Comparison of Nitrogen Fixation for North- and South-facing <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> Stands in Central Korea

The nitrogenase activity, root nodule biomass, and rates of nitrogen (N) fixation were measured in 25-year-old pure north- and south-facing Robinia pseudoacacia stands in an urban forest of Seoul (Kkachisan Mountain) in central Korea. The nitrogenase activity was estimated using an acetylene reduction (AR) assay, which showed an increasing trend during the early growing season, with sustained high rates from June through to September with a decrease thereafter. July had the highest nitrogenase activity rate (micromoles C₂H₄ per gram dry nodule per hour), averaging 95.8 and 115.1 for the north- and south-facing stands, respectively. The maximum root nodule biomass (kilograms per hectare) was 45.7 and 9.1 for the north- and south-facing stands in July, respectively. The AR rate appeared to be strongly correlated to the soil temperature (r ² = 0.68, P < 0.001) and soil pH (r ² = 0.59, P < 0.001) while root nodule biomass was correlated to the soil temperature (r ² = 0.36, P < 0.01) and water content (r ² = 0.35, P < 0.05). The soil temperature showed clear differences between seasons, while there was a significant difference in soil pH, organic matter, total N concentrations, and available phosphorus between the north- and south-facing stands. The N₂ fixation rates during the growing season varied from 0.1 to 37.5 kg N ha⁻¹ month⁻¹ depending on the sampling location and time. The annual N₂ fixation rate (kg N per hectare per year) was 112.3 and 23.2 for the north- and south-facing stands, respectively. The differences in N₂ fixation rate between the two stands were due mainly to the differences in total nodule biomass.     

Minimizing the effect of mineral nitrogen on biological nitrogen fixation in common bean by increasing nutrient levels

Although common bean (Phaseolus vulgaris L.) has good potential for N₂ fixation, some additional N provided through fertilizer usually is required for a maximum yield. In this study the suppressive effect of N on nodulation and N₂ fixation was evaluated in an unfertile soil under greenhouse conditions with different levels of soil fertility (low=no P, K and S additions; medium = 50, 63 and 10 mg kg^–1 soil and high = 200, 256 and 40 mg kg^–1 soil, respectively) and combined with 5, 15, 60 and 120 mg N kg^–1 soil of ¹⁵N-labelled urea. The overall average nodule number and weight increased under high fertility levels. At low N applications, nitrogen had a synergistic effect on N₂ fixation, by stimulating nodule formation, nitrogenase activity and plant growth. At high fertility and at the highest N rate (120 mg kg^–1 soil), the stimulatory effect of N fertilizer on N₂ fixation was still observed, increasing the amounts of N₂ fixed from 88 up to 375 mg N plant^–1. These results indicate that a suitable balance of soil nutrients is essential to obtain high N₂ fixation rates and yield in common beans.     

Effect of plant nutrient supply on nodule effectiveness and rhizobium strain competition for nodulation ofLotus pedunculatus

Summary The effect of nutrient supply on nodule formation and competition between Rhizobium strains for nodulation ofLotus pedunculatus was studied. Limiting plant growth by decreasing the supply of nutrients in an otherwise nitrogen-free medium, increased the size but decreased the number and the nitrogenase activity of nodules formed by a fast-growing strain of Lotus Rhizobium (NZP2037). In contrast decreasing nutrient supply caused only a small decline in the size, number and nitrogenase activity of nodules formed by a slow-growing strain (CC814s). Providing small quantities of NH₄NO₃ (50 to 250 g N) to plants grown with a normal supply of other nutrients stimulated nodule development by both Rhizobium strains and increased the nitrogenase activity of the NZP2037 nodules. Differences in the level of effectiveness (nitrogen-fixing ability) of nodules formed by different Rhizobium strains on plants grown with a normal supply of nutrients were less apparent when the plants were grown with decreased nutrient supply or when the plants were supplied with low levels of inorganic N.Inter-strain competition for nodulation ofL. pedunculatus between the highly effective slow-growing strain CC814s and 7 other fast- and slow-growing strains, showed CC814s to form 42 to 100% of the nodules in all associations. The greater nodulating competitiveness of strain CC814s prevailed despite changes in the nutrient supply to the host plant. A tendency was observed for partially effective Lotus Rhizobium strains to become more competitive in nodule formation when plant growth was supplemented with low levels of inorganic nitrogen.     

