Characterization of extracellular oxygen consumption by the green alga Selenastrum minutum

Cells of the green alga Selenastrum minutum display a high capacity for extra-mitochondrial O₂ consumption in the presence of effectors such as salicylhydroxamic acid and/or NADH. We provide evidence that this O₂ consumption is mediated by extracellular peroxidase. Peroxidase capacity, measured as the potential for stimulation of O₂ consumption by a combination of salicylhydroxamic acid and NADH, changed over a 10-day time course. Maximal stimulation of O₂ consumption occurred at day three, at which point the capacity for peroxidase-mediated O₂ consumption was three-to four-fold higher than that of the control O₂ consumption rate. Peroxidase-mediated O₂ consumption was sensitive to inhibition by 50 m M ascorbate and by cyanide. Cyanide titration curves indicated that O₂ consumption by peroxidase was much more sensitive to inhibition by cyanide than was O₂ consumption by cytochrome oxidase (I₅₀ < 1.6 μ M and I₅₀= 18.3 μ M cyanide, respectively). By using evidence from a combination of cyanide titration curves and ascorbate inhibition, we concluded that despite a large capacity for peroxidase-mediated O₂ consumption, peroxidase did not measurably contribute to control rates of O₂ consumption. In the absence of effectors, O₂ consumption was mediated primarily by cytochrome oxidase.     

EXTRACELLULAR PEROXIDASE-MEDIATED OXYGEN CONSUMPTION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

The unicellular green alga Chlamydomonas reinhardtii Dang. displays a high capacity for salicylhydroxamic acid (SHAM)—stimulated O₂ consumption, mediated by extracellular peroxidaie. Addition of exogenous NADH also resulted in stimulation of O₂ consumption. The SHAM-and NADH-stimulated peroxidase activity was partially sensitive to inhibition by exogenous superoxide dismutase, ascorbate, and gentisic acid. These compounds did not inhibit O₂ consumption in the absence of effectors. SHAM-and NADH-stimulated peroxidase activity also was sensitive to inhibition by cyanide, and cyanide titration curves indicated that O₂ consumption by peroxidase was more cyanide-sensitive than O₂ consumption by cytochrome oxidase. The differential sensitivity to cyanide was used to estimate partitioning of O₂ consumption between mitochondrial respiration and extracellular peroxidase. We suggest that, despite a large capacity for peroxidase-me-diated O₂ consumption, peroxidase did not consume O₂ at detectable rates in the absence of effectors. Therefore, in the absence of effectors, measured rates of O₂ consumption represented the rate of mitochondrial respiration .     

Respiration of barley roots: Assessment of activity of the alternative path using SHAM

The activity of the alternative path of O₂ consumption in detached and intact roots of barley [ Hordeum distichum (L.) Lam. cv. Maris Mink] was determined by titration with salicylhydroxamic acid (SHAM) in the presence and absence of cyanide. In the absence of cyanide, only high concentrations were inhibititory (> 5 m M ). whilst in its presence low SHAM concentrations (2.5–5.0 m M ) gave maximum inhibition: the resulting ϱ V_alt plots were non-linear. A SHAM-stimulated peroxidase could readily be washed from these roots, but non-linearity cannot be explained in terms of SHAM-stimulation of this peroxidase as it is not active in the absence of an exogenous supply of NADH. In detached roots the degree of inhibition of respiration with 25 m M SHAM was nearly double the capacity of the alternative path (measured as the degree of inhibition by SHAM in the presence of cyanide), suggesting non-specific inhibition. Effects of SHAM on cytochrome path activity in intact roots were examined by reverse titration with cyanide in the presence and absence of SHAM. At 5 m M SHAM had no effect on the cytochrome path, but at 25 m M it inhibited. We conclude that the only factor causing non-linearity of ϱV_alt plots in barley roots is non-specific inhibition of the cytochrome path by high concentrations of SHAM; consequently only low concentrations of SHAM (2.5–5.0 m M ) are suitable for estimating alternative path activity in barley roots.     

