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The use of metabolomics to dissect plant responses to abiotic stresses

The use of metabolomics to dissect plant responses to abiotic stresses Cell. Mol. Life Sci. (2012) 69:3225–3243 DOI 10.1007/s00018-012-1091-5 Cellular and Molecular Life Sciences MU LTI-A UT HO R R EVI E W The use of metabolomics to dissect plant responses to abiotic stresses Toshihiro Obata Alisdair R. Fernie Received: 7 July 2012 / Revised: 9 July 2012 / Accepted: 9 July 2012 / Published online: 12 August 2012 The Author(s) 2012. This article is published with open access at Springerlink.com Abstract Plant metabolism is perturbed by various Introduction abiotic stresses. As such the metabolic network of plants must be reconfigured under stress conditions in order to When plants face unfavourable growth conditions, abiotic allow both the maintenance of metabolic homeostasis and stress retards plant growth and productivity. Under most the production of compounds that ameliorate the stress. abiotic stress conditions, plant metabolism is perturbed The recent development and adoption of metabolomics and either because of inhibition of metabolic enzymes, shortage systems biology approaches enable us not only to gain a of substrate, excess demand for specific compounds or a comprehensive overview, but also a detailed analysis of combination of these factors and many other reasons. crucial components of the plant metabolic response to Therefore, the metabolic network must be reconfigured to abiotic stresses. In this review we introduce the analytical maintain essential metabolism and to acclimate by adopt- methods used for plant metabolomics and describe their ing a new steady state in light of the prevailing stress use in studies related to the metabolic response to water, conditions. This metabolic reprogramming is also neces- temperature, light, nutrient limitation, ion and oxidative sary to meet the demand for anti-stress agents including stresses. Both similarity and specificity of the metabolic compatible solutes, antioxidants and stress-responsive responses against diverse abiotic stress are evaluated using proteins. The accumulation of reactive oxygen species data available in the literature. Classically discussed stress (ROS) is another problem causing oxidation and dysfunc- compounds such as proline, c-amino butyrate and poly- tion of cellular components and in the worst case cell amines are reviewed, and the widespread importance of death. The optimisation of metabolic flux via the organellar branched chain amino acid metabolism under stress con- electron transport chains is, moreover, crucial in order to dition is discussed. Finally, where possible, mechanistic dampen ROS production. Maintenance of the redox state in insights into metabolic regulatory processes are discussed. the cell is thus another important task to provide the reducing power required for ROS scavenging. Despite such Keywords Metabolomics  Plants  Abiotic stress  important roles of metabolic regulation under stress con- Metabolic response  Branched chain amino acid  ditions, our current understanding of this process is Enzyme complex fragmented and far from complete. Metabolomics is a powerful tool by which to gain a comprehensive perspective of how metabolic networks are regulated and has indeed been applied by many researches in recent years. It can, additionally, be used to elucidate the Electronic supplementary material The online version of this functions of genes as a tool in functional genomics and article (doi:10.1007/s00018-012-1091-5) contains supplementary material, which is available to authorized users. systems biology approaches. The term ‘‘metabolomics’’ is defined as comprehensive and quantitative analysis of all T. Obata  A. R. Fernie (&) small molecules in a biological system [1]. The plant Max Planck Institute of Molecular Plant Physiology, kingdom may contain between 200,000 and 1,000,000 Am Muhlenberg 1, 14476 Potsdam-Golm, Germany metabolites, while for a single species the number may e-mail: fernie@mpimp-golm.mpg.de 123 3226 T. Obata, A.R. Fernie approach a few thousand (the estimate for Arabidopsis than 1 kDa) difficult. Due to these characteristics, GC-MS is ca. 5,000) [2–5]. Indeed the KNApSAck database (http:// facilitates the identification and robust quantification of a kanaya.naist.jp/KNApSAcK/) contains around 50,000 few hundred metabolites in plant samples including sugars, metabolite entries in plants described so far in the literature sugar alcohols, amino acids, organic acids and polyamines, [6]. Due to the large variety of chemical structures and resulting in fairly comprehensive coverage of the central properties of small molecules, there is so far no single pathways of primary metabolism. technique to identify and quantify all of them. Even the most comprehensive methods detect only between 1,000 and Liquid chromatography (LC)-MS 2,000 molecular features [7, 8]. Several techniques includ- ing gas chromatography-mass spectrometry (GC-MS), While GC has a limitation due to volatilisation of com- liquid chromatography (LC)-MS, capillary electrophoresis pounds, LC does not require prior sample treatment and (CE)-MS and nuclear magnetic resonance spectroscopy separates the components in a liquid phase. The choice of (NMR) are commonly used in plant metabolomics research. columns, including reversed phase, ion exchange and They are used sometimes in combination since they are hydrophobic interaction columns, provides the separation largely complimentary with each independent method of metabolites based on different chemical properties. having preferential coverage of diverse types of metabolite. Therefore, LC has the potential to analyse a wide variety of Here we briefly introduce the advantages and limitations of metabolites in plants. The recent development of ultra- each method. Thereafter studies employing the metabolo- performance liquid chromatography (UPLC) makes the mic approach to dissect plant response to abiotic stresses technique more powerful because of its higher resolution, will be discussed. Finally, using data already collected, sensitivity and throughput than conventional high-perfor- we attempt to elucidate the general and stress-specific mance liquid chromatography (HPLC) [16]. Electrospray responses. ionisation (ESI) is widely used for ionisation to connect LC and MS. Many types of MS including quadrupole (Q), TOF, qTOF, triple quadrupole (QqQ), ion trap (IT), linear Techniques used for plant metabolomic research trap quadrupole (LTQ)-Orbitrap and Fourier transform ion cyclotron resonance (FT-ICR)-MS are used depending on Gas chromatography-mass spectrometry (GC-MS) the sensitivity, mass-resolution and dynamic range required (see [17, 18] for the detail). The combination of these Gas chromatography-mass spectrometry is the most widely techniques allows us to identify and quantify a large variety used technique for plant metabolomics research to date. of metabolites even if they have high molecular mass, great polarity and low thermostability. On the other hand the Polar metabolites are derivatised to render them volatile and then separated by GC. Electron impact (EI) allows flexibility of the method also causes difficulty in estab- robust interfacing of GC with MS resulting in highly lishing large mass spectral libraries for peak identification reproducible fragmentation patterns. For detection, time- because of the instrument-type dependent retention time of-flight (TOF)-MS has become the method of choice and mass spectra [19], and forces each research group to because of advantages including fast scan times, which create its own ‘‘in-house’’ LC-MS reference library. That give rise to either improved deconvolution or reduced run said, there are a number of websites that aid in mass- times for complex mixtures, and high mass accuracy. The spectral analyses [20], and recent recommendations for crucial advantage of this technology is that it has long been metabolite reporting [7] should improve the transparency used for metabolite profiling, and thus there are stable of the methods used by researchers. Furthermore, isotope protocols for machine setup and maintenance, and chro- labelling as a means of confirming the identity of peaks has matogram evaluation and interpretation [9–11]. The recently been proposed and demonstrated to allow the robustness of the protocol means that libraries of retention identification of circa 1,000 metabolites using the FT-ICR- time and mass spectra data for standard compounds can be MS approach [8, 21]. To date, LC-MS is mainly used with shared among laboratories [12]. There are several metab- a reverse phase column to analyse secondary metabolites olite databases available including the NIST [13], FiehnLib because of its ability to separate compounds with similar [14] and Golm metabolic databases (GMD, [15]), which structure and to detect a wide range of metabolites. How- are useful tools for peak annotation. Additionally, the short ever, it is worth noting that specialised protocols for running time and relatively low running cost are also strong determining phosphorylated intermediates, which are not advantages of GC-MS. However the use of GC-MS is readily detected by GC-MS, have also begun to be devel- limited for thermally stable volatile compounds, making oped using this technology [22], as have methods for the the analysis of high molecular weight compounds (larger comprehensive analysis of phytohormones [23]. 123 Metabolomics of plant stress 3227 Capillary electrophoresis (CE)-MS vacuole from the rest of the cell cause distinctive signals from an identical metabolite and thus allows quantification Capillary electrophoresis separates polar and charged at the subcellular level [34, 35]. Thus analysing the compounds on the basis of their charge-to-mass ratio. CE is metabolite composition of a tissue extract, determining the able to separate a diverse range of chemical compounds structure of a novel metabolite, demonstrating the exis- and is a more powerful technique than LC with respect to tence of a particular metabolic pathway in vivo, isotope separation efficiency [24, 25]. ESI is commonly used for labelling experiment and localising the distribution of a ionisation as in LC-MS, with TOF-MS being the most metabolite in a tissue are all possible by NMR. For isotope commonly used detector in CE-MS-based metabolomics labelling, NMR has the advantage of providing facile studies. This combination provides high mass accuracy and access to atomic level labelling, which is highly laborious high resolution. The high scan speed of TOF-MS makes in the case of MS methods yet can be essential in flux this instrument very suitable for full scan analyses in estimation [35]. However, the number of compounds that metabolomics. One of the unique properties of CE-MS is can be detected in a single analysis is limited to one to the small amount of sample required for analysis; only several dozen [36, 37]. These properties of NMR make it nanolitres of sample are introduced into the capillary. the ideal tool for broad-range profiling of abundant Together with high electric fields and short separation metabolites whilst studying changes in non-annotated lengths, it can produce analysis within seconds. It also profiles is highly useful for metabolite fingerprinting of allows the metabolic analysis in volume-restricted samples. extensive sample collections [38, 39]. On the other hand, this leads to low concentration sensi- tivity requiring enrichment of metabolites within the samples [26]. Another drawback of CE is the poor migra- Metabolomic studies of plant stress responses tion time reproducibility and lack of reference libraries, which may only be partially overcome by the prediction of Metabolomics is becoming increasingly common in plant migration time [27]. Since CE and LC can both separate a physiology and biochemistry, and to date has been applied large variety of metabolites via fundamentally different to a staggering number of conditions. Here we will attempt mechanisms, they are often used in combination to provide a synthesis of the most prominent studies dealing with a wider coverage of metabolites [28–30]. That said, the use plant stress; however, the reader is also referred to two of CE-MS in plant studies remains relatively rare. previous reviews on this topic [40, 41]. In this section we independently review water stress, temperature stress, light Nuclear magnetic resonance (NMR) spectroscopy stress, ionic stress, nutrient limitation and oxidative stress before discussing stress combinations. We describe the Nuclear magnetic resonance spectroscopy offers an nature and symptoms of each stress, and then introduce entirely different analytical technique to that afforded by several metabolomic studies with the main metabolic MS-based techniques being based on atomic interaction. In changes observed in each study and the conclusion drawn a strong magnetic field, atoms with non-zero magnetic from the results. Following this survey we discuss com- 1 13 14 15 31 moment including H, C, N, N and P absorb and monalities and differences between the various stress re-emit electromagnetic radiation. The radiation is char- responses. acterised by its frequency (chemical shift), intensity, fine structure and magnetic relaxation properties, all of which Water stress reflect the precise environment of the detected nucleus. Therefore, atoms in a molecule give a specific spectrum of Water limitation is one of the major threats in crop pro- radiation that can be used for identification and quantifi- duction and this condition is projected to get considerably cation of metabolites within a complex biological sample. worse in coming decades [42]. For this reason considerable The sensitivity of this method is much lower than that of research effort has been expended to understand the MS-based techniques but the structural information con- response to this crucial and common stress. These studies tent, reproducibility and quantitative aspects can be have revealed an important role for metabolic regulation superior to them, and some journals require NMR spectra including regulation of photosynthesis and accumulation of as the final proof of chemical structure [31, 32]. Further- osmolytes in the drought stress response [43, 44]. Urano more the preparation of the sample is simple and even non- et al. [28] reported metabolomic changes in Arabidopsis destructive measurement is possible. In vivo NMR can leaves under drought condition. The accumulation of many further generate kinetic measurements and examine meta- metabolites was observed, including amino acids such as bolic responses on the same plant rather than on a set of proline, raffinose family oligosaccharides, c-amino buty- similar plants [33]. The different subcellular pHs of the rate (GABA) and tricarboxylic acid (TCA) cycle 123 3228 T. Obata, A.R. Fernie metabolites, which are known to respond to drought stress phosphate and glycerol-3-phosphate, were observed as well in plants. The authors also investigated the nc3-2 mutant, as changes in the levels of minor sugars and various which lacks the NCED3 gene involved in the dehydration- organic acids. When oxygen is decreased to 4 %, there is a inducible biosynthesis of abscisic acid (ABA), in order to general tendency for an increase in the levels of the assess the effect of ABA in the metabolic response to intermediates both of sucrose degradation and the TCA drought stress. By combination with transcriptome analysis cycle, and in the levels of most amino acids, whereas they they clearly demonstrated that the ABA-dependent tran- are decreased when the oxygen further decreased to 1 %, scriptional regulation is responsible to the activation of indicating the inhibition and reactivation of metabolic metabolic pathways including branched chain amino acid, activities. Together with the transcriptomic data showing a polyamine and proline biosynthesis, GABA shunt and general downregulation of energy-consuming processes, saccharopin metabolism, but is not involved in the regu- the results demonstrated a large-scale reprogramming of lation of the raffinose biosynthetic pathway during metabolism under oxygen-limited conditions. Rocha et al. dehydration. [51] examined the accumulation of alanine under anoxic In Arabidopsis research, drought tolerance is assessed conditions in Lotus japonicus, which is highly tolerant to predominantly under lethal conditions. However, in tem- water logging. In the roots of L. japonicus, succinate, perate climates, limited water availability rarely causes alanine and the direct co-substrates for alanine synthesis, plant death but does restrict biomass and seed yield. glutamate and GABA, were highly accumulated during Results of a recent elegant study experimentally demon- water logging, whereas the majority of amino acids that are strated that the survival rate under lethal conditions does derived from TCA cycle intermediate decreased. The not predict superior growth performance and biomass yield results are in agreement with the metabolic equilibriums gain under moderate drought [45], making this mild stress that are expected to drive the metabolic flux from glycol- condition more important. Skirycz et al. [46] conducted ysis, via