Distribution and expression of apyrases in pea and Arabidopsis

This dissertation describes the results of experiments aimed at defining the tissue distribution, regulated expression and nuclear function of apyases in both Pisum sativum and Arabidopsis thaliana. As an approach to tissue-specific localizations in Arabidopsis, promoter regions including 5’UTR of two Arabidopsis apyrases, Atapy1 and Atapy2, were linked with GUS and transformed into Arabidopsis thaliana. As evaluated by GUS staining, the expression patterns of the two apyrases were almost identical. The highest expression level for both apyrases was found in mature pollen, the stigma surface, root cap and columella cells, the abscission zone and the top part of stipules. Both apyrases were also expressed highly in selected cell types of roots other than root tip. However, expression in hypocotyls and in flowering stems was not detectable for either apyrase in light grown seedlings. In etiolated seedlings, both apyrases are expressed in the upper region of the hypocotyl. Semi-quantitative RT-PCR measurements showed that both apyrases are upregulated by blue light and red light. Quantitative GUS assay and RT-PCR also shows that Atapy1 and Atapy2 are differently regulated by physical injury. Possible functions of AtAPYs in secretion, wound responses and in phytochromemediated light signal transduction are proposed. Transgenic plants that express AtAPY1-GFP and the antisense of Atapy1 were also made. The AtAPY1-GFP hybrid protein was found to exist as dot-like structures primarily in the cytosol. These plants showed no visible phenotype differences to wild-type plants, in conformity to the observations that plants knocked out in Atapy1 had no apparent phenotypic differences from wild-type plants. In the nuclei of etiolated pea seedlings, expression of the apyrase protein is regulated by red light but not white light. The nuclear apyrase was found to exist in a large (MW ~ 400Kd-800Kd) protein complex bound to the nuclear matrix. Specific molecular mass proteins were found to associate with apyrase when it was separated on molecular sieve chromatography and immunoprecipitated with highly specific polyclonal antibodies. In Arabidopsis, attempts to use AtAPY1 as bait in the yeast two-hybrid system to search for its binding protein were not successful.