MEASUREMENT OF CA2-TRANSFORMED TOBACCO CELLS( FLUXES DURING ELICITATION OF THE OXIDATIVE BURST IN AEQUORIN)

We have employed suspension cultured aequorin-transformed tobacco cell s to examine the involvement of Ca2+ in signal transduction of the oxi dative burst. Use of cultured cells for this purpose was validated by demonstrating that the cells responded to cold shock quantitatively an d qualitatively similarly to the intact transgenic plants from which t hey were derived. Stimulation of the oxidative burst in the cell suspe nsion 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 am ylovora). The latter failed to promote any detectable increase ia cyto plasmic 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 thr ee elicitors were similar to 0.7, 1.4, and 1.3 mu M, respectively. Thr ee lines of evidence suggest that the observed Ca2+ pulses are essenti al to transduction of the oxidative burst signals by their respective elicitors: (i) inhibition of the Ca2+ transients with Ca2+ chelators o r Ca2+ channel blockers prevented expression of the oxidative burst, ( ii) introduction of exogenous Ca2+ into the same cells initiated the b urst 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 Ca2influx is required to communicate the burst signal but not maintain th e defense response. These data suggest that Ca2+ pulses serve frequent ly, but not invariably, to transduce an oxidative burst signal.