Journal of Experimental Botany, Vol. 51, No. 343, pp. 207-219,
February 2000
© 2000 Oxford University Press
Intracellular pH regulation in maize root tips exposed to ammonium at high external pH
1 Institute for Plant Nutrition and Soil Science, University of Kiel, Olshausenstraße 40, D-24118 Kiel, Germany
2 Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK
Received 21 June 1999; Accepted 25 October 1999
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Ammonium-induced changes in the cytoplasmic and vacuolar pH values of excised maize (Zea mays L.) root tips, measured by in vivo 31P nuclear magnetic resonance (NMR) spectroscopy, were correlated with the ammonium content of the tissue, determined by 14N NMR. Calculations based on these measurements indicated that the pH changes observed during exposure to 10 mM ammonium for 1 h at pH 9.0, and in the recovery following the removal of the external ammonium supply, were largely determined by the influx and efflux of the weak base NH3. Carboxylate synthesis, detected by both in vivo 13C NMR and the incorporation of [14C]bicarbonate, was stimulated by the ammonium-induced alkalinization of the root tips, but the contribution that this proton-generating process made to pH regulation during and after the ammonium treatment was quantitatively insignificant. Similarly, ammonium assimilation, which was shown to occur via the proton-generating glutamine synthetase/glutamate synthase pathway using in vivo 15N NMR, was also quantitatively insignificant in comparison with the large changes in ammonium content that occurred during the ammonium treatment and subsequent recovery. The results are discussed in relation to several recent studies in which ammonium was used to perturb intracellular pH values, and it is argued (i) that a new method for probing the subcellular compartmentation of amino acids, based on an ammonium-induced alkalinization of the cytoplasm may be difficult to implement in dense heterogeneous tissues; and (ii) that observations on the apparently proton-consuming effect of ammonium assimilation in rice root hairs may actually reflect unusually rapid assimilation.
Key words: Ammonium efflux, cytoplasmic pH, GS/GOGAT pathway, NMR spectroscopy, vacuolar pH.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The pH dependence of the structures and ionization states of many of the molecular constituents of the cell ensures that cellular processes are sensitive to pH. It follows that uncontrolled variations in pH, whether arising through normal cellular activity or through changes in the external environment, are potentially disruptive and, as a result, intracellular pH regulation is a characteristic and necessary feature of all living cells.
In plants one aspect of this general phenomenon relates to the acquisition and utilization of nitrogen (Raven and Smith, 1976
; Raven, 1985, 1986). Nitrate and ammonium are the major sources of nitrogen in the soil and, in principle, the uptake and assimilation of these ions could lead to changes in pH. Nitrate is taken up by a H+-cotransport system (Mistrik and Ullrich, 1996), and subsequent growth on nitrate is generally considered to be proton-consuming (Raven and Smith, 1976; Raven, 1985, 1986) while ammonium is taken up either by free diffusion of NH3 (Kleiner, 1981) or carrier-mediated diffusion of NH+4 (Ninnemann et al., 1994), both of which could influence the cytoplasmic pH, and subsequent growth on ammonium is generally considered to be proton-generating (Raven and Smith, 1976; Raven, 1985, 1986). An immediate consequence of the interaction between nitrogen nutrition and pH is that growth on nitrate is usually associated with an increase in root carboxylates and an increase in external pH; whereas growth on ammonium tends to be associated with a decrease in root carboxylates and a decrease in external pH (Marschner, 1995). In contrast to these well-documented responses, the effect of the nitrogen form on the cytoplasmic pH is much smaller, reflecting the effectiveness of the pH regulatory mechanisms under normal growth conditions (Gerendás et al., 1990).
Differences in the response of the intracellular pH values to growth on different nitrogen sources emerge when the pH of the growth medium is varied (Gerendás et al., 1990). In maize roots, it was found that the cytoplasmic and vacuolar pH values in ammonium-grown root tips were higher than the corresponding values in nitrate-grown tips at pH 8, and it was concluded that this was probably caused by the influx of free ammonia (Gerendás et al., 1990). The effect on the intracellular pH values is too small to explain the phenomenon of ammonium toxicity (Gerendás et al., 1997), but it remains of interest because of the availability of free ammonia in the natural environment. Thus the emission and reabsorption of gaseous ammonia (Pearson and Stewart, 1993; Yin et al., 1996; Yin and Raven, 1997; Hanstein et al., 1999) and the injection of high doses of ammonium fertilizer into soils, resulting in locally high pH and free ammonia concentrations (Izaurralde et al., 1990; Stehouwer et al., 1993), can be expected to lead to intracellular pH changes.
Several recent papers have sought to characterize the intracellular pH changes that occur in root tissues during exposure to ammonium (Brauer et al., 1997; Kosegarten et al., 1997; Wilson et al., 1998). These investigators used fluorescence techniques to probe the cytosolic (Kosegarten et al., 1997) and vacuolar (Brauer et al., 1997; Wilson et al., 1998) pH values in the root hair cells of rice and maize roots during ammonium uptake at alkaline pH values. In the present paper nuclear magnetic resonance (NMR) techniques (Ratcliffe, 1994) were used to investigate the effect of similar treatments on maize root tips, with the aim of identifying the pH regulatory mechanisms that operate during the recovery of the tissue from the alkalinization induced by exposure to ammonium. The correlation between the ammonium content of the tissue and the cytoplasmic and vacuolar pH values was explored using 14N and 31P NMR measurements, and the results highlight the importance of ammonium efflux as a pH regulatory mechanism.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material
Maize seeds (Zea maize L. var. Bastion) were soaked overnight in aerated 0.1 mM CaSO4 and then germinated in the dark between layers of filter paper soaked with 0.1 mM CaSO4 at 25 °C. After 2 d germination, 5 mm root tips were excised with a razor blade and transferred to an aerated ammonium-free buffer solution (see below).
