DOI: 10.

1093/jxb/erf092
Utilization of glycine and serine as nitrogen sources in the
roots of Zea mays and Chamaegigas intrepidus
W. Hartung
1
and R. G. Ratcliffe
2,3
1
Julius-von-Sachs Institut fu r Biowissenschaften, Lehrstuhl Botanik I, Universita t Wurzburg, Julius-von-Sachs
Platz 2, D-97082 Wu rzburg, Germany
2
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 [
15
N]glycine, [
15
N]serine and
[2-
13
C]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 [
15
N]ammonium during
[
15
N]-labelling experiments; and (ii) the detection of
a characteristic set of serine isotopomers in the
[2-
13
C]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-decient 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 of
plants and circumstances (Nasholm 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 (Nasholm
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 eld condi-
tions, suggesting that organic nitrogen may be a more
important source of nitrogen for plants under cultivation
than previously thought (Nasholm et al., 2000, 2001).
The observation that organic nitrogen compounds, such
as amino acids, can be absorbed by roots under eld
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
3
To whom correspondence should be addressed. Fax: +44 (0)1865 275074. E-mail: george.ratcliffe@plants.ox.ac.uk
Society for Experimental Biology 2002
Journal of Experimental Botany, Vol. 53, No. 379, pp. 23052314, December 2002

b
y

g
u
e
s
t

o
n

M
a
y

1
7
,

2
0
1
4
h
t
t
p
:
/
/
j
x
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

(Fischer et al., 1998), and indeed the use of dual-labelled
amino acids, for example, glycine labelled with both
13
C
and
15
N (Nasholm 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 signicance of the uptake process has been
the priority in most of these studies, with the result that the
subsequent metabolism of the absorbed nitrogenous com-
pounds 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 signicant
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 aminotrans-
ferases 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 environ-
ment 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
afnity transport system (K
m
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 identied, and so this
problem was investigated by analysing the metabolism of
[
15
N]-labelled glycine and serine with
15
N nuclear mag-
netic 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 experi-
ments were also performed on excised maize (Zea mays L.)
root tips using [
15
N]glycine, [
15
N]serine and [2-
13
C]
glycine. NMR analysis of the [
13
C]-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).
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 in
November 2000. The plants grew in dense mats rmly attached to
the soil, and air-dried slabs of approximately 200 cm
2
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
articial rock pool solution containing the low nutrient concentra-
tions typical of the natural habitat. This solution contained 50 mmol
m
3
KCl, 250 mmol m
3
NaCl, 200 mmol m
3
CaSO
4
, 50 mmol m
3
MgCl
2
, 5 mmol m
3
(NH
4
)
2
SO
4
, 0.2 mmol m
3
MnCl
2
, 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 23 d between layers of absorbent paper moistened with 0.1
mol m
3
CaSO
4
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 810, 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
CaSO
4
,
pH 6 (glucose-MES buffer), supplemented with either 5 mol m
3
[
15
N]glycine or 5 mol m
3
[
15
N]serine (98 atom%; Promochem,
Germany). After labelling, samples of roots were vacuum inltrated
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 2122 C, was recycled through the NMR tube at 67
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
[
15
N]glycine, 5 mol
m
3
[
15
N]serine, or 5 mol m
3
[2-
13
C]glycine (99 atom%;
Promochem, Germany). After labelling, the root tips were suspended
in MES buffer in the 10 mm NMR tube, but without vacuum
inltration 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.
2306 Hartung and Ratcliffe

