Cells expressing ENOD2 show differential spatial organization during the development of alfalfa root nodules

We have used in situ hybridization to examine the spatial organization of cells expressing the early nodulin gene (ENOD2) during the development of alfalfa root nodules. ENOD2 gene expression was found in the nodule parenchyma, uninfected cells surrounding the symbiotic region of both effective and ineffective nodules. However, in empty nodules, ENOD2 gene expression was found in a mass of parenchyma cells at the base of the nodule. Similar results were also observed in 11-day-old nodules that contained infected cells but that had not yet begun to express leghemoglobin. Although early events of nodulation result in the induction of ENOD2 expression in cells at the nodule base, the pattern of cells expressing ENOD2 during nodule growth appears to be correlated with the development of other peripheral tissues.     

Accumulation of extracellular proteins bearing unique proline-rich motifs in intercellular spaces of the legume nodule parenchyma

Summary. Nodulins encoding repetitive proline-rich cell wall proteins (PRPs) are induced during early interactions with rhizobia, suggesting a massive restructuring of the plant extracellular matrix during infection and nodulation. However, the proteins corresponding to these gene products have not been isolated or characterized, nor have cell wall localizations been confirmed. Posttranslational modifications, conformation, and interactions with other wall polymers are difficult to predict on the basis of only the deduced amino acid sequence of PRPs. PsENOD2 is expressed in nodule parenchyma tissue during nodule organogenesis and encodes a protein with distinctive PRP motifs that are rich in glutamate and basic amino acids. A database search for the ENOD2 signature motifs indicates that similar proteins may have a limited phylogenetic distribution, as they are presently only known from legumes. To determine the ultrastructural location of the proteins, antibodies were raised against unique motifs from the predicted ENOD2 sequence. The antibodies recognized nodule-specific proteins in pea (Pisum sativum), with a major band detected at 110 kDa, representing a subset of PRPs from nodules. The protein was detected specifically in organelles of the secretory pathway and intercellular spaces in the nodule parenchyma, but it was not abundant in primary walls. Similar proteins with an analogous distribution were detected in soybean (Glycine max). The use of polyclonal antibodies raised against signature motifs of extracellular matrix proteins thus appears to be an effective strategy to identify and isolate specific structural proteins for functional analysis. Correspondence and reprints: Delaware Biotechnology Institute, Newark, DE 19711, U.S.A.     

Expression of LjENOD40 genes in response to symbiotic and non-symbiotic signals: LjENOD40-1 and LjENOD40-2 are differentially regulated in Lotus japonicus

Nitrogen fixation in nodules provides leguminous plants with an ability to grow in nitrogen-starved soil. Infection of the host plants by microsymbionts triggers various physiological and morphological changes during nodule formation. In Lotus japonicus, expression of early nodulin (ENOD) genes is triggered by perception of bacterial signal molecules, nodulation factors (Nod factors). We examined the expression patterns of ENOD40 genes during the nodule formation process. Two ENOD40 genes of L. japonicus were specifically expressed in the nodule formation process, but they showed different expression patterns upon infection. Each ENOD40 gene demonstrates an individual specificity and regulation with regard to rhizobial infection.     

ENOD12, an early nodulin gene, is not required for nodule formation and efficient nitrogen fixation in alfalfa.

To demonstrate the importance of an extensively studied early nodulin gene ENOD12 in symbiotic nodule development, plants of different Medicago sativa subspecies were tested for the presence or absence of ENOD12 alleles. In M. s. ssp coerulea w2 (Mcw2), two ENOD12 genes were detected, whereas in M. s. ssp quasifalcata k93 (Mqk93) only one gene was present. In both plants, the ENOD12 genes were expressed in nodules induced by Rhizobium meliloti. The nucleotide sequence of the ENOD12 genes showed that the two Mcw2-specific genes were similar to the ENOD12A and ENOD12B genes of the tetraploid M. s. ssp sativa. ENOD12 from Mqk93 was similar to the corresponding gene found in M. truncatula. From the aligned ENOD12 sequences, an evolutionary tree was constructed. Genetic analysis of the progenies of a cross between Mqk93 and Mcw2 showed that several offspring in F1 carried a null allele originating from Mcw2, and among the F2 progenies, plants with the null allele only lacking the ENOD12 gene appeared. Surprisingly, the ENOD12-deficient plants were similar to their wild-type parents in viability, nodule development, nodule structure, and nitrogen fixation efficiency. Therefore, we concluded that in Medicago the ENOD12 gene is not required for symbiotic nitrogen fixation. Furthermore, we proposed that the heterozygous nature of these legumes can be exploited for the identification of mutated alleles of other known nodulin genes; this will permit the construction of plant mutants deficient in these genes.     

