Characterization of five CHASE-containing histidine kinase receptors from Populus × canadensis cv. Robusta sensing isoprenoid and aromatic cytokinins
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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.
KeywordsAromatic cytokinin Histidine kinase Hormone Isoprenoid cytokinin Poplar Topolin
N6-(cis-4-hydroxy-3-methyl-2-buten-1-yl)adenine, i.e. cis-zeatin
N-(1,2,3-thidiazol-5-yl)-N´-phenylurea, i.e. thidiazuron
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 http://www.jgi.doe.gov.
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Conflict of interest
The authors declare that they have no conflicts of interest with the contents of this article.
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