title: Plant membrane assays with cytokinin receptors underpin the unique role of free cytokinin bases as biologically active ligands.
abstract: Cytokinin receptors play a key role in cytokinin-dependent processes regulating plant growth, development, and adaptation; therefore, the functional properties of these receptors are of great importance. Previously the properties of cytokinin receptors were investigated in heterologous assay systems using unicellular microorganisms, mainly bacteria, expressing receptor proteins. However, within microorganisms receptors reside in an alien environment that might distort the receptor properties. Therefore, a new assay system has been developed allowing studies of individual receptors within plant membranes (i.e. closer to their natural environment). The main ligand-binding characteristics of receptors from Arabidopsis [AHK2, AHK3, and AHK4] and maize (ZmHK1) were refined in this new system, and the properties of full-length Arabidopsis receptor AHK2 were characterized for the first time. Ligand specificity profiles of receptors towards cytokinin bases were comparable with the profiles retrieved in bacterial assay systems. In contrast, cytokinin-9-ribosides displayed a strongly reduced affinity for receptors in the plant assay system, indicating that ribosides as the common transport form of cytokinins have no or very weak cytokinin activity. This underpins the central role of free bases as the sole biologically active cytokinin compounds. According to molecular modelling and docking studies, N (9)-ribosylation alters the bonding pattern in cytokinin-receptor interaction and prevents β6-β7 loop movement important for tight hormone binding. A common feature of all receptors was a greatly reduced ligand binding at low (5.0-5.5) pH. The particularly high sensitivity of ZmHK1 to pH changes leads to the suggestion that some cytokinin receptors may play an additional role as pH sensors in the lumen of the endoplasmic reticulum.
authors.affiliation: Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia.
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Lomin
authors.affiliation: Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia.
authors.forename: Dmitry M
authors.initials: DM
authors.lastname: Krivosheev
authors.affiliation: Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia.
authors.forename: Mikhail Yu
authors.initials: MY
authors.lastname: Steklov
authors.affiliation: Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia.
authors.forename: Dmitry V
authors.initials: DV
authors.lastname: Arkhipov
authors.affiliation: Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1/3, 119991 Moscow, Russia.
authors.forename: Dmitry I
authors.initials: DI
authors.lastname: Osolodkin
authors.affiliation: Institute of Biology/Applied Genetics, Dahlem Centre of Plant Sciences, Freie Universität Berlin, Albrecht-Thaer-Weg 6, D-14195 Berlin, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schmülling
authors.affiliation: Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory 1, 119992 Moscow, Russia gromanov@yahoo.com gar@ippras.ru.
authors.forename: Georgy A
authors.initials: GA
authors.lastname: Romanov
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003583:Cytokinins / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015870:Gene Expression; D000071677:Histidine Kinase; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D003313:Zea mays / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D003583:Cytokinins; D008024:Ligands; D010940:Plant Proteins; D011956:Receptors, Cell Surface; D011494:Protein Kinases; D000071677:Histidine Kinase; C000605492:AHK2 protein, Arabidopsis; C000605493:AHK3 protein, Arabidopsis; C421783:WOL protein, Arabidopsis
keywords: Arabidopsis thaliana; Zea mays.; cytokinin; cytokinin receptor; ligand specificity; pH sensor; plant assay system; receptor binding assay