Oxygen Diffusion Pathways and Nitrogen Fixation in Legume Root Nodules

The theoretical analysis presented in this paper suggests thatthe nature of the diffusion pathway from the surface of a noduleto the infected zone depends on the morphology of the nodule;in particular the cross-sectional area of the intercellularspaces in the inner cortex. If the diffusion barrier containscontinuous pathways there will be no pressure difference betweenthe atmosphere and the infected zone. The conditions under whichthe intercellular spaces of the inner cortex could be air-filled,water-filled, or a combination of both are explored. An experimentto resolve this issue is suggested. Information obtained usingcryo-scanning electron microscopy and oxygen electrode datahave been used to illustrate various points. Expressions arederived for the diffusion resistance of the nodule and its componentparts. To provoke further discussion a simple mechanism forthe control of diffusion is described in general terms. Oxygen, diffusion resistance, nodule, nitrogen fixation, soybean, Glycine max. (L.) Merr. cv. Fiskeby V, Trifolium repens L. cv. Blanca, lucerne, Medicago saliva L. cv. Europe     

Sucrose Synthase in Legume Nodules Is Essential for Nitrogen Fixation

The role of sucrose synthase (SS) in the fixation of N was examined in the rug4 mutant of pea (Pisum sativum L.) plants in which SS activity was severely reduced. When dependent on nodules for their N supply, the mutant plants were not viable and appeared to be incapable of effective N fixation, although nodule formation was essentially normal. In fact, N and C resources invested in nodules were much greater in mutant plants than in the wild-type (WT) plants. Low SS activity in nodules (present at only 10% of WT levels) resulted in lower amounts of total soluble protein and leghemoglobin and lower activities of several enzymes compared with WT nodules. Alkaline invertase activity was not increased to compensate for reduced SS activity. Leghemoglobin was present at less than 20% of WT values, so O₂ flux may have been compromised. The two components of nitrogenase were present at normal levels in mutant nodules. However, only a trace of nitrogenase activity was detected in intact plants and none was found in isolated bacteroids. The results are discussed in relation to the role of SS in the provision of C substrates for N fixation and in the development of functional nodules.     

Sucrose Catabolism in Clostridium pasteurianum and Its Relation to N2 Fixation

The growth constant and Y (sucrose) (grams of cells per mole of sucrose) for NH(3)-grown cultures of Clostridium pasteurianum were 1.7 times those of N(2)-grown cultures, whereas the rate of sucrose utilized per gram of cells per hour was similar for both conditions. The Y (sucrose) of chemostat cultures grown on limiting NH(3) under argon at generation times equal to those of N(2)-fixing cultures was less than that of cultures grown on excess NH(3), but cells of NH(3)-limited cultures contained the N(2)-fixing system in high concentration. The concentration of the N(2)-fixing system in whole cells, when measured with adenosine triphosphate (ATP) nonlimiting, was more than twofold greater than the amount needed for the N(2) actually fixed. Thus, energy production from sucrose, and not the concentration of the N(2)-fixing system nor the maximal rate at which N(2) could be fixed, was the limiting factor for growth of N(2)-fixing cells. Either NH(3) or some product of NH(3) metabolism partially regulated the rate of sucrose metabolism since, when cultures fixing N(2), growing on NH(3), or growing on limiting NH(3) in the absence of N(2) were deprived of their nitrogen source, the rate of sucrose catabplism decreased. Calculations showed that the rate of ATP production was the growth rate-limiting factor in cells grown on N(2), and that the increased sucrose requirement of N(2)-fixing cultures in part reflected the energy demand of N(2) fixation. Calculations indicated that whole cells require about 20 moles of ATP for the fixation of 1 mole of N(2) to 2 moles of NH(3).     

