Blue-light-induced phosphorylation of a plasma-membrane protein from phototropically sensitive tips of maize coleoptiles

Abstract

Tips of maize coleoptiles, which function as esential light sensors for the phototropic growth reaction, exhibit a rapid blue-light-induced phosphorylation of a plasma-membrane-associated 100-kDa protein. Characteristics of this reaction are as follows: (i) The functional unit involved in the light-dependent phosphorylation consists of a photoreceptor, a protein kinase and the 100-kDa protein. This complex is only localized in the plasma membrane of tips but not in other parts of the seedling, (ii) The photoreceptor is a cryptochrome-like compound, (iii) The pH optimum of the light-dependent phosphorylation on isolated plasma membranes is around pH 7.8 whereas the light-independent phosphorylation of other membrane proteins occurs at lower values (pH 6.2). (iv) The light-induced in-vitro phosphorylation of the 100-kDa protein is strongly inhibited by the protein-kinase inhibitor staurosporine (IC₅₀=4 nM). (v) The ³²P-moiety of a ³²P-[100 kDa]-protein complex generated after a light pulse with the aid of a membrane-associated protein kinase in the presence of [γ-³²P]ATP cannot be removed by a 100-fold higher level of (unlabelled) ATP. This fact indicates that protein and phosphate are covalently connected and that the complex is not a short-lived intermediate. (vi) The 100-kDa protein is not identical to the plasma-membrane H⁺-ATPase, as shown by immunostaining on Western blots. (vii) Irradiation-dependent in vivo phosphorylation of the 100-kDa protein in tips is already saturated by a light pulse of 5 s. In contrast, the de-phosphorylation of the protein in the dark is a slow reaction lasting about 30 min. It is suggested that the blue-light-triggered phosphorylated status of the 100-kDa protein is an early step in phototropism of the coleoptile, affecting the transport of auxin primarily in the irradiated flank.