Effects of constitutive expression of nitrate reductase in transgenic Nicotiana plumbaginifolia L. in response to varying nitrogen supply

Transformed Nicotiana plumbaginifolia plants with constitutive expression of nitrate reductase (NR) activity were grown at different levels of nitrogen nutrition. The gradients in foliar NO ₃ ^– content and maximum extractable NR activity observed with leaf order on the shoot, from base to apex, were much decreased as a result of N-deficiency in both the transformed plants and wild type controls grown under identical conditions. Constitutive expression of NR did not influence the foliar protein and chlorophyll contents under any circumstances. A reciprocal relationship between the observed maximal extractable NR activity of the leaves and their NO ₃ ^– content was observed in plants grown in nitrogen replete conditions at low irradiance (170 mol photons·m^–2 ·s^–1). This relationship disappeared at higher irradiance (450 mol photons·m^–2·S^–1) because the maximal extractable NR activity in the leaves of the wild type plants in these conditions increased to a level that was similar to, or greater than that found in constitutive NR-expressors. Much more NO ₃ ^– accumulated in the leaves of plants grown at 450 mol photons·m^–2·s^–1 than in those grown at 170 mol photons·m^–2·s^–1 in N-replete conditions. The foliar NO ₃ ^– level and maximal NR activity decreased with the imposition of N-deficiency in all plant types such that after prolonged exposure to nitrogen depletion very little NO ₃ ^– was found in the leaves and NR activity had decreased to almost zero. The activity of NR decreased under conditions of nitrogen deficiency. This regulation is multifactoral since there is no regulation of NR gene expression by NO ₃ ^– in the constitutive NR-expressors. We conclude that the NR protein is specifically targetted for destruction under nitrogen deficiency. Consequently, constitutive expression of NR activity does not benefit the plant in terms of increased biomass production in conditions of limiting nitrogen.Abbreviations Chl chlorophyll - N nitrogen - NR NADH-nitrate reductase - WT wild type     

Alteration in enrichment of NO 3 − and reduced-N in xylem exudate during and after extended plant exposure to15NO 3 −

Summary Distinctly different patterns of¹⁵N enrichment were observed in the nitrate and reduced-N fractions of xylem exudate from soybean plants during and after 5 to 6 days of exposure to¹⁵NO ₃ ^– . Within 1 d after changes in solution NO ₃ ^– label, more than 90% of the exudate NO ₃ ^– originated from the exogenous supply. Alterations in the enrichment of exudate reduced-N were much slower, however, and the enrichment reached only 40% even after 5 d of continuous exposure to¹⁵NO ₃ ^– . Taking into account possible reduction of endogenous NO ₃ ^– and delayed translocation of NO ₃ ^– reduction products, it was concluded that root reduction could have contributed only 30 to 42% of the reduced-N found in the exudate.     

The ability of several high arctic plant species to utilize nitrate nitrogen under field conditions

The ability to utilize NO _inf3 ^sup– in seven high arctic plant species from Truelove Lowland, Devon Island, Canada was investigated, using an in vivo assay of maximum potential nitrate reductase (NR) activity and applications of ¹⁵N. Plant species were selected on the basis of being characteristic of nutrient-poor and nutrient-rich habitats. In all species leaves were the dominant site of NR activity. Root NR activity was negligible in all species except Saxifraga cernua. NO _inf3 ^sup– availability per se did not appear to limit NR activity of the species typically found on nutrient-poor sites (Dryas integrifolia, Saxifraga oppositifolia, and Salix arctica), or in Cerastium alpinum, as leaf NR activities remained low, even after NO _inf3 ^sup– addition. ¹⁵NO _inf3 ^sup– uptake was limited in D. integrifolia and Salix arctica. However, the lack of field induction of NR activity in C. alpinum and Saxifraga oppositifolia was not due to restricted nitrate uptake, as ¹⁵NO _inf3 ^sup– labelled NO _inf3 ^sup– entered the roots and shoots of both species. Leaf NR activity rates were low in three of the species typical of nutrient-rich habitats (O. digyna, P. radicatum and Saxifraga cernua), sampled from a site containing low soil NO _inf3 ^sup– . Additions of NO _inf3 ^sup– significantly increased leaf NR activity in these latter species, suggesting that potential NR activity was limited by the availability of NO _inf3 ^sup– . ¹⁵N labelled NO _inf3 ^sup– was taken up by O. digyna. P. radicatum and Saxifraga cernua. Although two species (D. integrifolia and Salix arctica) showed little utilization of NO _inf3 ^sup– , we concluded that five of the seven selected high arctic plant species (C. alpinum, O. digyna, P. radicatum, Saxifraga cernua and Saxifraga oppositifolia) do have the potential to utilize NO _inf3 ^sup– as a nitrogen source under field conditions, with the highest potential to utilize NO _inf3 ^sup– occurring in three of the species typically found on fertile habitats.     

