, 251:1 | Cite as

Characterization of five CHASE-containing histidine kinase receptors from Populus × canadensis cv. Robusta sensing isoprenoid and aromatic cytokinins

  • Pavel Jaworek
  • Petr Tarkowski
  • Tomáš Hluska
  • Štěpán Kouřil
  • Ondřej Vrobel
  • Jaroslav Nisler
  • David KopečnýEmail author
Original Article


Main conclusion

Five poplar CHASE-containing histidine kinase receptors bind cytokinins and display kinase activities. Both endogenous isoprenoid and aromatic cytokinins bind to the receptors in live cell assays.


Cytokinins are phytohormones that play key roles in various developmental processes in plants. The poplar species Populus × canadensis, cv. Robusta, is the first organism found to contain aromatic cytokinins. Here, we report the functional characterization of five CHASE-containing histidine kinases from P. × canadensis: PcHK2, PcHK3a, PcHK3b, PcHK4a and PcHK4b. A qPCR analysis revealed high transcript levels of all PcHKs other than PcHK4b across multiple poplar organs. The ligand specificity was determined using a live cell Escherichia coli assay and we provide evidence based on UHPLC-MS/MS data that ribosides can be true ligands. PcHK2 exhibited higher sensitivity to iP-type cytokinins than the other receptors, while PcHK3a and PcHK3b bound these cytokinins much more weakly, because they possess two isoleucine residues that clash with the cytokinin base and destabilize its binding. All receptors display kinase activity but their activation ratios in the presence/absence of cytokinin differ significantly. PcHK4a displays over 400-fold higher kinase activity in the presence of cytokinin, suggesting involvement in strong responses to changes in cytokinin levels. trans-Zeatin was both the most abundant cytokinin in poplar and that with the highest variation in abundance, which is consistent with its strong binding to all five HKs and activation of cytokinin signaling via A-type response regulators. The aromatic cytokinins’ biological significance remains unclear, their levels vary diurnally, seasonally, and annually. PcHK3 and PcHK4 display the strongest binding at pH 7.5 and 5.5, respectively, in line with their putative membrane localization in the endoplasmic reticulum and plasma membrane.


Aromatic cytokinin Histidine kinase Hormone Isoprenoid cytokinin Poplar Topolin 







N6-(cis-4-hydroxy-3-methyl-2-buten-1-yl)adenine, i.e. cis-zeatin










Histidine kinase




Response regulators


N-(1,2,3-thidiazol-5-yl)-N´-phenylurea, i.e. thidiazuron




Zeatin riboside



This work was supported by the grant 15-16888S from the Czech Science Foundation, institutional support MZE-RO0418 and IGA_PrF_2018_033 (Palacký University, Olomouc, CZ). DK and PT were supported also from ERDF project “Plants as a tool for sustainable global development” (No. CZ.02.1.01/0.0/0.0/16_019/0000827). We acknowledge Zuzana Pěkná and Lukáš Spíchal for their help with the live cell competitive binding assay (Palacký University, Olomouc, CZ) and prof. Peter Hedden for proofreading the manuscript. Poplar calli were derived from dormant leaf buds provided by Dr. Jana Malá from Forestry and Game Management Research Institute (Jíloviště, CZ). The pINIIIΔEH vector and E. coli strain KMI001 were kindly provided by Dr. Zalabák (Palacký University, Olomouc, CZ). The authors are grateful to Sees-editing Ltd. (UK) for editing the manuscript. Populus deltoides sequence data were produced by the US Department of Energy Joint Genome Institute

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Supplementary material

425_2019_3297_MOESM1_ESM.docx (111 kb)
Supplementary material 1 (DOCX 110 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Pavel Jaworek
    • 1
    • 2
  • Petr Tarkowski
    • 1
    • 3
  • Tomáš Hluska
    • 3
  • Štěpán Kouřil
    • 1
  • Ondřej Vrobel
    • 1
    • 3
  • Jaroslav Nisler
    • 4
  • David Kopečný
    • 2
    Email author
  1. 1.Department of Phytochemistry, Faculty of Science, Centre of the Region Haná for Biotechnological and Agricultural ResearchPalacký University OlomoucOlomoucCzech Republic
  2. 2.Department of Protein Biochemistry and Proteomics, Faculty of Science, Centre of the Region Haná for Biotechnological and Agricultural ResearchPalacký University OlomoucOlomoucCzech Republic
  3. 3.Department of Genetic Resources for Vegetables, Medicinal and Special Plants, Centre of the Region Haná for Biotechnological and Agricultural ResearchCrop Research InstituteOlomoucCzech Republic
  4. 4.Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental BotanyAS CR & Palacký UniversityOlomoucCzech Republic

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