Uptake, metabolism and distribution of organic and inorganic nitrogen sources by Pinus sylvestris

Although an increasing number of studies show that many plant species have the capacity to take up amino acids from exogenous sources, the importance of such uptake for plant nitrogen nutrition is largely unknown. Moreover, little is known regarding metabolism and distribution of amino acid-N following uptake or of the regulation of these processes in response to plant nitrogen status. Here results are presented from a study following uptake, metabolism, and distribution of nitrogen from NO(3)(-) NH(4)(+), Glu, or Ala in Scots pine (Pinus sylvestris L). In a parallel experiment, Ala uptake, processing, and shoot allocation were also monitored following a range of pretreatments intended to alter plant C- and N-status. Uptake data, metabolite profiles, N fluxes through metabolite pools and tissues, as well as alanine aminotransferase activity are presented. The results show that uptake of the organic N sources was equal to or larger than NH(4)(+) uptake, while NO(3)(-) uptake was comparatively low. Down-regulation of Ala uptake in response to pretreatments with NH(4)NO(3) or methionine sulphoximine (MSX) indicates similarities between amino acid and inorganic N uptake regulation. N derived from amino acid uptake exhibited a rapid flux through the amino acid pool following uptake. Relative shoot allocation of amino acid-N was equal to that of NH(4)(+) but smaller than for NO(3)(-) Increased N status as well as MSX treatment significantly increased relative shoot allocation of Ala-N suggesting that NH(4)(+) may have a role in the regulation of shoot allocation of amino acid-N.     

High-affinity potassium transport in barley roots. Ammonium-sensitive and -insensitive pathways

In an attempt to understand the process mediating K(+) transport into roots, we examined the contribution of the NH(4)(+)-sensitive and NH(4)(+)-insensitive components of Rb(+) transport to the uptake of Rb(+) in barley (Hordeum vulgare L.) plants grown in different ionic environments. We found that at low external Rb(+) concentrations, an NH(4)(+)-sensitive component dominates Rb(+) uptake in plants grown in the absence of NH(4)(+), while Rb(+) uptake preferentially occurs through an NH(4)(+)-insensitive pathway in plants grown at high external NH(4)(+) concentrations. A comparison of the Rb(+)-uptake properties observed in roots with those found in heterologous studies with yeast cells indicated that the recently cloned HvHAK1 K(+) transporter may provide a major route for the NH(4)(+)-sensitive component. HvHAK1 failed to complement the growth of a yeast strain defective in NH(4)(+) transport, suggesting that it could not act as an NH(4)(+) transporter. Heterologous studies also showed that the HKT1 K(+)/Na(+)-cotransporter may act as a pathway for high-affinity Rb(+) transport sensitive to NH(4)(+). However, we found no evidence of an enhancement of Rb(+) uptake into roots due to Na(+) addition. The possible identity of the systems contributing to the NH(4)(+)-insensitive component in barley plants is discussed.     

Acquisition and assimilation of nitrogen as peptide-bound and D-enantiomers of amino acids by wheat

Nitrogen is a key regulator of primary productivity in many terrestrial ecosystems. Historically, only inorganic N (NH(4)(+) and NO(3)(-)) and L-amino acids have been considered to be important to the N nutrition of terrestrial plants. However, amino acids are also present in soil as small peptides and in D-enantiomeric form. We compared the uptake and assimilation of N as free amino acid and short homopeptide in both L- and D-enantiomeric forms. Sterile roots of wheat (Triticum aestivum L.) plants were exposed to solutions containing either (14)C-labelled L-alanine, D-alanine, L-trialanine or D-trialanine at a concentration likely to be found in soil solution (10 μM). Over 5 h, plants took up L-alanine, D-alanine and L-trialanine at rates of 0.9±0.3, 0.3±0.06 and 0.3±0.04 μmol g(-1) root DW h(-1), respectively. The rate of N uptake as L-trialanine was the same as that as L-alanine. Plants lost ca.60% of amino acid C taken up in respiration, regardless of the enantiomeric form, but more (ca.80%) of the L-trialanine C than amino acid C was respired. When supplied in solutions of mixed N form, N uptake as D-alanine was ca.5-fold faster than as NO(3)(-), but slower than as L-alanine, L-trialanine and NH(4)(+). Plants showed a limited capacity to take up D-trialanine (0.04±0.03 μmol g(-1) root DW h(-1)), but did not appear to be able to metabolise it. We conclude that wheat is able to utilise L-peptide and D-amino acid N at rates comparable to those of N forms of acknowledged importance, namely L-amino acids and inorganic N. This is true even when solutes are supplied at realistic soil concentrations and when other forms of N are available. We suggest that it may be necessary to reconsider which forms of soil N are important in the terrestrial N cycle.     

