.5, and 1.0 PM.…”

“…Chilling survival was estimated by relative TTC reduction (chilled/unchilled cells) and regrowth as described by Xin and Li (1992). For TTC reduction, 1 mL of unchilled or 7-dchilled cultures was incubated with 2 mL of 0.8% TTC in 50 ITLM potassium phosphate buffer (pH 7.5) for 24 h at room temperature in the dark.…”

“…The cell cultures were allowed to incorporate ~- [~~S].blet for 2 h at 28OC, were rinsed three times with fresh culture medium containing 1 mM unlabeled Met, and were then frozen in liquid nitrogen and stored at -7OOC. To determine the incorporation of ~-[~'SlMet into proteins, the method described previously by Xin and Li (1992) was used. Cells were ground with a liquid nitrogen-chilled mortar and pestle with 1 mL of O'Farrell lysis buffer without ampholyte (O'Farrell, 1975).…”