The Sequence of Change within the Photosynthetic Apparatus of Wheat following Short-Term Exposure to Ozone

The basis of inhibition of photosynthesis by single acute O₃ exposures was investigated in vivo using analyses based on leaf gas exchange measurements. The fully expanded second leaves of wheat plants (Triticum aestivum L. cv Avalon) were fumigated with either 200 or 400 nanomoles per mole O₃ for between 4 and 16 hours. This reduced significantly the light-saturated rate of CO₂ uptake and was accompanied by a parallel decrease in stomatal conductance. However, the stomatal limitation, estimated from the relationship between CO₂ uptake and the internal CO₂ concentration, only increased significantly during the first 8 hours of exposure to 400 nanomoles per mole O₃; no significant increase occurred for any of the other treatments. Analysis of the response of CO₂ uptake to the internal CO₂ concentration implied that the predominant factor responsible for the reduction in light-saturated CO₂ uptake was a decrease in the efficiency of carboxylation. This was 58 and 21% of the control value after 16 hours at 200 and 400 nanomoles per mole O₃, respectively. At saturating concentrations of CO₂, photosynthesis was inhibited by no more than 22% after 16 hours, indicating that the capacity for regeneration of ribulose bisphosphate was less susceptible to O₃. Ozone fumigations also had a less pronounced effect on light-limited photosynthesis. The maximum quantum yield of CO₂ uptake and the quantum yield of oxygen evolution showed no significant decline after 16 hours with 200 nanomoles per mole O₃, requiring 8 hours at 400 nanomoles per mole O₃ before a significant reduction occurred. The photochemical efficiency of photosystem II estimated from the ratio of variable to maximum chlorophyll fluorescence and the atrazine-binding capacity of isolated thylakoids demonstrated that photochemical reactions were not responsible for the initial inhibition of CO₂ uptake. The results suggest that the apparent carboxylation efficiency appears to be the initial cause of decline in photosynthesis in vivo following acute O₃ fumigation.     

Regulation of chloroplast photosynthetic activity by exogenous magnesium

Magnesium was most inhibitory to photosynthetic reactions by intact chloroplasts when the magnesium was added in the dark before illumination. Two millimolar MgCl₂, added in the dark, inhibited CO₂-dependent O₂ evolution by Hordeum vulgare L. and Spinacia oleracea L. (C₃ plants) chloroplasts 70 to 100% and inhibited (pyruvate + oxaloacetate)-dependent O₂ evolution by Digitaria sanguinalis L. (C₄ plant) mesophyll chloroplasts from 80 to 100%. When Mg²⁺ was added in the light, O₂ evolution was reduced only slightly. O₂ evolution in the presence of phosphoglycerate was less sensitive to Mg²⁺ inhibition than was CO₂-dependent O₂ evolution.

Magnesium prevented the light activation of several photosynthetic enzymes. Two millimolar Mg²⁺ blocked the light activation of NADP-malate dehydrogenase in D. sanguinalis mesophyll chloroplasts, and the light activation of phosphoribulokinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and fructose 1,6-diphosphatase in barley chloroplasts. The results suggest that Mg²⁺ inhibits chloroplast photosynthesis by preventing the light activation of certain enzymes.

     

