Journal of Experimental Botany, Vol. 53, No. 379, pp. 2305-2314,
December 1, 2002
© 2002 Oxford University Press
Utilization of glycine and serine as nitrogen sources in the roots of Zea mays and Chamaegigas intrepidus
Received 4 March 2002; Accepted 3 July 2002
1 Julius-von-Sachs Institut für Biowissenschaften, Lehrstuhl Botanik I, Universität Würzburg, Julius-von-Sachs Platz 2, D-97082 Würzburg, Germany
2 Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK
3 To whom correspondence should be addressed. Fax: +44 (0)1865 275074. E-mail: george.ratcliffe{at}plants.ox.ac.uk
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Glycine and serine are potential sources of nitrogen for the aquatic resurrection plant Chamaegigas intrepidus Dinter in the rock pools that provide its natural habitat. The pathways by which these amino acids might be utilized were investigated by incubating C. intrepidus roots and maize (Zea mays) root tips with [15N]glycine, [15N]serine and [2-13C]glycine. The metabolic fate of the label was followed using in vivo NMR spectroscopy, and the results were consistent with the involvement of the glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT) in the utilization of glycine. In contrast, the labelling patterns provided no evidence for the involvement of serine:glyoxylate aminotransferase in the metabolism of glycine by the root tissues. The key observations were: (i) the release of [15N]ammonium during [15N]-labelling experiments; and (ii) the detection of a characteristic set of serine isotopomers in the [2-13C]glycine experiments. The effects of aminoacetonitrile, amino-oxyacetate, and isonicotinic acid hydrazide, all of which inhibit GDC and SHMT to some extent, and of methionine sulphoximine, which inhibited the reassimilation of the ammonium, supported the conclusion that GDC and SHMT were essential for the metabolism of glycine. C. intrepidus was observed to metabolize serine more readily than the maize root tips and this may be an adaptation to its nitrogen-deficient habitat. Overall, the results support the emerging view that GDC is an essential component of glycine catabolism in non-photosynthetic tissues.
Key words: Glycine decarboxylase, nitrogen nutrition, NMR spectroscopy, non-photosynthetic glycine metabolism.
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Dissolved organic nitrogen in the soil provides an alternative to the usual inorganic forms in a wide range of plants and circumstances (Näsholm and Persson, 2001). In particular, when nitrogen mineralization is impaired, the concentration of inorganic nitrogen in the soil solution can be very low and, under such conditions, organic nitrogen, often in the form of amino acids, may be the major source of nitrogen that is available to the roots. This process has been shown to be an important factor in the nitrogen nutrition of plants in alpine and arctic habitats (Chapin et al. 1993; Kielland, 1994; Lipson and Monson, 1998; Raab et al., 1999), as well as for plants in heathlands (Schmidt and Stewart, 1997), boreal forests (Näsholm et al., 1998), grazed coastal marshland (Henry and Jefferies, 2002), and grassland communities (Falkengren-Grerup et al., 2000; Streeter et al., 2000; Thornton, 2001). Moreover, utilization of glycine has been demonstrated in agriculturally important species grown under field conditions, suggesting that organic nitrogen may be a more important source of nitrogen for plants under cultivation than previously thought (Näsholm et al., 2000, 2001).
The observation that organic nitrogen compounds, such as amino acids, can be absorbed by roots under field conditions at rates that can make a substantial contribution to the nitrogen requirement of plants, is good evidence for the involvement of such compounds in plant nitrogen nutrition. The existence of a range of amino acid transporters in plant roots suggests a likely mechanism (Fischer et al., 1998), and indeed the use of dual-labelled amino acids, for example, glycine labelled with both 13C and 15N (Näsholm et al., 1998, 2001), followed by the analysis of plant extracts using gas chromatography-mass spectrometry (GC-MS) provides a convenient method for investigating the contribution of direct and indirect uptake to the intracellular nitrogen pool. Demonstrating the quantitative significance of the uptake process has been the priority in most of these studies, with the result that the subsequent metabolism of the absorbed nitrogenous compounds has received much less attention, even though the pathways for their utilization may be unclear. Thus, while the uptake of glycine has been shown to be a significant source of plant nitrogen in many of these studies, the extent to which the glycine decarboxylase complex (GDC, EC 2.1.2.10) might complement the action of aminotransferases in the subsequent metabolism of the glycine has been investigated only infrequently. In one such study, it was concluded that glycine was metabolized in the roots and cluster roots of Hakea seedlings via aminotransferase activity (Schmidt and Stewart, 1999). This conclusion was consistent with earlier observations on the low GDC activity in pea root apices (Walton and Woolhouse, 1986), but more recent data suggest that glycine metabolism via GDC in heterotrophic tissues may actually occur quite readily (Mouillon et al., 1999).
