Journal of Experimental Botany, Vol. 59, No. 7, pp. 1685–1693, 2008
doi:10.1093/jxb/erm115 Advance Access publication 23 July, 2007
SPECIAL ISSUE OPINION PAPER
How stable isotopes may help to elucidate primary
nitrogen metabolism and its interaction with
(photo)respiration in C3 leaves
Guillaume Tcherkez1,2,* and Michael Hodges3
1
Plateforme Métabolisme-Métabolome, IFR 87, Bât. 630, Université Paris Sud-XI, F-91405 Orsay cedex, France
Laboratoire d’écophysiologie végétale, CNRS UMR 8079, Bât. 362, Université Paris Sud-XI, F-91405 Orsay
cedex, France
2
3
Institut de Biotechnologie des Plantes, CNRS UMR 8618, Bât. 630, Université Paris Sud-XI, F-91405 Orsay
cedex, France
Received 7 February 2007; Revised 6 April 2007; Accepted 27 April 2007
Abstract
Introduction
Intense efforts are currently devoted to elucidate the
metabolic networks of plants, in which nitrogen assimilation is of particular importance because it is strongly
related to plant growth. In addition, at the leaf level,
primary nitrogen metabolism interacts with photosynthesis, day respiration, and photorespiration, simply
because nitrogen assimilation needs energy, reductant, and carbon skeletons which are provided by
these processes. While some recent studies have
focused on metabolomics and genomics of plant
leaves, the actual metabolic fluxes associated with
nitrogen metabolism operating in leaves are not very
well known. In the present paper, it is emphasized that
12 13
C/ C and 14N/15N stable isotopes have proved to be
useful tools to investigate such metabolic fluxes and
isotopic data are reviewed in the light of some recent
advances in this area. Although the potential of stable
isotopes remains high, it is somewhat limited by our
knowledge of some isotope effects associated with
enzymatic reactions. Therefore, this paper should be
viewed as a call for more fundamental studies on
isotope effects by plant enzymes.
Plant growth and development depends upon N supply
and assimilation. In C3 plants lacking a N-fixing symbiotic organism, reduction of inorganic N and assimilation
to organic molecules can be operated by leaves and roots.
However, the contribution of leaves is usually more
important (although being species-dependent) and so the
mechanism(s) by which leaves reduce and integrate N into
primary metabolites and major amino acids is essential.
While stable isotopes (12C/13C and 14N/15N) have brought
a significant contribution to understanding N assimilation,
and the interactions with C allocation in the past (mainly
through 13C- and 15N-labelling experiments; Yoneyama
and Kumazawa, 1975; Layzell et al. 1981; Soussana and
Faurie, 1993; Maillard et al., 1994), or to elucidating
primary N metabolism (with 15N labelling and 15N natural
abundance; see the review of Yoneyama et al., 2003),
isotopic studies are now quite often neglected or rarely
quoted. However, stable isotopes are natural tracers
that are useful for the investigattion of metabolite
dynamics (i.e. turnover rates) and to identify the origin of
atoms. This lack of consideration could arise from
a dispersion of isotopic and metabolic/biochemical data in
the literature.
Nevertheless, some recent labelling studies analysing
13
C-distribution by nuclear magnetic resonance (NMR)
and gas chromatography coupled to mass spectrometry
(GC–MS) have shed some light on primary metabolic
Key words: Day respiration, fractionation, 2-oxoglutarate,
photorespiration, stable isotopes, transamination.
* To whom correspondence should be addressed at Laboratoire d’écophysiologie végétale, CNRS UMR 8079, Bât. 362, Université Paris Sud-XI,
91405 Orsay cedex, France. E-mail: [email protected]
Abbreviations: GO, glyoxylate; hPyr, hyrdoxy-pyruvate; 2OG, 2-oxoglutarate; OAA, oxaloacetate.
