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PubMed1999

Does root glutamine synthetase control plant biomass production in lotus japonicus L.?

To investigate the contribution of root cytosolic glutamine synthetase (GS) activity in plant biomass production, two different approaches were conducted using the model legume Lotus japonicus. In the first series of experiments, it was found that overexpressing GS activity in roots of transgenic plants leads to a decrease in plant biomass production. Using (15)N labelling it was shown that this decrease is likely to be due to a lower nitrate uptake accompanied by a redistribution to the shoots of the newly absorbed nitrogen which cannot be reduced due to the lack of nitrate reductase activity in this organ. In the second series of experiments, the relationship between plant growth and root GS activity was analysed using a series of recombinant inbred lines issued from the crossing of two different Lotus ecotypes, Gifu and Funakura. It was confirmed that a negative relationship exists between root GS expression and plant biomass production in both the two parental lines and their progeny. Statistical analysis allowed it to be estimated that at least 13% of plant growth variation can be accounted for by variation in GS activity.

Journal Article↗

PubMed2000

Lupine leghemoglobin I: expression in transgenic Lotus and tobacco tissues.

The proximal parts of the promoters of the genes for symbiotic-type hemoglobins are generally conserved, but the promoter of the lbI gene of lupine (LulbI) shows some unusual structural features. It lacks typical organ-specific elements characteristic of all the leghemoglobin gene promoters described thus far. We have analysed its functional activity in transgenic Lotus corniculatus. A fusion construct between the lbI promoter and the GUS reporter gene was expressed mainly in the central zone of the root nodule, but the product was also detected in the non-nodule root zone and in roots in tissue culture. In roots of transgenic tobacco, the activity of the promoter was only 24% lower than in Lotus nodules. LulbI promoter activity was also detected in tobacco leaves. Lupine hemoglobin I has a higher sequence identity to symbiotic-type hemoglobins and thus it groups within the "Class II" hemoglobins.

Base Sequence↗

PubMed1989

A multiwell lectin-binding assay using lotus tetragonolobus for measuring different glycosylated forms of haptoglobin.

Using the fucose-specific lectin, Lotus tetragonolobus, we recently isolated abnormally-glycosylated forms of haptoglobin (FHp) that are useful for monitoring cancer patients and for detecting active disease in rheumatoid arthritis. FHp is detected by electrophoresis and silver staining. In order to use FHp clinically, a better assay is required. A lectin-binding assay (LBA) is described, in which FHp is captured by lotus bound to multi-well plates, and the amount captured is measured by an enzyme-labelled antibody system. The LBA results correlate with those obtained with electrophoresis. The method also gives good precision and low background values. In the presence of 1 mol/l fucose the bound-FHp was reduced by between 60-100%. This confirms that the method is detecting abnormally-fucosylated forms of haptoglobin. This approach opens up exciting possibilities for investigating large numbers of pathological sera and it suggests that other combinations of lectin and antibody may be worth investigating in the future.

Arthritis, Rheumatoid↗

PubMed1990

A protein assay program for Lotus 1-2-3.

Protein assays have become a widespread procedure not only in biochemistry but in many disciplines. This paper describes a program that takes advantage of the capacity of a spreadsheet program such as Lotus 1-2-3 to produce a graph and perform the calculations necessary for such assays. Its simplicity allows users with little or no experience with Lotus 1-2-3 to run it once installed.

Mathematical Computing↗

PubMed1981

Identification of a subpopulation of chicken B lymphocytes by the lectin from Lotus tetragonolobus.

Lectin-reactive chicken lymphoid cells were detected by agglutination. The lectin from Lotus tetragonolobus agglutinated cells from bursa and spleen and did not agglutinate cells from thymus or peripheral blood of 28-day-old chickens. The percentages of Lotus tetragonolobus reactive cells were also measured by assays of lectin-induced rosette formation, binding of lectin-labelled latex beads, and binding of rhodamine-labelled lectin. The distribution of lectin reactive cells varied with the age of the chicken. The lectin appears to identify a unique subpopulation of chicken B lymphocytes.

