Chemical proteomic analysis of 6-benzylaminopurine molecular partners in wheat grains.

KEY MESSAGE: An affinity-based chemical proteomic technique enabled direct identification of BAP-interacting proteins in wheat, including the well-known cytokinin-binder, cytokinin-binding protein 1. In this work, we show the development of a chemical proteomic technique for the identification of proteins binding to natural aromatic cytokinins (CKs). 6-benzylaminopurine (BAP) and documented CK-binder, wheat germ-allocated cytokinin-binding protein 1 (CBP-1), were suggested as an ideal proof-of concept affinity pair. Therefore, wheat grains were chosen as a model plant material. The BAP affinity beads were prepared by the immobilization of synthesized BAP-derived ligand to a commercial, pre-activated resin and used to isolate target proteins. The proteomic analysis of complex plant extracts is often complicated by the presence of highly abundant background proteins; in this case, the omnipresent alpha-amylase inhibitors (AAIs). To cope with this problem, we included SDS-PAGE, in-gel trypsin digestion and fraction pooling prior to shotgun analysis, which brought about an obvious drop in the signals belonging to the obstructing proteins. This was accompanied by a sharp increase in the number of identified BAP targets in comparison to a conventional in-solution digestion approach. To distinguish specific CK-binding proteins from those having a general affinity for nucleotide-like compounds, competitive pull-downs with natural nucleotides and free BAP were included in every affinity experiment. By this approach, we were able to identify a group of BAP-interacting proteins, which were subsequently found to be related to biological processes affected by CKs. Moreover, the selected affinity enrichment strategy was verified by the detection of the aforementioned CK-interacting protein, CBP-1. We propose that the developed method represents a promising tool for appealing research of as yet unknown CK molecular partners in plants.

(E)-9-(But-2-en-1-yl)-6-chloro-9H-purine.

The asymmetric unit of the title compound, C9H9ClN4, contains two mol-ecules. In the crystal, the mol-ecules are ordered in a chain-like fashion along the a axis, and form layers offset relative to the C plane by approximately 30°. This ordering does not, however, appear to be directed by classical hydrogen bonding.The allylic side chains of both independent mol-ecules are disordered, with occupancies of 0.870 (4) and 0.934 (3) for the major components. The disorder components represent two possible spatial orientations of the atoms around the C=C double bond.