Effect of Applied Nitrogen on N2 Fixation, Assimilation of Nitrate and Ammonia in Nodules of Field-grown Moong (Vigna radiata)

A field experiment was conducted to study the effect of nitrogenapplication at 15, 30 and 45 kg ha^–1 of urea at pre-flowering(PF) and pod initiation (PI) stages on the activity of nitrogenase(N₂ase), nitrate reductase (NR) and other related parametersin the nodules of moong (Vigna radiata). Nitrogen applied atPF or PI stage was found to be inhibitory to N₂ase and glutaminesynthetase (GS) activities except at 15 kg N ha^–1 whenapplied at PF in the case of N₂ase. At both the stages therewas increase in NR and glutamate dehydrogenase (GDH) activitieswith the application of nitrogen. Seed yield increased by 18per cent with the application of 15 kg N ha^–1 at PI stagewhereas nitrogen application at PF stage only increased strawyield significantly. Nitrate reductase, nitrogenase, nitrogen application, ammonia assimilation, Vigna radiata     

Inverse Correlation of Thiol Proteinase with Nitrate Reductase Activities in Barley Leaves

Experiments conducted to determine the effects of leupeptin,a specific inhibitor of thiol proteinase, on extractable nitratereductase (NR) activity in leaves of Hordeum distichum duringdarkness revealed that leupeptin (0.01 mg.ml^–1) appliedto detached leaves significantly reduced the loss of NR activity.At the same time it also reduced the formation of small cytochromec reductase species, which is a degradation product of NR complex,Upon nitrate induction, extractable NR activity increased butthe content of thiol proteinase decreased. This inverse correlationwas also observed upon transfer of nitrate-grown barley seedlingsto nitrate-free nutrient solution. Furthermore, cycloheximide(0.1 mg.ml^–1) treatment of barley seedlings reduced thecontent of thiol proteinase and retarded the loss of NR activityunder noninducing conditions. These results suggest that invivo changes in NR content in leaves of Hordeum distichum arethe result of proteolysis by an endogenous thiol proteinase. (Received May 16, 1985; Accepted July 22, 1985)     

The Distribution of Nitrate Assimilation Between Root and Shoot in Vicia faba L.

Nitrate assimilation was examined in two cultivars (Banner Winterand Herz Freya) of Vicia faba L. supplied with a range of nitrateconcentrations. The distribution between root and shoot wasassessed. The cultivars showed responses to increased applied nitrateconcentration. Total plant dry weight and carbon content remainedconstant while shoot: root dry weight ratio, total plant nitrogen,total plant leaf area and specific leaf area (SLA) all increased.The proportion of total plant nitrate and nitrate reductase(NR) activity found in the shoot of both cultivars increasedwith applied nitrate concentrations as did NO₃^–: Kjeldahl-Nratios of xylem sap. The cultivars differed in that a greaterproportion of total plant NR activity occurred in the shootof cv. Herz Freya at all applied nitrate concentrations, andits xylem sap NO₃^–: Kjeldahl-N ratio and SLA were consistentlygreater. It is concluded that the distribution of nitrate assimilationbetween root and shoot of V. faba varies both with cultivarand with external nitrate concentration. Vicia faba L., field bean, nitrate assimilation, nitrate reductase, xylem sap analysis     

Triadimenol Increases Nitrate Levels and Nitrate Reductase Activity in Canola Leaves

Nitrate reductase activity in the first true leaves of canola(Brassica napus L.) seedlings grown in one-quarter strengthHoagland's solution from seeds pretreated with triadimenol (0.3or 30 g (a.i.) kg^–1 of seed) was higher than controlsduring the growth period of 15 to 25 d after planting. Triadimenolalso increased chlorophyll levels, the increase being more pronouncedat its lower concentration. The treatment also increased theweight and nitrate content of the leaves. When seedlings weregrown in nutrient solution containing 1 to 20 mM nitrate, theincrease in nitrate reductase activity by triadimenol was higherat lower rather than at higher nitrate concentrations. The nitratelevels and Kjeldahl nitrogen in the triadimenol-treated leaveswas higher than the controls at concentrations of added nitrateabove 2 mM. Addition of nitrate to plants grown in ammonium,increased nitrate reductase activity more in plants grown fromtriadimenol-treated seeds than controls. However, addition of10µM triadimenol for 24 h to ammonium-grown plants hadlittle effect on enzyme activity, both in the absence as wellas the presence of nitrate. This study demonstrates that triadimenolincreases nitrate reductase activity and nitrate accumulationin the leaves and at least part of the increased enzyme activityis independent of nitrate accumulation. Key words: Triazoles, nitrate content, nitrate reductase activity     

