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Regulation of Primary Metabolism in Response to Low Oxygen Availability as Revealed by Carbon and Nitrogen Isotope Redistribution1[OPEN] Carla António 2*, Carola Päpke 2, Marcio Rocha, Houssein Diab, Anis M. Limami, Toshihiro Obata, Alisdair R. Fernie, and Joost T. van Dongen Energy Metabolism Group (C.A., C.P., M.R., J.T.v.D.) and Central Metabolism Group (T.O., A.R.F.), Max Planck Institute of Molecular Plant Physiology, D–14476 Potsdam-Golm, Germany; Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier-Universidade Nova de Lisboa, 2780–157 Oeiras, Portugal (C.A.); Departamento de Produção Animal e Vegetal, Faculdade de Ciências Agrárias, Universidade Federal do Amazonas, Manaus, Amazonas 69082–653, Brazil (M.R.); University of Angers (H.D., A.M.L.) and Institut National de la Recherche Agronomique (A.M.L.), Unité Mixte de Recherche 1345 IRHS, SFR 4207 QUASAV, F–49045 Angers, France; and Institute for Biology I, RWTH Aachen University, D–52056 Aachen, Germany (J.T.v.D.) ORCID IDs: 0000-0001-6747-1243 (C.A.); 0000-0002-9985-2363 (A.M.L.); 0000-0001-7944-9289 (J.T.v.D.). Based on enzyme activity assays and metabolic responses to waterlogging of the legume Lotus japonicus, it was previously suggested that, during hypoxia, the tricarboxylic acid cycle switches to a noncyclic operation mode. Hypotheses were postulated to explain the alternative metabolic pathways involved, but as yet, a direct analysis of the relative redistribution of label through the corresponding pathways was not made. Here, we describe the use of stable isotope-labeling experiments for studying metabolism under hypoxia using wild-type roots of the crop legume soybean (Glycine max). [13C]Pyruvate labeling was performed to compare metabolism through the tricarboxylic acid cycle, fermentation, alanine metabolism, and the g-aminobutyric acid shunt, while [13C]glutamate and [15N]ammonium labeling were performed to address the metabolism via glutamate to succinate. Following these labelings, the time course for the redistribution of the 13C/15N label throughout the metabolic network was evaluated with gas chromatography-time of flight-mass spectrometry. Our combined labeling data suggest the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase, also known as complex II of the mitochondrial electron transport chain, providing support for the bifurcation of the cycle and the down-regulation of the rate of respiration measured during hypoxic stress. Moreover, up-regulation of the g-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia. 1 This work was supported by the Max Planck Society and the Federal Ministry of Education and Research (HydromicPro), by the Ministère de la Recherche et Technologies and the QUALISEM program funded by the Région des Pas de la Loire (to A.M.L. and H.D.), by the FCT Investigator program of the Fundação para a Ciência e a Tecnologia (grant no. IF/00376/2012/CP0165/CT0003 to C.A.), and by the Instituto de Tecnologia Química e Biológica António Xavier research unit GREEN-it, Bioresources for Sustainability (grant no. UID/Multi/04551/2013 to C.A.). 2 These authors contributed equally to the article. * Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Carla António ([email protected]). J.T.v.D. conceived the original screening and research plans; J.T.v.D. supervised the experiments; C.A., C.P., and J.T.v.D. designed the experiments; C.A. and C.P. performed the experiments; C.A. and C.P. analyzed the data; M.R. and H.D. provided assistance in data analysis; J.T.v.D. conceived the project; C.A. wrote the article with contributions of all the coauthors; A.M.L., A.R.F., and T.O. complemented the writing and data analysis. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.15.00266 Plants are sessile, unable to relocate when exposed to diverse environmental and seasonal stimuli, and hence must be able to respond rapidly to survive stress conditions. Flooding or waterlogging of the soil is a common environmental condition that can greatly affect crop production and quality by blocking the entry of oxygen into the soil so that roots and other belowground organs cannot maintain respiration. In recent decades, the number of extreme floodings has strongly increased, which is especially tragic because most arable land worldwide is located in regions that are threatened by regular flooding events (Voesenek and Bailey-Serres, 2015). In plant heterotrophic tissues, respiratory metabolism is composed of various pathways, including glycolysis, the mitochondrial tricarboxylic acid cycle, and the mitochondrial electron transport chain. Under normal conditions, the conversion of Glc to pyruvate in the cytosol involves an initial input of ATP and produces the reduced cofactor NADH. The reactions of the tricarboxylic acid cycle occur within the mitochondrial matrix and lead to the complete oxidation of pyruvate, moving electrons from organic acids to the oxidized Plant PhysiologyÒ, January 2016, Vol. 170, pp. 43–56, www.plantphysiol.org Ó 2016 American Society of Plant Biologists. All Rights Reserved. Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 43 António et al. redox cofactors NAD+ and FAD, forming the reducing equivalents NADH and FADH2 and concomitantly releasing carbon dioxide (Tovar-Méndez et al., 2003; Millar et al., 2011). Finally, the reduced cofactors generated during glycolysis and the tricarboxylic acid cycle are subsequently oxidized by the mitochondrial electron transport chain to fuel ATP synthesis by a process known as oxidative phosphorylation (Fernie et al., 2004; Plaxton and Podesta, 2006). The tricarboxylic acid cycle turnover rate depends greatly on the rate of NADH reoxidation by the mitochondrial electron transport chain and on the cellular rate of ATP utilization (Plaxton and Podesta, 2006). Besides supporting ATP synthesis, the reactions of the tricarboxylic acid cycle also contribute to the production of key metabolic intermediates for use in many other fundamental biosynthetic processes elsewhere in the cell (Fernie et al., 2004; Sweetlove et al., 2010; van Dongen et al., 2011; Araújo et al., 2012). Nevertheless, the control and regulation of the carbon flux through the tricarboxylic acid cycle are still poorly understood in plants, and noncyclic modes have been described to operate under certain circumstances (Rocha et al., 2010; Sweetlove et al., 2010; Araújo et al., 2012). Upon hypoxia, respiratory energy (ATP) production via oxidative phosphorylation by the mitochondrial electron transport chain goes down. To compensate for this, the glycolytic flux increases and Glc is consumed faster in an attempt to produce ATP via the glycolytic pathway, a process known as the Pasteur effect. To survive short-term hypoxia during flooding or waterlogging, plants must generate sufficient ATP and regenerate NADP+ and NAD+, which are required for glycolysis (Narsai et al., 2011; van Dongen et al., 2011). In addition to the accumulation of ethanol and lactate in oxygen-deprived plant tissues, metabolites such as Ala, succinate, and g-aminobutyric acid (GABA) have also been shown to accumulate (Sousa and Sodek, 2003; Kreuzwieser et al., 2009; van Dongen et al., 2009; Rocha et al., 2010; Zabalza et al., 2011), although hardly anything is known about the fate of these products of hypoxic metabolism. However, the relative abundance of these products of hypoxic metabolism varies between plant species, genotypes, and tissues and can change throughout the course of oxygen limitation stress as well (Narsai et al., 2011). A model describing metabolic changes during hypoxia has been described previously for waterlogged roots of the highly flood-tolerant model crop legume Lotus japonicus (Rocha et al., 2010): upon waterlogging, the rate of pyruvate production is enhanced due to the activation of glycolysis (Pasteur effect) and the concomitant production of ATP via substrate-level phosphorylation. At the same time, the fermentation pathway is activated with the accumulation of lactate via lactate dehydrogenase and ethanol via two subsequent reactions catalyzed by pyruvate decarboxylase and alcohol dehydrogenase (Tadege et al., 1999). The amount of pyruvate produced can be reduced via alanine aminotransferease (AlaAT), which catalyzes the reversible reaction interconverting pyruvate and Glu to Ala and 2-oxoglutarate (2OG). Concomitantly, 2OG was suggested to reenter the tricarboxylic acid cycle to be used to produce another ATP and also succinate, which accumulates in the cell (Rocha et al., 2010). This Ala pathway provides a means for the role of Ala accumulation during hypoxia via reorganization of the tricarboxylic acid cycle. Furthermore, given that the use of this strategy prevents pyruvate accumulation, the continued operation of glycolysis during waterlogging can occur. It should be noted, however, that measurements of metabolite levels alone do not provide information about the actual activity of the metabolic pathways involved. Furthermore, the previous studies did not reveal which enzymes of the tricarboxylic acid cycle change their activity that leads to reorganization of the tricarboxylic acid cycle. To overcome this, analysis of metabolism using isotope-labeled substrates has proven to be essential for understanding the control and regulation of metabolic networks, and it has often been observed that significant changes in carbon flow are sometimes associated with only small adjustments in metabolite abundance (Schwender et al., 2004; Ratcliffe and Shachar-Hill, 2006). Metabolomics studies that require extensive metabolite labeling utilize uniformly labeled stable isotope tracers. Alternatively, detailed analysis of central carbon metabolism can make use of positional labeling as well. Following the extraction of labeled metabolites, the 13C label redistribution is measured usually with NMR or gas chromatography-mass spectrometry methods (Jorge et al., 2015). Schwender and Ohlrogge (2002) used both labeling approaches to investigate embryo development in Brassica napus seeds. While uniformly labeled [13C6]Glc and [13C12]Suc were applied to determine the metabolic flux through the major pathways of carbon metabolism, positionally labeled [1,2-13C]Glc was used to specifically outline the glycolytic/oxidative pentose phosphate pathway network during embryo development (Schwender and Ohlrogge, 2002). Gas chromatography-mass spectrometry analysis was used in this study to evaluate the 13C enrichment and isotopomer composition. In earlier studies of hypoxic metabolism, positionally labeled [1-13C]Glc was used to specifically investigate energy metabolism and pH regulation in hypoxic maize (Zea mays) root tips (Roberts et al., 1992; Edwards et al., 1998). In this study, we performed stable isotope labeling experiments using wild-type soybean (Glycine max) roots in order to better understand the dynamics of metabolism in operation in plant cells under hypoxic conditions. For this, we used fully labeled 13C and 15N tracers rather than positional labeling, as this allowed us to cover a broad view of the central carbon and nitrogen metabolic network. The labeling pattern of metabolites was subsequently measured with gas chromatography-time of flight-mass spectrometry (GCTOF-MS). Our studies confirm the activity of Ala metabolism while revealing the parallel activity of the 44 Plant Physiol. Vol. 170, 2016 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Respiratory Metabolism during Low-Oxygen Stress GABA shunt. The results provide evidence that the bifurcation of the tricarboxylic acid cycle results from the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH), also known as complex II of the mitochondrial electron transport chain (mETC). RESULTS Metabolite Profiling and Isotope Redistribution Analysis To obtain an overview of the metabolic responses of soybean roots under our experimental conditions, we first carried out a broad metabolite profiling study with GC-TOF-MS (Lisec et al., 2006; Fig. 1). Using this approach, over 30 metabolites from the central primary metabolism were characterized, including sugars, amino acids, and intermediates of the tricarboxylic acid cycle. Figure 1 shows the changes of selected primary metabolites in soybean root extracts expressed as the ratio between values obtained with hypoxia and normoxia conditions. Significant accumulation (Student’s t test, P , 0.05) of Fru, Glc, lactate, Val, Leu, Ser, Phe, Ala, and Pro was observed in soybean roots after 6 h of hypoxic treatment (Fig. 1; Table I). Asp, Asn, and the organic acid fumarate were shown to decrease significantly after 6 h of hypoxic treatment (Student’s t test, P , 0.05; Fig. 1; Table I). These data showed that after 6 h, an increase in many amino acids was observed in hypoxic soybean roots while intermediates of the tricarboxylic acid cycle remained mostly unchanged, except for succinate, which accumulated. Isotope