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A pea cDNA clone representing the homologue of the soybean pGmENOD40-1 was isolated and characterized. At the nucleotide level both clones share 55% homology. Strikingly, the homology between the polypeptides derived from the pea and soybean ENOD40 cDNA sequences is only 14%. Despite this low homology Southern analyses revealed that the isolated pea cDNA clone represents the single pea ENOD40. In situ hybridizations showed that at early stages of nodule development and in mature nodules the expression pattern of pea ENOD40 is comparable to that of soybean ENOD40. Although ENOD40 show similar expression patterns in these two nodules, it is questionable whether the putative polypeptides have a similar function, since the homology is very low.

We used a semiquantitative root hair deformation assay for Vicia sativa (vetch) to study the activity of Rhizobium leguminosarum bv viciae nodulation (Nod) factors. Five to 10 min of Nod factor-root interaction appears to be sufficient to induce root hair deformation. The first deformation is visible within 1 h, and after 3 h about 80% of the root hairs in a small susceptible zone of the root are deformed. This zone encompasses root hairs that have almost reached their maximal size. The Nod factor accumulates preferentially to epidermal cells of the young part of the root, but is not restricted to the susceptible zone. In the interaction with roots, the glucosamine backbone of Nod factors is shortened, presumably by chitinases. NodRlv-IV(C18:4,Ac) is more stable than NodRlv-V(C18:4,Ac). No correlation was found between Nod factor degradation and susceptibility. Degradation occurs both in the susceptible zone and in the mature zone. Moreover, degradation is not affected by NH4NO3 and is similar in vetch and in the nonhost alfalfa (Medicago sativa).

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.

This paper shows that lipo-oligosaccharides (Nod factors) synthesized by Rhizobium bacteria elicit the induction of infection-related early nodulin genes (PsENOD5 and PsENOD12) in pea root hairs. R. leguminosarum bv. viciae secretes a mixture of Nod factors containing a C18 fatty acid chain with 4 (C18:4) or 1 double bond (C18:1). Purified Nod factors harbouring either a C18:4 or a C18:1 acyl moiety induce the expression of the pea early nodulin genes, PsENOD5 and PsENOD12, but the kinetics of induction are different. The expression of both early nodulin genes is induced in a transient manner by the purified Nod factors while a mixture of the Nod factors extends the period during which these genes are expressed. In spite of the host-specific nature of the infection process, heterologous Nod factors of R. meliloti also induce the expression of PsENOD5 and PsENOD12 genes, though with a marked delay compared with the homologous compounds.

Two cDNA clones of the soybean early nodulin GmENOD55 were characterized. These clones may represent two members of the soybean early nodulin gene family GmENOD55. GmENOD55 has an N-terminal signal peptide and it contains an internal domain consisting of proline and serine residues. Analyses of nodules lacking infection threads and intracellular bacteria suggest that the GmENOD55 gene is first expressed after release of Bradyrhizobium japonicum in plant cells. This conclusion is supported by in situ hybridization studies showing that the expression is restricted to the infected cell type.

In this paper, the soybean 'early nodulin' clone pGmENOD40 is characterized. The GmENOD40 encoded protein does not contain methionine and does not show homology to proteins identified so far. In situ hybridizations showed that this gene has a complex expression pattern during development of determinate soybean nodules. At early stages of development transcription is induced in dividing root cortical cells, the nodule primordium and the pericycle of the root vascular bundle. In mature soybean nodules, the gene is expressed in the uninfected cells of the central tissue and in the pericycle of the nodule vascular bundles. Studies on nodules devoid of intracellular bacteria and infection threads, showed that the expression of the gene in the nodule primordium is induced in these empty nodules, while the induction of the GmENOD40 gene in the nodule vascular bundle requires the presence of intracellular bacteria or infection threads. A pea cDNA clone homologous to GmENOD40 was isolated to enable in situ hybridization studies on indeterminate nodules. The expression patterns in both determinate and indeterminate nodules suggests that the ENOD40 protein might have a transport function.