alanine synthesis and oxoglutarate to succinate, metabolite profiling of Arabidopsis leaves that develop which prevents the accumulation of pyruvate activating under mild osmotic stress. They revealed that the stress fermentation and leading to ATP production by succinyl- response measured in growing and mature leaves was CoA ligase. largely distinct. Typical drought responses, namely accu- mulation of proline, erythritol and putrecine, were Temperature stress observed only in mature leaves, while many metabolites were decreased in expanding leaves, sharing the same Exposure to freezing environments leads to serious damage tendency with transcriptional response. When we com- of the plant cell by ice formation and dysfunction of cel- lular membranes [52]. Many plant species increase freezing pared the data from the studies [28, 46], 24 metabolites were detected in both. The decrease of aspartate and tolerance during exposure to non-freezing low temperature increase of proline are the only two responses shared by a process known as ‘‘cold acclimation’’. The molecular between mildly and severely desiccated leaves. Pro- basis of this process has been extensively studied, and the nouncedly, amino acid metabolism responds in opposite contribution of particular metabolites including compatible ways; most amino acids were accumulated in severely solutes [53] and the transcriptional regulatory network has desiccated leaves but decreased in mildly desiccated plants. been elucidated [54, 55]. The first metabolomic studies of These results highlight the variable response of plant cold acclimation were performed by two groups in 2004. metabolism in different developmental stages and degrees Cook et al. [56] compared metabolomic changes during of desiccation. Metabolite profiling has additionally been cold acclimation in two ecotypes of Arabidopsis thaliana, carried out in crop species exposed to water stress condi- Wassilewskija-2 (Ws-2) and Cape verde islands-1 (Cvi-1), tions. Intriguingly, common changes in the levels of which are relatively freezing tolerant and sensitive, metabolites including branched chain amino acids were respectively. The metabolome of Ws-2 plants was exten- observed in wheat, barley and tomato [47–49]. sively altered in response to low temperature. Seventy-five Too much water, as occurs in situations such as flooding percent of metabolites monitored were found to increase in or water-logging of the rhizosphere, also causes problems cold-acclimated plants including metabolites known to because of the reduced oxygen availability (hypoxia/ increase in Arabidopsis plants upon exposure to low tem- anoxia). Under anoxic conditions, ATP has to be produced perature, such as the amino acid proline and the sugars by fermentation, resulting in cytosolic acidification and the glucose, fructose, inositol, galactinol, raffinose and accumulation of toxic products. van Dongen et al. [50] sucrose. They also found novel changes—namely the analysed metabolic responses in Arabidopsis roots under increase of trehalose, ascorbate, putrescine, citrulline and anoxic conditions. The accumulation of amino acids, ala- some TCA cycle intermediates. There was considerable nine, proline and GABA, and the phosphoesters, glucose-6- overlap in the metabolite changes that occurred in the two 123 Metabolomics of plant stress 3229 ecotypes in response to low temperature; however, quan- effect. The esk1 mutant is isolated as freezing tolerant titative differences were evident. Kaplan et al. [57] without previous acclimation but the function of this gene conducted metabolome analysis of Arabidopsis over the had been unknown. Lugan et al. [64] tried to elucidate the time course following the shift to cold and heat conditions. basis of the freezing tolerance of esk1 by performing Surprisingly the majority of heat shock responses were metabolomic analysis under various environmental condi- shared with cold shock including the increase of pool sizes tions, namely cold, salinity and dehydration. Then the most of amino acids derived from pyruvate and oxaloacetate, specific metabolic responses to cold acclimation were not polyamine precursors and compatible solutes. The results phenocopied by esk1 mutation. However, esk1 accumu- of this study were analysed together with following tran- lated lower amount of Na in leaves than the wild type and script profiling data by the same group [58], and revealed its metabolic profile, and osmotic potential were only that the regulation of GABA shunt and proline accumula- slightly impacted under dehydration stress. These results tion under cold conditions are achieved by transcriptional suggested that ESK1 could rather be involved in water and post-transcriptional manners, respectively. Gray and homeostasis and as such highlighted the importance of Heath [59] examined the effects of cold acclimation on the cellular water status in stress tolerance. Arabidopsis metabolome using a non-targeted metabolic fingerprinting approach. It revealed a global reprogram- Light stress ming of metabolism as well as differential responses between the leaves that shifted to and those that developed Light is a highly energetic substrate driving photosynthesis in the cold. Hannah et al. [60] took advantage of the natural that can induce secondary destructive processes at the same genetic variation of Arabidopsis to elucidate the function of time. Therefore, too high light irradiance represents an metabolism in cold acclimation. Although there is no clear abiotic stress factor for plants. Wulff-Zottele et al. [65] relationship between global metabolite changes and dif- conducted metabolite profiling of Arabidopsis leaves for ferences in acclimation capacity or differences between the 6 days after transition to high light. Generally, most of the accessions in acclimated freezing tolerance, the probable metabolites of the glycolysis, TCA cycle and oxidative importance of central carbohydrate metabolism is indicated pentose phosphate pathway were altered in their content, by the identification of glucose, fructose and sucrose indicating that plants exposed to high light undergo a among metabolites positively correlating to freezing tol- metabolic shift and enhance the Calvin-Benson cycle to fix erance. Espinoza et al. [61] analysed the effect of diurnal more carbon. In addition, elevation of glycine indicated the gene/metabolite regulation during cold acclimation by activation of photorespiratory pathways. Caldana et al. [66] means of metabolomics and transcriptomics. Approxi- investigated the early metabolic response against high light mately 30 % of all analysed metabolites showed circadian as a part of a more comprehensive study. The accumulation oscillations in their pool size and low temperature affected of the photorespiratory intermediates, glycine and glyco- the cyclic pattern of metabolite abundance. These results late, were observed in the early phase (5–60 min after indicated that the interactions observed between circadian transition). Interestingly the response during the mid phase and cold regulation are likely highly relevant components (80–360 min) shares similar properties with low tempera- of cold acclimation. ture treatment, which includes the accumulation of Metabolomics was also used to reveal the functions of shikimate, phenylalanine and fructose, and the decrease of specific genes in cold acclimation. In the study described succinate; however, the physiological meaning of this above, Cook et al. [56] also investigated plants over- overlap is currently unknown. expressing CBF3, which is one of the C-repeat/dehydration Not only the quantity but also the quality of light affects responsive element-binding factor (CBF) transcriptional plant metabolism. In dense plant stands, such as crop fields activators induced rapidly under low temperature condi- or forests, individuals shade each other and create com- tions [62]. The metabolite profiles of non-acclimated CBF3 petition for light absorption [67]. Selective light absorption overexpressing lines were quite similar to those of the cold- by the upper leaf layers leads to an enrichment of far-red acclimated Ws-2 ecotype, suggesting a prominent role for wavelength [68], which induces excitation imbalances the CBF cold response pathway in configuring the low- between photosystem II and I disturbing both the redox temperature metabolome of Arabidopsis. Maruyama et al. chemistry in the transport chain and its coordination with [63] explored metabolic and transcript changes in Arabid- the Calvin-Benson cycle [69, 70]. For this reason, Bra ¨uti- opsis plants overexpressing CBF3/dehydration-responsive gam et al. [71] grew Arabidopsis plants under light, which element binding protein (DREB)1A and another DREB preferentially excited either photosystem I (PSI light) or II protein DREB2A. They observed similar changes of (PSII light) and then transferred it to the other light con- metabolites in CBF3-overexpressing plants like Cook et al. dition to analyse how plants acclimate to the light quality [56] but DREB2A overexpression showed only a minor shift. After long-term acclimation of 48 h, plants exhibited 123 3230 T. Obata, A.R. Fernie two distinct metabolic states. A PSI–II shift resulted in a Proline increased dramatically in both species as did decrease in primary products of photosynthesis, such as inositols, hexoses and complex sugars. The concentrations sugars, but an increase in important intermediates of sub- of metabolites were often several-fold higher in Thel- sequent metabolic pathways. By contrast, a PSII-I shift has lungiella and stress exacerbated the differences in some no effect on the sugar pools but leads to general down- metabolites. Transcript analyses supported the metabolic regulation of many subsequent metabolites, including results by suggesting that a Thellungiella is primed to amino acids and organic acids. Each of the metabolites anticipate such stresses. The difference in metabolites exhibited a different accumulation profile for establishing between Arabidopsis and Thellungiella under salt and the final pool size, indicating high complexity by which the osmotic stresses was more recently assessed for a broader two metabolic states were achieved. Comprehensive anal- range of metabolites [80]. Analysis of global physico- yses of these data alongside transcript profiles and other chemical properties of metabolites revealed a shift from physiological data suggested that photosynthesis and nonpolar to polar metabolites in both species but that this metabolism were under the control of a binary combination was much more pronounced in Thellungiella. Such a shift of inputs from the thioredoxin and plastoquinone systems. may contribute to keep the water potential during dehy- The dependency of plants upon sunlight also inevitably dration. Kim et al. [77] investigated the cellular level leads them into exposure to ultraviolet (UV) light, metabolic response using Arabidopsis T87 cultured cells. including in the wavelength range of 280–320 nm (UV-B). The results suggested that the methylation cycle for the This wavelength potentially damages DNA, RNA and supply of methyl groups, the phenylpropanoid pathway for proteins, and additionally increases the production of free lignin production and glycine betaine biosynthesis are radicals [72, 73]. Kusano et al. [74] treated Arabidopsis synergetically induced as a short-term response against plants with UV light and analysed the metabolic effect of salt-stress treatment. The results also suggest the UV light stress. Arabidopsis exhibits an apparent biphasic co-induction of glycolysis and sucrose metabolism as well response to UV-B stress, characterised by major changes in as co-reduction of the methylation cycle as long-term the levels of primary metabolites, including ascorbate responses to salt stress. derivatives. By contrast, mid- to late-term responses were Due to the importance of salinity stress in agriculture, observed in the classically defined UV-B protectants, such there are many metabolomic studies to assess the meta- as flavonoids and phenolics. The results suggested that in bolic effect of salinity in a variety of crop and related early stages of exposure to UV-B, the plant cell is ‘primed’ plant species including tomato [40, 81], grapevine [82], at the level of primary metabolism by a mechanism that poplar [83], sea lavender (Limonium latifolium,[84]) and involves reprogramming of the metabolism to efficiently rice [85]. Since these studies have been extensively divert carbon towards the aromatic amino acid precursors reviewed in [40, 86], we focus here on three recent studies of the phenylpropanoid pathway. It also suggested the on legume species [87–89]. These recent studies took a importance of ascorbate in the short-term response to functional genomic approach that integrated ionomic, UV-B. Further studies are, however, required to determine transcriptomic and metabolomic analyses of the glycopyte which of these metabolic changes are end responses to model legume Lotus japonicus and other Lotus species adapt to the enhanced exposure to UV-B and which are part subjected to long-term regimes of non-lethal levels of of the perception-signalling relay, which alerts the plant salinity. In Lotus japonicus the metabolic changes were cell that it needs to respond to the stress [75]. characterised by a general increase in the steady-state levels of many amino acids, sugars and polyols, with a Ion stress concurrent decrease in most organic acids [87]. The responses to salinity stress were compared between High levels of salinity in the soil hinder the growth and extremophile (L. creticus) and glycophytic (L. cornicula- development of crops and cause serious problems for world tus and L. tenuis), but the metabolic responses were food production [76]. High concentrations of NaCl may globally similar to each other [88]. These results suggest cause both hyperionic and hyperosmotic stress effects, that, in contrast to Thellungiella, the metabolic pre- which lead to a decline of turgor, disordered metabolism adaptation to salinity is not the major trait of L. creticus and the inhibition of uptake of essential ions, as well as contributing to the extramophile phenotype. However, by other problems in plant cells [77, 78]. Gong et al. [79] comparing six species displaying different salt tolerances, conducted metabolite profiling of salt-treated Arabidopsis they observed several genotype-specific features. One of thaliana and its relative Thellungiella halophila (salt them is the increase of asparagine levels in the more cress), which shows ‘extremophile’ characteristics mani- tolerant genotypes, suggesting that the roles of asparagine fested by extreme tolerance to a variety of abiotic stresses, metabolism in supporting core nitrogen metabolism may among them low humidity, freezing and high salinity. play a role in tolerance [89]. 