Abscisic Acid-Induced Freezing Resistance in Cultured Plant Cells

Abstract

ABSTlRACTThe effect of abscisic acid (ABA) on the cold hardiness of cell suspension was investigted. Cell (2,(15)(16)(17). Because of the involvement of ABA in many environmental stresses, ABA has been proposed as a common mediator for plant stress response (5). However, the role of ABA in the adaptation of plants to freezing stress is not clear. Irving and Lamphear (13) were among the first to suggest that ABA may be involved in the cold hardening process.Two lines of evidence support the involvement of ABA in plant cold hardening. First, an increase in the endogenous level of ABA has been observed when plants are exposed to low temperature (2,5,(14)(15)(16). Second, exogenous applications of ABA have been shown to increase the cold hardiness in certain plants (2,3,15,16). However, attempts to increase cold hardiness by exogenous applications of ABA have not always been successful (6, 9). In these studies ABA was applied either as a foliar spray (6) or through the roots in a hydroponics solution (10). The failure ofexogenously added ABA to increase cold hardiness in these studies may be due to the lack of uptake, or microbial and enzymic degradation. (20), and by regrowth.The procedures for measuring TTC reduction was essentially the same as that described by Towill and Mazur (18) with the following modifications: thawed cells (0.2 g fresh weight) were incubated in 3 ml TTC solution (0.08% TTC in 0.05 M sodium phosphate buffer, pH 7.4) for 18 h at 20°C in the dark. After TTC incubation, the cells were rinsed with 3 ml distilled H20 and the reduced TTC extracted with 5 ml of 95% ethanol. TTC reduction was determined by measuring A at 485 nm. Freezing injury is expressed as the percentage ofdecrease in TTC reduction as compared to a nonfrozen control. The temperature which resulted in a 50% decrease TTC reduction is defined as the 50% killing temperature (LT50).Viability as determined by regrowth was conducted under sterile conditions. Following the freeze test, cell aggregates approximately 1 mm in diameter were transferred to 0.7% agar containing the appropriate medium. After I month at 20°C in the dark, the growth of the callus was determined.

Two Homologous Low-Temperature-Inducible Genes from Arabidopsis Encode Highly Hydrophobic Proteins

“…This is in agreement with the observation that both drought and exogenous ABA treatments can mimic the effects of low temperatures and increase the freezing tolerance of several temperate species (Cloutier and Siminovitch, 1982;Chen and Gusta, 1983;Mantyla et al, 1995). Moreover, it has been shown that the endogenous levels of ABA increase in response to low temperature and water stress Lang et al, 1994).…”

“…We have also found that the levels of RCI2A and RC/2B transcripts increase in response to ABA, another treatment that enhances freezing tolerance (Chen and Gusta, 1983) and induces the expression of the majority of coldinducible genes (Hughes and Dunn, 1996). Endogenous levels of ABA have been shown to transiently increase in response to low temperature and water stress (Lang et al, 1994) with kinetics that parallel those of the cold-induced RC12 mRNA accumulation.…”

“…Furthermore, transcripts corresponding to RC/2A and RC/2B do not accumulate in response to salt stress, which differentiates them from other cold-responsive genes such as kin2 (Kurkela andBorg-Franck, 1992), lti78/rd29A/cor48, or Iti65/ rd29B (Yamaguchi-Shinozaki andShinozaki, 1994). However, the two RC/2 genes, like most of the cold-inducible genes (Thomashow, 1994;Hughes and Dunn, 1996), are induced by drought, a treatment that has been shown to increase plant freezing tolerance (Cloutier and Siminovitch, 1982;Chen and Gusta, 1983;Mantyla et al, 1995). Since both low temperature and drought can be considered to be dehydration stresses (Steponkus, 1984), it is therefore reasonable to imagine that they can induce common gene products.…”

Plant adaptation to temperature and photoperiod

“…ABA can also function as a drought related signal from the roots to the top of the plants (Davies and Zhang 1991). The role of ABA in regulating freezing stress tolerance has also been documented in a number of studies (Chen andGusta 1983, Reaney et al 1989).…”

Abscisic Acid-Induced Heat Tolerance in Bromus inermis Leyss Cell-Suspension Cultures (Heat-Stable, Abscisic Acid-Responsive Polypeptides in Combination with Sucrose Confer Enhanced Thermostability)

“…Heat tolerance was determined by incubating 0.40 g fresh weight of cells at 42.5OC for up to 160 min, with cells collected at 20-min intervals for viability assessment. Viability was assessed by regrowth, TTC reduction, and fluorescein diacetate staining (Chen and Gusta, 1983). In regrowth assays, 0.15 to 0.20 g fresh weight of stressed cells were transferred to fresh Erickson's niedium and increases in fresh weight were determined after 10 d at 25OC.…”