Dynamic and steady-state responses of inorganic nitrogen pools and NH(3) exchange in leaves of Lolium perenne and Bromus erectus to changes in root nitrogen supply

Short- and long-term responses of inorganic N pools and plant-atmosphere NH(3) exchange to changes in external N supply were investigated in 11-week-old plants of two grass species, Lolium perenne and Bromus erectus, characteristic of N-rich and N-poor grassland ecosystems, respectively. A switch of root N source from NO(-)(3)to NH(4)(+) caused within 3 h a 3- to 6-fold increase in leaf apoplastic NH(4)(+) concentration and a simultaneous decrease in apoplastic pH of about 0.4 pH units in both species. The concentration of total extractable leaf tissue NH(4)(+) also increased two to three times within 3 h after the switch. Removal of exogenous NH(4)(+) caused the apoplastic NH(4)(+) concentration to decline back to the original level within 24 h, whereas the leaf tissue NH(4)(+)concentration decreased more slowly and did not reach the original level in 48 h. After growing for 5 weeks with a steady-state supply of NO(-)(3)or NH(4)(+), L. perenne were in all cases larger, contained more N, and utilized the absorbed N more efficiently for growth than B. erectus, whereas the two species behaved oppositely with respect to tissue concentrations of NO(-)(3), NH(4)(+), and total N. Ammonia compensation points were higher for B. erectus than for L. perenne and were in both species higher for NH(4)(+)- than for NO(-)(3)-grown plants. Steady-state levels of apoplastic NH(4)(+), tissue NH(4)(+), and NH(3) emission were significantly correlated. It is concluded that leaf apoplastic NH(4)(+) is a highly dynamic pool, closely reflecting changes in the external N supply. This rapid response may constitute a signaling system coordinating leaf N metabolism with the actual N uptake by the roots and the external N availability.     

Regulation of amino acid uptake by carbon and nitrogen in<Emphasis Type="Italic"> Pinus sylvestris</Emphasis>

The simultaneous uptake of 13 different amino acids by Scots pine ( Pinus sylvestris L.) was characterized and its regulation investigated after pre-treatments with a range of C and N metabolites. The uptake system exhibited a broad substrate specificity, acquiring all tested amino acids at similar uptake rates. Uptake of all tested amino acids by excised roots was linear over a time period of 150 min and exhibited pH-dependency, showing a peak at pH 4.0-5.0. Uptake was increased following pre-treatments with glucose and sucrose, while ammonium pre-treatments had a negative effect on amino acid acquisition. Pre-treatments with the important Krebs cycle intermediates oxaloacetate or 2-oxoglutarate did not result in altered amino acid uptake. It is speculated that the up-regulation of uptake may be due to an increased flow of glucose through a sensor mechanism, such as hexokinase. The regulation of transport by N and C suggests a function for amino acid uptake in the N nutrition of the plant, thus further highlighting the importance of organic nitrogen for plant N nutrition suggested by an increasing amount of research.     