Interactions between respiration and inorganic phosphate uptake in phosphate-limited cells of Chlamydomonas reinhardtii

Phosphate addition to P-limited cells of Chlamydomonas reinhardtii resulted in an immediate increase in the rate of respiratory O₂ consumption. The respiration rate continued to increase for several minutes after the addition of P₁. Similar patterns of P₁ stimulation of respiratory O₂ consumption were observed in the presence of cyanide (cytochrome oxidase inhibitor) and propyl gallate (alternative oxidase inhibitor). Stimulation of O₂ consumption was accompanied by rapid changes in levels of glycolytic intermediates. These changes were consistent with activation of ATP-dependent phosphofructokinase and pyruvate kinase. The adenylate pool exhibited only minor perturbations, P₁, uptake resulted in extracellular acidification, which continued for several minutes after the exhaustion of added P₁, whereas exhaustion of extracellular P₁ resulted in a rapid decline in the O₂ consumption rate. These results are consistent with control of respiration in P-limited cells occurring largely at the level of glycolysis.     

Causes and location of non-specific effects of SHAM on O2 uptake by wheat roots

Uptake of O₂ by whole, detached, root systems of wheat ( Triticum aestivum L. cv. Alexandria) was titrated with salicylhydroxamic acid (SHAM) in the presence and absence of cyanide. The resulting Q_all plot was non-linear indicating that SHAM was acting non-specifically. The nature of the non-specific effects was investigated in reverse titration experiments. Uptake of O₂ was titrated with KCN in the presence and absence of SHAM at 1 m M and 25 m M , which yielded Q_cy1 values of < 1 and > 1, respectively. The results suggest that at 25 m M , SHAM inhibits the cytochrome pathway, but at 1 m M it stimulates an O₂-consuming process which is likely to be a peroxidase. A SHAM-stimulated peroxidase could easily be washed from these roots. In vitro, the peroxidase was stimulated to a similar extent by low (1 m M ) and high (25 m M ) concentrations of SHAM. Failure to inhibit with high concentrations of SHAM distinguishes this peroxidase from those bitherto eluted from root tissue. Reverse titration experiments in the presence and absence of 1 m M SHAM indicated that there were no significant side effects of SHAM in root tips. These data are supported by the negligible peroxidase activity that was washed from this root fraction. In contrast, significant side effects occurred in vivo, and substantial peroxidase activity was measured in vitro, from sections 4–6 cm and 18–20 cm behind the seminal root apex. The greatest activity was found with the 4–6 cm section which may be associated with high rates of cell wall lignification. The implications of these results for measurements of root respiration are discussed.     

Effects of salicylhydroxamate on respiration, seed germination and seedling growth in Avena fatua

The effects of inhibitors of alternative respiration [salicylhydroxamate (SHAM) and propyl gallate (PG)] on germination, seedling growth and O₂ uptake in Avena fatua L. (wild oats) were studied. SHAM did not inhibit germination or O₂ uptake prior to germination. SHAM-sensitive (alternative) respiration, therefore, cannot be a pre-requisite for germination. Following germination, both chemicals inhibited seedling growth with the root being more susceptible than the shoot. SHAM concentrations that inhibited root growth by 90 to 95%, inhibited O₂ uptake of 1 cm root apices by less than 15%. While sodium azide (a cytochrome-oxidase inhibitor; 1 m M ) alone inhibited O₂ uptake by only 40 to 50%, in the simultaneous presence of SHAM (or PG), O₂ uptake was inhibited by 90 to 99%. Thus: 1) respiration of wild oat seedling root apices is predominantly cytochrome-mediated and incomplete inhibition of O₂ uptake in the presence of azide alone is due to diversion of electrons to the alternative pathway and 2) even though these roots have little alternative respiration, they maintain the capacity to support a much greater flux of electrons via this path way. SHAM and PG at concentrations (0.05 to 0.4 m M ) which inhibited O₂ uptake significantly in the presence (but not in the absence) of azide had little effect on root growth suggesting that an effect(s) other than that on respiration is involved in the inhibition of root growth at higher concentrations. The effect of SHAM on wild oat root growth is not selective as it also inhibits growth of a number of crop species.     

Terminal oxidases of Crithidia oncopelti

Abstract Washed cell suspensions of Crithidia oncopelti oxidizing a variety of substrates gave complex plots for the inhibition of respiration by potassium cyanide or azide. The data indicated the presence of at least two and possibly three terminal oxidases on the basis of their differential sensitivity to these inhibitors. The oxidase most sensitive to cyanide, azide and CO accounted for approx. 65–70% of whole cell respiration and is probably cytochrome oxidase a/a₃. A second oxidase exhibiting low affinity for CO required high concentrations of KCN or azide for inhibition. This haemoprotein had the spectral characteristics of cytochrome o and accounted for 15–20% of cell respiration. Incomplete inhibition of respiration by high concentrations of KCN or azide suggested the presence of a third oxidase which was CO-unreactive.     

