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PubMed1990

Identification of Frankia strains in nodules by hybridization of polymerase chain reaction products with strain-specific oligonucleotide probes.

A set of oligonucleotides has been developed to study the competitivity of two Frankia strains in the nodulation of the roots of two host plant species: Alnus glutinosa and Alnus incana. Two 20 mer-oligonucleotides, complementary to highly conserved sequences inside the nifH gene, were used as primers for the polymerase chain reaction (PCR) system in order to amplify microsymbiont DNA extracted from actinorhizae. PCR products were analyzed using two strain-specific 15-mer oligonucleotides identified in the amplified region. Hybridization data indicate that strain ACoN24d is more competitive than strain ArI3 in the nodulation of both hosts.

Actinomycetales↗
PubMed2000

Role of malonate in chickpeas.

Analysis of the content and distribution of organic acids in chickpea plants (Cicer arietinum L.) showed that malonate was the most abundant acid in roots and nodules, whereas malate was the main acid in leaves and stems. The highest concentration of malonate in roots was in the apices. Malonate metabolism did not appear to be directly related to abiotic stress. We suggest that malonate has a role as a defensive chemical in roots and nodules of chickpeas.

Fabaceae↗
PubMed2004

Cytokinins play opposite roles in lateral root formation, and nematode and Rhizobial symbioses.

We used the cytokinin-responsive Arabidopsis response regulator (ARR)5 gene promoter fused to a beta-glucuronidase (GUS) reporter gene, and cytokinin oxidase (CKX) genes from Arabidopsis thaliana (AtCKX3) and maize (ZmCKX1) to investigate the roles of cytokinins in lateral root formation and symbiosis in Lotus japonicus. ARR5 expression was undetectable in the dividing initial cells at early stages of lateral root formation, but later we observed high expression in the base of the lateral root primordium. The root tip continues to express ARR5 during subsequent development of the lateral root. These results suggest a dynamic role for cytokinin in lateral root development. We observed ARR5 expression in curled/deformed root hairs, and also in nodule primordia in response to Rhizobial inoculation. This expression declined once the nodule emerged from the parent root. Root penetration and migration of root-knot nematode (RKN) second-stage larvae (L2) did not elevate ARR5 expression, but a high level of expression was induced when L2 reached the differentiating vascular bundle and during early stages of the nematode-plant interaction. ARR5 expression was specifically absent in mature giant cells (GCs), although dividing cells around the GCs continued to express this reporter. The same pattern was observed using a green fluorescent protein (GFP) reporter driven by the ARR5 promoter in tomato. Overexpression of CKX genes rendered the transgenic hairy roots resistant to exogenous application of the cytokinin [N6-(Delta2 isopentenyl) adenine riboside] (iPR). CKX roots have significantly more lateral roots, but fewer nodules and nematode-induced root galls per plant, than control hairy roots.

Animals↗
PubMed1997

Signalling in symbiotic root nodule formation.

Rhizobia form nodules on legume roots in which they carry out biological nitrogen fixation. The location and number of nodules is tightly regulated. Exchange of signal molecules that activate responses in the symbiotic partner takes place: plant flavonoids induce the production of rhizobial Nod factors; Nod factors induce gene expression and morphogenesis in the legume root. ENOD40 is a plant gene induced by Nod factors that is involved in root nodule formation. ENOD40 encodes a peptide of 12 amino acids in soya bean that can induce tolerance of high auxin concentrations in tobacco protoplasts.

Bacterial Proteins↗
PubMed1994

Factors Affecting the Acetylene to 15N2 Conversion Ratio in Root Nodules of Myrica gale L.

When nodules of actinorhizal plants are exposed to acetylene, there is often an initial peak rate of acetylene reduction followed by a decline and a partial recovery. Treatment of hydroponically grown Myrica gale L. with water deficiency or dark stress increased the magnitude of the acetylene-induced decline and decreased the extent of the recovery. When N2 fixation was measured with 15N2 in unstressed plants, the ratio of acetylene reduction (peak) to N2 fixation prior to acetylene exposure was 3.73 [plus or minus] 0.14 (mean [plus or minus] SE). This value does not differ significantly (P < 0.05) from the theoretical minimum value of 4.0. In water-stressed plants the conversion ratio for the peak rate was greater (4.32 [plus or minus] 0.10) and in dark-stressed plants it was lower (2.54 [plus or minus] 0.33) than 4.0. The conversion ratio for the recovered rate of acetylene reduction was much lower than 4.0 in all cases, with mean values ranging from 1.16 to 2.60. We conclude that the peak rate of acetylene reduction provides the most reliable estimate of N2 fixation. The recovered rate of acetylene reduction consistently underestimates N2 fixation, sometimes severely, and thus measurements of acetylene reduction made in closed systems also underestimate N2 fixation to varying degrees.

