JXB Advance Access originally published online on March 17, 2006
Journal of Experimental Botany 2006 57(6):1333-1340; doi:10.1093/jxb/erj110
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RESEARCH PAPER |
Glutamine transport and feedback regulation of nitrate reductase activity in barley roots leads to changes in cytosolic nitrate pools
1College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, PR China
2Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK
3Biological Chemistry Division, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK
* To whom correspondence should be addressed. E-mail: tony.miller{at}bbsrc.ac.uk
Received 10 August 2005; Accepted 28 December 2005
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Abstract |
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The size of tissue amino acid pools in plants may indicate nitrogen status and provide a signal that can regulate nitrate uptake and assimilation. The effects of treating barley roots with glutamine have been examined, first to identify the transport system for the uptake of the amino acid and then to measure root NR activity and cellular pools of nitrate. Treating N replete roots with glutamine elicited a change in the cell membrane potential and the size of this response was concentration dependent. In addition, the size of the electrical change depended on the previous exposures of the root to glutamine and was lost after a few cycles of treatment. Whole root tissue pools of glutamine and phenylalanine increased when roots were incubated in a nutrient solution containing 10 mM nitrate and 1 mM glutamine. Treating roots with 1 mM glutamine increased cytosolic nitrate activity from 3 mM to 7 mM and this change peaked after 2 h of treatment. Parallel measurements of root nitrate reductase activity during treatment with 1 mM glutamine showed a decrease. These measurements provide evidence for feedback regulation on NR activity that result in changes in cytosolic nitrate activity. After 6 h in glutamine both root NR activity and cytosolic nitrate activity returned to pretreatment values, while tissue concentrations of glutamine and phenylalanine remained elevated. The data are discussed in terms of the mechanisms that are most likely to be responsible for the changes in cytosolic nitrate.
Key words: Feedback regulation, glutamine transport, Hordeum vulgare L., nitrate reductase, nitrogen status
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The uptake and assimilation of nitrate by roots is known to change with supply in a manner that suggests that the nitrogen (N) status of plants is somehow sensed and can feedback to regulate these processes. As nitrate is assimilated via conversion to nitrite, ammonium, and then into amino acids, it has been suggested that the internal pools of amino acids within plants may indicate N status by providing a signal that can regulate N uptake and assimilation by the plant. In bacteria, cellular glutamine (Gln) concentrations signal N status through the PII protein and a similar model has been proposed for plants (Moorhead and Smith, 2003
).
Nitrate and ammonium uptake by plant roots can be negatively influenced by the external supply of amino acids or the tissue concentrations of amino acids (Lee et al., 1992; Muller et al., 1995; Rawat et al., 1999), but the nature of this feedback relationship is complicated and may not be present in some species (e.g. Brassica napus; Lainé et al., 1995). Glutamine has been found to be effective in inhibiting the expression of inducible high affinity nitrate transporters (Quesada et al., 1997; Krapp et al., 1998; Vidmar et al., 2000). Both nitrate-induced influx and transporter transcript abundance were decreased simultaneously in root tissue treated with exogenously applied amino acids (Vidmar et al., 2000). As amino acids can be inter-converted within plant tissues, chemical inhibitors of the conversion steps were used to identify Gln, rather than glutamate, as being responsible for down-regulating nitrate transporter expression (Vidmar et al., 2000). Physiological measurements have identified several different plant nitrate uptake systems (reviewed by Crawford and Glass, 1998) and feedback regulation by Gln on these uptake systems appears to be different. Lolium perenne plants grown in sterile culture and treated for 24 h with Gln showed decreased activity of the high, but not low, affinity nitrate uptake system (Thornton, 2004).
