Comparing Symbiotic Efficiency between Swollen versus Nonswollen Rhizobial Bacteroids

Symbiotic rhizobia differentiate physiologically and morphologically into nitrogen-fixing bacteroids inside legume host nodules. The differentiation is apparently terminal in some legume species, such as peas (Pisum sativum) and peanuts (Arachis hypogaea), likely due to extreme cell swelling induced by the host. In other legume species, such as beans (Phaseolus vulgaris) and cowpeas (Vigna unguiculata), differentiation into bacteroids, which are similar in size and shape to free-living rhizobia, is reversible. Bacteroid modification by plants may affect the effectiveness of the symbiosis. Here, we compare symbiotic efficiency of rhizobia in two different hosts where the rhizobia differentiate into swollen nonreproductive bacteroids in one host and remain nonswollen and reproductive in the other. Two such dual-host strains were tested: Rhizobium leguminosarum A34 in peas and beans and Bradyrhizobium sp. 32H1 in peanuts and cowpeas. In both comparisons, swollen bacteroids conferred more net host benefit by two measures: return on nodule construction cost (plant growth per gram nodule growth) and nitrogen fixation efficiency (H₂ production by nitrogenase per CO₂ respired). Terminal bacteroid differentiation among legume species has evolved independently multiple times, perhaps due to the increased host fitness benefits observed in this study.Legume-rhizobia interactions vary widely across a diverse paraphyletic group of soil bacteria known for symbiotic nitrogen fixation inside root nodules of over 18,000 species of legumes throughout the world (Lewis et al., 2005). In several legume species, rhizobial cells are induced to swell during their differentiation into nitrogen-fixing bacteroids (Oono et al., 2010). These legume species belong to five different major papilionoid clades (inverted repeat-lacking clade, genistoids, dalbergioids, mirbelioids, and millettioids), a pattern suggestive of convergent evolution. Swelling apparently leads to terminal differentiation; swollen bacteroids no longer divide normally (Zhou et al., 1985). In other legume host species, bacteroid differentiation is less extreme, leading to nonswollen bacteroids. Nonswollen bacteroids are similar in shape and size to free-living rhizobia and divide normally once outside of their nodules. The proximate mechanisms for host-imposed bacteroid swelling have been investigated (Van de Velde et al., 2010), but what drove the repeated evolution of this trait? The multiple independent origins of host traits causing bacteroids to swell suggest that swollen bacteroids may provide more net benefit to legumes. Could the swelling of bacteroids improve nitrogen fixation efficiency (e.g. nitrogen fixed relative to carbon cost)? In this study, we compare symbiotic efficiencies of rhizobia in legume hosts that are evolutionarily diverged but share a common effective rhizobial strain, whose bacteroids are swollen in one host and nonswollen in the other.Variations among host species in benefits and costs of symbiosis with rhizobia are not commonly explored (Thrall et al., 2000) because legume species typically nodulate with only one group of rhizobia (e.g. Sinorhizobium sp. in Medicago), although some legumes and some rhizobia are more promiscuous. Rhizobium sp. NGR234 has the largest known host range but does not fix nitrogen effectively with any legume species currently recognized to induce swelling of rhizobial bacteroids (Pueppke and Broughton, 1999). Some Sinorhizobium fredii strains apparently fix nitrogen in certain cultivars of soybean (Glycine max; hosting nonswollen bacteroids) and alfalfa (Medicago sativa; hosting swollen bacteroids; Hashem et al., 1997), but our efforts to replicate these results did not lead to successful nodulation. Therefore, we studied two strains, a transgenic strain that nodulates beans (Phaseolus vulgaris) and peas (Pisum sativum) and a second wild strain harvested from cowpeas (Vigna unguiculata) that also nodulates peanuts (Arachis hypogaea). Beans and cowpeas are both within the Phaseolid group and do not induce terminal differentiation of rhizobial bacteroids. Peas and peanuts both host