J. Exp. Bot.-2002-Hartung-2305-14

DOI: 10.1093/jxb/erf092

Utilization of glycine and serine as nitrogen sources in theroots of Zea mays and Chamaegigas intrepidus

W. Hartung1 and R. G. Ratcliffe2,3

1 Julius-von-Sachs Institut fuÈr Biowissenschaften, Lehrstuhl Botanik I, UniversitaÈ t WuÈrzburg, Julius-von-SachsPlatz 2, D-97082 WuÈrzburg, Germany2 Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK

Received 4 March 2002; Accepted 3 July 2002

Abstract

Glycine and serine are potential sources of nitro-

gen 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 pro-

vided 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 aminoa-

cetonitrile, 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 ammo-

nium, 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-de®cient habitat.

Overall, the results support the emerging view that

GDC is an essential component of glycine catabo-

lism in non-photosynthetic tissues.

Key words: Glycine decarboxylase, nitrogen nutrition, NMR

spectroscopy, non-photosynthetic glycine metabolism.

Introduction

Dissolved organic nitrogen in the soil provides an alter-native to the usual inorganic forms in a wide range ofplants and circumstances (NaÈsholm and Persson, 2001). Inparticular, when nitrogen mineralization is impaired, theconcentration of inorganic nitrogen in the soil solution canbe very low and, under such conditions, organic nitrogen,often in the form of amino acids, may be the major sourceof nitrogen that is available to the roots. This process hasbeen shown to be an important factor in the nitrogennutrition of plants in alpine and arctic habitats (Chapinet 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 (NaÈsholmet al., 1998), grazed coastal marshland (Henry andJefferies, 2002), and grassland communities (Falkengren-Grerup et al., 2000; Streeter et al., 2000; Thornton, 2001).Moreover, utilization of glycine has been demonstrated inagriculturally important species grown under ®eld condi-tions, suggesting that organic nitrogen may be a moreimportant source of nitrogen for plants under cultivationthan previously thought (NaÈsholm et al., 2000, 2001).

The observation that organic nitrogen compounds, suchas amino acids, can be absorbed by roots under ®eldconditions at rates that can make a substantial contributionto the nitrogen requirement of plants, is good evidence forthe involvement of such compounds in plant nitrogennutrition. The existence of a range of amino acidtransporters in plant roots suggests a likely mechanism

3 To whom correspondence should be addressed. Fax: +44 (0)1865 275074. E-mail: [email protected]

ã Society for Experimental Biology 2002

Journal of Experimental Botany, Vol. 53, No. 379, pp. 2305±2314, December 2002

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(Fischer et al., 1998), and indeed the use of dual-labelledamino acids, for example, glycine labelled with both 13Cand 15N (NaÈsholm et al., 1998, 2001), followed by theanalysis of plant extracts using gas chromatography-massspectrometry (GC-MS) provides a convenient method forinvestigating the contribution of direct and indirect uptaketo the intracellular nitrogen pool. Demonstrating thequantitative signi®cance of the uptake process has beenthe priority in most of these studies, with the result that thesubsequent metabolism of the absorbed nitrogenous com-pounds has received much less attention, even though thepathways for their utilization may be unclear. Thus, whilethe uptake of glycine has been shown to be a signi®cantsource of plant nitrogen in many of these studies, the extentto which the glycine decarboxylase complex (GDC, EC2.1.2.10) might complement the action of aminotrans-ferases in the subsequent metabolism of the glycine hasbeen investigated only infrequently. In one such study, itwas concluded that glycine was metabolized in the rootsand cluster roots of Hakea seedlings via aminotransferaseactivity (Schmidt and Stewart, 1999). This conclusion wasconsistent with earlier observations on the low GDCactivity in pea root apices (Walton and Woolhouse, 1986),but more recent data suggest that glycine metabolism viaGDC in heterotrophic tissues may actually occur quitereadily (Mouillon et al., 1999).