Effects of light quantity and quality and soil nitrogen status on nitrate reductase activity in rainforest species of the genus Piper

Summary We studied nitrate reductase (NR) activity in six species of the genus Piper (Piperaceae) growing under a broad range of light availabilities. Field measurements were made on plants growing naturally in rainforest at the Los Tuxtlas Tropical Biological Preserve, Veracruz, Mexico at high- and lowlight extremes for each species. Foliar nitrogen on an area basis was positively related to the average daily photosynthetically active photon flux density (PFD) received by the leaf (r=0.76, p<0.01). In vivo NR activity was highly correlated with PFD (r=0.95, p<0.001) and less so with total leaf nitrogen (r=0.68, p<0.05). In vivo NR activity was always higher in high-light plants than in low-light plants within a species. Similarly, gap species such as P. auritum had much higher in vivo NR activities than shade species such as P. aequale. Soil NO ₃ ^– and NH ₄ ⁺ pools and nitrogen-mineralization rates at Los Tuxtlas were similar between high- and low-light sites, indicating that the elevated NR activities in high-light plants were not the result of higher NO ₃ ^– availabilities in high-light microsites. We performed additional experiments at Stanford, California, USA on Piper plants grown at high- and low-light. Foliar NR was highly inducible by nitrate in the gap species (auritum) but not in the generalist (hispidum) or shade (aequale) species. Root NR activities were, in general, an order of magnitude lower than foliar activities. In total, these studies suggest that Piper gap species are inherently more competent to assimilate NO ₃ ^– and are better able to respond to sudden increases in NO ₃ ^– availability than are shade species.CJWDPB publication # 1097     

The impact of NO inf3 sup− loading on the freshwater macrophyte Littorella uniflora: N utilization strategy in a slow-growing species from oligotrophic habitats

The decline and disappearance of Littorella uniflora from oligotrophic waters which have become eutrophic has been associated with shading or reduced CO₂ supply. However NO _inf3 ^sup– concentrations can reach very high levels (100–2000 mmol m^–3 compared with <1–3 in oligotrophic habitats). To investigate the impact of NO _inf3 ^sup– loading alone, plants were grown under three NO _inf3 ^sup– regimes (very low, near-natural and high). The interactive effects of NO _inf3 ^sup– and photon flux density (low and high regimes) on N assimilation and accumulation, CO₂ concentrating mechanisms, C₃ photosynthesis and growth were also examined. The results were unexpected. Increased NO _inf3 ^sup– supply had very little effect on photosynthetic capacity, crassulacean acid metabolism (CAM) or lacunal CO₂ concentrations ([CO₂]_i), although there was considerable plasticity with respect to light regime. In contrast, increased NO _inf3 ^sup– supply resulted in a marked accumulation of NO _inf3 ^sup– , free amino acids and soluble protein in shoots and roots (up to 25 mol m^–3, 30 mol m^–3 and 9 mg g^–1 fresh weight respectively in roots), while fresh weight and relative growth rate were reduced. Total N content even under the very low NO _inf3 ^sup– regime (1.6–2.3%) was mid-range for aquatic and terrestrial species (and 3.1–4.3% under the high NO _inf3 ^sup– regime). These findings, together with field data, suggest that L. uniflora is not growth limited by low NO _inf3 ^sup– supply in natural oligotophic habitats, due not to an efficient photosynthetic nitrogen use but to a slow growth rate, a low N requirement and to the use of storage to avoid N stress. However the increased NO _inf3 ^sup– concentrations in eutrophic environments seem likely have detrimental effects on the long-term survival of L. uniflora, possibly as a consequence of N accumulation.     

