Scientic Correspondence

On the Discordance of Metabolomics with Proteomics and Transcriptomics: Coping with Increasing Complexity in Logic, Chemistry, and Network Interactions
Alisdair R. Fernie and Mark Stitt* r Molekulare Panzenphysiologie, 14476 Potsdam-Golm, Germany Max-Planck-Institut fu

We suspect that many biologists who have been following the development of functional omics and systems biology are wondering why, compared with the success in integrating information from large-scale studies of transcripts and, more recently, proteins, there is less success in integrating information about metabolism. Information about thousands of transcripts has been integrated into large databases that are used by thousands of scientists (Zimmermann et al., 2004). Rapid advances will soon allow a similar approach in quantitative proteomics. Strategies also exist to integrate information about the expression of genes, as transcripts, with information about changes in the levels of the encoded proteins. Even when changes in transcript levels do not lead to changes in the levels of the encoded proteins, this apparent discrepancy can often be explained by quantitative analysis of the data sets and information about translational regulation and usser protein turnover (Piques et al., 2009; Schwanha et al., 2011). Such advances are providing systemslevel information about transcriptional and signaling networks that regulate the transcript and protein abundance. However, another important goal of systems biology is a comprehensive understanding of the functional importance of these changes in transcript and protein abundance. Although there is sometimes an encouragingly simple relationship between changes of transcripts and proteins and changes in downstream biological functions, quite often there is a major discordance. In this scientic correspondence, we will discuss reasons for this apparent disconnect, which lie partly in technical issues and partly in the complexity and structure of metabolic networks, and then propose some priorities in metabolic research to combat these problems. As we climb up what has been described as the si and Oltvai, 2004) from pyramid of life (Baraba gene through RNA and protein to phenotype (which includes chemical composition), we are confronted with an increasing complexity both at the chemical level and in the logic that is needed to draw inferences from one level to the next. Nucleic acids consist of four
* Corresponding author; e-mail fernie@mpimp-golm.mpg.de www.plantphysiol.org/cgi/doi/10.1104/pp.112.193235

bases, joined by the same phosphoester bond and with pairwise interacting side chains. This simple structure allows a near-to-binary storage and transmission of information. Proteins are chemically much more complex, due to the functionalities of 20 different amino acid side chains. This large expansion in chemical complexity lies at the center of the exibility and complexity of biological systems, as it allows different proteins to perform an enormous range of different functions. Nevertheless, proteins still have a simple primary structure, with amino acids linked via a peptide bond in a xed sequence that, via the universal genetic code, can be unambiguously linked to the sequence of nucleic acids (Aebersold and Mann, 2003; Fernie et al., 2004; Baerenfaller et al., 2008). This logic breaks down when we consider entities that are generated by the activities of individual proteins or sets of proteins, like metabolites. It is impossible to infer the structures, chemicophysical properties, or biological activities of metabolites from the sequences of nucleic acids or proteins. This breakdown in the ladder of inference is compounded by the bewildering chemical diversity of the metabolite world (DAuria and Gershenzon, 2005). This diversity encompasses elemental composition, chemical bond functional groups, and physicochemical properties. The inherent complexity of networks also increases as we clamber up the pyramid of life. Transcript and protein networks are usually generated from information about relative abundances, with the rough but not completely unjustied assumption that when a transcript or protein increases in abundance, its biological activity will also increase. In metabolite networks, however, several complicating factors invalidate this simplifying assumption. (1) Metabolite networks include chemical transformation of one metabolite into another (i.e. the ow of atoms as well as regulatory interactions, which requires consideration of pathway structure and stoichiometry) and the laws of conservation and thermodynamics (Rolleston, 1972). (2) The extent to which a metabolite is transformed depends more on the kinetic properties of enzymes than on the concentration and properties of the metabolite itself (Cornish-Bowden, 1996). (3) The assumption that increased abundance is accompanied by increased biological activity often breaks down. In the simplest case,
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Plant Physiology, March 2012, Vol. 158, pp. 11391145, www.plantphysiol.org 2012 American Society of Plant Biologists. All Rights Reserved.