terminally differentiated bacteroids but are in distant clades and likely have different genetic origins for traits that induce terminal differentiation (Oono et al., 2010). Also, the swollen bacteroids in peas are branched while those in peanuts are spherical.Differences in symbiotic qualities between swollen and nonswollen bacteroids have been previously explored in peanuts and cowpeas by Sen and Weaver (1980, 1981, 1984), who also hypothesized that swollen bacteroids are more beneficial to the host plant than nonswollen ones. They found 1.5 to 3 times greater acetylene reduction by nitrogenase (as well as plant nitrogen) per nodule mass in peanuts than in cowpeas at multiple nodule ages (Sen and Weaver, 1980). Acetylene reduction per bacteroid was also greater in peanuts than in cowpeas when measuring whole nodules, but this difference disappeared when isolated bacteroids were assayed (Sen and Weaver, 1984). They concluded that swelling of peanut bacteroids per se was not responsible for the higher rate of nitrogen fixation per bacteroid. They suggested that in cowpea nodules, with greater numbers of smaller bacteroids per nodule volume, availability of oxygen to each bacteroid might be restricted such that the rate of oxidative phosphorylation, necessary for nitrogen fixation, is reduced. Fixation rates per bacteroid may be different between hosts due to nodule gas permeability or bacteroid crowding within nodules. However, fixation efficiency (nitrogen fixed per carbon respired) would not necessarily be affected by these and may be more important for the host than the rate of fixation.Rhizobial performances are often compared by measuring the symbiotic benefits, e.g. rates of acetylene reduction or plant growth (Sen and Weaver, 1984; Hashem et al., 1997; Lodwig et al., 2005), but rarely by measuring the symbiotic costs, e.g. carbon consumed or respired. Up to 25% of a legume’s net photosynthate may be required for nitrogen fixation by rhizobia (Minchin et al., 1981). Faster fixation rates (mol nitrogen per s) can be beneficial for hosts, but carbon costs can also be important. Rhizobia that fix more nitrogen per carbon respired could free more carbon for other functions, including the option of supporting more nodules with the same amount of photosynthate. If legumes are sometimes carbon limited, then improved carbon-use efficiency could enhance plant fitness. Measuring both benefits and costs is therefore key to an accurate understanding of the symbiotic performance of a rhizobial strain.While we recognize the many physiological differences between peas and beans or peanuts and cowpeas, the fact that terminal differentiation induced by host legumes evolved multiple times independently (Oono et al., 2010) suggests there may be some consistent host symbiotic benefit, such as improved fixation efficiency. Here, we measured the efficiency of each of two strains as swollen bacteroids in one host and nonswollen bacteroids in another. We measured nitrogenase activity as hydrogen (H₂) production in an N₂-free atmosphere (Layzell et al., 1984; Witty and Minchin, 1998), and compared it to carbon dioxide (CO₂) respiration to estimate return on nodule operation cost. We also compared host biomass growth per total nodule mass growth to estimate return on nodule construction cost. To further assess carbon allocation to the different types of bacteroids, we also measured the average amounts per bacteroid of polyhydroxybutyrate (PHB), an energy storage compound that can comprise up to 50% of bacteroid dry weight (Trainer and Charles, 2006). A greater PHB accumulation per bacteroid may require a decreased allocation of carbon for nitrogenase activity within the bacteroids, and hence, less plant growth per carbon invested in bacteroids. We demonstrate that peas and peanuts that host swollen bacteroids have higher fixation efficiency as well as greater plant return on nodule construction than beans and cowpeas, respectively, nodulated with the same rhizobial strains. PHB was not consistently correlated with plant:nodule growth efficiency with the tested strains. These findings show that swollen bacteroids can indeed provide greater benefits to their legume hosts.     