Comparison of the growth rates of dinitrogen fixing subterranean clover swards with those assimilating nitrate ions

Summary Subterranean clover plants were grown as swards (about 2000 plants/m²) under controlled conditions with N provided either by N₂-fixation (NO ₃ ^– withheld) or by assimilation of NO ₃ ^– (NO ₃ ^– supplied). Crop growth rates were measured by dry matter sampling over periods of up to 70 days at PPFD values of 400–1000 mole quanta/m²/s. When NO ₃ ^– was supplied from sowing the swards grew more rapidly than when the swards were not supplied with NO ₃ ^– and plants had to establish an N₂-fixing apparatus. When inter-plant competition was reduced within the sward, a difference in growth rate in favour of NO ₃ ^– -fed plants continued for at least 50 days. When however, a closed canopy was allowed to form, the NO ₃ ^– -fed swards had more dry weight than the N₂-fed swards at the time of canopy closure but thereafter the two swards grew at similar rates at light flux densities of above about 800 mole quanta/m²/s. At light flux densities of about 400 mole quanta/m²/s N₂-fed swards had a growth rate 70–80% of that of NO ₃ ^– -fed plants. NO ₃ ^– -fed plants had a higher organic N content than did N₂-fed plants under all conditions.     

Induction of nitrate reductase, nitrite reductase, and glutamine synthetase isoforms in sunflower cotyledons as affected by nitrate, light, and plastid integrity

Summary We investigated the inducibility of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), and glutamine synthetase (GS; EC 6.3.1.2) isoforms in cotyledons of 7-day-old seedlings of sunflower (Helianthus annuus L.) in relation to light, nitrogen source (NO ₃ ^– , NO ₂ ^– or NH ₄ ⁺ ), and the involvement of plastids. Nitrate was absolutely (and specifically) required for NR induction, and stimulated more effectively than NO ₂ ^– or NH ₄ ⁺ the synthesis of NiR and chloroplastic GS (GS₂) over the constitutive levels present in N-free-grown seedlings. In vivo inhibition of NR activity by tungsten application to seedlings and measurements of tissue NO ₃ ^– concentration indicate that NO ₃ ^– -dependent enzyme induction is elicited by NO ₃ ^– per se and not by a product of its assimilatory reduction, e.g., NO ₂ ^– or NH ₄ ⁺ . In the presence of NO ₃ ^– , light remarkably enhanced the appearance of NR, NiR, and GS₂, while the activity of the cytosolic GS isoform (GS₁) was adversely affected. Cycloheximide suppressed much more efficiently than chloramphenicol the light- and NO ₃ ^– -dependent increase of GS₂ activity, indicating that sunflower chloroplastic GS is synthesized on cytoplasmic 80S ribosomes. When the plastids were damaged by photooxidation in cotyledons made carotenoid-free by application of norflurazon, the positive action of light and NO ₃ ^– on the appearance of NR, NiR, and GS₂ isoform was greatly abolished. Therefore, it is suggested that intact chloroplasts are required for the inductive effect of light and NO ₃ ^– and/or for the accumulation of newly formed enzymes in the organelle.Abbreviations CAP chloramphenicol - CHX cycloheximide - GS glutamine synthetase - GS₁ cytosolic GS - GS₂ plastidic (chloroplastic) GS - NF norflurazon - NiR nitrite reductase - NR nitrate reductase     

Nitrogen status of cotton subjected to two short term periods of waterlogging of varying severity using a sloping plot water-table facility

Summary Cotton is reported to be susceptible to waterlogging, and there is evidence that some of the symptoms shown by waterlogged plants are due to impaired uptake of nitrogen. To investigate this for cotton, the nitrogen nutrition of a field-grown crop was monitored when the plants were subjected to two short term periods of waterlogging of varying severity using a sloping plot water-table facility. Growth of severely waterlogged cotton decreased after 4 days in the first and second floodings, and these plants were wilted by the end of the first flooding but not the second. Waterlogging resulted in decreased concentrations of total-N and especially NO ₃ ^– –N in the petiole and lamina of the youngest fully-expanded leaf. Uptake of N by waterlogged plants occurred, but was not as great as for well-aerated plants. The nitrate reductase activity of leaves was much lower in waterlogged plants. Stumps of detopped waterlogged plants did not exude sylem sap at the end of the first flooding, suggesting impaired solute uptake due to damaged roots. However, xylem exudate was obtained from stumps of waterlogged plants at the end of the second flooding, indicating adaptive changes to the root systems of these plants. Although cotton is reported to reduce little NO ₃ ^– –N in its roots, analysis of xylem exudate showed that about half of the N exported by roots was as amino compounds. The concentration of amino compounds in xylem exudate from severely waterlogged plants was higher than in well-aerated plants. It was concluded that the growth reduction in waterlogged cotton was due partly to induced N-deficiency.     

