A pump/leak/buffer model for plant nitrate uptake

A simple simulation model is described to account for the rates at which plants take up nitrate and reduce it to protein. It is based on the pump and leak principle, with the pump working at a constant rate per unit sap volume provided that there is an adequate concentration of nitrate at the root surface. The rate of leakage is assumed to be proportional to the concentration difference between the inside and the outside of the plant. Nitrogen is removed from the plant nitrate pool (the buffer) at a constant fraction of the photosynthesis rate. When applied to data for the diurnal variation in nitrate uptake by ryegrass, the model predicts an uptake pattern similar to that actually observed, with a time lag of about 5 hours between photosynthesis and uptake.     

Stable hydrogen isotope ratios of saponifiable lipids and cellulose nitrate from cam,c3 and c4 plants

Hydrogen and carbon isotope ratios of saponifiable lipids and cellulose nitrate from CAM, C₃, and C₄ plants that grew near one another were determined. The deuterium/protium (D/H) ratios of cellulose nitrate from CAM plants were much higher than those of cellulose nitrate from C₃ and C₄ plants, as has been observed previously. In contrast, the D/H ratios of saponifiable lipids from CAM plants did not differ from those of the same fraction from C₃ and C₄ plants. These observations indicate that deuterium enrichment in cellulose of CAM plants is not caused by any metabolic or physiological process which would lead to deuterium enrichment in all biochemical fractions.     

Is low rooting density of faba beans a cause of high residual nitrate content of soil at harvest ?

It was the aim of this study was to evaluate the hypothesis that low rooting density of faba beans is the major reason for the comparable low depletion of N_min-nitrogen from the rooted soil volume during the vegetation period. Therefore a simulation study was carried out using data from a two-year field experiment with faba beans and the reference crop oats. Since the nitrate dynamics in the soil is closely coupled with the water budget, the model simulated also the water uptake by plants, movement and content in the soil applying a numerical solution of the Richard's equation. The nitrogen budget part of the model includes calculation of vertical nitrate movement in the soil, mineralisation of nitrate from organic matter and nitrate uptake by the crop. Vertical nitrate movement was simulated with the convection-dispersion equation. Mineralisation was computed from a simple first order kinetic approach using only one fraction of mineralisable organic matter. Nitrate uptake was assumed to be determined either by the nitrogen demand of the crop, which was estimated from a logistic growth equation that was fitted to measured data of N-accumulation, or by the maximum nitrate transport rate towards the root surface. The latter was computed from a steady state solution of the diffusion - mass flow equation for cylindrical co-ordinates.For oats the model calculated a maximum nitrate transport rate towards roots that was quite close to the measured N-uptake of that crop. For faba beans, however, the calculated maximum nitrate transport towards roots was much lower than total N-uptake and lower than for oats. Consequently, simulated N_min-contents below faba beans were during the growing season about 20-30 kg N ha^–1 higher than below oats. This difference matches quite close with the observed differences between the two crops. Therefore it was concluded that low nitrate uptake resulting from low rooting density is the main reason for higher residual nitrate contents below faba beans at harvest time.     

Nitrate utilization in barley: relations to nitrate supply and light/dark cycles

Uptake and utilization of nitrate were investigated in Hordeum vulgare L. cvs Mette and Golf in the vegetative stage, 2 and 4 weeks after sowing. The plants were subjected to a light/dark cycle of 16/8 h (18/12°C). Results obtained with the two genotypes were essentially similar. In the light, xylem nitrate transport and shoot nitrate reduction approximately equalled the amount of nitrate absorbed by the root. A drastic decline in translocation to the shoot in darkness was entirely attributable to decreased transpiration since no major changes in xylem nitrate concentration were observed. Darkening caused only a slight decrease in nitrate uptake, while root nitrate reduction was enhanced. Nitrate starvation for 2 days did not significantlly affect dry matter increment, but resulted in a drastic drop in previously accumulated nitrate, indicating that the stored nitrate is accessible and can sustain unrestricted growth. Uptake increased upon re-addition of nitrate and after 8 h it was about twice that of non-starved plants. During recovery, restoration of root nitrate pools and root nitrate reduction took precedence over shoot nitrate accumulation and reduction. Net nitrate uptake and removal of nitrate from the root to the transpiration stream seem to be decisive for the rate of root nitrate reduction.     

