In-situ localization of nitrate reductase in maize roots

The localization of nitrate reductase (NR; EC 1.6.6.2) in cells of root tissues ofZea mays L. (W64A W182L) was determined using post-embedding immunogold labeling at the electron-microscopy level and using silver enhancement of the colloidal-gold signal for light microscopy. Nitrate reductase is located in the cytoplasm of root epidermal and cortical cells, and in the cells of the parenchyma and pericycle within the vascular cylinder. A weaker signal was also obtained in parenchymal cells of the pith lying next to the xylem. A positive signal for NR protein was seen in the chloroplast fraction of maize leaves and in the plastid fraction of roots. This signal was lost when affinity-purified antibodies were used. Sections of Lowicryl-embedded tissue were found to be suitable for the localization of the non-abundant NR protein when adequate controls and signal-enhancement procedures were used.Abbreviations IgG immunoglobulin G - NR nitrate reductase - PEPCase phosphoenolpyruvate carboxylase This research was funded by Natural Sciences and Engineering Research Council (NSERC) of Canada grants ISE0125461 (AO), OGP0106265 (JSG) and an NSERC Visiting Scientist Award to E.F.     

Correlation between nitrate uptake rate and nitrate inhibition of nitrogenase activity in Azotobacter chroococcum

In Azotobacter chroococcum cells exhibiting both nitrate (nitrite) assimilation ability and nitrogen fixation capability, the extent of nitrogenase activity inhibition by nitrate or nitrite positively correlated (r = 0.922) with the rate of nitrate (nitrite) taken up by the cells. These results corroborate our previous proposal that the anion must be assimilated to exert its inhibitory effect, and indicate that the inhibition is a graded rather than an all-or-none process.     

Plant nitrogen availability and crosstalk with phytohormones signallings and their biotechnology breeding application in crops

Nitrogen (N), one of the most important nutrients, limits plant growth and crop yields in sustainable agriculture system, in which phytohormones are known to play essential roles in N availability. Hence, it is not surprising that massive studies about the crosstalk between N and phytohormones have been constantly emerging. In this review, with the intellectual landscape of N and phytohormones crosstalk provided by the bibliometric analysis, we trace the research story of best-known crosstalk between N and various phytohormones over the last 20 years. Then, we discuss how N regulates various phytohormones biosynthesis and transport in plants. In reverse, we also summarize how phytohormones signallings modulate root system architecture (RSA) in response to N availability. Besides, we expand to outline how phytohormones signallings regulate uptake, transport, and assimilation of N in plants. Further, we conclude advanced biotechnology strategies, explain their application, and provide potential phytohormones-regulated N use efficiency (NUE) targets in crops. Collectively, this review provides not only a better understanding on the recent progress of crosstalk between N and phytohormones, but also targeted strategies for improvement of NUE to increase crop yields in future biotechnology breeding of crops.     

Characterisation of the glnK-amtB operon and the involvement of AmtB in methylammonium uptake in Azorhizobium caulinodans

This work reports the characterisation of the Azorhizobium caulinodans amtB gene, the deduced protein sequence of which shares similarity to those of several ammonium transporters. amtB is located downstream from glnK, a glnB-like gene. It is cotranscribed with glnK from an NtrC- and σ⁵⁴-dependent promoter. glnK and amtB insertion mutant strains have been isolated. Methylammonium uptake was assayed in these strains and in other mutant strains in which the regulation of nitrogen metabolism is impaired. Our data suggest that the AmtB protein is an ammonium transporter, which is mainly regulated by NtrC in response to nitrogen availability. Received: 2 February 1998 / Accepted: 20 March 1998     

The effect of endogenous and externally supplied nitrate on nitrate uptake and reduction in sugarbeet seedlings