Nitrate Reduction by Cassava

Nitrate reductase (NR) was assayed in vivo in cassava (Manihotesculenta Crantz). Activity in the leaves ranged from 0 to 2.51µmole of NO₃^– reduced g^–1 h^–1, withno activity in the younger leaves (leaf 1 on top). NR activitywas localized in the sides and toward the tip of the lobes ofthe leaf. (Received December 10, 1985; Accepted April 8, 1986)     

Effects of Nitrate Withdrawal and Resupply on the Assimilation of Nitrate in Leaves of Tomato

Nitrate reduction in leaves of tomato occurred at the same ratein plants grown in 8.0 mol m^–3 nitrate as in plants grownin 2.0 mol m^–3 nitrate, but at a much slower rate in plantsgrown in 0.1 mol m^–3 nitrate. However, the plants grownin 8.0 mol m^–3 nitrate had a larger leaf system than theplants grown in 2.0 mol m^–3 nitrate, and so the totalcapacity to assimilate nitrate was greater in the plants grownin the higher concentration. It was shown that plants grownin 8.0 mol m^–3 nitrate were better buffered against nitratewithdrawal than plants grown in 2.0 mol m^–3 nitrate asthe rate of nitrate reduction declined more slowly when plantswere transferred to 0.1 mol m^–3 nitrate from the higherconcentration than from the lower concentration. Furthermore,leaf expansion continued in the plants transferred from thehigher concentration, whereas it ceased abruptly in the plantstransferred from the lower concentration. It was concluded thatboth continuing expansion and continuing nitrate reduction wereaccompanied, and possibly caused by, a release of nitrate fromstorage pools in the lower part of the stem or the roots. Duringwithdrawal of nitrate the leaves were shown to maintain potentialactivity of the enzyme nitrate reductase although there wasno nitrate to be reduced. When nitrate was resupplied it couldbe reduced very quickly and reduction in the leaves was seento increase within 5 h of resupply. By 3 d after resupply furtherenzyme activity had been induced. Key words: Lycopersicon esculentum Mill, nitrate assimilation, nitrate reductase activity, nitrate withdrawal     

Purification and Characterization of Assimilatory Nitrate Reductase from the Cyanobacterium Plectonema boryanum

Assimilatory nitrate reductase (NR) was solubilized by acetonetreatment from Plectonema boryanum and was purified 7,700-foldby heat treatment, ammonium sulfate fractionation and chromatographyon DEAE-Sephacel and Sephadex G-150. Purified NR had a specificactivity of 85 µmol NO₂^– formed min^–1 mg^–1protein. The enzyme retained both ferredoxin (Fd)- and methylviologen (MV)-linked NR activities throughout the purificationprocedure. Molecular weight was 80,000. The pH optimum was 10.5in the MV-assay and 8.5 when assayed with enzymatically reducedFd as the electron donor. Apparent Km values for nitrate andMV were 700 µM and 2,500µM in the MVassay and 55µM and 75 µM for nitrate and Fd in the Fd-assay.The enzyme was inhibited by thiol reagents and metal-chelatingreagents. (Received October 1, 1982; Accepted March 8, 1983)     

Nitrogen Economy of the Main Shoot of Field-Grown Barley (Hordeum vulgare L.): II. IN VIVO NITRATE REDUCTASE ACTIVITY DURING GROWTH

