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PubMed1992

Direct detection of a globin-derived radical in leghaemoglobin treated with peroxides.

The root nodules of leguminous plants contain an oxygen-carrying protein which is somewhat similar to myoglobin. Reaction of the Fe3+ form of this protein (metleghaemoglobin; MetLb) with H2O2 is known to generate a ferryl [iron(IV)-oxo] species. This intermediate, which is analogous to Compound II of peroxidases and ferryl myoglobin, is one oxidizing equivalent above the initial level. In the present study it is shown that the second oxidizing equivalent from the peroxide is rapidly transferred into the surrounding protein, generating a protein radical which has been detected by e.p.r. spectroscopy; this reaction is analogous to that observed with metmyoglobin. An identical protein-derived species is observed with all three forms of MetLb tested (a, c1, c3) and with a number of other hydroperoxides and two-electron oxidants. This latter result, the observation that the concentration of this species is not affected by certain hydroxyl-radical scavengers, and the loss of the radical when the oxy or deoxy forms are used, demonstrate that this species is formed by electron transfer within the protein rather than by the generation and subsequent reaction of hydroxyl radicals (and related species from the other hydroperoxides). The e.p.r. signal of this species, which decays rapidly with a half-life of approx. 40 s, is consistent with the formation of a sterically constrained tyrosine-derived phenoxyl radical; protein-iodination experiments lend support to this assignment. Reaction between the radical and a number of other compounds has been observed, demonstrating that it is at least partially exposed on the surface of the protein. Analysis of the protein structure suggest that the radical may be centred on a tyrosine residue present at position 132 in the protein; this residue is close to the haem prosthetic group, which would facilitate rapid electron transfer.

Electron Spin Resonance Spectroscopy↗

PubMed1975

Identification of the rhizobium strains in pea root nodules using genetic markers.

Pea plants were inoculated jointly with pairs of genetically marked strains of Rhizobium leguminosarum. Out of 297 modules examined 56 contained both inoculant strains. The ratios of the strains in the inoculum did not affect the frequencies of mixed nodules. Generally one of the strains consistently occupied the majority of the nodules and ithe mixed nodules comprised the majority of bacteria. Transfer of the P-group R factor, RP4, between certain strains of Rhizobium within mixed nodules was detected. In some cases the non-parental progeny comprised 10% of the rhizobia isolated from such nodules.

Acetylene↗

PubMed1992

Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules.

Phosphoenolpyruvate carboxylase (PEPC) plays a key role in N2 fixation and ammonia assimilation in legume root nodules. The enzyme can comprise up to 2% of the soluble protein in root nodules. We report here the isolation and characterization of a cDNA encoding the nodule-enhanced form of PEPC. Initially, a 2945 bp partial-length cDNA was selected by screening an effective alfalfa nodule cDNA library with antibodies prepared against root nodule PEPC. The nucleotide sequence encoding the N-terminal region of the protein was obtained by primer-extension cDNA synthesis and PCR amplification. The complete amino acid sequence of alfalfa PEPC was deduced from these cDNA sequences and shown to bear striking similarity to other plant PEPCs. Southern blots of alfalfa genomic DNA indicate that nodule PEPC is a member of a small gene family. During the development of effective root nodules, nodule PEPC activity increases to a level that is 10- to 15-fold greater than that in root and leaf tissue. This increase appears to be the result of increases in amount of enzyme protein and PEPC mRNA. Ineffective nodules have substantially less PEPC mRNA, enzyme protein and activity than do effective nodules. Maximum expression of root nodule PEPC appears to be related to two signals. The first signal is associated with nodule initiation while the second signal is associated with nodule effectiveness. Regulation of root nodule PEPC activity may also involve post-translational processes affecting enzyme activity and/or degradation.

Amino Acid Sequence↗

PubMed1975

Demonstration of the isolation of non-infective Alnus crispa var. mollis Fern, nodule endophyte by morphological immunolabelling and whole cell composition studies.

