Nodule structure and nitrogenase activity ofCoriaria arborea in response to varying pO2

When excised root nodules ofCoriaria arborea are assayed for nitrogenase activity at various pO₂ they show a broad optimum between 20 and 40 kPa O₂, with some evidence for adaptation. Continuous flow assays of nodulated root systems of intact plants indicate that Coriaria shows an acetylene induced decline in nitrogenase activity. When root systems were subject to step changes in pO₂ nitrogenase activity responded with a steep decline followed by a slower rise in activity both at lower and higher than ambient pO₂. Thus Coriaria nodules are able to adapt rapidly to oxygen levels well above and well below ambient. Measurement of nodule diffusion resistance showed that the adaptation is accompanied by rapid increase in resistance at above ambient pO₂ and decrease in resistance at below ambient pO₂. Plants grown with root systems at pO₂ from 5–40 kPa O₂ did not differ in growth or nodulation. The anatomy of Coriaria nodules shows they have a dense periderm which encircles the nodule and also closely invests the infected zone. The periderm is both thicker and more heavily suberised in nodules grown at high pO₂ than at low pO₂. Vacuum infiltration of India ink indicates that oxygen diffusion is entirely through the lenticel and via a small gap adjacent to the stele.     

Oxygen protection of nitrogenase in Frankia sp. HFPArI3

O₂ protection of nitrogenase in a cultured Frankia isolate from Alnus rubra (HFPArI3) was studied in vivo. Evidence for a passive gas diffusion barrier in the vesicles was obtained by kinetic analysis of in vivo O₂ uptake and acetylene reduction rates in response to substrate concentration. O₂ of NH ₄ ⁺ -grown cells showed an apparent K _m O₂ of approximately 1M O₂. In N₂-fixing cultures a second K _m O₂ of about 215 M O₂ was observed. Thus, respiration remained unsaturated by O₂ at air-saturation levels. In vivo, the apparent K _m for acetylene was more than 10-fold greater than reported in vitro values. These data were inter oreted as evidence for a gas diffusion barrier in the vesicles but not vegetative filaments of Frankia sp. HFPArI3.     

Effect of oxygen on substrate utilization for nitrogen fixation and growth in Frankia spp.

Frankia, the actinomycete partner in the nitrogenfixing symbiosis of certain woody non-legumes, has been shown to fix nitrogen in pure culture under aerobic conditions. The sensitivity of in vivo nitrogen-fixation (acetylene reduction) to oxygen tension in the gas phase was measured in short-term assays with two Frankia isolates designated ARI3 and CcI3. The carbon source utilized had an effect on the optimum O₂ concentration for acetylene reduction. Cells utilizing an organic acid, e.g., propionate or pyruvate had maximum nitrogenase activity at an oxygen concentration of 15 to 20%. In contrast, cells respiring a sugar, e.g., trehalose or glucose, or endogenous reserves (glycogen or trehalose) had maximum acetylene reduction activity at 5 to 10% in the gas phase. Oxygen uptake kinetics showed that respiration in vesicle-containing cells utilizing trehalose had a biphasic response to oxygen concentration with a diffusion limited component at oxygen concentrations of 20 M to more than 300 M. These results suggested that trehalose was oxidized in the vesicles as well as in the vegetative hyphae. Oxygen concentration also had an effect on the trehalose-supported growth of cells (non nitrogenfixing, [⁺NH₄Cl]). Cells grown with 5–10% O₂ in the gas phase had a doubling time approximately half those grown with 20% O₂ (atmospheric). Propionate-grown cells showed similar growth rates at the two oxygen tensions, and grew faster (almost 2x) than the trehalose cells at 5–10% O₂. Trehalose also supported approximately 40% lower rates of oxygen uptake than propionate in vesicle-containing cells.     

