Regulation of Nitrate Assimilation and Nitrate Respiration in Aerobacter aerogenes

The influence of growth conditions on assimilatory and respiratory nitrate reduction in Aerobacter aerogenes was studied. The level of nitrate reductase activity in cells, growing in minimal medium with nitrate as the sole nitrogen source, was much lower under aerobic than anaerobic conditions. Further, the enzyme of the aerobic cultures was very sensitive to sonic disintegration, as distinct from the enzyme of anaerobic cultures. When a culture of A. aerogenes was shifted from anaerobic growth in minimal medium with nitrate and NH(4) (+) to aerobiosis in the same medium, but without NH(4) (+), the production of nitrite stopped instantaneously and the total activity of nitrate reductase decreased sharply. Moreover, there was a lag in growth of about 3 hr after such a shift. After resumption of growth, the total enzymatic activity increased again slowly and simultaneously became gradually sensitive to sonic disintegration. These findings show that oxygen inactivates the anaerobic nitrate reductase and represses its further formation; only after a de novo synthesis of nitrate reductase with an assimilatory function will growth be resumed. The enzyme in aerobic cultures was not significantly inactivated by air, only by pure oxygen. The formation of the assimilatory enzyme complex was repressed, however, by NH(4) (+), under both aerobic and anaerobic conditions. The results indicate that the formation of the assimilatory enzyme complex and that of the respiratory enzyme complex are regulated differently. We suggest that both complexes have a different composition, but that the nitrate reductase in both cases is the same protein.     

Herbicidal Effects on Water Uptake of Seeds of Selected Weed Species

Seeds of yellow foxtail, johnsongrass, pigweed and quackgrass were investigated to determine the effects of certain herbicides on water uptake by these species. Seeds of johnsongrass showed slight increase in water uptake over the 20-h period when treated with 1, 5, 10, and 500 mg/1 of 2,4-D. Tordon caused a most pronounced increase in water uptake at 0.1 and 1 mg/l as compared with the controls. Johnsongrass gave indications of either increases or decreases in water uptake when treated with herbicides during the 20-h period, but showed no effects during the 10-h period. Dormant seeds of yellow foxtail indicated an enhancement in water uptake at all 2,4-D concentrations during the 20 h period. The effects which the different herbicides had upon the uptake of water in yellow foxtail seeds seemed to be directly proportional to increase in imbibition time. In all cases, more water was imbibed after 20 h over that observed at 10 h. During the 10-h imbibition period, pigweed seeds treated with 2,4-D was noted to have a slight stimulating effect on water uptake at all concentrations employed except 5 mg/l, which indicated a depressing effect. It was also observed that 2,4-D caused a significant stimulating effect on water uptake of pigweed seeds over the 20-h period as compared with the controls. A stimulation in the total quantity of water imbibed after 20 h was noted at dacthal concentrations below 100 mg/l whereas at the 500 mg/l, a slight depression in water imbibition was observed. At dalapon concentrations of 1 to 100 mg/l, a marked stimulation in total water imbibed by quackgrass seeds was shown during the 10-h period.     

Regulation of Expression of Nitrate and Dinitrogen Assimilation by Anabaena Species

Anabaena sp. strain 7120 appeared more responsive to nitrogen control than A. cylindrica. Growth in the presence of nitrate strongly repressed the differentiation of heterocysts and fixation of dinitrogen in Anabaena sp. strain 7120, but only weakly in A. cylindrica. Nitrate assimilation by ammonium-grown cultures was strongly repressed in Anabaena sp. strain 7120, but less so in A. cylindrica. The repressive effect of nitrate on dinitrogen assimilation in Anabaena sp. strain 7120, compared to A. cylindrica, did not correlate with a greater rate of nitrate transport, reduction to ammonium, assimilation into amino acids, or growth. Although both species grew at similar rates with dinitrogen, A. cylindrica grew faster with nitrate, incorporated more ¹³NO₃⁻ into amino acids, and assimilated (transported) nitrate at the same rate as Anabaena sp. strain 7120. Full expression of nitrate assimilation in the two species occurred within 2.5 h (10 to 14% of their generation times) after transfer to nitrate medium. The induction and continued expression of nitrate assimilation was dependent on protein synthesis. The half-saturation constants for nitrate assimilation and for nitrate and ammonium repression of dinitrogen assimilation have ecological significance with respect to nitrogen-dependent growth and competitiveness of the two Anabaena species.     

