Co-localization of cytokinins with proteins related to cell proliferation in developing somatic embryos ofDactylis glomerataL.

Abstract

The present report provides evidence for co-localization of cytokinins with cell proliferation-associated nuclear proteins. Somatic embryos of Dactylis glomerata in two stages of development are used as a model system comprising both proliferating and initially differentiated cells. Cytokinins are localized using antibodies with marked specificity against isopentenyladenine/adenosine (2iP/2iPA) or zeatin/ riboside (Z/ZR). The proliferation-associated nuclear antigen, mitotin, is analysed using a specific monoclonal antibody. The nuclear protein BM28, required for the onset of DNA replication and for cell division, is identified by an affinity-purified polyclonal antibody. Using double immunofiuorescence labelling with the antibodies against cytokinins and against each of the nuclear proteins, immunoreaction is observed generally in the same nuclei of almost all cells in globular embryos and in the nuclei of cells in meristematic areas of the more developed embryos. Only small numbers of individual nuclei positive for both type of antibodies were found in the surrounding vacuolated parenchymatous cells. The occurrence of plant antigens homologous to BM28 and mitotin is confirmed by immunoblotting assay. In SDS-PAGE blots the anti-BM28 antibody reacts with a protein of 58 kDa. The anti-mitotin antibody recognizes several (160, 140, 125, 93, and 80 kDa) polypeptides. The data showing nuclear co-localization of cytokinins and proteins with a suggested role in the onset of DNA synthesis and in cell division provide a new base for further study on the mode of action of cytokinins in cell cycle regulation.