title: The specificity of cytokinin signalling in Arabidopsis thaliana is mediated by differing ligand affinities and expression profiles of the receptors.
abstract: Arabidopsis thaliana has three membrane-located cytokinin receptors (AHK2, AHK3 and CRE1/AHK4), which are sensor histidine kinases containing a ligand-binding CHASE domain. Despite their structural similarity the role of these receptors differs in planta. Here we have explored which parameters contribute to signal specification. In a bacterial assay, the CHASE domain of AHK2 has a similar ligand binding spectrum as CRE1/AHK4. It shows the highest affinity for isopentenyladenine (iP) and trans-zeatin (tZ) with an apparent K(D) of 1.4 and 4.0 nm, respectively. Real-time PCR analysis of cytokinin primary response genes in double mutants retaining only single receptors revealed that all receptors are activated in planta by cytokinin concentrations in the low nanomolar range. However, there are differences in sensitivity towards the principal cytokinins iP and tZ. The activation of the cytokinin-sensitive P(ARR5) :GUS reporter gene in three different double mutants shows specific, but also overlapping, spatial domains of activity, which were for all receptors predominantly in the shoot apical meristems and root cap columella. AHK2 and AHK3 signal specifically in leaf parenchyma cells, AHK3 in stomata cells, and CRE1/AHK4 in the root vasculature. Promoter-swap experiments demonstrate that CRE1/AHK4 can functionally replace AHK2 but not AHK3. However, the cytoplasmic AHK3 histidine kinase (Hk) domain can be replaced by the CRE1/AHK4 Hk domain, which suggests that functionality is mediated in this case by the extracytosolic domain. Together, the data show that both differential gene expression and ligand preference contribute to specify the receptor activity.
authors.affiliation: Institute of Biology/Applied Genetics, Dahlem Centre of Plant Sciences (DCPS), Freie Universität Berlin, Albrecht-Thaer-Weg 6, D-14195 Berlin, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Stolz
authors.forename: Michael
authors.initials: M
authors.lastname: Riefler
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Lomin
authors.forename: Katharina
authors.initials: K
authors.lastname: Achazi
authors.forename: Georgy A
authors.initials: GA
authors.lastname: Romanov
authors.forename: Thomas
authors.initials: T
authors.lastname: Schmülling
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D003583:Cytokinins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D000071677:Histidine Kinase; D008024:Ligands; D018519:Meristem / Q000378:metabolism; D009154:Mutation; D010937:Plant Growth Regulators / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018515:Plant Leaves / Q000378:metabolism; D018517:Plant Roots / Q000378:metabolism; D054046:Plant Stomata / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D018749:RNA, Plant / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D036226:Seedlings / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D019076:Transgenes / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D003583:Cytokinins; D018076:DNA, Complementary; D008024:Ligands; D010937:Plant Growth Regulators; D018749:RNA, Plant; D011956:Receptors, Cell Surface; D011494:Protein Kinases; D000071677:Histidine Kinase; C000605492:AHK2 protein, Arabidopsis; C000605493:AHK3 protein, Arabidopsis; C421783:WOL protein, Arabidopsis