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

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 C3 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 CO2 fixation rate in leaves, a novel in vivo 13C-labelling system was developed with 13CO2 for the kinetic determination of metabolic turnover that was the time-course of the 13C-labelling ratio in each metabolite. The system is equipped with a gas-exchange chamber that enables real-time monitoring of the CO2 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.

[1]  M. Stitt,et al.  Decreased-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana. Impact on mass-action ratios and fluxes to sucrose and starch, and estimation of Flux Control Coefficients and Elasticity Coefficients. , 1989, The Biochemical journal.

[2]  A. Fernie,et al.  Carbon assimilation and metabolism in potato leaves deficient in plastidial phosphoglucomutase , 2002, Planta.

[3]  Stephen M. Schrader,et al.  Carbon Balance and Circadian Regulation of Hydrolytic and Phosphorolytic Breakdown of Transitory Starch1 , 2006, Plant Physiology.

[4]  C. Raines,et al.  Photosynthetic capacity is differentially affected by reductions in sedoheptulose-1,7-bisphosphatase activity during leaf development in transgenic tobacco plants. , 2001, Plant physiology.

[5]  J. Berry,et al.  A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species , 1980, Planta.

[6]  M. Tamoi,et al.  Carbon metabolism in the Calvin cycle , 2005 .

[7]  M. Stitt,et al.  A Small Decrease of Plastid Transketolase Activity in Antisense Tobacco Transformants Has Dramatic Effects on Photosynthesis and Phenylpropanoid Metabolism , 2001, Plant Cell.

[8]  F. S. Rolleston.  A Theoretical Background to the Use of Measured Concentrations of Intermediates in Study of the Control of Intermediary Metabolism , 1972 .

[9]  Julie C. Lloyd,et al.  Reduced sedoheptulose-1,7-bisphosphatase levels in transgenic tobacco lead to decreased photosynthetic capacity and altered carbohydrate accumulation , 1997, Planta.

[10]  Alison M. Smith,et al.  The diurnal metabolism of leaf starch. , 2007, The Biochemical journal.

[11]  T. Sharkey,et al.  Rapid appearance of 13C in biogenic isoprene when 13CO2 is fed to intact leaves , 1993 .

[12]  Dimah Z. Habash,et al.  Reduction in phosphoribulokinase activity by antisense RNA in transgenic tobacco: effect on CO2 assimilation and growth in low irradiance , 1995 .

[13]  D. Fell,et al.  Modelling photosynthesis and its control. , 2000, Journal of experimental botany.

[14]  E. Fukusaki,et al.  Quantitative analysis of anionic metabolites for Catharanthus roseus by capillary electrophoresis using sulfonated capillary coupled with electrospray ionization-tandem mass spectrometry. , 2008, Journal of bioscience and bioengineering.

[15]  D. Robinson,et al.  Subcellular volumes and metabolite concentrations in spinach leaves , 1994, Planta.

[16]  J. Berry,et al.  Models of photosynthesis. , 2001, Plant physiology.

[17]  L. Willmitzer,et al.  Reduction of the chloroplastic fructose‐1,6‐bisphosphatase in transgenic potato plants impairs photosynthesis and plant growth , 1994 .

[18]  M. Stitt,et al.  A moderate decrease of plastid aldolase activity inhibits photosynthesis, alters the levels of sugars and starch, and inhibits growth of potato plants. , 1998, The Plant journal : for cell and molecular biology.

[19]  K. Koch,et al.  Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. , 2004, Current opinion in plant biology.

[20]  O. Fiehn,et al.  Metabolite profiling for plant functional genomics , 2000, Nature Biotechnology.

[21]  E. Harrison,et al.  Small decreases in SBPase cause a linear decline in the apparent RuBP regeneration rate, but do not affect Rubisco carboxylation capacity. , 2001, Journal of experimental botany.

[22]  C Giersch.  Mathematical modelling of metabolism. , 2000, Current opinion in plant biology.

[23]  B. M. Lange,et al.  Metabolite profiling of Calvin cycle intermediates by HPLC-MS using mixed-mode stationary phases. , 2008, The Plant journal : for cell and molecular biology.

[24]  F. Muñoz,et al.  Reappraisal of the currently prevailing model of starch biosynthesis in photosynthetic tissues: a proposal involving the cytosolic production of ADP-glucose by sucrose synthase and occurrence of cyclic turnover of starch in the chloroplast. , 2001, Plant & cell physiology.

[25]  A. Fernie,et al.  Expression of an Escherichia coli phosphoglucomutase in potato (Solanum tuberosum L.) results in minor changes in tuber metabolism and a considerable delay in tuber sprouting , 2005, Planta.

[26]  M. Van Montagu,et al.  Plant enolase: gene structure, expression, and evolution. , 1991, The Plant cell.

[27]  Masaru Tomita,et al.  Simultaneous determination of the main metabolites in rice leaves using capillary electrophoresis mass spectrometry and capillary electrophoresis diode array detection. , 2004, The Plant journal : for cell and molecular biology.

[28]  M. Badger,et al.  Specific reduction of chloroplast glyceraldehyde-3-phosphate dehydrogenase activity by antisense RNA reduces CO2 assimilation via a reduction in ribulose bisphosphate regeneration in transgenic tobacco plants , 2004, Planta.

[29]  G. Farquhar,et al.  Effect of temperature on the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the rate of respiration in the light , 1985, Planta.

[30]  Ute Roessner,et al.  Simultaneous analysis of metabolites in potato tuber by gas chromatography-mass spectrometry. , 2000 .

[31]  Yves Gibon,et al.  GC-EI-TOF-MS analysis of in vivo carbon-partitioning into soluble metabolite pools of higher plants by monitoring isotope dilution after 13CO2 labelling. , 2007, Phytochemistry.

[32]  S. Huber,et al.  Carbon Partitioning and Growth of a Starchless Mutant of Nicotiana sylvestris. , 1992, Plant physiology.

[33]  A. Fernie,et al.  The influence of cytosolic phosphoglucomutase on photosynthetic carbohydrate metabolism , 2002, Planta.

[34]  J. Zou,et al.  The plastidic phosphoglucomutase from Arabidopsis. A reversible enzyme reaction with an important role in metabolic control. , 2000, Plant physiology.

[35]  Thomas D. Sharkey,et al.  Photosynthesis in intact leaves of C3 plants: Physics, physiology and rate limitations , 2008, The Botanical Review.

[36]  T. Sharkey,et al.  Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity in Response to Changing Partial Pressure of O(2) and Light in Phaseolus vulgaris. , 1986, Plant physiology.

[37]  Xin-Guang Zhu,et al.  Optimizing the Distribution of Resources between Enzymes of Carbon Metabolism Can Dramatically Increase Photosynthetic Rate: A Numerical Simulation Using an Evolutionary Algorithm1[W][OA] , 2007, Plant Physiology.

[38]  Jörg Schwender,et al.  Metabolic flux analysis as a tool in metabolic engineering of plants. , 2008, Current opinion in biotechnology.

[39]  E. Neufeld,et al.  CARBOHYDRATE METABOLISM. , 1965, Annual review of biochemistry.