Carbon dioxide and nitrite photoassimilatory processes do not intercompete for reducing equivalents in spinach and soybean leaf chloroplasts

Previously, C Baysdorfer and JM Robinson (1985 Plant Physiol 77: 318-320) demonstrated that, in a reconstituted spinach chloroplast system, NADP photoreduction functioning at most maximal rate and reductant demand, was the successful competitor with NO₂⁻ photoreduction for reduced ferredoxin. This resulted in a repression of NO₂⁻ reduction until all NADP available had been almost totally reduced. Further experiments, employing isolated, intact spinach leaf plastids and soybean leaf mesophyll cells, were conducted to examine competition for reductant between CO₂ and NO₂⁻ photoassimilation, in situ. In isolated, intact plastid preparations, regardless of whether the demand for reductant by CO₂ photoassimilation was high (5 millimolar `CO<sub>2</sub>') with rates of CO<sub>2</sub> fixation in the range 40 to 90 micromoles CO<sub>2</sub> fixed per hour per milligram chlorophyll, low (0.5 millimolar `CO₂') with rates in the range 5 to 8 micromoles CO₂ per hour per milligram chlorophyll, or zero (no `CO<sub>2</sub>'), NO<sub>2</sub><sup>−</sup> photoreduction displayed equal rates in the range of 8 to 22 micromoles per hour per milligram chlorophyll. In the absence of `CO₂', but in the presence of saturating white light, 3-phosphoglycerate photoreduction at rates of 82 to 127 micromoles per hour per milligram chlorophyll did not repress, and occasionally stimulated concomitant rates of NO₂⁻ reduction which ranged from 23.4 to 38.5. Conversely, in plastid preparations, NO₂⁻ at levels of 50 to 100 micromolar, stimulated plastid CO₂ fixation when `CO<sub>2</sub>' was saturating with respect to carboxylation. Further, levels of NO<sub>2</sub><sup>−</sup> in the range 250 to 2500 micromolar, stimulated soybean leaf mesophyll cell net CO<sub>2</sub> fixation as much as 1.5-fold if `CO₂' was saturating with respect to CO₂ fixation. It appeared likely that, in high light in vivo, CO₂ and NO₂⁻ photoassimilatory processes are not forced to intercompete for reduced ferredoxin in the intact chloroplast.     

Spinach Leaf Chloroplast CO(2) and NO(2) Photoassimilations Do Not Compete for Photogenerated Reductant: Manipulation of Reductant Levels by Quantum Flux Density Titrations

Potential competition between CO₂ and NO₂⁻ photoassimilation for photogenerated reductant (e.g. reduced ferredoxin and NADPH) was examined employing isolates of mesophyll cells and intact chloroplasts derived from mature `source' spinach leaves. Variations in the magnitude of incident light energy were used to manipulate the supply of reductant in situ within chloroplasts. Leaf cell and plastid isolates were fed with saturating CO₂ and/or NO₂⁻ to produce the highest demand for reductant by CO₂ and/or NO₂⁻ assimilatory processes (enzymes). Even in the presence of CO₂ fixation, NO₂⁻ reduction in intact leaf cell isolates as well as plastid isolates was maximal at light energies as low as 50 to 200 microeinsteins per second per square meter. Simultaneously, 500 to 800 microeinsteins per second per square meter were required to support maximal CO₂ assimilation. Regardless of the magnitude of the incident light energy, CO₂ assimilation did not repress NO₂⁻ reduction, nor were these two processes mutually repressed. These observations have been interpreted to mean that reduced ferredoxin levels in situ in the plastids of mature source leaf mesophyll cells were adequate to supply the concurrent maximal demands exerted by enzymes associated with CO₂ as well as with inorganic nitrogen photoassimilation.     

