Cytokinin-binding protein from Arabidopsis thaliana leaves participating in transcription regulation

The 67 kDa cytokinin-binding protein (CBP) has been isolated from old rosette leaves of 9-week-old Arabidopsis thaliana plants. The procedure of CBP isolation included the protein purification by Sephadex G-50, hydrophobic chromatography on phenyl-Sepharose and affinity chromatography on zeatin riboside-Sepharose. Nuclear localization in leaves of 67 kDa CBP was demonstrated. The recognition of a natural cytokinin trans-zeatin by 67 kDa CBP from Arabidopsis leaves was detected by a very specific and sensitive assay based on trans-zeatin competition in ELISA with anti-idiotype antibodies (Ab(a-i)) for complex formation with CBP. Ab(a-i) were raised against antibodies against zeatin. trans-Zeatin competed with Ab(a-i) in a dose-dependent manner, demonstrating a high affinity for the protein. In combination with trans-zeatin, 67 kDa protein activated RNA synthesis in vitro in the transcription elongation systems containing chromatin-bound RNA polymerase I from barley leaves (heterologous system) or from Arabidopsis leaves (homologous system). These data permit a suggestion that the 67 kDa CBPs from monocots and dicots fulfill similar functions. A comparison of functional activity of 67 kDa CBP from young, growing rosette leaves of 3-week-old Arabidopsis plants, from mature leaves of 7-week-old plants and from senescent leaves of 9-week-old plants demonstrated age-dependent changes of the protein properties. The data are discussed in context of recent advances in the study of transcription regulation in eukaryotic cells.