Distribution of nitrate reductase activity in Vicia faba: Effect of nitrate and plant genotype

The short term effect of NO₃⁻ (12 mM) on nitrate reductase (NR. EC 1.6.6.1) activity has been studied in the roots, nodules and leaves of different genotypes of Vicia faba L. at the end of vegetative growth. Root and leaf NR activity responded positively to NO₃⁻ while nodule activity, where detected, proved to he strongly inhibited. The withdraw of this NO₃⁻ from the solution consistently reduced activity in the roots and leaves but surprising, promoted a significant increase in nodule activity, which matched or surpassed that of control plants On the other hand, nodules developed in the presence of 8 mM NO₃⁻ expressed an on average 141% higher level of NR activity than did controls. This effect was observed even in nodules with negligible control activity. In any case, a naturally occurring mutant (VF17) lacking root and nodule NR activity is described. The results indicate that in V. faba. the effects of NO₃⁻ and plant genotype on NR activity depended on plant organ and time of NO₃⁻ application, hut the distribution of NO₃⁻ reduction through the plain was mainly dependent on plant genotype, and to a lesser extent on NO: supply and plant age.     

Induction of nitrate reductase in needles of Scots pine seedlings by NOX and NO3−

The possibility to induce nitrate reductase (NR; EC 1.6.6.2) in needles of Scots pine ( Pinus sylvestris L.) seedlings was studied. The NR activity was measured by an in vivo assay. Although increased NR activities were found in the roots after application of NO₃⁻, no such increase could be detected in the needles. Detached seedlings placed in NO₃⁻ solution showed increasing NR activities with increasing NO₃⁻ concentrations. Exposure of seedlings to NO_x (70–80 ppb NO₂ and 8–12ppb NO) resulted in an increase of the NR activity from 10–20 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹ to about 400 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. This level was reached after 2–4 days of exposure, thereafter the NR activity decreased to about 200 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. Analyses of free amino acids showed low concentrations of arginine and glutamine in NO_x-fumigated seedlings compared to corresponding controls.     

Impact of gaseous nitrogen deposition on plant functioning

Dry deposition of NH₃ and NO_x (NO and NO₂) can affect plant metabolism at the cellular and whole-plant level. Gaseous pollutants enter the plant mainly through the stomata, and once in the apoplast NH₃ dissolves to form NH₄⁺, whereas NO₂ dissolves to form NO₃⁻ and NO₂⁻. The latter compound can also be formed after exposure to NO. There is evidence that NH₃-N and NO_x-N can be reversibly stored in the apoplast. Temporary storage might affect processes such as absorption rate, assimilation and re-emission. Once formed, NO₃⁻ and NO₂⁻ can be reduced, and NH₄⁺ can be assimilated via the normal enzymatic pathways, nitrate reductase (NR), nitrite reductase and the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Fumigation with low concentrations of atmospheric NH₃ increases in vitro glutamine synthetase activity, but whether this involves both or only one of the GS isoforms is still an open question. There seems to be no correlation between fumigation with low concentrations of NH₃ and in vitro GDH activity. The contribution of atmospheric NH₃ and NO₂ deposition to the N budget of the whole plant has been calculated for various atmospheric pollutant concentrations and relative growth rates ( RGRs ). It is concluded that at current ambient atmospheric N concentrations the direct impact of gaseous N uptake by foliage on plant growth is generally small.     

Response of nitrogen metabolism to boron toxicity in tomato plants

Boron (B) toxicity has become important in areas close to the Mediterranean Sea where intensive agriculture has been developed. The objective of this research was to study the effects of B toxicity (0.5 m m and 2.0 m m B) on nitrogen (N) assimilation of two tomato cultivars that are often used in these areas. Leaf biomass, relative leaf growth rate (RGR_L), concentration of B, nitrate (NO₃⁻), ammonium (NH₄⁺), organic N, amino acids and soluble proteins, as well as nitrate reductase (NR), nitrite reductase (NiR), glutamine synthase (GS), glutamate synthetase (GOGAT) and glutamate dehydrogenase (GDH) activities were analysed in leaves. Boron toxicity significantly decreased leaf biomass, RGR_L, organic N, soluble proteins, and NR and NiR activities. The lowest NO₃⁻ and NH₄⁺ concentration in leaves was recorded when plants were supplied with 2.0 m m B in the root medium. Total B, amino acids, activities of GS, GOGAT and GDH increased under B toxicity. Data from the present study prove that B toxicity causes inhibition of NO₃⁻ reduction and increases NH₄⁺ assimilation in tomato plants.     

