MAIN CONCLUSION: 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.

Department of Biochemistry Faculty of Science Palacký University Olomouc Šlechtitelů 27 78371 Olomouc Czech Republic

Department of Genetic Resources for Vegetables Medicinal and Special Plants Centre of the Region Hana for Biotechnological and Agricultural Research Crop Research Institute Šlechtitelů 29 78371 Olomouc Czech Republic

Department of Molecular Biology Faculty of Science Centre of the Region Hana for Biotechnological and Agricultural Research Palacký University Olomouc Šlechtitelů 27 78371 Olomouc Czech Republic

Department of Phytochemistry Faculty of Science Centre of the Region Hana for Biotechnological and Agricultural Research Palacký University Olomouc Šlechtitelů 27 78371 Olomouc Czech Republic

Department of Phytochemistry Faculty of Science Centre of the Region Hana for Biotechnological and Agricultural Research Palacký University Olomouc Šlechtitelů 27 78371 Olomouc Czech Republic Department of Genetic Resources for Vegetables Medicinal and Special Plants Centre of the Region Hana for Biotechnological and Agricultural Research Crop Research Institute Šlechtitelů 29 78371 Olomouc Czech Republic

Department of Phytochemistry Faculty of Science Centre of the Region Hana for Biotechnological and Agricultural Research Palacký University Olomouc Šlechtitelů 27 78371 Olomouc Czech Republic Department of Protein Biochemistry and Proteomics Faculty of Science Centre of the Region Hana for Biotechnological and Agricultural Research Palacký University Olomouc Šlechtitelů 27 78371 Olomouc Czech Republic

Department of Protein Biochemistry and Proteomics Faculty of Science Centre of the Region Hana for Biotechnological and Agricultural Research Palacký University Olomouc Šlechtitelů 27 78371 Olomouc Czech Republic

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