NMR spectroscopy
140 root tips were transferred to a 10 mm diameter NMR tube containing either 10 mM HEPES/0.1 mM CaSO4 at pH 8.0 or 8.5, or 10 mM TAPS/0.1 mM CaSO4 at pH 9.0 or 9.25. A potassium hydroxide solution was used to adjust the pH values. Oxygenated buffer was circulated through the NMR tube at 4 ml min-1, with the tissue maintained at 21–22 °C (Lee and Ratcliffe, 1983). The ammonium treatment was initiated by switching from the control buffer to a treatment buffer of the same composition, containing 5, 10 or 20 mM ammonium as (NH4)2SO4.
In vivo NMR spectra were recorded using a Bruker CXP 300 spectrometer (Bruker Analytische Messtechnik, Rheinstetten, Germany) with an Oxford Instruments (Oxford, UK) 7.05 T superconducting magnet.
1H-decoupled 31P NMR spectra were recorded at 121.49 MHz using a 10 mm double tuned 13C/31P probehead, a 45° pulse angle, a spectral width of 5200 Hz, a 0.5 s recycle time with broadband decoupling during the 0.07 s acquisition, and a total accumulation time of between 5 and 30 min. Chemical shifts were measured relative to the signal at -2.44 ppm from a capillary containing 70 mM phosphocreatine. Values of pHcyt and pHvac were determined from the chemical shift values of the cytoplasmic and vacuolar orthophosphate (Pi) signals using calibration curves corresponding to the approximate ionic conditions in the cytoplasm and vacuole (Martin et al., 1982).
1H-decoupled 14N NMR spectra were recorded at 21.67 MHz using a 10 mm diameter broadband probehead, a 70° pulse angle, a spectral width of 4500 Hz, a 3 s recycle time with broadband decoupling during the 0.23 s acquisition, and a total accumulation time of between 5 and 20 min. Chemical shifts were measured relative to the signal at -298.5 ppm from a capillary containing either 400 mM or 1.6 M NaOCN. The ammonium content of the tissue was determined by comparing the intensity of the tissue signal with the intensity of the signal from standard solutions of ammonium nitrate in artificial vacuolar sap (Gerendás et al., 1995).
1H-decoupled 13C NMR spectra were recorded at 75.46 MHz using a 10 mm diameter double tuned 13C/31P probehead, a 45° pulse angle, a spectral width of 16 700 Hz, a 2 s recycle time with low power broadband decoupling to maintain the nuclear Overhauser effect and normal decoupling during the 0.125 s acquisition, and a total accumulation time of 30 min. Label was supplied in the form of 5 mM sodium [13C]bicarbonate and the TAPS concentration was increased to 20 mM to increase the buffering capacity of the suspending medium. The prominent bicarbonate signal was used as a chemical shift reference.
1H-decoupled 15N NMR spectra were recorded at 30.42 MHz using a 10 mm broadband probehead, a 90° pulse angle, a spectral width of 4500 Hz, a 2 s recycle time with low power broadband decoupling to maintain the nuclear Overhauser effect and normal decoupling during the 0.25 s acquisition, and a total accumulation time of 20 min. Chemical shifts were measured relative to the signal at -354.7 ppm from 15NH4 on the scale that puts nitrate at 0 ppm.
When it was necessary to compare experiments of different time resolution, the more frequent resolution was adopted and the results from the other experiments were interpolated accordingly.
Efflux measurements
150, 5 mm root tips were transferred to a 2 ml polypropylene syringe and oxygenated 10 mM TAPS/0.1 mM CaSO4 at pH 9.0 was pumped through the sample at a rate of 4 ml min-1. The ammonium treatment was initiated by switching to a treatment buffer of the same composition containing 10 mM ammonium as (NH4)2SO4. After 1 h, the circulating medium was switched back to the control buffer and, after a further delay of 10 min to allow for the complete removal of any trace of the treatment buffer, the effluent was collected in 20 min aliquots and, after increasing the volume to 100 ml, analysed for ammonium using the autoanalyser method (described by Gerendás et al., 1995). The efflux during the first 10 min was calculated by linear extrapolation. The experiments were carried out at 21–22 °C and repeated three times.
14C incorporation
Fifty, 5 mm root tips were incubated at 21–22 °C in 100 ml beakers containing 50 ml 10 mM TAPS/5 mM NaHCO3/0.1 mM CaSO4 at pH 9. The NaHCO3 was included to reduce the effect of respiratory CO2: assuming a fresh weight of 300 mg, and a respiration rate comparable to the values reported elsewhere (700 µl O2 h-1 g-1 FW (Lee and Ratcliffe, 1983); 970 µl O2 h-1 g-1 FW (Saglio and Pradet, 1980)), endogenous CO2 production could have been 12.5 µmol h-1, and this was small in comparison with the initial bicarbonate concentration. Control experiments showed that the addition of 5 mM NaHCO3 to the buffer had no effect on the ammonium-induced changes in the 31P NMR spectra. Buffer solutions were continuously purged with air that had been acid-washed to trap atmospheric ammonia and base-washed to trap carbon dioxide. The experiment was carried out, similarly to the ammonium efflux measurements, but in a discontinuous mode with three replicates. Samples were incubated for 1 h in control buffer, then transferred to a buffer containing 10 mM ammonium as (NH4)2SO4 for 1 h. Thereafter, the tips were rinsed thoroughly with control buffer before being incubated in the same buffer for 3 h, with the solution being renewed at 1 h intervals. Control samples were incubated in ammonium-free buffer, which was also renewed at 1 h intervals.
