Lotus japonicus Contains Two Distinct ENOD40 Genes That Are Expressed in Symbiotic, Nonsymbiotic, and Embryonic Tissues

ENOD40, an early nodulin gene, has been postulated to play a significant role in legume root nodule ontogenesis. We have isolated two distinct ENOD40 genes from Lotus japonicus. The transcribed regions of the two ENOD40 genes share 65% homology, while the two promoters showed no significant homology. Both transcripts encode a putative dodecapeptide similar to that identified in other legumes forming determinate nodules. Both ENOD40 genes are coordinately expressed following inoculation of roots with Mesorhizobium loti or treatment with purified Nod factors. In the former case, mRNA accumulation could be detected up to 10 days following inoculation while in the latter case the accumulation was transient. High levels of both ENOD40 gene transcripts were found in nonsymbiotic tissues such as stems, fully developed flowers, green seed pods, and hypocotyls. A relatively lower level of both transcripts was observed in leaves, roots, and cotyledons. In situ hybridization studies revealed that, in mature nodules, transcripts of both ENOD40 genes accumulate in the nodule vascular system; additionally, in young seed pods strong signal is observed in the ovule, particularly in the phloem and epithelium, as well as in globular stage embryos.

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Responses of a Model Legume Lotus japonicus to Lipochitin Oligosaccharide Nodulation Factors Purified from Mesorhizobium loti JRL501

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.7.848 ◽

2001 ◽

Vol 14 (7) ◽

pp. 848-856 ◽

Cited By ~ 52

Author(s):

Shinobu Niwa ◽

Masayoshi Kawaguchi ◽

Haruko Imaizumi-Anraku ◽

Svetlana A. Chechetka ◽

Masumi Ishizaka ◽

...

Keyword(s):

High Performance ◽

Molecular Genetic ◽

Lotus Japonicus ◽

Cytoplasmic Bridge ◽

Cortical Cells ◽

Model Legume ◽

Mesorhizobium Loti ◽

Root Hair Deformation ◽

Early Nodulin ◽

Microbe Interactions

Lotus japonicus has been proposed as a model legume for molecular genetic studies of symbiotic plant-microbe interactions leading to the fixation of atmospheric nitrogen. Lipochitin oligosaccharides (LCOs), or Nod factors, were isolated from the culture of Mesorhizobium loti strain JRL501 (MAFF303099), an efficient microsymbiont of L. japonicus B-129 cv. Gifu. High-performance liquid chromatography and mass spectrometric analyses allowed us to identify at least five different structures of LCOs that were produced by JRL501. The major component was NodMl-V(C18:1, Me, Cb, AcFuc), an N-acetyl-glucosamine pentamer in which the nonreducing residue is N-acylated with a C18:1 acyl moiety, N-methylated, and carries a carbamoyl group and the reducing N-acetyl-glucosamine residue is substituted with 4-O-acetyl-fucose. Additional novel LCO structures bearing fucose instead of acetyl-fucose at the reducing end were identified. Mixtures of these LCOs could elicit abundant root hair deformation on L. japonicus roots at a concentration of 10-7 to 10-9 M. Spot inoculation of a few nanograms of LCOs on L. japonicus roots induced the formation of nodule primordia in which the early nodulin genes, ENOD40 and ENOD2, were expressed in a tissue-specific manner. We also observed the formation of a cytoplasmic bridge (preinfection thread) in the swollen outermost cortical cells. This is the first description of cytoplasmic bridge formation by purified LCOs alone in a legume-forming determinate nodules.

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087 Localization of ENOD2 Transcript Accumulation in Indeterminant Nodules of Maackia amurensis Rupr. & Maxim. (Amur Maackia)

HortScience ◽

10.21273/hortsci.34.3.456c ◽

1999 ◽

Vol 34 (3) ◽

pp. 456C-456

Author(s):

Harry T. Horner ◽

David J. Hannapel ◽

William R. Graves ◽

Carol M. Foster ◽

David J. Hannapel ◽

...

Keyword(s):

