JXB Advance Access originally published online on January 30, 2004
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Journal of Experimental Botany, Vol. 55, No. 397, pp. 761-769, March 1, 2004
© 2004 Oxford University Press
Plants and the Environment |
Inhibition of nitrate influx by glutamine in Lolium perenne depends upon the contribution of the HATS to the total influx
Received 20 May 2003; Accepted 11 November 2003
The Macaulay Institute, Craigiebuckler, Aberdeen AB15 8QH, UK
* Fax: +44 (0)1224 311556. E-mail: b.thornton{at}macaulay.ac.uk
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Abstract |
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Plants of Lolium perenne L. were grown in sterile solution culture supplied with 2 mol m–3 nitrogen as either nitrate or ammonium. Glutamine at 5 mol m–3 was added to the nutrient solution of half the plants for 24 h. Root nitrate influx (at external nitrate concentrations 0–2000 mmol m–3) and amino acid concentrations were determined. In a second experiment the concentration of the added glutamine was varied from 0–5 mol m–3 and nitrate influx determined at 250 and 2000 mmol m–3. The maximum rate of influx attributed to the high affinity transport system (HATS) was reduced by 66% by the presence of glutamine achieved through an 84% reduction in its constitutive component and a 59% reduction in its inducible component. Influx attributed to LATS was unaffected by the addition of glutamine. The inhibition of total nitrate influx by glutamine was positively related to the contribution of HATS to the total influx. In both nitrate- and ammonium-grown plants, the concentration of glutamine required to inhibit nitrate influx significantly was lower when influx was determined at 250 mmol m–3 compared with 2000 mmol m–3 nitrate. The addition of glutamine increased its concentrations in root tissue. However, the results cannot be attributed to changes in glutamine alone as its addition also resulted in increased concentrations of other amino acids. Implications for plants growing under field conditions are discussed.
Key words: Glutamine, HATS, LATS, Lolium perenne, nitrate influx.
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Introduction |
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Soil solutions contain complex mixtures of both inorganic and organic forms of nitrogen, with the organic forms including amino acids (Schmidt and Stewart, 1997; Henry and Jefferies, 2002; Shand et al., 2002). It has been shown that plants contain many transporters for amino acids, at least some of which play a role in the acquisition of amino acids by roots (Glass and Siddiqi, 1995; Fischer et al., 1998; Williams and Miller, 2001; Persson and Näsholm, 2003). Indeed root uptake of intact amino acids by plant roots has been proven (Näsholm et al., 2001; Thornton, 2001) and in some ecosystems this is considered to make a significant contribution to the overall nitrogen budget of plants (Kielland, 1997; Näsholm et al., 1998).
The root uptake of nitrate is regulated by many factors including nitrate itself and the products of its metabolism such as ammonium and amino acids (Glass and Siddiqi, 1995; Forde and Clarkson, 1999). The external application of amino acids to the roots of many plant species results in inhibition of nitrate uptake (Lee et al., 1992; Muller et al., 1995; Gessler et al., 1998). The regulatory role of amino acids on nitrate uptake is considered to be primarily through effects on nitrate influx (Muller et al., 1995). Therefore, in addition to effects from direct acquisition per se, amino acids in the soil solution may also influence the nitrogen budget of plants through inhibition of nitrate influx.
The total nitrate influx by roots comprises several component transport systems, these have been reviewed extensively elsewhere (Crawford and Glass, 1998; Forde and Clarkson, 1999; Glass et al., 2001) and so will only be described here in brief. At low nitrate concentrations (<1 mol m–3) two high affinity transport systems (HATS) operate which become saturated at high nitrate concentrations. One of these the constitutive high affinity transport system (cHATS) is expressed and operates without previous exposure of the roots to nitrate, the second system the inducible high affinity transport system (iHATS) operates only after prior exposure to nitrate. In addition, a low affinity transport system (LATS) operates, which exhibits linear non-saturable kinetics as external nitrate concentration increases. Based on membrane depolarization studies the nitrate LATS was considered mainly constitutive (Glass et al., 1992), however, an inducible component to nitrate LATS has been reported (Huang et al., 1996; Zhou et al., 1998).
