Frankia in acid soils of forests devoid of actinorhizal plants

The capacity of some acid forest soils to induce nodulation on a hybrid between Alnus incana (L.) Moench and A. glutinosa (L.) Gaertn. was investigated. Soil was sampled from tree stands devoid for decades of actinorhizal hosts. Seven-week-old Alnus seedlings growing m liquid culture were inoculated with soil dilutions. The nodules were counted after 6 weeks and classified as Sp⁻, if they lacked spores, or as Sp⁺. if spores were present, according to microscopy of microtome sections. Frankia was found in all the forest soils studied, apart from a soil from a Betula swamp. The highest nodulation capacities on Alnus , caused predominantly by Frankia of the Sp⁻ type. were observed in mineral soil sites with Betula stands — even higher than in soil from an A. incana stand. A positive correlation was found between the pH and the noduiation capacity of the soil.     

Restriction pattern analysis of deoxyribonucleic acid isolated from callus and cell suspension of actinorhizal and non-actinorhizal Betulaceae

Using cell suspensions, a method was elaborated to isolate high-molecular-weight genomic deoxyribonucleic acid (DNA; 65 MDa or more) from members of the Betulaceae: Alnus incana (L.) Moench, Alnus glutinosa (L.) Gaertn. and Betula papyrifera Marsh. The method was also effective for isolation of DNA from callus cells. Based on the chemical lysis of protoplasts, this procedure yielded 130 μg (callus) to 250 μg (cell suspension) of DNA (g fresh cells)⁻¹, with a ratio A₂₀₀/A₂₈ of 1.7–2.0. The purified DNA obtained, formed distinct bands when restricted fragments were electrophoresed. Among the 10 endonucleases used for restriction analysis of Alnus glutinosa, Alnus incana and Betula papyrifera genomes, PvuI1 (EC 3.1.23.33) was unique in giving identical patterns for the two Ainus species. An unusual pattern occurred when Al-2 DNA was restricted with Ava II (EC 3.1.23.4). It formed a ladder with a repeating fragment unit of 181 base pairs long. With the enzymes tested, no differences in restriction patterns were observed among clones of Alnus incana (AI-2 vs AI-2), Betula papyrifera (BP-4 vs BP-8) and subclones of Ainus glutinosa AG-1 (PLFJ709 vs LF1709), suggesting genetic stability of the Betulaceae cultures.     

Nitrogenase activity decay and energy supply in Frankia after addition of ammonium to the host plant Alnus incana

A clone of Alnus incana (L.) Moench was grown in symbiosis with a local source of Frankia or with Frankia Ar14. Seven to 9-week-old plants were given 20 m M NH₄Cl (20 m M KCl = control) for 3 days. Nitrogenase activity of intact plants decreased gradually within the 3 days of treatment to about 10% of the initial rates. Hydrogen evolution in air and total nitrogenase activity responded similarly to the treatment. Relative efficiency of nitrogenase thus remained the same throughout the study period. Control plants were not affected. Measurements of nitrogenase activity in root nodule homogenates (in vitro measurements) indicated loss of active nitrogenase rather than shortage of energy for nitrogenase activity in Frankia from ammonium-treated plants. Shoots were exposed to ¹⁴CO₂ and translocation of ¹⁴C to Frankia vesicle clusters prepared from root nodules was studied. Frankia vesicle clusters from ammonium-treated plants contained about half as much ¹⁴C as those of control plants during all 3 days studied. One explanation for the observed effects is that a reduced supply of carbon to Frankia vesicles in the root nodules caused a reduced metabolic rate, including reduced protein synthesis and synthesis of nitrogenase.     

Capacity for hexose respiration in symbiotic Frankia from Alnus incana

Frankia vesicle clusters were prepared from Alnus incana (L.) Moench root nodules containing a local source of Frankia by an improved homogenization-filtration procedure. The capacity of the vesicle clusters to metabolize hexoses was investigated by respirometric and enzymological studies. The vesicle clusters could utilize glucose, glucose-6-phosphate and 6-phosphogluconate provided that appropriate cofactors were added to the preparations. The enzymes hexokinase (EC 2.7.1.1), NADP⁺: glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and NAD⁺;6-phosphogluconate dehydrogenase (EC 1.1.1.44) were found in cell-free extracts of the vesicle clusters and kinetic constants for the enzymes were determined. Hexokinase had a lower K_m for glucose than for fructose. Extracts from both symbiotic and propionate grown Frankia AvcII also showed activity of these hexose-degrading enzymes, indicating that their presence is not necessarily dependent on sugars as carbon source. The NAD⁺- dependent 6-phosphogluconate dehydrogenase was only present in Frankia cells and not in alder root cells, which makes this enzyme a useful Frankia -specific marker in these symbiotic systems.     

