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     

Nodulation of Hydroponically Grown Soybean Plants and Inhibition of Nodule Development by Nitrate

A procedure is described for obtaining a large number of nodules(approximately 1000) on a hydroponically grown soybean plantafter a brief exposure to Rhizobium japonicum. This procedurewas used to study the effect of medium nitrate upon nodulationof the soybean plant. It was found that nitrate, at concentrationsequal to or greater than 2.0 mM, restricted nodule developmentbut did not inhibit appreciably the activity or synthesis ofnitrogenase in those few nodules that were formed. Key words: Soybean, Rhizobium japonicum, Nodulation     

Relationships Between Nitrate and Oxygen Supply in Symbiotic Nitrogen Fixation by White Clover

Exposure of mature, nodulated plants of white clover (Trifoliumrepens) cv. Blanca to 330 mg dm^–3 NO₃-N for 8 d causednitrogenase activity per plant to decrease by 80%. Total nodulatedroot respiration was not significantly affected but analysisof its components showed an 81% decrease in nitrogenase-linkedrespiration and a 340% increase in growth and maintenance respiration.Carbon costs of nitrogenase activity (mol CO₂ respired per molC₂H₄ produced) increased by 45% over the exposure period. Sucrosecontent of the nodules decreased, but the pattern of decreasedid not correlate with that of nitrogenase activity. The oxygendiffusion resistance of the nodules was increased by a factorof five. Characterization of this resistance increase suggestsan abnormal modification of the diffusion barrier and it isconcluded that alteration in the oxygen supply to the bacteroidsis involved in the effect of nitrate on nitrogenase activity. Key words: Nitrogenase activity, nitrate, oxygen     

Nitrogen Fixation, Assimilation and Transport During the Initial Growth Stage of Phaseolus vulgaris L

In nodulated common bean (Phaseolus vulgaris L.), there is typicallya period of N stress between 15 and 20 d after emergence (DAE),due to a lack of synchronization between the depletion of Nin the cotyledons and the beginning of N² fixation and transport.Screening trials identified some Rhizobium leguminosarum bv.phaseoli strains with which symptoms of N deficiency were notvisible (‘precocious’ strains). Cultivar Negro Argelwas then inoculated with two ‘traditional’ strains(C-05 and CIAT 727) and two ‘precocious’ strains(CNPAF 146 and CNPAF 512), and plants were harvested from 8to 30 DAE. There were no differences between the two groupsof strains in nodule dry weight or in the acetylene reductionrates between 8 and 16 DAE. However, nodules induced by the‘precocious’ strains showed earlier onset of glutaminesynthetase (GS) (EC 6.3.1.2 [EC] ) and glutamate synthase (GOGAT)(EC 1.4.1.14 [EC] ) activities, and ureide synthesis. The N concentrationin the nodules formed by ‘precocious’ strains variedfrom 4.2 to 4.5%, whereas with the ‘traditional’strains, it increased from 3.2% at 8 DAE to 65% at 18 DAE, atwhich time plants exhibited N-deficiency symptoms. By 21 DAE,GS and GOGAT activities in ‘traditional’ noduleswere increased, as well as the ureide-N-concentration in thexylem sap, nodule N content declined to 4.5% and the leavesbecame green. These results suggest that the N stress with ‘traditional’strains is not a limitation in early N² fixation activity butrather in the rates of expression of the processes of N assimilationand transport. Key words: Glutamate synthase, glutamine synthetase, nitrogen fixation, Phaseolus vulgaris, Rhizobium     