Two filamentous, branched, and septate actinomycetes were isolated from field-collected and from axenic in vitro produced root nodules of Alnus crispa var. mollis Fern. host plant. After their transfer to a chemically defined medium, these nodule isolates could not be distinguished from each other on the basis of morphology, cultural reactions, and whole cell composition and were considered to be the same species. They were morphologically similar to the root nodule endophyte, but were incapable of nodulating aseptic host plants growing in a nitrogen-deficient substrate. Whole cells of the nodule isolates were used for the production of rabbit antibodies. The resulting specific antiisolate antibodies were conjugated with fluorescein isothiocyanate and used in staining tests of the nodule endophyte. The immunofluorescence reactions demonstrated the homology of the nodule isolates with the nodule endophyte. After pectinase degradation of the endophyte capsule, the indirect immunoferritin method corroborated the fluorescent anti-body (FA) staining reactions. There was no antigenic relationship between the nodule isolates and 13 known strains of actinomycetes as determined by the FA techique. Fluorescent antibody reactions of adsorbed conjugates suggested that endophytes of both Alnus crispa var. mollis Fern. and Alnus rugosa (DuRoi) Spreng. root nodules belong to a common serotype. The LL and mesoisomers of diaminopimelic acid were present in similar proportions in the nodule endophyte and in the nodule isolates. Glucose, mannose, and an unknown sugar were the predominant whole cell sugars in the nodule isolates, although trace amounts of arabinose and rhamnose were also displayed. The unknown sugar found in the nodule isolates was also present in trace amounts in the endophyte-suspension hydrolysate.

Actinomycetales↗

PubMed1992

A Rhizobium meliloti lipopolysaccharide mutant altered in competitiveness for nodulation of alfalfa.

A transposon Tn5-induced mutant of Rhizobium meliloti Rm2011, designated Rm6963, showed a rough colony morphology on rich and minimal media and an altered lipopolysaccharide (LPS). Major differences from the wild-type LPS were observed in (i) hexose and 2-keto-3-deoxyoctonate elution profiles of crude phenol extracts chromatographed in Sepharose CL-4B, (ii) silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis patterns of crude and purified LPS fractions, and (iii) immunoreactivities otherwise present in purified LPS of the parental strain Rm2011. In addition, Rm6963 lost the ability to grow in Luria-Bertani medium containing the hydrophobic compounds sodium deoxycholate or SDS and showed a decrease in survival in TY medium supplemented with high calcium concentrations. The mutant also had altered symbiotic properties. Rm6963 formed nodules that fixed nitrogen but showed a delayed or even reduced ability to nodulate the primary root of alfalfa without showing changes in the position of nodule distribution profiles along the roots. Furthermore, 2 to 3 weeks after inoculation, plants nodulated by Rm6963 were smaller than control plants inoculated with wild-type bacteria in correlation with a transient decrease in nitrogen fixation. In most experiments, the plants recovered later by expressing a full nitrogen-fixing phenotype and developing an abnormally high number of small nodules in lateral roots after 1 month. Rm6963 was also deficient in the ability to compete for nodulation. In coinoculation experiments with equal bacterial numbers of both mutant and wild-type rhizobia, only the parent was recovered from the uppermost root nodules. A strain ratio of approximately 100 to 1 favoring the mutant was necessary to obtain an equal ratio (1:1) of nodule occupancy. These results show that alterations in Rm6963 which include LPS changes lead to an altered symbiotic phenotype during the association with alfalfa that affects the timing of nodule emergence, the progress of nitrogen fixation, and the strain competitiveness for nodulation.

Calcium↗

PubMed1992

Genetics of competition for nodulation of legumes.

An economically important problem in microbial ecology concerns the efficacy of rhizobial inoculants for the formation of nitrogen-fixing root nodules on legume crop plants such as soybean, alfalfa, and clover. Some strains of rhizobia can increase symbiotic nitrogen fixation under controlled conditions. However, attempts to improve nitrogen fixation under agricultural conditions with such strains often fail, usually as a result of the presence of indigenous rhizobia limiting nodulation by the inoculum strains. This problem is referred to as the Rhizobium competition problem, and molecular genetics is being used to address the problem from two perspectives. First, the host specificity of rhizobia is being characterized with the long term goal of developing strains that can nodulate a very strain-specific host-legume genotype. Second, the genetic basis of competitiveness in several strains is being examined. Genetic determinants of nodulation competitiveness have been isolated and mechanisms for their stable integration into the genome of superior nitrogen-fixing strains have been developed. Several phenotypes have been identified as playing an important role in nodulation competitiveness including antibiosis, motility, speed of nodulation, cell-surface characteristics, and nodulation efficiency. Several solutions to this problem are likely to result from these strategies and will be useful for certain legumes in specific locations.