Occurrence and activity of hydrogenase in symbiotic Frankia from field-collected Alnus incana

Occurrence and activity of the hydrogen uptake enzyme were studied in root nodule homogenates made from plants of Alnus incana (L.) Moench collected from field sites in the northern part of Sweden. Nitrogenase (EC 1.7.99.2) activity (estimated by acetylene reduction) and hydrogen evolution were studied in excised nodules. All Frankia sources showed acetylene reduction activity, and possessed a hydrogen uptake system. Hydrogen uptake in nodule homogenates from the Frankia sources measured at 23.8 μM H₂ ranged from 0.04 to 5.0 μmol H₂ (g fresh weight nodule)⁻¹ h⁻¹. The H₂ uptake capacity of nodule homogenates from one of the Frankia sources was almost 8 times higher than the hydrogen evolution from nitrogenase, both expressed on a nodule fresh weight basis. Frankia sources from field sites 6 and 11 showed K_m for H₂ of 13.0 and 23.6 μM H₂, respectively. This indicates similarities in the hydrogen uptake enzymes in the two Frankia sources. It is concluded that hydrogen uptake is a common characteristic in Frankia.     

Phylogenetic characterization of ineffective Frankia in Alnus glutinosa (L.) Gaertn. nodules from wetland soil inoculants

Ineffective Frankia endophytes were retrieved from various wet soils by using Alnus glutinosa clones as trapping plants. No pure cultures could be isolated from these ineffective nodules. Therefore, the phylogenetic position of these endophytes was determined by sequence analysis of cloned PCR products of bacterial 16S rDNA, derived from nodules. The results showed that all nodule endophytes belong to a hitherto undescribed cluster of the Frankia phylogenetic tree. The position of these uncultured ineffective Frankia nodule endophytes is different from that of the ineffective Frankia isolates derived from A. glutinosa nodules, even when originating from the same geographical location. This suggests a bias in current isolation techniques.     

The acetylene reduction assay inactivates root nodule uptake hydrogenase in some actinorhizal plants

Actinorhizal nodules do not usually evolve H₂ due to the action of an uptake hydrogenase. We have found that nodules of several Frankia symbioses evolved large amounts of H₂ gas when returned to air following exposure to 10 kPa C₂HT₂ during an acetylene reduction assay. Increased H₂ evolution in air persisted for several days when intact root systems of Alnus incana (L.) Moench (inoculated with Frankia UGL 011101) were treated with 10 kPa C.H₂ for 1 h. Full recovery of uptake hydrogenase activity required 4 to 8 days. Studies with crude homogenates of nodules of the same plants showed that hydrogenase (measured amperometrically with phenazine metho-sulfate as electron acceptor) was directly affected, since activity in treated nodules was only 10% of that in untreated nodules. A survey of actinorhizal symbioses revealed variation in the effect of an acetylene reduction assay on hydrogen metabolism. Nodules of three species, including Alnus rubra Bong, inoculated with Frankia HFPArD. showed complete inactivation of hydrogenase. H₂ evolution in air was 25% of the C₂H₂ reduction rate and H, evolution in Ar/O₂ was equal to the QH₂ reduction rate. Two symbioses, Ceanothus americanus L. (soil inoculant) and Batista glomerata Baill. (soil inoculant) showed no change following an acetylene reduction assay. A third group of symbioses showed an intermediate response.     