b
y

g
u
e
s
t

o
n

M
a
y

1
7
,

2
0
1
4
h
t
t
p
:
/
/
j
x
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

1
H-decoupled
15
N 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 acqui-
sition, 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
[
15
N]urea.
1
H-decoupled
13
C 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 acqui-
sition, 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 gures are representative examples from 36
independent labelling experiments.
Results
Metabolism of [
15
N]glycine and [
15
N]serine by
Chamaegigas intrepidus
Incubation experiments with [
15
N]glycine were performed
on whole plants and excised roots, and the redistribution of
the label was analysed using in vivo
15
N NMR spectros-
copy. Figure 1A shows the spectrum of a root sample taken
from plants that had been incubated in 5 mol m
3
[
15
N]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 [
14
C]glycine uptake data of Schiller et al. (1998b).
Comparison with the spectra obtained in an investigation
of the utilization of [
15
N]ammonium and [
15
N]urea by C.
intrepidus (Heilmeier et al., 2000), as well as the results of
[
15
N]-labelling experiments on other plants (Gerendas
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), gluta-
mate 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 [
15
N]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).
Spectra were also recorded from the roots of C.
intrepidus plants that had been incubated with
[
15
N]serine (Fig. 1B). Signals were observed from
glutamine 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 and
utilization of the amino acid.
Metabolism of [
15
N]glycine and [
15
N]serine by maize
root tips
Similar, but much stronger signals, were observed in the
in vivo
15
N NMR spectra of maize root tips that had been
incubated with [
15
N]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
rst hour of the incubation (data not shown). As with
C. intrepidus the detection of the glutamine amide-N
Fig. 1. In vivo
15
N NMR spectra of root systems from C. intrepidus
plants after pre-incubation with: (A) 5 mol m
3
[
15
N]glycine for 20 h;
and (B) 5 mol m
3
[
15
N]serine for 12 h. The spectra were accumulated
in 4 h, and the labelled signals can be assigned to: 2, glutamine
amide-N; 3, urea, from the capillary used for the chemical shift
reference; 4, glutamate and glutamine amino-N; 5, serine; and 6,
glycine.
Glycine metabolism in roots 2307

b
y

g
u
e
s
t

o
n

M
a
y

1
7
,

2
0
1
4
h
t
t
p
:
/
/
j
x
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

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 [
15
N]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).
Figure 3 shows the result of a series of inhibitor
experiments in which maize root tips were incubated with
5 mol m
3
[
15
N]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
15
N
NMR spectrum, apart from the peak from the unmetabo-
lized glycine (Fig. 3A). AAN, which is a structural
analogue of glycine that inhibits GDC (Usuda et al., 1980;
Gardestrom et al., 1981), eliminated or greatly reduced the
glutamine amide, asparagine amide, glutamate and gluta-
mine 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; Gardestrom 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
15
N NMR spectrum, leaving signals from
glutathione, serine, unmetabolized glycine, and ammo-
nium (Fig. 3D). The results of these experiments are
consistent with the involvement of GDC in the metabolism
Fig. 2. In vivo
15
N NMR spectra of maize root tips recorded during
the period corresponding to 812 h uptake in incubation experiments
with: (A) 5 mol m
3
[
15
N]glycine; and (B) 5 mol m
3
[
15
N]serine. The
assignments are the same as in Fig. 1, except for the additional
presence of: 1, the glycine N of glutathione; and 7, ammonium.
Resolution enhanced spectra (not shown) indicate that peak 2 contains
contributions from both glutamine and asparagine amide-N.
Fig. 3. In vivo
15
N NMR spectra of maize root tips recorded after
preincubation 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 the
same as in Figs 1 and 2.
2308 Hartung and Ratcliffe

