Hydrogen production and uptake by pea nodules as affected by strains of Rhizobium leguminosarum

Hydrogen evolution from root nodules has been reported to make N₂ fixation by some legume-Rhizobium symbiotic systems inefficient. We have surveyed the extent of H₂ evolution and estimated relative efficiencies of nodules of Austrian winter peas formed by 15 strains of R. leguminosarum. Their rates of H₂ evolution in air were about 30% of the rates of H₂ evolution under an atmosphere in which N₂ was replaced by Ar. Relative efficiency values based on C₂H₂ reduction rates ranged from 0.55 to 0.80. With some of the strains, hydrogenase activities were demonstrated in intact nodules and in bacteroids, but the levels of activity were insufficient to recycle all the H₂ evolved by the nitrogenase system. In both intact nodules and bacteroids the hydrogenase is less sensitive to O₂ damage than the nitrogenase system, so H₂ uptake capacity was observed in intact nodules by suppressing the nitrogenase-dependent H₂ evolution with an atmosphere containing a high O₂ concentration, and in bacteroids by using aerobically prepared bacteroid suspensions. The hydrogenase activity of both was dependent on O₂ consumption. A K _mfor H₂ of near 4 M was determined in suspension of bacteroids from nodules formed by strains 128C53 and 128C56.     

Hydrogen-stimulated CO2 fixation and coordinate induction of hydrogenase and ribulosebiphosphate carboxylase in a H2-uptake positive strain of Rhizobium japonicum

H₂-uptake positive strains (122 DES and SR) and H₂-uptake negative strains SR2 and SR3 of Rhizobium japonicum were examined for ribulosebisphosphate (RuBP) carboxylase and H₂-uptake activities during growth conditions which induced formation of the hydrogenase system. The rate of ¹⁴CO₂ uptake by hydrogenase-derepressed cells was about 6-times greater in the presence than in the absence of H₂. RuBP carboxylase activity was observed in free-living R. japonicum strains 122 DES or SR only when the cells were derepressed for their hydrogenase system. Hydrogenase and RuBP carboxylase activities were coordinately induced by H₂ and both were repressed by added succinate. Hydrogenase-negative mutant strains SR2 and SR3 derived from R. japonicum SR showed no detecyable RuBP carboxylase activities under hydrogenase derepression conditions. No detectable RuBP carboxylase was observed in bacteroids formed by H₂-uptake positive strains R. japonicum 122 DES or SR. Propionyl CoA carboxylase activity was consistently observed in extracts of cells from free-living cultures of R. japonicum but activity was not appreciably influenced by the addition of H₂. Neither phosphoenolpyruvate carboxylase nor phosphoenolpyruvate carboxykinase activity was detected in extracts of R. japonicum.Abbreviations RuBP Ribulose 1,5-bisphosphate - (Na₂EDTA) (Ethylenedinitrilo)-tetraacetic acid, disodium salt - (propionyl CoA) Propionyl coenzyme A - (PEP) Phosphoenolpyruvate - (GSH) Reduced glutathione - (Tricine) N-tris(hydroxymethyl)-methylglycine     

Hydrogen-Dependent Nitrogenase Activity and ATP Formation in Rhizobium japonicum Bacteroids

Rhizobium japonicum 122 DES bacteroids from soybean nodules possess an active H₂-oxidizing system that recycles all of the H₂ lost through nitrogenase-dependent H₂ evolution. The addition of 72 μM H₂ to suspensions of bacteroids increased O₂ uptake 300% and the rate of C₂H₂ reduction 300 to 500%. The optimal partial pressure of O₂ was increased, and the partial pressure of O₂ range for C₂H₂ reduction was extended by adding H₂. A supply of succinate to bacteroids resulted in effects similar to those obtained by adding H₂. Both H₂ and succinate provided respiratory protection for the N₂-fixing system in bacteroids. The oxidation of H₂ by bacteroids increased the steady-state pool of ATP by 20 to 40%. In the presence of 50 mM iodoacetate, which caused much greater inhibition of endogenous respiration than of H₂ oxidation, the addition of H₂ increased the steady-state pool of ATP in bacteroids by 500%. Inhibitor evidence and an absolute requirement for O₂ indicated that the H₂-stimulated ATP synthesis occurred through oxidative phosphorylation. In the presence of 50 mM iodoacetate, H₂-dependent ATP synthesis occurred at a rate sufficient to support nitrogenase activity. The addition of H₂ to H₂ uptake-negative strains of R. japonicum had no effect on ATP formation or C₂H₂ reduction. It is concluded that the H₂-oxidizing system in H₂ uptake-positive bacteroids benefits the N₂-fixing process by providing respiratory protection of the O₂-labile nitrogenase proteins and generating ATP to support maximal rates of C₂H₂ reduction by oxidation of the H₂ produced from the nitrogenase system.     

