Review articleCan stable isotope mass spectrometry replace radiolabelled approaches in metabolic studies?
Introduction
Metabolomics, the study of the metabolite complement of an organism or cell within a particular physiological condition, is a rapidly expanding research field with several applications ranging from broad phenotyping and diagnostic analysis to metabolic engineering and systems biology. Indeed, the metabolic network is one of the best characterized networks within biological systems. Plant metabolic studies were relatively neglected for a couple of decades, but they have received tremendous attention in the last decade [1], with significant developments in metabolite profiling considerably contributing to this renaissance. This is, at least in part, associated with such issues as the rising demand for bioenergy, concerns about crop yield stability particularly under the enhanced stress conditions anticipated with climate change, and bioprospecting for active natural products with significant efficacy against human diseases. Metabolomics is a relatively young science but there is a vast history of biochemistry and analytical chemistry studies underpinning it. In fact, pioneering studies which established key metabolic pathways such as the tricarboxylic acid (TCA) cycle [2], [3], [4], [5], as well as the glyoxylate [6] and the Calvin-Benson [7] cycles of plants were of pivotal importance in defining the structure of metabolic pathways. Thus, there is a vast body of descriptive and mechanistic data related to unravelling plant metabolic pathways and their regulation [8]. This fact notwithstanding, our ability to rationally engineer plant metabolism or to predict metabolic responses to stressful conditions as yet remains rather limited.
Given the pivotal role of metabolism in ultimately supporting both plant growth and development it is reasonable to assume that experiments which provide insights into metabolic flux coupled with its regulation would probably both improve our knowledge of biological systems and ultimately aid in the discovery of gene function in land plants. In this vein, a range of stable isotopic labeling experiments using for instance 13C and 15N-labelled precursors have been applied to investigate particular aspects of plant metabolism both in isolated organelles or cells [9], or in plant organs or whole plants [10], [11], [12]. Feeding experiments using stable isotopes (e.g., 13C and 15N-labelled precursors) have been also extensively used to delineate metabolic pathways but may equally be used to investigate and quantify metabolic flux and thus enhancing our understanding of the structure and regulation of metabolic pathways [13]. Labeling experiments with 14C-labelled substrates offer high sensitivity, and the fractionation of labelled metabolites and biomass components can readily indicate the fate of metabolized radiolabel [4]. Notably, the determination of radioisotope incorporation into biomass is not dependent of prior separation of metabolites per se and the presence of compounds which were not labeled does not impact the detection of radioactivity via scintillation counting or radiography [14].
In the past, radiolabelling was extensively used to discriminate metabolic intermediates belonging to a pathway and their respective order [15]. Thus, compounds that are more labelled are closest to the initial substrate, and the pathway can be defined according to the dilution of radiolabeling along the pathway. This is basically what allowed the differentiation of C3 and C4 modes of photosynthesis through the first labelled compound [16], [17]. Notably, short term experiments with labelled compounds were used qualitatively both to determine the sequence of intermediates of specific metabolic pathways and to quantify unidirectional fluxes. Historically speaking, such measurements were carried using spectrophotometric or simple chromatographic separation. By simply comparing the cost of those methodologies, one can expect that it should be less expensive than methods offering both high accuracy and sensitivity for analysis of highly complex mixture of compounds that have been developed during the last decades.
Quantifying the 14CO2 release by using different isotopomers of 14C-glucose provided compelling evidence for the relative activity of different metabolic pathways [4]. For instance, by following the pattern of 14CO2 evolution from various differentially labelled glucose supplied to plant tissues allowed the identification of alterations in metabolic flux involved in the oxidation of carbohydrates [18], as well as from the differential cycling through the oxidative pentose phosphate pathway (OPPP) or in pentan synthesis and TCA cycle [19], [20]. This comparison is based on the ratios of 14CO2 evolution from [1-14C]-, [2-14C]-, [3,4-14C]-, and [6-14C]glucose, that are typically used for understanding relative flux through different sections of the central pathways of carbohydrate oxidation [18], [20], [21]. In summary, by following the radioactivity distribution from different labelled substrates can be used as suitable method covering all major metabolic products (Fig. 1). Furthermore, it can be also applied as a sensitive method for determination of major flux in cells. Thus, although the utility of radiolabeling for the elucidation of metabolic pathways in plants has been extensively demonstrated in the past [22], [23], [24] it is still well suited to facilitate the elucidation of the metabolic behavior particularly following genetic or environmental perturbations.
