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 in 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 ~0.7, 1.4, and 1.3 μM, 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.