Maize Root Phytase (Purification, Characterization, and Localization of Enzyme Activity and Its Putative Substrate)

Abstract

Three phytase (EC 3.1.3.26) isoforms from the roots of 8-d-old maize (Zea mays 1. var Consul) seedlings were separated from phosphatases and purified to near homogeneity. The molecular mass of the native protein was 71 kD, and the isoelectric points of the three isoforms were pH 5.0, 4.9, and 4.8. Each of the three isoforms consisted of two subunits with a molecular mass of 38 kD.The temperature and pH optima (40"C, pH 5.0) of these three isoforms, as well as the apparent Michaelis constants for sodium inositol hexakisphosphate (phytate) (43, 25, and 24 p~) as determined by the release of inorganic phosphate, were only slightly different. Phytate concentrations higher than 300 p~ were inhibitory to all three isoforms. In contrast, the dephosphorylation of 4-nitrophenyl phosphate was not inhibited by any substrate concentration, but the Michaelis constants for this substrate were considerably higher (137-157 PM). Hydrolysis of phytate by the phytase isoforms is a nonrandom reaction. D/L-hOSitOl-l,2,3,4,5-pentakisphosphate was identified as the first and D/L-inOSitOl-1,2,5,6-tetrakisphosphate as the second intermediate in phytate hydrolysis. Phytase activity was localized in root slices. Although phosphatase activity was present in the stele and the cortex of the primary root, phytase activity was confined to the endodermis.Phytate was identified as the putative native substrate in maize roots (45 pg P g-' dry matter). It was readily labeled upon supplying [32Plphosphate to the roots.Phytate (inositolhexakisphosphate, InsP,) is the major storage form of phosphate in seeds and pollen. Although synthesis of phytate during seed filling and breakdown in the early phase of germination have been widely investigated, the role of phytate and phytase (EC 3.1.3.26) in nonreproducing plant tissues has not yet been elucidated. Saxena (1963) suggested that root phytase could be operative in the utilization of soil-borne phytate, which usually represents the bulk of organic phosphate in agricultura1 soils (Anderson, 1980). Severa1 authors (Tarafdar and Claassen, 1988; Adams and Pate, 1992) whether in these cases phytate was hydrolyzed by an extracellular or an intracellular phytase (Islam et al., 1979;Kraus, 1989 (Campbell et al., 1991). These findings are in line with earlier results by Roberts and Loewus (1968) and Scheiner et al. (1978), who demonstrated the accumulation of phytate in duckweed. Thus, in roots and shoots phytases might be involved in the breakdown of phytate, as in seeds.In addition to a possible role as a P-storage compound, phytate may also be effective as a chelator in the detoxification of heavy metal ions in plant tissue. Van Steveninck et al. (1993,1994 showed that roots of various plant species were able to immobilize excessively supplied zinc in globular, P-rich intracellular inclusions deposited in the root elongation zone. These deposits were similar to globoids that were found by Lott and Ockenden (1986) in seeds.The present work is a continuation of our investigations on phytases in maize root...

An Arabidopsis Purple Acid Phosphatase with Phytase Activity Increases Foliar Ascorbate

“…Phytase is a special type of phosphohydrolase with the capability of initiating dephosphorylation of phytate. Because phytate is the predominant inositol phosphate present in seeds and roots, phytase might be responsible for the hydrolysis of phytate in germinating seeds and in endodermis cells of primary roots (Hubel and Beck, 1996). Although phytases in seeds, leaves, and roots share remarkable similarity, the physiological function of phytase in nonreproductive tissue is unclear (Laboure et al, 1993;Hubel and Beck, 1996).…”

Regulation of Cell-Specific Inositol Metabolism and Transport in Plant Salinity Tolerance

“…Synthesis of the raffinose series sugars provides another specific example for translocation of inositol to be used in distant tissues and by other biochemical pathways (Bachmann et al, 1994;Bachmann and Keller, 1995). Similar examples are the conjugation of inositol to auxin for long-distance transport from shoot to root (Cohen and Bandurski, 1982) and phytase metabolism (Hübel and Beck, 1996).…”

“…It is part of at least four cycles in which it is either cycled or shunted to end products with slow turnover. First, inositol phosphates are essential to signaling in almost all organisms; in plants, inositol hexaphosphate provides for phosphate storage (Hübel and Beck, 1996). Second, inositol-containing lipids are components of membranes (Mathews and Van Holde, 1990).…”

Metabolic Processes During Seed Germination

“…Phytin in germinating seeds is hydrolyzed by an acid phosphatase enzyme called phytase [85], with releasing of phosphate, cations, and inositol which are utilized by the seedlings. It was found little changes in extractible P i in hazel seeds during chilling accompanied with IP6 mobilization that might be suggested the rapid conversion of P i into organic form [86].…”