Roland A. Werner

Correlations between the 13C Content of Primary and Secondary Plant Products in Different Cell Compartments and That in Decomposing Basidiomycetes

Relative carbon isotope ratio ([delta]13C values) of primary and secondary products from different compartments of annual plants, pine needles, wood, and decomposing Basidiomycetes have been determined. An enrichment in 13C was found for storage tissues of annual plants, because of the high level of the primary storage products sucrose and starch; however, the enrichment was even greater in leaf starch. All of these compounds had the same relative 13C enrichment in positions 3 and 4 of glucose. Secondary products in conifer needles (lignin, lipids) were depleted in 13C by 1 to 2 [per mille (thousand) sign] relative to carbohydrates from the same origin. Air pollution caused a small decrease in [delta]13C values; however, the relative content of plant products, especially of the soluble polar compounds, was also affected. Decomposing fungi showed a global accumulation of 13C by 4[per mille (thousand) sign] relative to their substrates in wood. Their chitin was enriched by 2[per mille (thousand) sign] relative to the cellulose of the wood. Hence, Basidiomycetes preferentially metabolize “light” molecules, whereas “heavy” molecules are preferentially polymerized. Our results are discussed on the basis of a kinetic isotope effect on the fructose-1,6-bisphosphate aldolase reaction and of metabolic branching on the level of the triose phosphates with varying substrate fluxes.

On-line δ18O measurement of organic and inorganic substances

A method for the automated sample conversion and on-line oxygen isotope ratio (delta(18)O) determination for organic and inorganic substances is presented. The samples are pyrolytically decomposed at 1400 degrees C in the presence of nickelized graphite. With the system presented organic as well as inorganic samples such as nitrates, sulphates and phosphates of 50-100 mg O can be analyzed for their delta(18)O values with a standard deviation usually better than 0.5 per thousand. Additionally, carbon isotope ratios of organic substances and nitrogen isotope ratios of inorganic nitrogenous compounds are available in the same sample run. Data for international and some inter-laboratory reference materials are presented to show the accuracy and reliability of the method. The effect of some additives on the CO yield was checked for substances which do not pyrolyze completely. Copyright 1999 John Wiley & Sons, Ltd.

Systematics of 2H patterns in natural compounds and its importance for the elucidation of biosynthetic pathways

The relative global 2H-content of natural plant products is correlated to that of the primary hydrogen source, i.e. water, to the site of their biosynthesis (C3-, C4- and CAM-plants; chloroplasts, cytosol), and to their biosynthetic pathways. A relative global 2H-content sequence can be established in the order phenylpropanoids > carbohydrates > bulk material > hydrolysable lipids > steroids. A detailed analysis of the 2H-patterns of the main groups of secondary compounds reveals regularities, in that they are correlated to the primary precursors and to the origin of hydrogen from four main pools with the mean δ2H-values [‰]V−SMOW: leaf H2O ∼+30; carbohydrates ∼−70; NADPH ∼−250; flavoproteins ∼−350. Aside from the 2H-discrimination between these pools, kinetic isotope effects on defined reactions only become effective in connection with metabolic branching events. So, the 2H-pattern of natural aromatic compounds can be correlated to the 2H-pattern of the precursor carbohydrates and a reduction step in the course of the shikimic acid pathway, furthermore to the implication of the NIH-shift. The pattern of aromatic compounds from the polyketide is different from that of the shikimate pathway. The alternating 2H-abundance of fatty acid chains is caused by the origin of their hydrogen atoms from carbohydrates and from NADPH, directly or via a flavoprotein, respectively. This is similar for isoprenoids, and the natural 2H-patterns permit their assignment to the mevalonate or non-mevalonate biosynthetic pathway. Generally, the correlations and regularities of the 2H-patterns of organic compounds found are a new reliable tool for the elucidation of biosynthetic pathways and origin assignments.