Effect of drought stress on net CO2 uptake by Zea leaves

The net CO₂ assimilation by leaves of maize (Zea mays L. cv. Adonis) plants subjected to slow or rapid dehydration decreased without changes in the total extractable activities of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH) and malic enzyme (ME). The phosphorylation state of PEPC extracted from leaves after 2–3 h of exposure to light was not affected by water deficit, either. Moreover, when plants which had been slowly dehydrated to a leaf relative water content of about 60% were rehydrated, the net CO₂ assimilation by leaves increased very rapidly without any changes in the activities of MDH, ME and PEPC or phosphorylation state of PEPC. The net CO₂-dependent O₂ evolution of a non-wilted leaf measured with an oxygen electrode decreased as CO₂ concentration increased and was totally inhibited when the CO₂ concentration was about 10%. Nevertheless, high CO₂ concentrations (5–10%) counteracted most of the inhibitory effect of water deficit that developed during a slow dehydration but only counteracted a little of the inhibitory effect that developed during a rapid dehydration. In contrast to what could be observed during a rapidly developing water deficit, inhibition of leaf photosynthesis by cis-abscisic acid could be alleviated by high CO₂ concentrations. These results indicate that the inhibition of leaf net CO₂ uptake brought about by water deficit is mainly due to stomatal closure when a maize plant is dehydrated slowly while it is mainly due to inhibition of non-stomatal processes when a plant is rapidly dehydrated. The photosynthetic apparatus of maize leaves appears to be as resistant to drought as that of C₃ plants. The non-stomatal inhibition observed in rapidly dehydrated leaves might be the result of either a down-regulation of the photosynthetic enzymes by changes in metabolite pool sizes or restricted plasmodesmatal transport between mesophyll and bundle-sheath cells.     

Nitrogenase activity, nodule respiration, and o(2) permeability following detopping of alfalfa and birdsfoot trefoil

Gas exchange measurements and noninvasive leghemoglobin (Lb) spectrophotometry (nodule oximetry) were used to monitor nodule responses to shoot removal in alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus). In each species, total nitrogenase activity, measured as H₂ evolution in Ar:O₂ (80:20), decreased to <50% of the initial rate within 1 hour after detopping, and net CO₂ production decreased to about 65% of the initial value. In a separate experiment in which nodule oximetry was used, nodule O₂ permeability decreased 50% within 5 hours in each species. A similar decrease in the O₂-saturated respiration rate (V_max) for the nodule central zone occurred within 5 hours in birdsfoot trefoil, but only after 24 hours in alfalfa. Lb concentration, also measured by oximetry, decreased after 48 to 72 hours. The decrease in permeability preceded the decrease in V_max in each species. V_max may depend mainly on carbohydrate availability in the nodule. If so, then the decrease in permeability could not have been triggered by decreasing carbohydrate availability. Both oximetry and gas exchange data were consistent with the hypothesis that, for the cultivars tested, carbohydrate availability decreased more rapidly in birdsfoot trefoil than in alfalfa nodules. Fractional Lb oxygenation (initially about 0.15) decreased during the first 24 hours after detopping but subsequently increased to >0.65 for a majority of nodules of each species. This increase could lead to O₂ inactivation of nitrogenase.     

Simultaneous Measurements of Steady State Chlorophyll a Fluorescence and CO(2) Assimilation in Leaves: The Relationship between Fluorescence and Photosynthesis in C(3) and C(4) Plants

Rates of CO₂ assimilation and steady state chlorophyll a fluorescence were measured simultaneously at different intercellular partial pressures of CO₂ in attached cotton (Gossypium hirsutum L. cv Deltapine 16) leaves at 25°C. Electron transport activity for CO₂ assimilation plus photorespiration was calculated for these experiments. Under light saturating (1750 microeinsteins per square meter per second) and light limiting (700 microeinsteins per square meter per second) conditions there was a good correlation between fluorescence and the calculated electron transport activity at 19 and 200 millibars O₂, and between fluorescence and rates of CO₂ assimilation at 19 millibars but not 200 millibars O₂. The values of fluorescence measured at about 220 microbars intercellular CO₂ were not greatly affected by increasing O₂ from 19 to 800 millibars. Fluorescence increased with light intensity at any one intercellular CO₂ partial pressure. But the values obtained for fluorescence, expressed as a ratio of the maximum fluorescence obtained in DCMU-treated tissue, over the same range of CO₂ partial pressure at 500 microeinsteins per square meter per second were similar to those obtained at 1000 and 2000 microeinsteins per square meter per second. There were two phases in the observed correlation between fluorescence and calculated electron transport activity: an initial inverse relationship at low CO₂ partial pressures which reversed to a positive correlation at higher values of CO₂ partial pressures. Similar results were observed in the C₃ species Helianthus annuus L., Phaseolus vulgaris L., and Brassica chinensis. In all C₄ species (Zea mays L., Sorghum bicolor L., Panicum maximum Jacq., Amaranthus edulis Speg., and Echinochloa frumentacea [Roxb.] Link) examined changes in fluorescence were directly correlated with changes in CO₂ assimilation rates. The nature and the extent to which Q (primary quencher) and high-energy state (q_E) quenching function in determining the steady state fluorescence obtained during photosynthesis in leaves is discussed.     