tracer experiments were next performed by assessing how 13C isotopes were redistributed among metabolites following the incubation of excised soybean roots in [13C]pyruvate or [13C]Glu under hypoxic conditions. The same experiment was performed using the corresponding 12C substrates as controls to assess the effect of substrate provision alone, and the labeling pattern of metabolites was subsequently measured with GC-TOF-MS. Metabolite profiling analysis revealed that the metabolic changes induced by low oxygen were independent from the supply of labeled/ unlabeled substrate. Similar levels of the characteristic hypoxia-responsive metabolites Ala, GABA, lactate, succinate, and Asp were observed in all treatments (Table I; Supplemental Table S1). The [13C]pyruvate labeling experiment revealed a rapid 13C label incorporation into lactate and Ala after 3 h of hypoxic treatment. Label incorporation doubled after 6 h (Fig. 2; Tables II and III; Supplemental Table S2). To a lesser extent, 13C label was also redistributed from pyruvate to citrate and 2OG, a process that was apparently independent of the oxygen concentration, since similar levels were observed under both normoxic and hypoxic conditions. In addition, considerable 13C label was incorporated into Glu (via 2OG). The [13C]Glu labeling experiment, on the other hand, revealed a 13C label incorporation mainly in GABA, succinate, and Asn (via Asp) immediately after 3 h of hypoxic treatment (Fig. 3; Tables IV and V; Supplemental Table S3). The accumulation of 13C label in succinate provides additional evidence that the GABA shunt is active during hypoxia. Furthermore, while almost undetectable 13C label was observed in fumarate under hypoxia, 13C label was observed in malate, presumably by incorporation via oxaloacetate (OAA), which is in agreement with an anticlockwise operation of the tricarboxylic acid cycle during hypoxia (Figs. 2 and 3). Our metabolite redistribution analysis was further investigated by performing a [15N]ammonium (15NH4+) labeling experiment for 36 h to study the redistribution of nitrogen through the pathways of both Glu and Ala synthesis. The 15NH4+ labeling experiment revealed an inhibition of the reversible reaction of aspartate aminotransferase (AspAT) activity upon hypoxia in the direction of Asp and Asn synthesis, as only negligible amounts of 15NH 4 + were incorporated into Asp and Asn (Fig. 4; Tables VI and VII; Supplemental Table S4). On the other hand, during the 24-h 15NH 4+ labeling period, considerable incorporation of 15N label into Ala and GABA in hypoxic soybean roots was observed. Respiration Rate Measurements The rate of respiratory oxygen consumption was measured on soybean root pieces in normoxic and hypoxic conditions (Fig. 5). Our data show a strong reduction of approximately 40% under hypoxic conditions compared with normoxic conditions. DISCUSSION Low-Oxygen Stress Induces a Highly Conserved Metabolic Response in Plants Metabolite profiling confirmed that, in soybean roots, a short-term hypoxic treatment (up to 6 h) already induces fermentation with an increase in lactate and Ala and several responses in most central metabolites, such as carbohydrates, glycolytic intermediates, and amino acids (Fig. 1; Sousa and Sodek, 2003; Bailey-Serres and Voesenek, 2008; Narsai et al., 2011; Bailey-Serres et al., 2012). Ser, derived from the glycolytic intermediate 3phosphoglycerate, increased; so did Phe (derived from phosphoenolpyruvate) and Val, Leu, and Ala (derived from pyruvate). The amino acids Glu and Pro, which are derived from the tricarboxylic acid cycle intermediate 2OG, increase during hypoxia. On the other hand, the amino acids Asp and Asn, derived from the tricarboxylic acid cycle intermediate OAA, decrease during hypoxia. We compared our metabolite profiling data with other previously reported studies of different hypoxic treatments applied to root material in several plant systems, namely pea (Pisum sativum; Zabalza et al., 2011), L. japonicus (Rocha et al., 2010), Arabidopsis (Arabidopsis thaliana; van Dongen et al., 2009), and the Plant Physiol. Vol. 170, 2016 45 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. António et al. Figure 1. Relative abundance of metabolites in soybean root pieces during a 6-h time course of hypoxia treatment determined with GC-TOF-MS. The relative metabolite levels are normalized to an internal standard (ribitol) and the fresh weight of the samples and are depicted on a primary metabolite map. The gray bars represent the ratio of metabolite levels between hypoxia and normoxia conditions at each time interval. The values are means 6 SE of six biological replicates. Asterisks indicate that these values showed significant differences from the control (normoxia) in Student’s t test (P , 0.05). AlaT, Ala aminotransferase; GABA-T, g-aminobutyric acid trans-aminase; GOGAT, Glu synthase; ICL, isocitrate lyase; ME, malic enzyme; MS, malate synthase; OGDH, 2-oxoglutarate dehydrogenase; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; SSADH, succinic semialdehyde dehydrogenase. hybrid Populus 3 canescens (Kreuzwieser et al., 2009). We observed that most of the known metabolic changes upon hypoxia are conserved across the different species analyzed, such as the activation of fermentation with an increase in lactate, Ala, and the accumulation of GABA and succinate (Supplemental Fig. S1). Therefore, we suggest that conclusions from soybean experiments are likely to be valid for many other plant species as well. Feeding Isotope-Labeled Substrate Does Not Affect the Metabolic Response to Hypoxia Even though metabolite changes in response to low oxygen concentrations suggest a regulation of primary metabolism through alternative pathways such as a noncyclic operation of the tricarboxylic acid cycle (Rocha et al., 2010), the existing data in the literature are mainly nonquantitative metabolite levels that do not provide final proof of the direction of the carbon flow 46 Plant Physiol. Vol. 170, 2016 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Respiratory Metabolism during Low-Oxygen Stress Table I. GC-TOF-MS primary metabolite profiling of soybean root tissue following incubation in different substrates Relative values are ratios between hypoxia and normoxia conditions and are represented as means 6 SE of six independent measurements. Values in boldface indicate significant differences from the control (normoxia) in Student’s t test (P , 0.05). False color imaging was performed on log10-transformed GC-TOF-MS metabolite data. A, Control (no substrate) versus pyruvate data. B, Control (no substrate) versus Glu data. The control data (no substrate) are the same and shown twice only for comparison purposes between pyruvate and Glu treatments. Plant Physiol. Vol. 170, 2016 47 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. António et al. through the metabolic pathways. To overcome this, metabolic flux analyses that make use of robust protocols for quantifying steady-state metabolic fluxes have been developed and applied to reveal novel aspects of the fluxes through the tricarboxylic acid cycle and associated pathways (Schwender et al., 2004, 2006; Ratcliffe and Shachar-Hill, 2006; Williams et al., 2008). Following a similar approach, we performed feeding experiments utilizing uniformly stable isotope-labeled precursors to evaluate the relative isotope redistribution within the different primary metabolic pathways in hypoxic soybean roots. To check if providing additional 13C-labeled substrates to the plant tissue affected the metabolic response to hypoxia, we first tested for changes in the metabolite levels (both 12C and 13C isotopes combined) in soybean roots under our hypoxic conditions (Table I; Supplemental Table S1). This experiment revealed that the metabolite responses to hypoxia were independent from the supply of isotope-labeled substrate, as the changes of metabolite levels were similar in all of the experimental treatments (Table I; Supplemental Table S1). Because in our experiments we used uniformly precursor substrates, all pathways in which the precursor is involved will be marked. Nevertheless, although uniformly stable isotope experiments will provide a more broad overview of changes in various metabolic pathways, it will simultaneously make it more difficult to specifically calculate the dynamics of the redistribution of label due to the mixing of pathways. Alternatively, the more expensive positional isotope tracers can be used. The latter will give more detailed information about the redistribution of label through specific pathways only. However, one problem with positional labeled molecules is that the label can get too rapidly lost due to decarboxylation reactions to provide effective information in some instances (Kruger and von Schaewen, 2003). Both Ala Metabolism and the GABA Shunt Are Activated during Hypoxia We first addressed the isotope redistribution from C-labeled pyruvate through the tricarboxylic acid cycle, fermentation, and Ala synthesis (Fig. 2). As expected by the induction of fermentation, the redistribution of label from pyruvate to lactate increased strongly during hypoxia. Similarly, the incorporation of 13 C in Ala increased during hypoxic conditions. In contrast, the redistribution of label into the tricarboxylic acid cycle intermediates citrate and 2OG was not affected by the oxygen concentration, whereas a progressive increase of label in succinate was observed during the course of the hypoxic treatment. The identical profiles of label incorporation in citrate and 2OG indicate that the metabolic pathway via aconitase and isocitrate dehydrogenase remains active during lowoxygen stress. This observation is opposed to our previous suggestion that these reactions were likely inhibited during hypoxia (Rocha et al., 2010). The 13 feeding experiments using [13C]Glu as precursor confirmed the suggested activation of AlaAT during hypoxia and the concomitant link between 2OG and succinate metabolism by the tricarboxylic acid cycle (Fig. 3). Apparently, hypoxia does not induce a complete shift between the two pathways that lead to 2OG production but, rather, activates the Ala pathway in addition to the existing tricarboxylic acid cycle reactions during hypoxia. In addition to the activation of the Ala pathway, both the [13C]pyruvate and [13C]Glu feeding experiments revealed considerable GABA shunt activity during hypoxia, also explaining the increase in the redistribution of 13C to succinate during hypoxia (Figs. 2 and 3). Interpretation of the fractional enrichment of the metabolites from the GABA shunt revealed the following sequence after [13C]pyruvate feeding for 6 h (Table III): Glu (15%) to GABA (11%) to succinate (7%); after [13C]Glu feeding for 6 h, the 13C enrichment was as follows (Table V): Glu (59%) to GABA (25%) to succinate (16%). The steadily decreasing fractional enrichment of the subsequent metabolites from the GABA shunt pathway are characteristic of a linear pathway and support the conclusion that the GABA shunt is active upon hypoxia, although a precise quantitative comparison of the activities of the Ala pathway and the GABA shunt is not possible from our data. As shown previously for Medicago truncatula roots (Limami et al., 2008), the reaction from 2OG to Glu is likely catalyzed by Glu synthase, which uses Gln along with [13C]2OG to generate a mixture of [13C]Glu and [12C]Glu while using NADH as reducing power, thus regenerating NAD+ (Figs. 2 and 3). Interpretation of the fractional enrichment sequence of the metabolites in the pathway from 2OG to succinate is more complex after [13C]Glu feeding for 6 h (Table V): Glu (59%) to 2OG (21%) to succinate (16%), indicating that a linear pathway from 2OG produced by AlaAT to succinate is possible. On the other hand, after 6 h of [13C]pyruvate feeding, the following fractional enrichment series was observed (Table III): pyruvate (89%) to citrate (3.07%) to 2OG (2.99%) to succinate (7.17%). This latter series indicates that the pathway via pyruvate to 2OG and succinate is not likely explained via a simple linear reaction pathway. Therefore, although it is not possible to calculate exactly the relative extent of both Ala and GABA pathways, this provides additional evidence that the GABA shunt is involved in the production of succinate during hypoxia. Quantitative comparison between the activity of both pathways will require the use of positional labeling in future experiments, since positional labeling of precursors allows us to better distinguish between specific metabolic pathways than do uniformly labeled substrates (Roberts et al., 1992; Edwards et al., 1998). Previously, a link between GABA and the tricarboxylic acid cycle was discussed to be unlikely during hypoxic stress because the reaction from GABA to succinate requires NAD+, which becomes limiting during hypoxic conditions (Rocha et al., 