| Current genetic evidence indicates that protein phosphatase and protein kinase genes are associated with seed dormancy and germination. Some of these genes are regulated by the phytohormone, abscisic acid (ABA). Here, protein phosphorylation in dormant and afterripened (germinable) wheat seeds was characterized at the biochemical level during the initial hours of seed imbibition. During the first minutes of hydration, phosphorylation of four proteins (68, 54, 51 and 42 kDa) was detected in both germinating (afterripened) and dormant seeds. Net protein phosphorylation of these four proteins remained high in germinating seeds, but decreased in dormant seeds by 30 minutes. Protein phosphorylation continued to decrease in imbibed, dormant seeds that remained growth-arrested. Drying of dormant seeds restored protein phosphorylation to initial levels. Application of the protein phosphatase inhibitor, okadaic acid, partially overcame seed dormancy, suggesting that decreased protein phosphorylation observed in dormant seeds was due in part to an okadaic acid-sensitive protein phosphatase.; The ABA-responsive protein kinase, PKABA1, originally cloned from dormant wheat embryos, was biochemically characterized. PKABA1 protein was expressed in Drosophila melanogaster. PKABA1 kinase activity was measured with a putative substrate, TaABF (Triticum aestivum ABA-response element binding factor), a basic leucine zipper (bZIP) transcription factor. Potential phosphorylation sites were identified in the TaABF sequence, and synthetic peptides were produced corresponding to those regions. Expressed PKABA1 exhibited kinase activity and phosphorylated synthetic peptides corresponding to sequences within the basic, central and amino-terminal regions of TaABF. PKABA1 kinase activity was not significantly affected by the presence or absence of calcium.; In summary, significant differences in net protein phosphorylation were demonstrated in imbibed dormant and germinating seeds. Potential intermediates in signaling processes involving reversible protein phosphorylation that lead to growth arrest of dormant seeds were also identified. These were an okadaic acid-sensitive protein phosphatase activity, and the ABA-responsive PKABA1 protein kinase and it putative substrate, TaABF, a bZIP transcription factor. |
