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Wheat (Triticum aestivum L.) possesses preformed phytase activity in the grain that is essential to make phosphate available to cell metabolism and in food and feed (Brejnholt S. et al., 2011). Cereals contain the purple acid phosphatase type of phytases, PAPhy (Dionisio G. et al., 2011a). Mature wheat grain is dominated by TaPAPhy_a which, after chromatographic purification, has been characterized by extensive peptide and glycopeptide sequencing by mass spectrometry. Seven N-linked glycosylation sites were found. Three of these sites were dominated by variant forms of the XylMan3FucGlcNAc2, i.e. the HRP-type of glycan. Complex-type glycans with one or two additional GlcNAc were observed, however in trace amount only. The mature protein is ca. 500 residues in size and appears to be truncated at the N- and C-termini (Dionisio G. et al., 2011b). References: Brejnholt S., Dionisio G., Glitsoe V., Skov L. Brinch-Pedersen H. (2011). The degradation of phytate by microbial and wheat phytases is dependent on the phytate matrix and the phytase origin. J. Sci. Food Agri. (in press). Dionisio G., Madsen C.K., Holm P.B., Welinder K.G., Jørgensen M., Stoger E., Arcalis E., Brinch-Pedersen H. (2011a) Cloning and Characterization of Purple Acid Phosphatase Phytases from Wheat (Triticum aestivum L.), Barley (Hordeum vulgare L.), Maize (Zea maize L.) and Rice (Oryza sativa L.). Plant Physiol. [in press, Jan 10, Epub ahead of print] Dionisio G., Brinch-Pedersen H., Welinder K.G., Jørgensen M. (2011b). Different site-specific N-glycan types in wheat (Triticum aestivum L.) PAP phytase. Phytochemistry (in press). Wheat (Triticum aestivum L.) possesses preformed phytase activity in the grain that is essential to make phosphate available to cell metabolism and in food and feed (Brejnholt S. et al., 2011). Cereals contain the purple acid phosphatase type of phytases, PAPhy (Dionisio G. et al., 2011a). Mature wheat grain is dominated by TaPAPhy_a which, after chromatographic purification, has been characterized by extensive peptide and glycopeptide sequencing by mass spectrometry. Seven N-linked glycosylation sites were found. Three of these sites were dominated by variant forms of the XylMan3FucGlcNAc2, i.e. the HRP-type of glycan. Complex-type glycans with one or two additional GlcNAc were observed, however in trace amount only. The mature protein is ca. 500 residues in size and appears to be truncated at the N- and C-termini (Dionisio G. et al., 2011b). References: Brejnholt S., Dionisio G., Glitsoe V., Skov L. Brinch-Pedersen H. (2011). The degradation of phytate by microbial and wheat phytases is dependent on the phytate matrix and the phytase origin. J. Sci. Food Agri. (in press). Dionisio G., Madsen C.K., Holm P.B., Welinder K.G., Jørgensen M., Stoger E., Arcalis E., Brinch-Pedersen H. (2011a) Cloning and Characterization of Purple Acid Phosphatase Phytases from Wheat (Triticum aestivum L.), Barley (Hordeum vulgare L.), Maize (Zea maize L.) and Rice (Oryza sativa L.). Plant Physiol. [in press, Jan 10, Epub ahead of print] Dionisio G., Brinch-Pedersen H., Welinder K.G., Jørgensen M. (2011b). Different site-specific N-glycan types in wheat (Triticum aestivum L.) PAP phytase. Phytochemistry (in press).

Barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) possess a significant phytase activity in the mature grains. Maize (Zea mays L.) and rice (Oryza sativa L.) possess little or virtually no pre-formed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here it’s demonstrated that wheat, barley, maize and rice all possess purple acid phosphatase genes which expressed in Pichia pastoris gives 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 and HvPAPhy_b1, HvPAPhy_b2; wheat: TaPAPhy_a1, TaPAPhy_a2 and TaPAPhy_b1, 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. In spite 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 (~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 are the origin of high phytase activity in mature grains. 77 Barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) possess a significant phytase activity in the mature grains. Maize (Zea mays L.) and rice (Oryza sativa L.) possess little or virtually no pre-formed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here it’s demonstrated that wheat, barley, maize and rice all possess purple acid phosphatase genes which expressed in Pichia pastoris gives 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 and HvPAPhy_b1, HvPAPhy_b2; wheat: TaPAPhy_a1, TaPAPhy_a2 and TaPAPhy_b1, 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. In spite 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 (~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 are the origin of high phytase activity in mature grains. 77

Potato tuber storage proteins were obtained from vacuoles isolated from field-grown starch potato tubers cv. Kuras. Vacuole sap proteins fractionated by gel filtration were studied by mass spectrometric analyses of trypsin and chymotrypsin digestions. The tuber vacuole appears to be a typical protein storage vacuole absent of proteolytic and glycolytic enzymes. The major soluble storage proteins included 28 Kunitz protease inhibitors, nine protease inhibitors 1, eight protease inhibitors 2, two carboxypeptidase inhibitors, eight patatins and five lipoxygenases (lox), which all showed cultivar-specific sequence variations. These proteins, except for lox, have typical endoplasmic reticulum (ER) signal peptides and putative vacuolar sorting determinants of either the sequence or structure specific type or the C-terminal type, or both. Unexpectedly, sap protein variants imported via the ER showed multiple molecular forms because of extensive and unspecific proteolytic cleavage of exposed N- and C-terminal propeptides and surface loops, in spite of the abundance of protease inhibitors. Some propeptides are potential novel vacuolar targeting peptides. In the insoluble vacuole fraction two variants of phytepsin (aspartate protease) were identified. These are most probably the processing enzymes of potato tuber vacuolar proteins. Database Proteome data have been submitted to the PRIDE database under accession number 17707.

Udgivelsesdato: FEB

Udgivelsesdato: OCT