Effects of substrate nitrate concentration on symbiotic nodule formation in actinorhizal plants

The response ofAlnus glutinosa, Casuarina cunninghamiana, Elaeagnus angustifolia andMyrica cerifera to a range of substrate nitrogen levels in solution, in relation to plant growth, infection, nodulation and root fine structure was studied. Nine concentrations of potassium nitrate ranging from 0.05 to 3.0 mM, were tested on each of the species. Plants were inoculated withFrankia pure cultures after a two week exposure to one of the nine levels of added nitrate. After six more weeks with constant exposure to nitrate, plants were harvested and assayed. With the exception of Myrica, regression analyses of whole plant dry weights as a function of added nitrate were highly significant. There was a tendency for nodulated plants grown at intermediate levels of added nitrate to exhibit higher relative growth rates, probably due to the additive effect of substrate nitrogen and fixation of atmospheric nitrogen. The mean numbers of nodules per plant were, with the exception of Alnus, significantly higher at intermediate levels of added nitrate, as were mean nodule dry weights. A highly significant inverse relationship between nodule weight as a percentage of whole plant weight was found in Elaeagnus and Myrica. The observed response of Elaeagnus to added nitrate compared to other actinorhizal plants appears to demonstrate that root hair infected plants are much more sensitive to the inhibitory effects of added nitrate than plants infected by intercellular penetration. A sharp reduction in the presence of root hairs at high concentrations of nitrate was observed. This indicates that the inhibition of nodulation in some actinorhizal plant species results from nitrate induced root hair suppression.     

The Respiratory Costs of Nitrogen Fixation in Soyabean, Cowpea, and White Clover: II. COMPARISONS OF THE COST OF NITROGEN FIXATION AND THE UTILIZATION OF COMBINED NITROGEN

Plants of soyabean, cowpea, and white clover were grown singlyin pots in Saxcil growth cabinets at 23/18 °C, 30/24 °C,and 20/15 °C, respectively, until seed maturation or for85 d (white clover). Two populations were produced within eachspecies: one population effectively nodulated and wholly dependentfor nitrogen on fixation in the root nodules, and a second populationcompletely lacking nodules but receiving abundant nitrate nitrogen.In each species, the two populations were compared in termsof rate of gross photosynthesis, rate of shoot respiration,and rate of root respiration. Source of nitrogen had littleor no effect on rate of photosynthesis or shoot respiration.In contrast, the rate of respiration of the nodulated rootsof plants fixing their own nitrogen was greater, sometimes two-foldgreater, than that of equivalent plants lacking nodules andutilizing nitrate nitrogen. This superiority in terms of rateof root respiration was generally confined to the period ofintense nitrogen fixation. An analysis of the magnitude of thisrespiratory burden in terms of daily photosynthesis indicatesthat, in all three legumes, plants fixing their own nitrogenrespire 11–13% more of their fixed carbon each day thanequivalent plants lacking nodules and utilizing nitrate nitrogen.     

The Development of Primary Root Nodules on Vicia faba L. Grown at Two Temperatures

The development of primary root nodules on the field bean (Viciafaba L.) grown at 10 and 18 °C was examined. The sequenceof anatomical changes observed was the same in both temperatureregimes. Nodules developed much more slowly at 10 °C andthe nodules were much larger when corresponding anatomical changesoccurred. Primary root nodules eventually ceased growth in bothtemperature regimes but ultimate nodule volume was nearly twiceas great as 10 °C as at 18 °C. The larger size did notcompensate for the lower specific nitrogenase activity of cold-grownnodules: nitrogen fixation (acetylene reduction) rates on awhole plant basis were much lower at 10 °C than at 18 °C.There was no difference in the total number of primary rootnodules in the two temperature regimes but their distributionwas biased towards the upper part of the root and the epicotylat 18 °C. Vicia faba L., field bean, nodulation, nitrogen fixation, temperature     

Effect of Low Temperature and Rh izospheric Application of Naringenin on Pea-Rhizobium leguminosarum biovar viciae Symbiosis

The production of Tsr factor by Rhizobium leguminosarum bv. viciae was influenced by low temperature (10°C) In the presence of seed exudate collected at 10°C and 25°C or naringenin (10fuM). Root exudate collected at 25°C and naringenin induced Tsr factor in R. leguminosarum causing thick and short root phenotype and root hair curling and deformation of host root. Root exudate collected at 10°C also induced root hair curling but Tsr activity was low. low temperature grown plants had poor nodulation, nitrogen fixation, nitrogen content and total blomass as compared to plants grown at 25°C. Rhizospheric application of naringenin partially alleviated the deleterious effect of low temperature on nodulation status and nodule efficiency.     

