Urea-derived inhibitors of cytokinin oxidase/dehydrogenase for agricultural applications

J. Nisler^1,2, D Kopečný³, Z Pěkná⁴, R Končitíková³, R Koprna⁴, N Murvanidze⁵, Stefaan P.O. Werbrouck⁵, L. Havlíček⁶, N. De Diego⁴, Z. Wimmer^2,6, P. Briozzo⁷, S. Moréra⁸, D. Zalabák⁹, L. Spíchal⁴, Miroslav Strnad¹

¹Laboratory of Growth Regulators, Institute of Experimental Botany of the Czech Academy of Sciences & Palacký University, Olomouc, Czech Republic

²University of Chemistry and Technology in Prague, Faculty of Food and Biochemical Technology, Department of Chemistry of Natural Compounds, Prague, Czech Republic

³Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic

⁴Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic

⁵Department Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium

⁶Isotope Laboratory, Institute of Experimental Botany, The Czech Academy of Sciences, Prague, Czech Republic

⁷Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, Versailles, France

⁸Institute for Integrative Biology of the Cell, CNRS-CEA-Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France

⁹Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic

david.kopecny@upol.cz

Numerous improvements in agriculturally important parameters of plants have been achieved by manipulating cytokinin content in plants. Cytokinins are plant hormones and their levels are regulated by a flavoenzyme family of FAD-dependent cytokinin oxidases/dehydrogenases (CKO/CKX). Thidiazuron (TDZ) and N-(2-chloro-pyridin-4-yl)-N´-phenylurea (CPPU) are well-known synthetic cytokinins and CKX inhibitors. These urea compounds compete with cytokinin metabolites, bases and ribosides, for the binding site of CKX. Development of novel potent CKX inhibitors reflects the ban on genetically engineered food and crops in EU. These inhibitors might increase the lifetime of endogenous cytokinins and affect different cytokinin functions, thereby having positive effects on seed filling, delayed senescence and stress tolerance toward biotic and abiotic stresses and thus improving crop yield.

Here, we report a development and biological activity of novel CKX inhibitors, which are derived mainly from diphenylurea. Several CKX isoforms from maize (Zea mays) were used to study the inhibitory strength of new inhibitors by analyzing enzyme kinetics as well as their binding mode by X-ray crystallography. We identified several compounds with IC₅₀ values in nanomolar range and solved crystal structure complexes up to 1.6 Å resolution. These compounds can alleviate stress responses to drought and salt as well as increase seed/grain yield in field trials on barley, wheat and barley. Hopefully, they can find place on the market of plant growth substances.

Supported by the grant 18-07563S from the Czech Science Foundation, ERDF grant project No. CZ.02.1.01/0.0/0.0/16_019/0000827 (Plants as a tool for sustainable global development) and grant No. CZ.02.2.69/0.0/0.0/16_027/0008482 from the Ministry of Education, Youth and Sports, Czech Republic. We acknowledge SOLEIL for provision of synchrotron radiation facilities (proposals ID 20160782 and 20170872) in using Proxima 1 and 2 beamlines at SOLEIL synchrotron.