ª The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
1686 Tcherkez and Hodges
fluxes in plant organs other than leaves (Bao et al., 2000;
Schwender and Ohlrogge, 2002; Roessner-Tunali et al.,
2004; Schwender et al., 2006). In leaves, such techniques
will probably be developed further in the future and
used to compare mutants affected in different metabolic
pathways (as in potato tubers, Roessner-Tunali et al.,
2004). Recent work in biochemistry and functional
genetics of leaf N assimilation has provided key information about gene expression, the abundance of
enzymes, and metabolite concentrations (see the reviews
of Stitt et al., 2002; Hodges, 2002; Coruzzi, 2003);
however, data on actual fluxes through metabolic pathways are scarce. Since stable isotopes can be followed as
tracers, their use should bring the missing pieces required
to complete the metabolic jigsaw puzzle. Thus, the aim of
the present paper is to re-interpret published isotopic
results in the light of new advances in biochemistry and
metabolomics of leaf N assimilation.
Basics of isotopic studies
Two complementary isotopic approaches have been used
so far: (i) labelling and (ii) analysis of natural abundance.
(i) Leaves or cells are labelled with 15N (e.g. with nitrate
or ammonium) or 13C (e.g. with CO2 or organic
molecules) and the atoms are simply followed as in
classical pulse/chase experiments (Robinson et al., 1991,
for 15N; Tcherkez et al., 2005, for 13C). The rate of
appearance and of disappearance of 13C or 15N allows the
turnover of metabolites or their distribution within the
plant to be followed. The detection of the isotopic tracers
in labelled compounds can be achieved with isotope-ratio
mass spectrometry (on purified metabolites or separated
from an extract by gas chromatography) or NMR
(on whole extracts or in vivo). Although NMR is well
adapted for 15N and 13C, 14N is less detectable and 12C
cannot be seen with this technique. Further information
about the technique may be found in Mesnard and
Ratcliffe (2005).
(ii) The analysis of natural abundance, with isotoperatio mass spectrometry, has the potential to be a more
powerful technique as it can give clues to both the origin
of atoms and the size of metabolic fluxes. However, its
interpretation requires more basic knowledge, such as
the isotope composition of products and substrates, the
isotope discrimination involved in enzymatic reactions,
and transport or uptake phenomena. The isotope composition is the relative difference of the isotopic
ratio (13C/12C or 15N/14N) between a given sample
and an international standard. The isotope composition,
or delta-value (denoted as d13C and d15N), is thus
defined by:
d¼
R Rst
Rst
where Rst is the isotopic ratio of the standard reference
material (Pee Dee Belemnite fossil for C and atmospheric
N2 for N). The delta-value is generally small and
expressed in per mil. By discrimination (or fractionation),
we mean the extent to which enzymes fractionate between
heavy (13C or 15N) and light (12C or 14N) isotopes as
a consequence of the faster reactivity of light isotopes. For
example, in vitro, nitrate reductase (NR) is known to
discriminate N isotopes by 15& (Ledgard et al., 1985),
that is, the d15N value of reduced nitrate (nitrite) is nearly
15& lower than that of nitrate because the reaction
favours the 14NO
3 isotopologues. It is important to note
that a given reaction or physico-chemical process cannot
fractionate between isotopes when it is complete, simply
because the enzyme (reaction) consumes all of the
substrate molecules and so it cannot choose between them
on an isotopic basis. This obvious principle has important
consequences (see below). The origin of isotopic discrimination is either a kinetic or a thermodynamic effect.
A kinetic isotope effect is simply related to the difference
in rate constants k between isotopomers, while a thermodynamic isotope effect applies to equilibria characterized
by different equilibrium constants K for the two isotopologues. Some enzymes involved in nitrogen metabolism
and NH2-exchanges catalyse irreversible reactions so that
isotope effects are often kinetic.
Nitrate uptake and reduction
Nitrogen can be absorbed as nitrate (NO
3 ) and ammonium (NH+4), although the latter is often absorbed to
a lower extent, but this depends on plant species. After
root uptake by transporters, the first step of nitrate
assimilation is the reduction to nitrite (NO
2 ) by nitrate
reductase (NR). While a significant part is reduced in the
leaves, a fraction of nitrate is nevertheless reduced in
roots. For example, 15N-labelling of nitrate and measurement of reduced and organic nitrogen within the plant
have indeed shown that in wheat (Triticum vulgare), up to
80% of absorbed nitrate is reduced within the leaves
(Ashley et al., 1975).
