Extracellular Nucleotides Elicit Cytosolic Free Calcium Oscillations in Arabidopsis    

Abstract

Extracellular ATP induces a rise in the level of cytosolic free calcium ([Ca 2+ ] cyt ) in plant cells. To expand our knowledge about the function of extracellular nucleotides in plants, the effects of several nucleotide analogs and pharmacological agents on [Ca 2+ ] cyt changes were studied using transgenic Arabidopsis (Arabidopsis thaliana) expressing aequorin or the fluorescence resonance energy transfer-based Ca 2+ sensor Yellow Cameleon 3.6. Exogenously applied CTP caused elevations in [Ca 2+ ] cyt that displayed distinct time-and dose-dependent kinetics compared with the purine nucleotides ATP and GTP. The inhibitory effects of antagonists of mammalian P2 receptors and calcium influx inhibitors on nucleotide-induced [Ca 2+ ] cyt elevations were distinct between CTP and purine nucleotides. These results suggest that distinct recognition systems may exist for the respective types of nucleotides. Interestingly, a mutant lacking the heterotrimeric G protein Gb-subunit exhibited a remarkably higher [Ca 2+ ] cyt elevation in response to all tested nucleotides in comparison with the wild type. These data suggest a role for Gb in negatively regulating extracellular nucleotide signaling and point to an important role for heterotrimeric G proteins in modulating the cellular effects of extracellular nucleotides. The addition of extracellular nucleotides induced multiple temporal [Ca 2+ ] cyt oscillations, which could be localized to specific root cells. The oscillations were attenuated by a vesicletrafficking inhibitor, indicating that the oscillations likely require ATP release via exocytotic secretion. The results reveal new molecular details concerning extracellular nucleotide signaling in plants and the importance of fine control of extracellular nucleotide levels to mediate specific plant cell responses.The calcium ion, Ca 2+

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

“…the premise that the sensitivity of different cells to extracellular purine nucleotides differs (Tanaka et al, 2010b). Suramin (a purinoceptor antagonist) inhibits Arabidopsis root elongation and extracellular ADPinduced [Ca 2+ ] cyt elevation (Demidchik et al, 2009).…”

“…D, Mean 6 SE fluorescence per pixel for 0.1 mM Ca 2+ buffer (n = 8), 1 mM Ca 2+ buffer (n = 17), 1 mM ADP with 1 mM Ca 2+ (n = 11), 1 mM ATP with 1 mM Ca 2+ (n = 11). lular AMP was found previously to be a very weak inducer of root Ca 2+ influx (Demidchik et al, 2009) and seedling [Ca 2+ ] cyt elevation (Tanaka et al, 2010b). Here, AMP did not elicit a K + efflux response even at 1 mM, in either the elongation zone or mature epidermis (elongation zone control; 237 6 34 nmol m 22 s 21 , n = 3: elongation zone + AMP, 231 6 26 nmol m 22 s 21 , n = 3; mature zone control; 219 6 13 nmol m 22 s 21 , n = 3: AMP, 210 6 18 nmol m 22 s 21 , n = 3).…”

“…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).…”