Journal Article↗
PubMedDate not supplied

[Investigation of lipopolysaccharides from Sinorhizobium meliloti SKHM1-188 and two of its mutants with decreased nodulation competitiveness].

A comparative study of the lipopolysaccharides (LPS) isolated from Sinorhizobium meliloti SKHM 1-188 and two its LPS-mutants (Th29 and Ts22) with sharply decreased nodulation competitiveness was conducted. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate revealed two forms of LPS in all the three strains: a higher molecular-weight LPS1, containing O-polysaccharide (O-PS), and a and lower molecular-weight LPS2 without O-PS. However, the LPS1 content in mutants was significantly smaller than in the parent strain. The LPS of the strains studied contained glucose, galactose, mannose, xylose, three nonidentified sugars--X1 (TGlc 0.53), X2 (TGlc 0.47), and X3 (TGlc 0.43), glucosamine, and ethanolamine, while the LPS of S. meliloti SKHM1-188 additionally contained galactosamine, glucuronic and galacturonic acids, and 2-keto-3-deoxyoctulosonic acid (KDO), as well as fatty acids, such as 3-OH C14:0, 3-OH C15:0, 3-OH C16:0, 3-OH C18:0, nonidentified hydroxy X (T3-OH C14:0 1.33), C18:0, and unsaturated C18:1 fatty acids. The LPS of both mutants were similar in the component composition but differed from the LPS of the parent strain by a lower X2, X3, and 3-OH C 14:0 content and a higher KDO, C18:0, and hydroxy X content. The LPS of all the strains were subjected to mild hydrolysis with 1% acetic acid and fractionated on a column with Sephadex G-25. The higher molecular weight fractions (2500-4000 Da) contained a set of sugars typical of intact LPS and, supposedly, corresponded to the LPS polysaccharide portion (PS1). In the lower molecular weight fractions (600-770 Da, PS2), glucose and uronic acids were the major components; galactose, mannose, and X1 were present in smaller amounts. The PS1/PS2 ratio for the two mutants was significantly lower than for strain SKHM1-188. The data obtained show that the amount of O-PS-containing molecules (LPS1) in the heterogeneous lipopolysaccharide complex of the mutants was smaller than in the SKHM1-188 LPS; this increases the hydrophobicity of the cell surface of the mutant bacteria. This supposedly contributes to their nonspecific adhesion on the roots of the host plant, thus decreasing their nodulation competitiveness.

Acetic Acid↗
PubMed2000

Disruption of a gene essential for sulfoquinovosyldiacylglycerol biosynthesis in Sinorhizobium meliloti has no detectable effect on root nodule symbiosis.

The sulfolipid sulfoquinovosyldiacylglycerol is commonly found in the thylakoid membranes of photosynthetic bacteria and plants. While there is a good correlation between the occurrence of sulfolipid and photosynthesis, a number of exceptions are known. Most recently, sulfoquinovosyldiacylglycerol was discovered in the non-photosynthetic, root nodule-forming bacterium Sinorhizobium meliloti. This discovery raised the questions of the phylogenetic origin of genes essential for the biosynthesis of this lipid in S. meliloti and of a function of sulfolipid in root nodule symbiosis. To begin to answer these questions, we isolated and inactivated the sqdB gene of S. meliloti. This gene and two other genes located directly 3' of sqdB are highly similar to the sqdB, sqdC, and sqdD genes known to be essential for sulfolipid biosynthesis in the photosynthetic, purple bacterium Rhodobacter sphaeroides. This observation confirms the close phylogenetic kinship between these two species. Furthermore, the reduced similarity of sqdB to the plant ortholog SQD1 of Arabidopsis thaliana does not support a previous sqd gene transfer from the plant as a consequence of close symbiosis. A sulfolipid-deficient mutant of S. meliloti disrupted in sqdB is capable of inducing functional nodules and does not show an obvious disadvantage under different laboratory culture conditions. Thus far, no specific function can be assigned to bacterial sulfolipid, in either nodule-associated or free-living cells. S. meliloti contains a rich set of polar membrane lipids some of which, including sulfolipid, may become critical only under growth conditions that still need to be discovered.