To determine CO2 incorporation during the 1 h intervals, 50 µl of sodium [14C]bicarbonate solution (Amersham International: 7.4 kBq µl-1 and 3.78 nmol µl-1) was added to the buffer and, after thorough mixing, 300 µl of the incubation medium was mixed with 1 ml tissue solubilizer (Soluene, Packard) and 4 ml liquid scintillation cocktail (Hionic Fluor, Packard) to give the initial radioactivity. At the end of the labelling period the tips were thoroughly rinsed with 0.1 mM CaSO4 and after weighing, 0.5 ml tissue solubilizer was added for overnight digestion (16 h) of the tissue. Thereafter 4 ml scintillation cocktail was added and the radioactivity measured in a Siemens liquid scintillation counter using the sample channel ratio mode for quench correction. Incorporation was calculated on a fresh weight basis taking the specific and initial activities into account.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Effect of high pH and ammonium on the intracellular pH values and NTP content
Maize root tips were incubated for periods of 1 h in solutions containing various concentrations of ammonium (0–20 mM) over a range of pH values (8.0–9.2). In vivo 31P NMR spectroscopy was used to assess the effect of these treatments on the intracellular pH and NTP content of the root tips, and Figs 1–3
show that the metabolic disruption of the tissue increased at higher ammonium concentrations and external pH values.
|
- and ß-phosphates of nucleoside triphosphate; 6, UDP-glucose and NAD(P)(H); and 7, UDP-glucose. The peak labelled with an asterisk (*) is the phosphocreatine signal from the reference capillary.
|
|
Figure 1 shows that treatment with 5 mM ammonium for 1 h at pH 8.5 had only a marginal effect on the 31P NMR spectra. There was a slight broadening of the vacuolar Pi signal during the treatment (Fig. 1b), but this was readily reversed during the subsequent recovery, and the spectrum in Fig. 1c, which shows small increases in phosphocholine and vacuolar Pi, is indistinguishable from control spectra (data not shown).
Increasing the ammonium concentration to 10 mM, and the pH to 9.0, had a much more substantial effect on the spectrum (Fig. 2). Figure 2b shows that the vacuolar Pi signal broadened and shifted to the left, merging with the cytoplasmic Pi signal. The position of the Pi signal is sensitive to the pH of the corresponding subcellular compartment, and the shift of the vacuolar Pi signal indicates that the influx of ammonium caused an alkalinization of the vacuole. Different vacuoles alkalinized at different rates, presumably reflecting the time taken for the ammonium to penetrate the tissue, and the resulting heterogeneity in the vacuolar pH caused severe broadening of the vacuolar Pi signal during the time-course (data not shown). Treatment with 10 mM ammonium at pH 9.0 also caused a marked reduction in the NTP signals (Fig. 2b), and this effect, like the effect on the vacuolar Pi signal was readily reversed following the removal of the ammonium from the suspending medium (Fig. 2c).
Increasing the ammonium concentration to 20 mM, and the pH to 9.25, increased the severity of the metabolic disruption still further (Fig. 3). Figure 3a shows that the high external pH caused no particular difficulty for the tissue, but exposure to 20 mM ammonium at this pH increased the cytoplasmic and vacuolar pH values to around 8.4 and all but eliminated the NTP pool (Fig. 3b). The pH gradient across the tonoplast and the NTP level returned to their normal values after this treatment (Fig. 3c), but the relatively large phosphocholine and cytoplasmic Pi signals, and the low glucose 6-phosphate and NDP-hexose signals, indicate that the metabolic recovery was far from complete after 3 h.
Figures 4 and 5 summarize the cytoplasmic and vacuolar pH values, and the NTP contents, observed in a series of NMR experiments in which the ammonium concentration was varied at pH 9.0 (Fig. 4) or the pH was varied at a constant 10 mM ammonium concentration (Fig. 5). Table 1 shows that the measured quantities were unaffected by the high external pH values alone, and thus emphasizes that the changes in Figs 4 and 5 were caused by the presence of the ammonium in the incubation medium.
|
), 5 (
), 10 (
), or 20 mM ammonium (•) for 1 h (40–100 min): (a) the cytoplasmic pH; (b) the vacuolar pH; and (c) the NTP content (typical experiments).
|
|
Figure 4 shows that 5 mM ammonium at pH 9.0 caused only a slight alkalinization of the cytoplasm and vacuole, and that a concentration of at least 10 mM was necessary for a substantial effect (Fig. 4a, b). The NTP content appeared to be more sensitive to low ammonium concentrations than the intracellular pH, and at 20 mM there was a considerable overshoot in the NTP during the recovery period (Fig. 4c). Figure 5 shows that an external pH of 9.0 was necessary to cause a substantial alkalinization of the cytoplasm and the vacuole in root tips exposed to 10 mM ammonium (Fig. 5a, b), with the pH of both subcellular compartments rising to around 8.2 at an external pH of 9.25. The effect on the NTP level also became more severe with increasing pH (Fig. 5c), and at the highest pH the NTP level recovered to more than its initial value following the removal of the ammonium.