That amino acids inhibit the iHATS has been established. Expression of putative inducible high affinity nitrate transporters has been shown to be increased in mutants of Arabidopsis thaliana and Nicotiana plumbaginifolia with low nitrate reductase activity, suggesting that their regulation was occurring due to reduced forms of nitrogen rather than by nitrate itself (Krapp et al., 1998; Filleur and Daniel-Vedele, 1999; Lejay et al., 1999). Indeed, addition of Gln to the root bathing solution reduced expression of the inducible high affinity nitrate transporters NpNRT2.1 (Quesada et al., 1997; Krapp et al., 1998) and HvNRT2 (Vidmar et al., 2000). In the case of HvNRT2 it was considered that Gln itself rather than other amino acids produced through its metabolism was the main regulatory factor (Vidmar et al., 2000). By contrast with nitrate influx by iHATS, nitrate influx by the LATS is considered insensitive to down-regulation by accumulated nitrogen (Glass et al., 2001). If different nitrate transport systems are differentially inhibited by the application of amino acids to roots, this may lead to the inhibition of total nitrate influx by amino acids being dependent on the degree to which various nitrate transport systems contribute to total influx at that moment.
Whilst changes in the expression of individual nitrate high affinity transporters can relate well to changes in high affinity nitrate influx and/or nitrate uptake (Amarasinghe et al., 1998; Vidmar et al., 2000; Faure-Rabasse et al., 2002) this is not exclusively the case (Amarasinghe et al., 1998). Both the HATS and LATS are encoded by multiple gene families (Glass et al., 2001). In addition to difficulties in comparing studies relating to different plant species, it is difficult to extrapolate the effects of added amino acids on the expression of individual transporters to effects on the nitrate cHATS, iHATS, LATS, and total influx at the level of whole plants. So whilst the effects of amino acids on some individual nitrate transporters are known, little information exists as to the effects of amino acids on the various transport systems contributing to the total nitrate acquisition of plants.
This study determined the contribution of nitrate cHATS, iHATS, and LATS to total influx in Lolium perenne L. over a range of external nitrate concentrations (0–2000 mmol m–3) sufficient to provide differences in the contribution of HATS and LATS to total nitrate influx. The effect of externally applying Gln to the roots on the activities of the individual nitrate transport systems and total influx was also determined. Lolium perenne was chosen as a grass species commonly found in pastures, the amide Gln was chosen because of its previously reported inhibitory effect on nitrate HATS when externally applied to roots (Quesada et al., 1997; Krapp et al., 1998; Vidmar et al., 2000). The hypotheses tested were: (1) that external application of Gln to roots of L. perenne inhibits the nitrate cHATS, iHATS, and LATS proportionally by the same extent and hence (2) that inhibition of total nitrate influx in L. perenne by Gln is independent of the degree to which individual transport systems contribute to total nitrate influx.
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Materials and methods |
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Experiment 1
Growth of plant material: Within a laminar flow cabinet, seeds of Lolium perenne L. cv. Parcour were vacuum infiltrated with sterile deionized water for 1 min then left to soak in the water for a further 2 h. The water was then poured off and the seeds surface-sterilized by soaking in 0.5% (v/v) peracetic acid for 15 min, seeds were then rinsed in several changes of sterile deionized water over a further 1 h period. Seeds were then transferred aseptically onto discs of Tygan mesh at a density of approximately 30 seeds per disc; individual discs were then placed over 1.0 l of deionized water within sterile culture vessels. Both the discs and the vessels have previously been described in detail (Thornton, 2001). Eighty culture vessels were placed within a controlled environment room (Conviron, Winnipeg, Canada) at 20 °C in the dark.
