Isotopic Studies of Nitrogen Fixation in Non-Legume Root Nodules

Isotopic studies are presented on six of the eight recognizednon-legume nodule-forming genera of Angiosperms. Evidence hasbeen obtained of the occurrence of fixation of atmospheric nitrogenin the root nodules of Casuarina, Ceanothus, and Shepherdia,now examined isotopically for the first time. In further studiesof the nitrogen-fixing nodules of Hippopha and Alnus it is shownthat the fixation continues for a longer period after detachmentfrom the plant than is the case with legume nodules, and thatthe enrichment in ¹³N which they (and also detached Casuarinanodules) finally achieve after exposure to excess of the freeisotope considerably exceeds that shown by detached legume nodules.Fixation in detached Myrica nodules was not clearly affectedby reduction in oxygen supplied until the proportion of oxygenwas less than 5 per cent.     

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     

Hydrogen Uptake and Exchange by Pea Root Nodules

Hydrogenase activity in pea root nodules was studied by followinggas exchanges of hydrogen and deuterium. It was found that thenodules did not evolve hydrogen but that hydrogen was takenup when it was provided in the gas mixture. When increasingpartial pressures of deuterium were supplied, hydrogen was evolvedat a rate which increased as the pressure of deuterium increased.Deuterium was taken up at the same time as this hydrogen wasevolved. Hydrogen evolution in the presence of deuterium wasinhibited by nitrogen, while the uptake of deuterium remainedunaffected. It was concluded that pea root nodules have at leasttwo separate hydrogenase system that are working in oppositedirections and must thus be situated in sites of different oxidation-reductionpotentials within the nodule.     

Carbon Dioxide Fixation by Lupin Root Nodules: I. Characterization, Association with Phosphoenolpyruvate Carboxylase, and Correlation with Nitrogen Fixation during Nodule Development

In vivo CO₂ fixation and in vitro phosphoenolpyruvate (PEP) carboxylase levels have been measured in lupin (Lupinus angustifolius L.) root nodules of various ages. Both activities were greater in nodule tissue than in either primary or secondary root tissue, and increased about 3-fold with the onset of N₂ fixation. PEP carboxylase activity was predominantly located in the bacteroid-containing zone of mature nodules, but purified bacteroids contained no activity. Partially purified PEP carboxylases from nodules, roots, and leaves were identical in a number of kinetic parameters. Both in vivo CO₂ fixation activity and in vitro PEP carboxylase activity were significantly correlated with nodule acetylene reduction activity during nodule development. The maximum rate of in vivo CO₂ fixation in mature nodules was 7.9 nmol hour⁻¹ mg fresh weight⁻¹, similar to rates of N₂ fixation and reported values for amino acid translocation.     

The Effect of Potassium on the Fixation of Molecular Nitrogen by Root Nodules of Vicia faba

The effect of potassium supply of Vicia faba on the fixation of molecular nitrogen by root nodules was studied by using ¹⁵N-labeled molecular nitrogen. Plants well supplied with potassium showed higher contents of ¹⁵N in the soluble amino fraction and in the protein fraction of various plant organs as compared with plants of a lower potassium status. This effect was evident particularly in the root nodules. Assimilation experiments, carried out with ¹⁴CO₂, revealed that the content of radioactivity in the sugars and amino acids of the root nodules was increased by the potassium supply of the host plants. In particular, the content of ¹⁴C amino acids in the root nodules was influenced beneficially by potassium, which means that potassium favored the provision of reduced nitrogen (NH₃). It is postulated that the better carbohydrate supply of nodules, by plants well supplied with potassium, results in a higher carbohydrate turnover in the nodules and thus the provision of ATP and reducing electrons required by the nitrogenase is enhanced.     

Nitrogen Fixation and Hydrogen Metabolism in Cyanobacteria

Summary: This review summarizes recent aspects of (di)nitrogen fixation and (di)hydrogen metabolism, with emphasis on cyanobacteria. These organisms possess several types of the enzyme complexes catalyzing N₂ fixation and/or H₂ formation or oxidation, namely, two Mo nitrogenases, a V nitrogenase, and two hydrogenases. The two cyanobacterial Ni hydrogenases are differentiated as either uptake or bidirectional hydrogenases. The different forms of both the nitrogenases and hydrogenases are encoded by different sets of genes, and their organization on the chromosome can vary from one cyanobacterium to another. Factors regulating the expression of these genes are emerging from recent studies. New ideas on the potential physiological and ecological roles of nitrogenases and hydrogenases are presented. There is a renewed interest in exploiting cyanobacteria in solar energy conversion programs to generate H₂ as a source of combustible energy. To enhance the rates of H₂ production, the emphasis perhaps needs not to be on more efficient hydrogenases and nitrogenases or on the transfer of foreign enzymes into cyanobacteria. A likely better strategy is to exploit the use of radiant solar energy by the photosynthetic electron transport system to enhance the rates of H₂ formation and so improve the chances of utilizing cyanobacteria as a source for the generation of clean energy.     