The nitrogen nutrition of the aquatic resurrection plant Chamaegigas intrepidus Dinter (syn. Lindernia intrepidus (Dinter) Oberm., Scrophulariaceae) in its natural environment is strongly dependent on the utilization of amino acids, particularly glycine and serine, and urea (Schiller et al., 1998b; Heilmeier et al., 2000; Heilmeier and Hartung, 2001). The existence of a pH-dependent high affinity transport system (Km 16 mmol m–3) is consistent with the utilization of glycine as a nitrogen source during the morning, when the pH of the rock pools in which the plant grows is mildly acidic (Schiller et al., 1998b). The pathways involved in the utilization of glycine and serine in C. intrepidus have not been identified, and so this problem was investigated by analysing the metabolism of [15N]-labelled glycine and serine with 15N nuclear magnetic resonance (NMR) spectroscopy. This approach allows the metabolism of the amino acids to be observed in vivo and it provides a convenient method for probing the pathways of plant nitrogen metabolism (Ratcliffe and Shachar-Hill, 2001). For comparison, labelling experiments were also performed on excised maize (Zea mays L.) root tips using [15N]glycine, [15N]serine and [2-13C] glycine. NMR analysis of the [13C]-labelling experiment provides a direct method for detecting the contribution of GDC and serine hydroxymethyl transferase (SHMT, EC 2.1.2.1) to glycine metabolism (Ashworth and Mettler, 1984) and this too has been applied previously to a range of plant tissues (Ratcliffe and Shachar-Hill, 2001). The experiments provide good metabolic evidence for the involvement of GDC in the utilization of glycine by both C. intrepidus and maize roots, and thus lend support to the emerging view that glycine catabolism by GDC is a characteristic feature of heterotrophic plant tissues (Mouillon et al., 1999).
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Plant material
Chamaegigas intrepidus DINTER (syn. Lindernia intrepidus (DINTER) OBERM., Scrophulariaceae) occurs endemically in Namibia (Hickel, 1967; Giess, 1969) and sampling took place near Omaruru in November 2000. The plants grew in dense mats firmly attached to the soil, and air-dried slabs of approximately 200 cm2 were collected and stored in darkness at room temperature for future use. Dried plants were rehydrated for at least 15 h at room temperature in an artificial rock pool solution containing the low nutrient concentrations typical of the natural habitat. This solution contained 50 mmol m–3 KCl, 250 mmol m–3 NaCl, 200 mmol m–3 CaSO4, 50 mmol m–3 MgCl2, 5 mmol m–3 (NH4)2SO4, 0.2 mmol m–3 MnCl2, and 10 mmol m–3 FeNaEDTA. Rehydrated plants were separated from each other, and after removing the sediment from the roots, they were kept in the same nutrient solution prior to labelling.
Maize seeds (Zea mays L. var. LG20.80) were germinated in the dark for 2–3 d between layers of absorbent paper moistened with 0.1 mol m–3 CaSO4 at 25 °C. After germination, 5 mm root tips were excised with a razor blade and transferred to an aerated buffer solution (see below).
Stable isotope labelling
Rehydrated C. intrepidus plants in groups of 8–10, and in some cases excised roots, were incubated for between 12 h and 30 h in 50 ml of aerated 50 mol m–3 glucose, 10 mol m–3 MES, 0.1 mol m–3 CaSO4, pH 6 (glucose-MES buffer), supplemented with either 5 mol m–3 [15N]glycine or 5 mol m–3 [15N]serine (98 atom%; Promochem, Germany). After labelling, samples of roots were vacuum infiltrated for 4 min in fresh MES buffer (Schiller et al., 1998a), before transfer to a 10 mm diameter NMR tube containing the same medium. Oxygenated MES buffer from a 250 or 500 ml reservoir, at a temperature of 21–22 °C, was recycled through the NMR tube at 6–7 ml min–1 throughout the subsequent measurements (Lee and Ratcliffe 1983).
