Nitrate Reductase Activity in Shoots and Roots of Maize Seedlings as Affected by the Form of Nitrogen Nutrition and the pH of the Nutrient Solution

The effect of nitrogen form (NH₄-N, NH₄-N + NO₃⁻, NO₃⁻) on nitrate reductase activity in roots and shoots of maize (Zea mays L. cv INRA 508) seedlings was studied. Nitrate reductase activity in leaves was consistent with the well known fact that NO₃⁻ increases, and NH₄⁺ and amide-N decrease, nitrate reductase activity. Nitrate reductase activity in the roots, however, could not be explained by the root content of NO₃⁻, NH₄-N, and amide-N. In roots, nitrate reductase activity in vitro was correlated with the rate of nitrate reduction in vivo. Inasmuch as nitrate reduction results in the production of OH⁻ and stimulates the synthesis of organic anions, it was postulated that nitrate reductase activity of roots is stimulated by the released OH⁻ or by the synthesized organic anions rather than by nitrate itself. Addition of HCO₃⁻ to nutrient solution of maize seedlings resulted in a significant increase of the nitrate reductase activity in the roots. As HCO₃⁻, like OH⁻, increases pH and promotes the synthesis of organic anions, this provides circumstantial evidence that alkaline conditions and/or organic anions have a more direct impact on nitrate reductase activity than do NO₃⁻, NH₄-N, and amide-N.     

Differential light induction of nitrate reductases in greening and photobleached soybean seedlings

Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO₃⁻, tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO₃⁻ and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO₃⁻ and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO₃⁻. The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO₃⁻, further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO₃⁻ requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO₃⁻ requirement for induction were quite different. K_m values for NO₃⁻ were identical for both nitrate reductases.     

The occurrence of nitrate reductase in leaves of prunus species

Nitrate reductase was found in leaves of apricot Prunus armeniaca, sour cherry P. cerasus, sweet cherry P. avium, and plum P. domestica, but not in peach P. persica, from trees grown in sand culture receiving a nitrate containing nutrient solution. Nitrate was found in the leaves of all species. Nitrate and nitrate reductase were found in leaves of field-grown apricot, sour cherry, and plum trees. The enzyme-extracting medium contained insoluble polyvinylpyrrolidone, and including dithiothreitol or mercaptobenzothiazole did not improve enzyme recovery. Inclusion of cherry leaf extract diminished, and peach leaf extract abolished, recovery of nitrate reductase from oat tissue. Low molecular weight phenols liberated during extraction were probably responsible for inactivation of the enzyme. The enzyme from apricot was two to three times as active as from the other species. Both nicotine adenine diphosphopyridine nucleotide and flavin mononucleotide were effective electron donors. The enzyme was readily induced in apricot leaves by 10 mm nitrate supplied through the leaf petiole.     

Nutrient inflows into apple roots

Summary The rates of uptake of nutrients from solution by apple roots were measured (a) in a root laboratory, using intact roots of mature trees growing under field conditions and (b) in controlled environment using young trees. Maximum nitrate inflows into Discovery/M.9 roots under field conditions were only slightly lower than those into roots of the same genotype in controlled environment, but up to 80 times lower than those into roots of Worcester Pearmain seedlings. At any given external P concentration, P inflows into roots of field-grown trees were about 2.5-times lower than those into the roots of young trees in controlled environment.Nitrate inflows were constant above a solution concentration of 20 mmol m^–3 in both field-grown and small trees. In both cases, phosphate inflows increased linearly with solution concentration up to 10 mmol m^–3.Among the various plant and environmental factors influencing nutrient uptake characteristics of apple roots were: the scion genotype, tissue nutrient levels, root origin, the form in which N is supplied, level of irradiance of the shoot, root temperature and the season of the year. The effects of these factors are illustrated with examples.     