After 7 d, when the seed had germinated, the water in the vessels was replaced aseptically with a complete nutrient solution sterilized by passing it through a 0.2 µm cellulose nitrate filter (Whatman, Maidstone, UK). The nutrient solution was as described by Thornton and Bausenwein (2000) except all nitrogen was supplied to 40 vessels each either as 2 mol m–3 KNO3 (nitrate-grown plants) or as 1 mol m–3 (NH4)2SO4 (ammonium-grown plants). At the same time, a 16 h photoperiod of 550 µmol m–2 s–1 PAR at plant height was introduced. The growth-room air temperature was adjusted to 12 °C during the light periods and to 20 °C during the dark periods. The lower room temperature during the light period was to counteract radiant heat from the lights; the plants within their culture vessels experienced a constant temperature of 20 °C. Plants were grown under these conditions for 17 d, then in continuous light for a further 4 d. Throughout the growth period, nutrient solutions were renewed aseptically every 7 d. At the end of the growth period, the nutrient solutions were replaced aseptically with a ‘pretreatment solution’. Half of the nitrate- and ammonium-grown plants continued to receive a nutrient solution identical to that during the growth period, the plants in the remaining vessels received an equivalent solution with the addition of 5 mol m–3 glutamine (Gln). There were, therefore, four treatments: (1) nitrate-grown plants, (2) nitrate-grown plants plus Gln, (3) ammonium-grown plants, and (4) ammonium-grown plants plus Gln. Plants continued to receive continuous light throughout the pretreatment period. Both nitrate influx and net uptake (Delhon et al., 1995; Macduff et al., 1997) and expression of nitrate transporters (Lejay et al., 1999) can show diurnal variation. As subsequent harvesting and influx determination (see below) occurred over a 6.5 h period, continuous light during the later stages of growth and during the pretreatment period was introduced to minimize any effects of the timing of harvest on the measured nitrate influx. Notwithstanding this, all treatments were harvested sequentially in replicate blocks. After 24 h in the pretreatment solution, plants were harvested, and roots were separated from shoot material. The roots of each vessel were separated into two equal aliquots and immediately placed within polypropylene mesh bags (300 µm mesh, 5 cm width x 8 cm depth, Plastok Associates Ltd, Birkenhead, UK) secured with a plastic paper clip. Roots within these bags were subsequently used for determination of either their rate of nitrate influx or amino acid contents.
Measurement of nitrate influx: Groups of four bags, one from each treatment, were sequentially dipped in 300 cm3 of 1 mol m–3 CaSO4 at 20 °C for 1 min, an ‘influx solution’ at 20 °C for 5 min and, finally, 300 cm3 of 1 mol m–3 CaSO4 at 4 °C for 1 min. The ‘influx solution’ was based on the solution used by Lainé et al. (1993) to estimate net nitrate uptake by depletion and comprised of 10 mol m–3 MES-KOH (2-[N-morpholino]ethanesulphonic acid) buffer at pH 5.5 and KNO3 with a 15N abundance of 99 atom%, at either 10, 50, 100, 150, 250, 500, 1000, 1500, or 2000 mmol m–3. The volume of the ‘influx solution’ was 500 cm3 for nitrate concentrations up to and including 250 mmol m–3 and 300 cm3 for nitrate concentrations greater than 250 mmol m–3. These volumes ensured a less than 0.5% reduction in nitrate concentration of the influx solutions during the 5 min influx period. After the final dip in CaSO4 roots were removed from the mesh bags, oven-dried at 65 °C for 3 d, then ball-milled (Retsch, Haan, Germany). The total N and 15N concentrations of a weighed aliquot of the ball-milled root material were determined using a TracerMAT continuous flow mass spectrometer (Finnigan MAT, Hemel Hempstead, UK). The influx of 15N-nitrate was determined using the equations of Millard and Nielsen (1989).