In addition to the possible fractionation associated
with nitrate uptake itself (Kohl and Shearer, 1980;
Mariotti et al., 1982), plant (and leaf) nitrogen isotope
composition is influenced by the fractionation associated
with nitrate reduction. The in vitro production of nitrite by
NR fractionates between N isotopes by 15& (Ledgard
et al., 1985). But surprisingly, the N isotope composition
of plant material does not commonly differ from that of
soil nitrate and does not show the expected offset of 15&
Stable isotopes and primary N metabolism 1687
Fig. 1. General (simplified) scheme of nitrate assimilation by plants.
The uptake of soil nitrate (the N isotope composition and the
concentration of which are d15Ne and ne, respectively) fractionates
between N isotopes (fractionation Du). The resulting internal nitrate (the
concentration of which is ni) is reduced and fixed to organic matter.
Nitrate reduction is accompanied by a fractionation Dr. The N isotope
composition of organic matter is d15Na.
(for a recent study, see Tcherkez and Farquhar, 2006).
Several reasons have been given to explain this phenomenon, but the simplest explanation relies on the model of
nitrate absorption and uptake depicted in Fig. 1. The
consequence of such a model, which assumes a steadystate for internal nitrate, is that the nitrogen isotope composition of assimilated N follows the general relationship
(note the analogy with the photosynthetic 12C/13C
fractionation):
ni
d15 Na ¼ d15 Ne Du þ ðDr Du Þ
ð1Þ
ne
where d15Na and d15Ne are the nitrogen isotope compositions of assimilated N and external (soil) nitrate, respectively. ni and ne are the internal and soil nitrate
concentrations respectively; Du and Dr is the fractionation
associated with uptake and assimilation (reduction). Note
that this equation can be converted to a flux-based one.
For this purpose, it is assumed that the gross uptake-flux
is written as fu¼G3ne and the efflux (root NO
3 loss) as
fi¼G#3ni where G and G# are conductances. The net
entering flux is then fa¼fu–fi. With the further assumption
that G¼G# and that uptake does not fractionate (Du¼0),
equation (1) rearranges to:
fa
ð2Þ
d15 Na ¼ d15 Ne Dr 1 fu
which is similar to the general equation of Evans (2001).
If nitrate availability is high and the resistance associated with nitrate uptake is low, then ni/ne is large and the
overall fractionation tends to d15Ne–d15Na¼Dr, that is, the
fractionation associated with reduction (by the NR). On
the other hand, if nitrate availability is low so that nitrate
uptake is limiting compared to reduction, then ni/ne is
small and the overall fractionation tends to Du (in other
words, nitrate reduction consumes all of the available
nitrate molecules and no 14N/15N discrimination is
possible during reduction). In such a framework, the
N isotope composition of plants is expected to be
positively related to soil nitrate concentration, and this is
indeed what is observed (for a review, see Evans, 2001).
However, a metabolic reason may also contribute to
eliminate the observed 14N/15N fractionation at the leaf
level; that is, if nitrate reduction in the leaf consumes
a significant part of the leaf nitrate pool, the fractionation
by leaf NR would be limited (since a complete reaction
cannot fractionate). This view is consistent with the results
obtained in tobacco (Nicotiana tabacum) where illuminated leaves accumulate ammonium and Gln and the leaf
nitrate concentration decreases by 50–70% (Scheible
et al., 2000). In other words, a significant proportion of
leaf nitrate is reduced by NR during the first part of
illumination (Stitt et al., 2002), and so the enzyme cannot
fractionate up to 15& (an estimate of the actual
fractionation would be closer to 5&).
Nitrate molecules not reduced in roots and exported
to the shoots should be enriched in 15N (because of the
fractionation by root NR), and a consequence of the
‘consumption’ effect in the leaf described above is a
further 15N-enrichment of leaf nitrate (the higher the
proportion of nitrate reduced to nitrite, the higher the
enrichment of the remaining nitrate molecules), as shown
in Yoneyama et al. (2003). This effect, that may
contribute to decrease nitrogen fractionation at the leaf
level, would correlate with the higher natural d15N values
of leaves compared to other organs (Evans et al., 1996).
