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.     

Ammonium and nitrate nutrition inPlantago lanceolata L. andPlantago major L. ssp.major

With the aims (1) to test whether the different natural occurrence of twoPlantago species in grasslands is explained by a different preference of the species for nitrate or ammonium; (2) to test whether the different occurrence is explained by differences in the flexibility of the species towards changes in the nitrogen form; (3) to find suitable parameters as a tool to study ammonium and nitrate utilization of these species at the natural sites in grasslands, plants ofPlantago lanceolata andP. major ssp.major were grown with an abundant supply of nitrate, ammonium or nitrate+ammonium as the nitrogen source (0.5 mM). The combination of ammonium and nitrate gave a slightly higher final plant weight than nitrate or ammonium alone. Ammonium lowered the shoot to root ratio inP. major. Uptake of nitrate per g root was faster than that of ammonium, but from the mixed source ammonium and nitrate were taken up at the same rate. In vivo nitrate reductase activity (NRA) was present in both shoot and roots of plants receiving nitrate. When ammonium was applied in addition to nitrate, NRA of the shoot was not affected, but in the root the activity decreased. Thus, a larger proportion of total NRA was present in the shoot than with nitrate alone. In vitro glutamate dehydrogenase activity (GDHA) was enhanced by ammonium, both in the shoot and in the roots.In vitro glutamine synthetase activity (GSA) was highest in roots of plants receiving ammonium. Both GDHA and GSA were higher inP. lanceolata than inP. major. The concentration of ammonium in the roots increased with ammonium, but it did not accumulate in the shoot. The concentration of amino acids in the roots was also enhanced by ammonium. Protein concentration was not affected by the form of nitrogen. Nitrate accumulated in both the shoot and the roots of nitrate grown plants. When nitrate in the solution was replaced by ammonium, the nitrate concentration in the roots decreased rapidly. It also decreased in the shoot, but slowly. It is concluded that the nitrogen metabolism of the twoPlantago species shows a similar response to a change in the form of the nitrogen source, and that differences in natural occurrence of these species are not related to a differential adaptation of nitrogen metabolism towards the nitrogen form. Suitable parameters for establishing the nitrogen source in the field are thein vivo NRA, nitrate concentrations in tissues and xylem exudate, and the fraction of total reduced nitrogen in the roots that is in the soluble form, and to some extent thein vitro GDHA and GSA of the roots. Grassland Species Research Group. Publ. no 118.     

Nitrate nutrition ofDeschampsia flexuosa (L.) Trin. in relation to nitrogen deposition in Sweden

Summary Current and maximally induced nitrate reductase activity (NRA), total-N, nitrate, K, P, Ca, Mg, Mo and sucrose in leaves ofDeschampsia flexuosa was measured three times during the vegetation period in forests along a deposition gradient (150 km) in south Sweden, in north Sweden where the nitrogen deposition is considerably lower, and at heavily N-fertilized plots. In addition, the interaction between nitrogen nutrition and light was studied along transects from clearings into forest in both south and north Sweden. Plants from sites with high nitrogen deposition had elevated current NRA compared to plants from less polluted sites, indicating high levels of available soil nitrate at the former. Current NRA and total N concentration in grass from sites with high deposition resembled those found at heavily N-fertilized plots. Under such circumstances, the ratio current NRA: maximally induced NRA as well as the concentration of nitrate was high, while the concentration of sucrose was low. This suggests that the grass at these sites was already utilizing a large portion of its capacity to assimilate nitrate. Light was found to play an important role in the assimilation of nitrate; leaf concentration of sucrose was found to be negatively correlated with both nitrate and total N. Consequently, grass growing under dense canopies in south Sweden is not able to dilute N by increasing growth. The diminished capacity of the grass to assimilate nitrate will increase leaching losses of N from forests approaching N saturation.     

nir1, a conditional-lethal mutation in barley causing a defect in nitrite reduction