In this paper, it has been investigated if cellular nitrate pools might be influenced by short-term treatment with exogenously supplied Gln. One underlying assumption for these experiments is that externally supplied amino acids can be directly taken up by roots and can directly influence the internal pools of these molecules. Uptake of radiolabelled amino acids by roots has been shown to occur (Watson and Fowden, 1975; Soldal and Nissen, 1978) and this treatment has been shown to increase some tissue pools of amino acids (Lee et al., 1992; Vidmar et al., 2000; Thornton, 2004). As the activity of plasma membrane transporters in individual cells can be detected by electrophysiological measurements (Miller et al., 2001) single barley root cells have been assayed for their amino acid transport activity by treating roots with Gln. The time-course of whole root amino acids pools was also measured to determine how treatment with Gln can influence these metabolite pools. As these pools can negatively feedback on nitrate uptake it has been tested if cellular pools of nitrate change under these conditions. Nitrate-selective microelectrodes were used to measure the cytosolic activities during treatment with Gln. Nitrate assimilation is an important sink for cytosolic nitrate so nitrate reductase (NR) activity of the barley roots was also measured during Gln treatment. These results suggest that feedback regulation may be occurring, not only by changing the activity of nitrate transporters but also by post-translational modification of NR.
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Materials and methods |
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Plant material
Seeds of barley (Hordeum vulgare L. cv. Klaxon) were germinated in the dark on filter paper soaked in CaS04 (0.2 mM) for 48 h. Seedlings were transferred to hydroponic culture (one-quarter Hoagland's solution) containing 10 mM nitrate, supplied as Ca(N03)2, in plastic containers that maintained the roots in the dark (Zhen et al., 1991
). The plants were allowed to grow for 3 d in a controlled environment cabinet at 20 °C with a 16 h photoperiod, commencing at 01.00 h, supplied at a photon flux density of 450–480 µmol m–2 s–1. Unless stated otherwise, all the Gln feeding experiments began 5–6 h into the light period.
Ethanolic extraction and amino acid determination for barley roots
Frozen root tissue (60 mg fresh weight) was ground in liquid N2 and then in 1.4 ml absolute ethanol and the homogenate transferred to a 2 ml microcentrifuge tube on ice. The homogenate was vigorously vortexed and 1 ml rapidly transferred to a tube containing 1 ml of an aqueous solution of the internal standard for amino acid determination (120 µM 
-aminobutyrate, which was undetectable in root extracts). The resulting mixture was immediately heated at 70 °C for 5 min, incubated at 4 °C overnight and pelleted by centrifugation for 10 min at 14 000 rpm at 4 °C. Next, 0.2 ml of the supernatant was vacuum-dried and the residue redissolved in 1.2 ml H2O. The solution was clarified by centrifugation for 20 min at 14 000 rpm at 4 °C and introduced through a 0.2 µm syringe filter into autosampler vials. The vials were loaded into Waters Alliance® (Waters Corporation, Milford, MA01757, USA) high performance liquid chromatography equipment and amino acids quantified in 10 µl by automated precolumn derivatization with o-phthaldialdehyde followed by fluorimetric detection, using a method modified from that described previously (Novitskaya et al., 2002). Peaks were quantified by reference to a linear standard curve generated using mixed amino acids. The amounts of amino acids were, in all cases, within the range of the standards, which varied from 0 to 250 pmol 10 µl–1. In total 17 different amino acids were measured, these included alanine, arginine, aspartate, asparagine, 
-aminobutryate, Gln, glutamate, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tyrosine, and valine.
Microelectrode measurements
All microelectrode measurements were made on roots of intact barley seedlings as described previously (Zhen et al., 1991). For measurements of the membrane potential changes associated with Gln transport, single barrel microelectrodes were used. These tips were pulled using single-barrel borosilicate glass with an inner diameter of 0.6 mm and an outer diameter 1.0 mm (Hilgenberg GmbH, Malsfeld, Germany), but without the final twisting step (Zhen et al., 1991). For single-barrel measurements of membrane potential the electrode tip was located in cortical cells as recordings of longer duration were more easily obtained from these cells. Cortical cell impalements were defined by the electrical recording of the second cell encountered by the tip moving through the tissue. Recordings from epidermal cells were defined as the first layer of cells encountered by the tip. Similar changes in membrane potential and cytosolic nitrate were obtained from both epidermal and cortical root cells when treated with Gln. Nitrate-selective microelectrodes were prepared, calibrated, and used as described previously (Zhen et al., 1991; van der Leij et al., 1998). Barley seedlings were grown hydroponically in a full nutrient solution containing 10 mM nitrate for 2 d and under these N-replete conditions two populations of nitrate electrode measurements, cytosol and vacuole were obtained (Zhen et al., 1991; van der Leij et al., 1998).
