Cell-specific expression of the promoters of two nonlegume hemoglobin genes in a transgenic legume, Lotus corniculatus

The promoters of the hemoglobin genes from the nitrogen-fixing tree Parasponia andersonii and the related nonnitrogen-fixing Trema tomentosa both confer beta-glucuronidase reporter gene expression to the central zone of the nodules of a transgenic legume, Lotos corniculatus. beta-Glucuronidase expression was high in the uninfected interstitial cells and parenchyma of the surrounding boundary layer and was low in the Rhizobium-infected cells. This contrasts with the expression of both the P. andersonii hemoglobin protein in P. andersonii nodules and the endogenous Lotos leghemoglobins that are expressed in the infected cells at very high levels. The expression pattern of the P. andersonii and T. tomentosa hemoglobin promoters in L. corniculatus resembles that of a nonsymbiotic hemoglobin gene from Casuarina glauca, which was introduced into this legume, and suggests that only the nonsymbiotic functions of the P. andersonii promoter are being recognized. Deletion of the distal segments of both the P. andersonii and T. tomentosa promoters identified regions important for the control of their tissue-specific and temporal activity in Lotus. Potential regulatory elements, which enhance nodule expression and suppress nonnodule expression, were also identified and localized to a distal promoter segment. A proximal AAGAG motif is present in the P. andersonii, T. tomentosa, and nonsymbiotic Casuarina hemoglobin genes. Mutation of this motif in the P. andersonii promoter resulted in a significant reduction in both the nodule and root expression levels in L. corniculatus. Some of the regulatory motifs characterized are similar to, but different from, the nodulin motifs of the leghemoglobins.