Superoxide dismutase, catalase and nitrogenase activities of symbiotic Frankia (Alnus incana) in response to different oxygen tensions

Presence and activity of the enzymes superoxide dismutase (SOD) and catalase were studied in Frankia in symbiosis with Alnus incana (L.) Moench. Analysis on native PAGE gels indicated that symbiotic Frankia contained an FeSOD and catalase. The activity of the enzymes was in the same range as reported for cultured Frankia . Attempts to characterize SOD by western blots with antisera from Escherichia coli and Azotobacter vinelandii did not give clear-cut results with the antibodies used. Alnus incana plants were grown with the root system in 5, 10, 21 or 40% O₂ for up to 6 days. Nitrogenase activity, measured as ARA (acetylene reducing activity) dropped within 3 h when roots were exposed to low or high oxygen. At 40% O₂ ARA was almost completely lost while at 5 and 10% O₂ ARA decreased to 69 and 74% of the inital value, respectively, Nitrogenase activity recovered at ail oxygen tensions. Recovery rates resembled the continuous increase in ARA in plants continuosly kept at 21% O₂, and suggests that new vesicles with envelopes of appropriate thickness were formed. The ARA measurements confirm results from an earlier study where nitrogenase activity was measured as H₂ evolution. There was a tendency for increased SOD and catalase activities in Frankia from root systems exposed to 40% O₂ for 24 h but not earlier or later than this. When data from all experimental times were pooled. SOD activity increased significantly with increased oxygen tension whereas catalase activity decreased. Although ARA per plant varied with oxygen tension, there was no statistically significant correlation between ARA and SOD or between ARA and catalase. It seems that being linked to nitrogenase activity is only one role of SOD and catalase in this symbiotic Frankia .     

Purity of Frankia preparations from root nodules of Alnus incana

Frankia vesicle clusters were prepared from Alnus incana (L.) Moench root nodules by a homogenization-filtration procedure. The preparation was examined by transmission electron microscopy and computerized picture analysis to quantify contamination from the host plant. Special attention was paid to plant mitochondria. Mitochondria were only found in 30% of the 50 sections of clusters examined. In sections containing mitochondria the mean number of mitochondria per cluster section was 1.5. The relative volume of all objects found in the vesicle clusters was calculated. More than 98% of the volume of a preparation consisted of Frankia vesicles and hyphae, while only 0.4% of the volume was host plant mitochondria. The frequency of mitochondria in a preparation could be further decreased by osmotic shock. It is concluded that Frankia vesicle clusters, prepared from Alnus incana by the homogenization-filtration technique used here, are sufficiently pure to be used for studies of Frankia metabolism.     

Host range differentiation of spore-positive and spore-negative strain types of Frankia in stands of Alnus glutinosa and Alnus incana in Finland

Host compatibility of different spore-positive (Sp⁺)and spore-negative (Sp^?) strain types of Frankia from alder stands in Finland was studied in Modulation tests with hydrocultures of Alnus glutinosa (L.) Gaertner, A. incana (L.) Moench and A. nitida Endl. Root nodules and soil samples from stands of A. incana (Lammi forest and Hämeenlinna forest) were dominated by Sp ⁺ types of Frankia (coded AiSp⁺ and AiSp⁺ H. respectively), which caused effective root nodules in test plants of A. incana, but failed to induce nodules in A. nitida. In A. glutinosa Frankia strain types AiSp ⁺ and AiSp ⁺ H caused small, ineffective root nodules with sporangia (coded Ineff ^?), which were recognized by the absence or near absence of vesicles in the nodule tissue. Ineffective nodules without sporangia (coded Ineff ^?) were induced on A. glutinosa with soil samples collected at Lammi swamp. The spore-negative strain type of Frankia was common in root nodules of A. glutinosa in Finland (Lammi swamp) and caused effective Sp^? type root nodules (coded AgSp ^?) in hydrocultures of A. incana, A. glutinosa and A. nitida. A different Sp ⁺ strain type of Frankia. coded AgSp⁺ Finland, was occasionally found in stands of A. glutinosa. It was clearly distinguished from strain type AiSp ⁺ by the ability to produce effective nodules on both A. glutinosa and A. incana. The nodulation capacities of soil and nodule samples were calculated from the nodulation response in hydrocutlure and served as a measure for the population density of infective Frankia particles. Sp ⁺ nodules from both strain types had equal and high nodulation capacities with compatible host species. The nodulation capacities of Sp type root nodules from A. glutinosa were consistently low. High frequencies of Frankia AiSp⁺ and AiSp⁺ H were found in the soil environment of dominant AiSp ⁺ nodule populations on A. incana. The numbers of infective particles of this strain type were insignificant in the soil environment of nearby Sp ^? nodule populations on A. glutinosa and in the former field at Hämeen-linna near the Sp⁺ nodule area in Hämeenlinna forest. Strain type AgSp^? had low undulation capacity in the soil environment of both A. incana and A. glutinosa stands, Explanations for the strong associations between Frankia strain types AiSp⁺ and AiSp ^? H and A. incana and between strain type AgSp^? and A. glutinosa are discussed in the light of host specificity and of some characteristics of population dynamics of both strain types. The possible need to adapt the concept of Frankia strain types Sp ⁺ and Sp ^? to strains with some variation in spore development was stressed by the low potentials of strain type AiSp ⁺ H to develop spores in symbioses with hydrocultures of A. incnna.     