Different effects of supplied ammonium on glutamine synthetase activity in mustard (Sinapis alba) and pine (Pinus sylvestris) seedlings

The activity of glutamine synthetase (GS) in mustard ( Sinapis alba L.) and Scots pine ( Pinus sylvestris L.) seedlings was used as an index to evaluate the capacity to cope with excessive ammonium supply. In these 2 species GS activity was differently affected by the application of nitrogen compounds (NH₄⁺ or NO₃⁻). Mustard seedlings older than 5 days showed a considerable increase in GS activity after NH₄⁺ or NO₃⁻ application. This response was independent of the energy flux, but GS activity in general was positively affected by light. Endogenous NH₄⁺ did not accumulate greatly after nitrogen supply. In contrast, seedlings of Scots pine accumulated NH₄⁺ in cotyledons and roots and showed no stimulation of GS activity after the application of ammonium. In addition, root growth was drastically reduced. Thus, the pine seedlings seem to have insufficient capacity to assimilate exogenously supplied ammonium. NO₃⁻, however, did not lead to any harmful effects.     

Regulation of K+ and NO3− fluxes in roots of sunflower (Helianthus annuus) after changes in light intensity

Shoot activity has been reported to affect rates of ion uptake by plant roots in other ways than merely through supply of assimilates. To elucidate the mechanisms by which a signal from the upper part of the plant controls the rate of K⁺ and NO₃⁻ uptake by roots, both uptake of K⁺ and NO₃⁻ and secretion into the xylem of young sunflower plants ( Helianthus annuus L.) were measured after changes in light intensity.
No close correlation was observed between the uptake of NO₃⁻ and that of K⁺; an increase in light intensity produced a much greater stimulation of NO₃⁻ uptake than of K⁺ uptake. On the other hand, secretion of NO₃⁻ into the xylem was tightly coupled to that of K⁺, and this coupling was strongly disturbed by excision of the root. The results suggest the involvement of the K₂-malate shuttle on the regulation by the shoot of K⁺ and NO₃⁻ secretion in the xylem, which is linked to NO₃⁻ uptake, while K⁺ uptake is independent of this regulation mechanism.     

Photosynthate costs associated with the utilization of different nitrogen–forms: influence on the carbon balance of plants and shoot–root biomass partitioning

The photosynthate costs of processes (amino acid and protein synthesis and turnover, and pH regulation) associated with the utilization of nitrate (NO₃⁻), ammonium (NH₄⁺) or glutamine (Gln) for plant growth were estimated. Based on these estimates, the effects of these forms of nitrogen (N) on the carbon balance of plants and on shoot–root biomass allocation were evaluated. The results indicated that NO₃⁻ as an N source for plant growth is not substantially more expensive to utilize than either NH₄⁺ or Gln, particularly in the long term when costs due to protein turnover dominate the total costs of N utilization. It is also suggested that the photosynthate use in processes associated with N assimilation has little impact on the carbon balance of plants, and hence on shoot–root biomass allocation.     

Control of Nitrate Uptake by Phloem-Translocated Glutamine in Zea mays L. Seedlings

Abstract: The putative role of glutamine, exported from leaves to roots, as a negative feedback signal for nitrate uptake was investigated in Zea mays L. seedlings. Glutamine (Gln) was supplied by immersion of the tip-cut leaves in a concentrated solution. Nitrate (NO₃⁻) uptake was measured by its depletion in amino acid-free medium. The treatment with Gln resulted in a strong inhibition of nitrate uptake rate, accompanied by a significant enrichment of amino compounds in root tissue. The effect of N-availability on NO₃⁻ uptake was determined in split-root cultures. The plants were subjected to complete or localized N supply. Inducible NO₃⁻ uptake systems were also induced in N-deprived roots when the opposite side of the root system was supplied with KNO₃. The inhibitory effect of Gln was unaffected by localized N supply on one side of the split-root. The potential role of Gln in the shoot-to-root control of NO₃⁻ uptake is discussed.     