Increase in the Formation and Nitrogen Fixation of Soybean Nodules by Vesicular-Arbuscular Mycorrhiza

Symbiotic interactions of the tripartite association of soybeanplant, vesiculararbuscular (VA) mycorrhizal fungus and Rhizobiumjaponicum were shown. Mycorrhizal plants absorbed more P, Ca and Mg and had higherP, Ca and Mg contents in their stems or leaves than non-mycorrhizalplants. Phosphorus concentration was also higher in the nodulesof mycorrhizal plants. VA mycorrhizae increased nodule number, nodule weight and acetylenereduction activity of nodules. Concomitantly seed productionand N content of leaves were enhanced. Both nodulating (A62-1) and non-nodulating (A62-2) cultivarsof soybean plants [Glycine max (L.) Merr.] were colonized byVA mycorrhizal fungi, identified as belonging to the genus Glomus. (Received August 12, 1985; Accepted January 14, 1986)     

Carbon Monoxide Promotes Lateral Root Formation in Rapeseed

Carbon monoxide (CO), an odorless, tasteless and colorless gas, has recently proved to be an important bioactive or signalmolecule in mammalian cells, with its effects mediated mainly by nitric oxide (NO). In the present report, we show thatexogenous CO induces lateral root (LR) formation, an NO-dependent process. Administration of the CO donor hematin torapeseed (Brassica napus L. Yangyou 6) seedlings for 3 days, dose-dependently promoted the total length and number ofLRs. These responses were also seen following the application of gaseous CO aqueous solutions of different saturatedconcentrations. Furthermore, the actions of CO on seedlings were fully reversed when the CO scavenger hemoglobin (Hb)or the CO-specific synthetic inhibitor zinc protoporphyrin-IX (ZnPPIX) were added. Interestingly, depletion of endogenousNO using its specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO)or the nitric oxide synthase (NOS) inhibitor N~G-nitro-L-arginine methyl ester (L-NAME),led to the complete abolition ofLR development, illustrating an important role for endogenous NO in the action of CO on LR formation. However, theinduction of LR development by 200 umol/L sodium nitroprusside (SNP),an NO donor, was not affected by the presenceor absence of ZnPPIX. Furthermore, using an anatomical approach combined with laser scanning confocal microscopywith the NO-specific fluorophore 4,5-diaminofluorescein diacetate, we observed that both hematin and SNP increased NOrelease compared with control samples and that the NO signal was mainly distributed in the LR primordia (LRP), especiallyafter 36 h treatment. The LRP were found to have similar morphology in control, SNP-and hematin-treated seedlings.Similarly, the enhancement of the NO signal by CO at 36 h was differentially quenched by the addition of cPTIO, L-NAME,ZnPPIX and Hb. In contrast, the induction of NO caused by SNP was not affected by the application of ZnPPIX. Therefore,we further deduced that CO induces LR formation probably mediated by the NO/NOS pathway and NO may act downstreamof CO signaling, which has also been shown to occur in animals.     

Stem Nodules in Aeschynomene indica and Their Capacity of Nitrogen Fixation

There has been no report on stem nodules with nitrogen fixing activity. Aeschynomene indica produce nodules on the aerial parts, and the stem nodules proved to be capable of considerable nitrogen fixation [C₂H₄ produced c. 4 μmol (g fresh weight)^-1 h^-1]. The stem nodules embed reddish tissues, and a rod-shaped bacterium was isolated from the tissues. The bacterium was ascertained to form root and stem nodules on seedlings of A. indica.     

