Cryptogein-induced initial events in tobacco BY-2 cells:: Pharmacological characterization of molecular relationship among cytosolic Ca2+ transients, anion efflux and production of reactive oxygen species

Ion fluxes and the production of reactive oxygen species (ROS) are early events that follow elicitor treatment or microbial infection. However, molecular mechanisms for these responses as well as their relationship have been controversial and still largely unknown. We here simultaneously monitored the temporal sequence of initial events at the plasma membrane in suspension-cultured tobacco cells (cell line BY-2) in response to a purified proteinaceous elicitor, cryptogein, which induced hypersensitive cell death. The elicitor induced transient rise in cytosolic Ca2+ concentration ([Ca2+](cyt)) showing two distinct peaks, followed by biphasic (rapid/transient and slow/prolonged) Cl- efflux and H+ influx. Pharmacological analyses suggested that the ;two phases of the [Ca2+](cyt) response correspond to Ca2+ influx through the plasma membrane and an inositol 1,4,5-trisphophate-mediated release of Ca2+ from intracellular Ca2+ stores, respectively, and the [Ca2+](cyt) transients and the Cl- efflux were mutually dependent events regulated by protein phosphorylation. The elicitor also induced production of ROS including O-.(2)- and H2O2, which initiated after the [Ca2+](cyt) rise and required Ca2+ influx, Cl- efflux and protein phosphorylation. An inhibitor of NADPH oxidase, diphenylene iodonium, completely inhibited the elicitor-induced production of O-.(2)- and H2O2, but did not affect the [Ca2+](cyt) transients. These results suggest that cryptogein-induced plasma membrane Ca2+ influx is independent of ROS, and NADPH oxidase dependent ROS production is regulated by these series of ion fluxes.