JXB Advance Access published online on December 14, 2006
Journal of Experimental Botany, doi:10.1093/jxb/erl238
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RESEARCH PAPER |
Nitrate supply affects ammonium transport in canola roots
1School of Earth and Geographical Sciences, M087, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009
2School of Mathematics and Physics, University of Tasmania, GPO Box 252-21, Hobart, Tasmania 7001
* To whom correspondence should be addressed. E-mail: olgab{at}cyllene.uwa.edu.au
Received 10 April 2006; Revised 17 October 2006 Accepted 18 October 2006
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Abstract |
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Plants may suffer from ammonium (NH4+) toxicity when NH4+ is the sole nitrogen source. Nitrate (NO3–) is known to alleviate NH4+ toxicity, but the mechanisms are unknown. This study has evaluated possible mechanisms of NO3– alleviation of NH4+ toxicity in canola (Brassica napus L.). Dynamics of net fluxes of NH4+, H+, K+ and Ca2+ were assessed, using a non-invasive microelectrode (MIFE) technique, in plants having different NO3– supplies, after single or several subsequent increases in external NH4Cl concentration. After an increase in external NH4Cl without NO3–, NH4+ net fluxes demonstrated three distinct stages: release (
1), return to uptake (2), and a decrease in uptake rate (3). The presence of NO3– in the bathing medium prevented the 1 release and also resulted in slower activation of the 3 stage. Net fluxes of Ca2+ were in the opposite direction to NH4+ net fluxes, regardless of NO3– supply. In contrast, H+ and K+ net fluxes and change in external pH were not correlated with NH4+ net fluxes. It is concluded that (i) NO3– primarily affects the NH4+ low-affinity influx system; and (ii) NH4+ transport is inversely linked to Ca2+ net flux. Key words: Ammonium toxicity, Brassica napus, Ca2+, H+, ion fluxes, nitrate
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Introduction |
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Ammonium affects plant development and growth (Walch-Liu et al., 2001; Britto and Kronzucker, 2002). Plants can experience ammonium toxicity when ammonium is the sole nitrogen source (Zhang and Rengel, 1999, 2000, 2003). Several hypotheses have been proposed to explain this phenomenon: (i) acidification of soil; (ii) acidification of the cytosol; (iii) NH4+-induced cation deficiency and cation versus anion imbalance; (iv) deficiency of carbon sources in the root zone; (v) stimulated nitrogen assimilatory capacity; and (vi) disturbed phytohormone and polyamine status (Redinbaugh and Campbell, 1993; Gerendas et al., 1997; Zhang and Rengel, 1999; Britto and Kronzucker, 2002). Britto et al. (2001) suggested that NH4+ toxicity in plants might occur because plants expend a large amount of energy on NH4+ efflux to keep a low NH4+ concentration in the cytoplasm.
The addition of NO3– to soil or bathing media is known to alleviate NH4+ toxicity (Ikeda and Osawa, 1981; Goyal et al., 1982). Generally, this alleviation could happen through NO3– interaction with any of the putative mechanisms of NH4+ toxicity mentioned above or through a more complicated feedback system involving growth and development processes.
Preventing a low root surface pH was proposed as a possible mechanism of NO3– alleviation of NH4+ toxicity, since the NO3– uptake involves H+ symport, which might alkalinize the root media (Imsande, 1986).
Nitrate can be involved in charge balancing, thus protecting root cells from NH4+-induced plasma membrane depolarization, as demonstrated in numerous studies (Ayling, 1993; Wang et al., 1993b), and can decrease the internal ratio of cations to anions in plants (Britto and Kronzucker, 2002). No direct effect of NO3– on nutrient deficiency can be envisaged; however, preventing acidification of the root media might play an important role in maintaining the plasma membrane potential and therefore maintaining physiological patterns of ion uptake, for example, preventing the increased rate of K+ release observed at low pH (Babourina et al., 2001). On the other hand, NH4+ can compete with K+ for entry through some K+ inward channels (those found permeable for NH4+), and through non-selective cation channels (Howitt and Udvardi, 2000; Kronzucker et al., 2001). Hence, NO3– might indirectly alleviate some nutrient deficiencies when plants have been grown with NH4+ as the sole nitrogen source.
