JXB Advance Access published online on January 8, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erl247
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Accumulation of ammonium in Norway spruce (Picea abies) seedlings measured by in vivo 14N-NMR
1Department of Biology, University of Oslo, PO Box 1066, Blindern, N-0316 Oslo, Norway
2Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315, Oslo, Norway
* To whom correspondence should be addressed. E-mail: halvor.aarnes{at}bio.uio.no
Received 26 July 2006; Revised 17 October 2006 Accepted 26 October 2006
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Abstract |
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14N-NMR and 31P-NMR have been used to monitor the in vivo pH in roots, stems, and needles from seedlings of Norway spruce, a typical ammonium-tolerant plant. The vacuolar and cytoplasmic pH measured by 31P-NMR was found to be c. pH 4.8 and 7.0, respectively, with no significant difference between plants growing with ammonium or nitrate as the N-source. The 1H-coupled 14NH
resonance is pH-sensitive: at alkaline pH it is a narrow singlet line and below pH 4 it is an increasing multiplet line with five signals. The pH values in ammonium-containing compartments measured by 14N-NMR ranged from 3.7 to 3.9, notably lower than the estimated pH values of the Pi pools. This suggests that, in seedlings of Norway spruce, ammonium is stored in vacuoles with low pH possibly to protect the seedlings against the toxic effects of ammonium (NH) or ammonia (NH3). It was also found that concentrations of malate were 3–6 times higher in stems than in roots and needles, with nitrate-grown plants containing more malate than plants grown with ammonium. Key words: Ammonium, cytoplasmic pH, in vivo 14N-NMR, in vivo 31P-NMR, malate, organic acids, Picea abies (L.) Karst, vacuolar pH
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Introduction |
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Conifers and ericaceous plants adapted to acid soils with low or no nitrification have a preference for NH
as the main nitrogen source. These plants can tolerate high concentrations of NH and have been found to possess a reduced capacity to use NO
(Kronzucker et al., 1995, 1997; Forde and Clarkson, 1999; Britto et al., 2001a; Britto and Kronzucker, 2002; Schjoerring et al., 2002), although in Picea abies and Vaccinium myrtillus short- and long-term field studies have shown the same capacity for NH and NO utilization (Persson et al., 2003). Sources of NH in soils are the ammonification of organic nitrogen and/or atmospheric wet- and dry-deposition of NH. High concentrations of NH can be harmful to non-adapted plants and toxicity can be avoided by rapidly converting NH to amino acids in the roots or by sequestering NH in a vacuolar reservoir. NH-toxicity syndromes are mostly found in NO-adapted plants and are still not well understood (Mehrer and Mohr, 1989; Britto et al., 2001a, b; Britto and Kronzucker, 2002). NH uptake by the roots is accompanied by the uptake of monovalent anions or the release of H+-ions resulting in acidification of the soil (Marschner et al., 1991; Forde and Clarkson, 1999; Schjoerring et al., 2002) which can be one of the causes of ammonium toxicity (Britto and Kronzucker, 2002). Most plants are susceptible to NH toxicity when NH is the only nitrogen source, an exception is, for instance, wetland rice that can tolerate very high concentrations of NH when growing on flooded anoxic soil with low nitrification (Britto et al., 2001b).
Primary assimilation of NH into amino acids is catalysed by the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT). Interestingly, although glutamine synthetase has a Km for NH at µmolar concentration, NH-adapted plants have been found to contain millimolar concentrations of NH (Lee and Ratcliffe, 1991; Aarnes et al., 1995; Britto et al., 2001a). We have earlier reported high concentrations of NH in all parts of spruce seedlings quantified by 14N-NMR and 15N-NMR and have found indications that NH can be stored in acidic compartments (Aarnes et al., 1995).
In vivo 31P-NMR (Ratcliffe, 1994) can be used to estimate intracellular vacuolar and cytoplasmic pH (Kime et al., 1982; Martin et al., 1982; Gerendás et al., 1990; Katsuhara et al., 1997; Espen et al., 2004; Pfeffer et al., 2004). In vivo 14N-NMR and 31P-NMR were used to compare the pH in vacuoles and cytoplasm of spruce seedlings growing with NH or NO as the nitrogen source. In addition, the amount of malate and citrate was monitored in different parts of the plants since these organic acids can play a role in cellular pH homeostasis (Britto and Kronzucker, 2005).
