Respiratory metabolism of illuminated leaves depends on CO2 and O2 conditions.

Day respiration is the process by which nonphotorespiratory CO2 is produced by illuminated leaves. The biological function of day respiratory metabolism is a major conundrum of plant photosynthesis research: because the rate of CO2 evolution is partly inhibited in the light, it is viewed as either detrimental to plant carbon balance or necessary for photosynthesis operation (e.g., in providing cytoplasmic ATP for sucrose synthesis). Systematic variations in the rate of day respiration under contrasting environmental conditions have been used to elucidate the metabolic rationale of respiration in the light. Using isotopic techniques, we show that both glycolysis and the tricarboxylic acid cycle activities are inversely related to the ambient CO2/O2 ratio: day respiratory metabolism is enhanced under high photorespiratory (low CO2) conditions. Such a relationship also correlates with the dihydroxyacetone phosphate/Glc-6-P ratio, suggesting that photosynthetic products exert a control on day respiration. Thus, day respiration is normally inhibited by phosphoryl (ATP/ADP) and reductive (NADH/NAD) poise but is up-regulated by photorespiration. Such an effect may be related to the need for NH2 transfers during the recovery of photorespiratory cycle intermediates.

In folio isotopic tracing demonstrates that nitrogen assimilation into glutamate is mostly independent from current CO2 assimilation in illuminated leaves of Brassica napus.

*Nitrogen assimilation in leaves requires primary NH(2) acceptors that, in turn, originate from primary carbon metabolism. Respiratory metabolism is believed to provide such acceptors (such as 2-oxoglutarate), so that day respiration is commonly seen as a cornerstone for nitrogen assimilation into glutamate in illuminated leaves. However, both glycolysis and day respiratory CO(2) evolution are known to be inhibited by light, thereby compromising the input of recent photosynthetic carbon for glutamate production. *In this study, we carried out isotopic labelling experiments with (13)CO(2) and (15)N-ammonium nitrate on detached leaves of rapeseed (Brassica napus), and performed (13)C- and (15)N-nuclear magnetic resonance analyses. *Our results indicated that the production of (13)C-glutamate and (13)C-glutamine under a (13)CO(2) atmosphere was very weak, whereas (13)C-glutamate and (13)C-glutamine appeared in both the subsequent dark period and the next light period under a (12)CO(2) atmosphere. Consistently, the analysis of heteronuclear ((13)C-(15)N) interactions within molecules indicated that most (15)N-glutamate and (15)N-glutamine molecules were not (13)C labelled after (13)C/(15)N double labelling. That is, recent carbon atoms (i.e. (13)C) were hardly incorporated into glutamate, but new glutamate molecules were synthesized, as evidenced by (15)N incorporation. *We conclude that the remobilization of night-stored molecules plays a significant role in providing 2-oxoglutarate for glutamate synthesis in illuminated rapeseed leaves, and therefore the natural day : night cycle seems critical for nitrogen assimilation.

Metabolic origin of the delta13C of respired CO2 in roots of Phaseolus vulgaris.

Root respiration is a major contributor to soil CO2 efflux, and thus an important component of ecosystem respiration. But its metabolic origin, in relation to the carbon isotope composition (delta13C), remains poorly understood. Here, 13C analysis was conducted on CO2 and metabolites under typical conditions or under continuous darkness in French bean (Phaseolus vulgaris) roots. 13C contents were measured either under natural abundance or following pulse-chase labeling with 13C-enriched glucose or pyruvate, using isotope ratio mass spectrometer (IRMS) and nuclear magnetic resonance (NMR) techniques. In contrast to leaves, no relationship was found between the respiratory quotient and the delta13C of respired CO2, which stayed constant at a low value (c. -27.5 per thousand) under continuous darkness. With labeling experiments, it is shown that such a pattern is explained by the 13C-depleting effect of the pentose phosphate pathway; and the involvement of the Krebs cycle fueled by either the glycolytic input or the lipid/protein recycling. The anaplerotic phosphoenolpyruvate carboxylase (PEPc) activity sustained glutamic acid (Glu) synthesis, with no net effect on respired CO2. These results indicate that the root delta13C signal does not depend on the availability of root respiratory substrates and it is thus plausible that, unless the 13C photosynthetic fractionation varies at the leaf level, the root delta13C signal hardly changes under a range of natural environmental conditions.

