Although being the cornerstone for nitrogen assimilation by plants, there remains much uncertainty about the origin of carbon skeletons required for Glu synthesis in illuminated leaves. Presumably, the carbon source for Glu production in the light comes from both newly synthesized (via photosynthates through the TCA cycle) and remobilized (derived from metabolites produced during the night) 2-oxoglutarate molecules. However, there is currently no corresponding assessment of the relative importance of such carbon sources. In this study, we took advantage of isotopic techniques and labelling with 13CO2 and 15N-ammonium nitrate, and carried out analyses with 13C- and 15N-NMR.
The TCA cycle in the light
The isotopic enrichment pattern of respiratory intermediates after 13CO2 feeding indicated that the TCA cycle was probably involved in Glu production in the light, as evidenced by the 13C enrichment in Glu and Gln (Fig. 3). The 13C enrichment pattern in citrate, Glu and Gln appeared to be quite similar (Fig. 4), suggesting that Glu and Gln are major carbon sinks for newly assimilated 13C atoms committed to the TCA cycle. Importantly, succinate was always poorly, if at all, labelled, strongly suggesting that the 13C label ([13C]-2-oxoglutarate) is directed to Glu/Gln synthesis at the expense of succinate. Such a conclusion is consistent with published biochemical data. Namely, 2-oxoglutarate dehydrogenation to succinyl-CoA is believed to be (1) impeded by the competition for Coenzyme A and E3 enzymatic subunits between pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase (Dry & Wiskich, 1987; Budde et al., 1991; Millar & Kunst, 1999), and (2) inhibited by large NADH/NAD ratios within the mitochondrial matrix (Igamberdiev & Gärdestrom, 2003). That is, the production of succinate is quite low in the light.
Within such a framework, the involvement of PEPc was critical for regenerating oxaloacetate and supplementing citrate synthesis (anaplerotic role). In fact, Mal appeared to be labelled in all C-atom positions, and the 13C enrichment in C-4 covaried with the C-atom positions of Glu (the path followed by atoms is given in Fig. S3, see Supporting Information). Although leaf fumarate content was low (not detected with 13C-NMR), an equilibrium between Mal and fumarate probably occurred, thereby explaining the very similar 13C enrichment pattern in C-1 and C-4 atom positions in Mal (Fig. 3). The in vivo involvement of PEPc, which is activated in the light by phosphorylation (Duff & Chollet, 1995), has been further acknowledged by other metabolic studies that used isotopes at natural abundance (reviewed in Tcherkez & Hodges, 2008).
Our results thus indicate that oxaloacetate molecules produced by PEPc are committed to both Mal and citrate production, and that the TCA cycle does not appear to operate like a proper cycle but, rather, involves two opposite branches fed by PEPc. Such a scenario is in agreement with previous results obtained on cocklebur (Xanthium strumarium) leaves, in which Glu and Mal were more 13C labelled by 13C-2-pyruvate than was succinate (Tcherkez et al., 2008). Furthermore, 13CO2 labelling followed by fluxomics calculations (flux coefficients) in X. strumarium leaves have provided evidence that oxaloacetate molecules have two competitive fates in the light, that is, fumarate/Mal synthesis (reversed left branch of the cycle) and Glu synthesis (right branch of the cycle) (Tcherkez et al., 2009).
Notwithstanding the above observations, the 13C flux into the TCA cycle to Glu (right-hand side of the cycle) was rather small, on the order of 25 mmol 13C-Glu per mole of assimilated 13CO2 (Fig. 2), that is, 2.5% × 14 = 0.35 μmol m−2 s−1. This value is similar (same order of magnitude) to that of day respiration measured by gas exchange on several C3 plants (Atkin et al., 2000). Such a value also matches the in vitro citrate synthase activity measured on leaf extracts (illuminated leaves) of c. 0.3 μmol m−2 s−1, whereas that of PEPc is as large as c. 5 μmol m−2 s−1 in Nicotiana tabacum (Scheible et al., 2000). We therefore conclude that the restriction of day respiration and of the TCA cycle by light (see the references already discussed in the Introduction) is sufficient to provide the necessary 13C flux to feed Glu synthesis.
