title: In planta analysis of a cis-regulatory cytokinin response motif in Arabidopsis and identification of a novel enhancer sequence.
abstract: The phytohormone cytokinin plays a key role in regulating plant growth and development, and is involved in numerous physiological responses to environmental changes. The type-B response regulators, which regulate the transcription of cytokinin response genes, are a part of the cytokinin signaling system. Arabidopsis thaliana encodes 11 type-B response regulators (type-B ARRs), and some of them were shown to bind in vitro to the core cytokinin response motif (CRM) 5'-(A/G)GAT(T/C)-3' or, in the case of ARR1, to an extended motif (ECRM), 5'-AAGAT(T/C)TT-3'. Here we obtained in planta proof for the functionality of the latter motif. Promoter deletion analysis of the primary cytokinin response gene ARR6 showed that a combination of two extended motifs within the promoter is required to mediate the full transcriptional activation by ARR1 and other type-B ARRs. CRMs were found to be over-represented in the vicinity of ECRMs in the promoters of cytokinin-regulated genes, suggesting their functional relevance. Moreover, an evolutionarily conserved 27 bp long T-rich region between -220 and -193 bp was identified and shown to be required for the full activation by type-B ARRs and the response to cytokinin. This novel enhancer is not bound by the DNA-binding domain of ARR1, indicating that additional proteins might be involved in mediating the transcriptional cytokinin response. Furthermore, genome-wide expression profiling identified genes, among them ARR16, whose induction by cytokinin depends on both ARR1 and other specific type-B ARRs. This together with the ECRM/CRM sequence clustering indicates cooperative action of different type-B ARRs for the activation of particular target genes.
authors.affiliation: Institute of Biology/Applied Genetics, Dahlem Centre of Plant Sciences (DCPS), Freie Universität Berlin, Germany.
authors.forename: Eswarayya
authors.initials: E
authors.lastname: Ramireddy
authors.forename: Wolfram G
authors.initials: WG
authors.lastname: Brenner
authors.forename: Andreas
authors.initials: A
authors.lastname: Pfeifer
authors.forename: Alexander
authors.initials: A
authors.lastname: Heyl
authors.forename: Thomas
authors.initials: T
authors.lastname: Schmülling
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003583:Cytokinins / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D018506:Gene Expression Regulation, Plant / Q000187:drug effects*; D008969:Molecular Sequence Data; D009154:Mutation; D059372:Nucleotide Motifs / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D010937:Plant Growth Regulators / Q000494:pharmacology; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D059467:Transcriptome / Q000187:drug effects
chemical_list: C117138:ARR1 protein, Arabidopsis; C117139:ARR2 protein, Arabidopsis; C586059:ARR6 protein, Arabidopsis; D029681:Arabidopsis Proteins; D003583:Cytokinins; D004268:DNA-Binding Proteins; D010937:Plant Growth Regulators; D014157:Transcription Factors
keywords: Arabidopsis thaliana; Cytokinin; Cytokinin response element; Cytokinin response motif; Response regulator; Transcriptional regulation