123 Metabolomics of plant stress 3231 Heavy metals such as cadmium (Cd), cesium (Cs), lead raffinose, glycerate and fatty acids, decreased. Central (Pb), zinc (Zn), nickel (Ni) and chromium (Cr) are major amino acids (glutamine, glutamate, aspartate and alanine) pollutants of the soil causing stress on plants. Even the and methionine, an S-containing amino acid, also essential nutrients including copper (Cu), iron (Fe) and decreased, indicating the inhibition of N and S assimila- manganese (Mn) can cause heavy metal stresses with tion, respectively. The increase of most other amino acids inappropriate concentration. Generally heavy metals indicates that proteolysis has commenced. Most of these induce enzyme inhibition, cellular oxidation and metabolic changes reverted rapidly after re-addition of sucrose into perturbation, resulting in growth retardation and in extreme the media. Usadel et al. [97] took advantage of extended instances in plant death [90]. Jahangir et al. [91] analysed dark treatment to induce C starvation under more natural the effects of Cu, Fe and Mn on the metabolite levels of conditions in the Arabidopsis rosettes. Intriguingly, how- Brassica rapa, which is a known metal accumulator. ever, the changes in metabolite levels were mostly Glucosinolates and hydroxycinnamic acids conjugated with comparable to those observed in liquid culture seedlings malates as well as primary metabolites such as carbohy- [96]. The marked decrease of carbohydrates within the first drates and amino acids were found to be the discriminating 4 h of extended night indicates that the treatment induced metabolites. Arabidopsis plants treated with Cd displayed C starvation very efficiently and that carbohydrates are increased levels of alanine, b-alanine, proline, serine, starting to acutely limit metabolism. On the other hand, putrescine, sucrose and other metabolites with compatible organic acids and other C-containing metabolites displayed solute-like properties, notably GABA, raffinose and tre- a rather gradual decrease. The prolonged dark treatment halose [92]. This study also indicated that concentrations of induced severe C starvation and leaf senescence by the end antioxidants (a-tocopherol, campesterol, ß-sitosterol and of the experiment. The metabolite profile of Arabidopsis isoflavone) also increased significantly. When taken toge- leaves subjected to prolonged darkness has been analysed ther these data indicate an important role of antioxidant in a series of studies to elucidate the metabolic bases of defences in the mechanisms of resistance to cadmium dark-induced senescence and the function of the mito- stress. Dubey et al. [93] conducted transcriptomic and chondrial alternative electron transport pathway during metabolomic analysis of rice roots treated with Cr. Under dark treatment [98–100]. Although a similar metabolic these conditions proline accumulated to levels three-fold phenotype as the two studies described above [96, 97] was those of the control as did ornithine, which can be used in observed during the first few days of dark treatment, a its synthesis. The content of several other metabolites subset of metabolites exhibits biphasic behaviour during including lactate, fructose, uracil and alanine increased prolonged exposure to darkness. This was particularly following exposure to Cr stress; these were taken to sug- notable for some TCA cycle intermediates including gest the modulation of the sucrose degradation pathway fumarate, isocitrate, malate and succinate, which accumu- involving the three main fermentation pathways operating lated after 7 days of dark treatment despite decreasing as a rescue mechanism when respiration is arrested. Further during the first 3 days of treatment. Additionally accumu- studies are however most likely warranted to gain a better lation of most amino acids including GABA became much understanding of the mechanisms underlying these more prominent. Metabolite profiles were also analysed in changes. a range of mutants deficient in the genes involved in mitochondrial alternative electron transport mediated Nutrient limitation by the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) Nutrient starvation also dramatically affects plant growth complex, namely ETFQO [98] and ETFb [99] as well as and metabolism. Especially limitation of macronutrients, enzymes involved in the provision of its substrates, namely namely carbon (C), nitrogen (N), phosphorus (P) and sul- IVDH, D2HGDH [101] and PSHX [100]. Although indi- phur (S), has direct effects on metabolism since most vidual genotypes showed similar responses during the first organic molecules comprise a combination of these ele- 3 days of dark treatment, there are subtle differences in ments. Changing environmental conditions continually their metabolic complements at the end of the experiment, alter the balance between C assimilation and utilisation. indicating an essential role of this alternative electron Even short periods of C starvation lead to an inhibition of transport machinery during dark-induced starvation [99]. growth, which is not immediately reversed when C Further detailed analysis revealed that the ETF/ETFQO becomes available again [94, 95]. Osuna et al. [96] complex is involved in both the branched chain amino investigated the metabolite profile of Arabidopsis seedlings acids and the lysine catabolism pathways, and acts as an in liquid culture under C starvation. In C-starved seedlings, electron donor to the mitochondrial ubiquinol pool [100, as could be anticipated, carbohydrates, organic acids and 101]. These studies suggest that more integrative analysis other C-containing metabolites, including myo-inositol, of the role of all aspects of protein degradation and 123 3232 T. Obata, A.R. Fernie consequent remobilisation should be performed within the as well as sugars, while organic acids were accumulated in context of understanding metabolic responses to stress. P-deficient nodules. Such a contrasting response may be Nitrogen is required for the synthesis of nucleotides and due to the N deficiency in P-starved nodules in which the amino acids, which are the building blocks of nucleic acids sole N supply from fixed N could be suppressed under and proteins, and for the synthesis of phospholipids and environmental limitations such as P starvation [107]. many secondary metabolites that have diverse roles in Huang et al. [108] profiled metabolites from both shoots and signalling, structure and adaptation. The effect of N defi- roots of P-deficient barley. Severe P deficiency increased ciency on the metabolite levels in tomato leaves were the levels of phosphorylated intermediates (glucose-6-P, investigated by Urbanczyk-Wochniak and Fernie [102]. As fructose-6-P, inositol-1-P and glycerol-3-P) and organic would perhaps be expected, amino acid levels generally acids (2-oxoglutarate, succinate, fumarate and malate). The decreased under nitrogen deficiency. The level of 2-oxo- results revealed that P-deficient plants modify carbohydrate glutarate, a key regulator of carbon and nitrogen metabolism initially to reduce P consumption and salvage P interactions [103], decreased under N starvation as well as from small P-containing metabolites, which consequently other TCA cycle intermediates including citrate, isocitrate, reduce the levels of organic acid in the TCA cycle [108]. succinate, fumarate and malate. Tschoep et al. [104] ana- Sulphur is another macronutrient essential for the syn- lysed the effect of mild but sustained N limitation in thesis of the S-containing amino acids cysteine and Arabidopsis. Malate and fumarate levels were strongly methionine as well as a wide range of S-containing metab- decreased in low N conditions like in tomato leaves [102]. olites including glutathione. There are some metabolomic However, their rosette protein content was unaltered and studies on the response to S starvation in Arabidopsis [109– total, and many individual amino acid levels increased 112], and they are nicely summarised in Hoefgen et al. compared with N-replete plants. The results revealed that [113]. At the time course of S-stress response, two metabolic Arabidopsis responds adaptively to low N condition. P is states can be distinguished. The short-term metabolic an essential component of intermediates in central and responses include the decrease of organic S-containing energy metabolism, signalling molecules and structural compounds on the S assimilation such as cysteine and glu- macromolecules like nucleic acids and phospholipids. tathione, which leads to the accumulation of their precursor Morcuende et al. [105] analysed the metabolite profile of O-acetyl-serine (OAS) as well as serine, and to the sub- Arabidopsis seedlings grown in liquid culture under P sequent re-channeling of the metabolic flow to glycine and starvation. The levels of sugar phosphates were very low tryptophan. Glucosinolate catabolism is activated to salvage but metabolites further down in glycolysis, glycerate-3- S from it. As a long-term response the lipid contents and a phosphate, glycerate-2-phosphate and phosphoenolpyr- S-containing molecule, S-adenosyl-methionine, decreased. uvate, increased in P-deficient seedlings. Pi-deficient Insufficient S supply leads to its disbalance with N and seedlings showed a marked accumulation of starch, sucrose further to the alterations in C1 metabolism that link photo- and reducing sugars as well as a general increase of organic respiration, S assimilation and dumping of N [113]. Results acids including citrate, fumarate, malate and oxoglutarate. of a very recent study on the Arabidopsis plants with The levels of most major amino acids did not alter or modified OAS levels suggest the importance of this increased slightly, whereas those of several minor amino metabolite since OAS plays a signalling role for a specific acids including the aromatic amino acids and histidine, part of the sulphate response as well as for the regulation of arginine and threonine. Together with transcriptomic data, the transcript levels of a specific gene set irrespective of the analysis of metabolites revealed that P deprivation leads to sulphur status of the plants [114]. a shift towards the accumulation of carbohydrates, organic Potassium (K) is not a component of organic molecules acids and amino acids. The effect of P starvation has also but plays essential roles as a major cation in plants and as a been studied on crop plants such as common bean and cofactor of enzymes [115]. Armengaud et al. [116] used barley. Hernandez et al. used metabolite profiling to assess metabolite profiling to identify metabolic targets of K the effect of P deficiency in the roots [106] and nodules stress. Metabolite profiles of low-K Arabidopsis plants [107] of the common bean. Most of the amino acids were were characterised by a strong increase in the concentra- increased in P-stressed roots. The accumulation of several tions of soluble sugars (sucrose, fructose and glucose) and sugars suggests that sugars may be partitioned preferen- a slight net increase of total protein content and the overall tially to P-stressed roots to support the expression of P amino acid level. Several basic or neutral amino acids stress-induced genes. The reduced amounts of organic accumulated during K deficiency, while acidic amino acids acids likely reflect exudation of these metabolites from the decreased. In addition a strong decrease of pyruvate and roots into the rhizosphere [106]. The metabolic response of organic acids was recorded only in the roots but not in the P-starved nodules is in contrast to that of roots. Amino shoots. They also measured enzyme activities and con- acids and other N-containing metabolites were decreased cluded that the primary effect of K deficiency induces an 123 Metabolomics of plant stress 3233 inhibition of glycolysis by the direct inhibition of enzymes removal of menadione from the culture media [121]. After [116]. menadione removal many of the stress-related changes reverted back to basal levels. However, each metabolic Oxidative stress pathway recovered in a differential time period, for instance, glycolytic carbon flow reverted to control level Oxidative stress is a key underlying component of most 18 h after menadione removal, although the TCA cycle and abiotic stresses and a major limiting factor of plant growth some amino acids such as aspartate and glutamate took in the field [117]. It occurs on the overproduction of longer to recover. It suggests the involvement of pathway- reactive oxygen species (ROS) in plant cells when plant specific regulatory processes for the oxidative stress metabolism is perturbed by various stresses. This conse- response. These metabolic responses to menadione- quently leads to oxidative damages of cellular components induced oxidative stress mentioned above seem to be such as DNA, proteins and lipids [118]. To cope with conserved among plant species and organs because quite oxidative stress, the metabolic network of plant cells must similar responses were observed both in Arabidopsis be reconfigured either to bypass damaged enzymes or to seedlings in liquid culture [122] and rice suspension cells support adaptive responses. In the study by Baxter et al. [123]. They are additionally at least partially similar to [119], heterotrophic Arabidopsis cells were treated with those observed when oxidative stress is mimicked by the menadione, which enhances the ROS production via elec- removal of enzymes involved in ameliorating against it, tron transport chains and changes in metabolite abundance, such as manganese superoxide dismutase [124]. In the and C-labelling kinetics were quantified. The accumula- Arabidopsis plants with suppressed expression of mito- tion of sugar phosphates related to glycolysis and oxidative chondrial manganese superoxide dismutase revealed a pentose phosphate pathways (OPPP) suggested the rerout- decrease of TCA cycle intermediates, probably because of ing of glycolytic carbon flow into the OPPP possibly to the inhibition of aconitase and isocitrate dehydrogenase provide NADPH for antioxidative effort. In addition the [124]. decrease of ascorbate and accumulation of its degradation product, threonate, indicated the activation of antioxidative Stress combination pathways in menadione-treated cells. The reduced glyco- lytic activity probably leads to the decrease of levels of Whilst convenient both for experiments and discussion at amino acids derived from glycolytic intermediates. The the single stress level, plants are actually subjected to a decrease of amino acids linked to TCA cycle intermediates combination of abiotic stress conditions in their natural and decrease of malate indicated a perturbation of TCA habitat. Even some abiotic stresses are already combina- cycle. These observations in metabolite levels were tions of stresses. For example high salt concentration emphasised by C-redistribution analysis, which indicated causes osmotic and ion stresses, and flooding results in increased carbon flux into OPPP intermediates and inhi- hypoxic and shading stresses. Although the metabolic bition of metabolic flux into all TCA cycle intermediates responses of plants under a single abiotic stress have been detected [119]. Lehmann et al. [120, 121] also conducted analysed extensively as shown above, there are only few both metabolite profiling and C-redistribution analysis of studies regarding to the effect of stress combinations on menadione-treated Arabidopsis roots and found that the plant metabolism. Rizhsky et al. [125] applied a combi- metabolic response of roots is distinct from that of het- nation of drought and heat stress to Arabidopsis plants and erotrophic cells in culture [120]. The redirection of analysed the metabolic profile. The metabolite profile of glycolytic carbon flow and inhibition of the TCA cycle plants subjected to a combination of drought and heat stress were suggested also in the roots. Especially the inhibition was more similar to that of plants subjected to drought than of the TCA cycle is more evident in roots as a perturbation to that of control plants or plants subjected to heat stress. of metabolite levels. In addition, roots showed pronounced However, the plants subjected to combined stresses accu- accumulation of some metabolites including GABA, OAS, mulated high levels of sucrose and other sugars instead of pyruvate, many amino acids and glucosinolates. It seems proline, which is highly accumulated to a very high level in likely that cellular oxidation inhibited S assimilation and plants subjected to drought but not under stress combina- caused OAS accumulation. A general increase of amino tion. They concluded that sucrose replaces proline as the acid levels is thought to be the result of enhanced protein major osmoprotectant in plants subjected to combined degradation. This is supported by C-labelling analysis in stress because the toxic effect of high level of proline is which the C-redistribution was not affected in most enhanced under heat stress, as they showed experimentally amino acids, indicating that the carbon in the increased [125]. Wulff-Zottele et al. [65] analysed the effect of amino acids was not from synthetic pathways [121]. They the combination of high light irradiance and S depletion, also followed the metabolic recovery process after the which can occur in the field simultaneously [126]. 