Content during Abscisic Acid Induced Freezing Tolerance in Bromegrass Cells1

Changes in water content and dry weight were determined in control cells and those induced to cold harden in response to abscisic acid (ABA) treatment (7.5 x 10-5 molar). Bromegrass (Bromus inermis Leyss cv Manchar) cells grown in suspension culture at room temperature (230C) for 7 days acclimated to -28°C (LT50) when treated with ABA, or to -50C when untreated. ABA significantly reduced cell growth rates at 5 and 7 days after treatment. Growth reduction was due to a decrease in cell number rather than cell size. When the cell water content was expressed as percent water (percent H20) or as grams water per gram dry weight (gram H20/gram dry weight [g DW]), the water content of hardy, ABA-treated cells decreased from 85% to 77% or from 6.4 to 3.3 g H20/g DW in 7 days. Control cell water content remained static at approximately 87% and 7.5 g H20/g DW. However, cell water content, expressed as milligrams water per million cells (milligram H20/106 cells), did not differ in ABA-treated or control cells. The dry matter content of ABA-treated cells, expressed as milligram DW/106 cells increased to 3.3 milligram/106 cells in 7 days, whereas the dry weight of the control cells remained between 1.4 to 2.1 milligrams/106 cells. The osmotic potential of ABA-treated cells decreased by the fifth day while that of control cells increased significantly and then decreased by day 7. Elevated osmotic potentials were not associated with increased ion uptake. In contrast to much published literature, these results suggest that cell water content does not decrease in ABA-treated cells during the induction of freezing tolerance, rather the dry matter mass per cell increased. Cell water content may be more accurately expressed as a function of cell number when accompanying changes to dry cell matter occur. During cold acclimation plants become resistant to freezing stress. Freezing injury is influenced by the site, rate, and extent of ice formation (1 1). Thus, a clear knowledge of the water status within the plant is important to understanding how plants acclimate. Many studies have shown that the water content of tissues declines as plants acclimate to low temperatures (3, 4, 10, 17). Also, plants accumulate dry matter under cold acclimating conditions (4, 5, 7). Despite the possibility that both water content and dry matter are changing during acclimation, technical difficulties particularly in whole plant measurements result in expression of water content that is not independent of dry matter accumulation: % H20: Oregon Agricultural Experiment Station Technical Paper No. 9005. 2 Present address: Department of Horticulture, University of Saskatchewan, Saskatoon, Saskatchewan, S7N OWO. 460 ([FW3 DW]/FW) x 100% (3, 10, 17); or g H20/g DW: (FW DW)/DW (6, 10). Erroneous interpretations can occur when two changing variables are presented as dependent functions. Although measurements of RWC and water deficit have been developed to account for this problem (17), changes associated with cold acclimation may alter the ability of cells to take up water to achieve full turgor. Confounding effects may result if full turgor is not attained. ABA-induction ofcold acclimation in cell cultures provides a useful tool for examining water content during frost acclimation: (a) ABA can substitute for low temperature in inducing cold acclimation and thus avoid plant responses to cold that are not related to acclimation (2, 8, 12-16); (b) ABA induction of hardiness is rapid and occurs in days rather than the weeks required for low temperature induced acclimation (2, 7, 8); (c) cell cultures offer a relatively homogeneous system where barriers to hormone uptake are removed; and (d) cell numbers can be readily estimated so data can be expressed as a function of cell number. This study compared three methods ofexpressing cell water content in bromegrass suspension cultures and characterized the dry matter status and growth of these cells during ABAinduced cold acclimation. MATERIALS AND METHODS