2010). Moreover, the drop in cytosolic pH that occurs during 48 Plant Physiol. Vol. 170, 2016 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Respiratory Metabolism during Low-Oxygen Stress Figure 2. Total 13C accumulation in primary metabolites in soybean roots following [13C]pyruvate feeding for a period of 6 h. Black and gray bars represent normoxia and hypoxia conditions, respectively. All data are given in nmol g21 fresh weight, and asterisks indicate that these values showed significant differences from the control (normoxia) in Student’s t test (P , 0.05). Red and blue color represent the transfer of carbon through the pathways of AlaATand the GABA shunt, respectively. The black dashed lines indicate that pyruvate and 2OG can participate in different reactions; however, the data are the same. The three carbons donated by pyruvate to Ala are highlighted in red. The two carbons donated by acetyl-CoA to citrate are highlighted in blue, at which point they become randomized and no longer can be traced. Abbreviations not defined in Figure 1 are as follows: GAD, Glu decarboxylase; LDH, lactate dehydrogenase. hypoxia (Felle, 2005) was expected to inhibit the activity of the enzyme succinate semialdehyde dehydrogenase (pH optimum of 9), which catalyzes the reaction converting GABA to succinate (Satya Narayan and Nair, 1989; Shelp et al., 1995). However, our results clearly indicate that the predicted inhibition of the GABA shunt activity does not affect the isotope between GABA and succinate. In contrast, the data support the hypothesis that the consumption of protons by Glu decarboxylase for the production of GABA can help to stabilize the cytosolic pH during exposure to different stress conditions, including hypoxia (Turano and Fang, 1998; Shelp et al., 2012). Hypoxia Leads to the Inhibition of SDH and Concomitantly of Respiration While label in succinate accumulated during hypoxia, very little 13C label was detected in fumarate in both the [13C]pyruvate and [13C]Glu feeding experiments (Figs. 2 and 3; Supplemental Fig. S2). In contrast, 13C incorporation in malate occurred in both experiments also during hypoxia. Of course, the interpretation of isotope accumulation in the organic acids of the tricarboxylic acid cycle can be complicated by the occurrence of sometimes large pools of unlabeled metabolites in different cellular compartments or the simultaneous occurrence of metabolites in various metabolic pathways, like malate and OAA that can also be produced from pyruvate and phosphoenolpyruvate via pyruvateorthophosphate dikinase and phosphoenolpyruvate carboxylase (Setién et al., 2014) or malate production via the glyoxylate cycle. Indeed, the expression of pyruvateorthophosphate dikinase genes was shown to be slightly up-regulated in low-oxygen experiments with rice (Oryza sativa) or Arabidopsis (Mustroph et al., 2010; Narsai et al., 2011), indicating that the reaction from pyruvate to phosphoenolpyruvate is an option. However, it should be noted that no conclusion can be drawn about the activity of these pathways based on these gene expression data, and further experiments are required to describe the pathways that lead to label accumulation in malate under hypoxia. Plant Physiol. Vol. 170, 2016 49 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. António et al. Table II. Total 13C accumulation in primary metabolites in soybean roots following incubation in [13C]pyruvate for 3 and 6 h Values are means 6 SE (nmol g21 fresh weight) of six independent measurements. Values in boldface showed significant differences from the control (normoxia) in Student’s t test (P , 0.05). Total Normoxia Fumarate Citrate 2OG GABA Succinate Ala Lactate Asp Glu Malate Pyruvate 13 C Label 3h Metabolite 0.016 0.182 0.240 0.532 0.595 0.619 0.645 0.866 1.122 2.464 17.346 6 6 6 6 6 6 6 6 6 6 6 0.000 0.006 0.028 0.016 0.013 0.052 0.048 0.057 0.086 0.134 0.788 6h Hypoxia 1.352E-05 0.176 0.207 0.889 0.816 1.054 2.180 0.506 1.482 0.611 19.188 6 6 6 6 6 6 6 6 6 6 6 Normoxia 0.000 0.011 0.025 0.024 0.020 0.048 0.113 0.033 0.075 0.015 0.526 Especially when label accumulation occurs very slowly (as for fumarate), the labeling efficiency should be assessed critically. For example, variations in the exchange of labeled compounds between different cellular compartments can conceal the redistribution of 13C through a metabolic pathway, especially when the concentration of labeled metabolites is low, such as in the case of mitochondrial fumarate. Having said so, the observation that 13C label in fumarate decreased to almost undetectable values during hypoxia (Tables III and V; Supplemental Fig. S2) can also be explained by the reduction of fumarate synthesis upon hypoxia as compared with the normoxic treatment, probably as a consequence of the inhibition during low-oxygen stress of the enzyme SDH, also known as complex II of the mETC. Inhibition of SDH will disrupt the tricarboxylic acid cycle, which leads to a noncyclic reaction pathway that has actually been described earlier to occur in plants and green algae in response to various conditions, including low oxygen availability (Vanlerberghe et al., 1989, 1990, 1991; Vanlerberghe and Turpin, 1990; 0.034 0.634 0.543 3.514 0.786 0.799 1.025 1.393 1.656 3.810 19.851 6 6 6 6 6 6 6 6 6 6 6 0.000 0.022 0.041 0.214 0.021 0.049 0.123 0.053 0.069 0.173 0.631 Hypoxia 8.003E-05 0.623 0.606 6.141 1.556 2.217 7.036 1.087 2.598 4.184 22.369 6 6 6 6 6 6 6 6 6 6 6 0.000 0.019 0.032 0.313 0.096 0.098 0.224 0.073 0.146 0.168 1.020 Hanning and Heldt, 1993; Schwender et al., 2006; Steuer et al., 2007; Boyle and Morgan, 2009; Tcherkez et al., 2009; Sweetlove et al., 2010; Grafahrend-Belau et al., 2013; Ma et al., 2014). Interestingly, similar observations were made on the relative levels of the metabolites associated with the tricarboxylic acid cycle in antisense SDH tomato (Solanum lycopersicum) plants, which were deficient in the expression of the iron-sulfur subunit of SDH (Araújo et al., 2011). In these transgenic lines, the activity of the tricarboxylic acid cycle was clearly reduced, with high accumulation of succinate in comparison with wild-type plants, while fumarate was not detected. The inhibition of SDH will not only affect the net output of redox equivalents by the tricarboxylic acid cycle reactions (NADH and FADH) as substrates for the mETC but also the direct input of electrons into the mETC via SDH itself. As a causal response, this is anticipated to result in a decrease of the activity of the mETC, leading to a reduction in the rate of respiratory oxygen consumption, as was shown previously in plants deficient in SDH expression (Araújo et al., 2011). Table III. Fractional enrichment of metabolites labeled in soybean root tissue following incubation in [13C]pyruvate for 3 and 6 h Values are means 6 SE (%) of six independent measurements. Values in boldface indicate significant differences from the control (normoxia) in Student’s t test (P , 0.05). 