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (mol C₂H₄g^–1 nodule fr. wt. h^–1), were both affected by the added nitrate.     

Differentiation of nodules of Glycine max

Plants of Glycine max var. Caloria, infected as 14 d old seedlings with a defined titre of Rhizobium japonicum 3Il b85 in a 10 min inoculation test, develop a sharp maximum of nitrogenase activity between 17 and 25 d after infection. This maximum (14±3 nmol C₂H₄ h^-1 mg nodule fresh weight^-1), expressed as per mg nodule or per plant is followed by a 15 d period of reduced nitrogen fixation (20–30% of peak activity). 11 d after infection the first bacteroids develop as single cells inside infection vacuoles in the plant cells, close to the cell wall and infection threads. As a cytological marker for peak multiplication of bacteroids and for peak N₂-fixation a few days later the association of a special type of nodule mitochondria with amyloplasts is described. 20 d after inoculation, more than 80% of the volume of infected plant cells is occupied by infection vacuoles, mostly containing only one bacteroid. The storage of poly--hydroxybutyrate starts to accumulate at both ends of the bacteroids. Non infected plant cells are squeezed between infected cells (25d), with infection vacuoles containing now more than two (up to five) bacteroids per section. Bacteroid development including a membrane envelope is also observed in the intercellular space between plant cells. 35 d after infection, more than 50% of the bacteroid volume is occupied by poly--hydroxybutyrate. The ultrastructural differentiation is discussed in relation to some enzymatic data in bacteroids and plant cell cytoplasm during nodule development.     

Drought-avoidant soybean germplasm maintains nitrogen-fixation capacity under water stress

Inoculated soybeans (Glycine max L. (Merrill)) were grown in controlled environments to evaluate the relationship between genotype and plant water status on nodule function, nitrogen assimilation, growth rates, and seed yield. Plants were grown under well-watered (WW) and water-stressed (WS) conditions during the linear pod-filling growth stage in sand culture using N-free nutrient solution. Dry matter and N accumulation were greater for the drought-adapted Plant Introduction 416937 (PI) than for Forrest, a commercially adapted genotype of similar phenology. These differences are attributed to: (i) more favorable internal water balance throughout the pod-filling period (higher total leaf water potential), (ii) higher photosynthetic function (more total leaf area and higher net carbon exchange rates), and (iii) stronger nodule function (larger nodule mass, greater specific and total nodule activity, and thus more nitrogen assimilation) for the PI than for Forrest. While Forrest out yielded the PI under WW conditions, the percentage reduction in seed mass per plant was less for the PI than for Forrest when both genotypes were exposed to desiccating conditions. The inference is that soybean germplasm with the capacity to maintain tissue turgidity, and thus leaf and nodule function, during reproductively-imposed desiccation may reduce the extent to which yield is compromised during drought. These findings have implications for the role of symbiotic nitrogen fixation in conserving yield under dry weather conditions.Abbreviations DAE Days After Emergence - NCE Net CO₂ Exchange - PI PI 416937 - SNA Specific Nodule Activity - TNA Total Nodule Activity - WS Water Stressed - WW Well Watered     

Utilization of nitrate by bacteroids of Bradyrhizobium japonicum in the soybean root nodule

Bacteroids of Bradyrhizobium japonicum strain CB1809, unlike CC705, do not have a high level of constitutive nitrate reductase (NR; EC 1.7.99.4) in the soybean (Glycine max. Merr.) nodule. Ex planta both strains have a high activity of NR when cultured on 5 mM nitrate at 2% O₂ (v/v). Nitrite reductase (NiR) was active in cultured cells of bradyrhizobia, but activity with succinate as electron donor was not detected in freshly-isolated bacteroids. A low activity was measured with reduced methyl viologen. When bacteroids of CC705 were incubated with nitrate there was a rapid production of nitrite which resulted in repression of NR. Subsequently when NiR was induced, nitrite was utilized and NR activity recovered. Nitrate reductase was induced in bacteroids of strain CB1809 when they were incubated in-vitro with nitrate or nitrite. Increase in NR activity was prevented by rifampicin (10 g· ml^-1) or chloramphenicol (50 g·ml^-1). Nitrite-reductase activity in bacteroids of strain CB1809 was induced in parallel with NR. When nitrate was supplied to soybeans nodulated with strain CC705, nitrite was detected in nodule extracts prepared in aqueous media and it accumulated during storage (1°C) and on further incubation at 25°C. Nitrite was not detected in nodule extracts prepared in ethanol. Thus nitrite accumulation in nodule tissue appears to occur only after maceration and although bacteroids of some strains of B. japonicum have a high level of a constitutive NR, they do not appear to reduce nitrate in the nodule because this anion does not gain access to the bacteroid zone. Soybeans nodulated with strains CC705 and CB1809 were equally sensitive to nitrate inhibition of N₂ fixation.Abbreviations NR nitrate reductase - NiR nitrite reductase - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Metabolism of poly-\-hydroxybutyric acid in bacteroids ofRhizobium lupini in connection with nitrogen fixation and photosynthesis

