In vivo Characterization of Nitrate Reductase Activity in Cotton

The in vivo nitrate reductase activity in leaf tissue of cotton (Gossypium hirsutum L.) was characterized. Enzymatic activity was linear with time up to 60 min. The assay for nitrate reductase activity was optimized in leaf slices 400 μm wide incubated in an anaerobic system at 30°C, in a 0.02 M KNO₃ medium at pH 7.0 with 1 % propanol. In vivo activity was highest in recently matured leaves at the top of the plant. Both light and nitrate enhanced in vivo enzymatic activity. The activity was highest after 9 hours in the light and then decreased steadily for several more hours even in the presence of light. The nitrate reductase activity was more strongly correlated to the levels of NO₃-N in the culture solution than to the NO₃-N level in the tissue. The utility of this technique in nitrate reductase assay in a tissue containing large amounts of phenolic compounds is discussed.     

Factors Involved in in Vitro Stabilization of Nitrate Reductase from Cotton (Gossypium hirsutum L.) Cotyledons

Experiments were conducted to determine if pretreatment of cotton (Gossypium hirsutum L.) plants resulted in differential in vitro stabilities of nitrate reductase (NR) activity. Although NR activity declines markedly during the second half of the daily light period, in vitro NR stability is not modified by time of harvest. Phenylmethylsulfonylfluoride, iodoacetamide, and N-ethylmaleimide do not influence in vitro NR stability, suggesting that serine or sulfhydryl proteases are not responsible for in vitro lability of NR from cotton cotyledons.

Imposition of water stress or artificial extension of the dark period lead to significant reductions in NR activity, but do not change in vitro NR stability.

Dilution of a crude extract leads to increasing lability of NR; hence the marked instability of NR cannot be attributed to an inactivator which follows simple enzyme kinetics. Since in vitro NR activity is much more stable in presence of both NADH and NO₃⁻, substrate availability must be considered as a possible factor influencing in vivo NR stability.

     

Changes in Leaf Nitrate Reductase Activity In Vivo and In Vitro During Light-Dark Transitions

Nitrate reductase activity in the leaves of a number of plants after transfer from light to dark was assayed both by in vivo and in vitro methods. The initial activity persisted during the dark phase for a considerable length of time and declined gradually. After exposure to light again, the NR activity increased rapidly. The possibility of nitrate assimilation in complete darkness is discussed.     

硝酸盐对球形棕囊藻生长和硝酸还原酶活性的影响

以我国南海海域分离的赤潮原因种——球形棕囊藻(Phaeocystisglobosa)为材料,研究了不同硝酸盐浓度下藻细胞生长及硝酸还原酶活性的变化。当培养基中不含硝酸盐时,藻细胞内硝酸还原酶的活性保持在非常低的水平,藻细胞的生长受到限制,不能形成正常的生长曲线:当培养基中硝酸盐浓度为3.62μmol.L-1时,藻细胞的硝酸还原酶活性和比生长速率达到最大。在含有硝酸盐的培养基中,接种培养后第9天藻细胞硝酸还原酶活性达到最大值,并且在4种不同硝酸盐浓度下,藻细胞硝酸还原酶活性的差异性达到极显著水平(P<0.01)。在接种培养第16天藻细胞密度达到最大值,并且4种不同硝酸盐浓度培养的藻细胞密度之间的差异性也达到极显著水平(P<0.01)。实验结果表明,在培养基中添加不同浓度的硝酸盐,对球形棕囊藻细胞硝酸还原酶的活性和藻细胞的生长有极显著的影响,含有较高硝酸盐的富营养化海域有利于球形棕囊藻细胞的持续生长。     

In Vitro Formation of Nitrate Reductase Using Extracts of the Nitrate Reductase Mutant of Neurospora crassa, nit-1, and Rhodospirillum rubrum