Nitrogen Fixation and Allantoin Formation in Soybean Plants

The activity of nitrogenase and the concentration of ammonia and allantoin (+ allantoic acid) in root nodules were measured throughout the growth period of soybean plants. Nitrogenase activity measured by acetylene reduction increased with plant growth and reached a maximum level at the flowering period. The level of ammonia and allantoin concentration in nodules was parallel with increased nitrogenase activity. At the late reproductive stage (pod-forming period), nitrogenase activity showed a marked decrease, but the ammonia and allantoin in the nodules remained at a constant level. Detached nodules from 56 day-old soybean plants were exposed to ¹⁵N₂ gas, and the distribution of ¹⁵N among nitrogen compounds was investigated. Enrichment of ¹⁵N in allantoin and allantoic acid reached a fairly high level after 90 min of nitrogen fixation; ca. 22% of ¹⁵N in acid-soluble nitrogen compounds was incorporated into allantoin + allantoic acid. In contrast, enrichment of ¹⁵N in amide nitrogen was relatively low. No significant ¹⁵N was detected in the RNA fraction. The data suggested that ureide formation in nitrogen-fixing root nodules did not take place through the breakdown of nucleic acids, but directly associated with the assimilating system of biologically fixed nitrogen.     

Cyclic Variations in Nitrogen Uptake Rate of Soybean Plants: Effects of External Nitrate Concentration

Net uptake of by non-nodulated soybean plants [Glycine max (L.) Merr.cv. Ransom] growing in flowing hydroponiccultures containing 0–5, 1.0 and 10-0 mol m^–3 was measured daily during a 24-d period of vegetativedevelopment to determine if amplitude of maximum and minimumrates of net uptake are responsive to external concentrations of . Removal of from the replenished solutions during each 24-h period was determinedby ion chromatography. Neither dry matter accumulation nor theperiodicity of oscillations in net uptakerate was altered bythe external concentrations. The maxima of the oscillations in net uptake rate, however, increased nearly3-fold in response to external concentrations. The maxima and minima, respectively, changed from 4.0 and 0–6mmol per gram root dry weight per day at an external solution level of 05 mol m^–3 to 15–2and -2–7 mmol per gram root dry weight per day at an external solution level of 10–0mol m^–3 . The negative values for minimum net uptake rate from10–0 mol m^–3 solutions show that net efflux was occurring andindicate thatthe magnitude of the efflux component of net uptake was responsiveto external concentration of . Key words: Nitrate uptake rate, carbon-nitrogen partitioning, flowing solution culture     

Inhibition of Nitrogen Fixation in Soybean Plants Supplied with Nitrate II. Accumulation and Properties of Nitrosylleghemoglobin in Nodules

The accumulation of nitrosylleghemoglobin (LbNO) in nodulesand the properties of LbNO in vitro were investigated in connectionwith the inhibition of nitrogen fixation in soybean nodulesby nitrate. The leghemoglobin extracted under argon gas from nodules ofplants supplied with nitrate consisted mainly of LbNO, as judgedfrom the spectrum which corresponded to that of LbNO formedin vitro by the reaction of leghemoglobin with nitrite in thepresence of dithionite or by the combination of ferrous leghemoglobin(Lb²⁺) with nitric oxide. Further, LbNO formed in vivo was easilydissociated by visible light, as was LbNO formed in vitro. Thus,authentic LbNO does actually accumulate in nodules. Most of the leghemoglobin was of the ferrous type in nodulesof plants supplied with nitrate. Some LbNO appeared to be derivedfrom LbO₂ which was deoxygenated by nitrite. The increase inlevels of LbNO in nodules paralleled the decrease in acetylenereducing activity. These results indicate that the decrease in nitrogenase activityin nodules of soybean plants supplemented with nitrate is causedby the decrease in levels of LbO₂ that carries oxygen into bacteroids,which results from the formation of LbNO (Received August 22, 1989; Accepted December 4, 1989)     

Increase in Alfalfa Nodulation, Nitrogen Fixation, and Plant Growth by Specific DNA Amplification in Sinorhizobium meliloti

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.     