Summary The darkening of lupin plants grown in a sand culture on a nitrogen-free medium at a stage of initial flowering led to a sharply decreased nitrogen fixation intensity which eventually ceased. Decreased intensity of nitrogen fixation in bacteroids was accompanied by an accumulation of poly--hydroxybutyric acid (PHB); in the course of 10–20 h (depending upon temperature) its content increased by 2.5–3.0 times. If, following darkening, the plants were once again exposed to light, an abrupt increase of nitrogen fixation intensity was observed and a simultaneous decrease of PHB content.It has been shown that lupin's exposure to light in¹⁴CO₂ atmosphere lasting 19 h resulted in the latter's incorporation into PHB, bacteroids and into the entire nodule; these processes developed almost in parallel.During the early period of vegetation growth prior to flowering, the PHB content of bacteroids decreased from 13–14 to 3–4% of dry weight, whereas the intensity of nitrogen fixation was raised. Concurrently increase of the activity of some enzymes connected with the PHB metabolism (acetoacetyl-CoA-reductase, acetyl-CoA-acetyl transferase, PHB-depolymerase, CoA-transferase of 3-ketoacids) occured. The plants' subsequent ageing and reduction of nitrogen fixation intensity led to a noticeable increase of PHB content and a decrease of the above mentioned enzymes' activity. The specific activity of \-hydroxybutyric dehydrogenase involved with PHB catabolism was high and was maintained at a constant level throughout the entire vegetative period.     

Monitoring watercourse vegetation, a synecological approach to dynamic gradients

Changes in vegetation under reduced control measures over 3 to 5 years in watercourses in a rural environment in The Netherlands were evaluated. A method to deal with slow changes on a steep gradient is presented. The gradient with various vegetation types between the middle of the watercourse and the bank-top was split up into zones. Species composition of each zone was evaluated using literature on syntaxonomy. Cover of character species, multiplied by the width of the zones, was used to quantify the contribution of various syntaxa in the vegetation. Changes in these contribution data were used to evaluate changes over the years. The method was applied to two experiments in which cleaning frequency was reduced. Submerged vegetation of Callitriche-Ranunculetum penicillati in one and of Potamogetonetalia pectinati in the other case, hardly changed. Emergent vegetation of Nasturtio-Glycerietalia or Sparganio-Glycerietum fluitans tended to expand into the submerged zone. Bank vegetation began to show signs of development into ruderal vegetation, as a shift from Molinio-Arrhenatheretea into Artemisietea was detected. The method allowed the conclusion that conditions were too eutrophic in both experiments for a diverse brook vegetation development without additional habitat improvement.     