Conservation in Soil of H2 Liberated from N2 Fixation by Hup- Nodules

Pigeon peas (Cajanus cajan) were grown in large soil columns (90-cm length by 30-cm diameter) and inoculated with four different strains of cowpea rhizobia, which varied with respect to hydrogen uptake activity (Hup). Despite the profuse liberation of H₂ from Hup^- nodules in vitro, H₂ gas was not detected in any of the soil columns. When H₂ was injected into the columns, the rates of consumption were highest in the treatments (including control) containing Hup^- nodules (218 and 177 nmol · h⁻¹ · cm⁻²) and lowest in the Hup⁺ treatments (158, 92, and 64 nmoles · h⁻¹ · cm⁻²). In situ H₂ uptake rates in small soil cores at fixed distances from the nodules decreased exponentially with distance from the nodule (R² = 0.99). This decrease in H₂ consumption was associated with a similar decrease in numbers of H₂-oxidizing chemolithotrophic bacteria as determined by the most-probable-number method. On the basis of two equations derived separately upon diffusive theory (Fix's Law) and kinetic theory (Michaelis-Menten), the empirically derived rate constants and coefficients indicated that all of the H₂ emitted from Hup^- nodules would be consumed by H₂-oxidizing bacteria within a 3- to 4.5-cm radius of the nodule surface. It is concluded that H₂ is not lost from the soil-plant ecosystem during N₂ fixation in C. cajan but is conserved by H₂-oxidizing bacteria.     

A Model of the Regulation of Nitrogenase Electron Allocation in Legume Nodules (I. The Diffusion Barrier and H2 Inhibition of N2 Fixation)

A mathematical model is presented to explain the regulation of nitrogenase electron allocation to N2 fixation (EAC) in legume nodules. The model is based on two assumptions: (a) that H2 inhibits N2 fixation in a competitive manner; and (b) that O2, H2, and N2 move into and out of nodules by diffusion and their movement is impeded by a diffusion barrier, the permeability of which is controlled to maintain a very low infected cell O2 concentration. When the model was used to simulate nodules displaying a range of values for total nitrogenase activity (TNA), maximum EAC values were predicted to be between 0.69 and 0.71, and a negative correlation was predicted to exist between EAC and TNA. These predictions were in good agreement with empirically derived values reported in the literature and support the suggestion that H2 inhibition of N2 fixation is a major determinant in the regulation of nitrogenase EAC in legume nodules. Two versions of the model were constructed. A closed-pore model assumed that the diffusion barrier consisted of a solid shell of water of variable thickness in the nodule cortex. An open-pore model assumed that a small number of gas-filled intercellular spaces connected the nodule central zone with the root atmosphere and these pores were opened or closed by water to provide variations in the nodule's permeability to gas diffusion. Because of differences in the diffusivity of gases in the gaseous and aqueous phases, the model predicted that, at a given infected cell O2 concentration, an open-pore diffusion barrier would result in less H2 accumulation in the infected cells than a closed-pore diffusion barrier. Therefore, the model may be used to test specific hypotheses about the physical structure of the barrier to gas diffusion in legume nodules.     

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.     

The Flux of 14C-Labelled Photosynthate through Soyabean Root Nodules during N2 Fixation

Experiments are described which examine the flux of photosyntheticassimilates from leaves to nodules of soyabean during N₂ fixation.The first part, where the respiratory efflux of ¹⁴CO₂ by noduleswas used as a means of assessing the import of labelled photosynthatefrom leaves, shows that most ¹⁴CO₂ loss from nodulated rootsis due to the metabolic activity of nodules. Much less photosynthatewas imported by nodules if the metabolic activity associatedwith N₂ fixation was inhibited by low O₂ concentration. The second part describes the chemical fate of current photosynthateas it is utilized by nodules. Labelled material was detectedin nodules within c.15 min of supplying ¹⁴CO₂ to the leaf. Thisrose to a maximum at c.70 min before declining by 85% withinthe following 4 h. Most (80%) ¹⁴carbon imported by nodules waseither lost as respiratory ¹⁴CO₂ or re-exported as productsof N₂ fixation. Ten per cent of imported carbon was found asstructural material and 10% as starch. Of the ¹⁴C soluble in ethanol, most was found in the neutralfraction (80% declining to 50% as sucrose) with smaller amountsas amino acids, organic acids (each category rising from 10%to 20%) and phosphate esters (<5%). Comparison of the distribution of ¹⁴C among amino acids, amidesand ureides in the nodules with that of xylem exudates indicatedthat selected compounds were exported from nodules. The ¹⁴Cdata indicate that c.80% of the nitrogen exported from noduleswas in the form of ureides (mainly allantoic acid) and only10–12% as amides. Key words: Nodules, ¹⁴C-photosynthate, Respiration, Carbon flux     