Nitrate deposition in northern hardwood forests and the nitrogen metabolism of Acer saccharum marsh

It is generally assumed that plant assimilation constitutes the major sink for anthropogenic Nitrate NO ₃ ^– deposited in temperate forests because plant growth is usually limited by nitrogen (N) availability. Nevertheless, plants are known to vary widely in their capacity for NO ₃ ^– uptake and assimilation, and few studies have directly measured these parameters for overstory trees. Using a combination of field and greenhouse experiments, we studied the N nutrition of Acer saccharum Marsh. in four northern hardwood forests receiving experimental NO ₃ ^– additions equivalent to 30 kg N ha^–1 year^–1. We measured leaf and fine-root nitrate reductase activity (NRA) of overstory trees using an in vivo assay and used ¹⁵N to determine the kinetic parameters of NO ₃ ^– uptake by excised fine roots. In two greenhouse experiments, we measured leaf and root NRA in A. saccharum seedlings fertilized with 0–3.5 g NO ₃ ^– –N m^–2 and determined the kinetic parameters of NO ₃ ^– and NH ₄ ⁺ uptake in excised roots of seedlings. In both overstory trees and seedlings, rates of leaf and fine root NRA were substantially lower than previously reported rates for most woody plants and showed no response to NO ₃ ^– fertilization (range = non-detectable to 33 nmol NO ₂ ^– g^–1 h^–1). Maximal rates of NO ₃ ^– uptake in overstory trees also were low, ranging from 0.2 to 1.0 mol g^–1 h^–1. In seedlings, the mean V _max for NO ₃ ^– uptake in fine roots (1 mol g^–1 h^–1) was approximately 30 times lower than the V _max for NH ₄ ⁺ uptake (33 mol g^–1 h^–1). Our results suggest that A. saccharum satisfies its N demand through rapid NH ₄ ⁺ uptake and may have a limited capacity to serve as a direct sink for atmospheric additions of NO ₃ ^– .     

Nitrate and nitrite uptake and reduction by intact sunflower plants

Nitrogen-starved sunflower plants (Helianthus annuus L. cv. Peredovic) cannot absorb NO ₃ ^– or NO ₂ ^– upon initial exposure to these anions. Ability of the plants to take up NO ₃ ^– and NO ₂ ^– at high rates from the beginning was induced by a pretreatment with NO ₃ ^– . Nitrite also acted as inducer of the NO ₂ ^– -uptake system. The presence of cycloheximide during NO ₃ ^– -pretreatment prevented the subsequent uptake of NO ₃ ^– and NO ₂ ^– , indicating that both uptake systems are synthesized de novo when plants are exposed to NO ₃ ^– . Cycloheximide also suppressed nitrate-reductase (EC 1.6.6.1) and nitrite-reductase (EC 1.7.7.1) activities in the roots. The sulfhydryl-group reagent N-ethylmaleimide greatly inhibited the uptake of NO ₃ ^– and NO ₂ ^– . Likewise, N-ethylmaleimide promoted in vivo the inactivation of nitrate reductase without affecting nitrite-reductase activity. Rates of NO ₃ ^– and NO ₂ ^– uptake as a function of external anion concentration exhibited saturation kinetics. The calculated K_m values for NO ₃ ^– and NO ₂ ^– uptake were 45 and 23 M, respectively. Rates of NO ₃ ^– uptake were four to six times higher than NO ₃ ^– -reduction rates in roots. In contrast, NO ₂ ^– -uptake rates, found to be very similar to NO ₃ ^– -uptake rates, were much lower (about 30 times) than NO ₂ ^– -reduction rates. Removal of oxygen from the external solution drastically suppressed NO ₃ ^– and NO ₂ ^– uptake without affecting their reduction. Uptake and reduction were also differentially affected by pH. The results demonstrate that uptake of NO ₃ ^– and NO ₂ ^– into sunflower plants is mediated by energy-dependent inducible-transport systems distinguishable from the respective enzymatic reducing systems.Abbreviations CHI cycloheximide - NEM N-ethylmaleimide - NiR nitrite reductase - NR nitrate reductase - pHME p-hydroxymercuribenzoate This research was supported by grant PB86-0232 from the Dirección General de Investigatión Científica y Técnica (Spain). One of us (E.A.) thanks the Consejeria de Educación y Ciencia de la Junta de Andalucia for the tenure of a fellowship. We thank Miss G. Alcalá and Miss C. Santos for their valuable technical and secretarial assistance.     

Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion and NO emission from leaf and root tissue

In wild-type Nicotiana plumbaginifolia Viv. and other higher plants, nitrate reductase (NR) is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. This inactivation is caused by phosphorylation of a conserved regulatory serine residue, Ser 521 in tobacco, and interaction with divalent cations or polyamines, and 14-3-3 proteins. The physiological importance of the post-translational NR modulation is presently under investigation using a transgenic N. plumbaginifolia line. This line expresses a mutated tobacco NR where Ser 521 has been changed into aspartic acid (Asp) by site-directed mutagenesis, resulting in a permanently active NR enzyme [C. Lillo et al. (2003) Plant J 35:566–573]. When cut leaves or roots of this line (S₅₂₁) were placed in darkness in a buffer containing 50 mM KNO₃, nitrite was excreted from the tissue at rates of 0.08–0.2 mol (g FW)^–1 h^–1 for at least 5 h. For the control transgenic plant (C1), which had the regulatory serine of NR intact, nitrite excretion was low and halted completely after 1–3 h. Without nitrate in the buffer in which the tissue was immersed, nitrite excretion was also low for S₅₂₁, although 20–40 mol (g FW)^–1 nitrate was present inside the tissue. Apparently, stored nitrate was not readily available for reduction in darkness. Leaf tissue and root segments of S₅₂₁ also emitted much more nitric oxide (NO) than the control. Importantly, NO emission from leaf tissue of S₅₂₁ was higher in the dark than in the light, opposite to what was usually observed when post-translational NR modulation was operating.Abbreviations NR Nitrate reductase - NO Nitric oxide - Ser Serine - WT Wild type     

Nitrate removal in a first-order stream: reconciling laboratory and field measurements

A combination of laboratory and field experiments were carried out to evaluate nitrate(NO ₃ ^t- ) removal during stream transport in a first-order agricultural drainage stream. Intact stream sediment cores overlain with stream and NO ₃ ^– -amended stream water indicated NO ₃ ^– losses averaging 93 — 353 mg m^–2 day^–1, with NO ₃ ^– concentration exerting a primary control on loss rate. Isotopic data indicated enrichment of NO ₃ ^– - ¹⁵N over time as NO ₃ ^– concentrations decreased, indicating a denitrification loss. Field experiments were designed to evaluate dilution of streamwater with low-NO ₃ ^– groundwater in addition to other NO ₃ ^– removal processes during transport. A series of bromide tracer and NO ₃ ^– - addition experiments were carried out in the field; groundwater dilution dominated the downstream NO ₃ ^– concentration trends, accounting for all observed decreases in NO ₃ ^– concentration. Isotopic data did not point to denitrification downstream as a major NO ₃ ^– removal process. This apparent disparity between simulated laboratory and in-situ stream removal rates appears to be a function of the hydrological processes controlling exchanges between stream bottom sediments and the overlying water. These results suggest that caution must be exercised in extrapolating potentials for NO ₃ ^– removal measured in laboratory experiments to the field, as these rates could be overestimated in some watersheds.     

