Key role of teichoic acids on aflatoxin B1 binding by probiotic bacteria

Aims:  To assess the ability of five probiotic bacteria to bind aflatoxin B₁ and to determine the key role of teichoic acids in the binding mechanism.
Methods and Results:  The strains were incubated in aqueous solutions containing aflatoxin B₁ (AFB₁). The amount of free toxin was quantified by HPLC. Stability of the bacteria–aflatoxin complex was evaluated by repeated washes with buffer. In order to understand the binding process, protoplasts, spheroplasts and cell wall components of two strains were analysed to assess their capacity to bind AFB₁. Additionally, the role of teichoic acids in the AFB₁ binding process was assessed. Lactobacillus reuteri strain NRRL14171 and Lactobacillus casei strain Shirota were the most efficient strains for binding AFB₁. The stability of the AFB₁–bacteria complex appears to be related to the binding ability of a particular strain; AFB₁ binding was also pH-dependent. Our results suggest that teichoic acids could be responsible for this ability.
Conclusions:  Our results provide information concerning AFB₁ binding by previously untested strains, leading to enhanced understanding of the mechanism by which probiotic bacteria bind AFB₁.
Significance and Impact of the Study:  Our results support the suggestion that some probiotic bacteria could prevent absorption of aflatoxin from the gastrointestinal tract.     

Effect of modification of storage atmosphere on phospholipids and ultrastructure of cauliflower mitochondria

Differences in mitochondrial membrane composition and ultrastructure were studied after storage of cauliflower ( Brassica oleracea , L., Botrytis group) for 5 days at 25°C in air or under controlled atmospheres: 3% O₂, 21% O₂+ 15% CO₂ or 3% O₂+ 15% CO₂. In air, postharvest senescence involved a 20% decrease in mitochondrial phospholipid content. A large reduction in the relative abundance of phosphati-dylcholine (PC) and in the degree of unsaturation of PC and phosphatidyl ethanolamine (PE) was observed. However, the degree of unsaturation increased in cardiolipin (CL). Storage under 3% O₂ did not prevent phospholipid breakdown. Low O₂ prevented the relative decrease in PC observed during storage in air and the loss of linoleic acid from PC, but not from PE. This relative protection offered by the low O₂ atmosphere was lost under 3% O₂+ 15% CO₂. The high CO₂ atmospheres caused twice as much loss in phospholipids as that observed during storage in air. Extensive loss of mitochondrial protein, a marked decrease in phospholipid to protein ratio, and electron micrograph observations suggest structural alterations in the presence of high CO₂.     

Properties of β-galactosidase of Lactobacillus kefiranofaciens K-1 isolated from kefir grains

β-Galactosidase from Lactobacillus kefiranofaciens K-1 was isolated and characterized. Optimal temperature and pH for the enzyme reaction were 50^°C and pH 6.5, respectively. Molecular weight was estimated to be approximately 311000. Glucose and galactose inhibited the activity, but the inhibition by galactose was rather weaker than observed in other β-galactosidases. MnCl₂ and MgCl₂ had no effect on the activity. FeSO₄, AgNO₃ and HgCl₂ acted as the inhibitor. β-Mercaptoethanol and L-cysteine activated the enzyme, while iodoacetamide inhibited the activity. The K _m values were 4.92 mmol/1 for ONPG and 1.27 mmol/1 for lactose.     

Effect of Hyperbaric Carbon Dioxide Pressure on the Microbial Flora of Pork Stored at 4 or 14°C

