The VfLb29 leghemoglobin gene promoter was polymerase chain reaction-amplified from a Vicia faba genomic library and was fused to the gusAint coding region. Expression of the chimeric gene was analyzed in transgenic hairy roots of the legumes V. faba, V. hirsuta, and Medicago truncatula as well as in transgenic Nicotiana tabacum plants. The VfLb29 promoter was found to be specifically active not only in the infected cells of the nitrogen-fixing zone of root nodules but also in arbuscule-containing cells of transgenic V. faba and M. truncatula roots colonized by the endomycorrhizal fungus Glomus intraradices. In addition to these two legumes, specific expression in arbuscule-containing cells was also observed in the nonlegume N. tabacum. All studies were done in comparison to the V. faba leghemoglobin gene promoter VfLb3 that as VfLb29 was expressed in the infected cells of root nodules but showed no activity in endomycorrhiza. An activation of the VfLb29 promoter due to hypoxia in metabolically active tissues was excluded. The conserved activation in arbuscule-containing cells of legumes and the nonlegume N. tabacum suggests a conserved trigger for this promoter in legume and nonlegume endomycorrhiza symbioses.

ENOD12 is one of the first nodulin genes expressed upon inoculation with Rhizobium and also purified Nod factors are able to induce ENOD12 expression. The ENOD12 gene family in pea (Pisum sativum) has two members. A cDNA clone representing PsENOD12A [26] and a PsENOD12B genomic clone [7] have been previously described. The isolation and characterization of a PsENOD12A genomic clone is presented in this paper. By using a Vicia hirsuta-Agrobacterium rhizogenes transformation system it is shown that both genes have a similar expression pattern in transgenic V. hirsuta root nodules. Promoter analyses of both PsENOD12 promoters showed that the 200 bp immediately upstream of the transcription start are sufficient to direct nodule-specific and Nod factor-induced gene expression.

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We recently reported on the broad bean gene VfENOD-GRP3 encoding a glycine-rich early nodulin. This gene was predominantly expressed in the interzone II-III region of Vicia faba root nodules. The VfENOD-GRP3 promoter contained several sequence motifs potentially involved in the regulation of gene expression. To investigate the molecular basis for the specific VfENOD-GRP3 expression, defined VfENOD-GRP3 promoter fragments were fused to an intron-containing gusAint gene. Agrobacterium rhizogenes ARqual strains carrying these fusions integrated into the TL DNA were used to generate hairy roots on Vicia hirsuta, which subsequently were nodulated. Histochemical analysis of transgenic nodules indicated that a strong gusAint expression in the interzone II-III region was mediated by the -1252/+10 VfENOD-GRP3 promoter region. This reporter gene expression in V. hirsuta was comparable to the location of VfENOD-GRP3 transcripts in V. faba nodules. An analysis of defined promoter fragments revealed that a strong gusAint expression in the interzone II-III region was also mediated by the -737/+10 promoter, whereas the -239/+10 promoter only mediated a weak gusAint expression in the interzone II-III region. Since the -239/+10 promoter fragment did not resemble published nodulin gene promoters, we propose that it contains new sequence motifs involved in mediating gene expression in the interzone II-III region of Vicia nodules.