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PubMed1981

Carbon Dioxide Fixation in Soybean Roots and Nodules: I. CHARACTERIZATION AND COMPARISON WITH N(2) FIXATION AND COMPOSITION OF XYLEM EXUDATE DURING EARLY NODULE DEVELOPMENT.

These studies demonstrate that soybean (Merr) roots and nodules possess an active system for fixing CO(2). The maximum rates of CO(2) fixation observed for roots and nodules of intact plants were 120 and 110 nanomoles CO(2) fixed per milligram dry weight per hour, respectively. Results of labeling studies suggest a primary role for phosphoenolpyruvate carboxylase in CO(2) assimilation in these tissues. After pulse-labeling with (14)CO(2) for 2 minutes, 70% of the total radioactivity was lost within 18 minutes via respiration and/or translocation out of nodules. During the vegetative stages of growth of soybeans grown symbiotically, CO(2) fixation in nodules increased at the onset of N(2) fixation but declined to a lower level prior to the decrease in N(2) fixation. This decrease coincided with a decrease in the transport of amino acids, especially asparagine, and an increase in the export of ureides. These findings are consistent with a dual role for CO(2) fixation, providing substrates for energy-yielding metabolism and supplying carbon skeletons for NH(4) (+) assimilation and amino acid biosynthesis.

Journal Article↗

PubMed1981

Dinitrogen Fixation by Cultures of Frankia sp CpIl Demonstrated by N(2) Incorporation.

The filamentous bacterium Frankia of the Actinomycetales, isolated from the nitrogen-fixing root nodules of certain woody plants, has shown nitrogenase activity in culture, using the acetylene reduction method. In the present work, nitrogenase activity in pure cultures of Frankia sp. CpIl is confirmed using mass spectrometric measurements of (15)N(2) incorporation. After addition of carrier NH(4) (+) to digested cultures, those exposed to (15)N(2) (25 atom%) had a (15)N content of 3.16 atom% compared to 0.354 atom% (15)N in the controls.

Journal Article↗

PubMed1990

Nitrogenase Activity and Nodule Gas Permeability Response to Rhizospheric NH(3) in Soybean.

This study was conducted on soybean (Glycine max L. Merr.) nodules to determine if exogenous NH(3) exerts a controlling influence over nitrogenase activity through changes in nodule gas permeability (P), and if decreasing carbohydrate availability, as a result of low-light treatment, increases the sensitivity of root nodules to NH(3). Nodulated root systems of intact plants were exposed to one of several NH(3) concentrations ranging from 0 to 821 microliters per liter for an 8-hour period. Treatments were conducted under high-light (2300 micromoles per square meter per second) or low-light (800 micromoles per square meter per second) conditions. Increasing the NH(3) concentration and length of exposure of NH(3) caused a progressive decline in acetylene reduction activity (ARA). There was generally a greater reduction in ARA under the low-light treatment compared to the high-light treatment at a particular NH(3) concentration. The NH(3) concentration necessary to decrease P was greater than that needed to decrease ARA, and there was no evidence of a causal relationship between P and ARA in response to NH(3).

Journal Article↗

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↗

PubMed2006

[A LuxR family regulator, ExpR regulates the expression of motC operon from Sinorhizobium meliloti].