Inhibition of Nitrogen Fixation in Soybean Plants Supplied with Nitrate II. Accumulation and Properties of Nitrosylleghemoglobin in Nodules

The accumulation of nitrosylleghemoglobin (LbNO) in nodulesand the properties of LbNO in vitro were investigated in connectionwith the inhibition of nitrogen fixation in soybean nodulesby nitrate. The leghemoglobin extracted under argon gas from nodules ofplants supplied with nitrate consisted mainly of LbNO, as judgedfrom the spectrum which corresponded to that of LbNO formedin vitro by the reaction of leghemoglobin with nitrite in thepresence of dithionite or by the combination of ferrous leghemoglobin(Lb²⁺) with nitric oxide. Further, LbNO formed in vivo was easilydissociated by visible light, as was LbNO formed in vitro. Thus,authentic LbNO does actually accumulate in nodules. Most of the leghemoglobin was of the ferrous type in nodulesof plants supplied with nitrate. Some LbNO appeared to be derivedfrom LbO₂ which was deoxygenated by nitrite. The increase inlevels of LbNO in nodules paralleled the decrease in acetylenereducing activity. These results indicate that the decrease in nitrogenase activityin nodules of soybean plants supplemented with nitrate is causedby the decrease in levels of LbO₂ that carries oxygen into bacteroids,which results from the formation of LbNO (Received August 22, 1989; Accepted December 4, 1989)     

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.     

In Vitro Nitrogen Fixation by Two Actinomycete Strains Isolated from Casuarina Nodules

Acetylene reduction activity was demonstrated in pure cultures of two actinomycete strains isolated from nodules of Casuarina equisetifolia. This activity was comparable to that of free-living Rhizobium strains, but appeared to be less sensitive to pO₂ and more sensitive to the presence of combined nitrogen.     

Inhibition of Nitrogen Fixation in Soybean Plants Supplied with Nitrate I. Nitrite Accumulation and Formation of Nitrosylleghemoglobin in Nodules

The accumulation of nitrite in nodules was investigated to elucidatethe mechanism of inhibition of nitrogen fixation in nodulesof soybean (Glycine max. [L.] Merr.) plants supplied with nitrate.Acetylene-reducing activity (ARA) in nodules fell within 24h as a result of the supply of exogenous nitrate, accompaniedby an increase in the accumulation of nitrite in the cytosolbut not in the bacteroids of nodules. Nitrate reductase (NR)activity in the nodule cytosol remained high, irrespective ofthe supply of nitrate. Nitrosylleghemoglobin (LbNO) was detectedspectrophotometrically in the extract from nodules in whichnitrogen fixation was inhibited by nitrate. In experiments invitro, it was found that LbNO was easily formed from leghemoglobinin the presence of nitrite and dithionite. Thus, it is suggested that nitrogen fixation was inhibited primarilyby a decrease in the function of leghemoglobin, attributableto the formation of LbNO, which was caused by the accumulationof nitrite generated from nitrate by NR in the nodule cytosol. (Received August 22, 1989; Accepted January 24, 1990)     

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     

Relationships between Acetylene Reduction Activity, Hydrogen Evolution and Nitrogen Fixation in Nodules of Acacia spp.: Experimental Background to Assaying Fixation by Acetylene Reduction under Field Conditions

Hansen, A. P., Pate, J. S. and Atkins, C. A. 1987. Relationshipsbetween acetylene reduction activity, hydrogen evolution andnitrogen fixation in nodules of Acacia spp.: Experimental backgroundto assaying fixation by acetylene reduction under field conditions.—J.exp. Bot. 38: 1–12 Glasshouse grown, symbiotically-dependent seedlings of Acaciaalata R.Br., .A. extensa Lindl., and A. pulchella R.Br. wereexamined for acetylene reduction in closed assay systems usingundisturbed potted plants, excavated whole plants, nodulatedroots or detached nodules. Nitrogenase activity declined sharplyover the first hour after exposure of detached nodules to acetylene(10% v/v in air), less steeply or not at all over a 3 h periodin assays involving attached nodules. Using detached nodules,rates of acetylene reduction, nitrogen (¹⁵N₂) fixation, andhydrogen evolution in air (¹⁵N₂) and acetylene-containing atmosphereswere measured in comparable 30 min assays. Total electron flowthrough nitrogenase in air was determined from rates of nitrogen(¹⁵N₂) fixation ( ? 3) plus hydrogen evolution, that in thepresence of acetylene from rates of acetylene reduction andhydrogen evolution in air: acetylene. Values for the ratio ofelectron flow in air: acetylene to that in air ranged from 0?43to 0?83 in A. pulcheila, from 0?44 to 0?66 in A. alala and from0?37 to 0?70 in A. extensa, indicating substantial inhibitionof electron flow through nitrogenase of detached nodules byacetylene. Relative efficiencies of nitrogenase functioningbased on hydrogen evolution and acetylene reduction were from0?15 to 0?79, those based on nitrogen (¹⁵N₂) fixation and hydrogenevolution from 0?53 to 0?87. Molar ratios of acetylene reducedto nitrogen (¹⁵N₂) fixed were 2?82 ? 0?24, 201 ? 0?15, and 1?91? 0?11 (?s.e.; n = 7) for A. pulcheila,A. extensa and A. alata respectively A standard 5–10 min acetylene reduction assay, conductedon freshly detached unwashed nodules in daytime (12.00–14.00h), was calibrated for field use by comparing total N accumulationof seedlings with estimated cumulative acetylene reduction overa 7-week period of glasshouse culture. Molar ratios for acetylenereduced: nitrogen fixed using this arbitrary method were 3?58for A. alata, 4?82 for A. extensa and 1?60 for A. pulchella.The significance of the data is discussed. Key words: Acacia spp, nitrogenase functioning     