In vitro formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase (NADPH: nitrate oxido-reductase, EC 1.6.6.2) has been attained by using extracts of the nitrate reductase mutant of Neurospora crassa, nit-1, and extracts of either photosynthetically or heterotrophically grown Rhodospirillum rubrum, which contribute the constitutive component. The in vitro formation of NADPH-nitrate reductase is characterized by the conversion of the flavin adenine dinucleotide (FAD) stimulated NADPH-cytochrome c reductase, contributed by the N. crassa nit-1 extract from a slower sedimenting form (4.5S) to a faster sedimenting form (7.8S). The 7.8S NADPH-cytochrome c reductase peak coincides in sucrose density gradient profiles with the NADPH-nitrate reductase, FADH(2)-nitrate reductase and reduced methyl viologen (MVH)-nitrate reductase activities which are also formed in vitro. The constitutive component from R. rubrum is soluble (both in heterotrophically and photosynthetically grown cells), is stimulated by the addition of 10(-4) M Na(2)MoO(4) and 10(-2) M NaNO(3) to cell-free preparations, and has variable activity over the pH range from 3.0 to 9.5. The activity of the constitutive component in some extracts showed a threefold stimulation when the pH was lowered from 6.5 to 4.0. The constitutive activity appears to be associated with a large molecular weight component which sediments as a single peak in sucrose density gradients. However, the constitutive component from R. rubrum is dialyzable and is insensitive to trypsin and protease. These results demonstrate that R. rubrum contains the constitutive component and suggests that it is a low molecular weight, trypsin- and protease-insensitive factor which participates in the in vitro formation of NADPH nitrate reductase.     

NO2 Suppression of Light-Induced Nitrate Reductase in Squash Cotyledons

NADH-nitrate reductase (NR) (EC 1.6.6.1 [EC] ) activity in the cotyledonsof squash (Cucurbita maxima Duch.) seedlings showed daily variationwhen the seedlings were subjected to an alternating light-darkcycle. When the seedlings were transferred into continuous darkness,NR activity rose at first and then decreased continuously. Irradiationafter continuous darkness induced a rapid increase in NR activity;this light induction of NR activity was inhibited completelyby fumigation with 4 ppm nitrogen dioxide (NO₂). This inhibitoryeffect of NO₂ was prominent even at 1 ppm and became more pronouncedas the concentration of NO₂ increased. NO₂ fumigation did notremarkably affect the content of reductant (NADH) in the cotyledons.The results of immunoblotting using anti-NR serum indicatedthat irradiation induced the increase in the NR-polypeptidecontent and NO₂ fumigation inhibited the increase, suggestingthat NO₂ put an inhibitory effect on the synthesis of NR inducedby irradiation. ⁴ Present address: College of Environmental Health, Azabu University,Fuchinobe, Sagamihara, Kanagawa 229, Japan ⁵ Present address: Faculty of Home Economics, Otuma Women'sUniversity, Sanban-cho, Chiyoda, Tokyo 102, Japan (Received October 21, 1987; Accepted January 13, 1988)     

Nitrogen Utilization in Lemna: I. Relations between Net Nitrate Flux, Nitrate Reduction, and in Vitro Activity and Stability of Nitrate Reductase

Cultures of Lemna gibba L. G3 were maintained at a constant, N-limited growth rate by adding nitrate daily in amounts calculated to sustain a rate of culture N increment of 0.20 day⁻¹. Nitrate added to the culture was consumed within 8 to 10 hours and the partitioning to reduction and accumulation during this phase corresponded to, on the average, 75 and 25% of net uptake, respectively. The calculated rate of nitrate reduction was stimulated by onset of net uptake without delay and decreased when net uptake ceased. NADH-nitrate reductase (NR) activity measured in vitro without inclusion of antiproteolytic agents more than doubled during the first hour after nitrate addition and then gradually fell to its original level over the rest of the 24 hour interval. In the presence of the proteinase inhibitor leupeptin during extraction, however, NR activity was in general much higher and without any apparent cycles. The relative stabilizing effect of leupeptin was greatest on NADH-NR and reduced flavin adenine mononucleotide-NR activities whereas the effect was less on NADH-cytochrome c reductase activity (diaphorase) and reduced methylviologen-NR activity. The constant nitrate reductase activity measured in the presence of proteinase inhibitors is assumed to reflect the physiological situation. It thus appeares that short-term changes in nitrate assimilation by N-limited Lemna is related to the flux of nitrate to the reducing site and not to changes in nitrate reductase activity.     

