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

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 1 ml 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.