Salicylhydroxamic acid-stimulated NADH oxidation by purified plasmalemma vesicles from wheat roots

Purified, right side-out plasmalemma vesicles were isolated from 7-day-old roots of dark-grown wheat ( Triticum aestivum L. cv. Drabant) by aqueous polymer two-phase partitioning. The oxygen consumption by these vesicles at pH 6.5 in the presence of 1 m M NADH [12–29 nmol (mg protein)⁻¹min⁻¹] was 66% inhibited by 1 m M KCN and ca 40% by 1 m M EDTA. It was unaffected by rotenone, antimycin A, carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), mersalyl, chlorotetracycline + Ca²⁺, and EGTA. Salicylhydroxamic acid (SHAM) and its analogue, m -chlorobenzhydroxamic acid, stimulated the rate of oxygen consumption 10–20 fold in the presence of 1 m M NAD(P)H with an apparent K_m (SHAM) of ca 40 μ M (with NADH). The dependence of O₂ consumption on NADH concentration in the presence of SHAM (2 m M ) was sigmoidal, possibly due to endogenous catalase activity, and half-maximal rate was obtained at 1.5 m M . In the absence of SHAM the rate increased with increasing acidity and no pH optimum was detectable between pH 4.5 and 8.5. In the presence of SHAM an optimum was observed at pH 6.5 and 0.8 mol of H₂O₂ was produced for every 1 mol O₂ consumed. Endogenous catalase converted this H₂O₂ to O₂ and after complete conversion the stoichiometry was 2 mol NADH consumed for every mol O₃. SHAM was not consumed in the reaction. The possible involvement of a cytochrome P-450/420 system is discussed.     

Importance of oxidative electron transport over oxidative phosphorylation in optimizing photosynthesis in mesophyll protoplasts of pea (Pisum sativum L.)

The role of mitochondrial respiration in optimizing photosynthesis was assessed in mesophyll protoplasts of pea ( Pisum sativum L., cv. Arkel) by using low concentrations of oligomycin (an inhibitor of oxidative phosphorylation), antimycin A (inhibits cytochrome pathway of electron transport) and salicylhydroxamic acid (SHAM, an inhibitor of alternative oxidase). All three compounds decreased the rate of photosynthetic O₂ evolution in mesophyll protoplasts, but did not affect chloroplast photosynthesis. The inhibition of photosynthesis by these mitochondrial inhibitors was stronger at optimal CO₂ (1.0 m M NaHCO₃) than that at limiting CO₂ (0.1 m M NaHCO₃). We conclude that mitochondrial metabolism through both cytochrome and alternative pathways is essential for optimizing photosynthesis at limiting as well as at optimal CO₂. The ratios of ATP to ADP in whole protoplast extracts were hardly affected, despite the marked decrease in their photosynthetic rates by SHAM. Similarly, the decrease in the ATP/ADP ratio by oligomycin or antimycin A was more pronounced at limiting CO₂ than at optimal CO₂. The mitochondrial oxidative electron transport, through both cytochrome and alternative pathways, therefore akppears to be more important than oxidative phosphorylation in optimizing photosynthesis, particularly at limiting CO₂ (when ATP demand is expected to be low). Our results also confirm that the alternative pathway has a significant role in contributing to the cellular ATP, when the cytochrome pathway is limited.     

The relationship between the in situ reduction level of the cytochrome c pool of Azorhizobium caulinodans growing in a chemostat with NH4+ or N2 as the N source and the total activity of cytochrome c oxidases

Abstract The in situ method for determination of reduction levels of cytochromes b and c pools during steady-state growth (Pronk et al., Anal. Biochem. 214, 149–155, 1993) was applied to chemostat cultures of the wild-type, a cytochrome aa₃ single mutant and a cytochrome aa₃/d double mutant of Azorhizobium caulinodans . For growth with NH₄⁺ as the N source, the results indicate that (i) the aa₃ mutant strains growing at a dissolved O₂ tension of 0.5% possess an active alternative cytochrome c oxidase, which is hardly present during fully aerobic growth, and assuming that (i) also pertains to the wild-type, (ii) the wild-type uses cytochrome aa₃ under fully aerobic conditions. For growth with N₂ as the N source, it was found that the aa₃ mutant strains growing at dissolved O₂ tensions ranging from 0.5 to 3.0% also contain an active alternative cytochrome c oxidase.     

