Cytokinin-binding protein (70 kDa): localization in tissues and cells of etiolated maize seedlings and its putative function

Abstract

The distribution pattern of a 70 kDa cytokinin-binding protein (CBP70) was studied in 4-d-old etiolated maize seedlings (Zea mays L., cv. Elbrus). CBP70 was detected in crude protein extracts of all root zones and shoot parts by western blotting and by the sandwich ELISA (enzyme-linked immunosorbent assay) technique, using a pair of monoclonal anti-CBP70 antibodies cross-reacting with non-overlapping protein epitopes. The highest amount of CBP70 was found in the root meristem, which corresponds to the concentration in the meristem of zeatin, its riboside, nucleotide, and 9N-glucoside. CBP70 accumulation was also detected in other zones of cell division: in the root cap, shoot apex, and vascular tissues, suggesting involvement of the protein in the processes related to cell proliferation. This suggestion was also supported by CBP70 distribution in the root meristem: mitotically inactive cells of the quiescent centre did not contain a detectable amount of the protein. Stem cells adjoining the quiescent centre contained less CBP70 than their daughter cells. Using monoclonal antibodies against CBP70 for immunocytochemistry, the presence of the protein in the cytoplasm and its accumulation in nuclei and especially in nucleoli was demonstrated; such a pattern was observed in all cell types of seedlings. The subcellular distribution pattern of CBP70 was analysed by immunogold electron microscopy of the meristem and leaf cells; CBP70 was localized in the cytoplasm and nucleoplasm, and its highest concentration was detected in nucleoli. CBP70 was not detected in the vacuole and cell wall. In the RNA polymerase I model system, purified CBP70 mediated a trans-zeatin-dependent activation of transcription in vitro, and anti-CBP70 monoclonal antibodies blocked this activation. Other natural and synthetic physiologically active cytokinins also activated transcript elongation in the model system in the presence of CBP70. Adenine and inactive analogues of cytokinins had no such effects. These data suggest that CBP70 is a transcript elongation factor or a modulator of elongation factor activity specifically mediating a cytokinin-dependent regulation of transcription.