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Materials and methods |
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Plant material
Seeds of Norway spruce [Picea abies (L.) Karst.] provenance B2 were grown for 3 weeks in vermiculite/perlite at an air temperature of 22 °C and a relative humidity of 60% in a climate-conditioned growth room in continuous light (250 µmol m–2 s–1, using metal halide lamps, Kolorarc 400 W from General Electric Co., USA) as described by Aarnes et al. (1995). Groups of 20 plants were removed from the growth medium, rinsed in distilled water, and transferred to drams glass containing 10 ml nutrient solution with different concentrations of nitrogen (5–25 mM N), supplied as (NH4)2SO4 or KNO3. The plants were grown for 2 d in the different nutrient solutions in the same light and temperature regime as described above. The pH of the nutrient solutions was determined with a glass electrode (Radiometer, Denmark).
NMR
One hundred seedlings were divided into roots, stems, and needles that were wrapped in Teflon tape and put into a 10 mm NMR tube with circulating air-bubbled buffer (25 mM MES pH 6.0, 0.01 mM CaSO4) at 20 °C (Lee and Ratcliffe,1983). 31P-NMR and 14N-NMR spectra were obtained by using a Bruker DPX300 spectrometer with a BBO 10 mm probe head. 31P-NMR spectra were recorded at 121.51 MHz, with an acquisition time of 0.85 s, a relaxation delay of 1 s, and a pulse angle of 90°. Chemical shifts were compared to 17 mM methylene diphosphonic acid in a capillary tube as external standard resonating at 16.9 ppm compared with H3PO3. The standard calibration curve that related chemical shifts of intracellular 31P resonances to different pH values was made according to Martin et al. (1982) and Roberts et al. (1981). The intracellular pH of the Pi pools was determined by using the chemical shifts of the 31P-NMR spectra. 14N-NMR spectra were recorded at 21.68 MHz, a 70° pulse angle, a recycle time of 4 s, and 1024 scans.
The pH value of the ammonium-containing compartments was determined by using the pH-dependent 1H-coupled line shape of 14NH-1H-coupled spectra. Solutions with 25 mM ammonium-acetate at different pH were used as pH standards. NO was used as a standard (0 ppm).
Extraction and determination of malate and citrate
Groups of 20 plants were washed in distilled water, divided into roots, stems, and needles and homogenized by grinding in a mortar with 6 ml ice-cold distilled water. The homogenates were centrifuged for 15 min at 10 000 g (4 °C). The supernatant was used to measure the pH and the concentrations of organic acids by a coupled enzymatic UV-assay (340 nm) for food analysis using test combinations for L-malic acid, citric acid, and oxalic acid from Boehringer Mannheim/Roche.
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Results |
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A typical in vivo 31P-spectrum of roots of Norway spruce seedlings with peaks for vacuolar and cytoplasmic Pi, sugar phosphates, and nucleoside phosphate is shown in Fig. 1. Similar spectra were obtained from stems and needles. The spectra give information about the pH in vacuoles and in the cytoplasm by comparing 31P-NMR Pi signals with a calibration curve showing the pH dependence of the Pi chemical shift (ppm). A pH of c. 4.8 in vacuoles and c. 7.0 in the cytoplasm were found, with no significant difference in vacuolar and cytoplasmic pH for plants growing with NO
or NH (Table 1).
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Figure 2 shows in vivo 14N-NMR spectra of NH
in roots, stems, and needles compared with spectra of different NH-pH standards. At increasing acidity intensity the five peak signal at around 355 ppm in the 14NH-NMR spectra becomes increasingly pronounced. Above pH 4 the 1H-coupled 14NH-NMR signal consists of only one peak. Using the right peak of the quintet signal as a reference, the pH in roots, stems, and needles was estimated to be 3.9, 3.9, and 3.7, respectively. The 14N-NMR spectra also showed a peak from the 
-amino nitrogen in amino acids at c. –335 ppm. The amino acid nitrogen peak was more pronounced in stems and needles than in roots.
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The amounts of malate and citrate in water extracts of roots, stems and needles are shown in Table 2. Concentrations of malate were 3–6 times higher in stems than in roots and needles. Malate concentrations were significantly lower in roots and stems of NH
-fed plants compared to plants grown with NO or without nitrogen. Except for stems of NO-fed plants, there were no significant differences between plants growing on NO or water (Table 2). Concentrations of oxalate were relatively high with 29.6±2.4; 19.8±0.9, and 40±4.8 µmol g–1 FW in needles, stems and roots, respectively. Oxalate concentrations did not appear to be affected by the nitrogen source. Based on a dry weight of 15% the concentrations of NH in spruce plants were recalculated from the 14NH NMR spectra to be 5–54 mM for plants growing in nutrient solutions with 0.5–50 mM NH. The highest content of ammonium was found in roots and needles of plants growing in nutrient solution containing 2.5 mM (NH4)2SO4. An increase in the nitrogen level of the nutrient solution up to 50 mM NH did not further affect the NH content of the plants. Similar results were obtained from 15N NMR spectra from plants growing on 15NH as shown previously (Aarnes et al., 1995). Recalculation of these data gave slightly lower amounts of 15NH in roots ranging from 9–23 mM, and up to 13 mM in the stems. However, very low concentrations of 15NH were found in needles. In this case the highest amount of NH was found in roots of plants growing in a nutrient solution with 2.5 mM 15NH4NO3.