The nitrogen nutrition of the aquatic resurrection plantChamaegigas intrepidus Dinter (syn. Lindernia intrepidus(Dinter) Oberm., Scrophulariaceae) in its natural environ-ment is strongly dependent on the utilization of aminoacids, particularly glycine and serine, and urea (Schilleret al., 1998b; Heilmeier et al., 2000; Heilmeier andHartung, 2001). The existence of a pH-dependent highaf®nity transport system (Km 16 mmol m±3) is consistentwith the utilization of glycine as a nitrogen source duringthe morning, when the pH of the rock pools in which theplant grows is mildly acidic (Schiller et al., 1998b). Thepathways involved in the utilization of glycine and serinein C. intrepidus have not been identi®ed, and so thisproblem was investigated by analysing the metabolism of[15N]-labelled glycine and serine with 15N nuclear mag-netic resonance (NMR) spectroscopy. This approachallows the metabolism of the amino acids to be observedin vivo and it provides a convenient method for probing thepathways of plant nitrogen metabolism (Ratcliffe andShachar-Hill, 2001). For comparison, labelling experi-ments 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 experimentprovides a direct method for detecting the contribution ofGDC and serine hydroxymethyl transferase (SHMT, EC2.1.2.1) to glycine metabolism (Ashworth and Mettler,1984) and this too has been applied previously to a range ofplant tissues (Ratcliffe and Shachar-Hill, 2001). Theexperiments provide good metabolic evidence for the

involvement of GDC in the utilization of glycine by bothC. intrepidus and maize roots, and thus lend support to theemerging view that glycine catabolism by GDC is acharacteristic feature of heterotrophic plant tissues(Mouillon et al., 1999).

Materials and methods

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 inNovember 2000. The plants grew in dense mats ®rmly attached tothe soil, and air-dried slabs of approximately 200 cm2 were collectedand stored in darkness at room temperature for future use. Driedplants were rehydrated for at least 15 h at room temperature in anarti®cial rock pool solution containing the low nutrient concentra-tions typical of the natural habitat. This solution contained 50 mmolm±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 mmolm±3 FeNaEDTA. Rehydrated plants were separated from each other,and after removing the sediment from the roots, they were kept in thesame nutrient solution prior to labelling.

Maize seeds (Zea mays L. var. LG20.80) were germinated in thedark for 2±3 d between layers of absorbent paper moistened with 0.1mol m±3 CaSO4 at 25 °C. After germination, 5 mm root tips wereexcised with a razor blade and transferred to an aerated buffersolution (see below).

Stable isotope labelling

Rehydrated C. intrepidus plants in groups of 8±10, and in some casesexcised roots, were incubated for between 12 h and 30 h in 50 ml ofaerated 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 in®ltratedfor 4 min in fresh MES buffer (Schiller et al., 1998a), before transferto a 10 mm diameter NMR tube containing the same medium.Oxygenated MES buffer from a 250 or 500 ml reservoir, at atemperature of 21±22 °C, was recycled through the NMR tube at 6±7ml min±1 throughout the subsequent measurements (Lee andRatcliffe 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 molm±3 [15N]serine, or 5 mol m±3 [2-13C]glycine (99 atom%;Promochem, Germany). After labelling, the root tips were suspendedin MES buffer in the 10 mm NMR tube, but without vacuumin®ltration since this was unnecessary. In some experiments thelabelling time-course was observed directly by incubating freshlyexcised root tips with a labelled amino acid in the NMR tube and inthis 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), isonicotinicacid hydrazide (INH), and methionine sulphoximine (MSX).Inhibitors were generally used at a concentration of 2 mol m±3, infreshly prepared solutions, and they were added at the start of theincubation with the labelled glycine.

NMR spectroscopy

In vivo NMR spectra were recorded using a Bruker CXP 300spectrometer (Bruker Analytische Messtechnik, Rheinstetten,Germany) with an Oxford Instruments (Oxford, UK) 7.05 Tsuperconducting magnet.

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1H-decoupled 15N NMR spectra were recorded at 30.42 MHzusing a 10 mm diameter broadband probehead, a 60° or 90° pulseangle, a spectral width of 4500 Hz, a 2 s recycle time, with lowpower broadband decoupling for 1.75 s to maintain the nuclearOverhauser enhancement and normal decoupling during the acqui-sition, and a minimum accumulation time of 1 h. Chemical shiftswere measured relative to the signal at ±298.8 ppm from a capillarycontaining 0.25 mol dm±3 [15N]urea.

1H-decoupled 13C NMR spectra were recorded at 75.46 MHzusing the same 10 mm diameter broadband probehead, a 90° pulseangle, a spectral width of 17 800 Hz, a 6 s recycle time, with lowpower broadband decoupling for 5 s to maintain the nuclearOverhauser enhancement and normal decoupling during the acqui-sition, and a minimum accumulation time of 1 h. Chemical shiftswere measured relative to the glycine C2 signal at 42.40 ppm.