Phloem Glutamine and the Regulation of O2 Diffusion in Legume Nodules

The aim of the present study was to test the hypothesis that the N content or the composition of the phloem sap that supplies nodulated roots may play a role in the feedback regulation of nitrogenase activity by increasing nodule resistance to O2 diffusion. Treating shoots of lupin (Lupinus albus cv Manitoba) or soybean (Glycine max L. Merr. cv Maple Arrow) with 100 [mu]L L-1 NH3 caused a 1.3-fold (lupin) and 2.6-fold (soybean) increase in the total N content of phloem sap without altering its C content. The increase in phloem N was due primarily to a 4.8-fold (lupin) and 10.5-fold (soybean) increase in the concentration of glutamine N. In addition, there was a decline in both the apparent nitrogenase activity and total nitrogenase activity that began within 4 h and reached about 54% of its initial activity within 6 h of the start of the NH3 treatment. However, the potential nitrogenase activity values in the treated plants were not significantly different from those of the control plants. These results provide evidence that changes in the N composition of the phloem sap, particularly the glutamine content, may increase nodule resistance to O2 diffusion and, thereby, down-regulate nodule metabolism and nitrogenase activity by controlling the supply of O2 to the bacteria-infected cells.     

N2 Fixation,Carbon Metabolism,and Oxidative Damage in Nodules of Dark-Stressed Common Bean Plants

Common beans (Phaseolus vulgaris L.) were exposed to continuous darkness to induce nodule senescence, and several nodule parameters were investigated to identify factors that may be involved in the initial loss of N2 fixation. After only 1 d of darkness, total root respiration decreased by 76% and in vivo nitrogenase (N2ase) activity decreased by 95%. This decline coincided with the almost complete depletion (97%) of sucrose and fructose in nodules. At this stage, the O2 concentration in the infected zone increased to 1%, which may be sufficient to inactivate N2ase; however, key enzymes of carbon and nitrogen metabolism were still active. After 2 d of dark stress there was a significant decrease in the level of N2ase proteins and in the activities of enzymes involved in carbon and nitrogen assimilation. However, the general collapse of nodule metabolism occurred only after 4 d of stress, with a large decline in leghemoglobin and antioxidants. At this final senescent stage, there was an accumulation of oxidatively modified proteins. This oxidative stress may have originated from the decrease in antioxidant defenses and from the Fe-catalyzed generation of activated oxygen due to the increased availability of catalytic Fe and O2 in the infected region.     

A Simulation Study of The Functional Requirements and Distribution of Leghaemoglobin in Relation to Biological Nitrogen Fixation in Legume Root Nodules

A simulation model incorporating structural, biochemical andphysiological features of root nodules of soyabean is described.The simulation is used to examine the effects of varying thelocation and kinetics of leghaemoglobin within infected cells.A striking feature is the capacity of the simulated nodule tomaintain its activity in the face of these changes, in spiteof relatively large changes in concentrations of free O₂, andleghaemoglobin oxygenation with the cells. These propertiesarise from the diffusion resistance and intracellular demandfor O₂, due to the respiratory activities of the bacteroids. Nitrogen fixation, diffusion, oxygen, model     

Microelectrode Measurements of Hydrogen Concentrations and Gradients in Legume Nodules