Effects of Root Temperature and Form of Nitrogen Nutrition on Nitrate Reductase Activity in Oilseed Rape (Brassica napus L.)

The effect of root temperature and form of inorganic nitrogensupply on in vitro nitrate reductase activity (NRA) was studiedin oilseed rape (Brassica napus L. cv. bien venu). Plants weregrown initially in flowing nutrient solution containing 10 µMNH₄NO₃ and then supplied with either nitrate or ammonium for15 d at root temperatures of 3, 7, 11 or 17 °C. Shoot temperatureregime was similar for all plants; 20/15 °C, day/night.Root NRA was highest when roots were grown at 3 and 7 °C.In laminae and petioles NRA was highest when roots were 11 or17 °C. The plants supplied with ammonium had much lowerlevels of NRA in roots after 5 d than the plants supplied onlywith nitrate. NRA in the laminae of plants supplied with ammoniumwas low relative to that in plants supplied with nitrate onlywhen root temperature was 11 or 17 °C. Values of the apparent activation energy (E_a) of NR, calculatedfrom the Arrhenius equation, in laminae and petioles were differentfrom roots suggesting difference in enzyme conformation. Evidencethat the temperature at which roots were growing affected E_awas equivocal. Oilseed rape, Brassica napus L., activation energy, ammonium, Arrhenius equation, nitrate, root temperature, nitrate reductase     

Nitrate induction of root hair density is mediated by TGA1/TGA4 and CPC transcription factors in Arabidopsis thaliana

Root hairs are specialized cells that are important for nutrient uptake. It is well established that nutrients such as phosphate have a great influence on root hair development in many plant species. Here we investigated the role of nitrate on root hair development at a physiological and molecular level. We showed that nitrate increases root hair density in Arabidopsis thaliana. We found that two different root hair defective mutants have significantly less nitrate than wild‐type plants, suggesting that in A. thaliana root hairs have an important role in the capacity to acquire nitrate. Nitrate reductase‐null mutants exhibited nitrate‐dependent root hair phenotypes comparable with wild‐type plants, indicating that nitrate is the signal that leads to increased formation of root hairs. We examined the role of two key regulators of root hair cell fate, CPC and WER, in response to nitrate treatments. Phenotypic analyses of these mutants showed that CPC is essential for nitrate‐induced responses of root hair development. Moreover, we showed that NRT1.1 and TGA1/TGA4 are required for pathways that induce root hair development by suppression of longitudinal elongation of trichoblast cells in response to nitrate treatments. Our results prompted a model where nitrate signaling via TGA1/TGA4 directly regulates the CPC root hair cell fate specification gene to increase formation of root hairs in A. thaliana.     

Improvement of the 15N dilution method for estimation of absorption of NOx by plants supplied with 15N-labelled fertilizer

To develop further the methods for estimation of NOx absorption by plants supplied with ¹⁵N-labelled fertilizer, we proposed a new calculation method, total N fixed method (TNF), and compared with the ¹⁵N dilution method and the classical mass balance method (MB).
Hydroponically grown soybean plants were supplied with ¹⁵N-labelled nitrate and exposed to 200–250 nl l⁻¹ NO₂ for 7 d. The proportions of the N derived from NO₂ to total N in exposed plants were estimated by the three methods.
The reported rates of NO₂ absorption by several plant species, estimated by the ¹⁵N dilution method, were recalculated using the TNF method. The results of the two methods were compared and showed that: (1) The ¹⁵N dilution method overestimated the content of NO₂-N in exposed plants compared with the MB method whilst the TNF method produced estimations of NO₂-N closer to those by the MB method when the plants were supplied with 5 m M nitrate. (2) The differences in estimations between the MB method and either the ¹⁵N dilution method or the TNF method increased with decreasing supply of ¹⁵N-labelled nitrate to roots.     

