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For this work, Lotus japonicustransgenic plants were constructed expressing a fusion reporter gene consisting of the genes β-glucuronidase(gus) and green fluorescent protein(gfp) under control of the soybean auxin-responsive promoter GH3. These plants expressed GUS and GFP in the vascular bundle of shoots, roots and leafs. Root sections showed that in mature parts of the roots GUS is mainly expressed in phloem and vascular parenchyma of the vascular cylinder. By detecting GUS activity, we describe the auxin distribution pattern in the root of the determinate nodulating legume L. japonicusduring the development of nodulation and also after inoculation with purified Nod factors, N-naphthylphthalamic acid (NPA) and indoleacetic acid (IAA). Differently than white clover, which forms indeterminate nodules, L. japonicuspresented a strong GUS activity at the dividing outer cortical cells during the first nodule cell divisions. This suggests different auxin distribution pattern between the determinate and indeterminate nodulating legumes that may be responsible of the differences in nodule development between these groups. By measuring of the GFP fluorescence expressed 21 days after treatment with Nod factors or bacteria we were able to quantify the differences in GH3 expression levels in single living roots. In order to correlate these data with auxin transport capacity we measured the auxin transport levels by a previously described radioactive method. At 48 h after inoculation with Nod factors, auxin transport showed to be increased in the middle root segment. The results obtained indicate that L. japonicustransformed lines expressing the GFP and GUS reporters under the control of the GH3 promoter are suitable for the study of auxin distribution in this legume.

A region of 16.8 kb of the Sym(biosis) plasmid pRL1JI of Rhizobium leguminosarum, consisting of the established 9.7 kb nodulation region which confers nodulation ability on Vicia hirsuta and a region of 7.1 kb which appeared to be necessary for nodulation on V. sativa and Trifolium subterraneum, was subcloned as fragments of maximally 2.5 kb in a newly developed IncQ transcriptional fusion vector. The expression of these fragments was studied in Rhizobium. One constitutive promoter, pr.nodD, and three plant-exudate inducible promoters were found, namely the known pr.nodA and pr.nodF as well as a new promoter designated pr.nodM. The latter promoters were localized within 114 bp, 330 bp and 630 bp respectively and they regulate the transcription of the operons nodA, B, C, I, J, nodF, E and of an operon of at least 2.5 kb located in the 7.1 kb region. Induction of the three inducible operons required plant exudate and a functional nodD product. The flavanone naringenin could replace plant exudate. Each of the three inducible promoters contained a nod-box. A consensus for the nod-box sequence, based on known sequences, is proposed. The 114 bp fragment which contains pr.nodA activity was used to localize pr.nodA by means of deletion mapping. The fragment which appeared necessary for complete pr.nodA activity is 72 bp in size, contains the complete nod-box and in addition a region of 21 bp downstream of the nod-box, in which the loosely conserved sequence AT(T)AG appears to be important for promoter activity.

Nodulation (root nodule formation) in legume roots is initiated by the induction of cell divisions and formation of root nodule primordia in the plant root cortex, usually in front of the protoxylem ridges of the central root cylinder. We isolated a factor from the central cylinder (stele) of pea roots which enhances hormone-induced cell proliferation in root cortex explants at positions similar to those of nodule primordia. The factor was identified as uridine. Uridine may act as a morphogen in plant roots at picomolar concentrations.

Rhizobium etli is a microsymbiont of plants of the genus Phaseolus. Using mass spectrometry we have identified the lipo-chitin oligosaccharides (LCOs) that are produced by R. etli strain CE3. They are N-acetylglucosamine pentasaccharides of which the non-reducing residue is N-methylated and N-acylated with cis-vaccenic acid (C18:1) or stearic acid (C18:0) and carries a carbamoyl group at C4. The reducing residue is substituted at the C6 position with O-acetylfucose. Analysis of their biological activity on the host plant Phaseolus vulgaris shows that these LCOs can elicit the formation of nodule primordia which develop to the stage where vascular bundles are formed. The formation of complete nodule structures, including an organized vascular tissue, is never observed. Considering the very close resemblance of the R. etli LCO structures to those of R. loti (I. M. López-Lara, J. D. J. van den Berg, J. E. Thomas Oates, J. Glushka, B. J. J. Lugtenberg, H. P. Spaink, Mol Microbiol 15: 627–638, 1995) we tested the ability of R. etli strains to nodulate various Lotus species and of R. loti to nodulate P. vulgaris. The results show that R. etli is indeed able to nodulate Lotus plants. However, several Lotus species are only nodulated when an additional flavonoid independent transcription activator (FITA) nodD gene is provided. Phaseolus plants can also be nodulated by R. loti bacteria, but only when the bacteria contain a FITA nodD gene. Apparently, the type of nod gene inducers secreted by the plants is the major basis for the separation of Phaseolus and Lotus into different cross inoculation groups.

A Rhizobium leguminosarum biovar viciae strain lacking a 40 kb DNA region of the Sym plasmid pRL1IJ to the left (3' side) of gene nodE failed to nodulate Vicia sativa plants. Therefore this DNA region was investigated for the presence of additional nodulation genes.

The flavonoid-inducible nod promoters of Rhizobium are positively regulated by the nodD gene which is highly conserved in various Rhizobium species. The nodD gene are functionally different in (i) their response to various exogenously added flavonoid inducers, (ii) the extent to which they mediate the activation of the flavonoid-inducible promoters, and (iii) the extent to which they repress their own constitutive transcription. In order to localize the regions of the nodD product which determine these differences, two series of nodD hybrid genes have been constructed. In one series the 5' moiety is derived from the R. meliloti nodD1 gene and the 3' moiety from the R. trifolii nodD gene. In the other series, the origins of the nodD moieties are reversed. Two regions of the nodD product appeared to be involved in autoregulation and it was also shown that the nodD promoters differ in their susceptibility to autoregulation. Many regions, dispersed over the entire nodD product, are involved in the specificity of activation by flavonoids. Several hybrid nodD genes were characterized which activate transcription with novel inducers. Furthermore, two classes of hybrid nodD genes were found from which the activation characteristics differ completely from those of the parental nodD genes. The first class activates the nodABCIJ promoter to the maximum level in the absence of flavonoid inducer. This level can no longer be influenced, positively or negatively, by the presence of (iso-)flavonoids. With the second class of hybrids, activation of the nodABCIJ promoter, even in the presence of flavonoid inducers, is no longer possible.