Nitrate reductase assays
For these assays all barley seedlings were 8 h into the light period in order to minimize any possible effects of diurnal changes in NR activity (Huber et al., 1992). Root tissue was analysed for NR activity as described previously (Kuo et al., 1982) with minor modifications. Root material (0.6 g) was removed from hydroponically grown barley plants, rapidly weighed, and ground to a fine powder with a pestle and mortar under liquid nitrogen. Extraction buffer (1 ml), comprising 0.25 M TRIS, pH 8.5, 10 mM DTT, 20 µM leupeptin, 10 µM FAD, and either 10 mM Na4EDTA or 10 mM MgCl2, was added and the frozen powder was allowed to thaw. After further brief grinding the resulting homogenate was centrifuged at 13 000 rpm for 10 min at 4 °C and the supernatant carefully aspirated and used without any further purification. Assays were performed in 250 µl reaction volumes of potassium phosphate buffer, (0.05 mM, pH 7.5) containing 10 mM KNO3, 1 mM NADH, and either 10 mM Na4EDTA or 10 mM MgCl2. Reactions were started by the addition of root supernatant and the tubes were incubated at 25 °C for 20 min in the dark. Either supernatant or KNO3 were omitted from the blank tubes and calibration curves were prepared without nitrate or NADH, but with final concentrations of KNO2 from 0–15 µM. The reaction was terminated by the addition of 50 µl of zinc acetate (1 M). The tubes were shaken and mixed vigorously and left for 20 min to oxidize excess NADH. Supernatant was added to the blanks and nitrite was estimated by adding 250 µ1 each of sulphanilamide (1% in 3 M HCl) and N-1-naphthylethylenediamine (0.02%, w/w). The solutions were clarified by centrifugation and the OD read at 540 nm after 15 min. Each treatment was repeated six to eight times, both in the absence and in the presence of MgCl2, using different plants for each measurement.
Measurement of nitrate concentration in roots
Total nitrate was measured as described previously (Gilliam et al., 1993) with nitrate extracted from the tissue by boiling fresh or frozen root material (20 mg) in distilled water (400 µl) for 20 min. NR (Aspergillus nidulans) was dissolved in deionized water (5 U ml–1) and allowed to reactivate for 30 min at 23 °C. The stock was diluted to 3.5 U ml–1 with 0.14 M KHPO3 buffer (pH 7.5) and stored on ice. Assays were performed for 1 h at room temperature in final volumes of 500 µl of assay medium comprising 0.056 M KHPO3 buffer (pH 7.5), FAD (2.5 µM), NADPH (100 µM) and activated NR (0.06 U ml–1). The reactions were started by the addition of 2.5 µl of root sample or NaNO3 solutions of concentrations ranging from 0 to 100 mM for the standard curve. The reactions were terminated by the addition of 500 µl each of sulphanilamide (1% in 3 M HCl) and N-1-naphthylethylenediamine (0.02%, w/w) and after standing for 30 min to allow the colour to develop, the nitrite concentrations in the samples were measured spectrophotometrically at 540 nm. Each measurement was made six times, using different plants on each occasion.
15N-nitrate influx assay
For measuring 15N enrichment, three roots were incubated in 50 ml nutrient solution containing 0.5 mM 99% atom 
for 5 min at 18 °C. The roots had been previously, and were subsequently, rinsed for 1 min in 0.1 mM CaSO4 solution and were finally weighed before being plunged into liquid nitrogen. The frozen tissue was then ground into a fine powder using a pestle and mortar and this was then dried for 3–4 d at 60 °C. The 15N/14N ratio of each single dried root was measured using an Isotope Ratio Mass Spectrometer (model Integra CN, PDZ Europa, Crewe, UK). The delta (
)-15N was calculated as the ratio of the sample in excess of air divided by the standard atmospheric 15N/14N ratio atom % (Mariotti, 1984). Samples with positive ()-15N values are enriched in 15N content with respect to the atmospheric standard.
Replication and statistical analysis of data
Unless stated otherwise all experiments were repeated with 6–8 replicates each using a different seedling. Means and SD were calculated using Excel (Microsoft 2000).