Summary Eleven green individuals were isolated when 95000 M₂ plants of barley (Hordeum vulgare L.), mutagenised with azide in the M₁, were screened for nitrite accumulation in their leaves after nitrate treatment in the light. The selected plants were maintained in aerated liquid culture solution containing glutamine as sole nitrogen source. Not all plants survived to flowering and some others that did were not fertile. One of the selected plants, STA3999, from the cultivar Tweed could be crossed to the wild-type cultivar and analysis of the F₂ progeny showed that leaf nitrite accumulation was due to a recessive mutation in a single nuclear gene, which has been designated Nir1. The homozygous nir1 mutant could be maintained to flowering in liquid culture with either glutamine or ammonium as sole nitrogen source, but died within 14 days after transfer to compost. The nitrite reductase cross-reacting material seen in nitrate-treated wild-type plants could not be detected in either the leaf or the root of the homozygous nir1 mutant. Nitrite reductase activity, measured with dithionite-reduced methyl viologen as electron donor, of the nitrate-treated homozygous nir1 mutant was much reduced but NADH-nitrate reductase activity was elevated compared to wild-type plants. We conclude that the Nir1 locus determines the formation of nitrite reductase apoprotein in both the leaf and root of barley and speculate that it represents either the nitrite reductase apoprotein gene locus or, less likely, a regulatory locus whose product is required for the synthesis of nitrite reductase, but not nitrate reductase. Elevation of NADH-nitrate reductase activity in the nir1 mutant suggests a regulatory perturbation in the expression of the Narl gene.     

A study on nitrate reductase activity (NRA) of geophytes from Mediterranean environment

Nitrate reductase activity (NRA) in different compartments of 14 Mediterranean geophytes (bulbous, tuberous and rhizomatous) and actual mineral nitrogen (NO₃⁻ and NH₄⁺) in their soils were investigated. The nitrate reduction capacities of each species were determined as NRA per total plant material. Differences among compartments for NRA were significant in all species. The highest NRA was found in leaves of tuberous species (Anemone coronaria, Cyclamen coum) and of most bulbous species (Allium flavum, Allium guttatum, Bellevelia sarmatica, Galanthus plicatus, Leucojum aestivum, Ornithogalum nutans, Tulipa sylvestris). Therefore, in this group of species the contribution of the leaves to total plant NRA was the highest. The other bulbous species (Allium scorodoprasum, Crocus chrysanthus, Fritillaria bithynica, Muscari neglectum) and one rhizomatous taxon (Iris suaveolens) have a different NRA distribution within the plants. In these species the highest values of NRA were found in different organs. For example, in Allium scorodoprasum the highest NRA was in tunics, and in flowers in M. neglectum. Although leaves are the main compartments reducing nitrate in most of the studied geophytes, other compartments also contribute to total plant nitrate reduction.Our results show that the nitrate reduction capacity is different among geophyte species. Even if it roughly reflects the nitrogen supply in a habitat, differences in nitrate reduction capacities of different species collected from same sites indicate that the nitrate reducing capacity is species-specific.     

Some effects of nitrate abundance and starvation on metabolism and accumulation of nitrogen in barley (Hordeum vulgare L. cv Sonja)

Nitrate and nitrite reductases were both induced by adding three concentrations of nitrate to the nutrient supply of nitrate-starved barley seedlings. Enzyme induction was not proportional to the amount of nitrate introduced. Glutamine synthetase also increased above a high endogenous activity but the increase did not differ significantly between any of the three nitrate treatments. Nitrate accumulated rapidly in leaves of plants given 4.0 mM or 0.5 mM nitrate but not with 0.1 mM nitrate. In all treatments, amino acids in leaves increased for 2 d, chiefly attributable to glutamine, then declined. Transferring plants from the three nitrate treatments to nitrate-free nutrient produced an immediate decline in nitrate reductase but nitrite reductase continued to increase for 2 d, before declining. Glutamine-synthetase activity was not affected by withdrawal of nitrate, nor did nitrate withdrawal retard plant growth during the 9-d period of the experiment. The disparity between accumulated nitrate and nitrate-reducing capacity and the rapid decrease in leaf nitrate when nutrient nitrate supply was removed, indicated the presence of a nitrate-storage pool that could be called upon to maintain amino-acid production in times of nitrogen starvation.Abbreviations GS glutamine synthetase - NR nitrate reductase - NiR nitrite reductase     

