In vivo nitrate reduction in relation to nitrate uptake, nitrate content, and in vitro nitrate reductase activity in intact barley seedlings

A study was done to relate the in vivo reduction of nitrate to nitrate uptake, nitrate accumulation, and induction of nitrate reductase activity in intact barley seedlings (Hordeum vulgare L. var. `Numar'). The characteristics of nitrate uptake in response to both time and ambient concentration of nitrate regulated reduction and accumulation. Uptake, accumulation, and in vivo reduction achieved steady state rates in 3 to 4 hours, whereas extractable (in vitro) nitrate reductase activity was still increasing at 12 hours. In vivo reduction of nitrate was better correlated exponentially than linearly over time with in vitro activity of nitrate reductase. A similar relationship occurred over increasing concentration of nitrate in the ambient solution. The results suggest that the rate of in vivo reduction of nitrate in barley seedlings may be regulated by the rate of uptake at the ambient concentrations of nitrate employed in the study.     

Studies of the uptake of nitrate in barley : v. Estimation of root cytoplasmic nitrate concentration using nitrate reductase activity-implications for nitrate influx

The cytoplasmic NO₃⁻ concentration ([NO₃⁻]_c) was estimated for roots of barley (Hordeum vulgare L. cv Klondike) using a technique based on measurement of in vivo nitrate reductase activity. At zero external NO₃⁻ concentration ([NO₃⁻]_o), [NO₃⁻]_c was estimated to be 0.66 mm for plants previously grown in 100 μm NO₃⁻. It increased linearly with [NO₃⁻]_o between 2 and 20 mm, up to 3.9 mm at 20 mm [NO₃⁻]_o. The values obtained are much lower than previous estimates from compartmental analysis of barley roots. These observations support the suggestion (MY Siddiqi, ADM Glass, TJ Ruth [1991] J Exp Bot 42: 1455-1463) that the nitrate reductase-based technique and compartmental analysis determine [NO₃⁻]_c for two separate pools; an active, nitrate reductase-containing pool (possibly located in the epidermal cells) and a larger, slowly metabolized storage pool (possibly in the cortical cells), respectively. Given the values obtained for [NO₃⁻]_c and cell membrane potentials of −200 to −300 mV (ADM Glass, JE Schaff, LV Kochian [1992] Plant Physiol 99: 456-463), it is very unlikely that passive influx of NO₃⁻ is possible via the high-concentration, low-affinity transport system for NO₃⁻. This conclusion is consistent with the suggestion by Glass et al. that this system is thermodynamically active and capable of transporting NO₃⁻ against its electrochemical potential gradient.     

Fate of nitrate during initial nitrate utilization by nitrogen-depleted dwarf bean

The fate of nitrate and nitrogen-15 was followed during the apparent induction phase (6h) for nitrate uptake by N-depleted dwarf bean (Phaseolus vulgaris L. ev. Witte Krombek). Experiments were done with intact plants and with detached root systems. Qualitatively and quantitatively, xylem exudation from detached roots was a bad estimate of the export of NO^?₃ or NO^?₃-¹⁵N from roots of intact plants. In vivo nitrate reductase activity (NRA) agreed well with in situ reduction, calculated as the difference between uptake and accumulation in whole plants, provided NRA was assayed with merely endogenous nitrate as substrate (‘actual’ NRA). The majority (75%) of the entering nitrate remained unmetabolized. Both nitrate reduction and nitrate accumulation occurred predominantly in the root system. Some (< 25%) of the root-reduced nitrate-N was translocated to the shoot. Nitrate uptake occurred against the concentration gradient between medium and root cells, and probably against the gradient of the electro-chemical potential of nitrate. Part of the energy expended for NO^?₃ absorption came from the tops, since decapitation and ringing at the stem base restricted nitrate uptake.     