Metabolomics studies are performed with the application of either nuclear magnetic resonance spectroscopy (NMR) or mass spectrometry (MS)-based methodologies. Briefly, NMR is quantitative and potentially non-destructive which offers the possibility to obtain real time metabolic concentrations in living cells. This fact apart, MS based platforms are more frequently used for metabolomics studies mostly due to its higher sensitivity and detection of larger number of metabolites (Fig. 1). In addition, isotope labeling offers advantages for both methods. The utilization of MS or NMR for detection of label incorporation allows the determination of isotopic enrichment (and in the case of NMR the exact isotope location) in individual metabolites (Fig. 1) without laborious chemical cleavage experiments such as those elegantly carried out previously following incubation with different 14C labelled substrates [25], [26], [27], [28], [29]. Furthermore, analysis of the isotopomer abundance of labelled metabolites offer a perspective in understanding the way by which the fluxes are distributed within a particular plant metabolic network. Thus, in the context of systems biology, metabolic networks deduced by labeling techniques provide several opportunities for reconstruction and validation of both dynamic and stoichiometric models of metabolism. As alluded to above one inherent disadvantage of stable isotope labeling approaches is that the detection of metabolites is highly dependent upon analytical capabilities. Although there are different methods of metabolite analysis such as liquid and gas chromatography coupled with MS, capillary electrophoresis, and NMR, here we will concentrate our discussion on the general pros and cons of MS approaches in comparison to radiolabelled approaches that are currently used for analyzing metabolite fates in plants. Before doing so it is important to note that despite its low sensitivity NMR has two strong advantages as a tool for flux analysis. First, it can be used in a non-destructive manner and plant cell metabolism can thus be monitored in vivo. Secondly, it provides positional information as to where within a molecular isotope accumulates which is highly useful for flux estimation since it facilitates the estimation of reverse fluxes [30]. However, these features and general properties of NMR are the subject of several excellent recent reviews [30], [31], [32] so we chose to focus this article on MS approaches.
In this review, we discuss the conceptual basis of these approaches, as well as their historical application and limitations, finally providing an update on recent technical developments. Current methodological challenges that limit the dissection of plant metabolism and future perspectives are briefly discussed and particular emphasis is placed on the importance of developing novel methods. We additionally attempt to discuss the importance of more sensitive flux profiling methodologies using stable isotopes to enable us to pursue new avenues of research in order to increase our understanding of complex metabolic networks governing plant metabolism.
Section snippets
Natural abundance studies
Natural abundance studies use the small differences in isotopic ratios that are observed in nature and are usually expressed as the difference delta (δ) to samples in which no further tracer has been applied. The natural abundance of radioactive species is usually negligible but it can be considerable for stable isotopes (e.g. 1.1% for 13C). The occurrence of isotope effects is mainly due to the preferential use of certain isotopes (e.g. 12C compared to 13C) in many biological (and chemical)
Metabolic application of labeling approaches: current status
The use of isotopes of different chemical elements is a useful tool for plant physiologists, and are currently used to generate insights into how exactly the metabolism behaves in response to environmental conditions [44], [81]. They provide insights into the isotopic composition of the plant material, which have great applications for instance in ecophysiology in analyzing plant water and nitrogen use efficiency [41]. Furthermore, isotopic labeling is powerful in determining metabolic fluxes,
Applications of stable and radioisotope labeling in plant metabolic studies
Labeling studies are directly dependent on administration of specific (radio)isotope labelled substrate to a tissue or organism and the capability to monitor the propagation of the tracer through the metabolic network. A pre requisite of this approach is that the structure of metabolic network should be known [105]. The application of labelled substrates in both prokaryote [82], [106] and eukaryote cell cultures [107], [108] is easily performed and widely accepted. However, its application to
Conclusions and perspectives
The importance of metabolic analysis coupled with analysis of flux is increasingly becoming an essential component of efforts to increase our understanding of the control and regulation of the metabolic networks. Studies involving both metabolic profiles and metabolic flux analysis are of high relevance particularly when used in conjunction with other post-genomic technologies and will additionally allow a better description of the metabolic network in several contexts. Likewise, the
Acknowledgements
We apologize to all colleagues whose work has not been mentioned due to space limitations and thank anonymous reviewers for their valuable comments and suggestions. This work was supported by funding from the Max Planck Society (to ARF, ANN and WLA) and the National Council for Scientific and Technological Development (CNPq-Brazil, Grant 306355/2012-4 to A.N.N and Grant 483525/2012-0 to W.L.A.) and the FAPEMIG (Foundation for Research Assistance of the Minas Gerais State, Brazil, Grant
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