123 3234 T. Obata, A.R. Fernie The combination of high light and S depletion gives rise to modulating both cell division and cell expansion. Growth similar metabolic pool modifications such as in high light. decreases rapidly upon stress onset, but it recovers and Proline accumulated in a differential time course under adapts once stress conditions become stable [127]. Accu- high light and stress combination. Other metabolites such mulated metabolites might be used as building blocks to as raffinose and putrescine seem to replace proline during support a recovery of growth. Figure 1 provides an over- the delay of proline accumulation in the plants subjected to view of the changes in the amount of selected metabolites. high light and S depletion. This replacement of proline Charts for all metabolites are found in the Supplementary with sugars is similar to that observed under the combi- data, Fig. S1. In general, changes in the amounts of nation of drought and heat stress [125]. Recently Caldana metabolites were stress-specific in contrast to the general et al. [66] reported a systematic study on the metabolomic responses observed in bacteria [128]. A stress-specific and transcriptomic response of Arabidopsis to eight envi- change in the metabolite level would be a result of an ronmental conditions including the combinations of inhibition/activation of a specific metabolic pathway changing light (darkness, high light) and/or temperature especially in the short term. It should mainly be related to (cold and heat). The analysis has demonstrated that dark- the properties of enzymes such as sensitivity to tempera- ness and high temperature have a synergistic effect, thus ture, oxidation and ion concentration. In addition, presenting a more extreme condition. The reconstructed rearrangement of the metabolic network should also result metabolic networks for this condition also revealed an in changes of metabolites, which are related to the regu- exclusive correlation between several amino acids, lated pathways. Therefore, such a metabolite must be a including GABA with intermediates of the TCA cycle, good candidate for an analysis to elucidate the specific notably succinate. These results suggested that in the effects of an abiotic stress and the adaptive processes absence of photosynthesis protein degradation occurs rap- against it. On the other hand, metabolites responding to idly and subsequent amino acid catabolism serves as the various stresses can be related to fundamental stress main cellular energy supply [66]. responses. In the present analysis, some metabolites can be seen to accumulate in most abiotic stress conditions although the time and extent of accumulation varied among Common and stress-specific metabolic responses conditions. Levels of sucrose were increased in most stress against diverse abiotic stress conditions in at least one time point (Fig. 1). Sucrose is a major transport sugar in most plant species and is known to As described above, plants show a variety of metabolic accumulate under stress conditions [129]. Compounds responses against diverse abiotic stresses. The question is defined as ‘‘compatible solutes’’ also accumulate under whether there are any common metabolic responses to all various abiotic stress conditions. They are very soluble in abiotic stresses or the responses are always specific to the water and are non-toxic at high concentrations and function stress factors. To evaluate the accumulation of these to sustain the ordered vicinal water around proteins by compounds under stress conditions and to search for the decreasing protein-solvent interactions at low water activ- novel metabolic fingerprints related to the stress responses, ities [52, 130]. This group of compounds includes betaines we analysed the published metabolite profiling data avail- and related compounds; polyols and sugars, such as man- able in the above-mentioned literature. Studies dealing nitol, sorbitol and trehalose; and amino acids, such as with Arabidopsis leaves were chosen (dehydration [28], proline [131, 132]. Recent studies have revealed that they salt [79], heat and cold [57], high light and sulphur limi- function to protect plants not only from osmotic stress but tation [65], UV [74], light quality change [71], low also from various stress factors [130, 131, 133, 134]. nitrogen [104] and potassium limitation [104]) to afford Therefore, the synthetic pathways of those metabolites greater comparability. Forty-five metabolites detected in have been of interest for metabolic engineering and some more than half of the studies were analysed and each datum interventions have indeed increased the tolerance of some was converted into the fold change values against control crop plants to abiotic stress [130, 131]. Raffinose is a sugar growth conditions and presented using the log scale synthesised from sucrose and known to protect plant cells (Supplementary data, Table S1). Table 1 shows the number as an osmoprotectant; it also accumulates under most stress of metabolites accumulated or decreased under each stress conditions especially at the later stages of stress treatment condition. This reveals that abiotic stresses generally (Fig. 1). Raffinose is also shown to function to protect induce accumulation of metabolites with only the light plants from oxidative damage [135], making the observa- quality change as an exception. The tendency of accumu- tion reasonable since oxidative damage likely underlies lation is probably related to a cessation of the growth- most stress conditions. Interestingly, myo-inositol, which is reducing consumption of metabolites. When subjected to closely related to raffinose biosynthesis, did not show abiotic stresses, plants actively re-program their growth by prominent changes other than under high light conditions 123 Metabolomics of plant stress 3235 Table 1 Number of metabolites that changed their abundance under possible role of BCAAs under stress conditions would be each stress condition that of an alternative electron donor for the mitochondrial electron transport chain. The mitochondrial electron Condition Increased Decreased transport chain is primary supplied by electrons from Dehydration 26 2 NADH and succinate to produce ATP. Additionally there is Salt 3 0 an alternative way to feed electrons from other substrates Heat 10 4 via electron transfer flavoprotein (ETF) complex. Recent Cold 27 9 studies highlighted the importance of the alternative path- High light 39 4 way under dark and stress conditions especially under Light quality 1 7 carbon starvation (see also the ‘‘Nutrient limitation’’ sec- UV 14 5 tion) [98–101]. A C-feeding experiment has proven that Low N 10 3 lysine and BCAA are converted into D-2-hydroxyglutarate -S 11 2 and isovaleryl-CoA in vivo to be a direct electron donor for -K 13 0 the ETF complex via the action of D-2-hydroxyglutarate dehydrogenase and isovaleryl-CoA dehydrogenase [101]. The changes greater than two fold were counted As such, BCAAs can provide electrons both directly to the electron transport chain via the ETF complex as well as (Fig. 1). The amino acid, proline, is known as a major indirectly because their catabolic products feed directly compatible solute in Arabidopsis [133] and also accumu- into the tricarboxylic acid (TCA) cycle [142] (Fig. 2b). The lated under stress conditions despite being detected only in source of accumulated BCAAs would be the protein deg- a limited number of studies (Fig. 1). On the other hand, radation product, which has recently been proposed to be trehalose accumulated only under specific conditions, an important alternative respiratory substrate especially suggesting that it displays functions other than being a under certain stress conditions [142], as well as the acti- compatible solute (Fig. 1). Indeed it is unlikely that tre- vated synthetic pathway, which is observed under drought halose contents in plants—other than resurrection plants— stress conditions [28, 141]. Thus, our analysis emphasised are high enough to be directly involved in stress protection the importance of BCAA metabolism generally under [136] and some trehalose metabolism mutants exhibit abiotic stress conditions. Interestingly, the pattern of potential negative effects on plant physiology [134]. The accumulation of GABA is similar to those of BCAAs amount of trehalose may reflect that of its precursor, tre- (Fig. 1), although the reason why they are related under halose-6-phosphate, which has been documented to act as a stress conditions remains unclear. Thus, clarifying the signal molecule in plants [137]. GABA is another metab- exact mechanistic role of BCAAs under various conditions olite discussed in a context of stress response since it is will be an important priority for the future. largely and rapidly produced in response to biotic and abiotic stresses [138–140]. Our analysis supported this observation (Fig. 1). There are many suggested functions Prospective: toward the elucidation of molecular of GABA and the GABA shunt, which protect plants to mechanisms underlying abiotic stress tolerance survive various stress conditions including regulation of cytosolic pH, protection against oxidative stress and A wealth of metabolomics data concerning the plant stress functions of GABA as an osmoregulator and as a signalling response has been accumulated and a large number of molecule [138]. However, whilst evidence for an important metabolic pathways are suggested to be regulated under metabolic role has been documented, that for a signalling stress. However, there are relatively few pathways and role in plants is still lacking. In the presented data set, metabolites that have been experimentally proven to branched chain amino acids (BCAAs), namely valine, function in abiotic stress tolerance. One problem is that a leucine and isoleucine, and other amino acids sharing metabolite profile does not tell exactly whether the related synthetic pathways with BCAA, including lysine, threonine metabolic pathway is up- or downregulated since both and methionine (Fig. 2a), were generally accumulated upregulation of upstream reaction and down-regulation of under abiotic stress conditions. These amino acids are a downstream reactions can lead to the accumulation of a novel group of metabolites that accumulated generally in metabolite. This can be solved by comparing the meta- response to stress conditions, although they have been bolomic data with those from transcriptomic or proteomic shown to accumulate under drought stress conditions [141]. analysis or activities of specific enzymes [143]. Hirai et al. Joshi et al. [141] suggested that they function as compat- [111] revealed gene to metabolite regulatory networks of ible osmolytes since BCAA showed a high fold increase glucosinolate synthesis and primary metabolism under under drought stress in various plant tissues. Another sulphur- and nitrogen-limited conditions by applying 123 3236 T. Obata, A.R. Fernie 4 10 3 Sucrose Raffinose myo-inositol -2 -1 -1 -4 Trehalose Proline -2 -4 -2 Isoleucine Valine Leucine 6 8 -2 -2 -2 6 6 Lysine Threonine Methionine 2 2 -2 -2 -2 GABA -2 Fig. 1 Changes of the levels of metabolites in Arabidopsis leaves (Low N) [104], sulphur limitation (-S) [65] and potassium limitation under various abiotic stress conditions. Each datum represents the (-K) [116] stresses. The data set used for the analysis is found in relative metabolite level in the fold change value against control Supplementary data, Table S1. The bars with different colours growth conditions at one time point. The values are taken from represent the values from different studies as shown in the figure. studies on dehydration [28], salt [79], heat and cold [57], high light Only the metabolites of interest are shown. The charts for all [65], light quality change [71], UV-B light (UV) [74], low nitrogen metabolites are presented as Supplementary data, Fig. S1 integrated analysis of transcriptome and metabolome data. [63] as described above. This approach is proven to be Integrated analyses of the transcriptome and the metabo- useful to elucidate the regulation of the pathway and also lome successfully demonstrated connections between the involvement of transcriptional regulation of the genes and metabolites, elucidating a wide range of signal pathway. The studies using proteomics together with output from ABA under dehydration [28] and the DREB1/ metabolomics are relatively rare in the plant stress response CBF transcription factors in response to low temperature field. One example is the study by Wienkoop et al. [144], Fold change (log2) Dehydration Salt Heat Cold High light light quality UV Low N -S -K Metabolomics of plant stress 3237 transcriptome and immunoprecipitation-based translatome a amino acid synthetic mode b protein degradation mode data sets have provided an important foundation for the Protein degradation Glycolysis Ile Leu Val analysis of the transcriptional and translational control of Met Thr Lys Ile Leu Val environmental responses in each tissue layer of the plant Met Thr Pyruvate [148, 149], the metabolomic studies are still rare because of Isovaleryl CoA HG Asp OAA the technical difficulties. Ebert et al. [150] applied single D2HGDH IVDH Lys cell sampling using microcapillaries to enable the cell- TCA cycle 2OG type-specific metabolic analysis of epidermal cell types in ETF ETFQO Arabidopsis thaliana pavement, basal and trichome cells. Succinate SDH Recently, Rogers et al. [151] demonstrated the feasibility - Ubiquinone of FACS-based metabolic profiling using high-resolution Complex I NADH mass spectrometry at cell type resolution in roots. ATP production The integration of the ‘‘omics’’ data revealed many Fig. 2 Metabolic modes leading to the accumulation of branched molecular mechanisms for metabolic regulation but also chain amino acids (BCAA) and related amino acids suggested under highlighted a complex relationship among the levels of abiotic stress conditions. a Amino acid synthetic mode. BCAAs are transcripts, metabolites and metabolic flux. It suggests the synthesised using pyruvate or oxaloacetate (OAA) as carbon skele- tons. b Protein degradation mode. Amino acids resulting from participation of post-transcriptional especially post-trans- degraded proteins would be direct and indirect electron donors to lational regulation of enzyme activity in the regulation of produce ATP. 2OG 2-oxoglutarate, SDH succinate dehydrogenase, primary metabolism. An important role of post-transcrip- HG hydroxyglutarate, D2HGDH D-2-hydroxyglutarate dehydroge- tional regulation in the stress response is also suggested nase, IVDH isovaleryl-CoA dehydrogenase, ETF electron transfer flavoprotein, ETFQO ETF-ubiquinone oxidoreductase, e electron by the poor statistical correlation of protein expression data with microarray results especially in the short-term which showed the importance of starch and raffinose response [152–156]. And there is an explicit indication family oligosaccharide metabolism during temperature that considerable metabolic control is executed on the stress by the metabolomic and proteomic analysis of the metabolite and on the protein level including protein starch-deficient Arabidopsis mutant lacking phosphoglu- modifications [157]. Metabolic enzymes are well known to comutase (pgm mutant). The number of such studies should be regulated allosterically by the substrates and/or the increase in the near future because of the improvement of products of the pathway [158, 159]. Many other post- analytical methods for proteomics. The activities of translational modifications of the enzyme proteins such as enzymes involved in a pathway should have a direct rela- phosphorylation, glutathionylation and nitrosylation could tionship with the amount of a metabolite and could be a be involved in metabolic regulation [160–162]. Among useful tool to assess the metabolic regulation. Changes in them, we discuss here the reconfiguration of enzyme pro- maximal enzyme activities were analysed together with tein complexes in this study. In the protein complex transcriptomic and metabolomic data in the study by ‘‘substrate channeling’’ can happen by which the interme- Armengaud et al. [116], which pinpointed that pyruvate diate produced by one enzyme is transferred to the next kinase activity was inhibited directly by K deficiency and enzyme without complete mixing with the bulk phase was primarily responsible for the metabolic disorders [163]. Especially the association of several sequential observed. Metabolic flux analysis is another powerful metabolic enzymes involved in one pathway is called a approach to study the regulation of metabolic pathways. metabolon [164]. Metabolite channeling can be envisioned Lehmann et al. [121] conducted C redistribution analysis as a means to improve catalytic efficiency by increasing to prove the downregulation of glycolysis under oxidative local substrate concentrations, regulating competition stress treatment, which is suggested by metabolic profiling between branch pathways for common metabolites, coor- [120]. Metabolic flux analysis is also useful to elucidate a dinating the activities of pathways with shared enzymes or metabolic regulation that cannot be detected by metabolic intermediates, and sequestering reactive or toxic interme- profiling since the carbon flow can be affected without any diates [165, 166]. The organisation of metabolic pathways apparent changes in the metabolite pool sizes [119, 145, by metabolic channeling has been discussed as the main 146]. Improvement of both experimental and theoretical molecular-scale organisation units to orchestrate the mul- mathematical approaches to flux estimation and parame- tiple metabolic processes and it is now supported by terisation will greatly aid our understanding here [147]. modelling as well as experimental evidence [167–170]. The analysis of cell-type-specific responses would reveal Recent bioinformatic study suggested that evolved protein more detailed mechanisms of the stress response that interactions may contribute significantly towards increas- are hidden in a mixture of the cells in a tissue. Although ing the efficiency of metabolic processes by permitting the fluorescence-activated cell sorting (FACS)-based higher metabolic fluxes [171]. Metabolite channeling is 123 3238 T. Obata, A.R. Fernie considered to be achieved not only when the enzyme information will provide an immense foundation for meta- association is stable but also when the association is bolic engineering and synthetic biology approaches to dynamic. Transient complexes offer the possibility of fast ensuring food security. exchange of some of the polypeptide components upon Open Access This article is distributed under the terms of the reassembly and thus can be a molecular basis for rapid fine Creative Commons Attribution License which permits any use, dis- tuning or redirection of metabolism. The reassembly of tribution, and reproduction in any medium, provided the original the metabolic enzyme complex therefore should have a author(s) and the source are credited. molecular basis underlying the metabolic regulation in mitochondria under short-term oxidative stress. Metabolic channeling including metabolon formation is reported in References many processes in plants such as glycolysis, cysteine synthesis, the Calvin-Benson cycle, cyanogenic glucoside 1. Fiehn O (2001) Combining genomics, metabolome analysis, and biosynthesis, the phenylpropanoid pathway, the glycine biochemical modelling to understand metabolic networks. 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The use of metabolomics to dissect plant responses to abiotic stresses

