15N2 Labeling Research Articles

Catalytic reduction of dinitrogen to ammonia using molybdenum porphyrin complexes.

Porphyrin complexes are well-known in O2 and CO2 reduction, but their application to N2 reduction is less developed. Here, we show that oxo and nitrido complexes of molybdenum supported by tetramesitylporphyrin (TMP) are effective precatalysts for catalytic N2 reduction to ammonia, verified by 15N2 labeling studies and other control experiments. Spectroscopic and electrochemical studies illuminate some relevant thermodynamic parameters, including the N-H bond dissociation free energy of (TMP)MoNH (43 ± 2 kcal mol-1). We place these results in the context of other work on homogeneous N2 reduction catalysis.

Soil texture and pH exhibit important effects on biological nitrogen fixation in paddy soil

Biological nitrogen fixation (BNF) is an important source of soil nitrogen. However, for dry-wet alternative paddy soil, the combined effects of multiple driving factors, including soil physicochemical properties and the soil microbiome, on BNF are not fully understood. In this study, three types of paddy soil were collected to determine the BNF amount by field-based 15N2 labeling experiments. Soil nitrogen fixing microbiome was analyzed using high-throughput sequencing based on the nifH gene. Random forest analysis, co-occurrence network analysis and hierarchical partitioning were performed to screen the key diazotrophic community and critical soil properties. The results revealed that rice nitrogen acquisition from BNF ranged from 1.62 % to 7.80 %. Three key diazotrophic genera, Rubrivivax, Frankia and Azospirillum, significantly affected BNF. The BNF amount in the 0–1 cm soil layer was significantly higher than that in the 1–15 cm soil layer, which was consistent with the relative abundance of Cyanobacteria. The soil silt content and pH were positively correlated with the BNF amount by mediating the key diazotrophic microbiome. These results indicated that the BNF amount plays an important role in rice nitrogen acquisition and paddy soil nitrogen sources, which might be regulated by soil texture and soil pH.

(Invited) ATP-Independent Electroenzymatic Ammonia Production Using an Organic Redox Polymer-Immobilized Enzymatic System

Consuming approximately 1% of the total fossil fuel of the world, the ammonia (NH3), a usable form to living organisms is produced by dinitrogen (N2) fixation. Currently, approximately 66% of NH3 is produced by the Haber–Bosch process using an iron catalyst with N2 and H2 at high temperatures and high pressures (400 °C and 20 MPa), which are estimated to be responsible for approximately 3% of CO2 emissions.1,2 As an alternative strategy, bioelectrosynthetic technique is studied for eco-friendly ammonia production using the nitrogenase, the only enzyme able to reduce N2 to NH3 at room temperature, neutral pH and ambient pressure. For the practical use of nitrogenase bioelectrocatalysis technology, it is indispensable to minimize the large input of chemical energy for the reduction of MoFe protein component of nitrogenase by hydrolysis of two ATP for each electron transferred (16 ATP per N2).3-5 Therefore, we have concentrated on approaches to bypass the reducing- and ATP-hydrolyzing properties of the dinitrogenase reductase by immobilizing the catalytic protein of nitrogenase, MoFe protein, on the electrode surface. One way to directly reduce the MoFe protein is a use of a redox polymer having thermodynamically strong reducing capability. Redox polymer immobilized bioelectrocatalysis facilitates an efficient electron transfer through self-exchange based conduction due to a high effective concentration of catalysts at the electrode surface and more efficient mediation. However, none of the previously reported redox polymers were capable of immobilized bioelectrocatalysis using nitrogenase.6-9 Thus, we report the MoFe nitrogenase immobilized at an electrode surface with a neutral red redox polymer which is used to reduce the MoFe protein (as mediator able to transfer the electrons to the MoFe independent of the Fe protein and of ATP hydrolysis) and support the mediated bioelectrocatalysis of N3 -, NO2 - and N2 to NH3 catalyzed by the MoFe protein. Representative Bulk bioelectrosynthetic experiments produced 209 ± 30 nmol NH3 nmol MoFe-1 h-1 from N2 reduction. 15N2 labeling experiments and NMR analysis were performed to confirm biosynthetic N2 reduction to NH3 . Reference K. Burgess and D. J. Lowe, Chem. Rev., 1996, 96, 2983–3012.Christiansen, D. R. Dean and L. C. Seefeldt, Annu. Rev. Plant Physiol. Plant Mol. Biol., 2001, 52, 269–295.E. Smith, Science, 2002, 297, 1654–1655.-Y. Yang, K. Danyal and L. C. Seefeldt, in Nitrogen Fixation, Methods in Molecular Biology 766, ed. M. W. Ribbe, Springer, New York, 2011, ch. 2. Milton, R. D.; Abdellaoui, S.; Khadka, N.; Dean, D. R.; Leech, D.; Seefeldt, L. C.; Minteer, S. D.Energy Environ. Sci., 2016, 9, 2550– 2554Hickey, D. P.,Cai, R., Yang, Z. Y., Grunau, K., Einsle, O., Seefeldt, L. C., Minteer, S. D., Am. Chem. Soc., 2019, 141, 17150– 17157Ackermann, D. A. Guschin, K. Eckhard, S. Shleev, W. Schuhmann, Electrochem Comm., 2010, 12, 640-643.Barrière, Y. Ferry, D. Rochefort, D. Leech, Electrochem Comm., 2004, 6, 237-241Yuan, S. D. Minteer, Curr. Opin. Electrochem., 2019, 15, 1-6

