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

Signaling patterns measured in large cell populations are the sum of differ ing signals from separate cells, and thus, the detailed kinetics of Ca2+ pu lses can often be masked. In an effort to evaluate whether the cytosolic Ca 2+ pulses previously reported in populations of elicitor- and stress-stimul ated tobacco cells accurately represent the pulses that occur in individual cells, a study of single cell Ca2+ fluxes in stress-stimulated tobacco cel ls was undertaken. Individual aequorin-transformed cells were isolated from a tobacco suspension culture and placed directly on a sensitive photo-mult iplier 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 sho ck 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 Ca 2+ flux patterns that were not previously seen in cell suspension studies. Additionally, because only 29% of the cells tested responded with measurabl e Ca2+ pulses to oligogalacturonic acid and 33% to the harpin protein, we c onclude 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 conclud e that in many cases the Ca2+ signaling patterns of single cells are accura tely represented in the signaling patterns of large populations, but that s ingle cell measurements are still required to characterize the Ca2+ fluxes of the less prominent cell populations. (C) 2001 Harcourt Publishers Ltd.