Wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) multiple inositol polyphosphate phosphatases (MINPPs) are phytases expressed during grain filling and germination.
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 degrees C, respectively, and the K(m) values for phytate were 246 and 334 microm 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.
Dionisio
Giuseppe
G
University of Aarhus, Faculty of Agricultural Sciences, Institute of Genetics and Biotechnology, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark.
Holm
Preben B
PB
Brinch-Pedersen
Henrik
H
D010832:6-Phytase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D018506:Gene Expression Regulation, Plant; D018525:Germination / Q000235:genetics*; D001467:Hordeum / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000201:enzymology / Q000502:physiology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014908:Triticum / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000502:physiology
D018076:DNA, Complementary; D010744:Phosphoric Monoester Hydrolases; D010832:6-Phytase; C064119:multiple inositol-polyphosphate phosphatase