Temporal dynamics of the carbon isotope composition in a Pinus sylvestris stand: from newly assimilated organic carbon to respired carbon dioxide

The 13C isotopic signature (C stable isotope ratio; δ13C) of CO2 respired from forest ecosystems and their particular compartments are known to be influenced by temporal changes in environmental conditions affecting C isotope fractionation during photosynthesis. Whereas most studies have assessed temporal variation in δ13C of ecosystem-respired CO2 on a day-to-day scale, not much information is available on its diel dynamics. We investigated environmental and physiological controls over potential temporal changes in δ13C of respired CO2 by following the short-term dynamics of the 13C signature from newly assimilated organic matter pools in the needles, via phloem-transported organic matter in twigs and trunks, to trunk-, soil- and ecosystem-respired CO2. We found a strong 24-h periodicity in δ13C of organic matter in leaf and twig phloem sap, which was strongly dampened as carbohydrates were transported down the trunk. Periodicity reappeared in the δ13C of trunk-respired CO2, which seemed to originate from apparent respiratory fractionation rather than from changes in δ13C of the organic substrate. The diel patterns of δ13C in soil-respired CO2 are partly explained by soil temperature and moisture and are probably due to changes in the relative contribution of heterotrophic and autotrophic CO2 fluxes to total soil efflux in response to environmental conditions. Our study shows that direct relations between δ13C of recent assimilates and respired CO2 may not be present on a diel time scale, and other factors lead to short-term variations in δ13C of ecosystem-emitted CO2. On the one hand, these variations complicate ecosystem CO2 flux partitioning, but on the other hand they provide new insights into metabolic processes underlying respiratory CO2 emission.

Comprehensive inter-laboratory calibration of reference materials for delta O-18 versus VSMOW using various on-line high-temperature conversion techniques

Internationally distributed organic and inorganic oxygen isotopic reference materials have been calibrated by six laboratories carrying out more than 5300 measurements using a variety of high-temperature conversion techniques (HTC)a in an evaluation sponsored by the International Union of Pure and Applied Chemistry (IUPAC). To aid in the calibration of these reference materials, which span more than 125 per thousand, an artificially enriched reference water (delta(18)O of +78.91 per thousand) and two barium sulfates (one depleted and one enriched in (18)O) were prepared and calibrated relative to VSMOW2b and SLAP reference waters. These materials were used to calibrate the other isotopic reference materials in this study, which yielded: Reference material delta(18)O and estimated combined uncertainty IAEA-602 benzoic acid+71.28 +/- 0.36 per thousand USGS 35 sodium nitrate+56.81 +/- 0.31 per thousand IAEA-NO-3 potassium nitrate+25.32 +/- 0.29 per thousand IAEA-601 benzoic acid+23.14 +/- 0.19 per thousand IAEA-SO-5 barium sulfate+12.13 +/- 0.33 per thousand NBS 127 barium sulfate+8.59 +/- 0.26 per thousand VSMOW2 water 0 per thousand IAEA-600 caffeine-3.48 +/- 0.53 per thousand IAEA-SO-6 barium sulfate-11.35 +/- 0.31 per thousand USGS 34 potassium nitrate-27.78 +/- 0.37 per thousand SLAP water-55.5 per thousand The seemingly large estimated combined uncertainties arise from differences in instrumentation and methodology and difficulty in accounting for all measurement bias. They are composed of the 3-fold standard errors directly calculated from the measurements and provision for systematic errors discussed in this paper. A primary conclusion of this study is that nitrate samples analyzed for delta(18)O should be analyzed with internationally distributed isotopic nitrates, and likewise for sulfates and organics. Authors reporting relative differences of oxygen-isotope ratios (delta(18)O) of nitrates, sulfates, or organic material should explicitly state in their reports the delta(18)O values of two or more internationally distributed nitrates (USGS 34, IAEA-NO-3, and USGS 35), sulfates (IAEA-SO-5, IAEA-SO-6, and NBS 127), or organic material (IAEA-601 benzoic acid, IAEA-602 benzoic acid, and IAEA-600 caffeine), as appropriate to the material being analyzed, had these reference materials been analyzed with unknowns. This procedure ensures that readers will be able to normalize the delta(18)O values at a later time should it become necessary.The high-temperature reduction technique for analyzing delta(18)O and delta(2)H is not as widely applicable as the well-established combustion technique for carbon and nitrogen stable isotope determination. To obtain the most reliable stable isotope data, materials should be treated in an identical fashion; within the same sequence of analyses, samples should be compared with working reference materials that are as similar in nature and in isotopic composition as feasible.