The effect of low osmotic potential on nitrite reduction in intact spinach chloroplasts

The effect of water stress (reduced osmotic potential) on photosynthetic nitrite reduction was investigated using intact, isolated spinach (Spinacia oleracea) chloroplasts. Nitrite-dependent O₂ evolution was inhibited 39% at −29.5 bars osmotic potential, relative to a control at −11 bars. In the presence of an uncoupler of photophosphorylation this inhibition was not seen. Reduced osmotic potential did not inhibit either methyl viologen reduction or photosynthetic O₂ reduction. These results indicate that an inhibition of electron transport to ferredoxin cannot account for the observed inhibition of nitrite-dependent O₂ evolution. In vitro assay of nitrite reductase activity showed that the interaction of the enzyme with nitrite was not affected by changes in the concentrations of ions or molecules that might be caused by water stress conditions. These results indicate that the most likely site for the effect of water stress on chloroplastic nitrite reduction is the interaction of ferredoxin with nitrite reductase.     

Decrease of Nitrate Reductase Activity in Spinach Leaves during a Light-Dark Transition

In leaves of spinach plants (Spinacia oleracea L.) performing CO₂ and NO₃⁻ assimilation, at the time of sudden darkening, which eliminates photosystem I-dependent nitrite reduction, only a minor temporary increase of the leaf nitrite content is observed. Because nitrate reduction does not depend on redox equivalents generated by photosystem I activity, a continuation of nitrate reduction after darkening would result in a large accumulation of nitrite in the leaves within a very short time, which is not observed. Measurements of the extractable nitrate reductase activity from spinach leaves assayed under standard conditions showed that in these leaves the nitrate reductase activity decreased during darkening to 15% of the control value with a half-time of only 2 minutes. Apparently, in these leaves nitrate reductase is very rapidly inactivated at sudden darkness avoiding an accumulation of the toxic nitrite in the cells.     

Selection of soybean plant leaves which yield mesophyll cell isolates with maximal rates of CO2 and NO inf2 sup− photoassimilation

A problem often encountered when assaying mesophyll cell isolates prepared from mature soybean leaves, was that of poor reproducibility in rates of net ¹⁴CO₂ photoassimilation and NO₂ ^– photoreduction. It was known that soybean source leaves repeatedly displayed their most active net CO₂ photoassimilation in the period from attainment of maximal leaf area to approximately two to five days subsequent to that point. Advantage was taken of the fact that when soybean leaflets of each leaf reach their maximal area they also have reached their maximal leaf length from base to tip. This facilitates a more rapid determination of the point in time in which leaflet areas had reached A_max. Soybean plants (Glycine max cv. Williams) were propagated in the growth chamber with a 12 h light-12 h dark cycle, 25C, 65% RH, and 700 microeinsteins per meter squared per second. At 24 d post-emergence, the third leaf (numbered acropetally from the unifoliates) of each plant had just attained maximum leaflet areas (110 cm²) and lengths (13 cm). For this study, leaf mesophyll cells were enzymatically isolated, using commercially prepared pectinase, from leaflet sets of leaves selected from each of the second, third, and fourth leaf positions. Maximal rates of net ¹⁴CO₂ photoassimilation (with 5 mM HCO₃ ^–) for the second, third and fourth leaf (leaflet) isolates were, respectively, 27.0, 57.0, and 41.7 mol ¹⁴CO₂ assimilated per milligram chlorophyll per hour; simultaneously maximal rates of NO _inf2 ^sup– photoreduction (1 mM NO _inf2 ^sup– ) were, respectively, 4.4, 8.1, and 0.0 mol NO _inf2 ^sup– reduced per milligram chlorophyll per hour. These studies made it clear that in order repeatedly to attain reproducible maximal rates of leaf cell isolate net ¹⁴CO₂ photoassimilation and NO _inf2 ^sup– photoreduction, it always was necessary to select the newest, fully expanded leaves (e.g. leaf number 3) for cell isolation. Leaves from several plants only were pooled if they were excised from identically the same node on each of the plants.Abbreviations A_max - maximum leaflet (trifoliolate) area attained during ontogeny - CO₂ - CO₂ gas dissolved in solution - HCO _inf3 ^sup– - bicarbonate - L_max - maximum leaf blade length (midvein) attained during ontogeny - NiRase - chloroplast nitrite reductase (reduced ferredoxin) - NiPR - nitrite photoreduction - PE - post-emergence - Pn - net CO₂ photoassimilation (for leaflets and mesophyll cell isolates) - PPRC - pentose phosphate reductive cycle     

Effect of photosynthetic inhibitors and uncouplers of oxidative phosphorylation on nitrate and nitrite reduction in barley leaves

The effects of several photosynthetic inhibitors and uncouplers of oxidative phosphorylation on NO₃⁻ and NO₂⁻ assimilation were studied using detached barley (Hordeum vulgare L. cv Numar) leaves in which only endogenous NO₃⁻ or NO₂⁻ were available for reduction. Uncouplers of oxidative phosphorylation greatly increased NO₃⁻ reduction in both light and darkness, while photosynthetic inhibitors did not.