The contribution of stored carbon as a source for Glu synthesis
The production of Glu was further fed by the remobilization of stored molecules synthesized from previously assimilated carbon (‘old’ C atoms). In fact, the synthesis of 13C-Glu in the light was low compared with that observed in the subsequent dark period and in the next light period under ordinary CO2 (natural 13C abundance) (Fig. 4). This was also observed for citrate, Gln and Mal (Fig. 4). Such weak contributions of recent 13C atoms to Glu, Gln and citrate synthesis in the light were certainly not caused by the isotopic dilution of label 13CO2 by 12CO2 from photorespiratory and day respiratory decarboxylations. First, the photorespiratory intermediates Ser and Gly are rapidly labelled (Canvin, 1979), and this leads to photorespired CO2 becoming totally labelled. Second, the decarboxylation rate associated with day respiration is usually close to 0.5 μmol m−2 s−1 (Atkin et al., 2006), and therefore accounts for only 3% and 12% of the net photosynthetic CO2 exchange flux under 400 and 100 μmol mol−1 CO2, respectively. In addition, the proportion of refixed CO2 with respect to total decarboxylated CO2 is estimated to be close to 20% (Gerbaud & Andre, 1987; Tcherkez et al., 2005), thereby leading to a rather small isotopic dilution of fixed 13CO2 between 0.6 and 2%.
Other isotopic studies have also suggested that the production of respiratory CO2 in the light at the leaf (Parnik et al., 2002) or mesocosm (Schnyder et al., 2003) level relies on stored carbon up to a contribution of c. 50%. Accordingly, Huege et al. (2007) have shown that shifting from a 13CO2 atmosphere to a 12CO2 atmosphere in the light for 2 h is associated with virtually no decrease in the 13C content in Glu and succinate, thereby indicating that the role of stored carbon is critical. Furthermore, Mal and fumarate exhibited 13C half-times of more than 15 and 24 h, respectively, suggesting a large buffering pool of organic acids.
Our study shows that Asp and Ala are 13C depleted after a 6 h exposure to ordinary CO2 (following 6 h 13CO2 and 30 min in the dark), in clear contrast with Mal and Glu (Fig. 3). In addition, we obtained a rather low 13C enrichment in Asp after 6 h with 13CO2 in the light (Fig. 4, first bar), thereby showing that the production of Asp remains low in the light. It is therefore plausible that, in addition to the natural metabolic turnover (production from current photosynthetically fixed carbon), Asp and Ala are consumed and contribute to feeding oxaloacetate (Mal), pyruvate and Glu production in the light, respectively. It has already been noted that Asp levels are higher in Arabidopsis leaves during the night (Lam et al., 1995), such that, during the light period, it is believed to be either exported to sink tissues or used for Glu production from 2-oxoglutarate via the Asp to Glu transamination equilibrium. This view would also agree with the natural isotopic abundance in 15N: as the Asp to Glu transamination fractionates against 15N by 1.7‰ (Macko et al., 1986), a naturally 15N-enriched Asp leaf pool is expected, and this is indeed the case (Tcherkez & Hodges, 2008). Thus, a part of Glu in the light plausibly originates from Asp transamination, with Asp also synthesized in the light but at a lower rate than its consumption rate (‘dynamic equilibrium’).The contribution of stored carbon can explain why 15N and 13C labelling are dissimilar (Fig. 5). Indeed, although virtually no 15N-Glu or 15N-Gln molecules were also 13C labelled, up to 40% of 13C-Gln molecules were also 15N labelled. Similarly, when the relative isotopic 13C and 15N commitments in Glu were plotted against each other, there was a visible 15N enrichment at zero 13C enrichment (Fig. 2). In other words, within the first hour of labelling, Gln and Glu were hardly 13C labelled, but appeared to be 15N labelled by nearly 5%, and. accordingly, the 15N-NMR signal was rapidly detected under both ordinary and high photorespiratory conditions (Figs S1 and S2, see Supporting Information). This strongly suggests that Glu and Gln synthesis mainly involves carbon remobilization, but captures recently assimilated nitrogen. On the other hand, a substantial amount of newly synthesized, 13C-enriched 2-oxoglutarate is committed to nitrogen assimilation into Glu and Gln. Consistently, when leaves were transferred from a 13CO2 to a 12CO2 (natural 13C abundance) atmosphere, the 13C enrichment in citrate, Glu and Gln increased, undoubtedly showing the contribution of 13C intermediates stored during the night (Fig. 4).