Changes in the microbial flora of pork stored at 4 or 14°C were studied in 5 atm CO₂, 1 atm CO₂ or 1 atm air. The time needed for the total aerobic count at 4°C to reach 5 × 10⁶ organisms/cm² was about three times longer in 5 atm CO₂ than in 1 atm CO₂, and about 15 times longer in 5 atm CO₂ than in air. At 14°C there was no difference in growth rate between 5 atm CO₂ and 1 atm CO₂. No off-odour was detected after storage in 5 atm CO₂ for 14 d, but the pork in 1 atm CO₂ (6 d) was organoleptically unacceptable.
The predominant organisms on the pork from the processing line were: Flavobacterium spp., Acinetobacter calcoaceticus, Pseudomonas spp., Micrococcus spp. and Moraxella spp. After aerobic storage at 4°C (8 d) or 14°C (3 d) more than 90% of the flora consisted of Pseudomonas spp. At 4°C all Pseudomonas spp. were of the non-fluorescent type, whilst at 14°C 32% were Ps. putida and Ps. fluorescens. After storage in 1 atm CO₂ Lactobacillus spp. represented 66% of the flora at 14°C (6 d) and 100% at 4°C (40 d), with L. xylosus dominating. After storage in 5 atm CO₂ Lactobacillus spp. constituted the total flora at both temperatures with L. lactis (14°C) and L. xylosus (4°C) dominating.
It was concluded that high partial pressures of CO₂ have a considerable shelf-life prolonging effect by (i) selecting the microflora towards Lactobacillus spp. and (ii) reducing the growth rate of these Lactobacillus spp. The controlling and growth inhibitory effect of CO₂ was promoted by reduced temperatures.     

Regulation by CO2 of 1-aminocyclopropane-1-carboxylic acid conversion to ethylene in climacteric fruits

Cheverry, J. L., Sy, M. O., Pouliquen, J. and Marcellin, P. 1988. Regulation by CO₂ of 1-aminocyclopropane-1-carboxylic acid conversion to ethylene in climateric fruits. - Physiol. Plant. 72: 535–540.
A high CO₂ concentration (20%) at 20°C rapidly and strongly inhibited the development of the climacteric ethylene burst in apple ( Malus domestica Borkh. cv. Granny Smith) and avocado ( Persea americana Mill. cv. Fuerte) fruits and did not change 1-aminocyclopropane-l-carboxylic acid (ACC) content. Treatment with 20% CO₂ markedly decreased ACC-dependent ethylene biosynthesis at 20°C in climacteric pericarp tissues. It is suggested, therefore, that high CO₂ levels inhibit conversion of ACC to ethylene.
Synthesis of the ethylene forming enzyme (EFE) was enhanced when intact preclimacteric apples or early climacteric avocados were pretreated for 40 h with 10 μ11^-1 ethylene. When CO₂ (20%) and ethylene were both applied, a reduced stimulatory effect of ethylene on EFE synthesis was observed. A high CO₂ concentration enhanced EFE acivity in excised tissues of apples and avocados incubated with ACC (2 m M ) and cycloheximide (1 m M ) or 2–5-norbornadiene (5 ml 1^-1). In the autocatalytic process, 20% CO₂ antagonized the stimulation of EFE synthesis by ethylene, but promoted EFE activity.     

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.     

Elevated atmospheric CO2 does not affect per se the preference for symbiotic nitrogen as opposed to mineral nitrogen of Trifolium repens L.

The objective of this investigation was to examine the effect of an elevated atmospheric CO₂ partial pressure ( p CO₂) on the N-sink strength and performance of symbiotic N₂ fixation in Trifolium repens L. cv. Milkanova. After initial growth under ambient p CO₂ in a nitrogen-free nutrient solution, T. repens in the exponential growth stage was exposed to ambient and elevated p CO₂ (35 and 60 Pa) and two levels of mineral N (N-free and 7·5 mol m^–3 N) for 36 d in single pots filled with silica sand in growth chambers. Elevated p CO₂ evoked a significant increase in biomass production from day 12 after the start of CO₂ enrichment. For plants supplied with 7·5 mol m^–3 N, the relative contribution of symbiotically fixed N (%N_sym) as opposed to N assimilated from mineral sources (¹⁵N-isotope-dilution method), dropped to 40%. However, in the presence of this high level of mineral N, %N_sym was unaffected by atmospheric p CO₂ over the entire experimental period. In plants fully dependent on N₂ fixation, the increase in N yield reflects a stimulation of symbiotic N₂ fixation that was the result of the formation of more nodules rather than of higher specific N₂ fixation. These results are discussed with regard to physiological processes governing symbiotic N₂ fixation and to the response of symbiotic N₂ fixation to elevated p CO₂ in field-grown T. repens .     