The pericarp of the dormant sugarbeet fruit acts as a storage reservoir for nitrate, ammonium and -amino-N. These N-reserves enable an autonomous development of the seedling for 8–10 d after imbibition. The nitrate content of the seed (1% of the whole fruit) probably induces nitrate-reductase activity in the embryo enclosed in the pericarp. Nitrate that leaks out of the pericarp is reabsorbed by the emerging radicle. Seedlings germinated from seeds (pericarp was removed) without external N-supply are able to take up nitrate immediately upon exposure via a low-capacity uptake system (v_max = 0.8 mol NO ₃ ^- ·(g root FW)^–1·h^–1; K_s = 0.12 mM). We assume that this uptake system is induced by the seed nitrate (10 nmol/seed) during germination. Induction of a high-capacity nitrate-uptake system (v_max = 3.4 mol NO ₃ ^- ·(g root FW)^–1·h^–1; K_s = 0.08 mM) by externally supplied nitrate occurs after a 20-min lag and requires protein synthesis. Seedlings germinated from whole fruits absorb nitrate via a highcapacity uptake mechanism induced by the pericarp nitrate (748 nmol/pericarp) during germination. The uptake rates of the high-capacity system depend only on the actual nitrate concentration of the uptake medium and not on prior nitrate pretreatments. Nitrate deprivation results in a decline of the nitrate-uptake capacity (t_1/2 of v_max = 5 d) probably caused by the decay of carrier molecules. Small differences in K_s but significant differences in v_max indicate that the low- and high-capacity nitrate-uptake systems differ only in the number of identical carrier molecules.Abbreviations NR nitrate reductase - pFPA para-fluorophenylalanine This work was supported by a grant from Bundesministerium für Forschung und Technologie and by Kleinwanzlebener Saatzucht AG, Einbeck.     

Regulation of mineral nitrogen uptake in plants

In the biosphere plants are exposed to different forms of N, which comprise mineral and organic N forms in soils as well as gaseous NH3, NOx, and molecular N2 in the atmosphere. The form of N uptake is mainly determined by its abundance and accessibility, which make and the most important N forms for plant nutrition under agricultural conditions. With minor importance, the form of N uptake is also subject to plant preferences, by which plants maintain their cation/anion balance during uptake. However, some species seem to have an obligatory preference which even prevents their growth on certain other N sources. In general, uptake of a certain N form closely matches the growth-related demand of the plant, at least when N transport to the root surface is not limiting. In addition, many plants accumulate large pools of N during vegetative growth which are remobilized in the generative stage. As a consequence, systems responsible for N transport need to be tightly regulated in their expression and activity upon sensing N availability and plant demand. Employing the tools of molecular genetics, the first plant genes encoding transporters for inorganic N have recently been isolated and characterized. These data can now complete the wealth of physiological and nutritional studies on N uptake. The present article will focus on the uptake of and into root cells and tries to link data derived from physiological, genetic and molecular studies.     

Nitrite reduction and carbohydrate metabolism in plastids purified from roots of Pisum sativum L.

Nitrate reduction in roots and shoots and exchange of reduced N between organs were quantitatively estimated in intact 13-d-old seedlings of two-row barley (Hordeum vulgare L. cv. Daisengold) using the ¹⁵N-incorporation model (A. Gojon et al. (1986) Plant Physiol. 82, 254–260), except that NH ₊ ⁴ was replaced by NO _- ² . N-depleted seedlings were exposed to media containing both nitrate (1.8 mM) and nitrite (0.2 mM) under a light-dark cycle of 12:12 h at 20°C; the media contained different amounts of ¹⁵N labeling. Experiments were started either immediately after the beginning (expt. 1) or immediately prior to the end (expt. 2) of the light period, and plants were sampled subsequently at each light-dark transition throughout 36 h. The plants effectively utilized ¹⁵NO _- ³ and accumulated it as reduced ¹⁵N, predominantly in the shoots. Accumulation of reduced ¹⁵N in both experiments was nearly the same at the end of the experiment but the accumulation pattern in roots and shoots during each 12-h period differed greatly depending on time and the light conditions. In expt. 1, the roots accounted for 31% (light), 58% (dark), and 9% (light) of nitrate reduction by the whole plants, while in expt. 2 the contributions of the root were 82% (dark), 20% (light), and 29% (dark), during each of the three 12-h periods. Xylem transport of nitrate drastically decreased in the dark, but that of reduced N rather increased. The downward translocation of reduced ¹⁵N increased while nitrate reduction in the root decreased, whereas upward translocation decreased while nitrate reduction in the shoot increased. We conclude that the cycling of reduced N through the plant is important for N feeding of each organ, and that the transport system of reduced N by way of xylem and phloem, as well as nitrate reduction by root and shoot, can be modulated in response to the relative magnitude of reduced-N demands by the root and shoot, with the one or the other predominating under different circumstances.Symbols Anl accumulation of reduced ¹⁵N from ¹⁵NO _- ³ in ¹⁴NO _- ³ -fed roots of divided root system - Ar accumulation in root of reduced ¹⁵N from ¹⁵NO _- ³ - As accumulation in shoot of reduced ¹⁵N from ¹⁵NO _- ³ - Rr ¹⁵NO _- ³ reduction in root - Rs ¹⁵NO _- ³ reduction in shoot - Tp translocation to root of shoot-reduced ¹⁵N from ¹⁵NO _- ³ in phloem - Tx translocation to shoot of root-reduced ¹⁵N from ¹⁵NO _- ³ in xylem     