Nitrate reduction and nitrate content in ash trees (Fraxinus excelsior L.): distribution between compartments,site comparison and seasonal variation

Summary Nitrate reductase activity (NRA), nitrate content and biomass components of leaflets, leaf stalks, old stem, current-year stem and roots of ash trees (Fraxinus excelsior L.) growing in their natural habitats were investigated. In addition, NRA, total nitrogen and nitrate concentration were analyzed in the leaves and roots of ash trees from four different field sites. The highest NRA per gram biomass and also per total compartment biomass was found in the leaflets, even though root biomass was much higher than total leaflet biomass. The highest nitrate concentrations were found in the leaf stalks. Correlations between nitrate availability in the soil and NRA in leaves were not significant due to high variability of the actual soil nitrate concentrations. The seasonal variation in foliar NRA, nitrate concentration and total nitrogen concentration is much smaller in F. excelsior than reported for herbaceous species and is mainly caused by changes in the actual soil nitrate availability and by senescence of the leaves.     

Influence of ammonium and nitrate nutrition on enzymatic activity in soybean and sunflower

Under conditions of controlled pH, nitrate and ammonium are equally effective in supporting the growth of young soybean (Glycine max var. Bansei) and sunflower (Helianthus annuus L. var., Mammoth Russian) plans. Soybean contains an active nitrate reductase in roots and leaves, but the low specific activity of this enzyme in sunflower leaves indicates a dependency upon the roots for nitrate reduction. Suppression of nitrate reductase activity in sunflower leaves may be due to high concentrations of ammonia received from the roots. Nitrate reductase activity in leaves of nitrate-supplied soybean and sunflower follows closely the distribution of nitrate reductase. For the roots of both species, glutamic acid dehydrogenase activity was greater with ammonium than with nitrate. The glutamic acid dehydrogenase of ammonium roots is wholly NADH-dependent, whereas that of nitrate roots is active with NADH and NADPH. In leaves, an NADPH-dependent glutamic acid dehydrogenase appears to be responsible for the assimilation of translocated ammonia and ammonia formed by nitrate reduction.     

Calcium transport in apple trees

⁴⁵Ca, applied to roots of apple seedlings, moved readily to the developing leaves. Kinetin, benzyladenine, and B sprays increased movement. NO₃⁻ as the source of N increased movement and accumulation of Ca into mature leaves; NH₄⁺ increased movement into new leaves. Translocation in the stem is effected by a nonspecific ion exchange. Any divalent cation can free Ca for ascent. The exchange may be a property of lignin. Genetic differences in the uptake and translocation of Ca exist among apple seedlings. Those which show juvenile leaf characteristics translocate Ca into mature leaves more readily. Translocation of Ca in woody species appears to be similar to that reported for herbaceous plants.     

Effects of sodium on mineral nutrition in rose plants

The effects of sodium (Na⁺) ion concentration on shoot elongation, uptake of ammonium (NH₄⁺) and nitrate (NO₃^?) and the activities of nitrate reductase (NR) and glutamine synthetase (GS) were studied in rose plants (Rosa hybrida cv. “Lambada”). The results showed that shoot elongation was negatively correlated with sodium concentration, although no external symptoms of toxicity were observed. Nitrate uptake decreased at high sodium levels, specifically at 30 meq litre4 of sodium. As flower development was normal under high saline conditions, this could suggest that nitrogen was being mobilised from shoot and leaf reserves. Ammonium uptake was not affected by any of the salt treatments applied probably because it diffuses through the cell membrane at low concentrations. Nitrate reductase activity was reduced by 50% at 30 meq litre 1 compared with control treatment, probably due to a decrease in the free nitrate related to nitrate uptake pattern. None of the salt treatments used affected total leaf GS activity (both chloroplastic and cytosolic isoforms) or leaf NPK mineral contents. Nitrate reductase activity in leaves increased at 10 meq litre^?1 of sodium and GS activity in roots (cytosolic isoform only) followed the same pattern as NR. It is suggested that the activation of both enzymes at low salt level could be attributed to the beneficial effect of increased sulphur in the nutrient solutions.     

