Martin Frühling

The promoter of the Vicia faba L. leghemoglobin gene VfLb29 is specifically activated in the infected cells of root nodules and in the arbuscule-containing cells of mycorrhizal roots from different legume and nonlegume plants

The VfLb29 leghemoglobin gene promoter was polymerase chain reaction-amplified from a Vicia faba genomic library and was fused to the gusAint coding region. Expression of the chimeric gene was analyzed in transgenic hairy roots of the legumes V. faba, V. hirsuta, and Medicago truncatula as well as in transgenic Nicotiana tabacum plants. The VfLb29 promoter was found to be specifically active not only in the infected cells of the nitrogen-fixing zone of root nodules but also in arbuscule-containing cells of transgenic V. faba and M. truncatula roots colonized by the endomycorrhizal fungus Glomus intraradices. In addition to these two legumes, specific expression in arbuscule-containing cells was also observed in the nonlegume N. tabacum. All studies were done in comparison to the V. faba leghemoglobin gene promoter VfLb3 that as VfLb29 was expressed in the infected cells of root nodules but showed no activity in endomycorrhiza. An activation of the VfLb29 promoter due to hypoxia in metabolically active tissues was excluded. The conserved activation in arbuscule-containing cells of legumes and the nonlegume N. tabacum suggests a conserved trigger for this promoter in legume and nonlegume endomycorrhiza symbioses.

The Vicia faba Leghemoglobin Gene VfLb29 Is Induced in Root Nodules and in Roots Colonized by the Arbuscular Mycorrhizal Fungus Glomus fasciculatum

To investigate similarities between symbiotic interactions of broad bean (Vicia faba) with rhizobia and mycorrhizal fungi, plant gene expression induced by both microsymbionts was compared. We demonstrated the exclusive expression of 19 broad bean genes, including VfENOD2, VfENOD5, VfENOD12 and three different leghemoglobin genes, in root nodules. In contrast, the leghemoglobin gene VfLb29 was found to be induced not only in root nodules, but also in broad bean roots colonized by the mycorrhizal fungus Glomus fasciculatum. In uninfected roots, none of the 20 nodulin transcripts investigated was detectable. VfLb29 has an unusually low sequence homology with all other broad bean leghemoglobins as well as with leghemoglobins from other legumes. It can be regarded as a novel kind of leghemoglobin gene not described until now and the induction of which is common to symbiotic interactions of broad bean with both Rhizobium and a mycorrhizal fungus.

The Broad Bean Gene VfNOD32 Encodes a Nodulin with Sequence Similarities to Chitinases That Is Homologous to ([alpha]/[beta])8-Barrel-Type Seed Proteins

Nodulin gene transcripts isolated from a broad bean (Vicia faba L.) root nodule cDNA library and designated VfNOD32 are detectable in the nitrogen-fixing zone III of nodules and in much smaller amounts in flowers. In nodules, these transcripts are detectable for the first time 7 d after inoculation, at least 1 d before leghemoglobin gene transcription starts. Two putative full-length cDNAs representing different transcript sequences of 92.5% identity were sequenced. The corresponding broad bean genes were termed VfNOD32-A1 and VfNOD32-A2, and the encoded proteins were termed Nvf32-A1 and Nvf32-A2. The derived amino acid sequences of the Nvf32 proteins are highly homologous to the Vicia narbonensis ([alpha]/[beta])8-barrel seed protein narbonin. Considering this homology, Nvf32 is assumed to have a similar structure consisting of [beta]-sheets forming a central barrel surrounded by [alpha]-helices. The two Nvf32 sequences also contain two conserved amino acid motifs that are characteristic of class-III chitinases. Several amino acids demonstrated to be essential for chitinase activity are conserved in both regions, whereas one essential glutamic acid was changed to glycine in the Nvf32-A1 isoform but not in the Nvf32-A2 isoform.

The nodule-specific VfENOD-GRP3 gene encoding a glycine-rich early nodulin is located on chromosome I of Vicia faba L. and is predominantly expressed in the interzone II-III of root nodules

A nodule-specific cDNA was isolated from a Vicia faba L. nodule cDNA library. Since time course experiments revealed an early expression of this transcript in the nodule, this cDNA coded for an early nodulin and was designated VfENOD-GRP3. Based on tissue print hybridizations, we found a predominant expression of VfENOD-GRP3 transcripts in the interzone II-III region of broad bean root nodules. The encoded early nodulin ENOD-GRP3 was characterized by an N-terminal signal peptide and a C-terminal domain displaying a glycine content of 31%. Sequence analysis of a genomic VfENOD-GRP3 clone revealed that the signal peptide and the glycine-rich domain were specified by two separate exons. Primer extension experiments identified two adjacent transcription start sites for VfENOD-GRP3 transcripts. The common nodulin sequences ‘AAAGAT’ and ‘CTCTT’ were present five and three times on both DNA strands of the putative VfENOD-GRP3 promoter, respectively. Additionally, three sequence motifs resembling organ-specific elements of the soybean lbc3 gene promoter and a sequence similar to the binding site 1 for the nodule trans-acting factor Nat2 were identified. From Southern blot data and from sequence analysis of genomic PCR fragments, the presence of a VfENOD-GRP3 gene family was inferred. By PCR experiments using sequence-specific primers and DNA of microisolated chromosomes as a template, this family was located on the long arm of chromosome I.