Effect of water deficit on photosynthetic oxygen exchange measured using 18O2 and mass spectrometry in Solanum tuberosum L. leaf discs

The effect of leaf dehydration on photosynthetic O₂ exchange of potato (Solanum tuberosum L., cv. Haig) leaf discs was examined using ¹⁸O₂ as a tracer and mass spectrometry. In normal air (350 μl·l^?1CO₂) and under an irradiance of 390 μmol photons·m^?2·s¹, a relative water deficit (RWD) of about 30% severely decreased net O₂ evolution and increased O₂ uptake by about 50%, thus indicating an enhancement of photorespiration. Increasing CO₂ concentrations diminished O₂ uptake and stimulated net O₂ evolution both in well-hydrated and in dehydrated (RWD of about 30%) leaves. Much higher CO₂ concentrations (up to 4%) were required to observe a complete effect of CO₂ in dehydrated leaves. The chloroplastic CO₂ concentration at the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) level (C_c) was calculated from O₂-exchange data in both well-hydrated and dehydrated leaves, assuming that the specificity factor of Rubisco was unaffected by desiccation. When plotting net O₂ photosynthesis as a function of C_c, a similar relationship was obtained for well-hydrated and waterstressed leaf discs, thus showing that the main effect of water deficit is a decrease of the chloroplastic CO₂ concentration. At saturating CO₂ levels, the non-cyclic electron-transport rate, measured either as gross O₂ photosynthesis or as the chlorophyll fluorescence ratio (F_m -F_s)/F_m, was insensitive to water deficit, provided RWD was below 40%. In this range of RWD, the decrease in gross O₂ photosynthesis observed in normal air was attributed to the inability of oxidative processes to sustain the maximal electron-flow rate at low chloroplastic CO₂ concentration. The maximal efficiency of photosystem II, estimated as the chlorophyll fluorescence ratio (F_m -F₀)/F_m measured in dark-adapted leaves, was not affected by water deficits up to 60%.     

Effect of Oxygen on the Contribution of Respiration to the CO(2) Compensation Point in Wheat and Bean Leaves

The CO₂ compensation point at 21% O₂ (Γ₂₁) and at 2% O₂ (Γ₂), and the rate of dark CO₂ efflux at 21% O₂ (R_n) were measured in adult wheat (Triticum aestivum L, cv Gabo) leaves at the end of the night and after a period of photosynthesis of 5 h at 800 μbar CO₂. The values of Γ₂₁ and R_n significantly increased after the light period, due to the stimulation of respiration by carbohydrates. In contrast, Γ₂ did not increase after the same period of photosynthesis, suggesting that the respiratory component of Γ₂ was not stimulated by carbohydrates. In a different experiment, Γ₂₁, Γ₂, and R_n were studied during the growth period of bean (Phaseolus vulgaris L, cv Hawkesbury Wonder) leaves. The values of Γ₂₁ and R_n were high in young leaves, and decreased rapidly in parallel during maturation. However, Γ₂ presented relatively low values in growing bean leaves, and a model predicted that the observed values of Γ₂ should have been considerably higher if their respiratory component was considered to be as large as that of Γ₂₁. The results suggest that the rate of respiration in the light contributing to the CO₂ compensation point in wheat and bean leaves is smaller at low O₂ levels than at ambient levels.     