Fernie and Stitt

increased use of a metabolite may be expected to result in a decrease in its level (stated at its simplest as the crossover theory [Newsholme and Crabtree, 1973]); however, due to the branching and circular nature of metabolic pathways, even this simple relation may not hold (Fell, 1996). (4) The nonlinearity of responses, which is exemplied most simply by ux control analysis (Kacser and Burns, 1973), explains why levels of enzymes can often be greatly changed without a major effect on metabolite levels and uxes. Increasing information about protein sequence and abundance will make it easier to draw inferences about metabolic responses. However, due to some major constraints, these inferences will probably always require checking in a case-by-case manner. It can be hoped that inferences from protein sequence to metabolic function will become increasingly powerful, as we learn more about the functionality of protein sequences and domains and the tools and the knowledge base for in silico structural modeling increase. However, this will be constrained by our capability to assign functional capacities to proteins. Similar sequences do not always encode for enzymes catalyzing the same reactions. While the combined use of sequence and (co)expression data can strengthen inferences (Mutwil et al., 2011), there remains much room for error. Indeed, there is an emerging suspicion that seemingly similar enzymes may often have fundamentally different roles, including scavenging of metabolites that are created due to indelities in catalysis by other enzymes (Hanson et al., 2010). Another constraint affects the use of information about protein abundance to draw inferences about metabolic events. The regulation of enzymes often occurs not by changing the amount of a protein (via changes in transcription and translation or degradation) but via posttranslational modication, which modies the activity and characteristics of an enzyme (e.g. the regulation of the Suc-to-starch transition or the tricarboxylic acid [TCA] jo cycle in sink organs; Geigenberger et al., 2004; Arau et al., 2012). Some problems are shared between network models at all levels. That said, many of these shared problems are more serious for metabolite networks. One example is missing or unreliable data. This issue is much larger for metabolites than for transcripts or proteins. Sheer chemical complexity and diversity means that it has not been, and probably never will be, possible to develop a generic method to extract and analyze all metabolites, and certainly not in a manner that is compatible with high-throughput analysis. Current coverage amounts to several hundred metabolites (Giavalisco et al., 2008; Iijima et al., 2008) and is probably only on the order of 1% of all the metabolites in an organism. The chemical complexity of metabolites also has major consequences for the quality of data; there are many more steps in harvest procedures, sample preparation, and analysis where artifacts (e.g. loss or transformation of metabolites; errors in identication or quantication) can occur. The kinds of
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artifacts that can occur will differ between different metabolites and types of metabolite and between different tissues and treatments. Another shared problem is compartmentation, either between parts of a cell or between cells. Compartmentation of metabolites is at least as complex, and more difcult to approach experimentally, than for transcripts and proteins (Stitt et al., 1980). The spatial location of transcripts and proteins can be analyzed using generic methods that are based on sequence information, molecular reporters, or specic molecule-molecule interactions (e.g. reporter genes, uorescence-tagged proteins, in situ hybridization, and immunolocalization). Analogous general methods for metabolites are lacking. Conventional biochemical procedures for subcellular fractionation and emerging methods for cell fractionation that have been developed to analyze transcript and protein localization are often unsuitable for metabolites, due to their rapid turnover (Stitt et al., 1980), while methods to visually localize metabolites are also much more challenging than those for nucleic acids or proteins (Chaudhuri et al., 2011). Another general issue in all types of networks is redundancy. While this occurs at the level of transcription, transcript processing, and translation, it can be argued that the extent of redundancy of isoforms and duplication of pathways is at least as great in central metabolism (e.g. the TCA cycle, the photorespiratory pathway, or Suc degradation; Timm jo et al., 2012). et al., 2008; Barratt et al., 2009; Arau Having discussed the problems we face, we will spend the rest of this article outlining some research priorities to address these issues and facilitate the collection of informative data sets about metabolites and metabolism that can be more effectively linked to responses at other functional levels. This is an important challenge. The depth of metabolic interactions and the fact that metabolites constitute an important operational phenotype (composition) and are closely related to other important phenotypes (e.g. growth) make them inherently biologically relevant, which cannot be stated de facto for coexpressed genes or protein-protein interactions, despite the fact that these can be very useful for understanding signaling networks (Usadel et al., 2009; Van Leene et al., 2010). It will not be possible to ameliorate two basic problems that are inherent in the structure of biological systems: the increasing difculty in inferring relationships as you move up the pyramid of life from transcripts to proteins to metabolites, and the accompanying explosion of chemical complexity. However, realizing that these are basic constraints allows us to highlight several challenges for the study of metabolism. One set of challenges revolves around technical issues that need to be addressed to cope with the chemical complexity of the metabolite world. A prime goal for metabolite analysis will be to extend its scope to cover more metabolites. Routine gas chromatographymass spectrometry or liquid chromatography-mass