Predicting the influence of fertilization regimes on potential N fixation through their effect on free-living diazotrophic community structure in double rice cropping systems

Free-living nitrogen fixation (FLNF) by diazotrophs is a ubiquitous renewable resource and may constitute an attractive viable solution to the problem of environmental degradation caused by nitrogen over-fertilization. However, the biotic and abiotic control mechanisms of free-living nitrogen fixation are poorly understood. Moreover, there is rare information to link the composition and functions of diazotrophic communities; additionally, there are insufficient or inadequate predictors of the potential N fixation rate (PNFR) to help improve fertilization strategies. Here, through high-throughput sequencing, network analysis, and 15N2 labelling methods, we investigated the long-term effects of partial substitution of mineral N by organic fertilizers on diazotroph abundance and composition in double rice cropping systems. The field trial was conducted for 34 years and included five treatments: control without fertilizer (CK); only mineral NPK fertilizer (NPK); 70% inorganic N + 30% organic N + PK (NPKM1); 50% inorganic N + 50% organic N + PK (NPKM2); 30% inorganic N + 70% organic N + PK (NPKM3). Our results revealed that at the same nutrient input level, increasing the percentage of organic N substitution reduced diazotrophic abundance. Compared with the CK and NPK treatments, the organic substitution regimes reduced the fluctuation range of the PNFR between the early and late rice growing seasons. NH4+-N was the primary factor that negatively correlated (p < 0.01) with diazotrophic abundance, while the potential N fixation rate was positively correlated (p < 0.01) with soil C/N. Among abiotic factors, the models incorporating soil C/N and Fe2+/Mo could accurately predict the potential N fixation rate. High-throughput sequencing and network analyses revealed that Bradyrhizobium and Geobacter were the dominant genera in diazotrophic co-occurrence and keystone taxa patterns under all fertilization regimes. Soil pH and NH4+-N were the most important factors in shaping the diazotrophic co-occurrence and keystone patterns in paddy soils, respectively. Diazotrophic keystone composition was more suitable for predicting and characterising the potential N fixation rate than the co-occurrence pattern of diazotrophs. Moreover, compared with the CK and NPK regimes, partial substitution of mineral N with organic N fertilizer improved the prediction accuracy of potential N fixation rate more effectively. Overall, we highlight the benefits of organic substitution fertilizer regime in stabilizing and improving the potential N fixation rate. Our findings provide relevant insights into the potential of key microbial taxa for predictive N fixation for optimising chemical N fertilizer application strategies in paddy soils.

Chemoheterotrophic diazotrophs contribute to nitrogen incorporation in a semi-arid desert

To explore the non-cyanobacteria diazotrophic populations and their microbial interactions, and further evaluate the N-fixing activities of chemoheterotrophic groups in deserts, we collected soil samples from the Mu Us Desert of northern China, and performed amplicon sequencing, targeting the functional gene nifH combined with the dark incubation with 15N2 labelling under laboratory conditions. A total of 4455 operational taxonomic units were clustered from 786,778 high-quality nifH reads, 96.57% of which were classified as Proteobacteria. Network analyses showed that three putative keystone taxa (i.e. the genera Azospirillum, Bradyrhizobium, and Mesorhizobium) were mostly responsible for connecting other diazotrophic populations, and that 98.03% of the associations between any two nodes were positive. The 15N2 labelling experiment showed that the average N-fixing rates added 0.03 mg N kg−1 day−1 as soil total N and 0.006 mg N kg−1 day−1 as total dissolved N over 28 days. Our results highlight the interactions of non-cyanobacteria diazotrophs and the potential of the chemoheterotrophic types in transforming atmospheric N2 into bio-available ammonium in desert soils.