Differences in nitrogen metabolism of Faidherbia albida and other N2-fixing tropical woody acacias reflect habitat water availability

The activities of nitrate reductase and glutamine synthetase were evaluated in young plants of Faidherbia albida , a tropical woody legume, fed with different N sources under hydroponic conditions. Results showed that assimilation of both NO₃⁻ and NH₄⁺ preferentially took place in shoots. A basal amount of nitrate reductase activity was detected in shoots of plants grown with an NO₃⁻-free solution or placed under N₂-fixing conditions, and also in nodules of N₂-fixing plants. This strongly suggests that constitutive nitrate reductase activity is present in these organs. Analyses of the soluble nitrogenous content showed that the major form of N in the different organs was α-amino acids (particularly amides), irrespective of the N status of the culture conditions. The same result was obtained for nodulated plants grown in local sandy soil. In this case, amide-N generally accounted for more than 40% of the total soluble N. This was especially true in nodules. Ureide-N never exceeded 9% of the total soluble N and did not appear to increase with increasing nodule nitrogenase activity. Amides were also predominant in three N₂-fixing Sahelian acacias ( Acacia seyal , A. nilotica and A. tortilis ), showing that F. albida does not differ from Sahelian Acacia in terms of the metabolism of fixed N. However, like another Sahelian acacia growing preferentially near water ( A. nilotica ), F. albida can be distinguished from acacias growing strictly in arid zones ( A. seyal and A. tortilis ) in terms of initial growth, water and nitrate management.     

The effect of nitrogen source on photosynthesis of carob at high CO2 concentrations

Carob seedlings ( Ceratonia siliqua L. cv. Mulata), fed with nitrate or ammonium, were grown in growth chambers containing two levels of CO₂ (360 or 800 μl l⁻¹), three root temperatures (15, 20 or 25°C), and the same shoot temperature (20/24°C, night/day temperature). The response of the plants to CO₂ enrichment was affected by environmental factors such as the type of inorganic nitrogen in the medium and root temperature. Increasing root temperature enhanced photosynthesis rate more in the presence of nitrate than in the presence of ammonium. Differences in photosynthetic products were also observed between nitrate- and ammonium-fed carob seedlings. Nitrate-grown plants showed an enhanced content of sucrose, while ammonium led to enhanced storage of starch. Increase in root temperature caused an increase in dry mass of the plants of similar proportions in both nitrogen sources. The enhancement of the rates of photosynthesis by CO₂ enrichment was proportionally much larger than the resulting increases in dry mass production when nitrate was the nitrogen source. Ammonium was the preferred nitrogen source for carob at both ambient and high CO₂ concentrations. The level of photosynthesis of a plant is limited not only by atmospheric CO₂ concentration but also by the nutritional and environmental conditions of the root.     

Oxygen-induced recovery from short-term nitrate inhibition of N2 fixation in white clover plants from spaced and dense stands

Nitrogenase (N₂ase; EC 1.18.6.1) activity (H₂ evolution) and root respiration (CO₂ evolution) were measured under either N₂:O₂ or Ar:O₂ gas mixtures in intact nodulated roots from white clover ( Trifolium repens L.) plants grown either as spaced or as dense stands. The short-term nitrate (5 m M ) inhibition of N₂-fixation was promoted by competition for light between clover shoots, which reduced CO₂ net assimilation rate. Oxygen-diffusion permeability of the nodule declined during nitrate treatment but after nitrate removal from the liquid medium its recovery parallelled that of nitrogenase activity. Rhizosphere pO₂ was increased from 20 to 80 kPa under N₂:O₂. A simple mono-exponential model, fitted to the nodule permeability response to pO₂, indicated NO⁻₃ induced changes in minimum and maximum nodule O₂-diffusion permeability. Peak H₂ production rates at 80 kPa O₂ and in Ar:O₂ were close to the pre-decline rates at 20 kPa O₂. At the end of the nitrate treatment, this O₂-induced recovery in nitrogenase activity reached 71 and 82%; for clover plants from spaced and dense stands, respectively. The respective roles of oxygen diffusion and phloem supply for the short-term inhibition of nitrogenase activity in nitrate-treated clovers are discussed.     