In Sinorhizobium meliloti strain Rm1021, ExpR+ mutation results in the overproduction of EPS II, which is required for efficient invasion of root nodules on the host plant alfalfa. When rhizobia were grown in LB/MC medium for 36-hour then placed at room temperature, most of ExpR+ mutant (Rm8530) cells aggregated at the bottom of the tubes, but ExpR+ sinR double mutant Rm11528 and wild type Rm1021 did not. The ExpR+ mutant was also found to swim slower than Rm1021 on swarming plates, but the ExpR+ sinR- mutant showed almost the same as wild type. The average diameter of swarming plaques for Rm8530, Rm11528 and Rm1021 was 13, 16 and 16 mm respectively, when bacteria were incubated at 28 degrees C for two days. After four days, the plaques enlarged to 17, 22 and 20 mm, respectively. These results indicate that ExpR+ mutation causes a serious defection of motility in this condition. By flagella staining with silver nitrate method, it was noted that all three strains had flagella. It suggests that the swimming behavior of Rm8530 may not result from the change in components and structures of flagella. Based on DNA microarray data, Hoang speculated that the Sin system seemed to regulate a multitude of genes in S. meliloti, including genes that participate in succinoglycan production, motility, and chemotaxis, as well as other cellular processes (Hoang et al. 2004). And most of the regulation by the Sin system was dependent on the presence of the ExpR regulator. Accordingly, the expression of the flaA, cheY1 and motC operon was determined using promoter: lacZ fusion in different genetic backgrounds. The results showed two fold lower of motC expression activity in ExpR+ mutant strain Rm8530 than in wild-type strain Rm1021 at early exponential phase and this decrease was hold back by further mutation of sinR. However, the beta-galactosidase activity of motC-lacZ fusion was almost the same in three strains at later exponential phase. These results suggest that ExpR may repress the expression of motC operon in a low cell density, but this repression can be deprived in a higher cell density. It may be a good explanation to the motility of Rm8530 on swarming plates, since it is lower cell density of bacteria on the swarming plates. Therefore, it may be concluded that ExpR both involved in the regulation of EPS II production and motility in S. meliloti.

Bacterial Proteins↗

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↗

PubMed2004

The promoters of two isoflavone synthase genes respond differentially to nodulation and defense signals in transgenic soybean roots.

Isoflavonoids are a group of secondary metabolites common to leguminous plants that play roles in nodulation and defense responses. Isoflavone synthase (IFS) catalyzes the key entry point step of isoflavone biosynthesis from the general phenylpropanoid pathway. We have cloned the 5' upstream regions of the genes encoding the two isoflavone synthase isoforms from soybean. We characterized the tissue-specific expression patterns of IFS1 and IFS2 genes of soybean by quantitative RT-PCR. Isoflavone synthase transcripts were detected primarily in the roots and seeds. In transgenic soybean plants in which the IFS1 promoter drove expression of the bacterial uidA (GUS) gene, the expression was localized to the root epidermis and root hairs. We detected differential tissue-specific expression of IFS1:GUS in response to nodulation and defense signals. Bradyrhizobium japonicum, a diazotropic symbiont of soybean, induced the expression of IFS1:GUS in root hairs and xylem poles in the young differentiation zone of the root. This observation suggests novel roles for isoflavonoids in nodulation. The defense related hormone salicylic acid induced the expression of IFS1:GUS in all cell types of the root. We also noticed corresponding alterations in the transcript levels of IFS1 and IFS2 in response to B. japonicum or salicylic acid as identified by quantitative RT-PCR. Using hairy root transformed soybean composite plants, we have identified regions of the IFS1 and IFS2 promoters mediating response to B. japonicum. We have also shown conserved root-specific expression of IFS1 in rice and Arabidopsis. The expression pattern of IFS1 in soybean is consistent with the physiological roles of isoflavonoids as defense compounds against pathogens and signal molecules to symbiotic bacteria in soybean.

5' Flanking Region↗

PubMed2003

The Lotus japonicus ndx gene family is involved in nodule function and maintenance.

To elucidate the function of the ndx homeobox genes during the Rhizobium-legume symbiosis, two Lotus japonicus ndr genes were expressed in the antisense orientation under the control of the nodule-expressed promoter Psenod12 in transgenic Lotus japonicus plants. Many of the transformants obtained segregated into plants that failed to sustain proper development and maintenance of root nodules concomitant with down-regulation of the two ndx genes. The root nodules were actively fixing nitrogen 3 weeks after inoculation, but the plants exhibited a stunted growth phenotype. The nodules on such antisense plants had under-developed vasculature and lenticels when grown on medium lacking nitrogen sources. These nodules furthermore entered senescence earlier than the wild-type nodules. Normal plant growth was resumed upon external addition of nitrogen. This suggests that assimilated nitrogen is not properly supplied to the plants in which the two ndx genes are down-regulated. The results presented here, indicate that the ndx genes play a role in the development of structural nodule features, required for proper gas diffusion into the nodule and/or transport of the assimilated nitrogen to the plant.

