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Journal of Experimental Botany, Vol. 59, No. 7, pp. 16851693, 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 tabolisme-Me tabolome, IFR 87, Ba t. 630, Universite Paris Sud-XI, F-91405 Orsay cedex, France Plateforme Me cophysiologie ve ge tale, CNRS UMR 8079, Ba t. 362, Universite Paris Sud-XI, F-91405 Orsay Laboratoire de cedex, France 3 t. 630, Universite Paris Sud-XI, F-91405 Orsay Institut de Biotechnologie des Plantes, CNRS UMR 8618, Ba cedex, France
2 1

Received 7 February 2007; Revised 6 April 2007; Accepted 27 April 2007

Abstract
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 uxes 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 uxes 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.
Key words: Day respiration, fractionation, 2-oxoglutarate, photorespiration, stable isotopes, transamination.

Introduction Plant growth and development depends upon N supply and assimilation. In C3 plants lacking a N-xing 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 signicant 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 (GCMS) have shed some light on primary metabolic

cophysiologie ve ge tale, CNRS UMR 8079, Ba t. 362, Universite Paris Sud-XI, * To whom correspondence should be addressed at Laboratoire de 91405 Orsay cedex, France. E-mail: guillaume.tcherkez@u-psud.fr Abbreviations: GO, glyoxylate; hPyr, hyrdoxy-pyruvate; 2OG, 2-oxoglutarate; OAA, oxaloacetate. The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

1686 Tcherkez and Hodges

uxes 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 uxes 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.

R Rst Rst

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 puried 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 uxes. 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 dened by:

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+ ), although the latter is often absorbed to 4 a lower extent, but this depends on plant species. After root uptake by transporters, the rst step of nitrate assimilation is the reduction to nitrite (NO 2 ) by nitrate reductase (NR). While a signicant 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 inuenced 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 (simplied) 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 xed 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 d15 Ne Du Dr Du 1 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 ux-based one. For this purpose, it is assumed that the gross uptake-ux is written as fuG3ne and the efux (root NO 3 loss) as fiG#3ni where G and G# are conductances. The net entering ux is then fafufi. With the further assumption that GG# and that uptake does not fractionate (Du0), equation (1) rearranges to:   fa 2 d15 Na 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 d15Ned15NaDr, 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 signicant 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 5070% (Scheible et al., 2000). In other words, a signicant proportion of leaf nitrate is reduced by NR during the rst 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 inuenced 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 xed 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 (Miin 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 xed 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 specic form in GOGAT functioning has not been proven (Ga lvez 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 dened (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 uxes (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 productionconsumption 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 (Ga lvez 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 (lled 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 articial 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 (lled 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 insufcient 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 xation by PEPC (Melzer and OLeary, 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 xation in the light, because the xation 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 uxes of which are not known with much precision. The analysis of compound-specic 15 N-abundance may help to determine uxes, because the distribution of 15N is not statistical, but results from the combination of several fractionating reactions occurring at given ux rates. Figure 3 summarizes the metabolic framework that may be considered to infer possible uxes 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 nding 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 conrmed 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 ux than primary N reduction (at least one order of magnitude). Thus the effect of photorespiration on the compound-specic 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 ux (CO2 efux) 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 simplied 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 ux 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 (12&) 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 ux 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 15 production of NH+ N 4 is likely to discriminate against 15 (D6, Fig. 3), the accumulation of N-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.

the inhibition of day respiration and the need to produce 2-oxoglutarate for N assimilation. However, it is striking that the actual ux 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 benecial 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 Universite Paris-Sud XI and the grants associated with the Projet Transversal of the Institut Fe de ratif de Recherche 87.

References
Abelson H, Hoering TC. 1961. Carbon isotope fractionation in formation of amino acids by photosynthetic organisms. Proceedings of the National Academy of Sciences, USA 47, 623632. Ashley DA, Jackson WA, Volk RJ. 1975. Nitrate uptake and assimilation by wheat seedlings during initial exposure to nitrate. Plant Physiology 55, 11021106. rdestrom P, Day DA. 2000. PhotosynAtkin OK, Millar AH, Ga thesis, carbohydrate metabolism and respiration in leaves of higher plants. In: Leegood RC, Sharkey TD, von Caemmerer S, eds. Photosynthesis, physiology and metabolism. London: Kluwer Academic Publishers, 153175. Bao XM, Focke M, Pollard M, Ohlrogge J. 2000. Understanding in vivo carbon precursor supply for fatty acid synthesis in leaf tissue. The Plant Journal 22, 3950.

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 (Ga lvez 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

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