Guillaume Tcherkez

Why are non-photosynthetic tissues generally 13C enriched compared with leaves in C3 plants? Review and synthesis of current hypotheses

Non-photosynthetic, or heterotrophic, tissues in C3 plants tend to be enriched in 13C compared with the leaves that supply them with photosynthate. This isotopic pattern has been observed for woody stems, roots, seeds and fruits, emerging leaves, and parasitic plants incapable of net CO2 fixation. Unlike in C3 plants, roots of herbaceous C4 plants are generally not 13C-enriched compared with leaves. We review six hypotheses aimed at explaining this isotopic pattern in C3 plants: (1) variation in biochemical composition of heterotrophic tissues compared with leaves; (2) seasonal separation of growth of leaves and heterotrophic tissues, with corresponding variation in photosynthetic discrimination against 13C; (3) differential use of day v. night sucrose between leaves and sink tissues, with day sucrose being relatively 13C-depleted and night sucrose 13C-enriched; (4) isotopic fractionation during dark respiration; (5) carbon fixation by PEP carboxylase; and (6) developmental variation in photosynthetic discrimination against 13C during leaf expansion. Although hypotheses (1) and (2) may contribute to the general pattern, they cannot explain all observations. Some evidence exists in support of hypotheses (3) through to (6), although for hypothesis (6) it is largely circumstantial. Hypothesis (3) provides a promising avenue for future research. Direct tests of these hypotheses should be carried out to provide insight into the mechanisms causing within-plant variation in carbon isotope composition.

Metabolic Origin of Carbon Isotope Composition of Leaf Dark-Respired CO2 in French Bean

The carbon isotope composition (δ13C) of CO2 produced in darkness by intact French bean (Phaseolus vulgaris) leaves was investigated for different leaf temperatures and during dark periods of increasing length. The δ13C of CO2 linearly decreased when temperature increased, from −19‰ at 10°C to −24‰ at 35°C. It also progressively decreased from −21‰ to −30‰ when leaves were maintained in continuous darkness for several days. Under normal conditions (temperature not exceeding 30°C and normal dark period), the evolved CO2 was enriched in 13C compared with carbohydrates, the most 13C-enriched metabolites. However, at the end of a long dark period (carbohydrate starvation), CO2 was depleted in 13C even when compared with the composition of total organic matter. In the two types of experiment, the variations of δ13C were linearly related to those of the respiratory quotient. This strongly suggests that the variation of δ13C is the direct consequence of a substrate switch that may occur to feed respiration; carbohydrate oxidation producing 13C-enriched CO2 and β-oxidation of fatty acids producing 13C-depleted CO2 when compared with total organic matter (−27.5‰). These results are consistent with the assumption that the δ13C of dark respired CO2 is determined by the relative contributions of the two major decarboxylation processes that occur in darkness: pyruvate dehydrogenase activity and the Krebs cycle.

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.

Theoretical considerations about carbon isotope distribution in glucose of C3 plants

The origin of the non-statistical intramolecular distribution of 13C in glucose of C3 plants is examined, including the role of the aldolisation of triose phosphates as proposed by Gleixner and Schmidt (1997). A modelling approach is taken in order to investigate the relationships between the intramolecular distribution of 13C in hexoses and the reactions of primary carbon metabolism. The model takes into account C-C bond-breaking reactions of the Calvin cycle and leads to a mathematical expression for the isotope ratios in hexoses in the steady state. In order to best fit the experimentally-observed intramolecular distribution, the values given by the model indicate that (i), the transketolase reaction fractionates against 13C by 4-7‰ and (ii), depending on the photorespiration rate used for estimations, the aldolase reaction discriminates in favour of 13C by 6‰ during fructose-1,6-bisphosphate production; an isotope discrimination by 2‰ against 13C is obtained when the photorespiration rate is high. Additionally, the estimated fractionations are sensitive to the flux of starch synthesis. Fructose produced from starch breakdown is suggested to be isotopically heavier than sucrose produced in the light, and so the balance between these two sources affects the average intramolecular distribution of glucose derived from stored carbohydrates. The model is also used to estimate photorespiratory and day respiratory fractionations that appear to both depend only weakly on the rate of ribulose-1,5-bisphosphate oxygenation.

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.