After germination, 130 maize root tips (approximately 0.5 g fr. wt) were incubated for between 12 h and 22 h in 50 ml aerated glucose-MES buffer, supplemented with either 5 mol m–3 [15N]glycine, 5 mol m–3 [15N]serine, or 5 mol m–3 [2-13C]glycine (99 atom%; Promochem, Germany). After labelling, the root tips were suspended in MES buffer in the 10 mm NMR tube, but without vacuum infiltration since this was unnecessary. In some experiments the labelling time-course was observed directly by incubating freshly excised root tips with a labelled amino acid in the NMR tube and in this case the volume of the recycling medium was reduced to 50 ml.
The effects of the following inhibitors were investigated: aminoacetonitrile (AAN), amino-oxyacetate (AOA), isonicotinic acid hydrazide (INH), and methionine sulphoximine (MSX). Inhibitors were generally used at a concentration of 2 mol m–3, in freshly prepared solutions, and they were added at the start of the incubation with the labelled glycine.
NMR spectroscopy
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 15N NMR spectra were recorded at 30.42 MHz using a 10 mm diameter broadband probehead, a 60° or 90° pulse angle, a spectral width of 4500 Hz, a 2 s recycle time, with low power broadband decoupling for 1.75 s to maintain the nuclear Overhauser enhancement and normal decoupling during the acquisition, and a minimum accumulation time of 1 h. Chemical shifts were measured relative to the signal at –298.8 ppm from a capillary containing 0.25 mol dm–3 [15N]urea.
1H-decoupled 13C NMR spectra were recorded at 75.46 MHz using the same 10 mm diameter broadband probehead, a 90° pulse angle, a spectral width of 17 800 Hz, a 6 s recycle time, with low power broadband decoupling for 5 s to maintain the nuclear Overhauser enhancement and normal decoupling during the acquisition, and a minimum accumulation time of 1 h. Chemical shifts were measured relative to the glycine C2 signal at 42.40 ppm.
The spectra in the figures are representative examples from 36 independent labelling experiments.
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Metabolism of [15N]glycine and [15N]serine by Chamaegigas intrepidus
Incubation experiments with [15N]glycine were performed on whole plants and excised roots, and the redistribution of the label was analysed using in vivo 15N NMR spectroscopy. Figure 1A shows the spectrum of a root sample taken from plants that had been incubated in 5 mol m–3 [15N]glycine for 20 h. Several signals were detected, including the signal from labelled glycine at –345.0 ppm, and the spectrum provides direct evidence for the uptake and metabolism of glycine by C. intrepidus, in agreement with the [14C]glycine uptake data of Schiller et al. (1998b). Comparison with the spectra obtained in an investigation of the utilization of [15N]ammonium and [15N]urea by C. intrepidus (Heilmeier et al., 2000), as well as the results of [15N]-labelling experiments on other plants (Gerendás et al., 1993; Carroll et al., 1994; Ford et al., 1996; Mesnard et al., 2000), indicates that the other signals in Fig. 1A can be assigned to glutamine amide-N (–263.4 ppm), glutamate and glutamine amino N (–334.6 ppm), and serine (–339.3 ppm). The observation of the glutamine amide-N signal is of particular interest, because, on the assumption that the most likely route to the labelling of the amide group is via glutamine synthetase (GS; EC 6.3.1.2), it indicates that labelled ammonium must be released either directly or indirectly from the [15N]glycine and this in turn points to the probable involvement of GDC. The glutamine amide-N signal was a prominent feature in the root spectrum irrespective of whether the roots were labelled as whole plants (Fig. 1A) or after excision (data not shown) indicating that it did not arise as a result of translocation of the labelled glycine to the shoots and the subsequent redistribution of the metabolic products to the roots. It is unlikely that the reassimilation of the ammonium released by GDC is solely responsible for the redistribution of the label observed in Fig. 1A and, in particular, the labelling of serine is likely to arise through the action of either SHMT or serine:glyoxylate aminotransferase (SGAT, EC 2.6.1.45).
