A strong constitutive positive element is essential for the ammonium-regulated expression of a soybean gene encoding cytosolic glutamine synthetase

In order to identify important promoter elements controlling the ammonium-r egulated expression of the soybean gene GS15 encoding cytosolic glutamine s ynthetase, a series of 5' promoter deletions were fused to the GUS reporter gene. To allow the detection of positive and negative regulatory elements, a series of 3' deletions were fused to a -90 CaMV 35S promoter fragment pl aced upstream of the GUS gene. Both types of construct were introduced into Lotus corniculatus plants and soybean roots via Agrobacterium rhizogenes-m ediated transformation. Both spectrophotometric enzymatic analysis and hist ochemical localization of GUS activity in roots, root nodules and shoots of transgenic plants revealed that a strong constitutive positive element (SC PE) of 400 bp, located in the promoter distal region is indispensable for t he ammonium-regulated expression of GS15. Interestingly, this SCPE was able to direct constitutive expression in both a legume and non-legume backgrou nd to a level similar to that driven by the CaMV 35S full-length promoter. In addition, results showed that separate proximal elements, located in the first 727 bp relative to the transcription start site, are essential for r oot- and root nodule-specific expression. This proximal region contains an AAAGAT and two TATTTAT consensus sequences characteristic of nodulin or nod ule-enhanced gene promoters. A putative silencer region containing the same TATTTAT consensus sequence was identified between the SCPE and the organ-s pecific elements. The presence of positive, negative and organ-specific ele ments together with the three TATTTAT consensus sequences within the promot er strongly suggest that these multiple promoter fragments act in a coopera tive manner, depending on the spatial conformation of the DNA for trans-act ing factor accessibility.