Ineffective utilization of nitrate by soybean during pod fill

The relative effectiveness of nitrate, allantoin, or nitrate plus allantoin as sources of nitrogen for the indeterminate soybean plant [ Glycine max (L.) Merr cv. Harper] was studied throughout vegetative and reproductive growth. All plants were provided with 3.0 m M nitrogen and were grown hydroponically in growth chambers. During vegetative and early reproductive growth, plants given nitrate or nitrate plus allantoin grew faster than plants provided allantoin only. However, during pod fill, plants provided with allantoin or allantoin plus nitrate gained weight more rapidly than plants receiving just nitrate. More importantly, at maturity plants that had been provided with allantoin or allantoin plus nitrate during pod fill were 30% heavier in total dry weight, 50% higher in nitrogen content, and 50% higher in seed yield than plants that had received just nitrate. At full bloom, all plants were inoculated with the same culture of Bradyrhizobium japonicum , and twice each week throughout pod fill each plant was assayed for nitrogen fixation (acetylene reduction). Correlation coefficients obtained by linear regression analysis show a strong positive correlation between the measured rate of nitrogen fixation and maximum plant fresh weight (r = 0.83), total plant nitrogen (r = 0.81), or seed yield (r = 0.76). The fact that nitrogen fixation during pod fill stimulates plant growth and seed yield, coupled with the facts that nitrate blocks nodulation and is not used efficiently during pod fill by the soybean plant, may explain why seed yield of field-grown soybeans usually does not respond to added fertilizer nitrogen. Thus, it is suggested that enhanced nitrogen fixation may be the key factor in improving soybean seed yield.     

Effect of dinitroaniline herbicides on rhizobia, nodulation and N2(C2H2) fixation of four groundnut cultivars

Field, greenhouse and laboratory investigations were conducted to determine the effect of four dinitroaniline herbicides on rhizobia, nodulation and nitrogen fixation of four groundnut cultivars. Benefin, dinitramine and nitralin used at recommended levels decreased nodule dry weight, nitrogenase activity and total nitrogen of groundnut tops and pod yield in three cultivars Kadiri 71-1, Kadiri-2, ICGS-11 and not for a fourth cultivar, Kadiri-3 of groundnut (Arachis hypogaea L.), but fluchloralin used at the recommended level increased the nodulation rate, nitrogenase activity and total nitrogen of groundnut tops and pod yield compared to untreated plants. Studies were conducted in vitro to determine the relative toxicity of the herbicides on four Rhizobium strains isolated from the nodules of four cultivars of groundnut. It was found that various strains of rhizobia differ in their sensitivity to different rates of the herbicides tested. Carbon dioxide exchange rate (CER) of all the cultivars which received herbicide treatment was measured at different time intervals to determine the relationship between photosynthesis and inhibition of nodulation. The lack of adverse effect on the CER of four cultivars when treated at recommended concentrations indicated that nitrogen fixation was affected in cultivars Kadiri 71-1, Kadiri-2 and ICGS-11 due to inhibition of nodulation.     

Enhanced Nitrogen Fixation Increases Net Photosynthetic Output and Seed Yield of Hydroponically Grown Soybean

Ten to 20% of the net photosynthetic output of a tropical grainlegume may be consumed by the nodulation-nitrogen-fixation process.If plant growth activities during the reproductive phase werelimited by photosynthetic output, enhanced nitrogen fixationwould seemingly lower total plant mass and seed yield. To testthis possibility, soybean [Glycine max (L.) Merr.] plants weregrown hydroponically on nutrient medium supplemented with minimalurea or with an excess of either nitrate or nitrate plus urea.Acetylene reduction activities (i.e. nitrogen fixation rates)and transpiration rates were measured twice weekly on theseplants through pod fill. Of the plants inoculated, those grownon minimal urea revealed significantly greater acetylene reductionactivities and transpiration rates. At maturity, plants thathad fixed nitrogen at a rapid rate during pod fill had a significantlygreater seed size, total plant mass (i.e. net photosyntheticrate) and nitrogen content than uninoculated or poorly nodulatedplants grown on an excess of nitrate. It is concluded, therefore,that a rapid rate of nitrogen fixation during pod fill enhancesboth transpiration and net photosynthetic output. The increasedavailability of usable nitrogen (i.e. ureides), coupled withenhanced transpiration and photosynthetic output, significantlyincreases total plant mass and seed yield. Thus, enhanced nitrogenfixation seems to be an inexpensive means of increasing seedyield of soybean and perhaps of other tropical grain legumes. Key words: Glycine max, nodulation, nitrate, urea     

Effect of different levels of mineral nitrogen on nodulation and N2 fixation of two cultivars of common bean (Phaseolus vulgaris L.)