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Results |
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Treating barley roots with Gln elicited a change in the membrane potential of root cells and this response could be repeated over several cycles (Fig. 1A). Glutamine elicited a change in membrane potential to a less negative value (a depolarization) and the size of the response to Gln treatment depended on the concentration applied to the root. Figure 1A shows the effects of repeated applications of Gln to the same barley root cortical cell. When the root was treated repeatedly with increasing concentrations of Gln eventually the response became smaller. For example, in Fig. 1A, the repeated treatments finally resulted in almost no response to an application of 2 mM Gln. To gather data for the kinetic characterization of the root cell amino acid transport system it was necessary to apply Gln as single pulses to roots that had not previously been exposed to the amino acid. At least two Gln uptake systems could be identified and Michaelis–Menten kinetics was fitted to the lower range transport system that appeared to saturate between 2 and 4 mM Gln. This amino acid transport had a Km=1.2 mM and Vmax=56 mV (Fig. 1B). There were not enough data points to characterize the putative second transport system adequately (Fig. 1B).
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Root amino acid pools were measured to check that treatment of the N-replete barley (growing in 10 mM nitrate) with Gln resulted in an increase in tissue amino acids. Figure 2 shows some of the amino acid pools measured in roots. Glutamine concentrations in the roots increased significantly by 3-fold after 4 h and were 2-fold after 10 h of treatment with 1 mM Gln and there was a decrease in the glutamate pool (see Fig. 2A). Figure 2B shows some changes in pools of phenylalanine, asparagine, and aspartate. Phenylalanine had significantly increased after 6 h and the other two amino acids decreased during the 10 h treatment. Tissue concentrations of other amino acids either showed small but significant decreases (threonine, serine) or no change during the 10 h treatment with Gln (data not shown). After 10 h treatment with Gln, of the tissue concentration of amino acids that were measured, only Gln and phenylalanine remained significantly increased.
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Nitrate-selective microelectrodes were used to investigate the effect of Gln treatment on root cell cytosolic nitrate pools. Treatment with 0.5 mM Gln did not change cytosolic nitrate activity (Fig. 3A), but application of 1 mM Gln resulted in an increase (Fig. 3B). As shown in Fig. 1A, feeding with both 0.5 and 1 mM Gln resulted in small depolarizations of the resting membrane potential of the cell (Fig. 3A, B). Treating roots continuously with 1 mM Gln did not give any longer term changes in resting membrane potential (Fig. 4A) and, as reported previously, more negative potentials were consistently measured in cortical cells when compared with epidermal cells (Zhen et al., 1991
). Cytosolic nitrate activity increased to a maximum of 7 mM after about 2 h, but by 6 h the activity was restored to around 3 mM (Fig. 4B). Similar patterns in these single cell responses were observed for both cortical and epidermal cells (Fig. 4A, B). Treating roots with Gln for 6 h did not significantly change whole root tissue nitrate (Fig. 4C), although at 6 h the mean value for the total tissue nitrate concentration was 12% higher than at the start of the experiment this change was not significantly different. This whole root tissue value represents a measure of vacuolar stored nitrate (Zhen et al., 1991; van der Leij et al., 1998).
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These changes in cytosolic nitrate may result from changes in uptake and as high affinity transport is particularly sensitive to tissue pools of Gln (Thornton, 2004
) influx was measured 2 h and 6 h after applying the amino acid to barley roots. Table 1 shows the nitrate influx rate for barley roots measured after 2 h and 6 h treatment with 1 mM Gln, and these values were not significantly different.