The beta-glucuronidase (GUS) activity expressed from the soybean early nodulin ENOD2(B) gene promoter was localized histochemically in nodules of Lotus corniculatus and Trifolium repens. In both the determinate Lotus nodules and the indeterminate Trifolium nodules, activity was found in the parenchyma cells and especially in cells close to the vascular tissue of nodules. The characteristic cell-specific expression of the soybean ENOD2 gene was therefore maintained by the ENOD2(B) promoter in the two developmentally different nodule types. Important DNA elements recognized in transgenic nodules were identified by deletion and hybrid promoter analysis in Lotus corniculatus. An indispensable positive element (PE) and a possible tissue specific element was defined between positions -1792 and -1582 from the transcription start site. Another qualitative control element located between -380 and -53 conferred the ENOD2 characteristic cell type expression on hybrid promoters. This element contains the conserved nodulin gene sequences CTCTT and AAAGAT. In contrast to the ENOD2(B) promoter a chimeric leghemoglobin Ibc3-GUS gene was expressed in the infected cells of both types of nodules. In the indeterminate nodules expression was restricted to the interzone II-III and the active nitrogen-fixing zone III. Interchange of the distal strong positive element (SPE) of Ibc3 and the ENOD2 positive element resulted in an expression pattern different from that observed for the Ibc3 and ENOD2 genes, indicating that different interactions of trans-acting factors are required for regulation of early as well as late nodulin genes.

A number of early nodulin genes are expressed in specific cell types as pea (Pisum sativum) root nodules develop. The Pisum sativum early nodulin PsENOD2 is detected only in the uninfected cells of the nodule parenchyma, whereas PsENOD12 is expressed at two spatially removed sites: in root hairs and adjacent cortical cells, both of which can be invaded by Rhizobium entering through infection threads, and in derivatives of newly divided root inner cortical cells that establish the nodule primordium. We tested whether Rhizobium infection is required for triggering PsENOD12 gene expression by inducing nodule-like structures on Afghanistan pea roots with the auxin transport inhibitor N-(1-naphthyl)phthalamic acid (NPA). These nodule-like structures lack infection threads but resemble Rhizobium-induced nodules in other aspects. For one, both PsENOD2 and PsENOD12 transcripts were detected in these structures. PsENOD2 mRNA was localized by in situ hybridization to a zone equivalent to the nodule parenchyma of Rhizobium-induced nodules, whereas PsENOD12 transcripts were detected in a group of cells comparable to the nodule primordium of developing nodules. In addition, PsENOD12 mRNA was detected in uninfected root hairs 48 h after NPA treatment. These results indicate that infection is not a trigger for PsENOD12 gene expression in Afghanistan pea and rather suggest that the expression of the PsENOD2 and PsENOD12 genes is correlated with the differentiation of specific cell types in the developing nodule.

Certain characteristics of late noduline gene from pea-Nod6 were investigated as part of works of characterization of higher plant genes taking part in symbiotic nitrogen fixation. The complete 450 b.p. long cDNA was sequenced, it's coding sequence includes the open reading frame. The part of DNA containing the corresponding gene from the genomic clone was also sequenced. The predicted Nod6 amino acid sequence has been analyzed and do not reveal the significant homology with any known protein.

The ENOD12 gene family in pea consists of two different members. The cDNA clone, pPsENOD12, represents the PsENOD12A gene. The second ENOD12 gene, PsENOD12B, was selected from a genomic library using pPsENOD12 as a probe and this gene was sequenced and characterized. The coding regions of the two genes are strikingly similar. Both encode proteins having a signal peptide sequence and a region with pentapeptide units rich in prolines. ENOD12A has a series of rather conserved repeating pentapeptide units, whereas in ENOD12B the number of pentapeptide units is less and these are less conserved. From the amino acid sequence it is obvious that the PsENOD12 genes encode proline-rich proteins which are closely related to proteins that have been identified as components of soybean cell walls (SbPRPs). Previously, Northern blot analyses had shown that ENOD12 genes are expressed in a tissue-specific manner. A high expression level is found in Rhizobium-infected roots and in nodules, whereas expression in flower and stem is lower. This raised the question of which gene is expressed where and when. The availability of the sequences of both ENOD12 genes allowed us to analyse the expression of the two genes separately. Specific oligonucleotides were used to copy the ENOD12 mRNAs and to amplify the cDNAs in a polymerase chain reaction. It was demonstrated that in all the tissues containing ENOD12 mRNA, both genes PsENOD12A and PsENOD12B are transcribed and that the relative amounts of PsENOD12A and PsENOD12B mRNA within each tissue are more or less equal. Moreover, the expression pattern during infection and nodule development is the same for the two genes.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of nitrogen fixing root nodules on the roots of leguminous plants is induced by soil bacteria (for example, from the genus Rhizobium). The formation of this plant organ involves specific activation of genes in both plant and bacterium. Analysis of these genes gives insight into the way in which plant and bacterium succeed in coordinating plant 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.

