Cycling of amino compounds in symbiotic lupin

The composition of amino acids was determined in the xylem andphloem sap of symbiotic lupins grown under a variety of treatmentsdesigned to alter the rate of nitrogen fixation. Asparaginewas the major amino acid in both xylem and phloem with glutamine,glutamate and aspartate also major components. GABA had a highconcentration in the xylem while valine was a major componentin the phloem. Exposure to combined nitrogen in the form ofeither ammonium or nitrate caused a reduction in specific nitrogenaseactivity and was associated with subsequent changes in bothof the translocated saps. Inhibiting nitrogen fixation by exposingnodules to oxygen produced a lower amide to amine ratio in thexylem sap (1.3:1) compared with control and nitrate ratios (2.6:1)and ammonium ratios (7.1:1). Similar ratios for amide aminewere also observed in the phloem sap. Labelling studies using¹⁵N₂ to follow nitrogen fixation, ammonium assimilation andamino acid transport have shown rapid accumulation of labelinto glutamine with subsequent enrichment in glutamate, aspartate,alanine, and GABA. Asparagine was found in high concentrationsin nodules and became slowly enriched. Labelled nitrogen fixedand assimilated in nodules was detected 40 min later in stemxylem extracts, largely as the amides glutamine and asparagine.These experiments provide evidence that large amounts of nitrogenouscompounds are cycled through the root nodules of symbiotic plants(contributing approximately 50% of xylem N) and that differencesin the composition of the phloem sap may influence nodule growthand activity. Key words: Nitrogen fixation, nitrogen translocation, isotope labelling, legumes, GC-MS     

Vascular transport and soybean nodule function: II. A role for phloem supply in product export*

Abstract The ureide content of soybean (Glycine max (L.) Merr.) nodules was unaffected by variations in the transpirational rate, while whole plant manipulations designed to decrease phloem supply to nodules resulted in lower rates of nitrogenase activity and an increase in the ureide content of the nodules. The rate of ureide export from the nodule was estimated from the exponential rate of decrease in the pool size of ureides in nodules, following exposure to an N₂-free atmosphere (Ar:O₂). Export was greatly reduced under treatments which reduced phloem supply to the nodule. A water budget for nodules suggested that the delivery of water to the nodule via mass flow in the phloem was comparable to that required for export of ureides from the nodule in the xylem from the nodule. Therefore, we suggest that xylem export from nodules is related to the phloem supply to the nodule rather than to the transpirational flux in the parent root. This suggestion is related to the reported decreases in nodule permeability to gases under conditions of phloem deprivation.     

大豆-根瘤菌共生体系光合与固氮关系研究

水培大豆和田间生长的大豆,接种根瘤菌 Rhizobium B16-11C 后植株全氮含量、叶片叶绿素含量和净光合速率及种子产量都明显增加。比较 Clark 大豆的结瘤品系和不结瘤品系获类似结果。摘除根瘤后3天内叶片净光合速率无明显变化。大豆植株遮阴、去叶或切掉地上部导致根瘤活性明显下降。但去豆荚不能提高根瘤固氮的比活性。根瘤活性的日变化不能用根瘤蔗糖、淀粉含量或周围温度的变化来解释,其控制因子尚待深入研究。     

Studies on the Relationship Between Photosynthesis and Nitrogen Fixation in the Symbiotic System of Soybean and Nodule Bacteria( Rhizobium)

The relationship between photosynthesis of soybean and nitrogen fixation of the nodules by symbiotic Rhizobium was studied. The contents of total nitrogen and chlorophyll, the net photosynthetic rate and seed yield of soybean were much higher in either hydroponically cultivated or field-grown plants inoculated with Rhizobium B16–11C (or Clark nodulating strain) than in control without inoculation (or Clark non-nodulating strain). These results show that the symbiotic nitrogen fixation has a beneficial effect on photosynthesis. However, the effect was indirect and slow so that there was no change in the net photosynthetic rate of the soybean leaves until three clays after removing nodules from the soybean roots. On the other hand, decreasing the photosynthate supply to nodule by shade, defoliation or shoot removal of the soybean, the nodule activity declined significantly. It seems that the supply of photosynthate to root nodule is a limiting factor for symbiotic nitrogen fixation. However, the diurnal variation of the nodule activity could not be explained by change neither in the contents of sucrose and starch of the root nodules nor in the ambient temperature. The factor controlling the diurnal variation deserves further study.     