The NO₂⁻ concentration in the control leaves was very low in both light and darkness; 98% or more of the NO₂⁻ formed from NO₃⁻ was further assimilated in control leaves. More NO₂⁻ accumulated in the leaves in light and darkness in the presence of photosynthetic inhibitors. Of this NO₂⁻, 94% or more was further assimilated. It appears that metabolites, either external or internal to the chloroplast, capable of reducing NADP (which, in turn, could reduce ferredoxin via NADP reductase) might support NO₂⁻ reduction in darkness and light when photosynthetic electron flow is inhibited by photosynthetic inhibitors.

Nitrite assimilation was much more sensitive to uncouplers in darkness than in light: in darkness, 74% or more of NO₂⁻ formed from NO₃⁻ was further assimilated, whereas in light, 95% or more of the NO₂⁻ was further assimilated.

     

Characterization of an Electron Transport Pathway Associated with Glucose and Fructose Respiration in the Intact Chloroplasts of Chlamydomonas reinhardtii and Spinach

The role of an electron transport pathway associated with aerobic carbohydrate degradation in isolated, intact chloroplasts was evaluated. This was accomplished by monitoring the evolution of ¹⁴CO₂ from darkened spinach (Spinacia oleracea) and Chlamydomonas reinhardtii chloroplasts externally supplied with [¹⁴C]fructose and [¹⁴C]glucose, respectively, in the presence of nitrite, oxaloacetate, and conventional electron transport inhibitors. Addition of nitrite or oxaloacetate increased the release of ¹⁴CO₂, but it was shown that O₂ continued to function as a terminal electron acceptor. ¹⁴CO₂ evolution was inhibited up to 30 and 15% in Chlamydomonas and spinach, respectively, by 50 μm rotenone and by amytal, but at 500- to 1000-fold higher concentrations, indicating the involvement of a reduced nicotinamide adenine dinucleotide phosphate-plastoquinone oxidoreductase. ¹⁴CO₂ release from the spinach chloroplast was inhibited 80% by 25 μm 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. ¹⁴CO₂ release was sensitive to propylgallate, exhibiting approximately 50% inhibition in Chlamydomonas and in spinach chloroplasts of 100 and 250 μm concentrations, respectively. These concentrations were 20- to 50-fold lower than the concentrations of salicylhydroxamic acid (SHAM) required to produce an equivalent sensitivity. Antimycin A (100 μm) inhibited approximately 80 to 90% of ¹⁴CO₂ release from both types of chloroplast. At 75 μm, sodium azide inhibited ¹⁴CO₂ evolution about 50% in Chlamydomonas and 30% in spinach. Sodium azide (100 mm) combined with antimycin A (100 μm) inhibited ¹⁴CO₂ evolution more than 90%. ¹⁴CO₂ release was unaffected by uncouplers. These results are interpreted as evidence for a respiratory electron transport pathway functioning in the darkened, isolated chloroplast. Chloroplast respiration defined as ¹⁴CO₂ release from externally supplied [1-¹⁴C]glucose can account for at least 10% of the total respiratory capacity (endogenous release of CO₂) of the Chlamydomonas reinhardtii cell.     