Effect of L-methionine-DL-sulphoximine, a specific inhibitor of glutamine synthetase, on ammonium and nitrate metabolism in the unicellular alga Cyanidium caldarium

In the unicellular alga Cyanidium caldarium nitrate utilization is strongly inhibited by ammonium and it is resumed when ammonium has been depleted. In the presence of L-methionine-DL-sulphoximine (MSX), which prevents ammonium assimilation through a specific irreversible inhibition of glutamine synthetase, nitrate reduction is no longer inhibited by ammonium, and most of the ammonium derived from nitrate reduction is excreted into the external medium. However, in the presence of MSX, nitrate reduction to ammonium proceeds at a reduced rate (45 to 70% of the control); this is particularly marked at low nitrate concentration. It is hypothesized that either MSX or accumulating ammonium bring about decrease in the rate of nitrate entry into the cell.     

Molecular mechanisms of urea transport in plants

Urea is a soil nitrogen form available to plant roots and a secondary nitrogen metabolite liberated in plant cells. Based on growth complementation of yeast mutants and “in-silico analysis”, two plant families have been identified and partially characterized that mediate membrane transport of urea in heterologous expression systems. AtDUR3 is a single Arabidopsis gene belonging to the sodium solute symporter family that cotransports urea with protons at high affinity, while members of the tonoplast intrinsic protein (TIP) subfamily of aquaporins transport urea in a channel-like manner. The following review summarizes current knowledge on the membrane localization, energetization and regulation of these two types of urea transporters and discusses their possible physiological roles in planta.     

Effects of rhizospheric bicarbonate on net nitrate uptake and partitioning between the main nitrate utilising processes in Populus canescens and Sambucus nigra

Increased levels of rhizospheric dissolved inorganic carbon have repeatedly been demonstrated to enhance plant growth by up to 80%, although carbon from dark fixation accounts for only 1–3% of total plant carbon gain. This study, therefore, aimed at investigating the effects of bicarbonate on nitrate uptake, assimilation and translocation to shoots. Clonal saplings of poplar (Populus canescens(Ait.) Sm.) and elder (Sambucus nigraL.) were grown hydroponically for 35 days in a nutrient solution containing 0, 0.5 and 1 mM bicarbonate and 2 mM nitrate as the sole nitrogen source at pH 7.0. Net nitrate uptake, root nitrate accumulation and reduction, and export of nitrogenous solutes to shoots were measured after incubating plants with ¹⁵N-labelled nitrate for 24 h. Net nitrate uptake increased non-significantly in plant species (19–61% compared to control plants) in response to 1 mM bicarbonate. Root nitrate reduction and nitrogen export to shoots increased by 80 and 95% and 15 and 44% in poplar and elder, respectively. With enhanced root zone bicarbonate, both species also exhibited a marked shift between the main nitrate utilising processes. Poplar plants increasingly utilised nitrate via nitrate reduction (73–88% of net nitrate uptake), whereas the proportions of export (20–9%) and storage in roots (7–3%) declined as plants were exposed to 1 mM external bicarbonate. On the other hand, elder plants exhibited a significant increase of root nitrate reduction (44–66%) and root nitrate accumulation (6–25%). Nitrate translocation to elder shoots decreased from 50 to 8% of net nitrate uptake. The improved supply of nitrogen to shoots did not translate into a significant stimulation of growth, relative growth rates increased by only 16% in poplar saplings and by 7% in elder plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sodium-dependent uptake of nitrate and urea by a marine diatom

Uptake of nitrate and urea by Phaeodactylum tricornutum is shown to be a sodium dependent process inhibited by lithium or potassium. The half-saturation constant for sodium (K_Na) was 2.6 mM for nitrate uptake and 71 mM for urea uptake. It is suggested that sodium dependent uptake mechanisms may be characteristic of marine plants.     

Ammonium and nitrate uptake by Eucalyptus nitens: effects of pH and temperature

Ammonium and nitrate uptake by roots of Eucalyptus nitens was characterised with respect to pH and temperature. Uptake of ammonium and nitrate was measured as depletion from solutions by roots of intact 11 week old solution-cultured seedlings. Uptake rates of ammonium were consistently higher than those of nitrate in all experiments. Uptake rates for ammonium were 200% higher at pH 4 than at pH 6, but for nitrate were unchanged. Uptake rates of ammonium and nitrate were both reduced to a similar extent (70%) with a decrease in temperature from 20 °C to 10 °C. For ammonium uptake, there was rapid (<24 hr) adaptation to a reduction in root temperature. The apparent preference shown here for ammonium over nitrate could be indicative of E. nitens growing in cold, acidic forest soils where ammonium is commonly more available than nitrate. These results suggest that N uptake rates of E. nitens may be maximised under a wide variety of conditions if N is supplied predominantly in the ammonium form. This revised version was published online in June 2006 with corrections to the Cover Date.     