Properties of the hydrogenase system in Rhizobium japonicum bacteroids

The hydrogenase system which catalyzes the oxyhydrogen reaction in soybean nodules produced by strains of $\text{Rhizobium japonicum}$ is located in the bacteroids. The hydrogenase complex in intact bacteroids has an apparent K_m for H₂ of 2.8 μM and an apparent K_m for O₂ of 1.3 μM. The addition of hydrogen to bacteroids increases oxygen uptake but decreases respiratory CO₂ production, indicating a conservation of endogenous substrates. After correction for the effect of hydrogen on endogenous respiration a ratio of 1.9 ± 0.1 for H₂ to O₂ uptake was determined. Bacteroids from greenhouse or field-grown soybeans that evolved hydrogen showed no measurable oxyhydrogen reaction activity whereas consistent activity was demonstrated by bacteroids from soybean nodules that evolved little or no H₂.     

Investigation of the H(2) Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids

The H₂-oxidizing complex in Rhizobium japonicum 122 DES bacteroids failed to catalyze, at a measurable rate, ²H¹H exchange from a mixture of ²H₂ and ¹H₂ in presence of ²H₂O and ¹H₂O, providing no evidence for reversibility of the hydrogenase reaction in vivo. In the H₂ oxidation reaction, there was no significant discrimination between ²H₂ and ¹H₂, indicating that the initial H₂-activation step in the over-all H₂ oxidation reaction is not rate-limiting. By use of improved methods, an apparent K_m for H₂ of 0.05 micromolar was determined. The H₂ oxidation reaction in bacteroids was strongly inhibited by cyanide (88% at 0.05 millimolar), theonyltrifluoroacetone, and other metal-complexing agents. Carbonyl cyanide m-chlorophenylhydrazone at 0.005 millimolar and 2,4-dinitrophenol at 0.5 millimolar inhibited H₂ oxidation and stimulated O₂ uptake. This and other evidence suggest the involvement of cytochromes and nonheme iron proteins in the pathway of electron transport from H₂ to O₂. Partial pressures of H₂ at 0.03 atmosphere and below had a pronounced inhibitory effect on endogenous respiration by bacteroid suspensions. The inhibition of CO₂ evolution by low partial pressures of H₂ suggests that H₂ utilization may result in conservation of oxidizable substrates and benefits the symbiosis under physiological conditions. Succinate, acetate, and formate at concentrations of 50 millimolar inhibited rates of H₂ uptake by 8, 29, and 25%, respectively. The inhibition by succinate was noncompetitive and that by acetate and formate was uncompetitive. A concentration of 11.6 millimolar CO₂ (initial concentration) in solution inhibited H₂ uptake by bacteroid suspensions by 18%. Further research is necessary to establish the significance of the inhibition of H₂ uptake by succinate, acetate, formate, and CO₂ in the metabolism of the H₂-uptake-positive strains of Rhizobium.     

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.     