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Barley plants can assimilate nitrate in the root and, therefore, have considerable root NR activity under N-replete (10 mM nitrate supply) conditions. Nitrate assimilation must be a sink for cytosolic nitrate and, therefore, the activity of NR was measured after treatment with 1 mM Gln. The activity of NR is regulated by reversible Mg-dependent phosphorylation, which has been shown to be responsible for the observed light-regulated fluctuations in its activity in leaf tissue (Tucker et al., 2004
). Phosphorylation enables a 110 kDa NR inhibitory protein to interact with the enzyme, and suppress its activity (reviewed by Kaiser and Huber, 2001). Consequently, in the presence of MgCl2, only the NR that is active in vivo is measured, whereas in the absence of MgCl2, a measurement of the total activity is obtained. For each experiment, batches of plants were transferred to one-quarter Hoagland's medium containing 1 mM glutamine and individual plants were harvested at time points between 0 h and 6 h after transfer and their roots immediately snap frozen in liquid nitrogen. NR activity was measured in the tissue, both in the absence and in the presence of MgCl2 throughout the extraction and assay procedures. In the absence of MgCl2 (Fig. 5A), no significant change in NR activity was observed over the time period, although there was a slight (12%) decrease in activity up to 3 h, followed by a progressive recovery to a level at 6 h equal to that at time 0 h. In the presence of MgCl2 (Fig. 5B), there was only 25% of the NR activity measured in its absence at time 0 h. This was further reduced to 10% after 2 h exposure to glutamine, reducing the activity to 45% of that measured at 0 h. After 3 h there was a recovery of activity and, at 6 h, levels had almost returned to those measured at the start of the experiment.
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These parallel measurements of root NR activity showed that the phosphorylation status, responsible for activation of the NR (Kaiser and Huber, 2001
; Kaiser et al., 2002) of the enzyme changed following a time-course that was very similar to the changes in cytosolic nitrate (see Fig. 5A, B). |
Discussion |
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The fact that repeated cycles of Gln application to barley roots resulted in a saturation of the electrophysiological response is important for two reasons. Firstly, as already discussed, the kinetics of the transport system could only be measured by single applications to roots that had not previously been treated with Gln (Fig. 1A). Secondly, this response suggests that there might be some type of saturation of the Gln uptake system that may result in decreased accumulation of the amino acid in the root tissue under these conditions. For this paper, N-replete barley seedlings, growing in 10 mM nitrate have been used to identify cytosolic nitrate microelectrode measurements (van der Leij et al., 1998
), so it was important to show that there was still a significant increase in root tissue Gln after external treatment with the amino acid (Fig. 2). The root tissue amino acid concentrations are similar to those reported previously for barley treated with 1 mM Gln (Vidmar et al., 2000). After treatment with 10 mM Gln maize roots had almost 10-fold higher concentrations of the more abundant amino acids when compared with those values shown in Fig. 2 (Lee et al., 1992). The previously reported positive correlation between tissue Gln and asparagine for maize roots (Lee et al., 1992) was not observed for barley roots (Fig. 2B). After 2 h of treatment with Gln there were significant alterations in root tissue amino acid pools (Fig. 2) and these could provide a feedback signal for the decreased electrical activity of the Gln transport system (Fig. 1A). More detailed measurements may be necessary to identify the relationship between N status of plants and the electrical activity of the Gln transport system. The barley root Gln transport system has a Km of 1.2 mM (Fig. 1B) and this affinity value seems an order of magnitude higher than most estimates of amino acid concentrations in the soil solution (Jones et al., 2002). The affinity of the amino acid transport system for Gln may depend on the N status of the plant or this may not be the usual transported substrate for this protein, for example, it may be a nitrate transporter (Zhou et al., 1998). The sizes of the Gln-elicited changes in root membrane potential are similar to those reported for oat coleoptiles fed with other amino acids (Kinraide et al., 1984). However, in coleoptiles, the shape and duration of the change in membrane potential depended on the net charge of the amino acid (Kinraide et al., 1984).
Supplying barley plants with 1 mM Gln altered root NR activity. The activity of NR is regulated by reversible Mg-dependent phosphorylation, which has been shown to be responsible for the observed diurnal fluctuations in its activity in leaf tissue (Kaiser et al., 1993). These changes in NR activity could provide a mechanism for the measured changes in cytosolic nitrate associated with treatment with 1 mM Gln. Microelectrode measurements demonstrated (Fig. 4B) that root cytosolic nitrate concentration underwent a transient increase in response to treatment with 1 mM Gln. However, total tissue nitrate concentration (vacuolar-stored nitrate) remained almost constant under these conditions (Fig. 4B). Measurements on Arabidopsis leaf cells (Cookson et al., 2005) and the results reported here would suggest that there is a strong dependence of cytosolic nitrate activity and NR activity. This finding does not agree with a recent paper reporting that nitrate uptake at the plasma membrane in tobacco leaves can supply an increased rate of nitrate reduction (Lea et al., 2004). The tobacco work used cut leaf pieces from NR mutants immersed in 1–50 mM KNO3 solutions for 5 h in darkness with nitrite excretion giving a measure of NR activity. However, in common with the microelectrode measurements, Lea et al. (2004) found that vacuolar-stored nitrate was not readily accessible to NR.