Nitrogen fixation and respiration by root nodules ofAlnus rubra Bong.: Effects of temperature and oxygen concentration

Summary Using a root nodule cuvette and a continuous flow gas exchange system, we simultaneously measured the rates of carbon dioxide evolution, oxygen uptake and acetylene reduction by nodules ofAlnus rubra. This system allowed us to measure the respiration rates of single nodules and to determine the effects of oxygen concentration and temperature on the energy cost of nitrogen fixation. Energy cost was virtually unchanged (2.8–3.5 moles of carbon dioxide or oxygen per mole of ethylene) from 16 to 26°C (pO₂=20 kPa) while respiration and nitrogenase activity were highly temperature dependent. At temperatures below 16°C, nitrogenase activity decreased more than did respiration and as a result, energy cost rose sharply. Acetylene reduction ceased below 8°C. Inhibition of nitrogenase activity at low temperatures was rapidly reversed upon return to higher temperatures. At high temperatures (above 30°C) nitrogenase activity declined irreversibly, while respiration and energy cost increased.Energy cost was nearly unchanged at oxygen partial pressures of 5 to 20 kPa (temperature of 20°C). Respiration and nitrogenase activity were strongly correlated with oxygen tension. Below 5 kPa, acetylene reduction and oxygen uptake decreased sharply while production of carbon dioxide increased, indicating fermentation. Fermentation alone was unable to support nitrogenase activity. Acetylene reduction was independent of oxygen concentration from 15 to 30 kPa. Nitrogenase activity decreased and energy cost rose above 30 kPa until nearly complete inactivation of nitrogenase at 70–80 kPa. Activity declined gradually, such that acetylene reduction at a constant oxygen concentration was stable, but showed further inactivation when oxygen concentration was once again increased. Alder nodules appear to consist of a large number of compartments that differ in the degree to which nitrogenase is protected from excess oxygen.Supported by United States Department of Agriculture Grant 78-59-2252-0-1-005-1     

A comparison of cultural characteristics and infectivity ofFrankia isolates from root nodules ofCasuarina species

Summary The isolations of three new strains ofFrankia were made from root nodules ofCasuarina cunninghamiana growing aeroponically. Two strains, HFPCCI1 and HFPCcI2 isolated by Lopez are typicalFrankia strains, producing sporangia among filamentous mats in culture and, in the absence of combined nitrogen, forming vesicles and showing acetylene reduction. They are red-pigmented and, although failing to nodulateCasuarina hosts, effectively nodulatedElaeagnus andHippophae. A third strain HFPCcI3 isolated by Zhang from the same source, also a typicalFrankia, can form sporangia and vesicles in culture and reduce acetylene, is unpigmented, fails to nodulateElaeagnus but effectively nodulatesC. cunninghamiana andC. equisetifolia. Comparisons are made among all of theCasuarina isolates in our collection from around the world (twelve in all) with regard to their cultural characteristics and capacity to infect host plant species. Questions are raised about the specificity of the various isolates and their possible affinities. Opportunities are suggested for inoculation of seedlings for forestry and field application using the infective, effective strains now available.     

The isolation,culture and infectivity of aFrankia strain fromGymnostoma papuanum (Casuarinaceae)

A strain ofFrankia was isolated fromGymnostoma papuanum(Casuarinaceae) nodules harvested from rooted cuttings which had been inoculated with a suspension of crushedCasuarina equisetifolia nodules. Designated HFPGpI1 (catalogue #HFP021801), this strain is pigmented and similar to other pigmentedFrankia strains in cultural characteristics. A previously unknown spiraled hyphal morphology was observed at very low frequency in some cultures of this strain. HFPGpI1 is infective and effective onG. papuanum but not on anyCasuarina species tested. It also infects members of the family Elaeagnaceae andMyrica gale. The host plantG. papuanum can be infected with a wide range ofFrankia isolates and thus can be considered a promiscuous host, unlike its close relatives in the genera Casuarina and Allocasuarina which are very restrictive as to which strains may nodulate them.     

