title: Phosphoproteomic profiling of wheat callus labelled in vivo.
abstract: Callus is an important intermediate tissue for the propagation of plants through tissue culture, as it can form new shoots, and hence plants, through somatic embryogenesis, a process whose underlying biochemical mechanisms are poorly understood. The involvement of kinases suggests that signalling through phosphorylation cascades may play an important role. In this report methods for labelling wheat (Triticum aestivum L.) callus phosphoproteins in vivo through the uptake of [(32)P]orthophosphate and subsequently revealing the phosphoproteome of this tissue by two-dimensional electrophoresis and autoradiography are described. Labelled proteins from 2D gels were identified by tandem mass spectrometry analysis using the 'Mascot' search engine, and by de novo sequencing in combination with BLAST and MS-BLAST, using a cross-species protein identification approach. Eight putative phosphoproteins from 10 spots were identified, confirming the potential utility of this method; three of these have not previously shown to be phosphorylated. Furthermore, the phosphorylation site for one of the proteins was identified. Protein synthesis functions were tentatively assigned to five of the phosphoproteins, one reactive oxygen species (ROS)-detoxifying enzyme ascorbate peroxidase, and two could not be identified reliably from current database matches.
authors.affiliation: Agriculture and Agrifood Canada, Cereal Research Centre, 195 Dafoe Road, Winnipeg, Man., Canada R3T 2M9. Electronic address: crampitsch@agr.gc.ca.
authors.forename: Christof
authors.initials: C
authors.lastname: Rampitsch
authors.affiliation: Agriculture and Agrifood Canada, Cereal Research Centre, 195 Dafoe Road, Winnipeg, Man., Canada R3T 2M9.
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Bykova
authors.affiliation: Agriculture and Agrifood Canada, Cereal Research Centre, 195 Dafoe Road, Winnipeg, Man., Canada R3T 2M9.
authors.forename: Wayne
authors.initials: W
authors.lastname: Mauthe
authors.affiliation: Agriculture and Agrifood Canada, Cereal Research Centre, 195 Dafoe Road, Winnipeg, Man., Canada R3T 2M9.
authors.forename: Nandadeva
authors.initials: N
authors.lastname: Yakandawala
authors.affiliation: Agriculture and Agrifood Canada, Cereal Research Centre, 195 Dafoe Road, Winnipeg, Man., Canada R3T 2M9.
authors.forename: Mark
authors.initials: M
authors.lastname: Jordan
keywords: Callus; Mass spectrometry; Phosphoproteomics; Somatic embryogenesis; Wali7