Nitrogen fixation and biomass production in symbioses between Alnus incana and Frankia strains with different hydrogen metabolism

Three different strains of Frankia , the pure cultures AvcI1 and CpI1 and a local strain (crushed nodule inoculum), were compared in symbiosis with one clone of Alnus incana (L.) Moench. Hydrogen metabolism, nitrogenase (EC 1.7.99.2) activity and relative efficiency of nitrogenase were studied as well as growth and nitrogen content of the plants. The local Frankia strain showed no measurable hydrogen uptake but high H₂-evolution. No H₂-evolution was detected in Frankia AvcI1 because of its hydrogenase activity. CpI1 also had hydrogenase, although only a very small H₂-evolution was detected at the end of the growth period. Hydrogenase activity was detected both in pure cultures and nodule homogenates of CpI1 and AvcI1. Growth, biomass production and nitrogen content were highest in alders inoculated with Frankia AvcI1 while the lowest values were found for alders living in symbiosis with the local Frankia strain. The presence of hydrogenase in Frankia seemed to be benefical for growth and biomass production in the alders. However, the strains also differed with respect to spore formation. The local strain, but not AvcI1 and CpI1, formed spores in the root nodules.     

Cloning of a full-length symbiotic hemoglobin cDNA and in situ localization of the corresponding mRNA in Casuarina glauca root nodule

We have characterized a full-length cDNA ( hb -Cg1F) that represents symbiotic mRNA hemoglobin ( hb ) from Casuarina glauca root nodules. In situ hybridization was used to examine the correlation between hb -Cg1F mRNA and the state of the Frankia infection process. The efficiency of in situ hybridization using DIG-labeled vs [³⁵S]-labeled probes was compared. The expression of hb -Cg1F gene is induced in young infected host cells prior to the detection of Frankia nif H mRNA. Since Frankia does not form vesicles in C. glauca nodules, it is proposed that Hb is necessary to reduce the O₂ concentration in the cytoplasm of the host cells before the nif genes are expressed.     

Nickel is essential for active hydrogenase in free-living Frankia isolated from Casuarina

Five free-living Frankia strains isolated from Casuarina were investigated for occurrence of hydrogenase activity. Nitrogenase activity (acetylene reduction) and hydrogen evolution were also evaluated. Acetylene reduction was recorded in all Frankia strains. None of the Frankia strains had any hydrogenase activity when grown on nickel-depleted medium and they released hydrogen in atmospheric air. After addition of nickel to the medium, the Frankia strains were shown to possess an active hydrogenase, which resulted in hydrogen uptake but no hydrogen evolution. The hydrogenase activity in Frankia strain KB5 increased from zero to 3.86 μ mol H₂ (mg protein)⁻¹ h⁻¹ after addition of up to 1.0 μ M Ni. It is likely that the hydrogenase activity could be enhanced even more as a response on further addition of Ni. It is indicated in this study that absence of hydrogenase activity in free-living Frankia isolated from Casuarina spp. is due to nickel deficiency. Frankia living in symbiosis with Casuarina spp. show hydrogenase activity. Therefore, the results also indicate that the hydrogenase to some extent is regulated by the host plant and/or that the host plant supplies the symbiotic microorganism with nickel. Moreover, the result shows that this Frankia is somewhat different from Frankia isolated from Alnus incana and Comptonia peregrina ., i.e., Frankia isolated from A. incana and C. peregrina showed a small hydrogen uptake activity even without addition of nickel.     