b
y

g
u
e
s
t

o
n

M
a
y

1
7
,

2
0
1
4
h
t
t
p
:
/
/
j
x
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

of glycine, and the effect of MSX highlights the role of GS
in the subsequent reassimilation of the labelled ammo-
nium. However, because of the overlapping specicity 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.
Metabolism of [2-
13
C]glycine by maize root tips
Excised maize root tips were incubated with [2-
13
C]glycine and the redistribution of the label was analysed
using in vivo
13
C NMR spectroscopy (Fig. 4). After an 18 h
incubation with 5 mol m
3
[2-
13
C]glycine, prominent
signals were detected from several labelled metabolites,
including [2-
13
C]glycine (42.40 ppm), glutathione (44.19
ppm), [2-
13
C]serine (57.33 ppm), [3-
13
C]serine (61.16
ppm), and an unidentied compound (63.92 ppm). The
labelling of [2-
13
C]serine is consistent with metabolism via
SHMT, while the labelling of [3-
13
C]serine indicates
metabolism via the combined action of GDC and SHMT.
Moreover, each of the serine signals is anked by the
doublet signal from [2,3-
13
C]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-
13
C]methionine.
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
Fig. 4. In vivo
13
C NMR spectra of maize root tips recorded after
preincubation for 18 h in the presence (A) and absence (B) of 5 mol
m
3
[2-
13
C]glycine. The spectra were recorded in 1 h and the insert
shows an expanded region of the spectrum recorded from the labelled
tissue. The labelled signals can be assigned to: 1, unknown; 2, a
central singlet peak from [3-
13
C]serine and a anking doublet from
carbon 3 of [2,3-
13
C]serine; 3, a central singlet peak from [2-
13
C]serine and a anking doublet from carbon 2 of [2,3-
13
C]serine; 4,
carbon 2 of the glycine in glutathione; and 5, [2-
13
C]glycine.
Fig. 5. In vivo
13
C NMR spectra of maize root tips recorded after
preincubation with 5 mol m
3
[2-
13
C]glycine for 18 h in the presence
of 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 are
the same as in Fig. 4. The insert on the right hand side of the gure
shows the glycine peak reduced by a factor of 4.
Glycine metabolism in roots 2309

b
y

g
u
e
s
t

o
n

M
a
y

1
7
,

2
0
1
4
h
t
t
p
:
/
/
j
x
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

while there was a marked reduction in the doublet signals
from [2,3-
13
C]serine there was an increase in the signal
from [2-
13
C]serine and no obvious effect on the signal
from [3-
13
C]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-
13
C]serine and four negative signals for [2,3-
13
C]serine. This result suggests that the AAN concentra-
tion was sufcient 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 (Gardestrom 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
13
C 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.
Discussion
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
[
15
N]-labelling and
15
N NMR (Neeman et al., 1985); while
the metabolism of the carbon skeleton can be followed
using [
13
C]-labelling and
13
C-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 particu-
larly 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 metab-
olism 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
[
15
N]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 amino-
transferase 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.
Fig. 6. Difference spectra derived from the spectra in Figs 4A and 5
showing the effects of (A) AOA, (B) AAN, (C) INH, and (D) MSX on
the redistribution of label from [2-
13
C]glycine. The assignments are
the same as in Fig. 4, and the spectra highlight the differential effects
of the various inhibitors on the labelling of the serine isotopomers.
2310 Hartung and Ratcliffe

b
y

g
u
e
s
t

o
n

M
a
y

1
7
,

2
0
1
4
h
t
t
p
:
/
/
j
x
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

First, the experiments with [
15
N]glycine provided direct
evidence for the release of [
15
N]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-
13
C]glycine (Fig. 4). More com-
plicated pathways for the release of ammonium can be
envisaged, for example, the oxidative deamination of
Fig. 7. Pathways for the metabolism of glycine showing the redistribution of label derived from [2-
13
C]glycine and [
15
N]glycine. The enzymes
are: GDC, glycine decarboxylase; GOGAT, glutamate synthase (EC 1.4.1.13); GS, glutamine synthetase; GSH-S, glutathione synthetase (EC
6.3.2.3); SGAT, serine:glyoxylate aminotransferase; and SHMT, serine hydroxymethyl transferase. Glutamate or alanine:glyoxylate
aminotransferase could provide an alternative route to labelled glyoxylate, although SGAT would appear to be more likely (Schmidt and Stewart,
1999).
Glycine metabolism in roots 2311