Functional analysis of an Arabidopsis T-DNA "knockout" of the high-affinity NH4(+) transporter AtAMT1;1

NH(4)(+) acquisition by plant roots is thought to involve members of the NH(4)(+) transporter family (AMT) found in plants, yeast, bacteria, and mammals. In Arabidopsis, there are six AMT genes of which AtAMT1;1 demonstrates the highest affinity for NH(4)(+). Ammonium influx into roots and AtAMT1;1 mRNA expression levels are highly correlated diurnally and when plant nitrogen (N) status is varied. To further investigate the involvement of AtAMT1;1 in high-affinity NH(4)(+) influx, we identified a homozygous T-DNA mutant with disrupted AtAMT1;1 activity. Contrary to expectation, high-affinity (13)NH(4)(+) influx in the amt1;1:T-DNA mutant was similar to the wild type when grown with adequate N. Removal of N to increase AtAMT1;1 expression decreased high-affinity (13)NH(4)(+) influx in the mutant by 30% compared with wild-type plants, whereas low-affinity (13)NH(4)(+) influx (250 microM-10 mM NH(4)(+)) exceeded that of wild-type plants. In these N-deprived plants, mRNA copy numbers of root AtAMT1;3 and AtAMT2;1 mRNA were significantly more increased in the mutant than in wild-type plants. Under most growth conditions, amt1;1:T-DNA plants were indistinguishable from the wild type, however, leaf morphology was altered. However, when grown with NH(4)(+) and sucrose, the mutant grew poorly and died. Our results are the first in planta evidence that AtAMT1;1 is a root NH(4)(+) transporter and that redundancies within the AMT family may allow compensation for the loss of AtAMT1;1.     

Conformational changes in Kir2.1 channels during NH4+-induced inactivation

We have shown previously that NH(4)(+) binding to the external pore of a Kir2.1 channel induces channel inactivation possibly through conformational changes. In this study, we performed further biophysical analyses of the NH(4)(+)-induced inactivation modeled by a refined kinetic scheme. Also, we investigated the conformational change hypothesis by examining whether the chemical modification of single-cysteine substitution of amino acids located at the internal pore alters the kinetics of the NH(4)(+)-induced inactivation. In addition, we examined whether the mutation of amino acids located at various parts of a Kir2.1 channel influences the NH(4)(+)-induced inactivation. Kir2.1 channels were expressed in Xenopus oocytes and studied using patch-clamp techniques. The gating of the NH(4)(+)-induced inactivation was affected by mutation of several amino acids located at various regions of the Kir2.1 channel. These results suggest that amino acids from different parts of a Kir2.1 channel are involved in the channel closure. Furthermore, internal chemical modification of several cysteine mutants resulted in the block of inward currents and changes in the on and off rate for the NH(4)(+)-induced inactivation, suggesting that the internal pore mouth is involved in the closure of a Kir2.1 channel. Taken together these results provide new evidence for conformational changes affecting the NH(4)(+)-induced inactivation in the Kir2.1 channel.     

Juvenile nitrogen uptake capacities and root architecture of two open-pollinated families of Picea abies. Effects of nitrogen source and ectomycorrhizal symbiosis

This study was carried out to find early physiological differences occurring in young seedlings between two contrasting Picea abies open-pollinated families (OPF), one with high- and one with low-growth performance in the field by, determining their N uptake capacities and their root architecture. We used three potential N-sources in forest soil solution, NO3-, NH4+ and amino acids, to establish N uptake rates by the plants, whether or not associated with a fungus isolated from the field and identified as Paxillus involutus. NO3- fluxes were determined locally at the root surface using NO3(-)-selective microelectrodes whereas NH4+ and amino acid (L-glutamate and L-aspartate) uptake rates were calculated from their depletion of the incubation solution by the whole root system. Root systems were digitised in order to determine the number and the length of different root types. In non-mycorrhizal plants, the results showed that the most distinguishing parameters between OPF were NO3- uptake rates measured in the white tip of the secondary roots and the root architecture, with higher values determined in high-growth than in low-growth field performance OPF. The presence of the mycorrhizal fungus decreased NO3- uptake rates in both OPF and had an opposite effect on root architecture by increasing it in low-growth and decreasing it in high-growth field performance OPF, respectively. In non-mycorrhizal plants, NH4+ and amino-acid uptake rates were not different between OPF. Mycorrhizal symbiosis did not change NH4+ uptake rates whereas it increased that of amino acids, specifically that of L-aspartate in the low-growth field performance OPF. Taken together these results suggest that the measurement of local fluxes in roots of young plants could be a good potential tool for the early evaluating of growth capacity of Picea abies OPF.     

