Extracellular ATP-induced NO production and its dependence on membrane Ca²⁺flux in Salvia miltiorrhiza hairy roots

Extracellular ATP (eATP) is a novel signalling agent, and nitric oxide (NO) is a well-established signal molecule with diverse functions in plant growth and development. This study characterizes NO production induced by exogenous ATP and examines its relationship with other important signalling agents, Ca2+and H2O2in Salvia miltiorrhiza hairy root culture. Exogenous ATP was applied at 10-500 μM to the hairy root cultures and stimulated NO production was detectable within 30 min. The NO level increased with ATP dose from 10-100 μM but decreased from 100-200 μM or higher. The ATP-induced NO production was mimicked by a non-hydrolysable ATP analogue ATPγS, but only weakly by ADP, AMP or adenosine. The ATP-induced NO production was blocked by Ca2+antagonists, but not affected by a protein kinase inhibitor. ATP also induced H2O2production, which was dependent on both Ca2+and protein kinases, and also on NO biosynthesis. On the other hand, ATP induced a rapid increase in the intracellular Ca2+level, which was dependent on NO but not H2O2. The results suggest that NO is implicated in ATP-induced responses and signal transduction in plant cells, and ATP signalling is closely related to Ca2+and ROS signalling.