b
y

g
u
e
s
t

o
n

M
a
y

1
7
,

2
0
1
4
h
t
t
p
:
/
/
j
x
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

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 [
15
N]glutamate (Fig. 3D). The experiments
with [
15
N]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-
13
C]glycine con-
rmed 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
13
C NMR
spectrum: [2-
13
C]serine is formed by the SHMT-mediated
reaction of unlabelled CH
2
-THF and [2-
13
C]glycine; [2,3-
13
C]serine arises from the reaction of [
13
C]-labelled CH
2
-
THF, generated by the action of GDC on [2-
13
C]glycine,
and [2-
13
C]glycine; and [3-
13
C]glycine arises from reac-
tion of [
13
C]-labelled CH
2
-THF and unlabelled glycine
(Fig. 7). The characteristic signals of the three isotopomers
are readily identiable and they provide unequivocal
evidence for the involvement of both GDC and SHMT in
the metabolism of glycine (Fig. 4). Moreover, the
13
C
spectra contained no signals that could be attributed to
transamination products of glycine, indicating that the
[
15
N]serine observed in the experiments with [
15
N]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
[
15
N]ammonium released by bacterial activity would
provide an alternative explanation for the labelling of the
glutamine amide nitrogen, but since the [2-
13
C]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 signicant 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 dene 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 [
13
C]-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 signicant contribution
to root nitrogen utilization under eld 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
2312 Hartung and Ratcliffe

b
y

g
u
e
s
t

o
n

M
a
y

1
7
,

2
0
1
4
h
t
t
p
:
/
/
j
x
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

comment. First, in vivo NMR signals from [
13
C]- and
[
15
N]-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
1
H 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 nd
applications in ux measurements. Secondly, the identity
of the substantial unassigned resonance observed at
63.92 ppm in the [2-
13
C]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 unas-
signed peaks may well correspond to the unidentied
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 metab-
olism, 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 sufcient to identify the com-
pound, 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-
13
C]glycine
and [
15
N]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
sufcient GDC activity in the roots to support the observed
uxes.
Acknowledgements
We thank the Deutsche Forschungsgemeinschaft (WH) and the UK
Biotechnology and Biological Sciences Research Council (RGR) for
nancial support. We also thank the anonymous referees for
constructive criticism.
References
Ashworth DJ, Mettler IJ. 1984. Direct observation of glycine
metabolism in tobacco suspension cells by carbon-13 NMR
spectroscopy. Biochemistry 23, 22522257.
Bird IF, Cornelius MJ, Keys AJ, Whittingham CP. 1972.
Oxidation and phosphorylation associated with the conversion of
glycine to serine. Phytochemistry 11, 15871594.
Bourguignon J, Vauclare P, Merand V, Forest E, Neuburger M,
Douce R. 1993. Glycine decarboxylase complex from higher
plants. Molecular cloning, tissue distribution and mass
spectrometry analysis of the T-protein. European Journal of
Biochemistry 217, 377386.
Bourguignon J, Rebeille F, Douce R. 1999. Serine and glycine
metabolism in higher plants. In: Singh BK, ed. Plant amino acids
New York: Marcel Dekker, 111146.
Brouquisse R, James F, Raymond P, Pradet A. 1991. Study of
glucose starvation in excised maize root tips. Plant Physiology
96, 619626.
Brouquisse R, James F, Pradet A, Raymond P. 1992. Asparagine
metabolism and nitrogen distribution during protein degradation
in sugar-starved maize root tips. Planta 188, 384395.
Carroll AD, Fox GG, Laurie S, Phillips R, Ratcliffe RG, Stewart
GR. 1994. Ammonium assimilation and the role of g-
aminobutyric acid in pH homeostasis in carrot suspension cells.
Plant Physiology 106, 513520.
Chapin FS, Moilanen L, Kielland K. 1993. Preferential use of
organic nitrogen for growth by a non-mycorrhizal arctic sedge.
Nature 361, 150153.
Dry IB, Wiskich JT. 1986. Comparative aspects of amino-
oxyacetate inhibition of glycine oxidation and aminotransferase
activity of pea leaf mitochondria. Plant Science 44, 2328.
Falkengren-Grerup U, Mansson KF, Olsson MO. 2000. Uptake
capacity of amino acids by ten grasses and forbs in relation to soil
acidity and nitrogen availability. Environmental and
Experimental Botany 44, 207219.
Fischer W-N, Andre B, Rentsch D, Krolkiewicz S, Tegeder M,
Breitkreuz K, Frommer WB. 1998. Amino acid transport in
plants. Trends in Plant Science 3, 188195.
Ford YY, Ratcliffe RG, Robins RJ. 1996. Phytohormone-induced
GABA production in transformed root cultures of Datura
stramonium: an in vivo
15
N NMR study. Journal of
Experimental Botany 47, 811818.
Gardestrom P, Bergman A, Ericson I. 1981. Inhibition of the
conversion of glycine to serine in spinach leaf mitochondria.
Physiologia Plantarum 53, 439444.
Gerendas J, Ratcliffe RG, Sattelmacher B. 1993. Relationship
between intracellular pH and N metabolism in maize roots. Plant
and Soil 155/156, 167170.
Giess W. 1969. Die Verbreitung von Lindernia intrepidus (Dinter)
Oberm. (Chamaegigas intrepidus Dinter) in Sudwestafrika.
Dinteria 2, 2327.
Heilmeier H, Hartung W. 2001. Survival strategies under extreme
and complex environmental conditions: the aquatic resurrection
plant Chamaegigas intrepidus. Flora 196, 245260.
Heilmeier H, Ratcliffe RG, Hartung W. 2000. Urea: a nitrogen
source of the aquatic resurrection plant Chamaegigas intrepidus
Dinter. Oecologia 123, 914.
Henry HAL, Jefferies RL. 2002. Free amino acid, ammonium and
nitrate concentrations in soil solutions of a grazed coastal marsh
in relation to plant growth. Plant, Cell and Environment 25, 665
676.
Hickel B. 1967. Zur Kenntnis einer xerophilen Wasserpanze:
Chamaegigas intrepidus Dtr. Aus Sudwestafrika. Internationale
Revue der gesamten Hydrologie 52, 361400.
Hodge A, Robinson D, Fitter A. 2000. Are micro-organisms more
Glycine metabolism in roots 2313

