The Plant Cell, Vol. 2, 973-986, October 1990 O 1990 American Society of Plant Physiologists Functional Analysis of the Sesbanía rostrata Leghemoglobin g/b3 Gene 5’-Upstream Region in Transgenic Lotus cornículatus and Nícotíana tabacum Plants LaszlÓ Szabados,’.’ Pascal RatetIav2 Britta Grunenberg,’ and Frans J. de BruijnaSbT3 a Max-Planck-lnstitutfür Züchtungsforschung, Carl-von-Linne-Weg1 O, 5000 Koln 30, Federal Republic of Germany East Lansing, Michigan 48824 Michigan State University-Departmentof Energy Plant Research Laboratory, Michigan State University, Expression of the Sesbania rostrata leghemoglobin gb3 gene was analyzed in transgenic Lotus corniculatus and tobacco plants harboring chimeric glb3-uidA ( gus) gene fusions to identify cis-acting elements involved in nodule- specific gene expression and general transcriptional control. A 1.9-kilobase fragment of the glb3 5’-upstream region was found to direct a high level of nodule-specific 8-glucuronidase (GUS) activity in L. corniculatus, restricted to the Rhizobium-infected cells of the nodules. The same fragment directed a low level of GUS activity in tobacco, restricted primarily to the roots and to phloem cells of the stem and petiole vascular system. A deletion analysis revealed that the region between coordinates -429 and -48 relative to the ATG was sufficient for nodule-specific expression. Replacement of the -161 to -48 region, containing the glb3 CAAT and TATA boxes, with the heterologous truncated promoters A-p35S and A-pnos resulted in a loss of nodule specificity and reduction of GUS activity in L. corniculatus but a significant increase in tobacco, primarily in the roots. The same fragment could not direct nodule-specific expression when fused to a heterologousenhancer in cis. This region contains DNA sequences required, but not sufficient, for nodule-specific expression in L. corniculatus that function poorly or may be involved in promoter silencing in tobacco. By fusing further upstream fragments to the A-p35S and A-pnos promoters, two positive regulatory regions were delimited between coordinates -1601 and -670, as well ‘as -429 and -162. The former region appears to function as a general enhancer because it significantly increased promoter activity in both orientations in L. corniculatus and tobacco. The latter region could enhance gene expression in both orientations in tobacco, but only in the correct orientation in L. corniculatus. These results show that efficient expression of the S. rostrata glb3 gene in nodules is mediated by an ATG-proximal, tissue-specific element, as well as further 5’- upstream positive elements; that the S. rostrata glb3 promoter is induced in a nodule-specific fashion in the heterologous legume L. corniculatus, suggesting a high degree of conservation of the relevant regulatory signals; and that the S. rostrata lb promoter is not silent in the nonlegume tobacco, but is expressed primarily in the roots. INTRODUCTION Regulated plant genes can be either inducible or tissue specific or both inducible and tissue specific. They may respond to environmental stimuli such as light, heat, an- aerobic stress, wounding, funga1 elicitors, circadian rhythms, hormones, or signals from pathogenic or sym- biotic bacteria. Alternatively, or in addition, they can be activated in green tissues, in tubers, in seeds, in flowers, ’ Current address: Biological Research Center lnstitute of Plant Physiology, P.O. Box 521,6701 Szeged, Hungary. * Current address: lnstitut des Sciences Vegetales, Centre Na- tional de Ia Recherche Scientifique, 91198 Gif-sur-Yvette Cedex, France. To whom correspondence should be addressed at Michigan State University . in pollen, or in nitrogen-fixing nodules induced by rhizobia. Members of both categories may also be subject to de- velopmental control processes (see Schell et al., 1988; Weising et al., 1988; Benfey and Chua, 1989). We are interested in studying the regulation of a class of plant genes that are specifically expressed during the process of rhizobial infection and nodule ontogeny or in mature nitrogen-fixing nodules. These genes have been termed nodulin genes (van Kammen, 1984) and appear to be induced directly or indirectly by rhizobial signals in a tissue-specific or nodule-specific fashion. In addition, their expression is developmentally controlled (see Govers et al., 1987; Verma and Delauney, 1988; Gloudemans and Bisseling, 1989; Long, 1989; de Bruijn et al., 1989, 1990).