Simultaneous Measurement of Nitrite and Nitrate Levels as Indices of Nitric Oxide Release in the Cerebellum of Conscious Rats

Abstract: We examined the modulation of nitric oxide production in vivo by measuring levels of nitrite (NO₂⁻) and nitrate (NO₃⁻) in the dialysate of the cerebellum in conscious rats, by using an in vivo brain microdialysis technique. The levels of both NO₂⁻ and NO₃⁻ were decreased by the intraperitoneal injection of N ^G-nitro- l -arginine methyl ester, an inhibitor of nitric oxide synthase, whereas N ^G-nitro- d -arginine methyl ester had no effect. l -Arginine by itself increased NO₂⁻ and NO₃⁻ levels and diminished the reduction of their levels caused by N ^G-nitro- l -arginine methyl ester. Direct infusion of l -glutamate, N -methyl- d -aspartate, or KCl into the cerebellum through a dialysis probe resulted in an increase in NO₂⁻ and/or NO₃⁻ levels. The effects of N -methyl- d -aspartate and KCl were dependent on extracellular calcium. Furthermore, the stimulatory effects of l -glutamate and N -methyl- d -aspartate were inhibited by N ^G-nitro- l -arginine methyl ester and (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), an N -methyl- d -aspartate receptor antagonist. These results suggest that NO₂⁻ and NO₃⁻ levels may be related to nitric oxide production in vivo.     

Soluble and membrane-associated nitrate reductases in the dinoflagellate Peridinium gatunense

An improved method of cell fractionation allowed the extraction of soluble (sNR) and membrane-associated (mNR) forms of nitrate reductase (NR) from a dinoflagellate, even though in previous studies only mNR had been found in these algae. Both activities were assayed in cell-free extracts of Peridinium gatunense from Lake Kinneret, Israel, after disruption of the cells and differential centrifugation. In the cultures used, sNR showed much higher NO₃⁻-reducing activity. Only a low proportion, 2.5–3% of NR activity, was found to be associated with mNR. Moreover, mNR comprised two forms as indicated by protein solubilization: a tightly membrane-bound and a more weakly attached NR. Ascorbate inhibited all NR activities, but that of mNR recovered after its removal. Polyvinyl pyrrolidone (PVP) and DTT also diminished sNR and mNR activities. For both enzymes, pH optima (7.65) and temperature optima (13–25°C) were similar, and agreed with those for optimum growth of P. gatunense both in culture and in the lake. The most efficient electron donor was NADH, though NADPH sustained low NR activities. Carboxylic anions such as succinate and malate did not support any reduction of NO₃⁻, nor did they cause any stimulation of sNR or mNR activities. Both forms of NR showed a high affinity for their substrates: K _m was c. 10 μM for NO₃⁻ and c. 5 μM for NADH. The high efficiency of NO₃⁻ assimilation by Peridinium seems to be limited mainly by energy under otherwise optimal nutritional conditions and, at low nitrate concentrations, the low K _m may be one of the main reasons for the high competitivity of this alga in Lake Kinneret.     