On-line determination of δ18O values of organic substances

Abstract A new method for the automated sample preparation and on-line isotope ratio determination of δ 18 O values of organic compounds is presented. The principle of the production of CO as measuring gas is a sudden pyrolysis in a helium carrier stream at a temperature of 1080 °C in the absence of surplus carbon. Results obtained with this method for carbohydrates and some aromatic substances show a very satisfactory agreement with δ 18 O values obtained by the Rittenberg-Ponticorvo method and with known δ 18 O values of standards; the standard deviation observed is usually better than 0.8%. The method is in principle also suitable for nitrogen containing compounds, as it implies a gas Chromatographic separation of CO and N 2 . Long-chained aliphatic alcohols, aldehydes, esters, and some nitrogen containing compounds did not give satisfactory results, probably because of an insufficient CO yield.

Preparation of starch and soluble sugars of plant material for the analysis of carbon isotope composition: a comparison of methods

Starch and soluble sugars are the major photosynthetic products, and their carbon isotope signatures reflect external versus internal limitations of CO(2) fixation. There has been recent renewed interest in the isotope composition of carbohydrates, mainly for use in CO(2) flux partitioning studies at the ecosystem level. The major obstacle to the use of carbohydrates in such studies has been the lack of an acknowledged method to isolate starch and soluble sugars for isotopic measurements. We here report on the comparison and evaluation of existing methods (acid and enzymatic hydrolysis for starch; ion-exchange purification and compound-specific analysis for sugars). The selectivity and reproducibility of the methods were tested using three approaches: (i) an artificial leaf composed of a mixture of isotopically defined compounds, (ii) a C(4) leaf spiked with C(3) starch, and (iii) two natural plant samples (root, leaf). Starch preparation methods based on enzymatic or acid hydrolysis did not yield similar results and exhibited contaminations by non-starch compounds. The specificity of the acidic hydrolysis method was especially low, and we therefore suggest terming these preparations as HCl-hydrolysable carbon, rather than starch. Despite being more specific, enzyme-based methods to isolate starch also need to be further optimized to increase specificity. The analysis of sugars by ion-exchange methods (bulk preparations) was fast but produced more variable isotope compositions than compound-specific methods. Compound-specific approaches did not in all cases correctly reproduce the target values, mainly due to unsatisfactory separation of sugars and background contamination. Our study demonstrates that, despite their wide application, methods for the preparation of starch and soluble sugars for the analysis of carbon isotope composition are not (yet) reliable enough to be routinely applied and further research is urgently needed to resolve the identified problems.

Metabolic fluxes, carbon isotope fractionation and respiration – lessons to be learned from plant biochemistry