Defoliation in White Clover: Regrowth, Photosynthesis and N2 Fixation

Single plants of white clover, grown in a controlled environmentand dependent for nitrogen on fixation in their root nodules,were defoliated once by removing approximately half their shoottissue. Their regrowth was compared with the growth of comparableundefoliated plants. Two similar experiments were carried out:in the first, plants were defoliated at 2.5 g, and in the secondat 1.2 g total plant d. wt. Defoliation reduced rate of N₂ fixation by > 70 per cent,rate of photosynthesis by 83–96 per cent, and rate ofplant respiration by 30–40 per cent. Nodule weights initiallydeclined following defoliation as a result of loss of carbohydratesand other unidentified components. No immediate shedding ofnodules was observed but nodules on the most severely defoliatedplants exhibited accelerated senescence. The original rates of N₂ fixation were re-attained after 5–6or 9 d regrowth, with increase in plant size at defoliation.In general, the rate of recovery of N₂ fixation was relatedto the re-establishment and increase of the plant's photosyntheticcapacity. Throughout the growth of both defoliated and undefoliatedplants nodule respiration (metabolism) accounted for at least23 ± 2 per cent of gross photosynthesis. The unit ‘cost’of fixing N₂ in root nodules, in terms of photosynthate, appearedto be unaffected by defoliation, except perhaps for plants veryrecently defoliated. Similarly, the percentage nitrogen contentsof shoot, root and nodules of defoliated plants became adaptedwithin a few days to those characteristic of undefoliated plants. Trifolium repens, white clover, N₂ fixation, defoliation, photosynthesis, respiration     

Nitrogen fixation and assimilation efficiency in Ethiopian and German pea varieties

Dinitrogen (N₂) fixation and assimilation efficiency in a German and two Ethiopian varieties of Pisum sativum L. was studied in a pot experiment during vegetative and reproductive growth. The objective of the study was to assess whether genotypes having contrasting growth habits showed differences in physiological processes that affect the efficiency of N₂ fixation and assimilation. Dry matter formation, nodulation and nitrogen assimilation were compared between two treatments where one depended solely on N₂ fixation while the other was nourished with nitrate. Moreover, carbon (C) costs of N₂ fixation and the capacity of different respiratory chains in roots and nodules were determined at vegetative and reproductive growth. As compared to the Ethiopian cultivars, the German variety displayed a more rapid vegetative growth with intensive N₂ fixation and assimilation and highly efficient individual nodules. However, during reproductive growth, N₂ fixation in the German variety declined sharply, while continuing in the Ethiopian varieties. Lowest C costs of N₂ fixation coincided with most efficient individual nodules in both growth intervals. C costs of N₂ fixation were lower during reproductive growth in all varieties which was accompanied by a shift in root/nodule respiratory capacity towards more C efficient respiratory pathways. The results provide further evidence that unreliable nitrogen fixation rates during reproductive growth of pea can be connected with restricted C supply to nodules. One strategy of pea plants to adapt to critical C availability is an increase in capacity of more C efficient root/nodule respiration.     

Nitrate levels affect the development of the black locust-Rhizobium symbiosis

Summary Experiments with black locust (Robinia pseudoacacia L.) seedlings grown under strictly controlled laboratory conditions indicated that the availability of nitrate has a marked impact on nitrogen fixation. When nitrate concentrations were very low, both nodulation and seedling growth were impaired, whereas nitrate concentrations high enough to promote plant growth strongly inhibited symbiotic nitrogen fixation. When nitrate was added to the growth medium after infection, nodulation and nitrogen fixation of the seedlings decreased. This effect was even more marked when nitrate was applied before infection with rhizobia. Higher nitrogen concentrations also reduced nodule number and nodule mass when applied simultaneously with the infecting bacteria. The contribution of symbiotic nitrogen fixation to black locust shoot mass by far exceeded its effects on shoot length and root mass. When nitrate availability was very low, specific nitrogen fixation (i. e. nitrogenase activity per nodule wet weight) was improved with increasing nitrogen supply, but rapidly decreased with higher nitrogen concentrations.     