The Interrelationship between Promoter Strength,Gene Expression,and Growth Rate

In exponentially growing bacteria, expression of heterologous protein impedes cellular growth rates. Quantitative understanding of the relationship between expression and growth rate will advance our ability to forward engineer bacteria, important for metabolic engineering and synthetic biology applications. Recently, a work described a scaling model based on optimal allocation of ribosomes for protein translation. This model quantitatively predicts a linear relationship between microbial growth rate and heterologous protein expression with no free parameters. With the aim of validating this model, we have rigorously quantified the fitness cost of gene expression by using a library of synthetic constitutive promoters to drive expression of two separate proteins (eGFP and amiE) in E. coli in different strains and growth media. In all cases, we demonstrate that the fitness cost is consistent with the previous findings. We expand upon the previous theory by introducing a simple promoter activity model to quantitatively predict how basal promoter strength relates to growth rate and protein expression. We then estimate the amount of protein expression needed to support high flux through a heterologous metabolic pathway and predict the sizable fitness cost associated with enzyme production. This work has broad implications across applied biological sciences because it allows for prediction of the interplay between promoter strength, protein expression, and the resulting cost to microbial growth rates.     

大豆叶面施氮量及氮素利用率的~(15)N标记

为研究大豆(Glycine max)叶面适宜施氮量及叶面氮素吸收与利用的规律,以黑龙江省三江平原大豆主栽品种合农64为实验材料,采用15N标记示踪法在大豆需氮关键时期R5期进行叶面施氮,分析大豆组织器官标记氮素的积累量及回收率。结果表明:在4.5 kg·hm–2(N3)施氮条件下,大豆组织器官干物质量及氮素积累量显著高于其它处理,其中籽粒干物质平均重22.7 g,总干物质平均重73.2 g,分别比不施氮处理(N0)高17.92%和16.38%;籽粒氮素积累量平均为134.4mg·plant–1,比不施氮处理(N0)高13.13%,说明4.5 kg·hm–2(N3)施氮条件是合农64在R5期的最适叶面施氮量。在不同施氮条件下,各组织器官标记氮积累量随着施氮量的增加呈先增加后降低的趋势,籽粒标记氮积累量在4.5 kg·hm–2(N3)施氮条件下最高,为9.96 mg·plant–1。这一结果同样说明了4.5 kg·hm–2(N3)是合农64在R5期的最适叶面施氮量,同时明确了叶面氮素是籽粒氮素积累增加的主要原因。在同一施氮水平下,各组织器官标记15N积累量顺序为籽粒茎叶荚皮叶柄根,且各器官间差异显著,说明在R5期叶面施氮籽粒积累的叶面氮素最多。从15N标记在各组织器官的贡献率来看,在3.5 kg·hm–2(N1)施氮条件下,籽粒氮素贡献率与植株氮素回收率最高,说明在叶面施氮量较小的条件下,氮素更容易被籽粒吸收利用,但净积累量却低于最适施氮量处理(N3)。在3.5 kg·hm–2(N1)施氮条件下,植株氮素回收率高于最适施氮量处理(N3)。     

Plant Growth-Promoting Rhizobacteria Inoculation to Enhance Vegetative Growth,Nitrogen Fixation and Nitrogen Remobilisation of Maize under Greenhouse Conditions

