Nodule Acetylene Reduction Activity Research Articles

The cin Quorum Sensing Locus of Rhizobium etli CNPAF512 Affects Growth and Symbiotic Nitrogen Fixation

Rhizobium etli CNPAF512 produces an autoinducer that inhibits growth of Rhizobium leguminosarum bv. viciae 248 and activates the Agrobacterium tumefaciens tra reporter system. Production of this compound in R. etli is dependent on two genes, named cinR and cinI, postulated to code for a transcriptional regulator and an autoinducer synthase, respectively. NMR analysis of the purified molecule indicates that the R. etli autoinducer produced by CinI is a saturated long chain 3-hydroxy-acyl-homoserine lactone, abbreviated as 3OH-(slc)-HSL. Using cin-gusA fusions, expression of cinI and cinR was shown to be growth phase-dependent. Deletion analysis of the cinI promoter region indicates that a regulatory element negatively controls cinI expression. Mutational analysis revealed that expression of the cinI gene is positively regulated by the CinR/3OH-(slc)-HSL complex. Besides 3OH-(slc)-HSL, R. etli produces at least six other autoinducer molecules, for which the structures have not yet been revealed, and of which the synthesis requires the previously identified raiI and raiR genes. At least three different autoinducers, including a compound co-migrating with 3OH-(slc)-HSL, are produced in R. etli bacteroids isolated from bean nodules. This is further substantiated by the observation that cinI and cinR are both expressed under symbiotic conditions. Acetylene reduction activity of nodules induced by the cin mutants was reduced with 60-70% compared with wild-type nodules, indicating that the R. etli 3OH-(slc)-HSL is involved in the symbiotic process. This was further confirmed by transmission electron microscopy of nodules induced by the wild type and the cinI mutant. Symbiosomes carrying cinI mutant bacteroids did not fully differentiate compared with wild-type symbiosomes. Finally, it was observed that the cinR gene and raiR control growth of R. etli.

Adequate range of boron nutrition is more restricted for root nodule development than for plant growth in young soybean plant

A pot experiment was conducted under growth chamber conditions to determine the lower and upper critical levels of boron (B) for plant growth, nodule development, and nodule acetylene reduction activity (ARA) in young soybean plants. Plants of a soybean cultivar, Tachinagaha, were grown in pots containing river sand to which a nutrient solution with different B levels was added and were inoculated with Bradyrhizobium japonicum A1017. At 8, 12, and 16 d after sowing (DAS), among the plants supplied with the solution at 0, 1.0, and 2.0 mg B L-1 , plants with 1.0 mg B L-1 showed the highest values for dry shoot and fresh root weight, root length, total number of developing nodules and meristematic nodules (DMN), and ARA. At 20 DAS plants grown with 11 B levels (0-2.0 mg L-1) were compared. The B critical deficiency levels for soybean dry shoot weight, fresh root weight, root length, DMN, number of complete nodules, and ARA were approximately 46, 35, 34, 57, 60, and 50 mg B kg-1 dry matter, and the critical toxicity levels were approximately 114, 137, 134, 97, 104, and 89 mg B kg-1 dry matter, respectively. The optimum B levels for the growth characters were approximately 34 to 137 mg B kg-1 dry matter. The optimum range of B levels for nodule formation and function was more restricted than that for the growth characters. Based on the results of treatments with various B concentrations, 0.4 mg B L-1 was found to be the concentration most beneficial for all the growth characters including nodule formation at the early stage (20 DAS) of development of soybean plants.