Competition for nitrate between denitrifying Pseudomonas stutzeri and nitrate ammonifying enterobacteria

Abstract Competition for nitrate between nitrate ammonifying enterobacteria and a denitrifying pseudomonad was studied in electron acceptor-limited chenostats. In pure cultures, using different carbon and energy sources, the C/N-ratio needed for denitrification is far lower than that required for nitrate ammonification. In mixed cultures of Citrobacter freundii and Pseudomonas stutzeri , competing for nitrate with l -lactate as electron donor, the nitrate ammonifying organism dominated at dilution rates of D ≤ 0.14 h⁻¹. Competition for both nitrate and l -lactate at a dilution rate of D = 0.05 h⁻¹ always resulted in the coexistence of both species. Using glucose as additional carbon source, the final ratio of nitrate ammonifying and denitrifying organism depended on the C/N-ratio as well as on the dilution rate. The results of the study are discussed with respect to field data.     

Endogenous nitrate loss as an assay for nitrate reduction in vivo

An in vivo assay method for nitrate reduction is proposed, based on the use of endogenous nitrate rather than on the accumulation of nitrite. Loss of endogenous nitrate and accumulation of nitrite were studied in barley (Hordeum vulgare L. cv. Gars Clipper ex Napier) leaves. Leaf sections were incubated in the dark in a gaseous environment of air or N₂. Nitrate disappeared under both conditions, the highest loss being observed in tissue under anaerobiosis. Nitrite accumulated only in leaf sections under anaerobiosis, but the amount of nitrite accumulated was much lower than the amount of nitrate lost. A comparative study of the capacity of barley leaf sections to use endogenous nitrate and accumulate nitrite showed that both activities were dependent on temperature in a manner characteristic of enzymatic reactions. Disappearance of endogenous nitrate increased with increasing levels of nitrate in the tissue.     

The Arabidopsis ATNRT2.7 nitrate transporter controls nitrate content in seeds

In higher plants, nitrate is taken up by root cells where Arabidopsis thaliana NITRATE TRANSPORTER2.1 (ATNRT2.1) chiefly acts as the high-affinity nitrate uptake system. Nitrate taken up by the roots can then be translocated from the root to the leaves and the seeds. In this work, the function of the ATNRT2.7 gene, one of the seven members of the NRT2 family in Arabidopsis, was investigated. High expression of the gene was detected in reproductive organs and peaked in dry seeds. beta-Glucuronidase or green fluorescent protein reporter gene expression driven by the ATNRT2.7 promoter confirmed this organ specificity. We assessed the capacity of ATNRT2.7 to transport nitrate in Xenopus laevis oocytes or when it is expressed ectopically in mutant plants deficient in nitrate transport. We measured the impact of an ATNRT2.7 mutation and found no difference from the wild type during vegetative development. By contrast, seed nitrate content was affected by overexpression of ATNRT2.7 or a mutation in the gene. Finally, we showed that this nitrate transporter protein was localized to the vacuolar membrane. Our results demonstrate that ATNRT2.7 plays a specific role in nitrate accumulation in the seed.     

Comparative effects of sodium nitrate and calcium nitrate on the potato

Summary Comparative trials with sodium nitrate and calcium nitrate on the potato indicate a certain superiority of the former, probably due to a greater utilization of potassium and hence improved carbohydrate metabolism.     

Bacterial nitrate reductases: Molecular and biological aspects of nitrate reduction

Nitrogen is a vital component in living organisms as it participates in the making of essential biomolecules such as proteins, nucleic acids, etc. In the biosphere, nitrogen cycles between the oxidation states +V and -III producing many species that constitute the biogeochemical cycle of nitrogen. All reductive branches of this cycle involve the conversion of nitrate to nitrite, which is catalyzed by the enzyme nitrate reductase. The characterization of nitrate reductases from prokaryotic organisms has allowed us to gain considerable information on the molecular basis of nitrate reduction. Prokaryotic nitrate reductases are mononuclear Mo-containing enzymes sub-grouped as respiratory nitrate reductases, periplasmic nitrate reductases and assimilatory nitrate reductases. We review here the biological and molecular properties of these three enzymes along with their gene organization and expression, which are necessary to understand the biological processes involved in nitrate reduction.     