Photosynthetic Capacities and Growth Characteristics of Nicotiana tabacum (cv. Xanthi) Cell Suspension Cultures

Photoheterotrophic growth of cell suspensions of Nicotiana tabacum L. (cv. Xanthi) in organic culture medium enriched in sucrose (30 g per liter) showed a classical sigmoid growth curve. The cells developed functional chloroplast structures during the exponential growth phase, when their chlorophyll content increased steadily. A limited drop (30%) in the chlorophyll amount and structural changes of the plastids (starch accumulation) were observed during the lag phase. The measurements of photosynthetic capacities (O₂ evolution and CO₂ fixation) during the growth cycle revealed changes in the photosynthetic ratio (O₂/CO₂), which was near 1 during the lag and stationary phases and near 2 during exponential growth. During exponential growth there was also a rapid NO₃^? uptake. Analysis of label distribution among the products of ¹⁴CO₂ fixation showed that both CO₂ assimilation pathways, linked to the ribulose-biphosphate carboxylase (the autotrophic pathway) and to phosphoenolpyruvate carboxylase (the non-autotrophic pathway) were operative with an important increase of the capacity of the latter during the exponential growth phase. Maximum rate of oxygen evolution, either endogenous or with p-benzoquinone as Hill reagent, as well as the increased CO₂ Fixation capacity via the non-autotrophic pathway during the exponential phase were concomitant with a high cyanide inhibited O₂ uptake.     

Differential Oxygen Response of Photosynthesis in Soybean and Panicum milioides

The carbon dioxide compensation concentration of Panicum milioides was less than that of soybean over the range of 15 to 35 C. In soybean (Glycine max [L.] Merr. cv. Wayne), the compensation concentration was directly proportional to O₂ concentration. In P. milioides, the compensation concentration was near zero up to 10% O₂ and then increased linearly with higher O₂, although the slope of the response was less than that in soybean. Leaf extracts of P. milioides contained 3-fold higher phosphoenolpyruvate carboxylase activity than soybean leaf extracts. Oxygen inhibition of photosynthesis and carboxy-lation efficiency was less in P. milioides than that observed in soybean. The affinity of P. millioides ribulose-1,5-di-P carboxylase for CO₂ appeared to be slightly greater than that of soybean. The affinity of both enzymes for O₂ was similar. The reduced response of the compensation concentration and photosynthesis to O₂ in P. milioides may be explained by photosynthetic phosphoenolpyruvate carboxylase fixation and by an apparent increased affinity of ribulose-1,5-di-P carboxylase for CO₂.     

Effects of slowly permeating osmotica on metabolism of vacuolated and nonvacuolated tissues

Vacuolated and nonvacuolated root tissues of Zea mays were exposed to low water potentials by addition of mannitol or glycerol. Temporary increases were observed for O₂ uptake, but CO₂ evolution remained steady. This increase in O₂ uptake ceased after 15 minutes. Further treatment induced decreases in respiration, with similar reductions in O₂ uptake and CO₂ evolution.     

Impacts of elevated CO2 on expression of plant defensive compounds in Bt‐transgenic cotton in response to infestation by cotton bollworm

• 1 The allocation of defensive compounds of transgenic Bt (cv. GK‐12) and nontransgenic cotton (cv. Simian‐3) grown in elevated CO₂ in response to infestation by cotton bollworm Helicoverpa armigera (Hübner) was studied in closed‐dynamics CO₂ chambers.
• 2 A significant reduction in foliar nitrogen content and Bt toxin protein occurred when transgenic Bt cotton grew under elevated CO₂. A significantly higher carbon/nitrogen ratio as well as condensed tannin and gossypol contents was observed for transgenic Bt (cv. GK‐12) and nontransgenic cotton in elevated CO₂, in partial support of the carbon nutrient balance hypothesis as a result of limiting nitrogen and excess carbon in cotton plants in response to elevated CO₂.
• 3 The CO₂ level and infestation time significantly affected the foliar nitrogen, condensed tannin, gossypol and Bt toxin protein contents of cotton plants after feeding by H. armigera. The interaction between CO₂ levels × cotton variety had a significant effect on foliar nitrogen content after injury by H. armigera.