Nevertheless, it is recognized that this effect, which
depends on the proportion of root versus leaf reduced
nitrate, is likely to be influenced by species, growth
conditions and environmental conditions (Radin, 1977,
1978).
The reactions of primary N metabolism
In leaves, nitrite is mainly reduced to ammonium by the
(chloroplastic) ferredoxin-dependent nitrite reductase
(NiR). Ammonium is then fixed to organic molecules.
The primary organic compounds produced via N assimilation are Gln and Glu. Several decades ago, it was
believed that Glu dehydrogenase had a major role in the
N assimilation process (Miflin and Lea, 1976). However,
this pathway has since been shown to contribute (very)
modestly to primary N assimilation (Lea et al., 1990), and
the use of isotopes has provided clear evidence of this.
1688 Tcherkez and Hodges
When 15N-nitrate is supplied to leaves, the main labelled
products are Gln and Glu, ammonium, Asp, and Asn
(Yoneyama et al., 2003). The 15N-labelling of Gln is
always quicker and stronger than that of Glu (Thorpe
et al., 1989), suggesting that Glu is produced from Gln.
Consistently, NMR tracing of assimilated 15N established
that the amide group of Gln are labelled before the amino
group of Glu, thus implying a negligible contribution of
Glu dehydrogenase to N assimilation (Thorpe et al., 1989;
Robinson et al., 1991).
It is now accepted that ammonium is fixed to a molecule
of Glu to give Gln by the action of a Gln synthetase (GS).
However, under certain circumstances ammonium can
also accumulate and it may be back-diffused to the
atmosphere (Schjoerring et al., 2000, and references
therein). Gln combines with 2-oxoglutarate, producing
two molecules of glutamate; this reaction is catalysed by
a Gln/2-oxoglutarate aminotransferase (GOGAT). In addition to the isotopic data outlined above, both the
involvement of (chloroplastic) GS and GOGAT steps in
primary N assimilation have been demonstrated by
enzymatic activities and the analysis of mutants (for
a review, see Coruzzi, 2003). To date, the enzymatic
origin of 2-oxoglutarate for N assimilation is still a matter
of debate. This 5-C organic acid is synthesized by the
activity of isocitrate dehydrogenases. Plants contain both
NAD- and NADP-dependent enzymes and the implication
of a specific form in GOGAT functioning has not been
proven (Gálvez et al., 1999).
Leaf N metabolism follows a diurnal cycle
The biochemical framework described above stems from
the reduction of nitrate which is a light-dependent
phenomenon in leaves, simply because NiR and GOGAT
are both ferredoxin-dependent and they require electrons
from the chloroplastic photosynthetic electron transfer
chain. Therefore, it is unsurprising that N metabolism and
namely, Gln synthesis, decreases as the light-to-dark
transition occurs, as revealed by 15N isotope labelling
(Bauer et al., 1977). In Solanaceae, metabolomic analyses
during a diurnal cycle (Scheible et al., 2000; UrbanczykWochniak et al., 2005) have shown that Gln levels
oscillate with a maximum in the light, while malate, Ala,
Gly, and Ser also accumulate during the light period. On
a metabolite and protein activity basis, three steps have
thus been defined (Stitt et al., 2002). First, nitrate
assimilation is large thereby reducing the leaf nitrate pool,
while NH+4 and Gln accumulate as well as malate, Gly,
and Ser (but Glu does not). Then in the late light period,
there is an increase in some C-metabolism enzymes
associated with ‘respiration’ (pyruvate kinase, citrate
synthase, and isocitrate dehydrogenase). This is associated
with a slight increase in isocitrate and citrate levels (but
surprisingly, no increase in 2-oxoglutarate is observed).
During the night, levels of Gln and the photorespiratory
molecules Gly and Ser decrease dramatically while the
citrate and nitrate pools are replenished. Although there
may be some lag phase between the different phases of
the N assimilating pathway (namely, between N reduction
and synthesis of acceptor molecules from respiration), this
is consistent with the reactional framework described above.