Isolation and symbiotic characterization of transposon Tn5-induced arginine auxotrophs of Sinorhizobium meliloti

Seventeen arginine auxotrophic mutants of Sinorhizobium meliloti Rmd201 were isolated by random transposon Tn5 mutagenesis using Tn5 delivery vector pGS9. Based on intermediate feeding studies, these mutants were designated as argA/argB/argC/argD/argE (ornithine auxotrophs), argF/argI, argG and argH mutants. The ornithine auxotrophs induced ineffective nodules whereas all other arginine auxotrophs induced fully effective nodules on alfalfa plants. In comparison to the parental strain induced nodule, only a few nodule cells infected with rhizobia were seen in the nitrogen fixation zone of the nodule induced by the ornithine auxotroph. TEM studies showed that the bacteroids in the nitrogen fixation zone of ornithine auxotroph induced nodule were mostly spherical or oval unlike the elongated bacteroids in the nitrogen fixation zone of the parental strain induced nodule. These results indicate that ornithine or an intermediate of ornithine biosynthesis, or a chemical factor derived from one of these compounds is required for the normal development of nitrogen fixation zone and transformation of rhizobial bacteria into bacteroids during symbiosis of S. meliloti with alfalfa plants.     

Characterization of Tn5-induced symbiotically defective mutants of cowpea rhizobia

Symbiotically defective mutants of cowpea rhizobia strain IRC256 were isolated by random Tn5 mutagenesis and characterized. One auxotroph (MS1) requiring adenine and thiamine was a non-nodulating mutant (Nod⁻) and three prototrophic mutants were Nod⁺ Fix⁻ which formed small and ineffective nodules on cowpeas (Vigna unguiculata). Acetylene reduction activity of the Nod⁺ Fix⁻ mutants was reduced to 80–94% of that of the wild-type strain. The non-nodulating mutant (MS1) induced root-hair curling but did not show any nodule initiation or nodule development. Ultrastructural examination of nodules formed by Fix⁻ mutants showed that these contained few bacteroids, indicating either early senescence or a reduction in bacterial release into the cytoplasm of the host cell. DNA hybridization of total DNAs from a representative number of Tn5 mutants showed that each of them had one copy of the transposon Tn5 which was randomly inserted into the genome of cowpea rhizobia.     

Failure to fix nitrogen by non-reproductive symbiotic rhizobia triggers host sanctions that reduce fitness of their reproductive clonemates

The legume-rhizobia symbiosis is a classical mutualism where fixed carbon and nitrogen are exchanged between the species. Nonetheless, the plant carbon that fuels nitrogen (N(2)) fixation could be diverted to rhizobial reproduction by 'cheaters'--rhizobial strains that fix less N(2) but potentially gain the benefit of fixation by other rhizobia. Host sanctions can decrease the relative fitness of less-beneficial reproductive bacteroids and prevent cheaters from breaking down the mutualism. However, in certain legume species, only undifferentiated rhizobia reproduce, while only terminally differentiated rhizobial bacteroids fix nitrogen. Sanctions were, therefore, tested in two legume species that host non-reproductive bacteroids. We demonstrate that even legume species that host non-reproductive bacteroids, specifically pea and alfalfa, can severely sanction undifferentiated rhizobia when bacteroids within the same nodule fail to fix N(2). Hence, host sanctions by a diverse set of legumes play a role in maintaining N(2) fixation.     

Nodulation competitiveness in the Rhizobium-legume symbiosis

Successful nodulation of legumes by rhizobia is a complex process that, in the open field, depends on many different environmental factors. Generally, legume productivity in an agricultural field may be improved by inoculation with selected highly effective N₂-fixing root nodule bacteria. However, field legume inoculation with Rhizobium and Bradyrhizobium spp. has often been unsuccessful because of the presence in the soil of native strains that compete with the introduced strain in nodule formation on the host plants. This ability to dominate nodulation is termed competitiveness and is critical for the successful use of inoculants.The author is with the Departmentode Microbiologia del Suelo y Sistemas Simbioticos, Estation Experimental del Zaidin, Consejo Superior de Investigaciones Cientificas, C/Professor Albareda 1, 18008 Granada, Spain     