Plant growth-promoting rhizobacteria (PGPR) may provide a biological alternative to fix atmospheric N₂ and delay N remobilisation in maize plant to increase crop yield, based on an understanding that plant-N remobilisation is directly correlated to its plant senescence. Thus, four PGPR strains were selected from a series of bacterial strains isolated from maize roots at two locations in Malaysia. The PGPR strains were screened in vitro for their biochemical plant growth-promoting (PGP) abilities and plant growth promotion assays. These strains were identified as Klebsiella sp. Br1, Klebsiella pneumoniae Fr1, Bacillus pumilus S1r1 and Acinetobacter sp. S3r2 and a reference strain used was Bacillus subtilis UPMB10. All the PGPR strains were tested positive for N₂ fixation, phosphate solubilisation and auxin production by in vitro tests. In a greenhouse experiment with reduced fertiliser-N input (a third of recommended fertiliser-N rate), the N₂ fixation abilities of PGPR in association with maize were determined by ¹⁵N isotope dilution technique at two harvests, namely, prior to anthesis (D₅₀) and ear harvest (D₆₅). The results indicated that dry biomass of top, root and ear, total N content and bacterial colonisations in non-rhizosphere, rhizosphere and endosphere of maize roots were influenced by PGPR inoculation. In particular, the plants inoculated with B. pumilus S1r1 generally outperformed those with the other treatments. They produced the highest N₂ fixing capacity of 30.5% (262 mg N₂ fixed plant⁻¹) and 25.5% (304 mg N₂ fixed plant⁻¹) of the total N requirement of maize top at D₅₀ and D₆₅, respectively. N remobilisation and plant senescence in maize were delayed by PGPR inoculation, which is an indicative of greater grain production. This is indicated by significant interactions between PGPR strains and time of harvests for parameters on N uptake and at. % ¹⁵N_e of tassel. The phenomenon is also supported by the lower N content in tassels of maize treated with PGPR, namely, B. pumilus S1r1, K. pneumoniae Fr1, B. subtilis UPMB10 and Acinetobacter sp. S3r2 at D₆₅ harvest. This study provides evidence that PGPR inoculation, namely, B. pumilus S1r1 can biologically fix atmospheric N₂ and provide an alternative technique, besides plant breeding, to delay N remobilisation in maize plant for higher ear yield (up to 30.9%) with reduced fertiliser-N input.     

Relationship Between Energy Substrate Utilization and Specific Growth Rate in Aspergillus nidulans

The maintenance coefficient of glucose-limited Aspergillus nidulans chemostat cultures at 30 C was 0.018 g per g (dry weight) per hr for glucose and 0.55 mmoles per g (dry weight) per hr for oxygen. These values can only be approximate because melanin was produced by the mold at low growth rates and because it is unlikely that this polymer contributed to the maintenance energy requirement although it contributed to the dry weight. Biomass (defined here as dry weight minus melanin) was used to calculate a more meaningful maintenance coefficient for glucose (0.029 g of glucose per g of biomass per hr). At the highest growth rates examined, a nonlinear relationship between growth rate and glucose utilization rate was obtained, suggesting a qualitative change in the metabolic activities of the mold at high growth rates. The oxidative capacity of the mold was highest at the highest growth rates. This observation indicates that the increased substrate utilization rate observed at the higher growth rates is a reflection of enhanced enzyme synthesis. This hypothesis was verified by assaying the specific activities of several enzymes at different growth rates. However, in contrast to all the other enzymes assayed, the activities of reduced nicotinamide adenine dinucleotide phosphate: (acceptor) oxido-reductases were highest at the lowest growth rates.     

Inhibition of Nitrogen Fixation in Soybean Plants Supplied with Nitrate I. Nitrite Accumulation and Formation of Nitrosylleghemoglobin in Nodules

The accumulation of nitrite in nodules was investigated to elucidatethe mechanism of inhibition of nitrogen fixation in nodulesof soybean (Glycine max. [L.] Merr.) plants supplied with nitrate.Acetylene-reducing activity (ARA) in nodules fell within 24h as a result of the supply of exogenous nitrate, accompaniedby an increase in the accumulation of nitrite in the cytosolbut not in the bacteroids of nodules. Nitrate reductase (NR)activity in the nodule cytosol remained high, irrespective ofthe supply of nitrate. Nitrosylleghemoglobin (LbNO) was detectedspectrophotometrically in the extract from nodules in whichnitrogen fixation was inhibited by nitrate. In experiments invitro, it was found that LbNO was easily formed from leghemoglobinin the presence of nitrite and dithionite. Thus, it is suggested that nitrogen fixation was inhibited primarilyby a decrease in the function of leghemoglobin, attributableto the formation of LbNO, which was caused by the accumulationof nitrite generated from nitrate by NR in the nodule cytosol. (Received August 22, 1989; Accepted January 24, 1990)     

氮处理对大豆根瘤固氮能力及GmLbs基因表达的影响

共生根瘤的固氮效率受外界氮素的严格调控。除固氮酶活性外,豆血红蛋白(Lb)浓度亦是反应固氮能力的重要指标。为明确氮水平对生物固氮作用的影响,以大豆(Glycine max)为材料,在低氮(0.53 mmol·L–1)条件下接种根瘤菌,30天后再进行高氮(5.3、10、20、30和40 mmol·L–1)处理7天,分析L...     

