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一种被称为apyrase的核苷酸(NTPs和NDPs)的水解酶是如何对植物的生长和发育产生巨大影响的。豌豆中的一个主要apyrase，像许多动物apyrase一样，是一种“外apyrase”，固定在质膜上，其活性部位面向植物的外壁。拟南芥中与豌豆apyrase最相似的两个apyrase，Aap y1和Aap y2，也可能是胞外apyrase，这与这两种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激素样物质。与这一解释一致的是，提高培养液中的胞外ATP浓度([xATP])和化学抑制apyrase的活性可以产生与基因敲除apyrase表达相同的生理和生长效应。Apyrase在植物中起着调节作用，这一点得到了以下观察的支持：在Aap y1和Aap y2中敲除突变的植物是矮小的，产生的花粉虽然有活力，但不能萌发。为了更好地理解这些发现的含义，该项目建议调查它们背后的分子基础。目前的证据表明，拟南芥apyrase和动物的胞外apyrase一样，是维持低[xATP]所必需的。直接测量表明，在叶片伤口处释放的液体中，[xATP]至少上升到0.2 mm。接近或低于这一水平的xATP浓度应用于未受伤的植物，可以启动与受伤时相同的信号步骤。为了了解[xATP]在各种植物组织中的含量有多高，该项目建议开发一种实时测定质膜外表面附近的[ATP]的方法。在动物体内，xATP信号是由专门的受体接收的。为了了解植物中是否存在类似的受体，该项目建议对最近选择的对xATP高度或低敏感的突变体进行表征。除了在植物中作为激素样剂的潜力之外，xATP还可以在调节转运蛋白的活性方面发挥作用，转运蛋白将各种生长调节物质输出到植物细胞之外。该提案进一步验证了这一假设，重点是apyrase对生长素和除草剂运输的影响。这项研究给社会带来的一个潜在的重要好处是，apyrase抑制剂虽然本身没有毒性，但似乎可以增强除草剂的效力。开发apyrase抑制剂作为除草剂增效剂，减少除草剂在环境中的使用，具有一定的商业意义。关于apyrase参与生长控制，本工作利用一种非常简便的方法抑制apyrase的表达，以研究apyrase对叶、茎和根生长的影响是否也延伸到花粉管的生长，而花粉管的生长特性是植物中研究得最好的之一。拟议中的研究将为研究生提供肥沃的培训场地。高中生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.