Rhizobium nod genes are involved in the induction of two early nodulin genes in Vicia sativa root nodules

Nodulin gene expresison was studied in Vicia sativa (common vetch) root nodules induced by several Rhizobium and Agrobacterium strains. An Agrobacterium transconjugant containing a R. leguminosarum symplasmid instead of its Ti-plasmid, that was previously shown to form empty nodules on pea, induced nodules on Vicia roots in which nodule cells were infected with bacteria. In the Vicia nodules induced by this transconjugant, two so-called early nodulin genes were found to be expressed, whereas in the nodules formed on pea the expression of only one early nodulin gene was detected. In both cases the majority of the nodulin genes was not expressed.Apparently, an intracellular location of the bacteria is not sufficient for the induction of the majority of the nodulin genes. All nodulin genes were expressed in nodules induced by cured Rhizobium strains containing cosmid clones that have a 10 kb nod region of the sym-plasmid in common. Since in tumours no nodulin gene expression was found at all, the Agrobacterium chromosome does not contribute to the induction of nodulin genes. Therefore it is concluded that the signal for the induction of the expression of the two Vicia early nodulin genes is encoded by the nod-region, and the signal involved in the induction of all other nodulin genes has to be located outside the sym-plasmid, on the Rhizobium chromosome. The apparent difference in early nodulin gene expression between pea and Vicia is discussed in the light of the usefulness of Agrobacterium transconjugants in the study of nodulin gene expression.     

The pea early nodulin gene PsENOD7 maps in the region of linkage group I containing sym2 and leghaemoglobin

The early nodulin gene, PsENOD7, is expressed in pea root nodules induced by Rhizobium leguminosarum bv. viciae, but not in other plant organs. In situ hybridization showed that this gene is transcribed during nodule maturation in the infected cells of the proximal part of the prefixation zone II. At the transition of zone II into interzone II–III, the level of PsENOD7 mRNA drops markedly. PsENOD7 has no significant homology to other genes. RFLP mapping studies have shown that PsENOD7 is located in linkage group I between the leghaemoglobin genes and sym2.     

The pea late nodulin gene PsNOD6 is homologous to the early nodulin genes PsENOD3/14 and is expressed after the leghaemoglobin genes

The pea late nodulin gene PsNOD6 has been cloned and sequenced. PsNOD6 is homologous to the pea early nodulin genes PsNOD3 and PsENOD14. In situ hybridization experiments showed that, like the PsENOD3 and PsENOD14 genes, the PsNOD6 gene is only expressed in the infected cell type. The PsNOD6 gene is first expressed at the transition of the pre-fixation zone II into the interzone II–III (the amyloplast-rich zone preceding the fixation zone III), whereas the early nodulin genes PsENOD3 and PsENOD14 are already induced in the pre-fixation zone II. Thus these nodulin genes encoding homologous proteins are induced at consecutive stages of nodule development.The expression of the late nodulin genes encoding leghaemoglobin precedes the expression of the late nodulin gene PsNOD6. Therefore these late nodulin genes have to be regulated by different mechanisms despite the fact they are expressed in the same cell type. This conclusion is consistent with the fact that PsNOD6 lacks one of the conserved regions occurring in the promoters of all other late nodulin genes studied.     

A complex ensemble of cis-regulatory elements controls the expression of a Vicia faba non-storage seed protein gene

We have identified cis-regulatory elements within the 5-upstream region of a Vicia faba non-storage seed protein gene, called usp, by studying the expression of usp-promoter deletion fragments fused to reporter genes in transgenic tobacco seeds. 0.4 kb of usp upstream sequence contain at least six, but probably more, distinct cis-regulatory elements which are responsible for seemingly all quantitative, spatial and temporal aspects of expression. Expression-increasing and-decreasing elements are interspersed and include an AT-rich sequence, a G-box element and a CATGCATG motif. The latter acts as a negative element in contrast to what has been found for the same motif in legumin-and vicilin-type seed storage protein gene promoters. Seed specificity of expression is mainly determined by the –68/+51 region which confers, however, only very low levels of expression. The data support the combinatiorial model of promoter function.     

Regulation of nodulin gene expression

The expression of plant genes specifically induced during rhizobial infection and the early stages of nodule ontogeny (early nodulin genes) and those induced in the mature, nitrogen-fixing nodule (late nodulin genes) is differentially regulated and tissue/cell specific. We have been interested in the signal transduction pathway responsible for symbiotic, temporal and spatial control of expression of an early (Enod2) and a late (Leghemoglobin;lb) nodulin gene from the stem-nodulated legumeSesbania rostrata, and in identifying thecis-acting elements andtrans-acting factors involved in this process (De Bruijn and Schell, 1992). By introducing chimericS. rostrata lb promoter-gus reporter gene fusions into transgenicLotus corniculatus plants, we have been able to show that thelb promoter directs an infected-cell-specific expression pattern inLotus nodules. We have been able to delimit thecis-acting element responsible for nodule-infected-cell-expression to a 78 pb region of thelb promoter (NICE Element) and have analyzed this element in detail by site-specific mutagenesis. We have studied the interaction of the NICE element, and further upstreamcis-acting elements, withtrans-acting factors of both plant- and rhizobial origin. We have obtained evidence for the involvement of rhizobial proteins in infected-cell-specific plant gene expression (Welters et al., 1993). We have purified one of the bacterial binding proteins from theS. rostrata symbiontAzorhizobium caulinodans (AcBBP1), and cloned and mutated the corresponding gene, in order to examine its symbiotic phenotype. We have also found that theS. rostrata Enod2 gene is rapidly induced by physiologically significant concentrations of cytokinins, suggesting the role of cytokinin as a potential secondary signal involved in nodulation (Dehio and De Bruijn, 1992). We are examining whether the observed cytokinin induction, as well as the nodule-specific expression pattern, are modulated by theSrEnod2 promoter.     

