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

Abstract

The 67 kDa cytokinin-binding protein (CBP) has been isolated from old rosette leaves of 9-week-oldArabidopsis thaliana plants. The procedure of CBP isolation included the protein puri?cation by SephadexG-50, hydrophobic chromatography on phenyl-Sepharose and a?nity chromatography on zeatin riboside-Sepharose. Nuclear localization in leaves of 67 kDa CBP was demonstrated. The recognition of a naturalcytokinin trans-zeatin by 67 kDa CBP from Arabidopsis leaves was detected by a very speci?c and sensitiveassay based on trans-zeatin competition in ELISA with anti-idiotype antibodies (Aba-i) for complex formationwith CBP. Aba-i were raised against antibodies against zeatin. trans-Zeatin competed with Aba-i in adose-dependent manner, demonstrating a high a?nity for the protein. In combination with trans-zeatin,67 kDa protein activated RNA synthesis in vitro in the transcription elongation systems containingchromatin-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 dicotsful?ll similar functions. A comparison of functional activity of 67 kDa CBP from young, growing rosetteleaves of 3-week-old Arabidopsis plants, from mature leaves of 7-week-old plants and from senescent leavesof 9-week-old plants demonstrated age-dependent changes of the protein properties. The data are discussedin context of recent advances in the study of transcription regulation in eukaryotic cells.