Early interactions between Alnus glutinosa and Frankia strain ArI3. Production and specificity of root hair deformation factor(s)

Actinomycetes from the genus Frankia are able to form symbiotic associations with more than 200 different species of woody angiosperms, so called actinorhizal plants. Many actinorhizal plants are infected via deformed root hairs. Factor(s) eliciting root hair deformation in actinorhizal symbioses have been found to be released into the culture medium, but the factor(s) has (have) not yet been characterized. In the present work, we describe the constitutive production of factor(s) by Frankia strain ArI3 causing root hair deformation on Alnus glutinosa . Deformation was detected after 4–5 h of incubation with both Frankia cultures and their cell-free culture filtrates. When culture filtrate was used, deformation was concentration dependent. A contact time of 2 min between culture filtrate and host roots was sufficient to induce subsequent root hair deformation. No root hair deformation on A. glutinosa could be detected with purified Nod factors from Rhizobium meliloti or R. leguminosarum biovar viciae . No correlation was found between Frankia strains belonging to different host specificity groups and their ability to deform root hairs on A. glutinosa. However, strains not able to deform root hairs on A. glutinosa were also unable to nodulate.     

Occurrence of hemoglobin in the nitrogen-fixing root nodules of Alnus glutinosa

A true hemoglobin (Hb) was shown to be present in the root nodules of Alnus glutinosa L. After purification by gel filtration and ion exchange, the Hb formed a stable complex with oxygen. This oxygen complex could then be converted to carboxyhemoglobin by treatment with CO. Optical absorption spectra typical of Hb were observed. The molecular weight was estimated to be 15 100 by gel filtration, and 18 300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Hb was largely insoluble when the initial homogenization was done in the absence of a detergent. Under these conditions much of the Hb appears to be associated with clusters of Frankia , the nitrogen-fixing actinomycete that infects plant cells within the nodules. The exact localization of the Hb in vivo is uncertain. The relatively low average concentration of Hb in Alnus nodules suggests that it is either confined to a relatively small fraction of total nodule volume, or has a function other than facilitation of O₂ transport.     

Correlations between the ascorbate-glutathione pathway and effectiveness in legume root nodules

Legume root nodules use the ascorbate-glutathione pathway to remove harmful H₂O₂. In the present study. effective and ineffective nodules from soybean and alfalfa were compared with regard to this pathway. Effective nodules had higher activity of all 4 enzymes (ascorbate peroxidase, EC 1. 11. 1. 11: monodehydroascorbate reductase, EC 1. 6. 5. 4: dehydroascorbate reductase, EC 1. 8. 5. 1: and glutathione reductase, EC 1. 6. 4. 2). The concentration of thiol tripeptides (primarily homoglutathione) was about 1 m M in effective nodules – a level 3–4-fold higher than in ineffective nodules. Effective nodules contained higher levels of NAD⁺. NADP⁺ and NADPH. but not of NADH or ascorbate. The increased capacity for peroxide scavenging in effective nodules as compared to ineffective nodules emphasizes the important protective role that this pathway may play in processes related to nitrogen fixation.     

Nodulation and nitrogenase activity in nitrogen-fixing woody plants stimulated by CO2 enrichment of the atmosphere

The responses of three species of nitrogen-fixing trees to CO₂ enrichment of the atmosphere were investigated under nutrient-poor conditions. Seedlings of the legume, Robinia pseudoacacia L. and the actinorhizal species, Alnus glutinosa (L.) Gaertn. and Elaeagnus angustifolia L. were grown in an infertile forest soil in controlled-environment chambers with atmospheric CO₂ concentrations of 350 μl ⁻¹ (ambient) or 700 μl ⁻¹. In R. pseudoacacia and A. glutinosa , total nitrogenase (N₂ reduction) activity per plant, assayed by the acetylene reduction method, was significantly higher in elevated CO₂, because the plants were larger and had more nodule mass than did plants in ambient CO₂. The specific nitrogenase activity of the nodules, however, was not consistently or significantly affected by CO₂ enrichment. Substantial increases in plant growth occurred with CO₂ enrichment despite probable nitrogen and phosphorus deficiencies. These results support the premises that nutrient limitations will not preclude growth responses of woody plants to elevated CO₂ and that stimulation of symbiotic activity by CO₂ enrichment of the atmosphere could increase nutrient availability in infertile habitats.     