A simple model of feedback regulation for nitrate uptake and N2 fixation in contrasting phenotypes of white clover

A simple three equation model is proposed for the feedback regulation of nitrate uptake and N2 fixation, based on the concentration of the organic N substrate pool within the plant and two parameters denoting the N substrate concentrations at which half-maximal inhibition occurs. This model simulated three contrasting phenotypes of white clover (Trifolium repens L.) inbred lines with (1) normal rates of nitrate uptake and N2 fixation (NNU); (2) low rates of nitrate uptake (LNU); and (3) very low rates of N2 fixation (VLF). The LNU phenotype was simulated by a decrease in the value of the inhibition parameter for nitrate uptake and the VLF phenotype was simulated by a decrease in the value of the N2 fixation inhibition parameter. The model was tested against nitrate uptake data obtained from white clover plants growing in flowing nutrient culture. There was an accurate prediction of the increase in nitrate uptake caused by N2 fixation activity of the NNU and LNU inbred lines being interrupted by a switch in gas phase from air to Ar : O2. The model was also tested against data for nitrate uptake, N2 fixation and %N from fixation for the three inbred clover lines grown in flowing nutrient culture at 0, 5 or 20 mmol m(-3) N(3-). Again there was accurate prediction of nitrate uptake, although simulated values for N2 fixation were more variable. The simple model has potential use as a sub-routine in larger models of legume growth under field conditions.     

Nitrogen assimilation and transport in carob plants

Most of the nitrate reductase activity (80%;) in carob ( Ceratonia siliqua L. cv. Mulata) is localised in the roots. The nitrate concentration in the leaves is relatively low compared to that in the roots, suggesting that nitrate influx into the leaf may be a major factor limiting the levels of nitrate reductase in the shoot. Transport of nitrate from root to shoot appears limited by the entrance of nitrate into the xylem. In order to study this problem, we determined the nitrate concentrations and nitrate reductase activities along the roots of nitrate-grown plants, as well as the composition of the xylem sap and the nitrate levels in the leaves. Some of the the bypocotyl, in order to bypass the loading of nitrate into the xylem of the roots. The results show that the loading of nitrate into the xylem is a limiting step.
The cation and anion concentrations of nitrate- and ammonium-fed plants were similar, showing almost no production of organic anions. In both nitrate- and ammonium-fed plants, the transport of nitrogen from root to shoot was in the form of organic nitrogen compounds. The nitrate reductase activity in the roots was more than sufficient to explain all the efflux of OH⁻ into the root medium of nitrate-fed plants. In carob plants the K-shuttle may thus be operative to a limited extent only, corresponding to between 11 and 27%; of the nitrate taken up. Potassium seems to be the cation accompanying stored nitrate in the roots of carob seedlings, since they accumulate nearly stoichiometric amounts of K⁺ and NO⁻₃.     

Elevated pCO2 Affects C and N Metabolism in Wild Type and Transgenic Tobacco Exhibiting Altered C/N Balance in Metabolite Analysis

Abstract: Diurnal changes in starch, sugar and amino acid concentrations in source leaves, sink leaves and roots of tobacco plants were determined. In addition to wild type tobacco, transformed plants deficient in root nitrate reductase and exhibiting decreased rates of growth were employed. Further, the growth rates of tobacco plants were modulated by exposure to elevated pCO₂. From the diurnal alterations in metabolite concentrations, the daily turnover of starch and amino N was estimated in order to: (i) elucidate whether turnover rates can be related to growth rates, and (ii) identify individual amino compounds with the potential to indicate nitrogen fluxes and the C/N status of plants. Elevated pCO₂ increased growth rates and daily turnover of starch in both wild type and transformed plants, indicating enhanced rates of photosynthesis. In wild type plants, elevated pCO₂ increased the turnover of amino N, notably glutamine and alanine, in mature source leaves, indicating enhanced nitrate reduction. By contrast, amino N turnover in source leaves of transformed plants was not affected by elevated pCO₂, although nitrate reduction was presumably enhanced. Apparently, export of amino N was increased from the source leaves of transformed plants. This assumption was supported by a significantly increased turnover of amino N in young sink leaves compared to mature source leaves, indicating a preference for acropetal amino N allocation and import into the young leaves of the transformed plants. Further, elevated pCO₂ increased the allocation of leaf‐derived amino N to the roots of transformed plants. This led to increased levels of amino compounds during the entire day, notably glutamate, but did not affect root growth of the transformed plants. The suitability of individual amino compounds as markers for major N fluxes, such as nitrate reduction, photorespiration, and amino N export and import is discussed.     