Effects of drought stress on legume symbiotic nitrogen fixation: physiological mechanisms

Drought stress is one of the major factors affecting nitrogen fixation by legume-rhizobium symbiosis. Several mechanisms have been previously reported to be involved in the physiological response of symbiotic nitrogen fixation to drought stress, i.e. carbon shortage and nodule carbon metabolism, oxygen limitation, and feedback regulation by the accumulation of N fixation products. The carbon shortage hypothesis was previously investigated by studying the combined effects of CO2 enrichment and water deficits on nodulation and N2 fixation in soybean. Under drought, in a genotype with drought tolerant N2 fixation, approximately four times the amount of 14C was allocated to nodules compared to a drought sensitive genotype. It was found that an important effect of CO2 enrichment of soybean under drought was an enhancement of photo assimilation, an increased partitioning of carbon to nodules, whose main effect was to sustain nodule growth, which helped sustain N2 rates under soil water deficits. The interaction of nodule permeability to O2 and drought stress with N2 fixation was examined in soybean nodules and led to the overall conclusion that O2 limitation seems to be involved only in the initial stages of water deficit stresses in decreasing nodule activity. The involvement of ureides in the drought response of N2 fixation was initially suspected by an increased ureide concentration in shoots and nodules under drought leading to a negative feedback response between ureides and nodule activity. Direct evidence for inhibition of nitrogenase activity by its products, ureides and amides, supported this hypothesis. The overall conclusion was that all three physiological mechanisms are important in understanding the regulation of N2 fixation and its response of to soil drying.     

Effects of calcium deficiency on symbiotic nitrogen fixation

The mechanism of the effect of mild calcium deficiency on nitrogen metabolism of the symbiotic plant was studied from the distribution of calcium and of nitrogen and carbohydrate fractions in plant organs.

Nitrogen concentrations of all plant organs decreased with calcium deficiency. Addition of either a nitrogen or a calcium salt increased nitrogen concentrations. For roots as well as whole plants the effects of one salt were largely replaced by the other. These effects establish that calcium deficiency decreased the supply of fixed nitrogen from nodules to other organs. As weight of nodules was independent of calcium it follows that nodular efficiency was impaired.

Since nitrogen concentrations of nodules decreased rather than increased with calcium deficiency it is suggested that calcium deficiency interferes with the rate of reduction of nitrogen in the nodule rather than with the export of reduced nitrogen. Distribution of α-amino, amide, and ammonium nitrogen are consistent with this suggestion.

Nitrogen fixation was not limited by translocation of carbohydrates to nodules as calcium deficiency had little effect on the concentration of soluble carbohydrates and actually increased the concentration of starch in nodules.

Calcium deficiency depressed the calcium content of nodules so that nitrogen fixation may have been impaired by inadequate calcium for nodular structure or metabolism.

     

环境因子对豆科共生固氮影响的研究进展

环境因子的限制一直是豆科植物一根瘤菌共生固氮体系没有在农业生产中充分发挥作用的重要原因之一。目前,研究涉及的环境因子主要行水分、矿质营养元素、温度、重金属、钠盐、CO2、土壤类型以及pH等。水分胁迫会导致豆科植物根瘤减少和固氮效率低下;矿质元素方面,除氮磷钾外,微量死素对固氮影响也很明显;不适的温度会对豆科植物的结瘤固氮产生一定的限制;重金属能从不同方面直接和间接地影响共生同氮,寻找适合作尾矿先锋植物的豆科植物是当前的一个研究热点。本文除详细阐述了这方面开展的研究以外,还浅析了这方而研究目前国内外存在的一些主要问题和发展趋势。     

Photosynthesis and nitrogen fixation in legume plants

Plants pass through a succession of growth phases at a rate largely controlled by environmental factors. The spatial arrangement and efficiency of plant organs are influenced by the fluxes of energy and matter in their environments. Thus, the successful integration of processes, such as photosynthesis and nitrogen fixation, occurring in the very different environments of the soil and the air requires a complex functional balance. Such a balance is particularly complex for legumes in which the genetic expressions of the host plant and Rhizobium influence the nitrogen economy. Progress towards improvements in symbiotic nitrogen fixation has been severely limited by the difficulty of distinguishing between the metabolic activities of the roots and nodules in whole plant studies. Recent improvements in experimental precision have revealed processes which govern gaseous diffusion in nodules and control their carbohydrate use. Furthermore, the application of quantitative models to problems of carbon and nitrogen nutrition is improving the understanding of plant growth.     