Lipid composition and nitrogenase activity of symbioticFrankia (Alnus incana) in response to different oxygen concentrations

Summary The role ofFrankia vesicle envelope lipids in regulating oxygen diffusion of symbiotic nitrogen fixation inAlnus incana was examined. Total lipids of symbioticFrankia (vesicle clusters) that had been adapted to oxygen tensions of 5,21, or 40 kPa were analyzed with a normal phase HPLC system. During the oxygen treatment, nitrogenase activity was measured as hydrogen evolution in an open flow-through system. When plants were transferred to low oxygen (5 kPa) or high oxygen (40 kPa), nitrogenase activity dropped initially. Activity recovered in both treatments with a rate comparable to the controls (21 kPa O₂). Both lipid content and lipid composition of vesicle clusters were affected by the oxygen treatments. With increasing oxygen tension, the vesicle cluster lipid content increased. This correlated with structural data (fluorescence microscopy and TEM) which showed a thicker vesicle envelope at higher oxygen tension. Three hopanoid lipids, bacteriohopanetetrol (bht) and two isomers of phenylacetyl monoester of bht, made up approximately 80% of the vesicle cluster lipids. With changing oxygen concentrations, the ratio of the two bht esters changed whereas the relative proportion of bht remained fairly constant. Therefore, in theFrankia-Alnus incana symbiosis, adaptation to different ambient oxygen tensions occurs at least partly by increasing the thickness of theFrankia vesicle envelope and by changing its lipid composition.Abbreviations dw dry weight - bht bacteriohopanetetrol - SE standard error - TEM transmission electron microscopy Dedicated to the memory of Professor John G. Torrey     

Studies of an effective strain ofFrankia fromAllocasuarina lehmanniana of the Casuarinaceae

Summary Seedlings ofCasuarina spp. andAllocasuarina spp. were grown from seed in the greenhouse and inoculated with a nodule suspension fromC. equisetifolia. Plants ofCasuarina spp. nodulated regularly and were effective in nitrogen-fixation. Only one species ofAllocasuariona, A. lehmanniana formed root nodules. Using these plants as source of inoculum, the isolation of a newFrankia sp. HFPA11I1 (HFP022 801) was made and the strain was grown in pure culture.Frankia sp. HFPA11I1 grows well in a defined medium and shows typical morphological characteristics. In media lacking combined nitrogen, the filamentours bacterium forms terminal vesicles in abundance and differentiaties large intrahyphal or terminal sporangia containing numerous spores. This strain, used as inoculum, nodulates effectively seedlings ofC. equisietifolia andC. cunninghamiana, forming nodules with verically-growing nodule roots. Although effective in acetylene reduction, the endophyte within the nodules is filamentous and lacks veiscles. When used to inoculated seedlings ofA llocasuarina lehmanniana, Frankia sp. HFPA11I1 induces root nodules which are coralloid and lacking nodule roots. The nodules are effective in acetylene reduction and the filamentous hyphae ofFrankia within the nodule lobes lack vesicles. Effective nodulation inA. Lehmanniana depends upon environmental conditions of the seedlings and proceeds much more slowly than in Casuariana.     

In vitro production of specialized reproductive torulose hyphae byFrankia strain ORS 021001 isolated fromCasuarina junghuhniana root nodules

Summary AFrankia strain (ORS 021001) isolated fromCasuarina junghuhniana root nodules was shown to produce four type of structuresin vitro: vegetative hyphae, sporangiospores within sporangia, N₂-fixing vesicles, and a fourth type of structure which is described in detail in this report. Structures of this latter type which we propose to call reproductive torulose hyphae: (RTH) result from enlargement and multiple segmentation of vegetative hyphae into torulose chaions of spore-like cells. RTH differ from sporangia in three major aspects: morphology, morphogenesis and outgrowth. RTH play an important role in survival and reproduction ofFrankia strain ORS 021001. Adding activated charcoal to the nutrient medium promotes the formation ofFrankia colonies orginating from RTH.     