Another explanation for NO3– alleviation of NH4+ toxicity is based on the ability of NO3– to increase the expression of enzymes that remove NH4+ from the cytoplasm, glutamine synthetase and ferredoxin-dependent glutamate synthase (Redinbaugh and Campbell, 1993; Kronzucker et al., 1999). It is difficult to envisage a direct NO3– interaction with other proposed causes of NH4+ toxicity, such as carbon skeleton deprivation or polyamine and phytohormone status. There might be a more complicated feedback system generated at the whole plant level (Loque and von Wiren, 2004).
An ammonium efflux system, which would be involved in NH4+ toxicity in plants, was proposed on the basis of the wash-out kinetics of 13NH4+ from seedlings (Britto et al., 2001). However, no specific transporter that functions as an ammonium efflux transporter has been cloned yet. There is a suggestion, still to be confirmed, that plants can transport NH4+ across the plasma membrane in both directions through transporters from the ammonium transporter (AMT) family or through non-selective cation channels (Loque and von Wiren, 2004).
Here direct electrophysiological assessment is provided of three possible mechanisms that have been proposed for the NH4+ toxicity syndrome, and its alleviation by NO3–, under high external NH4+ concentration: (i) a decrease in external pH caused by H+ efflux; (ii) changes in K+ and Ca2+ transport; and (iii) induction of NH4+ efflux to relieve toxicity. H+, K+, Ca2+, and NH4+ net fluxes were measured non-invasively by the MIFE® technique at the junction of the elongation/root hair zones of canola seedlings in the presence or absence of NO3–.
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Materials and methods |
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Plant material and growing conditions
Seeds of canola (Brassica napus L. cv. Monty) were surface-sterilized and planted in jars on 0.8% (w/v) agar made up in 1 mM KCl, 100 µM CaCl2, and 10 µM H3BO3 solution. Jars were placed in a cultivation room under fluorescent light at 75 µmol m–2 s–1 (14 h day/10 h night) at 25 °C day/20 °C night for 3 d.
The 10–15 cm long taproot of a seedling was fixed with a Parafilm®M strip in a Petri dish containing unbuffered bathing solution of 1 mM KCl, 100 µM CaCl2, and 100 µM NH4Cl for a non-NO3– treatment. For NO3– treatment, 1 mM KNO3 was included in this bathing solution. The Petri dish was then placed on the stage of an inverted microscope in the Faraday cage under the microscope light. Measurements started after 10 min (when ion fluxes had stabilized) to reduce possible transition effects of changes in the environmental conditions. During flux measurements, the pH of the bathing solution ranged between 5.5 and 5.8.
Ion flux measurements
Specific details of ion-selective microelectrode fabrication and calibration are given in a previous publication (Babourina et al., 2001). The theory of measurements using the MIFE® system (University of Tasmania, Hobart, Australia) has also been given in a review (Newman, 2001). MIFE measurements of ion net fluxes were made in the solution just outside the surface of the plant tissue: positive values indicate ion uptake and negative values indicate ion release (Newman, 2001).
Electrodes were pulled from borosilicate glass capillaries (GC150-10, Harvard Apparatus, Kent, UK), dried at 230 °C for 
5 h, and silanized with tributylchlorosilane (#90765, Fluka). The tips of dried and cooled electrode blanks were broken to a diameter of 5–10 µm and then back-filled. Back-filling solutions were 15 mM NaCl+40 mM KH2PO4 for the hydrogen electrode, 500 mM NH4Cl for the ammonium electrode, 500 mM KCl for the potassium electrode, and 500 mM CaCl2 for the calcium electrode. Immediately after back-filling, the electrode tips were front-filled with commercially available ionophore cocktails for measuring hydrogen (#95297, Fluka), ammonium (# 09882, Fluka; the selectivity coefficient against K+ determined by the fixed interference method is –1.0), potassium (#60031, Fluka; the selectivity coefficient against NH4+ determined by the fixed interference method is –2.0) and calcium (#21048, Fluka).
Net ion fluxes at the junction of the elongation and root hair zones were measured simultaneously (H+, NH4+, K+ in one group of experiments and H+, NH4+, Ca2+ in another) for 10–20 min prior to NH4Cl application from 10 mM stock solution to the bathing media indicated above. The actual NH4+ concentration in the media after each application was measured simultaneously with the ion net fluxes. In the first set of experiments, there was a one-step increase in NH4Cl to a designated external concentration. In the second set of experiments, the external NH4Cl concentration was increased in steps at successive 15 min intervals. After each NH4Cl addition, the bathing solution was thoroughly mixed by sucking and expelling it from a pipette 6–10 times during the first 1–2 min.
