Partitioning of Nitrogen Derived from N2 Fixation and Reserves in Nodulated Medicago sativa L. During Regrowth

Nodul{macron}ted alfalfa plants were grown hydroponically. Inorder to quantify N₂ fixation and remobilization of N reservesduring regrowth the plants were pulse-chase-labelled with ¹⁵N.Starch and ethanol-soluble sugar contents were analysed to examinechanges associated with those of N compounds. Shoot removalcaused a severe decline in N₂ fixation and starch reserves within6 d after cutting. The tap root was the major storage site formetabolizable carbohydrate compounds used for regrowth; initiallyits starch content decreased and after 14 d started to recoverreaching 50% of the initial value on day 24. Recovery of N₂fixation followed the same pattern as shoot regrowth. Afteran initial decline during the first 10 d following shoot removal,the N₂ fixation, leaf area and shoot dry weight increased sorapidly that their levels on day 24 exceeded initial values.Distribution of ¹⁵N within the plant clearly showed that a significantamount of endogenous nitrogen in the roots was used by regrowingshoots. The greatest use of N reserves (about 80% of N incrementin the regrowing shoot) occurred during the first 10 d and thencompensated for the low N₂ fixation. The distribution of N derivedeither from fixation or from reserves of source organs (taproots and lateral roots) clearly showed that shoots are thestronger sink for nitrogen during regrowth. In non-defoliatedplants, the tap roots and stems were weak sinks for N from reserves.By contrast, relative distribution within the plant of N assimilatedin nodules was unaffected by defoliation treatment. Key words: Medicago sativa L., N₂ fixation, N remobilization, N₂ partitioning, regrowth     

Ureide assay for measuring nitrogen fixation by nodulated soybean calibrated by N methods

We report experiments to quantify the relationships between the relative abundance of ureide-N in root-bleeding sap, vacuum-extracted sap, and hot water extracts of stems and petioles of nodulated soybean (Glycine max [L.] Merrill cv Bragg) and the proportion of plant N derived from nitrogen fixation. Additional experiments examined the effects of plant genotype and strain of rhizobia on these relationships. In each of the five experiments reported, plants of cv Bragg (experiment 1), cv Lincoln (experiments 3, 4, 5), or six cultivars/genotypes (experiment 2) were grown in a sand:vermiculite mixture in large pots in a naturally lit, temperature-controlled glasshouse during summer. Pots were inoculated at sowing with effective Bradyrhizobium japonicum CB1809 (USDA 136) or with one of 21 different strains of rhizobia. The proportions of plant N derived from nitrogen fixation were determined using ¹⁵N dilution. In one experiment with CB1809, plants were supplied throughout growth with either N-free nutrients or with nutrients supplemented with 1, 2, 4, or 8 millimolar ¹⁵N-nitrate and harvested on eight occasions between V6 and R7 for root-bleeding sap, vacuum-extracted sap, stems (including petioles), and whole plant dry matter. Analyses of the saps and stem extracts for ureides (allantoin plus allantoic acid), α-amino-N, and nitrate, and of dry matter for N and ¹⁵N, indicated a positive effect of nitrate supply on concentrations of nitrate in saps and extracts and a negative effect on ureides and on the proportion of plant N derived from nitrogen fixation. The relative abundance of ureide-N in root-bleeding sap, vacuum-extracted sap (100 [ureide-N]/[ureide-N+ α-amino-N + nitrate-N]) and stem extracts (100 [ureide-N]/[ureide-N + nitrate-N]) and the proportion of plant N, derived from nitrogen fixation between successive samplings were highly correlated (r = 0.97-1.00). For each variable, two standard curves were prepared to account for the shifts in the compositions of N solutes of xylem saps and extracts after flowering which were not related to a change in nitrogen fixation. Relationships between relative ureide-N and the proportion of plant N derived from nitrogen fixation were not affected by plant genotype or by strain of rhizobia. Therefore, assessment of nitrogen fixation by soybean using the ureide technique should now be possible with the standard curves presented, irrespective of genotype or strain of rhizobia occupying the nodules.     