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     

Soybean mutants lacking constitutive nitrate reductase activity : I. Selection and initial plant characterization

The objectives of this study were to select and initially characterize mutants of soybean (Glycine max L. Merr. cv Williams) with decreased ability to reduce nitrate. Selection involved a chlorate screen of approximately 12,000 seedlings (progeny of mutagenized seed) and subsequent analyses for low nitrate reductase (LNR) activity. Three lines, designated LNR-2, LNR-3, and LNR-4, were selected by this procedure.

In growth chamber studies, the fully expanded first trifoliolate leaf from NO₃⁻-grown LNR-2, LNR-3, and LNR-4 plants had approximately 50% of the wild-type NR activity. Leaves from urea-grown LNR-2, LNR-3, and LNR-4 plants had no NR activity while leaves from comparable wild-type plants had considerable activity; the latter activity does not require the presence of NO₃⁻ in the nutrient solution for induction and on this basis is tentatively considered as a constitutive enzyme. Summation of constitutive (urea-grown wild-type plants) and inducible (NO₃⁻-grown LNR-2, LNR-3, or LNR-4 plants) leaf NR activities approximated activity in leaves of NO₃⁻-grown wild-type plants. Root NR activities were comparable in wild-type and mutant plants grown on NO₃⁻, and roots of both plant types lacked constitutive NR activity when grown on urea. In both growth chamber- and field-grown plants, oxides of nitrogen [NO_(x)] were evolved from young leaves of wild-type plants, but not from leaves of LNR-2 plants, during in vivo NR assays. Analysis of leaves from different canopy locations showed that constitutive NR activity was confined to the youngest three fully expanded leaves of the wild-type plant and, therefore, on a total plant canopy basis, the NR activity of LNR-2 plants was approximately 75% that of wild-type plants. It is concluded that: (a) the NR activity in leaves of NO₃⁻-grown wild-type plants includes both constitutive and inducible activity; (b) the missing NR activity in LNR-2, LNR-3, and LNR-4 leaves is the constitutive component; and (c) the constitutive NR activity is associated with NO_(x) evolution and occurs only in physiologically young leaves.

     

Nitrate content and nitrate reductase activity in Rumex obtusifolius L.

Summary With Rumex obtusifolius L., the influence of some environmental conditions on nitrate uptake and reduction were investigated. Nitrate concentrations of plant material were determined by HPLC, the activity of nitrate reductase by an in vivo test. As optimal incubation medium, a buffer containing 0.04 M KNO₃; 0.25 M KH²PO₄; 1.5% propanol (v/v); pH 8.0 was found. Vacuum infiltration caused an increase of enzyme activity of up to 40%.High nitrate concentrations were found in roots and leaf petioles. Nitrate reductase activity of these organs, however, was low. On the other hand, the highest nitrate reductase activity was observed in leaf laminae, which contained lowest nitrate concentrations.In leaves, nitrate content and nitrate reductase activity exhibited inverse diurnal fluctuations. During darkness, decreasing activities of the enzyme were followed by increasing nitrate concentrations, while during light the contrary was true. In petioles diurnal fluctuations in nitrate content were observed, too. No significant correlations with illumination, however, could be found.Our results prove that Rumex obtusifolius is characterized by an intensive nitrate turnover. Theoretically, internal nitrate content of the plant would be exhausted within a few hours, if a supply via the roots would be excluded.     

Variation in cadmium accumulation potential and tissue distribution of cadmium in tobacco

Variation in Cd accumulation between Nicotiana species but not varieties has been observed in seedlings grown in solution culture with moderate-to-low levels of Cd. Nicotiana tabacum has been characterized as a leaf and root accumulator while Nicotiana rustica is shown to be primarily a root accumulator, having about half the leaf Cd per gram dry weight of N. tabacum. This phenotype is retained in the mature N. rustica plant. To characterize these two species which differ in their modes of Cd accumulation, tissue Cd distribution, partitioning of metal in soluble and insoluble fractions and the contribution of soluble Cd-binding proteins (peptides) to total plant Cd was assessed using mature solution cultured plants. Metal accumulation was highest in the most mature leaves and in young roots. The preponderance of young roots in N. rustica may, in part, account for low leaf/high root Cd accumulation in this species. While Cd-binding peptides appear to be a principal form of Cd in leaves and roots of seedlings and these also occur in mature leaves, Cd is equally distributed between soluble (about 80% as Cd-binding peptide) and uncharacterized insoluble forms in mature plant roots.     