Time-course of Nitrate Uptake and Nitrate Reductase Activity in Nitrogen-depleted Dwarf Bean

The rate of nitrate uptake by N-depleted French dwarf bean (Phaseolus vulgaris L. cv. Witte Krombek) increased steadily during the first 6 h after addition of NO₃ -After this initial phase the rale remained constant for many hours. Detached root systems showed the same time-course of uptake as roots of intact plants. In vivo nitrate reductase activity (NRA) was assayed with or without exogenous NO₃^- in the incubation medium and the result ing activities were denoted potential and actual level, respectively. In roots the difference between actual and potential NRA disappeared within 15 min after addition of nitrate, and NRA increased for about 15 h. Both potential and actual NRA were initially very low. In leaves, however, potential NRA was initially very high and was not affected by ambient nitrate (0.1–5 mol m^-3) for about 10 h. Actual and potential leaf NRA became equal after the same period of time. In the course of nitrate nutrition, the two nitrate reductase activities in leaves were differentially inhibited by cycloheximide (3.6 mmol m^-3) and tungstate (1 mol m^-3). We suggest that initial potential NRA reflects the activity of pre-existing enzyme, whereas actual NRA depends on enzyme assembly during NO₃^- supply. Apparent induction of nitrate uptake and most (85%) of the actual in vivo NRA occurred in the root system during the first 6 h of nitrate utilization by dwarf bean.     

Seedling Nitrate Reductase Activity and Nitrate Partitioning in Maize (Zea mays L.) Strains Divergently Selected For Post-anthesis Leaf-Lamina Nitrate Reductase Activity

Two experiments were conducted to evaluate the effects of phenotypicrecurrent selection for high and low post-anthesis leaf-laminain vivo NRA on nitrate uptake, nitrate partitioning and in vitroNRA of seedling roots and leaves. In Experiment 1, intact plantsof cycle 0, 4, and 6 of the high and low NRA strains were grownon NH₄-N for 11 d, then exposed to 1.0 mol m^–3 KNO₃, andcultures sampled at 6 h and 28 h (induction and post-inductionperiods). Nitrate uptake, tissue nitrate concentration and invitro NRA were determined. The pattern of response to selectionin seedling leaf NRA was similar to that observed for in vivoNRA of field grown plants. Leaf NRA increased between 6 h and28 h. Root NRA was not affected by selection or sampling time.Treatments differed in total fresh weight but not in reductionor uptake of nitrate per unit weight, indicating a lack of correspondencebetween NRA and reduction and supporting the idea that concomitantreduction by NR is not obligatorily linked to nitrate influxin the intact plant. In Experiment 2, dark-grown plants of cycle 0, and 6 of thehigh and low NRA strains were cultured without N, detopped onday 6, transferred the following day to 0-75 mol m^–3 KNO₃and sampled at 6 h and 28 h. In contrast to Experiment 1, selectionpopulations differed in nitrate reduction and root NRA, whichby 28 h reached higher average levels than root NRA of intactplants. Translocation and reduction were inversely related amongstrains within each sampling time. The high level of translocationin detopped plants of the low NRA strain was difficult to reconcilewith its low leaf NRA level of Experiment 1. It is suggestedthat nitrate transport in detopped roots is altered relativeto the intact system in a way which permits greater NRA inductionand nitrate reduction. The results indicate that nitrate partitioningby detopped root systems should be interpreted with caution. Key words: Zea, nitrate reductase activity, nitrate uptake, nitrate reduction, nitrate partitioning, selection     

In Vitro Stability of Nitrate Reductase from Wheat Leaves: III. Isolation and Partial Characterization of a Nitrate Reductase-inactivating Factor

A nitrate reductase (EC 1.6.6.1)-inactivating factor has been isolated from 8-day-old wheat leaves. The purification schedule involved ammonium sulfate precipitation, Sephadex G-100 filtration, DEAE-cellulose chromatography, and Sephadex G-150 filtration. No accurate assessment could be made as to the degree of purification relative to crude extract as the inactivating factor could not be detected in crude extract. However a 2,446-fold purification was achieved from the ammonium sulfate fraction to the pooled enzyme from the Sephadex G-150 step.     

