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.     

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.     

Comparison of nodule induction in legume and actinorhizal symbioses: the induction of actinorhizal nodules does not involve ENOD40

Two types of root nodule symbioses are known for higher plants, legume and actinorhizal symbioses. In legume symbioses, bacterial signal factors induce the expression of ENOD40 genes. We isolated an ENOD40 promoter from an actinorhizal plant, Casuarina glauca, and compared its expression pattern in a legume (Lotus japonicus) and an actinorhizal plant (Allocasuarina verticillata) with that of an ENOD40 promoter from the legume soybean (GmENOD40-2). In the actinorhizal Allocasuarina sp., CgENOD40-GUS and GmENOD40-2-GUS showed similar expression patterns in both vegetative and symbiotic development, and neither promoter was active during nodule induction. The nonsymbiotic expression pattern of CgENOD40-GUS in the legume genus Lotus resembled the nonsymbiotic expression patterns of legume ENOD40 genes; however, in contrast to GmENOD40-2-GUS, CgENOD40-GUS was not active during nodule induction. The fact that only legume, not actinorhizal, ENOD40 genes are induced during legume nodule induction can be linked to the phloem unloading mechanisms established in the zones of nodule induction in the roots of both types of host plants.     

VsENOD5, VsENOD12 and VsENOD40 expression during Rhizobium-induced nodule formation on Vicia sativa roots

We isolated ENOD5, ENOD12 and ENOD40 homologues from Vicia sativa and studied their expression pattern during Rhizobium-induced nodule formation. Comparison of the VsENOD40 nucleotide sequence with the pea, soybean and alfalfa ENOD40 sequences showed that the sequences contain two conserved regions, called region I and region II. Comparison of all the potential open reading frames (ORFs) showed that all the five different ENOD40 clones encode a highly conserved small polypeptide of 12 or 13 amino acids encoded by an ORF located in region I. Furthermore we studied with in situ hybridization the expression pattern of VsENOD5, VsENOD12 and VsENOD40 during Rhizobium-induced nodule formation. Although the expression of these genes is largely similar to that of the pea counterparts, differences where found for the expression of VsENOD12 and VsENOD40 in Vicia. VsENOD12 is expressed in the whole prefixation zone II, whereas in pea ENOD12 is only expressed in the distal part of this zone. VsENOD40 is expressed in the uninfected cells of interzone II–III; while in pea ENOD40 is expressed in both the uninfected and infected cells of this zone.     

The aberrant cell walls of boron-deficient bean root nodules have no covalently bound hydroxyproline-/proline-rich proteins.

B-deficient bean (Phaseolus vulgaris L.) nodules examined by light microscopy showed dramatic anatomical changes, mainly in the parenchyma region. Western analysis of total nodule extracts examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that one 116-kD polypeptide was recognized by antibodies raised against hydroxyproline-rich glycoproteins (HRGPs) from the soybean (Glycine max) seed coat. A protein with a comparable molecular mass of 116 kD was purified from the cell walls of soybean root nodules. The amino acid composition of this protein is similar to the early nodulin (ENOD2) gene. Immunoprecipitation of the soybean ENOD2 in vitro translation product showed that the soybean seed coat anti-HRGP antibodies recognized this early nodulin. Furthermore, we used these antibodies to localize the ENOD2 homolog in bean nodules. Immunocytochemistry revealed that in B-deficient nodules ENOD2 was absent in the walls of the nodule parenchyma. The absence of ENOD2 in B-deficient nodules was corroborated by performing hydroxyproline assays. Northern analysis showed that ENOD2 mRNA is present in B-deficient nodules; therefore, the accumulation of ENOD2 is not affected by B deficiency, but its assembly into the cell wall is. B-deficient nodules fix much less N2 than control nodules, probably because the nodule parenchyma is no longer an effective O2 barrier.     

The GmFWL1 (FW2‐2‐like) nodulation gene encodes a plasma membrane microdomain‐associated protein

The soybean gene GmFWL1 (FW2‐2‐like1) belongs to a plant‐specific family that includes the tomato FW2‐2 and the maize CNR1 genes, two regulators of plant development. In soybean, GmFWL1 is specifically expressed in root hair cells in response to rhizobia and in nodules. Silencing of GmFWL1 expression significantly reduced nodule numbers supporting its role during soybean nodulation. While the biological role of GmFWL1 has been described, its molecular function and, more generally, the molecular function of plant FW2‐2‐like proteins is unknown. In this study, we characterized the role of GmFWL1 as a membrane microdomain‐associated protein. Specifically, using biochemical, molecular and cellular methods, our data show that GmFWL1 interacts with various proteins associated with membrane microdomains such as remorin, prohibitins and flotillins. Additionally, comparative genomics revealed that GmFWL1 interacts with GmFLOT2/4 (FLOTILLIN2/4), the soybean ortholog to Medicago truncatula FLOTILLIN4, a major regulator of the M. truncatula nodulation process. We also observed that, similarly to MtFLOT4 and GmFLOT2/4, GmFWL1 was localized at the tip of the soybean root hair cells in response to rhizobial inoculation supporting the early function of GmFWL1 in the rhizobium infection process.     

Surprising developments in the legume root nodule

Root nodule morphogenesis involves the induction of mitotic activity in otherwise quiescent root cortical cells, giving rise to the nodule primordium. One gene expressed during nodule initiation, ENOD40, has been implicated in nodule growth and/or differentiation^(1,2). Interestingly, although the nucleotide sequence of ENOD40 genes from soybean^(3,4) and Medicago^(1,2) are highly homologous, they are unlikely to encode a similar protein product. In fact, a remarkable feature of these genes is their apparent lack of protein coding potential. Thus, ENOD40 is a member of the growing list of eukaryotic genes whose RNA product is implicated in control of cell growth and differentiation, the so called riboregulators⁽⁵⁾.     

Characterization of the pea ENOD12B gene and expression analyses of the two ENOD12 genes in nodule, stem and flower tissue

Summary 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 tissues-pecific 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. These results show that two closely related genes have the same tissue-specific expression pattern and that the gene that we have isolated is an actively transcribed gene. The 2.7 kb genomic region that contains the PsENODI2B gene has a 41 pb nearly direct repeat in the 5 flanking region of the gene (between -1447 and -1153) and another 14 by direct repeat 3' downstream (between 550 and 626). The region between the AGGA box and the TATA box has a striking homology with the same region in SbPRP genes.