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Spectra were also recorded from the roots of C. intrepidus plants that had been incubated with [15N]serine (Fig. 1B). Signals were observed from glutamine amide-N (–263.4 ppm), glutamate and glutamine amino N (–334.6 ppm), and serine (–339.3 ppm), again providing direct evidence for the uptake and utilization of the amino acid.
Metabolism of [15N]glycine and [15N]serine by maize root tips
Similar, but much stronger signals, were observed in the in vivo 15N NMR spectra of maize root tips that had been incubated with [15N]glycine (Fig. 2A). A substantial fraction of the glycine was converted to serine, but there were also smaller, but easily detectable, signals from glutathione (–258.0 ppm), glutamine amide-N (–263.4 ppm), asparagine amide-N (–264.0 ppm), glutamate and glutamine amino N (–334.8 ppm), and ammonium (–354.8 ppm). Time-course experiments showed that the glutamine amide and serine amino signals were detectable within the first hour of the incubation (data not shown). As with C. intrepidus the detection of the glutamine amide-N points to the release of ammonium by GDC and its reassimilation via GS, and the occurrence of a deamination step is strongly supported by the detection of the ammonium signal (Fig. 2A). Spectra recorded during the incubation of maize root tips with [15N]serine (Fig. 2B) showed that the substantial accumulation of serine was accompanied by only a minor redistribution of the label into glycine, ammonium, glutamine and asparagine amide-N and glutathione. The glycine signal was only observed after an incubation of at least 9 h and the accumulation and restricted metabolism of the labelled serine contrasted with the limited accumulation of the amino acid observed in C. intrepidus (Fig. 1B).
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Figure 3 shows the result of a series of inhibitor experiments in which maize root tips were incubated with 5 mol m–3 [15N]glycine and 2 mol m–3 concentrations of AOA (Fig. 3A), AAN (Fig. 3B), INH (Fig. 3C), and MSX (Fig. 3D). AOA, which is an inhibitor of GDC, SHMT and the aminotransferases (Sarojini and Oliver, 1985; Dry and Wiskich, 1986), eliminated all the signals from the 15N NMR spectrum, apart from the peak from the unmetabolized glycine (Fig. 3A). AAN, which is a structural analogue of glycine that inhibits GDC (Usuda et al., 1980; Gardeström et al., 1981), eliminated or greatly reduced the glutamine amide, asparagine amide, glutamate and glutamine amino, and ammonium signals, as well as reducing the size of the serine signal relative to the glycine peak (Fig. 3B). INH, which is another inhibitor of GDC (Bird et al., 1972; Gardeström et al., 1981), had a similar effect to AAN, but with an even greater reduction in the serine signal (Fig. 3C). Finally MSX, the inhibitor of GS (Tate and Meister, 1973), eliminated the glutamine amide, asparagine amide, and glutamate and glutamine amino signals from the 15N NMR spectrum, leaving signals from glutathione, serine, unmetabolized glycine, and ammonium (Fig. 3D). The results of these experiments are consistent with the involvement of GDC in the metabolism of glycine, and the effect of MSX highlights the role of GS in the subsequent reassimilation of the labelled ammonium. However, because of the overlapping specificity and varying effectiveness of AOA, INH and AAN, it remains unclear whether the labelled serine arises through SHMT activity or through the action of SGAT.
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Metabolism of [2-13C]glycine by maize root tips
Excised maize root tips were incubated with [2-13C]glycine and the redistribution of the label was analysed using in vivo 13C NMR spectroscopy (Fig. 4). After an 18 h incubation with 5 mol m–3 [2-13C]glycine, prominent signals were detected from several labelled metabolites, including [2-13C]glycine (42.40 ppm), glutathione (44.19 ppm), [2-13C]serine (57.33 ppm), [3-13C]serine (61.16 ppm), and an unidentified compound (63.92 ppm). The labelling of [2-13C]serine is consistent with metabolism via SHMT, while the labelling of [3-13C]serine indicates metabolism via the combined action of GDC and SHMT. Moreover, each of the serine signals is flanked by the doublet signal from [2,3-13C]serine, the doubly labelled isotopomer that is also expected to arise from the combined action of GDC and SHMT. Notable absences from Fig. 4B include: (i) any signal from labelled glyoxylate, or compounds that might be derived from it, arguing against a contribution to glycine metabolism from SGAT; and (ii) signals from labelled products of one carbon metabolism, such as [5-13C]methionine.