Nitrate Fluxes and Nitrate Reductase Activity of Suspension-Cultured Tobacco Cells (Effects of Internal and External Nitrate Concentrations)

Cell suspensions of tobacco (Nicotiana tabacum L., cv KY14) were used to determine the responses of NO3- uptake and NO3- reductase activity (NRA) to exogenous NO3- levels in the absence of long-distance NO3- transport. Tobacco cells grown with complete Murashige and Skoog medium for 7 d were subcultured for 3 d with NH4+-free media containing 0, 5, 10, 20, 30, and 40 mM NO3-. Cell NO3-, in vitro NRA, NO3- influx, and efflux of cell NO3- were determined. The NRA increased as cell NO3- increased. Cell NO3- efflux values increased as cell NO3- level increased. Cells with low intracellular NO3- had greater NO3- influx than cells with high intracellular NO3-. Woolf-Augustinsson-Hofstee transformations of the NO3- influx kinetic data revealed patterns characteristic of a high- and low-affinity two-component NO3- uptake system. Apparent Vmax values generally decreased and Km values increased as cell NO3- concentration increased. The NRA of cells supplied with 10 and 20 mM NO3- after 3-d growth in N- free medium increased about 5-fold within 2 h and then remained constant for the next 2 h, whereas NRA of cells supplied with 5 mM NO3- increased only 2-fold during the 4-h period. Intracellular NO3- and other N metabolites associated with cell NO3- levels exerted differential effects on the NO3- influx activity and NRA of tobacco cells cultured in suspension. Expression of high NRA was correlated with both high external and intracellular NO3-, whereas maximum NO3- influx activity required a low (depleted) level of cell NO3-.     

Blue Light-Induced In Vivo Absorbance Changes and In Vitro Activation of Nitrate Reductase in a Nitrate-Starved Chlorella Mutant

Illuminating a colorless mutant of Chlorella vulgaris 11h (M125)with blue light caused a reversible photoreduction of b-typecytochrome, i.e., absorbance increases at 423, 525 and 557 nm.This light-induced reduction of cytochrome b was most pronouncedin nitrate-starved cells, which showed some blue light responsesin carbon metabolism, including enhancement of respiration byblue light as reported previously. Prolonged illumination withblue light caused a decrease in the rate of the reduction. The photoactivation of nitrate reductase in the mutant cellswas studied in both cell-free crude extract and purified enzyme.The absorption spectrum of purified enzyme showed three peaksat 423, 525 and 557 nm after the addition of a reductant, indicatingthat the spectrum is that of cytochrome b associated with nitratereductase. Nitrate reductase activity was easily enhanced byblue light illumination after 1 min; red light had no effecton it. The blue light activation of nitrate reductase was notsignificant in growing cells, which showed its high activity. The relationship between the blue light-induced reduction ofcytochrome b and carbon metabolism is discussed. (Received September 30, 1987; Accepted February 9, 1988)     

Evaluation of Nitrate Reductase Activity in Rhizobium japonicum

Nitrate reductase activity was evaluated by four approaches, using four strains of Rhizobium japonicum and 11 chlorate-resistant mutants of the four strains. It was concluded that in vitro assays with bacteria or bacteroids provide the most simple and reliable assessment of the presence or absence of nitrate reductase. Nitrite reductase activity with methyl viologen and dithionite was found, but the enzyme activity does not confound the assay of nitrate reductase.     

Development of NAD(P)H: and NADH:Nitrate Reductase Activities in Soybean Cotyledons

The cotyledons of soybean begin to develop photosynthetic capacity shortly after emergence. The cotyledons develop nitrate reductase (NR) activity in parallel with an increase in chlorophyll and a decrease in protein. In crude extracts of 5- to 8-day-old cotyledons, NR activity is greatest with NADH as electron donor. In extracts of older cotyledons, NR activity is greatest with NADPH. Blue-Sepharose was used to purify and separate the NR activities into two fractions. When the blue-Sepharose was eluted with NADPH, NR activity was obtained which was most active with NADPH as electron donor. Assays of the NADPH-eluted NR with different concentrations of nitrate revealed that the highest activity was obtained in 80 millimolar KNO₃. Thus, this fraction has properties similar to the low nitrate affinity NAD(P)H:NR of soybean leaves. When 5- to 8-day-old cotyledons were extracted and purified, further elution of the blue-Sepharose with KNO₃, subsequent to the NADPH elution, yielded an NR fraction most active with NADH. Assays of this fraction with different nitrate concentrations revealed that this NR had a higher nitrate affinity and was similar to the NADH:NR of soybean leaves. The KNO₃-eluted NR fraction which was purified from the extracts of 9- to 14-day-old cotyledons, was most active with NADPH. The analysis of these fractions prepared from the extracts of older cotyledons indicated that residual NAD(P)H:NR contaminated the NADH:NR. Despite this complication, the pattern of development of the purified NR fractions was consistent with the changes observed in the crude extract NR activities. It was concluded that NADH:NR was most active in young cotyledons and that as the cotyledons aged the NAD(P)H:NR became more active.     