Effects of Various Concentrations of Carbon Dioxide on Respiration and Potassium Uptake in Barley Roots

Barley roots contain a CO₂ sensitive respiratory fraction which is inhibited in 50 per cent CO₂ and is partially restored upon subsequent exposure to air. The residual O₂ consumption occurring at CO₂ concentrations between 50 per cent and 95 per cent amounts to about 40 per cent of the O₂ uptake in air and can support K⁺ uptake for a limited time at a rate equal to or higher than occurs in air. Above 95 per cent CO₂ both O₂ and K⁺ uptakes decrease rapidly. 2,4-dinitrophenol (DNP), in the range of 10^?6 to 10^?5M, stimulates O₂ uptake by the roots in air. The stimulation is absent when roots are treated with DNP in 80 per cent CO₂, presumably because of the reduced demand for inorganic phosphate and phosphate acceptor at the lower respiratory level in high CO₂. In either air or CO₂, K⁺ uptake is strongly inhibited by DNP. A comparison of the respiratory and K⁺ uptake data indicates that O₂ consumption is a necessary requirement for the uptake process in high CO₂. Protoplasmic streaming in the root cells is rapidly stopped by high CO₂ although K⁺ uptake and O₂ consumption continue. The cation uptake mechanism in high CO₂ appears to be limited to the stationary cytoplasm. It is also possible that a similar mechanism may be involved in cation uptake in air.     

The Induction of Light Sensitivity in Weed Seeds by Burial

Seeds of a number of weed species were buried in soil for aperiod of 50 weeks. At the end of this period the soil was disturbedunder controlled illumination. In three species germinationappeared to be unaffected by light, prior to burial. Followingburial, however, germination took place only in the light, suggestingthe induction of light sensitivity. In eight other species,which showed varying degrees of light sensitivity when freshseeds were examined, germination, following burial, was completelydependent upon exposure to light. Seed germination has also been shown to be inhibited by burialunder laboratory conditions. The extent of this inhibition isdependent upon the depth of burial and the water content ofthe medium. Experiments using artificial aeration indicate thatit is the presence of a gaseous inhibitor in the soil atmospherewhich prevents germination. This inhibitor appears to arisefrom the seeds themselves and is not carbon dioxide.     

Movement of Weed Seeds in Reclamation Areas

The presence or absence of obstructions can affect seed dispersal. Reclamation activities often cause changes in the type and amount of such obstructions. The consequences of removing obstructions on the dispersal of undesirable species are unknown. In western North America, reclamation may often proceed in areas surrounded by the invasive Bromus tectorum L. (cheatgrass). The importance of preventing cheatgrass seed dispersal from surrounding landscapes is an unknown factor in reducing cheatgrass competition in these areas. I quantified cheatgrass seed dispersal in the early stages of reclamation, when soils were bare. Four groups of 100 sterilized, fluorescently marked cheatgrass seeds were released in each of three areas in NW Colorado, USA. Seeds were located at night using blacklights four times over 14 days, and the distance between each seed and the point of release was measured. Across sites, dispersal distance averaged 2.4 m, 5% of seeds traveled further than 7.6 m, and maximum recorded distance was 20.8 m. Maximum distances reported in this study are 50‐fold higher than previously reported for intact sagebrush ecosystems. I suggest that the difference is due to a lack of impediments to secondary dispersal on bare soil. When reclamation areas are surrounded by weeds such as cheatgrass, seeds dispersing from the perimeter may influence restoration outcome.     

Respiration of Mitochondria in Developing Sunflower Seeds

We studied the oxidative and phosphorylating activity of mitochondria in the seeds of three sunflower cultivars (Polevik, Peredovik, and Yubileinyi) during development of the seed embryo within 1 to 54 days after flowering. The rates of succinate oxidation by the mitochondria were 1.5–2 times those of malate or -ketoglutarate oxidation. The ratio of substrate oxidation rates underwent changes during the seed growth. The differences were recorded between cultivars as concerns the times when the maximum oxidation rates were reached. Oxidation was coupled with phosphorylation during the entire period of seed development: the value of respiratory control after Chance changed from 1.4 to 7. By the time of transition to maturation, the rates of oxidation of both substrates and the values of respiratory control and ADP/O decreased. The results we obtained suggest that by days 13–15 of seed embryo growth, the rate of ATP production decreases upon oxidation of Krebs cycle products.     