The Content of Alternative Oxidase of Euglena Mitochondria is Increased by Growth in the Presence of Cyanide and is not Cytochrome o.

ABSTRACT A study of the effect of respiratory inhibitors on O₂ uptake of Euglena gracilis mitochondria, isolated from cells grown in the presence of cyanide or with ethanol as carbon source, was undertaken. The contents of cytochrome c oxidase and alternative oxidase were also determined. Inhibition of respiration by antimycin and cyanide was only partial and it was dependent on the oxidizable substrate used. Succinate oxidation was the most sensitive to cyanide whereas lactate oxidation was the most resistant. Cell growth in the presence of cyanide or with ethanol as carbon source brought about an enhanced content of alternative oxidase without a concomitant increase in cytochrome aa₃ content. However, a correlation between cyanide-resistant respiration and alternative oxidase content was not found. Analysis of heme types in mitochondrial membranes revealed the absence of heme O. The data suggest the presence of an inducible alternative oxidase in Euglena mitochondria which has high resistance to cyanide and contains heme B. A close relationship between Euglena alternative oxidase and bacterial quinol oxidases containing B-type heme is proposed.     

Half reduction potentials and oxygen affinity of the cytochromes of Pseudomonas carboxydovorans

Abstract The midpoint redox potentials (E'₀) of the cytochromes of Pseudomonas carboxydovorans have been studied by means of coupled spectrum deconvolution and potentiometric analysis. Membranes of cells grown on different substrates (CO; H₂+ CO₂; or pyruvate) contained cytochromes with similar absorption peaks and redox potentials. The cytochromes of the CO-sensitive main electron pathway of the respiratory chain revealed redox potentials in the same range as mitochondrial cytochromes (cytochrome b -555, about −20 mV; cytochrome c and cytochrome a , about +220 mV). For the cytochromes of the CO-insensitive alternative electron pathway, which allows uninhibited growth and respiration in the presence of high concentrations of CO, redox potentials of approx. +50 mV (cytochrome b -558) and −11 to −215 mV (cytochrome b -561) were determined. Cytochrome [ib-561], earlier proposed as the alternative terminal oxidase o in this organism, was shown to possess the lowest half reduction potential of all the cytochromes present in the cells. Measurements of the apparent K _m value for oxygen revealed a low affinity of cytochrome a ( K _m/ 5 υ M O₂) and a very high affinity of the CO-insensitive oxidase ( K _m < 0.5 μ M O₂). The high affinity to oxygen might be responsible for the CO-insensitivity of this unusual cytochrome o .     

EFFECT OF DISSOLVED OXYGEN PARTIAL PRESSURE ON THE ACCUMULATION OF ASTAXANTHIN IN CHEMOSTAT CULTURES OF HAEMATOCOCCUS LACUSTRIS (CHLOROPHYTA)

The effect of dissolved oxygen partial pressure on the accumulation of astaxanthin in the green alga Haematococcus lacustris ( Gir.) Rostaf (UTEX16) was studied in N-limited continuous chemostat cultures. The steady-state astaxanthin content measured against culture volume, cell number, and biomass dry weigh of Haematococcus cultures was proportional to the dissolved O₂ partial pressure in the culture medium, over the range of 0–50% O₂ The steady-state biomass dry weight concentrations remained at between 0.52 and 0.57 g. L^-1 over the range of dissolved O₂ partial pressure studied. Steady-state cell densities at dissolved O₂ partial pressures above the air saturation level (1.13–1.58 × 10⁵ cells.mL^-1) were about half of that measured at lower dissolved O₂ partial pressures (2.42–2.63 × 10⁵ cells.mL^-1). Both biflagellated zoospores and nonmotile aplanospores were found at steady state. The fraction of nonmotile cells was higher at dissolved O₂ partial pressures above the air saturation level (94.44–98.01%) than at dissolved O₂ partial pressure below the air level (79.64–86.12 and 91.75% ).     

