Characterization of an AtPAP26-like protein (TrPAP26) from white clover (Trifolium repens L.) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Palmerston North, New Zealand

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Phosphate levels in soils are often in deficit in New Zealand agriculture systems, resulting in the need for phosphate supplements in the form of fertilizers. Plants are able to adapt to many environmental stresses and display a wide range of responses designed to cope with phosphate-deficiency, and the study of these may lead to the production of crop and pasture plants that can utilize added P more efficiently. One adaptive mechanism is to express purple acid phosphatase (PAP) genes, the protein products of which are able to generate, transport, and recycle inorganic phosphates from phosphate-rich compounds both intracellularly and extracellularly. Their general mechanism of action is to hydrolyze phosphate-rich esters that are found within cells, the cell wall or in the rhizosphere. One PAP, AtPAP26, has been extensively characterized in Arabidopsis thaliana and displays high levels of acid phosphatase activity during phosphate-starvation. AtPAP26 has been found to be the predominantly expressed PAP during phosphate-starvation and the enzyme plays a key role in supplying inorganic phosphate to the plant by hydrolyzing the organic phosphates present in the rhizosphere. An AtPAP26-like sequence has been identified previously in white clover and so this project firstly cloned the full-length TrPAP26 and then examined expression in response to phosphate-starvation. The protein product (TrPAP26) was also characterized and compared to AtPAP26 in terms of its putative biochemical functions. TrPAP26 was predicted to be a 55 kDa protein with three N-glycosylation sites, a signal peptide of 21 amino acid residues, and a metal-ligating motif typical of PAPs. Its observed mass was closer to 45 kDa, and preliminary experiments, using recombinant TrPAP26 partially purified from transgenic tobacco, suggested that it hydrolyzed a wide range of phosphate-rich esters including adenosine triphosphate (ATP), phosphoenolpyruvate (PEP), and pyrophosphate (PPi), but not inositol hexakisphosphate (phytate). TrPAP26 transcript levels were found to be constitutive in the roots of white clover, but correlated positively with phosphate supply in other tissues. The protein and activity levels were not directly correlated with the transcript levels suggesting other methods of regulation such as post- translational modifications, including N-glycosylation. TrPAP26 accumulated more in the mature leaves of white clover plants grown with a full supply of phosphates. Taken together, these results suggest that TrPAP26 may play a role in internal P remobilization, rather than P scavenging directly.
White clover, Genetics, Trifolium repens L., AtPAP26-like protein