In Situ Hybridization ◽

Periodic Acid ◽

Nodule Development ◽

Coding Region ◽

Transcript Accumulation ◽

Mrna Accumulation ◽

Peripheral Tissues ◽

Maackia Amurensis ◽

Early Nodulin

Early nodulin genes, such as ENOD2, play a role in the first stages of nodulation. Although ENOD2 is conserved among nodulating legumes studied to date, its occurrence and activity have not been studied among woody legumes such as Maackia amurensis Rupr. & Maxim. Our objective was to localize MaENOD2 transcripts during nodule development and describe the anatomy of nodules formed on the roots of M. amurensis in relation to ENOD2 mRNA accumulation. Nodules (<1 mm, 1-2 mm, >2 mm in diameter, and mature) were prepared for light microscopy, sectioned, and stained with safranin and fast green for structural contrast or with the periodic acid Schiff's reaction for starch. The location of ENOD2 transcripts was determined by using in situ hybridization with DIG-labeled sense and antisense RNAs transcribed from a 602-bp fragment of the coding region of MaENOD2. Mature nodules from M. amurensis possessed peripheral tissues, a distal meristem, and a central infected region characteristic of indeterminant development. In situ hybridization showed that MaENOD2 transcripts accumulated in the distribution layer and uninfected cells of the central symbiotic region. Amyloplasts that contained starch grains were identified in these tissues and in the inner parenchyma of the nodule. Throughout nodule development, transcripts were restricted to areas with high levels of stored starch that surrounded cells actively fixing N2. Our results suggest that ENOD2 in M. amurensis may be a cell wall component of tissues that regulate nutrient flow to and from sinks, such as symbiotic regions of a nodule. These data may lead to a better understanding of the role of the ENOD2 gene family during nodulation.

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Genome organization, functional analysis of biosynthetic genes and metabolic diversity of triterpenes in legumes

10.12681/eadd/35550 ◽

2013 ◽

Author(s):

Αφροδίτη Κροκιδά

Keyword(s):

Functional Analysis ◽

Arabidopsis Thaliana ◽

Genome Organization ◽

Nicotiana Benthamiana ◽

Lotus Japonicus ◽

Metabolic Diversity ◽

Biosynthetic Genes ◽

Mesorhizobium Loti

Τα τριτερπένια είναι φυτικοί δευτερογενείς μεταβολίτες που προέρχονται από την κυκλοποίηση του 2,3-οξειδοσκουαλενίου από εξειδικευμένα ένζυμα, τις συνθάσες ή κυκλάσες του οξειδοσκουαλενίου. Ο ρόλος της συνθάσης της λουπεόλης, που κωδικοποιείται από το γονίδιο OSC3, καθώς και το προϊόν του, λουπεόλη, διερευνήθηκε σε αναπτυσσόμενες ρίζες και φυμάτια του μοντέλου-ψυχανθούς Lotus japonicus. Τα πρότυπα έκφρασης του γονιδίου OSC3 σε διάφορα αναπτυξιακά στάδια μη μολυσμένων και μολυσμένων ριζών με το συμβιώτη Mesorhizobium loti καθορίστηκαν. Η ιστοειδική έκφραση του γονιδίου OSC3 καθορίστηκε με in situ υβριδισμό. Πραγματοποιήθηκε λειτουργική ανάλυση σε μετασχηματισμένες ρίζες που παρουσίαζαν αποσιώπηση του γονιδίου OSC3. Στις μετασχηματισμένες ρίζες καθορίστηκε η απουσία παραγωγής λουπεόλης με GC-MS. Τα μετασχηματισμένα φυτά παρουσίασαν το φαινότυπο της πιο ταχείας φυματιογένεσης σε σύγκριση με τα φυτά – μάρτυρες. Επιπλέον, η έκφραση του γονιδίου ENOD40, γονίδιο δείκτης της έναρξης σχηματισμού των καταβολών των φυματίων, αυξήθηκε σημαντικά στα μετασχηματισμένα φυτά. Η εξωγενής παροχή λουπεόλης σε αγρίου τύπου φυτά παρείχε επιπλέον ενδείξεις για τον αρνητικό ρυθμιστικό ρόλο της λουπεόλης στην έκφραση του γονιδίου ENOD40. Η βιοσύνθεση της λουπεόλης μπορεί να αποδωθεί αποκλειστικά στο γονίδιο OSC3. Λαμβάνοντας όλα αυτά υπ’όψιν, προτείνεται στη λουπεόλη ένας ρόλος στο σχηματισμό των φυματίων μέσω της ρύθμισης της έκφρασης του γονιδίου ENOD40. Επιπρόσθετα, τα γονίδια που συμμετέχουν σε μονοπάτια βιοσύνθεσης τριτερπενοειδών οργανώνονται σε γονιδιακές συστοιχίες στα φυτά της βρώμης και του Arabidopsis thaliana. Η παρουσία μιας ανάλογης γονιδιακής συστοιχίας στο φυτό L. japonicus ταυτοποιήθηκε. Στη γονιδιωματική περιοχή που εδράζεται το γονίδιο της συνθάσης της β-αμυρίνης, εντοπίστηκαν δύο γονίδια του κυτοχρώματος Ρ450 καθώς και μία ρεδουκτάση. Ο λειτουργικός χαρακτηρισμός της γονιδιακής συστοιχίας πραγματοποιήθηκε με ετερόλογη έκφραση σε φύλλα Nicotiana benthamiana. Μια καινούρια δομή τριτερπενοειδούς, η διυδρολουπεόλη, παράγεται από την συνθάση της β-αμυρίνης. Επιπλέον, ένα καινούριο ένζυμο του κυτοχρώματος Ρ450, το CYP71D353, χαρακτηρίστηκε, το οποίο καταλύει το σχηματισμό του 20-hydroxybetulinic acid μέσω τριών διαδοχικών βημάτων οξείδωσης της διυδρολουπεόλης. Τα πρότυπα έκφρασης των γονιδίων μελετήθηκαν σε διαφορετικές αναπτυξιακές και περιβαλλοντικές συνθήκες. Τα γονίδια της συστοιχίας συν-εκφράζονται κατά τη διάρκεια της ανάπτυξης των φυματίων και των ριζών, σε φυτά που υποβληθήκαν σε καταπονήσεις και σε φυτά που αναπτύχθηκαν παρουσία ορμονών. Ο φυσιολογικός ρόλος της γονιδιακής συστοιχίας στη φυματιογένεση και στην φυτική ανάπτυξη μελετήθηκε σε μετασχηματισμένα φυτά με αποσιώπηση. Επιπλέον, αποκαλύφθηκε ένας μηχανισμός επιγενετικής τροποποίησης που πιθανά εμπλέκεται στη ρύθμιση της γονιδιακής συστοιχίας. Μια γονιδιακή συστοιχία που αποτελείται από λειτουργικά συσχετιζόμενα γονίδια εμπλέκεται στη βιοσύνθεση τριτερπενοειδών στα ψυχανθή και πιθανά εμπλέκεται στη φυτική ανάπτυξη.

