Abstract: Current data indicate that that purinergic signaling can potentially affect every cell in the human body. Indeed, one theory is that extracellular ATP (eATP) is the oldest, extracellular signal involved in cell-cell communication. The mechanisms of purinergic signaling are well-established in animals and, indeed, support a multibillion dollar pharmaceutical industry. In contrast, relatively little is known about purinergic signaling in plants. Plants do not possess canonical P2X an P2Y purinoreceptors. Indeed, our lab previously identified a new class of purinoreceptors in plants, exemplified by plasma membrane lectin-receptor-like-kinases, P2K1 and P2K2. Our research indicates that purinergic signaling in plants is as ubiquitous and impactful as that found in mammals and, indeed, many of the downstream effects are similar. The differences seen, comparing plants and animals, are due largely to the unique biochemistry of the P2K receptors. These are unique receptors in that they possess both kinase and nucleotide cyclase activity. Hence, the primary aim of this proposal is to explore further the function of these receptors and, specifically, the relative contribution of these two activities to cellular response to eATP. Specific aim 1 will address the hypothesis that ATP released from plant cells activates both P2K1 nucleotide cyclase and kinase activity, which subsequently mediate distinct downstream, cellular responses. Study 1 will utilize mutant studies of P2K1 to define the relative contribution of these two activities to downstream signaling responses. Study 2 will explore the relative importance of P2K1 cyclase and kinase activity relative to the activation of cyclic nucleotide gated calcium channels (CNGC). Study 3 will examine whether activation of the cytoplasmic kinase, BIK1, via P2K1 phosphorylation leads to CNGC Ca2+ channel activation. Specific aim 2 will address the hypothesis that, as is the case in animals, plants likely have multiple purinoreceptors that may act in specific tissues, during specific stages of development or in response to specific stresses. Preliminary data argue that additional purinoreceptors exist in plants. Hence, Study 4 will seek to identify additional plant purinoreceptors using a variety of approaches. Most notable is our finding that mutations that suppress the phenotypes of p2k1 mutants do so in the apparent absence of any known purinoreceptor, clearly indicating that other mechanisms must exist. Preliminary data suggest that purinoreceptors mediate the negative effects of stress on plant growth. Study 5 will explore how wounding stress is coupled to a reduction in growth by exploring the role of an atypical basic-helix-loop-helix protein, previously implicated in growth regulation, that is a direct target of P2K1 phosphorylation. The net result of our work is to provide the comparative data to add to our overall understanding of purinergic signaling in higher eukaryotes, illustrating differenc...