Deposition of ammonium and nitrate in the roots of maize seedlings supplied with different nitrogen salts

This study measured total osmolarity and concentrations of NH(4)(+), NO(3)(-), K(+), soluble carbohydrates, and organic acids in maize seminal roots as a function of distance from the apex, and NH(4)(+) and NO(3)(-) in xylem sap for plants receiving NH(4)(+) or NO(3)(-) as a sole N-source, NH(4)(+) plus NO(3)(-), or no nitrogen at all. The disparity between net deposition rates and net exogenous influx of NH(4)(+) indicated that growing cells imported NH(4)(+) from more mature tissue, whereas more mature root tissues assimilated or translocated a portion of the NH(4)(+) absorbed. Net root NO(3)(-) influx under Ca(NO(3))(2) nutrition was adequate to account for pools found in the growth zone and provided twice as much as was deposited locally throughout the non-growing tissue. In contrast, net root NO(3)(-) influx under NH(4)NO(3) was less than the local deposition rate in the growth zone, indicating that additional NO(3)(-) was imported or metabolically produced. The profile of NO(3)(-) deposition rate in the growth zone, however, was similar for the plants receiving Ca(NO(3))(2) or NH(4)NO(3). These results suggest that NO(3)(-) may serve a major role as an osmoticant for supporting root elongation in the basal part of the growth zone and maintaining root function in the young mature tissues.     

Comprehensive screening of Arabidopsis mutants suggests the lysine histidine transporter 1 to be involved in plant uptake of amino acids

Plant nitrogen (N) uptake is a key process in the global N cycle and is usually considered a "bottleneck" for biomass production in land ecosystems. Earlier, mineral N was considered the only form available to plants. Recent studies have questioned this dogma and shown that plants may access organic N sources such as amino acids. The actual mechanism enabling plants to access amino acid N is still unknown. However, a recent study suggested the Lysine Histidine Transporter 1 (LHT1) to be involved in root amino acid uptake. In this study, we isolated mutants defective in root amino acid uptake by screening Arabidopsis (Arabidopsis thaliana) seeds from ethyl methanesulfonate-treated plants and seeds from amino acid transporter T-DNA knockout mutants for resistance against the toxic D-enantiomer of alanine (Ala). Both ethyl methanesulfonate and T-DNA knockout plants identified as D-Ala resistant were found to be mutated in the LHT1 gene. LHT1 mutants displayed impaired capacity for uptake of a range of amino acids from solutions, displayed impaired growth when N was supplied in organic forms, and acquired substantially lower amounts of amino acids than wild-type plants from solid growth media. LHT1 mutants grown on mineral N did not display a phenotype until at the stage of flowering, when premature senescence of old leaf pairs occurred, suggesting that LHT1 may fulfill an important function at this developmental stage. Based on the broad and unbiased screening of mutants resistant to D-Ala, we suggest that LHT1 is an important mediator of root uptake of amino acids. This provides a molecular background for plant acquisition of organic N from the soil.     

Transpiration, potassium uptake and flow in tobacco as affected by nitrogen forms and nutrient levels