Nodulation studies in legumes

Summary The influence of combined nitrogen (as ammonium nitrate) on the symbiotic performances of selected bacterial associations of four legumes was examined using sand culture.In barrel medic (Medicago tribuloides Desr.) and vetch (Vicia sativa L. andV. atropurpurea Desf.) bacterial partnerships of a host plant varied greatly in their nodulation responses to a range of amounts of nitrogen applied at sowing. Some bacterial strains exhibited varying degrees of stimulation of nodule number, growth and fixation by low or medium amounts of nitrogen. Higher levels of combined nitrogen depressed symbiosis. Other strain responses showed a severe restriction of symbiosis with any amount of added nitrogen.Seasonal influences conditioned symbiotic responses to combined nitrogen in an association of cowpea (Vigna sinensis End.) With a summer sowing small amounts of ammonium nitrate added at sowing benefited later symbiotic development. No such stimulation was evident in an autumn sowing and symbiotic injury from high levels of nitrogen was greater than in the summer sowing.The developing association of cowpea was found to be most sensitive to ammonium nitrate added just as the first leaves unfolded. Here damage was manifest in a permanent elevation of the top: root ratio with subnormal growth and functioning of nodules. Greatest benefit from added inorganic nitrogen followed applications made as the first nodules appeared on the primary root. In this case added combined nitrogen acted as an investment providing returns in additional fixation equivalent to 5–10 times the amount of nitrogen originally fed to the seedling and representing some 50 per cent greater total fixation than in minus-nitrogen controls.     

The Growth and Photosynthesis of Seedling Plants of White Clover at Low Temperature

The growth of white clover (Trifolium repens L.) in conditionstypical of April in Southern England (8 °C day/4 °Cnight, 12 h photoperiod of 90 J m^–2 s^–1 visibleradiation) was extremely slow, whether the plants were dependentfor nitrogen on fixation by their root nodules or were suppliedwith abundant nitrate; although growth was slower in the nodulatedplants. The reasons for slow growth were a large root: shootratio and a small leaf area, particularly in the nodulated plants,and a low photosynthetic rate in all plants. The probable effectsof these characteristics on the growth of white clover withgrasses in mixed pastures are discussed. Trifolium repens L, white clover, low temperature, leaf area, photosynthetic rate, nitrogen supply, growth     

Symbiotic effectiveness of Rhizobium meliloti at low root temperature

Laboratory, growth chamber and field experiments were conducted to select among 226 isolates of Rhizobium meliloti for the ability to grow, nodulate alfalfa (Medicago sativa L.) and support N₂-dependent plant growth between 9° and 12°C. There was wide variation in the abilities of R. meliloti isolates to grow and form nodules at 10°C. Culture doubling times (t_d) varied from 1 to 155h, and the number of nodules formed on alfalfa in growth pouches in 2 weeks varied from 0 to 3.8 nodules per plant. Nodulation occurred at 9°C, but there was no significant N₂-dependent plant growth at this temperature. However, several isolates of R. meliloti had the ability to nodulate alfalfa and produce N₂-dependent growth at root temperatures between 10° and 12°C root temperature than did 14 other isolates tested. In field experiments, inoculation with strain NRG-34 resulted in greater nodule numbers, nodule weight, proportion of nodules occupied by the inoculant strain and plant weight than did inoculation with a commercial strain (NRG-185). These results permitted selection of a strain with better low-temperature competitive abilities than the currently available commercial strains.     

Low Root Temperature Retardation of the Mineral Nitrogen Induced Decline in N2Fixation by a Northern Ecotype of White Clover

Nodulated white clover plants (Trifolium repensL.) of a Norwegian ecotype from Pasvik (70°N) were grown in flowing solution culture. Root temperature was 17°C until 51d after sowing, when it was lowered decrementally over 5d to 7°C in four of the eight plant culture units. After a further 24h, mineral N was supplied automatically at 20μMNH₄NO₃in three culture units at each root temperature (7 and 17°C) over 17d. The remaining two units provided control plants solely dependent on N₂fixation at 7 and 17°C.The supply of NH₄NO₃greatly reduced the nodule biomass per plant at 17°C over 17d compared with control plants, but had little effect at 7°C. The nodule decline at 17°C accompanied an acute and progressive decrease in specific rate of N₂fixation, from 9mmolN d^-1g^-1nodule d.wt on day 0 to zero by day 10. Whilst initial rates of N₂fixation were lower at 7°C, the mineral N-induced decrease in fixation rates was also less severe than at 17°C and specific fixation rates recovered after reaching a minimum on day 11. N₂fixation accounted for 36% of the total uptake of N by +min.N plants during the treatment period at 7°C as opposed to only 13% at 17°C. The total N₂fixed at 7°C was 86% of that fixed at 17°C, although the specific growth rate (d.wt) at 7°C was only 55% of that at 17°C. Addition of NH₄NO₃at 7°C had little effect on the gross amount of N₂fixed subsequently. In contrast, total N₂fixation by +min.N plants at 17°C was only 24% of that fixed by the corresponding controls. The possible mechanisms by which mineral N affects N₂fixation are discussed.     