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Figures 5 and 6 show the results of experiments in which 2 mol m–3 concentrations of various inhibitors were added to the incubation medium. Incubation with AOA prevented almost all the metabolism and the spectrum was dominated by the large signal from the unmetabolized glycine (Figs 5A, 6A). AAN reduced the overall labelling of serine, but while there was a marked reduction in the doublet signals from [2,3-13C]serine there was an increase in the signal from [2-13C]serine and no obvious effect on the signal from [3-13C]serine (Figs 5B, 6B). The differential effect on the labelling of the serine isotopomers is highlighted in the difference spectrum (Fig. 6B) which shows a positive signal for [2-13C]serine and four negative signals for [2,3-13C]serine. This result suggests that the AAN concentration was sufficient to cause appreciable inhibition of GDC and very little inhibition of SHMT, a result that is in agreement with earlier observations on the effect of the inhibitor (Gardeström et al., 1981). INH had a stronger effect on glycine metabolism than AAN, causing a marked reduction in all the serine resonances and the unassigned signal at 63.92 ppm (Figs 5C, 6C). Finally MSX, which prevented the reassimilation of the ammonium released by GDC (Fig. 3D), caused a marked increase in the incorporation of 13C into serine and glutathione, resulting in a smaller glycine signal (Figs 5D, 6D). MSX also reduced the intensity of the unassigned signal at 63.92 ppm.
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Stable isotope labelling coupled with in vivo NMR detection of the redistribution of the label provides a convenient method for detecting the uptake and utilization of glycine and serine by plant cells and excised plant tissues (Ratcliffe and Shachar-Hill, 2001). The metabolic fate of the amino group can be observed directly using [15N]-labelling and 15N NMR (Neeman et al., 1985); while the metabolism of the carbon skeleton can be followed using [13C]-labelling and 13C-NMR (Ashworth and Mettler, 1984; Neeman et al., 1985; Prabhu et al., 1996, 1998; Mouillon et al., 1999). The two approaches provide complementary information, and they have been particularly useful in demonstrating the involvement of the mitochondrial GDC system in the metabolism of glycine. The expected redistribution of glycine label through this pathway has been observed in both photosynthetic (Neeman et al., 1985; Prabhu et al., 1996, 1998) and non-photosynthetic (Ashworth and Mettler, 1984; Mouillon et al., 1999) tissues. Moreover, it has been argued on the basis of the results obtained with a heterotrophic Acer pseudoplatanus cell culture that metabolism through the GDC pathway must be an essential feature of heterotrophic plant metabolism (Mouillon et al., 1999), despite the generally low levels of extractable GDC activity in such tissues (Walton and Woolhouse, 1986; Bourguignon et al., 1993).
Labelled glycine was readily metabolized by the roots of both C. intrepidus and maize, and the main aim of the investigation was to obtain evidence for the pathway of glycine utilization in the roots, with the intention of distinguishing between the involvement of GDC, SHMT and the aminotransferases (Fig. 7). As argued elsewhere (Schmidt and Stewart, 1999), the aminotransferase route might be expected to be the preferred pathway and, indeed, the effect of inhibitors on the redistribution of label from [15N]glycine in Hakea roots was interpreted in terms of a dominant role for SGAT (Schmidt and Stewart, 1999). However, this conclusion depended on the assumption that AOA could be used as a selective inhibitor of aminotransferase activity, whereas it is known from other work (Sarojini and Oliver, 1985; Dry and Wiskich, 1986) that AOA also inhibits GDC and SHMT through reaction with their pyridoxal phosphate cofactors. In contrast, the experiments reported here provide two lines of evidence in favour of an important role for GDC and SHMT, and against the involvement of aminotransferases, in the metabolism of glycine by root tissues.