Determination of nitrogen fixation effectiveness in selected <Emphasis Type="Italic">Medicago truncatula</Emphasis> isolates by measuring nitrogen isotope incorporation into pheophytin

Effectiveness is a term used to describe the input that a bacterial nitrogen-fixing symbiosis makes to plant nitrogen metabolism. In legumes, effectiveness is considered a polymorphic trait where specific interactions between the plant and symbiotic rhizobia contribute to the success of the interaction. Evaluation of effectiveness using model legumes like Medicago truncatula may open new avenues for genetic studies. In previous work, an isotope dilution mass spectrometry method, which uses the effect of nitrogen fixation on the nitrogen isotope composition of chlorophyll in plants grown on ¹⁵N fertilizer as a measure of effectiveness, was developed for estimating the contribution of symbiotic nitrogen fixation to plant nitrogen content. This ¹⁵N-dilution assay was used to evaluate the level of nitrogen fixation effectiveness in three Medicago truncatula lines that have been used as parents in generating recombinant inbred lines. Three Sinorhizobium meliloti strains, USDA 1600, 102F51 and MK506, differ in this measure of effectiveness on three lines of M. truncatula: Jemalong A17, DZA315.16 and F83005.5. Plant–rhizobia combinations grown in two different conditions showed comparable differences in effectiveness.     

Comparison of isotope techniques and non-nodulating isolines to study the effect of ammonium fertilization on dinitrogen fixation in soybean,Glycine max

Summary Two experiments were carried out with two nodulating and non-nodulating soybean isolines, with three different levels of N as (¹⁵NH₄)₂SO₄ at the equivalent of 0, 25 and 50 kg N/ha. In the first experiment three seeds were sown in each pot and the plants harvested at 35, 55 and 75 days. In the second experiment only one seed was sown per pot and harvested at 75 days.Isotope dilution technique and in certain cases natural isotope variation (¹⁵N) was used to determine directly the origin of nitrogen in the plant, whether from soil, fertilizer or biological N₂-fixation. The use of nodulating and non-nodulating isolines enabled comparison with the classical method of estimating N₂-fixation by difference from total plant N. Results at the 75 day harvest were similar for either method, but at the earlier harvests, particularly at 35 days, the total-N method was inadequate. The isotope method appeared more sensitive while the total-N method suffered from greater variability with correspondingly high standard errors and significant differences.It was found that by the 35 and 55 day harvests hardly any N₂-fixation had taken place, plant nitrogen being almost entirely derived from soil or fertilizer N. Plants in competition used up soil fertilizer N more rapidly, thus stimulating symbiotic nitrogen fixation. When only one plant was grown in each pot it had a greater proportion of N derived from soil or fertilizer, and less N derived from fixation. In general the¹⁵N data showed that only about 25% of the applied fertilizer N was absorbed by the plant.The nodulating isoline absorbed more N than the non-nodulating plants. This suggests a possible synergistic effect of N₂-fixation on N derived from other sources, giving an increase in total-N content of nudulated plants. The N derived from N₂-fixation was scarcely detectable in the roots but appeared to be translocated almost entirely to shoots and pods.With 25 kg N/ha the greater proportion of the nitrogen in the pods was derived from N₂-fixation. Even with 50 kg N/ha the nitrogen in the pods derived from fixation remained high, that being derived from fertilizer being less than 15%. About 80% of the nitrogen in the nodules was due to fixation.In the present experiment the application of 25 kg N/ha appeared sufficient to give maximum N absorption by both isolines. At this level symbiotic fixation by Rhizobium remained high in nodulating plants, while the proportion of total N due to fixation was reduced with 50 kg N/ha.UNDP/IAEA Project BRA 78/006.     