Salinity effects on growth analysis and nutrient composition in four grain legumes‐rhizobiumsymbiosis

Abstract The effect of salinity on growth response, nitrogen (N) fixation and tissue mineral content was investigated for four legumes: faba bean (Vicia faba L), pea (Pisum sativum L), soybean (Glycine max L), and common bean (Phaseolus vulgaris L). Plants were grown in a vermiculite culture system supplied with a N‐free nutrient solution with the addition of 0, 50, and 100 mM NaCl. Plants were harvested at the beginning of the flowering period and the dry weights of shoots and roots and acetylene reduction activity (ARA) were evaluated at the same time plant tissues were analysed for N, potassium (K), calcium (Ca), magnesium (Mg), and sodium (Na) contents. The depressive effect of saline stress on ARA of nodules was directely related to the salt induced decline in dry weight and N content in shoots. Growth inhibition by NaCl treatments was greater for the pea than for other legumes, whereas the soybean was the most salt‐tolerant Saline stress also affected the N content in shoots and roots. In general th...

Effects of salt stress on growth and nitrogen fixation by pea, faba-bean, common bean and soybean plants

The effects of salinity on growth, nodulation, acetylene reduction activity (ARA), nodule leghemoglobin (Lb) content and respiratory capacity of bacteroids from pea ( Pisum sativwn cv. Lincoln), faba-bean ( Vicia faba L. var. minor cv. Alborea), bean ( Phaseolus vulgaris cv. Contender) and soybean ( Glycine max L. var. Williams) were determined. The depressive effect of saline stress (50 and 100 mM NaCl) on dry weight and ARA of nodules was directly related to the salt-induced decline in dry weight and N content in shoots. The results indicate that P. savitum was the legume most severely affected by salinity, whereas G. max was the most salt-tolerant species. Saline stress was also responsible for a decrease in cytosolic protein of nodules, specifically Lb; this effect was more pronounced in pea and bean nodules than in soybean and faba-bean nodules. Bacteroids isolated from soybean and bean nodules exhibited higher respiratory activity in the absence of energy-yielding substrates than those isolated from pea and faba-bean plants. Salt added directly to the incubation mixture of bacteroids inhibited O 2 uptake of bacteroids isolated from nodules of each legume. However, salt treatment of plants decreased respiratory capacity only in pea and faba-bean bacteroids. Inhibition of ARA under moderate saline stress may be related to the drop in bacteroid respiration. Under severe stress both the reduction in Lb content and inhibition of bacteroid respiration may be involved in salt-induced inhibition of nitrogen fixation.

Interaction between root and stem nodulation in hydroponically grown plants ofSesbania rostrata

Abstract Plant growth parameters, nitrogen content, root and stem nodulation, and acetylene reduction activity of nodules after root and shoot inoculation of hydroponically grown plants of Sesbania rostrata were studied. Stem nodulation reduced the number of root nodules whether stem inoculation was done simultaneously before or after root inoculation. Stem inoculation seven days after root inoculation gave best plant growth. Acetylene reduction was greatest in the root nodules. No significant difference in nitrogen % was observed with the different types of inoculation.

Water stress induced changes in concentrations of proline and total soluble sugars in nodulated alfalfa (Medicago sativa) plants

Susceptibility of alfalfa (Medicago sativa L. cv. Aragón) nodules and leaves to water stress has been investigated. Nodule acetylene reduction activity (ARA), leaf CO2 exchange rate (CER) as well as soluble protein, proline and total soluble sugar (TSS) contents were determined during drought. Water status was estimated as water potential (Ψw) and Relative water content (RWC) of the respective tissues. Maximum rates of ARA required higher Ψw than CER. Nodules had lower RWC for a given Ψw than leaves. Water stress reduced soluble protein content in both tissues; however, the decline in soluble protein content was detected at greater Ψw in nodules than in leaves. Proline and TSS increased in leaves and nodules, and again the threshold Ψw triggering such accumulation was higher in nodule tissues. Oior results suggest that alfalfa nodules are more susceptible to water shortage than leaves. Effects of accumulated TSS and proline upon leaf and nodule physiology are discussed in relation to protein stability (proline), pH control (proline) and osmotic adjustment (proiine and TSS). The TSS accumulation induced by water stress suggests that substrate shortage would not be the primary effect of drought on nodule activity.

