A model of photosynthetic CO2 assimilation in C3 leaves accounting for respiration and energy recycling by the plastidial oxidative pentose phosphate pathway.

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 (Ca ). 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 Ca ≈ 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 Ca . 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/Ci curve drop at high Ca . Furthermore, we critically examine the models and parameterisation and derive recommendations for follow-up studies.

Compartment-specific energy requirements of photosynthetic carbon metabolism in Camelina sativa leaves.

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%. 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.

Reimport of carbon from cytosolic and vacuolar sugar pools into the Calvin-Benson cycle explains photosynthesis labeling anomalies.

SignificancePhotosynthesis 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.

Metabolism is a major driver of hydrogen isotope fractionation recorded in tree-ring glucose of Pinus nigra.

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.

Anaplerotic flux into the Calvin-Benson cycle: hydrogen isotope evidence for in vivo occurrence in C3 metabolism.

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.

Intramolecular carbon isotope signals reflect metabolite allocation in plants.

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.

Carbon flux around leaf-cytosolic glyceraldehyde-3-phosphate dehydrogenase introduces a 13C signal in plant glucose.

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 versus fructose 1,6-bisphosphate metabolism; and (ii) the contribution of non-phosphorylating versus phosphorylating glyceraldehyde-3-phosphate dehydrogenase to catalysing the glyceraldehyde 3-phosphate to 3-phosphoglycerate forward reaction of glycolysis. The theory is supported by 13C fractionation modelling. Modelling results provide the 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.

CO2 fertilization of Sphagnum peat mosses is modulated by water table level and other environmental factors.

Sphagnum mosses account for most accumulated dead organic matter in peatlands. Therefore, understanding their responses to increasing atmospheric CO2 is needed for estimating peatland C balances under climate change. A key process is photorespiration: a major determinant of net photosynthetic C assimilation that depends on the CO2 to O2 ratio. We used climate chambers to investigate photorespiratory responses of Sphagnum fuscum hummocks to recent increases in atmospheric CO2 (from 280 to 400 ppm) under different water table, temperature, and light intensity levels. We tested the photorespiratory variability using a novel method based on deuterium isotopomers (D6S /D6R ratio) of photosynthetic glucose. The effect of elevated CO2 on photorespiration was highly dependent on water table. At low water table (-20 cm), elevated CO2 suppressed photorespiration relative to C assimilation, thus substantially increasing the net primary production potential. In contrast, a high water table (~0 cm) favored photorespiration and abolished this CO2 effect. The response was further tested for Sphagnum majus lawns at typical water table levels (~0 and -7 cm), revealing no effect of CO2 under those conditions. Our results indicate that hummocks, which typically experience low water table levels, benefit from the 20th century's increase in atmospheric CO2 .

Global CO2 fertilization of Sphagnum peat mosses via suppression of photorespiration during the twentieth century.

Natural peatlands contribute significantly to global carbon sequestration and storage of biomass, most of which derives from Sphagnum peat mosses. Atmospheric CO2 levels have increased dramatically during the twentieth century, from 280 to > 400 ppm, which has affected plant carbon dynamics. Net carbon assimilation is strongly reduced by photorespiration, a process that depends on the CO2 to O2 ratio. Here we investigate the response of the photorespiration to photosynthesis ratio in Sphagnum mosses to recent CO2 increases by comparing deuterium isotopomers of historical and contemporary Sphagnum tissues collected from 36 peat cores from five continents. Rising CO2 levels generally suppressed photorespiration relative to photosynthesis but the magnitude of suppression depended on the current water table depth. By estimating the changes in water table depth, temperature, and precipitation during the twentieth century, we excluded potential effects of these climate parameters on the observed isotopomer responses. Further, we showed that the photorespiration to photosynthesis ratio varied between Sphagnum subgenera, indicating differences in their photosynthetic capacity. The global suppression of photorespiration in Sphagnum suggests an increased net primary production potential in response to the ongoing rise in atmospheric CO2, in particular for mire structures with intermediate water table depths.

Intramolecular 13C analysis of tree rings provides multiple plant ecophysiology signals covering decades.

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.

2 H-fractionations during the biosynthesis of carbohydrates and lipids imprint a metabolic signal on the δ2 H values of plant organic compounds.

Hydrogen (H) isotope ratio (δ2 H) analyses of plant organic compounds have been applied to assess ecohydrological processes in the environment despite a large part of the δ2 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 δ2 H values of plant organic compounds. The experiments demonstrate that information recorded in the δ2 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 2 H-fractionations occur in the biosynthesis of plant organic compounds and how these 2 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.