Effect of changes in nitrogen nutrition on the polyamine content of Alnus glutinosa

Abstract. The principal polyamines in Alnus glutinosa roots, nodules and root pressure sap, putrescine, spermidine and spermine, were quantified by reversed-phase, high-performance liquid chromatography with fluorescence detection following precolumn derivatization with 9-fluorenylmethyl chloroformate and 1-ada-mantanamine. Putrescine was the major component of all tissues and sap. It comprised 70% or more of the polyamine pool except in roots of KNO₃-fed plants, in which similar amounts of putrescine and spermidine were present at levels five-fold lower than plants fed (NH₄)₂SO₄. Polyamine levels in nodules were 50% greater than in roots. The polyamine content of roots and nodules was not altered significantly when the nitrogen nutrition was changed from sole reliance on nitrogen fixation to partial or complete utilization of (NH₄)₂SO₄. However, the polyamine content of root pressure sap from nodulated plants increased almost four-fold when they were fed with increasing concentrations of NH₄NO₃, although the total polyamine content remained low (5mmol m⁻³ sap). The polyamine content of the Alnus root system changed with plant age. In particular, the spermidine content of both roots and nodules was higher in 10- as compared to 16-week-old plants.     

Effects of phosphorus and nitrogen on nodulation are seen already at the stage of early cortical cell divisions in Alnus incana

BACKGROUND AND AIMS: The present work aimed to study early stages of nodulation in a chronological sequence and to study phosphorus and nitrogen effects on early stages of nodulation in Alnus incana infected by Frankia. A method was developed to quantify early nodulation stages in intact root systems in the root hair-infected actinorhizal plant A. incana. Plant tissue responses were followed every 2 d until 14 d after inoculation. Cortical cell divisions were already seen 2 d after inoculation with Frankia. Cortical cell division areas, prenodules, nodule primordia and emerging nodules were quantified as host responses to infection. METHODS: Seedlings were grown in pouches and received different levels of phosphorus and nitrogen. Four levels of phosphorus (from 0.03 to 1 mM P) and two levels of nitrogen (0.71 and 6.45 mM N) were used to study P and N effects on these early stages of nodule development. KEY RESULTS: P at a medium concentration (0.1 mM) stimulated cell divisions in the cortex and a number of prenodules, nodule primordia and emerging nodules as compared with higher or lower P levels. A high N level inhibited early cell divisions in the cortex, and this was particularly evident when the length of cell division areas and presence of the nodulation stages were related to root length. CONCLUSIONS: Extended cortical cell division areas were found that have not been previously shown in A. incana. The results show that effects of P and N are already expressed at the stage when the first cortical cell divisions are induced by Frankia.     

Effects of elevated oxygen tensions on acetylene reduction in Alnus incana-Frankia symbioses

An open flow-through gas system was used to determine the effect of C₂H₂ and elevated O₂ on acetylene reduction activity (ARA) and respiration of the intact, potted root system of Alnus incana (L.) Moench in symbiosis with Frankia Avcll or with a local source of Frankia . Both symbiotic systems responded to C₂H₂ by an immediate plateau range in ARA. The Plateau in ARA was in some cases followed by a decline of less extent than reported for many legumes. A concurrent decline in net respiration of the root system was on average 8% of the CO₂ efflux prior to C₂H₂ introduction.
Respiration of the root systems in both symbioses responded to elevated oxygen levels in the 10 kPa C₂H₂ atmosphere by an increase of up to 17% of the net respiration prior to C₂H₂ introduction in 21 kPa O₂. In contrast, the elevated oxygen levels resulted in an immediate drop in ARA followed by a minor increase to a stable level lower than that at the preceding, lower oxygen tension. The symbiosis with the local Frankia had lost all ARA when the partial pressure of O₂ exceeded 50 kPa, whereas the symbiosis with Avcll still had some activity at 80 kPa O₂. This difference in tolerance of elevated O₂ clearly shows that the oxygen exclusion mechanisms may be controlled by the microsymbiont in Alnus-Frankia symbioses. The symbiotic systems recovered ARA to a similar extent when returned from elevated O₂ levels to 21 kPa O₂.