Effect of Acetylene on Root Respiration and Acetylene Reducing Activity in Nodulated Soya Bean

Acetylene decreased root and nodule respiration, as measured by CO(2) evolution of nodulated or non-nodulated Glycine max. An inhibition of 25 to 35% in 15 to 30 minutes occurred when 13% C(2)H(2) was introduced in the gas flux which aerated the root nutrient solution. When the light intensity was doubled to 800 microeinsteins per square meter per second, the inhibition increased to 50% and nodule acetylene reduction activity was inhibited 50%.

Nitrogen Assimilating Enzyme Activities and Enzyme Protein during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules

Effective (N(2)-fixing) alfalfa (Medicago sativa L.) and plant-controlled ineffective (non-N(2)-fixing) alfalfa recessive for the in(1) gene were compared to determine the effects of the in(1) gene on nodule development, acetylene reduction activity (ARA), and nodule enzymes associated with N assimilation and disease resistance. Effective nodule ARA reached a maximum before activities of glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), asparagine synthetase (AS), and phosphoenolpyruvate carboxylase (PEPC) peaked. Ineffective nodule ARA was only 5% of effective nodule ARA. Developmental profiles of GS, GOGAT, AAT, and PEPC activities were similar for effective and ineffective nodules, but activities in ineffective nodules were lower and declined earlier. Little AS activity was detected in developing ineffective nodules. Changes in GS, GOGAT, AAT, and PEPC activities in developing and senescent effective and ineffective nodules generally paralleled amounts of immunologically detectable enzyme polypeptides. Effective nodule GS, GOGAT, AAT, AS, and PEPC activities declined after defoliation. Activities of glutamate dehydrogenase, malate dehydrogenase, phenylalanine ammonia lyase, and caffeic acid-o-methyltransferase were unrelated to nodule effectiveness. Maximum expression of nodule N-assimilating enzymes appeared to require the continued presence of a product associated with effective bacteroids that was lacking in in(1) effective nodules.

Nitrogen fixation by subterranean clover ( Trifolium subterraneum L.) in an acid soil in response to moisture deficits

Subterranean clover cv. Mt Barker was grown in intact cores of (i) undisturbed acid soil (pH 4.3), (ii) cultivated acid soil (pH 4.3) and (iii) lime-amended soil (pH 5.5) for 12 weeks after which half of the plants were watered from the base with a sub-optimal amount of water. Normal plant-water relations were maintained by plants in basally-irrigated treatments although the soil at the top of the profile began to slowly dry. Nodules in the drying soil also began to slowly lose moisture. Loss of moisture from nodules was very rapid once the permanent wilting point of the soil (−1.5 MPa) had been reached. Large nodules produced on the roots of plants grown in undisturbed acid soil recorded a water potential (ψ nod) of −1.3 MPa after the soil had reached the permanent wilting point whereas the smaller nodules produced on plants grown in lime-amended soil were more severely desiccated (ψ nod of −1.8 MPa) after the permanent wilting point had been reached. Most of the root nodules occurred in the top 9 cm of the soil cores and acetylene reduction activity of nodulated roots in this region of the soil profile (divided into 0–4 and 4–9 cm segments) was measured in a flow-through system. Moisture deficits reduced the acetylene reduction activity of nodules. In moist soil, nitrogenase activity was similar for plants in all treatments. However, irrespective of nodule size, highest rates of nitrogenase activity occurred at a ψ nod > −0.4 MPa (RWC nod of 82%), and acetylene reduction activity declined rapidly when ψ nod fell below this level.

