Extracellular ATP Induces the Accumulation of Superoxide via NADPH Oxidases in Arabidopsis

Abstract

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 (O2−) 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 ATPγS. 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 O2−, indicating that NADPH oxidases are responsible for the induced O2− accumulation. Inhibitors of mammalian P2-type ATP receptors abolished ATP-induced O2− 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 O2− 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 μm, well above the level needed to induce O2− accumulation and gene expression changes. Transgenic plants overexpressing an apyrase gene had reduced O2− 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.

Receptor-Like Activity Evoked by Extracellular ADP in Arabidopsis Root Epidermal Plasma Membrane

“…1A), very similar to that characterized in response to 20 mM extracellular ATP in this cell type (Demidchik et al, 2009). The concentration used is representative of wounding levels (Song et al, 2006). The increase in inwardly directed current at hyperpolarized (inside negative) voltage was observed 1 to 3 min after extracellular ADP addition (a similar time course to the extracellular ATP response reported by Demidchik et al, 2009;Shang et al, 2009) and was evident over the physiological voltage range for this cell type (Maathuis and Sanders, 1993).…”

“…In Arabidopsis root hairs, extracellular ATP also causes intracellular ROS accumulation by activating NADPH oxidases (Clark et al, 2010b). Leaves also support extracellular ATP-induced transcription via PM NADPH oxidases (Song et al, 2006). Nitric oxide is now also established as a product of extracellular purine nucleotide perception.…”

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…Indeed, a number of downstream effects of P2 receptor activation, such as expression of MUC5AC, MMP-9 or IL-8 and stimulated cell migration, have been linked to activation of DUOX1 (87,105,107,108), suggesting a common association of DUOX with epithelial purinergic signaling. Intriguingly, NOX activation has similarly been associated with ATP-mediated purinergic signaling in several other mammalian cell types (160)(161)(162)(163)(164), and in NADPH oxidase-mediated wound responses in plants (165,166), which suggests that the general signaling pathway illustrated in Fig. 4 may be widely conserved as a common biological response mechanism to environmental stress.…”

Apyrases (Nucleoside Triphosphate-Diphosphohydrolases) Play a Key Role in Growth Control in Arabidopsis

“…As yet there are insufficient data to answer this question. The fact that submicromolar levels of eATP can induce signaling changes in plants (Demidchik et al, 2003;Song et al, 2006) would suggest that plants, like animals, have purinoceptors that can bind eATP with a high affinity and transduce that binding into transduction pathways. However, as discussed by Demidchik et al (2003), Roux et al (2006), and others, as yet no purinoceptor has been identified in plants.…”

“…Plasma membrane-associated apyrases in plants could, in principle, function as ectoapyrases because plant cells, like animal cells, release significant quantities of ATP into their ECM when they are mechanically stimulated (Jeter et al, 2004), when they are wounded , and when they are engaged in activities that involve active secretion, such as growth (Kim et al, 2006). Moreover, control of this eATP could be important because plant cells have significant signaling responses to submicromolar ATP (Demidchik et al, 2003;Song et al, 2006) and extensive depletion of eATP can result in loss of cell viability (Chivasa et al, 2005).…”