Biochemical characterization of nitrate and nitrite reduction in the wild-type and a nitrate reductase mutant of soybean

Enzyme activities involved in nitrate assimilation were analyzed from crude leaf extracts of wild-type (cv. Williams) and mutant ( nr₁ ) soybean [ Glycine max (L.) Merr.] plants lacking constitutive nitrate reductase (NR) activity. The nr₁ soybean mutant (formerly LNR-2), had decreased NADH-NR, FMNH₂-NR and cytochrome c reductase activities, all of which were associated with the loss of constitutive NR activity. Measurement of FMNH₂-NR activity, by nitrite determination, was accurate since nitrite reductase could not use FMNH₂ as a reductant source. Nitrite reductase activity was normal in the nr₁ plant type in the presence of reduced methyl viologen. Assuming that constitutive NR is similar in structure to nitrate reductases from other plants, presence of xanthine dehydrogenase activity and loss of cytochrome c reductase activity indicated that the apoprotein and not the molybdenum cofactor had been affected in the constitutive enzyme of the mutant. Constitutive NR from urea-grown wild-type plants had 1) greater ability to use FMNH₂ as an electron donor, 2) a lower pH optimum, and 3) decreased ability to distinguish between NO₃⁻ and HCO₃⁻, compared with inducible NR from NO₃⁻-grown nr₁ plants. The presence in soybean leaves of a nitrate reductase with a pH optimum of 7.5 is contrary to previous reports and indicates that soybean is not an exception among higher plants for this activity.     

Nitrate and nitrite reduction in the plant fraction of alfalfa root nodules

The plant fraction of alfalfa ( Medicago sativa L. cv. Aragon) nodules contained both nitrate reductase (NR) and nitrite reductase (NiR). Specific activity of NADH-NR from the cytosol of nodules not treated with NO₃- was about 30 nmol (mg protein)^-1-h^-1 and was not basically affected by NO₃ addition. In contrast, typical specific activity for cytosolic NiR was 1.5 umol (mg protein)^-1h^-1 using methyl viologen as electron donor. This activity strongly increased with NO₃ concentration, probably due to substrate induction. Maximal activity was 3.5 μmol (mg protein)^-1h^-1 at 50 to 200 mM NO₃.
Estimates indicate that the contribution of cytosol to the overall NR and NiR activities of alfalfa nodules is distinctly different: less than 10% and about 70%, respectively. The increasing amounts of NO₂ accumulating in the cytosol upon NO₃, supply, and the different response to NO₃ of bacteroid and cytosolic NRs support the concept that most of this NO₂ comes from the bacteroids.     

Regulation of nitrogenase activity in Bradyrhizobium japonicum/soybean symbiosis by plant N status as determined by shoot C:N ratio

Regulation of nitrogenase is not sufficiently understood to engineer symbioses that achieve a high N₂ fixation rate under high levels of soil N. In the present hydroponic growth chamber study we evaluated the hypothesis that nitrogenase activity and the extent of its inhibition by NO₃⁻ may be related to both N and carbohydrate levels in plant tissues. A wide range of C:N ratios in various plant tissues (8.5 to 41.0, 1.9 to 3.7, and 0.8 to 1.8, respectively, in shoots, roots, and nodules) was generated through a combination of light and CO₂ levels, using two soybean genotypes differing in C and N acquisition rates. For both genotypes, N concentration in shoots was negatively correlated to nitrogenase activity and positively correlated to the extent of nitrogenase inhibition by NO₃⁻. Furthermore, nitrogenase activity was positively correlated to total nonstructural carbohydrates (TNC) and C:N ratio in shoot and nodules for both genotypes. Nitrogenase inhibition by NO₃⁻ was negatively correlated to TNC and C:N ratio in shoots, but not in nodules for both genotypes. At the onset of nitrogenase inhibition by NO₃⁻, C:N ratio declined in shoots but not in nodules. These results indicate that both C and N levels in plant tissues are involved in regulation of nitrogenase activity. We suggest that the level of nitrogenase activity may be determined by (1) N needs (as determined by shoot C:N) and (2) availability of carbohydrates in nodules. Modulation of the nitrogenase activity may occur through sensing changes in plant N, i.e. changes in shoot C:N ratio, possibly through some phloem translocatable compound(s).     

