A transient isotopic labeling methodology for 13C metabolic flux analysis of photoautotrophic microorganisms

Shastri, Avantika A.; Morgan, John A.

doi:10.1016/j.phytochem.2007.03.042

Cited by 92 publications

(63 citation statements)

References 47 publications

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“…We have observed a similar lack of coupling between flux and pool size estimates in previous studies (29), which appears to be a general characteristic of INST-MFA models. INST-MFA provides a comprehensive approach to map the flow and fate of carbon throughout autotrophic metabolic networks (26). This method enables quantitative studies of integrated metabolic pathways, rather than individual reactions or nodes in isolation.…”

Section: Adp-glucose and Udp-glucose Reveal Metabolic Compartmentationmentioning

confidence: 99%

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Isotopically nonstationary ¹³ C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation

Jazmin²,

Young

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

202

221

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Improving plant productivity is an important aim for metabolic engineering. There are few comprehensive methods that quantitatively describe leaf metabolism, although such information would be valuable for increasing photosynthetic capacity, enhancing biomass production, and rerouting carbon flux toward desirable end products. Isotopically nonstationary metabolic flux analysis (INST-MFA) has been previously applied to map carbon fluxes in photoautotrophic bacteria, which involves model-based regression of transient 13 C-labeling patterns of intracellular metabolites. However, experimental and computational difficulties have hindered its application to terrestrial plant systems. We performed in vivo isotopic labeling of Arabidopsis thaliana rosettes with . Approximately 1,400 independent mass isotopomer measurements obtained from analysis of 37 metabolite fragment ions were regressed to estimate 136 total fluxes (54 free fluxes) under each condition. The results provide a comprehensive description of changes in carbon partitioning and overall photosynthetic flux after longterm developmental acclimation of leaves to high light. Despite a doubling in the carboxylation rate, the photorespiratory flux increased from 17 to 28% of net CO 2 assimilation with high-light acclimation (Vc/Vo: 3.5:1 vs. 2.3:1, respectively). This study highlights the potential of 13 C INST-MFA to describe emergent flux phenotypes that respond to environmental conditions or plant physiology and cannot be obtained by other complementary approaches.isotopomer modeling | metabolic flux analysis | photosynthesis | 13 C-labeling | primary metabolism P hotosynthetic organisms assimilate more than 100 billion tons of carbon, ∼15% of the atmospheric total, each year and generate organic compounds for food and renewable chemicals (1). However, photosynthesis is a complex process that responds to heterotrophic tissue demands and environmental stimuli such as drought, temperature, and light intensity (2, 3). The light incident on the plant varies with intensities in the range of 0-2,000 μmol photons·m −2 ·s −1 and can change dramatically because of passing clouds, shading, and the position of the sun. Thus, plants adjust light harvesting and carbon assimilation steps to accommodate many fluctuations, resulting in changes in plant morphology, physiology, and metabolism (4).For 95% of all terrestrial plants (i.e., C3 plants), the reductive pentose phosphate (Calvin-Benson-Bassham, or CBB) cycle directly links light and dark reactions and sustains anabolic activities (5). RuBisCO (ribulose-1,5-bisphosphate carboxylase oxygenase) plays a central role in the cycle by carboxylating ribulose-1,5-bisphosphate (RUBP) with CO 2 to form two 3-phosphoglycerate (3PGA) molecules. The other 10 enzymes in the CBB cycle regenerate the RUBP substrate to repeat this process. RuBisCO has a low turnover rate (∼3/s; ref. 6) and also performs a competitive oxygenation side reaction that limits carboxylation activity. The binding of RuBisCO to oxygen produces 2-phosphoglycolat...

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Section: Adp-glucose and Udp-glucose Reveal Metabolic Compartmentationmentioning

confidence: 99%

“…However, leaves exhibit diurnal patterns of metabolism with limited metabolic steady states (24,25). Furthermore, autotrophic tissues produce uniform steady-state 13 C-labeling patterns that are largely uninformative (26). Therefore, transient 13 CO 2 labeling studies are necessary to quantify leaf metabolic fluxes.…”

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confidence: 99%

Isotopically nonstationary ¹³ C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation

Jazmin²,

Young

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

202

221

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“…Chlorophyll (Chl) fluorescence, which is sensitive to environmental changes and stress conditions that induce alterations in photosynthetic components (Iwai et al, 2008), can monitor the efficiency of linear electron flow (Baker et al, 2007) and has indicated that in N. oceanica (Simionato et al, 2013) and other algae, including C. reinhardtii (Berges et al, 1996;Li et al, 2010;Blaby et al, 2013), photosynthetic efficiency falls after N deprivation. Photosynthetically driven metabolic fluxes can also be probed by supplying 13 C-labeled bicarbonate/CO 2 and quantifying 13 C incorporation into metabolite pools (Shastri and Morgan, 2007;Feng et al, 2010;Young et al, 2011).…”

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confidence: 99%

The Regulation of Photosynthetic Structure and Function during Nitrogen Deprivation in Chlamydomonas reinhardtii

