Protein Synthesis in Bromegrass (Bromus inermis Leyss) Cultured Cells during the Induction of Frost Tolerance by Abscisic Acid or Low Temperature

Abstract

Bromus inermis Leyss cell cultures treated with 75 micromolar abscisic acid (ABA) at both 23 and 3°C developed more freezing resistance than cells cultured at 3°C. Protein synthesis in cells induced to become freezing tolerant by ABA and low temperature was monitored by I'4Cileucine incorporation. Protein synthesis continued at 3°C, but net cell growth was stopped. Most of the major proteins detected at 23°C were synthesized at 3WC. However, some proteins were synthesized only at low temperatures, whereas others were inhibited. ABA showed similar effects on protein synthesis at both 23 and 3°C. Comparative electrophoretic analysis of I'4Cileucine labeled protein detected the synthesis of 19, 21 and 47 kilodalton proteins in less than 8 hours after exposure to exogenous ABA. Proteins in the 20 kilodalton range were also synthesized at 3°C. In addition, a 31 kilodalton protein band showed increased expression in freezing resistant ABA treated cultures after 36 hours growth at both 3 and 23°C. Quantitative analysis of I'4Clleucine labeled polypeptides in two-dimensional gels confirmed the increased expression of the 31 kilodalton protein. Two-dimensional analysis also resolved a 72 kilodalton protein enriched in ABA treated cultures and identified three proteins (24.5, 47, and 48 kilodaltons) induced by low temperature growth.Freezing resistance is induced in some plants (20,27) and plant suspension cultures (6) by exposure to low temperatures.Endogenous levels of ABA have been shown to increase during high and low temperature growth conditions (8). Following application of exogenous ABA, improved cold resistance has been observed in alfalfa seedlings (22), potatoes (5), and suspension cultures of some plant species (4,19) and callus cultures (14). A novel aspect of these studies is that ABA can substitute for the cold requirement in inducing freezing resistance (4, 5).Cycloheximide inhibits cold hardening in winter wheat (26), winter rape (12), and potato stem cultures (5), which suggests protein synthesis is required for the induction of freezing resistance. However, little is understood about the effects of low temperature or ABA on protein synthesis or gene expression. Electrophoretic analysis ofproteins synthesized during cold hardening of winter wheat, rye (7, 23), rape (13,17), and mulberry trees (28) has shown increased or decreased synthesis of certain proteins in hardier varieties. The relationship of most of these proteins to freezing resistance in plants is probably equivocal, since other complicating processes such as vernalization, dormancy, adjustment to low temperature growth and development occur concomitantly.We have used bromegrass suspension cultures as a model system to study cold acclimation and ABA induced freezing tolerance. Our approach has been to compare protein changes occurring during ABA induced hardening responses at 23°C with those during low temperature hardening. The correlation of specific protein changes occurring during ABA induced freezing tolerance at both low (3°C) an...

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

“…The first pattern (A) includes transcripts that were detected primarily after ABA treatment. This type of response is similar to that of the cold-responsive genes identified in other species (Robertson et al, 1987;Mohapatra et al, 1988Mohapatra et al, , 1989Cattivelli and Bartels, 1990;Hajela et al, 1990;Kurkela and Franck, 1990;Lu0 et al, 1992). With the second pattem (B) of gene expression, transcripts were detected in the controls but were up-regulated by ABA or cold treatment.…”

“…With bromegrass (Bromus inermis) cell cultures, it has been demonstrated that 4 to 5 d of ABA treatment increases freezing tolerance by 23OC, with a change in from -7 to -3OOC (Chen and Gusta, 1983;Lee et al, 1991). Associated with a rapid increase in hardiness were qualitative changes in the population of polypeptides (Robertson et al, 1987) and translatable RNAs (Lee et al, 1991). Deacclimation of ABA-hardened bromegrass cells is a relatively slow process (Reaney et al, 1989;Lee et al, 1991) compared to other coldhardy species (Guy et al, 1985;Hajela et al, 1990;Tseng and Li, 1990).…”

Cell-Wall Changes and Cell Tension in Response to Cold Acclimation and Exogenous Abscisic Acid in Leaves and Cell Cultures

“…ABA is known to accumulate during cold acclimation of plants . Also, there have been many studies that suggest that ABA may be a mediator of cold tolerance in plants (Robertson et al, 1987;Thomashow, 1990). In addition, exogenous application of ABA has been shown to increase cold tolerance in whole plants and cell cultures, to substitute the cold-acclimation treatment, and to cause the expression of proteins associated with cold acclimation (Chen and Gusta, 1983;Robertson et al, 1987).…”

“…However, it is not known how these cell-wall changes affect the physical properties of cell walls in relation to their ability to undergo deformation during freezing. Also, cold acclimation is known to be associated with the accumulation of ABA, which is believed to be responsible for induction of changes associated with cold acclimation in plants Reaney and Gusta, 1987;Robertson et al, 1987).…”

Chilling-Induced Inactivation and Its Recovery of Tonoplast H+-ATPase in Mung Bean Cell Suspension Cultures

“…Upon exposure to low temperature, both activities of the tonoplast H+-ATPase, substrate hydrolysis and proton transport declined rapidly, reaching a minimum within 10 h. This effect appears far earlier than irreversible cell injury. On the contrary, no cold-induced inactivation was observed in the tonoplast H+-ATPase in suspension cultured cells of bromegrass (Table I), which are reported to be capable of cold hardening (12,13). Therefore, there seems to be a marked difference in the cold stability of tonoplast H+-ATPase in vivo between chilling-sensitive and -resistant cultured cells.…”