     

Photoreduction of O(2) Primes and Replaces CO(2) Assimilation

A mass spectrometer with a membrane inlet system was used to monitor directly gaseous components in a suspension of algae. Using labeled oxygen, we observed that during the first 20 seconds of illumination after a dark period, when no net O₂ evolution or CO₂ uptake was observed, O₂ evolution was normal but completely compensated by O₂ uptake. Similarly, when CO₂ uptake was totally or partially inhibited, O₂ evolution proceeded at a high (near maximal) rate. Under all conditions, O₂ uptake balanced that fraction of the O₂ evolution which could not be accounted for by CO₂ uptake.     

O(2)-insensitive photosynthesis in c(3) plants : its occurrence and a possible explanation

Leaves of C₃ plants which exhibit a normal O₂ inhibition of CO₂ fixation at less than saturating light intensity were found to exhibit O₂-insensitive photosynthesis at high light. This behavior was observed in Phaseolus vulgaris L., Xanthium strumarium L., and Scrophularia desertorum (Shaw.) Munz. O₂-insensitive photosynthesis has been reported in nine other C₃ species and usually occurred when the intercellular CO₂ pressure was about double the normal pressure. A lack of O₂ inhibition of photosynthesis was always accompanied by a failure of increased CO₂ pressure to stimulate photosynthesis to the expected degree. O₂-insensitive photosynthesis also occurred after plants had been water stressed. Under such conditions, however, photosynthesis became O₂ and CO₂ insensitive at physiological CO₂ pressures. Postillumination CO₂ exchange kinetics showed that O₂ and CO₂ insensitivity was not the result of elimination of photorespiration.

It is proposed that O₂ and CO₂ insensitivity occurs when the concentration of phosphate in the chloroplast stroma cannot be both high enough to allow photophosphorylation and low enough to allow starch and sucrose synthesis at the rates required by the rest of the photosynthetic component processes. Under these conditions, the energy diverted to photorespiration does not adversely affect the potential for CO₂ assimilation.

     

Estimation of Photorespiration Based on the Initial Rate of Postillumination CO(2) Release: II. Effects of O(2), CO(2), and Temperature

An open system associated with an infrared gas analyzer was employed to study transients in CO₂ exchange generated upon darkening preilluminated leaf discs of tobacco (Nicotiana tabacum vars John Williams Broadleaf and Havana Seed). An empirical formula presented previously enabled prediction of the analyzer response under nonsteady state conditions as a function of time and of the leaf CO₂ exchange rate. A computer was used to evaluate parameters of the leaf CO₂ release rate to provide an estimate of the initial rate of postillumination CO₂ evolution and to produce maximal agreement between predicted and observed analyzer responses. In 21% O₂, the decline in rate of CO₂ evolution upon darkening followed first order kinetics. Initial rates of CO₂ evolution following darkening were relatively independent of the prior ambient CO₂ concentrations. However, rates of photorespiration expressed as a fraction of net photosynthesis declined rapidly with increasing external CO₂ concentration at 21% O₂. Under normal atmospheric conditions, photorespiration was 45 to 50% of the net CO₂ fixation rate at 32°C and high irradiance. The rapid initial CO₂ evolution observed upon darkening at 21% O₂ was absent in 3% O₂. Rates of photorespiration under normal atmospheric concentrations of CO₂ and O₂ as measured by the postillumination burst were highly dependent upon temperature (observed activation energy = 30.1 kilocalories per mole). The results are discussed with respect to previously published estimates of photorespiration in C₃ leaf tissue.     

Kinetics and Apparent K(m) of Oxygen Cycle under Conditions of Limiting Carbon Dioxide Fixation

A mass spectrometer with a membrane inlet was used to monitor light-driven O₂ evolution, O₂ uptake, and CO₂ uptake in suspensions of algae (Scenedesmus obliquus). We observed the following. (a) The rate of O₂ uptake, which, in the presence of iodoacetamide, replaces the uptake of CO₂, showed a distinct plateau (V_max) beyond ~30% O₂ and was half-maximal at ~8% O₂. We concluded that this light-driven O₂ uptake process, which does not involve carbon compounds, is saturated at lower O₂ concentrations than are photorespiration and glycolate formation. (b) In the absence of inhibitor, O₂ evolution was relatively unaffected by the presence or absence of CO₂. During the course of CO₂ depletion, electron flow to CO₂ was replaced by an equivalent flow to O₂. (c) There was a distinct delay between the cessation of CO₂ uptake and the increase in O₂ uptake. We ascribe this delay to the transient utilization of another electron acceptor—possibly bicarbonate or another bound form of CO₂.     