In Vivo Blue-Light Activation of Chlamydomonas reinhardii Nitrate Reductase

Chlamydomonas reinhardii cells, growing photoautotrophically under air, excreted to the culture medium much higher amounts of NO₂⁻ and NH₄⁺ under blue than under red light. Under similar conditions, but with NO₂⁻ as the only nitrogen source, the cells consumed NO₂⁻ and excreted NH₄⁺ at similar rates under blue and red light. In the presence of NO₃⁻ and air with 2% CO₂ (v/v), no excretion of NO₂⁻ and NH₄⁺ occurred and, moreover, if the bubbling air of the cells that were currently excreting NO₂⁻ and NH₄⁺ was enriched with 2% CO₂ (v/v), the previously excreted reduced nitrogen ions were rapidly reassimilated. The levels of total nitrate reductase and active nitrate reductase increased several times in the blue-light-irradiated cells growing on NO₃⁻ under air. When tungstate replaced molybdate in the medium (conditions that do not allow the formation of functional nitrate reductase), blue light activated most of the preformed inactive enzyme of the cells. Furthermore, nitrate reductase extracted from the cells in its inactive form was readily activated in vitro by blue light. It appears that under high irradiance (90 w m⁻²) and low CO₂ tensions, cells growing on NO₃⁻ or NO₂⁻ may not have sufficient carbon skeletons to incorporate all the photogenerated NH₄⁺. Because these cells should have high levels of reducing power, they might use NO₃⁻ or, in its absence, NO₂⁻ as terminal electron acceptors. The excretion of the products of NO₂⁻ and NH₄⁺ to the medium may provide a mechanism to control reductant level in the cells. Blue light is suggested as an important regulatory factor of this photorespiratory consumption of NO₃⁻ and possibly of the whole nitrogen metabolism in green algae.     

Photosynthesis and ion content of leaves and isolated chloroplasts of salt-stressed spinach

Spinach (Spinacia oleracea) plants were subjected to salt stress by adding NaCl to the nutrient solution in increments of 25 millimolar per day to a final concentration of 200 millimolar. Plants were harvested 3 weeks after starting NaCl treatment. Fresh and dry weight of both shoots and roots was decreased more than 50% compared to control plants but the salt-stressed plants appeared healthy and were still actively growing. The salt-stressed plants had much thicker leaves. The salt-treated plants osmotically adjusted to maintain leaf turgor. Leaf K⁺ was decreased but Na⁺ and Cl⁻ were greatly increased.

The potential photosynthetic capacity of the leaves was measured at saturating CO₂ to overcome any stomatal limitation. Photosynthesis of salt-stressed plants varied only by about 10% from the controls when expressed on a leaf area or chlorophyll basis. The yield of variable chlorophyll a fluorescence from leaves was not affected by salt stress. Stomatal conductance decreased 70% in response to salt treatment.

Uncoupled rates of electron transport by isolated intact chloroplasts and by thylakoids were only 10 to 20% below those for control plants. CO₂-dependent O₂ evolution was decreased by 20% in chloroplasts isolated from salt-stressed plants. The concentration of K⁺ in the chloroplast decreased by 50% in the salt-stressed plants, Na⁺ increased by 70%, and Cl⁻ increased by less than 20% despite large increases in leaf Na⁺ and Cl⁻.

It is concluded that, for spinach, salt stress does not result in any major decrease in the photosynthetic potential of the leaf. Actual photosynthesis by the plant may be reduced by other factors such as decreased stomatal conductance and decreased leaf area. Effective compartmentation of ions within the cell may prevent the accumulation of inhibitory levels of Na⁺ and Cl⁻ in the chloroplast.

     

In Vitro Reconstitution of Electron Transport from Glucose-6-Phosphate and NADPH to Nitrite

An NADPH-dependent NO₂⁻-reducing system was reconstituted in vitro using ferredoxin (Fd) NADP⁺ oxidoreductase (FNR), Fd, and nitrite reductase (NiR) from the green alga Chlamydomonas reinhardtii. NO₂⁻ reduction was dependent on all protein components and was operated under either aerobic or anaerobic conditions. NO₂⁻ reduction by this in vitro pathway was inhibited up to 63% by 1 mm NADP⁺. NADP⁺ did not affect either methyl viologen-NiR or Fd-NiR activity, indicating that inhibition was mediated through FNR. When NADPH was replaced with a glucose-6-phosphate dehydrogenase (G6PDH)-dependent NADPH-generating system, rates of NO₂⁻ reduction reached approximately 10 times that of the NADPH-dependent system. G6PDH could be replaced by either 6-phosphogluconate dehydrogenase or isocitrate dehydrogenase, indicating that G6PDH functioned to: (a) regenerate NADPH to support NO₂⁻ reduction and (b) consume NADP⁺, releasing FNR from NADP⁺ inhibition. These results demonstrate the ability of FNR to facilitate the transfer of reducing power from NADPH to Fd in the direction opposite to that which occurs in photosynthesis. The rate of G6PDH-dependent NO₂⁻ reduction observed in vitro is capable of accounting for the observed rates of dark NO₃⁻ assimilation by C. reinhardtii.     

