Immunocytological Localization of a Wheat Embryo Cytokinin Binding Protein and its Homology with Proteins in other Cereals

Abstract

A protein (CBF-1) isolated from wheat embryos was shown several years ago to bind in a non-covalent manner and with relatively high affinity N⁶-substituted purines which have cytokinin activity in standard assays (7,8). Although the subunit composition of the protein and the kinetics of the binding have been the subject of some controversy in the literature (6,11,12), recent studies in our laboratory (1) demonstrate that the native protein consists of three identical subunits, each with a molecular mass near 54,000 and confirm our earlier finding (6) that one molecule of the cytokinin, N⁶-benzylaminopurine (bzlADE) is bound per molecule of the native protein. CBF-1 appears in the developing wheat embryo about two weeks after anthesis and increases rapidly during the next 14 day period reaching a level of approximately 47 μg per embryo, or about 9% of the soluble protein in mature wheat embryos (3). A number of cDNA clones have been isolated for the CBF-1 gene (2). An amino acid sequence for the binding domain and for much of the protein has been obtained and will be reported elsewhere. In this paper we present evidence which demonstrates that CBF-1 is highly tissue specific, being localized to the scutellum, the coleoptile and the coleorhiza and that within the cells of these tissues, the cytokinin-binding protein is packaged in membrane bound protein bodies. In addition we demonstrate that cytokinin-binding moieties which are immunologically related to CBF-1 occur in certain other cereals, confirming preliminary reports on this subject (9,10)