Simultaneous measurement of denitrification and nitrogen fixation using isotope pairing with membrane inlet mass spectrometry analysis

A method for estimating denitrification and nitrogen fixation simultaneously in coastal sediments was developed. An isotope-pairing technique was applied to dissolved gas measurements with a membrane inlet mass spectrometer (MIMS). The relative fluxes of three N(2) gas species ((28)N(2), (29)N(2), and (30)N(2)) were monitored during incubation experiments after the addition of (15)NO(3)(-). Formulas were developed to estimate the production (denitrification) and consumption (N(2) fixation) of N(2) gas from the fluxes of the different isotopic forms of N(2). Proportions of the three isotopic forms produced from (15)NO(3)(-) and (14)NO(3)(-) agreed with expectations in a sediment slurry incubation experiment designed to optimize conditions for denitrification. Nitrogen fixation rates from an algal mat measured with intact sediment cores ranged from 32 to 390 microg-atoms of N m(-2) h(-1). They were enhanced by light and organic matter enrichment. In this environment of high nitrogen fixation, low N(2) production rates due to denitrification could be separated from high N(2) consumption rates due to nitrogen fixation. Denitrification and nitrogen fixation rates were estimated in April 2000 on sediments from a Texas sea grass bed (Laguna Madre). Denitrification rates (average, 20 microg-atoms of N m(-2) h(-1)) were lower than nitrogen fixation rates (average, 60 microg-atoms of N m(-2) h(-1)). The developed method benefits from simple and accurate dissolved-gas measurement by the MIMS system. By adding the N(2) isotope capability, it was possible to do isotope-pairing experiments with the MIMS system.     

15N2 incorporation by rhizosphere soil influence of rice variety,organic matter and combined nitrogen

Summary Heterotrophic nitrogen fixation by rhizosphere soil samples from 20 rice cultivars grown under uniform field conditions was estimated employing¹⁵N-tracer technique. Rhizosphere soil samples from different rice cultivars showed striking differences with regard to their ability to incorporate¹⁵N₂. Rhizosphere samples from rice straw-amended (3 and 6 tons/ha) soil exhibited more pronounced nitrogen-fixing activity than the samples from unamended soil; while the activity of the rhizosphere samples from soils receiving combined nitrogen (40 and 80 kg N/ha) was relatively low. However, the inhibitory effect of combined nitrogen was not expressed in the presence of rice straw at 6 tons/ha. Results suggest that plant variety, application of combined nitrogen and organic matter influence the rhizosphere nitrogen fixation.     

Acetylene reduction (nitrogen fixation) associated with corn inoculated with Spirillum.

Sorghum and corn breeding lines were grown in soil in field and greenhouse experiments with and without an inoculum of N2-fixing in Spirillum strains from Brazil. Estimated rates of N2 fixation associated with field-grown corn and sorghum plants were less than 4 g of N2/ha per day. The mean estimated N2-fixation rates determined on segments of roots from corn inoculated with Spirillum and grown in the greenhouse at 24 to 27 degrees C were 15 g of N2/ha per day (16 inbreds), 25 g of N2/ha per day (six hybrids), and 165 g of N2/ha per day for one hybird which was heavily inoculated. The corresponding mean rates determined from measurements of in situ cultures of the same series of corn plants (i.e., 16 inbreds, six hybrids, and one heavily inoculated hybrid) were 0.4, 2.3, and 1.1 g of N2/ha per day, respectively. Lower rates of C2H2 reduction were associated with control corn cultures which had been treated with autoclaved Spirillum than with cultures inoculated with live Spirillum. No C2H2 reduction was detected in plant cultures treated with ammonium nitrate. Numbers of nitrogen-fixing bacteria on excised roots of corn plants increased an average of about 30-fold during an overnight preincubation period, and as a result acetylene reduction assays of root samples after preincubation failed to serve as a valid basis for estimating N2 fixation by corn in pot cultures. Plants grown without added nitrogen either with or without inoculum exhibited severe symptoms of nitrogen deficiency and in most cases produced significantly less dry weight than those supplied with fixed nitrogen. Although substantial rates of C2H2 reduction by excised corn roots were observed after preincubation under limited oxygen, the yield and nitrogen content of inoculated plants and the C2H2-reduction rates by inoculated pot cultures of corn, in situ, provided no evidence of appreciable N2 fixation.     