However, some questions remain to be answered, such
as why does the level of a key-metabolite like Glu stay
constant throughout the day cycle? A simple interpretation
of this would be that Glu-production (GS reaction) and
Glu-consumption fluxes (GOGAT and other transaminations) simultaneously increase to the same extent, so that
the amount of Glu remains virtually constant. This view
would agree with the central role of Glu to provide aminogroups for transamination reactions that lead to the
production of other amino acids in the cell. However,
when illuminated leaves are fed with 15NO
3, the labelling
observed in Glu is lower than that of Gln (Bauer et al.,
1977). This would indicate that the net synthesis of Glu
molecules from newly assimilated nitrate molecules is
lower so that, presumably, the recycling of (unlabelled)
nitrogen might contribute to feeding leaf Glu synthesis.
The origin of such N atoms remains a mystery even
though it can be assumed that the turnover of proteins and
free amino acids may be involved. Still, as the measured
‘leaf’ Glu integrates Glu molecules from all cell compartments, it is possible that the diurnal cycle of Glu occurs
uniquely in the chloroplasts, but this is buffered by the
production–consumption via Glu cycling in other cell
compartments. Furthermore, Glu diurnal oscillations
appear to be species-dependent: while Glu levels stay
nearly constant in tobacco, they cycle in Arabidopsis (Lam
et al., 1995). Future 15N-labelling studies in Arabidopsis
should give some answers to this Glu mystery. However,
natural 13C- and 15N-abundances already provide some
clues, as explained below.
The relationships with carbon metabolism as
studied with 13C
Respiration is obviously a key process for N metabolism,
namely because it can provide carbon skeletons needed
for Glu synthesis (as well as NADH for the NR and ATP
for the GS reactions). The observed increase of leaf night
respiration when leaves are incubated with NO
3 , a fact
known for half a century (Moyse, 1950), is then viewed as
unsurprising. However, the metabolic relationships between respiration and N assimilation are complex.
First, the 2-oxoglutarate molecules used for Glu
synthesis are thought to be provided by the Krebs cycle
through the decarboxylation of isocitrate via the NADdependent isocitrate dehydrogenase (Gálvez et al., 1999).
Stable isotopes and primary N metabolism 1689
Fig. 2. Comparison of natural 13C and 15N isotope compositions of
amino acids. d13C data are from Abelson and Hoering (1961) for
Chlorella (filled symbols) or other photosynthetic organisms except red
algae (empty symbols). d13C values are corrected so that source-CO2
isotope composition equals 8& (atmospheric value) instead of the
artificial value of 0& used in the experiment. d15N data are from Bol
et al. (2002) and correspond to the mean of values obtained in Juncus
and Lolium leaves (filled symbols) or from Hofmann et al. (1991) on
Triticum leaves (empty symbols). As the d15N of the nitrogen source is
unknown in the latter data set, values were corrected so that the d15N
value of Glu equals that obtained in Bol et al. (2002).
This agrees with the relative 13C-depletion of Glu as
compared to Asp [which originates from oxaloacetate
(OAA) generated from phosphoenolpyruvate carboxylase
(PEPC) activity – see below] (Fig. 2). The Krebs cycle
intermediates are indeed 13C-depleted because the C-2 and
C-3 atoms of pyruvate are 13C-depleted and decarboxylases discriminate against 13C (Tcherkez and Farquhar,
2005). However, as N-metabolism, leaf respiration also
follows a diurnal cycle. It has been repeatedly shown that
leaf ‘mitochondrial’ respiration (or day respiration), as
measured by non-photorespiratory CO2 production in the
light, is lower than night respiration, the ratio being near
0.5 (Atkin et al., 2000). The mechanism(s) by which
respiratory decarboxylations are inhibited in the light is
slowly emerging: the pyruvate decarboxylase reaction is
partly inhibited in the light (Budde and Randall, 1990)
and the Krebs cycle decarboxylations are down-regulated,
as revealed by the lower Krebs-cycle dependent NADH
production in extracts of illuminated leaves (Hanning and
Heldt, 1993) and the very small 13CO2 production from
13
C-labelled intermediates supplied to detached illuminated leaves (Tcherkez et al., 2005). It has been shown
that some respiratory intermediates such as citrate accumulate during the night in tobacco leaves and decrease
during the day (Scheible et al., 2000; UrbanczykWochniak et al., 2005). It is thus plausible that a reserve
of (vacuolar) citrate produced during the night is used
during the subsequent light period for Glu synthesis. This
would involve the concerted action of cytosolic aconitase
and NADP-dependent isocitrate dehydrogenase as proposed by Chen and Gadal (1990). However, this is
probably not the unique 2-oxoglutarate producing pathway for Glu synthesis because (i) the apparent quantity of
remobilized citrate (estimated using the drop of citrate
levels) in tobacco leaves appears to be insufficient to feed
the total of Glu synthesis (Stitt et al., 2002) and
(ii) transgenic potato plants with only 8% of normal
NADP-isocitrate dehydrogenase activity do not exhibit
changes that can be associated with an altered N-assimilation
(Kruse et al., 1998). Further experiments are necessary to
test this assumption.
