Nitrate Reductase Activity in Maize (Zea mays L.) Leaves: I. Regulation by Nitrate Flux

The roles that leaf nitrate content and nitrate flux play in regulating the levels of nitrate reductase activity (NRA) were investigated in 8- to 14-day old maize (Zea mays L.) plants containing high nitrate levels while other environmental and endogenous factors were constant. The nitrate flux of intact plants was measured from the product of the transpiration rate and the concentration of nitrate in the xylem. NRA decreased when the seedlings were deprived of nitrate. The nitrate flux and the leaf nitrate content also decreased. When nitrate was resupplied to the roots, all three parameters increased.     

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

Nitrate reductase activity (in vivo method, substrate non-limiting)in unshaded leaves from the top of the canopy has been determinedfor field-grown potato plants over the course of the growingseason. The pattern of change was almost identical for plantsreceiving no added fertilizer and those receiving 24 g N m^–2.Activity increased to a peak at about 90 days after plantingand declined thereafter. On a fresh weight basis activity wasalways higher in fertilized plants. Nitrate reductase activitywas positively and significantly correlated with leaf proteincontent in high N plants (r² = 0.71; P = 0.05), but poorly correlatedwith both the nitrate content of the leaf lamina and the nitrateconcentration in petiole sap. Up until 90 days after planting(mid-July) there appeared to be a positive relationship betweenincreased activity of nitrate reductase and solar radiation.However, results obtained over two seasons showed that the declinein activity after this time was not consistently linked witha fall in the level of solar radiation. Remobilization of reduced-Nand stored nitrate from leaves and stems accompanied this declinein nitrate reductase activity and in the latter part of theseason appeared to account for all of the N gained by growingtubers. In unfertilized plants nitrate-N accounted for 5 per cent orless of total plant N. Fertilized plants contained up to 25per cent nitrate-N. While nitrate availability limited growthin unfertilized plants, sub-optimal rates of nitrate assimilationin fertilized plants, particularly during the early stages ofpost-emergence growth, may contribute to inefficient use ofacquired nitrate. The carbohydrate status of leaf lamina and petiole sap weremodified by N supply. The soluble sugar and starch contentsof low N leaves were higher than in their high N counterparts.By contrast, the concentration of soluble sugars in petiolesap increased to a higher value in high N samples. Althoughsap sugar levels declined in both treatments towards the endof the season, N application delayed this decline for severalweeks. Solanum tuberosum, nitrate reductase, nitrate assimilation, senescence     

Diurnal Fluctuations in the Activity of the Enzyme Nitrate Reductase in Lemna paucicostata

Diurnal fluctuations were observed in nitrate reducta.se activity in Lemna paucicostatu Hegelm, grown under 16-h photoperiod. The enzyme activity showed a single peak in the middle of the photo period, i.e. 8 h after the onset of light. The activity decreased during the latter half of the photoperiod and reached a minimum level in the middle of the dark period. The peak in enzyme activity persists in continuous light as well as continuous dark, at least for a couple of days, suggesting the existence of endogenous rhythmicity     

Nitrogen Utilization in Lemna: II. Studies of Nitrate Uptake Using NO(3)

¹³N-labeled nitrate was used to trace short-term nitrate influx into Lemna gibba L. G3 in experiments where disappearance of both radioactivity and total nitrate from the incubation medium was measured continuously and simultaneously. In plants performing net nitrate uptake from an initial nitrate concentration of 40 to 60 micromolar, there was no discrepancy between net uptake and influx, irrespective of the N status of the plants, indicating that concomitant nitrate efflux was low or nil. Plants treated with tungstate to inactivate nitrate reductase were able to take up nitrate following induction of the uptake system by exposure to a low amount of nitrate. Also, in this case, net uptake was equivalent to influx. In tungstate-treated plants preloaded with nitrate, both net uptake and influx were nil. In contrast to these observations, a clear discrepancy between net uptake and influx was observed when the plants were incubated at an initial nitrate concentration of approximately 5 micromolar, where net uptake is low and eventually ceases. It is concluded that plasmalemma nitrate transport is essentially unidirectional in plants performing net uptake at a concentration of 40 to 60 micromolar, and that transport is nil when internal nitrate sinks (vacuole, metabolism) are eliminated. The efflux component becomes increasingly important when the external concentration approaches the threshold value for net nitrate uptake (the nitrate compensation point) where considerable exchange between internal and external nitrate occurs.     

Relationship between Nitrate Uptake, Flux, and Reduction and the Accumulation of Reduced Nitrogen in Maize (Zea mays L.): I. GENOTYPIC VARIATION

The study presented here was an extension of a preceding field project concerned with changes in N metabolism of four maize hybrids during grain development. The objectives were to relate uptake, flux, and reduction of nitrate to accumulation of reduced N in growth-chamber-grown seedlings of the same four hybrids and to compare these results with those obtained in the field study.     

