Isoprenoid and aromatic cytokinins occur in poplar as free compounds and constituents of tRNA, poplar isopentenyltransferases are involved in the production of isoprenoid cytokinins, while biosynthesis of their aromatic counterparts remains unsolved. Cytokinins are phytohormones with a fundamental role in the regulation of plant growth and development. They occur naturally either as isoprenoid or aromatic derivatives, but the latter are quite rare and less studied. Here, the spatial expression of all nine isopentenyl transferase genes of Populus × canadensis cv. Robusta (PcIPTs) as analyzed by RT-qPCR revealed a tissue preference and strong differences in expression levels for the different adenylate and tRNA PcIPTs. Together with their phylogeny, this result suggests a functional diversification for the different PcIPT proteins. Additionally, the majority of PcIPT genes were cloned and expressed in Arabidopsis thaliana under an inducible promoter. The cytokinin levels measured in the Arabidopsis-overexpressing lines as well as their phenotype indicate that the studied adenylate and tRNA PcIPT proteins are functional in vivo and thus will contribute to the cytokinin pool in poplar. We screened the cytokinin content in leaves of 12 Populus species by ultra-high performance-tandem mass spectrometry (UHPLC-MS/MS) and discovered that the capacity to produce not only isoprenoid, but also aromatic cytokinins is widespread amongst the Populus accessions studied. Important for future studies is that the levels of aromatic cytokinins transiently increase after daybreak and are much higher in older plants.

• Klíčová slova
• Cytokinin, Expression, Isopentenyltransferase, Poplar, Topolin, tRNA,
• MeSH
• alkyltransferasy a aryltransferasy genetika   metabolismus   MeSH
• Arabidopsis genetika   metabolismus   MeSH
• cytokininy biosyntéza   MeSH
• fylogeneze MeSH
• geneticky modifikované rostliny MeSH
• listy rostlin genetika   metabolismus   MeSH
• Populus genetika   metabolismus   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenylate isopentenyltransferase MeSH Prohlížeč
• alkyltransferasy a aryltransferasy MeSH
• cytokininy MeSH
• regulátory růstu rostlin MeSH
• rostlinné proteiny MeSH

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.

• Klíčová slova
• Affinity purification, Chemical proteomics, Cytokinin, Molecular target identification, Plant proteomics, Wheat,
• MeSH
• benzylové sloučeniny metabolismus   MeSH
• chromatografie kapalinová MeSH
• cytokininy metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• jedlá semena metabolismus   MeSH
• proteom metabolismus   MeSH
• proteomika metody   MeSH
• pšenice metabolismus   MeSH
• puriny metabolismus   MeSH
• rostlinné proteiny metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• benzylaminopurine MeSH Prohlížeč
• benzylové sloučeniny MeSH
• cytokininy MeSH
• proteom MeSH
• puriny MeSH
• rostlinné proteiny MeSH