Cellular and Molecular Life Sciences , Volume 69 (19) – Aug 12, 2012

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Abstract

Cell. Mol. Life Sci. (2012) 69:3225–3243 DOI 10.1007/s00018-012-1091-5 Cellular and Molecular Life Sciences MU LTI-A UT HO R R EVI E W The use of metabolomics to dissect plant responses to abiotic stresses Toshihiro Obata Alisdair R. Fernie Received: 7 July 2012 / Revised: 9 July 2012 / Accepted: 9 July 2012 / Published online: 12 August 2012 The Author(s) 2012. This article is published with open access at Springerlink.com Abstract Plant metabolism is perturbed by various Introduction abiotic stresses. As such the metabolic network of plants must be reconfigured under stress conditions in order to When plants face unfavourable growth conditions, abiotic allow both the maintenance of metabolic homeostasis and stress retards plant growth and productivity. Under most the production of compounds that ameliorate the stress. abiotic stress conditions, plant metabolism is perturbed The recent development and adoption of metabolomics and either because of inhibition of metabolic enzymes, shortage systems biology approaches enable us not only to gain a of substrate, excess demand for specific compounds or a comprehensive overview, but also a detailed analysis of combination of these factors and many other reasons. crucial components of the plant metabolic response to Therefore, the metabolic network must be reconfigured to abiotic stresses. In this review we introduce the analytical maintain essential metabolism and to acclimate by adopt- methods used for plant metabolomics and describe their ing a new steady state in light of the prevailing stress use in studies related to the metabolic response to water, conditions. This metabolic reprogramming is also neces- temperature, light, nutrient limitation, ion and oxidative sary to meet the demand for anti-stress agents including stresses. Both similarity and specificity of the metabolic compatible solutes, antioxidants and stress-responsive responses against diverse abiotic stress are evaluated using proteins. The accumulation of reactive oxygen species data available in the literature. Classically discussed stress (ROS) is another problem causing oxidation and dysfunc- compounds such as proline, c-amino butyrate and poly- tion of cellular components and in the worst case cell amines are reviewed, and the widespread importance of death. The optimisation of metabolic flux via the organellar branched chain amino acid metabolism under stress con- electron transport chains is, moreover, crucial in order to dition is discussed. Finally, where possible, mechanistic dampen ROS production. Maintenance of the redox state in insights into metabolic regulatory processes are discussed. the cell is thus another important task to provide the reducing power required for ROS scavenging. Despite such Keywords Metabolomics  Plants  Abiotic stress  important roles of metabolic regulation under stress con- Metabolic response  Branched chain amino acid  ditions, our current understanding of this process is Enzyme complex fragmented and far from complete. Metabolomics is a powerful tool by which to gain a comprehensive perspective of how metabolic networks are regulated and has indeed been applied by many researches in recent years. It can, additionally, be used to elucidate the Electronic supplementary material The online version of this functions of genes as a tool in functional genomics and article (doi:10.1007/s00018-012-1091-5) contains supplementary material, which is available to authorized users. systems biology approaches. The term ‘‘metabolomics’’ is defined as comprehensive and quantitative analysis of all T. Obata  A. R. Fernie (&) small molecules in a biological system [1]. The plant Max Planck Institute of Molecular Plant Physiology, kingdom may contain between 200,000 and 1,000,000 Am Muhlenberg 1, 14476 Potsdam-Golm, Germany metabolites, while for a single species the number may e-mail: fernie@mpimp-golm.mpg.de 123 3226 T. Obata, A.R. Fernie approach a few thousand (the estimate for Arabidopsis than 1 kDa) difficult. Due to these characteristics, GC-MS is ca. 5,000) [2–5]. Indeed the KNApSAck database (http:// facilitates the identification and robust quantification of a kanaya.naist.jp/KNApSAcK/) contains around 50,000 few hundred metabolites in plant samples including sugars, metabolite entries in plants described so far in the literature sugar alcohols, amino acids, organic acids and polyamines, [6]. Due to the large variety of chemical structures and resulting in fairly comprehensive coverage of the central properties of small molecules, there is so far no single pathways of primary metabolism. technique to identify and quantify all of them. Even the most comprehensive methods detect only between 1,000 and Liquid chromatography (LC)-MS 2,000 molecular features [7, 8]. Several techniques includ- ing gas chromatography-mass spectrometry (GC-MS), While GC has a limitation due to volatilisation of com- liquid chromatography (LC)-MS, capillary electrophoresis pounds, LC does not require prior sample treatment and (CE)-MS and nuclear magnetic resonance spectroscopy separates the components in a liquid phase. The choice of (NMR) are commonly used in plant metabolomics research. columns, including reversed phase, ion exchange and They are used sometimes in combination since they are hydrophobic interaction columns, provides the separation largely complimentary with each independent method of metabolites based on different chemical properties. having preferential coverage of diverse types of metabolite. Therefore, LC has the potential to analyse a wide variety of Here we briefly introduce the advantages and limitations of metabolites in plants. The recent development of ultra- each method. Thereafter studies employing the metabolo- performance liquid chromatography (UPLC) makes the mic approach to dissect plant response to abiotic stresses technique more powerful because of its higher resolution, will be discussed. Finally, using data already collected, sensitivity and throughput than conventional high-perfor- we attempt to elucidate the general and stress-specific mance liquid chromatography (HPLC) [16]. Electrospray responses. ionisation (ESI) is widely used for ionisation to connect LC and MS. Many types of MS including quadrupole (Q), TOF, qTOF, triple quadrupole (QqQ), ion trap (IT), linear Techniques used for plant metabolomic research trap quadrupole (LTQ)-Orbitrap and Fourier transform ion cyclotron resonance (FT-ICR)-MS are used depending on Gas chromatography-mass spectrometry (GC-MS) the sensitivity, mass-resolution and dynamic range required (see [17, 18] for the detail). The combination of these Gas chromatography-mass spectrometry is the most widely techniques allows us to identify and quantify a large variety used technique for plant metabolomics research to date. of metabolites even if they have high molecular mass, great polarity and low thermostability. On the other hand the Polar metabolites are derivatised to render them volatile and then separated by GC. Electron impact (EI) allows flexibility of the method also causes difficulty in estab- robust interfacing of GC with MS resulting in highly lishing large mass spectral libraries for peak identification reproducible fragmentation patterns. For detection, time- because of the instrument-type dependent retention time of-flight (TOF)-MS has become the method of choice and mass spectra [19], and forces each research group to because of advantages including fast scan times, which create its own ‘‘in-house’’ LC-MS reference library. That give rise to either improved deconvolution or reduced run said, there are a number of websites that aid in mass- times for complex mixtures, and high mass accuracy. The spectral analyses [20], and recent recommendations for crucial advantage of this technology is that it has long been metabolite reporting [7] should improve the transparency used for metabolite profiling, and thus there are stable of the methods used by researchers. Furthermore, isotope protocols for machine setup and maintenance, and chro- labelling as a means of confirming the identity of peaks has matogram evaluation and interpretation [9–11]. The recently been proposed and demonstrated to allow the robustness of the protocol means that libraries of retention identification of circa 1,000 metabolites using the FT-ICR- time and mass spectra data for standard compounds can be MS approach [8, 21]. To date, LC-MS is mainly used with shared among laboratories [12]. There are several metab- a reverse phase column to analyse secondary metabolites olite databases available including the NIST [13], FiehnLib because of its ability to separate compounds with similar [14] and Golm metabolic databases (GMD, [15]), which structure and to detect a wide range of metabolites. How- are useful tools for peak annotation. Additionally, the short ever, it is worth noting that specialised protocols for running time and relatively low running cost are also strong determining phosphorylated intermediates, which are not advantages of GC-MS. However the use of GC-MS is readily detected by GC-MS, have also begun to be devel- limited for thermally stable volatile compounds, making oped using this technology [22], as have methods for the the analysis of high molecular weight compounds (larger comprehensive analysis of phytohormones [23]. 123 Metabolomics of plant stress 3227 Capillary electrophoresis (CE)-MS vacuole from the rest of the cell cause distinctive signals from an identical metabolite and thus allows quantification Capillary electrophoresis separates polar and charged at the subcellular level [34, 35]. Thus analysing the compounds on the basis of their charge-to-mass ratio. CE is metabolite composition of a tissue extract, determining the able to separate a diverse range of chemical compounds structure of a novel metabolite, demonstrating the exis- and is a more powerful technique than LC with respect to tence of a particular metabolic pathway in vivo, isotope separation efficiency [24, 25]. ESI is commonly used for labelling experiment and localising the distribution of a ionisation as in LC-MS, with TOF-MS being the most metabolite in a tissue are all possible by NMR. For isotope commonly used detector in CE-MS-based metabolomics labelling, NMR has the advantage of providing facile studies. This combination provides high mass accuracy and access to atomic level labelling, which is highly laborious high resolution. The high scan speed of TOF-MS makes in the case of MS methods yet can be essential in flux this instrument very suitable for full scan analyses in estimation [35]. However, the number of compounds that metabolomics. One of the unique properties of CE-MS is can be detected in a single analysis is limited to one to the small amount of sample required for analysis; only several dozen [36, 37]. These properties of NMR make it nanolitres of sample are introduced into the capillary. the ideal tool for broad-range profiling of abundant Together with high electric fields and short separation metabolites whilst studying changes in non-annotated lengths, it can produce analysis within seconds. It also profiles is highly useful for metabolite fingerprinting of allows the metabolic analysis in volume-restricted samples. extensive sample collections [38, 39]. On the other hand, this leads to low concentration sensi- tivity requiring enrichment of metabolites within the samples [26]. Another drawback of CE is the poor migra- Metabolomic studies of plant stress responses tion time reproducibility and lack of reference libraries, which may only be partially overcome by the prediction of Metabolomics is becoming increasingly common in plant migration time [27]. Since CE and LC can both separate a physiology and biochemistry, and to date has been applied large variety of metabolites via fundamentally different to a staggering number of conditions. Here we will attempt mechanisms, they are often used in combination to provide a synthesis of the most prominent studies dealing with a wider coverage of metabolites [28–30]. That said, the use plant stress; however, the reader is also referred to two of CE-MS in plant studies remains relatively rare. previous reviews on this topic [40, 41]. In this section we independently review water stress, temperature stress, light Nuclear magnetic resonance (NMR) spectroscopy stress, ionic stress, nutrient limitation and oxidative stress before discussing stress combinations. We describe the Nuclear magnetic resonance spectroscopy offers an nature and symptoms of each stress, and then introduce entirely different analytical technique to that afforded by several metabolomic studies with the main metabolic MS-based techniques being based on atomic interaction. In changes observed in each study and the conclusion drawn a strong magnetic field, atoms with non-zero magnetic from the results. Following this survey we discuss com- 1 13 14 15 31 moment including H, C, N, N and P absorb and monalities and differences between the various stress re-emit electromagnetic radiation. The radiation is char- responses. acterised by its frequency (chemical shift), intensity, fine structure and magnetic relaxation properties, all of which Water stress reflect the precise environment of the detected nucleus. Therefore, atoms in a molecule give a specific spectrum of Water limitation is one of the major threats in crop pro- radiation that can be used for identification and quantifi- duction and this condition is projected to get considerably cation of metabolites within a complex biological sample. worse in coming decades [42]. For this reason considerable The sensitivity of this method is much lower than that of research effort has been expended to understand the MS-based techniques but the structural information con- response to this crucial and common stress. These studies tent, reproducibility and quantitative aspects can be have revealed an important role for metabolic regulation superior to them, and some journals require NMR spectra including regulation of photosynthesis and accumulation of as the final proof of chemical structure [31, 32]. Further- osmolytes in the drought stress response [43, 44]. Urano more the preparation of the sample is simple and even non- et al. [28] reported metabolomic changes in Arabidopsis destructive measurement is possible. In vivo NMR can leaves under drought condition. The accumulation of many further generate kinetic measurements and examine meta- metabolites was observed, including amino acids such as bolic responses on the same plant rather than on a set of proline, raffinose family oligosaccharides, c-amino buty- similar plants [33]. The different subcellular pHs of the rate (GABA) and tricarboxylic acid (TCA) cycle 123 3228 T. Obata, A.R. Fernie metabolites, which are known to respond to drought stress phosphate and glycerol-3-phosphate, were observed as well in plants. The authors also investigated the nc3-2 mutant, as changes in the levels of minor sugars and various which lacks the NCED3 gene involved in the dehydration- organic acids. When oxygen is decreased to 4 %, there is a inducible biosynthesis of abscisic acid (ABA), in order to general tendency for an increase in the levels of the assess the effect of ABA in the metabolic response to intermediates both of sucrose degradation and the TCA drought stress. By combination with transcriptome analysis cycle, and in the levels of most amino acids, whereas they they clearly demonstrated that the ABA-dependent tran- are decreased when the oxygen further decreased to 1 %, scriptional regulation is responsible to the activation of indicating the inhibition and reactivation of metabolic metabolic pathways including branched chain amino acid, activities. Together with the transcriptomic data showing a polyamine and proline biosynthesis, GABA shunt and general downregulation of energy-consuming processes, saccharopin metabolism, but is not involved in the regu- the results demonstrated a large-scale reprogramming of lation of the raffinose biosynthetic pathway during metabolism under oxygen-limited conditions. Rocha et al. dehydration. [51] examined the accumulation of alanine under anoxic In Arabidopsis research, drought tolerance is assessed conditions in Lotus japonicus, which is highly tolerant to predominantly under lethal conditions. However, in tem- water logging. In the roots of L. japonicus, succinate, perate climates, limited water availability rarely causes alanine and the direct co-substrates for alanine synthesis, plant death but does restrict biomass and seed yield. glutamate and GABA, were highly accumulated during Results of a recent elegant study experimentally demon- water logging, whereas the majority of amino acids that are strated that the survival rate under lethal conditions does derived from TCA cycle intermediate decreased. The not predict superior growth performance and biomass yield results are in agreement with the metabolic equilibriums gain under moderate drought [45], making this mild stress that are expected to drive the metabolic flux from glycol- condition more important. Skirycz et al. [46] conducted ysis, via alanine synthesis and oxoglutarate