Review Insights into plant metabolic networks from steady-state metabolic flux analysis Nicholas J. Kruger * , R. George Ratcliffe * Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK article info Article history: Received 28 November 2008 Accepted 15 January 2009 Available online 27 January 2009 Keywords: Flux map Metabolomics Network performance Primary metabolism Stable isotope labelling abstract Steady-state metabolic flux analysis (MFA) is an experimental approach that allows the measurement of multiple fluxes in the core network of primary carbon metabolism. It is based on isotopic labelling experiments, and although well established in the analysis of micro-organisms, and some mammalian systems, the extension of the method to plant cells has been challenging because of the extensive subcellular compartmentation of the metabolic network. Despite this difficulty there has been substantial progress in developing robust protocols for the analysis of heterotrophic plant metabolism by steady-state MFA, and flux maps have now been published that reflect the metabolic phenotypes of excised root tips, developing embryos and cotyledons, hairy root cultures, and cell suspensions under a variety of physiological conditions. There has been a steady improvement in the quality, extent and statistical reliability of these analyses, and new information is emerging on the performance of the plant metabolic network and the contributions of specific pathways. The principles of steady-state MFA are outlined here, the current status of the technique for character- izing primary metabolism in plants is described, and its complementary relationship to metabolomic analysis based on metabolite composition is discussed. It is argued that there is still considerable scope for further development of the technique, either by implementing refinements that have already been adopted in microbial investigations, or by developing techniques that are particularly relevant to the problems posed by plant tissues. If successful, these developments will lead to a more powerful phe- notyping tool that will be faster to implement, and which will provide the basis for fully predictive mechanistic models of the network. This in turn will lead to an improved understanding of the regulation of plant metabolic networks, as well as a firm foundation for rational metabolic engineering. Ó 2009 Elsevier Masson SAS. All rights reserved. 1. Introduction In plants, as in all living organisms, the inputs that sustain life are transformed into biomass and other outputs by metabolic pathways that support the necessary fluxes of material. These fluxes provide a dynamic description of the metabolic phenotype of the plant [1], and so methods that allow fluxes to be measured across the metabolic network at either cell or tissue level are of considerable interest. Metabolic flux analysis (MFA) encompasses a suite of methods for achieving this objective, and in plants there is particular interest in using MFA to determine multiple fluxes through the pathways of primary carbon metabolism [2]. The hope is that such measurements will lead to a greater understanding of the regulation of the metabolic network, and hence to the devel- opment of improved strategies for metabolic engineering [3,4]. The importance of flux measurements for the characterization of the plant metabolic network is commonly recognised [5–8]. A special issue of the journal Phytochemistry was devoted to the topic in 2007 [9], and flux analysis is a major component of a forthcoming book on the plant metabolic network [10]. So-called steady-state MFA is currently the method of choice for analyzing the flux distribution in heterotrophic plant tissues, and since the pioneering application of steady-state MFA to central carbon metabolism in maize root tips [11], the development of the field has been reviewed extensively [2–4,12–16]. Here the intention is: (i) to consider the current status of steady-state MFA as an analytical technique for characterizing primary carbon metabolism in plants; and (ii) to identify some of the future developments that are required if MFA is to fulfil its potential as a tool for analyzing plant metabolic phenotypes. 2. Steady-state MFA The principles and practice of steady-state MFA have been fully described in the context of the plant metabolic network elsewhere * Corresponding authors. Tel.: þ44 1865 275000; fax: þ44 1865 275074. E-mail addresses: nick.kruger@plants.ox.ac.uk (N.J. Kruger), george.ratcliffe@ plants.ox.ac.uk (R.G. Ratcliffe). Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi 0300-9084/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2009.01.004 Biochimie 91 (2009) 697–702