Dataset Information

Metabolic turnover analysis by a combination of in vivo 13C-labelling from 13CO2 and metabolic profiling with CE-MS/MS reveals rate-limiting steps of the C3 photosynthetic pathway in Nicotiana tabacum leaves.

ABSTRACT: Understanding of the control of metabolic pathways in plants requires direct measurement of the metabolic turnover rate. Sugar phosphate metabolism, including the Calvin cycle, is the primary pathway in C(3) photosynthesis, the dynamic status of which has not been assessed quantitatively in the leaves of higher plants. Since the flux of photosynthetic carbon metabolism is affected by the CO(2) fixation rate in leaves, a novel in vivo (13)C-labelling system was developed with (13)CO(2) for the kinetic determination of metabolic turnover that was the time-course of the (13)C-labelling ratio in each metabolite. The system is equipped with a gas-exchange chamber that enables real-time monitoring of the CO(2) fixation rate and a freeze-clamp that excises a labelled leaf concurrently with quenching the metabolic reactions by liquid nitrogen within the photosynthesis chamber. Kinetic measurements were performed by detecting mass isotopomer abundance with capillary electrophoresis-tandem mass spectrometry. The multiple reaction monitoring method was optimized for the determination of each compound for sensitive detection because the amount of some sugar phosphates in plant cells is extremely small. Our analytical system enabled the in vivo turnover of sugar phosphates to be monitored in fresh tobacco (Nicotiana tabacum) leaves, which revealed that the turnover rate of glucose-1-phosphate (G1P) was significantly lower than that of other sugar phosphates, including glucose-6-phosphate (G6P). The pool size of G1P is 12 times lower than that of G6P. These results indicate that the conversion of G6P to G1P is one of the rate-limiting steps in the sugar phosphate pathway.

SUBMITTER: Hasunuma T

PROVIDER: S-EPMC2826653 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Metabolic turnover analysis by a combination of in vivo 13C-labelling from 13CO2 and metabolic profiling with CE-MS/MS reveals rate-limiting steps of the C3 photosynthetic pathway in Nicotiana tabacum leaves.

Hasunuma Tomohisa T Harada Kazuo K Miyazawa Shin-Ichi S Kondo Akihiko A Fukusaki Eiichiro E Miyake Chikahiro C

Journal of experimental botany 20091221 4

Understanding of the control of metabolic pathways in plants requires direct measurement of the metabolic turnover rate. Sugar phosphate metabolism, including the Calvin cycle, is the primary pathway in C(3) photosynthesis, the dynamic status of which has not been assessed quantitatively in the leaves of higher plants. Since the flux of photosynthetic carbon metabolism is affected by the CO(2) fixation rate in leaves, a novel in vivo (13)C-labelling system was developed with (13)CO(2) for the ki ...[more]

PMID: 20026474

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Project description:Our understanding of biogenic volatile organic compound (BVOC) emissions improved substantially during the last years. Nevertheless, there are still large uncertainties of processes controlling plant carbon investment into BVOCs, of some biosynthetic pathways and their linkage to CO2 decarboxylation at central metabolic branching points. To shed more light on carbon partitioning during BVOC biosynthesis, we used an innovative approach combining δ13CO2 laser spectroscopy, high-sensitivity proton-transfer-reaction time-of-flight mass spectrometry and a multiple branch enclosure system in combination with position-specific 13C-metabolite labelling. Feeding experiments with position-specific 13C-labelled pyruvate, a central metabolite of BVOC synthesis, enabled online detection of carbon partitioning into 13C-BVOCs and respiratory 13CO2. Measurements of trace gas emissions of the Mediterranean shrub Halimium halimifolium revealed a broad range of emitted BVOCs. In general, [2-13C]-PYR was rapidly incorporated into emitted acetic acid, methyl acetate, toluene, cresol, trimethylbenzene, ethylphenol, monoterpenes and sesquiterpenes, indicating de novo BVOC biosynthesis of these compounds. In contrast, [1-13C]-pyruvate labelling substantially increased 13CO2 emissions in the light indicating C1-decarboxylation. Similar labelling patterns of methyl acetate and acetic acid suggested tightly connected biosynthetic pathways and, furthermore, there were hints of possible biosynthesis of benzenoids via the MEP-pathway. Overall, substantial CO2 emission from metabolic branching points during de novo BVOC biosynthesis indicated that decarboxylation of [1-13C]-pyruvate, as a non-mitochondrial source of CO2, seems to contribute considerably to daytime CO2 release from leaves. Our approach, combining synchronised BVOC and CO2 measurements in combination with position-specific labelling opens the door for real-time analysis tracing metabolic pathways and carbon turnover under different environmental conditions, which may enhance our understanding of regulatory mechanisms in plant carbon metabolism and BVOC biosynthesis.

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Dataset Information

Metabolic turnover analysis by a combination of in vivo 13C-labelling from 13CO2 and metabolic profiling with CE-MS/MS reveals rate-limiting steps of the C3 photosynthetic pathway in Nicotiana tabacum leaves.

Publications

Metabolic turnover analysis by a combination of in vivo 13C-labelling from 13CO2 and metabolic profiling with CE-MS/MS reveals rate-limiting steps of the C3 photosynthetic pathway in Nicotiana tabacum leaves.

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