Growth, Nitrogen Fixation, and Spectral Attenuation in Cultivated Trichodesmium Species

Physiological studies of Trichodesmium species have been hindered by difficulties in maintaining actively growing, nitrogen-fixing cultures. Previous cultivation successes have not been widely duplicated. We present here a simple modified seawater medium and handling techniques which have been used to maintain actively growing Trichodesmium thiebautii in laboratory culture for over 1 year. The cultured population, isolated from coastal Atlantic waters, has a growth rate of 0.23 division day^-1 and exhibits light-dependent nitrogen fixation during exponential growth. Various morphologies, including solitary trichomes, and aggregates (spherical puffs and fusiform tufts) are present during growth. Spectral and scalar irradiance were measured within naturally occurring (coastal Atlantic) aggregates with small (diameter, 50 to 70 μm) spherical fiber-optic sensors. In contrast to naturally occurring puffs, cultivated Trichodesmium aggregates exhibited spectral properties consistent with low-light adaptation. Cultivated puff-type aggregates were also examined by using oxygen microelectrodes. The simple medium and approach used for cultivation should be easily reproducible and amenable to further manipulations and modifications useful for physiological studies of Trichodesmium spp. in culture.     

Plant Growth Promoting Rhizobacteria and Soybean [ Glycine max (L.) Merr.] Nodulation and Nitrogen Fixation at Suboptimal Root Zone Temperatures

Co-inoculation of plant growth promoting rhizobacteria (PGPR)withBradyrhizobium has been shown to increase legume nodulationand nitrogen fixation at optimal soil temperatures. Nine rhizobacteriaco-inoculated withBradyrhizobium japonicum532C were tested fortheir ability to reduce the negative effects of low root zonetemperature (RZT) on soybean [Glycine max(L.) Merr.] nodulationand nitrogen fixation. Three RZTs were tested: 25 (optimal),17.5 (somewhat inhibitory), and 15°C (very inhibitory).At each temperature some PGPR strains increased the number ofnodules formed and the amount of fixed nitrogen when co-inoculatedwithB. japonicum,but the stimulatory strains varied with temperatures.The strains that were most stimulatory varied among temperaturesand were as follows: 15°C,Serratia proteamaculans 1-102;17.5°C,S. proteamaculans 1-102andAeromonas hydrophilaP73;25°C,Serratia liquefaciens2-68. Bradyrhizobium japonicum ; Glycine max; plant growth promoting rhizobacteria; suboptimal root zone temperatures     

Cyclic Variations in Nitrogen Uptake Rate in Soybean Plants: Uptake During Reproductive Growth

Net uptake of by non-nodulated soybean plants [Glycme max(L ) Merr cv Ransom] growing in flowing hydroponicculture was measured daily during a 63 d period of reproductivedevelopment between the first florally inductive photopenodand late seed growth Removal of from a replenished solution containing 10 mol m^– was determined by ion chromatography Uptake of continued throughout reproductive development The net uptakerate of cycled between maxima and minima with a periodicity of oscillation of 3 to 7 d during the floralstage and about 6 d during the fruiting stage. Coupled withincreasing concentrations of carbon and C:N ratios in tissues,the oscillations in net uptake rates of are evidence that the demand for carbohydrate by reproductiveorgans is contingent on the availability of nitrogen in theshoot pool rather than that the demand for nitrogen followsthe flux of carbohydrate into reproductive tissues. Key words: Nitrate uptake rate, carbon-nitrogen partitioning, Glycme max (L ) Merrill     

Effect of Nitrogen Supply on the Grass and Clover Components of Simulated Mixed Swards Grown under Favourable Environmental Conditions II. Nitrogen Fixation and Nitrate Uptake