13 Normoxia Fumarate Citrate 2OG Malate GABA Succinate Asp Glu Lactate Ala Pyruvate C Enrichment 3h Metabolite 0.842 0.959 1.259 1.296 3.258 4.458 5.875 8.756 10.976 14.576 89.586 6 6 6 6 6 6 6 6 6 6 6 0.004 0.027 0.060 0.075 0.133 0.187 0.136 0.050 0.125 0.074 0.167 6h Hypoxia 0.001 1.013 1.195 1.155 2.495 4.986 5.463 8.654 11.592 14.316 87.576 6 6 6 6 6 6 6 6 6 6 6 0.000 0.021 0.073 0.052 0.214 0.114 0.121 0.054 0.103 0.084 0.145 Normoxia 1.173 2.990 2.557 4.088 8.761 6.274 8.205 11.754 17.275 19.429 88.386 6 6 6 6 6 6 6 6 6 6 6 0.004 0.111 0.346 0.068 0.118 0.210 0.235 0.061 0.053 0.049 0.195 Hypoxia 0.004 3.074 2.992 4.025 11.322 7.173 10.184 14.748 18.492 20.327 89.420 50 6 6 6 6 6 6 6 6 6 6 6 0.000 0.019 0.241 0.095 0.159 0.226 0.083 0.090 0.109 0.037 0.030 Plant Physiol. Vol. 170, 2016 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Respiratory Metabolism during Low-Oxygen Stress Figure 3. Total 13C accumulation in primary metabolites in soybean roots following [13C]Glu feeding for a period of 6 h. Black and gray bars represent normoxia and hypoxia conditions, respectively. All data are given in nmol g21 fresh weight, and asterisks indicate that these values showed significant differences from the control (normoxia) in Student’s t test (P , 0.05). Red and blue color represent the transfer of carbon through the pathways of AlaATand the GABA shunt, respectively. The black dashed line indicates that 2OG can participate in different reactions; however, the data are the same. The five carbons donated by Glu to 2OG are highlighted in red. Abbreviations are as defined in Figures 1 and 2. Indeed, the inhibition of respiratory activity was measured during hypoxic conditions as compared with normoxic conditions (in a buffer solution that is in equilibrium with air; Fig. 5). It should be noted that the oxygen concentration in the hypoxic solution is about 2 orders of magnitude higher than the Km for oxygen of cytochrome c oxidase (0.1–0.15 mM ). These results thus provide a mechanistic explanation for the proactive down-regulation of respiratory oxygen consumption during hypoxia that was discussed previously (Geigenberger et al., 2000; Geigenberger, 2003; Gupta et al., 2009; Zabalza et al., Table IV. Total 13C accumulation in primary metabolites in soybean roots following incubation in [13C]Glu for 3 and 6 h Values are means 6 SE (nmol g21 fresh weight) of six independent measurements. Values in boldface indicate significant differences from the control (normoxia) in Student’s t test (P , 0.05). Total Metabolite C Label 3h Normoxia Fumarate Pro Ala 2OG Succinate Asp GABA Glu Malate Asn 13 0.011 0.026 0.029 0.097 0.150 0.535 0.789 1.196 2.433 3.726 6 6 6 6 6 6 6 6 6 6 0.000 0.016 0.006 0.017 0.034 0.086 0.048 0.388 0.078 0.153 6h Hypoxia 9.864E-06 0.115 0.072 0.533 1.655 0.365 5.547 8.764 1.167 17.862 6 6 6 6 6 6 6 6 6 6 Normoxia 0.000 0.024 0.011 0.033 0.115 0.075 0.113 0.426 0.065 0.065 0.016 0.099 0.089 0.115 0.381 1.626 4.695 8.191 8.974 21.974 6 6 6 6 6 6 6 6 6 6 0.000 0.014 0.022 0.023 0.053 0.169 0.123 0.331 0.241 1.036 Hypoxia 2.316E-04 0.813 0.588 0.752 3.497 2.848 13.480 10.394 14.489 34.674 Plant Physiol. Vol. 170, 2016 6 6 6 6 6 6 6 6 6 6 0.000 0.113 0.019 0.073 0.068 0.146 0.224 0.570 0.432 1.011 51 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. António et al. Table V. Fractional enrichment of metabolites labeled in soybean root tissue following incubation in [13C] Glu for 3 and 6 h Values are means 6 SE (%) of six independent measurements. Values in boldface indicate significant differences from the control (normoxia) in Student’s t test (P , 0.05). 13 Normoxia Fumarate Ala Pro Succinate Malate Asn 2OG Asp GABA Glu C Enrichment 3h Metabolite 0.598 0.680 1.046 1.124 1.283 2.020 2.896 3.599 4.793 9.345 6 6 6 6 6 6 6 6 6 6 0.004 0.020 0.211 0.038 0.054 0.215 0.119 0.033 0.140 0.054 6h Hypoxia 0.001 0.975 2.487 10.055 2.215 2.872 16.870 3.947 15.574 51.211 6 6 6 6 6 6 6 6 6 6 2009; Armstrong and Beckett, 2011; Nikoloski and van Dongen, 2011). Ala Metabolism and the GABA Shunt Are Activated at the Expense of the Production of Other Amino Acids The 15NH4+ labeling experiment was performed to study isotope redistribution through the pathways of Normoxia 0.000 0.017 0.199 0.068 0.036 0.099 0.311 0.041 0.098 0.074 0.727 2.169 4.010 2.997 9.630 3.488 3.209 9.588 11.655 58.177 6 6 6 6 6 6 6 6 6 6 0.005 0.099 0.231 0.059 0.290 0.198 0.328 0.076 0.050 0.036 Hypoxia 0.009 5.412 11.576 16.135 13.955 4.681 20.542 26.703 24.857 59.028 6 6 6 6 6 6 6 6 6 6 0.000 0.025 0.179 0.074 0.325 0.326 0.075 0.088 0.124 0.048 both Glu and Ala synthesis. Ammonium is assimilated into Gln and sequentially converted into Glu. The synthesis or regeneration of Glu is an important issue in hypoxic tissues to maintain AlaAT and Glu decarboxylase pathways. These two pathways contribute to mitigate the damaging effects of hypoxia. AlaAT contributes to save carbon derived from glycolysis by using pyruvate competitively with fermentation, because Figure 4. Total 15N accumulation in primary metabolites in soybean roots following 15NH4+ feeding for a period of 36 h. Black and gray bars represent normoxia and hypoxia conditions, respectively. All data are given in nmol g21 fresh weight, and asterisks indicate that these values showed significant differences from the control (normoxia) in Student’s t test (P , 0.05). Red color represent the transfer of nitrogen through the pathways of GOGAT and the GABA shunt. The black dashed line indicates that 2OG can participate in different reactions. GS, Gln synthetase. Other abbreviations are as defined in Figures 1 and 2. 