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• Understanding isotope fractionation mechanisms is fundamental for analyses of plant ecophysiology and paleoclimate based on tree-ring isotope data.
• To gain new insights into isotope fractionation, we analysed intramolecular ¹³C discrimination in tree-ring glucose (Δ_i', i = C-1 to C-6) and metabolic deuterium fractionation at H¹ and H² (ε_met) combinedly. This dual-isotope approach was used for isotope-signal deconvolution.
• We found evidence for metabolic processes affecting Δ₁' and Δ₃', which respond to air vapour pressure deficit (VPD), and processes affecting Δ₁' and Δ₃', and ε_met, which respond to precipitation but not VPD. These relationships exhibit change points dividing a period of homeostasis (1961–1980) from a period of metabolic adjustment (1983–1995). Homeostasis may result from sufficient groundwater availability. Additionally, we found Δ₅' and Δ₆' relationships with radiation and temperature, which are temporally stable and consistent with previously proposed isotope fractionation mechanisms.
• Based on the multitude of climate covariables, intramolecular carbon isotope analysis has a remarkable potential for climate reconstruction. While isotope fractionation beyond leaves is currently considered to be constant, we propose significant parts of the carbon and hydrogen isotope variation in tree-ring glucose originate in stems (precipitation-dependent signals). As basis for follow-up studies, we propose mechanisms introducing Δ₁', Δ₂', Δ₃', and ε_met variability.

• Recently, we reported estimates of anaplerotic carbon flux through the oxidative pentose phosphate pathway (OPPP) in chloroplasts into the Calvin–Benson cycle. These estimates were based on intramolecular hydrogen isotope analysis of sunflower leaf starch. However, the isotope method is believed to underestimate the actual flux at low atmospheric CO₂ concentration (C_a).
• Since the OPPP releases CO₂ and reduces NADP⁺, it can be expected to affect leaf gas exchange under both rubisco- and RuBP-regeneration-limited conditions. Therefore, we expanded Farquhar-von Caemmerer–Berry models to account for OPPP metabolism. Based on model parameterisation with values from the literature, we estimated OPPP-related effects on leaf carbon and energy metabolism in the sunflowers analysed previously.
• We found that flux through the plastidial OPPP increases both above and below C_a ≈ 450 ppm (the condition the plants were acclimated to). This is qualitatively consistent with our previous isotope-based estimates, yet gas-exchange-based estimates are larger at low C_a.
• We discuss our results in relation to regulatory properties of the plastidial and cytosolic OPPP, the proposed variability of CO₂ mesophyll conductance, and the contribution of day respiration to the A/C_i curve drop at high C_a. Furthermore, we critically examine the models and parameterisation and derive recommendations for follow-up studies.

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 CO2 concentrations, Ca. Additionally, we modelled deuterium fractionations expected for the anaplerotic pathway and compared modelled with measured fractionations.

We report deuterium fractionation signals at H1 and H2 of starch glucose. Below a Ca change point, these signals increase with decreasing Ca consistent with modelled fractionations by anaplerotic flux. Under standard conditions (Ca = 450 ppm corresponding to intercellular CO2 concentrations, Ci, of 328 ppm), we estimate negligible anaplerotic flux. At Ca = 180 ppm (Ci = 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 1,5-bisphosphate under source-limited growth conditions to facilitate photorespiratory nitrogen assimilation required to build-up source strength; and counteract oxidative stress.

Main conclusion: The oxidative pentose phosphate pathway provides cytosolic NADPH yet reduces carbon and energy use efficiency. Repressing this pathway and introducing cytosolic NADPH-dependent malate dehydrogenase may increase crop yields by ≈5%.