Fabaceae↗

PubMed1975

Insecticides and soil microorganisms. III. Fate of 14C-labelled Dipterex as affected by two nodule-forming rhizobium spp. and roots of their respective leguminous host plants.

Chromatographic analysis led to the identification of monomethyl- and dimethyl-phosphates as metabolites resulting from the enzymatic degradation of 14C-labelled Dipterex in the buffer solutions and root tissues of broad bean and clover plants, as well as in the culture media of rhizobium leguminosarum and Rhizobium trifolii. The formation of 14CO2 from rhizobial cultures containing radioactive Dipterex suggests that some of the liberated methanol groups (during breakdown of Dipterex) are oxidatively degraded by the two Rhizobium spp.

Buffers↗

PubMed1992

Activation of flavonoid biosynthesis in roots of Vicia sativa subsp. nigra plants by inoculation with Rhizobium leguminosarum biovar viciae.

Infective (nodulating) Rhizobium leguminosarum biovar viciae (R.l. viciae) bacteria release Nod factors which stimulate the release of nodulation gene-inducing flavanones and chalcones from roots of the host plant Vicia sativa subsp. nigra (K. Recourt et al., Plant Mol Biol 16: 841-852; H.P. Spaink et al., Nature 354: 125-130). The hypothesis that this release results from increased synthesis of flavonoids was tested by studying the effect of inoculation of V. sativa with infective and uninfective R.l. viciae bacteria on (i) activity of L-phenylalanine ammonia-lyase, (ii) level of chalcone synthase mRNA, and (iii) activity of (eriodictyol) methyltransferase in roots. Consistent with the hypothesis, each of these parameters was found to increase 1.5 to 2-fold upon inoculation with infective R.l. viciae bacteria relative to the situation for uninoculated roots and for roots inoculated with uninfective rhizobia.

Acyltransferases↗

PubMed1978

Rhizobium strain identification in Arachis hypogaea nodules by enzyme-linked immunosorbent assay (ELISA).

The technique of enzyme-linked immunosorbent assay (ELISA) was used for serological identification of peanut Rhizobium strains both in cell suspension of pure culture and in single root nodules of groundnut (Arachis hypogaea) plants. Antisera of three peanut Rhizobium strains were tested against eight different Rhizobium isolates. Three serogroups identified by agglutination and immunodiffusion tests were confirmed by ELISA. In this experiment ELISA was more sensitive by four to six orders of magnitude than the agglutination and immunodiffusion tests and enabled the detection of Rhizobium antigens in cell suspensions of 10(4)-10(5) cells per millilitre. The reactions of culture and nodule antigens were identical for all strains investigated. ELISA enabled the precise typing of rhizobial isolates in single small root nodules. The minimum fresh weight of nodule tissue necessary to perform the ELISA test was 0.4 mg crushed in 1 ml of phosphate-buffered saline (PBS). ELISA was also successfully used for strain identification in mixed inoculated plants. One of the strains in each pair formed most of the nodules examined.

Antigens, Bacterial↗

PubMed1976

Control of synbiotic nitrogen fixation in Rhizobia. Regulation of NH4+ assimilation.