Animals↗

PubMed2006

Superhydrophobic behavior of a perfluoropolyether lotus-leaf-like topography.

We demonstrate the fabrication of 2-D arrays of nanopillars made from perfluoropolyether derivatives using a porous anodic aluminum oxide membrane as a template. Pretexturing the aluminum prior to anodization enables one to engineer multiple morphological length scales and thereby synthesize a lotus-leaf-like topography. Both nanopillars on a flat surface and on a lotus-leaf-like topology exhibit superhydrophobicity, low contact angle hysteresis, and self-cleaning.

Aluminum Oxide↗

PubMed1997

Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Le(x) determinant.

A defined set of oligosaccharides and glycopeptides containing alpha-linked fucose were used to examine the specificity of the immobilized fucose-binding lectin Lotus tetragonolobus agglutinin (LTA1), also known as lotus lectin. Glycans containing the Lewis x determinant (Le(x)) Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAc beta 1-4[Fuc alpha 1-3]GlcNAc. The lectin did not bind glycans containing either sialylLe(x) or VIM-2 determinants, nor did it bind the isomeric Le(x), Gal beta 1-3[Fuc alpha 1-4]GlcNAc-R. Although 2'-fucosyllactose Fuc alpha 1-2Gal beta 1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fuc alpha 1-2Gal beta 1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Le(x) antigen and is useful in analyzing specific fucosylation of glycoconjugates.

Agglutinins↗

PubMed2001

Characterisation and expression studies of a root cDNA encoding for ferredoxin-nitrite reductase from Lotus japonicus.

A full-length cDNA encoding for ferredoxin-nitrite reductase (NiR, EC 1.7.7.1), has been isolated from a root cDNA library from the legume Lotus japonicus and characterised. The NiR gene (Nii) is present as a single copy in this plant, and encodes a protein of 582 amino acids. The Lotus NiR protein is synthesised as a precursor with an amino-terminal transit peptide consisting of 25 amino acid residues. Sequence comparisons with leaf NiRs from different plant species and with other related redox proteins identified in the root NiR the same highly conserved residues involved in the cofactor binding than previously reported for leaves. Besides, a putative binding site for ferredoxin was also found in the N-terminal region of the protein. The NiR gene is expressed in roots and leaves, although the level of expression is much higher in roots, in accordance with the fact that L. japonicus assimilates nitrate mainly in roots. NiR mRNA, protein and activity are induced by nitrate in roots and leaves, while ammonium-grown plants only showed basal levels. No oscillations of NiR mRNA, protein and activity were observed during the day/night cycle, neither in roots nor leaves, making an interesting difference with rhythms observed in other plant species.

Journal Article↗

PubMed1987

Expression of a complete soybean leghemoglobin gene in root nodules of transgenic Lotus corniculatus.

The complete soybean leghemoglobin lbc(3) gene was transferred into the legume Lotus corniculatus using an Agrobacterium rhizogenes vector system. Organ-specific expression of the soybean gene was observed in root nodules formed on regenerated transgenic plants after infection with Rhizobium loti. The primary transcript was processed in the same way as in soybean nodules and the resulting mRNA was translated into Lbc(3) protein. Quantitative determination of the Lbc(3) protein in nodules of transgenic plants indicated that the steady-state level of the soybean protein is comparable to that of endogenous Lotus leghemoglobin.

Journal Article↗

PubMed2005

Short photoperiod induces dormancy in Lotus (Nelumbo nucifera).