to succinate, metabolite profiling of Arabidopsis leaves that develop which prevents the accumulation of pyruvate activating under mild osmotic stress. They revealed that the stress fermentation and leading to ATP production by succinyl- response measured in growing and mature leaves was CoA ligase. largely distinct. Typical drought responses, namely accu- mulation of proline, erythritol and putrecine, were Temperature stress observed only in mature leaves, while many metabolites were decreased in expanding leaves, sharing the same Exposure to freezing environments leads to serious damage tendency with transcriptional response. When we com- of the plant cell by ice formation and dysfunction of cel- lular membranes [52]. Many plant species increase freezing pared the data from the studies [28, 46], 24 metabolites were detected in both. The decrease of aspartate and tolerance during exposure to non-freezing low temperature increase of proline are the only two responses shared by a process known as ‘‘cold acclimation’’. The molecular between mildly and severely desiccated leaves. Pro- basis of this process has been extensively studied, and the nouncedly, amino acid metabolism responds in opposite contribution of particular metabolites including compatible ways; most amino acids were accumulated in severely solutes [53] and the transcriptional regulatory network has desiccated leaves but decreased in mildly desiccated plants. been elucidated [54, 55]. The first metabolomic studies of These results highlight the variable response of plant cold acclimation were performed by two groups in 2004. metabolism in different developmental stages and degrees Cook et al. [56] compared metabolomic changes during of desiccation. Metabolite profiling has additionally been cold acclimation in two ecotypes of Arabidopsis thaliana, carried out in crop species exposed to water stress condi- Wassilewskija-2 (Ws-2) and Cape verde islands-1 (Cvi-1), tions. Intriguingly, common changes in the levels of which are relatively freezing tolerant and sensitive, metabolites including branched chain amino acids were respectively. The metabolome of Ws-2 plants was exten- observed in wheat, barley and tomato [47–49]. sively altered in response to low temperature. Seventy-five Too much water, as occurs in situations such as flooding percent of metabolites monitored were found to increase in or water-logging of the rhizosphere, also causes problems cold-acclimated plants including metabolites known to because of the reduced oxygen availability (hypoxia/ increase in Arabidopsis plants upon exposure to low tem- anoxia). Under anoxic conditions, ATP has to be produced perature, such as the amino acid proline and the sugars by fermentation, resulting in cytosolic acidification and the glucose, fructose, inositol, galactinol, raffinose and accumulation of toxic products. van Dongen et al. [50] sucrose. They also found novel changes—namely the analysed metabolic responses in Arabidopsis roots under increase of trehalose, ascorbate, putrescine, citrulline and anoxic conditions. The accumulation of amino acids, ala- some TCA cycle intermediates. There was considerable nine, proline and GABA, and the phosphoesters, glucose-6- overlap in the metabolite changes that occurred in the two 123 Metabolomics of plant stress 3229 ecotypes in response to low temperature; however, quan- effect. The esk1 mutant is isolated as freezing tolerant titative differences were evident. Kaplan et al. [57] without previous acclimation but the function of this gene conducted metabolome analysis of Arabidopsis over the had been unknown. Lugan et al. [64] tried to elucidate the time course following the shift to cold and heat conditions. basis of the freezing tolerance of esk1 by performing Surprisingly the majority of heat shock responses were metabolomic analysis under various environmental condi- shared with cold shock including the increase of pool sizes tions, namely cold, salinity and dehydration. Then the most of amino acids derived from pyruvate and oxaloacetate, specific metabolic responses to cold acclimation were not polyamine precursors and compatible solutes. The results phenocopied by esk1 mutation. However, esk1 accumu- of this study were analysed together with following tran- lated lower amount of Na in leaves than the wild type and script profiling data by the same group [58], and revealed its metabolic profile, and osmotic potential were only that the regulation of GABA shunt and proline accumula- slightly impacted under dehydration stress. These results tion under cold conditions are achieved by transcriptional suggested that ESK1 could rather be involved in water and post-transcriptional manners, respectively. Gray and homeostasis and as such highlighted the importance of Heath [59] examined the effects of cold acclimation on the cellular water status in stress tolerance. Arabidopsis metabolome using a non-targeted metabolic fingerprinting approach. It revealed a global reprogram- Light stress ming of metabolism as well as differential responses between the leaves that shifted to and those that developed Light is a highly energetic substrate driving photosynthesis in the cold. Hannah et al. [60] took advantage of the natural that can induce secondary destructive processes at the same genetic variation of Arabidopsis to elucidate the function of time. Therefore, too high light irradiance represents an metabolism in cold acclimation. Although there is no clear abiotic stress factor for plants. Wulff-Zottele et al. [65] relationship between global metabolite changes and dif- conducted metabolite profiling of Arabidopsis leaves for ferences in acclimation capacity or differences between the 6 days after transition to high light. Generally, most of the accessions in acclimated freezing tolerance, the probable metabolites of the glycolysis, TCA cycle and oxidative importance of central carbohydrate metabolism is indicated pentose phosphate pathway were altered in their content, by the identification of glucose, fructose and sucrose indicating that plants exposed to high light undergo a among metabolites positively correlating to freezing tol- metabolic shift and enhance the Calvin-Benson cycle to fix erance. Espinoza et al. [61] analysed the effect of diurnal more carbon. In addition, elevation of glycine indicated the gene/metabolite regulation during cold acclimation by activation of photorespiratory pathways. Caldana et al. [66] means of metabolomics and transcriptomics. Approxi- investigated the early metabolic response against high light mately 30 % of all analysed metabolites showed circadian as a part of a more comprehensive study. The accumulation oscillations in their pool size and low temperature affected of the photorespiratory intermediates, glycine and glyco- the cyclic pattern of metabolite abundance. These results late, were observed in the early phase (5–60 min after indicated that the interactions observed between circadian transition). Interestingly the response during the mid phase and cold regulation are likely highly relevant components (80–360 min) shares similar properties with low tempera- of cold acclimation. ture treatment, which includes the accumulation of Metabolomics was also used to reveal the functions of shikimate, phenylalanine and fructose, and the decrease of specific genes in cold acclimation. In the study described succinate; however, the physiological meaning of this above, Cook et al. [56] also investigated plants over- overlap is currently unknown. expressing CBF3, which is one of the C-repeat/dehydration Not only the quantity but also the quality of light affects responsive element-binding factor (CBF) transcriptional plant metabolism. In dense plant stands, such as crop fields activators induced rapidly under low temperature condi- or forests, individuals shade each other and create com- tions [62]. The metabolite profiles of non-acclimated CBF3 petition for light absorption [67]. Selective light absorption overexpressing lines were quite similar to those of the cold- by the upper leaf layers leads to an enrichment of far-red acclimated Ws-2 ecotype, suggesting a prominent role for wavelength [68], which induces excitation imbalances the CBF cold response pathway in configuring the low- between photosystem II and I disturbing both the redox temperature metabolome of Arabidopsis. Maruyama et al. chemistry in the transport chain and its coordination with [63] explored metabolic and transcript changes in Arabid- the Calvin-Benson cycle [69, 70]. For this reason, Bra ¨uti- opsis plants overexpressing CBF3/dehydration-responsive gam et al. [71] grew Arabidopsis plants under light, which element binding protein (DREB)1A and another DREB preferentially excited either photosystem I (PSI light) or II protein DREB2A. They observed similar changes of (PSII light) and then transferred it to the other light con- metabolites in CBF3-overexpressing plants like Cook et al. dition to analyse how plants acclimate to the light quality [56] but DREB2A overexpression showed only a minor shift. After long-term acclimation of 48 h, plants exhibited 123 3230 T. Obata, A.R. Fernie two distinct metabolic states. A PSI–II shift resulted in a Proline increased dramatically in both species as did decrease in primary products of photosynthesis, such as inositols, hexoses and complex sugars. The concentrations sugars, but an increase in important intermediates of sub- of metabolites were often several-fold higher in Thel- sequent metabolic pathways. By contrast, a PSII-I shift has lungiella and stress exacerbated the differences in some no effect on the sugar pools but leads to general down- metabolites. Transcript analyses supported the metabolic regulation of many subsequent metabolites, including results by suggesting that a Thellungiella is primed to amino acids and organic acids. Each of the metabolites anticipate such stresses. The difference in metabolites exhibited a different accumulation profile for establishing between Arabidopsis and Thellungiella under salt and the final pool size, indicating high complexity by which the osmotic stresses was more recently assessed for a broader two metabolic states were achieved. Comprehensive anal- range of metabolites [80]. Analysis of global physico- yses of these data alongside transcript profiles and other chemical properties of metabolites revealed a shift from physiological data suggested that photosynthesis and nonpolar to polar metabolites in both species but that this metabolism were under the control of a binary combination was much more pronounced in Thellungiella. Such a shift of inputs from the thioredoxin and plastoquinone systems. may contribute to keep the water potential during dehy- The dependency of plants upon sunlight also inevitably dration. Kim et al. [77] investigated the cellular level leads them into exposure to ultraviolet (UV) light, metabolic response using Arabidopsis T87 cultured cells. including in the wavelength range of 280–320 nm (UV-B). The results suggested that the methylation cycle for the This wavelength potentially damages DNA, RNA and supply of methyl groups, the phenylpropanoid pathway for proteins, and additionally increases the production of free lignin production and glycine betaine biosynthesis are radicals [72, 73]. Kusano et al. [74] treated Arabidopsis synergetically induced as a short-term response against plants with UV light and analysed the metabolic effect of salt-stress treatment. The results also suggest the UV light stress. Arabidopsis exhibits an apparent biphasic co-induction of glycolysis and sucrose metabolism as well response to UV-B stress, characterised by major changes in as co-reduction of the methylation cycle as long-term the levels of primary metabolites, including ascorbate responses to salt stress. derivatives. By contrast, mid- to late-term responses were Due to the importance of salinity stress in agriculture, observed in the classically defined UV-B protectants, such there are many metabolomic studies to assess the meta- as flavonoids and phenolics. The results suggested that in bolic effect of salinity in a variety of crop and related early stages of exposure to UV-B, the plant cell is ‘primed’ plant species including tomato [40, 81], grapevine [82], at the level of primary metabolism by a mechanism that poplar [83], sea lavender (Limonium latifolium,[84]) and involves reprogramming of the metabolism to efficiently rice [85]. Since these studies have been extensively divert carbon towards the aromatic amino acid precursors reviewed in [40, 86], we focus here on three recent studies of the phenylpropanoid pathway. It also suggested the on legume species [87–89]. These recent studies took a importance of ascorbate in the short-term response to functional genomic approach that integrated ionomic, UV-B. Further studies are, however, required to determine transcriptomic and metabolomic analyses of the glycopyte which of these metabolic changes are end responses to model legume Lotus japonicus and other Lotus species adapt to the enhanced exposure to UV-B and which are part subjected to long-term regimes of non-lethal levels of of the perception-signalling relay, which alerts the plant salinity. In Lotus japonicus the metabolic changes were cell that it needs to respond to the stress [75]. characterised by a general increase in the steady-state levels of many amino acids, sugars and polyols, with a Ion stress concurrent decrease in most organic acids [87]. The responses to salinity stress were compared between High levels of salinity in the soil hinder the growth and extremophile (L. creticus) and glycophytic (L. cornicula- development of crops and cause serious problems for world tus and L. tenuis), but the metabolic responses were food production [76]. High concentrations of NaCl may globally similar to each other [88]. These results suggest cause both hyperionic and hyperosmotic stress effects, that, in contrast to Thellungiella, the metabolic pre- which lead to a decline of turgor, disordered metabolism adaptation to salinity is not the major trait of L. creticus and the inhibition of uptake of essential ions, as well as contributing to the extramophile phenotype. However, by other problems in plant cells [77, 78]. Gong et al. [79] comparing six species displaying different salt tolerances, conducted metabolite profiling of salt-treated Arabidopsis they observed several genotype-specific features. One of thaliana and its relative Thellungiella halophila (salt them is the increase of asparagine levels in the more cress), which shows ‘extremophile’ characteristics mani- tolerant genotypes, suggesting that the roles of asparagine fested by extreme tolerance to a variety of abiotic stresses, metabolism in supporting core nitrogen metabolism may among them low humidity, freezing and high salinity. play a role in tolerance [89]. 