Urate oxidase of Chlamydomonas reinhardii

Urate oxidase (EC 1.7.3.3) of Chlamydomonas reinhardii cells grown on purines and purine derivatives has been partially characterized. Crude enzyme preparations have a pH optimum of 9.0, require O₂ for activity, have an apparent K_m of 12 μ M for urate, and are inhibited by high concentrations of this substrate. Enzyme activity was particularly sensitive to metal ion chelating agents like cyanide, cupferron, diethyldithiocarbamate and o -phenanthroline, and to structural analogues of urate like hypoxanthine and xanthine. Chlamydomonas cells grow phototrophically on adenine, guanine, hypoxanthine, xanthine, urate, allantoin or allantoate as sole nitrogen source, indicating that in this alga the standard pathway of aerobic degradation of purines of higher plants, animals and many microorganisms operates. As deduced from experiments in vivo , urate oxidase from Chlamydomonas is repressed in the presence of ammonia or nitrate.     

Hydrogen Peroxide Production by Rat Brain In Vivo

Abstract: H₂ O₂ production by rat brain in vivo was observed with a method based on the measurement of brain catalase. The administration to the rat of 3-amino-1, 2, 4-triazole, an H₂ O_2- dependent inhibitor of catalase, caused progressive inhibition of brain catalase activity in both the supernatant and pellet fractions of homogenates of the striatum and prefrontal cortex. The prevention of catalase inhibition by prior administration of ethanol confirmed that catalase inhibition in vivo was dependent upon H₂ O₂. A significant portion of the catalase (30-33%) appeared in the supernatant fraction from a slow-speed homogenization procedure and was not significantly contaminated by either erythrocytes or capillaries. In the whole homogenate, less than 6% of the catalase activity was attributed to erythrocytes. Modification of intracellular monoamine oxidase activity by either pargyline or reserpine did not change the rate of inhibition of catalase by aminotriazole. A probable interpretation of these data is that H₂ O₂ generated by mitochondrial monoamine oxidase does not reach the catalase compartment; the catalase is contained in particles described by other investigators as the microperoxisomes of brain. In studies in vitro , the production of H₂ O₂ by rat brain mitochondria with either dopamine or serotonin as substrate was confirmed.     

OXYGEN CONSUMPTION ASSOCIATED WITH FERRIC REDUCTASE ACTIVITY AND IRON UPTAKE BY IRON-LIMITED CELLS OF CHLORELLA KESSLERI (CHLOROPHYCEAE)

Plasma membrane ferric reductase activity was enhanced 5-fold under iron limitation in the unicellular green alga Chlorella kessleri Fott et Nováková. Furthermore, ferric reductase activity in iron-limited cells was approximately 50% higher in the light than in the dark. In contrast, iron uptake rates of iron-limited cells were unaffected by light versus dark treatments. Rates of iron uptake were much lower than rates of ferric reduction, averaging approximately 2% of the dark ferric reduction rate. Ferric reduction was associated with an increased rate of O₂ consumption in both light and dark, the increase in the light being approximately 1.5 times as large as in the dark. The increased rate of O₂ consumption could be decreased by half by the addition of catalase, indicating that H₂O₂ is the product of the O₂ consumption and that the increased O₂ consumption is nonrespiratory. The stimulation of O₂ consumption was almost completely abolished by the addition of bathophenanthroline disulfonate, a strong chelator of Fe^{2 +} . Anaerobic conditions or the presence of exogenous superoxide dismutase affected neither ferric reduction nor iron uptake. We suggest that the O₂ consumption associated with ferric reductase activity resulted from superoxide formation from the aerobic oxidation of Fe^{2 +} , which is the product of ferric reductase activity. At saturating concentrations of Fe^{3 +} chelates, ferric reductase activity is much greater than the iron uptake rate, leading to rapid oxidation of Fe^{2 +} and superoxide generation. Therefore, O₂ consumption is not an integral part of the iron assimilation process.     

A direct demonstration of "co-respiration" of oxygen and nitrogen oxides by Pseudomonas nautica: some spectral and kinetic properties of the respiratory components

Abstract: Pseudomonas nautica grown anaerobically is capable of simultaneously utilizing oxygen and nitrate or its reduced products (nitrite and nitrous oxide). Evidence for this 'co-respiration' came from kinetic studies on oxygen consumption depending on oxygen concentration and from spectral studies which revealed changes in the cytochromes composition of the electron transport chain under aerobic or anaerobic conditions. A constitutive o -type cytochrome oxidase was detected either aerobically or anaerobically with an apparent K _m for O₂ evaluated at 315 μM. Two oxidases were induced only in anaerobic conditions. One of these two enzymes identified as a cd -type cytochrome oxidase shows a relatively high affinity for oxygen with an apparent K _m value of 25 μM.     