With graphs of total nitrate influx versus nitrate concentration for each treatment, influx attributed to LATS was subtracted from the total influx, to reveal the influx attributed to HATS (see Results). The results of the HATS influx were fitted to Michaelis–Menten equations using the Enzyme Kinetics Module 1.1 of SigmaPlot 2002 for Windows Version 8.0 (SPSS UK Ltd, Woking, UK). Ammonium ions have previously been shown not to induce expression of genes encoding for nitrate iHATS (Gansel et al., 2001). Plants grown in nitrate were, therefore, assumed to have nitrate influx by HATS due to both cHATS and iHATS, whereas plants grown in ammonium were assumed only to have nitrate influx due to the cHATS. Therefore, for a given treatment (either minus or plus Gln), the difference in HATS nitrate influx, on a per mass of root basis, between plants grown in nitrate and those grown in ammonium was due to the iHATS.
Amino acid determination: Groups of four bags, one from each treatment, were dipped in 300 cm3 of 1 mol m–3 CaSO4 at 20 °C for 1 min. Roots were removed from the bags, weighed fresh, then frozen and stored at –80 °C. Amino acids in the roots were subject to ethanol extraction and purification by ion exchange chromatography as described by Thornton (2001). Amino acids in the resultant extracts were converted to their t-butyldimethylsilyl derivatives and determined by gas chromatography–mass spectrometry (GC–MS) as described by Millard et al. (1998).
Experiment 2
Plants of L. perenne cv. Parcour were grown in 40 sterile solution culture vessels, 20 receiving nitrogen as 2 mol m–3 KNO3 (nitrate-grown plants) and 20 receiving nitrogen as 1 mol m–3 (NH4)2SO4 (ammonium-grown plants), as previously described. At the end of the growth period, in four vessels of each treatment, the nutrient solutions were replaced aseptically with a ‘pretreatment solution’ containing a nutrient solution identical to that during the growth period with the addition of either 0, 5, 50, 500, or 5000 mmol m–3 Gln. After 24 h in the ‘pretreatment solution’ the roots of plants in a single vessel were used to determine the nitrate influx at nitrate concentrations of 250 and 2000 mmol m–3 as previously described, with the one exception that a 10 min influx labelling period was used. The two nitrate concentrations were chosen to give contrasting contributions of the HATS to total nitrate influx.
Statistics
The replication used to determine values of nitrate influx and amino acid concentrations is given in the appropriate Figure and/or Table legend. Differences between treatments were assessed by analysis of variance using GenStat® for Windows Sixth Edition, version 6.1.0.200
[EC]
, © Lawes Agricultural Trust (IACR-Rothamsted). For data of concentrations of amino acids it was necessary to log10(x+0.001) transform the data prior to analysis in order to stabilize the variance. With the data of total nitrate influx against nitrate concentration, linear contrasts were made over the nitrate concentration range 500–2000 mmol m–3 and evidence of interactions between either form of nitrogen supplied during growth or presence of Gln in the pretreatment solution with the effect of external nitrate concentration on nitrate influx were investigated. The regressions made were weighted according to 1/fitted values in order to stabilize the variance.
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Results |
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Experiment 1
For all treatments at external nitrate concentrations from 500 mmol m–3 to 2000 mmol m–3, positive linear relationships existed between the rate of total nitrate influx and the concentration of nitrate in the influx solution (P <0.001: Fig. 1A). The gradient of these lines was assumed to be influx attributable to the LATS. The gradient of the LATS were (mean ±SE): nitrate-grown plants=1.10±0.41 µmol g–1 FW h–1 (mol m–3)–1; nitrate-grown plants plus Gln=1.06±0.23 µmol g–1 FW h–1 (mol m–3)–1; ammonium-grown plants=0.79±0.11 µmol g–1 FW h–1 (mol m–3)–1, and ammonium-grown plants plus Gln=0.63±0.16 µmol g–1 FW h–1 (mol m–3)–1. In the nitrate concentration range 500–2000 mmol m–3, there was weak evidence of an interaction between the effect of nitrate concentration with form of nitrogen supplied upon nitrate influx (P=0.07: Fig. 1A), suggesting a greater rate of LATS influx in nitrate-compared with ammonium-grown plants. No such interaction between the effect of nitrate concentration with the presence of Gln in the pretreatment solution upon nitrate influx was observed (P >0.05: Fig. 1A). Using the gradients of the fitted lines (Fig. 1A), the rate of nitrate influx attributed to the LATS alone was calculated for all nitrate concentrations at which influx was determined (Fig. 1B), then subtracted from the total nitrate influx to reveal the influx attributed to HATS (Fig. 2).
