Analysis of metabolic flux using dynamic labeling and metabolic modeling

Metabolic fluxes and the capacity to modulate them are a crucial component of the ability of the plant cell to react to environmental perturbations. Our ability to quantify them and to attain information concerning the regulatory mechanisms which control them is therefore essential to understand and influence metabolic networks. For all but the simplest of flux measurements labelling methods have proven to be the most informative. Both steady-state and dynamic labelling approaches having been adopted in the study of plant metabolism. Here the conceptual basis of these complementary approaches, as well as their historical application in microbial, mammalian and plant sciences are reviewed and an update on technical developments in label distribution analyses is provided. This is supported by illustrative cases studies involving the kinetic modelling of secondary metabolism. One issue that is particularly complex in the analysis of plant fluxes is the extensive compartmentation of the plant cell. This problem is discussed from both theoretical and experimental perspectives and the current approaches used to address it are assessed. Finally, current limitations and future perspectives of kinetic modelling of plant metabolism are discussed

Benthic Foraminiferal Oxygen Isotope Offsets Over The Last Glacial-Interglacial Cycle

The oxygen isotope (?18O) offset between contemporaneous benthic foraminiferal species is often assumed constant with time and geographic location. We present an inventory of benthic foraminiferal species ?18O offsets from the major ocean basins covering the last glacial-interglacial cycle, showing that of the twenty down-core records investigated, twelve show significant temporal changes in ?18O offsets that do not resemble stochastic variability. Some of the temporal changes may be related to kinetic fractionation effects causing deglacial/interglacial enrichment or glacial depletion in mainly infaunal species, but additional research is needed to confirm this. In addition to stratigraphic implications the finding of temporally varying offsets between co-existing benthic foraminiferal species could have implications for sea-level, deep water temperature, and regional deep water ?18O estimates

Carbon allocation and carbon isotope fluxes in the plant-soil-atmosphere continuum: a review

The terrestrial carbon (C) cycle has received increasing interest over the past few decades, however, there is still a lack of understanding of the fate of newly assimilated C allocated within plants and to the soil, stored within ecosystems and lost to the atmosphere. Stable carbon isotope studies can give novel insights into these issues. In this review we provide an overview of an emerging picture of plant-soil-atmosphere C fluxes, as based on C isotope studies, and identify processes determining related C isotope signatures. The first part of the review focuses on isotopic fractionation processes within plants during and after photosynthesis. The second major part elaborates on plant-internal and plant-rhizosphere C allocation patterns at different time scales (diel, seasonal, interannual), including the speed of C transfer and time lags in the coupling of assimilation and respiration, as well as the magnitude and controls of plant-soil C allocation and respiratory fluxes. Plant responses to changing environmental conditions, the functional relationship between the physiological and phenological status of plants and C transfer, and interactions between C, water and nutrient dynamics are discussed. The role of the C counterflow from the rhizosphere to the aboveground parts of the plants, e.g. via CO<sub>2</sub> dissolved in the xylem water or as xylem-transported sugars, is highlighted. The third part is centered around belowground C turnover, focusing especially on above- and belowground litter inputs, soil organic matter formation and turnover, production and loss of dissolved organic C, soil respiration and CO<sub>2</sub> fixation by soil microbes. Furthermore, plant controls on microbial communities and activity via exudates and litter production as well as microbial community effects on C mineralization are reviewed. A further part of the paper is dedicated to physical interactions between soil CO<sub>2</sub> and the soil matrix, such as CO<sub>2</sub> diffusion and dissolution processes within the soil profile. Finally, we highlight state-of-the-art stable isotope methodologies and their latest developments. From the presented evidence we conclude that there exists a tight coupling of physical, chemical and biological processes involved in C cycling and C isotope fluxes in the plant-soil-atmosphere system. Generally, research using information from C isotopes allows an integrated view of the different processes involved. However, complex interactions among the range of processes complicate or currently impede the interpretation of isotopic signals in CO<sub>2</sub> or organic compounds at the plant and ecosystem level. This review tries to identify present knowledge gaps in correctly interpreting carbon stable isotope signals in the plant-soil-atmosphere system and how future research approaches could contribute to closing these gaps

Listening In on the Past: What Can Otolith δ18O Values Really Tell Us about the Environmental History of Fishes?

Oxygen isotope ratios from fish otoliths are used to discriminate marine stocks and reconstruct past climate, assuming that variations in otolith δ18O values closely reflect differences in temperature history of fish when accounting for salinity induced variability in water δ18O. To investigate this, we exploited the environmental and migratory data gathered from a decade using archival tags to study the behaviour of adult plaice (Pleuronectes platessa L.) in the North Sea. Based on the tag-derived monthly distributions of the fish and corresponding temperature and salinity estimates modelled across three consecutive years, we first predicted annual otolith δ18O values for three geographically discrete offshore sub-stocks, using three alternative plausible scenarios for otolith growth. Comparison of predicted vs. measured annual δ18O values demonstrated >96% correct prediction of sub-stock membership, irrespective of the otolith growth scenario. Pronounced inter-stock differences in δ18O values, notably in summer, provide a robust marker for reconstructing broad-scale plaice distribution in the North Sea. However, although largely congruent, measured and predicted annual δ18O values of did not fully match. Small, but consistent, offsets were also observed between individual high-resolution otolith δ18O values measured during tag recording time and corresponding δ18O predictions using concomitant tag-recorded temperatures and location-specific salinity estimates. The nature of the shifts differed among sub-stocks, suggesting specific vital effects linked to variation in physiological response to temperature. Therefore, although otolith δ18O in free-ranging fish largely reflects environmental temperature and salinity, we counsel prudence when interpreting otolith δ18O data for stock discrimination or temperature reconstruction until the mechanisms underpinning otolith δ18O signature acquisition, and associated variation, are clarified