Mitochondrial Respiration Can Support NO(3) and NO(2) Reduction during Photosynthesis : Interactions between Photosynthesis, Respiration, and N Assimilation in the N-Limited Green Alga Selenastrum minutum

Mass spectrometric analysis shows that assimilation of inorganic nitrogen (NH₄⁺, NO₂⁻, NO₃⁻) by N-limited cells of Selenastrum minutum (Naeg.) Collins results in a stimulation of tricarboxylic acid cycle (TCA cycle) CO₂ release in both the light and dark. In a previous study we have shown that TCA cycle reductant generated during NH₄⁺ assimilation is oxidized via the cytochrome electron transport chain, resulting in an increase in respiratory O₂ consumption during photosynthesis (HG Weger, DG Birch, IR Elrifi, DH Turpin [1988] Plant Physiol 86: 688-692). NO₃⁻ and NO₂⁻ assimilation resulted in a larger stimulation of TCA cycle CO₂ release than did NH₄⁺, but a much smaller stimulation of mitochondrial O₂ consumption. NH₄⁺ assimilation was the same in the light and dark and insensitive to DCMU, but was 82% inhibited by anaerobiosis in both the light and dark. NO₃⁻ and NO₂⁻ assimilation rates were maximal in the light, but assimilation could proceed at substantial rates in the light in the presence of DCMU and in the dark. Unlike NH₄⁺, NO₃⁻ and NO₂⁻ assimilation were relatively insensitive to anaerobiosis. These results indicated that operation of the mitochondrial electron transport chain was not required to maintain TCA cycle activity during NO₃⁻ and NO₂⁻ assimilation, suggesting an alternative sink for TCA cycle generated reductant. Evaluation of changes in gross O₂ consumption during NO₃⁻ and NO₂⁻ assimilation suggest that TCA cycle reductant was exported to the chloroplast during photosynthesis and used to support NO₃⁻ and NO₂⁻ reduction.     

Nitrite assimilation and amino nitrogen synthesis in isolated spinach chloroplasts

The assimilation of nitrite leading to de novo synthesis of amino nitrogen in a chloroplast-enriched fraction isolated from freshly harvested young spinach (Spinacia oleracea L.) leaves was demonstrated. The preparations showed approximately 55% intact chloroplasts as determined by light scattering properties and fixed CO₂ at rates of approximately 100 μmoles hr⁻¹ mg chlorophyll⁻¹.     

Regulation of Photosynthetic Electron Transport in Intact Spinach Chloroplasts: I. INFLUENCE OF EXOGENOUS SALTS ON OXALOACETATE REDUCTION

Relatively high concentrations of monovalent salts (150 millimolar) stimulated light-saturated uncoupled rates of O₂ evolution linked to oxaloacetic acid (OAA) reduction by intact chloroplasts 2-to 3-fold. In contrast, monovalent salts partially inhibited light-saturated rates of O₂ evolution coupled to CO₂ fixation and uncoupled rates of nitrite reduction. In the presence of high salt concentration, light-saturated rates of electron transport were about equivalent for all three terminal electron acceptors. It is inferred that exogenous monovalent salts have at least two effects on photosynthetic electron transport, independent of photophosphorylation and CO₂ metabolism: a partial inhibitory effect common to OAA, NO₂⁻ and CO₂ reduction and a marked stimulatory effect unique to the photoreduction of OAA.     