Arabidopsis nitrate transporter NRT1.9 is important in phloem nitrate transport

This study of the Arabidopsis thaliana nitrate transporter NRT1.9 reveals an important function for a NRT1 family member in phloem nitrate transport. Functional analysis in Xenopus laevis oocytes showed that NRT1.9 is a low-affinity nitrate transporter. Green fluorescent protein and β-glucuronidase reporter analyses indicated that NRT1.9 is a plasma membrane transporter expressed in the companion cells of root phloem. In nrt1.9 mutants, nitrate content in root phloem exudates was decreased, and downward nitrate transport was reduced, suggesting that NRT1.9 may facilitate loading of nitrate into the root phloem and enhance downward nitrate transport in roots. Under high nitrate conditions, the nrt1.9 mutant showed enhanced root-to-shoot nitrate transport and plant growth. We conclude that phloem nitrate transport is facilitated by expression of NRT1.9 in root companion cells. In addition, enhanced root-to-shoot xylem transport of nitrate in nrt1.9 mutants points to a negative correlation between xylem and phloem nitrate transport.     

Orchid seed sensitivity to nitrate reflects habitat preferences and soil nitrate content

• Orchids are distributed around the world, however, the factors shaping their specific distribution and habitat preferences are largely unknown. Moreover, many orchids are at risk of becoming threatened as landscapes change, sometimes declining without apparent reason. One important factor affecting plant distribution is nutrient levels in the environment. Nitrates can inhibit not only orchid growth and persistence, but also seed germination.
• We used in vitro axenic cultures to exactly determine the germination sensitivity of seven orchid species to nitrates and correlated this with soil properties of the natural sites and with the species’ habitat preferences.
• We found high variation in response to nitrate between species. Orchids from oligotrophic habitats were highly sensitive, while orchids from more eutrophic habitats were almost insensitive. Sensitivity to nitrate was also associated with soil parameters that indicated a higher nitrification rate.
• Our results indicate that nitrate can affect orchid distribution via direct inhibition of seed germination. Nitrate levels in soils are increasing rapidly due to intensification of agricultural processes and concurrent soil pollution, and we propose this increase could cause a decline in some orchid species.

     

Effects of electron donors on nitrate removal by nitrate and nitrite reductases

Effects of artificial electron donors to deliver reducing power on enzymic denitrification were investigated using nitrate reductase and nitrite reductase obtained fromOchrobactrum antropi. The activity of nitrite reductase in the soluble portion was almost the same as that in the precipitated portion of the cell extract. Nitrate removal efficiency was higher with benzyl viologen than with methyl viologen or NADH as an artificial electron donor. The turn-over numbers of nitrate and nitrite reductase were 14.1 and 1.9 μmol of nitrogen reduced/min·mg cell extracts, respectively when benzyl viologen was used as an electron donor.     

Silver nitrate prophylaxis.

In many countries the statutory use of silver nitrate prophylaxis as soon as possible after birth has recently been reviewed from both a human rights and a medical standpoint. It has been argued that silver nitrate does not prevent all cases of gonococcal ophthalmia neonatorum (GON) and that it causes chemical conjunctivitis, pain and visual impairment, which may interfere with parent-infant bonding. Furthermore, the low incidence of GON, better methods of prenatal diagnosis, and the availability of suitable alternative prophylactic medication and of effective methods of treatment of GON have prompted recommendations that alternative prophylaxis be legally allowed or that mandatory prophylaxis be eliminated altogether. This paper reviews the situation and provides updated recommendations.     