The relationship between nodulin gene expression and the Rhizobium nod genes in Vicia sativa root nodule development

The role of the Rhizobium nod genes in the induction of nodulin gene expression was examined by analyzing nodules formed on vetch roots by bacterial strains containing only the nod region. Introduction of an 11-kb cloned nod region of the R. leguminosarum sym plasmid pRL1JI into sym plasmid-cured rhizobia conferred on the recipient strains the ability to induce nodules in which all nodulin genes were expressed. This proves that from the sym plasmid only the nod region is involved in the induction of nodulin gene expression. A transconjugant of Agrobacterium carrying the same nod region induces nodules in which only early nodulin gene expression is detected. Thus, the nod region is essential for the induction of early nodulin gene expression. In this case, nodule cytology may indicate that a defense response of the plant interferes with the induction of late nodulin gene expression. Indirect evidence is presented that indeed the Rhizobium nod genes are also in some way involved in the induction of the expression of late noduling genes. The combination between histological data and pattern of nodulin gene expression furthermore reveals a correlation between nodule structure and nodulin gene expression. This correlation may aid in speculations about the functions of nodulins.     

Comparative electrophoretical analysis of plasmid-like mitochondrial DNAs in Vicia faba and in some other legumes

Minicircular DNAs found in mitochondria of 6-d-old etiolated seedlings of Vicia faba L. were also present in mitochondria isolated from cell suspension cultures and from green leaves of this plant. These results support the suggestion that the plasmid-like molecules found in mitochondria of V. faba are an essential component of the mitochondrial genome. The minicircular DNAs were, apparently, peculiar for the species V. faba since they were found in all three cultivars of this species which were studied. The distribution pattern of plasmid-like DNAs in Vicia villosa L. was completely different and mitochondria from Medicago sativa L. also contained specific minicircular DNAs. Thus, minicircular DNAs are typical for the mitochondrial genomes of several legumes and different plant species have their specific mitochondrial plasmid-like DNAs     

The expression of a chimeric Phaseolus vulgaris nodulin 30-GUS gene is restricted to the rhizobially infected cells in transgenic Lotus corniculatus nodules

In Phaseolus vulgaris there is a nodulin family, Npv30, of ca. 30 kDa, as detected in an in vitro translation assay [2]. We isolated a gene (npv30-1) for one of the members of this family. The nucleotide sequence of the promoter of npv30-1 contains nodule-specific motifs common to other late nodulin genes. The promoter was fused to the GUS reporter gene; this chimeric fusion was introduced into Lotus corniculatus via Agrobacterium rhizogenes transformation. GUS activity was only detected in the infected cells of the nodules of transgenic plants. By contrast, the expression of a 35S-GUS construct was restricted to the uninfected cells and the vascular tissue.     

Nodulin gene expression during soybean (Glycine max) nodule development

In vitro translation products of total RNA isolated from soybean nodules at successive stages of nodule development were analyzed by two-dimensional gel electrophoresis. In that way the occurrence of over 20 mRNAs specifically transcribed from nodulin genes was detected. The nodulin genes could be divided into two classes according to the time of expression during nodule development. Class A comprises at least 4 nodulin mRNAs which are found when a globular meristem is present in the root cortex. These class A nodulin genes have a transient expression. Class B nodulin genes are expressed when the formation of a nodule structure has been completed. Bradyrhizobium japonicum nod ⁺ fix^-mutants, with large deletions spanning the nif H,DK region, still induced nodules showing normal expression of all nodulin genes, indicating that the nif H,DK region is not involved in the induction of nodulin genes. In nodules induced by Bradyrhizobium japonicum nod ⁺ fix^-mutant HS124 the bacteria are rarely released from the infection thread and the few infected cells appear to be collapsed. All class A and class B nodulin genes are expressed in HS124 nodules with the exception of 5 class B genes.     

Studying Early Nodulin Gene ENOD40 Expression and Induction by Nodulation Factor and Cytokinin in Transgenic Alfalfa

ENOD40, an early nodulin gene, is expressed following inoculation with Rhizobium meliloti or by adding R. meliloti-produced nodulation (Nod) factors or the plant hormone cytokinin to uninoculated roots. We isolated two MsENOD40 clones, designated MsENOD40–1 and MsENOD40–2, with distinct promoters from an alfalfa (Medicago sativa cv Chief) genomic library. The promoters were fused to the reporter gene uidA (gus), and the constructs were introduced into alfalfa. We observed that the MsENOD40–1 construct was expressed almost exclusively under symbiotic conditions. The MsENOD40–2 construct was transcribed under both symbiotic and nonsymbiotic conditions and in nonnodular and nodular tissues. Both MsENOD40 promoter-gus constructs were similarly expressed as nodules developed, and both were expressed in roots treated with 6-benzylaminopurine or purified Nod factor. However, no blue color was detected in nodule-like structures induced by the auxin transport inhibitor N-1-(naphthyl)phthalamic acid on roots of plants containing the MsENOD40–1 promoter construct, whereas pseudonodules from plants containing the MsENOD40–2 promoter construct stained blue. A 616-bp region at the distal 5′ end of the promoter is important for proper spatial expression of MsENOD40 in nodules and also for Nod-factor and cytokinin-induced expression.