Simulated mixed swards of Perennial Ryegrass (Lolium perenneL.) cv. S23 and White clover (Trifolium repens L.) cv. S100were grown from seed under a constant 20 °C day/15 °Cnight temperature regime and harvested at intervals over and88 d growht period. The swards received a nutrient solutiondaily, which was either High (220 mg l^–1) or Low (10 mgl^–1) in nitrate N. The nitrate was labelled with the ¹⁵Nisotope. An acetylene reduction assay was carried out on eachsward just prior to harvest. Rates of acetylene reduction agreed qualitatively with the ^l5Nanalyses but absolute values did not match (assuming a 4:1 C₂H₄:N₂ratio) and errors in the acetylene assay are discussed. In theLow-N swards clover relied almost entirely on symbioticallyfixed N₂, fixing more than ten times as much as the High-N cloverplants. In the Low-N treatment the grass was N-deficient despiteobtaining much more nitrate per unit root dry weight than clover.In the High-N swards, however, clover took up more nitrate perunit root weight than grass. The High-N clover plants also fixedsome N₂ and maintained a higher total-N content than grass throughoutthe period. There was no evidence of transfer of symbioticallyfixed N from the clover to the grass in either treatment. Trifolium repens, Lolium perenne, nitrate, nitrogen fixation, ¹⁵N, acetylene reduction     

Nitrogen and Nitrate Accumulation in Species Having Different Relationships Between Nitrate Uptake and Reduction

Species differ in the relationship of nitrate reductase activityto nitrate uptake. In Capsicum annuum different diurnal patternsof leaf nitrate reductase activity and nitrate uptake have beenreported. As a consequence, the relationship of free nitratein the plant to nitrate supplied has a higher level of significancethan has reduced nitrogen to nitrate supplied. In Zea mays ithas been reported that leaf nitrate reductase activity respondsdirectly to nitrate translocation to the leaf and in this speciesthe relationship of greatest significance is reduced nitrogencontent to nitrate supplied. In both species, and also in Cucumis melo, the proportion oftotal plant free nitrate and reduced nitrogen in the roots decreases,and in the stem increases, with increasing nitrate supplied. The accumulation of free nitrate in leaves is accompanied bya quantitatively different relationship between reduced nitrogenand dry weight compared to leaves not accumulating nitrate. Capsicum annuum. L., Cucumis melo L., melon, Zea mays L., maize, sweet corn, nitrate reductase, nitrate uptake     

Nitrogen Utilization in Lemna: I. Relations between Net Nitrate Flux, Nitrate Reduction, and in Vitro Activity and Stability of Nitrate Reductase

Cultures of Lemna gibba L. G3 were maintained at a constant, N-limited growth rate by adding nitrate daily in amounts calculated to sustain a rate of culture N increment of 0.20 day⁻¹. Nitrate added to the culture was consumed within 8 to 10 hours and the partitioning to reduction and accumulation during this phase corresponded to, on the average, 75 and 25% of net uptake, respectively. The calculated rate of nitrate reduction was stimulated by onset of net uptake without delay and decreased when net uptake ceased. NADH-nitrate reductase (NR) activity measured in vitro without inclusion of antiproteolytic agents more than doubled during the first hour after nitrate addition and then gradually fell to its original level over the rest of the 24 hour interval. In the presence of the proteinase inhibitor leupeptin during extraction, however, NR activity was in general much higher and without any apparent cycles. The relative stabilizing effect of leupeptin was greatest on NADH-NR and reduced flavin adenine mononucleotide-NR activities whereas the effect was less on NADH-cytochrome c reductase activity (diaphorase) and reduced methylviologen-NR activity. The constant nitrate reductase activity measured in the presence of proteinase inhibitors is assumed to reflect the physiological situation. It thus appeares that short-term changes in nitrate assimilation by N-limited Lemna is related to the flux of nitrate to the reducing site and not to changes in nitrate reductase activity.