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spectrometry metabolite proling typically provides information about a minute fraction of the complete plant metabolome (see above). Recent advances have facilitated the detection of up to 1,000 metabolites using a single protocol (Giavalisco et al., 2008; Iijima et al., 2008). A number of specialized platforms have been established for metabolites that require specic attention (Arrivault et al., 2009; Kojima et al., 2009; Tohge and Fernie, 2010). It will not be enough to just measure more metabolites. It will also be essential that the research community establishes robust, yet functional and pragmatic, methodologies and protocols to document the reliability of metabolite measurements and to ensure comparability between laboratories. The compilation of large transcriptome databases was enabled by the use of a relatively small number of standardized array platforms to generate the data and by the development of standardized biostatistical routines for quality control. This has only begun for studies of metabolism, as can be seen in a few studies in which standardized protocols are additionally being supported by the development of databases to house the data in an analogous manner to those well established for transcriptomics (Kopka et al., 2005; Schauer et al., 2005; Bais et al., 2010). This task will be complicated by the vast variability of the chemical matrices in different species and by the large number of analytic protocols and machines that are needed to achieve adequate coverage of metabolites. We can expect that there will be a need for continuous effort as method development progresses. However, probably the enabling step will be the establishment of accepted and widely used protocols that ensure the comparability of metabolite data collected in different laboratories, at different times, and even by different analytic machines and methods. This will probably require much closer attention to quantication and the routine use of chemical standards or standardized biological matrices as controls (Fernie et al., 2011). Ideally, this will include the provision of quantitative data rather than relative values. Unless this is done, most studies of metabolism will remain as stand-alone data sets that cannot be rigorously combined in databases and used for comprehensive data mining and meta-analysis. A second set of challenges revolve around the need to obtain spatial information about metabolites. As already outlined, analysis of the cellular and subcellular compartmentation of metabolites is far more difcult than for transcripts and proteins. Nevertheless, methods do exist, and their robustness has been fully documented (Stitt et al., 1980; Chaudhuri et al., 2011). The key issue is that these methods have been underused. This is probably because they are technically challenging and extremely labor intensive. The issue of compartmentation needs to be addressed by more widespread use of existing methods for tissue fractionation, be they cellular based (e.g. laser-capture microdissection, trichome isolation, differential grinding of deep-frozen material) or subcellular based
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(nonaqueous fractionation, silicon oil fractionation, genetically encoded sensors), and the development of novel methods must be prioritized. There is an urgent need to increase the ease by which these methods can be applied, for example, by robotization or by the development of other less time-consuming and novel approaches. Ideally, these would be coupled to more tightly controlled methods of genetic intervention allowing cellular and temporal control. A complementary approach would be to use quantitative analysis of labeling kinetics to distinguish between active and dormant (i.e. metabolic pools that are and are not directly involved in a given metabolic ux). This would be a highly complementary approach, as it would prioritize metabolites and treatments to time-consuming spatial studies. A related area in which information is still lacking, even for major plant pathways such as photorespiration, is the identication of all the required membrane transport proteins (Reumann and Weber, 2006); however, it is conceivable that reconstitution of candidate proteins en masse into liposomes may allow their identication. The vital importance of evaluating the cellular and subcellular compartmentation of metabolism is one that we can no longer afford to ignore, and community acknowledgment of and insistence on this point will be crucial to ensure its future coverage. Use of the available tools for compartmental analysis should become a requirement for studies of metabolism. It should be noted that the suitability of a given method will depend on the turnover time of the metabolite in question. This underlines the importance of information about metabolite turnover times as a vital input in designing experimental protocols. Metabolite turnover times can be calculated from knowledge of pool sizes, pathway ux, and pathway stoichiometry (Arrivault et al., 2009) or determined by isotope labeling experiments (see below). While the fastest turning-over transcripts and proteins in eukaryotes have turnover times on the order of 5 to 10 min, many metabolites have turnover times of less than 1 min, and sometimes even less than 1 s, especially in central metabolism (Arrivault et al., 2009). A third set of challenges revolves around the need to relate measurements of metabolites to pathway ux. Flux is the ultimate output of metabolism. Despite the fact that they are commonly treated as such, metabolites are not all equal. While many are intermediates, some are quasi-products and some are entry-point substrates. As at other levels of the molecular hierarchy of the cell, ux often does not directly correlate with changes in metabolite pool sizes (Cornish rdenas, 2001; see above). This must Bowden and Ca be taken into account in analyzing metabolite data and attempting to integrate them with information about transcript and protein abundance, on the one hand, and biological responses, on the other hand. Many of the issues that complicate the analysis of metabolic networks, which were noted in the rst part of this