Chloroplast Respiration : A MEANS OF SUPPLYING OXIDIZED PYRIDINE NUCLEOTIDE FOR DARK CHLOROPLASTIC METABOLISM

A spinach (Spinacia oleracia var. America) chloroplast particle fortified with ferredoxin, fructose-1,6-bisphosphate, or ribose-5-phosphate and NADP has been shown to generate NADPH by the oxidation of glyceraldehyde-3 phosphate to glycerate-3-phosphate (PGA) and to reduce ferredoxin with the NADPH. The resulting reduced ferredoxin can reduce O₂ to H₂O₂, nitrite to ammonia, or protons to H₂. Hydrogen production was the result of adding hydrogenase from Chlamydomonas reinhardii to the chloroplast preparation. The predicted stoichiometry of 1 PGA:1 O₂ in the absence of and 2 PGA:1 O₂ in the presence of catalase was observed indicating H₂O₂ as the end product of O₂ reduction. The predicted stoichiometry of 3 PGA:1 nitrite:1 ammonia was also observed. A scheme is presented to account for a sustained generation of NADP and ATP necessary for the dissimilation of starch in the darkened chloroplast. The unifying term chloroplast respiration is introduced to account for those reactions in which reduced ferredoxin interacts with physiological acceptors other than NADP or nitrite, hydrogen, or O₂ respiration when nitrite, protons, or O₂ is the ultimate electron acceptor.     

Nitrate Reduction in Response to CO(2)-Limited Photosynthesis : Relationship to Carbohydrate Supply and Nitrate Reductase Activity in Maize Seedlings

The effects of CO₂-limited photosynthesis on ¹⁵NO₃⁻ uptake and reduction by maize (Zea mays, DeKalb XL-45) seedlings were examined in relation to concurrent effects of CO₂ stress on carbohydrate levels and in vitro nitrate reductase activities. During a 10-hour period in CO₂-depleted air (30 microliters of CO₂/ per liter), cumulative ¹⁵NO₃⁻ uptake and reduction were restricted 22 and 82%, respectively, relative to control seedlings exposed to ambient air containing 450 microliters of CO₂ per liter. The comparable values for roots of decapitated maize seedlings, the shoots of which had previously been subjected to CO₂ stress, were 30 and 42%. The results demonstrate that reduction of entering nitrate by roots as well as shoots was regulated by concurrent photosynthesis. Although in vitro nitrate reductase activity of both tissues declined by 60% during a 10-hour period of CO₂ stress, the remaining activity was greatly in excess of that required to catalyze the measured rate of ¹⁵NO₃⁻ reduction. Root respiration and soluble carbohydrate levels in root tissue were also decreased by CO₂ stress. Collectively, the results indicate that nitrate uptake and reduction were regulated by the supply of energy and carbon skeletons required to support these processes, rather than by the potential enzymatic capacity to catalyze nitrate reduction, as measured by in vitro nitrate reductase activity.     

Differential light induction of nitrate reductases in greening and photobleached soybean seedlings

Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO₃⁻, tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO₃⁻ and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO₃⁻ and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO₃⁻. The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO₃⁻, further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO₃⁻ requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO₃⁻ requirement for induction were quite different. K_m values for NO₃⁻ were identical for both nitrate reductases.     

Kinetics of Denitrifying Growth by Fast-Growing Cowpea Rhizobia

Two fast-growing strains of cowpea rhizobia (A26 and A28) were found to grow anaerobically at the expense of NO₃⁻, NO₂⁻, and N₂O as terminal electron acceptors. The two major differences between aerobic and denitrifying growth were lower yield coefficients (Y) and higher saturation constants (K_s) with nitrogenous oxides as electron acceptors. When grown aerobically, A26 and A28 adhered to Monod kinetics, respectively, as follows: K_s, 3.4 and 3.8 μM; Y, 16.0 and 14.0 g · cells eq⁻¹; μ_max, 0.41 and 0.33 h⁻¹. Yield coefficients for denitrifying growth ranged from 40 to 70% of those for aerobic growth. Only A26 adhered to Monod kinetics with respect to growth on all three nitrogenous oxides. The apparent K_s values were 41, 270, and 460 μM for nitrous oxide, nitrate, and nitrite, respectively; the K_s for A28 grown on nitrate was 250 μM. The results are kinetically and thermodynamically consistent in explaining why O₂ is the preferred electron acceptor. Although no definitive conclusions could be drawn regarding preferential utilization of nitrogenous oxides, nitrite was inhibitory to both strains and effected slower growth. However, growth rates were identical (μ_max, 0.41 h⁻¹) when A26 was grown with either O₂ or NO₃⁻ as an electron acceptor and were only slightly reduced when A28 was grown with NO₃⁻ (0.25 h⁻¹) as opposed to O₂ (0.33 h⁻¹).     