b
y

g
u
e
s
t

o
n

M
a
y

1
7
,

2
0
1
4
h
t
t
p
:
/
/
j
x
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

effective than plants at competing for nitrogen? Trends in Plant
Science 5, 304308.
Kielland K. 1994. Amino acid absorption by arctic plants:
implications for plant nutrition and nitrogen cycling. Ecology
75, 23732383.
Lee RB, Ratcliffe RG. 1983. Development of an aeration system
for use in plant tissue NMR experiments. Journal of Experimental
Botany 34, 12131221.
Lipson DA, Monson RK. 1998. Plantmicrobe competition for soil
amino acids in the alpine tundra: effects of freezethaw and dry
rewet events. Oecologia 113, 406414.
Mesnard F, Azaroual N, Marty D, Fliniaux M-A, Robins RJ,
Vermeersch G, Monti J-P. 2000. Use of
15
N reverse gradient
two-dimensional nuclear magnetic resonance spectroscopy to
follow metabolic activity in Nicotiana plumbaginifolia cell
suspension cultures. Planta 210, 446453.
Meyer AJ, May MJ, Fricker MD. 2001. Quantitative in vivo
measurement of glutathione in Arabidopsis cells. The Plant
Journal 27, 6778.
Mouillon J-M, Aubert S, Bourguignon J, Gout E, Douce R,
Rebeille F. 1999. Glycine and serine catabolism in non-
photosynthetic higher plant cells: their role in C1 metabolism.
The Plant Journal 20, 197205.
Nasholm T, Ekblad A, Nordin A, Giesler R, Hogberg M,
Hogberg P. 1998. Boreal forest plants take up organic nitrogen.
Nature 392, 914916.
Nasholm T, Huss-Danell K, Hogberg P. 2000. Uptake of organic
nitrogen in the eld by four agriculturally important plant species.
Ecology 81, 11551161.
Nasholm T, Huss-Danell K, Hogberg P. 2001. Uptake of glycine
by eld-grown wheat. New Phytologist 150, 5963.
Nasholm T, Persson J. 2001. Plant acquisition of organic nitrogen
in boreal forests. Physiologia Plantarum 111, 419426.
Neeman M, Aviv D, Degani H, Galun E. 1985. Glucose and
glycine metabolism in regenerating tobacco protoplasts followed
non-destructively by nuclear magnetic resonance spectroscopy.
Plant Physiology 77, 374378.
Prabhu V, Chatson KB, Abrams GD, King J. 1996.
13
C nuclear
magnetic resonance detection of interactions of serine
hydroxymethyltransferase with C1-tetrahydrofolate synthase and
glycine decarboxylase complex activities in Arabidopsis. Plant
Physiology 112, 207216.
Prabhu V, Chatson KB, Lui H, Abrams GD, King J. 1998.
Effects of sulfanilamide and methotrexate on
13
C uxes through
the glycine decarboxylase/serine hydroxymethyltransferase
enzyme system in Arabidopsis. Plant Physiology 116, 137144.
Raab TK, Lipson DA, Monson RK. 1999. Soil amino acid
utilization among species of the Cyperaceae: Plant and soil
processes. Ecology 80, 24082419.
Rabenstein DL, Brown DW, McNeil CJ. 1985. Determination of