Table 2 summarizes the ammonium-induced alkalinization of the cytoplasm for the full range of conditions investigated, and it can be seen that the pH change increased with increasing external pH and ammonium concentration. One set of conditions (10 mM ammonium at pH 9.0) was chosen for more detailed investigation, and Fig. 6 shows the changes in cytoplasmic pH and NTP under these conditions. The cytoplasmic pH and the NTP pool were stable at pH 9.0, but exposure to 10 mM ammonium for 1 h caused an increase in the pH from 7.57 to 7.82 and a decrease in the NTP pool to 50% of its initial value. Removal of the ammonium lead to the rapid recovery of both parameters, although the cytoplasmic pH stabilized at 7.62, 0.05 pH units above its control value (Fig. 6).
|
|
Uptake of ammonium
In vivo 14N NMR was used to monitor the ammonium content of the maize root tips during experiments with 10 mM ammonium at pH 9.0 (Fig. 7). These measurements were simplified by the pH-dependence of the 14N ammonium NMR signal, the external signal was shifted upfield of the intracellular signal, allowing the intracellular ammonium pool to be quantified directly. Figure 7 shows that there was a rapid accumulation of ammonium during exposure to 10 mM ammonium at pH 9.0, with the tissue content reaching 34.2±1.2 µmol g-1 FW after 1 h. The ammonium content continued to increase if the treatment was prolonged, but if the external ammonium was removed then there was a steady fall to 8.4±0.7 µmol g-1 FW (n=4) after 3 h in an ammonium-free medium. This reduction in the ammonium content (25 µmol g-1 FW) could be caused by both efflux and assimilation, and so these processes were investigated next.
|
Efflux of ammonium
Figure 8 shows the efflux of ammonium from maize root tips that had been incubated for 1 h in 10 mM ammonium at pH 9.0. The initial efflux rate was 15 µmol h-1 g-1 FW, and this gradually declined to less than 4 µmol h-1 g-1 FW. In contrast control tissue lost only small amounts of ammonium (Fig. 8). Integrating the ammonium efflux curve between 100 and 300 min indicates that the tips lost 25.4±0.7 µmol g-1 FW, in good agreement with the observed decrease in the tissue ammonium (Fig. 7). This indicates that ammonium efflux is the dominant mechanism for reducing the ammonium content under these conditions, and that this is likely to make an important contribution to the recovery of the intracellular pH values.
|
Assimilation of ammonium
Ammonium assimilation also occurred following exposure to ammonium at pH 9.0, and this was monitored using [15N]ammonium and in vivo 15N NMR spectroscopy (Fig. 9). In accordance with the operation of the glutamine synthetase (GS)/glutamate synthase (GOGAT) pathway, the 15N label was first detected in the amide N of glutamine (Fig. 9a) and only subsequently in the amino N of glutamate and other amino acids (Fig. 9b). Interestingly, preloading the tissue with [14N] ammonium for 40 min before switching to [15N]ammonium also resulted in the amide N of glutamine being labelled first (Fig. 9c). In this latter experiment, the increase in the ammonium content (Fig. 7 indicates a tissue content of about 23 mmol kg-1 FW after 40 min) and the decrease in the NTP level (Fig. 6b) might have been expected to favour the assimilation of ammonium by glutamate dehydrogenase (GDH), since GDH has a higher Km for ammonium than GS and no requirement for ATP, but Fig. 9c shows that the labelling pattern was unaffected by the pretreatment and therefore still consistent with the GS/GOGAT pathway. Figure 9 provides a qualitative demonstration that ammonium assimilation occurred during the exposure to ammonium at pH 9, but it could only have made a minor contribution to the reduction of the tissue ammonium content after the switch to the ammonium-free medium because of the substantial efflux recorded in Fig. 8. However, it should be noted that incubating root tips with 1 mM methionine sulphoximine (MSO) at pH 9.0 caused an accumulation of endogenous ammonium (1.5 µmol g-1 FW after 1.6 h and 4.4 µmol g-1 FW after 5.6 h; 14N NMR measurements; data not shown), emphasizing the role played by the GS/GOGAT pathway in recycling ammonium within the root.
|
Synthesis of carboxylates
The synthesis of organic acids often increases in response to an alkalinization of the cytoplasm and the in vivo 13C NMR spectra in Fig. 10 show that exposure to 10 mM ammonium at pH 9.0 increased the incorporation of [13C]bicarbonate into malate. A quantitative analysis of the incorporation of [14C]bicarbonate showed that dark CO2 fixation was substantially higher during the ammonium treatment (Fig. 11), although the observed difference of 0.44 µmol h-1 g-1 FW was negligible compared to the influx of ammonium (34 µmol g-1 FW) that occurred during the 1 h treatment.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Changes in intracellular pH and NTP during ammonium uptake
As expected from earlier work on cell suspensions (Fox and Ratcliffe, 1990
), varying the external pH in the range 8 to 9.25 in the absence of ammonium had little effect on the intracellular pH values and NTP content of the maize root tips (Table 1). However, significant changes in these quantities were observed in the presence of ammonium, with ammonium causing an increase in the cytoplasmic and vacuolar pH values, and a decrease in NTP (Figs 1–6; Table 2). The perturbing effects of ammonium became more pronounced with increasing external pH and ammonium concentration, and they could be readily reversed by removing the ammonium from the suspending medium.
These observations can be compared with the results of a number of other recent studies on maize roots. For example, 13C and 31P NMR measurements on maize root tips showed that incubation with up to 10 mM ammonium at an external pH in the range pH 6.5–7, had no effect on the cytoplasmic pH but caused a substantial increase in the vacuolar pH (Roberts and Pang, 1992). These experiments also provided evidence for the heterogeneity of the vacuolar response since the line broadening of the vacuolar malate signal indicated that different vacuoles alkalinized at different rates. In another NMR study, 31P NMR spectra of maize root segments showed that exposure to 10 mM ammonium at pH 9 caused a substantial increase in the pH of most of the vacuoles and a decrease in NTP, but there was apparently no effect on the cytoplasmic pH (Brauer et al., 1997). Fluorescence ratio measurements of the cytoplasmic (Kosegarten et al., 1997) and vacuolar (Brauer et al., 1997; Wilson et al., 1998) pH values in maize root hairs have also been reported, and these investigations have shown ammonium-induced increases in pH, including transient changes in the cytoplasmic pH (Kosegarten et al., 1997), under various conditions.
