Relation between nitrogenase synthesis and activity in a marine unicellular diazotrophic strain, Gloeothece sp. 68DGA (Cyanophyte), grown under different light/dark regimens

The relationship between the abundance of nitrogenase and its activity was studied in the marine unicellular cyanobacterium Gloeothece sp. 68DGA cultured under different light/dark regimens. The Fe‐ and MoFe‐protein of nitrogenase and nitrogen (N₂)‐fixing (acetylene reduction) activity were detected only during the dark phase when the cells were grown under a 12 h light/12 h dark cycle (12L/12D). Nitrogenase activity appeared about 4 h after entering the dark phase. Maximum nitrogenase activity occurred at around the middle of the dark phase, and the activity rapidly decreased to zero before the start of the light phase. The rapid decrease of nitrogenase activity and the Fe‐protein of nitrogenase near the end of the dark phase in 12L/12D were partly recovered by the addition of l ‐methionine‐sulfoximine, an inhibitor of glutamine synthetase. Diurnal oscillation of the abundance of nitrogenase was maintained in the first subjective dark phase (i.e. the period corresponding to the dark phase) after the cells were transferred from 12L/12D to continuous illumination. However, enzyme activity was detected only when photosynthetic oxygen (O₂) evolution was completely suppressed by reducing the light intensity or by the addition of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea. Nitrogenase always appeared in the cells about 16 h after starting the light phase, even when the 12L/12D cycle was modified by the addition or subtraction of a single 6 h period of light or dark. These results suggest the following: (i) N₂‐fixation by Gloeothece sp. 68DGA is primarily regulated by an endogenous circadian oscillator at the level of nitrogenase synthesis. (ii) The endogenous circadian rhythm resets on a shift of the timing of the light phase. (iii) Nitrogenase activity is not always reflected in the presence of nitrogenase. (iv) The activity of nitrogenase is negatively regulated by fixed nitrogen and the concentration of ambient O₂.     

Kinetics of synthesis of nitrogenase in batch and continuous culture of Anabaena flos-aquae

Summary Nitrogenase of Anabaena flos-aquae was inactivated by oxygen and recovery of activity was measured in batch and iron, phosphate and urea-limited continuous cultures. In batch culture, canavanine, chloramphenicol, methylamine, proflavine, puromycin and urea inhibited the recovery process. The rate of recovery of nitrogenase activity in continuous cultures was dependent on light intensity, concentration of urea, ammonium salts and nitrate, and independent of growth rate. Oxygen and urea caused an inactivation of nitrogenase in continuous cultures.     

Glycogen content and nitrogenase activity in Anabaena variabilis

Nitrogenase (=acetylene-reducing activity) was followed during photoautotrophic growth of Anabaena variabilis (ATCC 29413). When cell density increased during growth, (1) inhibition of light-dependent activity by DCMU, an inhibitor of photosynthesis, increased, and (2) nitrogenase activity in the dark decreased. Addition of fructose stabilized dark activity and alleviated the DCMU effect in cultures of high cell density.The resistance of nitrogenase towards oxygen inactivation decreased after transfer of autotrophically grown cells into the dark at subsequent stages of increasing culture density. The inactivation was prevented by addition of fructose. Recovery of acetylene-reducing activity in the light, and in the dark with fructose present, was suppressed by ammonia or chloramphenicol. In the light, also DCMU abolished recovery.To prove whether the observed effects were related to a lack of photosynthetic storage products, glycogen of filaments was extracted and assayed enzymatically. The glycogen content of cells was highest 10 h after inoculation, while light-dependent nitrogenase activity was at its maximum about 24 h after inoculation. Glycogen decreased markedly as growth proceeded and dropped sharply when the cells were transferred to darkness. Thus, when C-supply (by photosynthesis or added fructose) was not effective, the glycogen content of filaments determined the activity of nitrogenase and its stability against oxygen. In cells lacking glycogen, nitrogenase activity recovered only when carbohydrates were supplied by exogenously added fructose or by photosynthesis.Abbreviations Chl chlorophyll a - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Cyclic variations of photosynthetic activity under nitrogen fixing conditions in Synchococcus RF-1

