The main objective of this research is to learn more about how an enzyme that hydrolyzes nucleotides (NTPs and NDPs), called apyrase, exerts its dramatic effects on plant growth and development. A major apyrase in peas is, like many animal apyrases, an "ectoapyrase", anchored in the plasma membrane with its active site facing out into the plant wall. The two Arabidopsis apyrases that most resemble the pea apyrase, Atapy1 and Atapy2, are also likely to be ectoapyrases, consistent with the hypothesis that the function of these enzymes is to regulate the concentration of extracellular ATP and ADP, which are both hormone-like agents in vertebrates. Consistent with this interpretation, raising the extracellular ATP concentration ([xATP]) in the medium and chemically inhibiting apyrase activity can produce the same physiological and growth effects as genetically knocking out apyrase expression. That apyrases play a regulatory role in plants is supported by the observation that plants that have knock out mutations in Atapy1 and Atapy2 are dwarf and produce pollen that, though viable, cannot germinate. To better understand the implications of these findings this project proposes to investigate the molecular bases underlying them.
Current evidence indicates that the Arabidopsis apyrases, like animal ectoapyrases, are needed to maintain low [xATP]. Direct measurements show that [xATP] rises to at least 0.2 mM in the fluid released at leaf wound sites. Concentrations of xATP near to or below this level applied to unwounded plants can turn on the same signaling steps that are turned on by wounding. To learn just how high [xATP] rises in various plant tissues, this project proposes to develop a real-time assay for measuring the [ATP] near the external face of the plasma membrane.
In animals xATP signals are received by specialized receptors. To learn whether there are similar receptors in plants, this project proposes to characterize recently selected mutants that are hyper- or hyposensitive to xATP. Independent of its potential to be a hormone-like agent in plants, xATP could also play a role in regulating the activity of transporters that export various growth-regulating substances out of plant cells. The proposal further tests this hypothesis, with a focus on apyrase effects on the transport of auxin and of herbicides. One potentially important benefit to society from this study is that apyrase inhibitors, though not toxic by themselves, appear to enhance the potency of herbicides. There is some commercial interest in developing apyrase inhibitors as herbicide synergizers, allowing a decreased use of the herbicides in the environment. Regarding apyrase involvement in growth control, the proposed work takes advantage of a remarkably facile method to suppress apyrase expression to study whether apyrase effects on leaf, stem and root growth extend also to growth in pollen tubes, whose growth characteristics are among the best studied in plants.
The proposed research will provide a fertile training ground for graduate students. High school and minority undergraduate students will also actively participate in the research.