An experiment was conducted under greenhouse conditions to evaluate the effect of mineral nitrogen on N₂ fixation of two cultivars of Phaseolus vulgaris L., Puebla 152 and Negro Argel. Nitrogen application was 0, 2.5, 12.5 and 25 mg N Kg^–1 of a vermiculite-sand-mixture at planting time. Shoot and root growth were elevated by nitrogen application at all growth stages. During vegetative growth (V 5) nodule dry weight and nitrogenase activity (acetylene reducing activity) per plant were reduced by nitrogen supply in both cultivars, but less in Negro Argel than in Puebla 152. At later stages nodulation in nitrogen-treated Puebla 152 did not differ from that in non-treated plants, whereas increased nodule number was found in Negro Argel at high nitrogen levels. The influence of mineral N on the total amount of nitrogen fixed in the two bean cultivars was only slightly different.     

The regulation of nodulation,nitrogen fixation and ammonium assimilation under a carbohydrate shortage stress in the Discaria trinervis-Frankia symbiosis

N₂-fixation is sensitive to limitation in the availability of newly synthesised carbohydrates for the nodules. We decided to explore the response of the D. trinervis - Frankia symbiosis to a transient decrease in carbohydrate supply to nodules. Feedback inhibition of nodulation as well as nodule growth was not released by a 6-day dark stress in D. trinervis nodulated plants. However, nitrogen fixation and assimilation were affected by the imposed stress. Nitrogenase activity was totally inhibited after 4 days of darkness although high levels of nitrogenase components were still detected at this time. Degradation of FeMo and Fe nitrogenase subunits – both at similar rates – was observed after 6 days of dark stress, revealing the need for inactivation to precede enhancement of protein turnover. Glutamine synthetase (GS), malate dehydrogenase (MDH) and asparagine synthetase (AS) polypeptides were also degraded during the dark stress, although at a lower rate than nitrogenase. ARA and nitrogenase were totally recovered 8 days after resuming normal illumination. It seems that current nitrogenase activity and ammonium assimilation are not, or are only weakly linked with the feedback control of nodulation in D. trinervis. These observations give support to the persistence of an autoregulatory signal in mature nodules that is not sensitive to transient shortages of carbon supply and sustains the inhibition of nodulation in the transient absence of N₂ fixation.     

Effects of the herbicides Lindex and Simazine on chloroplast and nodule development,nodule activity,and grain yield inLupinus albus L.

Field and laboratory investigations were carried out to determine the influence of two commercial herbicides Lindex and Simazine on symbiotic N₂ fixation, the photosynthetic apparatus, percentage of proteins, and grain yield of lupins (Lupinus albus L. cv. Multolupa). The herbicides were added (3 kg commercial product in 9001 per ha) two weeks after sowing. The nodulated roots were tested for nitrogenase activity by the acetylene reduction assay (ARA) at the first and second flowering. The ARA values decreased with herbicide application. The decrease was smaller in the Simazine treatments with inoculated seeds. The ARA for plants treated with Lindex did not increase with inoculation. Nitrogenase activity was greater in the non-inoculated plants growing together with weeds. The effect of Simazine on the photosynthetic apparatus proved to be more toxic than that of Lindex, not only considerably reducing the size of the chloroplasts but also affecting the grana structure. The latter appeared as an electron-dense compact mass. There were no visible alterations in the photosynthetic apparatus of plants treated with Lindex. However starch, which was not present in the control leaves, accumulated. No ultrastructural changes were observed in the nodule cells treated with Lindex. Simazine altered nodule cells by causing vesicle formation, degeneration of bacteria, and by decreasing the number of N₂-fixing bacteroids. The grain yield decreased with both herbicide treatments, the decrease being lower in the inoculated plants, especially in the Simazine treatment. Our results suggest that each herbicide had a different effect on N₂ fixation, the photosynthetic apparatus and grain yield. Laboratory studies indicated that cyanazine and simazine did not adversely affect the growth of Bradyrhizobium.     