High yield isolation of mesophyll protoplasts from wheat, barley and rye

Efficient procedures are described for high-yield isolation of mesophyll protoplasts from spring wheat ( Triticum aestivum L. cv. Glenlea), winter wheat ( Triticum aestivum L. cv. Frederick), barley ( Hordeum vulgare L. cv. Bruce) and rye ( Secale cereale L. cv. Puma). Factors such as plant age, composition of the incubation medium during isolation, purification procedures and culture medium affect protoplast yield, viability and metabolic competence, as measured by light-dependent CO₂ fixation. Optimal osmolarity of the isolation medium was equivalent to 1.8 times that measured in the leaves of all plant material used. The presence of 2 m M ascorbic acid in the preincubation and isolation medium increased the yield by 50% and conserved viability and metabolic competence. The protoplasts were stable for up to 48 h without loss of either viability or of original activity of CO₂ fixation, which was in the order of 100 μmol CO₂ (mg chl)⁻¹h⁻¹.
In our MC-56 liquid medium these protoplasts regenerated cell walls within 72 h and a few divided.     

CATABOLISM OF PROSTAGLANDIN ENDOPEROXIDES INTO PROSTAGLANDIN E2 AND F2x BY THE RAT BRAIN

Abstract— Tritium labeled prostaglandin (PG) endoperoxides PGG₂ and PGH₂ were rapidly transformed (2 min, 37°C) in good yield (> 50%) by homogenates of whole rat brain into a mixture of products including prostaglandin E₂ and F_2x: under similar conditions (10min. 37°C) tritium labeled arachidonic acid remained essentially unoxidised. The ratio of PGE-like products: PGF_2x formed was approx 0.5 as determined by radio thin layer chromatography. This ratio changed to unity when homogenates of cerebral cortex or cerebral hemispheres were employed. On the other hand cerebellar homogenates formed PGF_2x in much greater amounts. The structures of the products were confirmed by mass spectrometry and were further supported by experiments using octadeuterio-endoperoxides. In the latter experiments the resulting PGE₂ and PGF_2x contained the expected seven and eight deuterium atoms respectively. Evidence for the formation of heptadeuterio PGD₂. heptadeuterio-6-keto-PGF₁, and hexadeuterio 12-hydroxyheptadecatrienoic acid was also obtained by mass spectrometry. These experiments demonstrate for the first time in brain tissue the biosynthesis of labeled prostaglandins from exogenous tritiated and deuterated precursors.     

Genetic analysis of seed size, yield and days to flowering in a chickpea recombinant inbred line population derived from a Kabuli × Desi cross

Quantitative traits, seed size, yield and days to flowering were studied in a chickpea intraspecific recombinant inbred line (RIL) population (F_6:7) derived from a Kabuli × Desi cross. The population was evaluated in two locations over 2 years. Days to flowering was also evaluated in the greenhouse under short-day conditions. Seed size was the most heritable trait (0.90), followed by days to flowering (0.36) and yield (0.14). Negative and significant correlation was found between yield and seed size in the second year where environmental homogeneity was tested by analysing the controls included in each assay. During the first year, the environment was not considered homogeneous for yield in either location. Quantitative trait loci (QTLs) for the three characters were detected in linkage group (LG) 4. In relation to seed size, two QTLs were located in LG4 (QTL_SW1) and LG8 (QTL_SW2). QTL_SW1 accounted 20.3% of the total phenotypic variation and QTL_SW2 explained 10.1%. A QTL for yield (QTL_YD) was located in LG4 explaining around 13% of variation. QTL_YD might be pleiotropic with QTL_SW1. For days to flowering, a QTL (QTL_DF1) was located in LG4 for all environments analysed explaining around 20% of variation. QTL_DF1 was closely linked to QTL_SW1 and QTL_YD in LG4.     