Nitrate levels affect the development of the black locust-Rhizobium symbiosis

Summary Experiments with black locust (Robinia pseudoacacia L.) seedlings grown under strictly controlled laboratory conditions indicated that the availability of nitrate has a marked impact on nitrogen fixation. When nitrate concentrations were very low, both nodulation and seedling growth were impaired, whereas nitrate concentrations high enough to promote plant growth strongly inhibited symbiotic nitrogen fixation. When nitrate was added to the growth medium after infection, nodulation and nitrogen fixation of the seedlings decreased. This effect was even more marked when nitrate was applied before infection with rhizobia. Higher nitrogen concentrations also reduced nodule number and nodule mass when applied simultaneously with the infecting bacteria. The contribution of symbiotic nitrogen fixation to black locust shoot mass by far exceeded its effects on shoot length and root mass. When nitrate availability was very low, specific nitrogen fixation (i. e. nitrogenase activity per nodule wet weight) was improved with increasing nitrogen supply, but rapidly decreased with higher nitrogen concentrations.     

Isolation of pea nodule bacteroids utilizing silica sol (Percoll) density gradient centrifugation

Isolation of bacteroids from effective (Fix⁺) and ineffective (Fix^–) pea nodules, inoculated withRhizobium leguminosarum K, were performed by a density gradient centrifugation method using silica sol (Percoll). Only one zone (=1.064–1.072; n-zone) was recognized in the Fix⁺ nodule which contained typical Y-shaped bacteroids while two zones (n-zone and =1.125–1.145; n'-zone) were obtained from the Fix^– nodule. The cells in the n'-zone, which are long rods differed morphologically from free-living cells at any growth phase (=1.108–1.125; f-zone and =1.074–1.078; f'-zone), and differed from Y-shaped bacteroids by cell density. The esterase isozyme pattern of bacteroids in the n-and n'-zones also showed clear differences from that of f-and f'-zone of free-living cells.     

Enzymes of sucrose,maltose, and α,α-trehalose catabolism in soybean root nodules

Crude, Sephadex-filtered extracts of soybean (Glycine max (L.) Merr.) root nodules contained invertase (E.C. 3.2.1.26) activity with pH optima at 5.4 and 7.8, ,-trehalase (E.C. 3.2.1.28) activity with pH optima at 3.8 and 6.6, and maltase (E.C. 3.2.1.20) activity with a broad pH optimum between 4.5 and 5.0. Bacteroids and cytosol were separated using Percoll density gradients. Cellulase and pectinase were employed to separate protoplasts from the infected region from the nodule cortex, which remained intract. Assays of disaccharidases from these nodule fractions indicated the following localization of enzymes: (1) Bacteroids lack invertase activity (pH 5.4 and 7.8). (2) Much, if not most, of the invertase activity may be localized in the nodule cortex; this is especially likely for acid invertase. However, there was substantial invertase activity in cytosol from the infected region. (3) Most of the maltase activity (pH 5.0) and trehalase activity (pH 3.8 and 6.6) were localized in the cytosol. It is likely that most of these disaccharidase activities are in the cytosol of the infected region, in contrast to invertase. (4) Bacteroids contain maltase (pH 5.0) and trehalase (pH 3.8 and 6.6), but the amount of these enzyme activities was less than 15% of total activity in nodules. Bacteroids and nodule cortex were capable of in-vivo hydrolysis of [¹⁴C]trehalose and [¹⁴C]maltose. These disaccharides were also hydrolyzed by soybean roots and hypocotyls. Therefore, while ,-trehalose in soybean nodules is probably synthesized by the bacteroids, the capability for utilization of trehalose was not restricted to the bacteroids.Approved for publication as Journal Article 74–81 of the Ohio Agricultural Research and Development Center     

Potential and pitfalls of trying to extend symbiotic interactions of nitrogen-fixing organisms to presently non-nodulated plants,such as rice

It has been a long-standing goal in the field of biological nitrogen fixation to extend nitrogen-fixing symbioses to presently non-nodulated cereal plants, such as rice. A number of researchers have recently described the induction of nodule-like structures on the roots of cereals primarily by rhizobia, in either the presence or absence of plant cell-wall-degrading enzymes or plant hormones. We briefly review this research and discuss the potential problems associated with the introduction of nitrogen-fixing microbes in novel physiological environments, such as rice roots. The results of experiments carried out in China on the induction of nodule-like structures on rice roots by rhizobia are highlighted. In addition, we present preliminary results of a series of experiments designed to repeat and evaluate these results using a variety of microscopic techniques and molecular genetic approaches.     