Pea Efficiency of Post-drought Recovery Relies on the Strategy to Fine-Tune Nitrogen Nutrition.

As drought is increasingly frequent in the context of climate change it is a major constraint for crop growth and yield. The ability of plants to maintain their yield in response to drought depends not only on their ability to tolerate drought, but also on their capacity to subsequently recover. Post-stress recovery can indeed be decisive for drought resilience and yield stability. Pea (Pisum sativum), as a legume, has the capacity to fix atmospheric nitrogen through its symbiotic interaction with soil bacteria within root nodules. Biological nitrogen fixation is highly sensitive to drought which can impact plant nitrogen nutrition and growth. Our study aimed at dynamically evaluating whether the control of plant N status after drought could affect nodulated pea plant’s ability to recover. Two pea genotypes, Puget and Kayanne, displaying different drought resilience abilities were compared for their capacity to tolerate to, and to recover from, a 2-weeks water-deficit period applied before flowering. Physiological processes were studied in this time-series experiment using a conceptual structure–function analysis framework focusing on whole plant carbon, nitrogen, and water fluxes combined to two 13CO2 and 15N2 labeling experiments. While Puget showed a yield decrease compared to well-watered plants, Kayanne was able to maintain its yield. During the recovery period, genotype-dependent strategies were observed. The analysis of the synchronization of carbon, nitrogen, and water related traits dynamics during the recovery period and at the whole plant level, revealed that plant growth recovery was tightly linked to N nutrition. In Puget, the initiation of new nodules after water deficit was delayed compared to control plants, and additional nodules developed, while in Kayanne the formation of nodules was both rapidly and strictly re-adjusted to plant growth needs, allowing a full recovery. Our study suggested that a rapid re-launch of N acquisition, associated with a fine-tuning of nodule formation during the post-stress period is essential for efficient drought resilience in pea leading to yield stability.

Paddy System with a Hybrid Rice Enhances Cyanobacteria Nostoc and Increases N2 Fixation

Biological nitrogen (N) fixation (BNF) plays a significant role in maintaining soil fertility in paddy field ecosystems. Rice variety influences BNF, but how different rice varieties regulate BNF and associated diazotroph communities has not been quantified. Airtight, field-based 15N2-labelling growth chamber experiments were used to assess the BNF capacity of different rice varieties. In addition, both the 16S rRNA and nifH genes were sequenced to assess the influence of different rice varieties on bacterial and diazotrophic communities in paddy soils. After subjecting a rice-soil system to 74 d of continuous airtight, field-based 15N2 labelling in pots in a growth chamber, the amounts of fixed N were 22.3 and 38.9 kg ha–1 in inbred japonica (W23) and hybrid indica (IIY) rice cultivars planted in the rice-soil systems, respectively, and only 1%–2.5% of the fixed N was allocated to the rice plants and weeds. A greater abundance of diazotrophs was found in the surface soil (0–1 cm) under IIY than under W23. Sequencing of the 16S rRNA gene showed significantly greater abundances of the cyanobacterial genera Nostoc, Anabaena, and Cylindrospermum under IIY than under W23. Sequencing of the nifH gene also showed a significantly greater abundance of Nostoc under IIY than under W23. These results indicate that the hybrid rice cultivar (IIY) promoted BNF to a greater extent than the inbred rice cultivar (W23) and that the increase in BNF might have been due to the enhanced heterocystous cyanobacteria Nostoc.

Soil aluminum oxides determine biological nitrogen fixation and diazotrophic communities across major types of paddy soils in China

Biological nitrogen fixation (BNF) contributed greatly in maintaining the fertility of paddy soils without the application of chemical fertilizers for thousands of years. However, factors affecting the BNF and composition of diazotrophs in paddy soils across large geographic scales are still largely unknown. In this study, the field-based 15N2 labeling technique and nifH gene high-throughput sequencing were applied to four types of paddy soils collected from northeast to southwest China, representing major rice-growing areas. Results showed that the BNF amount had distinct geographical patterns and ranged from 2.2 ± 0.5 to 20.1 ± 3.7 kg N ha−1 during 42 days 15N2 labeling period, which was positively correlated with the abundance of Nostocales species. Among the soil properties analyzed, the contents of amorphous aluminum oxides may play a key role in determining the BNF amount and major N2-fixers across all paddy soils using a random forest model. Overall, our study demonstrates the potential biotoxic effects of aluminum oxides on the BNF in paddy soils in southern China and may has important implications for the enhancement of BNF in acidic Ultisols and Oxisols.

