JXB Advance Access originally published online on December 14, 2006
Journal of Experimental Botany 2007 58(3):651-658; doi:10.1093/jxb/erl238
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 (NH
) toxicity when NH is the sole nitrogen source. Nitrate (NO
) is known to alleviate NH toxicity, but the mechanisms are unknown. This study has evaluated possible mechanisms of NO
alleviation of NH toxicity in canola (Brassica napus L.). Dynamics of net fluxes of NH, H+, K+ and Ca2+ were assessed, using a non-invasive microelectrode (MIFE) technique, in plants having different NO supplies, after single or several subsequent increases in external NH4Cl concentration. After an increase in external NH4Cl without NO, NH net fluxes demonstrated three distinct stages: release (
1), return to uptake (2), and a decrease in uptake rate (3). The presence of NO 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 NH net fluxes, regardless of NO supply. In contrast, H+ and K+ net fluxes and change in external pH were not correlated with NH net fluxes. It is concluded that (i) NO primarily affects the NH low-affinity influx system; and (ii) NH 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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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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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) NH
-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 NH toxicity in plants might occur because plants expend a large amount of energy on NH efflux to keep a low NH concentration in the cytoplasm.
The addition of NO to soil or bathing media is known to alleviate NH toxicity (Ikeda and Osawa, 1981; Goyal et al., 1982). Generally, this alleviation could happen through NO interaction with any of the putative mechanisms of NH 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 NO alleviation of NH toxicity, since the NO uptake involves H+ symport, which might alkalinize the root media (Imsande, 1986).
Nitrate can be involved in charge balancing, thus protecting root cells from NH-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 NO 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, NH can compete with K+ for entry through some K+ inward channels (those found permeable for NH), and through non-selective cation channels (Howitt and Udvardi, 2000; Kronzucker et al., 2001). Hence, NO might indirectly alleviate some nutrient deficiencies when plants have been grown with NH as the sole nitrogen source.
Another explanation for NO alleviation of NH toxicity is based on the ability of NO to increase the expression of enzymes that remove NH 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 NO interaction with other proposed causes of NH 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 NH toxicity in plants, was proposed on the basis of the wash-out kinetics of 13NH 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 NH 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 NH toxicity syndrome, and its alleviation by NO, under high external NH concentration: (i) a decrease in external pH caused by H+ efflux; (ii) changes in K+ and Ca2+ transport; and (iii) induction of NH efflux to relieve toxicity. H+, K+, Ca2+, and NH 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 NO.
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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-NO treatment. For NO 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 NH 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+, NH, K+ in one group of experiments and H+, NH, Ca2+ in another) for 10–20 min prior to NH4Cl application from 10 mM stock solution to the bathing media indicated above. The actual NH 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 NH 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 NH
application, using the MIFE electrometer. Electrodes had a tip diameter <1 µm and were back-filled with 0.5 M KCl. |
Results |
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Changes in NH
transport after a single increase of external NH4Cl from 0.1 mM to 1 mM are shown in Fig. 1. In plants without KNO3, NH release (negative net flux) was observed almost immediately (1 stage), demonstrating an active state of the efflux system in plants without NO supply and the plant's ability to maintain cytoplasmic NH at a certain level. Plants with a 1 mM NO supply did not show NH release; however, they had a lag phase, also designated 1, before exhibiting a rise in NH net flux. This initial release or lag stage was followed by a return to NH uptake (positive slope of net flux graph, 2), which peaked 12 min after the NH4Cl addition (1+2). Thereafter, the NH uptake rate decreased (3). This decrease in NH uptake rate indicates a decrease in the activity of an NH influx system or an increase in the activity of an NH efflux system. The threshold points for the three stages of NH 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 NH
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, NH
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 NH concentrations, the relationship between concentrations and net fluxes was similar for both types of media, regardless of the presence of NO (Fig. 4). Hence, NO did not change the kinetic traits of the NH transporters: external NH concentrations required for reaching the same peak net flux were similar for both NO and non-NO media, as were the limitations on NH uptake rate (by inactivating NH influx systems or activating NH efflux systems). However, the concentrations needed to cause a given net flux were higher in plants when NH 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 NO did not alter this behaviour, but there is evidence for saturation of the NH net flux peak at the first application in the presence of NO (Fig. 4).
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The time required for NH
uptake to stop rising (1+2) in experiments with the multiple NH applications was significantly shorter in NO-free plants than in plants supplied with NO (Fig. 5a). For the first or single NH application, the time for stopping NH uptake (12 min, Fig. 5a) was independent of the presence of NO in the media. On the other hand, absence of NO from the media caused a significant acceleration in reaching 3 in experiments with multiple NH applications at NH concentrations 
1 mM (1–2 mM, Fig. 5b).