Arabidopsis Proteins↗
PubMed1986

Production, characterization, and applications of monoclonal antibodies reactive with soybean nodule xanthine dehydrogenase.

Seven monoclonal antibodies were produced against soybean nodule xanthine dehydrogenase, an enzyme involved in ureide synthesis. Specificity of the seven monoclonal antibodies for xanthine dehydrogenase was demonstrated by immunopurifying the enzyme to homogeneity from a crude nodule extract using antibodies immobilized to Sepharose 4B beads. Each monoclonal antibody was covalently bound to Sepharose 4B beads for the preparation of immunoaffinity columns for each antibody. All seven antibodies were found to be of the IgG1,K subclass. A competitive, indirect enzyme-linked immunosorbent assay demonstrated that two of the seven antibodies shared a common epitope while the remaining five antibodies defined unique determinants on the protein. Rapid, large scale purification of active xanthine dehydrogenase to homogeneity was performed by immunoaffinity chromatography. The presence of xanthine dehydrogenase activity and protein in every organ of the soybean plant was determined. Crude extracts of nodules, roots, stems, and leaves cross-reacted with all seven monoclonal antibodies in an indirect enzyme-linked immunosorbent assay. A positive correlation was observed between the degree of cross-reactivity of a given organ and the level of enzyme activity in that organ. These data demonstrate that xanthine dehydrogenase is not nodule specific. Antigenic variability of xanthine dehydrogenase present in crude extracts from nodules of soybean, wild soybean, cowpea, lima bean, pea, and lupin were detected in the indirect enzyme-linked immunosorbent assay which corresponded to six binding patterns for xanthine dehydrogenase from these plant species. These results correspond well with the epitope determination data which showed that the seven antibodies bind to six different binding determinants on the enzyme.

Journal Article↗
PubMed1994

In situ hybridization of two nodule-specific gene products from yellow lupin.

We have applied in situ hybridization technique to examine the expression of two tissue-specific plant genes represented by cDNA clones: pLN-13 and pLN-50 during the symbiosis of Lupinus luteus with Bradyrhizobium lupini. Both genes are expressed in root nodules and their transcripts are restricted to the infected zone of the nodule as revealed with 35S-or 3H-labelled antisense RNA probes. The number of grains over bacteroid-containing cells after hybridization with 3H-labelled probes differed during three tested phases of nodule development: at the 14th, 21st and 28th day after the infection with B. lupini. As control, apical root meristems and segments of root hair zones of uninfected plants were used for incubation with RNA of either sense or antisense orientation preceded by RNAse A digestion. The data obtained confirm that both genes are organ-specific and reveal their developmentally regulated expression pattern during the course of root nodule morphogenesis.

Antisense Elements (Genetics)↗
PubMed1987

Effect of localized nitrogen availability to soybean half-root systems on photosynthate partitioning to roots and nodules.

Soybean (Glycine max [L.] Merr. cv Davis) was grown in a split-root growth system designed to maintain control of the root atmosphere. Two experiments were conducted to examine how 80% Ar:20% O(2) (Ar:O(2)) and air (Air) atmospheres affected N assimilation (NH(4)NO(3) and N(2) fixation) and the partitioning of photosynthate to roots and nodules. Application of NH(4)NO(3) to nonnodulated half-root systems enhanced root growth and root respiration at the site of application. A second experiment applied Ar:O(2) or air to the two sides of nodulated soybean half-root systems for 11 days in the following combinations: (a) Air to both sides (Air/Air); (b) Air to one side, Ar:O(2) to the other (Air/Ar:O(2)), and (c) Ar:O(2) to both sides (Ar:O(2)/Ar:O(2)). Results indicated that dry matter and current photosynthate ((14)C) were selectively partitioned to nodules and roots where N(2) was available. Both root and nodule growth on the Air side of Air/Ar:O(2) plants was significantly greater than the Ar:O(2) side. The relative partitioning of carbon and current photosynthate between roots and nodules on a half-root system was also affected by N(2) availability. The Ar:O(2) sides partitioned relatively more current photosynthate to roots (57%) than nodules (43%), while N(2)-fixing root systems partitioned 36 and 64% of the carbon to roots and nodules, respectively. The Ar:O(2) atmosphere decreased root and nodule respiration by 80% and nitrogenase activity by 85% compared to half-root systems in Air while specific nitrogenase activity of nodules in Ar:O(2) was 50% of nodules supplied Air. Results indicated that nitrogen assimilation, whether from N(2) fixation or inorganic sources, had a localized effect on root development. Nodule development accounted for the major decrease in total photosynthate partitioning to non-N(2)-fixing nodules. Soybean compensates for ineffective nodulation by controlling the flux of carbon to ineffective nodules and their associated roots.