Growth,nodulation and nitrogen fixation in soybean as affected by air humidity and root temperature

Growth, nodulation and N₂ fixation inGlycine max L. Merr., cv. Biison as affected by the relative humidity of air (RH) during the dark period (95 or 50 – 65 %) and day/night root temperature (T_r) (28/28, 25/25, 18/18, 22/28, 22/18 °C) were studied. The growth parameters (plant fresh and dry mass, yield), nodulation (nodule number and fresh mass) and N₂ fixation abilities (total nitrogen content, nitrogenase activity) increased significantly with the increasing T_r. In addition, at the same T_r during the day all studied parameters were increased at the higher T_r during the dark period. Growth, nodulation and N₂ fixation were significantly enhanced at low RH. The findings indicate that all studied parameters could be regulated by environmental factors during the dark period.     

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.     

Ontogenetic Variation of Nitrogenase, Nitrate Reductase, and Glutamine Synthetase Activities in Oryza sativa

The relationship between the rates of nitrogenase, nitrate reductase, and glutamine synthetase activities, and plant ontogeny in rice (Oryza sativa L.), cultivar `M9', grown in salt marsh sediment with and without nitrate treatment was studied. In both treatments, nitrogenase activity measured as the immediate linear rate of acetylene reduction by bacteria associated with the roots varied with plant age. In control plants, the nitrogenase activity developed during the vegetative stage, peaked during early reproductive growth and then declined. The application of 10 kilograms N per hectare as KNO₃ once every 2 weeks delayed the development of and decreased the nitrogenase activity. The nitrogenase activity in both treatments developed as leaf nitrate reductase activity declined. The per cent nitrogen of roots was negatively correlated with the rates of acetylene reduction during the life cycles of control and nitrate-treated plants. This suggests that the concentration of combined nitrogen in the plants controlled the development and rate of root-associated nitrogenase activity. During reproductive growth, no nitrate reductase activity was detected in the roots from either treatment. In control plants, the patterns of nitrogenase activity and glutamine synthetase activity in the roots were similar. Thus, rice roots have the potential to assimilate ammonia while fixing N₂. During the vegetative and early reproductive stages of growth, the development of maximal rates of nitrogenase activity coincided with an increase of total nitrogen of the plants in both treatments.     

Nitrogenase Inhibition in Nodules from Pea Plants Grown Under Salt Stress Occurs at the Physiological Level and can be Alleviated by B and Ca

In order to shed new light on the mechanisms of salt-mediated symbiotic N₂-fixation inhibition, the effect of salt stress (75 mM) on N₂-fixation in pea root nodules induced by R. leguminosarum was studied at the gene expression, protein production and enzymatic activity levels. Acetylene reduction assays for nitrogenase activity showed no activity in salt-stressed plants. To know whether salt inhibits N₂-fixing activity at a molecular or at a physiological level, expression of the nifH gene, encoding the nitrogenase reductase component of the nitrogenase enzyme was analyzed by RT-PCR analysis of total RNA extracted from nodulated roots. The nifH messenger RNA was present both in plants grown in the presence and absence of salt, although a reduction was observed in salt-stressed plants. Similar results were obtained for the immunodetection of the nitrogenase reductase protein in Western-blot assays, indicating that nitrogen fixation failed mainly at physiological level. Given that nutrient imbalance is a typical effect of salt stress in plants and that Fe is a prosthetic component of nitrogenase reductase and other proteins required by symbiotic N₂-fixation, as leghemoglobin, plants were analyzed for Fe contents by atomic absorption and the results confirmed that Fe levels were severely reduced in nodules developed in salt-stressed plants. In a previous papers (El-Hamdaoui et al., 2003b), we have shown that supplementing inoculated legumes with boron (B) and calcium (Ca) prevents nitrogen fixation decline under saline conditions stress. Analysis of salt-stressed nodules fed with extra B and Ca indicated that Fe content and nitrogenase activity was similar to that of non-stressed plants. These results indicate a linkage between Fe deprivation and salt-mediated failure of nitrogen fixation, which is prevented by B and Ca leading to increase of salt tolerance.     