While there is general agreement between these studies, to the extent that it is clear that incubation with ammonium can lead to intracellular alkalinization, it is also apparent that the precise response depends on the nature of the tissue and the way in which the conditions are imposed. Particular points that emerge from the NMR studies include the effect of the ammonium treatments on the NTP level, the heterogeneous response of the vacuolar pH, and the differential effects on the cytoplasmic and vacuolar pH values. The decrease in the NTP level (Figs 4c, 5c, 6b) probably reflects impaired ATP synthesis as a result of the ammonium-induced uncoupling of oxidative phosphorylation (Wakiuchi et al., 1971), rather than an inherent inability to respond to an increased demand for ATP that might arise, for example, through increased ammonium assimilation or malate synthesis.
The differential effect of the ammonium treatment on the cytoplasmic and vacuolar pH values is of particular interest in relation to a recently proposed 13C NMR method for probing the subcellular compartmentation of amino acids (Aubert et al., 1998, 1999). The method depends on pretreating the tissue with an ammonium solution at high pH in order to induce an alkalinization of the cytoplasm that is sufficient to generate separate 13C NMR signals from the cytoplasmic and vacuolar pools of amino acids. When the ammonium pretreatment method was applied to Acer pseudoplatanus cells (Aubert et al., 1998) and the leaves of Pringlea antiscorbutica (Aubert et al., 1999), it was possible to generate a significant increase in the cytoplasmic pH in only 30 min using just 0.5 mM ammonium at pH 9. Thus in the case of the sycamore cells, the cytoplasmic pH increased from 7.5 to 8.5, and the vacuolar pH from 5.7 to 6.5, under these conditions and as a result the C3 signal from the homoserine pool split into two separate signals corresponding to the cytoplasmic and vacuolar pools. However, the generality of this method is called into question by the data in Fig. 4, where it can be seen that it was necessary to use a 10 mM ammonium solution at pH 9 to generate even a small increase in the cytoplasmic pH of maize root tips, and this treatment had an immediate effect on the vacuolar pH. Ultimately, the success of the ammonium pretreatment method depends on being able to impose an alkalinization of the cytoplasm before the inevitable accumulation of the ammonium in the more acidic vacuole leads to a significant increase in the vacuolar pH (Pick et al., 1991). It follows that the method is likely to be critically dependent on such factors as the accessibility of the cells to ammonium, the cytoplasmic to vacuolar volume ratio, and the buffering capacity of the two compartments. The results presented here suggest that it may be difficult to satisfy these criteria in a dense heterogeneous tissue such as the maize root tip since a significant alkalinization of the cytoplasm was only observed under conditions which had an immediate and variable effect on the vacuoles.
Cause and extent of ammonium-induced alkalinization
The free base accounts for about 36% of the total ammonium concentration at pH 9, assuming a pKa of 9.25, and since membranes are highly permeable to NH3 (Kleiner, 1981), it is reasonable to attribute the ammonium-induced intracellular alkalinization to the influx of uncharged NH3 and its subsequent protonation. The extent of the alkalinization can be calculated from the observed accumulation of ammonium (Fig. 7) and the results are summarized in Table 3.
|
The predicted pH values in Table 3 depend on several assumptions. First, it is assumed that there is negligible pH regulation and that the observed pH change is entirely the result of the subcellular accumulation of ammonium. Secondly, it is assumed that the cytoplasm and vacuole each occupy 45% of the tissue volume in 5 mm maize root tips; these volumes have been measured in 5 mm pea root tips (Patel et al., 1990
) and protein content measurements suggest that the volumes are similar in 5 mm maize root tips (Spickett et al., 1993). Thirdly, it is necessary to have an estimate of the buffering capacity for each compartment, and the values used for the calculations in Table 3 fall into the measured range. Thus the cytoplasmic buffering capacity in plant cells lies in the range 20–100 µmol H+ ml-1 pH-1 unit (Kurkdjian and Guern, 1989); while reported values of the vacuolar buffering capacity include 7 µmol H+ g-1 tissue pH-1 unit in 2 mm maize root tips (Roberts et al., 1992) and 36 µmol H+ ml-1 pH-1 unit in vacuoles isolated from Catharanthus roseus cells (Mathieu et al., 1989). Fourth, while the calculations allow for the contribution to the buffering capacity from the ammonium at high pH values, it is otherwise assumed that the buffering capacity is constant, i.e. no allowance has been made for the expected pH-dependent variation in the buffering capacity of the cytoplasm and vacuole. Finally, in the absence of direct measurements of the subcellular ammonium pools, it is necessary to make an assumption about the distribution of the ammonium between the cytoplasm and the vacuole. In general, as demonstrated in maize root tips (Lee and Ratcliffe, 1991), the lower pH of the vacuole would favour the vacuolar accumulation of most of the ammonium, but this tendency will diminish as the influx of NH3 raises the vacuolar pH. While the assumptions in the pH calculations, and particularly the uncertainties in the buffering capacities and the subcellular distribution of the ammonium, limit the accuracy of the predicted pH values in Table 3, the general agreement with the results in Figs 4–6 support the view that the observed pH changes are largely determined by the influx of NH3. Thus the predicted values show that the influx of 34 µmol ammonium g-1 FW is capable of generating a cytoplasmic pH in the range 7.8–8, assuming an intermediate buffering capacity and a cytoplasmic : vacuolar ammonium distribution ratio of 1 : 2 or 1 : 4; and that the same treatment can increase the vacuolar pH to the same range as the cytoplasmic pH. The calculations also show that the efflux of most of the ammonium from the tissue, coupled with the concentration of most of the remaining ammonium in the vacuole, can restore the cytoplasmic pH to the observed value. Thus the calculations emphasize the importance of ammonium efflux in the recovery of the pH.
