Investigations into extracellular nucleotide-based signaling mechanisms in plants

Nucleoside tri- and diphosphates, ubiquitous molecules inside all living cells, function as essential intracellular metabolites and are also known to perform extracellular signaling functions in animals. Apyrases, also known as ENTPDases, are key regulatory enzymes modulating the concentration of NTPs and NDPs in the extracellular matrix, thereby altering the activity of specialized P2- receptors that respond to these signaling agents. P2-receptor activation induces calcium-dependent downstream responses in animals. We present data showing that Arabidopsis thaliana possesses apyrase homologues, and responds to exogenously applied ATP and ADP analogs by increasing [Ca2+]cyt via an unknown class of receptor. vii Our results show that xATP and xADP and their analogues. ATPγS and ADPβS applied to whole Arabidopsis seedlings transformed with apo-aequorin induce a dose-dependent and saturable increase in [Ca2+]cyt. Pretreatment with agents such as BAPTA and Gd3+ block the response to ADPβS, providing evidence that the ECM is the source of the Ca2+ contributing to the observed ∆[Ca2+]cyt. Oligogalacturonides, cell wall fragments released by wounding, and ADPβS, induce a higher increase in [Ca2+]cyt together than either alone, suggesting a physiological significance in wound responses. We cloned the first Arabidopsis apyrase, Atapy1 a potential regulatory enzyme with NTPDase activity. The sequence of Atapy1 led to the identification of other apyrases in Arabidopsis, some of which are likely to function in the ECM, candidate regulatory enzymes of ATP-based signaling in plants. We used a sequence-based approach to search for a plant P2-receptor homologue. A thorough search of the A. thaliana proteome has failed to reveal a plant P2-like receptor. The absence of an apparent plant P2-receptor, in spite of the rapid, specific and concentration-dependent increase in [Ca2+]cyt suggests plants perceive xATP (and xADP) by a novel type of receptor. These data suggest plants, like animals, employ extracellular nucleotidebased signaling mechanisms. Taken together with further data obtained in collaboration with other Roux lab members, xATP-based signaling is proposed to function in mediating stress and wound responses in a calcium-dependent manner.