glutathione in intact and hemolyzed erythrocytes by titration with
tert-butyl hydroperoxide with end-point detection by
1
H nuclear
magnetic resonance spectrometry. Analytical Chemistry 57,
22942299.
Ratcliffe RG, Shachar-Hill Y. 2001. Probing plant metabolism
with NMR. Annual Review of Plant Physiology and Plant
Molecular Biology 52, 499526.
Russell J, Spickett CM, Reglinski J, Smith WE, McMurray J,
Abdullah IB. 1994. Alteration of the erythrocyte glutathione
redox balance by N-acetylcysteine, captopril and exogenous
glutathione. FEBS Letters 347, 215220.
Sarojini G, Oliver DJ. 1985. Inhibition of glycine oxidation by
carboxymethoxylamine, methoxylamine and acethydrazide. Plant
Physiology 77, 786789.
Schiller P, Hartung W, Ratcliffe RG. 1998a. Intracellular pH
stability in the aquatic resurrection plant Chamaegigas intrepidus
in the extreme environmental conditions that characterize its
natural habitat. New Phytologist 140, 17.
Schiller P, Heilmeier H, Hartung W. 1998b. Uptake of amino
acids by the aquatic resurrection plant Chamaegigas intrepidus
and its implication for N-nutrition. Oecologia 117, 6369.
Schmidt S, Stewart GR. 1997. Waterlogging and re impacts on
nitrogen availability and utilization in a subtropical wet heathland
(wallum). Plant, Cell and Environment 20, 12311241.
Schmidt S, Stewart GR. 1999. Glycine metabolism by plant roots
and its occurrence in Australian plant communities. Australian
Journal of Plant Physiology 26, 253264.
Streeter TC, Bol R, Bardgett RD. 2000. Amino acids as a nitrogen
source in temperate upland grasslands: the use of dual labelled
(
13
C,
15
N)glycine to test for direct uptake by dominant grasses.
Rapid Communications in Mass Spectrometry 14, 13511355.
Tate SS, Meister A. 1973. Glutamine synthetases of mammalian
liver and brain. In: Prusiner S, Stadtman ER, eds. The enzymes of
glutamine metabolism. New York: Academic Press, 77127.
Thornton B. 2001. Uptake of glycine by non-mycorrhizal Lolium
perenne. Journal of Experimental Botany 52, 13151322.
Usuda H, Arron GP, Edwards GE. 1980. Inhibition of glycine
decarboxylation by aminoacetonitrile and its effect on
photosynthesis in wheat. Journal of Experimental Botany 31,
14771483.
Walton NJ, Woolhouse HW. 1986. Enzymes of serine and glycine
metabolism in leaves and non-photosynthetic tissues of Pisum
sativum L. Planta 167, 119128.
Whatley FR, Greenaway W, Dunstan RH. 1986. Glycine
metabolism in carrot suspension culture cells investigated by
gas chromatography/mass spectrometry. Physiologie Vegetale 24,
313.
2314 Hartung and Ratcliffe

b
y

g
u
e
s
t

o
n

M
a
y

1
7
,

2
0
1
4
h
t
t
p
:
/
/
j
x
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m