Dinitrogen fixation by field grown soybeans: statistical analysis of variations in δ15N and proposed sampling procedureand proposed sampling procedure

Summary This paper reports a field study for the assessment of the variation in¹⁵N natural abundance method to estimate nitrogen fixation. For two successive years (1979 and 1980) at the same site, distribution of the variable δ¹⁵N in populations of nodulating and non-nodulating soybeans (Glycine max) has been studied. In 1980, the population structure was studied in order to detect a neighbour effect which could explain bimodal trend observed in the distribution. There was no evidence for such an effect. A sampling procedure is proposed: with 15 to 30 nodulating plants chosen randomly, a confidence interval of ±0.5δ can be obtained, while less than 10 non-nodulating plants are sufficient to achieve a very good precision. Some other aspects are studied: effect of pooling plants which considerably reduce the experimental work; the use of a non leguminous reference plant is possible. All our results show that fixing capacity introduces major variability in the δ¹⁵N measured in the plants, while the absence of fixation leads to a homogenous population, so the percentage of fixed N can be evaluated with a rather good precision. The higher the reference value, the better the accuracy of this evaluation, for a given precision of a δ¹⁵N measurement.     

15N同位素稀释法评估甘蔗的生物固氮量

为评估甘蔗生物固氮量,采用15N同位素稀释法,以木薯为参比植物,进行温室桶栽试验.结果表明:甘蔗全生育期植株固氮11.3514％ Ndfa,固氮量每桶0.9269 g.甘蔗根、茎、叶的固氮百分率和固氮量大小依序为叶＞茎＞根.叶的固氮百分率(13.2668％ Ndfa)略高于植株,但两者差异不显著.甘蔗植株全氮量中来自空气氮(生物固氮)、肥料氮和土壤氮的比例分别为11.3514％、7.6857％、80.9629％.甘蔗的氮肥利用率为58.7583％.甘蔗根、茎、叶各部位均有固氮现象,生产上可以用叶代替植株来评估甘蔗的生物固氮量.     

Nitrogen fixation byAlnus viridis (Chaix) DC.

Summary InAlnus viridis nodule growth relative to plant growth was inversely related to the quantity of nitrate added to nutrient solutions. Nodulated plants showed maximum growth when grown independently of supplied nitrogen and made better growth in its absence than unnodulated plants at any level of added nitrogen. Low levels of nitrate caused a depression of growth of nodulated plants, apparently by suppressing both nitrogen fixation and nodule growth. Nodules in nitrogen-free sand culture fixed atmospheric nitrogen at a rate of 6.6 mg/day/g nodule. Phosphorus deficiency was induced by low levels of phosphate and resulted in small plants with dark-green foliage. Root and nodule growth as a percentage of total plant growth and the percentage of total accumulated plant nitrogen below ground were greater at a root temperature of 11°C than 21°C. Thus at low root temperature processes other than nitrogen fixation were limiting to plant growth. Excised nodules were exposed to an N ₂ ¹⁵ -enriched atmosphere. A positive correlation between rate of nitrogen fixation and temperature was obtained, with optimum fixation occurring at about 20°C. It was shown that in spite of decreasing mean temperatures with increase in altitude, rate of nitrogen fixation by nodules of plants growing in the field increased with increase in altitude. This latter trend was deduced to be a reflection of the extent to which the field sites were nitrogen deficient in relation to climatically possible growth.     

Weather and nodule mediated variations in delta 13C and delta 15N values in field-grown soybean (Glycine max L.) with special interest in the analyses of xylem fluids

