A Novel Phytase with Increased Tolerance to Arsenate In Pteris Vittata.

Phytic acid (myo-Inositol hexakisphosphate, phytate) is a phosphorus-rich organic acid, which is present in plant seeds, roots, fruits and pollens. Phytase is one type of acid phosphatase implicated releasing P from soil organic P-esters. Phytase activity can be inhibited by a variety of metal ions but is especially sensitive to arsenic. Arsenic has a deleterious effect on numerous enzymes, disabling their normal function. Studies show phytase activity (i.e. Pisum sativum, Trifolium subterraneum) is reduced by as much as 95% in the presence of 1 mM arsenate. This is not surprising due to the shared homology between phosphorus and arsenic.

Pteris vittata, a vascular fern within the Pteridaceae family, can accumulate up to 2% arsenic (dry weight) in its aboveground biomass. Since P. vittata can tolerate high concentrations of arsenic, the enzymes may be uniquely resistant to arsenic. To test this hypothesis, phytase activity from P. vittata and Pteris ensiformis (a non-hyperaccumulator) tissues were assayed following three days of exposure to arsenate and/or P_i stress. Plant tissues were separated into frond, rhizome, and root for maceration and protein collection. The extracts were subject to gel filtration at 4°C on Sephadex G-25 and analyzed for activity by incubating in 15 mM MES buffer (pH 5.0) at 37°C amended with 5 mM phytate containing 0 or 1 mM arsenate. Activity was expressed as the amount of P_i released per unit of protein per minute.

Phytase activity associated with P. vittata frond, root, and rhizome responded (increased or decreased) in all treatments of arsenate and phosphate while P. ensiformis phytase activity responded only to phosphate treatments from the frond. For example, activity in the root of P. vittata was ~6.4 nmol P_i protein^-1 min^-1 when grown with adequate P and increased to ~44.0 nmol P_i protein^-1 min^-1 when grown with additional arsenate. Root extract of P. ensiformis remained ~4.0 nmol P_i protein^-1 min^-1 regardless of treatment. The same experiments were repeated with 1 mM arsenate in the reaction mixture to determine arsenic resistance. In P. vittata, enzyme activity was unaltered by additions of arsenate up to 1 mM, decreasing by 20% as arsenate concentrations increased to 5 mM. Activity from P. ensiformis was reduced by 70% in the presence of 0.5 mM arsenate and up to 90% as concentrations increased to 5 mM.

To gauge the role of phytase utilization in P acquisition, spores from P. vittata were grown asceptically on 0.5X Murashige & Skoog media amended with phosphate, phytic acid, and/or arsenate. Following 6 weeks, gametophytes on plates amended with phytic acid (0.25 – 1.0 mM) exhibited more vigorous growth compared to phosphate (0.06 – 0.6 mM) only plates. Furthermore, gametophytes were larger and denser on plates with arsenate (1 mM) in addition to P.

The results suggest an intrinsic role of phytase in P homeostasis and arsenic tolerance by P. vittata, which potentially makes P. vittata a model plant in understanding the role of phytase in arsenic tolerance and organic P acquisition.