The temporal and spatial transcription pattern in root nodules of Vicia faba nodulin genes encoding glycine-rich proteins

Four different transcript sequences encoding gene products with an unusually high glycine content were identified in Vicia faba root nodules. Northern blot analysis revealed a strong nodule specific expression of the corresponding genes. Time course experiments showed that two of these genes were transcribed before the onset of leghemoglobin expression and hence were designated VfENOD-GRP2 and VfENOD-GRP5, whereas the first detection of VfNOD-GRP1 and VfNOD-GRP4 transcripts coincided with the appearance of leghemoglobin transcripts in V. faba root nodules. A characteristic feature of all encoded nodulins was a hydrophobic N-terminus, which in the case of the nodulins ENOD-GRP2 and ENOD-GRP5 has the characteristics of a signal peptide. Such a structure is comparable to other plant glycine-rich proteins described as components of the plant cell wall. Based on tissue print hybridizations, we found that VfNOD-GRP1, VfENOD-GRP2 and VfNOD-GRP4 were expressed in the interzone II-III and in the whole nitrogen-fixing zone III. In contrast to VfENOD-GRP2 and VfNOD-GRP4, the signal intensity of hybridizing VfNOD-GRP1 transcripts was slightly reduced in the more proximal part of broad bean root nodules. Apart from the interzone II-III and the nitrogen fixing zone III, VfENOD-GRP5 RNA was also detected in large areas of the prefixing zone II.

A small gene family of broad bean codes for late nodulins containing conserved cysteine clusters

Five transcripts encoding different members of a nodulin family with conserved cysteine clusters (Cys-X-4-Asp-Cys and Cys-X-4-Cys) were identified in broad bean root nodules. They displayed homologies to the early nodulins PsENOD3 and PsENOD14 and the late nodulin PsNOD6 from pea. In addition to the occurence of putative secretory signal peptides, the spatial distribution of the cysteine residues was comparable in both the broad bean and the pea nodulins. Based on tissue print hybridizations, we found that the corresponding broad bean genes VfNOD-CCP1, VfNOD-CCP3 and VfNOD-CCP5 were expressed in the interzone II-III and the nitrogen fixing zone III of mature nodules whereas the gene VfNOD-CCP4 was first induced in the prefixing zone II. A strong expression of the VfNOD-CCP2 gene only could be detected the interzone II-III region. Sequence analysis of a genomic VfNOD-CCP1 clone isolated revealed the presence of one intron seperating a first exon encoding the signal peptide from a second exon encoding the cysteine cluster domain of this nodulin. Apart from the multiple presence of the common nodulin motifs AAAGAT and CTCTT on both DNA strands of the putative VfNOD-CCP1 promoter region a sequence element resembling the organ specific element of the soybean lbc3 gene promoter was identified

The Vicia faba lipoxygenase gene VfLOX1 is expressed in the root nodule parenchyma

A full-length cDNA encoding the broad bean lipoxygenase VfLOX1 was isolated from a nodule cDNA library. The VfLOX1 gene was strongly expressed in nodules, and only weakly in roots. VfLOX1 transcripts were localized in the nodule parenchyma and in the cells surrounding the root stele.

Genomic organization and expression properties of the VfENOD5 gene from broad bean (Vicia faba L.).

A full-length cDNA encoding the broad bean (Vicia faba L.) early nodulin VfENOD5 was isolated from a nodule cDNA library. In addition to the ENOD5 homologues from other legumes the derived VfENOD5 amino acid sequence also displayed homologies to the phytocyanin-related nodulins GmENOD55-2, MtENOD16, and MtENOD20. A close inspection of the ENOD5 proteins from broad bean, pea and vetch indicated that all these nodulins possess a putative C-terminal GPI-anchor signal sequence. This novel finding supports the hypothesis that ENOD5 is an arabinogalactan protein. Tissue print hybridizations revealed that the broad bean ENOD5 gene was not only expressed in the central tissues of root nodules. In contrast to other legumes hybridizing transcripts were also be detected in a narrow zone within the peripheral nodule tissues. Sequence analysis of a genomic clone indicated the presence of a single intron interrupting the VfENOD5 coding region at a position precisely corresponding to the MtENOD16 and MtENOD20 introns.

The asparagine synthetase gene VfAS1 is strongly expressed in the nitrogen-fixing zone of broad bean (Vicia faba L.) root nodules

A full-length transcript sequence encoding the broad bean (Vicia faba L.) asparagine synthetase (AS) VfAS1 was isolated from a nodule cDNA library. Sequence homologies indicated that VfAS1 belonged to the glutamine-dependent type of AS enzymes. The corresponding gene was highly expressed in root nodules and at a three-fold lower level in uninfected roots. Additionally, traces of VfAS1 transcripts were detected in epicotyl and stem tissues. Tissue-print hybridizations revealed that the VfAS1 gene was strongly expressed in the nitrogen-fixing zone III of root nodules. VfAS1 transcripts were absent from the meristem, the prefixing zone IE as well as from peripheral nodule tissues

The modular nodulins Nvf-28/32 of broad bean (Vicia faba L.): alternative exon combinations account for different modular structures

The broad bean late nodulins, Nvf-28/32, are composed of two types of repetitively occurring sequence modules flanked by unique N- and C-terminal modules. Six isoforms of these nodulins were characterized by a specific modular structure resulting from a different individual order of repetitive sequence modules. A detailed analysis of genomic PCR fragments revealed that the repetitive modules and the N-terminal unique module exactly corresponded to exons, whereas the C-terminal module was specified by two exons. Since those exons encoding the repetitive modules missing in specific Nvf-28/32 isoforms were consistently present within genomic sequences, a post-transcriptional generation of VfNOD28/32 transcripts specifying six Nvf-28/32 nodulins was concluded. Using tissue-print hybridizations, these transcripts were localized in the interzone II–III and the nitrogen-fixing zone III of root nodules. From this and from cDNA-cDNA hybridizations demonstrating a comparable timing of expression of VfNOD28/32 and of leghemoglobin transcripts in root nodules, a function of the modular nodulins Nvf-28/32 in late developmental stages of broad bean nodules was inferred.