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article, revolve around the dichotomy between metabolites as measured parameter and metabolites as components of a pathway and a metabolic ux. We are currently hampered by the overreliance of the plant metabolism research eld on snapshot metabolomics data, wherein metabolic ux is not considered. This shortcoming is tied to partly unsolved technical and conceptual issues as well as cost. The development of kinetic data sets is a clear research priority for the understanding of metabolic networks. The wider employment of ux analysis would be one way to improve this. There are several excellent recent reviews on this topic (Kruger and Ratcliffe, 2009; Schwender, 2011), so we will not retread the same ground; sufce it to say that while measurements of ux are often complicated at the experimental, technical, and theoretic levels, they often provide greater insight into metabolic regulation than steady-state metabolite levels can offer (Baxter et al., 2007). Flux analyses are currently only carried out at a broad scale by a handful of groups worldwide. Nevertheless, these studies have already allowed important advances in our understanding of metabolic compartmentation, the directionality of enzyme operation, and the assignment of enzymes and proteins to hitherto undened processes (Schwender et al., 2004, 2006; Kruger et al., 2007). We can expect that they will also provide important information about the compartmentation of metabolites (although it should be noted that this has been available for over 40 years [see the classic study of Lips and Beevers, 1966]), compartment-specic regulation of uxes (Stitt et al., 1983), and metabolite turnover times. A fourth set of challenges is to combine analyses of metabolites and uxes with information concerning posttranslational modications of proteins and their kinetic properties. This information is needed to close the gap between proteomic and metabolite data sets, due to the major role played by posttranslational mechanisms in the regulation of metabolism (DaranLapujade et al., 2007). However, while such data sets are eminently desirable, they are not facile to produce. Tools to track such modications on a large scale are only on the horizon (Blagoev et al., 2003, Reiland et al., 2011; Taylor et al., 2011; Wu et al., 2011). Pioneering studies have shown that it is possible to analyze posttranslational modications in a comprehensive manner for specied biological systems, as in the mammalian epidermal growth factor signaling pathway (Blagoev et al., 2003). It will be a major challenge to adapt such methods to obtain an integrated overview of posttranslational regulation, metabolite levels, and uxes in major metabolic pathways in plants. The development of routine pipelines to analyze how they affect the activities and characteristics of proteins will be even more challenging. Mass spectrometry-based analyses always face a dilemma between the use of broad and untargeted analyses, which often lack sensitivity and specicity, and targeted analyses of a small number of prioritized
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proteins. Measurements of metabolites can play an important role in prioritizing candidate proteins that are targeted for detailed analyses. In this context, it will be important to give a higher priority to experiments that study transients in which the system is moving from one steady state to another. Studies of transients reveal what happens when systems are knocked out of balance, and how they reestablish the original balance or establish a new balance, thus uncovering links that may be masked in steady-state conditions, including sites at which regulatory loops are operating. Establishing the time scale of changes also immediately highlights if the responses of metabolites and uxes can be explained, or at least solely explained, by transcriptional or translational regulation, or whether posttranslational mechanisms are required (Stitt et al., 1982; Krapp and Stitt, 1995; Gibon et al., 2004, 2006). Time-resolved analyses of transients were a hallmark of many studies of metabolic regulation in the 1960s to 1990s (Krause and Bassham, 1969; Rolleston, 1972; Krapp and Stitt, 1995), but they have not received as much attention as they should have in the last 10 years of plant metabolomics, when the emphasis has been on comparison of steady states. This may be partly because network analyses based primarily on statistical approaches (especially correlations) are less easily applied to non-steady-state data sets. However, dynamic metabolite changes have also been modeled in groundbreaking work on the primary metabolism in Escherichia coli (Yuan et al., 2009). Recent examples include studies in Arabidopsis (Arabidopsis thaliana) that tracked kinetic changes of transcript and metabolite levels after supply of nutrients (Hirai et al., 2004) or after changing the photoperiod or both the photoperiod and temperature (Caldana et al., 2011). These examples illustrate how important information can be derived from combined proling studies not only in identifying clear functional connections between transcripts and metabolites (UrbanczykWochniak et al., 2003; Hannah et al., 2010) but also in instances wherein elements of the networks are disconnected, since these likely indicate that metabolic regulation is unlikely to be carried out at a simple transcriptional or translational level. A nal set of challenges revolve about the need to develop appropriate metabolic models and to integrate these with experimental studies. There is a fundamental reason why models will play an essential role in integrating information about metabolic parameters. In the rst part of this article, we outlined the inherent complexity of metabolic networks. Unless we use models that capture these different elements of the network, it will be impossible to obtain any kind of predictive understanding of the relations between different metabolites or metabolites and proteins (let alone transcripts). Individual focused studies may be able to link different sorts of information in an intuitive manner. However, integration of metabolite data into robust models will probably be a precondition for any large-scale integration with other