Nitrate induction in spruce: an approach using compartmental analysis

Using ¹³NO ₃ ^– -efflux analysis, the induction of nitrate uptake by externally supplied nitrate was monitored in roots of intact Picea glauca (Moench) Voss. seedlings over a 5-d period. In agreement with our earlier studies, efflux analysis revealed three compartments, which have been identified as surface adsorption, apparent free space, and cytoplasm. While induction of nitrate uptake was pronounced, NO ₃ ^– fluxes in induced plants were decidedly lower and the induction response was slower than in other species. Influx rose from 0.1 mol·g^–1·h^–1 (measured at 100 M [NO ₃ ^– _o) in uninduced plants to a maximum of 0.5 mol·g^–1h^–1 after 3 d of exposure to 100 M [NO ₃ ^– _o and declined to 0.3–0.4 mol·g^–1h^–1 at the end of the 5-d period. Efflux remained relatively constant around 0.02-0.04 mol·g^–1h^–1, but its percentage with respect to influx declined from initially high values (around 30%) to steady-state values of 4–7%. Cytoplasmic [NO ₃ ^– ] ranged from the low micromolar in uninduced plants to a maximum of 2 mM in plants fully induced at 100 M [NO ₃ ^– ]_o. In-vivo root nitrate reductase activity (NRA) was measured over the same time period, and was found to follow a similar pattern of induction as influx. The maximum response in NRA slightly preceded that of influx. It increased from 25 nmol·g^–1·h^–1 without prior exposure to NO ₃ ^– to peak values around 150 nmol· g^–1h^–1 after 2 d of exposure to 100 M [NO ₃ ^– ]_o. Subsequently, NRA declined by about 50%. The dynamics of flux partitioning to reduction, to the vacuole, the xylem, and to efflux during the induction process are discussed.The research was supported by an Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and by a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia campus for providing ¹³N, to Drs. R.D. Guy and S. Silim for providing plant material, and to Dr. M.Y. Wang, Mr. J. Bailey, Mr. J. Mehroke and Mr. J. Vidmar for essential assistance in experiments.     

Nitrate or ammonium nutrition in french bean

Summary Bean Plants were grown in a greenhouse in sand irrigated with nutrient solutions containing either 2 mM NO ₃ ^– or 2 mM NH ₄ ⁺ . After 45 days fresh weight of NH ₄ ⁺ plants was half that of NO ₃ ^– plants. Cation concentration in NH ₄ ⁺ plants was 30% less than in NO ₃ ^– plants. Amino acids (SER, ASN, GLN) accummulated 3 to 10 times more in NH ₄ ⁺ plants. The concentration of organic acids (malic, malonic, citric) was 10 to 30 times higher in NO ₃ ^– plants. The ATP-costings for the synthesis of amino acids and organic acids in NH ₄ ⁺ plants was half that of NO ₃ ^– ones: therefore it could not account for the reduction of growth in the ammonium-fed plants.     

Inhibition of N2 fixation by white clover (Trifolium repens L.) at low concentrations of NO inf3 sup− in flowing solution culture

The impact of sustained low external concentrations of NO ₃ ^– (0, 10, 100 and 1000 mmol m^–3) on plant growth and the relative acquisition of N through N₂ fixation and NO ₃ ^– uptake by established, nodulated white clover (Trifolium repens L. cv. Blanca) was studied over 28 days in flowing solution culture. Nitrogen fixation was measured by N difference and ¹⁵N dilution methods. Plants supplied with NO ₃ ^– achieved higher relative growth rates (% MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGabmiEayaara% aaaa!3702!\[\bar x\]=0.091 d^–1) compared with control plants dependent on N₂ fixation (0.073 d^–1). Nitrate plants showed progressive increases in shoot: root d.w. ratios from 4 to 6.5–7.6 between days 0–28, compared with 5.1 on day 28 for control plants. Increases in both nodule d.w. and numbers per plant were inhibited after day seven at all concentrations of NO ₃ ^– . The severity of inhibition of N₂ fixation increased with increasing NO ₃ ^– concentration and with time. The total amounts of N₂ fixed per plant between days 0–7 after supplying 10, 100 and 1000 mmol m^–3 NO ₃ ^– , respectively, were 37–39, 28–30 and 0–13%, of the total N acquired. Between days 7–28 the proportional contributions of N₂ fixation to total N acquisition declined to 3, 0.5 and 0%, respectively, in these treatments. The corresponding mean specific rates of N₂ fixation between days 0–7 were, respectively, 5.4, 3.2, and 2.0 mmol N d^–1 g^–1 nodule d.w., compared with 7.9 mmol N d^–1 g^–1 nodule d.w. for zero NO ₃ ^– plants. There was no evidence of a transitory increase in N₂ fixation following the addition of NO ₃ ^– , even at the lowest supply concentration.     