BACKGROUND AND AIMS: Ammonium can result in toxicity symptoms in many plants when it is supplied as the sole source of N. In this work, influences of different nitrogen forms at two levels (2 and 15 mm N) on growth, water relations and uptake and flow of potassium were studied in plants of Nicotiana tabacum 'K 326'. METHODS: Xylem sap from different leaves was collected from 106-d-old tobacco plants cultured in quartz sand by application of pressure to the root system. Whole-shoot transpiration for each of the treatments was measured on a daily basis by weight determination. KEY RESULTS: Total replacement of NO(3)(-)N by NH(4)(+)-N caused a substantial decrease in dry weight gain, even when plants grew under nutrient deficiency. Increasing nutrient concentration resulted in a greater net dry weight gain when nitrogen was supplied as NO(3)(-) or NH(4)NO(3), but resulted in little change when nitrogen was supplied as NH(4)(+). NH(4)(+)-N as the sole N-source also caused reduction in transpiration rate, changes in plant WUE (which depended on the nutrient levels) and a decrease in potassium uptake. However, the amount of xylem-transported potassium in the plants fed with NH(4)(+) was not reduced: it was 457 % or 596 % of the potassium currently taken up at low or high nutrient level, respectively, indicating a massive export from leaves and cycling of potassium in the phloem. CONCLUSIONS: Ammonium reduces leaf stomatal conductance of tobacco plants. The flow and partitioning of potassium in tobacco plants can be changed, depending on the nitrogen forms and nutrient levels.     

Changes in growth and nutrient uptake in Brassica oleracea exposed to atmospheric ammonia

BACKGROUND AND AIMS: Plant shoots form a sink for NH3, and are able to utilize it as a source of N. NH3 was used as a tool to investigate the interaction between foliar N uptake and root N uptake. To what extent NH3 can contribute to the N budget of the plant or can be regarded as a toxin, was investigated in relation to its concentration and the N supply in the root environment. METHODS: Brassica oleracea was exposed to 0, 4 and 8 microL L(-1) NH3, with and without nitrate in the nutrient solution. Growth, N compounds, nitrate uptake rate, soluble sugars and cations were measured. KEY RESULTS: In nitrate-sufficient plants, biomass production was not affected at 4 microL L(-1) NH3, but was reduced at 8 microL L(-1) NH3. In nitrate-deprived plants, shoot biomass was increased at both concentrations, but root biomass decreased at 8 microL L(-1) NH3. The measured nitrate uptake rates agreed well with the plant's N requirement for growth. In nitrate-sufficient plants nitrate uptake at 4 and 8 microL L(-1) NH3 was reduced by 50 and 66 %, respectively. CONCLUSIONS: The present data do not support the hypothesis that NH3 toxicity is caused by a shortage of sugars or a lack of capacity to detoxify NH3. It is unlikely that amino acids, translocated from the shoot to root, are the signal metabolites involved in the down-regulation of nitrate uptake, since no relationship was found between changes in nitrate uptake and root soluble N content of NH3-exposed plants.     

Root-zone acidity and nitrogen source affects Typha latifolia L. growth and uptake kinetics of ammonium and nitrate

The NH(4)(+) and NO(3)(-) uptake kinetics by Typha latifolia L. were studied after prolonged hydroponics growth at constant pH 3.5, 5.0, 6.5 or 7.0 and with NH(4)(+) or NO(3)(-) as the sole N-source. In addition, the effects of pH and N source on H(+) extrusion and adenine nucleotide content were examined. Typha latifolia was able to grow with both N sources at near neutral pH levels, but the plants had higher relative growth rates, higher tissue concentrations of the major nutrients, higher contents of adenine nucleotides, and higher affinity for uptake of inorganic nitrogen when grown on NH(4)(+). Growth almost completely stopped at pH 3.5, irrespective of N source, probably as a consequence of pH effects on plasma membrane integrity and H(+) influx into the root cells. Tissue concentrations of the major nutrients and adenine nucleotides were severely reduced at low pH, and the uptake capacity for inorganic nitrogen was low, and more so for NO(3)(-)-fed than for NH(4)(+)-fed plants. The maximum uptake rate, V(max), was highest for NH(4)(+) at pH 6.5 (30.9 micro mol h(-1) g(-1) root dry weight) and for NO(3)(-) at pH 5.0 (31.7 micro mol h(-1) g(-1) root dry weight), and less than 10% of these values at pH 3.5. The affinity for uptake as estimated by the half saturation constant, K((1/2)), was lowest at low pH for NH(4)(+) and at high pH for NO(3)(-). The changes in V(max) and K((1/2)) were thus consistent with the theory of increasing competition between cations and H(+) at low pH and between anions and OH(-) at high pH. C(min) was independent of pH, but slightly higher for NO(3)(-) than for NH(4)(+) (C(min)(NH(4)(+)) approximately 0.8 mmol m(-3); C(min)(NO(3)(-)) approximately 2.8 mmol m(-3)). The growth inhibition at low pH was probably due to a reduced nutrient uptake and a consequential limitation of growth by nutrient stress. Typha latifolia seems to be well adapted to growth in wetland soils where NH(4)(+) is the prevailing nitrogen compound, but very low pH levels around the roots are very stressful for the plant. The common occurrence of T. latifolia in very acidic areas is probably only possible because of the plant's ability to modify pH-conditions in the rhizosphere.     