Effects of Cysteine on Cellular Activity of Dissimilatory Nitrate Reductase in Pseudomonas denitrificans

Dissimilatory nitrate reductase activity in Pseudomonas denitrificans grown under denitrifying conditions was markedly decreased upon incubation of the cells with Cys or GSH. The activity of the enzyme was also low in the membrane fraction isolated from the Cys-treated cells, indicating that the decrease in enzyme activity was due to the inactivation of the enzyme. Cys appeared to exert its effect, at least in part, after conversion to GSH as judged by the stimulation of the Cys effect by concomitant addition of Glu, although even a trace of GSH could not be detected in the cells presumably owing to the high activity of GSH consumption. The inactivating effect of Cys on the enzyme was found to compete with the activating effect of Na⁺ that had been reported previously, suggesting the presence of a natural mechanism controlling nitrite accumulation in the environment.     

Chlorate Toxicity and Nitrate Reductase Activity in Tomato Plants

Chlorate damage was studied in tomato plants ( Lycopersicum esculentum cv. Moneymaker) that were supplied with a nitrogen-free nutrient solution or with a nutrient solution, containing either nitrate or ammonium as a nitrogen source. Damage was low in ammonium-fed plants and high in nitrate-fed plants and in nitrogen-less plants. Nitrate reductase activity could be detected in all treatments, although the activity was highest in the nitrate-fed plants.
The hypothesis that chlorate can be used as a substrate by the enzyme nitrate reductase in higher plants, was studied and proved to be true for the tomato plants, as was found earlier for Escherichia and Chlorella . The affinity of the enzyme for chlorate was lower than for nitrate, the K _m being 4 m M and 0.15 m M respectively. Induction of the enzyme by chlorate could not be detected. The enzyme activity was lowered in leaf discs after a 7 h treatment with chlorate and the inhibition was proportional to the chlorate concentration of the medium.
The results were discussed in terms of competition between nitrate and chlorate at the uptake and the enzyme site and with regard to a possible influence of chlorate on synthesis and breakdown of the enzyme.     

15N Studies on the In Vivo Assay of Nitrate Reductase in Leaves: Occurrence of Underestimation of the Activity Due to Dark Assimilation of Nitrate and Nitrite

The reduction of nitrate and nitrite in leaf disks from sevendi- and two monocotyledonous species under in vivo nitrate reductaseassay conditions was studied using 15N-labeled substrates. Significantreduction of both nitrate and nitrite into ammonia and aminoacids was detected under aerobic conditions (in an atmosphereof air): in some cases, the amount of nitrate-N reduced to ammoniaand amino acids was more than that remaining as nitrite. Anaerobicincubation (under an atmosphere of N₂ gas) enhanced the accumulationof nitrite, but the subsequent reduction to the basic nitrogencompounds was 40 to 180% of the aerobic rates. The present examinationindicates that in vivo assays of nitrate reductase under aerobicconditions may give greatly underestimated results due to nitritereduction and that exclusion of oxygen from the in vivo assaymixture is desirable in terms of the quantity of nitrite formed.n-Propanal treatment increased nitrite accumulation under aerobicbut not under anaerobic conditions, and depressed the incorporationof nitrate-N into basic fractions under both conditions. Therefore,addition of n-propanol may be desirable for assays under aerobicconditions. No significant difference in the reduction of nitratesupplied as sodium and potassium salts was observed on the nitriteformation and on the incorporation of nitrate-N into basic fractions. ¹⁵N experiments on dark assimilation of nitrate, nitrite andammonia into amino acids in wheat leaves showed that these threenitrogen sources were assimilated through the same route andthat the glutamine synthetase/glutamate synthase pathway wasthe major route. With anaerobic treatment, the incorporationof nitrogen into alanine and serine remained at relatively high,but the incorporation into aspartate and asparagine was muchlower than in the cases of aerobic treatment. (Received July 11, 1981; Accepted October 3, 1981)