Critical evaluation of thein situ nitrate reductase assay

Anin situ method, derived from anin vivo method, was used to determine nitrate reductase activity (NRA) in:i) excised barley and corn shoots and excised soybean leaves during a N-depletion experiment and; ii) roots and shoots of N-depleted barley and corn seedlings during induction of nitrate, reductase (NR). Nitrate reduction, calculated from thesein situ RNA measurements, was compared with estimates of each organ's nitrate reduction in light aerobic conditions from NO ₃ ⁻ consumption and a¹⁵N model (Gojonet al., 1986b). Thein situ RNA of roots strongly underestimated their¹⁵NO ₃ ⁻ reduction. In contrast, in barley and corn shoots and in the first trifoliolate leaves from 26-day-old, soybean, thein situ NRA assay gave a fair approximation of the true NO ₃ ⁻ reduction rate (relative differences ranging from −14 to +32%). In young soybean leaves (from 20-day-old plants), however, thein situ NRA strongly underestimated the actual NO ₃ ⁻ reduction. The physiological significance of thein situ NRA assay in shoots and roots, and its value for field studies are discussed from these results.     

Enhancement of Nitrate Reductase Activity by Benzyladenine in Agrostemma githago

Nitrate reductase activity in excised embryos of Agrostemma githago increases in response to both NO₃⁻ and cytokinins. We asked the question whether cytokinins affected nitrate reductase activity directly or through NO₃⁻, either by amplifying the effect of low endogenous NO₃⁻ levels, or by making NO₃⁻ 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 NO₃⁻ and benzyladenine, additive responses were obtained. The effects of NO₃⁻ 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 NO₃⁻, as compared to embryos treated with benzyladenine. Casein hydrolysate inhibited the development of nitrate reductase activity. The response to NO₃⁻ was more susceptible to inhibition by casein hydrolysate than the response to the hormone. When NO₃⁻ 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 NO₃⁻. At the time of the second exposure to benzyladenine, no NO₃⁻ was detectable in extracts of Agrostemma embryos. This is taken as evidence that cytokinins enhance nitrate reductase activity directly and not through induction by NO₃⁻.     

Synthesis and degradation of barley nitrate reductase

Nitrate and light are known to modulate barley (Hordeum vulgare L.) nitrate reductase activity. The objective of this investigation was to determine whether barley nitrate reductase is regulated by enzyme synthesis and degradation or by an activation-inactivation mechanism. Barley seedling nitrate reductase protein (cross-reacting material) was determined by rocket immunoelectrophoresis and a qualitative immunochemical technique (western blot) during the induction and decay of nitrate reductase activity. Nitrate reductase cross-reacting material was not detected in root or shoot extracts from seedlings grown without nitrate. Low levels of nitrate reductase activity and cross-reacting material were observed in leaf extracts from plants grown on nitrate in the dark. Upon nitrate induction or transfer of nitrate-grown etiolated plants to the light, increases in nitrate reductase activity were positively correlated with increases in immunological cross-reactivity. Root and shoot nitrate reductase activity and cross-reacting material decreased when nitrate-induced seedlings were transferred to a nitrate-free nutrient solution or from light to darkness. These results indicate that barley nitrate reductase levels are regulated by de novo synthesis and protein degradation.     