Ontogenic variation in nitrogen assimilation,nodulation and nitrogen fixation in groundnut (Arachis hypogaea) genotypes*

Seasonal pattern of acetylene reduction (AR) and shoot nitrogen accumulation was studied in nine groundnut cultivars. Shoot N accumulation by all the cultivars was maintained until shortly before maturity and it occurred faster over the reproductíve growth phase than over the earlier phases. In all cultivars plant AR (PAR) did not reflect this pattern of N accumulation, being greater over the vegetative and pod initiation phases. This suggests that the commonly observed low PAR values for groundnut over the reproductive growth phase may be the result of factors other than sink competition. There were significant interactions of cultivar with stage of crop growth for PAR, nodule mass, and specific nitrogenase activity (SNA). Virginia types generally showed better nodulation, higher N₂-fixing capacity (both PAR and SNA) than valencias, and significant differences were observed between cultivars within a botanical type.     

Some characteristics of symbiotic nitrogen fixation,yield, protein and oil accumulation in irrigated peanuts (Arachis hypogaea L.)

Summary The potential of peanuts for symbiotic nitrogen fixation is considerable and under optimal edaphic and climatic conditions it reached 222 kg N₂/ha, which was 58% of the nitrogen accumulated in the plants. The effect of the Rhizobium inoculation on crude protein accumulation in the yield (kg/ha) was 3–4 times greater than its effect on the yield of pods and hay. There was an inverse relationship between the protein and oil content in the kernels.Seasonal changes in nitrogenase activity in the nodules were determined by the acetylene reduction method during two growing seasons. Under favorable conditions the specific activity of the nitrogenase reached a very high level (up to 975 moles C₂H₂ g dry wt nod/h) and the total activity (moles C₂H₄/plant/h) was also high in spite of the relatively poor nodulation (weight and number). The high activity was drastically reduced (to 75 moles C₂H₄ g dry wt nod/h) due to exceptionally hot and dry weather, which occurred in the middle of the second half of the growing season. It appears that N₂-fixation (nitrogenase activity) is more sensitive to these unfavorable conditions, than is nodule growth. Maximum nitrogenase activity was observed during the podfilling stage; until 50–60 days after planting, nitrogenase activity was very low.     

Effect of Rhizobium sp. BARIRGm901 inoculation on nodulation,nitrogen fixation and yield of soybean (Glycine max) genotypes in gray terrace soil

Soybean plants require high amounts of nitrogen, which are mainly obtained from biological nitrogen fixation. A field experiment was conducted by soybean (Glycine max) genotypes, growing two varieties (Shohag and BARI Soybean6) and two advanced lines (MTD10 and BGM02026) of soybean with or without Rhizobium sp. BARIRGm901 inoculation. Soybean plants of all genotypes inoculated with Rhizobium sp. BARIRGm901 produced greater nodule numbers, nodule weight, shoot and root biomass, and plant height than non-inoculated plants. Similarly, inoculated plants showed enhanced activity of nitrogenase (NA) enzyme, contributing to higher nitrogen fixation and assimilation, compared to non-inoculated soybean plants in both years. Plants inoculated with Rhizobium sp. BARIRGm901 also showed higher pod, stover, and seed yield than non-inoculated plants. Therefore, Rhizobium sp. BARIRGm901 established an effective symbiotic relationship with a range of soybean genotypes and thus increased the nodulation, growth, and yield of soybean grown in gray terrace soils in Bangladesh.     