The method for assay of in vivo nitrate reductase (NR) activitywas standardized for barley (Hordeum vulgare L.). NR activitywas determined in the various organs of the main shoot of field-grownJyoti barley at 40 kg N ha_–1. Total nitrate reductaseactivity (TNRA) of each organ for the period it was metabolicallyactive was calculated. The NR activity was highest in the laminae,followed by the sheaths, reproductive organs; and internodes.The NR activity was high in the first-formed laminae and itshowed a decline in the ones formed subsequently. The valuesvaried from 43.2 ± 4.33 to 7.2 ± 1.49 µmolNO^–₃ reduced g^–1 dry wt. h^–1. Maximum TNRAin the laminae, sheath, and internodes was at 49, 84, and 84–93d after sowing, respectively. The TNRA of the main shoot asa whole showed three peaks, one around 49 d, a second around63 d, and a third around 84 d after sowing. Correlation coefficient(r) between NR and NO3 concentration was highly significantin the laminae and sheath viz. 0.76^*** and 0.62^***, respectively.The results are discussed in relation to alteration in managementpractices to maximize nitrate assimilatory activity and theamount of reduced N harvested.     

The Occurrence of Nitrate Reduction in the Leaves of Woody Plants

Nitrate reductase activities greater than 02 µmol h^–1g^–1 f. wt, measured by an in vivo assay, occurred in 41per cent of a large sample (555 species) of woody plants. Ifseveral taxonomic groups (Gymnosperms, Ericaceae and Proteaceae)with consistently low activities were discounted activitiesgreater than 02 µmol h^–1 g^–1 f. wt occurredin 73 per cent of the species. This compares with 93 per centin herbaceous species, suggesting that leaf nitrate reductionis of common occurrence in woody plants. In a small sample ofspecies leaf nitrate reductase activity correlated with nitrateconcentration in the xylem sap. Low activities occurred consistentlyin the Gymnosperms, Ericaceae and Proteaceae. Feeding cut shootsof representatives of these groups with nitrate caused inductionof leaf nitrate reductase activity in the Gymnosperms and Proteaceae,but only limited induction in the Ericaceae. The Ericaceae,with the exception of two species, had low activities and lownitrate reductase inducibility. Root assimilation may predominatein the Gymnosperms and Proteaceae. It is suggested that nitratereduction generally occurs in the leaves of trees from a varietyof plant communities and that this may be related to the lowerenergy cost of leaf, as opposed to root, nitrate assimilation. Nitrate reductase, trees and shrubs, leaves, nitrate assimilation, nitrate translocation, nitrate reductase induction, energy cost, plant ecology     

Responses of nitrate assimilation and N translocation in tomato (Lycopersicon esculentum Mill) to reduced ambient air humidity

The impact of low humidity in ambient air on water relations,nitrate uptake, and translocation of recently absorbed nitrogen,was investigated in 5-week-old tomato (Lycopersicon esculentumMill cv. Ailsa Craig) plants grown hydroponically in a completenutrient solution. Plants were subjected to dry air (relativehumidity 2–4% for 6 h. The transpiration rate increasedseveral-fold and the shoot water content decreased by almost20%, whereas root water content was unaffected. No effect onin vitro nitrate reductase (NR) activity was detected when usingan EDTA-contraining assay buffer. Replacement of EDTA with Mg²⁺revealed a significant decline in shoot NR activity, which suggestsphosphorylation of the enzyme during the stress treatment. Plantswere grown in a split-root system, in which one root half wasfed ¹⁵N-nitrate during the treatment, in order to determinenitrate uptake and translocation of recently absorbed nitrogenin the plants. Uptake of nitrate was substantially inhibited,but the proportion of absorbed ¹⁵N that was translocated tothe shoots was only slightly affected. In untreated plants,71% of the ¹⁵N recovered in roots had been retranslocated fromthe shoots, whereas in plants subjected to stress the deliveryof ¹⁵N from shoots to roots appeared to be completely inhibited.The data show that lowered humidity in air has significant effectson both uptake of nitrate as well as translocation of nitrogenwithin the plants. Some of these effects appear to be commonwith those observed in plants subjected to reduced water potentialsin the root environment and point to the possibility of theshoot water relations being highly influential on nitrogen uptakeand translocation. Key words: Air humidity, nitrate assimilation, nitrate reductase activity, nitrogen translocation, tomato, water stress     

