title: Identification of AHK2- and AHK3-like cytokinin receptors in Brassica napus reveals two subfamilies of AHK2 orthologues.
abstract: Cytokinin (CK) signalling is known to play key roles in the regulation of plant growth and development, crop yields, and tolerance to both abiotic stress and pathogen defences, but the mechanisms involved are poorly characterized in dicotyledonous crops. Here the identification and functional characterization of sensor histidine kinases homologous to Arabidopsis CK receptors AHK2 and AHK3 in winter oilseed rape are presented. Five CHASE-containing His kinases were identified in Brassica napus var. Tapidor (BnCHK1-BnCHK5) by heterologous hybridization of its genomic library with gene-specific probes from Arabidopsis. The identified bacterial artificial chromosome (BAC) clones were fingerprinted and representative clones in five distinct groups were sequenced. Using a bioinformatic approach and cDNA cloning, the precise gene and putative protein domain structures were determined. Based on phylogenetic analysis, four AHK2 (BnCHK1-BnCHK4) homologues and one AHK3 (BnCHK5) homologue were defined. It is further suggested that BnCHK1 and BnCHK3, and BnCHK5 are orthologues of AHK2 and AHK3, originally from the B. rapa A genome, respectively. BnCHK1, BnCHK3, and BnCHK5 displayed high affinity for trans-zeatin (1-3nM) in a live-cell competitive receptor assay, but not with other plant hormones (indole acetic acid, GA3, and abscisic acid), confirming the prediction that they are genuine CK receptors. It is shown that BnCHK1 and BnCHK3, and BnCHK5 display distinct preferences for various CK bases and metabolites, characteristic of their AHK counterparts, AHK2 and AHK3, respectively. Interestingly, the AHK2 homologues could be divided into two subfamilies (BnCHK1/BnCK2 and BnCHK3/BnCHK4) that differ in putative transmembrane domain topology and CK binding specificity, thus implying potential functional divergence.
authors.affiliation: Functional Genomics and Proteomics of Plants, Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: Alena
authors.initials: A
authors.lastname: Kuderová
authors.affiliation: Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71 Olomouc, Czech Republic.
authors.forename: Lucia
authors.initials: L
authors.lastname: Gallová
authors.affiliation: Functional Genomics and Proteomics of Plants, Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: Katarína
authors.initials: K
authors.lastname: Kuricová
authors.affiliation: Functional Genomics and Proteomics of Plants, Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: Eliška
authors.initials: E
authors.lastname: Nejedlá
authors.affiliation: Functional Genomics and Proteomics of Plants, Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: Anna
authors.initials: A
authors.lastname: Čurdová
authors.affiliation: Faculty of Medicine, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Micenková
authors.affiliation: Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71 Olomouc, Czech Republic.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Plíhal
authors.affiliation: Faculty of Medicine, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: David
authors.initials: D
authors.lastname: Šmajs
authors.affiliation: Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71 Olomouc, Czech Republic.
authors.forename: Lukáš
authors.initials: L
authors.lastname: Spíchal
authors.affiliation: Functional Genomics and Proteomics of Plants, Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic hejatko@sci.muni.cz.
authors.forename: Jan
authors.initials: J
authors.lastname: Hejátko
mesh_terms: D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins; D029688:Brassica napus / Q000235:genetics* / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D000071677:Histidine Kinase; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D015398:Signal Transduction
chemical_list: D029681:Arabidopsis Proteins; D010940:Plant Proteins; D011494:Protein Kinases; D000071677:Histidine Kinase; C000605492:AHK2 protein, Arabidopsis; C000605493:AHK3 protein, Arabidopsis
keywords: CHASE-containing His kinase; E. coli-based live-cell competitive receptor assay; JBnB library; gene structure; modular protein architecture; phylogenetic analysis