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Isolation and characterization of a cDNA clone encoding polyubiquitin from the root nodule ofElaeagnus umbellata

Canadian Journal of Botany ◽

10.1139/b99-084 ◽

1999 ◽

Vol 77 (9) ◽

pp. 1270-1278

Author(s):

Ho Bang Kim ◽

Chung Sun An

Keyword(s):

Amino Acids ◽

In Situ Hybridization ◽

Amino Acid ◽

Cdna Clone ◽

Root Nodule ◽

Vascular System ◽

Northern Hybridization ◽

Elaeagnus Umbellata ◽

Isolation And Characterization

A cDNA clone encoding polyubiquitin was isolated from a root nodule cDNA library of Elaeagnus umbellata Thunb. by differential hybridization, and its molecular aspects were characterized. The polyubiquitin clone pEuNOD-PUB1 has an insert size of 1642 bp and has the capacity to code for a 458 amino acid residue polyubiquitin protein. The derived amino acid sequence indicates that pEuNOD-PUB1 encodes a polyprotein consisting of six repeats of ubiquitin monomer, except for the last repeat, which has two additional amino acids (Asp-Phe) to be removed in the course of polyubiquitin processing into monomer. The molecular mass of ubiquitin monomer, consisting of 76 amino acids, was predicted to be 8524 Da, and the pI value was predicted to be 7.57. The nucleotide sequence of ubiquitin monomers from pEuNOD-PUB1 showed 73.1-86.0% sequence similarity with those from other organisms. The polyubiquitin mRNA content was four to six times higher in the root nodules than in the leaves and roots. In situ hybridization results showed polyubiquitin transcripts were strongly detected in the meristem zone, in infected cells of the fixation zone, and in the central vascular system. Genomic Southern hybridization revealed that polyubiquitin genes are present as a small multigene family in the genome of E. umbellata.Key words: Elaeagnus umbellata, root nodule, cDNA, polyubiquitin, Northern hybridization, in situ hybridization.

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Nitric Oxide Detoxification by Mesorhizobium loti Affects Root Nodule Symbiosis with Lotus japonicus

Microbes and Environments ◽

10.1264/jsme2.me21038 ◽

2021 ◽

Vol 36 (3) ◽

pp. n/a

Author(s):

Mitsutaka Fukudome ◽

Yuta Shimokawa ◽

Shun Hashimoto ◽

Yusuke Maesako ◽

Nahoko Uchi-Fukudome ◽

...

Keyword(s):

Nitric Oxide ◽

Root Nodule ◽

Lotus Japonicus ◽

Mesorhizobium Loti ◽

Root Nodule Symbiosis ◽

Nitric Oxide Detoxification ◽

Nodule Symbiosis

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Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162545 ◽

1996 ◽

Vol 54 ◽

pp. 16-17

Author(s):

J. R. Hully ◽

K. R. Luehrsen ◽

K. Aoyagi ◽

C. Shoemaker ◽

R. Abramson

Keyword(s):

Nucleic Acids ◽

Viral Infections ◽

Anatomical Location ◽

Rt Pcr ◽

Reverse Transcription Pcr ◽

Cellular Source ◽

Gene Transcripts ◽

Initial Description ◽

In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

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Comparison of Nodule Induction in Legume and Actinorhizal Symbioses: The Induction of Actinorhizal Nodules Does Not Involve ENOD40

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2003.16.9.808 ◽

2003 ◽

Vol 16 (9) ◽

pp. 808-816 ◽

Cited By ~ 22

Author(s):

Carole Santi ◽

Uritza von Groll ◽

Ana Ribeiro ◽

Maurizio Chiurazzi ◽

Florence Auguy ◽

...