In Vitro Stability of Nitrate Reductase from Wheat Leaves: II. Isolation of Factors from Crude Extract Which Affect Stability of Highly Purified Nitrate Reductase

When a crude extract from 8-day-old wheat (Triticum aestivum L. cv. Olympic) leaves was fractionated by a combination of ammonium sulfate precipitation and Sephadex G-100 chromatography the presence of three factors which have a marked effect on the stability of highly purified nitrate reductase was revealed. Two of these factors (I and III) have a positive effect and the other factor (II) has a negative effect on stability. Factors I and III can each overcome the instability-promoting effect of II; however, this was apparently not due to a direct effect on factor II.     

Relationships between Nitrate Reductase Activity, Plant Weight and Nitrogen Content in Seedlings of Triticum, Aegilops and Triticale

Seedlings of 12 genotypes were grown in pots and watered withnutrient solutions providing 0, 1, 6 and 20 mg equivalents ofnitrate per I. Increasing the external nitrate supply broughtabout increases in plant weight, nitrate, reduced nitrogen concentrationsand in vivo nitrate reductase activity. When given solutioncontaining 6 mg equivalents of nitrate per litre, the plantscontained approximately 0.1 per cent nitrate, a concentrationsimilarto that found in field-grown plantsat thesamestage of growth.At the 6 mg equivalent level nitrate supply, nitrate reductaseactivity was strongly positively correlated with the concentrationsof nitrate and reduced nitrogen and negatively correlated withplant weight. Similar, though weaker, correlations were foundat the lower and higher levels of nitrate supply. The two Triticalegenotypes however, had higher than average plant weights andnitrate reductase activities, while plants of the two Aegilopsspecies weighed much less, especially at the higher levels ofnitrate supply, than the average of all 12 genotypes and generallyhad correspondingly greater nitrate and reduced nitrogen concentrationsand nitrate reductase activities. For individual genotypes,plant weight at a given level of nitrate supply was stronglycorrelated with weight at all other levels. In a second experiment seedlings of 150 genotypes were grownin compost watered with 10 mM Ca(NO₃)₂ Nitrate and reduced nitrogenconcentrations were negatively correlated with plant weightbut there was no significant correlation between nitrate reductaseactivityand either plant weight, nitrate or reduced nitrogen concentration. The results are taken to indicate that genetic factors, otherthan those determining the supply of reduced nitrogen, werelimiting growth and that as a consequence small plants accumulatednitrate and reduced nitrogen compounds in greater concentrationsthan large ones. The greater nitrate concentrations in smallplants may have induced the increased nitrate reductase activityfound in these, as compared with larger plants. Because plantweight varied more than did reduced nitrogen concentration,variation in reduced nitrogen per plant was more highly correlatedwith plant weight than with per cent reduced nitrogen.     

Soybean Mutants Lacking Constitutive Nitrate Reductase Activity : II. Nitrogen Assimilation, Chlorate Resistance, and Inheritance

Nitrogen assimilation in three nitrate reductase (NR) mutants of soybean (Glycine max L. Merr. cv Williams) was studied in the growth chamber and in the field. These mutants, LNR-2, LNR-3, and LNR-4, lack the non-NO₃⁻-inducible or constitutive fraction of leaf NR activity found in wild-type plants, but this had no effect on the concentration of nitrogen accumulated when grown on NO₃⁻ in the growth chamber. Dry weight accumulation of two of the mutants (LNR-3 and LNR-4) was decreased relative to LNR-2 and wild type. In the field, LNR-2 had dry weights and nitrogen concentrations similar to the wild type at 34 and 61 days after planting, and at maturity. Acetylene reduction activities were also similar at 61 days.     

Nitrate Absorption by Barley: II. Influence of Nitrate Reductase Activity

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.     

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.     

Effects of Energy Substrates on Nitrate Reduction and Nitrate Reductase Activity in a Ruminal Bacterium, Selenomonas ruminantium

The factors affecting the rate of nitrate reduction and the nitrate reductase content in Selenomonas ruminantium were examined. The rate of nitrate reduction per cell mass was higher when S. ruminantium was grown on lactate than when grown on glucose, and the rate was further enhanced when grown on succinate. The nitrate reduction rate was parallel to the nitrate reductase content in cells, suggesting that the amount of nitrate reductase limits the rate of nitrate reduction. The amount of nitrate reductase was inversely related to growth rate. The growth rate was related to the level of intracellular ATP, which was inversely related to the levels of ADP and AMP. The ratio of NADH to NAD⁺was related to the rate of nitrate reduction and to the amount of nitrate reductase. From these results, it is conceivable that the synthesis of nitrate reductase is regulated in response to the sufficiency of energy and electron supply. Intracellular concentrations of adenine nucleotides and pyridine nucleotides may be the regulating factors. The amount of nitrate reductase was increased by the presence of nitrate, suggesting that the synthesis of nitrate reductase is enhanced by nitrate. In addition, nitrate reduction altered the fermentation pattern as a result of electron consumption.     