The spectra in the ®gures are representative examples from 36independent labelling experiments.

Results

Metabolism of [15N]glycine and [15N]serine byChamaegigas intrepidus

Incubation experiments with [15N]glycine were performedon whole plants and excised roots, and the redistribution ofthe label was analysed using in vivo 15N NMR spectros-copy. 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 uptakeand metabolism of glycine by C. intrepidus, in agreementwith the [14C]glycine uptake data of Schiller et al. (1998b).Comparison with the spectra obtained in an investigationof 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 (GerendaÂset al., 1993; Carroll et al., 1994; Ford et al., 1996; Mesnardet al., 2000), indicates that the other signals in Fig. 1A canbe assigned to glutamine amide-N (±263.4 ppm), gluta-mate and glutamine amino N (±334.6 ppm), and serine(±339.3 ppm). The observation of the glutamine amide-Nsignal is of particular interest, because, on the assumptionthat the most likely route to the labelling of the amidegroup is via glutamine synthetase (GS; EC 6.3.1.2), itindicates that labelled ammonium must be released eitherdirectly or indirectly from the [15N]glycine and this in turnpoints to the probable involvement of GDC. The glutamineamide-N signal was a prominent feature in the rootspectrum irrespective of whether the roots were labelled aswhole plants (Fig. 1A) or after excision (data not shown)indicating that it did not arise as a result of translocation ofthe labelled glycine to the shoots and the subsequentredistribution of the metabolic products to the roots. It isunlikely that the reassimilation of the ammonium releasedby GDC is solely responsible for the redistribution of thelabel observed in Fig. 1A and, in particular, the labelling ofserine is likely to arise through the action of either SHMTor serine:glyoxylate aminotransferase (SGAT, EC2.6.1.45).

Spectra were also recorded from the roots of C.intrepidus plants that had been incubated with[15N]serine (Fig. 1B). Signals were observed fromglutamine amide-N (±263.4 ppm), glutamate and gluta-mine amino N (±334.6 ppm), and serine (±339.3 ppm),again providing direct evidence for the uptake andutilization of the amino acid.

Metabolism of [15N]glycine and [15N]serine by maizeroot tips

Similar, but much stronger signals, were observed in thein vivo 15N NMR spectra of maize root tips that had beenincubated with [15N]glycine (Fig. 2A). A substantialfraction of the glycine was converted to serine, but therewere also smaller, but easily detectable, signals fromglutathione (±258.0 ppm), glutamine amide-N (±263.4ppm), asparagine amide-N (±264.0 ppm), glutamate andglutamine amino N (±334.8 ppm), and ammonium (±354.8ppm). Time-course experiments showed that the glutamineamide and serine amino signals were detectable within the®rst hour of the incubation (data not shown). As withC. intrepidus the detection of the glutamine amide-N

Fig. 1. In vivo 15N NMR spectra of root systems from C. intrepidusplants after pre-incubation with: (A) 5 mol m±3 [15N]glycine for 20 h;and (B) 5 mol m±3 [15N]serine for 12 h. The spectra were accumulatedin 4 h, and the labelled signals can be assigned to: 2, glutamineamide-N; 3, urea, from the capillary used for the chemical shiftreference; 4, glutamate and glutamine amino-N; 5, serine; and 6,glycine.

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points to the release of ammonium by GDC and itsreassimilation via GS, and the occurrence of a deaminationstep is strongly supported by the detection of theammonium signal (Fig. 2A). Spectra recorded during theincubation of maize root tips with [15N]serine (Fig. 2B)showed that the substantial accumulation of serine wasaccompanied by only a minor redistribution of the labelinto glycine, ammonium, glutamine and asparagine amide-N and glutathione. The glycine signal was only observedafter an incubation of at least 9 h and the accumulation andrestricted metabolism of the labelled serine contrasted withthe limited accumulation of the amino acid observed in C.intrepidus (Fig. 1B).