Growth of Trifolium subterraneum L. Selected for Sparse and Abundant Nodulation as Affected by Root Temperature and Rhizobium Strain

Root temperature greatly affected plant growth whether or notplants depended on symbiotic nitrogen fixation. The two plantselections responded differently to the three strains of Rhizobiumand this response was differentially affected by root temperature. Plant yield was significantly decreased by each fall of 4 °Cin temperature from 19 to 7 °C by amounts that dependedboth on the host and Rhizobium strain. Symbiosis with strainTA1, originally isolated from a cold environment, was most tolerantof a root temperature of 11 °C; TA1 produced as much ormore plant material of the abundantly nodulating host in 40days growth at 7 and 11 °C as did the uninoculated plantsgiven KNO³. Root temperature affected the number, rate of formation, anddistribution of nodules on the root system. At 7 °C fewernodules formed than between 11 and 19 °C. At 7 °C nodulesdid not form on secondary roots by 40 days but at 11 °Cthe secondary roots nodulated rapidly between 30 and 40 days.Nodule formation at 19 °C was almost completed at 20 days,when secondary root nodules accounted for 60 per cent of thetotal. Within the range 15 to 19 °C, at which the originalselections for sparse and abundant nodulation were made, plantsnodulated true to selection, but not at 11 °C. At 7 and11 °C plants nodulated with TA1 yielded more with increasingnumber of nodules.     

Expression of nir, nor and nos denitrification genes from Bradyrhizobium japonicum in soybean root nodules

Expression of Bradyrhizobium japonicum wild-type strain USDA110 nirK , norC and nosZ denitrification genes in soybean root nodules was studied by in situ histochemical detection of β -galactosidase activity. Similarly, P_nirK- lacZ , P_norC- lacZ , and P_nosZ- lacZ fusions were also expressed in bacteroids isolated from root nodules. Levels of β -galactosidase activity were similar in both bacteroids and nodule sections from plants that were solely N₂-dependent or grown in the presence of 4 m M KNO₃. These findings suggest that oxygen, and not nitrate, is the main factor controlling expression of denitrification genes in soybean nodules. In plants not amended with nitrate, B. japonicum mutant strains GRK308, GRC131, and GRZ25, that were altered in the structural nirK , norC and nosZ genes, respectively, showed a wild-type phenotype with regard to nodule number and nodule dry weight as well as plant dry weight and nitrogen content. In the presence of 4 m M KNO₃, plants inoculated with either GRK308 or GRC131 showed less nodules, and lower plant dry weight and nitrogen content, relative to those of strains USDA110 and GRZ25. Taken together, the present results revealed that although not essential for nitrogen fixation, mutation of either the structural nirK or norC genes encoding respiratory nitrite reductase and nitric oxide reductase, respectively, confers B. japonicum reduced ability for nodulation in soybean plants grown with nitrate. Furthermore, because nodules formed by each the parental and mutant strains exhibited nitrogenase activity, it is possible that denitrification enzymes play a role in nodule formation rather than in nodule function.     

Relationships between nitrogen fixation and photosynthesis as the main components of the productivity in alfalfa

Alfalfa (Medicago sativa L.) plants were inoculated with Sinorhizobium meliloti Tn-5 mutants featuring various nitrogen-fixing effectiveness and then grown in sand culture to study relations between CO₂ exchange, plant productivity, and nitrogen fixation. At the flowering stage, the relationship between nitrogen fixation and photosynthesis of whole alfalfa plants was described with the logarithmic curve. At the same stage of plant development, a close relationship was observed between nitrogen fixation rate and plant weight; this relationship showed a trend toward saturation at high rates of nitrogen fixation. The increase in nitrogenase activity of root nodules was accompanied by stimulation of root respiration; the relation of respiration to nitrogen-fixing activity was manifested stronger than its relation to the total root weight. It is concluded that highly effective strains of root nodule bacteria can realize their potential only in combination with complementary plant genotypes featuring active photosynthesis that provides a balanced supply of assimilates for both the symbiotic apparatus and growth processes in the macrosymbiont.     

