Molecular and Biochemical Characterization of AtPAP15, a Purple Acid Phosphatase with Phytase Activity,in Arabidopsis

Purple acid phosphatase (PAP) catalyzes the hydrolysis of phosphate monoesters and anhydrides to release phosphate within an acidic pH range. Among the 29 PAP-like proteins in Arabidopsis (Arabidopsis thaliana), AtPAP15 (At3g07130) displays a greater degree of amino acid identity with soybean (Glycine max; GmPHY) and tobacco (Nicotiana tabacum) PAP (NtPAP) with phytase activity than the other AtPAPs. In this study, transgenic Arabidopsis that expressed an AtPAP15 promoter∷β-glucuronidase (GUS) fusion protein showed that AtPAP15 expression was developmentally and temporally regulated, with strong GUS staining at the early stages of seedling growth and pollen germination. The expression was also organ/tissue specific, with strongest GUS staining in the vasculature, pollen grains, and roots. The recombinant AtPAP purified from transgenic tobacco exhibited broad substrate specificity with moderate phytase activity. AtPAP15 T-DNA insertion lines exhibited a lower phytase and phosphatase activity in seedling and germinating pollen and lower pollen germination rate compared with the wild type and their complementation lines. Therefore, AtPAP15 likely mobilizes phosphorus reserves in plants, particularly during seed and pollen germination. Since AtPAP15 is not expressed in the root hair or in the epidermal cells, it is unlikely to play any role in external phosphorus assimilation.At pH in the range of 4 to 7, purple acid phosphatases (PAPs) catalyze the hydrolysis of a wide range of activated phosphoric acid monoesters and diesters and anhydrides (Klabunde et al., 1996). They are distinguished from the other phosphatases by their insensitivity to l-(+) tartrate inhibition and therefore are also known as tartrate-resistant acid phosphatases. Their characteristic pink or purple color derives from a charge transfer transition between a Tyr residue and the “chromophoric” ferric ion in the binuclear Fe(III)-Me(II) center, where the metal (Me) is iron, zinc, or manganese (Schenk et al., 1999). PAP proteins are also characterized by seven conserved amino acid residues (shown in boldface) in the five conserved motifs DXG, GDXXY, GNH(D/E), VXXH, and GHXH, which are involved in the coordination of the dimetal nuclear center (Li et al., 2002).PAPs are widespread in mammals, fungi, bacteria, and plants. Interestingly, while only a few copies of PAP-like genes are present in mammalian and fungal genomes (Mullaney and Ullah, 2003; Flanagan et al., 2006), multiple copies are present in plant genomes (Schenk et al., 2000). For example, 29 PAP-like genes have been identified in the Arabidopsis (Arabidopsis thaliana) genome (Li et al., 2002). It is intriguing that so many PAP-like genes are required for plant metabolism; this diverse portfolio of PAP-like genes implies differential functions for them. Plant PAPs are generally considered to mediate phosphorus acquisition and redistribution based on their ability to hydrolyze phosphorus compounds (Cashikar et al., 1997; Bozzo et al., 2004; Lung et al., 2008). However, additional biological roles have been reported for some plant PAPs. For example, the PAPs AtACP5 (AtPAP17), SAP1, and SAP2 (del Pozo et al., 1999; Bozzo et al., 2002) display not only phosphatase but also peroxidase activity, suggesting their involvement in the removal of reactive oxygen compounds in plant organs. GmPAP3, isolated from salted-stressed soybean (Glycine max), reportedly mediates salt tolerance via NaCl and oxidative stress inductions but not by phosphorus starvation (Liao et al., 2003).Some PAP members can hydrolyze phytic acid (myoinositol hexakisphosphate InsP6]) to inorganic phosphate and free or lower phosphoric esters of myoinositol. Since the major storage form of phosphorus in plant seeds and pollen grains is phytate, PAPs with phytase activity may play a role in seed and pollen germination. However, not all PAPs exhibit phytase activity. The first plant phytase PAP, GmPHY, was isolated from the cotyledon of germinating soybean seedlings (Hegeman and Grabau, 2001). A tobacco (Nicotiana tabacum) root PAP phytase was identified more recently that is likely involved in mobilizing external organic phosphorus in soil (Lung et al., 2008).Relatively little is known about the biochemical properties and physiological roles of the 29 PAP-like Arabidopsis genes (del Pozo et al., 1999; Veljanovski et al., 2006). An enzyme assay involving the glutathione S-transferase (GST)-AtPAP23 fusion protein revealed that the Arabidopsis PAP AtPAP23 exhibits phytase activity (Zhu et al., 2005). A GUS study showed that AtPAP23 is exclusively expressed in the flower of the Arabidopsis plant. In a recent report, a recombinant AtPAP15 expressed in Escherichia coli was also found to exhibit phytase activity; this PAP potentially modulates plant ascorbate synthesis through supply of myoinositol from the phytate hydrolysis reaction (Zhang et al., 2008). However, the possible physiological roles of AtPAP15 in phosphorus mobilization have not been examined.In this study, AtPAP15 expressed in a plant (tobacco) system was biochemically characterized, and its temporal and spatial expression patterns in Arabidopsis were examined. The physiological roles of AtPAP15 in phosphorus mobilization were also delineated.