Figure 3 shows the result of a series of inhibitorexperiments in which maize root tips were incubated with5 mol m±3 [15N]glycine and 2 mol m±3 concentrations ofAOA (Fig. 3A), AAN (Fig. 3B), INH (Fig. 3C), and MSX(Fig. 3D). AOA, which is an inhibitor of GDC, SHMT andthe aminotransferases (Sarojini and Oliver, 1985; Dry and

Wiskich, 1986), eliminated all the signals from the 15NNMR spectrum, apart from the peak from the unmetabo-lized glycine (Fig. 3A). AAN, which is a structuralanalogue of glycine that inhibits GDC (Usuda et al., 1980;GardestroÈm et al., 1981), eliminated or greatly reduced theglutamine amide, asparagine amide, glutamate and gluta-mine amino, and ammonium signals, as well as reducingthe size of the serine signal relative to the glycine peak(Fig. 3B). INH, which is another inhibitor of GDC (Birdet al., 1972; GardestroÈm et al., 1981), had a similar effectto AAN, but with an even greater reduction in the serinesignal (Fig. 3C). Finally MSX, the inhibitor of GS (Tateand Meister, 1973), eliminated the glutamine amide,asparagine amide, and glutamate and glutamine aminosignals from the 15N NMR spectrum, leaving signals fromglutathione, serine, unmetabolized glycine, and ammo-nium (Fig. 3D). The results of these experiments areconsistent with the involvement of GDC in the metabolism

Fig. 2. In vivo 15N NMR spectra of maize root tips recorded duringthe period corresponding to 8±12 h uptake in incubation experimentswith: (A) 5 mol m±3 [15N]glycine; and (B) 5 mol m±3 [15N]serine. Theassignments are the same as in Fig. 1, except for the additionalpresence of: 1, the glycine N of glutathione; and 7, ammonium.Resolution enhanced spectra (not shown) indicate that peak 2 containscontributions from both glutamine and asparagine amide-N.

Fig. 3. In vivo 15N NMR spectra of maize root tips recorded afterpreincubation with 5 mol m±3 [15]glycine: (A) for 12 h plus 2 mol m±3

AOA; (B) for 15 h plus 2 mol m±3 AAN; (C) for17 h plus 2 mol m±3

INH; and (D) for 13 h plus 2 mol m±3 MSX. The assignments are thesame as in Figs 1 and 2.




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of glycine, and the effect of MSX highlights the role of GSin the subsequent reassimilation of the labelled ammo-nium. However, because of the overlapping speci®city andvarying effectiveness of AOA, INH and AAN, it remainsunclear whether the labelled serine arises through SHMTactivity or through the action of SGAT.

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 analysedusing in vivo 13C NMR spectroscopy (Fig. 4). After an 18 hincubation with 5 mol m±3 [2-13C]glycine, prominentsignals were detected from several labelled metabolites,including [2-13C]glycine (42.40 ppm), glutathione (44.19ppm), [2-13C]serine (57.33 ppm), [3-13C]serine (61.16ppm), and an unidenti®ed compound (63.92 ppm). Thelabelling of [2-13C]serine is consistent with metabolism viaSHMT, while the labelling of [3-13C]serine indicatesmetabolism via the combined action of GDC and SHMT.Moreover, each of the serine signals is ¯anked by thedoublet signal from [2,3-13C]serine, the doubly labelledisotopomer that is also expected to arise from thecombined action of GDC and SHMT. Notable absencesfrom Fig. 4B include: (i) any signal from labelledglyoxylate, or compounds that might be derived from it,arguing against a contribution to glycine metabolism from

SGAT; and (ii) signals from labelled products of onecarbon metabolism, such as [5-13C]methionine.

Figures 5 and 6 show the results of experiments in which2 mol m±3 concentrations of various inhibitors were addedto the incubation medium. Incubation with AOA preventedalmost all the metabolism and the spectrum was dominatedby the large signal from the unmetabolized glycine (Figs5A, 6A). AAN reduced the overall labelling of serine, but

Fig. 4. In vivo 13C NMR spectra of maize root tips recorded afterpreincubation for 18 h in the presence (A) and absence (B) of 5 molm±3 [2-13C]glycine. The spectra were recorded in 1 h and the insertshows an expanded region of the spectrum recorded from the labelledtissue. The labelled signals can be assigned to: 1, unknown; 2, acentral singlet peak from [3-13C]serine and a ¯anking doublet fromcarbon 3 of [2,3-13C]serine; 3, a central singlet peak from [2-13C]serine and a ¯anking doublet from carbon 2 of [2,3-13C]serine; 4,carbon 2 of the glycine in glutathione; and 5, [2-13C]glycine.

