Elizabeth Gout

In vivo respiratory metabolism of illuminated leaves

Day respiration of illuminated C3 leaves is not well understood and particularly, the metabolic origin of the day respiratory CO2 production is poorly known. This issue was addressed in leaves of French bean (Phaseolus vulgaris) using 12C/13C stable isotope techniques on illuminated leaves fed with 13C-enriched glucose or pyruvate. The 13CO2 production in light was measured using the deviation of the photosynthetic carbon isotope discrimination induced by the decarboxylation of the 13C-enriched compounds. Using different positional 13C-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 CO2 in the light, simply suggesting that they can rarely enter glycolysis. Nuclear magnetic resonance analysis confirmed this view; when leaves are fed with 13C-glucose, leaf sucrose and glucose represent nearly 90% of the leaf 13C 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.

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 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, 13C 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 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.

Changes in Kennedy pathway intermediates associated with increased triacylglycerol synthesis in oil-seed rape

The rates of triacylglycerol synthesis in maturing oil-seed rape (Brassica napus cv Shiralee) were manipulated by light/dark treatments. Under conditions of high lipid accumulation the amounts of the Kennedy pathway intermediates, phosphatidate and particularly, diacylglycerol were increased significantly. At the same time there were no significant changes in the activities of the four pathway enzymes, of which diacylglycerol acyltransferase had the lowest detectable activity. The alteration in carbon flux was accompanied by some changes in the acyl quantity of the diacylglycerol pool but not in that of other intermediates. The results provide additional evidence for our proposal that diacylglycerol acyltransferase can exert significant flux control at times of high lipid accumulation in oil-seed rape.

Metabolic origin of the δ13C 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.

Effect of glyphosate on plant cell metabolism. 31P and 13C NMR studies

The effect of glyphosate (N-phosphonomethyl glycine; the active ingredient of Roundup herbicide) on plant cells metabolism was analysed by 31P and 13C NMR using suspension-cultured sycamore (Acer pseudoplatanus L) cells. Cells were compressed in the NMR tube and perfused with an original arrangement enabling a tight control of the circulating nutrient medium. Addition of 1 mM glyphosate to the nutrient medium triggered the accumulation of shikimate (20-30 mumol g-1 cell wet weight within 50 h) and shikimate 3-phosphate (1-1.5 mumol g-1 cell wet weight within 50 h). From in vivo spectra it was demonstrated that these two compounds were accumulated in the cytoplasm where their concentrations reached potentially lethal levels. On the other hand, glyphosate present in the cytoplasmic compartment was extensively metabolized to yield aminomethylphosphonic acid which also accumulated in the cytoplasm. Finally, the results presented in this paper indicate that although the cell growth was stopped by glyphosate the cell respiration rates and the level of energy metabolism intermediates remained unchanged.

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.

Formation of Autophagic Vacuoles and Accumulation of Deacylation Products of Membrane Polar Lipids During the Course of Sucrose Starvation in Higher Plant Cells

Transfer of sycamore (Acer pseudoplatanus L.) cells into flasks containing sucrose-free culture medium triggered the following cascade of reactions: a) initially cellular carbohydrate reserves (vacuolar sucrose and starch present in plastids) were consumed; b) when almost all the intracellular carbohydrate pools had disappeared the cell fatty acids deriving from membrane polar lipids such as phosphatidylcholine were utilized as oxidizable substrates for ATP production. Using electron microscopy we demonstrated the presence of a very active autophagic activity during sucrose starvation in sycamore cells. Autophagic vacuoles (double membrane-bounded vesicles) were formed in the peripheral cytoplasm and were expelled into the large central vacuole as single membrane-bounded vesicles. Our results shed new light on the regression of the mitochondrial system previously observed during the course of sucrose deprivation in plant cells [1]: following 5 days of sucrose starvation the total mitochondrial volume within the cell cytoplasm decreased to less than 15% of that of the normal cells. The degradation of intracellular membrane polar lipids by autophagy led to a transient accumulation of phosphodiesters and to a steady accumulation of phosphocholine in the cell cytoplasm which is not further metabolized. The total amount of phosphocholine present within the cell should reflect the extent of intracellular membrane degradation during the course of sucrose starvation. Finally during the course of leaf senescence phosphocholine molecules, derived from phosphatidylcholine degradation, may escape out of the cell and be exported toward the xylem vessel where they accumulate. Under these conditions, phosphocholine would be transported in the xylem fluids to meristematic cells or fast growing tissues where it could be used as a source of choline for membrane synthesis. We have demonstrated that phosphocholine was rapidly hydrolyzed outside the cell and that choline and Pi entered the cytosolic compartment where choline kinase re-forms phosphocholine.