Keyword(s):

Expression Pattern ◽

Host Plants ◽

Root Nodule ◽

Higher Plants ◽

Expression Patterns ◽

Lotus Japonicus ◽

Casuarina Glauca ◽

Actinorhizal Plant ◽

Allocasuarina Verticillata ◽

Actinorhizal Nodules

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 (GmENOD402). 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.

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Developmental expression of mouse stromelysin-3 mRNA

Development ◽

10.1242/dev.121.4.947 ◽

1995 ◽

Vol 121 (4) ◽

pp. 947-955 ◽

Cited By ~ 2

Author(s):

O. Lefebvre ◽

C. Regnier ◽

M.P. Chenard ◽

C. Wendling ◽

P. Chambon ◽

...

Keyword(s):

Spinal Cord ◽

Mesenchymal Cells ◽

Developmental Expression ◽

Spatiotemporal Pattern ◽

Commissural Axons ◽

Embryonic Tissues ◽

Trophoblastic Cells ◽

Neuroepithelial Cells ◽

Stromelysin 3

We have used northern blot analysis and in situ hybridization to study the spatial distribution of stromelysin-3 (ST3) expression during mouse embryogenesis. ST3 mRNA was observed in trophoblastic cells at the site of embryonic implantation (7.5-8.5 days) and in a variety of developing embryonic tissues. In these tissues, the highest ST3 expression levels were observed during the development of the external features of limb, tail and snout, and during bone and spinal cord morphogenesis. In limb, tail and snout, ST3 expression was specifically detected in mesenchymal cells lining the basement membrane at the junction of primitive dermis and epidermis, and adjacent to epithelial cells undergoing proliferation and/or apoptosis. In bone, ST3 was expressed in invasive mesenchymal cells and, in the spinal cord in neuroepithelial cells of the floor plate, at the time that this structure is crossed by commissural axons. Altogether, these observations suggest a role for ST3 during embryonic morphogenesis, in tissue remodeling processes associated with cell proliferation, death and/or invasion. Moreover, when compared to urokinase and tissue plasminogen activators, the spatiotemporal pattern of ST3 expression shows some similarities, but was not completely superimposable, suggesting that these genes may cooperate in some developing tissues and have specific functions in others.

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Novel tenascin variants with a distinctive pattern of expression in the avian embryo

Development ◽

10.1242/dev.120.3.637 ◽

1994 ◽

Vol 120 (3) ◽

pp. 637-647

Author(s):

R.P. Tucker ◽

J. Spring ◽

S. Baumgartner ◽

D. Martin ◽

C. Hagios ◽

...

Keyword(s):

Genomic Library ◽

Chicken Embryo Fibroblast ◽

Cdna Probe ◽

Avian Embryo ◽

Degenerate Primers ◽

Type Iii ◽

Embryonic Tissues ◽

Fibronectin Type Iii ◽

Fibronectin Type

Previous studies have shown that several forms of the glycoprotein tenascin are present in the embryonic extracellular matrix. These forms are the result of alternative splicing, which generates tenascin variants with different numbers of fibronectin type III repeats. We have used degenerate primers and PCR to isolate a novel tenascin exon from an avian genomic library. Genomic clones contained a sequence encoding a fibronectin type III repeat that corresponds to repeat ‘C’ from the variable domain of human tenascin. To demonstrate that tenascin containing repeat ‘C’ is actually synthesized by avian cells, a monospecific antiserum was raised against a repeat ‘C’ fusion protein. This antiserum recognized a novel high-molecular-weight variant on immunoblots of tenascin isolated from chicken embryo fibroblast-conditioned medium, and stained tendons on frozen sections of chicken embryos. A cDNA probe specific for mRNA encoding repeat ‘C’ was used for in situ hybridization. This probe hybridized in a subset of the embryonic tissues labelled with a universal tenascin probe, including tendons, ligaments and mesenchyme at sites of epithelial-mesenchymal interactions. Finally, we provide evidence that additional fibronectin type III repeats, one corresponding to a recently discovered human repeat as well as one entirely novel sequence, also exists in chicken tenascin mRNA. These data indicate that tenascin is present in the embryonic matrix in a multitude of forms and that these forms have distinctive distributions that may reflect more than one function for tenascin in development.

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