Viability of Bradyrhizobium japonicum bacteriods

Homogenates from soybean nodules, formed by 12 strains of Bradyrhizobium japonicum, were plated into yeast-extract mannitol agar containing 3 or 37 g mannitol 1^-1. Viable counts ranged from 8.298 to 11.265 log₁₀ cells-gram nodule^-1. When monitored over the life cycle of the symbiosis, the viability of strains USDA 110 and USDA 123 increased with days after planting (DAP), and at 70 DAP was 95% and 81%, respectively. By contrast, the viability of USDA 38 bacteroids decreased with time, and at 70 DAP was only 1.9%. At 49 DAP, nodules induced by USDA 38 had significantly fewer bacteroids per peribacteroid membrane than those formed by USDA 110 or USDA 123, and at 70 DAP, 27% of the USDA 38 bacteroids showed some degree of degeneration. Viable counts of USDA 123 and USDA 110 bacteroids, isolated from the nodules of 12 different cultivars, ranged from 10.963 to 11.463 and from 10.683 to 11.117 log₁₀ viable cells-gram nodule^-1, respectively. Varying the osmolarity of the medium had no predictable effect on bacteroid viability. When surface-sterilized nodules of IPAGO 587 (high bacteroid viability) and USDA 38 (low bacteroid viability) were inoculated into a nonsterile silt loam soil, at rates equivalent to 5.0×10⁸ and 5.0×10⁶ viable bacteroids g^-1 soil, respectively, and then incubated at 28° C for 60 days, 4.3×10⁴ and 1.5×10⁴ surviving cells g^-1 soil, respectively, were recovered. Thus, despite differences due to host and strain variation, bacteroid viability appears to be unrelated to persistence of individual strains following an annual legume crop cycle.Journal paper No. 14930, Agricultural Experiment Station University of Minnesota, St. Paul, MN 55108, USA     

Symbiotic Legume Nodules Employ Both Rhizobial Exo- and Endo-Hydrogenases to Recycle Hydrogen Produced by Nitrogen Fixation

Background

In symbiotic legume nodules, endosymbiotic rhizobia (bacteroids) fix atmospheric N₂, an ATP-dependent catalytic process yielding stoichiometric ammonium and hydrogen gas (H₂). While in most legume nodules this H₂ is quantitatively evolved, which loss drains metabolic energy, certain bacteroid strains employ uptake hydrogenase activity and thus evolve little or no H₂. Rather, endogenous H₂ is efficiently respired at the expense of O₂, driving oxidative phosphorylation, recouping ATP used for H₂ production, and increasing the efficiency of symbiotic nodule N₂ fixation. In many ensuing investigations since its discovery as a physiological process, bacteroid uptake hydrogenase activity has been presumed a single entity.

Methodology/Principal Findings

Azorhizobium caulinodans, the nodule endosymbiont of Sesbania rostrata stems and roots, possesses both orthodox respiratory (exo-)hydrogenase and novel (endo-)hydrogenase activities. These two respiratory hydrogenases are structurally quite distinct and encoded by disparate, unlinked gene-sets. As shown here, in S. rostrata symbiotic nodules, haploid A. caulinodans bacteroids carrying single knockout alleles in either exo- or-endo-hydrogenase structural genes, like the wild-type parent, evolve no detectable H₂ and thus are fully competent for endogenous H₂ recycling. Whereas, nodules formed with A. caulinodans exo-, endo-hydrogenase double-mutants evolve endogenous H₂ quantitatively and thus suffer complete loss of H₂ recycling capability. More generally, from bioinformatic analyses, diazotrophic microaerophiles, including rhizobia, which respire H₂ may carry both exo- and endo-hydrogenase gene-sets.

Conclusions/Significance

In symbiotic S. rostrata nodules, A. caulinodans bacteroids can use either respiratory hydrogenase to recycle endogenous H₂ produced by N₂ fixation. Thus, H₂ recycling by symbiotic legume nodules may involve multiple respiratory hydrogenases.     

Oxyleghemoglobin-mediated Hydrogen Oxidation by Rhizobium japonicum USDA 122 DES Bacteroids

Oxyleghemoglobin was used to supply low concentrations of O₂ to H₂-oxidizing bacteroids from Rhizobium japonicum USDA 122 DES. The H₂ oxidation system of these bacteroids was capable of effectively utilizing O₂ at the low concentrations of O₂ expected to be found in soybean nodules. Apparent K_m values of approximately 10 nanomolar O₂ have been calculated for the oxyhydrogen reaction. These values include the K_m values for both H₂ oxidation and endogenous substrate oxidation. Even in the presence of oxyleghemoglobin, H₂ additions stimulated C₂H₂ reduction, reduced the rate of endogenous respiration and maintained the ATP contents of bacteroids. In our reconstituted oxyleghemoglobin and bacteriod system, we estimate that the H₂ oxidation system is capable of recycling all of the H₂ evolved during the N₂ fixation process.     

