Temperature and Abscisic Acid Can Be Used to Regulate Survival, Growth, and Differentiation of Cultured Guard Cell Protoplasts of Tree Tobacco

Abstract

Cuard cell protoplasts isolated from leaves of Nicofiana g h c a (Craham) were cultured. Conditions were sought that would maximize survival and maintain cells i n their differentiated state. Temperature was an important determinant of survival, growth, and differentiation. As temperatures were increased from 24 to 32'C, survival for 1 week in culture increased from approximately 20% to approximately 80% of cells used to initiate cultures. At all of these temperatures, approximately 90% of surviving cells divided to form callus tissue. "Footprint" areas of cells cultured for 1 week at 32°C increased almost 30-fold. Cells cultured for 1 week at 34 t o 40°C also survived i n high percentages (approximately 80%), but they retained a morphology similar to that of guard cells and they did not divide. Footprint areas of cells cultured for 1 week at 38°C increased 6-fold. Cells cultured at 36 t o 4OoC in media containing 0.1 or 1.0 W M abscisic acid survived in high percentages and did not divide. At 38°C their footprint areas did not increase, but cells so cultured increased in diameter when treated with fusicoccin. Morphologies and electrophoretic profiles of total sodium dodecyl sulfate-extractable proteins suggest that cells cultured at 38°C in media containing abscisic acid remain differentiated. ~-a-(2-Aminoethoxyviny1)-glycine reduced survival t o <1 O/O at 26 or 32°C but had no effect at 38OC. At lower temperatures, cell growth and survival appear to be ethylene dependent.

Heat Suppresses Activation of an Auxin-Responsive Promoter in Cultured Guard Cell Protoplasts of Tree Tobacco

“…GCP cultured at 38°C survive in the same high percentages as at 32°C, but neither exogenous NAA nor BAP is required for, nor do 1-aminoethoxyvinylglycine or ABA reduce, cell survival at 38°C (Roberts et al, 1995;Gushwa et al, 2003). GCP cultured at 38°C in NAA and BAP expand only 5-to 6-fold (Roberts et al, 1995).…”

“…GCP cultured at 38°C in NAA and BAP expand only 5-to 6-fold (Roberts et al, 1995). They do not regenerate cell walls, nor do they make the G1-to-S phase cell cycle transition (Gushwa et al, 2003).…”

“…At 32°C, in a medium containing an auxin (1-naphthaleneacetic acid [NAA]) and a cytokinin (6-benzylaminopurine [BAP]), GCP survive in high percentages (70%-80%) for up to 1 month (Roberts et al, 1995). Under the influence of NAA and BAP, GCP expand 20-to 30-fold, regenerate cell walls, dedifferentiate, and divide to form callus from which plants can be regenerated (Sahgal et al, 1994).…”

“…Cultured guard cell protoplasts (GCP) of tree tobacco (Nicotiana glauca) 'Graham' are a novel system for investigating the cell signaling mechanisms that lead to acquired thermotolerance and thermoinhibition (Roberts et al, 1995;Gushwa et al, 2003;Tallman, 2005). At 32°C, in a medium containing an auxin (1-naphthaleneacetic acid [NAA]) and a cytokinin (6-benzylaminopurine [BAP]), GCP survive in high percentages (70%-80%) for up to 1 month (Roberts et al, 1995).…”

“…Both NAA and BAP must be supplied exogenously before GCP will survive and undergo the extensive cellular remodeling that accompanies dedifferentiation and division (Gushwa et al, 2003). 1-Aminoethoxyvinyl-glycine, an inhibitor of ethylene biosynthesis, and abscisic acid (ABA) are each lethal at 32°C (Roberts et al, 1995).…”