The Carbon Balance of a Legume and the Functional Economy of its Root Nodules

Budgets for carbon and nitrogen in shoot, root, and nodulesof garden pea (Pisum sativum L.) are drawn up for a 9-d intervalin the life cycle, from data on nitrogen fixation, carbon accumulationin dry matter, respiratory output of plant organs, and organicsolute exchange between shoot and nodulated root. Of the carbon gained photosynthetically by the shoot from theatmosphere 26 per cent is incorporated directly into its drymatter, 32 per cent translocated to the nodules, and 42 percent to the supporting root. Of the nodules’ share, 5per cent is consumed in growth, 12 per cent in respiration,and 15 per cent returned to the shoot via the xylem, as aminocompounds generated in nitrogen fixation. Growth and respirationof the root utilize, respectively, 7 and 35 per cent. The respiratory efficiency of a nodulated root in terms of nitrogenfixation (5.9mg C per mg N₂-N fixed) is found to be very similarto that of an uninoculated root assimilating nitrate (6.2 mgC per mg NO₃-N reduced). The nodules require in growth, respiration,and export 4.1 mg C ( 10.3 mg carbohydrate) for each mg N whichthey fix. The nodules consume 3 ml O₂ for every 1 ml N₂ utilized in fixation. In exporting a milligram of fixed nitrogen the nodules requireat least 0.35 ml of water. Almost half of this requirement mightbe met by mass flow into the nodules via the phloem.     

Evidence supporting a non-phloem source of water for export of solutes in the xylem of soybean root nodules

The vascular anatomy of soybean nodules [Glycine max (L.) Merr.] suggests that export of solutes in the xylem should be dependent on influx of water in the phloem. However, after severing of stem xylem and phloem by shoot decapitation, export of ureides from nodules continued at an approximately linear rate for 5h. This result was obtained with decapitated roots remaining in the sand medium, but when roots were disturbed by removal from the rooting medium prior to shoot decapitation, export of ureides from nodules was greatly reduced. Stem exudate could not be collected from disturbed roots, indicating that flow in the root xylem had ceased. Thus, ureide export from nodules appeared to be dependent on a continuation of flow in the root xylem. When seedlings were fed a mixture of ³H₂O and ¹⁴C-inulin for periods of 14–21 min, nodules had higher ³H/¹⁴C ratios than roots from which they were detached. The combined results are not consistent with the proposal that export of nitrogenous compounds from nodules is dependent on import of water via the phloem. The results do support the view that a portion of the water required for xylem export from soybean nodules is supplied via a symplastic route from root cortex to nodule cortex to the nodule vascular apoplast.     

Ontogenetic Interactions between Photosynthesis and Symbiotic Nitrogen Fixation in Legumes

Photosynthetic data collected from Pisum sativum L. and Phaseolus vulgaris L. plants at different stages of development were related to symbiotic N₂ fixation in the root nodules. The net carbon exchange rate of each leaf varied directly with carboxylation efficiency and inversely with the CO₂ compensation point. Net carbon exchange of the lowest leaves reputed to supply fixed carbon to root nodules declined in parallel with H₂ evolution from root nodules. The decrease in H₂ evolution also coincided with the onset of flowering but preceded the peak in N₂ fixation activity measured by acetylene-dependent ethylene production. A result of these changes was that the relative efficiency of N₂ fixation in peas increased to 0.7 from an initial value of 0.4. The data reveal that attempts to identify photosynthetic contributions of leaves to root nodules will require careful timing and suggest that the relative efficiency of N₂ fixation may be influenced by source-sink relationships.     

The application of ascorbate or its immediate precursor, galactono-1,4-lactone, does not affect the response of nitrogen-fixing pea nodules to water stress

Nitrogen fixation in legumes is dramatically inhibited by abiotic stresses, and this reduction is often associated with oxidative damage. Although ascorbate (ASC) has been firmly associated with antioxidant defence, recent studies have suggested that the functions of ASC are related primarily to developmental processes. This study examines the hypothesis that ASC is involved in alleviating the oxidative damage to nodules caused by an increase in reactive oxygen species (ROS) under water stress. The hypothesis was tested by supplying 5 mM ASC to pea plants (Pisum sativum L.) experiencing moderate water stress (ca. −1 MPa) and monitoring plant responses in relation to those experiencing the same water stress without ASC. A supply of exogenous ASC increased the nodule ASC+dehydroascorbate (DHA) pool compared to water-stressed nodules without ASC, and significantly modulated the response to water stress of the unspecific guaiacol peroxidase (EC 1.11.1.7) in leaves and nodules. However, ASC supply did not produce recovery from water stress in other nodule antioxidant enzymes, nodule carbon and nitrogen enzymes, or nitrogen fixation. The supply of the immediate ASC precursor, galactono-1,4-lactone (GL), increased the nodule ASC+DHA pool, but also failed to prevent the decline of nitrogen fixation and the reduction of carbon flux in nodules. These results suggest that ASC has a limited role in preventing the negative effects of water stress on nodule metabolism and nitrogen fixation.     

