Plant extracellular ATP signalling by plasma membrane NADPH oxidase and Ca²⁺channels

Abstract

SUMMARYExtracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca 2+ can be elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots of Arabidopsis thaliana to determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca 2+ is determined by ATP perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca 2+-permeable channels (determined using patch-clamp electrophysiology), which contribute to the elevation of cytosolic free Ca 2+ . Disruption of this pathway in the AtrbohC mutant impaired the extracellular ATP-induced increase in reactive oxygen species (ROS), the activation of Ca 2+ channels, and the transcription of the MAP kinase3 gene that is known to be involved in stress responses. This study shows that higher plants, although bereft of purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma membrane.

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

“…activated by increasingly negative voltage) Ca 2+ -permeable conductance (Fig. 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 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). Mean 6 SE current-voltage (IV) relationships are shown in Figure 1B; negative voltages are physiologically relevant.…”

“…Extracellular ADP elevates [Ca 2+ ] cyt in both roots and leaves of Arabidopsis and it can be equally or more effective than extracellular ATP (Demidchik et al, 2003a(Demidchik et al, , 2009Jeter et al, 2004;Tanaka et al, 2010b). In common with extracellular ATP, extracellular ADP can elicit oscillations of [Ca 2+ ] cyt in Arabidopsis seedlings (Tanaka et al, 2010b).…”

“…It remains possible that plants evolved structurally different systems for purine sensing to animals but antagonists of animal purinoceptors are effective against plant extracellular ATP responses. Despite the apparent absence of classical purinoceptors in higher plants, extracellular purine nucleotides can cause transient elevations of cytosolic free Ca 2+ ([Ca 2+ ] cyt ), in common with animals (Demidchik et al, 2003a(Demidchik et al, , 2009Jeter et al, 2004;Tanaka et al, 2010b). In Arabidopsis roots, extracellular ATP most likely triggers intracellular Ca 2+ release, resulting in stimulation of PM NADPH oxidase activity (Demidchik et al, 2009).…”

“…Extracellular purine nucleotides have been shown to be involved in the regulation of plant cell viability, membrane permeability, immunity, symbiosis, stress responses, and growth (Lew and Dearnaley, 2000;Tang et al, 2003;Chivasa et al, 2005Chivasa et al, , 2009Kim et al, 2006Kim et al, , 2009Roux and Steinebrunner, 2007;Wu et al, 2007;Riewe et al, 2008a;Wu and Wu, 2008;Yi et al, 2008;Demidchik et al, 2009;Govindarajulu et al, 2009;Kim et al, 2009;Clark et al, 2010aClark et al, , 2010bTanaka et al, 2010aTanaka et al, , 2010bTerrile et al, 2010;Tonó n et al, 2010). Purine nucleotide release from plant cells may occur through wounding, exocytosis, or through the activity of plasma membrane (PM) ATP-binding cassette transporters (Thomas et al, 2000;Kim et al, 2006).…”