Nitrate reductase and nitrite reductase activity in free-living cells and bacteroids of Rhizobium loti

Cells of Rhizobium loti strains T1 and U226 cultured in defined medium with glutamate as the only nitrogen source and bacteroids isolated from root nodules of Lotus corniculatus, L. pedunculatus and L. tenuis did not show constitutive (non-nitrate induced) nitrate reductase activity (NRA). In contrast, nitrite reductase activity (NiRA) was present in both free-living cells and bacteroids of either strain T1 or U226. Constitutive NRA and NiRA were detected in the cytosol fraction from nodules of all three symbioses examined. An induced NRA was expressed in bacteroids after a 10 h incubation in the presence of nitrate.     

Partitioning of inorganic nitrogen assimilation between the roots and shoots of cerrado and forest trees of contrasting plant communities of South East Brasil

Summary Woody plants growing in cerrado and forest communities of south-east Brasil were found to have low levels of nitrate reductase activity in their leaves suggesting that nitrate ions are not an important nitrogen source in these communities. Only in the leaves of species growing in areas of disturbance, such as gaps and forest margins, were high levels of nitrate reductase present. When pot-grown plants were supplied with nitrate, leaves and roots of almost all species responded by inducing increased levels of nitrate reductase. Pioneer or colonizing species exhibited highest levels of nitrate reductase and high shoot: root nitrate reductase activities. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase were present in leaves and roots of the species examined.¹⁵N-labelled nitrate and ammonium were used to compare the assimilatory characteristics of two species:Enterolobium contortisiliquum, with a high capacity to reduce nitrate, andCalophyllum brasiliense, of low capacity. The rate of nitrate assimilation in the former was five times that of the latter. Both species had similar rates of ammonium assimilation. Results for eight species of contrasting habitats showed that leaf nitrogen content increased in parallel with xylem sap nitrogen concentrations, suggesting that the ability of the root system to acquire, assimilate or export nitrate determines shoot nitrogen status. These results emphasise the importance of nitrogen transport and metabolism in roots as determinants of whole plant nitrogen status.     

Effect of two contrasted N fertilisations on rapeseed growth and nitrate metabolism

Winter oilseed rape was grown under two nitrogen fertilisation conditions. The N1-plants and N5-plants were respectively supplied with 4.5 g N per plant (N-limiting condition) and 22.5 g N per plant (non-N-limiting condition). Growth parameters and nitrate reducing capacity were monitored at five sampling stages interspersed with ammonium nitrate applications. N5-plants showed a higher growth rate producing more leaves and stems, early flower and silique formation and delayed leaf senescence. They also contained more nitrate and a higher nitrate reductase activity (NRA) especially in leaves which represented the main site of nitrate reduction before flowering. However, stems and siliques contributed to NRA especially in nitrogen-limited plants that lost their leaves early. This present study outlines the importance of siliques as individual sinks reducing nitrate essentially in the pod walls. The soluble protein content decreased in senescing leaves which was indicative of the reallocation of proteinic nitrogen towards stems and siliques. In non-limiting conditions, other nitrogen compounds of leaves may account for such a reallocation. Hence, the timing of leaf fall could contribute to the low nitrogen recovery in rapeseed.     

Nitrate concentration and nitrate reductase activity in the leaves of three legumes and three cereals*

Nitrate concentration and nitrate reductase activity (NRA) were studied in the leaves of soybean (Glycine max), groundnut (Arachis hypogaea and cowpea (Vigna unguiculata) and sorghum (Sorghum bicolor), pearl millet (Pennisetum americanum) and maize (Zea mays) at three nitrogen fertiliser levels in two field experiments. Higher nitrate concentrations were detected in the leaves of groundnut, cowpea and pearl millet than in sorghum and maize. Nitrate content in the leaves and leaf NRA were not related across crop species, nor was a generalised pattern of leaf NRA and leaf nitrate observed within legumes or within cereals. Nitrogen application resulted in higher nitrate availability in the leaves, with varied leaf NRA.     

