Measurement of Ca2+ fluxes during elicitation of the oxidative burst in aequorin-transformed tobacco cells

We have employed suspension cultured aequorin-transformed tobacco cells to examine the involvement of Ca2+ in signal transduction of the oxidative burst. Use of cultured cells for this purpose was validated by demonstrating that the cells responded to cold shock quantitatively and qualitatively similarly to the intact transgenic plants from which they were derived. Stimulation of the oxidative burst in the cell suspension was achieved by administration of oligogalacturonic acid, Mas-7 (a peptide known to activate G proteins and Ca2+ fluxes), hypo-osmotic stress, or harpin (a protein from the pathogenic bacterium Erwinia amylovora). The latter failed to promote any detectable increase ia cytoplasmic Ca2+ concentration, whereas each of the former three triggered a rapid rise in cytosolic Ca2+ followed by a return within seconds to basal Ca2+ levels. Peak Ca2+ concentrations induced by the former three elicitors were similar to 0.7, 1.4, and 1.3 mu M, respectively. Three lines of evidence suggest that the observed Ca2+ pulses are essential to transduction of the oxidative burst signals by their respective elicitors: (i) inhibition of the Ca2+ transients with Ca2+ chelators or Ca2+ channel blockers prevented expression of the oxidative burst, (ii) introduction of exogenous Ca2+ into the same cells initiated the burst even in the absence of other inducers of the response, and (iii) the observed Ca2+ transients often returned to near basal levels well before any H2O2 synthesis could be detected; suggesting that the Ca2+ influx is required to communicate the burst signal but not maintain the defense response. These data suggest that Ca2+ pulses serve frequently, but not invariably, to transduce an oxidative burst signal.