Development Of Nitrate Reductase Activity Research Articles

Nitrate reduction in cotyledons of Cicer arietinum L.: regulatory role of cytokinins

Application of different concentrations of kinetin to chick-pea seeds inhibits the nitrate reductase ativity detected in cotyledons when the seeds are sown without a hormone. However, kinetin concentrations between 10 −6 and 10 −5 M, applied to excised cotyledons are able to induce nitrate reductase activity, which also becomes manifest in an active metabolism of nitrate and nitrite in the cotyledons. The development of nitrate reductase activity in excised cotyledons is due to synthesis of the enzyme, except when induction is carried out by applying low concentrations of cytokinins. In this case, apart from synthesis, an inactive nitrate reductase becomes activated.

Further Studies on the Induction of Nitrate Reductase by Arginine in the Filamentous Cyanobacterium Oscillatoria chalybea

In an earlier publication we reported on the role of arginine for the development of nitrate reductase activity in cells of the filamentous cyanobacterium Oscillatoria chalybea (Bednarz and Schmid, Z. Naturforsch. 46c, 591 (1991)). In the present paper we present further evidence that arginine is the natural inducer for nitrate reductase activity. Thus, we show that the induction is regulated by transcription, probably related to the apoprotein or the molybdenumcofactor. We also examined the influence of arginine on nitrate reductase activity in the filamentous cyanobacterium Anabaena PCC7120. In contrast to Oscillatoria chalybea this cyanobacterium forms heterocysts and shows nitrogen fixation activity. Like in Oscillatoria chalybea nitrate reductase activity in Anabaena PCC7120 is stimulated in the presence of arginine as the sole nitrogen source. However, this stimulation is limited to an early growth stage. Subsequently, nitrogen fixation activity appears and nitrate reductase activity decreases.

Induction of Nitrate Reductase Activity by Arginine in the Filamentous Cyanobacterium Oscillatoria chalybea

Abstract In Oscillatoria chalybea, as in other cyanobacteria, nitrate reduction depends directly on photosynthetic activity. Hence, nitrate reduction occurs only in the light and appears inhibited when photosynthesis is inhibited by the herbicide DCMU . Growth of Oscillatoria cells is possible on a variety of exogenous nitrogen sources in the medium and appears largely independent on the type of nitrogen source. However, if citrulline is the exogenous nitrogen source or if no exogenous nitrogen source is given in the medium, growth appears almost fully inhibited. Nitrate reductase activity measured in French-press particles of nitrate-grown cells is depend­ent on the age of the culture with maximum nitrate reductase activity being reached on the 5th day. Thereafter activity decreases steeply to less than 20% of the maximal activity within 10 days. Besides the growth stage it is the type of exogenous nitrogen source used in the medium which is important for the development of nitrate reductase activity. It appears that in the presence of nitrate, nitrite and arginine, nitrate reductase activity is induced whereas in the presence of ammonia or amino acids like alanine nitrate reductase activity is not induced, as already reported in the literature. Nitrate reductase is also induced if arginine and ammonia are simultaneously offered as exogenous nitrogen source. Arginine metabolism in Oscillatoria cells is characterized by the fact that thylakoid preparations of Oscillatoria catalyze the trans­ formation of arginine to give ornithine and ammonium. The arginine-metabolizing enzyme differs from the usual arginine-induced arginase. The enzyme seems to be constitutive, not manganese-dependent, exhibiting an approximately 5 times higher substrate affinity to arginine than the known arginase. In the present paper we propose that in Oscillatoria it is arginine which induces the synthesis of nitrate reductase.

The Influence of Nitrogen Supply on the Development of Nitrate Reductase Activity in Leaves of Potato (Solanum tuberosum L.)

Changes in nitrate reductase activity in the youngest expanded leaves of the potato plant have been monitored in the field under a range of N regimes. Highest activity ( in vivo assay, substrate non-limiting) occurred with the highest rate of N application, as did the highest leaf protein content. In high-N leaves the onset of the decline in nitrate reductase activity and protein content, which commenced around 80 days after planting, was not prevented by the application of high levels of nitrogen. This could not be explained by a fall in the concentration of nitrate in the petiole xylem sap supplying the leaves in question. The ratio of nitrate-N to amino acid-N in petiole xylem sap remained relatively constant throughout the growing period in plants receiving 8 g N m −2 at 14 day intervals, but declined under lower N inputs. Only in the highest N treatment did the ratio remain greater than unity. The reasons for these observations are discussed. Nitrate reductase activity was also determined in leaves of field-grown plants with all of the N (20 g m −2 ) applied pre-planting. A comparison of in vivo assays carried out with and without nitrate in the assay medium showed that nitrate reductase was likely to limit nitrate assimilation only during the earliest stages of post-emergence growth.