Nodule protein synthesis and nitrogenase activity of soybeans exposed to fixed nitrogen

Nitrate or ammonium was added to soybean (Glycine max L. Merrill cv Corsoy) plants grown in plastic pouches 10 days after nodules first appeared. By the third day of treatment with 10 millimolar nitrate, nitrogenase specific activity (per unit nodule weight) had decreased to 15% to 25% of that of untreated plants. Longer incubations and higher concentrations of nitrate had no greater effect. In addition, exogenous nitrate or ammonium resulted in slower nodule growth and decreased total protein synthesis in both the bacterial and the plant portion of the nodule (as measured by incorporation of ³⁵S). Two-dimensional gel electrophoresis revealed that the nitrogenase components were not repressed or degraded relative to other bacteroid proteins. In the presence of an optimal carbon source, the nitrogenase specific activity of nodules detached from nitrate-treated plants was equivalent to that of nodules from untreated plants. These results are consistent with models that propose decreased availability or utilization of photosynthate in root nodules when legumes are exposed to fixed nitrogen.     

Sensitivity of the common bean (Phaseolus vulgaris L.) symbiosis to high soil temperature

Summary Experiments were done to test whether N fixation is more sensitive to high soil temperatures in common bean than in cowpea or soybean. Greenhouse experiments compared nodulation, nitrogenase activity, growth and nitrogen accumulation of several host/strain combinations of common bean with the other grain legumes and with N-fertilization, at various root temperatures. Field experiments compared relative N-accumulation (in symbiotic relative to N-fertilized plants) of common bean with cowpea under different soil thermal regimes. N-fertilized beans were unaffected by the higher temperatures, but nitrogen accumulation by symbiotic beans was always more sensitive to high root temperatures (33°C, 33/28°C, 34/28°C compared with 28°C) than were cowpea and soybean symbiosis. Healthy bean nodules that had developed at low temperatures functioned normally in acetylene reduction tests done at 35°C. High temperatures caused little or no suppression of nodule number. However, bean nodules produced at high temperatures were small and had low specific activity. ForP. vulgaris some tolerance to high temperature was observed among rhizobium strains (e.g., CIAT 899 was tolerant) but not among host cultivars. Heat tolerance ofP. acutifolius andP. lunatus symbioses was similar to that of cowpea and soybean. In the field, high surface soil temperatures did not reduce N accumulation in symbiotic beans more than in cowpea, probably because of compensatory nodulation in the deeper and cooler parts of the soil.     

Effectiveness of Lotus Root Nodules: III. EFFECT OF COMBINED NITROGEN ON NODULE EFFECTIVENESS AND FLAVOLAN SYNTHESIS IN PLANT ROOTS

When grown in a nutrient solution containing combined nitrogen(NH₄NO₃), Lotus pedunculatus and L. tenuis seedlings inoculatedwith a fast-growing strain of Rhizoblum (NZP2037) did neitherdevelop root nodules nor develop flavolans in their roots. Incontrast, the roots of nodulated seedlings growing in a nitrogen-freenutrient solution contained flavolans. Flavolan synthesis coincidedwith root nodule development on these plants. When added as a single dose, high concentrations of NH₄NO₃ (5and 10 mg N per plant) stimulated the growth of L. pedunculatusplants but suppressed nodulation and nitrogen fixation. In contrastthe continued supply of a low concentration of NH₄NO₃ (1?0 mgN d^–1 per plant) stimulated nitrogen fixation by up to500%. This large increase in nitrogen fixation was associatedwith a large increase in nodule fresh weight per plant, a doublingof nodule nitrogenase activity, and a lowering of the flavolancontent of the plant roots. The close relationship between nitrogendeficiency, nodule development, and flavolan synthesis in L.pedunculatus meant that it was not possible (by nitrogen pretreatmentof plants) to alter the ineffective nodule response of a Rhizobiumstrain (NZP2213) sensitive to the flavolan present in the rootsof this plant.     

