Composite structure of auxin response elements.

Abstract

The auxin-responsive soybean GH3 gene promoter is composed of multiple auxin response elements (AuxREs), and each AuxRE contributes incrementally to the strong auxin inducibility of the promoter. Two independent AuxREs of 25 bp (Dl) and 32 b p (D4) contain the sequence TGTCTC. Results presented here show that the TGTCTC element in D1 and D4 is required but not sufficient for auxin inducibility in carrot protoplast transient expression assays. Additional nucleotides upstream of TGTCTC are also required for auxin inducibility. These upstream sequences showed constitutive activity and no auxin inducibility when part or all of the TGTCTC element was mutated or deleted. In D1, the constitutive element overlaps the 5' portion of TGTCTC; in D4, the constitutive element is separated from TGTCTC. An 11-bp element in D1, CCTCGTGTCTC, conferred auxin inducibility to a minimal cauliflower mosaic virus 35s promoter in transgenic tobacco seedlings as well as in carrot protoplasts (i.e., transient expression assays). 60th constitutive elements bound specifically to plant nuclear proteins, and the constitutive element in D1 bound to a recombinant soybean basic leucine zipper transcription factor with G-box specificity. To demonstrate further the composite nature of AuxREs and the ability of the TGTCTC element to confer auxin inducibility, we created a novel AuxRE by placing a yeast GAL4 DNA binding site adjacent to the TGTCTC element. Expression of a GAL4-c-Rel transactivator in the presence of this novel AuxRE resulted in auxin-inducible expression. Our results indicate that at least some AuxREs have a composite structure consisting of a constitutive element adjacent to a conserved TGTCTC element that confers auxin inducibility.

Structural basis for specific gene regulation by Auxin Response Factors

“…Strikingly, the preferred binding site of both ARF1 and ARF5 appears to be TGTCGG (Fig. 4F,G), rather than the "canonical" AuxRE TGTCTC 30 . It should be noted however, that the latter is found as an enriched motif, although the former is strongly preferred (Fig.…”

“…Based on promoter analysis of an auxin-responsive gene in soybean, a canonical auxinresponse element has been defined as TGTCTC 30 . ARF1 was first identified in a screen for factors binding this motif 29 .…”

“…Introduction of their target genes to activate or repress transcription 29,30 , but in the absence of auxin they are bound by Aux/IAA proteins that inhibit their activity 24,31 . Auxin binds to both the SCF(TIR1/AFB) receptor complex and the Aux/IAA protein, increasing the binding affinity between the two, leading to ubiquitination and degradation of the Aux/IAA proteins 32,33 .…”

“…Most ARFs tested have been shown to bind a generic auxin response element (AuxRE) ; 127 that was identified based on its occurrence in auxin-dependent promoters 30 . However, since target sites have not been screened exhaustively, it is unknown whether different ARFs prefer distinct binding sites and if so, what the molecular basis for such differences is.…”

“…The AuxRE 6mer has the form of TGTCNN. In the 1990's, TGTCTC was described as the recognition sequence for ARFs 30 . TGTCTC was found in the promoter region of the soybean GH3 auxin responsive gene, and was next used as bait to isolate the first members of the ARF family.…”