White Clover N2-Fixation in Response to Root Temperature and Nitrate

Established, nodulated white clover plants were transferred to eight tanks of a flowing culture apparatus with solution temperatures of 5, 11, 17, and 25 °C (two tanks per temperature). Shoot temperature and light environment were common to all plants. After 7 d, NO3- (10 mmol m−3) was continuously supplied to one tank at each temperature while in the remaining four tanks (one at each temperature) the plants were completely dependent on nodule N2-fixation. Plants were randomly selected at intervals during the following 14 d period in order to measure root and nodule respiration and acetylene reduction activity (ARA) in a flow-through system set at the adapted root temperature. Additional plants were assayed for in vitro nitrate reductase activity in leaves, roots, and nodules. Apparent nitrogenase activity (ARA) and respiration associated with it were each markedly affected by temperature in two ways; (1) Activity per unit weight of nodule was reduced at lower temperatures; (2) Development of the plant, and thus also nodule mass, was restricted at lower temperatures which, in turn, restricted total nodule activity per plant. The presence of nitrate significantly reduced ARA of nodules, particularly at higher temperatures. However, significant discrepancies were found when N2-fixation rates, estimated from the acetylene reduction assay, were compared with N2-fixation rates calculated from curves fitted to N accumulation data (minus the rate of NO3- uptake in the case of nitrate-treated plants). Carbon use efficiency (CO2 respired per C2H4 produced) was not significantly affected by temperature or the presence of nitrate. Nitrate reductase activity (NRA) developed in all plant parts at the three highest temperatures, but not at 5 °C. We calculate that leaf NRA may account for 82, 75, and 68% of total nitrate reduction at 11, 17, and 25 °C respectively.

Effect of Source Removal on Functioning of the Nitrogen Fixation of Pigeonpea ( Cajanus cajan L.) nodules

Seven week old pigeonpea (Cajanus cajan L.) plants grown in sand culture were detopped (stem excised just above the sand surface) at the 6th, 10th,14th, and 18th h of the day and sampled after every 4 hours up to a total period of 12 hours. Irrespective of the time of detopping, acetylene reduction activity of nodules showed a sharp decrease within 4 hours of the treatment. Detopping at the 10th and 14th hours resulted in a 90 % decrease in the nitrogenase activity over the control. However, the same treatments resulted in only a 10–15 % decrease in nodule soluble carbohydrates, reducing sugars and starch content. Nodule respiration, however, showed a decline almost parallel to nitrogen fixation (correlation coefficient = 0.9) upon detopping. In a short term experiment, decapitation and stem girdling reduced the ARA by 50 and 66 % of the control within 1 hour, respectively. It, therefore, appears from the present investigation that certain unknown mechanism operates which puts a constraint on utilization of carbohydrates for nodule respiration which supports nitrogen fixation.

Evaluation of three mediterranean forage legumes for biological nitrogen fixation

Abstract Continuous grain cropping in many areas of the world has reduced soil N levels to a degree such that agricultural production is now largely dependent upon nitrogen fertilizer. Through the identification of highly effective legume and Rhizobium strain combinations, however, increased production of protein‐rich livestock forage and green manure is possible. Three common Mediterranean legumes, Lupinus albus, Medicago truncatula, and Trifolium alexandrium were chosen to be tested for symbiotic N‐fixation ability with various available strains of Rhizobium. Greenhouse and growth chamber experiments were conducted for six weeks to evaluate shoot, root, and nodule dry weight, nodule number, nodule acetylene reduction activity, and total plant N. For L. albus, four single strains, 96A5, 96A19, 96B15, and 96B23, and one commercially available multiple strain ‘H’, produced acetylene reduction activity from 100 to 190 μmoles ethylene evolved/h/gdw of nodules. Since plant dry weights and total plant N values...

Isolation and characterization of symbiotic mutants of bradyrhizobium sp. (Arachis) strain NC92: mutants with host-specific defects in nodulation and nitrogen fixation.