52 Plant Physiol. Vol. 170, 2016 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Respiratory Metabolism during Low-Oxygen Stress Table VI. Total 15 N accumulation in primary metabolites in soybean roots following incubation in 15 21 NH4+ for 3, 12, 24, and 36 h Values are means 6 SE (nmol g fresh weight) of five independent measurements. Values in boldface indicate significant differences from the control (normoxia) in Student’s t test (P , 0.05). Total Metabolite 3h Normoxia Ser Gly Ala Asp GABA Gln Glu Asn 0.150 0.244 0.412 0.760 1.552 1.602 3.762 7.049 6 6 6 6 6 6 6 6 N Label 12 h Hypoxia 0.001 0.001 0.000 0.001 0.002 0.001 0.000 0.002 15 0.118 0.189 0.650 0.258 2.011 0.181 3.277 7.360 6 6 6 6 6 6 6 6 Normoxia 0.001 0.475 0.002 0.907 0.001 0.920 0.001 2.054 0.002 2.436 0.000 1.973 0.000 4.588 0.003 58.114 6 6 6 6 6 6 6 6 24 h Hypoxia 0.001 0.001 0.000 0.001 0.003 0.001 0.001 0.002 0.242 0.443 2.738 0.396 3.501 0.209 3.982 5.282 6 6 6 6 6 6 6 6 Normoxia 0.001 0.792 0.001 1.247 0.001 1.563 0.001 3.557 0.001 2.880 0.001 3.388 0.001 5.854 0.001 26.618 ethanol is released to the rhizosphere and the carbon is lost for the plant (Bailey-Serres and Voesenek, 2008). 15 NH4+ labeling revealed that the reversible reaction of AspAT activity is strongly inhibited upon hypoxia in the direction of Asp synthesis, confirmed by the very low 15N label redistributed to Asp and Asn, in favor of an increased metabolic activity of AlaAT and Glu decarboxylase, both of which use Glu as an amino donor. This observation is supported by the high 15N enrichment patterns observed in Ala and GABA synthesis during hypoxia (Fig. 4). Furthermore, the slow 15N enrichment in Asn is in agreement with its function as nitrogen storage and transport, resulting in lower nitrogen assimilation under low oxygen availability. A slow 15N enrichment was also observed in Ser and Gly during hypoxia, suggesting that the Glu:glyoxylate aminotransferase activity might be higher under normoxia (Ricoult et al., 2006; Limami et al., 2008); however, further directed studies will be required to verify this hypothesis. 6 6 6 6 6 6 6 6 36 h Hypoxia 0.001 0.001 0.000 0.001 0.002 0.001 0.000 0.002 0.282 0.533 6.576 0.509 4.904 0.309 4.025 3.782 6 6 6 6 6 6 6 6 Normoxia 0.001 1.326 0.002 2.491 0.001 3.010 0.001 5.365 0.002 3.248 0.000 6.163 0.000 6.629 0.003 33.835 6 6 6 6 6 6 6 6 Hypoxia 0.001 0.001 0.000 0.001 0.003 0.001 0.001 0.002 0.559 0.879 8.330 0.574 7.368 0.368 3.367 6.009 6 6 6 6 6 6 6 6 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 subsequent question that arises is how this regulation is mediated. It was discussed previously that hypoxiamediated changes in the redox status of the cell could be involved in regulating low-oxygen stress responses (Considine and Foyer, 2014). Although it is not possible to fully answer the question of how the tricarboxylic acid cycle is controlled upon hypoxia with our current experimental data, our results provide interesting indications that give rise to the hypothesis that redox-mediated control of the tricarboxylic acid cycle might be involved. Recently, mitochondrial thioredoxins were identified as master regulators of the carbon flow through the tricarboxylic acid cycle in plants (Daloso et al., 2015). Based on 13C metabolic flux analysis in Arabidopsis thioredoxin mutants and in vitro activity studies on enzymes of the tricarboxylic acid cycle, it was shown that the activity of SDH and fumarase is deactivated by reduced thioredoxin, while ATP-citrate lyase (ACL) is activated. Via the enzyme ACL, citrate is converted to acetyl-CoA and OAA in the cytosol (Klinman and Rose, 1971). Here, OAA reacts to Asp. This pathway was shown previously to exist in soybean (Allen and Young, 2013) and is up-regulated upon hypoxia in mammalian cells (Metallo et al., 2011). However, while ACL might be involved in the synthesis of malate and Asp under hypoxia, it cannot explain the labeling of these metabolites, because the two carbons that are derived from labeled pyruvate in citrate are released as the twocarbon moiety by the action of ACL (Klinman and Is the Redox Status of the Cell Involved in the Regulation of Tricarboxylic Acid Cycle Activity? Evidence from our results here, as well as from other publications (Vanlerberghe et al., 1989), point out that the activity of the tricarboxylic acid cycle is modulated when the oxygen availability to a cell decreases. The Table VII. Fractional enrichment of metabolites labeled in soybean root tissue following incubation in 15 NH4+ for 3, 12, 24, and 36 h Values are means 6 SE (%) of five independent measurements. Values in boldface indicate significant differences from the control (normoxia) in Student’s t test (P , 0.05). 15 N Enrichment Metabolite 3h Normoxia Ser Gly Asn Ala Asp GABA Gln Glu 3.317 4.620 4.992 7.366 15.143 23.128 64.167 69.943 6 6 6 6 6 6 6 6 0.159 0.080 0.159 0.057 0.095 0.441 0.050 0.029 12 h Hypoxia 2.653 3.357 5.237 7.431 5.070 28.379 12.070 60.858 6 6 6 6 6 6 6 6 0.054 0.136 0.224 0.067 0.060 0.275 0.058 0.049 Normoxia 10.137 19.528 25.658 17.675 37.024 45.956 67.725 77.435 6 6 6 6 6 6 6 6 0.083 0.071 0.178 0.029 0.078 0.331 0.080 0.141 24 h Hypoxia 5.337 8.151 8.965 36.894 7.751 58.608 16.538 74.686 6 6 6 6 6 6 6 6 0.070 0.082 0.105 0.058 0.051 0.108 0.107 0.147 Normoxia 15.143 22.239 39.167 29.037 65.471 54.768 70.732 81.015 6 6 6 6 6 6 6 6 0.109 0.090 0.159 0.037 0.095 0.241 0.070 0.019 36 h Hypoxia 6.248 10.350 14.239 78.806 9.861 71.036 17.725 64.342 6 6 6 6 6 6 6 6 0.064 0.196 0.284 0.087 0.060 0.175 0.060 0.038 Plant Physiol. Vol. 170, 2016 Normoxia 22.239 43.460 44.798 54.075 87.500 60.236 79.955 87.615 6 6 6 6 6 6 6 6 0.093 0.081 0.198 0.039 0.058 0.231 0.030 0.101 Hypoxia 12.575 17.678 20.173 86.075 10.980 81.949 22.495 46.660 6 6 6 6 6 6 6 6 0.070 0.082 0.105 0.058 0.051 0.108 0.082 0.147 53 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. António et al. (5 mM KNO3, 0.5 mM KCl, 0.25 mM KH2PO4, 0.25 mM K2HPO4, 1 mM MgSO4, 0.05 mM FeEDTA, and trace elements 9.1 mM MnCl2, 0.046 mM H3BO3, 0.765 mM ZnCl2, 0.56 mM NaMoO4, and 0.32 mM CuCl2) as described previously (Hoagland and Arnon, 1950). After 4 weeks, mature nodules were observed. Introduction of Label and Sampling Figure 5. Respiratory oxygen consumption rates of soybean roots during normoxia and hypoxia conditions. Values are means 6 SE of at least 45 independent measurements (of freshly excised roots 50 min in buffer). FW, Fresh weight. Rose, 1971). Taken altogether, it is reasonable to suggest a hypothesis that the tricarboxylic acid cycle activity is modulated upon hypoxia via a redox-mediated mechanism in which thioredoxin is involved. CONCLUSION Upon hypoxia, a series of drastic metabolic adaptations are initiated in plants. Of these, the best known is the activation of fermentation and