Rhizobium meliloti Mutants Defective in Symbiotic Nitrogen Fixation Affect the Oxygen Gradient in Alfalfa (Medicago sativa) Root Nodules

Rhizobium meliloti bacteroids carrying mutations in either fdxNor fixX isolated from alfalfa root nodules were shown to containthe nitrogenase proteins NifH, NifD and NifK. In contrast toan in vitro system of N₂-fixation based on R. meliloti wild-typebacteroids, nitrogenase activity could not be restored in crudeextracts of these mutant bacteroids by the addition of an artificialelectron donor, indicating that the nitrogenase proteins werepresent but not functional. ESR-studies revealed that both mutantslacked the FeMo-cofactor of nitrogenase. To analyse the roleof free O₂ on the damage of the nitrogenase components and theFeMo-cofactor in these mutant bacteroids, microelectrode studiesof O₂ concentrations and gradients within alfalfa root noduleswere carried out. R. meliloti mutants defective in other genesnecessary for symbiotic N₂-fixation were also included in thisstudy. Four distinct types of O₂ gradients were defined by theapparent presence or absence of an O₂ diffusion barrier andby the minimum internal O₂ concentration. These data clearlydemonstrated the influence of the microsymbiont on the O₂ gradientswithin the nodules. Nodules induced by Rm0540, an R. melilotimutant with altered exopolysaccharide production, which is notable to infect plant cells, did not contain an O₂ diffusionbarrier. In contrast, nodules containing a mutant defectivein dicarboxylate transport (dctA-), produced an O₂ gradientsimilar to the wild-type. Microelectrode measurements revealedH₂ concentrations in alfalfa wild-type nodules comparable tosoyabean, whereas no hydrogen could be detected in nodules harbouringthe dctA mutant or any other mutant strain. Key words: Nitrogen fixation, Rhizobium meliloti bacteroids, ferredoxin-like proteins, microelectrode studies     

Uptake of Carbon Monoxide and Hydrogen at Environmentally Relevant Concentrations by Mycobacteria†

Liquid culture assays revealed a previously unreported capacity for Mycobacterium bovis BCG, M. gordonae, and M. marinum to oxidize CO and for M. smegmatis to consume molecular hydrogen. M. bovis BCG, M. gordonae, M. smegmatis, and M. tuberculosis H37Ra oxidized CO at environmentally relevant concentrations (<50 ppm); H₂ oxidation by M. gordonae and M. smegmatis also occurred at environmentally relevant concentrations (<10 ppm). CO was not consumed by M. avium or M. microti, although the latter appeared to possess CO dehydrogenase (CODH) genes based on PCR results with primers designed for the CODH large subunit, coxL. M. smegmatis and M. gordonae oxidized CO under suboxic (10 and 1% atmospheric oxygen) and anoxic conditions in the presence of nitrate; no oxidation occurred under anoxic conditions without nitrate. Similar results were obtained for H₂ oxidation by M. smegmatis. Phylogenetic analyses of coxL PCR products indicated that mycobacterial sequences form a subclade distinct from that of other bacterial coxL, with limited differentiation among fast- and slow-growing strains.