Random transposon Tn5 mutagenesis of Bradyrhizobium sp. (Arachis) strain NC92, a member of the cowpea cross-inoculation group, was carried out, and kanamycin-resistant transconjugants were tested for their symbiotic phenotype on three host plants: groundnut, siratro, and pigeonpea. Two nodulation (Nod- phenotype) mutants were isolated. One is unable to nodulate all three hosts and appears to contain an insertion in one of the common nodulation genes (nodABCD); the other is a host-specific nodulation mutant that fails to nodulate pigeonpea, elicits uninvaded nodules on siratro, and elicits normal, nitrogen-fixing nodules on groundnut. In addition, nine mutants defective in nitrogen fixation (Fix- phenotype) were isolated. Three fail to supply symbiotically fixed nitrogen to all three host plants. Surprisingly, nodules elicited by one of these mutants exhibit high levels of acetylene reduction activity, demonstrating the presence of the enzyme nitrogenase. Three more mutants have partially effective phenotypes (Fix +/-) in symbiosis with all three host plants. The remaining three mutants fail to supply fixed nitrogen to one of the host plants tested while remaining partially or fully effective on the other two hosts; two of these mutants are Fix- in pigeonpea and Fix +/- on groundnut and on siratro, whereas the other one is Fix- on groundnut but Fix+ on siratro and on pigeonpea. These latter mutants also retain significant nodule acetylene reduction activity, even in the ineffective symbioses. Such bacterial host-specific fixation (Hsf) mutants have not previously been reported.

Variation in Nitrate Tolerance of Nitrogen Fixation in the Pea/Rhizobium Symbiosis

AbstractThe short‐term effects of nitrate on symbiotic nitrogen fixation were studied in six cultivars of pea (Pisum sativum L.) using two strains of Rhizobium leguminosarum. Plants grown symbiotically for three weeks, in order to eliminate the effect of NO3 on nodule formation, were exposed to nil, 5 or 15 mM NO3 for a period of seven days. Nodule mass, nitrogenase activity by acetylene reduction and the NO2 and NO3 concentrations in nodules were determined.Exposure to nitrate reduced the total acetylene reduction activity (ARA) in all cultivars, and a significant cultivar × nitrate treatment interaction was found. Nitrate influenced the components of N2 fixation, nodule mass and specific ARA (ARA/mg nodule), differently within and among cultivars. Nodule mass was reduced in all cultivars, but a variation among cultivars in the degree of reduction was found. Nitrate also reduced the specific ARA to different extents in the cultivars‐ The specific ARA of nodules from NO3‐treated plants, expressed as per cent of the specific ARA of nodules from plants grown without NOT3 was negatively correlated with the concentration of NO3 in nodules. No relationship was found between the reduction of N2 fixation and the nitrite concentration in nodules.The results indicate that it is possible to improve the nitrate tolerance of symbiotic N2 fixation of the established pea/Rhizobium symbiosis by selection of suitable host plants.

Characteristics of glutamine synthetase I and II mutants of Rhizobium.

Mutants of Rhizobium meliloti 41 and AK631 deficient in either glutamine synthetase I or II were characterized for growth and acetylene reduction activity. Comparison of the various mutants indicates that either GSI or GSII is competent to provide glutamine for cellular requirements with ammonia as the nitrogen source. The acetylene reduction activity of nodules induced by some GSI- or GSII- mutants was equal to the wild- type activity indicating that neither GS is required for symbiotic effectiveness and that either form is sufficient for effectiveness. No effect of purines on growth was found in the GS mutants of this strain.

