Measurements of carbon isotope contents of plant organic matter provide important information in diverse fields such as plant breeding, ecophysiology, biogeochemistry and paleoclimatology. They are currently based on 13C/12C ratios of specific, whole metabolites, but we show here that intramolecular ratios provide higher resolution information. In the glucose units of tree-ring cellulose of 12 tree species, we detected large differences in 13C/12C ratios (>10‰) among carbon atoms, which provide isotopically distinct inputs to major global C pools, including wood and soil organic matter. Thus, considering position-specific differences can improve characterisation of soil-to-atmosphere carbon fluxes and soil metabolism. In a Pinus nigra tree-ring archive formed from 1961 to 1995, we found novel 13C signals, and show that intramolecular analysis enables more comprehensive and precise signal extraction from tree rings, and thus higher resolution reconstruction of plants’ responses to climate change. Moreover, we propose an ecophysiological mechanism for the introduction of a 13C signal, which links an environmental shift to the triggered metabolic shift and its intramolecular 13C signature. In conclusion, intramolecular 13C analyses can provide valuable new information about long-term metabolic dynamics for numerous applications.

Terrestrial vegetation currently absorbs approximately a third of anthropogenic CO 2 emissions, mitigating the rise of atmospheric CO 2 . However, terrestrial net primary production is highly sensitive to atmospheric CO 2 levels and associated climatic changes. In C 3 plants, which dominate terrestrial vegetation, net photosynthesis depends on the ratio between photorespiration and gross photosynthesis. This metabolic flux ratio depends strongly on CO 2 levels, but changes in this ratio over the past CO 2 rise have not been analyzed experimentally. Combining CO 2 manipulation experiments and deuterium NMR, we first establish that the intramolecular deuterium distribution (deuterium isotopomers) of photosynthetic C 3 glucose contains a signal of the photorespiration/photosynthesis ratio. By tracing this isotopomer signal in herbarium samples of natural C 3 vascular plant species, crops, and a Sphagnum moss species, we detect a consistent reduction in the photorespiration/photosynthesis ratio in response to the ∼100-ppm CO 2 increase between ∼1900 and 2013. No difference was detected in the isotopomer trends between beet sugar samples covering the 20th century and CO 2 manipulation experiments, suggesting that photosynthetic metabolism in sugar beet has not acclimated to increasing CO 2 over >100 y. This provides observational evidence that the reduction of the photorespiration/photosynthesis ratio was ca. 25%. The Sphagnum results are consistent with the observed positive correlations between peat accumulation rates and photosynthetic rates over the Northern Hemisphere. Our results establish that isotopomers of plant archives contain metabolic information covering centuries. Our data provide direct quantitative information on the "CO 2 fertilization" effect over decades, thus addressing a major uncertainty in Earth system models.A tmospheric CO 2 levels have increased from ∼200 ppm during the last ice age to currently 400 ppm, and they may, according to pessimistic scenarios, exceed 1,000 ppm in the year 2100 (1). Understanding plant responses to increasing CO 2 is currently hampered by two fundamental limitations: First, it is unknown how well manipulation experiments represent responses to the gradual CO 2 increase over decades and centuries. In Free-Air CO 2 Enrichment (FACE) experiments, which most closely mimic natural conditions, increases in [CO 2 ] generally increase plant growth, but this "CO 2 fertilization" effect often declines after a few years of enrichment (2). Such transient responses may be related to the step increases in [CO 2 ] used in the experiments, their limited duration (2), or factors other than CO 2 becoming limiting (3). Second, in response to the [CO 2 ] increase since industrialization, genetic (4) and phenotypic plant responses (5-7) have been observed. Although century-scale changes have been detected in carbon isotopes (δ 13 C) and attributed to [CO 2 ], these responses are tied to differences in intercellular substrate concentrations that reflect several metabolic fluxes and dif...

Mitigation of N-nitrosodimethylamine (NDMA) and other hazardous water disinfection byproducts (DBP) is currently hampered by a limited understanding of DBP formation mechanisms. Because variations of the stable isotope composition of NDMA can potentially reveal reaction pathways and precursor compounds, we developed a method for the compound-specific isotope analysis (CSIA) of (13)C/(12)C, (15)N/(14)N, and (2)H/(1)H ratios of NDMA by gas chromatography coupled to isotope ratio mass spectrometry (GC/IRMS). Method quantification limits for the accurate isotope analysis of NDMA, N-nitrosodiethyl-, -dipropyl-, and -dibutylamine as well as N-nitrosopyrrolidine were between 0.18 to 0.60 nmol C, 0.40 to 0.80 nmol N, and 2.2 to 5.8 nmol H injected on column. Coupling solid phase extraction (SPE) to GC/IRMS enabled the precise quantification of C, N, and H isotope ratios of NDMA in aqueous samples at concentrations of 0.6 μM (45 μg L(-1)). We validated the proposed method with a laboratory experiment, in which NDMA was formed with stoichiometric yield (97 ± 4%) through chloramination of the pharmaceutical ranitidine (3 μM). δ(13)C and δ(2)H values of NDMA remained constant during NDMA formation while its δ(15)N increased due to a reaction at a N atom in the rate-limiting step of NDMA formation. The δ(2)H value of NDMA determined by SPE-GC/IRMS also corresponded well to the δ(2)H value of the N(CH3)2-group of ranitidine measured by quantitative deuterium nuclear magnetic resonance spectroscopy. This observation implies that the N(CH3)2-moiety of ranitidine is transferred to NDMA without being chemically altered and illustrates the accuracy of the proposed method.

Summary Stable isotope abundances convey valuable information about plant physiological processes and underlying environmental controls. Central gaps in our mechanistic understanding of hydrogen isotope abundances impede their widespread application within the plant and biogeosciences. To address these gaps, we analysed intramolecular deuterium abundances in glucose of Pinus nigra extracted from an annually resolved tree‐ring series (1961–1995). We found fractionation signals (i.e. temporal variability in deuterium abundance) at glucose H1 and H2 introduced by closely related metabolic processes. Regression analysis indicates that these signals (and thus metabolism) respond to drought and atmospheric CO2 concentration beyond a response change point. They explain ≈ 60% of the whole‐molecule deuterium variability. Altered metabolism is associated with below‐average yet not exceptionally low growth. We propose the signals are introduced at the leaf level by changes in sucrose‐to‐starch carbon partitioning and anaplerotic carbon flux into the Calvin–Benson cycle. In conclusion, metabolism can be the main driver of hydrogen isotope variation in plant glucose.

- Stable isotope abundances convey valuable information about plant physiological processes and underlying environmental controls. Central gaps in our mechanistic understanding of hydrogen isotope abundances impede their widespread application within the plant and Earth sciences. - To close these gaps, we analysed intramolecular deuterium abundances in glucose of Pinus nigra extracted from an annually resolved tree-ring series (1961 to 1995). - We found fractionation signals at glucose H1 and H2 introduced by closely related metabolic processes. These signals (and thus metabolism) respond to drought and atmospheric CO2 concentration beyond a response change point. They explain ≈60% of the whole-molecule deuterium variability. Altered metabolism is associated with below-average yet not exceptionally low growth. - We propose the signals are introduced at the leaf-level by changes in sucrose-to-starch carbon partitioning and anaplerotic carbon flux into the Calvin-Benson cycle. In conclusion, metabolism can be the main driver of hydrogen isotope variation in plant glucose.