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Transgenic alfalfa plants harboring a gene fusion between the soybean leghemoglobin (lbc3) promoter region and the chloramphenicol acetyl transferase (cat) gene were used to determine the influence of rhizobial mutants on lb gene expression in nodules. The promoter region of the Sesbania rostrata glb3 (Srglb3) leghemoglobin gene was examined for the presence of conserved motifs homologous to binding site 1 and 2 of the soybean lbc3 promoter region, found to interact with a trans-acting factor present in soybean nodule nuclear extracts (Jensen EO, Marcker KA, Schell J, de Bruijn FJ, EMBO J 7:1265-1271, 1988). Subfragments of the S. rostrata glb3 (Srglb3) promoter region were examined for binding to trans-acting factors from nodule nuclear extracts. In addition to the binding sites previously identified (Metz BA, Welters P, Hoffmann HJ, Jensen EO, Schell J, de Bruijn FJ, Mol Gen Genet 214: 181-191), several other sites were found to interact with trans-acting factors. In most cases the same trans-acting factor(s) were shown to be involved. One fragment (202) was found to bind specifically to a different factor (protein) which was extremely heat-resistant (100 degrees C). The appearance of this factor was shown to be developmentally regulated since the expected protein-DNA complexes were first observed around 12 days after infection, concomitant with the production of leghemoglobin proteins. Fragments of the Srglb3 5' upstream region were fused to the beta-glucuronidase reporter gene with its own CAAT and TATA box region or those of the cauliflower mosaic virus 35S and nopaline synthase (nos) promoters.(ABSTRACT TRUNCATED AT 250 WORDS)