Glutamine synthetase,glutamate synthase and glutamate dehydrogenase in Rhizobium japonicum strains grown in cultures and in bacteroids from root nodules of Glycine max

The growth yields of three strains of Rhizobium japonicum (CB 1809, CC 723, CC 705) in culture solutions containing L-glutamate were about twice those grown with ammonium. The activities of glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.4) were dependent on the nitrogen source in the medium and also varied with growth. Both NADPH-and NADH-dependent glutamate synthase (GOGAT; EC 1.4.1.13) and NADPH-dependent GDH were found in strains grown with either glutamate or ammonium but NADH-linked GDH was only detected in glutamate-grown cells. Glutamine synthetase was adenylylated in cells grown with NH ₄ ⁺ (90%) and to lesser extent in those grown with L-glutamate (50%). In root nodules produced by the three strains in Glycine max (L.) Merr., the bulk of GS was located in the nodule cytosol (60–85%). The enzyme was adenylylated in bacteroids (43–75%) and in the nodule tissues (52–68%). The enzyme in cell-free extracts of Rh. japonicum (CC 705) grown in culture solutions containing glutamate and in bacteroids (CC 705) was deadenylylated by snake-venom phosphodiesterase. L-methionine-DL-sulfoximine restricted the incoporation of ¹⁵N-labelled (NH₄)₂SO₄ into cells of strains CB 1809 and CC 705, as well as in bacteroids of strain CC 705. It is noteworthy that appreciable activities for GDH were found in the free-living rhizobia grown on glutamate. Thus the presence of an enzyme does not necessarily imply that a particular pathway is operative in assimilating ammonium into cell nitrogen. Based on ¹⁵N studies, the GS-GOGAT pathway of rhizobia (strains CB 1809 and CC 705) is important when grown in culture solutions as well as in bacteroids from root nodules of G. max.     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

Impacts of invading N2-fixing Acacia species on patterns of nutrient cycling in two Cape ecosystems: evidence from soil incubation studies and 15N natural abundance values

This study examines the impacts of woody, N₂-fixing invasive Acacia spp. on the patterns of nutrient cycling in two invaded ecosystems of differing nutrient status in the Cape floristic region. Patterns of soil nutrient mineralization were measured by a field incubation method while the significance of the fixation process in altering nutrient cycling was assessed by the ¹⁵N natural abundance technique. The results confirm earlier reports that invasion by woody shrubs results in organic matter and nutrient enrichment of surface soils of both ecosystems. However, patterns of nutrient availability (phosphorus and nitrogen) were not necessarily enhanced. In the more fertile strandveld both phosphorus and nitrogen (significant at P<0.10) showed trends towards enhanced annual mineralization rates upon invasion, while in the low nutrient fynbos system only phosphorus followed this trend. It is unclear whether this differential response is a consequence of plant- or soil-derived feedbacks on the decomposition processes in each system. The ¹⁵N values of the soils from the invaded sites of both ecosystems indicated a strong influence of the alien species on the soil nitrogen component. However, as with other studies of natural ecosystems, the contribution of nitrogen from fixation could not be readily quantified with the ¹⁵N natural abundance method because of problems in selecting suitable non-N₂-fixing reference plants. A technique of disrupting nodule structure and function, by fumigation with O₂, to obtain the ¹⁵N value of a non-N₂-fixing speciment of the study species was tried and found to overcome some of the problems associated with the lack of suitable reference plants. With this technique it was possible to detect the almost total dependence of A. saligna on N₂-fixation in the fynbos soils with their low nitrogen mineralization rates. In the strandveld ecosystem with much higher soil nitrogen release rates A. cyclops was only partly dependent on fixation (about half) for its nitrogen. The nutrient enrichment of both ecosystems and trends towards enhanced rates of nutrient mineralization could have profound implications on the long-term success of alien invader clearing operations and the restoration of the indigenous flora at these sites.     