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First, the experiments with [15N]glycine provided direct evidence for the release of [15N]ammonium (Fig. 2), and its subsequent incorporation into glutamine and glutamate via the GS/GOGAT pathway (Figs 1A, 2A). The simplest explanation for the ammonium release is that glycine is metabolized by GDC (Fig. 7) and, indeed, this explanation is supported both by the inhibitor experiments (Fig. 3) and the experiments with [2-13C]glycine (Fig. 4). More complicated pathways for the release of ammonium can be envisaged, for example, the oxidative deamination of glutamate, labelled as a result of glutamate:glyoxylate aminotransferase (EC 2.6.1.4) activity, but it is not necessary to do so and this pathway can be ruled out because incubation with MSX, which would not be expected to affect an aminotransferase, eliminated the signal from [15N]glutamate (Fig. 3D). The experiments with [15N]glycine also showed substantial labelling of serine (Figs 1A, 2A), which could arise either via SHMT or the aminotransferases (Fig. 7), and, in principle, this could also be a source of free ammonium via the activity of serine dehydratase (EC 4.2.1.13). However, by contrast with GDC, this enzyme is poorly characterized and its role in the catabolism of serine is uncertain (Bourguignon et al., 1999).
Secondly, the experiments with [2-13C]glycine confirmed the involvement of GDC and showed that SHMT, both alone and in concert with GDC, was responsible for the conversion of glycine to serine (Fig. 4). These conclusions can be deduced from the labelling patterns observed for the serine isotopomers in the 13C NMR spectrum: [2-13C]serine is formed by the SHMT-mediated reaction of unlabelled CH2-THF and [2-13C]glycine; [2,3-13C]serine arises from the reaction of [13C]-labelled CH2-THF, generated by the action of GDC on [2-13C]glycine, and [2-13C]glycine; and [3-13C]glycine arises from reaction of [13C]-labelled CH2-THF and unlabelled glycine (Fig. 7). The characteristic signals of the three isotopomers are readily identifiable and they provide unequivocal evidence for the involvement of both GDC and SHMT in the metabolism of glycine (Fig. 4). Moreover, the 13C spectra contained no signals that could be attributed to transamination products of glycine, indicating that the [15N]serine observed in the experiments with [15N]glycine (Figs 2A, 3) must have arisen from SHMT activity rather than aminotransferase activity. The effect of the GDC and SHMT inhibitors on the labelling of the serine isotopomers (Figs 5, 6) is consistent with this conclusion, and it is clear that the effectiveness of the inhibitors decreases in the order AOA, INH, AAN.
A further point that needs to be considered is the possibility of microbial activity during the labelling experiments. Although the root tissues were not sterile, the observed NMR signals must have originated in the plant tissue because the bacterial biomass would have been too small to give detectable signals. In principle, the observed metabolites could have been generated by bacterial pathways, but it seems improbable that there would then have been a substantial transfer of metabolites such as serine and glutathione to the plant tissue. Uptake of [15N]ammonium released by bacterial activity would provide an alternative explanation for the labelling of the glutamine amide nitrogen, but since the [2-13C]glycine experiments show that the ammonium was released through the combined action of GDC and SHMT it would still be necessary to envisage the transfer of the labelled serine to the plant tissue. The data in any case show that glycine is taken up by the root tissue and in this situation it is reasonable to assume that the spectroscopic changes are dominated by metabolic events in the root.
