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

本研究的目的是了解更多关于一种新的腺苷三磷酸双磷酸酶的功能意义的植物生长和发育。在豌豆中，这种酶的mRNA表达在经历快速生长和发育的区域最高。豌豆cDNA编码的酶的基因已被分离和表征。它的翻译序列包括一个信号肽，该信号肽将其靶向质膜，并将其鉴定为腺苷三磷酸双磷酸酶家族的成员，该家族通常在细胞外，被称为外腺苷三磷酸双磷酸酶。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、生长和发育的意义尚不清楚。拟议的研究扩展了PI目前的腺苷三磷酸双磷酸酶项目中出现的两个关键观察结果：豌豆ectoapyrase补充了磷酸盐转运缺陷的酵母突变体，并增强了拟南芥根中的磷酸盐摄取，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转运蛋白的多药耐药性功能。 这些惊人的发现对于理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大意义，并且它们引起了人们对细胞外ATP（xATP）在植物生长和发育中的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验证。为此，提出了三组实验。 第一组实验旨在更好地定义腺苷三磷酸双磷酸酶促进的磷酸盐摄取的结构基础。虽然豌豆胞外腺苷三磷酸双磷酸酶促进磷酸盐转运，但尚不清楚腺苷三磷酸双磷酸酶的一级结构如何支持这种功能，因为它只有一个明显的跨膜结构域。最近的研究表明，人外腺苷三磷酸双磷酸酶，其类似于豌豆酶在其一级结构和质膜位置，具有类似于已知的转运蛋白的结构的质膜中的四聚体结构，因此具有转运功能的结构潜力。第二组实验将进一步研究腺苷三磷酸双磷酸酶在植物和酵母中的过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种模型，该模型假定外腺苷三磷酸双磷酸酶在毒素抗性中发挥作用的能力取决于其通过水解xATP来帮助维持细胞内外之间ATP梯度的陡度的能力。 第三组实验解决的问题是ATP是否可以在植物中发挥信号作用，就像它在动物中一样。该小组开发了一种功能筛选，开始测试植物质膜上是否有ATP受体。 所有提出的实验将大大有助于腺苷三磷酸双磷酸酶抗体，特异性腺苷三磷酸双磷酸酶抑制剂，和过表达的酶或抑制其表达的转基因植物的可用性。拟议的研究将产生有价值的和新的见解如何水解xATP的外腺苷三磷酸双磷酸酶影响植物生长和发育的各个方面。

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.