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Discussion |
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In the present study the pH in different parts of cells in roots, stems, and needles of Norway spruce seedlings has been measured by in vivo NMR methods. Cytoplasmic and vacuolar pH in roots, stems, and needles was found to be 7.0 and 4.8, respectively, while 14N-NMR spectra of NH
showed a pH of about 3.8. The estimated cytoplasmic pH (around 7.0) is lower than reported pH values of 7.7 for roots of Pinus sylvestris and 7.5 in leaves of Pisum sativum (Gerlitz and Werk, 1994; Bligny et al., 1997). This may be due to mild hypoxia in the samples (Felle, 2005), most probably because of slow buffer circulation and aeration instead of oxygenation. Hydroponics were used for growth of the seedlings, a process that differs from the natural situation involving ectomycorrhiza.
The position of the Pi signal is insensitive to a pH below 5, allowing only approximate estimates of pH in this range by 31P-NMR. To circumvent these limitations the 13C-NMR signal of malate (Chang and Roberts, 1989) and 19F-NMR (Pfeffer et al., 2004) have been used previously. In contrast to the 31P-NMR signal, the line-shape of the ammonium signal is clearly sensitive to pH changes in the acidic region, allowing a more accurate estimation of the pH in the ammonium-containing compartment. At low pH the nitrogen atoms will always carry four hydrogen atoms splitting the shape of the coupled- 14NH resonance into a five-peak signal. The amount of NH3 will increase with increasing pH, but the three hydrogen atoms will not stay long enough at the nitrogen atom for the NMR response to be quartet. Because hydrogen jumps on and off, the nitrogen nucleus has no time to see the effect of hydrogen, and at a pH above pH 4 the signal becomes a single peak, as for a decoupled signal, eventually changing to a narrow single peak between pH 4 and 6.
The 31P-NMR method used here to determine the pH of cytoplasm and vacuole only estimates the pH in phosphate compartments which, according to the 14N-NMR results, seem to be less acidic than the NH compartments. As NH and NH3 (ammonia) are in a pH-dependent equilibrium an acidic NH compartment will keep the pH considerably lower than the pKa value ensuring an NH/NH3 equilibrium displaced towards NH. Acidic NH compartments separated from the main phosphate storage pools could explain the pH values found here. However, it is also possible that Pi is stored in the NH-storing vacuoles because the position of the Pi signal in 31P-NMR is relatively insensitive to pH changes below 5 (Pfeffer et al., 2004). Belton et al. (1985) have also proposed NH compartments in their study of inorganic nitrogen metabolism in NH-fed barley roots with the 14N-NMR method, but pH values as low as those reported here have only been described for vacuoles of some hyperacidifying plants (Felle, 2005). Neither the intra-thylakoidal space of chloroplasts under full illumination, nor vesicles of the endosomal Golgi-related complex, have been shown to have such low pH.
Roberts and Pang (1992) has used both the 31P-NMR and 13C-NMR method to examine the NH distribution between cytoplasm and vacuoles in maize root tips. They found rapid movement and accumulation of NH in the vacuole, but no significant effects of NH treatments on cytoplasmic pH with the 31P-NMR method. The more sensitive 13C-method showed an increase in short-term NH-induced vacuolar pH (Roberts and Pang, 1992). Gerendás et al. (1990) and Gerendás and Ratcliffe (2000) have used in vivo 31P-NMR spectroscopy and have shown ammonium-induced changes in both cytoplasmic and vacuolar pH values in root tissues from maize seedlings grown in alkaline or acidic nutrient solution with NH. In spruce, which is a gymnosperm, the situation might be different from maize where Espen et al. (2004) have found that nitrate uptake is an acidifying process. In this study, no effect of different nitrogen sources on the pH in cytoplasm and vacuoles could be measured by 31P-NMR. This could be due to NH compartmentation and might represent an effective system for homeostasis of cellular pH. However, in our studies, the K+-levels in the nutrient solution are allowed to vary widely between treatment since KNO3 was used as nitrate source, and both the K and N levels are high compared with nutrient levels actually present in the natural environment. Separate acid compartments could allow high concentrations of NH in cells without harmful effects for the plant.