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The rate of nitrate influx of the HATS against nitrate concentration exhibited Michaelis–Menten kinetics for plants grown in either nitrate or ammonium without Gln and for nitrate-grown plants plus Gln (Fig. 2; Table 1). For ammonium-grown plants plus Gln, a linear relationship (y=0.000048x) was sufficient to explain the data (Fig. 2). In plants that did not receive Gln in the pretreatment solution (Fig. 2A), nitrate influx by iHATS was 2.6-fold greater than that by cHATS, each respectively contributing 72% and 28% of the HATS Imax (Table 1; Fig. 2A). The Imax attributed to HATS was reduced overall by 66% by the presence of Gln in the pretreatment solution (P < 0.05: Fig. 2; Table 1). This was achieved through an 84% reduction in cHATS (at 2000 mmol m–3 nitrate) and a 59% reduction in iHATS (P <0.05 in each case, Fig. 2; Table 1). The Michaelis constant Km of the HATS (cHATS plus iHATS) was unaffected by the addition of Gln (P > 0.05: Fig. 2; Table 1).
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The contribution of the HATS to the total plant nitrate influx reduced as the external nitrate concentration increased (Fig. 3A). For nitrate-grown plants, the contribution of HATS to total influx was 88% at 25 mmol m–3 nitrate, this had fallen to 79% at 250 mmol m–3 nitrate and only 44% at 2000 mmol m–3 nitrate (Fig. 3A). In ammonium-grown plants that only expressed cHATS, the contribution of HATS to total influx was lower than in nitrate-grown plants that possessed both cHATS and iHATS (Fig. 3A). However, the contribution of HATS to the total influx of ammonium-grown plants also fell with increasing external nitrate concentrations, from 70% at 25 mmol m–3, to 56% at 250 mmol m–3 and only 23% at 2000 mmol m–3 (Fig. 3A).
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The inhibition of the total nitrate influx by Gln was also dependent on the nitrate concentration (Fig. 3B). The addition of Gln caused greater inhibition of nitrate influx in ammonium- compared with nitrate-grown plants, especially at lower nitrate concentrations (Fig. 3B). The inhibition of nitrate influx by Gln in both nitrate and ammonium-grown plants was reduced as nitrate concentration increased (Fig. 3B), being 52% and 74%, respectively, at 25 mmol m–3 nitrate, but only by 30% and 34% at 2000 mmol m–3 nitrate (Fig. 3B).
The inhibition of total nitrate influx by Gln was positively related to the contribution of HATS to the total influx (Fig. 3C). This relationship was linear for ammonium-grown plants (Fig. 3C), i.e. plants in which cHATS alone contributed to the HATS influx. For nitrate-grown plants, deviation from linearity occurred, but especially when HATS was making its largest contributions to total influx (Fig. 3C); it is possible that this deviation was due to changes in the relative contribution of cHATS and iHATS to the HATS. At a given contribution of HATS to total influx, a greater inhibition of total influx by Gln in ammonium-grown compared with nitrate-grown plants was observed (Fig. 3C) this reflects the stronger inhibition by Gln on cHATS compared with iHATS.
