Abstract
Steady state 13C-MFA is classically used to measure fluxes in complex metabolic networks. However, the modeling of steady state labeling allows the quantification of internal fluxes only and requires the estimation, by other methods, of the external fluxes, corresponding to substrate uptake (carbon input into the network) and to the production rate of compounds that accumulate within plant cells (network output). Additionally, it is not always possible to discriminate between different pathways that lead to the same label distribution. Methods to measure fluxes, based on direct measurements of pool size and on 14C short-time labeling experiments, are described in this chapter. To illustrate this approach, we focus on the quantification of sucrose and starch turnovers.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Salon C, Raymond P, Pradet A (1988) Quantification of carbon fluxes through the tricarboxylic acid cycle in early germinating lettuce embryos. J Biol Chem 263:12278–12287
Dieuaide-Noubhani M, Raffard G, Canioni P, Pradet A, Raymond P (1995) Quantification of compartmented metabolic fluxes in maize root tips using isotope distribution from 13C- or 14C-labeled glucose. J Biol Chem 270:13147–13159
Alonso AP, Raymond P, Hernould M et al (2007) A metabolic flux analysis to study the role of sucrose synthase in the regulation of the carbon partitioning in central metabolism in maize root tips. Metab Eng 9:419–432
Rontein D, Dieuaide-Noubhani M, Dufourc EJ, Raymond P, Rolin D (2002) The metabolic architecture of plant cells. Stability of central metabolism and flexibility of anabolic pathway during the growth cycle of tomato cells. J Biol Chem 277:43948–43960
Williams TCR, Miguet L, Masakapalli SK et al (2008) Metabolic network fluxes in heterotrophic Arabidopsis cells: stability of the flux distribution under different oxygenation conditions. Plant Physiol 148:704–718
Masakapalli SK, Kruger NJ, Ratcliffe RG (2013) The metabolic flux phenotype of heterotrophic Arabidopsis cells reveals a complex response to changes in nitrogen supply. Plant J. doi:10.1111/tpj.12142
Sriram G, Fulton DB, Iyer VV et al (2004) Quantification of compartmented metabolic fluxes in developing soybean embryos by employing biosynthetically directed fractional 13C labeling, two dimensional [13C, 1H] nuclear magnetic resonance, and comprehensive isotopomer balancing. Plant Physiol 136:3043–3057
Lonien J, Schwender J (2009) Analysis of metabolic flux phenotypes for two Arabidopsis mutants with severe impairment in seed storage lipid synthesis. Plant Physiol 151:1617–1634
Alonso AP, Dale VL, Shachar-Hill Y (2010) Understanding fatty acid synthesis in developing maize embryos using metabolic flux analysis. Metab Eng 12:488–497
Spielbauer G, Margl L, Hannah LC et al (2006) Robustness of central carbohydrate metabolism in developing maize kernels. Phytochemistry 67:1460–1475
Alonso AP, Val DL, Shachar-Hill Y (2011) Central metabolic fluxes in the endosperm of developing maize seeds and their implications for metabolic engineering. Metab Eng 13:96–107
Keeling PL, Wood JR, Tyson RH, Bridges IG (1988) Starch biosynthesis in developing wheat grain. Evidence against the direct involvement of triose phosphates in the metabolic pathway. Plant Physiol 87:311–319
Hatzfeld WD, Stitt M (1990) A study of the rate of recycling of triose phosphates in heterotrophic Chenopodium rubrum cells, potato tubers and maize endosperm. Planta 180:198–204
Viola R, Davies HV, Chudeck AR (1991) Pathways of starch and sucrose biosynthesis in developing tubers of potato (Solanum tuberosum L.) and seeds of faba bean (Vicia faba L.). Planta 183:202–208
Alonso AP, Vigeolas H, Raymond P, Rolin D, Dieuaide-Noubhani M (2005) A new substrate cycle in plants. Evidence for a high glucose phosphate-to-glucose turnover from in vivo steady-state and pulse labeling experiments with [13C]glucose and [14C]glucose. Plant Physiol 138:2220–2232
Marx A, de Graaf AA, Wiechert W et al (1996) Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing. Biotechnol Bioeng 49:111–129
Hargreaves JA, ap Rees T (1988) Turnover of starch and sucrose in roots of Pisum sativum. Phytochemistry 27:1627–1629
Dancer J, Hatzfeld WD, Stitt M (1990) Cytosolic cycle regulate the turnover of sucrose in heterotrophic cell-suspension cultures of Chenopodium rubrum L. Planta 182:223–231
Geigenberger P, Stitt M (1991) A “futile” cycle of glucose synthesis and degradation is involved in regulating partitioning between sucrose, starch and respiration in cotyledons of germinating Ricinus communis L. seedlings when phloem transport is inhibited. Planta 185:81–90
Hill SA, ap Rees T (1994) Fluxes of carbohydrate metabolism in ripening bananas. Planta 192:52–60
N'tchobo H, Dali N, Nguyen-Quoc B et al (1999) Starch synthesis in tomato remains constant throughout fruit development and is dependent on sucrose supply and sucrose synthase activity. J Exp Bot 50:1457–1463
Trethewey RN, Riesmeier JW, Willmitzer L, Stitt M, Geigenberger P (1999) Tuber-specific expression of a yeast invertase and a bacterial glucokinase in potato leads to an activation of sucrose phosphate synthase and the creation of a sucrose futile cycle. Planta 208:227–238
Saglio PH, Pradet A (1980) Soluble sugar, respiration, and energy charge during aging of excised maize root tips. Plant Physiol 66:516–519
Stit M, ap Rees T (1978) Pathways of carbohydrate oxidation in leaves of Pisum sativum and Triticum aestivum. Phytochemistry 18:1905–1911
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Pianelli, K., Monier, A., Andrieu, MH., Beauvoit, B., Dieuaide-Noubhani, M. (2014). 14C Pulse Labeling to Estimate External Fluxes and Turnovers in Primary Metabolism. In: Dieuaide-Noubhani, M., Alonso, A. (eds) Plant Metabolic Flux Analysis. Methods in Molecular Biology, vol 1090. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-688-7_3
Download citation
DOI: https://doi.org/10.1007/978-1-62703-688-7_3
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-687-0
Online ISBN: 978-1-62703-688-7
eBook Packages: Springer Protocols