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.     

Nitrate Reductase Activity in Maize (Zea mays L.) Leaves: II. Regulation by Nitrate Flux at Low Leaf Water Potential

Experiments were conducted to determine whether the nitrate flux to the leaves or the nitrate content of the leaves regulated the nitrate reductase activity (NRA) in leaves of intact maize (Zea mays L.) seedlings having low water potentials (ψ_w) when other environmental and endogenous factors were constant. In seedlings that were desiccated slowly, the nitrate flux, leaf nitrate content, and NRA decreased as ψ_w decreased. The decrease in nitrate flux was caused by a decrease in both the rate of transpiration and the rate of nitrate delivery to the transpiration stream. Upon rewatering, the recovery in NRA was correlated with the nitrate flux but not the leaf nitrate content.     

Mitochondrial Complex I Impairment and Nitrate Reductase Activity in Leaves of Nicotiana sylvestris

Loss of major mitochondrial complex I subunit Nad 7 in the leaves of CMS II mutant of Nicotiana sylvestris caused increase in both in vivo and in vitro nitrate reductase activities and the per cent activation state of NR was also higher in CMS II as against wild type. The differences suggest a possibility for export of mitochondrial NADH due to impairment of complex I, a major and high affinity sink for NADH. Loss of complex I subunit also resulted in constitutive expression of alternate oxidase (Aox) thereby providing a mechanism for continuous supply of carbon skeletons required for nitrate assimilation. The possibility of close coordination between mitochondrial redox perturbation and nitrate reduction and its further assimilation is discussed.     

Salicylic Acid Influences Net Photosynthetic Rate,Carboxylation Efficiency,Nitrate Reductase Activity,and Seed Yield in Brassica juncea

Aqueous solutions of salicylic acid (SA) were applied to the foliage of 30-d-old plants of mustard (Brassica juncea Czern & Coss cv. Varuna). The plants sprayed with the lowest used concentration (10₋₅ M) of SA were healthier than those sprayed with water only or with higher concentrations of SA (10⁻⁴ or 10⁻³ M). 60-d-old plants possessed 8.4, 9.8, 9.3, 13.0 and 18.5 % larger dry mass, net photosynthetic rate, carboxylation efficiency, and activities of nitrate reductase and carbonic anhydrase over the control, respectively. Moreover, the number of pods and the seed yield increased by 13.7 and 8.4 % over the control. This revised version was published online in August 2006 with corrections to the Cover Date.     

In Vitro Studies of Nitrate Reductase Activity in Cotton Cotyledons: Effects of Dowex 1-Cl and BSA

Germinating cotton (Gossypium hirsutum L. cv. Deltapine 16) cotyledons developed two peaks of in vitro nitrate reductase activity; the first was stable in vitro and appeared 24 hours after imbibition; and the second, which was extremely labile in vitro, began to develop after the seedlings had emerged and developed chlorophyll. Nitrite reductase activity peaked only after the seedlings had emerged. Dowex 1-Cl (10%, w/v) and bovine serum albumin (3%, w/v) significantly improved the activity of extracted enzyme; greater improvement occurred as expansion of the cotyledons progressed. The major effect of bovine serum albumin on nitrate reductase activity in cotyledon extracts appeared to be that of making the extracted enzyme more active rather than increasing the amount of nitrate reductase extracted or improving the stability of the extracted enzyme.     

Influence of Ethephon on Nitrate Reductase, Protein Amino Nitrogen and Nitrate Content of Solanum tuberosum L.

Nitrate reductase activity was stimulated in roots and stems,but suppressed in leaves of potato plants grown in nutrientculture by 30 mg.liter^–1 ethephon applied to the culturesolution. In stems, nitrate reductase activity was stimulatedafter 5 h and by 24 h it was more than two fold that of thecontrol. The magnitude of stimulation by ethephon was less inroots compared to stems. Ethephon treatment enhanced ethyleneproduction by roots, stems and leaves but the level of productionwas not significantly different in these organs. The stimulationof nitrate reductase activity was prevented by cycloheximideand cordycepin suggesting the involvement of new protein synthesis. Ethephon enhanced TCA precipitable protein levels in both rootsand stems while that in leaves was not significantly affected.Amino nitrogen content increased in parallel with protein contentin response to ethephon, with roots exhibiting substantial stimulation.Nitrate accumulation in stem tissues was not affected by ethephontreatment but was increased in roots at 24 and 48 h. Leaf NO³content declined with time in both ethephon-treated and controlplants and after 24 h significantly less NO³ accumulated intreated leaves. These results are explained in terms of ethephonstimulated protein synthesis and increase in cellular metabolismand permeability. (Received August 21, 1984; Accepted January 7, 1985)     

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.