Measurement of stress-induced Ca2+pulses in single aequorin-transformed tobacco cells

Abstract

Signaling patterns measured in large cell populations are the sum of differing signals from separate cells, and thus, the detailed kinetics of Ca2+pulses can often be masked. In an effort to evaluate whether the cytosolic Ca2+pulses previously reported in populations of elicitor- and stress-stimulated tobacco cells accurately represent the pulses that occur in individual cells, a study of single cell Ca2+fluxes in stress-stimulated tobacco cells was undertaken. Individual aequorin-transformed cells were isolated from a tobacco suspension culture and placed directly on a sensitive photo-multiplier tube mounted in a dark chamber. Ca2+-dependent luminescence was then monitored after stimulation with hypo- or hyper-osmotic shock, cold shock, or defense elicitors (oligogalacturonic acid and harpin). Hypo-osmotic shock induced a biphasic Ca2+transient in 67% of the single cells tested that exhibited similar kinetics to the biphasic pulses measured repeatedly in 1ml cell suspensions. In contrast, 33% of the stimulated cells displayed Ca2+flux patterns that were not previously seen in cell suspension studies. Additionally, because only 29% of the cells tested responded with measurable Ca2+pulses to oligogalacturonic acid and 33% to the harpin protein, we conclude that not all cells in a suspension are simultaneously sensitive to stimulation with defense elicitors. In contrast, all cells tested responded with an immediate Ca2+influx after cold or hyperosmotic shock. We conclude that in many cases the Ca2+signaling patterns of single cells are accurately represented in the signaling patterns of large populations, but that single cell measurements are still required to characterize the Ca2+fluxes of the less prominent cell populations.