Six to eight seedlings were measured for each different treatment. In some figures, only representative examples are shown to illustrate the character of the ion flux responses. Statistical information on the time for activation of the NH4+ efflux system is presented in Fig. 5. Significance of difference between means was based on Student's t-test.
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Membrane potential measurements
Membrane potentials of epidermal cells at the junction of the elongation and root hair zones of canola roots were measured with glass microelectrodes (GC 150-10F, Clark Electromedical Instruments, Pangbourne, Berks, UK) before and during NH4+ application, using the MIFE electrometer. Electrodes had a tip diameter <1 µm and were back-filled with 0.5 M KCl.
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Results |
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Changes in NH4+ transport after a single increase of external NH4Cl from 0.1 mM to 1 mM are shown in Fig. 1. In plants without KNO3, NH4+ release (negative net flux) was observed almost immediately (
1 stage), demonstrating an active state of the efflux system in plants without NO3– supply and the plant's ability to maintain cytoplasmic NH4+ at a certain level. Plants with a 1 mM NO3– supply did not show NH4+ release; however, they had a lag phase, also designated 1, before exhibiting a rise in NH4+ net flux. This initial release or lag stage was followed by a return to NH4+ uptake (positive slope of net flux graph, 2), which peaked 12 min after the NH4Cl addition (1+2). Thereafter, the NH4+ uptake rate decreased (3). This decrease in NH4+ uptake rate indicates a decrease in the activity of an NH4+ influx system or an increase in the activity of an NH4+ efflux system. The threshold points for the three stages of NH4+ flux dynamics (1, 2, and 3) are indicated in Fig. 1 and their values for each plant were used for further calculations of peak net fluxes (at the start of 3) and time intervals.
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Since the resins used in the microelectrodes are not completely selective, data are presented for K+ and NH4+ net fluxes measured simultaneously in one experiment (Fig. 2). K+ net fluxes did not show any change in response to the addition of 1 mM NH4Cl to the media.
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In another set of experiments, NH4+ external concentration was increased incrementally every 15 min. Final concentrations after each application are indicated in Fig. 3. The multiple subsequent applications of NH4Cl led to a gradual increase of the peak net flux at the start of
3 (Fig. 3). Generally, flux dynamics looked similar to those observed for the single application experiments (Fig. 1) with well-pronounced 2 and 3 stages.
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When net fluxes at the start of
3 were averaged and plotted against the external NH4+ concentrations, the relationship between concentrations and net fluxes was similar for both types of media, regardless of the presence of NO3– (Fig. 4). Hence, NO3– did not change the kinetic traits of the NH4+ transporters: external NH4+ concentrations required for reaching the same peak net flux were similar for both NO3– and non-NO3– media, as were the limitations on NH4+ uptake rate (by inactivating NH4+ influx systems or activating NH4+ efflux systems). However, the concentrations needed to cause a given net flux were higher in plants when NH4+ was applied once or for the first time in the multiple experiments, compared with net fluxes following incremental concentration increases (Fig. 4). The presence of NO3– did not alter this behaviour, but there is evidence for saturation of the NH4+ net flux peak at the first application in the presence of NO3– (Fig. 4).
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The time required for NH4+ uptake to stop rising (
1+2) in experiments with the multiple NH4+ applications was significantly shorter in NO3–-free plants than in plants supplied with NO3– (Fig. 5a). For the first or single NH4+ application, the time for stopping NH4+ uptake (12 min, Fig. 5a) was independent of the presence of NO3– in the media. On the other hand, absence of NO3– from the media caused a significant acceleration in reaching 3 in experiments with multiple NH4+ applications at NH4+ concentrations 
1 mM (1–2 mM, Fig. 5b). Simultaneous measurement of other ion net fluxes in NO3–-free media elucidated an interaction between NH4+ and other cations (H+, K+, and Ca2+). There was no effect on K+ and H+ net fluxes either of application of NH4+ in the range 150–250 µM (Fig. 6a) or of a single application of 1–2 mM NH4Cl (data not shown). Ca2+ was different. In experiments with a single NH4+ application, the abrupt increase in external NH4+ concentration led to high negative correlation between NH4+ and Ca2+ net fluxes (Table 1). This opposite direction of NH4+ and Ca2+ net fluxes was also observed in experiments with multiple NH4+ applications without NO3– (Fig. 6b). The relationships (or lack of them) between net fluxes of H+, K+, and Ca2+ were independent of plants’ exposure to NO3– (data not shown).