Triiodothyronine Is a High-Affinity Inhibitor of Amino Acid Transport System L1 in Cultured Astrocytes

Abstract: The relationship between the transport of thyroid hormones and that of amino acids was examined by measuring the uptake of amino acids that are characteristic substrates of systems L, A, and N, and the effect of 3,3',5-triiodo-L-thyronine (T₃) on this uptake, in cultured astrocytes. Tryptophan and leucine uptakes were rapid, Na⁺-independent, and efficiently inhibited by T₃ (half-inhibition at ∼ 2 μ M ). Two Na⁺-independent L-like systems (L₁ and L₂), common to leucine and aromatic amino acids, were characterized kinetically. System L₂ had a low affinity for leucine and tryptophan ( K _m= 0.3–0.9 m M ). The high-affinity system L₁ ( K _m∼ 10 μ M for both amino acids) was competitively inhibited by T₃ with a K _i of 2–3 μ M (close to the T₃ transport K _m). Several T₃ analogues inhibited system L₁ and the T₃ transport system similarly. Glutamine uptake and α-(methylamino)isobutyric acid uptake were, respectively, two and 200 times lower than tryptophan and leucine uptakes. T₃ had little effect on the uptakes of glutamine and α-(methylamino)isobutyric acid. The results indicate that the T₃ transport system and system L₁ are related.     

Effect of temperature on swimming performance of sea bass juveniles

At four temperatures ( T= 15, 20, 25 and 28° C) swimming performance of Dicentrarchus labrax was significantly correlated with total length (23–43 mm L _T); r²=0.623–0.829). The relative critical swimming speed ( RU _crit= U _crit L _T⁻¹), where U _crit is the critical swimming speed, was constant throughout the L _T range studied. The significant effect of temperature on the relative critical swimming speed was described binomially: RU _crit=−0.0323T²+ 1.578 T −10.588 (r²=1). The estimated maximum RU _crit (8.69 L _T s⁻¹) was achieved at 24.4° C, and the 90% performance level was estimated between 19.3 and 29.6° C.     

The comparative effects of nitrogen and oxygen on the microflora of beef steaks in carbon dioxide-containing modified atmosphere vacuum skin-packaging (MA-VSP) systems

The effects of 80% oxygen–20% carbon dioxide (O₂–CO₂) and 80% nitrogen–20% carbon dioxide (N₂–CO₂) atmospheres were compared with respect to the microbial and sensory characteristics of vacuum skin-packaged grain-fed beef steaks stored at −1 and 4 °C. In both N₂–CO₂ and O₂–CO₂ atmospheres, lactobacilli were predominant over Brochothrix , pseudomonads, enterobacteria and yeasts and moulds. The results of the current investigation showed that the O₂–CO₂ atmospheres did not yield total viable counts in excess of 10⁵ cfu cm⁻² on beef steaks after 4 weeks of storage. However, the sensory analysis and thiobarbituric acid (TBA) values (as a measure of oxidative rancidity) of the products were unacceptable at this time. In contrast, the N₂–CO₂ atmospheres yielded maximum total viable counts of approximately 10⁷ cfu cm⁻² and the sensory analysis and TBA values of the product were judged to be acceptable after 4 weeks of storage at −1 °C. These results indicate that sensory effects of the product were influenced to a greater extent by the chemical effects of high concentration of O₂ on rancidity than by the high levels of lactobacilli.     

Flooding, gas exchange and hydraulic root conductivity of highbush blueberry

Highbush blueberry plants ( Vaccinium corymbosum L. cv. Bluecrop) growing in containers were flooded in the laboratory for various durations to determine the effect of flooding on carbon assimilation, photosynthetic response to varying CO₂ and O₂ concentrations and apparent quantum yield as measured in an open flow gas analysis system. Hydraulic conductivity of the root was also measured using a pressure chamber. Root conductivity was lower and the effect of increasing CO₂ levels on carbon assimilation less for flooded than unflooded plants after short-(i-2 days), intermediate-(10–14 days) and long-term (35–40 days) flooding. A reduction in O₂ levels surrounding the leaves from 21 to 2% for unflooded plants increased carbon assimilation by 33% and carboxylation efficiency from 0.012 to 0.021 mol CO₂ fixed (mol CO₂)⁻¹. Carboxylation efficiency of flooded plants, however, was unaffected by a decrease in percentage O₂, averaging 0.005 mol CO₂ fixed (mol CO₂)⁻¹. Apparent quantum yield decreased from 2.2 × 10⁻¹ mol of CO₂ fixed (mol light)⁻¹ for unflooded plants to 2.0 × 10⁻³ and 9.0 × 10⁻⁴ for intermediate- and long-term flooding durations, respectively. Shortterm flooding reduced carbon assimilation via a decrease in stomatal conductance, while longer flooding durations also decreased the carboxylation efficiency of the leaf.     

