An Arabidopsis Purple Acid Phosphatase with Phytase Activity Increases Foliar Ascorbate

Abstract

Ascorbate (AsA) is the most abundant antioxidant in plant cells and a cofactor for a large number of key enzymes. However, the mechanism of how AsA levels are regulated in plant cells remains unknown. The Arabidopsis (Arabidopsis thaliana) activationtagged mutant AT23040 showed a pleiotropic phenotype, including ozone resistance, rapid growth, and leaves containing higher AsA than wild-type plants. The phenotype was caused by activation of a purple acid phosphatase (PAP) gene, AtPAP15, which contains a dinuclear metal center in the active site. AtPAP15 was universally expressed in all tested organs in wild-type plants. Overexpression of AtPAP15 with the 35S cauliflower mosaic virus promoter produced mutants with up to 2-fold increased foliar AsA, 20% to 30% decrease in foliar phytate, enhanced salt tolerance, and decreased abscisic acid sensitivity. Two independent SALK T-DNA insertion mutants in AtPAP15 had 30% less foliar AsA and 15% to 20% more phytate than wild-type plants and decreased tolerance to abiotic stresses. Enzyme activity of partially purified AtPAP15 from plant crude extract and recombinant AtPAP15 expressed in bacteria and yeast was highest when phytate was used as substrate, indicating that AtPAP15 is a phytase. Recombinant AtPAP15 also showed enzyme activity on the substrate myoinositol-1-phosphate, indicating that the AtPAP15 is a phytase that hydrolyzes myoinositol hexakisphosphate to yield myoinositol and free phosphate. Myoinositol is a known precursor for AsA biosynthesis in plants. Thus, AtPAP15 may modulate AsA levels by controlling the input of myoinositol into this branch of AsA biosynthesis in Arabidopsis.

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

“…The biochemical properties of the recombinant AtPAP15 from plant were very different from that presented by Zhang et al (2008), who reported the specific activities of a GST:AtPAP15 toward a few substrates. In our study, the GST:AtPAP15 produced from E. coli was inactive.…”

“…In our study, the GST:AtPAP15 produced from E. coli was inactive. Zhang et al (2008) reported the specific activity of GST:AtPAP15 toward pNPP to be only 0.546 units mg 21 , which was far lower than the plant-derived AtPAP15 level (165 units mg 21 ; Table III) and that reported for the other plant PAPs (222-368 units mg 21 for tomato [Solanum lycopersicum] PAPs [Bozzo et al, 2002] and 266 units mg 21 for KbPAP [Vogel et al, 2002]). Regarding specific activities toward phytate, the two recombinant AtPAPs gave 10.0 (plant) and 0.7 (bacterial) units mg 21 , respectively.…”

“…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.…”

Overexpressing AtPAP15 Enhances Phosphorus Efficiency in Soybean

“…Overexpression of AtPAP15 in Arabidopsis significantly decreased phytate content in leaves (Zhang et al, 2008). Sequence analysis indicates that AtPAP15 exhibits 74% similarity to the soybean (Glycine max) phytase gene, GmPhy (Hegeman and Grabau, 2001).…”

“…AtPAP15, a purple APase with confirmed phytase activity from Arabidopsis, can hydrolyze myoinositol hexakisphosphate, yielding myoinositol and Pi (Zhang et al, 2008). Overexpression of AtPAP15 in Arabidopsis significantly decreased phytate content in leaves (Zhang et al, 2008).…”