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.

The expression of plant genes involved in the pea-Rhizobium symbiosis was studied by analysing mRNA from root nodules. The RNA was translated in vitro and the translation products were separated by two-dimensional gel electrophoresis. The results show differential expression of nodulin genes during root nodule development. One gene encoding N-40' is expressed at a significant level 5 days before the leghemoglobin genes. Most other nodulin genes are expressed more of less concomitantly with the leghemoglobin genes whereas the N-21 mRNA is only present late during the development. In the development of ineffective root nodules induced by infection with different nod+fix- mutants of R. leguminosarum all nodulin genes are expressed except for the N-21 gene. The results suggest that neither bacteroid development, heme excretion nor nitrogen fixation are essential for the induction of nodulin gene expression in the host plant. Further, it appears that the amount of leghemoglobin in ineffective nodules is regulated at a post-transcriptional level.

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.

A cDNA library prepared from pea nodule poly(A)(+) RNA was screened by differential hybridization with cDNA probes synthesized from root and nodule RNA respectively. From the cDNA clones that hybridized exclusively with the nodule probe five clones, designated pPsNod 6, 10, 11, 13 and 14 and each containing unique sequences, were further characterized together with one leghemoglobin and one "root-specific" cDNA clone. In vitro translation of RNA selected by the pPsNod clones showed that the corresponding genes encode nodulins with molecular weights ranging from 5 800 to 19 000. During pea root nodule development expression of the five PsNod genes starts more or less concomitantly with the onset of nitrogen fixing activity in the nodules and the time course of appearance and accumulation of the nodulin mRNAs is similar to that of leghemoglobin mRNA. In ineffective pea root nodules expression of the PsNod genes is induced but the final accumulation levels of the mRNAs are markedly reduced to various degrees. The expression of another nodulin gene, designated ENOD2, was followed using a heterologous soybean cDNA clone as probe. In pea root nodules the ENOD2 gene is expressed at least five days before the PsNod and leghemoglobin genes, and in contrast to the PsNod mRNAs the concentration of the ENOD2 mRNA is the same in wild type and fix (-) nodules. The results described suggest that in root nodules several regulatory mechanisms exist which determine the final nodulin mRNA amounts accumulating in the root nodule.

The mRNA population in pea root hairs was characterized by means of in vitro translation of total root hair RNA followed by 2-dimensional gel electrophoresis of the translation products. Root hairs contain several mRNAs not detectable in total RNA preparations from roots. Most of these root hair-specific mRNAs occur in elongating root hairs at higher levels than in mature root hairs. The expression of some genes in pea root hairs is typically affected by inoculation with Rhizobium leguminosarum. One gene, encoding RH-42, is specifically induced while the expression of another gene, encoding RH-44, is markedly enhanced. Using R. leguminosarum mutants it was shown that the nodC gene is required for the induction and enhancement of expression of the RH-42 and RH-44 genes, respectively, while the Rhizobium chromosomal gene pss1, involved in exopolysaccharide synthesis, is not essential. After induction of the nod genes with apigenin the bacteria excrete into the culture medium a factor that causes root hair deformation. This deformation factor stimulates the expression of the RH-44 gene but does not induce the expression of the gene encoding RH-42.