Water balance of N2-fixing root nodules: Can phloem and xylem transport explain it?

Abstract Analysis of nodule inputs via the phloem and outputs via the xylem leads to the conclusion that water fluxes along those conduits alone would give a xylem sap osmolality in excess of that of sieve tubes and would thus plasmolyse the latter unless N₂ fixation involved a very high respiratory consumption of organic C entering in the phloem, or there is significant water influx from soil through the nodule surface. Whether N₂ fixation by attached nodules not in contact with an external water supply is energetically inefficient (and hence also, at a whole plant level, inefficient in terms of water-use) is as yet untested. However, the hypothesis which we prefer involves the shortfall in water entry via the phloem being made up the parenchymatic water flux from the root to the nodule.     

Improvement of symbiotic nitrogen fixation in plants: molecular-genetic approaches and evolutionary models

Efficiency of symbiotic nitrogen fixation in legumes depends on bringing together the processes of N₂ fixation, assimilation of its products, supply of nitrogenase with energy, and development of nodule tissue and cellular structures. Coordination of these processes could arise from the evolutionary old functions of the nodules associated with deposition of the products of photosynthesis governed by systemic signals traveling between the above-ground organs and the roots. Further increase in symbiotic efficiency was associated with a pronounced ability to fix N₂ by intracellular bacteroids that lost capability to propagate (as observed in galegoid legumes from the tribes Viciae, Trifolieae, and Galegae producing indeterminate nodules). However, efficiency of these symbioses is restricted by a slow removal from the nodules of the products of N₂ fixation, which are assimilated along the same amide pathway as nitrogen compounds arriving from the soil. In legumes from the tribe Phaseoleae, such a restriction was overcome owing to a particular way of nitrogen assimilation via its incorporation into ureides (in determinate nodules). Development of symbioses where specialization of bacteroids in symbiotic fixation of atmospheric nitrogen is combined with its ureide assimilation will make it possible to produce new forms of plants highly efficient in symbiotic nitrogen fixation.     

Nodule growth and activity may be regulated by a feedback mechanism involving phloem nitrogen*

We present a mechanism of regulation of growth and activity of legume root nodules which is consistent with published experimental observations. The concentration of reduced nitrogen compounds, probably amino acids, flowing into the nodules from the phloem, is sensed by the nodules; growth and activity of the nodules is adjusted accordingly. In many legumes this response may involve changes in the oxygen diffusion resistance of the nodule cortex. A straightforward feedback mechanism in which nodule activity is lowered when reduced N in the phloem is high and increased when it is low is envisaged. Almost all import into nodules is via the phloem sap originating in the lower leaves. As a plant develops, these mature leaves no longer utilize nitrogen delivered in the xylem and so export it in the phloem. In plants with an adequate nitrogen supply (from nodules or combined nitrogen in soil), a high concentration of nitrogen containing compounds in the phloem from the lower leaves may inhibit nodule growth as well as activity. This suggestion is an alternative to the hypotheses of carbohydrate deprivation or nitrate inhibition which are commonly used to explain the effects of combined nitrogen on nodule growth and activity.     

Does GABA increase the efficiency of symbiotic N2 fixation in legumes?