Development of Nitrate Reductase Activity in Expanding Leaves of Nicotiana tabacum in Relation to the Concentration of Nitrate and Potassium

Up to 80% of the total nitrate reductase activity (NRA) determined in vivo in different parts of vegetative tobacco plant (Nicotiana tabacum) was located in the leaves. The NRA reached a peak when a leaf had expanded to 27% of its final weight and 33% of its final area. Thereafter, with advancing expansion and age of the leaf, the activity declined. This pattern of development of NRA during the ontogenesis of leaves was not influenced by raising the supply of NO(3) (-) from 3 to 6 milliequivalent per cubic decimeter in the substrate solution. The concentration of NO(3) (-) in leaves, stem and root was inversely related to NRA at both NO(3) (-) levels. Raising the supply of K(+) from 1 to 6 milliequivalent per cubic decimeter at either concentration of NO(3) (-) slowed down the development of NRA in the initial stages of expansion, but promoted it subsequently. The peak of the activity which developed in a leaf of 62% of its final area was higher at the higher supply of K(+). The higher activity was maintained thereafter in the expanding and in matured and older leaves. It was concluded that NRA and the pattern of its development in expanding leaves is related to the availability of metabolites and their incorporation into enzyme proteins. Both these processes are influenced by: (a) the vertical profile of concentration of K(+) in the shoot and (b) the concentration of K(+) in a leaf, which depend upon its supply.

Nitrate Reduction in Solanum tuberosum L.: Development of Nitrate Reductase Activity in Field-grown Plants

Nitrate Reduction in Solanum tuberosum L.: Development of Nitrate Reductase Activity in Field-grown Plants

REQUIREMENT FOR A LONG LAG PERIOD FOR THE INDUCTION OF NITRATE REDUCTASE IN WHEAT (TRITICUM AESTIVUM) EMBRYOS DURING GERMINATION

SummaryThe induction of nitrate reductase (NR) in the embryos of wheat required a period of 20 h of incubation in nitrate during the early stages of seed germination. The lag phase in the development of NR activity was not due to the non‐availability of nitrate in the embryos, the presence of a nitrate reductase inactivating factor, or the synthesis of inactive NR. At the onset of the inducible phase the enzyme was synthesized de novo, but was not dependent on the synthesis of fresh m‐RNA. Protein synthesis during the lag phase was essential for the subsequent induction of NR in the inducible phase.

Quantitative transfer of the molybdenum cofactor from xanthine oxidase and from sulphite oxidase to the deficient enzyme of the nit-1 mutant of Neurospora crassa to yield active nitrate reductase

An assay method is described for measurement of absolute concentrations of the molybdenum cofactor, based on complementation of the defective nitrate reductase ('apo nitrate reductase') in extracts of the nit-1 mutant of Neurospora crassa. A number of alternative methods are described for preparing, anaerobically, molybdenum-cofactor-containing solutions from sulphite oxidase, xanthine oxidase and desulpho xanthine oxidase. For assay, these were mixed with an excess of extract of the nit-1 mutant, incubated for 24 h at 3.5 degrees C then assayed for NADPH:nitrate reductase activity. In all cases, the specific activity of the molybdenum cofactor, expressed as mumol of NO2-formed/min per ng-atom of Mo added from the denatured molybdoenzyme , was 25 +/- 4, a value that agrees with the known catalytic activity of the nitrate reductase of wild-type Neurospora crassa. This indicates that, under our conditions, there was quantitative transfer of the molybdenum cofactor from denatured molybdoenzyme to yield fully active nitrate reductase. Comparable cofactor assay methods of previous workers, apparently indicating transfer efficiencies of at best a few per cent, have never excluded satisfactorily the possibility that cofactor activity arose, not from stoichiometric constituents of the molybdoenzymes , but from contaminants. The following factors were investigated separately in developing the assay:the efficiency of extraction of the cofactor from the original enzyme, the efficiency of the complementation reaction between cofactor and apo nitrate reductase, and the assay of the resultant nitrate reductase, which must be carried out under non-inhibitory conditions. Though the cofactor is unstable in air (t1/2 about 15 min at 3.5 degrees C), it is stable when kept anaerobic in the presence of sodium dithionite, in aqueous solution or in dimethyl sulphoxide (activity lost at the rate of about 3%/24 h at 20-25 degrees C). Studies of stabilities, and investigations of the effect of added molybdate on the assay, permit conclusions to be drawn about the ligation of molybdenum to the cofactor and about steps in incorporation of the cofactor into the apoenzyme. Though the development of nitrate reductase activity is slow at 3.5 degrees C (t1/2 1.5-3 h) the complementation reaction may be carried out in high yield, aerobically. This is ascribed to rapid formation of an air-stable but catalytically inactive complex of the cofactor, as a precursor of the active nitrate reductase.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of Plant Growth Regulators on Nitrate Utilization by Roots of Nitrogen-Depleted Dwarf Bean

