Proteomic Identification of a Candidate Sequence of Wheat Cytokinin-Binding Protein 1

Abstract

Our knowledge on the principle mechanisms of cytokinin action has been significantly deepened over the last years, but several weakly explored areas still remain on the map of cytokinin cellular physiology. Cytokinin-binding proteins could also be included in this pending field of cytokinin research. Probably, the best explored representative of this group is the wheat cytokinin-binding protein 1 (CBP-1). The role of this germ-allocated protein as a presumable regulator of free aromatic cytokinin levels during grain germination has been discussed intensively. To dig deeper into this interesting protein, this study was aimed at the identification of the CBP-1 amino acid sequence. A combination of in silico BLAST search, classical biochemical CBP-1 purification based on isoelectric point precipitation and ion-exchange chromatography, and proteomic analysis of the isolated protein by ultra-high resolution tandem mass spectrometry allowed us to uncover and validate two CBP-1 subunit candidate sequences with molecular masses of 56.2 and 55.0 kDa, respectively. Interestingly, we found the latter sequence alternated in two amino acids in the putative cytokinin-binding motive in comparison to the composition of this domain reported in the original studies. A BLAST search for the amino acid sequence of the binding region among plant proteomes revealed several highly related protein sequences, all originating from the Poaceae family. This piece of information could give support to the elucidation of the role of CBP-1 in physiological processes mediated by aromatic cytokinins.