Fig. 5. In vivo 13C NMR spectra of maize root tips recorded afterpreincubation with 5 mol m±3 [2-13C]glycine for 18 h in the presenceof 2 mol m±3 concentrations of: (A) AOA, (B) AAN, (C) INH, and(D) MSX. The spectra were recorded in 1 h and the assignments arethe same as in Fig. 4. The insert on the right hand side of the ®gureshows the glycine peak reduced by a factor of 4.




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while there was a marked reduction in the doublet signalsfrom [2,3-13C]serine there was an increase in the signalfrom [2-13C]serine and no obvious effect on the signalfrom [3-13C]serine (Figs 5B, 6B). The differential effect onthe labelling of the serine isotopomers is highlighted in thedifference spectrum (Fig. 6B) which shows a positivesignal for [2-13C]serine and four negative signals for [2,3-13C]serine. This result suggests that the AAN concentra-tion was suf®cient to cause appreciable inhibition of GDCand very little inhibition of SHMT, a result that is inagreement with earlier observations on the effect of theinhibitor (GardestroÈm et al., 1981). INH had a strongereffect on glycine metabolism than AAN, causing a markedreduction in all the serine resonances and the unassignedsignal at 63.92 ppm (Figs 5C, 6C). Finally MSX, whichprevented the reassimilation of the ammonium releasedby GDC (Fig. 3D), caused a marked increase in the

incorporation of 13C into serine and glutathione, resultingin a smaller glycine signal (Figs 5D, 6D). MSXalso reduced the intensity of the unassigned signal at63.92 ppm.

Discussion

Stable isotope labelling coupled with in vivo NMRdetection of the redistribution of the label provides aconvenient method for detecting the uptake and utilizationof glycine and serine by plant cells and excised planttissues (Ratcliffe and Shachar-Hill, 2001). The metabolicfate of the amino group can be observed directly using[15N]-labelling and 15N NMR (Neeman et al., 1985); whilethe metabolism of the carbon skeleton can be followedusing [13C]-labelling and 13C-NMR (Ashworth andMettler, 1984; Neeman et al., 1985; Prabhu et al., 1996,1998; Mouillon et al., 1999). The two approaches providecomplementary information, and they have been particu-larly useful in demonstrating the involvement of themitochondrial GDC system in the metabolism of glycine.The expected redistribution of glycine label through thispathway has been observed in both photosynthetic(Neeman et al., 1985; Prabhu et al., 1996, 1998) andnon-photosynthetic (Ashworth and Mettler, 1984;Mouillon et al., 1999) tissues. Moreover, it has beenargued on the basis of the results obtained with aheterotrophic Acer pseudoplatanus cell culture that metab-olism through the GDC pathway must be an essentialfeature of heterotrophic plant metabolism (Mouillon et al.,1999), despite the generally low levels of extractable GDCactivity in such tissues (Walton and Woolhouse, 1986;Bourguignon et al., 1993).

Labelled glycine was readily metabolized by the roots ofboth C. intrepidus and maize, and the main aim of theinvestigation was to obtain evidence for the pathway ofglycine utilization in the roots, with the intention ofdistinguishing between the involvement of GDC, SHMTand the aminotransferases (Fig. 7). As argued elsewhere(Schmidt and Stewart, 1999), the aminotransferase routemight 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 adominant role for SGAT (Schmidt and Stewart, 1999).However, this conclusion depended on the assumption thatAOA could be used as a selective inhibitor of amino-transferase activity, whereas it is known from other work(Sarojini and Oliver, 1985; Dry and Wiskich, 1986) thatAOA also inhibits GDC and SHMT through reaction withtheir pyridoxal phosphate cofactors. In contrast, theexperiments reported here provide two lines of evidencein favour of an important role for GDC and SHMT, andagainst the involvement of aminotransferases, in themetabolism of glycine by root tissues.

Fig. 6. Difference spectra derived from the spectra in Figs 4A and 5showing the effects of (A) AOA, (B) AAN, (C) INH, and (D) MSX onthe redistribution of label from [2-13C]glycine. The assignments arethe same as in Fig. 4, and the spectra highlight the differential effectsof the various inhibitors on the labelling of the serine isotopomers.




