Heat Stress Responses in Cultured Plant Cells

Abstract

The response of suspension-cultured pear (Pyrus communes cv Bartlett) cells to heat stress was studied using three viability tests: regrowth (culture growth during 10 days after stress); triphenyltetrazolium chloride reduction; and electrolyte leakage. Critical (50% injury) temperatures for a 20-minute exposure were 420, 52°, and 560C, respectively, for these viability tests. Electrolyte Plant responses to superoptimal temperature include inhibition of photosynthesis (2, 4), pollination (5), protein synthesis (1, 4, 11), translocation (6); promotion ofcallose synthesis (6,8), and leakage of cell contents (2)(3)(4). This diversity of responses complicates efforts to study specific mechanisms and to identify meaningful markers of improved heat tolerance. We have begun a study of heat hardening and injury in suspension-cultured plant cells. Beyond its usefulness in clarifying the heat response of whole plant systems, information on cultured cells is important because it may be possible to select for heat tolerance in vitro. Before in vitro techniques can be used to improve heat tolerance, the basic parameters of cellular response to heat must be understood. In the first phase of this project, described here, we examined pear cell culture growth after heat stress treatment and assessed the validity of other viability tests. The availability of a suitable viability test for cultured cells will facilitate: (a) comparison of acclimation by elevated growth temperature to that induced by heat shock (14); (b) correlation of biochemical changes (e.g. fatty acid saturation and heat shock proteins) to changes in heat tolerance; and (c) development of criteria for selection and characterization of cell lines with increased heat tolerance. 30 ml of cell suspension in 125-ml Erlenmeyer flasks; they were aerated with gyrotary shaking at 85 to 90 cycles/min. The normal growing temperature was 220 C. Stock cultures were 110 ml in 500-ml Erlenmeyer flasks; they were batch-propagated by transferring 10 ml of cell suspension to 100 ml fresh medium at 7-d intervals. MATERIALS AND METHODSHeat Stress Treatment. All heat stress treatments were applied to cells which were from 9-d-old cultures. In all cases, cells were separated from culture medium by filtration through Miracloth and washed with 7 volumes of a solution containing 0.22 M sucrose and 4.52 /AM 2,4-D. Washed cells were then suspended in the same solution; there were approximately 12.5 mg (cell dry wt) per ml. To measure electrolyte leakage, it was necessary to suspend the cells in a solution of low conductivity. To standardize the cells' environment for all viability tests, the same solution was used throughout this study. Preliminary experiments (data not shown) indicated that sucrose in the medium had little effect on heat injury.We imposed heat stress to 3-ml samples of the washed cells in 1-x 10-cm test tubes, using a heated water bath. When testing different stress temperatures, the duration of exposure was 20 min. In other experiments, time at a constant tempe...

Heat Stress Responses in Cultured Plant Cells

“…Knowledge initial study (15). The culture medium and most methods (culture maintenance, growth conditions and measurement, heat stress treatment, and viability tests) were as described previously (15 (13,14).…”

“…The culture medium and most methods (culture maintenance, growth conditions and measurement, heat stress treatment, and viability tests) were as described previously (15 (13,14). Our procedure (15) involved pipetting aliquots to test tubes for imposition of heat stress. We found that handling (transfer by pipette) influenced heat sensitivity (Fig.…”

Heat Stress Responses in Cultured Plant Cells

“…In the experiments for Figure 2, zero time measurements could not be obtained. However, without the handling involved in HS treatment, 22°C-grown cells showed less than 15% survival of a 43°C, 20-min stress (13). Heat tolerance was fully maintained for 2 d at 22°C and then began to gradually decline (Fig.…”

“…All experiments were conducted with suspension-cultured cells of pear (Pyrus communis cv Bartlett) which were used in our previous studies (13,14). The culture medium and most methods (culture maintenance, growth conditions and measurement, heat stress treatment, and viability tests) were as described before (13).…”

“…The culture medium and most methods (culture maintenance, growth conditions and measurement, heat stress treatment, and viability tests) were as described before (13). All stock cultures and experimental controls were grown at 22C.…”