Essentiality of Boron for Dinitrogen Fixation in Anabaena sp. PCC 7119

The relationship between the requirement for boron and the form of N supplied in nutrient media to cyanobacterium Anabaena sp. PCC 7119 was investigated. When cells were grown in a medium which contained nitrate or ammonium-N, boron deficiency in the nutrient media did not inhibit growth or change cell composition. However, when cells were dependent on N₂ fixation, the lack of boron inhibited growth (i.e. growth ceased after 96 hours under these conditions). Additionally, boron-deficient cells showed a significant decrease in their content of phycobiliproteins and chlorophyll and accumulated carbohydrates within 24 hours of removing boron from the nutrient media. Inhibition of photosynthetic O₂ evolution accompanied the decrease in photosynthetic pigments. Boron deficiency symptoms were relieved when either boron or combined N was added to boron-deficient cultures. The degree of recovery depended upon the age of the cultures. Assays of nitrogenase activity showed that, after 2 hours of growth, nitrogenase activity of boron-deficient cells was inhibited by 40%. After 24 hours a total inactivation of nitrogenase activity was observed in boron-deficient cells. These results strongly suggest an involvement of boron in N₂ fixation in cyanobacteria.     

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (mol C₂H₄g^–1 nodule fr. wt. h^–1), were both affected by the added nitrate.     

Effects of Brassmosteroids on Growth, Nodulation, Phytohormone Content and Nitrogenase Activity in French Bean Under Water Stress

Effect of pre-treatments of 1 and 5 M epibrassinolide or homobrassinolide prior to water stress induction on changes in root nodulation and contents of endogenous abscisic acid (ABA) and cytokinin trans-zeatin riboside (ZR), and nitrogenase activity was investigated in the nodulated roots of Phaseolus vulgaris L. cv. Arka Suvidha. Brassinosteroids in the unstressed plants increased root nodulation, ZR content and nitrogenase activity, and also ameliorated their stress-induced decline in the nodulated roots. The ABA contents in the nodules of control or stressed plants were not altered by brassinosteroids treatment. There was an increase in pod yield by brassinosteroids treatment (5 M) in the irrigated control as well as stressed plants without influencing pod number or pod length. Among the brassinosteroids, epibrassinolide was relatively more effective.     

N(2)-Fixation by Freshly Isolated Nostoc from Coralloid Roots of the Cycad Macrozamia riedlei (Fisch. ex Gaud.) Gardn

Nitrogenase (EC 1.7.99.2) activity (acetylene reduction) and nitrogen fixation (¹⁵N₂ fixation) were measured in cyanobacteria freshly isolated from the coralloid roots of Macrozamia riedlei (Fisch. ex Gaud.) Gardn. Light and gas phase oxygen concentration had marked interactive effects on activity, with higher (up to 100-fold) rates of acetylene reduction and ¹⁵N₂ fixation in light. The relationship between ethylene formation and N₂-fixation varied in the freshly isolated cyanobacteria from 4 to 7 nanomoles of C₂H₄ per nanomole ¹⁵N₂. Intact coralloid roots, incubated in darkness and ambient air, showed a value of 4.3. Maximum rates of nitrogenase activity occurred at about 0.6% O₂ in light, while in darkness there was a broad optimum around 5 to 8% O₂. Inhibition of nitrogenase, in light, by pO₂ above 0.6% was irreversible. Measurements of light-dependent O₂ evolution and ¹⁴CO₂ fixation indicated negligible photosynthetic electron transport involving photosystem II and, on the basis of inhibitor studies, the stimulatory effect of light was attributed to cyclic photophos-phorylation. Nitrogenase activity of free-living culture of an isolate from Macrozamia (Nostoc PCC 73102) was only slightly inhibited by O₂ levels above 6% O₂ and the inhibition was reversible. These cells showed rates of light-dependent O₂ evolution and ¹⁴CO₂ fixation which were 100- to 200-fold higher than those by the freshly isolated symbiont. Furthermore, nitrogenase activity was dependent on both photosynthetic electron transport and photophosphorylation. These data indicate that cyanobacteria within cycad coralloid roots are differentiated specifically for symbiotic functioning in a microaerobic environment. Specializations include a high heterocyst frequency, enhanced permeability to O₂, and a direct dependence on the cycad for substrates to support nitrogenase activity.     