Nodule and Leaf Nitrate Reductases and Nitrogen Fixation in Medicago sativa L. under Water Stress

The effect of water stress on patterns of nitrate reductase activity in the leaves and nodules and on nitrogen fixation were investigated in Medicago sativa L. plants watered 1 week before drought with or without NO₃⁻. Nitrogen fixation was decreased by water stress and also inhibited strongly by the presence of NO₃⁻. During drought, leaf nitrate reductase activity (NRA) decreased significantly particularly in plants watered with NO₃⁻, while with rewatering, leaf NRA recovery was quite important especially in the NO₃⁻-watered plants. As water stress progressed, the nodular NRA increased both in plants watered with NO₃⁻ and in those without NO₃⁻ contrary to the behavior of the leaves. Beyond −15.10⁵ pascal, nodular NRA began to decrease in plants watered with NO₃⁻. This phenomenon was not observed in nodules of plants given water only.     

Role of Nitrate and Nitrite in the Induction of Nitrite Reductase in Leaves of Barley Seedlings

The role of NO₃⁻ and NO₂⁻ in the induction of nitrite reductase (NiR) activity in detached leaves of 8-day-old barley (Hordeum vulgare L.) seedlings was investigated. Barley leaves contained 6 to 8 micromoles NO₂⁻/gram fresh weight × hour of endogenous NiR activity when grown in N-free solutions. Supply of both NO₂⁻ and NO₃⁻ induced the enzyme activity above the endogenous levels (5 and 10 times, respectively at 10 millimolar NO₂⁻ and NO₃⁻ over a 24 hour period). In NO₃⁻-supplied leaves, NiR induction occurred at an ambient NO₃⁻ concentration of as low as 0.05 millimolar; however, no NiR induction was found in leaves supplied with NO₂⁻ until the ambient NO₂⁻ concentration was 0.5 millimolar. Nitrate accumulated in NO₂⁻-fed leaves. The amount of NO₃⁻ accumulating in NO₂⁻-fed leaves induced similar levels of NiR as did equivalent amounts of NO₃⁻ accumulating in NO₃⁻-fed leaves. Induction of NiR in NO₂⁻-fed leaves was not seen until NO₃⁻ was detectable (30 nanomoles/gram fresh weight) in the leaves. The internal concentrations of NO₃⁻, irrespective of N source, were highly correlated with the levels of NiR induced. When the reduction of NO₃⁻ to NO₂⁻ was inhibited by WO₄²⁻, the induction of NiR was inhibited only partially. The results indicate that in barley leaves NiR is induced by NO₃⁻ directly, i.e. without being reduced to NO₂⁻, and that absorbed NO₂⁻ induces the enzyme activity indirectly after being oxidized to NO₃⁻ within the leaf.     

Interaction of ferredoxin and ferredoxin-NADP reductase with thylakoids

Ferredoxin-NADP reductase accounts for about 50% of the NADPH diaphorase activity of spinach leaf homogenates. The enzyme is bound to thylakoid membranes, but can be slowly extracted by aqueous buffers. Ferredoxin-NADP reductase can be extracted from the membranes by a 1- to 2-min treatment with a low concentration of trypsin. This treatment completely inactivates NADP photoreduction but does not affect electron transport from water to ferredoxin. It is shown that the inactivation is due to solubilization of ferredoxin-NADP reductase: the activity can be restored by addition of a very large excess of soluble enzyme in pure form. When ferredoxin-NADP reductase is added as a soluble enzyme after extraction or inactivation (by a specific antibody) of the membrane-bound enzyme, NADP photoreduction requires a very large excess of this enzyme, and the apparent K_m for ferredoxin is also increased. These observations are discussed as related to the interactions of thylakoids with ferredoxin-NADP reductase.