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First, the experiments with [15N]glycine provided directevidence for the release of [15N]ammonium (Fig. 2), and itssubsequent incorporation into glutamine and glutamate viathe GS/GOGAT pathway (Figs 1A, 2A). The simplestexplanation for the ammonium release is that glycine is

metabolized by GDC (Fig. 7) and, indeed, this explanationis supported both by the inhibitor experiments (Fig. 3) andthe experiments with [2-13C]glycine (Fig. 4). More com-plicated pathways for the release of ammonium can beenvisaged, for example, the oxidative deamination of

Fig. 7. Pathways for the metabolism of glycine showing the redistribution of label derived from [2-13C]glycine and [15N]glycine. The enzymesare: GDC, glycine decarboxylase; GOGAT, glutamate synthase (EC 1.4.1.13); GS, glutamine synthetase; GSH-S, glutathione synthetase (EC6.3.2.3); SGAT, serine:glyoxylate aminotransferase; and SHMT, serine hydroxymethyl transferase. Glutamate or alanine:glyoxylateaminotransferase could provide an alternative route to labelled glyoxylate, although SGAT would appear to be more likely (Schmidt and Stewart,1999).




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glutamate, labelled as a result of glutamate:glyoxylateaminotransferase (EC 2.6.1.4) activity, but it is notnecessary to do so and this pathway can be ruled outbecause incubation with MSX, which would not beexpected to affect an aminotransferase, eliminated thesignal from [15N]glutamate (Fig. 3D). The experimentswith [15N]glycine also showed substantial labelling ofserine (Figs 1A, 2A), which could arise either via SHMT orthe aminotransferases (Fig. 7), and, in principle, this couldalso be a source of free ammonium via the activity ofserine dehydratase (EC 4.2.1.13). However, by contrastwith GDC, this enzyme is poorly characterized and its rolein the catabolism of serine is uncertain (Bourguignon et al.,1999).

Secondly, the experiments with [2-13C]glycine con-®rmed the involvement of GDC and showed that SHMT,both alone and in concert with GDC, was responsible forthe conversion of glycine to serine (Fig. 4). Theseconclusions can be deduced from the labelling patternsobserved for the serine isotopomers in the 13C NMRspectrum: [2-13C]serine is formed by the SHMT-mediatedreaction 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 reac-tion of [13C]-labelled CH2-THF and unlabelled glycine(Fig. 7). The characteristic signals of the three isotopomersare readily identi®able and they provide unequivocalevidence for the involvement of both GDC and SHMT inthe metabolism of glycine (Fig. 4). Moreover, the 13Cspectra contained no signals that could be attributed totransamination products of glycine, indicating that the[15N]serine observed in the experiments with [15N]glycine(Figs 2A, 3) must have arisen from SHMT activity ratherthan aminotransferase activity. The effect of the GDC andSHMT inhibitors on the labelling of the serine isotopomers(Figs 5, 6) is consistent with this conclusion, and it is clearthat the effectiveness of the inhibitors decreases in theorder AOA, INH, AAN.

A further point that needs to be considered is thepossibility of microbial activity during the labellingexperiments. Although the root tissues were not sterile,the observed NMR signals must have originated in theplant tissue because the bacterial biomass would have beentoo small to give detectable signals. In principle, theobserved metabolites could have been generated bybacterial pathways, but it seems improbable that therewould then have been a substantial transfer of metabolitessuch as serine and glutathione to the plant tissue. Uptake of[15N]ammonium released by bacterial activity wouldprovide an alternative explanation for the labelling of theglutamine amide nitrogen, but since the [2-13C]glycineexperiments show that the ammonium was releasedthrough the combined action of GDC and SHMT itwould still be necessary to envisage the transfer of the

labelled serine to the plant tissue. The data in any caseshow that glycine is taken up by the root tissue and in thissituation it is reasonable to assume that the spectroscopicchanges are dominated by metabolic events in the root.

Thus it can be seen that GDC and SHMT are directlyinvolved in the metabolism of glycine by C. intrepidus andmaize root tissues, and this provides further support for theemerging view that GDC plays an essential part inheterotrophic metabolism (Mouillon et al., 1999). Thisconclusion is also signi®cant in relation to the nitrogennutrition of C. intrepidus in its natural habitat, sinceglycine is the second most abundant nitrogen source, afterurea, 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 canact as a direct source of ammonium for the GS/GOGATpathway and thus de®ne 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 abundantamino acid in the rock pools, also appears to be a directsource of ammonium, but whether this occurs via SHMTand GDC or serine hydratase has yet to be established. InAcer cells, the catabolism of serine involved the glycolyticpathway as well as the action of SHMT and GDC, andexperiments with [13C]-labelled serine will be required toestablish the relative importance of the different pathwaysin root tissues. In contrast to C. intrepidus, maize root tipsmetabolized serine only to a limited extent under theconditions used here (Fig. 2B) and future work needs totest whether this can be explained within a framework inwhich serine catabolism is governed by the demands ofone carbon metabolism (Mouillon et al., 1999).

The physiological relevance of the metabolism ofglycine via GDC and SHMT in the roots has yet to beestablished. Glucose starvation in excised maize root tipshas a profound effect on carbon and nitrogen metabolism(Brouquisse et al., 1991, 1992) and this will only havebeen partly reduced by the provision of glucose at 50 moldm±3 in the experiments reported here. Future work needsto address this point directly, both by investigating theeffect of varying the exogenous carbohydrate supply on theutilization of glycine by excised root tips and by carryingout labelling experiments on intact maize seedlings.Subsequently, it will be necessary to establish whetherthe GDC/SHMT pathway makes a signi®cant contributionto root nitrogen utilization under ®eld conditions. This isby no means certain, given the competition for organicnitrogen in the rhizosphere (Hodge et al., 2000), althoughinvestigations of plant species, including C. intrepidus(Schiller et al., 1998b; Heilmeier et al., 2000), in a range ofhabitats point to the importance of this process.

While the main interest in the spectroscopic data lies inthe evidence for the importance of GDC and SHMT in themetabolism of glycine, two other observations deserve




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comment. First, in vivo NMR signals from [13C]- and[15N]-labelled glutathione have not been reported before instudies of plant tissues and, in principle, this mightcomplement existing methods for the quantitative analysisof glutathione in vivo (Meyer et al., 2001). In fact anestablished in vivo 1H NMR method that appears not tohave been applied to plant tissues, and which allows theratio of oxidized to reduced glutathione to be measureddirectly (Rabenstein et al., 1985; Russell et al., 1994)would probably be the most appropriate NMR method forquantitative analysis, and the in vivo detection of labelledglutathione signals reported here is more likely to ®ndapplications in ¯ux measurements. Secondly, the identityof the substantial unassigned resonance observed at63.92 ppm in the [2-13C]glycine experiments (Fig. 4)remains unclear. An apparently similar peak can be seen inspectra obtained from tobacco cells following glycineuptake (Ashworth and Mettler, 1984), and these unas-signed peaks may well correspond to the unidenti®edglycine derivative detected in a mass spectrometricanalysis of glycine metabolism in carrot cells (Whatleyet al., 1986). The intensity of the unassigned signal wasreduced by AAN, INH and AOA (Figs 5, 6), which mightpoint to the involvement of GDC and the subsequentaccumulation of a labelled product of one carbon metab-olism, and the metabolite is unlikely to be a derivative ofserine, because the different isotopomers of serine mightlead to more than one unassigned signal. Unfortunately,these constraints are not suf®cient to identify the com-pound, although it is possible to rule out a number ofplausible candidates on the basis of the chemical shift ofthe signal, including threonine, glycerate, ethanolamine,and choline.

In conclusion, labelling experiments with [2-13C]glycineand [15N]glycine point to the involvement of GDC andSHMT in the metabolism of glycine by the root tissues ofC. intrepidus and maize. The increasing awareness of thecontribution that organic nitrogen forms make to plantnitrogen nutrition in a wide range of habitats, includingones of agricultural importance, suggest that more detailedinvestigations of the pathways of both glycine and serinemetabolism would be worthwhile. Such investigationsmight focus on the regulation and compartmentation of thepathways in roots, as well as seeking evidence, throughimmunoassays and enzymic measurements, that there issuf®cient GDC activity in the roots to support the observed¯uxes.

Acknowledgements

We thank the Deutsche Forschungsgemeinschaft (WH) and the UKBiotechnology and Biological Sciences Research Council (RGR) for®nancial support. We also thank the anonymous referees forconstructive criticism.

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