Regulation of Respiration in the Leaves and Roots of Two Lolium perenne Populations with Contrasting Mature Leaf Respiration Rates and Crop Yields

Measurements of O₂ uptake were made on leaves and roots of two populations of Lolium perenne L. cv S23 (GL66 and GL72), previously shown to have contrasting rates of CO₂ evolution and yields of dry matter. O₂ uptake was faster in the mature leaves of GL66 than those of GL72, but no difference was observed in the respiratory rates of meristematic leaf bases or mature roots. The growth rate of GL72 was faster than that of GL66. Cyanide resistance was substantial in mature leaves but the alternative path did not contribute to O₂ uptake in the dark. In both populations, adding malate and glycine stimulated O₂ uptake, but exogenous sucrose only stimulated when uncoupler was also present. The difference between the respiratory rates of the two populations was maintained under all investigated conditions. We conclude that the rate of mature leaf respiration in the dark in L. perenne is limited by adenylate control of glycolysis. The difference between the fast (GL66) and slow (GL72) respiring populations reflected a greater respiratory capacity and higher turnover of ATP in GL66. Alternative path capacity was also high in the roots of both and contributed substantially to O₂ uptake, as indicated by inhibition by salicylhydroxamic acid in the absence of KCN. The alternative path capacity of meristematic leaf bases was considerably less than that in mature leaves.

Transverse and cross-sections were made of mature leaves of both populations to study anatomical features which might explain the differences in ATP turnover, suggested by the biochemical experiments. Leaves of GL72 were thicker but did not show a different anatomy when compared with GL66. The increased thickness was not due to more or larger cells but entirely to a larger intercellular volume.

     

Respiration in Adipocytes is Inhibited by Reactive Oxygen Species

It is a desirable goal to stimulate fuel oxidation in adipocytes and shift the balance toward less fuel storage and more burning. To understand this regulatory process, respiration was measured in primary rat adipocytes, mitochondria, and fat‐fed mice. Maximum O₂ consumption, in vitro, was determined with a chemical uncoupler of oxidative phosphorylation (carbonylcyanide p‐trifluoromethoxyphenylhydrazone (FCCP)). The adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio was measured by luminescence. Mitochondria were localized by confocal microscopy with MitoTracker Green and their membrane potential (Δψ_M) measured using tetramethylrhodamine ethyl ester perchlorate (TMRE). The effect of N‐acetylcysteine (NAC) on respiration and body composition in vivo was assessed in mice. Addition of FCCP collapsed Δψ_M and decreased the ATP/ADP ratio. However, we demonstrated the same rate of adipocyte O₂ consumption in the absence or presence of fuels and FCCP. Respiration was only stimulated when reactive oxygen species (ROS) were scavenged by pyruvate or NAC: other fuels or fuel combinations had little effect. Importantly, the ROS scavenging role of pyruvate was not affected by rotenone, an inhibitor of mitochondrial complex I. In addition, mice that consumed NAC exhibited increased O₂ consumption and decreased body fat in vivo. These studies suggest for the first time that adipocyte O₂ consumption may be inhibited by ROS, because pyruvate and NAC stimulated respiration. ROS inhibition of O₂ consumption may explain the difficulty to identify effective strategies to increase fat burning in adipocytes. Stimulating fuel oxidation in adipocytes by decreasing ROS may provide a novel means to shift the balance from fuel storage to fuel burning.     

Role of Menaquinone in Nitrate Respiration in Staphylococcus aureus

Two menaquinone-deficient and one aromatic-deficient mutants of Staphylococcus aureus were unable to reduce nitrate to nitrite. Reinitiation of menaquinone synthesis in the aromatic-deficient mutant by growing it with shikimic acid restored its nitrate respiratory activity. The results clearly demonstrate a role for menaquinone in nitrate respiration in Staphylococcus aureus.     

The Photocontrol of Respiration in Light-Sensitive Lettuce Seeds

The rate of respiration of positively photoblastic lettuce (Lactucasativa L. cv. Cannington Forcing) at 25 °C is increasedfollowing brief exposure to red light and is under phytochromecontrol. The higher rate is maintained until radicle emergencebegins, at which time a further increase takes place. At 30°C the rate of respiration is higher than at 25°C butno red light promotion occurs and very few-seeds germinate.     

