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

University Of Texas At Austin, Austin TX

Investigators

Abstract

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.