Nitrogenase activity and N2 fixation are stimulated by elevated CO2 in a tropical N2-fixing tree

 Seeds of Gliricidia sepium, a fast-growing woody legume native to seasonal tropical forests of Central America, were inoculated with N₂-fixing Rhizobium bacteria and grown in environmentally controlled glasshouses for 67–71 days under ambient CO₂ (35 Pa) and elevated CO₂ (70 Pa) conditions. Seedlings were watered with an N-free, but otherwise complete, nutrient solution such that bacterial N₂ fixation was the only source of N available to the plant. The primary objective of our study was to quantify the effect of CO₂ enrichment on the kinetics of photosynthate transport to nodules and determine its subsequent effect on N₂ fixation. Photosynthetic rates and carbon storage in leaves were higher in elevated CO₂ plants indicating that more carbon was available for transport to nodules. A ¹⁴CO₂ pulse-chase experiment demonstrated that photosynthetically fixed carbon was supplied by leaves to nodules at a faster rate when plants were grown in elevated CO₂. Greater rates of carbon supply to nodules did not affect nodule mass per plant, but did increase specific nitrogenase activity (SNA) and total nitrogenase activity (TNA) resulting in greater N₂ fixation. In fact, a 23% increase in the rate of carbon supplied to nodules coincided with a 23% increase in SNA for plants grown in elevated CO₂, suggesting a direct correlation between carbon supply and nitrogenase activity. The improvement in plant N status produced much larger plants when grown in elevated CO₂. These results suggest that Gliricidia, and possibly other N₂-fixing trees, may show an early and positive growth response to elevated CO₂, even in severely N-deficient soils, due to increased nitrogenase activity. Received: 27 February 1996 / Accepted: 19 June 1996     

Does oxygen limit nitrogenase activity in soybean exposed to elevated CO2?

Soybean (Glycine max L. Merr) plants grown under control (360 µmol mol^?1) or elevated CO₂ concentration (800 µmol mol^?1) from 33 to 42 d after sowing were assayed for various components of in vivo nitrogenase activity to test the hypothesis that increasing carbohydrate supply to nodules would increase the potential (i.e. O₂ saturated) nitrogenase activity and impose a more severe O₂ limitation on both nodule metabolism and total nitrogenase activity. Within 51 h of elevated CO₂ treatment, significant increases relative to control plants were seen in total nitrogenase activity expressed per plant. After 6 d of elevated CO₂, the total nitrogenase activity per plant was 18% higher than that in control. This was attributed to an initial increase in nodule size, and a subsequent increase in nodule number following plant exposure to elevated CO₂. However, after 9 d of elevated CO₂, the potential and total nitrogenase activities per gram nodule dry weight were lower, not higher than corresponding values in plants in the control treatment. These results did not support the hypothesis. It was concluded that the metabolic capacity of the control nodules were not limited by carbohydrate supply, at least at the assay temperatures employed here.     