Regulation of nitrogenase activity in Bradyrhizobium japonicum/soybean symbiosis by plant N status as determined by shoot C:N ratio

Regulation of nitrogenase is not sufficiently understood to engineer symbioses that achieve a high N₂ fixation rate under high levels of soil N. In the present hydroponic growth chamber study we evaluated the hypothesis that nitrogenase activity and the extent of its inhibition by NO₃⁻ may be related to both N and carbohydrate levels in plant tissues. A wide range of C:N ratios in various plant tissues (8.5 to 41.0, 1.9 to 3.7, and 0.8 to 1.8, respectively, in shoots, roots, and nodules) was generated through a combination of light and CO₂ levels, using two soybean genotypes differing in C and N acquisition rates. For both genotypes, N concentration in shoots was negatively correlated to nitrogenase activity and positively correlated to the extent of nitrogenase inhibition by NO₃⁻. Furthermore, nitrogenase activity was positively correlated to total nonstructural carbohydrates (TNC) and C:N ratio in shoot and nodules for both genotypes. Nitrogenase inhibition by NO₃⁻ was negatively correlated to TNC and C:N ratio in shoots, but not in nodules for both genotypes. At the onset of nitrogenase inhibition by NO₃⁻, C:N ratio declined in shoots but not in nodules. These results indicate that both C and N levels in plant tissues are involved in regulation of nitrogenase activity. We suggest that the level of nitrogenase activity may be determined by (1) N needs (as determined by shoot C:N) and (2) availability of carbohydrates in nodules. Modulation of the nitrogenase activity may occur through sensing changes in plant N, i.e. changes in shoot C:N ratio, possibly through some phloem translocatable compound(s).     

Nitrate metabolism in the cyanobacterium Anabaena cycadeae: Regulation of nitrate uptake and reductase by ammonia

Nitrogen regulation of nitrate uptake and nitrate reductase (EC 1.7.99.4) was studied in the cyanobacterium Anabaena cycadeae Reinke and its glutamine auxotroph. Development of the nitrate uptake system preceded, and was independent of, the development of the nitrate reductase system. The levels of both systems were several-fold higher in the glutamine auxotroph lacking glutamine synthetase (EC 6.3.1.2) than in the wild type strain having normal glutamine synthetase activity. The nitrate uptake system was found to be NH4-repressible and the nitrate reductase system NO₃⁻-inducible. NH₄⁺ was the initial repressor signal for the uptake process which was involved in the control of the NO₃⁻inducible reductase system.     

The role of sulphur compounds for breeding success of Ips typographus L. (Col., Scolytidae) on Norway Spruce (Picea abies [L.] Karst.)

Bark beetles, especially Ips typographus L. represent a severe biotic threat for spruce ( Picea abies [L.] Karst.) at low altitudes in Europe. We compared sulphur (total S, SO₄²⁻, glutathione, cysteine, methionine), nitrogen (total N, NO₃⁻, total protein, free amino acids), carbon, total phosphorus and PO₄³⁻, tree vigour index (TVI) and water content of the phloem after felling, and their dependent changes (tdc) with the breeding success of I. typographus . Twenty trees were classified according to age (34/90 years) and crown density (high/intermediate/low). Water content was higher in young trees than in old trees, higher in the crown than at breast height, and decreased significantly within the 8-week study period. In old trees, breeding success, length of mother galleries and SO₄²⁻ were significantly higher, while total protein, NO₃⁻ and water content were significantly lower than in young trees. Trees with intermediate crown density provided the best breeding success for I. typographus and had significantly higher arginine content and C/N ratio as well as low amounts of phosphate and glutamine. During the period of bark beetle breeding, total sulphur, glutathione, protein, NO₃⁻, aspartate, glutamine, glutamate, arginine and γ-aminobutyrate decreased significantly. The results support previous investigations that I. typographus develops best in physiologically weakened trees.     