Our scenario is also in agreement with the metabolic results obtained so far with mutants. First, tomato lines (Lycopersicon esculentum) with impaired fumarase activity showed no alterations in leaf Glu, pyro-Glu, succinate and glutarate contents in the light, whereas Mal and isocitrate tended to be larger (Nunes-Nesi et al., 2007). This indicates that fumarase is not essential for the regeneration of 2-oxoglutarate through the TCA cycle, and other carbon sources feed Glu production, such as stored organic acids and/or the anaplerotic PEPc activity, or the remaining fumarase activity is sufficient not to alter the carbon flow to nitrogen assimilation. Second, tomato lines with a reduced aconitase activity exhibited unchanged Glu and Gln levels when compared with wild-type plants in the light (but were much lower in darkness), whereas aspartate, succinate and fumarate contents were significantly lower and (iso)citrate accumulated (Carrari et al., 2003). Clearly then, the production of 2-oxoglutarate and Glu is independent of the variations in other TCA intermediates, indicating that the recycling of stored carbon skeletons to Glu occurs. Third, potato lines (Solanum tuberosum) with increased PEPc activity (2.7- to 4.7-fold) showed a larger Glu to Gln ratio, as well as pyruvate and 2-oxoglutarate contents (Rademacher et al., 2002), suggesting that the anaplerotic input by PEPc is essential for providing 2-oxoglutarate and Glu. In addition, in tobacco lines (Nicotiana sylvestris) in which the nitrate reductase activity is increased, the Gln to Glu ratio has been found to be exacerbated, whereas the Glu level is unaffected, so that it is probable that the production of 2-oxoglutarate is not limiting (Gojon et al., 1998).
Taken as a whole, such a lack of correlation between TCA intermediates and Glu levels points to a significant role for night-stored carbon (such as organic acids) in nitrogen assimilation and Glu synthesis in the light. This view is fully consistent with fluxomics calculations made in X. strumarium leaves, in which citrate synthase activity appears to be limiting, so that Glu synthesis in the light is associated with citrate remobilization (Tcherkez et al., 2009). In fact, the amount of organic acids, such as citrate, is likely to be sufficient to sustain Glu synthesis in the light. According to the fluxes found by Tcherkez et al. (2005), the rate associated with day respiratory CO2 evolution is c. 0.5 μmol m−2 s−1, whereas that associated with TCA activity is c. 0.05 μmol m−2 s−1. Therefore, Glu production is within the 0.05–0.5 μmol m−2 s−1 range (a value of 0.35 μmol m−2 s−1 was estimated here, see above). This would represent a carbon skeleton requirement of c. 0.35 × 6 × 3600 = 7.5 mmol m−2 during a 6 h illumination period. Under our experimental conditions, the Mal content was close to 7 mmol m−2 after 6 h illumination (data not shown), which is adequate to complete a light period of 12 h. Although we recognize that the Mal content may vary during the night (Gerhardt & Heldt, 1984), it thus seems that the sum of Mal and citrate contents available at the very beginning of the light period is sufficient to provide nearly all the 2-oxoglutarate molecules required for Glu production.