Nitrate reductase activity, nitrogenase activity and photosynthesis of black alder exposed to chilling temperatures

Actinorhizal ( Frankia -nodulated) black alder [ Alnus glutinosa (L.) Gaertn.] seedlings fertilized with 0.36 m M nitrate (low nitrate fertilizer treatment) or 7.14 m M nitrate (high nitrate fertilizer treatment) and acclimated in a growth chamber for 2 weeks were exposed to 2.5 h of night-time chilling temperatures of −1 to 4°C. Cold treatment decreased nitrogenase activity (acetylene reduction activity) 33% for low nitrate fertilized plants and 41% for high nitrate fertilized plants. Recovery of nitrogenase activity occurred within 7 days after chilling treatment. In contrast, in vivo nitrate reductase (NR) activities of leaves and fine roots increased immediately after chilling then decreased as nitrogenase activities recovered. Fine roots of alder seedlings exhibited NR activities proportional to the amounts of nitrate in the rooting medium. In contrast, the NR activities of leaves were independent of substrate and tissue nitrate levels and corresponded to nitrogenase activity in the root nodules. In a separate experiment, net photosynthesis (PS) of similarly treated black alder seedlings was measured before and after chilling treatments. Net PS declined in response to chilling by 17% for plants receiving low nitrate fertilizer and 19% for plants receiving high nitrate fertilizer. After chilling, stomatal conductance (g_s) decreased by 39% and internal CO₂ concentration (c_i) decreased by 5% in plants receiving the high nitrate fertilizer, whereas plants receiving the low nitrate fertilizer showed no change in g_s and a 13% increase in c_i. Results indicate that chilling stimulates stomatal closure only at the high nitrate level and that interference with biochemical functions is probably the major impact of chilling on PS.     

Analysis of raphidophyte assimilatory nitrate reductase reveals unique domain architecture incorporating a 2/2 hemoglobin

Eukaryotic assimilatory nitrate reductase (NR) is a multi-domain protein that catalyzes the rate-limiting step in nitrate assimilation. This protein is highly conserved and has been extensively characterized in plants and algae. Here, we report hybrid NRs (NR2-2/2HbN) identified in two microalgal species, Heterosigma akashiwo and Chattonella subsalsa, with a 2/2 hemoglobin (2/2Hb) inserted into the hinge 2 region of a prototypical NR. 2/2Hbs are a class of single-domain heme proteins found in bacteria, ciliates, algae and plants. Sequence analysis indicates that the C-terminal FAD/NADH reductase domain of NR2-2/2HbN retains identity with eukaryotic NR, suggesting that the 2/2Hb domain was inserted interior to the existing NR domain architecture. Phylogenetic analysis supports the placement of the 2/2Hb domain of NR2-2/2HbN within group I (N-type) 2/2Hbs with high similarity to mycobacterial 2/2HbNs, known to convert nitric oxide to nitrate. Experimental data confirms that H. akashiwo is capable of metabolizing nitric oxide and shows that HaNR2-2/2HbN expression increases in response to nitric oxide addition. Here, we propose a mechanism for the dual function of NR2-2/2HbN in which nitrate reduction and nitric oxide dioxygenase reactions are cooperative, such that conversion of nitric oxide to nitrate is followed by reduction of nitrate for assimilation as cellular nitrogen.     

Osmoregulation and role of nitrate during regrowth after cutting of ryegrass (Lolium perenne)