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。     

Nitrogen dynamics in grain crop and legume pasture systems under elevated atmospheric carbon dioxide concentration: A meta‐analysis

Understanding nitrogen (N) removal and replenishment is crucial to crop sustainability under rising atmospheric carbon dioxide concentration ([CO₂]). While a significant portion of N is removed in grains, the soil N taken from agroecosystems can be replenished by fertilizer application and N₂ fixation by legumes. The effects of elevated [CO₂] on N dynamics in grain crop and legume pasture systems were evaluated using meta‐analytic techniques (366 observations from 127 studies). The information analysed for non‐legume crops included grain N removal, residue C : N ratio, fertilizer N recovery and nitrous oxide (N₂O) emission. In addition to these parameters, nodule number and mass, nitrogenase activity, the percentage and amount of N fixed from the atmosphere were also assessed in legumes. Elevated [CO₂] increased grain N removal of C₃ non‐legumes (11%), legumes (36%) and C₄ crops (14%). The C : N ratio of residues from C₃ non‐legumes and legumes increased under elevated [CO₂] by 16% and 8%, respectively, but the increase for C₄ crops (9%) was not statistically significant. Under elevated [CO₂], there was a 38% increase in the amount of N fixed from the atmosphere by legumes, which was accompanied by greater whole plant nodule number (33%), nodule mass (39%), nitrogenase activity (37%) and %N derived from the atmosphere (10%; non‐significant). Elevated [CO₂] increased the plant uptake of fertilizer N by 17%, and N₂O emission by 27%. These results suggest that N demand and removal in grain cropping systems will increase under future CO₂‐enriched environments, and that current N management practices (fertilizer application and legume incorporation) will need to be revised.     

Long‐term non‐invasive and continuous measurements of legume nodule activity

Symbiotic nitrogen fixation is a process of considerable economic, ecological and scientific interest. The central enzyme nitrogenase reduces H⁺ alongside N₂, and the evolving H₂ allows a continuous and non‐invasive in vivo measurement of nitrogenase activity. The objective of this study was to show that an elaborated set‐up providing such measurements for periods as long as several weeks will produce specific insight into the nodule activity's dependence on environmental conditions and genotype features. A system was developed that allows the air‐proof separation of a root/nodule and a shoot compartment. H₂ evolution in the root/nodule compartment can be monitored continuously. Nutrient solution composition, temperature, CO₂ concentration and humidity around the shoots can concomitantly be maintained and manipulated. Medicago truncatula plants showed vigorous growth in the system when relying on nitrogen fixation. The set‐up was able to provide specific insights into nitrogen fixation. For example, nodule activity depended on the temperature in their surroundings, but not on temperature or light around shoots. Increased temperature around the nodules was able to induce higher nodule activity in darkness versus light around shoots for a period of as long as 8 h. Conditions that affected the N demand of the shoots (ammonium application, Mg or P depletion, super numeric nodules) induced consistent and complex daily rhythms in nodule activity. It was shown that long‐term continuous measurements of nodule activity could be useful for revealing special features in mutants and could be of importance when synchronizing nodule harvests for complex analysis of their metabolic status.     

Model of gas exchange and diffusion in legume nodules

A mathematical model is described which allows the estimation of rates of O₂, CO₂, N₂, and H₂ exchange from legume nodules under steady state conditions of N₂ fixation. Calculated rates of gas exchange under defined conditions of nodule size, relative growth rate (RGR), specific total nitrogenase activity (TNA), nitrogenase electron allocation coefficient (EAC), uptake-hydrogenase activity (HUP) and nature of the N export product compared favorably with experimentally-obtained rates reported in the literature. Therefore the model was used to predict the effects of varying each of these nodule characteristics on the rates of gas exchange, and on the apparent respiratory cost (CO₂/NH₃) and sucrose cost (sucrose consumed/NH₃) of N₂ fixation.The model predicted that, all other characters being equal, ureide-producing nodules would consume 8% less sucrose per N fixed than asparagine-producing nodules, but would display an apparent respiratory cost which would be 5% higher than that in asparagine-producing nodules. In both ureide-producing and asparagine-producing nodules, the major factor affecting the apparent respiratory cost of N₂ fixation was predicted to be EAC, followed by TNA, nodule RGR and nodule size. The relative importance of HUP in improving the apparent respiratory cost of N₂ fixation was predicted to be largely dependent upon its potential role in the regulation of EAC. Abbreviations: See Appendix 1.     