Uptake of intact amino acids by plants depends on soil amino acid concentrations

Studies in different ecosystems have shown that plants take up intact amino acids directly but little is known about the influence of free amino acid concentrations in the soil on this process. We investigated the effect of three different soil amino acid N concentrations (0.025, 0.13 and 2.5 μg N g^?1 soil) on direct uptake of four dual labelled (¹⁵N, ¹³C) amino acids (glycine, tyrosine, lysine, valine) in a greenhouse experiment using Anthoxantum odoratum as a model plant.Our results revealed that 8–45% of applied ¹⁵N was incorporated into plant root and shoot tissue 48 h after labelling. Additional ¹³C enrichment showed that 2–70% of this incorporated ¹⁵N was taken up as intact amino acid. Total ¹⁵N uptake and ¹⁵N uptake as intact amino acids were significantly affected by soil amino acid N concentrations and significantly differed between the four amino acids tested.We found a positive effect of soil amino acid concentrations on uptake of mineralized ¹⁵N relative to amino acid concentrations for all amino acids which was presumably due to higher diffusion rates of mineralized tracer to the root surface. However, intact amino acid uptake relative to amino acid concentrations as well as the proportion of total ¹⁵N taken up directly decreased with increasing soil amino acid N concentrations for all amino acids, irrespective of their microbial degradability. This effect is most likely controlled by the mineral N concentration in soil and perhaps in plants which inhibits direct amino acids uptake.Overall, we conclude that plant internal regulation of amino acid uptake controlled by mineral N is the main mechanism determining direct uptake of amino acids and thus a lower contribution of intact amino acid uptake to the plants N nutrition has to be expected for higher amino acid concentrations accompanied by mineralization in soil.     

The in vivo nitrogen isotope discrimination among organic plant compounds

The bulk delta 15 N-value of plant (leaf) biomass is determined by that of the inorganic primary nitrogen sources NO(3)(-), NH(4)(+) and N(2), and by isotope discriminations on their uptake or assimilation. NH(4)(+) from these is transferred into "organic N" mainly by the glutamine synthetase reaction. The involved kinetic nitrogen isotope effect does not become manifest, because the turnover is quantitative. From the product glutamine any further conversion proceeds in a "closed system", where kinetic isotope effects become only efficient in connection with metabolic branching. The central and most important corresponding process is the GOGAT-reaction, involved in the de novo nitrogen binding and in recycling processes like the phenylpropanoid biosynthesis and photorespiration. The reaction yields relatively 15N-depleted glutamate and remaining glutamine, source of 15N-enriched amide-N in heteroaromatic compounds. Glutamate provides nitrogen for all amino acids and some other compounds with different 15N-abundances. An isotope equilibration is not connected to transamination; the relative delta 15 N-value of individual amino acids is determined by their metabolic tasks. Relative to the bulk delta 15 N-value of the plant cell, proteins are generally 15N-enriched, secondary products like chlorophyll, lipids, amino sugars and alkaloids are depleted in 15N. Global delta 15 N-values and 15N-patterns of compounds with several N-atoms can be calculated from those of their precursors and isotope discriminations in their biosyntheses.