In illuminated leaves, the fate of pyruvate molecules
that are not processed by pyruvate dehydrogenase (which
is partly inhibited in the light, see above) may be, to some
extent, used in Ala synthesis, catalysed by an Ala aminotransferase that transfers NH2- from Glu to pyruvate.
Indeed, Ala has been shown to accumulate in the light in
Arabidopsis (Lam et al., 1995) and an NMR analysis of
bean leaves supplied with 13C-pyruvate exhibited Ala
labelling (Tcherkez et al., 2005).
Second, the production of organic acids is supplemented
by PEPC activity. This enzyme compensates for the
consumption of organic acids such as 2-oxoglutarate by
Glu synthesis, by providing OAA (or indirectly malate) to
feed the Krebs cycle (the so-called anapleurotic function
of PEPC). In addition, some OAA molecules may be
directly aminated to Asp so that PEPC activity may be
viewed as a primary producer of NH2-acceptors (Huppe
and Turpin, 1994). Such a relationship between PEPC and
N metabolism is supported by the higher PEPC transcript
abundance and enzymatic activity during the light period
(Scheible et al., 2000; Urbanczyk-Wochniak et al., 2005).
Furthermore, transgenic C3 plants overexpressing PEPC
or with a higher PEPC activity redirect more carbon to
several amino acids (Rademacher et al., 2002; Miyao and
Fukayama, 2003). However, the most obvious evidence of
the PEPC involvement in N metabolism is, maybe, the
measure of the 13C natural abundance (d13C) in carbon
atoms of Asp. In tobacco leaves, the C-4 atom of Asp is
consistently 13C-enriched, so that nearly 50% of the Asp
pool has been assumed to originate from HCO
3 fixation
by PEPC (Melzer and O’Leary, 1987; see also Fig. 2).
Although N metabolism may be somewhat different in
unicellular algae as compared to plants, it has also been
shown in Selenastrum minutum that N assimilation is
accompanied by a decrease in the carbon isotope discrimination (D13C) associated with CO2 fixation in the light,
because the fixation by PEPC is triggered (Guy et al.,
1989). It was also noted that, although PEPC activity
correlates with N metabolism, other biological functions
may be emphasized, such as pH homeostasis: the synthesis
of organic acids is required to balance alkalinization caused
by nitrate assimilation (for a discussion on this topic in
higher plants, see Scheible et al., 2000).
1690 Tcherkez and Hodges
Major transamination reactions
N metabolism follows a complex network of transamination reactions, the fluxes of which are not known with
much precision. The analysis of compound-specific
15
N-abundance may help to determine fluxes, because the
distribution of 15N is not statistical, but results from the
combination of several fractionating reactions occurring at
given flux rates. Figure 3 summarizes the metabolic
framework that may be considered to infer possible fluxes
from 15N-abundance.
Because there is likely to be a kinetic isotope fractionation associated (i) with the GOGAT reaction (probably of
around 20&; Werner and Schmidt, 2002; D2 in Fig. 3)
and (ii) with NH2-transfer from Glu to glyoxylate,
enriching the Glu molecules available for the GS reaction
(which discriminates against 15N by ;16&; Yoneyama
et al., 1993; D1 in Fig. 3), the leaf Gln pool is enriched in
15
N compared with Glu. The d15N value of Glu+Gln is
indeed higher than that of Glu alone, and the same is
observed for the couple Asp+Asn (the amido-NH2 of the
latter comes from Gln) (Yoneyama and Tanaka, 1999).
