The signaling role of extracellular ATP and its dependence on Ca²⁺flux in elicitation of Salvia miltiorrhiza hairy root cultures

The application of a polysaccharide elicitor from yeast extract, YE, to Salvia miltiorrhiza hairy root cultures induced transient release of ATP from the roots to the medium, leading to a dose-dependent increase in the extracellular ATP (eATP) level. The eATP level rose to a peak (about 6.5 nM with 100 mg l-1YE) at about 10 h after YE treatment, but dropped to the control level 6 h later. The elicitor-induced ATP release was dependent on membrane Ca2+influx, and abolished by the Ca2+chelator EGTA or the channel blocker La3+. The YE-induced H2O2production was strongly inhibited by reactive blue (RB), a specific inhibitor of membrane purinoceptors. On the other hand, the application of exogenous ATP at 10-100 μM to the cultures also induced rapid and dose-dependent increases in H2O2production and medium pH, both of which were effectively blocked by RB and EGTA. The non-hydrolyzable ATP analog ATPγS was as effective as ATP, but the hydrolyzed derivatives ADP or AMP were not so effective in inducing the pH and H2O2increases. Our results suggest that ATP release is an early event and that eATP plays a signaling role in the elicitation of plant cell responses; Ca2+is required for activation of the elicitor-induced ATP release and the eATP signal transduction. This is the first report on ATP release induced by a fungal elicitor and its involvement in the elicitor-induced responses in plant cells.