When nitrogen fixing cell cultures of Synechococcus RF-1 were subjected to an alternating lightdark regime (12 h:12 h), a cyclic decrease in the photosynthetic oxygen evolution potential was observed during the dark periods. This rhythm of net photosynthesis rate was maintained for at least two days after transition to continuous light. The decrease in net photosynthesis was accompanied by a stimulation of dark respiration. However, the magnitude of oxygen uptake was considerably smaller than the observed decrease in oxygen evolution. The photosynthetic activity of cells taken from the dark period was characterized by (i) a significantly lower quantum yield and (ii) a strong reduction in the light-saturated rate of photosynthesis. Growing the cultures on nitrate or under continuous light completely suppressed this rhythm. Protein synthesis was not necessary for the recovery of the light-saturated rate of photosynthesis during the light period. The cellular content of chlorophyll a and of phycobiliproteins did not vary between light and dark period, indicating that quantitative changes in the composition of the photosynthetic apparatus are not the basis for the observed oscillations. Regulatory modifications of the photosynthetic efficiency are proposed as an adaptation mechanism to adjust the intracellular oxygen concentration to the needs for nitrogenase activity.Abbreviation Chl chlorophyll     

Regulation of nitrogenase activity by light in the azolla-anabaena symbiosis

Nitrogenase activity and the rate of photosynthesis were measured simultaneously in Azolla by a continuous gas flow system. The mode of interaction between light, photosynthesis and nitrogenase activity was analysed.Nitrogenase activity dropped off when either Azolla plants or the cyanobiont Anabaena were transferred from light to dark. This decline was immediate and was independent of length or intensity of the prior light phase. Reillumination restored nitrogenase activity.Nitrogenase activity did not depend on the rate of photosynthesis at light intensities below 10 μE m⁻² s⁻¹. Its activity was saturated at 200 μE m⁻² s⁻¹ while CO₂ fixation was saturated at a light intensity of 850 μE m⁻² s⁻¹. Azolla photosynthetic activity followed the absorption spectrum of chlorophyll a, while nitrogenase activity markedly increased between 690 and 710 nm. Inhibition of photosynthesis by DCMU was accompanied by an increase in nitrogenase activity. These results suggest direct light regulation of nitrogenase activity in Azolla independent of CO₂ fixation, and a possible inhibition of nitrogenase activity by the oxygen produced in photosynthesis.     

Oxygen and the light–dark cycle of nitrogenase activity in two unicellular cyanobacteria

Cyanobacteria capable of fixing dinitrogen exhibit various strategies to protect nitrogenase from inactivation by oxygen. The marine Crocosphaera watsonii WH8501 and the terrestrial Gloeothece sp. PCC6909 are unicellular diazotrophic cyanobacteria that are capable of aerobic nitrogen fixation. These cyanobacteria separate the incompatible processes of oxygenic photosynthesis and nitrogen fixation temporally, confining the latter to the dark. Although these cyanobacteria thrive in fully aerobic environments and can be cultivated diazotrophically under aerobic conditions, the effect of oxygen is not precisely known due to methodological limitations. Here we report the characteristics of nitrogenase activity with respect to well‐defined levels of oxygen to which the organisms are exposed, using an online and near real‐time acetylene reduction assay combined with sensitive laser‐based photoacoustic ethylene detection. The cultures were grown under an alternating 12–12 h light–dark cycle and acetylene reduction was recorded continuously. Acetylene reduction was assayed at 20%, 15%, 10%, 7.5%, 5% and 0% oxygen and at photon flux densities of 30 and 76 μmol m^?2 s^?1 provided at the same light–dark cycle as during cultivation. Nitrogenase activity was predominantly but not exclusively confined to the dark. At 0% oxygen nitrogenase activity in Gloeothece sp. was not detected during the dark and was shifted completely to the light period, while C. watsonii did not exhibit nitrogenase activity at all. Oxygen concentrations of 15% and higher did not support nitrogenase activity in either of the two cyanobacteria. The highest nitrogenase activities were at 5–7.5% oxygen. The highest nitrogenase activities in C. watsonii and Gloeothece sp. were observed at 29°C. At 31°C and above, nitrogenase activity was not detected in C. watsonii while the same was the case at 41°C and above in Gloeothece sp. The differences in the behaviour of nitrogenase activity in these cyanobacteria are discussed with respect to their presumed physiological strategies to protect nitrogenase from oxygen inactivation and to the environment in which they thrive.     

Effect of light nitrogenase function and synthesis in Rhodopseudomonas capsulata.