We examined the effect of pretreatments (18 h at 5 //mol dm-3) with abscisic acid, the ethylene-releasing substance 'Ethephon', gibberellic acid, indoleacetic acid, kinetin and zeatin on nitrate uptake and in vivo nitrate reductase activity (NRA) in roots of nitrogen-depleted Phaseolus vulgaris L. Nitrate uptake showed an apparent induction pattern with a steady state after about 6 h, in all treatments. The nitrate uptake rate after 6 h was unaffected or at most 30% lower after treatments with the plant growth regulators. Gibberellic acid, kinetin and zeatin induced substantial NRA in roots in the absence of nitrate, whereas Ethephon enhanced NRA only during nitrate nutrition. Kinetin-induced NRA (Ki-NRA) was maximal after a pretreatment at 1 //mol dm~3, and showed a lag phase of 6-8 h. Ki-NRA was additive to nitrate-induced NRA (NOj-NRA) for at least 24 h, independent of the induction sequence. After full induction, Ki-NRA approximated 20% of NO7-NRA. Abscisic acid counteracted the development of Ki-NRA, but not of NOj-NRA. Cycloheximide and tungstate were equally effective to suppress the development of nitrate reductase activity after supply of kinetin or NO3. Our data are consistent with the operation of two independent enzyme fractions (Ki-NRA and NO7-NRA) with apparently identical properties but with separate control mechanisms. The absence of major effects of plant growth regulators on the time-course and rate of nitrate uptake suggests that exogenous regulators, and possibly endogenous phytohormones are of minor importance for initial nitrate uptake. The differential effect of some regulators on nitrate uptake and root NRA furthermore indicates that the processes of uptake and reduction of NOj are not obligatory or exclusively coupled to each other.

Development of Nitrate Reductase Activity in Green Tissues of Soybean Seedlings (Glycine max. L. Merr.)

The pattern of in vivo activity of nitrate reductase was determined in chlorophyllous tissues of soybean seedlings cultivated under constant temperature and continuous light. Enzyme activity was detected in the cotyledons after approximately 20 hours of light exposure, the values being proportional to the concentration of nitrate in the irrigating solutions. In cotyledons watered with 15 mM of nitrate the peak of activity was reached after 120 hours of illumination. Chlorophyll accumulation preceded and approximately paralleled nitrate reductase activity in the cotyledons indicating that nitrate reduction is associated with the development of anabolic processes. Nitrate reductase activity in light-grown cotyledons was inhibited by cycloheximide when the inhibitor was added at the beginning of the illumination period. The effect progressively disappeared as the tissues were treated at later stages after having being exposed to light. On the other hand, actinomycin D did not affect enzyme activity, suggesting that the process does depend in part on de novo protein synthesis but does not require RNA synthesis. The appearance of enzyme activity in the primary leaves coincided with the time of maximum activity in the cotyledons. By the time nitrate reductase activity started decreasing in the primary leaves, the first trifoliate leaf showed increasing enzyme activity. The patterns determined in leaves showed low values of activity in the early stages of development, reached a peak when the leaves were almost expanded and declined thereafter. Maximum nitrate reductase activities were calculated as 2.3, 3.2 and 2.2 times higher in leaves infiltrated with nitrate than in the controls without exogenous nitrate, in the cotyledons, primary and first trifoliolate leaves, respectively.

On the mechanism of development of nitrate reductase activity in tobacco cells

An observation is described whihc suggests that the initial development of nitrate reductase activity in logarithmic-phase XD cells of tobacco is independent of nitrate. The cells can develop nitrate reductatse activity upon removal of the nitrogen source from the medium. The development of enzyme activity depends on RNA synthesis. The process seems to be analogous to derepression. Nitrate provides a positive feedback signal.

Development of in vivo Nitrate Reductase Activity in Tuber Slices of Solanum tuberosum L.

Tuber tissue of the potato, Solanum tuberosum L. contain low endogenous nitrate reductase activity and nitrate level. Wounding and subsequent aging did not enhance the activity of this enzyme in the absence of exogenous nitrate. In the presence of nitrate aged slices exhibited enhanced nitrate accumulation and reduction compared to fresh slices. Both fresh and aged slices showed maximum enzyme activity at a nitrate concentration of 50 mM. Results are discussed in accordance with the importance of substrate to the development of nitrate reductase activity.