Experiment 2
In general, as the concentration of Gln in the pretreatment solution was increased it resulted in progressively greater inhibition of nitrate influx (Fig. 4). An exception to this was for the influx of nitrate at 2000 mmol m–3 in plants grown with ammonium as their source of nitrogen, where inhibition from the appropriate control only occurred at the greatest Gln concentration supplied (Fig. 4D). The concentration of Gln required to inhibit nitrate influx was dependent on both the form of N on which the plants had grown and on the concentration of nitrate at which influx was determined (Fig. 4). For plants grown both on nitrate and ammonium, the concentration of Gln required to reduce nitrate influx significantly was lower for the influx of nitrate at 250 mmol m–3 compared with influx at 2000 mmol m–3 (P <0.05 in both cases, Fig. 4). This is consistent with the applied Gln inhibiting nitrate HATS but not LATS (Figs 1, 2) as the greater sensitivity to applied Gln occurred when HATS contributed a greater proportion of total nitrate influx. When nitrate influx was measured at 250 mmol m–3, the concentration of Gln required to reduce influx significantly was lower in ammonium-grown plants compared with nitrate-grown plants (P <0.05 in both cases, Fig. 4A, B). This is again consistent with previous results (Fig. 2) which indicated Gln inhibited cHATS to a greater extent than iHATS.
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The total concentration of amino acid nitrogen was greater in roots of plants supplied ammonium compared with those supplied nitrate, but only when Gln was not included in the pretreatment solution (P <0.05; Table 2). This was mainly brought about by changes in the concentration of Asn, as ammonium-grown plants without the addition of Gln had a 35-fold increase in root Asn concentration compared with the equivalent nitrate-grown plants (P <0.001; Table 2). In both nitrate- and ammonium-grown plants, the addition of Gln to the pretreatment solution did result in a greatly increased concentration of Gln in the root tissue (P <0.001). The addition of Gln also resulted in increased concentrations of Asn, Asp, and Gaba in the roots of both nitrate- and ammonium-grown plants (P <0.05 in all cases; Table 2) and of Glu and Ala in roots of nitrate-grown plants only (P <0.05 in both cases; Table 2). A significant increase in the concentration of total amino acid nitrogen in roots, resultant from the presence of Gln in the pretreatment solution only occurred in plants supplied with nitrate (P <0.05; Table 2).
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Discussion |
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Although the Imax for the HATS of L. perenne plants grown on nitrate without the addition of Gln (1.88 µmol g–1 FW h–1) is at the lower end of reported values for a range of plant species (Forde and Clarkson, 1999), it is larger than previously reported values for L. perenne (0.5–3.0 µmol g–1 DW h–1, (Louahlia et al., 2000). Current results tentatively support the belief that the LATS, in addition to HATS, comprises both constitutive and inducible components (Huang et al., 1996; Zhou et al., 1998).
In the current study, the external application of Gln did increase the root concentration of Gln. The increase was not, however, exclusive to Gln, and the effects of Gln application on nitrate influx cannot be ascribed to Gln alone. The close association between the root concentrations of Gln and Asn observed in Zea mays (Lee et al., 1992) was not apparent in L. perenne. Whilst the current results agree with the general concept of increased concentrations of root amino acids inhibiting nitrate uptake/influx in L. perenne this is not universally observed for all species. In Brassica napus, whilst an inverse relationship between root amino acids and nitrate influx/uptake can occur, this is not the case in all situations (Lainé et al., 1995; Faure-Rabasse et al., 2002).
The fact that the nitrate HATS influx became fully saturated with increasing nitrate concentrations, whilst over the same concentration range the rate of LATS influx did not, resulted in the contribution of HATS to total nitrate influx being dependent on the external nitrate concentration, increasing as external nitrate concentration decreased. Currently, L. perenne plants were grown supplied 2 mol m–3 N, with nitrate influx measured following an instantaneous switch to an equal or range of lower concentrations for 5 min. The possibility exists that L. perenne plants, experiencing slower change in nitrate concentration, up- and down-regulate their nitrate HATS and LATS to different degrees.