Journal Article↗
PubMed2002

HAR1 mediates systemic regulation of symbiotic organ development.

Symbiotic root nodules are beneficial to leguminous host plants; however, excessive nodulation damages the host because it interferes with the distribution of nutrients in the plant. To keep a steady balance, the nodulation programme is regulated systemically in leguminous hosts. Leguminous mutants that have lost this ability display a hypernodulating phenotype. Through the use of reciprocal and self-grafting studies using Lotus japonicus hypernodulating mutants, har1 (also known as sym78), we show that the shoot genotype is responsible for the negative regulation of nodule development. A map-based cloning strategy revealed that HAR1 encodes a protein with a relative molecular mass of 108,000, which contains 21 leucine-rich repeats, a single transmembrane domain and serine/threonine kinase domains. The har1 mutant phenotype was rescued by transfection of the HAR1 gene. In a comparison of Arabidopsis receptor-like kinases, HAR1 showed the highest level of similarity with CLAVATA1 (CLV1). CLV1 negatively regulates formation of the shoot and floral meristems through cell-cell communication involving the CLV3 peptide. Identification of hypernodulation genes thus indicates that genes in leguminous plants bearing a close resemblance to CLV1 regulate nodule development systemically, by means of organ-organ communication.

Arabidopsis Proteins↗
PubMed2005

Changes in ascorbate peroxidase, catalase, guaiacol peroxidase and superoxide dismutase activities in common bean (Phaseolus vulgaris) nodules under salt stress.

To analyse nodular antioxidant enzyme expression in response to salt stress, Phaseolus vulgaris genotype BAT477 was inoculated with reference strain CIAT899, and treated with 50 mM NaCl. Plant growth, nodulation and nitrogen fixing activity were analysed. Results showed that: (1) all parameters, particularly in nodules, were affected by salt treatments, and (2) confirmed preferential growth allocation to roots. The ARA was significantly decreased by salt treatments. Protein dosage confirmed that nodules were more affected by salt treatment than were roots. We analysed superoxide dismutase, catalase, ascorbate peroxidase and peroxidase in nodules, roots and a free rhizobial strain. Our results indicated that SOD and CAT nodular isozymes had bacterial and root origins. The SOD expressed the same CuZn, Fe and Mn SOD isoforms in nodules and roots, whereas in free rhizobia we found only one Fe and Mn SOD. APX and POX nodule and root profiles had only root origins, as no rhizobial band was detected. Under salt stress, plant growth, nitrogen fixation and activities of antioxidant defense enzymes in nodules were affected. Thus, these enzymes appear to preserve symbiosis from stress turned out that NaCl salinity lead to a differential regulation of distinct SOD and POX isoenzyme. So their levels in nodules appeared to be consistent with a symbiotic nitrogen fixing efficiency hypothesis, and they seem to function as the molecular mechanisms underlying the nodule response to salinity.

Ascorbate Peroxidases↗
PubMed1987

Bacterial heme synthesis is required for expression of the leghemoglobin holoprotein but not the apoprotein in soybean root nodules.

In Bradyrhizobium japonicum/soybean symbiosis, the leghemoglobin (legume hemoglobin) apoprotein is a plant product, but the origin of the heme prosthetic group is not known. B. japonicum strain LO505 is a transposon Tn5-induced cytochrome-deficient mutant; it excreted the oxidized heme precursor coproporphyrin III into the growth medium. Mutant strain LO505 was specifically deficient in protoporphyrinogen oxidase (protoporphyrinogen-IX:oxygen oxidoreductase, EC 1.3.3.4) activity, and thus it could not catalyze the penultimate step in heme biosynthesis. Soybean root nodules formed from this mutant did not contain leghemoglobin, but the apoprotein was synthesized nevertheless. Data show that bacterial heme synthesis is required for leghemoglobin expression, but the heme moiety is not essential for apoleghemoglobin synthesis by the plant. Soybean leghemoglobin, therefore, is a product of both the plant and bacterial symbionts.