Distribution of nitrate reductase in ageing bean seedlings

The in vivo activity of nitrate reductase (NR, E.C. 1.6.6.1 [EC] )in the roots, stem and leaves of bean (Phaseolus vulgaris L.)was measured at different ages of seedlings. The leaves alwayshad higher levels of the enzyme than the roots or stem. Thelevel of the enzyme in the very young leaves were low, increasingto a maximum by day 10 to 11 of seedling growth at 26°C,after which it start to decline. The level of the enzyme in7 dayold decotyledonized leaves was about 2.5 times higher thanthat in leaves from intact seedlings. A supply of exogenousnitrate caused a considerable increase in the total organicnitrogen in the leaf only after day 9, when the nitrogen supplyfrom the cotyledons presumably is low. (Received March 22, 1975; )     

An in situ approach for measuring root-associated respiration and nitrate uptake of forest trees

The ecophysiological characteristics of fine roots of mature forest plants are poorly understood because of difficulties of measurement. We explored a root in-growth approach to measure respiration and nitrate uptake of woody plant roots in situ. Roots of seven species were grown into sand-filled chambers. Root-associated respiration was measured as CO ₂ emission on four dates and nitrate uptake was quantified using ¹⁵N. All the roots were younger than 3 months at the time of measurement. Fine root respiration measured over the temperature range of 14.5–15.5 °C averaged 18.9–36.5 nmol gDM ^–1 s ^–1 across species. Nitrate uptake rates by these fine roots (1.3–6.8 nmol gDM ^–1 s ^–1) were comparable to other studies of forest trees. The root respiration rates were several times higher than measurements on detached roots of mature trees, concurring with literature observations that young roots respire much more rapidly than older roots. The root in-growth approach appears promising for providing information on the metabolic activity of fine roots of mature forest trees growing in soil.     

Nitrate Reduction in Roots and Shoots of Barley (Hordeum vulgare L.) and Corn (Zea mays L.) Seedlings: I. N Study

Nitrate reduction in roots and shoots of 7-day-old barley seedlings, and 9-day-old corn seedlings was investigated. The N-depleted seedlings were transferred for 24 h or 48 h of continuous light to a mixed nitrogen medium containing both nitrate and ammonium. Total nitrate reduction was determined by ¹⁵N incorporation from ¹⁵NO₃⁻, translocation of reduced ¹⁵N from the roots to the shoots was estimated with reduced ¹⁵N from ¹⁵NH₄⁺ assimilation as tracer, and the translocation from the shoots to the roots was measured on plants grown with a split root system. A model was proposed to calculate the nitrate reduction by roots from these data. For both species, the induction phase was characterized by a high contribution of the roots which accounted for 65% of the whole plant nitrate reduction in barley, and for 70% in corn. However, during the second period of the experiment, once this induction process was finished, roots only accounted for 20% of the whole plant nitrate reduction in barley seedlings, and for 27% in corn. This reversal in nitrate reduction localization was due to both increased shoot reduction and decreased root reduction. The pattern of N exchanges between the organs showed that the cycling of reduced N through the plant was important for both species. In particular, the downward transport of reduced N increased while nitrate assimilation in roots decreased. As a result, when induction was achieved, the N feeding of the roots appeared to be highly dependent on translocation from the leaves.     

Effect of potassium on growth and nitrate reductase during water stress and recovery in maize

Abstract The effect of potassium (0,50, 100 and 200 mg/pot) was studied on growth characteristics and nitrate reductase activity in maize (Zea mays) seedlings during water stress and subsequent recovery. In irrigated plants K⁺ increased the rate of leaf area expansion, leading to increased leaf area per plant. Increased leaf area was associated with decreased chlorophyll content. Water stress (–15 bars) enhanced the stomatal resistance of leaves which was further accentuated by K⁺ application. Nitrate reductase activity rose in irrigated plants 24 h after K⁺ application. Subsequently, as water stress developed, K⁺ helped to maintain higher NR activity for the first two days. However, K⁺ had no effect on half life of NR in light or darkness. During recovery from stress K⁺ aided to maintain the higher leaf expansion rate, the chlorophyll content and the stomatal resistance. The results above are discussed in relation to the ability of K⁺ to maintain better growth under water stress.