This paper describes the construction and operation of H₂ specificmicroelectrodes and their application to measurements of H₂concentrations, H₂ gradients and H₂ inhibition of N₂-fixationin legume root nodules. Electrode construction was similar to that of O₂ specific microelectrodespreviously reported. They comprise an outer casing drawn toa 2–20 µm tip plugged with silicone rubber and aconcentric inner electrode made from glass coated platinum wire.The exposed tip of the Pt wire was placed close to the siliconeplug and polarized positively at 0?4 V with respect to an internalAg reference electrode. With an internal electrolyte of KC1/HC1current flow through the electrode was proportional to H₂ concentrationand independent of CO₂ and O₂. With appropriate amplificationand screening the detection limit for this system was 0?0001atm H₂ (4?0 µmol m^–3). Within newly detached nodulesof Hup^–ve symbioses of soyabean, pea and clover H₂ concentrationvaried from 0?009 to 0?014atm compared with 0?021 atm in lupinnodules. In nodules formed by the Hup^–ve soyabean/RCR3442symbiosis internal pH₂ increased from 0?012 atm to 0?09 atmwhen external pO₂ was raised to 0?60 atm. Hydrogen could notbe detected within nodules of the Hup^+ve Clarke/RCR3407 symbiosiseven when N₂ in the gas phase was replaced with Ar and externalpO₂ was increased to 0?60 atm. An assessment of H₂ inhibition of nitrogen fixation in the soyabean(Clarke/RCR3442) symbioses involved measurements of H₂ productionat increasing internal H₂ levels, induced by stepped increasesin gas phase H₂ concentration. The initial relative efficiencyof 0?66 (calculated from the pH₂ of nodules exposed to air andAr/O₂ mixtures) started to decrease at an internal pH₂ of 0?02to 0?03 atm and fell by 80% to 0?18 at an internal pH₂ of 0?1atm. This threshold value for inhibition is above the measuredmean H₂ concentration for this symbiosis of 0?01 atm. Hydrogen gradients through the nodule showed a sharp increasein the region of the inner cortex, which was reciprocal to adecrease in O₂ concentration, and a shallow gradient throughthe infected zone. These results indicate that the inner airspaces in the nodule are interconnected and confirm that thebarrier to O₂ diffusion is located in the inner cortex. Key words: Root nodules, hydrogen, hydrogenase, oxygen     

Characterization of the Resistance to Oxygen Diffusion in Legume Nodules

A method for characterizing the resistance to oxygen diffusionin legume nodules has been developed. This is based on the assumptionsthat diffusion can be described using a simple resistance analogueand that the respiratory response of the bacteriod-containingcells to external oxygen concentrations can be analysed as adiffusion-limited process. Applying this analysis to experimentaldata from infact white clover plants allowed the total diffusionresistance to be separated into (a) a minimum resistance and(b) the extent to which this resistance can be increased. Whenthe carbohydrate status of the nodules was reduced by dark treatments,the minimum diffusion resistance increased, and after 24–28h darkness equalled the maximum resistance. At the same timethe ability to control this resistance was lost. White clover, nitrogen fixation, oxygen diffusion, nodule respiration     

Assimilate Partitioning in Red and White Clover Either Dependent on N2 Fixation in Root Nodules or Utilizing Nitrate Nitrogen

