Cloning and characterization of a cDNA encoding a maize seedling phytase

Abstract

During germination, maize seedlings express a phytase able to hydrolyse the large amount of phytin stored in the dry seed. Previous studies allowed purification and characterization of this enzyme as a homodimer of 38 kDa subunits [Laboure, Gagnon and Lescure, Biochem. J. (1993) 295, 413-419]. In the present work, an antibody against the purified maize phytase has been used to screen a maize seedling cDNA expression library. Several positive clones containing an insert of about 1400 bp were isolated. The nucleotide sequence of the insert of one of these clones has been established. This cDNA, called phy S11, was 1335 bp long and contained an open reading frame of 387 amino acids. The sequence of N-terminal residues (23 amino acids) of the purified phytase has been established. These residues are found at positions 19-41 of the amino acid sequence encoded by phy S11. This confirms that this cDNA codes for the maize phytase. The deduced amino acid sequence appears to be very different from those of published Aspergillus niger phytases; however, an homologous region of 33 amino acids was detected. This region of the fungal sequence contains the RHGxRxP consensus motif found in various high molecular mass acid phosphatases and believed to be the acceptor site for phosphate. Expression of the phy S11 cDNA in Escherichia coli allowed the production of the phytase subunit and its assembly to give a protein of the same size as the native phytase. The time course of phy S11 mRNA accumulation during germination showed that no transcript was present in dry seeds. The mRNA accumulated during the first day of germination, to reach a maximum after 2 days (radicle protrusion), and then decreased in young seedlings. Genomic Southern blot analyses suggest the existence of at least two genes and genetic mapping reveals two loci separated by 1 cM on chromosome 3 of maize. The cloning of this first cDNA coding for a plant phytase, will allow the isolation of the corresponding genes and the study of their regulation during germination.

Transgenic expression of a novel M. truncatula phytase gene results in improved acquisition of organic phosphorus by Arabidopsis

“…The cloning and characterization of plant phytase genes have been reported for maize (Maugenest et al 1997(Maugenest et al , 1999 and soybean (Hegeman and Grabau 2001). However, phytases encoded by the soybean or the maize genes do not appear to be secreted or involved in P acquisition of roots from external phytate (Hu¨bel and Beck 1996;Maugenest et al 1999;Hegeman and Grabau 2001).…”

The genetics of phytate and phosphate accumulation in seeds and leaves of Arabidopsis thaliana, using natural variation

“…There probably are multiple phytases in seed and pollen, with different patterns of expression and regulation, the molecular biology and genetics of which is relatively undeveloped (Loewus and Murthy 2000). There is significant progress in the study of microbial phytase genes (Maugenest et al 1997).…”

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

“…A HAP phytase was identified and characterized in lily (Lilium longiflorum) pollen (Mehta et al, 2006). Maize has been reported to possess two genomic HAP-encoding sequences (PHYTI and PHYT2;Maugenest et al, 1997Maugenest et al, , 1999. Both maize genes were expressed preferentially in the rhizodermis, endodermis, and pericycle layers of the adult root.…”