COMPOSITE STRUCTURE OF AUXIN RESPONSE ELEMENTS

The auxin-responsive soybean GH3 gene promoter is composed of multiple auxin response elements (AuxREs), and each AuxRE contributes incremen tally to the strong auxin inducibility of the promoter. Two independen t AuxREs of 25 bp (D1) and 32 bp (D4) contain the sequence TGTCTC. Res ults presented here show that the TGTCTC element in D1 and D4 is requi red but not sufficient for auxin inducibility in carrot protoplast tra nsient expression assays. Additional nucleotides upstream of TGTCTC ar e also required for auxin inducibility. These upstream sequences showe d constitutive activity and no auxin inducibility when part or all of the TGTCTC element was mutated or deleted. In D1, the constitutive ele ment overlaps the 5' portion of TGTCTC; in D4, the constitutive elemen t is separated from TGTCTC. An 11-bp element in D1, CCTCGTGTCTC, confe rred auxin inducibility to a minimal cauliflower mosaic virus 35S prom oter in transgenic tobacco seedlings as well as in carrot protoplasts (i.e., transient expression assays). Both constitutive elements bound specifically to plant nuclear proteins, and the constitutive element i n D1 bound to a recombinant soybean basic leucine zipper transcription factor with G-box specificity. To demonstrate further the composite n ature of AuxREs and the ability of the TGTCTC element to confer auxin inducibility, we created a novel AuxRE by placing a yeast GAL4 DNA bin ding site adjacent to the TGTCTC element. Expression of a GAL4-c-Rel t ransactivator in the presence of this novel AuxRE resulted in auxin-in ducible expression. Our results indicate that at least some AuxREs hav e a composite structure consisting of a constitutive element adjacent to a conserved TGTCTC element that confers auxin inducibility.