title: In Vivo and in Vitro Phosphorylation of the Phosphoenolpyruvate Carboxylase from Wheat Seeds during Germination.
abstract: Phosphoenolpyruvate carboxylase (PEPC) activity was detected in the aleurone endosperm of wheat (Triticum aestivum cv Chinese Spring) seeds, and specific anti-Sorghum C4 PEPC polyclonal anti-bodies cross-reacted with 103- and 100-kD polypeptides present in dry seeds and seeds that had imbibed; in addition, a new, 108-kD polypeptide was detected 6 h after imbibition. The use of specific anti-phosphorylation-site immunoglobulin G (APS-IgG) identified the presence of a phosphorylation motif equivalent to that found in other plant PEPCs studied so far. The binding of this APS-IgG to the target protein promoted changes in the properties of seed PEPC similar to those produced by phosphorylation, as previously shown for the recombinant Sorghum leaf C4 PEPC. In desalted seed extracts, an endogenous PEPC kinase activity catalyzed a bona fide phosphorylation of the target protein, as deduced from the immunoinhibition of the in vitro phosphorylation reaction by the APS- IgG. In addition, the major, 103-kD PEPC polypeptide was also shown to be radiolabeled in situ 48 h after imbibition in [32P]orthophosphate. The ratio between optimal (pH 8) and suboptimal (pH 7.3 or 7.1) PEPC activity decreased during germination, thereby suggesting a change in catalytic rate related to an in vivo phosphorylation process. These collective data document that the components needed for the regulatory phosphorylation of PEPC are present and functional during germination of wheat seeds.
authors.affiliation: Departamento de Biologia Vegetal, Facultad de Biologia, Universidad de Sevilla, Avenida Reina Mercedes no. 6, 41012 Seville, Spain (L.O., C.E.).
authors.forename: L.
authors.initials: L
authors.lastname: Osuna
authors.forename: M. C.
authors.initials: MC
authors.lastname: Gonzalez
authors.forename: F. J.
authors.initials: FJ
authors.lastname: Cejudo
authors.forename: J.
authors.initials: J
authors.lastname: Vidal
authors.forename: C.
authors.initials: C
authors.lastname: Echevarria