Evidence that the barrier to the penetration of oxygen into heterocysts depends upon two layers of the cell envelope

Mutants of Anabaena sp. PCC 7120 with O₂-sensitive acetylene-reducing activity were studied to identify envelope components that contribute to the barrier limiting diffusion of oxygen into the heterocyst. Mutant strain EF114, deficient in a heterocyst-specific glycolipid, reduced acetylene only under strictly anaerobic conditions. Analysis of in vivo O₂ uptake as a function of dissolved pO₂ showed that EF114 has lost the low affinity, diffusion-limited respiratory component associated with heterocysts in wild-type filaments. The low affinity respiratory activity was also lost in EF116, a mutant in which the cohesiveness of the outer polysaccharide layer was reduced. Restoration of aerobic nitrogen fixation in a spontaneous revertant of EF116 and in a strain complemented with cosmid 41E11 was associated with restoration of the low affinity component of respiratory activity. The results provide evidence that the barrier to diffusion of gas into heterocysts depends upon both the glycolipid layer and the polysaccharide layer of the heterocyst envelope.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

A comparison of two methods and different media for isolatingFrankia from Casuarina root nodules

Four species of Casuarina were raised in the glasshouse and inoculated with nodules collected from nine different geographical areas within Australia. Isolations ofFrankia were attempted from 10 of the Casuarina-Frankia nodule combinations using two methods, a nodule dissection and a filtration method. With both techniquesFrankia isolates were obtained from four of the 10Frankia sources. Spores were not observed in sections of nodules from the four sources from whichFrankia was isolated, whereas spores were observed in the remaining six nodule sources. For selected nodule sources a range of isolation media were tried, but no improvement in the isolation success rate was achieved. The effect of host species on ease of isolation was studied. The results obtained suggested it was theFrankia strain and not the host plant species which determined the ease of isolation from Casuarina nodules.     

Transient responses of nitrogenase to acetylene and oxygen in actinorhizal nodules and cultured frankia

Nitrogenase activity in root nodules of four species of actinorhizal plants showed varying declines in response to exposure to acetylene (10% v/v). Gymnostoma papuanum (S. Moore) L. Johnson. and Casuarina equisetifolia L. nodules showed a small decline (5-15%) with little or no recovery over 15 minutes. Myrica gale L. nodules showed a sharp decline followed by a rapid return to peak activity. Alnus incana ssp. rugosa (Du Roi) Clausen. nodules usually showed varying degrees of decline followed by a slower return to peak or near-peak activity. We call these effects acetylene-induced transients. Rapid increases in oxygen tension also caused dramatic transient decreases in nitrogenase activity in all species. The magnitude of the transient decrease was related to the size of the O₂ partial pressure (pO₂) rise, to the proximity of the starting and ending oxygen tensions to the pO₂ optimum, and to the time for which the plant was exposed to the lower pO₂. Oxygen-induced transients, induced both by step jumps in pO₂ and by O₂ pulses, were also observed in cultures of Frankia. The effects seen in nodules are purely a response by the bacterium and not a nodule effect per se. Oxygen-induced nitrogenase transients in actinorhizal nodules from the plant genera tested here do not appear to be a result of changes in nodule diffusion resistance.     

Morphology,physiology and infectivity of twoFrankia isolates An 1 and An 2 from root nodules ofAlnus nitida

Summary Two different strains, An 1 and An 2, were obtained from root nodules ofAlnus nitida Endl., collected from one locality in the area of its natural habitat near Bahrin, District Swat, Pakistan. The light and electron microscopy of the isolates revealed the occurrence of septate and branched hyphae bearing sporangia and vesicles. The strains differed in their growth requirements, nitrogen-fixing ability and production of extracellular pigments, thus indicating the existence of more than oneFrankia strain in the same locality. In the absence of combined nitrogen in the medium strain An 1 formed vesicles and fixed N₂ (up to 200 nmol C₂H₄. mg protein^–1.h^–1), while strain An 2 under the experimental conditions formed only few vesicles and fixed N₂ at a very low rate (ca 10 nmol C₂H₄. mg protein^–1 .h^–1). The nitrogenase activity of strain An 1 was strongly affected by the O₂ concentration.Frankia An 1 and An 2 were infective and effective onA. nitida andA. glutinosa but not onDatisca cannabina andElaeagnus umbellata. Both An 1 and An 2 strains were more infective and effective onA. glutinosa thanFrankia strains AvcIl and CpI1.     