Nitrogen fixation in several grain legume species with contrasting sensitivities to copper nutrition

The nitrogen fixation response to copper nutrition in faba bean, yellow lupin and soybean was studied. Copper nutrition significantly increased the pod yields of all tested grain legumes but faba bean gave the greatest Cu-use efficiency for pod and grain production. The accumulation of dry matter in vegetative parts, nodules, N and leghemoglobin concentration in nodules and nitrogen accumulation in the whole plants were increased by copper supply in faba bean and yellow lupin in contrast with soybean. Cu nutrition significantly increased the Cu concentrations in nodules of all cultivated plants. The differential sensitivity of N₂ fixation in tested grain legume species to copper nutrition could be connected with the level of phenols in nodules and depended on both the host plants and strains of rhizobia, which differ in their ability to produce catechol-like siderophores. Copper requirements by symbiotic N₂ fixation could also depend on the nature of phenols in nodules (presence of o-dihydroxyphenols or number of hydroxyls in molecule).     

The response and recovery of nitrogen fixation activity in soybean to water deficit at different reproductive developmental stages

Soybean (Glycine max [L.] Merr.) N₂ fixation is a primary plant mechanism responsible for meeting plant-N demand during seed development. Nitrogen fixation is recognized as a drought-sensitive mechanism; however, N₂ fixation response to water deficit and N₂ fixation recovery at different reproductive stages are not well documented. We tested the hypothesis that water deficit during late reproductive stages would inhibit N₂ fixation and lead to the breakdown of essential leaf proteins and an inability to recover N₂ fixation. Acetylene reduction activity (ARA) and N redistribution response to a 5-d drought period at flowering (R2), early seed fill (R5), and late seed fill (R6) were evaluated in one genotype (Hendricks, maturity group 0). Control plants maintained high rates of nodule activity until late seed fill. Plants drought stressed at R2 and R5 recovered ARA after rewatering and in some cases had higher nitrogenase activity than control plants during mid-seed fill. Recovery of ARA on plants stressed at R2 and R5 was associated with higher shoot N concentration than control plants at maturity. Drought stress at R6 reduced ARA, and the inability to recover ARA after stress alleviation at R6 resulted in decreased individual seed mass, which was likely caused by an acceleration of leaf N redistribution and a shorter seed-fill period. Results emphasized the importance of soybean N₂ fixation during late seed development on seed yield and that the ability to recover N₂ fixation following drought is dependent upon crop developmental stage.     

Effect of Altered pO(2) in the Aerial Part of Soybean on Symbiotic N(2) Fixation

Dry matter accumulation, nitrogen content and N₂ fixation rates of soybean (Glycine max [L.] Merr. cv. Wye) plants grown in chambers in which the aerial portion was exposed to a pO₂ of 5, 10, 21, or 30% and a pCO₂ of 300 μl CO₂/l or a pO₂ of 21% and a pCO₂ of 1200 μl CO₂/l during the complete growth cycle were measured. Total N₂[C₂H₂] fixed was increased by CO₂/O₂ ratios greater than those in air and was decreased by ratios smaller than those in air; the effects on N₂ fixation of decreased pO₂ or elevated pCO₂ were quantitatively similar during the period of vegetative growth. Decreased pO₂ produced a smaller increase then elevated pCO₂ during the reproductive period, presumably because of the decreased sink activity of the arrested reproductive growth under subambient pO₂. At a pO₂ of 5% and a pCO₂ of 300 μl CO₂/l total N₂ fixed was increased 125% and per cent nitrogen content in the vegetative parts was increased relative to air while that in the seed was decreased. Dry matter production was increased and reproductive growth was arrested as previously reported for plants receiving only fertilizer nitrogen. At a pO₂ of 30% and a pCO₂ of 300 μl CO₂/l total N₂ fixed was decreased 50% and per cent nitrogen content in the vegetative part was increased relative to air while that in the reproductive structures was unaffected. Dry matter production was similarly decreased in both vegetative and reproductive structures. These effects of altered pO₂ in the aerial part on N₂ fixation are consistent with the hypothesis that the amount of photosynthate available to the nodule may be the most significant primary factor limiting N₂ fixation while sink activity of the reproductive structures may be a secondary factor.