Phosphorus deficiency increases the argon-induced decline of nodule nitrogenase activity in soybean and alfalfa

Open-flow assays of H₂ evolution in Ar:O₂ (80:20, v/v) by nodulated roots were performed in situ with soybean [Glycine max (L.) Merr.] and alfalfa [Medicago sativa L.) grown in sand with orthophosphate (Pi) nutrition either limiting (low-P) or non-limiting (control) for plant growth. Nodule growth was more limited than shoot growth by P deficiency. Phosphorus concentration was less affected in nodules than in other parts of the low-P plants. During assays, nitrogenase activity declined a few minutes after exposure of the nodulated roots to Ar. The magnitude of this argon-induced decline (Ar-ID) was less in alfalfa than in soybean. In both symbioses the magnitude of the Ar-ID was larger in low-P than control plants. Moreover, the minimum H₂ evolution after the Ar-ID, was reached earlier in low-P plants. The Ar-ID was partly reversed by raising the external partial pressure of O₂ in the rhizosphere. The magnitude of the Ar-ID in soybean was correlated negatively to nodule and shoot mass per plant, individual nodule mass, H₂ evolution in air prior to the assay, and nodule N and P concentrations. Possible reasons, including nodule size and nodule O₂ permeability, for the increase in Ar-ID in P-deficient plants are discussed and an interpretation of the P effect on nodule respiration and energetic metabolism is proposed. Received: 17 May 1996 / Accepted: 16 September 1996     

Cinnamate toxicity expression on phenylalanine ammonia-lyase activity,germination and growth of cucumber (Cucumis sativis) seedlings

Low pH (5.2) decreased nodule number and acetylene reduction. Aluminium further depressed those parameters in theRhizobium leguminosarum-Pisum sativum associations examined. In the Al-treated plants nodule formation by strains 128C53 and 128C30 was not affected by 3 or 15 and 30 or 60 μM Al, respectively, as compared with the number of nodules on plants grown at pH 5.2 in the absence of Al. However, improved nodulation rates by those strains did not enhance plant dry weight or reduced nitrogen content. No differences in nitrogenase activity were found among strains of nodulating plants grown at the same aluminium level. These results suggest that Al-ions affected specifically nitrogenase activity and that this effect was primarily responsible for the reduction in plant growth.     

Manganese tolerance in subterranean clover (Trifolium subterraneum L.) genotypes grown with ammonium nitrate or symbiotic nitrogen

Summary A wide range of clover accessions were screened for reaction to manganese (Mn) in solution culture. Growth was supported with ammonium nitrate (NH₄NO₃) or symbiotic nitrogen to assess Mn effects on symbiosis and the suitability of NH₄NO₃ dependent growth for assessing Mn tolerance in clover. Reduction of dry matter at Mn 45 ppm varied 0–70%, at Mn 90 ppm, 38–92%, the extent depending on genotype. Tolerant clovers tended to restrict the movement of Mn from roots to shoots. Several previously untested lines were the most tolerant while some commercial lines possessed poor tolerance. Ranks of tolerance for the two nitrogen (N) sources at Mn 45 ppm were correlated suggesting no dominant, discriminatory effects of N source on Mn tolerance; but inclusion of symbiotic effectiveness in a multiple correlation improved the relation between relative tolerances of genotypes under different N sources. Mn affected some aspects of symbiosis. Total nodule nitrogenase activity mainly reflected effects of Mn on nodule number but nitrogenase activity per nodule also contributed. To establish relative tolerances of subterranean clover to Mn growth with NH₄NO₃ is suitable and useful when symbiotic effectiveness is unknown.     