Post-carboxylation carbon kinetic isotope fractionation (KIF) is in the focus of interest because it alters the isotopic signal imprinted on newly assimilated organic matter by photosynthetic isotope discrimination in downstream metabolic processes (Badeck et al., 2005; Gessler et al., 2009). KIF during respiration and the dynamic change of the isotopic composition (i.e. dC) of respired CO2 is providing insight into the carbon flow through metabolic pathways (Ghashghaie et al., 2003; Pataki, 2005) and thus serves as an important tool to characterise plant physiological adaptations to environmental conditions. The isotopic signature of respired CO2 (d Cres) is determined by the following factors: (1) carbon source and its dC with (2) a heterogeneous intramolecular C-distribution and ‘molecule fragmentation’ (fragmentation fractionation; Tcherkez et al., 2004), (3) a possible C kinetic isotope effect (KIE) of respiratory enzymes in connection with (4) specific metabolic turnover rates (‘commitment values’) of respiratory substrates. The C-enrichment in respired CO2 as often observed in leaves is in general attributed to the release of CO2 from the ‘heavy’ (+ 4.1‰ relative to glucose mean dC-value) C3 and C4 positions of glucose (Rossmann et al., 1991; Gleixner & Schmidt, 1997) by the pyruvate dehydrogenase (PDH) reaction. In addition, the PDH reaction has a C-KIE on the C1 atom of pyruvate (former C3 ⁄ C4 of glucose; Melzer & Schmidt, 1987). Recently, an intensive and interesting discussion in the Forum of New Phytologist (Tcherkez, 2010; Werner, 2010) took place about the interpretation of respiratory isotope signals formed by metabolic fluxes and pathways and how to identify the factors determining dCres. In this correspondence the following main issues were discussed: KIF during respiration (cf. Schmidt, 2003; Tcherkez et al., 2005); and the phenomenon of light enhanced dark respiration (LEDR), both affecting dCres. With LEDR it is assumed that a change in dC is due to transient substrate switches and reorganisation of the tricarboxylic acid (TCA, citric acid or Krebs) cycle (Barbour et al., 2007; Gessler et al., 2009) when light-acclimated leaves experience darkness. This discussion shows the potential of respiratory isotope signals to identify plant metabolic responses, but also the need for a careful interpretation of such data combined with further specific research. Here, we want to draw the attention to the following biochemical facts, which have not been taken into account sufficiently in the general discussion of respiratory isotope fractionation: the impermeability of the inner mitochondrial membrane for acetyl-coenzyme A (acetyl-CoA), which influences the KIF of citrate synthase (CS) during respiration, and the interplay between mitochondrial malate dehydrogenase (mtMDH) and mitochondrial malic enzyme (mtME) during the light–dark transition, and its effects on LEDR.

Multi-factorial in vivo stable isotope fractionation: causes, correlations, consequences and applications

Many physical and chemical processes in living systems are accompanied by isotope fractionation on H, C, N, O and S. Although kinetic or thermodynamic isotope effects are always the basis, their in vivo manifestation is often modulated by secondary influences. These include metabolic branching events or metabolite channeling, metabolite pool sizes, reaction mechanisms, anatomical properties and compartmentation of plants and animals, and climatological or environmental conditions. In the present contribution, the fundamentals of isotope effects and their manifestation under in vivo conditions are outlined. The knowledge about and the understanding of these interferences provide a potent tool for the reconstruction of physiological events in plants and animals, their geographical origin, the history of bulk biomass and the biosynthesis of defined representatives. It allows the use of isotope characteristics of biomass for the elucidation of biochemical pathways and reaction mechanisms and for the reconstruction of climatic, physiological, ecological and environmental conditions during biosynthesis. Thus, it can be used for the origin and authenticity control of food, the study of ecosystems and animal physiology, the reconstruction of present and prehistoric nutrition chains and paleaoclimatological conditions. This is demonstrated by the outline of fundamental and application-orientated examples for all bio-elements. The aim of the review is to inform (advanced) students from various disciplines about the whole potential and the scope of stable isotope characteristics and fractionations and to provide them with a comprehensive introduction to the literature on fundamental aspects and applications.

Standardization for oxygen isotope ratio measurement - still an unsolved problem.

Numerous organic and inorganic laboratory standards were gathered from nine European and North American laboratories and were analyzed for their delta(18)O values with a new on-line high temperature pyrolysis system that was calibrated using Vienna standard mean ocean water (VSMOW) and standard light Antartic precipitation (SLAP) internationally distributed reference water samples. Especially for organic materials, discrepancies between reported and measured values were high, ranging up to 2 per thousand. The reasons for these discrepancies are discussed and the need for an exact and reliable calibration of existing reference materials, as well as for the establishment of additional organic and inorganic reference materials is stressed. Copyright 1999 John Wiley & Sons, Ltd.