Simultaneous measurement of other ion net fluxes in NO-free media elucidated an interaction between NH and other cations (H+, K+, and Ca2+). There was no effect on K+ and H+ net fluxes either of application of NH 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 NH application, the abrupt increase in external NH concentration led to high negative correlation between NH and Ca2+ net fluxes (Table 1). This opposite direction of NH and Ca2+ net fluxes was also observed in experiments with multiple NH applications without NO (Fig. 6b). The relationships (or lack of them) between net fluxes of H+, K+, and Ca2+ were independent of plants’ exposure to NO (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 NO
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 NO (26±15 mV depolarization for non-NO 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 NH
and multiple applications to observe the dynamics of NH 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 NO and NH transport systems in eucalyptus (Garnett et al., 2003). Garnett et al. (2003) noticed that at concentrations >100 µM NH4Cl, NH net fluxes did not follow Michaelis–Menten kinetics for uptake. Those authors suggested that the reduction in NH net fluxes at high external concentrations was due to an increase in unidirectional NH efflux. This suggestion is in agreement with the earlier results by Britto et al. (2001) demonstrating that plants activate NH efflux to cope with high NH influx under high external concentrations of NH.
From the present results, concentration ranges for both high and low affinity NH transport systems can be estimated. After the first increase in external NH concentration from the 100 µM in the bathing solution (Figs 1, 3), NH net flux demonstrated two well-defined stages: net flux increasing (2) to a peak and then decreasing (3). However, when the peaks of NH net fluxes were averaged, grouped, and plotted against the corresponding external NH concentrations, the peak net flux increased linearly with concentration (Fig. 4a). This is similar to what has been demonstrated for low-affinity NH transporters (Wang et al., 1993a; Kronzucker et al., 1996; Rawat et al., 1999).
Generally similar time-courses for NH dynamics have been reported for the two methods used: the electrophysiological MIFE measurements and other workers’ influx measurements using labelled NH. 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 NH than the next 5 min. In its time-course, this decreased NH 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 NH concentrations in that and other studies on NH influx equals 15 min. This time interval covers the 1 and 2 stages for plants without NO and covers 1, 2, and the start of 3 in plants with 1 mM KNO3, presuming that NH 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 NH net flux to be discerned (Fig. 1a), which was hidden in earlier influx experiments with labelled NH (Kronzucker et al., 1996). In more recent elution experiments, Britto and Kronzucker (2001) observed increased NH efflux within the first 10 min after plants were transferred to a 100-fold higher external concentration of NH. 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 NH net flux during 3 in Fig. 1 could result from the inactivation of an NH influx system or the activation of an NH efflux system. However, all plants without NO in the bathing media demonstrated not only a decrease in NH net flux, but also an NH release (negative net flux values) during 3 (Fig. 1). This observation supports the hypothesis that the 3 phase of NH flux dynamics occurs primarily due to the activation of an NH efflux system.
During the first NH application, a given peak of NH uptake into tissue required a higher external concentration than was required under multiple increases in external NH 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 NH efflux system. In summary, 1+2 is the suppression of that NH efflux system, with 3 its recovery.
Effect of NO supply on NH dynamics
In the present study, NO affected NH transport in three ways. First, the presence of NO in the bathing media inhibited the release of NH during 1, the first minute after the sudden addition of NH to the external medium (Fig. 1). This NO effect, preventing the initial release, cannot be explained by a direct NO influence on Em because the measurements of Em during the first minutes of NH application demonstrated that depolarization did not differ significantly between treatments. Instead the NO effect could be an action on NH4 transporters involved in cytosolic NH homeostasis or a more complicated feedback response via plant metabolism. This feedback response could be based on the increased activity of NH assimilation enzymes (Redinbaugh and Campbell, 1993). The increased activity of NH transporters, regulated at the post-transcriptional level, could lead to a higher rate of NH uptake into internal compartments (vacuole or plastids) or further transport to the xylem. All these processes would lead to a lower NH concentration in the cytoplasm of root cells.
Secondly, plants without NO started to release NH 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 NO, under external NH4Cl concentrations 
1 mM, plants started to release NH after 5 min, sooner than the 10 min when in the presence of NO (Fig. 5b). These two observations can be considered together: without NO, critical NH uptake values (at the end of 2) to activate the NH efflux system were achieved quicker than if NO was present in the bathing solution, especially when the NH efflux system was already in an ‘active’ mode (i.e. following the second and subsequent NH4Cl applications). Similarly, plants without NO in the bathing media also release NH for a shorter time (3 is shorter) than plants supplied with NO. 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, NO delayed the onset of NH release (Fig. 5b) at concentrations >1 mM. Thus NO appears to affect mostly low-affinity NH transporters.
The present observations are in good agreement with conclusions reached by Kronzucker et al. (1999), where co-provision of NO with NH caused lower NH release, which is also observed in the present study, and enhanced xylem loading of NH. The latter can maintain a low NH concentration in the cytoplasm of root cells, which is also considered to be the most likely mechanism of NO alleviation of NH toxicity.
Interaction between NH fluxes and H+ and Ca2+ fluxes
It has been proposed that for high-affinity NH 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 NH and are suggested to be uniporters because NH 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 NH (Fig. 6).
The results clearly demonstrate a linkage between NH and Ca2+ transport (Fig. 6b; Table 1): canola root cells release Ca2+ when they are taking up NH 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 NH 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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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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