Nodule Physiology of a Supernodulating Soybean (Glyine max) Mutant

The nodule physiology of a supernodulating, nitrate tolerant symbiosis soybean (Glycine max (L.) Merr.) mutant (nts382) was compared to that of its wild-type parent, cv. Bragg. Nodule number and mass were greater in nts382 than cv. Bragg and individual nodule mass, bacteroid and haem content, and acetylene reduction activity per g nodule were less. Acetylene reduction activity expressed per mg bacteroid protein was the same in the two genotypes. In median sections, the ratio of infected to total nodule area was smaller in nts382, infected cell size was smaller and there were fewer bacteroids per peribacteroid envelope. When inoculum dose was decreased from 109 to 103 viable cells per pot, nodule number on nts382 decreased approximately to that on cv. Bragg; nodule size, bacteroid and haem contents increased as did nodule acetylene reduction activity. Application of moderate levels of nitrate, which did not significantly affect symbiotic parameters of cv. Bragg or high inoculum nts382, stimulated nodule growth and nitrogenase activity of low inoculum nts382. A combination of nitrate and low inoculum levels enhanced nodule parameters of nts382 to the level usually seen with cv. Bragg. When supernodulated, plant dry weight of nts382 was less than that of Bragg; decreasing inoculum dose had no significant effect but nitrate application increased plant growth; nitrate plus low inoculum induced similar plant growth to that of cv. Bragg. Nodule carbohydrate content was similar in both genotypes but nodule and xylem sap ureide contents were higher in nts382. In general, nodules of supernodulated nts382 resembled under-developed cv. Bragg nodules; when supernodulation was avoided by using low inoculum doses, ,nts382 nodules resembled those of cv. Bragg. Nitrogen metabolism in the mutant seems to be disturbed, resulting in ureide accumulation.

The influence of temperature and nitrate on vegetative growth and nitrogen accumulation by nodulated soybeans

Inoculated soybeans [Glycine max (L.) Merrill] were grown in controlled environments to evaluate the relationship between temperature and applied NO3−N on growth rates, N accumulation, and acetylene reduction activity during the vegetative growth stage. Soybeans were grown at day/night temperatures of 22/18 and 26/22°C in sand culture with daily applications of 21.4 mM (high) and 2.1 mM (low) NO3−N in a complete nutrient solution for durations of 14, 21, and 42 days after emergence and with an N-free solution. Dry matter and N accumulation were greater at 26/22 than 22/18°C. In general, both increased as the level and duration of applied NO3−N was increased. These increases were attributable to an abbreviation in the interval between emergence and onset of rapid growth. The presence and assimilation of NO3−N, even at the high level, did not inhibit development of functional nodules. Neither mass nor acetylene reduction activity of nodules was reduced by high NO3−N; however, the root mass was increased by NO3−N more than the nodule mass. There was an interaction between temperature and NO3−N on specific nodule activity as measured by acetylene reduction. The specific nodule activity was unaffected by NO3−N at 22/18°C, but at 26/22°C the specific activity was lower in the absence of NO3−N than when NO3−N was present. Apparently, rapid early growth at 26/22°C depleted cotyledonary reserves of N before nodules became active and, thereafter, the plants were unable to develop adequate leaf area to support nodule development and functioning. This result has implications in N fertilization of late-planted soybeans.

Carbon Dioxide Fixation by Lupin Root Nodules

In vivo CO(2) fixation and in vitro phosphoenolpyruvate (PEP) carboxylase levels have been measured in lupin (Lupinus angustifolius L.) root nodules of various ages. Both activities were greater in nodule tissue than in either primary or secondary root tissue, and increased about 3-fold with the onset of N(2) fixation. PEP carboxylase activity was predominantly located in the bacteroid-containing zone of mature nodules, but purified bacteroids contained no activity. Partially purified PEP carboxylases from nodules, roots, and leaves were identical in a number of kinetic parameters. Both in vivo CO(2) fixation activity and in vitro PEP carboxylase activity were significantly correlated with nodule acetylene reduction activity during nodule development. The maximum rate of in vivo CO(2) fixation in mature nodules was 7.9 nmol hour(-1) mg fresh weight(-1), similar to rates of N(2) fixation and reported values for amino acid translocation.The results suggest that the oxaloacetete used as the primary "carbon skeleton" acceptor for ammonia assimilation and amino acid synthesis in lupin nodules is provided via the PEP carboxylase reaction rather than through the tricarboxylic acid cycle. The source of PEP is presumably glycolysis, while the major source of CO(2) is inferred to be respiration.