Abstract: Detailed knowledge about plant energy metabolism may aid crop improvements. Using published estimates of flux through central carbon metabolism, we phenotype energy metabolism in illuminated Camelina sativa leaves (grown at 22 °C, 500 µmol photons m−2 s−1) and report several findings. First, the oxidative pentose phosphate pathway (OPPP) transfers 3.3% of the NADPH consumed in the Calvin–Benson cycle to the cytosol. NADPH supply proceeds at about 10% of the rate of net carbon assimilation. However, concomitantly respired CO2 accounts for 4.8% of total rubisco activity. Hence, 4.8% of the flux through the Calvin–Benson cycle and photorespiration is spent on supplying cytosolic NADPH, a significant investment. Associated energy requirements exceed the energy output of the OPPP. Thus, autotrophic carbon metabolism is not simply optimised for flux into carbon sinks but sacrifices carbon and energy use efficiency to support cytosolic energy metabolism. To reduce these costs, we suggest bioengineering plants with a repressed cytosolic OPPP, and an inserted cytosolic NADPH-dependent malate dehydrogenase tuned to compensate for the loss in OPPP activity (if required). Second, sucrose cycling is a minor investment in overall leaf energy metabolism but a significant investment in cytosolic energy metabolism. Third, leaf energy balancing strictly requires oxidative phosphorylation, cofactor export from chloroplasts, and peroxisomal NADH import. Fourth, mitochondria are energetically self-sufficient. Fifth, carbon metabolism has an ATP/NADPH demand ratio of 1.52 which is met if ≤ 21.7% of whole electron flux is cyclic. Sixth, electron transport has a photon use efficiency of ≥ 62%. Last, we discuss interactions between the OPPP and the cytosolic oxidation–reduction cycle in supplying leaf cytosolic NADPH.

Stable isotopes at natural abundance are key tools to study physiological processes occurring outside the temporal scope of manipulation and monitoring experiments. Whole-molecule carbon isotope ratios (13C/12C) enable assessments of plant carbon uptake yet conceal information about carbon allocation. Here, we identify an intramolecular 13C/12C signal at tree-ring glucose C-5 and C-6 and develop experimentally testable theories on its origin. More specifically, we assess the potential of processes within C3 metabolism for signal introduction based (inter alia) on constraints on signal propagation posed by metabolic networks. We propose that the intramolecular signal reports carbon allocation into major metabolic pathways in actively photosynthesizing leaf cells including the anaplerotic, shikimate, and non-mevalonate pathway. We support our theoretical framework by linking it to previously reported whole-molecule 13C/12C increases in cellulose of ozone-treated Betula pendula and a highly significant relationship between the intramolecular signal and tropospheric ozone concentration. Our theory postulates a pronounced preference for leaf cytosolic triose-phosphate isomerase to catalyse the forward reaction in vivo (dihydroxyacetone phosphate to glyceraldehyde 3-phosphate). In conclusion, intramolecular 13C/12C analysis resolves information about carbon uptake and allocation enabling more comprehensive assessments of carbon metabolism than whole-molecule 13C/12C analysis.

• 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.

Photosynthesis metabolites are quickly labeled when 13CO2 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.

Leaf cytosol contains non-phosphorylating and phosphorylating glyceraldehyde 3-phosphate dehydrogenase (np-GAPDH and p-GAPDH, respectively). From the viewpoint of carbon metabolism, np-GAPDH is redundant. However, mutants lacking np-GAPDH show significant metabolic adjustments and decreased growth suggesting that np-GAPDH has central functions in plant metabolism. Here, a cytosolic oxidation-reduction (COR) cycle is proposed. In its forward direction, np-GAPDH supplies NADPH. In its backward direction, phosphoglycerate kinase and p-GAPDH consume ATP and NADH. Thus, COR cycling may constitute a central hub in energy metabolism.

Within the plant and earth sciences, stable isotope analysis is a versatile tool conveying information (inter alia) about plant physiological and paleoclimate variability across scales. Here, we identify a ¹³C signal (i.e., systematic ¹³C/¹²C variation) at tree-ring glucose C-4 and report an experimentally testable theory on its origin. We propose the signal is introduced by glyceraldehyde-3-phosphate dehydrogenases in the cytosol of leaves. It conveys two kinds of (potentially convoluted) information: (i) commitment of glyceraldehyde 3-phosphate to 3-phosphoglycerate vs. fructose 1,6-bisphosphate metabolism, and (ii) the contribution of non-phosphorylating vs. phosphorylating glyceraldehyde-3-phosphate dehydrogenase to catalysing the glyceraldehyde 3-phosphate to 3-phosphoglycerate forward reaction of glycolysis. Theory is supported by ¹³C fractionation modelling. Modelling results provide first evidence in support of the cytosolic oxidation-reduction (COR) cycle, a carbon-neutral mechanism supplying NADPH at the expense of ATP and NADH which may help to maintain leaf-cytosolic redox balances. In line with expectations related to COR cycling, we found a positive correlation between air vapour pressure deficit and ¹³C discrimination at glucose C-4. Overall, ¹³C-4 signal analysis may enable an improved understanding of leaf carbon and energy metabolism.