Thus it can be seen that GDC and SHMT are directly involved in the metabolism of glycine by C. intrepidus and maize root tissues, and this provides further support for the emerging view that GDC plays an essential part in heterotrophic metabolism (Mouillon et al., 1999). This conclusion is also significant in relation to the nitrogen nutrition of C. intrepidus in its natural habitat, since glycine is the second most abundant nitrogen source, after urea, in the rock pools that support the plant (Schiller et al., 1998b; Heilmeier et al., 2000; Heilmeier and Hartung, 2001). The data show that the metabolism of glycine can act as a direct source of ammonium for the GS/GOGAT pathway and thus define the pathway that permits C. intrepidus to utilize glycine as a nitrogen source. Serine, which was also readily metabolized by C. intrepidus (Fig. 1B) and which is typically the second most abundant amino acid in the rock pools, also appears to be a direct source of ammonium, but whether this occurs via SHMT and GDC or serine hydratase has yet to be established. In Acer cells, the catabolism of serine involved the glycolytic pathway as well as the action of SHMT and GDC, and experiments with [13C]-labelled serine will be required to establish the relative importance of the different pathways in root tissues. In contrast to C. intrepidus, maize root tips metabolized serine only to a limited extent under the conditions used here (Fig. 2B) and future work needs to test whether this can be explained within a framework in which serine catabolism is governed by the demands of one carbon metabolism (Mouillon et al., 1999).
The physiological relevance of the metabolism of glycine via GDC and SHMT in the roots has yet to be established. Glucose starvation in excised maize root tips has a profound effect on carbon and nitrogen metabolism (Brouquisse et al., 1991, 1992) and this will only have been partly reduced by the provision of glucose at 50 mol dm–3 in the experiments reported here. Future work needs to address this point directly, both by investigating the effect of varying the exogenous carbohydrate supply on the utilization of glycine by excised root tips and by carrying out labelling experiments on intact maize seedlings. Subsequently, it will be necessary to establish whether the GDC/SHMT pathway makes a significant contribution to root nitrogen utilization under field conditions. This is by no means certain, given the competition for organic nitrogen in the rhizosphere (Hodge et al., 2000), although investigations of plant species, including C. intrepidus (Schiller et al., 1998b; Heilmeier et al., 2000), in a range of habitats point to the importance of this process.
While the main interest in the spectroscopic data lies in the evidence for the importance of GDC and SHMT in the metabolism of glycine, two other observations deserve comment. First, in vivo NMR signals from [13C]- and [15N]-labelled glutathione have not been reported before in studies of plant tissues and, in principle, this might complement existing methods for the quantitative analysis of glutathione in vivo (Meyer et al., 2001). In fact an established in vivo 1H NMR method that appears not to have been applied to plant tissues, and which allows the ratio of oxidized to reduced glutathione to be measured directly (Rabenstein et al., 1985; Russell et al., 1994) would probably be the most appropriate NMR method for quantitative analysis, and the in vivo detection of labelled glutathione signals reported here is more likely to find applications in flux measurements. Secondly, the identity of the substantial unassigned resonance observed at 63.92 ppm in the [2-13C]glycine experiments (Fig. 4) remains unclear. An apparently similar peak can be seen in spectra obtained from tobacco cells following glycine uptake (Ashworth and Mettler, 1984), and these unassigned peaks may well correspond to the unidentified glycine derivative detected in a mass spectrometric analysis of glycine metabolism in carrot cells (Whatley et al., 1986). The intensity of the unassigned signal was reduced by AAN, INH and AOA (Figs 5, 6), which might point to the involvement of GDC and the subsequent accumulation of a labelled product of one carbon metabolism, and the metabolite is unlikely to be a derivative of serine, because the different isotopomers of serine might lead to more than one unassigned signal. Unfortunately, these constraints are not sufficient to identify the compound, although it is possible to rule out a number of plausible candidates on the basis of the chemical shift of the signal, including threonine, glycerate, ethanolamine, and choline.
In conclusion, labelling experiments with [2-13C]glycine and [15N]glycine point to the involvement of GDC and SHMT in the metabolism of glycine by the root tissues of C. intrepidus and maize. The increasing awareness of the contribution that organic nitrogen forms make to plant nitrogen nutrition in a wide range of habitats, including ones of agricultural importance, suggest that more detailed investigations of the pathways of both glycine and serine metabolism would be worthwhile. Such investigations might focus on the regulation and compartmentation of the pathways in roots, as well as seeking evidence, through immunoassays and enzymic measurements, that there is sufficient GDC activity in the roots to support the observed fluxes.
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Acknowledgements |
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We thank the Deutsche Forschungsgemeinschaft (WH) and the UK Biotechnology and Biological Sciences Research Council (RGR) for financial support. We also thank the anonymous referees for constructive criticism.
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References |
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