The metabolic versatility of the purple nonsulfur photosynethetic bacterial permits the expression of either a phototrophic or a dark aerobic mode of growth. These organism also possess nitrogenase activity which may function under semiaerboic conditions. On the basis of these important properties, the light dependence of nitrogenase function and synthesis in Rhodopseudomonas capsulata was investigated. Nitrogenase activity was strictly dependent on light; no activity was observed in the dark, even when energy (ATP) was supplied by oxidative phosphorylation. It was concluded that the low-potential reducing agent required by the nitrogenase-catalyzed reaction could only be generated by a photochemical reaction. Nitrogenase biosynthesis was also largely dependent on light; however, a small amount of synthesis was observed in resting cells incubated in the dark. Resting cells prepared from dark-grown cultures synthesized nitrogenase at high rates upon illumination. The highest stability of nitrogenase in these resting cells was observed when suspensions were exposed to a diurnal pattern of illumination rather than continuous light. Although nitrogenase function and synthesis are closely coupled to photosynthetic activity, the biosyntheses of bacteriochorophyll and nitrogenase are independent of each other and are most probably subject to different regulatory mechanisms by light.     

Stimulation of nitrogenase activity and photosynthesis in some cyanobacteria by glyoxylate

Abstract The effect og glyoxylate on nitrogenase activity (C₂H₂ reduction) and photosynthesis (H¹⁴CO₃ fixation and O₂ evolution) was in vestigated in the three heterocystous cyanobacteria Anabaena cylindrica, A. variabiltis and N. muscorum. Glyoxylate had virtually no effect on the rate of dark respiration and was unable to sustain photoheterotrophic growth, though some slight stimulation (= 30%) of photorophic growth was noted. A considerable stimulation of both nitrogenase and photosynthetic activities was observed in presence of glyoxylate. In the light the stimulation increased with time up to about 15-25 h after adding optimal concentrations of 4–6 mM glyoxylate. Placing glyoxylate treated samples in the dark or adding DCMU (30 μM) in the light, showed that glyoxylate initially supported significantly higher nitrogenase activity than did samples in absence of glyoxylate. However, after a prolonged incubation in the dark or in presence of DCMU glyoxylate is unable to relieve the adverse effects of such conditions. The stimulation of the nitrogenase activity was even more pronounced when the glyoxylate was added to cells preincubated in the dark (“carbon starved”) than for cells kept constantly in light. The results suggest that glyoxylate, or a metabolite, may act as an inhibitor of cyanobacterial photorespiration and this hypothesis is discussed.     

Regulation of nitrogenase activity in Rhodobacter capsulatus under dark microoxic conditions

Rhodobacter capsulatus modulates its in vivo nitrogenase activity in the light in response to the addition of NH4+ in a variety of ways: with ADP-ribosylation of the Fe-protein of nitrogenase, with a switch-off response that is independent of ADP-ribosylation, and with a "magnitude response." In the light, these responses are differentially shown by cultures that differ in the degree of their nitrogen limitation. Here we examined the response of these culture types to the addition of NH4+ under dark, microoxic conditions and found that all three responses can be observed under these conditions. However, the magnitude response was much more sensitive to the ammonium concentration, and the ADP-ribosylation response correlated only poorly with activity changes, similar to results obtained in the light. In contrast to previous reports, Fe-protein was not ADP-ribosylated in response to the presence of oxygen.     

Phosphorylation and nitrogenase activity in isolated heterocysts from Anabaena variabilis (ATCC 29413)

Adenylate-pool composition, energy charge, and nitrogenase activity were examined in isolated heterocysts from Anabaena variabilis (ATCC 29413). ATP formation was detected as a light- or oxygen-induced increase in ATP concentration. No cofactors or substrates had to be added for photophosphorylation to occur, whereas oxidative phosphorylation was dependent on hydrogen and oxygen (Knallgas reaction). The increase in ATP concentration was reflected by a decrease in AMP concentration, accompanied by small changes in ADP levels. Thus, a regulation of the adenylate pool by a myokinase (adenylate kinase) has to be assumed. Upon dark-light transitions, the energy charge in heterocysts increased from values below 0.4 to values approaching 0.8. High energy-charge values, reached in the light only, allowed for high rates of acetylene reduction in the presence of hydrogen. The increase in the energy charge in the dark to approx. 0.64 by addition of oxygen (5% ($\text{v}\text{v}$) in the presence of hydrogen) resulted in low nitrogenase activities, generally not exceeding 1–3% of the light-induced rates. In the dark, oxygen concentrations above 10% were inhibitory to both ATP formation and acetylene reduction. Increasing light intensities led to a steep increase in energy charge followed by an increase in nitrogenase activity. Plotting enzyme activity versus energy charge, a nonlinear, asymptotic relationship was observed.     