Unusual regulatory nitrate reductase activity in cotyledons of Brassica napus seedlings: enhancement of nitrate reductase activity by ammonium supply

The effect of supplying either nitrate or ammonium on nitrate reductase activity (NRA) was investigated in Brassica napus seedlings. In roots, nitrate reductase activity (NRA) increased as a function of nitrate content in tissues and decreased when ammonium was the sole nitrogen source. Conversely, in the shoots (comprising the cotyledons and hypocotyl), NRA was shown to be independent of nitrate content. Moreover, when ammonium was supplied as the sole nitrogen source, NRA in the shoots was surprisingly higher than under nitrate supply and increased as a function of the tissue ammonium content. Under 15 mM of exogenous ammonium, the NRA was up to 2.5-fold higher than under nitrate supply after 6 d of culture. The NR mRNA accumulation under ammonium nutrition was 2-fold higher than under nitrate supply. The activation state of NR in shoots was especially high compared with roots: from nearly 80% under nitrate supply it reached 94% under ammonium. This high NR activation state under ammonium supply could be the consequence of the slight acidification observed in the shoot tissue. The effect of ammonium on NRA was only observed in cotyledons and when more than 3 mM ammonium was supplied. No such NRA increase was evident in the roots or in foliar discs. Addition of 1 mM nitrate under ammonium nutrition halved NRA and decreased the ammonium content in shoots. Thus, this unusual NRA was restricted to seedling cotyledons when nitrate was lacking in the nitrogen source.     

Nitrate assimilation in coexisting vascular plants in mire and swamp forest habitats in Central Sweden

Summary In order to monitor the nitrate assimilation capability of mire plants, in vivo current and maximally induced nitrate reductase activity (NRA) were investigated in 14 species of vascular plants from four different sites in a central Swedish mire. One of the sites was a swamp forest. The plants studied included species with both wide and restricted ecological ranges, and the mire sites selected covered a wide range of plant productivity. At the most productive site, current NRA differed among coexisting species. This differentiation in the use of nitrate as a source of nitrogen suggested the possibility of resource partitioning with regard to nitrogen acquisition. Maximally induced NRA, measured 3 days after an addition of nitrate, was highest at the most productive sites and differed among coexisting species. Plant species characteristic of rich fens had the highest maximally induced NRA. In all species, there was a positive correlation between the ability to assimilate peaks of available nitrate and total leaf nitrogen concentration.     

The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii

The regulation of the development of nitrate reductase (NR) activity in Chlamydomonas reinhardii has been compared in a wild-type strain and in a mutant (nit-A) which possesses a modified nitrate reductase enzyme that is non-functional in vivo. The modified enzyme cannot use NAD(P)H as an electron donor for nitrate reduction and it differs from wild-type enzyme in that NR activity is not inactivated in vitro by incubation with NAD(P)H and small quantities of cyanide; it is inactivated when reduced benzyl viologen or flavin mononucleotide is present. After short periods of nitrogen starvation mutant organisms contain much higher levels of terminal-NR activity than do similarly treated wild-type ones. Despite the inability of the mutant to utilize nitrate, no nitrate or nitrite was found in nitrogen-starved cultures; it is therefore concluded that the appearance of NR activity is not a consequence of nitrification. After prolonged nitrogen starvation (22 h) the NR level in the mutant is low. It increases rapidly if nitrate is then added and this increase in activity does not occur in the presence of ammonium, tungstate or cycloheximide. Disappearance of preformed NR activity is stimulated by addition of tungstate and even more by addition of ammonium. The results are interpreted as evidence for a continuous turnover of NR in cells of the mutant with ammonium both stimulating NR breakdown and stopping NR synthesis. Nitrate protects the enzyme from breakdown. Reversible inactivation of NR activity is thought to play an insignificant rôle in the mutant.Abbreviations NR nitrate reductase - BV benzyl viologen     