On-line high-precision carbon position-specific stable isotope analysis: a review

Since the first commercial availability of gas chromatographs coupled with a combustion furnace and an isotope ratio mass spectrometry in 1990, compound-specific stable isotope analysis of organic molecules has been at the origin of scientific breakthroughs in a wide range of research fields. The presence of non-reacting atoms, however, can mask changes in molecular stable isotopic signatures; position-specific isotope analysis (PSIA) is the study of intramolecular isotopic variations. After a brief consideration of the potential and prospect of this new level in isotopic studies, in this study, we review few existing custom-built systems for on-line PSIA using continuous flow isotope ratio mass spectrometry instrumentation and how researchers have addressed issues related to this technique. Molecular fragmentation processes observed and optimised for various molecules are also discussed for on-line PSIA, as it would provide information on their application to new compounds

Temperature influence on the carbon isotopic composition of Orbulina universa and Globigerina bulloides (planktonic foraminifera)

Laboratory experiments with the planktonic foraminifera Orbulina universa (symbiotic) and Globigerina bulloides (nonsymbiotic) were used to examine the effects of temperature, irradiance (symbiont photosynthesis), [CO32-], [HPO42-], and ontogeny on shell d13C values. In ambient seawater ([CO32-] = 171 mmol kg-1), the d13C of O. universa shells grown under low light (LL) levels is insensitive to temperature and records the d13C value of seawater TCO2. In contrast, the d13C of high light (HL) shells increases ~0.4‰ across 15-25°C (+0.050‰/°C). This suggests that the d13C enrichment due to symbiont photosynthetic activity is temperature-dependent. A comparison of HL O. universa grown in elevated [CO32-] seawater with ambient specimens shows that temperature does not affect the slope of the d13C/[CO32-] relationship previously described [Spero et al., 1997]. The d13C of G. bulloides shells decreases across the 15-24°C temperature range and d13C:temperature slopes decrease with increasing shell size (-0.13, -0.10, and -0.09‰/°C in 11- 12-, and 13-chambered shells, respectively). The pattern of lower d13C values at higher temperatures likely results from the incorporation of more respired CO2 into the shell at higher metabolic rates. The d13C of HL O. universa increases with increased seawater [HPO42-]

Nitrogen isotopic fractionation during abiotic synthesis of organic solid particles

The formation of organic compounds is generally assumed to result from abiotic processes in the Solar System, with the exception of biogenic organics on Earth. Nitrogen-bearing organics are of particular interest, notably for prebiotic perspectives but also for overall comprehension of organic formation in the young solar system and in planetary atmospheres. We have investigated abiotic synthesis of organics upon plasma discharge, with special attention to N isotope fractionation. Organic aerosols were synthesized from N2-CH4 and N2-CO gaseous mixtures using low-pressure plasma discharge experiments, aimed at simulating chemistry occurring in Titan s atmosphere and in the protosolar nebula, respectively. Nitrogen is efficiently incorporated into the synthesized solids, independently of the oxidation degree, of the N2 content of the starting gas mixture, and of the nitrogen speciation in the aerosols. The aerosols are depleted in 15N by 15-25 permil relative to the initial N2 gas, whatever the experimental setup is. Such an isotopic fractionation is attributed to mass-dependent kinetic effect(s). Nitrogen isotope fractionation upon electric discharge cannot account for the large N isotope variations observed among solar system objects and reservoirs. Extreme N isotope signatures in the solar system are more likely the result of self-shielding during N2 photodissociation, exotic effect during photodissociation of N2 and/or low temperature ion-molecule isotope exchange. Kinetic N isotope fractionation may play a significant role in the Titan s atmosphere. We also suggest that the low delta15N values of Archaean organic matter are partly the result of abiotic synthesis of organics that occurred at that time

Evolution of <i>E. coli</i> on [U^-13C] Glucose Reveals a Negligible Isotopic Influence on Metabolism and Physiology

13C-Metabolic flux analysis (13C-MFA) traditionally assumes that kinetic isotope effects from isotopically labeled compounds do not appreciably alter cellular growth or metabolism, despite indications that some biochemical reactions can be non-negligibly impacted. Here, populations of Escherichia coli were adaptively evolved for ~1000 generations on uniformly labeled 13C-glucose, a commonly used isotope for 13C-MFA. Phenotypic characterization of these evolved strains revealed ~40% increases in growth rate, with no significant difference in fitness when grown on either labeled (13C) or unlabeled (12C) glucose. The evolved strains displayed decreased biomass yields, increased glucose and oxygen uptake, and increased acetate production, mimicking what is observed after adaptive evolution on unlabeled glucose. Furthermore, full genome re-sequencing revealed that the key genetic changes underlying these phenotypic alterations were essentially the same as those acquired during adaptive evolution on unlabeled glucose. Additionally, glucose competition experiments demonstrated that the wild-type exhibits no isotopic preference for unlabeled glucose, and the evolved strains have no preference for labeled glucose. Overall, the results of this study indicate that there are no significant differences between 12C and 13C-glucose as a carbon source for E. coli growth