Contribution of ammonium assimilation to pH regulation
Although the calculations summarized in Table 3 indicate that the cytoplasmic and vacuolar pH changes are largely determined by the influx and efflux of the weak base NH3, there are several other mechanisms that might influence pH during these experiments. Figure 9 shows that the root tips are able to assimilate ammonium via the GS/GOGAT pathway and, while the amount of assimilation is small, the potential contribution to pH regulation is worth discussing in the light of a recent paper in which it was argued that ammonium assimilation could be considered to be a proton-consuming process (Kosegarten et al., 1997). This conclusion was reached on the basis of (i) experimental evidence that showed that inhibition of ammonium assimilation with MSO resulted in a smaller cytosolic alkalinization in rice root hairs during ammonium uptake and (ii) an analysis of the stoichiometry of the GS/GOGAT pathway. Irrespective of any consideration of whether the fluorescent probe was reporting cytosolic pH changes (Kosegarten et al., 1997; Wilson et al., 1998) or vacuolar pH changes (Brauer et al., 1997), the argument that the GS/GOGAT pathway consumes protons is surprising since it is generally agreed that using ammonium as a nitrogen source leads to the release of protons (Raven and Smith, 1976; Raven, 1986). One well-known consequence is an acidification of the external medium when plants are grown on ammonium (Marschner and Römheld, 1983; Tolley-Henry and Raper, 1986). Moreover it has also been argued that the endogenous proton production associated with ammonium assimilation can lead to lower cytoplasmic pH values in the root tips of ammonium-grown maize seedlings in acidic media (Gerendás et al., 1993).
In order to explain the discrepancy between these two views it is necessary to look at the stoichiometry of the GS/GOGAT pathway in some detail. One balanced equation that describes the pathway takes the form:
 |
 |
). However, in assessing the overall proton balance of the GS/GOGAT pathway it is also necessary to consider the source of the electrons and the 2-oxoglutarate for the balanced equation given above. The electrons are likely to be derived from the oxidation of glucose:
 |
 |
 |
 |
 |
 |
The situation changes if these conditions are not satisfied. For example, if the 2-oxoglutarate pool is replenished by the metabolism of stored malate, via the combined effects of malic enzyme and malate dehydrogenase, then
 |
 |
 |
 |
This analysis shows that the assimilation of ammonium is only likely to consume protons under rather specific metabolic conditions, i.e. under conditions where there is inadequate mobilization of reduced carbon to maintain a supply of reducing power (electrons) and carbon skeletons for the GS/GOGAT pathway. This situation is most likely to arise under conditions of very rapid ammonium assimilation, and this would appear to be the most likely explanation for the observations reported by Kosegarten et al. on rice root hairs (Kosegarten et al., 1997). In fact, pretreatment with MSO had no effect on the ammonium-induced alkalinization in maize root hairs (Kosegarten et al., 1997) and so it would be interesting to measure the ammonium assimilation rates in the two species.
More generally, ammonium assimilation is expected to release protons and since Fig. 9 shows that assimilation can occur during the influx of ammonium at pH 9, this process would have tended to counteract the observed alkalinization of the cytoplasm. However, the contribution of ammonium assimilation to the observed pH changes could only have been marginal, since the production of labelled glutamine and glutamate was limited over the short time-scale of these experiments. Thus, in contrast to earlier work on carrot cells where ammonium assimilation was observed to cause an acidification of the cytoplasm under conditions of reduced oxygen supply (Carroll et al., 1994), there was no obvious manifestation of the proton-releasing character of ammonium assimilation in the experiments described here.
One final point in this discussion concerns the NTP level. The analysis of the GS/GOGAT pathway presented here assumes that the ATP required by the GS reaction is regenerated, and under these conditions the GS reaction can be considered to be proton neutral. In fact the NTP level dropped by up to 50% in these experiments (Fig. 6), and the contribution of the associated release of protons to reducing the alkalinization of the cytoplasm during the 1 h treatment with ammonium was probably comparable to the contribution from the limited amount of ammonium assimilation that occurred over the same period.
Contribution of carboxylate synthesis to pH regulation
Increased malate synthesis, following the pH-dependent activation of PEP carboxylase, is a common response to alkalinization of the cytoplasm (Davies, 1986; Gout et al., 1992). The pathway generates protons and, in principle, it can contribute to pH regulation through the operation of a biochemical pH-stat (Davies, 1986), although the significance of this contribution to pH homeostasis is debatable (Gout et al., 1992). In the experiments reported here, malate synthesis in the maize root tips increased during exposure to 10 mM ammonium at pH 9 (Figs 10, 11), but the increase in CO2 fixation (0.44 µmol h-1 g-1 FW) was only modest in comparison with the influx of ammonium (34 µmol g-1 FW). It can be concluded that the proton production associated with malate synthesis would have done little to offset the ammonium-induced alkalinization.
Contribution of ion transport processes to pH regulation
The pH of a solution is determined by the buffering capacity, which is a function of all the weak acids and bases in the system, and by the strong ion difference (SID), the net unbalanced charge on the fully dissociated ions in the solution (Stewart, 1983; Ullrich and Novacky, 1992; Gerendás and Schurr, 1999). For intracellular solutions, these properties are determined by the effects of metabolism and membrane transport, and any change in pH must be caused by some combination of the biochemical and biophysical events that can alter the composition of the solution. A key feature of this way of describing pH is that intracellular pH changes cannot be explained simply in terms of the H+ fluxes generated by electrogenic proton pumps; such fluxes only occur under conditions where there is a stoichiometric flux of other ions across the same membrane and the change in pH should be attributed to the resulting change in SID (Gerendás and Schurr, 1999).