The effect of nitrogen refeeding on starved cells of Platymonas striata Butcher

A rapid uptake of nitrogen was observed in nitrogen-starved cells of Platymonas striata after refeeding with ammonium or nitrate ions. This was followed by a net loss of nitrogen per cell. Cells initially grown in and then starved in a regime of continuous light showed greater increases in average cell nitrogen on refeeding with ammonium or nitrate ions than did cells initially grown in and then starved in a regime of alternating light and darkness. A particulate subcellular location was observed for nitrate reductase (EC 1.6.6.1) in broken cell suspensions prepared by sonication. Nitrite reductase (EC 1.6.6.4) was located in the soluble fraction of these cell suspensions. Broken cell preparations displayed a lowered nitrate reductase activity as compared with the particulate component of these preparations. This was shown not to be due to heat-stable inhibitors present in the soluble phase of the cell. It appeared to be an artefact produced by the high nitrite reductase activity of the broken cell preparations, which removed much of the nitrite as it was formed. Nitrogen starvation of nitrate-grown cultures produced cellular increases in nitrate reductase and nitrite reductase activities which were further increased after the addition of nitrate. The results are discussed.Abbreviations ASP2 complete culture medium - ASP2 INF medium lacking in inorganic nitrogen - ASP2 NF medium lacking all nitrogen - NAR nitrate reductase - NIR nitrite reductase - EDTA Ethylenediaminetetracetic acid - PVP Polyvinylpyrollidone, M.W. 44,000     

Heterotrophic nitrification in a health soil demonstrated by anin situ method

Summary Nitrate reductase activity (NRA) in the leaves of two subspecies ofHypochaeris radicata was taken as a parameter for nitrate production in the soilin situ. Ammonium addition to the soil ofH. radicata ssp.radicata (soil pH 6.2) resulted in an increase of NRA, thus indicating nitrate formation by chemolithotrophic nitrifiers after a certain time-lag. Addition of ammonium to the soil ofH. radicata ssp.ericetorum (soil pH 4.3) dit not affect NRA in the leaves. Tests based on the MPN method failed to demonstrate the occurrence of chemolithotrophic nitrifiers in this soil. However, the addition of peptone led to an increase of NRA within seven days, which indicates the presence of heterotrophic nitrifying organisms. The results obtainedin situ were confirmed in a laboratory experiment, where soil samples were incubated in the presence and absence of (NH₄)₂SO₄ or peptone. The addition of ammonium led to a decrease in the production of nitrate to zero as compared with the control in the acid soil of ssp.ericetorum, whereas the addition of peptone resulted in nitrate levels amounting to about twice the control value. In the soil of ssp.radicata nitrate formation showed a rapid increase, compared with the control, after the addition of ammonium as well as after the addition of peptone.Grassland species research group, publication no27     

Responses of nitrogen metabolism in N-sufficient barley primary leaves to plant growth in elevated atmospheric carbon dioxide

Effects of atmospheric carbon dioxide enrichment on nitrogen metabolism were studied in barley primary leaves (Hordeum vulgare L. cv. Brant). Seedlings were grown in chambers under ambient (36 Pa) and elevated (100 Pa) carbon dioxide and were fertilized daily with complete nutrient solution providing 12 millimolar nitrate and 2.5 millimolar ammonium. Foliar nitrate and ammonium were 27% and 42% lower (P ≤ 0.01) in the elevated compared to ambient carbon dioxide treatments, respectively. Enhanced carbon dioxide affected leaf ammonium levels by inhibiting photorespiration. Diurnal variations of total nitrate were not observed in either treatment. Total and Mg²⁺inhibited nitrate reductase activities per gram fresh weight were slightly lower (P ≤ 0.01) in enhanced compared to ambient carbon dioxide between 8 and 15 DAS. Diurnal variations of total nitrate reductase activity in barley primary leaves were similar in either treatment except between 7 and 10 h of the photoperiod when enzyme activities were decreased (P ≤ 0.05) by carbon dioxide enrichment. Glutamate was similar and glutamine levels were increased by carbon dioxide enrichment between 8 and 13 DAS. However, both glutamate and glutamine were negatively impacted by elevated carbon dioxide when leaf yellowing was observed 15 and 17 DAS. The above findings showed that carbon dioxide enrichment produced only slight modifications in leaf nitrogen metabolism and that the chlorosis of barley primary leaves observed under enhanced carbon dioxide was probably not attributable to a nutritionally induced nitrogen limitation. This revised version was published online in June 2006 with corrections to the Cover Date.     