Efficiency of nitrate uptake in spinach: impact of external nitrate concentration and relative growth rate on nitrate influx and efflux

Regulation of nitrate influx and efflux in spinach (Spinacia oleracea L., cv. Subito), was studied in short-term label experiments with 13N- and 15N-nitrate. Nitrate fluxes were examined in relation to the N demand for growth, defined as relative growth rate (RGR) times plant N concentration. Plants were grown at different nitrate concentrations (0.8 and 4 mM), with mineral composition of growth and uptake solutions identical. Nitrate influx, efflux and net nitrate uptake rate (NNUR) were independent of the external nitrate concentration, despite differences in internal nitrate concentration. At both N regimes, NNUR was adequate to meet the N demand for growth. RGR-related signals predominantly determined the nitrate fluxes. At high RGR (0.25 g g-1 day-1), nitrate influx was 20 to 40% lower and nitrate efflux was 50 to 70% lower than at lower RGR (0.17 g g-1 day-1); efflux:influx ratio (E:I) declined from 0.5 at low RGR to 0.2 at higher RGR. Thus, the efficiency of NNUR substantially increased with increasing RGR. Differences in nitrate translocation between morning and afternoon coincided with differences in nitrate efflux, which is in accordance with the suggested regulation of nitrate efflux by the root cytoplasmic nitrate concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrite uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.Abbreviations EDTA ethylenediaminetetraacetic acid - FAD flavine-adenine dinucleotide - IgG immunoglobulin G - NR nitrate reductase - PM plasma membrane - TX-100 Triton X-100     

Nitrate reductase and nitrate accumulation in relation to nitrate toxicity in Boronia megastigma

Moderate levels of N were toxic to the native Australian plant boronia (Boronia megastigma Nees). As NO^-₃ is the major N form available for plants under cultivated conditions, NO^-₃ reduction and accumulation patterns in boronia were examined following the supply of various levels of NO^-₃ to understand the physiological basis of this toxicity. At a low level of supplied NO^-₃ [15 mmol (plant)^-1], NO^-₃ was reduced without any detectable accumulation and without nitrate reductase activity (NRA) reaching its maximum capacity. When higher NO^-₃ levels [≥25 mmol (plant)^-1] were supplied, both NRA and NO^-₃ accumulation increased further. However, NRA increased to a maximum of ca 500 nmol NO^-₃ (g fresh weight)^-1 h^-1, both in the roots and leaves, irrespective of a 4-fold difference in the levels of supplied NO^-₃, whereas NO^-₃ continued to accumulate in proportion to the level of supplied NO^-₃. Chlorotic toxicity symptoms appeared on the leaves at an accumulation of ca 32 μmol NO^-₃ (g fresh weight)^-1. High endogenous NO^-₃ concentrations inhibited NRA. The low level of NRA in boronia was not limited by NO^-₃ or electron donor availability. It is concluded that the low NR enzyme activity is a genetic adaptation to the low NO^-₃ availability in the native soils of boronia. Thus, when NO^-₃ supply is high, the plat cannot reduce it at high rates, leading to large and toxic accumulations of the ion in the leaf tissues.     

Oxygen inhibition of nitrate uptake is a general regulatory mechanism in nitrate respiration

It has recently been demonstrated that oxygen inhibits nitrate uptake by denitrifying Pseudomonas aeruginosa. The purpose of the present investigation was to determine if this novel mechanism of regulation is universal for the regulation of nitrate respiration in other widely divergent species of bacteria. Nitrate transport by whole cell suspensions was completely and reversibly inhibited in 11 out of 12 species tested, whereas nitrate reduction by cell-free extracts was not affected by oxygen or was only partially inhibited in some cases. These results indicate that oxygen inhibition of nitrate uptake is a general regulatory phenomenon.     

Effect of nitrate on the synthesis and decay of nitrate reductase of Neurospora

1. A method was developed to examine the turnover of nitrate reductase by the use of tungstate. 2. Evidence is presented which suggests that the disappearance of nitrate reductase activity from Neurospora mycelia exposed to non-inducing conditions is due to the disappearance of the enzyme protein(s) from the mycelia, and not merely due to the disappearance of its (their) catalytic power. 3. The presence of NO(3) (-) in the culture medium slows down the rate of degradation of nitrate reductase in Neurospora in vivo.