NO2‐induced nitrate reductase activity in needles of Norway spruce (Picea abies) under laboratory and field conditions

The induction of activity of the enzyme nitrate reductase (NR, EC 1.6.6.1, 1.6.6.2) in needles of Norway spruce ( Picea abies [L.] Karst.) by nitrogen dioxide (NO₂) was studied under laboratory and field conditions. In fumigation chambers an increase in nitrate reductase activity (NRA) was detected 4 h after the start of the NO₂ treatment. During the first 2 days with 100 µg NO₂ m⁻³, NRA reached a constant level and did not change during the following 4 days. At the same level of NO₂, NRA was lower in needles from trees grown on NPK‐fertilized soil than on non‐fertilized soil. After the transfer of spruce trees from fertilized soil to NPK‐rich nutrient solution, NRA was transiently increased. This effect was assigned to root injuries causing nitrate transport to the shoot and subsequent induction of NRA. Neither trees on fertilized soil nor trees transferred to NPK‐poor nutrient solution had increased NRA unless NO₂ was provided. The NO₂ gradient in the vicinity of a highway was used to test the long‐term effect of elevated levels of NO₂ on needle NRA of potted and field‐grown spruce trees. Compared with less polluted sites, permanently increased NRAs were detected when NO₂ concentrations were above 20 µg m⁻³. Controls of field measurements some 10 years after the introduction of catalytic converters in cars showed no significant change neither in NO₂ levels nor in the decreasing NRA of spruce needles with the distance from the highway.     

Isolation, characterization, and nitrification potential of a methylotroph and two heterotrophic bacteria from a consortium showing methane-dependent nitrification

Abstract A methanotrophic nitrifying consortium was previously obtained from a humisol which showed CH₄-dependent nitrification. Although the methanotroph could not be obtained in pure culture, three other members of the consortium have been isolated: An obligately methylotrophic Methylobacillus (Is-1) which grows only on CH₃OH and does not nitrify; a Pseudomonas (Is-2) which grows on Is-1 culture filtrate and produces NO₂⁻, NO₃⁻ and N₂O from NH₂OH, and NO₃⁻ from NO₂⁻; and a second Pseudomonas (Is-3) which produces NO₃⁻ from NH₄⁺ or NO₂⁻, and N₂O from NH₂OH. A model is proposed for the trophic relations and nitrogen transformations in the consortium which may apply to some natural systems.     

Methane-dependent nitrate production by a microbial consortium enriched from a cultivated humisol

Abstract A consortium was enriched from a humisol incubated with 3.6 kPa CH₄ and NH₄⁺. This consortium oxidized NH₄⁺ to NO₂⁻ and NO₃⁻ (NO₃⁻/NO₂⁻ ratio about 20) with smaller amounts of N₂O. This oxidation stopped in the stationary phase after depletion of CH₄. CH₃OH or CO₂ did not support oxidation. Growth and resting cell experiments suggested that nitrification was associated with methanotrophic activity and that chemoautotrophic nitrifiers were absent.     

Cytosolic and chloroplastic glutamine synthetase of sugarbeet (Beta vulgaris) respond differently to organ ontogeny and nitrogen source

Changes in the activity and subunit composition of cytosolic glutamine synthetase (GS 1; EC 6.3.1.2) and chloroplastic GS (GS 2) were studied in response to an internal (organ ontogeny) and external signal (N source: NO₃⁻ or NH₄⁺). Maximum GS 1 activity of all organs examined was measured in the fibre roots, irrespective of the N source. The response of GS 1 to the N source was, however, organ specific. In the fibre roots, NH₄⁺ nutrition resulted in a 2- to 7-fold (based on protein or freshweight, respectively) increase of GS 1 activity compared to NO₃⁻-grown plants. In contrast to the roots, GS 1 activity in the leaf blades was 2-fold lower with NH₄⁺ nutrition, whereas only minor changes occurred in the petioles. GS 2 activity was highest in the mature and senescing leaf blade; activity was 2-fold higher with NH₄⁺ than with NO₃⁻ nutrition. Not only activity, but also subunit composition of GS 1 changed during organ ontogeny as well as in response to the N source. In contrast to GS 1, only minor changes were evident in GS 2 subunit composition, despite significant changes in GS 2 activity. Up to 5 different GS 1 subunits of ≈41–43 kDa were separated; they were identical in all organs examined. GS 2 was composed of 4 different subunits of ≈48 kDa.