The nodulating soybean (Enrei) and its non-nodulating mutant (EN 1282) were grown in outdoor plots for 2 years (1994: extraordinary dry, high temperature, 1995: ordinary year). Carbon and nitrogen accumulation, delta 13C and delta 15N values in plant parts and xylem fluids and delta 15N values in the water-extractable soil N were analysed throughout the growing period. Plant growth in 1994 was rapid during the early growth stages, but no pods were produced. In 1995, plant growth was normal and pods were formed. The delta 13C values of the leaves were less negative in 1994 than in 1995 and the nodulated plants showed less negative delta 13C values than non-nodulated plants in both years. The delta 13C values of the leaves during the vegetative phase were positively correlated to the leaf N concentrations. Leaf N concentrations in their turn were influenced by nodulation and weather conditions and/or soil available N. The delta 15N values in the plants and xylem fluids were lower in the nodulated soybean than in non-nodulated soybean in both years, and estimates of the contribution of N2 fixation in nodulated plants based on plant top delta 15N values were 7-14% in 1994 and 37-63% in 1995. The delta 13C values of xylem fluids did not differ between nodulated and non-nodulated plants. Thus, the expected contribution by phosphopenolpyruvate carboxylase-mediated CO2 fixation in the root nodules to plant C-incorporation could not have been significant.     

Measurements of genetic variability for symbiotic dinitrogen fixation in fieldgrwon fababean (Vicia faba L.) using a low level15N-tracer technique

Before starting a breeding program aimed at improving the nitrogen nutrition ofVicia faba, the authors tried an alternative technique to the acetylene reduction assay, to measure some genetic variability in the plant material. The quantity of dinitrogen fixed by several cultivars ofVicia faba was estimated using a low enrichment¹⁵N tracer method and high precision¹⁵N mass spectrometry. The fababeans were cultivated for two years in two different soils. The percentage of fixed dinitrogen in the seed varied between genotypes from 40 to 83% of the total nitrogen and was positively correlated with the total seed nitrogen (r=0.64 to 0.86). A highly significant positive correlation was also found between the total seed nitrogen and the quantity of fixed dinitrogen in the seed (r=0.95 to 0.99). The technique used to measure dinitrogen fixation proved to be useful and reliable enough to discriminate between various genotypes, grown over a period of two years in two different soils. However, several non-fixing control plants showed significant differences in their¹⁵N enrichment and the problem of choosing a good reference plant was raised and discussed.     

The Effect of NaCl on growth, N2 fixation (acetylene reduction), and percentage total nitrogen in Leucaena leucocephala (Leguminosae) var. K-8

Leucaena leucocephala var. K-8 is a fast-growing, tropical leguminous tree that has multiple economic uses. This study was conducted to evaluate the effect(s) of varying NaCl concentrations on growth, N(2) fixation, and percentage of total tissue nitrogen in different organs in L. leucocephala. Seeds were germinated and grown for 10 wk with a nitrogen-free fertilizer applied every 2 wk. At 10 wk, plants were treated for either 0, 7, 14, 21, or 28 wk with either deionized water (control), 0.00625 mol/L, 0.0125 mol/L, 0.025 mol/L, 0.05 mol/L, or 0.1 mol/L NaCl in addition to the fertilizer every 2 wk. Growth was measured as plant height, nodule number and mass, and dry tissue mass. N(2) fixation was measured by the acetylene reduction assay. Percentage of tissue nitrogen was determined using Kjeldahl analysis. In younger plants (7-wk treatment), major fluctuations in NaCl tolerance were observed in the different plant organs. As plants matured (14- and 21-wk treatment) NaCl concentrations of 0.025 mol/L and higher caused the greatest reduction in growth and tissue nitrogen. We conclude that NaCl concentrations of 0.025 mol/L and greater caused a major decrease in growth, N(2) fixation, and percentage of tissue nitrogen in L. leucocephala plants that were less than 1 yr old.     