Effect of O2 on vesicle formation, acetylene reduction, and O2-uptake kinetics in Frankia sp. HFPCcI3 isolated from Casuarina cunninghamiana

The effect of the partial pressure of oxygen (PO2) on the formation of vesicles, which are thought to be the site of N2 fixation in Frankia, was studied in HFPCcI3, an effective isolate from Casuarina cunninghamiana. Unlike other actinorhizal root nodules, vesicles are not produced by the endophyte in Casuarina nodules. However, in culture under aerobic conditions, large, phase-bright vesicles are formed in HFPCcI3 within 20 h following removal of NH+4 from the culture medium and reach peak numbers within 72 to 96 h. In vivo acetylene reduction activity parallels vesicle formation. Optimum rates of acetylene reduction in short-term assays occurred at 20% O2 (0.2 atm (1 atm = 101.325 kPa] in the gas phase. O2 uptake (respiration) determined polarographically showed diffusion-limited kinetics and remained unsaturated by O2 until 300 microM O2. In contrast, respiration in NH+4-grown cells was saturated by O2 between 8 and 10 microM O2. These results indicate the presence of a diffusion barrier associated with the vesicles. Vesicle development was repressed in cells incubated in N-free media sparged with gas mixtures with PO2 between 0.001 and 0.003 atm. Nitrogenase was induced under these conditions, but acetylene reduction was extremely O2 sensitive. The kinetics of O2 uptake as a function of dissolved O2 concentration in avesicular cells were similar to those in NH+4-grown cells indicating the lack of a diffusion barrier. These results demonstrate that vesicle formation and the development of the O2 protection mechanisms of nitrogenase are regulated by ambient PO2 in HFPCcI3.     

Infectivity and effectivity of Frankia strains from the Rhamnaceae family on different actinorhizal plants

Ten strains of Frankia isolated from root nodules of plant species from five genera of the host family Rhamnaceae were assayed in cross inoculation assays. They were tested on host plants belonging to four actinorhizal families: Trevoa trinervis (Rhamnaceae), Elaeagnus angustifolia (Elaeagnaceae), Alnus glutinosa (Betulaceae) and Casuarina cunninghamiana (Casuarinaceae). All Frankia strains from the Rhamnaceae were able to infect and nodulate both T. trinervis and E. angustifolia. Strain ChI4 isolated from Colletia hystrix was also infective on Alnus glutinosa. All nodules showed a positive acetylene reduction indicating that the microsymbionts used as inoculants were effective in nitrogen fixation. The results suggest that Frankia strains from Rhamnaceae belong to the Elaeagnus-infective subdivision of the genus Frankia.     

Gas diffusion pathway in nodules ofCasuarina cunninghamiana

The gas diffusion pathway in nodules was traced by vacuum infiltration with India ink or aniline blue and by electron microscopy. India ink infiltration was observed in the outermost and the innermost cortex in sliced nodules, but not in intact nodules. With aniline blue infiltration, it was observed that intercellular air spaces in the outermost and the innermost cortex were connected to those in nodule roots. No air spaces were in contact with walls of infected cells, although intercellular air spaces existed in some groups of uninfected cells within the infected zone. Infiltration with either India ink or aniline blue could not be observed in the infected zone in essentially all cases. Thus it is suggested that the discontinuity of the intercellular air spaces represents a major resistance to O₂ diffusion in nodules ofCasuarina cunninghamiana.     

Frankia vesicles provide inducible and absolute oxygen protection for nitrogenase

When Frankia HFPCcI3 was grown in culture at oxygen O₂ levels ranging from 2 to 70 kilopascals O₂, under nitrogen fixing conditions, nitrogenase activity adapted to ambient pO₂ and showed a marked optimum close to growth pO₂. Vesicles were thin walled at low pO₂ and very thick walled at high pO₂. Freeze fracture transmission electron microscopy confirmed that Frankia produces vesicles with outer walls thickened by multiple lipid-like monolayers, in proportion to ambient pO₂.     