In soil, nutrients including nitrate are heterogeneous in nature, varying both spatially and temporally (Afzal and Adams, 1992; Jackson and Caldwell, 1993; Schmidt and Stewart, 1997). If, under field conditions, an individual L. perenne plant experiences a range of nitrate concentrations, the contribution of HATS to total nitrate influx will also vary. In addition to nitrate heterogeneity at a given site, differences in average nitrate concentrations occur between sites. In a fertilized sward, Louahlia et al. (2000) showed that soil nitrate concentrations were sufficient to saturate the nitrate HATS of L. perenne most of the time; total nitrate influx under these conditions making significant use of the LATS. However, in more extensive grasslands, soil nitrate concentrations are lower compared with fertilized sites. Shand et al. (2002) reported that the nitrate concentration of soil solution, obtained by centrifugation, from an extensive grass pasture was in the order of 20 mmol m–3. Current results show that, under these conditions, the HATS contribution to total nitrate influx by L. perenne would be greater than 88%. The contribution of HATS to total nitrate influx of L. perenne will therefore differ between plants growing on sites of contrasting fertility.
It has been shown that the cHATS itself may be induced by nitrate (Aslam et al., 1992; Kronzucker et al., 1995), therefore, in the present study, for plants previously exposed to nitrate the estimated iHATS will comprise of iHATS plus the induced portion of the cHATS. Notwithstanding this, with the external addition of Gln a relatively greater inhibition of cHATS compared with iHATS was observed. This contrasts with the effect of fluctuations in the external nitrate concentration where a relatively greater control of iHATS compared with cHATS is observed (Aslam et al., 1992; Kronzucker et al., 1995). One possible function of the cHATS is considered to be cell homeostasis, allowing external nitrate, when it is available, to be absorbed in sufficient quantities to induce the nitrate iHATS (Behl et al., 1988; Forde and Clarkson, 1999). Current results would suggest that, in plants experiencing high external concentrations of Gln, the capability of the cHATS to fulfil this role might be compromized. This may be relevant for plants growing under field conditions, especially if, in addition to Gln, other amino acids also inhibit the cHATS.
Current results clearly indicate that Gln applied externally to the roots of L. perenne inhibited the nitrate HATS but not LATS. This was manifest in two ways: (1) the inhibition of total nitrate influx by 5 mol m–3 Gln increased as external nitrate concentration was reduced and (2) as the external nitrate concentration was reduced the concentration of Gln required to cause significant inhibition of total nitrate influx was also reduced. In consequence, the inhibition of total nitrate influx by Gln was greater as the HATS contribution to total nitrate influx increased. This implies that a given concentration of Gln in the soil solution would cause relatively greater inhibition of the total nitrate influx to L. perenne, both (1) in an individual plant experiencing a transient lower concentration of nitrate and (2) in plants growing in extensive systems compared with plants growing in more nitrate-rich environments. In L. perenne plants previously exposed to nitrate, 50 mmol m–3 Gln was sufficient to inhibit the influx of 250 mmol m–3 nitrate significantly. At the lower nitrate concentrations 
20 mmol m–3 reported in extensive grasslands (Shand et al., 2002), Gln concentrations considerably less than 50 mmol m–3 would be expected to cause an inhibition of nitrate influx of L. perenne. Coupling this with the fact that soils from more extensive grassland systems contain greater concentrations of water-extractable amino acids than improved grasslands (Streeter et al., 2000), would indicate that such inhibition of nitrate influx by soil solution amino acids is probable under certain field conditions. Other situations leading to low soil solution concentrations of nitrate, and hence increased sensitivity of nitrate influx to external amino acids, can be coupled to increases in soil solution amino acid concentrations. For example, waterlogging of a heathland soil reduced concentrations of nitrate concomitant with increased amino acid concentrations (Schmidt and Stewart, 1997).
The original hypotheses tested were disproved; (1) that the external application of Gln to roots of L. perenne differentially inhibited the various nitrate transport systems; cHATS was inhibited to a greater extent than iHATS whilst LATS was unaffected; and (2) that inhibition of total nitrate influx in L. perenne by Gln was positively related to the contribution of the nitrate HATS to the total nitrate influx.
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Acknowledgements |
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I thank Mrs M Tyler for skilled technical assistance, Dr D Elston and Miss EI Duff of the Biomathematics and Statistics Scotland (BioSS) for statistical advice, the Analytical Group of the Macaulay Institute for 15N analysis, the Scottish Executive Environmental and Rural Affairs Department for funding this work, and anonymous referees for helpful comments.
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