Hydroponic growth and the nondestructive assay for dinitrogen fixation

Hydroponic growth medium must be well buffered if it is to support sustained plant growth. Although 1.0 millimolar phosphate is commonly used as a buffer for hydroponic growth media, at that concentration it is generally toxic to a soybean plant that derives its nitrogen solely from dinitrogen fixation. On the other hand, we show that 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid, pK_a 6.1, has excellent buffering capacity, and it neither interferes with nor contributes nutritionally to soybean plant growth. Furthermore, it neither impedes nodulation nor the assay of dinitrogen fixation. Hence, soybean plants grown hydroponically on a medium supplemented with 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid and 0.1 millimolar phosphate achieve an excellent rate of growth and, in the absence of added fixed nitrogen, attain a very high rate of dinitrogen fixation. Combining the concept of hydroponic growth and the sensitive acetylene reduction technique, we have devised a simple, rapid, reproducible assay procedure whereby the rate of dinitrogen fixation by individual plants can be measured throughout the lifetime of those plants. The rate of dinitrogen fixation as measured by the nondestructive acetylene reduction procedure is shown to be approximately equal to the rate of total plant nitrogen accumulation as measured by Kjeldahl analysis. Because of the simplicity of the procedure, one investigator can readily assay 50 plants individually per day.     

Soil moisture and potassium affect the performance of symbiotic nitrogen fixation in faba bean and common bean

Potassium (K) is reported to improve plant's resistance against environmental stress. A frequently experienced stress for plants in the tropics is water shortage. It is not known if sufficient K supply would help plants to partially overcome the effects of water stress, especially that of symbiotic nitrogen fixation which is often rather low in the tropics when compared to that of temperate regions. Thus, the impact of three levels of fertilizer potassium (0.1, 0.8 and 3.0 mM K) on symbiotic nitrogen fixation was evaluated with two legumes under high (field capacity to 25% depletion) and low (less than 50% of field capacity) water regimes. Plants were grown in single pots in silica sand under controlled conditions with 1.5 mM N (¹⁵N enriched NH₄NO₃). The species were faba bean (Vicia faba L.), a temperate, amide producing legume and common bean (Phaseolus vulgaris L.), a tropical, ureide producing species. In both species, 0.1 mM K was insufficient for nodulation at both moisture regimes, although plant growth was observed. The supply of 0.8 or 3.0 mM K allowed nodulation and subsequent nitrogen fixation which appeared to be adequate for respective plant growth. High potassium supply had a positive effect on nitrogen fixation, on shoot and root growth and on water potential in both water regimes. Where nodulation occurred, variations caused by either K or water supply had no consequences on the percentage of nitrogen derived from the symbiosis. The present data indicate that K can apparently alleviate water shortage to a certain extent. Moreover it is shown that the symbiotic system in both faba bean and common bean is less tolerant to limiting K supply than plants themselves. However, as long as nodulation occurs, N assimilation from the symbiotic source is not selectively affected by K as opposed to N assimilation from fertilizer.     

Nitrogen fixation in annual and perennial legume-grass mixtures across a fertility gradient

Background and aims

The selection of legume species and species mixtures influences agroecosystem nitrogen (N) and carbon cycling. We utilized a fertility gradient to investigate the effects of plant species interactions on biological N fixation of an annual and perennial legume in response to shifting soil resource availability.

Methods

Legume N fixation of annual field pea (Pisum sativum) and perennial red clover (Trifolium pratense) grown in monoculture and mixtures with oats (Avena sativa) or orchardgrass (Dactylis glomerata) was estimated using the ¹⁵N natural abundance method across 15 farm fields and we measured six soil N pools ranging from labile to more recalcitrant.

Results

Evidence of complementary and facilitative species interactions was stronger for the perennial red clover-orchardgrass mixture than for the annual field pea-oat mixture (N Land Equivalency Ratios were 1.6 and 1.2, respectively). We estimated that the transfer of fixed N from red clover to orchardgrass increased aboveground N fixation estimates by 15% from 33 to 38?kg?N ha^?1. Despite a more than 2-fold range in soil organic matter levels and more than 3-fold range in labile soil N pools across field sites, the N fertility gradient was not a strong predictor of N fixation. While grass N assimilation was positively correlated with soil N pools, we found only weak, inverse correlations between legume N fixation and soil N availability. In grass-legume mixtures, soil N availability indirectly influenced N fixation through plant competition.

Conclusions

These results suggest that increasing diversity of cropping systems, particularly through the incorporation of perennial mixtures into rotations, could improve overall agroecosystem N cycling efficiency.     