BACKGROUND AND AIMS: Lotus (Nelumbo nucifera) has been cultivated as an ornamental and food plant in Japan for more than 1000 years. As large areas are required for its cultivation (approximately 2 m2 per plant), physiological research, such as into the effect of environmental factors on dormancy, has not been well studied until recently. In this paper, seedlings were used to examine environmental factors affecting dormancy induction. METHODS: In a first experiment, seeds were sown from 6 April to 6 October at 2-month intervals, and cultivated for 2 months in an unheated greenhouse. In a second experiment, seeds were prepared for germination on 16 November and 16 May and the seedlings were grown at 25 or 30 degrees C under natural daylength in phytotron growth rooms. After 1 month, the seedlings were cultivated at 20, 25 or 30 degrees C for a further month. The number of leaves and rhizome branches on the main stem were counted, and growth of rhizomes on the main stem was calculated using a rhizome enlargement index (= maximum internode diameter/internode length) after 2 months of culture in both experiments. KEY RESULTS: Rhizomes elongated without enlargement when the seeds were sown in April and June. Sowing the seeds in August and October resulted in rhizome enlargement from the tenth and fifth internodes, respectively. Rhizomes enlarged in the November-sowing but elongated in the May-sowing irrespective of temperature treatments under natural daylength in the phytotron rooms. The seedlings cultivated from May at 25-30 degrees C for 2 months had more leaves, and more rhizome branches and nodes than those cultivated from November. CONCLUSIONS: Short days led to induced dormancy in lotus.

Nelumbo↗

PubMed1996

Lotus lectin labels subpopulation of olfactory receptor cells.

Experiments were performed to test the hypothesis that subsets of olfactory receptor cells could be recognized based on their lectin binding and that mapping of their projections onto the olfactory bulb would reveal details of anatomic organization of the olfactory nerve projection to the olfactory bulb. The results from one lectin, Lotus, were examined in detail. Olfactory receptor cells in the lateral part of the main epithelium were labeled, as well as scattered cells in the remainder of the epithelium. Glomeruli labeled by Lotus were concentrated primarily in the region of the olfactory bulb that receives its input from the lateral epithelium, although scattered glomeruli could be identified in other regions. Within the terminal field of these axons there was a mosaic pattern, with some glomeruli densely labeled, some lightly labeled and others unlabeled. These findings support the notion that there are biochemically distinct populations of olfactory receptor cells having localized distributions in the epithelium, with axons that coalesce to terminate in specific glomeruli, rather than diffusely over their projection field.

Animals↗

PubMed2000

Isolation of two different phenotypes of mycorrhizal mutants in the model legume plant Lotus japonicus after EMS-treatment.

Lotus japonicus has been proposed as a model plant for the molecular genetic study of plant-microbe interaction including Mesorhizobium loti and arbuscular mycorrhizal (AM) fungi. Non-mycorrhizal mutants of Lotus japonicus were screened from a collection of 12 mutants showing non-nodulating (Nod-), ineffectively nodulating (Fix-) and hypernodulating (Nod++) phenotypes with monogenic recessive inheritance induced by EMS (ethylmethane sulfonate) mutagenesis. Three mycorrhizal mutant lines showing highly reduced arbuscular mycorrhizal colonization were obtained. All of them were derived from Nod- phenotypes. In Ljsym72, the root colonization by Glomus sp. R-10 is characterized by poor development of the external mycelium, formation of extremely branched appressoria, and the blocking of hyphal penetration at the root epidermis. Neither arbuscules nor vesicles were formed in Ljsym72 roots. Fungal recognition on the root surface was strongly affected by the mutation in the LjSym72 gene. Unique characteristics in mutant lines Ljsym71-1 and Ljsym71-2 were the overproduction of deformed appressoria and arrested hyphal penetration of the exodermis. Small amounts of internal colonization including degenerated arbuscule formation occurred infrequently in these types of mutants. Not only fungal development on the root surface but also that in the root exodermis and cortex was affected by the mutation in LjSym71 gene. These mutants represent a key advance in molecular research on the AM symbiosis.

Ethyl Methanesulfonate↗

PubMed1997

Classical and molecular genetics of the model legume Lotus japonicus.