Short-Term Nitrate Effects on Hydroponically-Grown Soybean cv. Bragg and its Supernodulating Mutant: I. CARBON, NITROGEN AND MINERAL ELEMENT DISTRIBUTION, RESPIRATION AND THE EFFECT OF NITRATE ON NITROGENASE ACTIVITY

A comparison between two hydroponically-grown soybean genotypes(Glycine max [L.] Merr.) cv. Bragg and the supernodulating mutantnts 1007 was made in terms of dry matter accumulation, carbon,nitrogen, and mineral element distribution, ¹⁵N natural abundanceand the effect of short-term treatment with 4·0 mol m^–3KNO₃ on nitrogenase activity and respiration. Differences weremost pronounced in nodule dry weight and plant nitrogen content,both of which were recorded to be substantially elevated inthe mutant. Mineral element concentrations in different plantparts proved to be rather similar with the exception of Ca,found to be lower in leaves of the mutant, and Mn concentrationswhich were twice as high in roots of nts 1007. The values of¹⁵N natural abundance showed that both genotypes were equallydependent on nitrogen fixation when nitrate was absent. Theresults of the acetylene reduction assays indicated similarspecific nodule activity, while on a per plant basis nitrogenaseactivity of the mutant proved to be more than twice the amountof Bragg. This effect was also reflected in higher nodule respirationwhile root respiration remained below that of Bragg. Nitrate induced a substantial reduction in nitrogenase activitynot only in Bragg, but also in nts 1007. Nodule respiratoryactivity of Bragg was reduced by nitrate from 1·27 to0·34 mg C h^–1 plant^–1. In nts 1007 correspondingvalues were 2·70 to 1·52 mg C h^–1 plant^–1.Starch concentration in nodules was decreased in both genotypes,but nevertheless remained higher in nts 1007. Values for solublesugars in nodules even increased in the mutant in response tonitrate while the same treatment caused a reduction in Bragg.The data indicate that nitrogenase activities of Bragg and nts1007 are equally sensitive to short-term application of nitrate. Key words: Glycine max, C and N distribution, nitrate, root respiration, ¹⁵N natural abundance     

The Uptake of Gaseous Ammonia by the Leaves of Italian Ryegrass

Lockyer, D. R. and Whitehead, D. C. 1986. The uptake of gaseousammonia by the leaves of Italian ryegrass.—J. exp. Bot.37: 919–927. Plants of Italian ryegrass (Lolium multiflorum Lam.) grown insoil with two rates of added ¹⁵N-labelled nitrate were exposed,in chambers, for 40 d to NH₃ in the air at concentrations of16, 118 and 520 µg m^–3. At the highest concentrationof NH₃, this source provided 47?3% of the total nitrogen inplants grown with the lower rate of nitrate addition (100mgN kg^–1 dry soil) and 35?2% with the higher rate (200mgN kg^–1 dry soil) At the intermediate concentration ofNH₃, the contributions to total plant N were 19?6% and 10?8%,respectively, at low and high nitrate while, at the lowest concentrationof NH₃, they were 5?1% and 32%. Most of the N derived from theNH₃ remained in the leaves, but some was transported to theroots. The amount of N derived from the NH³ that was presentin the leaves was not reduced by washing the leaves in waterat pH 5?0 before harvesting, indicating that the N was assimilatedby the plant and not adsorbed superficially. Rates of uptakeof NH₃ per unit leaf area ranged from 1?7 µg dm^–2h^–1 at a concentration of 16 µg m^–3 to 29?0µg dm^–2 h^–1 at a concentration of 520 µgm^–3 and with the lower rate of nitrate addition. Increasingthe supply of nitrate to the roots slightly reduced the rateof uptake of NH₃ per unit leaf area. Uptake of N from the higherrate of nitrate was reduced at the highest concentration ofNH₃ in the air. Key words: Ammonia, nitrogen, leaf sorption, Lolium multiflorum     