The ability to regulate the rates of metabolic processes in response to changes in the internal and/or external environment is a fundamental feature which is inherent in all organisms. This adaptability is necessary for conserving the stability of the intercellular environment (homeostasis) which is essential for maintaining an efficient functional state in the organism. Symbiotic nitrogen fixation in legumes is an important process which establishes from the complex interaction between the host plant and microorganism. This process is widely believed to be regulated by the host plant nitrogen demand through a whole plant N feedback mechanism in particular under unfavorable conditions. This mechanism is probably triggered by the impact of shoot-borne, phloem-delivered substances. The precise mechanism of the potential signal is under debate, however, the whole phenomenon is probably related to a constant amino acid cycling within the plant, thereby signaling the shoot nitrogen status. Recent work indicating that there may be a flow of nitrogen to bacteroids is discussed in light of hypothesis that such a flow may be important to nodule function. Large amount of γ-aminobutyric acid (GABA) are cycled through the root nodules of the symbiotic plants. In this paper some recent evidence concerning the possible role of GABA in whole-plant-based upregulation of symbiotic nitrogen fixation will be reviewed.Key words: γ-aminobutyric acid, nitrogen fixation, nodule, symbiosis, translocation, signalingNitrogen (N) is major limiting nutrient for the growth of most plant species in different ecosystems. Acquisition and assimilation of N is second in importance only to photosynthesis for plant growth and development. Elemental N is a key constituent of protein, nucleic acids and other vital cellular components. Most plants acquire N from the soil solution either as nitrate or ammonium ions. In addition, some plants can utilize the atmospheric gaseous nitrogen pool through symbiotic associations with species of bacteria, cyanobacteria or actinomycetes that contain the N₂ fixing enzyme, nitrogenase. Clearly, the crucial role that symbiotic plants play in plant growth requires that physiologists understand the biochemical and molecular events that regulate fixation and subsequent metabolism of nitrogen.Symbiotic N₂ fixation is an important process for increasing the plant available N and thereby the growth capacity of legumes. This process results from the complex interaction between the host plant and microorganism.¹ The host plant provides the microorganism with carbon and a source of energy for growth and functions while the microorganism fixes atmospheric N₂ and provides the plant with a source of reduced nitrogen in the form of ammonium. An adequate supply of carbohydrates is an essential requirement of nodule functioning as N₂ fixation is expensive in terms both of energy and carbon for the synthesis of N-products. Sucrose synthesized in photosynthesis and exported to the nodules via the phloem, is the primary fuel for N₂ fixation.² Sucrose can be metabolized in the cytoplasm of infected, uninfected or interstitial cells with organic acids as the end products. Malate is strongly believed to be the major respiratory substrate for bacteroids.³ This dicarboxylic acid is the major energy source for the bacteroids and plant mitochondria, and is used for NH₄⁺ assimilation as carbon skeleton in the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway.⁴ The products of symbiotic N₂ fixation are exported from the nodules to the rest of the host plant where they are incorporated into essential macro-molecules such as amino acids, proteins that drive plant growth, development and yields. According to the fixation products, root nodules are generally divided into two major groupings:¹ (1) indeterminate nodules that are elongate-cylindrical activity that transport fixed N as amides such as alfalfa, pea and clover; and (2) determinate nodules that are spherical with determinate internal meristematic activity that transport fixed N as ureides, such as soybean and common bean. The complex series of events leading to the formation and functioning of the fixation machinery required controlled coordinated expression of both bacterial and host plant genes.     

Translocation of amino acids from stem nodules of Sesbania rostrata demonstrated by GC-MS in planta 15N isotope dilution

We report a novel use of the ¹⁵N dilution technique to detail the translocation of amino compounds in the legume Sesbania rostrata . The conventional ¹⁵N dilution technique follows the dilution of ¹⁵N within a labelled plant, as ¹⁴N₂ is fixed by symbiotic bacteria. In our experiments, stem-nodulated Sesbania rostrata were enriched by feeding with ¹⁵N ammonium nitrate for 2 weeks, followed by a 1 week period where the only N available to the plants was via nitrogen fixation of atmospheric N₂. We measured the composition, concentration and ¹⁵N enrichment of amino compounds in various plant tissues, both above and below the stem nodules, using GC-MS and isotopic abundance mass spectrometry techniques. Approximately 28% of the total N in the stem nodules was derived from internal plant sources. The ureides allantoic acid and allantoin were not abundant in xylem, leaf or nodule tissues. The amides asparagine and glutamine were the major export products from stem nodules although a wide range of other amino compounds are also synthesized. Amino acids within the nodules had a low level of enrichment, demonstrating that a small fraction (≈ 11%) was derived from outside the nodules, and significant cycling of N (28% of xylem N) through the root system was revealed by measurements of ¹⁵N distribution and amino acid concentrations.     