White Clover N2-Fixation in Response to Root Temperature and Nitrate: II. N2-FIXATION, RESPIRATION, AND NITRATE REDUCTASE ACTIVITY

Established, nodulated white clover plants were transferredto eight tanks of a flowing culture apparatus with solutiontemperatures of 5, 11, 17, and 25 ?C (two tanks per temperature).Shoot temperature and light environment were common to all plants.After 7 d, (10 mmol m^–3) was continuouslysupplied to one tank at each temperature while in the remainingfour tanks (one at each temperature) the plants were completelydependent on nodule N₂-fixation. Plants were randomly selected at intervals during the following14 d period in order to measure root and nodule respirationand acetylene reduction activity (ARA) in a flow-through systemset at the adapted root temperature. Additional plants wereassayed for in vitro nitrate reductase activity in leaves, roots,and nodules. Apparent nitrogenase activity (ARA) and respiration associatedwith it were each markedly affected by temperature in two ways;(1) Activity per unit weight of nodule was reduced at lowertemperatures; (2) Development of the plant, and thus also nodulemass, was restricted at lower temperatures which, in turn, restrictedtotal nodule activity per plant. The presence of nitrate significantly reduced ARA of nodules,particularly at higher temperatures. However, significant discrepancieswere found when N₂-fixation rates, estimated from the acetylenereduction assay, were compared with N₂-fixation rates calculatedfrom curves fitted to N accumulation data (minus the rate of uptake in the case of nitrate-treated plants). Carbon use efficiency (CO² respired per C₂H₄ produced) was notsignificantly affected by temperature or the presence of nitrate. Nitrate reductase activity (NRA) developed in all plant partsat the three highest temperatures, but not at 5 ?C. We calculatethat leaf NRA may account for 82, 75, and 68% of total nitratereduction at 11, 17, and 25 ?C respectively. Key words: Trifolium repens, white clover, N₂ fixation, root temperature, acetylene reduction assay, nitrate, nitrate reductase     

The effect of source,concentration and time of application of nitrogen on the growth,nodulation and nitrogen fixation ofLotus pedunculatus andTrifolium repens

Summary Studies under growth cabinet conditions investigated the effect of source and concentration of nitrogen and timing of nitrogen application on the growth and nitrogen fixation byLotus pedunculatus cv. Maku andTrifolium repens cv. S184. KNO₃, NaNO₃ and NH₄NO₃ were added at transplanting at the following rates: 3.33, 7.78 and 13.33 mg N/plant. KNO₃ was added at 3.33 and 7.78 mg N/plant at 0, 6, 12, 18, 24 or 30 days after transplanting.Lotus shoot weight increased with all increasing nitrogen sources but clover only responded to KNO₃ and NaNO₃. The root weight of both species increased with increasing KNO₃ and NH₄NO₃. The percentage increase in lotus and clover shoot growth was greater than that of root growth when KNO₃ was added within a week of transplanting. Increases in growth by both species resulted from added nitrogen except with lotus when NaNO₃ was applied where increased nitrogen fixation also contributed to increased growth.Weight and number of effective nodules on both species were increased with 3.33 mg N per plant as KNO₃ but nitrogen fixation was not affected. Addition of 13.33 mg N as NaNO₃ reduced weight and number of effective nodules in both species and also nitrogen fixation by lotus.KNO₃ increased growth and nodulation of both species when applied within one week after transplanting. Nodulated lotus plants responded to KNO₃ by increasing growth but not nodulation.KNO₃ appeared to affect infection and development of nodules on lotus and may affect the growth of existing nodules on clover.     

Root and nodule growth in Pisum sativum L. in relation to photosynthesis: analysis using 13C-labelling

The effect of the nitrogen source (gaseous nitrogen, N2, or nitrate ions, NO3-) on the use of carbon (C) for root and nodule growth of pea (Pisum sativum L.) was investigated using 13C-labelling of assimilated CO2 at various stages of growth. Nitrate supply and growing conditions (sowing dates, air CO2 concentration) were varied to alter photosynthetic rates. Nodules are the sink with the highest demand for C in both the vegetative and flowering stages, growing at the expense of shoot and root in the vegetative stage, but only at the expense of roots at flowering. Until flowering, the addition of C into root and nodule biomass was linearly related to pre-existing biomass, thus determining net sink strengths which decreased with root and nodule age. Nodule growth patterns did not depend on the N source, whereas root growth was increased by nitrate when nodule biomass was low. At seed filling, the increase in C of biomass of the root system was no longer related to pre-existing biomass and C was preferentially diverted to roots of plants assimilating nitrate, or to nodules for plants fixing N2.