Cell Nucleus↗

PubMed1974

The inhibition of soybean metabolism by cadmium and lead.

Lead (300 mum) and cadmium (18 mum) inhibit pod fresh weight in soybeans (Glycine max L.) by 35%. Eighteen micromolar cadmium caused a 30% decline in nitrogenase activity by day 52 (the day on which maximum activity was measured) and a 71% inhibition by day 59. The heavy metals depressed photosynthetic rates; when photosynthesis was depressed by 60%, as measured on the day of peak photosynthesis activity, carbohydrate did not accumulate in the nodules. The reduction of pod fresh weight correlated with the effect of lead and cadmium on several other aspects of plant metabolism (shoot, root, leaf, and nodule dry weight; nodule ammonia, protein and carbohydrate content).

Journal Article↗

PubMed1980

Identification of "nodule-specific" host proteins (nodoulins) involved in the development of rhizobium-legume symbiosis.

Infection of legume roots with Rhizobium species results in the development of a root nodule structure in which the bacteria form an intracellular symbiosis with the plant. We report here that the infection of soybean (Glycine max L.) roots with Rhizobium japonicum results in the synthesis by the plant of at least 18-20 polypeptides other than leghemoglobin during the development of root nodules. Identification of these "nodule-specific" host polypeptides (referred to as nodulins) was accomplished by two-dimensional gel analysis of the immunoprecipitates formed by a "nodule-specific" antiserum with in vitro translation products of root-nodule polysomes that are free of bacteroidal contaminations. Nodulins account for 7-11% of the total 35S-methionine-labeled protein synthesized in the host cell cytoplasm, and the majority of them are of 12,000-20,000 molecular weight. These proteins are absent from the uninfected roots, bacteroids and free-living Rhizobium, and appear to be coded for the plant genes that may be obligatory for the development of symbiosis in the legume root nodules. Analysis of nodulins in ineffective (unable to fix nitrogen) nodules developed due to Rhizobium strains SM5 and 61A24 showed that their synthesis is reduced and their expression differentially influenced by mutations in rhizobia. Two polypeptides of bacterial origin were also found to be cross-reactive with the "nodule-specific" antiserum, suggesting that they are secreted by Rhizobium into the host cell cytoplasm during symbiotic nitrogen fixation.

Fabaceae↗

PubMed2004

Biochemical characterization of symbiosome membrane proteins from Medicago truncatula root nodules.

The symbiosome membrane represents a specialized plant membrane that forms both a structural and a functional interface between the legume plant and its bacterial counterpart. In this study, the symbiosome membrane protein profile from the model system Medicago truncatula and the corresponding bacterium Sinorhizobium meliloti was examined using two-dimensional electrophoresis and microcapillary high-performance liquid chromatography (HPLC) tandem mass spectrometry. The identities of 51 proteins were obtained and these proteins were categorized into functional classes to indicate biochemical roles. Symbiosome membrane proteins include an H(+)-ATPase, ENOD16, ENOD8, nodulin-25, BiP, HSP70, PDI, multifunctional aquaporin, a putative syntaxin, and other proteins of known and unknown identity and function. The majority of the proteins identified were involved with protein destination and storage. These results allow us to understand better the biochemical composition of the symbiosome membrane and thus provide a basis to hypothesize mechanisms of symbiosome membrane formation and function.

Chromatography, High Pressure Liquid↗

PubMed2005

Beta-rhizobia from Mimosa pigra, a newly discovered invasive plant in Taiwan.