Approaches for enhancement of N2 fixation efficiency of chickpea (Cicer arietinum L.) under limiting nitrogen conditions

Chickpea (Cicer arietinum) is an important pulse crop in many countries in the world. The symbioses between chickpea and Mesorhizobia, which fix N₂ inside the root nodules, are of particular importance for chickpea's productivity. With the aim of enhancing symbiotic efficiency in chickpea, we compared the symbiotic efficiency of C‐15, Ch‐191 and CP‐36 strains of Mesorhizobium ciceri in association with the local elite chickpea cultivar ‘Bivanij’ as well as studied the mechanism underlying the improvement of N₂ fixation efficiency. Our data revealed that C‐15 strain manifested the most efficient N₂ fixation in comparison with Ch‐191 or CP‐36. This finding was supported by higher plant productivity and expression levels of the nifHDK genes in C‐15 nodules. Nodule specific activity was significantly higher in C‐15 combination, partially as a result of higher electron allocation to N₂ versus H⁺. Interestingly, a striking difference in nodule carbon and nitrogen composition was observed. Sucrose cleavage enzymes displayed comparatively lower activity in nodules established by either Ch‐191 or CP‐36. Organic acid formation, particularly that of malate, was remarkably higher in nodules induced by C‐15 strain. As a result, the best symbiotic efficiency observed with C‐15‐induced nodules was reflected in a higher concentration of the total and several major amino metabolites, namely asparagine, glutamine, glutamate and aspartate. Collectively, our findings demonstrated that the improved efficiency in chickpea symbiotic system, established with C‐15, was associated with the enhanced capacity of organic acid formation and the activities of the key enzymes connected to the nodule carbon and nitrogen metabolism.     

Translocation of Nitrogenous Compounds in Symbiotic and Nitrate-Fed Amide-Exporting Legumes

Peoples, M. B., Sudin, M. N. and Herridge, D. F. 1987. Translocationof nitrogenous compounds insymbiotic and nitrate-fed amide-exportinglegumes.–J. exp. Bot. 38: 567–579. The transport of nitrogen from the roots and nodules of chickpea(Cicer anetinum L.), lentil (Lens culinaris Medic), faba bean(Vicia faba L.) and pea (Pisum sativum L.) was examined in glasshouse-grownplants supplied either with nitrate-free nutrients or with nutrientssupplemented with 1,2,4 or 8 mol m^-3153N-nitrate. A sixth treatmentcomprised uninoculated plants supplied with 8–0 mol m^-31513N-nitrate. For each species, more than 75% of the nitrogenwas exported from the nodules as the amides, asparagine andglutamine. In fully symbiotic plants, the amides also dominatednitrogen transport to the shoot When N2 fixation activity wasdecreased by the addition of nitrate to the rooting medium,the N-composition of xylem exudate and stem solutes changedconsiderably. The relative concentrations of asparagine tendedto increase in the xylem whilst those of glutamine were reduced;the levels of nitrate increased in both xylem exudate and thesoluble nitrogen pool of the stem with a rise in nitrate supply.The changes in relative nitrate contents reflected generallythe contributions of root and shoot to overall nitrate reductaseactivity at the different levels of nitrate used. The relationshipsbetween the relative contents of xylary or stem nitrate andamino nitrogen and the plants' reliance on N2 fixation (determinedby the ¹⁵N isotope dilution procedure) were examined. Data suggestthat compositional relationships based on nitrate may be reasonableindicators of symbiotic dependence for all species under studyexcept faba bean when greater than 25% of plant nitrogen wasderived from N2 fixation. Key words: Nitrogen, translocation, legumes     

Symbiotic N2 fixation activity in relation to C economy of Pisum sativum L. as a function of plant phenology

The relationships between symbiotic nitrogen fixation (SNF) activity and C fluxes were investigated in pea plants (Pisum sativum L. cv. Baccara) using simultaneous 13C and 15N labelling. Analysis of the dynamics of labelled CO2 efflux from the nodulated roots allowed the different components associated with SNF activity to be calculated, together with root and nodule synthetic and maintenance processes. The carbon costs for the synthesis of roots and nodules were similar and decreased with time. Carbon lost by turnover, associated with maintenance processes, decreased with time for nodules while it increased in the roots. Nodule turnover remained higher than root turnover until flowering. The effect of the N source on SNF was investigated using plants supplied with nitrate or plants only fixing N2. SNF per unit nodule biomass (nodule specific activity) was linearly related to the amount of carbon allocated to the nodulated roots regardless of the N source, with regression slopes decreasing across the growth cycle. These regression slopes permitted potential values of SNF specific activity to be defined. SNF activity decreased as the plants aged, presumably because of the combined effects of both increasing C costs of SNF (from 4.0 to 6.7 g C g-1 N) and the limitation of C supply to the nodules. SNF activity competed for C against synthesis and maintenance processes within the nodulated roots. Synthesis was the main limiting factor of SNF, but its importance decreased as the plant aged. At seed-filling, SNF was probably more limited by nodule age than by C supply to the nodulated roots.