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Although there was a shift towards acidic pH in long running experiments (up to 3 h), a difference in the medium pH between treatments, without or with NO3– supply, was not observed (Table 2). Similarly, plasma membrane potential measurements demonstrated that changes in Em from pre-treatment to the end of
2 were not significantly dependent on NO3– (26±15 mV depolarization for non-NO3– plants and 22±17 mV depolarization for plants exposed to 1 mM KNO3).
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Discussion |
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Influx and efflux ammonium transport systems
The activation of AMT systems and their relationship to other cation transport was assessed in two types of experiments in the present study: a single application of NH4+ and multiple applications to observe the dynamics of NH4+ uptake. It has previously been shown that the measurement of ion net fluxes under a sequence of short-time increases of external concentrations of the ion allowed evaluation of the kinetic parameters for chloride in oat (Babourina et al., 1998) and for high-affinity NO3– and NH4+ transport systems in eucalyptus (Garnett et al., 2003). Garnett et al. (2003) noticed that at concentrations >100 µM NH4Cl, NH4+ net fluxes did not follow Michaelis–Menten kinetics for uptake. Those authors suggested that the reduction in NH4+ net fluxes at high external concentrations was due to an increase in unidirectional NH4+ efflux. This suggestion is in agreement with the earlier results by Britto et al. (2001) demonstrating that plants activate NH4+ efflux to cope with high NH4+ influx under high external concentrations of NH4+.
From the present results, concentration ranges for both high and low affinity NH4+ transport systems can be estimated. After the first increase in external NH4+ concentration from the 100 µM in the bathing solution (Figs 1, 3), NH4+ net flux demonstrated two well-defined stages: net flux increasing (2) to a peak and then decreasing (3). However, when the peaks of NH4+ net fluxes were averaged, grouped, and plotted against the corresponding external NH4+ concentrations, the peak net flux increased linearly with concentration (Fig. 4a). This is similar to what has been demonstrated for low-affinity NH4+ transporters (Wang et al., 1993a; Kronzucker et al., 1996; Rawat et al., 1999).
Generally similar time-courses for NH4+ dynamics have been reported for the two methods used: the electrophysiological MIFE measurements and other workers’ influx measurements using labelled NH4+. Both approaches used non-steady-state conditions or ‘perturbation’ experiments (Wang et al., 1993a; Kronzucker et al., 1996). During radioisotope studies, Kronzucker et al. (1996) demonstrated that the first 5 min of labelling time gave greater influx values for NH4+ than the next 5 min. In its time-course, this decreased NH4+ influx looked very similar to the 3 stage in the present study (Fig. 1b), since labelling was preceded by a 5 min pre-wash at the same concentration as was used for labelling. Thus the real time of plant exposure to higher NH4+ concentrations in that and other studies on NH4+ influx equals 15 min. This time interval covers the 1 and 2 stages for plants without NO3– and covers 1, 2, and the start of 3 in plants with 1 mM KNO3, presuming that NH4+ transport in canola seedlings is similar to that found in spruce, rice, and other plants (Wang et al., 1993a; Kronzucker et al., 1996, 1998). However, the MIFE measurements allowed the multiphasic character of NH4+ net flux to be discerned (Fig. 1a), which was hidden in earlier influx experiments with labelled NH4+ (Kronzucker et al., 1996). In more recent elution experiments, Britto and Kronzucker (2001) observed increased NH4+ efflux within the first 10 min after plants were transferred to a 100-fold higher external concentration of NH4+. This efflux was suggested to have a cytosolic origin (Britto and Kronzucker 2003). This observation led the authors to warn the scientific community about the interpretation of earlier influx experiments based on concentration shifts. However, elution experiments alone, which show the tracer efflux, do not allow estimation of the concurrent unlabelled influx to provide the net flux (in which influx and efflux may dominate in turn), which is provided by the MIFE technique and shown in the present study.
Given that net flux was measured, the observed decrease in NH4+ net flux during 3 in Fig. 1 could result from the inactivation of an NH4+ influx system or the activation of an NH4+ efflux system. However, all plants without NO3– in the bathing media demonstrated not only a decrease in NH4+ net flux, but also an NH4+ release (negative net flux values) during 3 (Fig. 1). This observation supports the hypothesis that the 3 phase of NH4+ flux dynamics occurs primarily due to the activation of an NH4+ efflux system.