In the case of the ammonium-induced alkalinization of the maize root tips, influx of NH3 increases the concentration of a weak base, and the subsequent protonation increases the intracellular pH values. In principle, biophysical events, as well as such biochemical events as the assimilation of ammonium and the stimulation of malate synthesis, could counteract the alkalinization of the intracellular solutions, since enhanced cation efflux or enhanced anion influx would both result in a decreased SID and a lower pH. Potassium efflux (Thibaud et al., 1986) and chloride influx (Smith, 1980) are the two most likely candidates for ion transport, but potassium efflux is of greater interest here because chloride was not included in the suspending medium. Efflux of potassium would require depolarization of the plasma membrane, and ammonium-induced depolarizations have been observed, for example, in Riccia cells during exposure to only 20 µM ammonium at pH 5.6 (Felle, 1980). The high cytoplasmic pH and the low ATP content observed in the maize root tips during exposure to 10 mM ammonium at pH 9 would both favour depolarization, since the activity of the plasma membrane H+-ATPase decreases with increasing pH above its pH optimum of 6–6.5 (Fischer-Schliebs et al., 1994) and its Km value (0.3–1.4 mM; Michelet and Boutry, 1995) is comparable with the normal cytoplasmic ATP concentration (Lee and Ratcliffe, 1993). If potassium efflux did occur in these experiments, then it could provide an explanation for the discrepancy between the predicted vacuolar pH at the end of the experiment (for example, pH 6.06, assuming a cytoplasmic:vacuolar ammonium distribution of 1:9 and a vacuolar buffering capacity of 20 µmol H+ g-1 tissue pH-1 unit, Table 3) and the observed value of 5.35 (Fig. 5). Calculations suggest that the efflux of around 7.1 µmol K+ g-1 FW would account for this discrepancy, which is close to the ammonium content at the end of the experiment (8.5 µmol NH+4 g-1 FW). It would be interesting to measure this putative flux in future work, but such measurements would be unlikely to alter the main conclusion from Table 3, which is that the observed pH changes are mainly determined by the influx and efflux of NH3.
|
Acknowledgments |
|---|
We thank the Deutsche Forschungsgemeinschaft (JG) and the BBSRC (RGR) for financial support. We also thank B Sattelmacher for stimulating discussions and his interest in this work.
|
Notes |
|---|
3 To whom correspondence should be addressed. Fax: +49 431 880 1625. E-mail: jgerendas{at}plantnutrition.uni\|[hyphen]\|kiel.de
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Aubert S, Curien G, Bligny R, Gout E, Douce R. 1998. Transport, compartmentation and metabolism of homoserine in higher plant cells. Carbon-13 and phosphorus-31 nuclear magnetic resonance studies. Plant Physiology 116, 547–557.
Aubert S, Hennion F, Bouchereau A, Bligny R, Dorne AJ. 1999. Subcellular compartmentation of proline in the leaves of the subantarctic Kerguelen cabbage Pringlea antiscorbutica R. Br. In vivo 13C NMR study. Plant, Cell and Environment 22, 255–259.
Brauer D, Uknalis J, Triana R, Tu SI. 1997. Effects of external pH and ammonium on vacuolar pH in maize roothair cells. Plant Physiology and Biochemistry 35, 31–39.
Carroll AD, Fox GG, Laurie S, Phillips R, Ratcliffe RG, Stewart GR. 1994. Ammonium assimilation and the role of 
-aminobutyric acid in pH homeostasis in carrot cell suspensions. Plant Physiology 106, 513–520.[Abstract]
Davies DD. 1986. The fine control of cytosolic pH. Physiologia Plantarum 67, 702–706.
Felle H. 1980. Amine transport at the plasmalemma of Riccia fluitans. Biochimica et Biophysica Acta 602, 181–195.[Medline]
Fischer-Schliebs E, Varanini Z, Lüttge U. 1994. Isolation of H+-transport-competent plasma membrane vesicles from corn roots by discontinuous sucrose gradient centrifugation: effect of membrane protectant agents. Journal of Plant Physiology 144, 505–512.
Fox GG, Ratcliffe RG. 1990. 31P NMR observations on the effect of the external pH on the intracellular pH values in plant cell suspension cultures. Plant Physiology 93, 512–521.
Gerendás J, Ratcliffe RG, Sattelmacher B. 1990. 31P nuclear magnetic resonance evidence for differences in intracellular pH in the roots of maize seedlings grown with nitrate or ammonium. Journal of Plant Physiology 137, 125–128.[ISI]
Gerendás J, Ratcliffe RG, Sattelmacher B. 1993. Relationship between intracellular pH and N metabolism in maize (Zea mays L.) roots. Plant and Soil 155/156, 167–170.
Gerendás J, Ratcliffe RG, Sattelmacher B. 1995. The influence of nitrogen and potassium supply on the ammonium content of maize (Zea mays L.) leaves including a comparison of measurements made in vivo and in vitro. Plant and Soil 173, 11–20.
Gerendás J, Schurr U. 1999. Physicochemical aspects of ion relations and pH regulation in plants—a quantitative approach. Journal of Experimental Botany 50, 1101–1114.
Gerendás J, Zhu Z, Bendixen R, Ratcliffe RG, Sattelmacher B. 1997. Physiological and biochemical processes related to ammonium toxicity in higher plants. Zeitschrift für Pflanzenernährung und Bodenkunde 160, 239–251.
Gout E, Bligny R, Douce R. 1992. Regulation of intracellular pH values in higher plant cells. Carbon-13 and phosphorus-31 nuclear magnetic resonance studies. Journal of Biological Chemistry 267, 13903–13909.