Nitrate Absorption by Barley

The influence of protein synthesis and nitrate reductase activity on nitrate absorption by barley (Hordeum vulgare L.) was investigated. Cycloheximide decreased nitrate absorption. Pretreatment studies showed that cycloheximide affects either energy transfer or nitrate reductase activity or both.Illumination increased plant capacity for nitrate absorption, possibly through increased energy supply and/or increased nitrate reductase activity. There was a positive correlation between nitrate reductase activity and light. Inhibiting the development of nitrate reductase activity by tungstate decreased nitrate absorption.AT LEAST TWO NITRATE TRANSPORT SYSTEMS ARE THUS PROPOSED IN BARLEY: one operating in the dark, with little nitrate reductase activity detectable; and one closely correlated with nitrate reductase activity. Total absorption is the sum of dark absorption and absorption facilitated by nitrate reductase.

Nitrogen Metabolism in Plant Cell Suspension Cultures

Certain amino acids inhibit growth of tobacco (Nicotiana tabacum L. var. xanthi), tomato (Lycopersicon esculentum) carrot (Daucus carota), and soybean (Glycerine max L. co. Mandarin) cell cultures when nitrate or urea are the nitrogen sources but not when ammonia is the nitrogen source. These amino acids also inhibit development of nitrate reductase activity (NADH:nitrate oxidoreductase EC 1.6.6.1) in tobacco and tomato cultures. Threonine, the most inhibitory amino acid, also inhibits nitrate uptake in tobacco cells. Arginine, and some other amino acids, abolish the inhibition effects caused by other amino acids. We suggest that amino acids inhibit assimilation of intracellular ammonium into amino acids in cells grown on nitrate or urea.

Control of Nitrite Reductase Activity in Excised Embryos of Agrostemma githago

When excised embryos of Agrostemma githago were incubated with nitrate, the activities of both nitrate reductase and nitrite reductase were enhanced. By contrast, benzyladenine induced nitrate reductase only. Our data suggest that nitrate affected nitrite reductase activity directly, without first being reduced to nitrite. When the endogenous nitrite production was increased by raising the level of nitrate reductase through simultaneous treatment with nitrate and benzyladenine, the activity of nitrite reductase was not higher than in embryos treated with nitrate alone. On the other hand, tungstate given together with nitrate drastically inhibited the development of nitrate reductase activity without reducing the enhancement of nitrite reductase activity. Nitrite enhanced nitrite reductase activity, though less efficiently than nitrate.

Distribution and Development of Nitrate Reductase Activity in Germinating Cotton Seedlings

Activity of nitrate reductase in roots and cotyledons of cotton seedings (Gossypium hirsutum L. cv. Deltapine 16) increased rapidly on germination, reaching a maximum after 1 day of imbibition. Thereafter, activity declined until emergence and greening of the cotyledons, when it again began to increase steadily. Germinating soybean (Glycine max (L.) Merrill cv. Merit) and sunflower (Helianthus annuus L. cv. Peredovic) seedlings did not show the early peak of activity. The early peak depended on nitrate and was sensitive to cycloheximide, but not to actinomycin D or other inhibitors of RNA synthesis. The second, light-dependent increase was sensitive to actinomycin D. In roots, the early peak of activity occurred before any growth. After emergence of the root tip from the seed coat, activity was localized in the terminal 2 millimeters, whether expressed on a fresh weight, protein, or root basis. The difference in activity between the apical (0-2 millimeter) and subapical (2-4 millimeter) segments did not result from differences in nitrate availability, energy supply, or turnover rates of nitrate reductase. Root activity was similar to that of the cotyledons after emergence, in that both were sensitive to actinomycin D.