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     

Nitrite reduction in Bacteroids of Rhizobium “hedysari” strain HCNT 1

Ex planta, bacteroids of the sulla-symbiont Rhizobium hedysari strain HCNT 1 terminated reduction of nitrite at nitrous oxide irrespective of the presence or absence of acetylene. Nitrate was not reduced during the experimental period, but slight nitrate reductase activity occurred if incubation with nitrate was prolonged (up to 15 h). As was observed in free-living cells, exposure of the bacteroids to the metal chelator, diethyldithiocarbamate, prevented reduction of nitrite, indicating the presence of a copper-containing nitrite reductase. Pulses of 10–75 M nitrite transiently impeded O₂ uptake in bacteroids, which resumed consumption of O₂ when the nitrite had been reduced. Exposure to >1.0 mM nitrite for 24h greatly inhibited nitrogenase activity (assayed as acetylene reduction activity) of bacteroids in planta. Exposure to the same concentrations of nitrite after 1h of incubation in the presence of acetylene almost completely stopped ongoing ethylene production in bacteroids of strain HCNT 1 extracted from nodules. Free cells of the non-nitrite-reducing R. hedysari strain CC 1335 were lacking in nitrogenase (acetylene-reduction) activity, whereas identically cultured (low-oxygen) strain HCNT 1 cells reduced both nitrite and acetylene.Abbreviations PMS phenazine methosulfate - DDC diethyldithiocarbamate     

High-affinity oxygen uptake byBifidobacterium bifidum

Oxygen uptake by washed cell suspensions ofBifidobacterium bifidum DSM 20082 was studied by using spectrophotometric measurements of the degree of oxygenation of added myoglobin as a measure of the concentration of dissolved O₂. The absorbance changes during consumption of O₂ in a closed reaction vessel were analysed by computer to obtain estimates of the changes in dissolved O₂ concentration. The cell were then used to calculate the rate of O₂ uptake as a function of the dissolved O₂ concentration. The cell suspensions showed Michaelis-Menten kinetics with an apparent K_m value of 0.06 M O₂. Cell-free extracts contained a soluble NADH oxidase activity with a stoichiometry corresponding to the reduction of O₂ to H₂O and with a high affinity for O₂.     

Morphological and structural adaptation of nodules of cowpea to functioning under sub- and supra-ambient oxygen pressure

Nodules of cowpea (Vigna unguiculata (L.) Walp. cv. Vita 3:Bradyrhizobium CB 756) from 28-d-old plants cultured for 23 d with their root systems maintained in O₂ levels from 1 to 80% (v/v, in N₂) in the external gas phase showed a range of structural changes which have been interpreted in relation to an over- or under-supply of O₂. A response to the partial pressure of O₂ in the gas phase (pO₂) was noted with respect to nodule size, lenticel development, the relative distributions of cortical and infected central tissue, the differentiation of cortex, especially the inner cortex, the frequency and size of infected and uninfected interstitial cells, the volume of extracellular spaces both in cortex and infected tissue, and in the frequency of bacteroids. As a consequence of these changes the surface area of inner cortex relative to the nitrogenase-containing units of fixing tissue (infected cells or bacteroids) was increased by as much as 20-fold. Effectiveness of bacteroid functioning increased from 0.10 ± 0.02 · 10^-9 μmol acetylene reduced per bacteroid in air-grown nodules to 0.9 ± 0.16 · 10^-9 (same units) per bacteroid in those cultured in 1% O₂. This work was supported by a grant from the Australian Research Council (to C.A.A.) and an Australian International Development Assistance Bureau postgraduate fellowship (to F.D.D.). The authors wish to thank Dr. W.F.C. Blumer for his considerable help with morphometric analysis, Dr. J. Kuo for guidance in the use of histological techniques, and to Dr. J.S. Pate for the suggestion that lenticel development might be quantified by surface staining of nodules.     