123 Metabolomics of plant stress 3231 Heavy metals such as cadmium (Cd), cesium (Cs), lead raffinose, glycerate and fatty acids, decreased. Central (Pb), zinc (Zn), nickel (Ni) and chromium (Cr) are major amino acids (glutamine, glutamate, aspartate and alanine) pollutants of the soil causing stress on plants. Even the and methionine, an S-containing amino acid, also essential nutrients including copper (Cu), iron (Fe) and decreased, indicating the inhibition of N and S assimila- manganese (Mn) can cause heavy metal stresses with tion, respectively. The increase of most other amino acids inappropriate concentration. Generally heavy metals indicates that proteolysis has commenced. Most of these induce enzyme inhibition, cellular oxidation and metabolic changes reverted rapidly after re-addition of sucrose into perturbation, resulting in growth retardation and in extreme the media. Usadel et al. [97] took advantage of extended instances in plant death [90]. Jahangir et al. [91] analysed dark treatment to induce C starvation under more natural the effects of Cu, Fe and Mn on the metabolite levels of conditions in the Arabidopsis rosettes. Intriguingly, how- Brassica rapa, which is a known metal accumulator. ever, the changes in metabolite levels were mostly Glucosinolates and hydroxycinnamic acids conjugated with comparable to those observed in liquid culture seedlings malates as well as primary metabolites such as carbohy- [96]. The marked decrease of carbohydrates within the first drates and amino acids were found to be the discriminating 4 h of extended night indicates that the treatment induced metabolites. Arabidopsis plants treated with Cd displayed C starvation very efficiently and that carbohydrates are increased levels of alanine, b-alanine, proline, serine, starting to acutely limit metabolism. On the other hand, putrescine, sucrose and other metabolites with compatible organic acids and other C-containing metabolites displayed solute-like properties, notably GABA, raffinose and tre- a rather gradual decrease. The prolonged dark treatment halose [92]. This study also indicated that concentrations of induced severe C starvation and leaf senescence by the end antioxidants (a-tocopherol, campesterol, ß-sitosterol and of the experiment. The metabolite profile of Arabidopsis isoflavone) also increased significantly. When taken toge- leaves subjected to prolonged darkness has been analysed ther these data indicate an important role of antioxidant in a series of studies to elucidate the metabolic bases of defences in the mechanisms of resistance to cadmium dark-induced senescence and the function of the mito- stress. Dubey et al. [93] conducted transcriptomic and chondrial alternative electron transport pathway during metabolomic analysis of rice roots treated with Cr. Under dark treatment [98–100]. Although a similar metabolic these conditions proline accumulated to levels three-fold phenotype as the two studies described above [96, 97] was those of the control as did ornithine, which can be used in observed during the first few days of dark treatment, a its synthesis. The content of several other metabolites subset of metabolites exhibits biphasic behaviour during including lactate, fructose, uracil and alanine increased prolonged exposure to darkness. This was particularly following exposure to Cr stress; these were taken to sug- notable for some TCA cycle intermediates including gest the modulation of the sucrose degradation pathway fumarate, isocitrate, malate and succinate, which accumu- involving the three main fermentation pathways operating lated after 7 days of dark treatment despite decreasing as a rescue mechanism when respiration is arrested. Further during the first 3 days of treatment. Additionally accumu- studies are however most likely warranted to gain a better lation of most amino acids including GABA became much understanding of the mechanisms underlying these more prominent. Metabolite profiles were also analysed in changes. a range of mutants deficient in the genes involved in mitochondrial alternative electron transport mediated Nutrient limitation by the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) Nutrient starvation also dramatically affects plant growth complex, namely ETFQO [98] and ETFb [99] as well as and metabolism. Especially limitation of macronutrients, enzymes involved in the provision of its substrates, namely namely carbon (C), nitrogen (N), phosphorus (P) and sul- IVDH, D2HGDH [101] and PSHX [100]. Although indi- phur (S), has direct effects on metabolism since most vidual genotypes showed similar responses during the first organic molecules comprise a combination of these ele- 3 days of dark treatment, there are subtle differences in ments. Changing environmental conditions continually their metabolic complements at the end of the experiment, alter the balance between C assimilation and utilisation. indicating an essential role of this alternative electron Even short periods of C starvation lead to an inhibition of transport machinery during dark-induced starvation [99]. growth, which is not immediately reversed when C Further detailed analysis revealed that the ETF/ETFQO becomes available again [94, 95]. Osuna et al. [96] complex is involved in both the branched chain amino investigated the metabolite profile of Arabidopsis seedlings acids and the lysine catabolism pathways, and acts as an in liquid culture under C starvation. In C-starved seedlings, electron donor to the mitochondrial ubiquinol pool [100, as could be anticipated, carbohydrates, organic acids and 101]. These studies suggest that more integrative analysis other C-containing metabolites, including myo-inositol, of the role of all aspects of protein degradation and 123 3232 T. Obata, A.R. Fernie consequent remobilisation should be performed within the as well as sugars, while organic acids were accumulated in context of understanding metabolic responses to stress. P-deficient nodules. Such a contrasting response may be Nitrogen is required for the synthesis of nucleotides and due to the N deficiency in P-starved nodules in which the amino acids, which are the building blocks of nucleic acids sole N supply from fixed N could be suppressed under and proteins, and for the synthesis of phospholipids and environmental limitations such as P starvation [107]. many secondary metabolites that have diverse roles in Huang et al. [108] profiled metabolites from both shoots and signalling, structure and adaptation. The effect of N defi- roots of P-deficient barley. Severe P deficiency increased ciency on the metabolite levels in tomato leaves were the levels of phosphorylated intermediates (glucose-6-P, investigated by Urbanczyk-Wochniak and Fernie [102]. As fructose-6-P, inositol-1-P and glycerol-3-P) and organic would perhaps be expected, amino acid levels generally acids (2-oxoglutarate, succinate, fumarate and malate). The decreased under nitrogen deficiency. The level of 2-oxo- results revealed that P-deficient plants modify carbohydrate glutarate, a key regulator of carbon and nitrogen metabolism initially to reduce P consumption and salvage P interactions [103], decreased under N starvation as well as from small P-containing metabolites, which consequently other TCA cycle intermediates including citrate, isocitrate, reduce the levels of organic acid in the TCA cycle [108]. succinate, fumarate and malate. Tschoep et al. [104] ana- Sulphur is another macronutrient essential for the syn- lysed the effect of mild but sustained N limitation in thesis of the S-containing amino acids cysteine and Arabidopsis. Malate and fumarate levels were strongly methionine as well as a wide range of S-containing metab- decreased in low N conditions like in tomato leaves [102]. olites including glutathione. There are some metabolomic However, their rosette protein content was unaltered and studies on the response to S starvation in Arabidopsis [109– total, and many individual amino acid levels increased 112], and they are nicely summarised in Hoefgen et al. compared with N-replete plants. The results revealed that [113]. At the time course of S-stress response, two metabolic Arabidopsis responds adaptively to low N condition. P is states can be distinguished. The short-term metabolic an essential component of intermediates in central and responses include the decrease of organic S-containing energy metabolism, signalling molecules and structural compounds on the S assimilation such as cysteine and glu- macromolecules like nucleic acids and phospholipids. tathione, which leads to the accumulation of their precursor Morcuende et al. [105] analysed the metabolite profile of O-acetyl-serine (OAS) as well as serine, and to the sub- Arabidopsis seedlings grown in liquid culture under P sequent re-channeling of the metabolic flow to glycine and starvation. The levels of sugar phosphates were very low tryptophan. Glucosinolate catabolism is activated to salvage but metabolites further down in glycolysis, glycerate-3- S from it. As a long-term response the lipid contents and a phosphate, glycerate-2-phosphate and phosphoenolpyr- S-containing molecule, S-adenosyl-methionine, decreased. uvate, increased in P-deficient seedlings. Pi-deficient Insufficient S supply leads to its disbalance with N and seedlings showed a marked accumulation of starch, sucrose further to the alterations in C1 metabolism that link photo- and reducing sugars as well as a general increase of organic respiration, S assimilation and dumping of N [113]. Results acids including citrate, fumarate, malate and oxoglutarate. of a very recent study on the Arabidopsis plants with The levels of most major amino acids did not alter or modified OAS levels suggest the importance of this increased slightly, whereas those of several minor amino metabolite since OAS plays a signalling role for a specific acids including the aromatic amino acids and histidine, part of the sulphate response as well as for the regulation of arginine and threonine. Together with transcriptomic data, the transcript levels of a specific gene set irrespective of the analysis of metabolites revealed that P deprivation leads to sulphur status of the plants [114]. a shift towards the accumulation of carbohydrates, organic Potassium (K) is not a component of organic molecules acids and amino acids. The effect of P starvation has also but plays essential roles as a major cation in plants and as a been studied on crop plants such as common bean and cofactor of enzymes [115]. Armengaud et al. [116] used barley. Hernandez et al. used metabolite profiling to assess metabolite profiling to identify metabolic targets of K the effect of P deficiency in the roots [106] and nodules stress. Metabolite profiles of low-K Arabidopsis plants [107] of the common bean. Most of the amino acids were were characterised by a strong increase in the concentra- increased in P-stressed roots. The accumulation of several tions of soluble sugars (sucrose, fructose and glucose) and sugars suggests that sugars may be partitioned preferen- a slight net increase of total protein content and the overall tially to P-stressed roots to support the expression of P amino acid level. Several basic or neutral amino acids stress-induced genes. The reduced amounts of organic accumulated during K deficiency, while acidic amino acids acids likely reflect exudation of these metabolites from the decreased. In addition a strong decrease of pyruvate and roots into the rhizosphere [106]. The metabolic response of organic acids was recorded only in the roots but not in the P-starved nodules is in contrast to that of roots. Amino shoots. They also measured enzyme activities and con- acids and other N-containing metabolites were decreased cluded that the primary effect of K deficiency induces an 123 Metabolomics of plant stress 3233 inhibition of glycolysis by the direct inhibition of enzymes removal of menadione from the culture media [121]. After [116]. menadione removal many of the stress-related changes reverted back to basal levels. However, each metabolic Oxidative stress pathway recovered in a differential time period, for instance, glycolytic carbon flow reverted to control level Oxidative stress is a key underlying component of most 18 h after menadione removal, although the TCA cycle and abiotic stresses and a major limiting factor of plant growth some amino acids such as aspartate and glutamate took in the field [117]. It occurs on the overproduction of longer to recover. It suggests the involvement of pathway- reactive oxygen species (ROS) in plant cells when plant specific regulatory processes for the oxidative stress metabolism is perturbed by various stresses. This conse- response. These metabolic responses to menadione- quently leads to oxidative damages of cellular components induced oxidative stress mentioned above seem to be such as DNA, proteins and lipids [118]. To cope with conserved among plant species and organs because quite oxidative stress, the metabolic network of plant cells must similar responses were observed both in Arabidopsis be reconfigured either to bypass damaged enzymes or to seedlings in liquid culture [122] and rice suspension cells support adaptive responses. In the study by Baxter et al. [123]. They are additionally at least partially similar to [119], heterotrophic Arabidopsis cells were treated with those observed when oxidative stress is mimicked by the menadione, which enhances the ROS production via elec- removal of enzymes involved in ameliorating against it, tron transport chains and changes in metabolite abundance, such as manganese superoxide dismutase [124]. In the and C-labelling kinetics were quantified. The accumula- Arabidopsis plants with suppressed expression of mito- tion of sugar phosphates related to glycolysis and oxidative chondrial manganese superoxide dismutase revealed a pentose phosphate pathways (OPPP) suggested the rerout- decrease of TCA cycle intermediates, probably because of ing of glycolytic carbon flow into the OPPP possibly to the inhibition of aconitase and isocitrate dehydrogenase provide NADPH for antioxidative effort. In addition the [124]. decrease of ascorbate and accumulation of its degradation product, threonate, indicated the activation of antioxidative Stress combination pathways in menadione-treated cells. The reduced glyco- lytic activity probably leads to the decrease of levels of Whilst convenient both for experiments and discussion at amino acids derived from glycolytic intermediates. The the single stress level, plants are actually subjected to a decrease of amino acids linked to TCA cycle intermediates combination of abiotic stress conditions in their natural and decrease of malate indicated a perturbation of TCA habitat. Even some abiotic stresses are already combina- cycle. These observations in metabolite levels were tions of stresses. For example high salt concentration emphasised by C-redistribution analysis, which indicated causes osmotic and ion stresses, and flooding results in increased carbon flux into OPPP intermediates and inhi- hypoxic and shading stresses. Although the metabolic bition of metabolic flux into all TCA cycle intermediates responses of plants under a single abiotic stress have been detected [119]. Lehmann et al. [120, 121] also conducted analysed extensively as shown above, there are only few both metabolite profiling and C-redistribution analysis of studies regarding to the effect of stress combinations on menadione-treated Arabidopsis roots and found that the plant metabolism. Rizhsky et al. [125] applied a combi- metabolic response of roots is distinct from that of het- nation of drought and heat stress to Arabidopsis plants and erotrophic cells in culture [120]. The redirection of analysed the metabolic profile. The metabolite profile of glycolytic carbon flow and inhibition of the TCA cycle plants subjected to a combination of drought and heat stress were suggested also in the roots. Especially the inhibition was more similar to that of plants subjected to drought than of the TCA cycle is more evident in roots as a perturbation to that of control plants or plants subjected to heat stress. of metabolite levels. In addition, roots showed pronounced However, the plants subjected to combined stresses accu- accumulation of some metabolites including GABA, OAS, mulated high levels of sucrose and other sugars instead of pyruvate, many amino acids and glucosinolates. It seems proline, which is highly accumulated to a very high level in likely that cellular oxidation inhibited S assimilation and plants subjected to drought but not under stress combina- caused OAS accumulation. A general increase of amino tion. They concluded that sucrose replaces proline as the acid levels is thought to be the result of enhanced protein major osmoprotectant in plants subjected to combined degradation. This is supported by C-labelling analysis in stress because the toxic effect of high level of proline is which the C-redistribution was not affected in most enhanced under heat stress, as they showed experimentally amino acids, indicating that the carbon in the increased [125]. Wulff-Zottele et al. [65] analysed the effect of amino acids was not from synthetic pathways [121]. They the combination of high light irradiance and S depletion, also followed the metabolic recovery process after the which can occur in the field simultaneously [126]. 