Symbiosis revisited: phosphorus and acid buffering stimulate N&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt; fixation but not &amp;lt;i&amp;gt;Sphagnum&amp;lt;/i&amp;gt; growth

Abstract. In pristine Sphagnum-dominated peatlands, (di)nitrogen (N2) fixing (diazotrophic) microbial communities associated with Sphagnum mosses contribute substantially to the total nitrogen input, increasing carbon sequestration. The rates of symbiotic nitrogen fixation reported for Sphagnum peatlands, are, however, highly variable, and experimental work on regulating factors that can mechanistically explain this variation is largely lacking. For two common fen species (Sphagnum palustre and S. squarrosum) from a high nitrogen deposition area (25 kg N ha−1 yr−1), we found that diazotrophic activity (as measured by 15 − 15N2 labeling) was still present at a rate of 40 nmol N gDW−1 h−1. This was surprising, given that nitrogen fixation is a costly process. We tested the effects of phosphorus availability and buffering capacity by bicarbonate-rich water, mimicking a field situation in fens with stronger groundwater or surface water influence, as potential regulators of nitrogen fixation rates and Sphagnum performance. We expected that the addition of phosphorus, being a limiting nutrient, would stimulate both diazotrophic activity and Sphagnum growth. We indeed found that nitrogen fixation rates were doubled. Plant performance, in contrast, did not increase. Raised bicarbonate levels also enhanced nitrogen fixation, but had a strong negative impact on Sphagnum performance. These results explain the higher nitrogen fixation rates reported for minerotrophic and more nutrient-rich peatlands. In addition, nitrogen fixation was found to strongly depend on light, with rates 10 times higher in light conditions suggesting high reliance on phototrophic organisms for carbon. The contrasting effects of phosphorus and bicarbonate on Sphagnum spp. and their diazotrophic communities reveal strong differences in the optimal niche for both partners with respect to conditions and resources. This suggests a trade-off for the symbiosis of nitrogen fixing microorganisms with their Sphagnum hosts, in which a sheltered environment apparently outweighs the less favorable environmental conditions. We conclude that microbial activity is still nitrogen limited under eutrophic conditions because dissolved nitrogen is being monopolized by Sphagnum. Moreover, the fact that diazotrophic activity can significantly be upregulated by increased phosphorus addition and acid buffering, while Sphagnum spp. do not benefit, reveals remarkable differences in optimal conditions for both symbiotic partners and calls into question the regulation of nitrogen fixation by Sphagnum under these eutrophic conditions. The high nitrogen fixation rates result in high additional nitrogen loading of 6 kg ha−1 yr−1 on top of the high nitrogen deposition in these ecosystems.

Heterotrophic and phototrophic 15N2 fixation and distribution of fixed 15N in a flooded rice–soil system

A range of different N2 fixing systems can contribute to the nitrogen (N) economy of flooded rice fields, but the direct evidence of N2 fixation and the proportional contribution of heterotrophic and phototrophic N2 fixation are difficult to assess. Here we report on the development and application of a field-based 15N2 labelling technique in which a flooded rice–soil system was exposed to a 15N2-enriched atmosphere to assess the biological nitrogen fixation (BNF) in paddy fields. One month old rice seedlings (Oryza sativa L.) were transplanted to non N-fertilized pots of flooded soil and exposed to a 15N2-enriched atmosphere (approx. 7 atom% 15N) in a gas-tight growth chamber for 70 days. The highest 15N-enrichment 3.7551 atom% was observed in blue green algae in unplanted pots. After 70 days incubation, 49% of the total 15N fixed in flooded rice–soil system was found in the plants (rice and weeds), while 51% was found in the flooded soils. Rice planting enhanced both phototrophic and heterotrophic BNF and increased the proportion of heterotrophic BNF to phototrophic BNF from 0.50 in treatment without rice (fallow) to 0.99 in treatment with rice. The findings suggested that rice benefits greatly from BNF processes and a major improvement for quantification of BNF in flooded rice fields based on field 15N2 labelling technique.

