Plant Biotechnology Journal (2007) 5, pp. 325–338 doi: 10.1111/j.1467-7652.2007.00244.x © 2007 Blackwell Publishing Ltd 325 Blackwell Publishing, Ltd. Oxford, UK PBI Plant Biotechnology Journal 1467-7644 © 2007 Blackwell Publishing Ltd ? 2007 5 ? Original Article Wheat and barley MINPPs Giuseppe Dionisioet al. Wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) multiple inositol polyphosphate phosphatases (MINPPs) are phytases expressed during grain filling and germination † Giuseppe Dionisio, Preben B. Holm and Henrik Brinch-Pedersen* University of Aarhus, Faculty of Agricultural Sciences, Institute of Genetics and Biotechnology, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark Summary At present, little is known about the phytases of plant seeds in spite of the fact that this group of enzymes is the primary determinant for the utilization of the major phosphate storage compound in seeds, phytic acid. We report the cloning and characterization of complementary DNAs (cDNAs) encoding one of the groups of enzymes with phytase activity, the multiple inositol phosphate phosphatases (MINPPs). Four wheat cDNAs (TaPhyIIa1, TaPhyIIa2, TaPhyIIb and TaPhyIIc) and three barley cDNAs (HvPhyIIa1, HvPhyIIa2 and HvPhyIIb) were isolated. The open reading frames ranged from 1548 to 1554 bp and the level of homology between the barley and wheat proteins ranged from 90.5% to 91.9%. All cDNAs contained an N-terminal signal peptide encoding sequence, and a KDEL-like sequence, KTEL, was present at the C-terminal, indicating that the enzyme was targeted to and retained within the endoplasmic reticulum. Expression of TaPhyIIa2 and HvPhyIIb in Escherichia coli revealed that the MINPPs possessed a significant phytase activity with narrow substrate specificity for phytate. The pH and temperature optima for both enzymes were pH 4.5 and 65 °C, respectively, and the K m values for phytate were 246 and 334 μ M for the wheat and barley recombinant enzymes, respectively. The enzymes were inhibited by several metal ions, in particular copper and zinc. The cDNAs showed significantly different temporal and tissue-specific expression patterns during seed development and germination. With the exception of TaPhyIIb , the cDNAs were present during late seed development and germination. We conclude that MINPPs constitute a significant part of the endogenous phytase potential of the developing and germinating barley and wheat seeds. Received 15 September 2006; revised 18 December 2006; accepted 20 December 2006. *Correspondence (fax +4589993501; e-mail Henrik.BrinchPedersen@agrsci.dk) †G EN B ANK accession numbers: TaPhyIIa1, DQ995971; TaPhyIIa2, DQ995972; TaPhyIIb, DQ995973; TaPhyIIc, DQ995974; HvPhyIIa1, DQ995968; HvPhyIIa2, DQ995969; HvPhyIIb, DQ995970. Keywords: cereals, multiple inositol polyphosphate phosphatases (MINPPs), phosphate, phytase, phytate. Introduction Phytases (myo-inositol hexakisphosphate phosphohydrolases) (EC 3.1.3.26 and EC 3.1.3.8) are phosphatases that initiate the degradation of phytic acid (phytate, InsP 6 , myo- inositolphosphate 1,2,3,4,5,6-hexakisphosphate) by the removal of one or more phosphate groups. Thereafter, less specific phosphatases can take over and remove the remain- ing phosphate groups. Phytases are therefore important for the provision of phosphate, inositol phosphates and inositol that are required for a large number of activities, including signal transduction, in the cell (Brinch-Pedersen et al ., 2002). In plants, phytases are particularly important as they are the primary determinants for the mobilization of the phosphate reserves in the seed, with phytic acid typically accounting for 80% of these reserves. Monogastric animals have little or no phytase activity in their digestive tract and, in order to facilitate the utilization of the phosphate reserves in phytic acid and reduce the level of phosphate excretion from these animals, microbial phytases are today added to conventional seed-based feed in a number of regions with intense livestock production.