Effects of carbon dioxide in anther cultures

In anther cultures of Anemone canadensis L., Anemone dichotoma L., Anemone hupehensis Lemoine, Clematis viticella L. and Papaver setigerum DC. a positive relationship between incubation in 2% CO₂ and the production of microspore-derived embryos was observed. In anther cultures of Nicotiana tabacum L., Anemone hupehensis and Clematis viticella a combination of cold treatment (7°C) and incubation in 2% CO₂ increased embryo production. In Anemone canadensis cold treatment increased the number of proembryos, whereas incubation in 2% CO₂ had no effect. In Anemone hupehensis 5% CO₂ increased embryo production by more than 2%. In Anemone dichotoma and Papaver setigerum 2% CO₂ was the more efficient level. CO₂ had no significant effect on pH in the culture medium in anther cultures of Anemone canadensis.     

Adaptation to high CO2 concentration in an optimal environment: radiation capture, canopy quantum yield and carbon use efficiency

The effect of elevated [CO₂] on wheat (Triticum aestivum L. Veery 10) productivity was examined by analysing radiation capture, canopy quantum yield, canopy carbon use efficiency, harvest index and daily C gain. Canopies were grown at either 330 or 1200 μ mol mol^–1[CO₂] in controlled environments, where root and shoot C fluxes were monitored continuously from emergence to harvest. A rapidly circulating hydroponic solution supplied nutrients, water and root zone oxygen. At harvest, dry mass predicted from gas exchange data was 102·8 ± 4·7% of the observed dry mass in six trials. Neither radiation capture efficiency nor carbon use efficiency were affected by elevated [CO₂], but yield increased by 13% due to a sustained increase in canopy quantum yield. CO₂ enrichment increased root mass, tiller number and seed mass. Harvest index and chlorophyll concentration were unchanged, but CO₂ enrichment increased average life cycle net photosynthesis (13%, P < 0·05) and root respiration (24%, P < 0·05). These data indicate that plant communities adapt to CO₂ enrichment through changes in C allocation. Elevated [CO₂] increases sink strength in optimal environments, resulting in sustained increases in photosynthetic capacity, canopy quantum yield and daily C gain throughout the life cycle.     

Age, growth and reproduction of the barrelfish Hyperoglyphe perciformis (Mitchill) in the western North Atlantic

Otoliths ( n = 847) and gonads ( n = 817) were collected from barrelfish Hyperoglyphe perciformis that were captured by commercial fishermen in the waters off South Carolina and Georgia in 1995, 1997 and 2001–2006. Of the otoliths collected, 97% were aged successfully, and specimens sampled ranged from 5 to 85 years, with a median age of 12 years. The von Bertalanffy growth parameters yielded the equation: L_t = 857·8{1 − e^{−0·0985[ t −(−8·95)]}}, where L_t is fork length ( L _F) at time t . Through histological examination, 94% of the gonads assessed were assigned to a sex and reproductive class. Females spawned from September to May with a peak from November to January. Males spawned year round, but had a peak from September to April. The sex ratio (M:F) for this population was 1:1·34. The smallest mature female was 605 mm L _F and the youngest immature female was 697 mm L _F. Estimates of L _F and age at 50% maturity ( L ₅₀ and A ₅₀) for females were 660 mm L _F (95% CI = 633–667 mm L _F) and 6·08 years (95% CI = 3·50–7·27 years), respectively. The youngest mature male was 575 mm L _F and the oldest immature male was 762 mm L _F, and no estimates of L ₅₀ or A ₅₀ were made for males. It was determined that barrelfish exhibit the typical characteristics of long life span, slow growth and high age at maturity seen in other deepwater fishes, and that care should be taken to manage this species accordingly.     