This communication is concerned with physiological, biochemical, and genetic studies of the regulation of ammonium (NH4+) assimilation by Rhizobia (root nodule bacteria) that infect leguminous plants. The major conclutions are (i) physiological studies show that Rhizobia are able to assimilate NH4+ for growth only when supplemented with certain organic nitrogen sources (e.g., L-aspartate, L-leucine, L-serine). Addition of as little as 2 mug/ml of L-aspartate supported growth on NH4+ as nitrogen source. In contrast, addition of glutamate in combination with NH4+-blocked NH4+ utilization; (ii) biochemical analysis show that glutamate synthase activity (NADP- and NAD-linked) is always present in cells capable of assimilating NH4+; also cells without glutamate synthase activity were found to be incapable of NH4+ utilization. Glutamate synthase levels were observed to fluctuate markedly depending on the available nitrogen source and on the growth stage of the culture; (iii) mutants were selected in which assimilation of NH4+ is no longer subject to inhibition (repression?) by glutamate. The levels of glutamate synthase activity (NADP-linked) (in the presence of glutamate) show approximately a two-fold increase over the level in the parent strain. The mutants no longer require supplementation with small amounts of organic nitrogen for growth in medium containing inorganic nitrogen (e.g., NH4+ or NO3-); (iv) these findings are discussed in relation to the working model of symbiotic nitrogen fixation recently proposed (O'Gara and Shanmugam (1976), Biochim. Biophys. Acta 437, 313--321).

Aspartic Acid↗

PubMed1976

The purification and properties of the glutamine synthetase from the cytosol of Soya-bean root nodules.

The major portion of glutamine synthetase activity in root nodules of soya-bean plants is associated with the cytosol rather than with Rhizobium japonicum bacteroids. Glutamine synthetase accounts for about 2% of the total soluble protein in nodule cytosol. Glutamine synthetase from nodule cytosol has been purified by a procedure involving fractionation with protamine sulphate, ammonium sulphate and polypropylene glycol, chromatography on DEAE-Bio-Gel A and Bio-Gel A-5m and affinity chromatography on glutamate-agarose columns. The purified preparation appeared to be homogeneous in the analytical ultracentrifuge. From sedimentation-equilibrium experiments a mol. wt. of about 376000 was determined for the native enzyme and 47300 for the enzyme in guanidinium chloride. From these data and measurements of electron micrographs, we have concluded that glutamine synthetase from nodule cytosol consists of eight subunits arranged in two sets of planar tetramers which form a cubical configuration with dimensions of about 10 nm (100 A) across each side. Glutamine synthetase from nodule cytosol has a higher glycine and proline content and a lower content of phenylalanine than the glutamine synthetase that has been prepared from pea seed. The cytosol enzyme contains four half-cystine molecules per subunit, which is in contrast with two reported for the enzyme from pea seed. Enzyme activity is striking influenced by the relative proportion of Mg2+ and Mn2+ in the assay medium. Activity is inhibited by feedback inhibitors and is influenced by energy charge.

Amino Acids↗

PubMed1976

Effect of some pesticides on the efficiency of the inoculated Rhizobium associated with broad bean plants.

The effect of some pesticides on the efficiency of the inoculated rhizobium in broad bean plants (Vicia faba) was studied in greenhouse experiments. Different pesticides were used in this work, including insecticides, herbicides, nematocides, and a molecide in different concentrations. The study revealed the following conclusions. Application of insecticides in different concentrations generally affected the formation of efficient nodules on the root system of broad bean plants. The inhibitory effect was also reflected to some extent on the dry weight of the plants and total nitrogen fixed by rhizobia in association with the mentioned plants. Birlane (insecticide) was found to be the most inhibitory insecticide on the inoculated rhizobium. Some of the insecticides, such as Temik and Dursban, at lower concentration were found to be a stimulant to symbiotic N-fixation, giving higher amounts of nitrogen fixed in the macrosymbiont.

Antinematodal Agents↗

PubMed1978

Inhibitory effects of the herbicide trifluralin on the establishment of the clover root nodule symbiosis.

Trifluralin, a widely used herbicide (Rahman, 1977), may act via interaction with plant microtubule protein (Hess and Bayer, 1974, 1977). In trifluralin treated seedlings of Trifolium repens, root tips increased in diameter and decreased in length, cell elongation was decreased, cell wall deposition was abnormal, root hairs were deformed, and a marked reduction was seen in the number of infection threads induced by the bacterial symbionts. Our observations support the hypothesis that trifluralin (at low concentrations, from 2 x 10(-6) M) interferes with microtubule-based morphogenetic processes in developing seedlings and adversely affects the establishment of the bacteria-legume symbiosis.

Morphogenesis↗

PubMed1979

Development of the nitrogen-fixing and protein-synthesizing apparatus of bacteroids in pea root nodules.