Apoproteins↗
PubMed2001

Actin monoubiquitylation is induced in plants in response to pathogens and symbionts.

Most dramatic examples of actin reorganization have been described during host-microbe interactions. Plasticity of actin is, in part, due to posttranslational modifications such as phosphorylation or ubiquitylation. Here, we show for the first time that actins found in root nodules of Phaseolus vulgaris are modified transiently during nodule development by monoubiquitylation. This finding was extended to root nodules of other legumes and to other plants infected with mycorrhiza or plant pathogens such as members of the genera Pseudomonas and Phytophthora. However, neither viral infections nor diverse stressful conditions (heat shock, wounding, or osmotic stress) induced this response. Additionally, this phenomenon was mimicked by the addition of a yeast elicitor or H2O2 to Phaseolus vulgaris suspension culture cells. This modification seems to provide increased stability of the microfilaments to proteolytic degradation and seems to be found in fractions in which the actin cytoskeleton is associated with membranes. All together, these data suggest that actin monoubiquitylation may be considered an effector mechanism of a general plant response against microbes.

Actins↗
PubMed1989

Production of root hair deformation factors by Rhizobium meliloti nodulation genes in Escherichia coli: HsnD (NodH) is involved in the plant host-specific modification of the NodABC factor.

The role of the hsnD (nodH) gene in the determination of the host-specific nodulation ability of Rhizobium meliloti was studied by expressing the common nodulation genes (nodABC) with or without the hsnD gene in Escherichia coli and testing for biological activity on various leguminous plants. In this way, four categories of plants were established. Upon infection with E. coli carrying the nodABC construct, root hair deformation (Had) was detected on clovers while the hsnD gene was additionally needed for the elicitation of the same response on alfalfa and sweet clover. A weak root hair deformation was seen on siratro by inoculation with E. coli harbouring the nodABC genes and was highly increased when hsnD was also introduced. Cowpea and Desmodium did not respond to any of the E. coli strains constructed. Exudates or cytosolic fractions of the respective E. coli derivatives elicited the same root hair deformation as the intact bacteria. These data indicate that not only the nodABC gene products but also the hsnD product are involved in the synthesis of Had factors. Subclones expressing only the nodA, nodB, or nodC genes or the same genes in pairs (nodAB, nodBC, nodAC) did not provide a compound with activity comparable to the NodABC factor, suggesting that all three genes are required for the production of the Had factor which is active on clover. Coinoculation of alfalfa plants with two strains of E. coli, one carrying the nodABC genes and the other expressing only hsnD, or combining exudates or cytosolic fractions from these strains did not result in root hair deformation on alfalfa. These data indicate that the HsnD protein itself or its product is not an additional alfalfa-specific extracellular signal but more likely is enzymatically involved in the modification of the basic compound determined by the nodABC genes.

DNA, Bacterial↗
PubMed1982

Simultaneous measurement of acetylene reduction and respiratory gas exchange of attached root nodules.

A method was developed for the simultaneous measurement of acetylene reduction, carbon dioxide evolution and oxygen uptake by individual root nodules of intact nitrogen-fixing plants (Alnus rubra Bong.). The nodules were enclosed in a temperature-controlled leak-tight cuvette. Assay gas mixtures were passed through the cuvette at a constant, known flow rate and gas exchange was measured by the difference between inlet and outlet gas compositions. Gas concentrations were assayed by a combination of an automated gas chromatograph and a programmable electronic integrator. Carbon dioxide and ethylene evolution were determined with a coefficient of variation which was less than 2%, whereas the coefficient of variation for oxygen uptake measurements was less than 5%. Nodules subjected to repeated removal from and reinsertion into the cuvette and to long exposures of 10% v/v acetylene showed no irreversible decline in respiration or acetylene reduction. This system offers long-term stability and freedom from disturbance artifacts plus the ability to monitor continuously, rapidly and specifically the changes in root nodule activity caused by environmental perturbation.

Journal Article↗