title: Cloning and characterization of purple acid phosphatase phytases from wheat, barley, maize, and rice.
abstract: Barley (Hordeum vulgare) and wheat (Triticum aestivum) possess significant phytase activity in the mature grains. Maize (Zea mays) and rice (Oryza sativa) possess little or virtually no preformed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here, it is demonstrated that wheat, barley, maize, and rice all possess purple acid phosphatase (PAP) genes that, expressed in Pichia pastoris, give fully functional phytases (PAPhys) with very similar enzyme kinetics. Preformed wheat PAPhy was localized to the protein crystalloid of the aleurone vacuole. Phylogenetic analyses indicated that PAPhys possess four conserved domains unique to the PAPhys. In barley and wheat, the PAPhy genes can be grouped as PAPhy_a or PAPhy_b isogenes (barley, HvPAPhy_a, HvPAPhy_b1, and HvPAPhy_b2; wheat, TaPAPhy_a1, TaPAPhy_a2, TaPAPhy_b1, and TaPAPhy_b2). In rice and maize, only the b type (OsPAPhy_b and ZmPAPhy_b, respectively) were identified. HvPAPhy_a and HvPAPhy_b1/b2 share 86% and TaPAPhya1/a2 and TaPAPhyb1/b2 share up to 90% (TaPAPhy_a2 and TaPAPhy_b2) identical amino acid sequences. despite of this, PAPhy_a and PAPhy_b isogenes are differentially expressed during grain development and germination. In wheat, it was demonstrated that a and b isogene expression is driven by different promoters (approximately 31% identity). TaPAPhy_a/b promoter reporter gene expression in transgenic grains and peptide mapping of TaPAPhy purified from wheat bran and germinating grains confirmed that the PAPhy_a isogene set present in wheat/barley but not in rice/maize is the origin of high phytase activity in mature grains.
authors.affiliation: Faculty of Agricultural Sciences, Department of Genetics and Biotechnology, Research Centre Flakkebjerg, Aarhus University, DK-4200 Slagelse, Denmark.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Dionisio
authors.forename: Claus K
authors.initials: CK
authors.lastname: Madsen
authors.forename: Preben B
authors.initials: PB
authors.lastname: Holm
authors.forename: Karen G
authors.initials: KG
authors.lastname: Welinder
authors.forename: Malene
authors.initials: M
authors.lastname: Jørgensen
authors.forename: Eva
authors.initials: E
authors.lastname: Stoger
authors.forename: Elsa
authors.initials: E
authors.lastname: Arcalis
authors.forename: Henrik
authors.initials: H
authors.lastname: Brinch-Pedersen
mesh_terms: D010832:6-Phytase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D018525:Germination; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology / Q000235:genetics; D010802:Phylogeny; D010944:Plants / Q000201:enzymology* / Q000235:genetics*; D012639:Seeds / Q000166:cytology / Q000201:enzymology / Q000648:ultrastructure; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D014908:Triticum / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000648:ultrastructure; D003313:Zea mays / Q000201:enzymology / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D006023:Glycoproteins; C091137:purple acid phosphatase; D000135:Acid Phosphatase; D010832:6-Phytase