Nitrogenase activity of two genotypes of Acacia mangium as affected by phosphorus nutrition

The specific nodulation, nitrogenase activity (acetylene reduction) and budgets of carbon allocation to respiration by nodulated roots were examined in two provenances of Acacia mangium Willd. grown in a glasshouse for 17 weeks to investigate the effects of soil phosphorus and genotypes of the host plant on symbiotic nitrogen fixation. Application of phosphorus (0–80 mg P kg^-1 soil) increased specific nodulation (g nodule dry weight g^-1 plant dry weight) of provenance Ma11 by two-fold and the percentage of nodulated root respiration allocated to nitrogenase by 50%, but had no effect on specific activity of nitrogenase or specific respiration coupled with nitrogenase activity. Improved phosphorus nutrition increased the specific nitrogenase activity of provenance Ma9 by 2-fold, the percentage of nodulated root respiration allocated to nitrogenase, and specific nitrogenase-linked respiration by 50%, respectively, but had no effect on the specific nodulation. The percentage of respiration coupled with nitrogenase activity in nodulated root respiration by provenance Ma9 was 60–70% higher than that in provenance Ma11, regardless of phosphorus levels applied. At the optimal level of phosphorus addition (10 mg P kg^-1 soil), provenance Ma9 had a lower dry mass than provenance Ma11. This was accompanied by a lower nodulated root respiration and a higher percentage of nodulated root respiration allocated to nitrogenase activity in provenance Ma9.     

Gas-exchange activity,carbohydrate status,and protein turnover in root nodule subpopulations of field pea (Pisum sativum L. cv. Century)

Root nodule ontogeny was followed in different parts of the root system of field peas (Pisum sativum L. cv. Century) to investigate the contribution to total nitrogen fixation by different nodule subpopulations. Seed-inoculated plants were grown to maturity in controlled-environment growth chambers. In a flow-through system nitrogenase activity (H₂-evolution in air) and nodulated-root respiration (net CO₂-evolution) were measured weekly or biweekly in different parts (top and mid) of the root system. Root nodule extracts were assayed for total soluble cytosolic protein, total heme, proteolytic capacity (at pH 7.0), soluble carbohydrates and starch. Total nitrogenase activity and nodule respiration were higher in the top zone, which was explained by differences in root and nodule mass. Nodule specific nitrogenase activity was similar in both zones, and gradually declined throughout the experiment. No differences were found between nodule subpopulations in the dry-matter specific concentrations of glucose, fructose, sucrose or starch. Neither did nodule concentrations of protein or leghemoglobin differ between the zones. Throughout reproductive growth, no decline was found in total or nodule specific nitrogenase activity, in any of the nodule subpopulations. Growth of the root nodules continued throughout the experiment, though growth of shoot and roots had ceased. The data gives no support for carbohydrate limitation in root nodules during pod-filling, since nodule respiration remained high, the concentration of soluble carbohydrates increased significantly, and the amount of starch was not reduced. We conclude that when this symbiosis is grown under controlled conditions, nitrogenase activity in nodules sub-sampled from the crown part of the root system is representative for the whole nodule population.     

Interaction of Combined Nitrogen with the Expression of Root-Associated Nitrogenase Activity in Grasses and with the Development of N(2) Fixation in Soybean (Glycine max L. Merr.)

Soluble root N concentrations of corn, sorghum, pearl millet, rice, wild rice, and soybeans were determined and related to measurements of nitrogenase activity and changes in availability of combined N to plants. In corn, sorghum, and pearl millet, applications of fertilizer N increased soluble root N concentrations, but root-associated nitrogenase activity was negligible in control and treated plants. Applications of NH₄NO₃ to rice increased the water soluble root N concentrations and inhibited root-associated nitrogenase activity. In wild rice, root-associated nitrogenase activity was absent during vegetative growth and developed at the reproductive growth stage. The soluble root N concentration decreased progressively as wild rice grew indicating that the availability of combined N in the root environment declined. Therefore, development of nitrogenase activity in wild rice is associated with the change in availability of combined N in the root environment. The development of nitrogenase activity in wild rice was probably not due to colonization of roots by N₂-fixing bacteria because most probable numbers of recovery did not significantly vary throughout the plants' growth cycle. In field-grown soybeans with or without fertilizer N application, we also observed a relationship between a decrease in soluble root N concentration and the development of nitrogenase activity.     