Use of a 15N2 labelling technique to estimate exudation by white clover and transfer to companion ryegrass of symbiotically fixed N

N rhizodeposition by legumes leads to enrichment of N in soils and in companion plants. N rhizodeposition can be divided into two major components, root exudation and root senescence. Our aim was to quantify N root exudation in white clover (Trifolium repens L.) through an estimation of short-term N rhizodeposition and to assess its impact on N transfer to companion perennial ryegrass (Lolium perenne L.) grown in mixture with clover. 15N2 provided in the root atmosphere for 3 days was used to estimate transfer of symbiotically fixed nitrogen (SFN) to the growing medium by clover grown in pure stand and to ryegrass by clover grown in mixture for 2 months. The proportion of N rhizodeposited over the 3 days increased from 3.5 % of SFN in pure stand to 5.3 % in mixture. The 15N-enrichment of ammonium from the adhering substrate shows that a part of the rhizodeposited N was released in the form of ammonium. 4 % of the rhizodeposited N was taken up by ryegrass during the labelling period. This study showed a significant contribution of root N exudation to the total N rhizodeposition of legumes and in the transfer of N to grasses.

Nitrogen fixation in the western equatorial Pacific: Rates, diazotrophic cyanobacterial size class distribution, and biogeochemical significance

A combination of 15N2 labeling, Tyramide Signal Amplification–Fluorescent in Situ Hybridization (TSA‐FISH) assay, and chemical analyses were performed along a trophic gradient (8000 km) in the equatorial Pacific. Nitrogen fixation rates were low (0.06 ± 0.02 to 2.8 ± 2.1 nmol L−1 d−1) in HNLC waters, higher in the warm pool (0.11 ± 0.0 to 18.2 ± 2.8 nmol L−1 d−1), and extremely high close to Papua New Guinea (38 ± 9 to 610 ± 46 nmol L−1 d−1). Rates attributed to the &lt;10‐μm fraction accounted for 74% of total activity. Both unicellular and filamentous diazotrophs were detected and reached 17 cells mL−1 and 1.85 trichome mL−1. Unicellular diazotrophs were found to be free‐living in &lt;10‐μm fraction or in association with mucilage, particles, or eukaryotes in the &gt;10‐μm fraction, leading to a possible overestimation of this fraction to total N2 fixation. In oceanic waters, 98% of the unicellular diazotrophs were picoplanktonic. Finally, we found a clear longitudinal pattern of niche partitioning between diazotroph groups: while unicellular diazotrophs were present all along the transect, Trichodesmium spp. were detected only in coastal waters, where nitrogen fixation associated to both size fractions was greatly stimulated.

Nonsymbiotic nitrogen fixation in 3-year-old Jeffrey pines and the role of elevated [CO2

Increased belowground labile C inputs under elevated [CO2] could stimulate nonsymbiotic N2 fixation, thereby enhancing growth responses of vegetation to elevated [CO2] on nutrient-poor sites. To test this hypothesis, nonsymbiotic N2 fixation rates in soils planted with 3-year-old Jeffrey pine (Pinus jeffreyi Grev. &amp; Balf.) trees grown under 365 and 700 µL·L–1 atmospheric [CO2] were measured by exposing the soil to 15N2-enriched air for 78 d. Nitrogen fixation rates were estimated by measuring 15N content of trees and soil. Compared with the ambient CO2 treatment, the elevated CO2 treatment did not affect biomass, N content, or δ15N of individual plant parts and soils, indicating that elevated [CO2] did not stimulate nonsymbiotic N2 fixation. Because belowground C inputs did not increase under elevated [CO2], the initial hypothesis could not be accepted or rejected. The results from the 15N2 labeling study agree with other studies showing that nonsymbiotic N2 fixation is not likely to provide a large input of N in forest ecosystems. The 15N2 labeling technique was promising for studying N2 fixation in plant–soil systems, but the preliminary nature of this study did not allow for firm conclusions with regard to the effects of elevated [CO2].

