Figure 10.

Effect of ABA on the in situ phosphorylation of PEPC during imbibition of wheat seeds. Eight deembryonated seeds were allowed to imbibe (72 h) in a buffer (100 mm Tris-Cl, pH 7.4) containing 200 μCi of [³²P]phosphate and 10 mm CaCl₂ in the absence (−) or presence (+) of 40 μm ABA at room temperature and in the dark. Proteins from the aleurone-endosperm tissue were extracted in a buffer (as described in “Materials and Methods”) containing 1 mm nonradioactive ATP, and PEPC was immunoprecipitated (immunoprecipitation as described by Osuna et al. [1996]) with polyclonal sorghum-leaf PEPC IgG (134 μg of protein) and analyzed by SDS-PAGE (8% acrylamide). The proteins were electrotransferred to nitrocellulose, autoradiographed, and probed with 300 μg of polyclonal sorghum leaf PEPC antibodies. An immunoblot from an aleurone-endosperm immunoprecipitated sample and the corresponding autoradiograph are shown.