Hydrogen Production by Rumen Holotrich Protozoa: Effects of Oxygen and Implications for Metabolic Control by In Situ Conditions

Experiments with washed suspensions of holotrich protozoa (Isotricha spp. and Dasytricha ruminantium ) showed that both organisms have an efficient 0,-scavenging capability (apparent K_m values 2.3 and 0.3 μM, respectively). Reversible inhibition of H₂, production increased almost linearly with increasing O₂ up to 1.5 μM; higher levels of O₂ gave irreversible inhibition. In situ determinations of H, CH₄, O₂, and CO₂, in ovine rumen liquor, using a membrane inlet mass spectrometer probe, indicated that O₂, was present before feeding at 1-1.5 μM and decreased to undetectable levels (<0.25 μM) within 25 min after feeding. A transient increase in O₂. concentration after feeding occurred only in defaunated animals and resulted in suppression of CH₄ and CO₂ production. The presence of washed holotrich protozoa decreases the O₂ sensitivity of CH₄ production by suspensions of a cultured methanogenic bacterium Methanosarcina barkeri . It is concluded that holotrich protozoa play a role in ruminal O₂ utilization as well as in the production of fermentation end products (especially short-chain volatile fatty acids) utilized by the ruminant and H, utilized by methanogenic bacteria. These hydrogenosome-containing protozoa thus both control patterns of fermentation by influencing O₂ levels, and are themselves regulated by the low ambient O₂ concentrations they experience in the rumen.     

Oxygen Perception and O2 Toxicity in the Freshwater Ciliated Protozoon Loxodes

ABSTRACT. Loxodes reached peak abundance close to the oxic-anoxic boundary (O₂ 5% atm) in two lakes, in test tube cultures, and in glass chambers with horizontal O₂ gradients. Vertical profiles of CO₂, pH, sulfide, and Fe²⁺ in a lake were not closely related to Loxodes abundance. In a laboratory experiment, Loxodes followed a retreating source of O₂ and was repelled by a high pO₂. This behavior was sustained when cells simultaneously swam up or down gradients of both CO₂ and pH. Aggregation of cells was abolished by KCN (10^-4-10^-6 M). Sodium azide (10^-1-10^-4 M) had no effect and 2,4-DNP sharpened the aggregation. Rotenone, Antimycin A, and HOQNO had no obvious effect. Cytochrome oxidase is probably the oxygen receptor. Loxodes striatus contained low activities of superoxide dismutase and catalase. Extracellular production of superoxide (O_-²) and hydrogen peroxide (H₂O₂) were probably not responsible for the exclusion of Loxodes from water with a high pO₂. Continuous exposure of Loxodes to oxygen at normal atmospheric pressure at 10°C led to 50% mortality in 10 days. Cells left free to swim in an oxygen gradient doubled their number in the same period. Light exacerbated the toxic effects of O₂. Behavioral responses to the dissolved oxygen tension probably controlled the spatial distribution of Loxodes.     

Oxygen control prevents denitrifiers and barley plant roots from directly competing for nitrate

Abstract Two denitrifying bacteria ( Pseudomonas chlororaphis and P. aureofaciens ) and a plant (barley, Hordeum vulgare ) were used to study the effect of O₂ concentration on denitrification and NO₃⁻ uptake by roots under well-defined aeration conditions. Bacterial cells in the early stationary phase were kept in a chemostat vessel with vigorous stirring and thus a uniform O₂ concentration in the solution. Both Pseudomonads lacked N₂O reductase and so total denitrification could be directly measured as N₂O production.
Denitrification decreased to 6–13% of the anaerobic rate at 0.01% O₂ saturation (0.14 μM O₂) and was totally inhibited at 0.04% O₂ saturation (0.56 μM O₂). In this well-mixed system denitrification was 10-times more oxygen sensitive than stated in earlier reports. Uptake of nitrate by plants was measured in the same system under light. The NO₃⁻ uptake rate decreased gradually from a maximum in 21% O₂-saturated medium (air saturated) to zero at 1.6% O₂ saturation (22.4 μM O₂). Owing to the very different non-overlapping oxygen requirements of the two processes, direct competition for nitrate between plant roots and denitrifying bacteria cannot occur.