Carbon and nitrogen metabolism in Rhizobium

One of the paradigms of symbiotic nitrogen fixation has been that bacteroids reduce N2 to ammonium and secrete it without assimilation into amino acids. This has recently been challenged by work with soybeans showing that only alanine is excreted in 15N2 labelling experiments. Work with peas shows that the bacteroid nitrogen secretion products during in vitro experiments depend on the experimental conditions. There is a mixed secretion of both ammonium and alanine depending critically on the concentration of bacteroids and ammonium concentration. The pathway of alanine synthesis has been shown to be via alanine dehydrogenase, and mutation of this enzyme indicates that in planta there is likely to be mixed secretion of ammonium and alanine. Alanine synthesis directly links carbon catabolism and nitrogen assimilation in the bacteroid. There is now overwhelming evidence that the principal carbon sources of bacteroids are the C4-dicarboxylic acids. This is based on labelling and bacteroid respiration data, and mutation of both the dicarboxylic acid transport system (dct) and malic enzyme. L-malate is at a key bifurcation point in bacteroid metabolism, being oxidized to oxaloacetate and oxidatively decarboxylated to pyruvate. Pyruvate can be aminated to alanine or converted to acetyl-CoA where it either enters the TCA cycle by condensation with oxaloacetate or forms polyhydroxybutyrate (PHB). Thus regulation of carbon and nitrogen metabolism are strongly connected. Efficient catabolism of C4-dicarboxylates requires the balanced input and removal of intermediates from the TCA cycle. The TCA cycle in bacteroids may be limited by the redox state of NADH/NAD+ at the 2-ketoglutarate dehydrogenase complex, and a number of pathways may be involved in bypassing this block. These pathways include PHB synthesis, glutamate synthesis, glycogen synthesis, GABA shunt and glutamine cycling. Their operation may be critical in maintaining the optimum redox poise and carbon balance of the TCA cycle. They can also be considered to be overflow pathways since they act to remove or add electrons and carbon into the TCA cycle. Optimum operation of the TCA cycle has a major impact on nitrogen fixation.

Rapid assimilation of recently fixed N2 in root nodules of Myrica gale

In Myrica gale L. plants the assimilation of ammonia released by symbiotic Frankia was observed by 15N2 labelling and subsequent analysis of the isotopic enrichment of nodule amino acids over time by single ion monitoring gas chromatography‐mass spectrometry. In detached nodules of Myrica, glutamine was the first amino acid labelled at 30 s and subsequently the amino acids glutamate, aspartate, alanine and γ‐amino butyric acid (GABA) became labelled. This pattern of labelling is consistent with the incorporation of ammonium via glutamine synthetase [GS; EC 6.3.1.2]. No evidence for the ammonium assimilation via glutamate dehydrogenase [GDH; EC 1.4.1.2] was observed as glutamate became labelled only after glutamine. Using attached nodules and pulse‐chase labelling, we observed synthesis of glutamine, glutamate, aspartate, alanine, GABA and asparagine, and followed the transport of fixed nitrogen in the xylem largely as glutamine and asparagine. Estimation of the cost of nitrogen fixation and asparagine synthesis in Myrica nodules suggests a minimum of one sucrose required per asparagine produced. Rapid translocation of recently fixed nitrogen was observed in Myrica gale nodules as 80% of the nitrogen fixed during a 1‐h period was translocated out of the nodules within 9 h. The large pool of asparagine that is present in nodules may buffer the transport of nitrogen and thus act to regulate nitrogen fixation via a feedback mechanism.

Identification of amino compounds synthesized and translocated in symbiotic Parasponia

ABSTRACTWe investigated the synthesis and translocation of amino compounds in Parasponia, a genus of the Ulmaceae that represents the only non‐legumes known to form a root nodule symbiosis with rhizohia. In the xylem sap of P. andersonii we identified asparagine. aspartate. glutamine, glutamated significant quantities of a non‐protein amino acid. 4‐methylglutamte(2‐amino‐4‐methylpentanedioic acid). This identification was confirmed by two methods, capillary gas chromatography (GC) electron ionization (El) mass spectrometry (MS) and reverse phase high pressure liquid chromatography (HPLC) analysis of derivatized compounds. In leaf, root and nodule samples from P. andersonii and P. parviflora we also identified the related compounds 4‐methyleneglutamate and 4‐methyleneglulamine. Using 15N2 labelling and GC‐Ms analysis of root nodule extracts we followed N2 fixation and ammonia assimilation in P. andersonii root nodules and observed Label initially in glutamine and subsequently in glutamate, suggesting operation of the glutamine synthetase/glutamine:2‐oxoglutarate aminotransferase (GS/GOGAT) pathway. Importantly, we observed the incorporation of significant quantities of 15N into 4‐methylglutamate in nodules, demonstrating the de nova synthesis of this non protein amino acid and suggesting a role in the translation of N in symbioticParasponia.