An evaluation of the stoichiometry ofin vitro nitrate assimilation inZea mays

Summary Reported non-stoichiometry in the reduction of nitrate to nitrite was investigated in leaves ofZea mays L.. The nitrogen balance sheet forin vitro nitrate assimilation was influenced by enzyme protectants in the extraction media and by the method employed to terminate the reaction. A number of limitations were found in the generally acceptedin vitro nitrate reductase assay, in particular the presence of endogenous components which interfered with the assay of nitrite were considered. A stoichiometric balance for thein vitro reduction of nitrate to nitrite was obtained when interfering factors were minimized. The absence of back reactions from ammonia in the assay was confirmed.     

Seasonal changes in nitrate use by three woody species: the importance of the leaf-expansion period

Seasonal changes in plant NO₃ ⁻-N use were investigated by measuring leaf nitrate reductase activity (NRA), leaf N concentration, and leaf expansion in one evergreen woody species (Quercus glauca Thunb.) and two deciduous woody species [Acer palmatum Thunb. and Zelkova serrata (Thunb.) Makino]. Leaf N concentration was highest at the beginning of leaf expansion and decreased during the expansion process to a steady state at the point of full leaf expansion in all species. The leaf NRA of all species was very low at the beginning of leaf expansion, followed by a rapid increase and subsequent decrease. The highest leaf NRA was observed in the middle of the leaf-expansion period, and the lowest leaf NRA occurred in summer for all species. Significant positive correlations were detected between leaf NRA and leaf expansion rates, while leaf N concentrations were negatively correlated with leaf area. In the evergreen Q. glauca, the N concentration in current buds increased before leaves opened; concurrently, the N concentration in 1-year-old leaves decreased by 25%. Our results show that the leaf-expansion period is the most important period for NO₃ ⁻-N assimilation by broadleaf tree species, and that decreases in leaf N concentration through the leaf-expansion period are at least partly compensated for by newly assimilated NO₃ ⁻-N in current leaves.     

The response ofPlantago lanceolata L. andP. major L. spp.major to nitrate depletion

To study aspects of the ecology of grassland species, in a comparative experiment, plants ofP. lanceolata andP. major were grown in pots in a greenhouse, and subjected to a gradual nitrate depletion for several weeks. Control plants were weekly supplied with nitrate. Growth, leaf appearance and disappearance, concentrations of cations and inorganic anions, soluble and insoluble reduced nitrogen concentrations,in vivo nitrate reductase activity (NRA) and the concentration of non-structural carbohydrates in several parts of the plants were followed. Depletion of nitrate caused a reduction of shoot growth, both in biomass and number of leaves. Withering of leaves increased. Accumulation of root dry matter was little (P. lanceolata), or not (P. major) affected. The concentration of reduced nitrogen in all tissues also decreased, both that of the soluble and that of the insoluble fraction. As a result, nitrogen use efficiency (NUE, g dry matter produced per mmol N incorporated) increased by nitrate depletion. NRA was higher in the roots than in the leaves, and decreased with increasing nitrate depletion. In control plants, nitrate became also limiting. This resulted in decreasing nitrate concentrations in leaves and roots. In the leaves, the decrease in nitrate concentration was preceded by a decrease in NRA. The decrease of the nitrate concentration was parallelled by an increase in the concentration of soluble sugar. No major differences in the response towards nitrate depletion were observed between the two species. Grassland Species Research Group, publication no. 129