Direct and indirect effects of Cd2+ on photosynthesis in sugar beet (Beta vulgaris)

Sugar-beet plants ( Beta vulgaris L. cv. Monohill) were cultivated for 4 weeks in a complete nutrient solution. Indirect effects of cadmium were studied by adding 5, 10 or 20 μ M CdCl₂ to the culture medium while direct effects were determined by adding 1, 5, 20, 50 or 2 000 μ M CdCl₂ to the assay media. The photosynthetic properties were characterized by measurement of CO₂ fixation in intact plants, fluorescence emission by intact leaves and isolated chloroplasts, photosystem (PS) I and PSII mediated electron transport of isolated chloroplasts, and CO₂-dependent O₂ evolution by protoplasts. When directly applied to isolated leaves, protoplasts and chloroplasts. Cd²⁺ impeded CO₂ fixation without affecting the rates of electron transport of PSI or PSII or the rate of dark respiration. When Cd²⁺ was applied through the culture medium the capacity for, and the maximal quantum yield of CO₂ assimilation by intact plants both decreased. This was associated with: (1) decreased total as well as effective chlorophyll content (PSII antennae size), (2) decreased coupling of electron transport in isolated chloroplasts, (3) perturbed carbon reduction cycle as indicated by fluorescence measurements. Also, protoplasts isolated from leaves of Cd²⁺-cultivated plants showed an increased rate of dark respiration.     

Interaction of enriched CO2 and water stress on the physiology of and biomass production in sweet potato grown in open-top chambers

Abstract. The objective of this study was to investigate the effects of water stress in sweet potato ( Ipomoea batatas L. [Lam] 'Georgia Jet') on biomass production and plant-water relationships in an enriched CO₂ atmosphere. Plants were grown in pots containing sandy loam soil (Typic Paleudult) at two concentrations of elevated CO₂ and two water regimes in open-top field chambers. During the first 12 d of water stress, leaf xylem potentials were higher in plants grown in a CO₂ concentration of 438 and 666 μmol mol⁻¹ than in plants grown at 364 μmol mol⁻¹. The 364 μmol mol⁻¹ CO₂ grown plants had to be rewatered 2 d earlier than the high CO₂-grown plants in response to water stress. For plants grown under water stress, the yield of storage roots and root: shoot ratio were greater at high CO₂ than at 364 μmol mol⁻¹; the increase, however, was not linear with increasing CO₂ concentrations. In well-watered plants, biomass production and storage root yield increased at elevated CO₂, and these were greater as compared to water-stressed plants grown at the same CO₂ concentration.     

Oxygen-induced recovery from short-term nitrate inhibition of N2 fixation in white clover plants from spaced and dense stands

Nitrogenase (N₂ase; EC 1.18.6.1) activity (H₂ evolution) and root respiration (CO₂ evolution) were measured under either N₂:O₂ or Ar:O₂ gas mixtures in intact nodulated roots from white clover ( Trifolium repens L.) plants grown either as spaced or as dense stands. The short-term nitrate (5 m M ) inhibition of N₂-fixation was promoted by competition for light between clover shoots, which reduced CO₂ net assimilation rate. Oxygen-diffusion permeability of the nodule declined during nitrate treatment but after nitrate removal from the liquid medium its recovery parallelled that of nitrogenase activity. Rhizosphere pO₂ was increased from 20 to 80 kPa under N₂:O₂. A simple mono-exponential model, fitted to the nodule permeability response to pO₂, indicated NO⁻₃ induced changes in minimum and maximum nodule O₂-diffusion permeability. Peak H₂ production rates at 80 kPa O₂ and in Ar:O₂ were close to the pre-decline rates at 20 kPa O₂. At the end of the nitrate treatment, this O₂-induced recovery in nitrogenase activity reached 71 and 82%; for clover plants from spaced and dense stands, respectively. The respective roles of oxygen diffusion and phloem supply for the short-term inhibition of nitrogenase activity in nitrate-treated clovers are discussed.