The model legume Lotus japonicus was demonstrated to be amenable to classical and molecular genetic analysis, providing the basis for the genetic dissection of the plant processes underlying nodulation and nitrogen fixation. We have developed an efficient method for the sexual hybridization of L. japonicus and obtained F1 progeny derived from a cross of L. japonicus B-129-S9 Gifu x B-581 Funakura. Over half of the cross-pollinations resulted in fertile hybrid seed, which were confirmed morphologically and by single arbitrary primer DNA amplification polymorphisms using the DAF technique. Molecular and morphological markers segregated in true Mendelian fashion in a F2 population of 100 plants. Several DAF loci were linked using the MAPMAKER software to create the first molecular linkage groups of this model legume. The mapping population was advanced to generate a set of immortal recombinant inbred lines (F6; RILs), useful for sharing plant material fixed genetically at most genomic regions. Morphological loci for waved stem shape (Ssh), dark leaf color (Lco), and short flowering period (Fpe) were inherited as single dominant Mendelian loci. DAF markers were dominant and were detected between Gifu and Funakura at about one per primer, suggesting that the parents are closely related. One polymorphism (270G generated by single octomer primer 8.6m) was linked to a morphological locus controlling leaf coloration. The results demonstrate that (i) Lotus japonicus is amenable to diploid genetic analysis, (ii) morphological and molecular markers segregate in true diploid fashion, (iii) molecular polymorphisms can be obtained at a reasonable frequency between the related Gifu and Funakura lines, and iv) the possibility exists for map-based cloning, marker assisted selection and mapping of symbiotic mutations through a genetic and molecular map.

Base Sequence↗

PubMed2001

Lotus japonicus forms early senescent root nodules with Rhizobium etli.

Mesorhizobium loti and Rhizobium etli are microsymbionts of the Lotus and Phaseolus spp., respectively, and secrete essentially the same Nod factors. Lotus japonicus efficiently formed root nodules with R. etli CE3, irrespective of the presence or absence of a flavonoid-independent transcription activator nodD gene. On a nitrogen-free medium, however, the host plant inoculated with R. etli showed a severe nitrogen deficiency symptom. Initially, the nodules formed with R. etli were pale pink and leghemoglobin mRNA was detectable at significant levels. Nevertheless, the nodules became greenish with time. Acetylene-reduction activity of nodules formed with R. etli was comparable with that formed by M. loti 3 weeks postinoculation, but thereafter it decreased rapidly. The nodules formed with R. etli contained much more starch granules than those formed with M. loti. R. etli developed into bacteroids in the L. japonicus nodules, although the density of bacteroids in the infected cells was lower than that in the nodules formed with M. loti. The nodules formed with R. etli were of the early senescence type, in that membrane structures were drastically disintegrated in the infected cells of the greenish nodules. Thus, L. japonicus started and then ceased a symbiotic relationship with R. etli at the final stage.

Cytoplasm↗

PubMed1998

Lotus corniculatus nodulation specificity is changed by the presence of a soybean lectin gene

Plant lectins have been implicated as playing an important role in mediating recognition and specificity in the Rhizobium-legume nitrogen-fixing symbiosis. To test this hypothesis, we introduced the soybean lectin gene Le1 either behind its own promoter or behind the cauliflower mosaic virus 35S promoter into Lotus corniculatus, which is nodulated by R. loti. We found that nodulelike outgrowths developed on transgenic L. corniculatus plant roots in response to Bradyrhizobium japonicum, which nodulates soybean and not Lotus spp. Soybean lectin was properly targeted to L. corniculatus root hairs, and although infection threads formed, they aborted in epidermal or hypodermal cells. Mutation of the lectin sugar binding site abolished infection thread formation and nodulation. Incubation of bradyrhizobia in the nodulation (nod) gene-inducing flavonoid genistein increased the number of nodulelike outgrowths on transgenic L. corniculatus roots. Studies of bacterial mutants, however, suggest that a component of the exopolysaccharide surface of B. japonicum, rather than Nod factor, is required for extension of host range to the transgenic L. corniculatus plants.