A total of 191 rhizobial isolates from the root nodules of three geographically separate populations of the invasive plant Mimosa pigra in Taiwan were examined using amplified rDNA restriction analysis, 16S rDNA sequences, protein profiles and ELISA. Of these, 96% were identified as Burkholderia and 4% as Cupriavidus taiwanensis. The symbiosis-essential genes nodA and nifH were present in two strains of Burkholderia (PAS44 and PTK47), and in one of C. taiwanensis (PAS15). All three could nodulate M. pigra. Light and electron microscopy studies with a green fluorescent protein transconjugant variant of strain PAS44 showed the presence of fluorescent bacteroids in M. pigra nodules. These bacteroids expressed the nifH protein, hence this is the first confirmation that Burkholderia is a genuine symbiont of legume nodules. The predominance of Burkholderia in Taiwanese M. pigra suggests that this species may have brought its symbionts from its native South America, rather than entering into association with the Taiwanese Mimosa symbiont C. taiwanensis which so successfully nodulates Mimosa pudica and Mimosa diplotricha.

Burkholderia↗

PubMed2003

Local and systemic effects of phosphorus and nitrogen on nodulation and nodule function in Alnus incana.

Phosphorus (P) and nitrogen (N) effects on nodulation, nitrogenase activity and plant growth were studied in the root-hair-infected actinorhizal plant Alnus incana (L.) Moench. A split-root experiment, as well as a short-term experiment with entire root systems and a broader range of P concentrations, showed that P effects were specific on nodulation and not a general stimulation via a plant growth effect. These results indicate that nodule initiation and nodule growth have a high P demand. The split-root assay, comprising seven combinations of two N and two P levels, showed that P could counteract systemic N inhibition of nodulation, but did not counteract N inhibition of nitrogenase activity.

Alnus↗

PubMed1988

Early Infection and Competition for Nodulation of Soybean by Bradyrhizobium japonicum 123 and 138.

Interactions of soybean with Bradyrhizobium japonicum 123 (serogroup 123) and 138 (serogroup c1) were used to examine the relationship between early infection rates, competition for nodulation, and patterns of nodule occupancy. Both strains formed more infections in autoclaved soil (sterile soil) than in untreated soil (unsterile soil). Inoculation did not increase numbers of infection threads in unsterile soil-grown plants, where infection of proximal portions of primary roots was complete by 5 days after planting. Both strains infected and nodulated at similar rates in sterile soil. Nodules were always clustered on the upper root system, regardless of inoculation and soil treatment. Sixty-seven percent of the nodules of uninoculated plants grown in unsterile soil were occupied by rhizobia belonging to serogroups other than 123 or c1. Inoculation with strain 123 or 138 increased occupancy by that strain at the expense of residency by other rhizobia. Eighty-three percent of all nodules on plants dually inoculated with both strains in sterile soil contained strain 138. The corresponding value for plants inoculated in unsterile soil was 31%. Neither inoculum strain dominated occupancy of first-formed nodules in unsterile soil. It appears that north central Missouri soil may not have populations of highly competitive serogroup 123 and that early infection and nodulation rates do not contribute to the competitive success of strain 138.

Journal Article↗

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↗

PubMed2003

Rhizobium leguminosarum biovar viciae 1-aminocyclopropane-1-carboxylate deaminase promotes nodulation of pea plants.

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.

Carbon-Carbon Lyases↗

PubMed1995

Symbiotic induction of a MADS-box gene during development of alfalfa root nodules.

In response to infection by Rhizobium, highly differentiated organs called nodules form on legume roots. Within these organs, the symbiotic association between the host plant and bacteria is established. A putative plant transcription factor, NMH7, has been identified in alfalfa root nodules. nmh7 contains a MADS-box DNA-binding region and shows homology to flower homeotic genes. This gene is a member of a multigene family in alfalfa and was identified on the basis of nucleic acid homology to plant regulatory protein genes (MADS-box-containing genes) from Antirrhinum and Arabidopsis. RNA analysis and in situ hybridization showed that expression of this class of regulatory genes is limited to the infected cells of alfalfa root nodules and is likely to be involved in the signal transduction pathway initiated by the bacterial symbiont, Rhizobium meliloti. The expression of nmh7 in a root-derived organ is unusual for this class of regulatory genes.

Amino Acid Sequence↗