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.     

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.     

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.     

Characterization of nodule-specific cDNA clones from Sesbania rostrata and expression of the corresponding genes during the initial stages of stem nodules and root nodules formation

We have constructed a Sesbania rostrata stem nodule-specific cDNA library. By screening with heterologous probes from pea and soybean, we have isolated several nodulin cDNA clones. On the basis of nucleotide and amino acid sequence homology, two nearly full-length cDNA clones coding for two different leghemoglobin-like proteins have been identified. The inserts of two other clones reveal a high degree of amino acid sequence homology (81% and 72%) to the early nodulin Enod2 from soybean; the characteristic heptapeptide repeat units PPHEKPP and PPYEKPP of the soybean Enod2 are conserved in the proteins encoded by these Sesbania cDNA clones. The time course of Enod2 and leghemoglobin mRNA appearance during the formation of stem nodules and root nodules on S. rostrata was analyzed by northern blot hybridization. Significant differences were found for the initiation of mRNA accumulation of these nodulins between S. rostrata and soybean.     

Tissue- and stage-specific expression of a soybean (Glycine max L.) seed-maturation,biotinylated protein

A cDNA clone GmPM4 which encodes mRNA species in mature or dry soybean seeds was characterized. DNA sequence analysis shows that the deduced polypeptides have a molecular mass of 68 kDa. GmPM4 proteins have a relatively high amino acid sequence homology with a major biotinylated protein isolated from pea seeds, SBP65, but both of these proteins differ markedly from that of presently known biotin enzymes. The accumulation of GmPM4 mRNA is detectable in the leaf primodium and the vascular tissues of the hypocotyl-radicle axis of mature seeds, and the GmPM4 proteins are present at high levels in dry and mature soybean seeds, but not in fresh immature seeds. It degrades rapidly at the early stage of seed germination. These proteins are boiling-soluble and biotinylated when they are present endogenously in soybean seeds; however, the same recombinant protein expressed in Escherichia coli is boiling-soluble, but it is not biotinylated.     

Repression of the pea lipoxygenase gene loxg is associated with carpel development

A cDNA clone (loxg) corresponding to a gene repressed during carpel development has been isolated from a cDNA library of unpollinated carpels induced to grow by treatment with gibberellic acid (GA₃). The sequences of loxg cDNA and the deduced polypeptide have a high similarity with legume type 2 lipoxygenases, especially with Phaseolus lox1 (78.5% similarity at the protein level) and pea and soybean lox3 (83.6% and 85.4%, respectively). loxg expression is constant in unstimulated carpels but it decreases in carpels induced to keep growing by fertilization or hormone treatment. A similar pattern of repression was observed in lipoxygenase activity of pea and tomato carpels. In situ hybridization studies showed that loxg mRNAs are present in the endocarp and the mesocarp of pea pods; no loxg expression was detectable either in the pod exocarp or in the ovules. Loxg is also expressed in other young growing tissues, especially in flower organs. Nevertheless, the natural pattern of flower and fruit development is associated with loxg repression.     

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.