Temperature dependence of inorganic nitrogen uptake and assimilation in Antarctic sea-ice microalgae

Summary The influence of temperature on NO ₃ ^- and NH ₄ ⁺ uptake, and the activity of the assimilatory enzyme NO ₃ ^- reductase (NR) was compared to inorganic C uptake (photosynthesis) in natural assemblages of Antarctic sea-ice microalgae. NO ₃ ^- and NH ₄ ⁺ uptake reached a maximum between 0.5°–2.0°C and 2.0°–3.0°C, respectively, which was close to that for photosynthesis (2.5°–3.0°C). NR showed a distinctly higher temperature maximum (10.0°–12.0°C) and a lower Q₁₀ value than inorganic N and C transport. Our data imply that, owing to differential temperature characteristics between N transport and N assimilation at in situ temperature (-1.9°C), the incorporation of extracellular NO ₃ ^- into cellular macromolecules, may be limited by transport of NO ₃ ^- into the cell rather than the intracellular reduction of NO ₃ ^- to NH ₄ ⁺ . Despite differences in temperature maxima between N transport and N assimilation, the overall low temperature maxima of inorganic N metabolism characterizes Antarctic sea-ice microalgae as psychrophilic. Our study is the first to examine the temperature dependence of inorganic N uptake and assimilation in sea-ice microbial communities.     

Short-term studies of NO 3 − uptake in Pisum using 13NO 3 −

Influx, efflux and net uptake of NO ₃ ^– was studied in Pisum sativum L. cv. Marma in short-term experiments where ¹³NO ₃ ^– was used to trace influx. The influx rate in N-limited plants was similar both during net uptake at external concentrations of around 50 M, and at low external NO ₃ ^– concentrations (4–6 M) when net uptake was practically zero. Efflux could be inferred from discrepancies between influx and net uptake but was never very high in the N-limited plants during net uptake. Close to the threshold concentration for not NO ₃ ^– uptake, efflux was high and equalled influx. Thus, the threshold concentration can be regarded as a NO ₃ ^– compensation point. The inclusion of NH ₄ ⁺ in the outer medium decreased influx by about 40% but did not significantly affect efflux. The roles of NO ₃ ^– fluxes and nitrate-reductase activity in regulating/limiting NO ₃ ^– utilization are discussed.Abbreviations DW dry weight - FW fresh weight - R_N relative nitrogen addition rate     

Nitrate use by tobacco cells in response to N-stress and ammonium nutrition

Summary Characterization of NO ₃ ^– use by suspension cultured tobacco cells during a culture cycle is needed to take advantage of cell cultures for further study of the biochemical regulation of NO ₃ ^– uptake induction and decay processes. Tobacco (Nicotiana tabacum L., cv. Ky14) cells were cultured with media containing different N sources. Cells cultured with a mixture of NO ₃ ^– and NH ₄ ⁺ (40 mM NO ₃ ^– plus 20 mM NH ₄ ⁺ , in Murashige and Skoog media) initially grew slightly faster but attained the same maximum cell culture density as those cultured with 40 mM NO ₃ ^– only. Cells subcultured with N-free media grew at a similar rate for the first 3 d as those cells grown with N, then ceased further growth. The cessation of growth of cells subcultured with N-free media coincided with depletion of cell NO ₃ ^– . The NO ₃ ^– influx of cells subcultured with N-free media increased eleven-fold and those grown with N increased four- to five-fold before declining. Maximal NO ₃ ^– influx rates occurred at the onset of the stationary growth phase for N-stressed cells, while cells grown with N reached maximums prior to the stationary phase of cell growth. Cells grown with a mixture of NO ₃ ^– and NH ₄ ⁺ had lower NO ₃ ^– reductase (NR) activity and higher cell NO ₃ ^– levels than those of cells grown with NO ₃ ^– only. The NR activity of cells subcultured with N-free media peaked within 1 d after subculture before declining to a constitutive level when cell NO ₃ ^– was depleted. The level of cell NO ₃ ^– plays a critical role in the expression of the NO ₃ ^– uptake and reduction processes. The transitions in the expression of NO ₃ ^– uptake and reduction activities of tobacco cell suspension cultures should prove valuable for further study of the biochemical and molecular basis for the regulation of these processes.Abbreviations DTT DL-dithiothreitol - EDTA ethylenediamine tetraacetate - FW fresh weight - MS media Murashige & Skoog media - NADH ß-nicotinamide adenine dinucleotide reduced form - PMSF phenylmethyl-sulfonyl fluoride     

Nitrate (15NO3) limitation affects nitrogen partitioning between metabolic and storage sinks and nitrogen reserve accumulation in chicory (Cichorium intybus L.)