Hanstein S, Mattsson M, Jaeger HJ, Schjoerring JK. 1999. Uptake and utilization of atmospheric ammonia in three native Poaceae species: leaf conductances, composition of apoplastic solution and interactions with root nitrogen supply. New Phytologist 141, 71–83.
Izaurralde RC, Kissel DE, Cabrera ML. 1990. Simulation model of banded ammonia in soils. Soil Science Society of America Journal 54, 917–922.
Kleiner D. 1981. The transport of NH3 and NH+4 across biological membranes. Biochimica et Biophysica Acta 639, 41–52.
Kosegarten H, Grolig F, Wieneke J, Wilson G, Hoffmann B. 1997. Differential ammonia-elicited changes of cytosolic pH in root hair cells of rice and maize as monitored by 2',7'-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein fluorescence ratio. Plant Physiology 113, 451–461.[Abstract]
Kurkdjian A, Guern J. 1989. Intracellular pH: measurement and importance in cell activity. Annual Reviews of Plant Physiology 40, 271–303.[ISI]
Lee RB, Ratcliffe RG. 1983. Development of an aeration system for use in plant tissue NMR experiments. Journal of Experimental Botany 34, 1213–1221.
Lee RB, Ratcliffe RG. 1991. Observation on the subcellular distribution of the ammonium ion in maize root tissue using in vivo 14N nuclear magnetic resonance spectroscopy. Planta 183, 359–367.[ISI]
Lee RB, Ratcliffe RG. 1993. Subcellular distribution of inorganic phosphate, and levels of nucleoside triphosphate, in mature maize roots at low external phosphate concentrations: measurements with 31P NMR. Journal of Experimental Botany 44, 587–598.
Marschner H. 1995. Mineral nutrition of higher plants, 2nd edn. London: Academic Press.
Marschner H, Römheld V. 1983. In vivo measurement of root-induced pH changes at the soil-root interface: effect of plant species and nitrogen source. Zeitschrift für Pflanzenphysiologie 111, 241–251.
Martin JB, Bligny R, Rebeille F, Douce R, Leguay JJ, Mathieu Y, Guern J. 1982. A 31P nuclear magnetic resonance study of intracellular pH of plant cells cultivated in liquid medium. Plant Physiology 70, 1156–1161.
Mathieu Y, Guern J, Kurkdjian A, Manigault P, Zielinska T, Gillet B, Beloeil JC, Lallemand JY. 1989. Regulation of vacuolar pH of plant cells. 1. Isolation and properties of vacuoles suitable for 31P NMR studies. Plant Physiology 89, 19–26.
Michelet B, Boutry M. 1995. The plasma membrane H+-ATPase. A highly regulated enzyme with multiple physiological functions. Plant Physiology 108, 1–6.[ISI][Medline]
Mistrik I, Ullrich CI. 1996. Mechanism of anion uptake in plant roots: quantitative evaluation of H+/NO-3 and H+/H2PO-4 stoichiometries. Plant Physiology and Biochemistry 34, 629–636.
Ninnemann O, Jauniaux JC, Frommer WB. 1994. Identification of a high affinity NH++ transporter from plants. EMBO Journal 13, 3464–3471.
Patel DD, Barlow PW, Lee RB. 1990. Development of vacuolar volume in the root tips of pea. Annals of Botany 65, 159–169.
Pearson J, Stewart GR. 1993. The deposition of atmospheric ammonia and its effects on plants. New Phytologist 125, 283–305.[ISI]
Pick U, Zeelon O, Weiss M. 1991. Amine accumulation in acidic vacuoles protects the halotolerant alga Dunaliella salina against alkaline stress. Plant Physiology 97, 1226–1233.
Ratcliffe RG. 1994. In vivo NMR-studies of higher plants and algae. Advances in Botanical Research 20, 43–123.
Raven JA. 1985. Regulation of pH and generation of osmolarity in vascular plants: a cost-benefit analysis in relation to efficiency of use of energy, nitrogen and water. New Phytologist 101, 25–77.[ISI]
Raven JA. 1986. Biochemical disposal of excess H+ in growing plants? New Phytologist 104, 175–206.
Raven JA, Smith FA. 1976. Nitrogen assimilation and transport in vascular land plants in relation to intracellular pH regulation. New Phytologist 76, 415–431.[ISI]
Roberts JKM, Hooks MA, Miaullis AP, Edwards S, Webster C. 1992. Contribution of malate and amino acid metabolism to cytoplasmic pH regulation in hypoxic maize root tips studied using nuclear magnetic resonance spectroscopy. Plant Physiology 98, 480–487.
Roberts JKM, Pang MKL. 1992. Estimation of ammonium ion distribution between cytoplasm and vacuole using nuclear magnetic resonance spectroscopy. Plant Physiology 100, 1571–1574.
Saglio PH, Pradet A. 1980. Soluble sugars, respiration, and energy charge during ageing of excised maize root tips. Plant Physiology 66, 516–519.
Smith FA. 1980. Comparison of the effects of ammonia and methylamine on chloride transport and intracellular pH in Chara corallina. Journal of Experimental Botany 31, 597–606.
Spickett CM, Smirnoff N, Ratcliffe RG. 1993. An in vivo nuclear magnetic resonance investigation of ion transport in maize (Zea mays) and Spartina anglica roots during exposure to high salt concentrations. Plant Physiology 102, 629–638.[Abstract]
Stehouwer RC, Traina SJ, Johnson JW. 1993. Potassium adsorption and exchange selectivity within an anhydrous ammonia fertilizer band. Soil Science Society of America Journal 57, 346–350.
Stewart PA. 1983. Modern quantitative acid-base chemistry. Canadian Journal of Physiology and Pharmacology 61, 1444–1461.[I