Relationship between Respiration and Photosynthesis in Guard Cell and Mesophyll Cell Protoplasts of Commelina communis L

A mass spectrometric method combining ¹⁶O/¹⁸O and ¹²C/¹³C isotopes was used to quantify the unidirectional fluxes of O₂ and CO₂ during a dark to light transition for guard cell protoplasts and mesophyll cell protoplasts of Commelina communis L. In darkness, O₂ uptake and CO₂ evolution were similar on a protein basis. Under light, guard cell protoplasts evolved O₂ (61 micromoles of O₂ per milligram of chlorophyll per hour) almost at the same rate as mesophyll cell protoplasts (73 micromoles of O₂ per milligram of chlorophyll per hour). However, carbon assimilation was totally different. In contrast with mesophyll cell protoplasts, guard cell protoplasts were able to fix CO₂ in darkness at a rate of 27 micromoles of CO₂ per milligram of chlorophyll per hour, which was increased by 50% in light. At the onset of light, a delay observed for guard cell protoplasts between O₂ evolution and CO₂ fixation and a time lag before the rate of saturation suggested a carbon metabolism based on phosphoenolpyruvate carboxylase activity. Under light, CO₂ evolution by guard cell protoplasts was sharply decreased (37%), while O₂ uptake was slowly inhibited (14%). A control of mitochondrial activity by guard cell chloroplasts under light via redox equivalents and ATP transfer in the cytosol is discussed. From this study on protoplasts, we conclude that the energy produced at the chloroplast level under light is not totally used for CO₂ assimilation and may be dissipated for other purposes such as ion uptake.     

Light-Stimulated Changes in the Acidity of Suspensions of Oat Protoplasts: Dependence upon Photosynthesis

Light induced an alkalinization and stimulated a subsequent acidification of the medium surrounding oat (Avena sativa L. cv Garry) leaf protoplasts. Blue light was less effective than would be predicted from photosynthetic action spectra. Nonetheless, 3-(3,4-dichlorophenyl)-1,1-dimethylurea prevented alkalinization and reduced acidification to the dark rate for protoplast suspensions exposed to all light regimes tested.

Alkalinization increased in parallel with initial rates of O₂ evolution as the quantum flux density of white light was raised to 75 microeinsteins per square meter per second. Alkalinization was accompanied by a decrease in the CO₂ content of the medium; therefore, it was attributed to photosynthetically induced CO₂ uptake. The effect of CO₂ depletion on the acidity of the medium appeared to be mainly restricted to the first 15 minutes of exposure to light. Consequently, subsequent pH changes primarily reflected a constant net proton efflux. Acidification occurred in the dark, but rates of acidification increased in response to increased light approximately in parallel with changes in a concomitant net O₂ efflux. The results indicated that protoplasts could acidify the medium in response to nonphotosynthetic activity, but that photosynthesis mediated light stimulation of acidification.

     

Effects of pH and Oxygen on Photosynthetic Reactions of Intact Chloroplasts

Oxygen inhibition of photosynthesis was studied with intact spinach (Spinacia oleracea L.) chloroplasts which exhibited very high rates of photosynthetic CO₂ reduction and were insensitive to additions of photosynthetic intermediates when CO₂ was available at saturating concentrations. Photosynthetic rates were measured polarographically as O₂ evolution, and the extent of the reduction of substrate was estimated from the amount of O₂ evolved. With CO₂ as substrate, inhibition of photosynthesis by O₂ was dependent on pH. At pH values above 8, rates of O₂ evolution were strongly inhibited by O₂ and only a fraction of the added bicarbonate was reduced before O₂ evolution ceased. The extent of O₂ evolution declined with increasing O₂ concentration and decreasing initial bicarbonate concentration. At pH 7.2, the initial photosynthetic rate was inhibited about 30% at high O₂ levels, but the extent of O₂ evolution was unaffected and most of the added bicarbonate was reduced. Photosynthetic O₂ evolution with 3-phosphoglycerate as substrate was similarly dependent on pH and O₂ concentration. In contrast, there was little effect of O₂ and pH on oxaloacetate-dependent oxygen evolution. Acid-base shift experiments with osmotically shocked chloroplasts showed that ATP formation was not affected by O₂. The results are discussed in terms of a balance between photosynthetic O₂ evolution and O₂ consumption by the ribulose diphosphate oxygenase reaction.     

