ABSTRACT
In plants, the ERF/EREBP family of transcriptional regulators plays a key role in adaptation to various biotic and abiotic stresses. These proteins contain a conserved AP2 DNA-binding domain and several uncharacterized motifs. Here, we describe a short motif, termed 'EDLL', that is present in AtERF98/TDR1 and other clade members from the same AP2 sub-family. We show that the EDLL motif, which has a unique arrangement of acidic amino acids and hydrophobic leucines, functions as a strong activation domain. The motif is transferable to other proteins, and is active at both proximal and distal positions of target promoters. As such, the EDLL motif is able to partly overcome the repression conferred by the AtHB2 transcription factor, which contains an ERF-associated amphiphilic repression (EAR) motif. We further examined the activation potential of EDLL by analysis of the regulation of flowering time by NF-Y (nuclear factor Y) proteins. Genetic evidence indicates that NF-Y protein complexes potentiate the action of CONSTANS in regulation of flowering in Arabidopsis; we show that the transcriptional activation function of CONSTANS can be substituted by direct fusion of the EDLL activation motif to NF-YB subunits. The EDLL motif represents a potent plant activation domain that can be used as a tool to confer transcriptional activation potential to heterologous DNA-binding proteins.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Gene Expression Regulation, Plant , Transcriptional Activation , Amino Acid Motifs , Amino Acid Sequence , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Cloning, Molecular , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Flowers/metabolism , Flowers/physiology , Genes, Plant , Genes, Reporter , Molecular Sequence Data , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Plants, Genetically Modified/physiology , Promoter Regions, Genetic , Protein Structure, Tertiary , Protoplasts/cytology , Protoplasts/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Time Factors , Transcription Factors/genetics , Transcription Factors/metabolism , TransfectionABSTRACT
CONSTANS is an evolutionarily-conserved central component of the genetic pathway that controls the onset of flowering in response to daylength. However, the specific biochemical mechanism by which the CONSTANS protein regulates the expression of its target genes remains largely unknown. *By using a combination of cell-based expression analysis and in vitro DNA binding studies, we have demonstrated that CONSTANS possesses transcriptional activation potential and is capable of directly binding to DNA. *CONSTANS was found to bind DNA via a unique sequence element containing a consensus TGTG(N2-3)ATG motif. This element is present in tandem within the FLOWERING LOCUS T promoter and is sufficient for CO binding and activity. The conserved CCT (CONSTANS, CONSTANS-like and TOC1) domain of CONSTANS was shown to be required for its recruitment to the DNA motif and other CCT-containing proteins were also found to have the ability to regulate gene expression via this element. *The CCAAT box, which has been previously hypothesized as a recruitment site for complexes containing the CONSTANS protein, potentiated CONSTANS-mediated activation but was not essential for CONSTANS recruitment to a target promoter or for its activity as a transcriptional factor.