Ammonium assimilation in root nodules of actinorhizal Discaria trinervis. Regulation of enzyme activities and protein levels by the availability of macronutrients (N,P and C)

Asparagine was found to be the main N compound exported from Discaria trinervis nodules. Aspartate (Asp), glutamate (Glu), alanine (Ala) and serine (Ser) were also detected in root xylem sap, but at lower concentrations. A comparable picture is found in nodulated alfalfa. We hypothesized that a similar set of enzymes for Asn synthesis was present in D. trinervis nodules. We demonstrate the expression of most of the enzymes involved in the synthesis of Asn from NH⁺ ₄ and oxoacids, in nodules – but not in roots – of fully symbiotic D. trinervis. By complementation of enzyme assays (^A) and immunodetection (^I) we detected glutamane-synthetase (GS^{A, I}), Asp-aminotransferase (AAT^A), malate-dehydrogenase (MDH^{A, I}, at least two isoforms), Glu-dehydrogenase (GDH^A), Glu-synthase (GOGAT^I) and Asn-synthetase (AS^I). PEP-carboxylase (PEPC) activity was not detected. We previously shown that N acts as a negative regulator of nodulation and nodule growth, while P is a strong stimulator for nodule growth. We present data on the regulation of nodule N metabolism by altering, during 4 weeks, the availability of N, P and light in symbiotic D. trinervis. NH₄NO₃ (2 mM) induced inactivation and degradation of nodule GS, MDH and AS, but activation of GDH and AAT; the amount of nitrogenase components was not affected. A 10-fold increase in P supply did not greatly affect activity and amount of enzymes, suggesting that N metabolism is not P-limited in nodules. On the other hand, suppression of P supply induced an important reduction of nodule GS, GOGAT, MDH and AS protein levels, although nitrogenase was not affected. GDH was the only measured activity that was stimulated by limiting P supply. Shading plants did result in complete degradation of nitrogenase and partial degradation of GS, AS and nodule-specific MDH isoform, but GDH and AAT were activated. These results are discussed in connection with the regulation of nodulation and nodule growth in D. trinervis.     

Asparagine as a major factor in the N‐feedback regulation of N2 fixation in Medicago truncatula

The objective of this study was to assess whether a whole plant N‐feedback regulation impact on nitrogen fixation in Medicago truncatula would manifest itself in shifts of the composition of the amino acid flow from shoots to nodules. Detected shifts in the phloem amino acid composition were supposed to be mimicked through artificial phloem feeding and concomitant measurement of nodule activity. The amino acid composition of the phloem exudates was analyzed from plants grown under the influence of treatments (limiting P supply or application of combined nitrogen) known to reduce nodule nitrogen fixation activity. Plants in nutrient solution were supplied with sufficient (9 µM) control, limiting (1 µM) phosphorus or 3 mM NH₄NO₃ (downregulated nodule activity). Low phosphorus and the application of NH₄NO₃ reduced per plant and specific nitrogenase activity (H₂ evolution). At day 64 of growth, phloem exudates were collected from cuts of the shoot base. The amount of amino acids was strongly increased in both phloem exudates and nodules of the treatments with downregulated nodule activity. The increase in the downregulated treatments was almost exclusively the result of a higher proportion of asparagine in both phloem exudates and nodules. Leaf labeling with ¹⁵N showed that nitrogen from the leaves is retranslocated to nodules. An artificial phloem feeding with asparagine resulted in an increased concentration of asparagine in nodules and a decreased nodule activity. A possible role of asparagine in an N‐feedback regulation of nitrogen fixation in M. truncatula is discussed.     