Difference in delta(15)N signatures between nodulated roots and shoots of soybean is indicative of the contribution of symbiotic N(2) fixation to plant N

Symbiotic N(2) fixation has a variable effect on the (15)N abundance of different parts of legumes. Increases in fixation result in (15)N enrichment of nodules, while decreases, in combination with an increased uptake of mineral N, result in (15)N depletion of the root system. The difference between soybean shoot and below-ground delta(15)N (Deltadelta(15)N=delta(15)N(shoot)-delta(15)N(belowground)) was assessed in hydroponic culture over a range of rates of supply of mineral N. The fractional contribution of N(2) fixation to N uptake (%Ndfa) was determined using the natural abundance (NA) technique with ryegrass as a reference plant. Deltadelta(15)N and %Ndfa were highly correlated, and the relationship was tested using the same soybean cultivar grown in pots in N-rich soil. Estimates of %Ndfa derived from the NA method and from the Deltadelta(15)N approach yielded near-identical values. A literature survey showed similar relationships between %Ndfa and Deltadelta(15)N with different growth stages of soybeans grown under glasshouse and field conditions, different cowpea (Vigna unguiculata) cultivars in the field, and tagasaste (Chamaecytisus proliferus) in hydroponic culture. Possible confounding and species-specific (either plant or Rhizobium spp.) influences are discussed. The difference in delta(15)N signatures between nodulated roots and shoots is confirmed as a robust means of quantifying %Ndfa: it is independent of reference plants and offers the possibility of estimating %Ndfa in soils where the isotope composition of mineral N closely matches that of atmospheric N(2).     

Why and how to estimate the cost of symbiotic N2 fixation? A progressive approach based on the use of14C and15N isotopes

The process of symbiotic nitrogen fixation, though of obvious advantage to legumes in situations in which nitrogen is limiting, results in substantial penalty to the host plant in terms of cost of maintenance, synthesis and nitrogen reduction. Accurate estimates of costs are difficult to obtain because of the lack of simple methods to measure N₂ fixation and associated energy consumption. In relation to these difficulties, a multiple-step approach involving isotopes (¹⁴CO₂–¹⁵N₂) methodologies is described.The estimation of net respiratory cost associated with the N₂ reduction activity in near-natural conditions was achieved using simultaneous¹⁴CO₂ and¹⁵N₂ labelling. It gives a minimum value of 2.5 mg C/mg N fixed. This value was corrected by the estimation of the amount of carbon saved through the process of CO₂ fixation by the PEP carboxylase of the nodules, using¹⁴CO₂ in the soil atmosphere. This gives a real respiratory cost of 4 mg C/mg N fixed.     

Nitrogen from senescing lower leaves of common bean is re-translocated to nodules and might be involved in a N-feedback regulation of nitrogen fixation

The objective of the present study was to elucidate whether remobilized N from lower leaves is involved in causing the drop in N(2) fixation during pod-filling in common bean (Phaseolus vulgaris L). Moreover, we addressed the question of whether remobilized N from lower leaves would reach the nodules. Nodulated common bean plants were grown in a growth chamber in quartz sand. During a 2-week period, at vegetative and at reproductive growth, 50% of the leaves (lower part) were either excised or individually darkened, thereby removing the same photosynthetic capacity yet allowing N to be remobilized from the darkened leaves. Moreover, at the vegetative growth period, three lower leaves per plants were (15)N labelled by applying (15)NH(4)NO(3) prior to imposing the darkening treatment. Leaf darkening at vegetative growth induced N remobilization as well as reduced N(2)-fixation rates and growth. Leaf excision at reproductive growth enhanced N(2) fixation. Changes in N(2)-fixation rates were in all cases the result of altered growth rates, while the % N in the whole plant and in various plant parts remained conserved. Directly after leaf labelling, but also at the end of the vegetative growth period, substantial amounts of (15)N from the leaves could be recovered in nodules in the control, and in higher amounts in the leaf-darkening treatment. It is proposed that nitrogen from leaves circulates within the plant via nodules, and that the strength or composition of this circular flow may be the signal for a putative N-feedback effect.