Alterations in nitrogen assimilation and partitioning in nitrogen stressed plants

Although nutrient stress is known to alter partitioning between shoots and roots, the physiological basis for the phenomenon is unresolved. Experiments were conducted to examine assimilation of ¹⁵NO₃ by N-stressed plants and to determine whether apparent changes in assimilation in the root contributed to alterations in whole-plant partitioning of reduced-N. Tobacco plants (Nicotiana tabacum L. cv. NC 2326) were exposed to a low concentration of NO₃^? in solution (80 μM) for 9 days to effect a N-stress response. Exposure of plants to 1000 μM¹⁵NO₃^? for 12 h on selected days revealed that roots of N-stressed plants developed an increased capacity to absorb NO₃^?, and accumulation of reduced-¹⁵N in the root increased to an even greater extent. When plants were exposed to 80 or 1000 μM¹⁵NO₃^? in steady-state, ¹⁵NO₃^? uptake over a 12 h period was noticeably restricted at the lower concentration, but a larger proportion of the absorbed ¹⁵N still accumulated as reduced-¹⁵N in the root. The alteration in reduced-¹⁵N partitioning was maintained in N-stressed plants during the subsequent 3-day “chase” period when formation of insoluble reduced-¹⁵N in the root was quantitatively related to the disappearance of ¹⁵NO₃^? and soluble reduced-¹⁵N. The results indicate that increased assimilation of absorbed NO₃^?, in the root may contribute significantly to the altered reduced-N partitioning which occurs in N-stressed plants.     

Regulation of the expression of ferredoxin-glutamate synthase in barley

We have investigated the regulation of ferredoxin–glutamate synthase (Fd-GOGAT) in leaves of barley (Hordeum vulgare L. cv. Maris Mink) at the mRNA, protein and enzyme activity levels. Studies of the changes in Fd-GOGAT during plant development showed that the activity in shoots increases rapidly after germination to reach a maximum (on a fresh-weight basis) at day 10 and then declines markedly to less than 50% of the maximal activity by day 30, this decline being correlated with an equivalent loss of Fd-GOGAT protein. Growing the plants in darkness reduced the maximum activity attained in the shoots, but did not affect the overall pattern of the changes or their timing. The activity of Fd-GOGAT increased two- to three-fold within 48 h when etiolated leaves were exposed to light, and Northern blots indicated that the induction occurred at the mRNA level. However, whilst a carbon source could at least partially substitute for light in the induction of nitrate reductase activity, no induction of Fd-GOGAT activity was seen when etiolated leaves were treated with either sucrose or glucose. Interestingly, the levels of Fd-GOGAT mRNA and activity remained high up to a period of 16 h or 72 h darkness, respectively. Compared with plants grown in N-free medium, light-grown plants supplied with nitrate had almost two-fold higher Fd-GOGAT activities and increased Fd-GOGAT mRNA levels, but nitrate had no effect on the abundance of the enzyme or its mRNA in etiolated plants, indicating that light is required for nitrate induction of barley Fd-GOGAT. Received: 23 April 1997 / Accepted: 28 May 1997     

An updated model for nitrate uptake modelling in plants. II. Assessment of active root involvement in nitrate uptake based on integrated root system age: measured versus modelled outputs

Background and Aims

An updated version of a mechanistic structural–functional model was developed to predict nitrogen (N) uptake throughout the growth cycle by a crop of winter oilseed rape, Brassica napus, grown under field conditions.

Methods

The functional component of the model derives from a revisited conceptual framework that combines the thermodynamic Flow–Force interpretation of nitrate uptake isotherms and environmental and in planta effects on nitrate influx. Estimation of the root biomass (structural component) is based upon a combination of root mapping along the soil depth profile in the field and a relationship between the specific root length and external nitrate concentration. The root biomass contributing actively to N uptake was determined by introduction of an integrated root system age that allows assignment of a root absorption capacity at a specific age of the root.

Key Results

Simulations were well matched to measured data of N taken up under field conditions for three levels of N fertilization. The model outputs indicated that the two topsoil layers (0–30 and 30–60 cm) contained 75–88 % of the total root length and biomass, and accounted for 90–95 % of N taken up at harvest.

Conclusions

This conceptual framework provides a model of nitrate uptake that is able to respond to external nitrate fluctuations at both functional and structural levels.     