Some properties of carbohydrate and C4-dicarboxylic acid utilization negative mutants ofRhizobium leguminosarum biovarphaseoli strain P121

After NTG treatment of the very effective wild type strain P121 ofRhizobium leguminosarum biovarphaseoli, mutants defective in the utilization of sugars or organic acids were obtained. All the mutants nodulated the cultivar Goldie ofPhaseolus vulgaris. The arabinose, fructose, glucose and pyruvate utilization mutants formed nodules similar in shape and size to the nodules formed by the wild type strain. These mutants exhibited an acetylene reduction activity significantly lower than the activity observed with the wild type strain. All the C₄-dicarboxylic acid utilization mutatns, formed ineffective nodules that did not show a significant acetylene reduction activity. The C₄-dicarboxylic acids uptake system is apparently inducible in the free-living bacteria of strain P121. When P121 cells were grown on glucose in the presence of 2.5 mM malate, the rate of glucose-dependent O₂ consumption significantly decreased suggesting the presence of a catabolite repression-like phenomenon. Isolated bacteroids of strain P121, under the experimental conditions used, were able to oxidize succinate, fumarate or malate but did not oxidize pyruvate, glucose, fructose or sucrose.     

木麻黄和桤木离体根瘤和立地土壤的固氮酶与N2O还原酶活性的研究

为了解非豆科固氮树种的固氮酶和N_2O还原酶(Nos)活性,采用乙炔还原法和乙炔抑制技术对细枝木麻黄(Casuarina cunninghamiana)和江南桤木(Alnus trabeculosa)离体根瘤及立地土壤的两种酶活性进行了研究。结果表明,离体根瘤只在厌氧条件下有固氮酶活性,在好氧条件下有Nos活性。根瘤区根际土和非根瘤区根际土的固氮酶活性在好氧条件大于厌氧条件,Nos活性只表现在厌氧条件下。在好氧条件下,根瘤区根际土和非根瘤区根际土的固氮酶活性无显著差异;根瘤区根际土的Nos活性显著大于非根瘤区根际土。除离体根瘤在好氧条件下不表现固氮酶活性外,细枝木麻黄和桤木的离体根瘤、根瘤区根际土和非根瘤区根际土的固氮酶活性均都大于Nos活性。好氧条件下根瘤区根际土的固氮酶活性与非根瘤区根际土的呈极显著正相关,而厌氧条件下根瘤的固氮酶活性与好氧条件下根瘤区根际土和非根瘤区根际土固氮酶活性、好氧条件下根瘤的Nos活性与厌氧条件下根瘤区根际土和非根瘤区根际土Nos活性均呈极显著负相关。这为研究弗兰克氏菌结瘤植物共生固氮体系对N2O汇强度的影响和调控奠定基础。     

Variation with soil depth,topographic position and host species in the capacity of soils from an Australian locale to nodulateCasuarina andAllocasuarina seedlings

Sandy alluvial soils in a floodplain supporting a native stand ofCasuarina cunninghamiana Miq. produced about three times as many nodulated seedlings and more than twice as many nodules per nodulated seedling on roots of baitedCasuarina spp. than did clay loam red earth soils from the adjacent valley slope. Moist and well-aerated subsurficial alluvial sands had the greatest nodulation capacity of all the soils sampled. For all topographic positions, soil samples from depths greater than 20 cm promoted 76% more nodulated Casuarina seedlings than samples from the surficial 20 cm.Seedlings of three provenances ofC. cunninghamiana, together with seedlings ofC. glauca Sieb. ex Spreng.,C. cristata F. Muell ex Miq. andC. obesa Miq. developed significantly more nodules per pot and nodules per nodulated seedling in soils from this locale than seedlings of twoCasuarina equisetifolia Forst. provenances. Seedlings of two provenances ofAllocasuarina torulosa (Ait.) L. Johnson had fewer than 1% nodulated seedlings, a significantly lower level by far than that ofCasuarina seedlings.A. torulosa provenances also had significantly fewer nodulated seedlings per pot and nodules per nodulated seedling than all Casuarina hosts excepting one poorly-nodulated provenance ofC. equisetifolia.Nodulated seedlings of allCasuarina species had the capacity to fix atmospheric N₂, as indicated by acetylene-reduction capability. The presence of yellow cladodes and low rates of acetylene reduction per plant forC. cristata Miq. suggest that this association was poorly effective.