Interdependence and nodule specificity of cis-acting regulatory elements in the soybean leghemoglobin lbc 3 and N23 gene promoters

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Abstract
The qualitative and quantitative contributions of four separate cis-acting DNA elements controlling the root nodule-specific soybean leghemoglobin lbc3 gene were analyzed in transgenic Lotus corniculatus plants. Expression from internal deletions in the 5′ region between positions −49 and −1956 was monitored from a CAT reporter gene. The strong positive element (SPE; −1090, −947) responsible for high-level expression was demonstrated to be an organ-specific element by deleting proximal nodule-specific control elements. Deletion of the downstream qualitative organ-specific element (OSE; −139, −102) containing the putative nodulin consensus sequences 5′AAAGAT and 5′CTCTT resulted in a low expression level. Efficient SPE enhancement is therefore dependent on the organ-specific element, which by itself does not enhance expression. This quantitative effect of the immediate upstream region carrying the consensus sequences was also found in hybrid promoter studies using the soybean nodulin N23 gene promoter, suggesting the involvement of these motifs in a regulatory mechanism for nodulin genes. Deletion of the lbc3negative element (NE, −102, t-49) linking the SPE and OSE onto the TATA box did not lead to unregulated expression. These results indicate that interaction between positive, negative and neutral qualitative elements controls lbc3 expression. Binding of the nuclear protein NAT2 at the lbc3 weak positive element (WPE; −230, −170) is probably not directly required for this mechanism.