The overall commitment of the 13C label into Glu was c. 25–30 mmol per mole of net assimilated CO2, regardless of the photorespiratory conditions (Fig. 2), that is, on the order of 3%. Previous 14C-labelling experiments gave similar results in Arabidopsis thaliana and spinach (Spinacia oleracea), with values of c. 6 and 3%, respectively (Lawyer et al., 1981; Carrari et al., 2003). Photorespiration had a stimulating effect on such a 13C commitment, that is, on 13C-Glu neosynthesis (Figs 2,3). Accordingly, labelling experiments with 14CO2 and 13C-pyruvate have shown that Glu synthesis is promoted under photorespiratory conditions (Lawyer et al., 1981; Tcherkez et al., 2008): Glu and/or Gln represented a larger amount of isotopic label and a higher isotopic specific activity after labelling in ordinary conditions relative to nonphotorespiratory conditions. Still, the contribution of stored carbon to Glu synthesis was apparent (i.e. weakly sensitive to photorespiration), with the clear production of 13C-Glu when the experimental conditions turned to 12CO2 conditions, regardless of the CO2 mole fraction (Fig. 4).
Rationale and perspectives
Interactions between current carbon assimilation, day respiration and photorespiration are quite complex because photorespiration, PEPc activity and the TCA cycle are associated with amino acid metabolism (Gly, Ser, Asp, Glu, Gln). Although Glu and 2-oxoglutarate are involved in a cycle (the photorespiratory Glu recovery cycle), and may thus be assumed to have stationary levels, the shift from steady ordinary (380 μmol mol−1 CO2; 21% O2) to large values of both the oxygenation to carboxylation ratio (vo/vc) and nitrogen assimilation into Glu require net 2-oxoglutarate synthesis. Our results indicate that the remobilization of night-stored molecules, such as organic acids (e.g. citrate, see Scheible et al., 2000 and Niedziela et al., 1993) or amino acids (e.g. Asp and Ala), plays a major role in feeding 2-oxoglutarate synthesis for nitrogen assimilation. Indeed, our results show that the natural day : night cycle is critical for nitrogen assimilation, as intermediates produced in the dark are required during the subsequent light period for nitrogen reduction and assimilation in leaves (Canvin & Atkins, 1974; Pilgrim et al., 1993; Scheible et al., 2000; Lillo et al., 2001).
Within such a framework, the well-recognized relationship between nitrogen assimilation and maintenance respiration in darkness (for a review, see Thornley & Cannell, 2000) comes as no surprise. Similarly, although the nitrogen content in leaves may vary strongly between species (up to two-fold), leaf night respiration per nitrogen content unit remains constant (at c. 10 nmol CO2 nmol−1 N s−1), again suggesting that nitrogen assimilation correlates with night respiratory metabolism (Poorter et al., 1990).
We nevertheless recognize that such a metabolic compromise along the day : night cycle may depend on environmental conditions, such as temperature or light, that influence respiration, which may, in turn, change nitrate reduction and assimilation rates. For example, during growth under a CO2-enriched atmosphere and nitrogen-limited conditions, there is a marked decrease in nitrate reductase activity and amino acid concentration (Geiger et al., 1999), as well as plant nitrogen concentration (percentage of nitrogen), with both maintenance and basal dark-adapted respiration decreasing accordingly (Gifford & Bayer, 1995). That said, the whole-plant respiratory cost R : P is weakly, if at all, affected by CO2 conditions (Gifford & Bayer, 1995; Albrizio & Steduto, 2003), indicating that the control on the commitment of assimilates to respiration is critical for nitrogen assimilation. Furthermore, some evidence has been provided that nitrogen uptake and assimilation in roots are dependent on the input of carbon assimilates that are, in turn, used as respiratory substrates (Gojon et al., 1991). Therefore, the control of nitrogen assimilation by respiration at the whole-plant scale is further complicated by allocation patterns and shoot : root ratios, and, consequently, there remains much uncertainty as to whether our results are still valid on a long-term basis when environmental conditions vary; this will be addressed in a subsequent study.