During the first NH4+ application, a given peak of NH4+ uptake into tissue required a higher external concentration than was required under multiple increases in external NH4+ concentration (Fig. 4). A longer time interval to reach peak net flux (1+2=12 min) was also required for the first application, compared with 6 min only for subsequent applications (Fig. 5a). The simplest interpretation is that plants take time to suppress activity of a constantly working NH4+ efflux system. In summary, 1+2 is the suppression of that NH4+ efflux system, with 3 its recovery.
Effect of NO3– supply on NH4+ dynamics
In the present study, NO3– affected NH4+ transport in three ways. First, the presence of NO3– in the bathing media inhibited the release of NH4+ during 1, the first minute after the sudden addition of NH4+ to the external medium (Fig. 1). This NO3– effect, preventing the initial release, cannot be explained by a direct NO3– influence on Em because the measurements of Em during the first minutes of NH4+ application demonstrated that depolarization did not differ significantly between treatments. Instead the NO3– effect could be an action on NH4 transporters involved in cytosolic NH4+ homeostasis or a more complicated feedback response via plant metabolism. This feedback response could be based on the increased activity of NH4+ assimilation enzymes (Redinbaugh and Campbell, 1993). The increased activity of NH4+ transporters, regulated at the post-transcriptional level, could lead to a higher rate of NH4+ uptake into internal compartments (vacuole or plastids) or further transport to the xylem. All these processes would lead to a lower NH4+ concentration in the cytoplasm of root cells.
Secondly, plants without NO3– started to release NH4+ in 6 min during multiple incremental applications of NH4Cl, more quickly than the 12 min for a single addition (Fig. 5a). Thirdly, also in the absence of NO3–, under external NH4Cl concentrations 
1 mM, plants started to release NH4+ after 5 min, sooner than the 10 min when in the presence of NO3– (Fig. 5b). These two observations can be considered together: without NO3–, critical NH4+ uptake values (at the end of 2) to activate the NH4+ efflux system were achieved quicker than if NO3– was present in the bathing solution, especially when the NH4+ efflux system was already in an ‘active’ mode (i.e. following the second and subsequent NH4Cl applications). Similarly, plants without NO3– in the bathing media also release NH4+ for a shorter time (3 is shorter) than plants supplied with NO3–. The NH4Cl concentration of 0.5 mM is considered to be a threshold above which plants start using low-affinity transporters (Britto et al., 2001). In the present experiments, NO3– delayed the onset of NH4+ release (Fig. 5b) at concentrations >1 mM. Thus NO3– appears to affect mostly low-affinity NH4+ transporters.
The present observations are in good agreement with conclusions reached by Kronzucker et al. (1999), where co-provision of NO3– with NH4+ caused lower NH4+ release, which is also observed in the present study, and enhanced xylem loading of NH4+. The latter can maintain a low NH4+ concentration in the cytoplasm of root cells, which is also considered to be the most likely mechanism of NO3– alleviation of NH4+ toxicity.
Interaction between NH4+ fluxes and H+ and Ca2+ fluxes
It has been proposed that for high-affinity NH4+ transport, plants use transporters from the AMT family, whereas low-affinity transport occurs through non-selective cation channels or K+ channels (Howitt and Udvardi, 2000; Kronzucker et al., 2001; Loque and von Wiren, 2004). The AMTs are highly selective for NH4+ and are suggested to be uniporters because NH4+ transport through them was not affected by external pH (Ludewig et al., 2002). This is consistent with 1H-NMR (nuclear magnetic resonance) measurements showing hardly any disturbance of the cytoplasmic pH (Bligny et al., 1997). It is supported by the present studies, where H+ net fluxes were found to be unaffected by the addition of NH4+ (Fig. 6).
The results clearly demonstrate a linkage between NH4+ and Ca2+ transport (Fig. 6b; Table 1): canola root cells release Ca2+ when they are taking up NH4+ and vice versa. Earlier, Plieth and co-authors (2000) found a cytoplasmic Ca2+ increase in Arabidopsis in response to a sudden increase in external NH4Cl; however, this phenomenon was observed only at high pH and therefore was linked with NH3 influx. Given that the present experiments were performed in solutions with pH 5.5–5.8, where the NH3 concentration is negligible, the observed link between NH4+ and Ca2+ fluxes is different from the one reported by Plieth et al. (2000). Future pharmacological studies might provide details on the nature of this link.
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Acknowledgements |
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This work was supported by grants from the Australian Research Council.
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Abbreviations |
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AMT, ammonium transporter.
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References |
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