Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience. Although their Histidine Kinase receptors are substantially localised to the endoplasmic reticulum, cellular sites of cytokinin perception and importance of spatially heterogeneous cytokinin distribution continue to be debated. Here we show that cytokinin perception by plasma membrane receptors is an effective additional path for cytokinin response. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP) closely matches intracellular cytokinin content in roots, yet we also find cytokinins in extracellular fluid, potentially enabling action at the cell surface. Cytokinins covalently linked to beads that could not pass the plasma membrane increased expression of both TCSn::GFP and Cytokinin Response Factors. Super-resolution microscopy of GFP-labelled receptors and diminished TCSn::GFP response to immobilised cytokinins in cytokinin receptor mutants, further indicate that receptors can function at the cell surface. We argue that dual intracellular and surface locations may augment flexibility of cytokinin responses.

CEITEC Central European Institute of Technology and NCBR Faculty of Science Masaryk University CZ 62500 Brno Czech Republic

Chr Hansen 2630 Taastrup Denmark

Department of Chemical Biology and Genetics Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science of Palacký University CZ 78371 Olomouc Czech Republic

Department of Life Sciences Imperial College London London SW7 2AZ UK

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

Department of Plant and Environmental Sciences Copenhagen University 2630 Taastrup Denmark

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

Institute of Science and Technology Austria 3400 Klosterneuburg Austria

Laboratory of Growth Regulators Institute of Experimental Botany of the Czech Academy of Sciences and Faculty of Science of Palacký University CZ 78371 Olomouc Czech Republic

Umeå Plant Science Centre Department of Forest Genetics and Plant Physiology Swedish University of Agricultural Sciences SE 901 83 Umeå Sweden

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