During vegetative growth in controlled environments, the patternof distribution of ¹⁴C-labelled assimilates to shoot and root,and to the meristems of the shoot, was measured in red and whiteclover plants either wholly dependent on N₂ fixation in rootnodules or receiving abundant nitrate nitrogen but lacking nodules. In experiments where single leaves on the primary shoot wereexposed to ¹⁴CO₂, nodulated plants of both clovers generallyexported more of their labelled assimilates to root (+nodules),than equivalent plants utilizing nitrate nitrogen, and thiswas offset by reduced export to branches (red clover) or stolons(white clover). The intensity of these effects varied with experiment.The export of labelled assimilate to growing leaves at the terminalmeristem of the donor shoot was not influenced by source ofnitrogen. Internode elongation in the donor shoot utilized nolabelled assimilate. Whole plants of white clover exposed to ¹⁴CO₂ on seven occasionsover 32 days exhibited the same effect on export to root (+nodules),which increased slightly in intensity with increasing plantage. Nodulated plants had larger root: shoot ratios than theirequivalents utilizing nitrate nitrogen. Trifolium repens, Trifolium pratense, red clover, white clover, nitrogen fixation, nitrate utilization, assimilate partitioning     

Direct Evidence for Changes in the Resistance of Legume Root Nodules to O2 Diffusion

Indirect evidence suggests that legumes can adjust rapidly theresistance of their root nodules to O₂ diffusion. Here we describeexperiments using O₂ specific micro-electrodes and dark fieldmicroscopy to study directly the operation of this diffusionbarrier. The O₂ concentration sensed by the electrode decreasedsharply in the region of the inner cortex and was less than1.0 mmol m^–3 throughout the infected tissue in nodulesof both pea (Pisum sativum) and french bean (Phaseolus vulgaris).In a number of experiments the ambient O₂ concentration wasincreased to 40% while the electrode tip was just inside theinner cortex. In 13 out of 21 cases the O₂ concentration atthis position either remained low and unchanged or increasedirreversibly to near ambient values. In the remaining casesthe O₂ concentration increased after 1 to 2.5 min and then decreasedto its former value. These results are ascribed to an increasein resistance of the barrier in response to increased O₂ fluxinto the nodule. It was shown microscopically that air spacesboth at the boundary between the infected zone and the innercortex, and within the infected zone started to disappear 3min after nodules were exposed to high ambient O₂ concentrationsand had disappeared completely after 8 min. These spaces werenot changed by exposure of the nodule for 10 min to either N₂or air. Key words: Oxygen, root nodules, air spaces     

Catalase Activity of Nodules in the Ureide- and Amide-transporting Legume Plants

The relations of catalase activity to the efficiency of symbiotic dinitrogen fixation and leghemoglobin (Lb) content were investigated in roots and nodules of several legume plant species together with the catalase distribution between the inner bacteroidal and the outer cortical nodule tissues. The catalase activity in the nodules exceeded that of the roots of the amide- and ureide-synthesizing plant species by one and two orders of magnitude. During the growth period, catalase activity and Lb content changed in parallel and reached their highest levels early in the stage of flowering or fruit formation, depending on plant species. In the case of effective symbiosis, catalase activity in the nodules was 2.5–5 times higher than in the case of ineffective symbiosis. Catalase activity in the bacteroidal zone of the nodules was several times higher than that of the cortical tissue, and two nodule tissues differed in catalase activity more notably in the plant species exporting ureides. The authors suggest that high catalase activity in the nodules, especially in their bacteroidal zone, is essential for the efficient functioning of the symbiotic system of dinitrogen fixation in both ureide- and amide-transporting plants.     

The Rarity of Survival to Old Age Does Not Drive the Evolution of Senescence

The evolution of senescence is often explained by arguing that, in nature, few individuals survive to be old and hence it is evolutionarily unimportant what happens to organisms when they are old. A corollary to this idea is that extrinsically imposed mortality, because it reduces the chance of surviving to be old, favors the evolution of senescence. We show that these ideas, although widespread, are incorrect. Selection leading to senescence does not depend directly on survival to old age, but on the shape of the stable age distribution, and we discuss the implications of this important distinction. We show that the selection gradient on mortality declines with age even in the hypothetical case of zero mortality, when survivorship does not decline. Changing the survivorship function by imposing age independent mortality has no affect on the selection gradients. A similar result exists for optimization models: age independent mortality does not change the optimal result. We propose an alternative, brief explanation for the decline of selection gradients, and hence the evolution of senescence.