Enhancement of Nitrate Reductase Activity by Benzyladenine in Agrostemma githago

Nitrate reductase activity in excised embryos of Agrostemma githago increases in response to both NO(3) (-) and cytokinins. We asked the question whether cytokinins affected nitrate reductase activity directly or through NO(3) (-), either by amplifying the effect of low endogenous NO(3) (-) levels, or by making NO(3) (-) available for induction from a metabolically inactive compartment. Nitrate reductase activity was enhanced on the average by 50% after 1 hour of benzyladenine treatment. In some experiments, the cytokinin response was detectable as early as 30 minutes after addition of benzyladenine. Nitrate reductase activity increased linearly for 4 hours and began to decay 13 hours after start of the hormone treatment. When embryos were incubated in solutions containing mixtures of NO(3) (-) and benzyladenine, additive responses were obtained. The effects of NO(3) (-) and benzyladenine were counteracted by abscisic acid. The increase in nitrate reductase activity was inhibited at lower abscisic acid concentrations in embryos which were induced with NO(3) (-), as compared to embryos treated with benzyladenine. Casein hydrolysate inhibited the development of nitrate reductase activity. The response to NO(3) (-) was more susceptible to inhibition by casein hydrolysate than the response to the hormone. When NO(3) (-) and benzyladenine were withdrawn from the medium after maximal enhancement of nitrate reductase activity, the level of the enzyme decreased rapidly. Nitrate reductase activity increasd again as a result of a second treatment with benzyladenine but not with NO(3) (-). At the time of the second exposure to benzyladenine, no NO(3) (-) was detectable in extracts of Agrostemma embryos. This is taken as evidence that cytokinins enhance nitrate reductase activity directly and not through induction by NO(3) (-).

Regulation of nitrite reductase and its relationship to the regulation of nitrate reductase in cultured tobacco cells

1. 1. The assay for nitrite reductase (EC 1.6.6.4) activity of Ramirez et al. (Biochim. Biophys. Acta, 140 (1966) 58), utilizing methyl viologen reduced by sodium hydrosulfite as electron donor, was optimized for the enzyme from the XD line of cultured tobacco cells. The enzyme reduced nitrite to ammonia under the assay conditions. 2. 2. Nitrite reductase activity was low in cells which had been starved for nitrogen, or which had been grown on the nitrogen in urea or casein hydrolysate. 3. 3. Both nitrate reductase (NADH: nitrate oxidoreductase, EC 1.6.6.1) and nitrite reductase activities developed in cells exposed to either nitrate or nitrite. Nitrate reductase developed more rapidly in response to nitrite than nitrate. 4. 4. Tungstate, a specific inhibitor of development of nitrate reductase activity, and hence of nitrite synthesis, did not inhibit development of nitrite reductase activity in response to nitrate. This is taken as evidence that nitrate does not have to be reduced to nitrite to cause an increase in nitrite reductase activity. 5. 5. Casein hydolysate enhanced the development of both enzyme activities in response to nitrite, but inhibited development of both activities in response to nitrate. 6. 6. Nitrite reductase is more stable and its decay kinetics are more complex in vivo, compared to nitrate reductase activity. After removal of nitrate from the medium, nitrite reductase at first decayed with a half-life of 28 h, but later with a half-life of 124 h, while nitrate reductase decayed with a half-life of approximately 6 h.

The development of nitrate reductase in Chlorella and its repression by ammonium

Chlorella vulgaris, grown with ammonium sulphate as nitrogen source, contains very little nitrate reductase activity in contrast to cells grown with potassium nitrate. When ammonium-grown cells are transferred to a nitrate medium, nitrate reductase activity increases rapidly and the increase is partially prevented by chloramphenicol and by p-fluorophenylalanine, suggesting that protein synthesis is involved. The increase in nitrate reductase activity is prevented by small quantities of ammonium; this inhibition is overcome, in part, by raising the concentration of nitrate. Although nitrate stimulates the development of nitrate reductase activity, its presence is not essential for the formation of the enzyme since this is formed when ammonium-grown cells are starved of nitrogen and when cells are grown with urea or glycine as nitrogen source. It is concluded that the formation of the enzyme is stimulated (induced) by nitrate and inhibited (repressed) by ammonium.

Nitrate Reductase Activity in Corn Seedlings as Affected by Light and Nitrate Content of Nutrient Media

Young corn plants placed in complete darkness for 48 hours lost 90% of their nitrate reductase activity. The activity was quickly restored when the plants were returned to the light. In other experiments, corn plants were grown under artificial shade in the greenhouse. The nitrate reductase activity in these plants decreased roughly in proportion to the amount of shading. It was shown that both light and nitrate are necessary for the formation of nitrate reductase in quantities required by the plant for normal growth. Corn seedlings germinated in the dark in Hoagland solution showed a fourfold increase in nitrate reductase within 8 hours after they were transferred into the light. Plants that were germinated in the dark without nitrate, and given nitrate at the same time they were transferred to the light, showed a slower rate of development of nitrate reductase activity. Green house experiments in which the nitrate content of nutrient media was varied showed that the level of nitrate reductase activity was dependent in part upon the substrate concentration. There was a positive correlation between the nitrate reductase activity and the growth and protein content of both strains. 18 references, 5 figures, 4 tables.