Nitrate Reduction in Roots as Affected by the Presence of Potassium and by Flux of Nitrate through the Roots

Dark-grown, detopped corn seedlings (cv. Pioneer 3369A) were exposed to treatment solutions containing Ca(NO₃)₂, NaNO₃, or KNO₃; KNO₃ plus 50 or 100 millimolar sorbitol; and KNO₃ at root temperatures of 30, 22, or 16 C. In all experiments, the accelerated phase of NO₃⁻ transport had previously been induced by prior exposure to NO₃⁻ for 10 hours. The experimental system allowed direct measurements of net NO₃⁻ uptake and translocation, and calculation of NO₃⁻ reduction in the root. The presence of K⁺ resulted in small increases in NO₃⁻ uptake, but appreciably stimulated NO₃⁻ translocation out of the root. Enhanced translocation was associated with a marked decrease in the proportion of absorbed NO₃⁻ that was reduced in the root. When translocation was slowed by osmoticum or by low root temperatures, a greater proportion of absorbed NO₃⁻ was reduced in the presence of K⁺. Results support the proposition that NO₃⁻ reduction in the root is reciprocally related to the rate of NO₃⁻ transport through the root symplasm.     

Regulation of Nitrate Reductase Activity by Nitrate during Light-Dark Transition in Detached Oat Leaves

In oat (Avena sativa L. cv. Suregrain) leaf segments, light-darkmodulation of nitrate reductase (NR) activity could be observedonly when segments were kept in –NO₃ conditions. We presenthere evidence that nitrate would regulate NR activity by modulatingthe phosphorylation status of the enzyme. (Received June 19, 1995; Accepted August 14, 1995)     

Feedback Regulation of Nitrate Influx in Barley Roots by Nitrate,Nitrite, and Ammonium

The short-lived radiotracer 13N was used to study feedback regulation of nitrate influx through the inducible high-affinity transport system of barley (Hordeum vulgare L. cv Steptoe) roots. Both wild-type plants and the mutant line Az12:Az70 (genotype nar1a;nar7w), which is deficient in the NADH-specific and NAD(P)H-bispecific nitrate reductases (R.L. Warner, R.C. Huffaker [1989] Plant Physiol 91: 947-953) showed strong feedback inhibition of nitrate influx within approximately 5 d of exposure to 100 fmu]M nitrate. The result with the mutant, in which the flux of nitrogen into reduced products is greatly reduced, indicated that nitrate itself was capable of exercising feedback regulation upon its own influx. This conclusion was supported by the observation that feedback in wild-type plants occurred in both the presence and absence of L-methionine sulfoximine, an inhibitor of ammonium assimilation. Nitrite and ammonium were also found to be capable of exerting feedback inhibition upon nitrate influx, although it was not determined whether these ions themselves or subsequent metabolites were responsible for the effect. It is suggested that feed-back regulation of nitrate influx is potentially mediated through several nitrogen pools, including that of nitrate itself.     

Nitrate Reduction in Different Grass Species

Optimal extraction conditions, assay conditions, and levels of nit rate reductase activity (NRA) were determined for eight forage grass species adaptable to growing conditions in western North Dakota. Optimal pH for extraction of the enzyme nitrate reductase (NADH: nitrate oxidoreductase) for these species ranged from 7.0 to 9.5, whereas assay pH was 7.6 in all eight species. Substrate concentrations ranged from 1.0 to 10.0 mM for maximum NRA with higher concentrations (100 mM) significantly inhibiting NRA. The enzyme was NADH₂ (0.1 to 0.2 mM for maximum activity) specific. Enhancement of maximum activity with the addition of cysteine during extraction was species dependent; six species required high cysteine concentrations between 5 mM and 10 mM and one species required only a 2.5 mM concentration. The degree of sulfhydryl protection offered by cysteine also varied. Comparisons were made between in vivo and in vitro assay methods. Ratios of in vitro to in vivo NRA ranged from 2.2. to 10.8. Use of bovine serum albumin as a protein stabilizer during extraction increased the measurable NRA in some species. Applications of nitrate reductase assay techniques to field work will be discussed.