Does GABA increase the efficiency of symbiotic N2 fixation in legumes?

The ability to regulate the rates of metabolic processes in response to changes in the internal and/or external environment is a fundamental feature which is inherent in all organisms. This adaptability is necessary for conserving the stability of the intercellular environment (homeostasis) which is essential for maintaining an efficient functional state in the organism. Symbiotic nitrogen fixation in legumes is an important process which establishes from the complex interaction between the host plant and microorganism. This process is widely believed to be regulated by the host plant nitrogen demand through a whole plant N feedback mechanism in particular under unfavorable conditions. This mechanism is probably triggered by the impact of shoot-borne, phloem-delivered substances. The precise mechanism of the potential signal is under debate, however, the whole phenomenon is probably related to a constant amino acid cycling within the plant, thereby signaling the shoot nitrogen status. Recent work indicating that there may be a flow of nitrogen to bacteroids is discussed in light of hypothesis that such a flow may be important to nodule function. Large amount of γ-aminobutyric acid (GABA) are cycled through the root nodules of the symbiotic plants. In this paper some recent evidence concerning the possible role of GABA in whole-plant-based upregulation of symbiotic nitrogen fixation will be reviewed.Key words: γ-aminobutyric acid, nitrogen fixation, nodule, symbiosis, translocation, signalingNitrogen (N) is major limiting nutrient for the growth of most plant species in different ecosystems. Acquisition and assimilation of N is second in importance only to photosynthesis for plant growth and development. Elemental N is a key constituent of protein, nucleic acids and other vital cellular components. Most plants acquire N from the soil solution either as nitrate or ammonium ions. In addition, some plants can utilize the atmospheric gaseous nitrogen pool through symbiotic associations with species of bacteria, cyanobacteria or actinomycetes that contain the N₂ fixing enzyme, nitrogenase. Clearly, the crucial role that symbiotic plants play in plant growth requires that physiologists understand the biochemical and molecular events that regulate fixation and subsequent metabolism of nitrogen.Symbiotic N₂ fixation is an important process for increasing the plant available N and thereby the growth capacity of legumes. This process results from the complex interaction between the host plant and microorganism.¹ The host plant provides the microorganism with carbon and a source of energy for growth and functions while the microorganism fixes atmospheric N₂ and provides the plant with a source of reduced nitrogen in the form of ammonium. An adequate supply of carbohydrates is an essential requirement of nodule functioning as N₂ fixation is expensive in terms both of energy and carbon for the synthesis of N-products. Sucrose synthesized in photosynthesis and exported to the nodules via the phloem, is the primary fuel for N₂ fixation.² Sucrose can be metabolized in the cytoplasm of infected, uninfected or interstitial cells with organic acids as the end products. Malate is strongly believed to be the major respiratory substrate for bacteroids.³ This dicarboxylic acid is the major energy source for the bacteroids and plant mitochondria, and is used for NH₄⁺ assimilation as carbon skeleton in the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway.⁴ The products of symbiotic N₂ fixation are exported from the nodules to the rest of the host plant where they are incorporated into essential macro-molecules such as amino acids, proteins that drive plant growth, development and yields. According to the fixation products, root nodules are generally divided into two major groupings:¹ (1) indeterminate nodules that are elongate-cylindrical activity that transport fixed N as amides such as alfalfa, pea and clover; and (2) determinate nodules that are spherical with determinate internal meristematic activity that transport fixed N as ureides, such as soybean and common bean. The complex series of events leading to the formation and functioning of the fixation machinery required controlled coordinated expression of both bacterial and host plant genes.