cDNA cloning and developmental expression of pea nodulin genes

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.     

Comparison of soybean and pea ENOD40 cDNA clones representing genes expressed during both early and late stages of nodule development

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.     

Expression of the early nodulin, ENOD40, in soybean roots in response to various lipo-chitin signal molecules

The lipo-chitin (LCO) nodulation signal (nod signal) purified from Bradyrhizobium japonicum induced nodule primordia on soybean (i.e. Glycine soja) roots. These primordia were characterized by a bifurcated vascular connection, cortical cell division, and the accumulation of mRNA of the early nodulin gene, ENOD40. A chemically synthesized LCO identical in structure to the Nod signal purified from B. japonicum cultures showed the same activity when inoculated on to soybean roots. Surprisingly, synthetic LCO or chitin pentamer, inactive in inducing root hair curling (HAD) or cortical cell division (NOI) in G. soja, induced the transient accumulation of ENOD40 mRNA. In roots inoculated with such LCO, ENOD40 mRNA was abundant at 40 h after inoculation but decreased to the background levels 6 days after inoculation. In contrast, nod signals active in inducing HAD and NOI induced high levels of ENOD40 accumulation at 40 h and 6 days after inoculation. In situ hybridization analysis showed that ENOD40 mRNA accumulated in the pericycle of the vascular bundle at 24 h after root inoculation with nod signal. At 6 days post-inoculation with nod signal, ENOD40 expression was seen in dividing subepidermal cortical cells. These results provide morphological and molecular evidence that nodule induction in soybean in response to purified or synthetic nod signal is similar, if not identical, to nodule formation induced by bacterial inoculation. Surprisingly, ENOD40 mRNA accumulation occurs in response to non-specific chitin signals. This suggests that, in the case of ENOD40, nodulation specificity is not determined at the level of initial gene expression.     

Nuclear factors interact with conserved A/T-rich elements upstream of a nodule-enhanced glutamine synthetase gene from French bean.

The gln-gamma gene, encoding the gamma subunit of glutamine synthetase in French bean (Phaseolus vulgaris), is strongly induced during nodule development. We have determined the nucleotide sequence of a 1.3-kilobase region at its 5' end and have identified several sequences common to the promoter regions of late nodulin genes from other legume species. The 5'-flanking region was analyzed for sequence-specific interactions with nuclear factors from French bean. A factor from nodules (PNF-1) was identified that binds to multiple sites between -860 and -154, and a related but distinct factor (PRF-1) was detected in extracts from uninfected roots. PNF-1 and PRF-1 bound strongly to a synthetic oligonucleotide containing the sequence of an A/T-rich 21-base pair imperfect repeat found at positions -516 and -466. The same factors also had a high affinity for a protein binding site from a soybean leghemoglobin gene and appeared to be closely related to the soybean nodule factor NAT2, which binds to A/T-rich sequences in the lbc3 and nodulin 23 genes [Jacobsen et al. (1990). Plant Cell 2, 85-94]. Comparison of NAT2/PNF-1 binding sites from a variety of nodulin genes revealed the conservation of the short consensus core motif TATTTWAT, and evidence was obtained that this sequence is important for protein recognition. Cross-recognition by PNF-1 of a protein binding site in a soybean seed protein gene points to the existence of a ubiquitous family of factors with related binding affinities. Our data suggest that PNF-1 and PRF-1 belong to an evolutionarily conserved group of nuclear factors that interact with specific A/T-rich sequences in a diverse set of plant genes. We consider the possible role of these factors in coregulating the expression of gln-gamma and other late nodulin genes.