An interesting finding is that Asp is slightly 15N-enriched
compared with Glu (Fig. 2; Hare et al., 1991; Yoneyama
et al., 1998), suggesting that the isotope effect associated
with OAA-Glu aminotransferase (D4, Fig. 3) is thermodynamic rather than kinetic. In other words, this reaction
is near equilibrium rather than a unidirectional irreversible
reaction in the leaf. This is possible as the enzyme is
known to catalyse the reaction in both the forward and
reverse directions (Velick and Vavra, 1962). The in vitro
thermodynamic isotope discrimination is 6.6& for the
equilibrium corresponding to Asp production (Macko
et al., 1986) and 5.6& in the porcine heart enzyme
(Rishavy and Cleland, 2000). It was noted that Asp levels
are higher in Arabidopsis leaves during the night (Lam
et al., 1995). During the light period, the amounts of Asp
decrease and it is possible that it is exported to sink
tissues as well as being used for Glu production from
2-oxoglutarate via the reverse reaction of the equilibrium.
Since the Asp-to-Glu conversion fractionates against 15N
by 1.7& (Macko et al., 1986), the result would be a
15
N-enriched Asp leaf pool. Thus, a part of Glu in the
light plausibly comes from Asp (and at least a fraction of
that is necessary to start the GS/GOGAT cycle). If true,
one might expect a slow turnover of Asp in the light as
the reverse reaction (Asp to Glu) is favoured, and this has
been confirmed by 15N-labelling (Bauer et al., 1977). The
resulting production of OAA probably helps feeding
malate synthesis which is indeed prevalent in illuminated
tobacco leaves (see above).
Photorespiration and NH+4 recycling
The N-assimilatory system interacts with the photorespiratory cycle operating in illuminated leaves. In the
peroxisome, the conversion of glycolate (the product of
the RuBisCo oxygenation reaction) to glycine involves
transamination, the amino-donor being Glu. In the
mitochondria, the decarboxylation of Gly to Ser by Gly
decarboxylase (GDC) produces large amounts of NH+4 that
is believed to be mainly reassimilated (recycled) in the
chloroplast to produce Gln. This photorespiratory cycling
has a much higher flux than primary N reduction (at least
one order of magnitude). Thus the effect of photorespiration on the compound-specific 15N natural abundances
may be large, and this question is addressed below.
Although nitrogen isotopes are adapted tools to study the
N-containing metabolites involved in the photorespiratory
pathway, the photorespiratory flux (CO2 efflux) associated
with glycine decarboxylation has been investigated at the
leaf level using 13C, taking advantage of the 12C/13C
fractionation by this enzyme (Igamberdiev et al., 2004; for
a review, see Tcherkez and Farquhar, 2006).
Glycine production and deamination
Fig. 3. A simplified scheme of the most important reactions of primary
N metabolism associated with a 14N/15N isotope effect. For each
reaction, the isotopic fractionation is indicated as Di. See the text for the
details on fractionation values. Arrow thickness indicates the plausible
flux rate.
Although the 14N/15N isotope effect has not been
measured in vitro, the NH2-transfer from Glu to glyoxylate
(D3, Fig. 3) to produce Gly is presumed to discriminate to
the same degree as other transamination reactions (Werner
and Schmidt, 2002). As a consequence, this depletes
Gly in 15N compared with Glu. On the other hand, the
GDC-catalysed loss of NH+4 to produce Ser (D6, Fig. 3)
also probably discriminates against 15N, leading to a
15
N-enrichment of the remaining Gly molecules. So the
15
N abundance in Gly should simply be the result of the
balance between the two reactions. However, (i) a recycling of photorespiratory NH+4 occurs by the interplay of
Glu/Gln (GS/GOGAT) and (ii) Ser acts as an amino donor
for the glyoxylate-to-Gly conversion, a reaction that
probably fractionates against 15N (D7, Fig. 3).
Stable isotopes and primary N metabolism 1691
As a result of this small N-cycle between Gly and Ser, it
is the entry of N (from Glu) and the loss (deamination of
Gly by GDC) of NH+4 that determines the isotope
composition of photorespiratory N-compounds. Depending on the balance between the fractionations of these
steps, Gly may be enriched, depleted or have the same
15
N-abundance as Glu.
