Significance of a Novel Light-Regulated Apyrase for Plant Growth and Development

本研究的目的是进一步了解一种新型吡脲酶在植物生长发育中的功能意义。在豌豆中，这种酶的mRNA的表达在快速生长发育的区域是最高的。已分离并鉴定了该酶基因的豌豆cDNA。它的翻译序列包括一个信号肽，该信号肽将其定位于质膜，并将其识别为apyrase家族的成员，该家族通常是细胞外的，被称为外链apyrase。在动物中，这些酶在终止ATP/ adp诱导的信号转导（如ATP诱导的细胞凋亡）中起关键作用。该酶的生化功能是水解NTPs和ndp，但该功能对植物生理、生长发育的意义尚不清楚。拟议的研究扩展了PI当前apyrase项目中出现的两个关键观察结果：豌豆外apyrase补充了酵母中缺乏磷酸盐运输的突变体，并增加了拟南芥根部的磷酸盐吸收；它赋予酵母和拟南芥植物毒素抗性，可能是通过增强这些生物体中ABC转运蛋白的多重耐药功能。这些惊人的发现对于了解植物中磷酸盐营养和毒素抗性的替代模式具有潜在的重要意义，并引起人们对细胞外ATP （xATP）在植物生长发育中先前未被怀疑的作用的关注。为了更好地理解它们的含义，有必要研究它们背后的分子基础，并测试基于它们的预测是否可以被验证。为此，提出了三组实验。第一组实验旨在更好地定义淀粉酶促进磷酸吸收的结构基础。尽管豌豆外酯脲酶促进磷酸盐运输，但由于它只有一个明显的跨膜结构域，目前尚不清楚酯酶的初级结构如何支持这一功能。近年来的研究表明，人外胰酶在质膜上具有与已知转运蛋白结构相似的四聚体结构，其初级结构和质膜位置与豌豆酶相似，因此具有转运功能的结构潜力。第二组实验将进一步研究apyrase在植物和酵母中的过度表达如何赋予它们对某些有毒化合物的抗性。特别是，这些实验将测试一个模型，该模型假设外腺苷酶在毒素抗性中的功能取决于其通过水解xATP来帮助维持细胞内外ATP梯度的陡度的能力。第三组实验解决了ATP在植物中是否能像在动物中一样发挥信号作用的问题。该小组开发了一种功能筛选，开始测试植物的质膜上是否存在ATP受体。所有提出的实验都将在很大程度上得益于apyrase抗体、特异性apyrase抑制剂的可用性，以及过度表达或抑制其表达的转基因植物。所提出的研究将对外链酶水解xATP如何影响植物生长发育的各个方面产生有价值的和新颖的见解。

The objective of this research is to learn more about the functional significance of a novel apyrase for plant growth and development. In peas the expression of the mRNA for this enzyme is highest in regions undergoing rapid growth and development. A pea cDNA encoding the gene for the enzyme has been isolated and characterized. Its translated sequence includes a signal peptide, which targets it to the plasma membrane, and identifies it as a member of the family of apyrase enzymes that are typically extracellular and are called ectoapyrases. In animals these enzymes play a key role in terminating ATP/ADP-induced signal transduction, such as ATP-induced apoptosis. The biochemical function of this enzyme is to hydrolyze NTPs and NDPs, but the significance of this function for plant hysiology, growth and development is not known. The proposed research expands on two key observations that emerge from the PI's current apyrase project: the pea ectoapyrase complements a yeast mutant deficient in phosphate transport and augments phosphate uptake in Arabidopsis roots, and it confers toxin resistance to yeast and to Arabidopsis plants, possibly by enhancing the Multi-Drug-Resistance function of an ABC transporter in those organisms. These startling findings have potentially great significance for understanding alternate modes of phosphate nutrition and toxin resistance in plants, and they call attention to a previously unsuspected role for extracellular ATP (xATP) in the growth and development of plants. To better understand their implications, it will be necessary to investigate the molecular bases underlying them and to test whether predictions based on them can be validated. Toward this end three groups of experiments are proposed. The first group of experiments is designed to better define the structural basis of apyrase-facilitated phosphate uptake. Although the pea ectoapyrase facilitates phosphate transport, it is not clear how the primary structure of apyrase would support this function, since it has only one evident membrane-spanning domain. Recent studies show that the human ectoapyrase, which resembles the pea enzyme in its primary structure and plasma membrane locale, has a tetrameric structure in the plasma membrane similar to the structure of known transport proteins, and thus has the structural potential for a transport function. The second group of experiments will further investigate how overexpression of apyrase in plants and in yeast confers on them resistance to certain toxic compounds.In particular these experiments will test a model which postulates that the ability of ectoapyrase to function in toxin resistance is dependent on its ability to help maintain the steepness of the ATP gradient between the inside and outside of cells by hydrolyzing xATP. The third group of experiments addresses the question of whether ATP can serve a signaling role in plants as it does in animals. This group develops a functional screen to begin testing whether there are ATP receptors on the plasma membrane of plants. All proposed experiments will be greatly aided by the availability of apyrase antibodies, specific apyrase inhibitors, and transgenic plants that overexpress the enzyme or that are suppressed in its expression. The proposed studies will generate valuable and novel insights into how the hydrolysis of xATP by ectoapyrases influences diverse aspects of plant growth and development.