Identifying abnormalities in symbiotic development between Trifolium spp. and Rhizobium leguminosarum bv. trifolii leading to sub-optimal and ineffective nodule phenotypes

Background and Aims

Legumes overcome nitrogen limitations by entering into a mutualistic symbiosis with N₂-fixing bacteria (rhizobia). Fully compatible associations (effective) between Trifolium spp. and Rhizobium leguminosarum bv. trifolii result from successful recognition of symbiotic partners in the rhizosphere, root hair infection and the formation of nodules where N₂-fixing bacteroids reside. Poorly compatible associations can result in root nodule formation with minimal (sub-optimal) or no (ineffective) N₂-fixation. Despite the abundance and persistence of strains in agricultural soils which are poorly compatible with the commercially grown clover species, little is known of how and why they fail symbiotically. The aims of this research were to determine the morphological aberrations occurring in sub-optimal and ineffective clover nodules and to determine whether reduced bacteroid numbers or reduced N₂-fixing activity is the main cause for the Sub-optimal phenotype.

Methods

Symbiotic effectiveness of four Trifolium hosts with each of four R. leguminosarum bv. trifolii strains was assessed by analysis of plant yields and nitrogen content; nodule yields, abundance, morphology and internal structure; and bacteroid cytology, quantity and activity.

Key Results

Effective nodules (Nodule Function 83–100 %) contained four developmental zones and N₂-fixing bacteroids. In contrast, Sub-optimal nodules of the same age (Nodule Function 24–57 %) carried prematurely senescing bacteroids and a small bacteroid pool resulting in reduced shoot N. Ineffective-differentiated nodules carried bacteroids aborted at stage 2 or 3 in differentiation. In contrast, bacteroids were not observed in Ineffective-vegetative nodules despite the presence of bacteria within infection threads.

Conclusions

Three major responses to N₂-fixation incompatibility between Trifolium spp. and R. l. trifolii strains were found: failed bacterial endocytosis from infection threads into plant cortical cells, bacteroid differentiation aborted prematurely, and a reduced pool of functional bacteroids which underwent premature senescence. We discuss possible underlying genetic causes of these developmental abnormalities and consider impacts on N₂-fixation of clovers.     

Some Enzymes of Hydrogen Peroxide Metabolism in Leaves and Root Nodules of Medicago sativa

Leaves and nodules (bacteroids and cytosol) of alfalfa (Medicago sativa L. cv Aragon) plants inoculated with Rhizobium meliloti strain 102F51 have been analyzed for the presence of the enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (EC 1.11.1.6), and peroxidase (EC 1.11.1.7). All three fractions investigated (leaves, bacteroids, and nodular cytosol) show Cu,Zn-SOD activity. Besides, the bacteroids and cytosol of nodules possess CN⁻-insensitive SOD activities. Studies of SOD inactivation with H₂O₂ indicate that, very likely, a Mn-SOD is present in the bacteroids, and suggest that the cytosol contain both Mn-SOD and Fe-SOD. Bacteroids show high catalase activity but lack peroxidase. By contrast, the nodule cytosol exhibits an elevated peroxidase activity as compared with the foliar tissue; this activity was completely inhibited by 50 to 100 micromolar KCN. The significantly lower contents of H₂O₂ and malondialdehyde (a product of lipid peroxidation) in nodules with respect to those in leaves reveal that the above-mentioned bacteroid and cytosol enzymes act in an efficient and combined manner to preserve integrity of nodule cell membranes and to keep leghemoglobin active.     

Identification and characterization of plasmids in hydrogen uptake positive and hydrogen uptake negative strains ofRhizobium japonicum

Modifications were made of published procedures to allow routine isolation of plasmids fromRhizobium japonicum. The plasmid profiles of a series of H₂ uptake positive and H₂ uptake negative strains were compared. None of the strains ofR. japonicum with high H₂ uptake activities exhibited discernible plasmids, while most of the strains, with little or no H₂ uptake activity, showed plasmids with molecular weights ranging from approximately 49–290 x10⁶. An examination of H₂ uptake negative mutants derived from an H₂ uptake positive parent revealed two discernible plasmid bands in nonrevertible mutants but no detectable plasmids in revertible mutants or in the parent strain from which mutants were derived.     

