Cryptogein-induced anion effluxes: electrophysiological properties and analysis of the mechanisms through which they contribute to the elicitor-triggered cell death.

Anion effluxes are amongst the earliest reactions of plant cells to elicitors of defence responses. However, their properties and their role in disease resistance remain almost unknown. We previously demonstrated that cryptogein, an elicitor of tobacco defence responses, induces a nitrate (NO(3) (-)) efflux. This efflux is an early prerequisite to the cryptogein-triggered hypersensitive response (HR). Here, we analyzed the electrophysiological properties of the elicitor-mediated NO(3) (-) efflux and clarified the mechanisms through which it contributes to cell death. Application of the discontinuous single electrode voltage-clamp technique in tobacco cells elicited with cryptogein enabled us to record the activation of slow-type deactivating anion channel currents. Cryptogein-induced plasma membrane depolarization and Ca(2+) influx, an essential component of elicitor signalling for HR cell death, were prevented by inhibiting the NO(3) (-) efflux. Similarly, pharmacological blocking of the anion efflux suppressed vacuolar collapse, a hallmark of cell death. The role of NO(3) (-) efflux in mediating proteases activation was further assessed. It is shown that cryptogein induced the activation of three proteases with apparent molecular masses of 95, 190 and 240 kDa. Their activation occurred independently on the anion efflux and, together with cell death, was strongly reduced by cycloheximide and the protease inhibitor PMSF. In contrast, the NO(3) (-) efflux was shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases previously reported to contribute to the disruption of vacuole integrity observed during the HR. Collectively, our data indicate that anion efflux is an early prerequisite to morphological and biochemical events participating to cell death.