Some aspects of root nodule development of Pisum sativum inoculated with Rhizobium leguminosarum were examined. 1. Nitrogenase activity (measured as acetylene reduction) appears to be preceded by leghemoglobin synthesis (measured immunologically). 2. Syntheses of component I and component II of nitrogenase are not strictly coordinated. Synthesis of component I starts before component II. 3. Plant and bacteroid protein synthesis (measured by [35S]sulfate labeling) in root nodules declines rapidly during nodule development. Corresponding with this decline is a decrease in quantity and quality of rRNA.

Fabaceae↗

PubMed1976

A search for a leghaemoglobin-like compound in root nodules of Trema cannabina Lour.

Root nodules collected from the non-leguminous plant Trema cannabina Lour. grown under natural field conditions were examined for the presence of leghaemoglobin. No trace of any soluble haemoglobin-like compound could be detected by spectrophotometric analysis or molecular weight comparison studies, although a small amount of soluble haemoprotein with peroxidase activity was isolated. Possible alternatives to the possession of a leghaemoglobin in non-legume/Rhizobium N2-fixing associations are discussed.

Binding Sites↗

PubMed1978

Nodule infection by bean yellow mosaic virus in Phaseolus vulgaris.

Infection of root nodules of beans, Phaseolus vulgaris L., by bean yellow mosaic virus (BYMV) and the effect of the disease on the specific activity of the nodule are reported. Infectivity and serological microprecipitin assays with two sources of BYMV antiserum demonstrated that nodules from bean plants whose leaves had been inoculated with BYMV contain BYMV antigen. The disease reduced the fresh weights of tops, roots, and root nodules and induced premature nodule decay and/or nodule drop. The disease also reduced leghemoglobin content, on a plant weight basis, and N2 fixation rate, on an individual plant basis, as measured by the acetylene reduction assay. The increased leghemoglobin content per gram-nodule in BYMV-infected nodules relative to healthy nodules might be associated with multiplication of the virus in the nodule and/or unknown cellular effects derived from the BYMV-Rhizobium interaction.

Antigens, Viral↗

PubMed1992

Signaling and host range variation in nodulation.

Rhizobium, Bradyrhizobium, and Azorhizobium strains, collectively referred to as rhizobia, elicit on their leguminous hosts, in a specific manner, the formation of nodules in which they fix nitrogen. Rhizobial nod genes, which determine host specificity, infection, and nodulation, are involved in the exchange of low molecular weight signal molecules between the plant and the bacteria as follows. Transcription of the nod operons is under the control of NodD regulatory proteins, which are specifically activated by plant flavonoid signals. The common and species-specific structural nod genes are involved in turn in the synthesis of specific lipo-oligosaccharides that signal back to the plant to elicit root-hair deformations, cortical-cell divisions, and nodule-meristem formation.

Fabaceae↗

PubMed1992

Forcing expression of a soybean root glutamine synthetase gene in tobacco leaves induces a native gene encoding cytosolic enzyme.

Glutamine synthetase (GS; EC 6.3.1.2) is present in different subcellular compartments in plants. It is located in the cytoplasm in root and root nodules while generally present in the chloroplasts in leaves. The expression of GS gene(s) is enhanced in root nodules and in soybean roots treated with ammonia. We have isolated four genes encoding subunits of cytosolic GS from soybean (Glycine max L. cv. Prize). Promoter analysis of one of these genes (GS15) showed that it is expressed in a root-specific manner in transgenic tobacco and Lotus corniculatus, but is induced by ammonia only in the legume background. Making the GS15 gene expression constitutive by fusion with the CaMV-35S promoter led to the expression of GS in the leaves of transgenic tobacco plants. The soybean GS was functional and was located in the cytoplasm in tobacco leaves where this enzyme is not normally present. Forcing this change in the location of GS caused concomitant induction of the mRNA for a native cytosolic GS in the leaves of transgenic tobacco. Shifting the subcellular location of GS in transgenic plants apparently altered the nitrogen metabolism and forced the induction in leaves of a native GS gene encoding a cytosolic enzyme. The latter is normally expressed only in the root tissue of tobacco. This phenomenon may suggest a hitherto uncharacterized metabolic control on the expression of certain genes in plants.

Base Sequence↗