Cloning and Characterization of Purple Acid Phosphatase Phytases from Wheat, Barley, Maize, and Rice

Giuseppe Dionisio,Karen G Welinder,Claus K Madsen,Elsa Arcalis,Preben B Holm,Eva Stoger,Henrik Brinch-Pedersen,Malene Jørgensen

doi:10.1104/pp.110.164756

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.

Highlights

Barley (Hordeum vulgare) and wheat (Triticum aestivum) possess significant phytase activity in the mature grains
Wheat and barley, where preformed phytase activity is present in the mature grain, as well as maize and rice, with little or no preformed phytase activity in the mature grain, possess PAPhy genes encoding fully functional phytases with very similar enzyme kinetics
The PAPhy clade shares four consensus motifs that can be used for initial PAPhy identification followed by assaying after heterologous expression in P. pastoris

Summary

Introduction

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. 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. Increased phytase activities in transgenic soybean (Glycine max) and canola (Brassica napus) seeds reduced phosphorus secretion by 50% and 48% when fed to broilers and piglets, respectively (Denbow et al, 1998; Zhang et al, 2000) Despite of their importance for basic plant processes and their significance for human and livestock nutrition, little is known about the molecular mechanisms regulating phytase formation during grain development and germination. Phytase activities have been detected in two Arabidopsis (Arabidopsis thaliana) proteins termed AtPAP15 and AtPAP23 (Zhu et al, 2005; Kuang et al, 2009)

Methods

Results

Discussion

Conclusion