Journal Article↗

PubMed1987

Identification of lotus rhizobia by direct DNA hybridization of crushed root nodules.

Hybridization of crushed Lotus pedunculatus root nodules with P-labeled total genomic DNA probes was used to identify Rhizobium loti and Bradyrhizobium sp. (Lotus rhizobia). Probes always hybridized with homologous target DNA and frequently with DNAs of other strains from the same genus. Intergeneric hybridization did not occur. Results were comparable to those from colony hybridization.

Journal Article↗

PubMed1989

Unaltered Nodulation Competitiveness of a Strain of Bradyrhizobium sp. (Lotus) after a Decade in Soil.

A Bradyrhizobium sp. (Lotus) strain that formed a soil population that was highly competitive for nodulation of Lotus pedunculatus 11 years after its introduction into a field soil and a culture of the same strain stored lyophilized were compared with an antibiotic-resistant mutant in respect of their nodulation competitiveness. The mutant was less competitive than the wild-type strain it was isolated from and had to be present at a cell ratio of 5.76:1 in mixed inoculum in sand culture to form 50% of the nodules on L. pedunculatus (50% nodulation value, 5.76). The 50% nodulation values for a soil population of the mutant mixed with soil populations of the lyophilized and field soil strain were, respectively, 6.83 and 5.77, indicating that the field soil strain was not significantly different from the lyophilized strain in nodulation competitiveness. A 50% nodulation value of 11.18 obtained when soil containing a recently established mutant population was mixed with the field soil containing the population established 11 years before, indicating that the plant infection technique underestimated cell numbers of the field soil population by 100%. Nodulation competitiveness was unaffected by the size of the strain populations in the range of 100 to 1,000 cells per g of soil; at 10 cells per g a significant correlation between strain ratios in nodules and in soil was still evident. The results indicated that apparently superior nodulation competitiveness of a well-established soil population relative to that of a subsequently introduced strain may not necessarily reflect the intrinsic competitive abilites of the strain(s) involved. The soil strain did not differ from laboratory-maintained cultures in antigenic properties, effectiveness, or whole cell protein electrophoresis profiles.

Journal Article↗

PubMed1996

Four unnamed species of nonsymbiotic rhizobia isolated from the rhizosphere of Lotus corniculatus.

Previously, we found that genetically diverse rhizobia nodulating Lotus corniculatus at a field site devoid of naturalized rhizobia had symbiotic DNA regions identical to those of ICMP3153, the inoculant strain used at the site (J. T. Sullivan, H. N. Patrick, W. L. Lowther, D. B. Scott, and C. W. Ronson, Proc. Natl. Acad. Sci. USA 92:8985-8989, 1995). In this study, we characterized seven nonsymbiotic rhizobial isolates from the rhizosphere of L. corniculatus. These included two from plants at the field site sampled by Sullivan et al. and five from plants at a new field plot adjacent to that site. The isolates did not nodulate Lotus species or hybridize to symbiotic gene probes but did hybridize to genomic DNA probes from Rhizobium loti. Their genetic relationships with symbiotic isolates obtained from the same sites, with inoculant strain ICMP3153, and with R. loti NZP2213T were determined by three methods. Genetic distance estimates based on genomic DNA-DNA hybridization and multilocus enzyme electrophoresis were correlated but were not consistently reflected by 16S rRNA nucleotide sequence divergence. The nonsymbiotic isolates represented four genomic species that were related to R. loti; the diverse symbiotic isolates from the site belonged to one of these species. The inoculant strain ICMP3153 belonged to a fifth genomic species that was more closely related to Rhizobium huakuii. These results support the proposal that nonsymbiotic rhizobia persist in soils in the absence of legumes and acquire symbiotic genes from inoculant strains upon introduction of host legumes.

Base Sequence↗