Temporal separation of nitrogen fixation and photosynthesis in the filamentous,non-heterocystous cyanobacterium Oscillatoria sp.

When growing in laternating light-dark cycles, nitrogenase activity (acetylene reduction) in the filamentous, non-heterocystous cyanobacterium Oscillatoria sp. strain 23 (Oldenburg) is predominantly present during the dark period. Dark respiration followed the same pattern as nitrogenase. Maximum activities of nitrogenase and respiration appeared at the same time and were 3.6 mol C₂H₄ and 1.4 mg O₂ mg Chl a ^-1·h^-1, respectively. Cultures, adapted to light-dark cycles, but transferred to continuous light, retained their reciprocal rhythm of oxygenic photosynthesis and nitrogen fixation. Moreover, even in the light, oxygen uptake was observed at the same rate as in the dark. Oxygen uptake and nitrogenase activity coincided. However, nitrogenase activity in the light was 6 times as high (22 mol C₂H₄ mg Chl a ^-1·h^-1) as compared to the dark activity. Although some overlap was observed in which both oxygen evolution and nitrogenase activity occurred simultaneously, it was concluded that in Oscillatoria nitrogen fixation and photosynthesis are separated temporary. If present, light covered the energy demand of nitrogenase and respiration very probably fulfilled a protective function.     

Activity and expression of nitrogenase in Rhodobacter capsulatus under aerobiosis in the dark and in the light

Rhodobacter capsulatus was grown chemotrophically in the dark in oxygen-regulated chemostat culture and in the presence of limiting amounts of fixed N. When the oxygen partial pressure was varied, in situ nitrogen fixation occurred only at 1% of air saturation of the medium. By contrast, nitrogenase proteins and their activity measured in the absence of oxygen could be detected up to 30% of air saturation. This revealed that expression of nitrogenase is much less sensitive toward oxygen than the in situ function of the enzyme. At oxygen partial pressures > 1% of air saturation, the degree of modification of the Fe protein of nitrogenase was increased. Light was of no stimulatory effect on both the activity and the expression of nitrogenase. This holds true for growth at 1% or 5% of air saturation. At 5% of air saturation, however, high illumination enhanced the inhibitory effect of oxygen on nitrogenase formation.     

The effect of pyruvate on nitrogenase activity in the blue-green alga Anabaena cyclindrica

Exogenous pyruvate added to cultures of the bluegreen alga, Anabaena cylindrica stimulated nitrogenase activity (measured by acetylene reduction) only in the dark under low pO₂ (0.05 atmospheres). Under aerobic conditions or in the light, stimulation was absent and replaced by an inhibition of activity above 5 mM added pyruvate. The curve of nitrogenase activity versus oxygen concentration had a similar maximal value of ethylene production with or without added pyruvate, but in the presence of pyruvate this maximum occurred at 0.05 atmospheres O₂, whilst in the absence of pyruvate the maximum occurred at 0.10 atmospheres O₂. Malate, citrate, α-ketoglutarate, glucose and fructose were tested also, but none gave a similar effect to pyruvate. Addition of ¹⁴C-pyruvate and autoradiography indicated that exogenous pyruvate is metabolized through the interrupted Krebs cycle. These results are explained in terms of the activity of pyruvate: ferredoxin oxidoreductase and the ATP-induced oxygen sensitivity of nitrogenase.     

Stimulation by light of nitrogenase synthesis in cells of Rhodopseudomonas capsulata growing in N-limited continuous cultures

