An investigation into the role of oxalate in the leaves of Pisonia grandis

"Pisonia grands R. Br. (Nyctaginaceae) is a large tropical tree whose distribution is restricted to coral cays, in particular those with high seasonal nesting seabird populations. These trees are often the dominant plant species found on coral cays. This is the case for the chosen study site on North West Island, a coral cay which lies off the Central Queensland coast within the Mackay Capricorn section of the Great Barrier Reef. Coral cays are almost entirely composed of coral rubble and sand and as a result the substrate pH (c. 9.0) and many ions are found as insoluble salts. The high pH, abundance of calcium, lack of water and high seasonal input of nitrates from nesting seabirds, are possible influences on the biosynthesis of oxalates which are found in high concentrations in P. grandis. The aims of this study are twofold. The first aim was to investigate the influence of environmental factors, such as pH and nitrate concentration, on the biosynthesis of oxalate in Pisonia grandis leaf. The second aim was to purify and characterise the enzyme glycolic acid oxidase (E.C. 1.1.3.1) (GAO), an enzyme involved in the biosynthesis of oxalate..." --abstract.. Pisonia grandis R. Br. (Nyctaginaceae) is a large tropical tree whose distribution is restricted to coral cays, in particular those with high seasonal nesting seabird populations. These trees are often the most dominant plant species found on coral cays. This is the case for the chosen study site on North West Island, a coral cay which lies off the Central Queensland coast within the Mackay Capricorn section of the Great Barrier Reef. Coral cays are almost entirely composed of coral rubble and sand and as a result the substrate pH is high (c. 9.0) and many ions are found as insoluble salts. The high pH, abundance of calcium, lack of water and high seasonal input of nitrates from nesting seabirds, are possible influences on the biosynthesis of oxalates which are found in high concentrations in P. grandis. The aims of this study were twofold. The first aim was to investigate the influence of environmental factors, such as pH and nitrate concentration, on the biosynthesis of oxalate in Pisonia grandis leaf. The second aim was to purify and characterise the enzyme glycolic acid oxidase (E.C. 1.1 .3.1) (GAO), an enzyme involved in the biosynthesis of oxalate. P. grandis was grown in a hydroponic system which was constructed in a glasshouse. The effect of variations in light intensity, pH and the concentrations of nitrate, calcium and sodium chloride in the nutrient sol ution on leaf oxalate concentration was investigated. A small diurnal variation in leaf soluble oxalate concentration was observed (O.5mmol/g DW at 07:00hrs and 0.62 mmol/g/DW at 19:00hrs) which was consistent with the notion that an intermediate in the formation of oxalate was derived from photosynthesis or photorespiration. In all other experimental regimes leaf oxalate concentrations were shown to vary in response to changes in the excess cation content of the leaves. These hydroponic studies suggested that changes in leaf excess cation concentration, and associated changes in intracellular pH, stimulate changes in the biosynthesis of oxalate in Pisonia. GAO was isolated to purity from the leaves of P. grandis and was found to be similar to GAO from other sources. The holoenzyme was found to be a hexamer (M.Wt. 250000) of subunits with molecular weight 44000. In vitro, with glycolate substrate, GAO had a pH optima of 7.0 and a Km of O.4mM. The enzyme exhibited partial competitive inhibition by oxalate (Ki 2.75mM) and was irreversibly inhibited by phydroxymercuribenzoate. Phenyllactate and iodoacetate did not inhibitthe enzyme preparation. These studies were repeated for glyoxylate and lactate as the enzyme substrates. The pH optima determined for GAO in vitro with glycolate and glyoxylate substrates, in conjunction with the pH optima reported for aminotransferase enzymes, supports the notion that leaf oxalate participates in a biochemical pH stat within the leaves of Pisonia grandis.