The osmotic role of nitrate during aftermath growth of Lolium perenne L. cv. Réveille was investigated. Plants were grown from seed in a controlled environment using a liquid medium with 1.0 m M NH₄NO₃ as nitrogen source.
Eight-week-old plants were cut 4.0 cm above the root system and then harvested over a 14-day period of regrowth on the same initial nutrient solution, except that nitrate was ¹⁵N labelled. Throughout the experimental period, nitrate storage and reduction in roots were low. In stubble and especially in leaves, nitrate accumulated during the first 6 days of regrowth whereas nitrate reduction mainly occurred after this period. Analyses of carbohydrate, chloride and potassium contents in stubble and leaves showed that the accumulation of nitrate osmotically compensated for the decrease in soluble sugars during the first 6 days of regrowth.
The cumulative osmotic potential of sugars, chloride and nitrate in differently treated plants was studied in stubble and leaves. Compared with uncut plants, the lower carbohydrate concentrations found in cut plants regrowing on 1.0 m M NH₄NO₃ were compensated for by an accumulation of nitrate. During aftermath growth on low nitrogen nutrition (0.2 m M NH₄NO₃), chloride replaced nitrate, supporting the proposed osmotic function of nitrate.
It is concluded that nitrate is involved in the osmotic adjustment of ryegrass during regrowth after cutting.     

A model relating the maximum nitrate inflow of lettuce (Lactuca satvia L.) to the growth of roots and shoots

The net inflow of nitrate can be calculated from the nitrate concentration at the root surface by means of the Michaelis-Menten equation. Because of maximum inflow (Imax) is not constant but varies with plant age and growing conditions, a model for calculating Imax during plant growth was derived. Lettuce was grown in nutrient solution. Variations in temperature, radiation and plant age were used to vary growth rates and N-demand of plants. There was a linear relationship between relative growth rates (RGR) and maximum nitrate inflow (Imax), that could be described by the following regression function: Imax = 0.24 + 6.57 RGR. A residual analysis showed a further influence on Imax from the root:shoot-ratio (RSR), the effects of which could be accounted for by including an e-function in the relationship: Imax = (0.27 + 10.63 RGR) e^{(–0.0017 RSR)}. This model for calculating Imax was validated in two further experiments.     

Regulation of nitrate metabolism through anion polycompartmentation in plant roots

A new concept illustrated by a corresponding mathematical model of nitrate metabolism regulation is proposed. The model is based on root nitrate compartmentation in several functional pools: storage, metabolic and a mobile pool which is intended for translocation to shoots. Data on nitrate uptake, compartmentation, reduction in intact roots and translocation to shoots were obtained on steady-state wheat seedlings grown at 25 and 12 degrees C in the root zone. The net uptake, influx/efflux ratio, mobile pool size and translocation changed depending on the medium temperature. The oscillations of the net uptake rate, nitrate tissue concentration were revealed and the effect of temperature on these changes was demonstrated. The scheme of regulation is based on the idea that net uptake through nitrate influx/efflux is under the control of the nitrate the mobile pool whose size was dependent on the nitrate translocation into shoots. The mathematical model is represented by a system of ordinary differential equations simplified according to the time hierarchy of reactions. It has a limit cycle at definite values of the parameters. The model postulates the mechanism of a positive feed-back regulation of the transfer of newly absorbed nitrate into translocated pool formed in the root cortex. Theoretical results are verified experimentally.     

Dynamic changes in root hydraulic properties in response to nitrate availability

Changes in root hydraulic resistance in response to alterations in nitrate supply were explored in detail as a potential mechanism that allows plants to respond rapidly to changes in their environment. Sunflower (Helianthus annuus cv. Holiday) plants grown hydroponically with limited nitrate availability (200 micromol l(-1)) served as our model system. Experimental plants were 6-9-weeks-old with total dry mass of 2-4 g. Root pressurization of intact plants and detached root systems was used to elucidate the temporal dynamics of root hydraulic properties in sunflower plants following changes in external nitrate availability. The response was rapid, with a 20% decrease in hydraulic resistance occurring within the first hour after the addition of 5 mM nitrate and the magnitude of the effect was dependent on nitrate concentration. The change in root hydraulic resistance was largely reversible, although the temporal dynamics of the response to nitrate addition versus nitrate withdrawal was not symmetric (a gradual decrease in resistance versus its fast increase), raising the possibility that the underlying mechanisms may also differ. Evidence is presented that the observed changes in root hydraulic properties require the assimilation of nitrate by root cells. The hydraulic resistance of roots, previously stimulated by the addition of nitrate, increased more than in control plants in low nitrate under anoxia and that suggests a key role of aquaporin activity in this response. It is proposed that a rapid decrease in root hydraulic resistance in the presence of increased nitrate availability is an important trait that could enhance a plant's ability to compete for nitrate in the soil.