123 3234 T. Obata, A.R. Fernie The combination of high light and S depletion gives rise to modulating both cell division and cell expansion. Growth similar metabolic pool modifications such as in high light. decreases rapidly upon stress onset, but it recovers and Proline accumulated in a differential time course under adapts once stress conditions become stable [127]. Accu- high light and stress combination. Other metabolites such mulated metabolites might be used as building blocks to as raffinose and putrescine seem to replace proline during support a recovery of growth. Figure 1 provides an over- the delay of proline accumulation in the plants subjected to view of the changes in the amount of selected metabolites. high light and S depletion. This replacement of proline Charts for all metabolites are found in the Supplementary with sugars is similar to that observed under the combi- data, Fig. S1. In general, changes in the amounts of nation of drought and heat stress [125]. Recently Caldana metabolites were stress-specific in contrast to the general et al. [66] reported a systematic study on the metabolomic responses observed in bacteria [128]. A stress-specific and transcriptomic response of Arabidopsis to eight envi- change in the metabolite level would be a result of an ronmental conditions including the combinations of inhibition/activation of a specific metabolic pathway changing light (darkness, high light) and/or temperature especially in the short term. It should mainly be related to (cold and heat). The analysis has demonstrated that dark- the properties of enzymes such as sensitivity to tempera- ness and high temperature have a synergistic effect, thus ture, oxidation and ion concentration. In addition, presenting a more extreme condition. The reconstructed rearrangement of the metabolic network should also result metabolic networks for this condition also revealed an in changes of metabolites, which are related to the regu- exclusive correlation between several amino acids, lated pathways. Therefore, such a metabolite must be a including GABA with intermediates of the TCA cycle, good candidate for an analysis to elucidate the specific notably succinate. These results suggested that in the effects of an abiotic stress and the adaptive processes absence of photosynthesis protein degradation occurs rap- against it. On the other hand, metabolites responding to idly and subsequent amino acid catabolism serves as the various stresses can be related to fundamental stress main cellular energy supply [66]. responses. In the present analysis, some metabolites can be seen to accumulate in most abiotic stress conditions although the time and extent of accumulation varied among Common and stress-specific metabolic responses conditions. Levels of sucrose were increased in most stress against diverse abiotic stress conditions in at least one time point (Fig. 1). Sucrose is a major transport sugar in most plant species and is known to As described above, plants show a variety of metabolic accumulate under stress conditions [129]. Compounds responses against diverse abiotic stresses. The question is defined as ‘‘compatible solutes’’ also accumulate under whether there are any common metabolic responses to all various abiotic stress conditions. They are very soluble in abiotic stresses or the responses are always specific to the water and are non-toxic at high concentrations and function stress factors. To evaluate the accumulation of these to sustain the ordered vicinal water around proteins by compounds under stress conditions and to search for the decreasing protein-solvent interactions at low water activ- novel metabolic fingerprints related to the stress responses, ities [52, 130]. This group of compounds includes betaines we analysed the published metabolite profiling data avail- and related compounds; polyols and sugars, such as man- able in the above-mentioned literature. Studies dealing nitol, sorbitol and trehalose; and amino acids, such as with Arabidopsis leaves were chosen (dehydration [28], proline [131, 132]. Recent studies have revealed that they salt [79], heat and cold [57], high light and sulphur limi- function to protect plants not only from osmotic stress but tation [65], UV [74], light quality change [71], low also from various stress factors [130, 131, 133, 134]. nitrogen [104] and potassium limitation [104]) to afford Therefore, the synthetic pathways of those metabolites greater comparability. Forty-five metabolites detected in have been of interest for metabolic engineering and some more than half of the studies were analysed and each datum interventions have indeed increased the tolerance of some was converted into the fold change values against control crop plants to abiotic stress [130, 131]. Raffinose is a sugar growth conditions and presented using the log scale synthesised from sucrose and known to protect plant cells (Supplementary data, Table S1). Table 1 shows the number as an osmoprotectant; it also accumulates under most stress of metabolites accumulated or decreased under each stress conditions especially at the later stages of stress treatment condition. This reveals that abiotic stresses generally (Fig. 1). Raffinose is also shown to function to protect induce accumulation of metabolites with only the light plants from oxidative damage [135], making the observa- quality change as an exception. The tendency of accumu- tion reasonable since oxidative damage likely underlies lation is probably related to a cessation of the growth- most stress conditions. Interestingly, myo-inositol, which is reducing consumption of metabolites. When subjected to closely related to raffinose biosynthesis, did not show abiotic stresses, plants actively re-program their growth by prominent changes other than under high light conditions 123 Metabolomics of plant stress 3235 Table 1 Number of metabolites that changed their abundance under possible role of BCAAs under stress conditions would be each stress condition that of an alternative electron donor for the mitochondrial electron transport chain. The mitochondrial electron Condition Increased Decreased transport chain is primary supplied by electrons from Dehydration 26 2 NADH and succinate to produce ATP. Additionally there is Salt 3 0 an alternative way to feed electrons from other substrates Heat 10 4 via electron transfer flavoprotein (ETF) complex. Recent Cold 27 9 studies highlighted the importance of the alternative path- High light 39 4 way under dark and stress conditions especially under Light quality 1 7 carbon starvation (see also the ‘‘Nutrient limitation’’ sec- UV 14 5 tion) [98–101]. A C-feeding experiment has proven that Low N 10 3 lysine and BCAA are converted into D-2-hydroxyglutarate -S 11 2 and isovaleryl-CoA in vivo to be a direct electron donor for -K 13 0 the ETF complex via the action of D-2-hydroxyglutarate dehydrogenase and isovaleryl-CoA dehydrogenase [101]. The changes greater than two fold were counted As such, BCAAs can provide electrons both directly to the electron transport chain via the ETF complex as well as (Fig. 1). The amino acid, proline, is known as a major indirectly because their catabolic products feed directly compatible solute in Arabidopsis [133] and also accumu- into the tricarboxylic acid (TCA) cycle [142] (Fig. 2b). The lated under stress conditions despite being detected only in source of accumulated BCAAs would be the protein deg- a limited number of studies (Fig. 1). On the other hand, radation product, which has recently been proposed to be trehalose accumulated only under specific conditions, an important alternative respiratory substrate especially suggesting that it displays functions other than being a under certain stress conditions [142], as well as the acti- compatible solute (Fig. 1). Indeed it is unlikely that tre- vated synthetic pathway, which is observed under drought halose contents in plants—other than resurrection plants— stress conditions [28, 141]. Thus, our analysis emphasised are high enough to be directly involved in stress protection the importance of BCAA metabolism generally under [136] and some trehalose metabolism mutants exhibit abiotic stress conditions. Interestingly, the pattern of potential negative effects on plant physiology [134]. The accumulation of GABA is similar to those of BCAAs amount of trehalose may reflect that of its precursor, tre- (Fig. 1), although the reason why they are related under halose-6-phosphate, which has been documented to act as a stress conditions remains unclear. Thus, clarifying the signal molecule in plants [137]. GABA is another metab- exact mechanistic role of BCAAs under various conditions olite discussed in a context of stress response since it is will be an important priority for the future. largely and rapidly produced in response to biotic and abiotic stresses [138–140]. Our analysis supported this observation (Fig. 1). There are many suggested functions Prospective: toward the elucidation of molecular of GABA and the GABA shunt, which protect plants to mechanisms underlying abiotic stress tolerance survive various stress conditions including regulation of cytosolic pH, protection against oxidative stress and A wealth of metabolomics data concerning the plant stress functions of GABA as an osmoregulator and as a signalling response has been accumulated and a large number of molecule [138]. However, whilst evidence for an important metabolic pathways are suggested to be regulated under metabolic role has been documented, that for a signalling stress. However, there are relatively few pathways and role in plants is still lacking. In the presented data set, metabolites that have been experimentally proven to branched chain amino acids (BCAAs), namely valine, function in abiotic stress tolerance. One problem is that a leucine and isoleucine, and other amino acids sharing metabolite profile does not tell exactly whether the related synthetic pathways with BCAA, including lysine, threonine metabolic pathway is up- or downregulated since both and methionine (Fig. 2a), were generally accumulated upregulation of upstream reaction and down-regulation of under abiotic stress conditions. These amino acids are a downstream reactions can lead to the accumulation of a novel group of metabolites that accumulated generally in metabolite. This can be solved by comparing the meta- response to stress conditions, although they have been bolomic data with those from transcriptomic or proteomic shown to accumulate under drought stress conditions [141]. analysis or activities of specific enzymes [143]. Hirai et al. Joshi et al. [141] suggested that they function as compat- [111] revealed gene to metabolite regulatory networks of ible osmolytes since BCAA showed a high fold increase glucosinolate synthesis and primary metabolism under under drought stress in various plant tissues. Another sulphur- and nitrogen-limited conditions by applying 123 3236 T. Obata, A.R. Fernie 4 10 3 Sucrose Raffinose myo-inositol -2 -1 -1 -4 Trehalose Proline -2 -4 -2 Isoleucine Valine Leucine 6 8 -2 -2 -2 6 6 Lysine Threonine Methionine 2 2 -2 -2 -2 GABA -2 Fig. 1 Changes of the levels of metabolites in Arabidopsis leaves (Low N) [104], sulphur limitation (-S) [65] and potassium limitation under various abiotic stress conditions. Each datum represents the (-K) [116] stresses. The data set used for the analysis is found in relative metabolite level in the fold change value against control Supplementary data, Table S1. The bars with different colours growth conditions at one time point. The values are taken from represent the values from different studies as shown in the figure. studies on dehydration [28], salt [79], heat and cold [57], high light Only the metabolites of interest are shown. The charts for all [65], light quality change [71], UV-B light (UV) [74], low nitrogen metabolites are presented as Supplementary data, Fig. S1 integrated analysis of transcriptome and metabolome data. [63] as described above. This approach is proven to be Integrated analyses of the transcriptome and the metabo- useful to elucidate the regulation of the pathway and also lome successfully demonstrated connections between the involvement of transcriptional regulation of the genes and metabolites, elucidating a wide range of signal pathway. The studies using proteomics together with output from ABA under dehydration [28] and the DREB1/ metabolomics are relatively rare in the plant stress response CBF transcription factors in response to low temperature field. One example is the study by Wienkoop et al. [144], Fold change (log2) Dehydration Salt Heat Cold High light light quality UV Low N -S -K Metabolomics of plant stress 3237 transcriptome and immunoprecipitation-based translatome a amino acid synthetic mode b protein degradation mode data sets have provided an important foundation for the Protein degradation Glycolysis Ile Leu Val analysis of the transcriptional and translational control of Met Thr Lys Ile Leu Val environmental responses in each tissue layer of the plant Met Thr Pyruvate [148, 149], the metabolomic studies are still rare because of Isovaleryl CoA HG Asp OAA the technical difficulties. Ebert et al. [150] applied single D2HGDH IVDH Lys cell sampling using microcapillaries to enable the cell- TCA cycle 2OG type-specific metabolic analysis of epidermal cell types in ETF ETFQO Arabidopsis thaliana pavement, basal and trichome cells. Succinate SDH Recently, Rogers et al. [151] demonstrated the feasibility - Ubiquinone of FACS-based metabolic profiling using high-resolution Complex I NADH mass spectrometry at cell type resolution in roots. ATP production The integration of the ‘‘omics’’ data revealed many Fig. 2 Metabolic modes leading to the accumulation of branched molecular mechanisms for metabolic regulation but also chain amino acids (BCAA) and related amino acids suggested under highlighted a complex relationship among the levels of abiotic stress conditions. a Amino acid synthetic mode. BCAAs are transcripts, metabolites and metabolic flux. It suggests the synthesised using pyruvate or oxaloacetate (OAA) as carbon skele- tons. b Protein degradation mode. Amino acids resulting from participation of post-transcriptional especially post-trans- degraded proteins would be direct and indirect electron donors to lational regulation of enzyme activity in the regulation of produce ATP. 2OG 2-oxoglutarate, SDH succinate dehydrogenase, primary metabolism. An important role of post-transcrip- HG hydroxyglutarate, D2HGDH D-2-hydroxyglutarate dehydroge- tional regulation in the stress response is also suggested nase, IVDH isovaleryl-CoA dehydrogenase, ETF electron transfer flavoprotein, ETFQO ETF-ubiquinone oxidoreductase, e electron by the poor statistical correlation of protein expression data with microarray results especially in the short-term which showed the importance of starch and raffinose response [152–156]. And there is an explicit indication family oligosaccharide metabolism during temperature that considerable metabolic control is executed on the stress by the metabolomic and proteomic analysis of the metabolite and on the protein level including protein starch-deficient Arabidopsis mutant lacking phosphoglu- modifications [157]. Metabolic enzymes are well known to comutase (pgm mutant). The number of such studies should be regulated allosterically by the substrates and/or the increase in the near future because of the improvement of products of the pathway [158, 159]. Many other post- analytical methods for proteomics. The activities of translational modifications of the enzyme proteins such as enzymes involved in a pathway should have a direct rela- phosphorylation, glutathionylation and nitrosylation could tionship with the amount of a metabolite and could be a be involved in metabolic regulation [160–162]. Among useful tool to assess the metabolic regulation. Changes in them, we discuss here the reconfiguration of enzyme pro- maximal enzyme activities were analysed together with tein complexes in this study. In the protein complex transcriptomic and metabolomic data in the study by ‘‘substrate channeling’’ can happen by which the interme- Armengaud et al. [116], which pinpointed that pyruvate diate produced by one enzyme is transferred to the next kinase activity was inhibited directly by K deficiency and enzyme without complete mixing with the bulk phase was primarily responsible for the metabolic disorders [163]. Especially the association of several sequential observed. Metabolic flux analysis is another powerful metabolic enzymes involved in one pathway is called a approach to study the regulation of metabolic pathways. metabolon [164]. Metabolite channeling can be envisioned Lehmann et al. [121] conducted C redistribution analysis as a means to improve catalytic efficiency by increasing to prove the downregulation of glycolysis under oxidative local substrate concentrations, regulating competition stress treatment, which is suggested by metabolic profiling between branch pathways for common metabolites, coor- [120]. Metabolic flux analysis is also useful to elucidate a dinating the activities of pathways with shared enzymes or metabolic regulation that cannot be detected by metabolic intermediates, and sequestering reactive or toxic interme- profiling since the carbon flow can be affected without any diates [165, 166]. The organisation of metabolic pathways apparent changes in the metabolite pool sizes [119, 145, by metabolic channeling has been discussed as the main 146]. Improvement of both experimental and theoretical molecular-scale organisation units to orchestrate the mul- mathematical approaches to flux estimation and parame- tiple metabolic processes and it is now supported by terisation will greatly aid our understanding here [147]. modelling as well as experimental evidence [167–170]. The analysis of cell-type-specific responses would reveal Recent bioinformatic study suggested that evolved protein more detailed mechanisms of the stress response that interactions may contribute significantly towards increas- are hidden in a mixture of the cells in a tissue. Although ing the efficiency of metabolic processes by permitting the fluorescence-activated cell sorting (FACS)-based higher metabolic fluxes [171]. Metabolite channeling is 123 3238 T. Obata, A.R. Fernie considered to be achieved not only when the enzyme information will provide an immense foundation for meta- association is stable but also when the association is bolic engineering and synthetic biology approaches to dynamic. Transient complexes offer the possibility of fast ensuring food security. exchange of some of the polypeptide components upon Open Access This article is distributed under the terms of the reassembly and thus can be a molecular basis for rapid fine Creative Commons Attribution License which permits any use, dis- tuning or redirection of metabolism. The reassembly of tribution, and reproduction in any medium, provided the original the metabolic enzyme complex therefore should have a author(s) and the source are credited. molecular basis underlying the metabolic regulation in mitochondria under short-term oxidative stress. Metabolic channeling including metabolon formation is reported in References many processes in plants such as glycolysis, cysteine synthesis, the Calvin-Benson cycle, cyanogenic glucoside 1. Fiehn O (2001) Combining genomics, metabolome analysis, and biosynthesis, the phenylpropanoid pathway, the glycine biochemical modelling to understand metabolic networks. 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Published: Aug 12, 2012

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