THE PLANT CELL, Vol 2, Issue 10 973-986, Copyright © 1990 by American Society of Plant Biologists
Functional Analysis of the Sesbania rostrata Leghemoglobin glb3 Gene 5[prime] -Upstream Region in Transgenic Lotus corniculatus and Nicotiana tabacum Plants
L. Szabados, P. Ratet, B. Grunenberg and F. J. de Bruijn
Max-Planck-Institut fur Zuchtungsforschung, Carl-von-Linne-Weg 10, 5000 Koln 30, Federal Republic of Germany
Expression of the Sesbania rostrata leghemoglobin glb3 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[prime]-upstream region was found to
direct a high level of nodule-specific [beta]-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 [delta]-p35S and [delta]-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 heterologous
enhancer 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 [delta]-p35S and [delta]-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[prime]-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.
