Functional analyses of two arabidopsis apyrases
ATP can serve as a signal molecule in the extracellular space to regulate biological processes and physiological effects in plant and animal cells. In mammalian cells, the level of extracellular ATP (eATP) is regulated by ectoapyrases, which can hydrolyze extracellular ATP to ADP and ADP to AMP. In this dissertation, I describe the important role of two Arabidopsis apyrases in the regulation of plant growth. Seven apyrases have been identified in Arabidopsis thaliana. The genes for two of these seven apyrases, APY1 and APY2, which have high sequence similarity, were cloned and characterized previously. The function of APY1 and APY2 was analyzed by T-DNA insertional mutant lines. The double knockout (DKO) apyrase pollen displayed a complete block of pollen germination, which implicated this step as the cause of the lethality of apyrase double knockout mutants. The vast majority of the mutant pollen grains were identical to wild-type in their nuclear state, and were viable as assayed by metabolic activity and plasma membrane integrity. Pollen tube elongation was inhibited by suppression of apyrase activity using anti-apyrase antibodies or by chemical inhibitors of apyrases. Etiolated hypocotyls overexpressing APY1 (with expression driven by a constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter) exhibited faster growth rates compared to wild-type plants. Because of the lethality of apy1apy2 double mutants, RNA interference (RNAi) was performed as an alternative approach to posttranscriptionally silence the expression of apyrases. The suppression of apyrases in the RNAi lines resulted in a dwarf phenotype in overall vegetative growth and dramatically reduced growth in primary root and etiolated hypocotyls. In addition, the RNAi mutant plants lacked a well-defined meristematic zone and had a greatly reduced elongation zone in the primary root. Previously, promoter-GUS fusions showed that high expression of apyrase was associated with areas of rapid growth and regions with high auxin levels. Abnormal auxin accumulation was found in the proximal regions of the primary roots of RNAi mutant plants, which demonstrated that the absence of apyrase results in disrupted auxin distribution. Other phenotypes in RNAi mutant plants, such as less lateral root formation and more adventitious roots, could also be associated with abnormal auxin distribution. The investigation of the subcellular localization of apyrases showed that some fraction of apyrase was localized on cell periphery. These results suggest that the expression of APY1 and APY2 is essential for plant growth. They favor the hypothesis that Arabidopsis apyrases, like their homologs in animals, control the levels of ATP in the extracelluar space, and this control allows them to act as key regulators in growth.