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.

Significance Photosynthesis metabolites are quickly labeled when 13 CO 2 is fed to leaves, but the time course of labeling reveals additional contributing processes involved in the metabolic dynamics of photosynthesis. The existence of three such processes is demonstrated, and a metabolic flux model is developed to explore and characterize them. The model is consistent with a slow return of carbon from cytosolic and vacuolar sugars into the Calvin–Benson cycle through the oxidative pentose phosphate pathway. Our results provide insight into how carbon assimilation is integrated into the metabolic network of photosynthetic cells with implications for global carbon fluxes.

Climate reconstructions using stable isotopes from tree-rings are steadily increasing. The investigations concentrate mostly on cellulose due to its high stability. In recent years the available amount of cellulose has steadily decreased, mainly because micro-structures of plant material have had to be analyzed. Today, the amounts of cellulose being studied are frequently in the milligram and often in the microgram range. Consequently, homogeneity problems with regard to the stable isotopes of carbon and oxygen from cellulose have occurred and these have called for new methods in the preparation of cellulose for reliable isotope analyses. Three different methods were tested for preparing isotopically homogenous cellulose, namely mechanical grinding, freezing by liquid nitrogen with subsequent milling and ultrasonic breaking of cellulose fibres. The best precision of isotope data was achieved by freeze-milling and ultrasonic breaking. However, equipment for freeze-milling is expensive and the procedure is labour-intensive. Mechanical grinding resulted in a rather high loss of material and it is also labour-intensive. The use of ultrasound for breaking cellulose fibres proved to be the best method in terms of rapidity of sample throughput, avoidance of sample loss, precision of isotope results, ease of handling, and cost.

Hydrogen (H) isotope ratio (δ H) analyses of plant organic compounds have been applied to assess ecohydrological processes in the environment despite a large part of the δ H variability observed in plant compounds not being fully elucidated. We present a conceptual biochemical model based on empirical H isotope data that we generated in two complementary experiments that clarifies a large part of the unexplained variability in the δ H values of plant organic compounds. The experiments demonstrate that information recorded in the δ H values of plant organic compounds goes beyond hydrological signals and can also contain important information on the carbon and energy metabolism of plants. Our model explains where H-fractionations occur in the biosynthesis of plant organic compounds and how these H-fractionations are tightly coupled to a plant's carbon and energy metabolism. Our model also provides a mechanistic basis to introduce H isotopes in plant organic compounds as a new metabolic proxy for the carbon and energy metabolism of plants and ecosystems. Such a new metabolic proxy has the potential to be applied in a broad range of disciplines, including plant and ecosystem physiology, biogeochemistry and palaeoecology.

As the central carbon uptake pathway in photosynthetic cells, the Calvin-Benson cycle is among the most important biochemical cycles for life on Earth. A carbon flux of anaplerotic origin (i.e. through the chloroplast-localized oxidative branch of the pentose phosphate pathway) into the Calvin-Benson cycle was proposed recently.Here, we measured intramolecular deuterium abundances in leaf starch of Helianthus annuus grown at varying ambient CO 2 concentrations, C a . Additionally, we modelled deuterium fractionations expected for the anaplerotic pathway and compared modelled with measured fractionations.We report deuterium fractionation signals at H 1 and H 2 of starch glucose. Below a C a change point, these signals increase with decreasing C a consistent with modelled fractionations by anaplerotic flux. Under standard conditions (C a = 450 ppm corresponding to intercellular CO 2 concentrations, C i , of 328 ppm), we estimate negligible anaplerotic flux. At C a = 180 ppm (C i = 140 ppm), more than 10% of the glucose-6-phosphate entering the starch biosynthesis pathway is diverted into the anaplerotic pathway.In conclusion, we report evidence consistent with anaplerotic carbon flux into the Calvin-Benson cycle in vivo. We propose the flux may help to: maintain high levels of ribulose 1,5bisphosphate under source-limited growth conditions to facilitate photorespiratory nitrogen assimilation required to build-up source strength; and counteract oxidative stress.