Short-Term Nitrate Effects on Hydroponically-Grown Soybean cv. Bragg and its Supernodulating Mutant: I. CARBON, NITROGEN AND MINERAL ELEMENT DISTRIBUTION, RESPIRATION AND THE EFFECT OF NITRATE ON NITROGENASE ACTIVITY

A comparison between two hydroponically-grown soybean genotypes(Glycine max [L.] Merr.) cv. Bragg and the supernodulating mutantnts 1007 was made in terms of dry matter accumulation, carbon,nitrogen, and mineral element distribution, ¹⁵N natural abundanceand the effect of short-term treatment with 4·0 mol m^–3KNO₃ on nitrogenase activity and respiration. Differences weremost pronounced in nodule dry weight and plant nitrogen content,both of which were recorded to be substantially elevated inthe mutant. Mineral element concentrations in different plantparts proved to be rather similar with the exception of Ca,found to be lower in leaves of the mutant, and Mn concentrationswhich were twice as high in roots of nts 1007. The values of¹⁵N natural abundance showed that both genotypes were equallydependent on nitrogen fixation when nitrate was absent. Theresults of the acetylene reduction assays indicated similarspecific nodule activity, while on a per plant basis nitrogenaseactivity of the mutant proved to be more than twice the amountof Bragg. This effect was also reflected in higher nodule respirationwhile root respiration remained below that of Bragg. Nitrate induced a substantial reduction in nitrogenase activitynot only in Bragg, but also in nts 1007. Nodule respiratoryactivity of Bragg was reduced by nitrate from 1·27 to0·34 mg C h^–1 plant^–1. In nts 1007 correspondingvalues were 2·70 to 1·52 mg C h^–1 plant^–1.Starch concentration in nodules was decreased in both genotypes,but nevertheless remained higher in nts 1007. Values for solublesugars in nodules even increased in the mutant in response tonitrate while the same treatment caused a reduction in Bragg.The data indicate that nitrogenase activities of Bragg and nts1007 are equally sensitive to short-term application of nitrate. Key words: Glycine max, C and N distribution, nitrate, root respiration, ¹⁵N natural abundance     

Influence of Flavonoids on Nodulation and Carbon Partitioning in Pea-Rhizobium Symbiosis

Rhizospheric application of nod regulators influenced the nodulation status, nodule efficiency and partitioning of assimilated CO₂ in Pisum sativum-Rhizobium leguminosarum symbiosis. Depending upon the plant growth stage, naringenin and flavone enhanced nodule number (22.8–87.6%), nodule weight (19.2–35.2%) and nitrogenase activity (17.8–108.6%). Syringaldehyde had negative effect on various parameters of symbiosis. Proportion of ¹⁴C-assimilates was higher in nodules of plants from naringenin and flavone treatments as compared to the control or syringaldehyde treatments. The enhanced nodule efficiency in naringenin and flavone treatments was reflected in the increased N content (12.7–50.4%) and biomass (4.3–35.1%) of plants.     

Nitrogenase activity in root nodule homogenates ofAlnus incana

Summary Root nodule homogenates of actinorhizal plants may representFrankia in a symbiotic stage but released from environmental influence of the host plant. Anaerobic homogenization with a blender in buffer supplied with sucrose, polyvinylpyrrolidone and reducing substances gave three times higher yields of nitrogenase activity (C₂H₂-reduction) than crushing the nodules in liquid nitrogen. The activity in the homogenates was very reproducible and was, on average, nearly twice as high as the activity in excised nodules and c. 10% of the activity in intact plants. The difference in activity between excised nodules and intact plants was, roughly by halves, due to removal of the root system from the pot and to excision of the nodules. The nitrogenase activity in the homogenates was slightly higher when nodule excision was done in Ar or under water as well as after treatment of the homogenate with toluene or Triton X-100 or osmotic shock. These gains in activity were considered too small to outweigh the increased complications of preparing homogenates for routine use. Due to the reproducible recovery of nitrogenase in the homogenates the technique seems useful for physiological studies on nitrogen fixation inAlnus incana.