Uptake and evolution of H2 and reduction of C2H2 by root nodules and nodule homogenates ofAlnus glutinosa

Summary In the growing season no net H₂ evolution is detected when root nodules ofAlnus glutinosa are incubated in air or in argon containing 20% O₂. Due to the hydrogenase activity, N₂-fixing root nodules consume added H₂ at a rate of about 1.4 moles H₂.g fresh nodule^–1.h^–1. The uptake of H₂ is only found in summer. At the end of the season, in autumn, nodules evolve significant quantities of H₂ although the nodules still continue to fix nitrogen. In-vitro studies with fractionated homogenates of summer-harvested nodules show that the recovery of the hydrogenase is high when using methylene-blue or phenazine metasulfate as electron acceptors. No hydrogenase activity is detected in homogenates of autumn-harvested nodules.The hydrogenase is localised in the microsymbiont.     

Nitrogen nutrition and the development of biochemical functions associated with nitrogen fixation and ammonia assimilation of nodules on cowpea seedlings

During early development (up to 18 d after sowing) of nodules of an effective cowpea symbiosis (Vigna unguiculata (L.) Walp cv. Vita 3: Rhizobium strain CB756), rapidly increasing nitrogenase (EC 1.7.99.2) activity and leghaemoglobin content were accompanied by rapid increases in activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), enzymes of denovo purine synthesis (forming inosine monophosphate) xanthine oxidoreductase (EC 1.2.3.2), urate oxidase (EC 1.7.3.3), phosphoenolpyruvate carboxylase (EC 4.1.1.31) and led to increased export of ureides (allantoin and allantoic acid) to the shoot of the host plant in the xylem. Culturing plants with the nodulated root systems maintained in the absence of N₂ (in 80 Ar: 20 O₂, v/v) had little effect on the rates of induction and increase in nitrogenase activity and leghaemoglobin content but, in the absence of N₂ fixation and consequent ammonia production by bacteroids, there was no stimulation of activity of enzymes of ammonia assimilation or of the synthesis of purines or ureides. Addition of NO ₃ ^- (0.1–0.2 mM) relieved host-plant nitrogen deficiency caused by the Ar: O₂ treatment but failed to increase levels of enzymes of N metabolism in either the bacteroid or the plant-cell fractions of the nodule. Premature senescence in Ar: O₂-grown nodules occurred at 18–20 d after sowing, and resulted in reduced levels of nitrogenase activity and leghaemoglobin but increased the activity of hydroxybutyrate oxidoreductase (EC 1.1.1.30).     

Specificity and regulation of the dicarboxylate carrier on the peribacteroid membrane of soybean nodules

Malate and succinate were taken up rapidly by isolated, intact peribacteroid units (PBUs) from soybean (Glycine max (L.) Merr.) root nodules and inhibited each other in a competitive manner. Malonate uptake was slower and was severely inhibited by equimolar malate in the reaction medium. The apparent K_m for malonate uptake was higher than that for malate and succinate uptake. Malate uptake by PBUs was inhibited by (in diminishing order of severity) oxaloacetate, fumarate, succinate, phthalonate and oxoglutarate. Malonate and butylmalonate inhibited only slightly and pyruvate,isocitrate and glutamate not at all. Of these compounds, only oxaloacetate, fumarate and succinate inhibited malate uptake by free bacteroids. Malate uptake by PBUs was inhibited severely by the uncoupler carbonylcyanidem-chlorophenyl hydrazone and the respiratory poison KCN, and was stimulated by ATP. We conclude that the peribacteroid membrane contains a dicarboxylate transport system which is distinct from that on the bacteroid membrane and other plant membranes. This system can catalyse the rapid uptake of a range of dicarboxylates into PBUs, with malate and succinate preferred substrates, and is likely to play an important role in symbiotic nitrogen fixation. Energization of both the bacteroid and peribacteroid membranes controls the rate of dicarboxylate transport into peribacteroid units.     

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.