A highly active synthetic auxin response element (AuxRE), referred to as DR5, was created by performing site-directed mutations in a natural composite AuxRE found in the soybean GH3 promoter. DR5 consisted of tandem direct repeats of 11 bp that included the auxin-responsive TGTCTC element. The DR5 AuxRE showed greater auxin responsiveness than a natural composite AuxRE and the GH3 promoter when assayed by transient expression in carrot protoplasts or in stably transformed Arabidopsis seedlings, and it provides a useful reporter gene for studying auxin-responsive transcription in wild-type plants and mutants. An auxin response transcription factor, ARF1, bound with specificity to the DR5 AuxRE in vitro and interacted with Aux/IAA proteins in a yeast two-hybrid system. Cotransfection experiments with natural and synthetic AuxRE reporter genes and effector genes encoding Aux/IAA proteins showed that overexpression of Aux/IAA proteins in carrot protoplasts resulted in specific repression of TGTCTC AuxRE reporter gene expression.

The plant hormone auxin regulates plant physiology by modulating the interaction of transcription factors with auxin response elements (AuxREs) of the affected genes. A transcription factor, Auxin Response Factor 1 (ARF1), that binds to the sequence TGTCTC in AuxREs was cloned from Arabidopsis by using a yeast one-hybrid system. ARF1 has an amino-terminal DNA-binding domain related to the carboxyl terminus of the maize transactivator Viviparous-1. Sequence requirements for ARF1 binding in vitro are identical to those that confer auxin responsiveness in vivo. The carboxyl terminus of ARF1 contains two motifs found in the Aux/IAA class of proteins and appears to mediate protein-protein interactions.

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