氮素水平对花生氮素代谢及相关酶活性的影响

 在大田高产条件下研究了氮素水平对花生(Arachis hypogaea)可溶性蛋白质、游离氨基酸含量及氮代谢相关酶活性的影响, 结果表明, 适当提高氮素水平既能增加花生各器官中可溶性蛋白质和游离氨基酸的含量, 又能提高硝酸还原酶、谷氨酰胺合成酶和谷氨酸脱氢酶等氮素同化酶的活性, 使其达到同步增加; 氮素水平过高虽能提高硝酸还原酶和籽仁蛋白质含量, 但谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(GDH)的活性下降; N素施肥水平不改变花生植株各器官中可溶性蛋白质、游离氨基酸含量以及硝酸还原酶(NR)、谷氨酰胺合成酶、谷氨酸脱氢酶活性的变化趋势, 但适量施N (A2和A3处理)使花生各营养器官中GS、GDH活性提高; 氮素水平对花生各叶片和籽仁中GS、GDH活性的高低影响较大, 但对茎和根中GDH活性大小的影响较小。     

Soyabean Stem In Vivo Nitrate Reductase Activity

Stem from three- and four-week-old Soyabean [Glycine max (L.)Merr. cv. Tracy] plants reduced from 0.3 to 0.7 µmol nitrateh^–l g^–l f. wt. Leaf activity was 4.7–7.6 µmolnitrate h^–l g^–l f. wt. Outer stem was two to fourtimes more active at reducing nitrate than was inner stem. Plantnitrate nutrition had a strong effect upon the ratio of activitypresent in stem and leaf. More nitrate increased the proportionpresent in leaves. Glycine max L., soyabean, nitrate assimilation, nitrogen metabolism, Rhizobium japonicum     

Purification and Characterization of Thiol Proteinase as a Nitrate Reductase-Inactivating Factor from Leaves of Hordeum distichum L.

A thiol proteinase was purified 6,400-fold from leaves of Hordeumdistichum L. by a sequence of ammonium sulfate fractionation,gel filtration, ion exchange chromatography, hydrophobic chromatographyand chromatofocusing. This enzyme also had nitrate reductase(NR)-inactivating activity, which was associated with proteolyticactivity in almost constant proportions during the purificationprocedures. Its molecular weight was estimated as 74,000 bygel filtration, and it had an isoelectric point of 4.05 andan apparent K_m of 0.83 mg ml^–1 for azocasein. The respectiveoptimum pH for proteolytic and NR-inactivating activities were6.0 and 7.0. On electrophoresis, the proteinase gave a majorband that coincided with both activities; it also produced afaint band associated with no activity. Our purified thiol proteinase inactivated FMNH₂-NR and MVH-NRas well as NADH-NR, but it had only a slight effect on NADHcytochrome c reductase activity. This enzyme also inactivatedglutamine synthetase. (Received September 16, 1983; Accepted January 26, 1984)     

The Effect of Abscisic Acid on Amino Nitrogen and Protein Content of Potato Plants in Relation to the Inhibition of Nitrate Reductase Activity

Treatment of potato plants grown in nutrient solution with 3.8µM ABA resulted in reduced soluble protein in roots andin leaves at 24 h, but not in stems. This treatment reducedin vivo nitrate reductase activity in all organs for about 48h with the most pronounced reduction occurring in the roots.Excised root and leaf segments from plants treated with ABAfor 24, 48 and 72 h absorbed significantly more ¹⁴C leucine,compared to the control but the percent incorporation into proteinwas not altered in roots. In response to ABA total free amino nitrogen in leaves was lowerat 5 and 72 h and in stems at 72 h. Amino nitrogen content ofroots was enhanced by ABA at 5, 24 and 72 h due to generallyhigher levels of aspartate, serine, glutamate, proline and ammonia.There was no consistent relationship between ABA suppressionof nitrate reductase activity and ammonia or specific aminoacid (except proline) levels in leaves and stems. The increasedfree amino nitrogen levels in response to the hormone may bethe result of impaired NO₃– reduction rather than thecause. The results of protein synthesis studies and solubleprotein content suggest that ABA inhibition of nitrate reductaseis not due to general inhibition of protein synthesis and mayinvolve specific inhibition of nitrate reductase protein synthesis. ¹ Contribution No. 684, Department of Plant Science, Universityof Manitoba.     