In chicory, we examined how NO₃ ⁻ supply affected NO₃ ⁻ uptake, N partitioning between shoot and root and N accumulation in the tuberized root throughout the vegetative period. Plants were grown at two NO₃ ⁻ concentrations: 0.6 and 3 mM. We used ¹⁵N-labelling/chase experiments for the quantification of N fluxes between shoot and root and for determining whether N stored in the tuberized root originates from N remobilized from the shoot or from recently absorbed NO₃ ⁻. The rate of ¹⁵NO₃ ⁻ uptake was decreased by low NO₃ ⁻ availability at all stages of growth. In young plants (10–55 days after sowing; DAS), in both NO₃ ⁻ treatments the leaves were the strongest sink for ¹⁵N. In mature (tuberizing) plants, (55–115 DAS), the rate of ¹⁵NO₃ ⁻ uptake increased as well as the amount of exogenous N allocated to the root. In N-limited plants, N allocation to the tuberized root relied essentially on recent N absorption, while in N-replete plants, N remobilized from the shoot contributed more to N-reserve accumulation in the root. In senescing plants (115–170 DAS) the rate of ¹⁵NO₃ ⁻ uptake decreased mainly in N-replete plants whereas it remained almost unchanged in N-limited plants. In both NO₃ ⁻ treatments the tuberized root was the strongest sink for recently absorbed N. Remobilization of previously absorbed N from shoot to tuberized root increased greatly in N-limited plants, whereas it increased slightly in N-replete plants. As a consequence, accumulation of the N-storage compounds vegetative storage protein (VSP) and arginine was delayed until later in the vegetative period in N-limited plants. Our results show that although the dynamics of N storage was affected by NO₃ ⁻ supply, the final content of total N, VSP and arginine in roots was almost the same in N-limited and N-replete plants. This indicates that chicory is able to build up a store of available N-reserves, even when plants are grown on low N. We also suggest that in tuberized roots there is a maximal capacity for N accumulation, which was reached earlier (soon after 100 DAS) in N-replete plants. This hypothesis is supported by the fact that in N-replete plants despite NO₃ ⁻ availability, N accumulation ceased and significant amounts of N were lost due to N efflux. Received: 14 October 1996 / Accepted: 4 February 1997     

Impact of nitrogen form on iron uptake and distribution in maize seedlings in solution culture

Comparative studies on the effect of nitrogen (N) form on iron (Fe) uptake and distribution in maize (Zea mays L. cv Yellow 417) were carried out through three related experiments with different pretreatments. Experiment 1: plants were precultured in nutrient solution with 1.0×10^–4 M FeEDTA for 6 d and then exposed to NO₃–N or NH₄–N solution with 1.0×10^–4 M FeEDTA or without for 7 d. Experiment 2: plants were precultured with ⁵⁹FeEDTA for 6 d and were then transferred to the solution with different N forms, and 0 and 1.0×10^–4 M FeEDTA for 8 d. Experiment 3: half of roots were supplied with ⁵⁹FeEDTA for 5 d and then cut off, with further culturing in treatment concentrations for 7 d. In comparison to the NH₄-fed plants, young leaves of the NO₃-fed plants showed severe chlorosis under Fe deficiency. Nitrate supply caused Fe accumulation in roots, while NH₄–N supply resulted in a higher Fe concentration in young leaves and a lower Fe concentration in roots. HCl-extractable (active) Fe was a good indicator reflecting Fe nutrition status in maize plants. Compared with NO₃-fed plants, a higher proportion of ⁵⁹Fe was observed in young leaves of the Fe-deficient plants fed with NH₄–N. Ammonium supply greatly improved ⁵⁹Fe retranslocation from primary leaves and stem to young leaves. Under Fe deficiency, about 25% of Fe in primary leaves of the NH₄-fed plants was mobilized and retranslocated to young leaves. Exogenous Fe supply decreased the efficiency of such ⁵⁹Fe retranslocation. The results suggest that Fe can be remobilized from old to young tissues in maize plants but the remobilization depends on the form of N supply as well as supply of exogenous Fe.