In cells of Rhodopseudomonas capsulata growing in nitrogen-limited continuous culture the nitrogenase-specific activity was found to be closely dependent on the light intensity. As light intensity, measured with a photodiode immersed in the culture, was varied stepwise from 1000 to 7000 lux, the nitrogenase activity, measured at steady state, increased gradually up to 5-fold. Shifting light intensity from 1200 to 7000 lux resulted in a sharp rise in nitrogenase activity which doubled within the first two hours. The determination by immunoassays of the intracellular levels of each nitrogenase component revealed that the light-dependent stimulation of nitrogenase activity was correlated with the accumulation of the nitrogenase enzyme inside the cells. Under high illumination, nitrogenase represented up to 40% of the cytoplasmic proteins. The specific activities of each component in intact cells, calculated on the basis of their relative concentration in the cells and on in vivo nitrogenase assays, appeared roughly constant and hardly affected by changes of light intensity. The specific activity of the Fe protein was about 7-fold higher in intact cells than in the purified state. The ratio of the two nitrogenase components remained fairly constant and close to one, irrespective of the light intensity to which cells were exposed. These results demonstrate that in nitrogen-limited grown cells of Rps. capsulata light brings about an induction or a derepression of nitrogenase synthesis the extent of which is dependent on light intensity.     

The Influence of Light and Oxygen on Nitrogenase Activity in the Lichen Stereocaulon paschale

Thalli of the lichen Slereocaulon paschale (L.) Fr. were prctreated in the light (light activated) or in the dark (dark starved). In short-time experiments with both light activated arid dark starved thalli, the nitrogenasc activity was higher in the light than in the dark, Light activated thalli had a very much higher rale of C₂H₂ reduction than dark starved thalli, both in the light and in the dark. The dark starved lhalli showed increasing nilrogenase activity when incubated in the light. Either light or oxygen was necessary for nitrogenase activity in light activated thalli. and up to about 10kPa oxygen they showed additive effects. Both in the light and in the dark the nitrogenase activity decreased when the oxygen partial pressure was lower than in normal air. The experimental data thus showed a short-term effect of light on nilrogenase activity by provision of ATP and reductant, and a long term effect probably by build up of reserves that were later utilized. Any immediate effect of photorespiration on nitrogenase activity could not be found in light activated thalli.     

Organic acid utilization,succinate excretion,encystation and oscillating nitrogenase activity inAzospirillum brasilense under microaerobic conditions

Oscillating nitrogenase activity in long lasting batch cultures ofAzospirillum brasilense ATCC 29145 is independent of the carbon source malate. With fumarate, succinate or pyruvate as sole carbon source nitrogenase activity is also oscillating. Cultivation in a medium with 20-fold the buffer concentration also results in oscillating nitrogenase activity. Nitrogen-fixing cultures ofAzospirillum brasilense ATCC 29145 excrete ammonia into the culture medium varying between 0.02 and 0.04 mM concentrations. This is not sufficient to cause a drop of nitrogenase activity inAzospirillum brasilense after the first maximum. During growth under nitrogen-fixing conditions with malate as carbon source, the cells excrete significant quantities of succinate into the culture medium. Cultures with only 0.05% malate reutilized the excreted succinate as soon as malate disappeared from the medium. Azospirillum brasilense ATCC 29145 is shown to have the capability of encystation. Encysted cells are different from vegetative cells in their resistance to desiccation, by the spherical shape and by immotility. The results indicate that oscillating nitrogenase activity in long lasting cultures reflects the development from vegetative cells to cysts and again to vegetative cells under microaerobic conditions.     

Diurnal Nitrogenase Modification in the Cyanobacterium Anabaena variabilis*

The nitrogen-fixing cyanobacterium Anabaena variabilis (ATCC 29413) was cultivated as continuous culture under a 12 h: 12 h light-dark cycle. In the light, photosynthetic activity resulted in a continuous increase in cellular glycogen content, followed by an almost complete dissimilation of the polysaccharide during the dark period. Nitrogenase activity, assayed by the acetylene reduction technique, was low at the end of the dark period and increased quickly upon illumination to reach a maximum after 4 to 6 h of light. The activity rapidly declined after darkening the culture. Increase and decrease of activity were accompanied by a change in the electrophoretic mobility of the Fe-protein of nitrogenase (dinitrogenase reductase) indicative of enzyme modification being involved in the diurnal control of nitrogenase activity. Modification and demodification of the Fe-protein were not coupled to the cell cycle since they followed darkening and illumination when the light or dark periods were changed. Addition of fructose increased nitrogenase activity even in darkness and caused demodification of the Fe-protein. Ammonium chloride supplied at the onset of illumination slowed down the increase of nitrogenase activity. A delayed inhibition of the enzyme was accompanied by partial Feprotein modification only. The reaction was completed after transfer to darkness. The function of enzyme modification in maintaining a constant C: N ratio is discussed and a dominating role of carbohydrate supply in this regulation is indicated by the reported findings.     

