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

Abstract

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 H₂O₂ in 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 H₂O₂ production, 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.