Xylem exudation is related to nitrate assimilation pathway in detopped maize seedlings: use of nitrate reductase and glutamine synthetase inhibitors as tools

The xylem exudation of detopped 7-d-old seedlings of Zea maysL. doubled when KCI was present in the root medium comparedto seedlings maintained on water. It was further enhanced whenKCI was replaced by nitrogen compounds such as nitrate, ammoniumand glutamine. The role of the nitrate assimilation pathwayon the enhancement of xylem exudation rate was investigatedusing tungstate, an inhibitor of nitrate reductase (NR) activity,and phosphinothricin or methionine sulphoximine, inhibitorsof glutamine synthetase (GS) activity. The sap levels of NO₃^–,NH₄⁺, glutamine, and asparagine was used to ascertain the invivo inhibition of both enzymes. The tungstate effects werealso checked by measuring leaf in vitro NA activity and NR proteincontent. Xylem exudation rate of detopped seedlings fed withKNO₃ decreased when the nitrate assimilation pathway was blockedeither at the NR or at GS sites. This decrease was preventedwhen urea (acting as NH₄⁺ supply) was given simultaneously withtungstate. KNO₃ does not act directly on exudation, but throughthe involvement of NH₄⁺. The involvement of glutamine was alsoshown since GS inhibition resulted in a cancellation of theenhancing effect of KNO₃ on exudation. As change of exudationrate was not linked to change in sap osmolarity, it is assumedthat the assimilation chain could modify root water conductance.The role of glutamine was discussed. Key words: Exudation, maize, nitrate, conductance, NR, GS     

Flavine nucleotide nitrate reductase from broad bean leaves

Hydrosulfite-reduced FMN served as an electron donor for nitratereductase purified from broad bean leaves. FMN was successfullyreplaced with BV. The flavine nucleotide nitrate reductase hadits pH optima at about 7.8 with phosphate buffer and at about7.4 with Tris-HCl buffer. The Km's for nitrate and FMN were3.7 ? 10^–4 M and 3.7 ? 10^–5 M, respectively. NADH₂: nitrate reductase activity was completely inhibited by0.1 mM p-CMB, whereas FMNH₂: nitrate reductase activity wasnot. Inhibited activity was restored by the addition of cysteine.A sulfhydryl enzyme is involved in the NADH₂: nitrate reductasesystem but not in the FMNH₂ : nitrate reductase system. NADH₂and FMNH₂ probably feed electrons into the electron transportchain at different sites. The nitrate reductase preparationhad an NADH₂-specific diaphorase activity which was almost completelyinhibited by 0.1 mM p-CMB. The NADH₂-specific diaphorase mayform the sulfhydryl enzyme which mediates electron transferbetween NADH₂ and nitrate. (Received May 6, 1969; )     

In vivo Nitrate Reductase Activity in Barley (Hordeum vulgare L.) during Ear Development

Experiments were conducted during the 1974–75 and 1975–76winter season with the barley (Hordeum vulgare L.) cultivarJyoti. From amongst the various plant parts, the flag leaf bladehad higher in vivo nitrate reductase (NR) activity than thelower two leaf blades, glumes, and grains. However, the potentialof a plant part to reduce NO₃^– is a function of its freshweight and the NR per unit fresh weight. On this basis, thesecond and third leaf blades could reduce more NO₃^– thanthe flag leaf blade. N fertilizer application resulted in enhancementof the activity of the leaf blades alone. N fertilizer appliedduring the reproductive phase was taken up and assimilated bythe various plant parts. The studies suggest that, even whenthe fertilizer is applied at optimum levels for obtaining maximumyields, the upper leaf blades have sub-optimal NR activity andthat there is a likelihood of either a preferential flow ofNO₃^– to the leaf blades or transnational barriers to NO₃^–movement to the ear.