Role of bacterial polysaccharides in the derepression of ex-planta nitrogenase activity with rhizobia

Abstract Since bacterial polysaccharides may limit the availability of oxygen to the cells, we have investigated the role of rhizobial extracellular polysaccharides (EPS) and the non-rhizobial polyscharide, xanthan, in the depression of ex-planta nitrogenase activity with rhizobia in liquid medium. Two rhizobial strains known to exhibit ex-planta nitrogenase activity on solid media were used; the slow-growing Bradyrhizobium japonicum USDA 110 and the arctic Rhizobium strain N31, both being prolific EPS producers. In low nitrogen mannitol (LNM) liquid medium strain N31 exhibited nitrogenase activity only after 15 days, when sufficient EPS had accumulated in the medium, and activity was correlated with EPS production. When rhizobial EPS from an old culture was added to the LNM medium, nitrogenase activity was detected after 48 h incubation, indicating that EPS of the medium decreased oxygen diffusion to cells to a level that depressed nitrogenase activity. In modified LNM medium with xanthan nitrogenase activity was readily depressed. In both strains activity increased with increased xanthan concentration, but decreased sharply at higher concentrations. Strain N31 exhibited a narrower range of polysaccharide concentration for nitrogenase activity than the slow strain USDA 110. Thus, the condition for derepression of nitrogenase might be a careful balancing of the oxygen concentration surrounding the cells, and this condition is met when a balancing of polsaccharide, either synthesized by the rhizobia or added to the medium, can permit oxygen diffusion to within the narrow range required for the depression and expression of nitrogenase.     

Control by light of whole-cell nitrogenase activity and of nitrogenase and bacteriochlorophyll a formation in Rhodobacter capsulatus strain B10S

Control of nitrogenase and bacteriochlorophyll a (BChl) by light was studied under steady-state conditions with continuous cultures of Rhodobacter capsulatus B10S supplied with malate and growth-limiting amounts of ammonium. Consumption of malate and, correspondingly, the C/N ratio at which malate and ammonium were consumed increased when illumination was increased from 3 to approximately 20 klx and became constant at higher illuminations of up to 40 klx. Essentially the same kinetics were observed with respect to nitrogenase activity of cells, contents of nitrogenase polypeptides, and nifH promoter activity. Substrate consumption was half-maximal at 8 klx and was independent of the presence of nitrogenase. Therefore, it is concluded that light controls the C/N ratio (a quantitative measure of the nitrogen status of cells), which in turn is involved in the control of nitrogenase at the level of nif promoter activity. Post-translational regulation of nitrogenase activity by ADP-ribosylation was not observed under steady-state conditions, but it took place when illumination was suddenly decreased to the range where malate consumption and, consequently, the C/N ratio decreased. Irrespective of the presence or absence of nitrogenase, specific BChl contents of the cultures were constant above 20 klx, and they increased at lower illuminations. These results do not confirm a recently proposed link between nitrogen fixation and photosynthesis as represented by BChl. Received: 29 October 1998 / Accepted: 30 December 1998     

Effects of water stress on nitrogenase activity in Abuts incana

Tolerance to water stress was studied in plants of grey alder, Alnus incana (L.) Moench, grown in a climate chamber in pots of sand supplied with a nitrogen-free nutrient solution. The plants were subjected to a single drying and recovery cycle, during which acetylene reduction, transpiration and stomatal resistance were measured. At different stress levels the plants were placed in a closed system to equilibrate the water potential in the plant-soil system. The water potential of the plants was determined, after which they were watered and their recovery studied. Nitrogenase activity showed low tolerance to water deficit. At moderate stress (−0.6 to −0.8 MPa) acetylene reduction was reduced by half, and at more severe stress, (< −1 MPa) activity was near zero. There was a rapid decrease in nitrogenase activity coincident with stomatal closure, which indicates a continuous need for photoassimilates for nitrogenase activity. Nodules or nitrogenase activity seemed to be weak sinks for assimilates compared with root pressure bleeding. Measurements of nitrogenase activity in root nodule homogenates supplied with ATP and reductant suggested a loss of active nitrogenase in the nodules in response to water stress. The recovery from moderate stress or long dark treatment took several days, and recovery from severe stress took still longer. Shortage of assimilates and disturbances in oxygen and nitrogen balances in the nodules are discussed as reasons for the reduced nitrogenase activity in response to water stress.