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functional levels, both below (e.g. transcript and protein abundance) and above (e.g. changes in composition or growth rates) metabolism. As a rst step, modeling will be required at the level of the metabolites themselves and is most probably best supported by the analysis of kinetic labeling data (Arita, 2003; Huege et al., 2007; Young et al., 2011). Collected experimental data may be (1) at metabolic steady state and isotopic steady state, (2) at metabolic steady state but not at isotopic steady state, or (3) at neither metabolic nor isotopic steady state. The rst of these is relatively trivial to handle mathematically but requires the assumption of a closed system and as such is applicable in only a limited number of scenarios in the life cycle of a plant. Dynamic metabolite changes have also been modeled in groundbreaking work on the primary metabolism in E. coli (Yuan et al., 2009). Mathematical methods are also now available to extract information from non-steady-state labeling kinetics and have been applied in studies of plant secondary metabolism (Heinzle et al., 2007) and photosynthesis (Young et al., 2011). The use of stable isotopes to analyze ux is likely to go hand in hand with the further development of mathematical models to extract information from non-steady-state labeling kinetics. Future developments may even allow important information to be gleaned from conditions under which neither metabolic nor isotopic steady state is achieved. It seems likely that large-scale stoichiometric, kinetic, and thermodynamic supported metabolic modeling, coupled with a clear understanding of the benets and limitations of each type of model, will further aid in our understanding of the systems biology underlying plant phenotypes (Sweetlove and Ratcliffe, 2011). Groundbreaking work of the Palsson group has already layered gene-regulatory models on top of ux-balance models to considerable effect (Schellenberger et al., 2011). Even when used independently of gene-regulatory models, metabolic models can provide important insights, for example, into the operation of Rubisco in the absence of the Calvin cycle, the wide variety of ux modes of the TCA cycle, and the inherent stability of central metabolism to environmental perturbation (Schwender et al., 2004; Williams et al., 2008; Sweetlove et al., 2010), while genome-wide metabolic reconstructions have been used to dene the crucial metabolic reactions that are needed to maintain growth (Sweetlove and Ratcliffe, 2011). Taking this one step further by using the approach pioneered by Palsson (Schellenberger et al., 2011) and layering gene-regulatory models or other such levels of information on top of ux-balance models (Cho et al., 2011) will certainly prove an important step forward in plant systems biology. Simple stoichiometric models are independent of data quality and are the most reliable, at least when evaluated from this criteria alone. However, models based on pathway stoichiometry alone can only be used to ask questions that do not depend on more
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complex features, like the thermodynamic structure of a pathway and the kinetic and regulatory properties of enzymes. Evaluation of the utility of other kinds of plant metabolic models is currently difcult, since our community experience is lacking. It will also require the collection of more information. Research priorities in this area include (1) the identication of missing reactions (including transport steps) within the metabolic network and (2) the establishment of a more physiologically relevant and systems-wide enzyme kinetic characterization, as already dened by the STRENDA consortium (Apweiler et al., 2005). It is also our opinion that models of metabolism can be greatly enhanced by the incorporation of information about metabolite levels at a subcellular resolution. As already discussed, in addition to tissue fractionation, labeling kinetics can provide a valuable source of information regarding the likelihood of there being more than one metabolic pool and even the relative sizes of pools within the cell (Roessner-Tunali et al., 2004; Kruger et al., 2007). Indeed, this is already being implemented in some plant metabolic models (M. Szecowka, R. Heisse, Z. Nikoloski, M. Stitt, A.R. Fernie, and S. Arrivault, unpublished data). Such data sets will be of increasing importance with the enhanced scope of models being generated, since large-scale models will likely require an elevated quantity and quality of data for effective parameterization. However, it is perhaps pertinent to highlight that, as noted previously, model quality can be improved by precise information about the subcellular concentrations of even a single metabolite (Wiechert et al., 2007). Thus, it is highly likely that the experimental approaches currently available, if comprehensively applied, will be sufcient to provide the information required to accurately model metabolism.
Received January 2, 2012; accepted January 16, 2012; published January 17, 2012.

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