AbstractExtracellular ATP induces a rise in the level of cytosolic free calcium ([Ca2+]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 [Ca2+]cyt changes were studied using transgenic Arabidopsis (Arabidopsis thaliana) expressing aequorin or the fluorescence resonance energy transfer-based Ca2+ sensor Yellow Cameleon 3.6. Exogenously applied CTP caused elevations in [Ca2+]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 [Ca2+]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 Gβ-subunit exhibited a remarkably higher [Ca2+]cyt elevation in response to all tested nucleotides in comparison with the wild type. These data suggest a role for Gβ 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 [Ca2+]cyt oscillations, which could be localized to specific root cells. The oscillations were attenuated by a vesicle-trafficking 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.