Molecular mechanisms of potassium and sodium uptake in plants

Potassium (K⁺) is an essential nutrient and the most abundant cation in plants, whereas the closely related ion sodium (Na⁺) is toxic to most plants at high millimolar concentrations. K⁺ deficiency and Na⁺ toxicity are both major constraints to crop production worldwide. K⁺ counteracts Na⁺ stress, while Na⁺, in turn, can to a certain degree alleviate K⁺ deficiency. Elucidation of the molecular mechanisms of K⁺ and Na⁺ transport is pivotal to the understanding – and eventually engineering – of plant K⁺ nutrition and Na⁺ sensitivity. Here we provide an overview on plant K⁺ transporters with particular emphasis on root K⁺ and Na⁺ uptake. Plant K⁺-permeable cation transporters comprise seven families: Shaker-type K⁺ channels, `two-pore' K⁺ channels, cyclic-nucleotide-gated channels, putative K⁺/H⁺ antiporters, KUP/HAK/KT transporters, HKT transporters, and LCT1. Candidate genes for Na⁺ transport are the KUP/HAK/KTs, HKTs, CNGCs, and LCT1. Expression in heterologous systems, localization in plants, and genetic disruption in plants will provide insight into the roles of transporter genes in K⁺ nutrition and Na⁺ toxicity.     

Electrophysiological factors in the influence of nitrate and ammonium ions on calcium uptake and translocation in tomato plants

Abstract Tomato plants (Lycopersicon esculentum Mill. cv. San Marzano), grown in dilute nutrient solutions containing (in meq ˙ 1^-1) 0.5 NaNO₃, 0.5 NH₄NO₃ or 0.25 (NH₄)₂ SO₄ as the nitrogen source, were detopped for collection of xylem sap and measurement of trans-root electrical potentials. The plant parts and the xylem exudate were subsequently analysed for mineral content. The commonly observed effects of NH₄⁺ were noted, including reduction of calcium concentration in the xylem sap, and of calcium content in stems and leaves, compared with NO₃^-fed plants. This effect was attributed principally to the less negative trans-root electrical potential measured in NH₄⁺-fed plants, and the resultant reduction of inward driving force on passively moving divalent cations.     

Short-term nitrous oxide emissions from pasture soil as influenced by urea level and soil nitrate

Nitrogen excreted by cattle during grazing is a significant source of atmospheric nitrous oxide (N₂O). The regulation of N₂O emissions is not well understood, but may vary with urine composition and soil conditions. This laboratory study was undertaken to describe short-term effects on N₂O emissions and soil conditions, including microbial dynamics, of urea amendment at two different rates (22 and 43 g N m⁻²). The lower urea concentration was also combined with an elevated soil NO ₃ ⁻ concentration. Urea solutions labelled with 25 atom%¹⁵N were added to the surface of repacked pasture soil cores and incubated for 1, 3, 6 or 9 days under constant conditions (60% WFPS, 14 °C). Soil inorganic N (NH ₄ ⁺ , NO ₂ ⁻ and NO ₃ ⁻ ), pH, electrical conductivity and dissolved organic C were quantified. Microbial dynamics were followed by measurements of CO₂ evolution, by analyses of membrane lipid (PLFA) composition, and by measurement of potential ammonium oxidation and denitrifying enzyme activity. The total recovery of¹⁵N averaged 84%. Conversion of urea-N to NO ₃ ⁻ was evident, but nitrification was delayed at the highest urea concentration and was accompanied by an accumulation of NO ₂ ⁻ . Nitrous oxide emissions were also delayed at the highest urea amendment level, but accelerated towards the end of the study. The pH interacted with NH ₄ ⁺ to produce inhibitory concentrations of NH₃(aq) at the highest urea concentration, and there was evidence for transient negative effects of urea amendment on both nitrifying and denitrifying bacteria in this treatment. However, PLFA dynamics indicated that initial inhibitory effects were replaced by increased microbial activity and net growth. It is concluded that urea-N level has qualitative, as well as quantitative effects on soil N transformations in urine patches.     

植物吸收转运无机氮的生理及分子机制

氮是植物生长必需的营养元素。植物从土壤中吸收的氮素主要是NO3-和NH4 等无机氮源。植物吸收NO3-和NH4 的系统均有高亲和转运系统(high-affinity transport system,HATS)和低亲和转运系统(low-affinity transport system,LATS)之分。近10多年的研究已对这些转运系统的分子基础有了较好的理解,本文着重对近年来植物吸收无机氮分子机制的研究进展进行了综述。