Although the changes in root NR activity (Fig. 5A, B) show a similar pattern and time-course to the microelectrode measured changes in cytosolic nitrate activity (Fig. 4B), this does not prove a direct relationship between these two data sets. The interpretation of these data sets is complicated by the fact that one was obtained from whole roots while the other was measured in single cells. Changes in cytosolic pH could provide a mechanism that explains the effects of 1 mM Gln on whole root NR activity (Fig. 5). Glutamine is uncharged but if it is co-transported with protons (H+) this may result in an acidification of the cytosol. Such a change in cytosolic pH could result in increased NR activity (Kaiser and Huber, 2001; Kaiser et al., 2002), but an acidification of this compartment would also hyperpolarize the membrane potential of the cell as the activity of the plasma membrane H+ pump would be stimulated (Portillo, 2000; Ullrich and Novacky, 1990). No such changes were observed in resting membrane potential during the glutamine feeding experiments (Figs 3, 4A). It is believed that the lack of any changes in the cell membrane potential make an acidification of cytosolic pH an unlikely mechanism to explain the changes in NR activity (Fig. 5), although this might be worthy of further study.
Treatment with Gln may also alter membrane nitrate fluxes that could result in a transient increase in cytosolic nitrate activity. However, no significant change in high affinity nitrate influx was found at the plasma membrane after 2 h and 6 h of treatment with Gln (Table 1). Lolium perenne plants treated with Gln showed that the main inhibitory effect of Gln was on the contribution of the high affinity transport system (HATS) to the total influx (Thornton, 2004). Although in these experiments with barley the plants had previously been grown in 10 mM nitrate and so HATS had a minor contribution to uptake, there may have been some changes in the low affinity transport system. Nitrate efflux at the plasma membrane was not measured, but a decrease in this flux could also result in an increase in cytosolic nitrate activity. The vacuolar pool of nitrate does not show any significant changes during the time-course of 6 h treatment with Gln (Fig. 4B) suggesting that altered tonoplast fluxes are unlikely to be responsible for the increase in cytosolic nitrate. However, whole tissue analysis may not be sufficiently sensitive to detect more subtle changes in fluxes across the tonoplast.
In conclusion, it has been shown that increases in cellular pools of Gln can alter NR activity by regulating the phosphorylation status of the enzyme. These changes in NR activity may be responsible for a transient increase in cytosolic nitrate.
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Acknowledgements |
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Rothamsted Research is grant-aided by the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK. Funding from the National Basic Research Program (973) of China (grant No. 2005CB120903) and the National Natural Science Foundation of China (grant No. 30390082) also supported this work. Thanks to Dr Josirley Carvalho for her advice and help with the extraction and measurement of the amino acids and to Sue Smith and Maureen Birdsey (Agriculture and Environment Division, Rothamsted Research) for their help with the 15N analysis.
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Footnotes |
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Abbreviations: Gln, glutamine; N, nitrogen; NR, nitrate reductase.
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References |
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A. J. Miller, X. Fan, M. Orsel, S. J. Smith, and D. M. Wells Nitrate transport and signalling J. Exp. Bot., July 1, 2007; 58(9): 2297 - 2306. [Abstract] [Full Text] [PDF]
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X. Fan, L. Jia, Y. Li, S. J. Smith, A. J. Miller, and Q. Shen Comparing nitrate storage and remobilization in two rice cultivars that differ in their nitrogen use efficiency J. Exp. Bot., May 1, 2007; 58(7): 1729 - 1740. [Abstract] [Full Text] [PDF]
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 M. Orsel, F. Chopin, O. Leleu, S. J. Smith, A. Krapp, F. Daniel-Vedele, and A. J. Miller Characterization of a Two-Component High-Affinity Nitrate Uptake System in Arabidopsis. Physiology and Protein-Protein Interaction Plant Physiology, November 1, 2006; 142(3): 1304 - 1317. [Abstract] [Full Text] [PDF]
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