Significance of Apyrase Activity for Plant Growth and Development

这项研究的主要目的是了解更多关于水解核苷酸（NTPs和NDP）的酶，称为腺苷三磷酸双磷酸酶，如何对植物生长和发育产生显着影响。豌豆中的一种主要腺苷三磷酸双磷酸酶与许多动物腺苷三磷酸双磷酸酶一样，是一种“外腺苷三磷酸双磷酸酶”，锚定在质膜上，其活性位点面向植物壁。最类似于豌豆腺苷三磷酸双磷酸酶的两种拟南芥腺苷三磷酸双磷酸酶Atapy1和Atapy2也可能是胞外腺苷三磷酸双磷酸酶，这与这些酶的功能是调节细胞外ATP和ADP浓度的假设一致，这两种酶都是脊椎动物中的类腺苷三磷酸试剂。与这种解释一致，提高培养基中的细胞外ATP浓度（[xATP]）和化学抑制腺苷三磷酸双磷酸酶活性可以产生与遗传敲除腺苷三磷酸双磷酸酶表达相同的生理和生长效应。三磷酸腺苷双磷酸酶在植物中发挥调节作用得到以下观察结果的支持，即在Atapy1和Atapy2中具有敲除突变的植物是矮小的，并且产生尽管有活力但不能发芽的花粉。为了更好地理解这些发现的含义，该项目建议调查其背后的分子基础。 目前的证据表明，拟南芥腺苷三磷酸双磷酸酶，像动物外腺苷三磷酸双磷酸酶，需要保持低[xATP]。 直接测量表明，[xATP]上升到至少0.2毫米的液体中释放在叶伤口部位。将接近或低于该水平的xATP浓度应用于未受伤的植物可以开启与受伤所开启的相同的信号步骤。为了了解[xATP]在各种植物组织中的升高程度，该项目提出开发一种实时测定方法，用于测量质膜外表面附近的[ATP]。 在动物中，xATP信号由专门的受体接收。为了了解植物中是否存在类似的受体，该项目建议表征最近选择的对xATP超敏感或低敏感的突变体。 xATP除了在植物中可能是一种类似于细菌的物质外，还可以在调节转运蛋白的活性方面发挥作用，这些转运蛋白将各种生长调节物质输出到植物细胞外。该提案进一步验证了这一假设，重点是腺苷三磷酸双磷酸酶对生长素和除草剂运输的影响。这项研究对社会的一个潜在的重要益处是，腺苷三磷酸双磷酸酶抑制剂虽然本身无毒，但似乎可以增强除草剂的效力。 在开发腺苷三磷酸双磷酸酶抑制剂作为除草剂增效剂方面存在一些商业利益，从而允许减少环境中除草剂的使用。关于腺苷三磷酸双磷酸酶参与生长控制，建议的工作利用一个非常简单的方法来抑制腺苷三磷酸双磷酸酶的表达，研究腺苷三磷酸双磷酸酶对叶，茎和根生长的影响是否也延伸到花粉管中的生长，其生长特性是最好的研究植物。 拟议中的研究将为研究生提供一个肥沃的训练基地。 高中和少数民族本科生也将积极参与研究。

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.