On the resilience of nitrogen assimilation by intact roots under starvation, as revealed by isotopic and metabolomic techniques.

The response of root metabolism to variations in carbon source availability is critical for whole-plant nitrogen (N) assimilation and growth. However, the effect of changes in the carbohydrate input to intact roots is currently not well understood and, for example, both smaller and larger values of root:shoot ratios or root N uptake have been observed so far under elevated CO(2). In addition, previous studies on sugar starvation mainly focused on senescent or excised organs while an increasing body of data suggests that intact roots may behave differently with, for example, little protein remobilization. Here, we investigated the carbon and nitrogen primary metabolism in intact roots of French bean (Phaseolus vulgaris L.) plants maintained under continuous darkness for 4 days. We combined natural isotopic (15)N/(14)N measurements, metabolomic and (13)C-labeling data and show that intact roots continued nitrate assimilation to glutamate for at least 3 days while the respiration rate decreased. The activity of the tricarboxylic acid cycle diminished so that glutamate synthesis was sustained by the anaplerotic phosphoenolpyruvate carboxylase fixation. Presumably, the pentose phosphate pathway contributed to provide reducing power for nitrate reduction. All the biosynthetic metabolic fluxes were nevertheless down-regulated and, consequently, the concentration of all amino acids decreased. This is the case of asparagine, strongly suggesting that, as opposed to excised root tips, protein remobilization in intact roots remained very low for 3 days in spite of the restriction of respiratory substrates.

In folio respiratory fluxomics revealed by 13C isotopic labeling and H/D isotope effects highlight the noncyclic nature of the tricarboxylic acid "cycle" in illuminated leaves.

While the possible importance of the tricarboxylic acid (TCA) cycle reactions for leaf photosynthesis operation has been recognized, many uncertainties remain on whether TCA cycle biochemistry is similar in the light compared with the dark. It is widely accepted that leaf day respiration and the metabolic commitment to TCA decarboxylation are down-regulated in illuminated leaves. However, the metabolic basis (i.e. the limiting steps involved in such a down-regulation) is not well known. Here, we investigated the in vivo metabolic fluxes of individual reactions of the TCA cycle by developing two isotopic methods, (13)C tracing and fluxomics and the use of H/D isotope effects, with Xanthium strumarium leaves. We provide evidence that the TCA "cycle" does not work in the forward direction like a proper cycle but, rather, operates in both the reverse and forward directions to produce fumarate and glutamate, respectively. Such a functional division of the cycle plausibly reflects the compromise between two contrasted forces: (1) the feedback inhibition by NADH and ATP on TCA enzymes in the light, and (2) the need to provide pH-buffering organic acids and carbon skeletons for nitrate absorption and assimilation.

In vivo respiratory metabolism of illuminated leaves.

Day respiration of illuminated C(3) leaves is not well understood and particularly, the metabolic origin of the day respiratory CO(2) production is poorly known. This issue was addressed in leaves of French bean (Phaseolus vulgaris) using (12)C/(13)C stable isotope techniques on illuminated leaves fed with (13)C-enriched glucose or pyruvate. The (13)CO(2) production in light was measured using the deviation of the photosynthetic carbon isotope discrimination induced by the decarboxylation of the (13)C-enriched compounds. Using different positional (13)C-enrichments, it is shown that the Krebs cycle is reduced by 95% in the light and that the pyruvate dehydrogenase reaction is much less reduced, by 27% or less. Glucose molecules are scarcely metabolized to liberate CO(2) in the light, simply suggesting that they can rarely enter glycolysis. Nuclear magnetic resonance analysis confirmed this view; when leaves are fed with (13)C-glucose, leaf sucrose and glucose represent nearly 90% of the leaf (13)C content, demonstrating that glucose is mainly directed to sucrose synthesis. Taken together, these data indicate that several metabolic down-regulations (glycolysis, Krebs cycle) accompany the light/dark transition and emphasize the decrease of the Krebs cycle decarboxylations as a metabolic basis of the light-dependent inhibition of mitochondrial respiration.