On the metabolic origin of the carbon isotope composition of CO2 evolved from darkened light‐acclimated leaves in Ricinus communis

The (13)C isotopic signature (delta(13)C) of CO(2) respired from plants is widely used to assess carbon fluxes and ecosystem functioning. There is, however, a lack of knowledge of the metabolic basis of the delta(13)C value of respired CO(2). To elucidate the physiological mechanisms driving (12)C/(13)C fractionation during respiration, the delta(13)C of respired CO(2) from dark-acclimated leaves during the night, from darkened leaves during the light period, and from stems and roots of Ricinus communis was analysed. The delta(13)C of potential respiratory substrates, the respiratory quotient and the activities of phosphoenolpyruvatecarboxylase (PEPc) and key respiratory enzymes were also measured. It is shown here that the CO(2) evolved from darkened light-acclimated leaves during the light period is (13)C-enriched, and that this correlates with malate accumulation in the light and rapid malate decarboxylation just after the onset of darkness. Whilst CO(2) evolved from leaves was generally (13)C-enriched (but to a lesser extent during the night), CO(2) evolved from stems and roots was depleted compared with the putative respiratory substrates; the difference was mainly caused by intensive PEPc-catalysed CO(2) refixation in stems and roots. These results provide a physiological explanation for short-term variations of delta(13)C in CO(2), illustrating the effects of variations of metabolic fluxes through different biochemical pathways.

Experimental evidence for diel variations of the carbon isotope composition in leaf, stem and phloem sap organic matter in Ricinus communis

Carbon isotope fractionation in metabolic processes following carboxylation of ribulose-1,5-bisphosphate (RuBP) is not as well described as the discrimination during photosynthetic CO(2) fixation. However, post-carboxylation fractionation can influence the diel variation of delta(13)C of leaf-exported organic matter and can cause inter-organ differences in delta(13)C. To obtain a more mechanistic understanding of post-carboxylation modification of the isotopic signal as governed by physiological and environmental controls, we combined the modelling approach of Tcherkez et al., which describes the isotopic fractionation in primary metabolism with the experimental determination of delta(13)C in leaf and phloem sap and root carbon pools during a full diel course. There was a strong diel variation of leaf water-soluble organic matter and phloem sap sugars with relatively (13)C depleted carbon produced and exported during the day and enriched carbon during the night. The isotopic modelling approach reproduces the experimentally determined day-night differences in delta(13)C of leaf-exported carbon in Ricinus communis. These findings support the idea that patterns of transitory starch accumulation and remobilization govern the diel rhythm of delta(13)C in organic matter exported by leaves. Integrated over the whole 24 h day, leaf-exported carbon was enriched in (13)C as compared with the primary assimilates. This may contribute to the well-known--yet poorly explained--relative (13)C depletion of autotrophic organs compared with other plant parts. We thus emphasize the need to consider post-carboxylation fractionations for studies that use delta(13)C for assessing environmental effects like water availability on ratio of mole fractions of CO(2) inside and outside the leaf (e.g. tree ring studies), or for partitioning of CO(2) fluxes at the ecosystem level.

Respiratory carbon metabolism following illumination in intact French bean leaves using 13C/12C isotope labeling

The origin of the carbon atoms in the CO2 respired by French bean (Phaseolus vulgaris) leaves in the dark has been studied using 13C/12C isotopes as tracers. The stable isotope labeling was achieved through a technical device that uses an open gas-exchange system coupled online to an elemental analyzer and linked to an isotope ratio mass spectrometer. The isotopic analysis of the CO2 respired in the dark after a light period revealed that the CO2 was labeled, but the labeling level decreased progressively as the dark period increased. The pattern of disappearance depended on the amount of carbon fixed during the labeling and indicated that there were several pools of respiratory metabolites with distinct turnover rates. We demonstrate that the carbon recently assimilated during photosynthesis accounts for less than 50% of the carbon in the CO2 lost by dark respiration and that the proportion is not influenced by leaf starvation in darkness before the labeling. Therefore, most of the carbon released by dark respiration after illumination does not come from new photosynthates.

Respiratory carbon fluxes in leaves.

Leaf respiration is a major metabolic process that drives energy production and growth. Earlier works in this field were focused on the measurement of respiration rates in relation to carbohydrate content, photosynthesis, enzymatic activities or nitrogen content. Recently, several studies have shed light on the mechanisms describing the regulation of respiration in the light and in the dark and on associated metabolic flux patterns. This review will highlight advances made into characterizing respiratory fluxes and provide a discussion of metabolic respiration dynamics in relation to important biological functions.