The effect of feeding on CO2 production and energy expenditure in ponies measured by indirect calorimetry and the 13C-bicarbonate technique

Energy expenditure (EE) can be estimated based on respiratory gas exchange measurements, traditionally done in respiration chambers by indirect calorimetry (IC). However, the ¹³C-bicarbonate technique (¹³C-BT) might be an alternative minimal invasive method for estimation of CO₂ production and EE in the field. In this study, four Shetland ponies were used to explore the effect of feeding on CO₂ production and EE measured simultaneously by IC and ¹³C-BT. The ponies were individually housed in respiration chambers and received either a single oral or intravenous (IV) bolus dose of ¹³C-labelled sodium bicarbonate (NaH¹³CO₃). The ponies were fed haylage 3 h before (T₋₃), simultaneously with (T₀) or 3 h after (T₊₃) administration of ¹³C-bicarbonate. The CO₂ produced and O₂ consumed by the ponies were measured for 6 h with both administration routes of ¹³C-bicarbonate at the three different feeding times. Feeding time affected the CO₂ production (P<0.001) and O₂ consumption (P<0.001), but not the respiratory quotient (RQ) measured by IC. The recovery factor (RF) of ¹³C in breath CO₂ was affected by feeding time (P<0.01) and three different RF were used in the calculation of CO₂ production measured by ¹³C-BT. An average RQ was used for the calculations of EE. There was no difference between IC and ¹³C-BT for estimation of CO₂ production. An effect of feeding time (P<0.001) on the estimated EE was found, with higher EE when feed was offered (T₀ and T₊₃) compared with when no feed was available (T₋₃) during measurements. In conclusion, this study showed that feeding time affects the RF and measurements of CO₂ production and EE. This should be considered when the ¹³C-BT is used in the field. IV administration of ¹³C-bicarbonate is recommended in future studies with horses to avoid complex ¹³C enrichment-time curves with maxima and shoulders as observed in several experiments with oral administration of ¹³C-bicarbonate.     

Effect of pO(2) on Growth and Nodule Functioning of Symbiotic Cowpea (Vigna unguiculata L. Walp.)

Nodulated cowpea (Vigna unguiculata L. Walp. cv Vita 3:Bradyrhizobium CB 756) plants were cultured with their whole root system or crown root nodulation zone maintained for periods from 5 to 69 days after planting in atmospheres containing a range of pO₂ (1-80%, v/v) while the rest of the plant grew in normal air. Growth (dry matter yield) and N₂ fixation were largely unaffected by pO₂ from 10 to 40%. Decrease in fixation at pO₂ below 5% was due to lower nodulation and nodule mass and, at pO₂ above 60%, to a fall in specific N₂-fixing activity of nodules. Root:shoot ratios were significantly lower at pO₂ below 2.5%. The effect of pO₂ on nitrogenase activity (acetylene reduction), both of whole nodulated root systems and crown root nodulation zones, varied with plant age but was generally lower at supra- and subambient extremes of O₂. H₂ evolution showed a sharp optimum at 20% O₂ but was at most 4% of total nitrogenase activity. The ratio of CO₂ evolved to substrate (C₂H₂+H⁺) reduced by crown root nodulation zones was constant (6 moles CO₂ per mole substrate reduced) from 2.5 to 60% O₂ but at levels below 2.5 and above 80% O₂ reached values between 20 and 30 moles CO₂ per mole substrate reduced. Effects of long-term growth with nonambient pO₂ on adaptation and efficiency of functioning of nodules are discussed.