We have earlier reported high levels of NH (4–46 µmol NH g–1 FW in roots, stems, and needles of young Norway spruce seedlings growing in nutrient solutions with NH-N ranging from 5–50 mM as measured by 14N- and 15N-NMR (Aarnes et al., 1995). In these analyses the 15N-estimates of NH levels were slightly lower than the 14N-estimates.
In vivo 14N-NMR methods allow direct determination of ammonium concentrations and its subcellular distribution in cells (Mesnard and Ratcliffe, 2005). Comparing decoupled and coupled in vivo 14N-NMR spectra, Lee and Ratcliffe (1991) estimated vacuolar and cytoplasmic concentrations of NH in maize roots and found up to 15 mM NH vacuoles. Britto et al. (2001b) used the short-lived radiotracer 13N for the estimation of cytosolic concentrations of NH in root cells of barley and rice. Notably high levels of NH were found, 358 mM and 232 mM for barley and rice, respectively. Similarly, high NH levels were reported in the cytosol of root cells of aspen (134 mM), Douglas-fir (77 mM), and white spruce (33 mM) (Kronzucker et al., 2003), all in line with the high NH levels we have found in roots of Norway spruce (Aarnes et al., 1995).
Millimolar concentrations of NH were also found in the xylem sap and leaf apoplast of oilseed rape and tomato (Schjoerring et al., 2002), and in the cytosol of both NH-sensitive and -tolerant plants grown in root medium with high NH-concentrations (Kronzucker et al., 2003). Interestingly, the highest cytosolic NH concentrations were found in NH-sensitive plant species, indicating an uncontrolled accumulation of NH in these plants. One explanation for NH toxicity could be an increased energetic burden for the sensitive plants (Britto et al., 2001b; Kronzucker et al., 2003).
In addition to fluxes, assimilation of NH is important for the regulation at the cytosolic level. It was concluded earlier that, by inhibiting GS by methionine sulphoximine, spruce plants use the GS/GOGAT NH-assimilation pathway (Aarnes et al., 1995). By the use of 15N-NMR spectroscopy, high concentrations of alanine, glutamine, and arginine have been found, the latter two being efficient NH-storage and NH-transport forms. Spruce plants can also use urea as a nitrogen source, and the arginine and ornithine cycle are important in the further assimilation of NH (Thorpe et al., 1989; Aarnes et al., 1995). When K15NO3 was used in the nutrient solution, a small signal from NH was obtained in the roots, in stems a very small signal, and in needles no signal was found at all. The lack of 15N signals in needles, when plants were grown in 5 mM K15NO3, can be a dilution effect where it was not possible to label NH100% (Aarnes et al., 1995) However, the low amount of NH found in the NO-fed plants is the reason why only data from NH-grown plants are presented in this study.
The import of NH is coupled to the export of protons, explaining the low pH in the root medium of plants growing on NH. H+-ATPase can generate pH gradients that mediate secondary transport and play a role in pH homeostasis. Chang and Roberts (1989) suggested that malate can provide H+ that can be stored in compartments in the cell. Davies (1986) has proposed a biochemical pH-stat based on the formation and removal of carboxyl groups from malate or oxalate in order to maintain pH homeostasis. A revision of the classical malate biochemical pH-stat hypothesis has been suggested (Britto and Kronzucker, 2002, 2005), emphasizing the importance of the membrane proton system and reaffirming the anaplerotic role of PEP carboxylase within the context of N metabolism, assimilation, and storage. High concentrations of malate were found in the stem, suggesting a role of malate in the transport of nitrogen from root to shoots in spruce seedlings. High malate concentrations in the stem might also lead to the accumulation of malate in xylem parenchyma cells, even at low malate concentrations in the xylem sap. High concentrations of water-soluble oxalate, which is probably present in vacuoles or cell walls, was also found. Organic acids were extracted with water, and a denaturing extraction agent to reduce the possible action of enzymes during homogenization of tissues would have been better.
In summary, the 14N-NMR data suggest that, in Norway spruce seedlings, large amounts of NH are stored in acidic vacuoles thereby reducing the harmful effects of NH3 and NH on cells.
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Acknowledgements |
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The authors thank Tim Southon for kindly introducing us to the in vivo NMR technique, and Dr Uwe Klein and two anonymous referees for valuable suggestions.
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References |
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