Deshpande²,

et al. 2014

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The accumulation of carbon storage compounds by many unicellular algae after nutrient deprivation occurs despite declines in their photosynthetic apparatus. To understand the regulation and roles of photosynthesis during this potentially bioenergetically valuable process, we analyzed photosynthetic structure and function after nitrogen deprivation in the model alga Chlamydomonas reinhardtii. Transcriptomic, proteomic, metabolite, and lipid profiling and microscopic time course data were combined with multiple measures of photosynthetic function. Levels of transcripts and proteins of photosystems I and II and most antenna genes fell with differing trajectories; thylakoid membrane lipid levels decreased, while their proportions remained similar and thylakoid membrane organization appeared to be preserved. Cellular chlorophyll (Chl) content decreased more than 2-fold within 24 h, and we conclude from transcript protein and 13 C labeling rates that Chl synthesis was down-regulated both pre-and posttranslationally and that Chl levels fell because of a rapid cessation in synthesis and dilution by cellular growth rather than because of degradation. Photosynthetically driven oxygen production and the efficiency of photosystem II as well as P700 + reduction and electrochromic shift kinetics all decreased over the time course, without evidence of substantial energy overflow. The results also indicate that linear electron flow fell approximately 15% more than cyclic flow over the first 24 h. Comparing Calvin-Benson cycle transcript and enzyme levels with changes in photosynthetic 13 CO 2 incorporation rates also pointed to a coordinated multilevel down-regulation of photosynthetic fluxes during starch synthesis before the induction of high triacylglycerol accumulation rates.

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“…Guy et al, 2008), only a few metabolomic approaches have been published using microbial (e.g. Koek et al, 2006) or cyanobacterial models (Yang et al, 2002;Shastri and Morgan, 2007).…”

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Metabolome Phenotyping of Inorganic Carbon Limitation in Cells of the Wild Type and Photorespiratory Mutants of the CyanobacteriumSynechocystissp. Strain PCC 6803

et al. 2008

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The amount of inorganic carbon represents one of the main environmental factors determining productivity of photoautotrophic organisms. Using the model cyanobacterium Synechocystis sp. PCC 6803, we performed a first metabolome study with cyanobacterial cells shifted from high CO 2 (5% in air) into conditions of low CO 2 (LC; ambient air with 0.035% CO 2 ). Using gas chromatography-mass spectrometry, 74 metabolites were reproducibly identified under different growth conditions. Shifting wild-type cells into LC conditions resulted in a global metabolic reprogramming and involved increases of, for example, 2-oxoglutarate (2OG) and phosphoenolpyruvate, and reductions of, for example, sucrose and fructose-1,6-bisphosphate. A decrease in Calvin-Benson cycle activity and increased usage of associated carbon cycling routes, including photorespiratory metabolism, was indicated by synergistic accumulation of the fumarate, malate, and 2-phosphoglycolate pools and a transient increase of 3-phosphoglycerate. The unexpected accumulation of 2OG with a concomitant decrease of glutamine pointed toward reduced nitrogen availability when cells are confronted with LC. Despite the increase in 2OG and low amino acid pools, we found a complete dephosphorylation of the PII regulatory protein at LC characteristic for nitrogen-replete conditions. Moreover, mutants with defined blocks in the photorespiratory metabolism leading to the accumulation of glycolate and glycine, respectively, exhibited features of LC-treated wild-type cells such as the changed 2OG to glutamine ratio and PII phosphorylation state already under high CO 2 conditions. Thus, metabolome profiling demonstrated that acclimation to LC involves coordinated changes of carbon and interacting nitrogen metabolism. We hypothesize that Synechocystis has a temporal lag of acclimating carbon versus nitrogen metabolism with carbon leading.

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A transient isotopic labeling methodology for 13C metabolic flux analysis of photoautotrophic microorganisms

Cited by 92 publications

References 47 publications

Isotopically nonstationary ¹³ C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation

Isotopically nonstationary ¹³ C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation

The Regulation of Photosynthetic Structure and Function during Nitrogen Deprivation in Chlamydomonas reinhardtii

Metabolome Phenotyping of Inorganic Carbon Limitation in Cells of the Wild Type and Photorespiratory Mutants of the CyanobacteriumSynechocystissp. Strain PCC 6803

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A transient isotopic labeling methodology for 13C metabolic flux analysis of photoautotrophic microorganisms

Cited by 92 publications

References 47 publications

Isotopically nonstationary 13 C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation

Isotopically nonstationary 13 C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation

The Regulation of Photosynthetic Structure and Function during Nitrogen Deprivation in Chlamydomonas reinhardtii

Metabolome Phenotyping of Inorganic Carbon Limitation in Cells of the Wild Type and Photorespiratory Mutants of the CyanobacteriumSynechocystissp. Strain PCC 6803

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Isotopically nonstationary ¹³ C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation

Isotopically nonstationary ¹³ C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation