Extracellular ATP induces the accumulation of superoxide via NADPH oxidases in Arabidopsis

Extracellular ATP can serve as a signaling agent in animal cells, and, as suggested by recent reports, may also do so in plant cells. In animal cells it induces the production of reactive oxygen species through the mediation of NADPH oxidase. Similarly, here we report that in leaves of Arabidopsis (Arabidopsis thaliana), applied ATP, but not AMP or phosphate, induces the accumulation of superoxide (O-2(-)) in a biphasic, dose-dependent manner, with a threshold at 500 nM ATP. This effect did not require ATP hydrolysis for it was mimicked by ATPgS. ATP also induced increased levels of Arabidopsis respiratory burst oxidase homolog D (AtrbohD) mRNA, but ATP-treated plants that had disrupted AtrbohD and AtrbohF genes did not accumulate O-2(-), indicating that NADPH oxidases are responsible for the induced O-2(-) accumulation. Inhibitors of mammalian P2-type ATP receptors abolished ATP-induced O-2(-) production, suggesting that the ATP effects may be mediated through P2-like receptors in plants. Cytosolic Ca2+ and calmodulin are likely to help transduce the ATP responses, as they do in animal cells, because a Ca2+ channel blocker, a Ca2+ chelator, and calmodulin antagonist all reduced ATP-induced O-2(-) accumulation. Furthermore, ATP treatment enhanced the expression of genes that are induced by wounds and other stresses. The ATP measured at wound sites averaged 40 mM, well above the level needed to induce O-2(-) accumulation and gene expression changes. Transgenic plants overexpressing an apyrase gene had reduced O-2(-) production in response to applied ATP and wounding. Together, these data suggest a possible role for extracellular ATP as a signal potentially in wound and stress responses.