Clearly, Gly is constantly depleted in 15N compared to
Glu (Fig. 3), indicating that the amino-transfer from Glu
fractionates against 15N, maybe more than the production
of NH+4 during conversion to Ser. In addition, while
Ser and Gly have approximately the same 15N-isotope
composition, Ser is on average (1–2&) enriched compared with Gly in (Fig. 2). This indicates that the aminotransfer from Ser to glycolate can still fractionate to
a (very) small extent so that the resulting Gly molecules
are 15N-depleted compared with Ser. In other words, the
conversion of Ser to hydroxypyruvate is nearly stoichiometric, but a small flux of, say, 5% (that is, the 1&
depletion divided by the average transamination discrimination of 20&) escapes. Since photorespiratory Ser
production is high in the light (near 2.5 lmol m2 s1 in
tobacco under usual ‘ambient’ conditions), the integrated
quantity may be large. This explains the observed
Ser accumulation in illuminated leaves (Scheible et al.,
2000).
Photorespiratory recycling of ammonium
Much uncertainty remains as to whether photorespiratory
NH+4 is completely recycled through N-assimilation to
Gln and Glu in the light. Illuminated leaves can produce
NH3 which diffuses out of the plant (Farquhar et al.,
1980; Schjoerring et al., 2000) and NH+4 can accumulate
in illuminated tobacco leaves (Scheible et al., 2000).
Obviously, from a mass-balance point of view, as the
production of NH+4 is likely to discriminate against 15N
(D6, Fig. 3), the accumulation of 15N-depleted NH+4
enriches all other N-containing molecules in 15N. The
subsequent diffusion of NH3, although very small, may
then contribute to eliminate the original discrimination by
NR in leaf organic matter (see above). Nevertheless, the
nitrogen isotope composition of this NH3 is currently
unknown and future studies that use NH3 trapping and
subsequent 14N/15N isotope ratio measurements are thus
needed.
Perspectives
Because there is no N-atom in 2-oxoglutarate, N isotopes
cannot solve the uncertainty concerning the origin of this
molecule which feed Glu synthesis in the light (Gálvez
et al., 1999; Hodges, 2002). Measurements of the d13C of
leaf 2-oxoglutarate has not been done yet, and progress on
this topic can be expected in the near future, to reconcile
the inhibition of day respiration and the need to produce
2-oxoglutarate for N assimilation. However, it is striking
that the actual flux through the Krebs cycle of illuminated
leaves is near 0.05 lmol m2 s1 while that of average
N-assimilation over the life span of a leaf is in the same
order of magnitude (;4.5 mol N m2 divided by 15 d of
16 h light needed for the development of one leaf; this
gives ;0.05 lmol m2 s1). Although such numbers
may be only a coincidence, whether or not there is
a mystery regarding 2-oxoglutarate production for N
assimilation may be the question that isotopes will
answer.
Isotope experiments would also be useful to help in
specifying the role of photorespiration in primary
N metabolism. While photorespiration is often considered
as a wasteful process, some studies have suggested that
photorespiration may be beneficial to N-assimilation
(Rachmilevitch et al., 2003). Isotopic 15N-labelling under
different O2 conditions (21% O2 or non-photorespiratory
conditions) would help to clarify this aspect of C/N
interactions.
An aim of the present paper was to show that stable
isotopes are a powerful tool to elucidate several aspects of
N metabolism, from enzymatic reactional mechanisms
(Tcherkez and Farquhar, 2006) to metabolic pathways
(Yoneyama et al., 2003). However, the progress in
classical biochemistry of isotope effects has slowed down
for nearly a decade and the isotopic fractionation
associated with many key enzymes are still missing (such
as D3, D6, D7 in Fig. 3). On the other hand, ecological
applications of stable isotopes are growing exponentially,
therefore the need of obtaining fundamental information
concerning isotope effects is more important than ever.
Acknowledgements
Both authors acknowledge the support of the CNRS and the
Université Paris-Sud XI and the grants associated with the ‘Projet
Transversal of the Institut Fédératif de Recherche 87’.
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