the up-regulation of glycolytic activity to increase the yield of ATP from the glycolytic pathway. Our evaluation of isotope redistribution following 13C and 15N feeding demonstrates the existence of an alternative carbon flux that explains the accumulation of Ala, GABA, and succinate upon hypoxia via pathways mediated by an Ala and GABA shunt. These alternative pathways go hand-in-hand with the bifurcation of the tricarboxylic acid cycle into separate oxidative and reductive pathways. Concomitantly, the net production of redox equivalents in the mitochondria decreases, which could explain the reduction of respiratory oxygen consumption during hypoxic conditions. Future analysis of the regulation of this part of respiratory metabolism might include positional labeling experiments to provide more detailed information about the relative activity of the different alternative pathways that are identified here. MATERIALS AND METHODS Plant Material and Growth Conditions Wild-type soybean (Glycine max ‘IAC-17’) plants were grown from seeds in the greenhouse under natural light and temperature. After 2 weeks of germination in the greenhouse, soybean roots were inoculated with Bradyrhizobium elkanii strain DSM 11554 before being transferred into pots containing hydroponic culture substrate granules (Lecaton Original; Fibo Exclay Deutschland). B. elkanii were grown in liquid culture using a medium solution composed of 0.5 g L21 KH2PO4, 0.8 g L21 MgSO40$7H2O, 0.1 g L21 NaCl, 0.01 g L21 FeCl30$6H2O, 0.8 g L21 yeast extract, 10 g L21 mannitol, and 5 mL of 0.5% (w/v) Bromotimol Blue in ethanol (pH 6.8) prior to inoculation. Plants were watered daily and supplied twice per week with 200 mL of nitrogen-containing nutrient solution The introduction of 13C label was performed by adding [U-13C3]pyruvate (99 atom % 13C) or [U-13C5]Glu (98 atom % 13C; Euriso-Top) at a final concentration of 20 mM to 25 mL of buffer solution (10 mM MES + KOH, pH 6.5). Unlabeled control samples were prepared by adding [12C]pyruvate or [12C]Glu at a final concentration of 20 mM. Nodules were removed, and root pieces of a pool of six independent plants were incubated for 45 min in buffer solution, without label, in order to adapt the root pieces to the experimental normoxic (approximately 250 mM oxygen) and hypoxic (approximately 50 mM oxygen) conditions. Normoxic and hypoxic conditions were obtained by supplying air and an oxygen/ nitrogen mixture to the flasks, respectively. Samples were then harvested at different time points (0, 3, and 6 h) after the addition of 13C label or 12C unlabeled substrate. A control experiment without the addition of substrate was also performed. Root pieces were washed three times with buffer (10 mM MES + KOH, pH 6.5) and snap frozen in liquid nitrogen. Samples were stored at 280°C prior to extraction and GC-TOF-MS analysis. The introduction of 15N label was performed by adding 15NH4+ (98 atom % or greater 15N; Euriso-Top) at a final concentration of 20 mM to 25 mL of buffer solution (10 mM MES + KOH, pH 6.5). Nodules were removed, and root pieces of a pool of five independent plants were incubated for 45 min in buffer solution, without label, in order to adapt the root pieces to the experimental normoxic (approximately 250 mM oxygen) and hypoxic (approximately 50 mM oxygen) conditions. Normoxic and hypoxic conditions were obtained by supplying air and an oxygen/nitrogen mixture to the flasks, respectively. Samples were then harvested at different time points (0, 3, 24, and 36 h) after the addition of 15NH4+. Root pieces were washed three times with buffer (10 mM MES + KOH, pH 6.5) and snap frozen in liquid nitrogen. Samples were stored at 280°C prior to extraction and GC-TOF-MS analysis. Extraction of Metabolites and GC-TOF-MS Metabolite Profiling Analysis of 13C/15N Labeling Primary metabolites were extracted using a methanol/chloroform extraction procedure as described previously in the literature (Lisec et al., 2006). Briefly, a total of 20 mg (fresh weight) of homogenized soybean root material was extracted in 280 mL of 100% (v/v) methanol with 12 mL of ribitol (0.2 mg mL–1 ribitol in water) as an internal standard. Extracts were incubated for 15 min at 70°C on a shaker (950 rpm) and then centrifuged at room temperature at 12,000g for 10 min. The supernatant was transferred to a new tube, mixed with 150 mL of chloroform and 300 mL of water, and vortex mixed. Extracts were centrifuged at room temperature at 12,000g for 15 min. Aliquots (150 mL) of the polar (upper) phase were evaporated to dryness using a centrifugal concentrator, and metabolites were subsequently derivatized and analyzed using an established GC-TOF-MS protocol (Lisec et al., 2006). GC-TOF-MS chromatograms were evaluated using TagFinder (Luedemann et al., 2008). Analytes were manually identified using the TargetFinder plug-in of the TagFinder software and a reference library of ambient and 13C- or 15N-labeled mass spectra and retention indices from the Golm Metabolome Database (http://gmd.mpimp-golm.mpg. de/; Kopka et al., 2005; Schauer et al., 2005). A peak intensity matrix containing all available mass isotopomers of characteristic mass fragments that represented the primary metabolites under investigation was generated by TagFinder. This matrix was processed using the CORRECTOR software tool, a TagFinder-based high-throughput tool for the mass isotope correction of GC-TOF-MS flux profiling experiments (http://www.mpimp-golm.mpg.de/ 10871/Supplementary_Materials). Fractional 15N enrichments of mass fragments were calculated using this processing tool as described previously (Huege et al., 2011). Fractional 13C enrichments were evaluated by determination of the intensities of the 12C spectral fragments, and the isotopic spectral fragments of unlabeled controls were compared with the fragmentation patterns of metabolites detected in the chromatograms of the 13C-fed soybean roots as described by Roessner-Tunali et al. (2004). The total 13C and 15N label present in a metabolite pool (expressed as nmol 13C- or 15N-labeled metabolite g21 fresh weight) was calculated by multiplying the absolute concentration of that metabolite determined after GC-TOF-MS analysis by its mean 13C fractional enrichment (Roessner-Tunali et al., 2004). 54 Plant Physiol. Vol. 170, 2016 Downloaded from on June 14, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Respiratory Metabolism during Low-Oxygen Stress Respiratory Oxygen Consumption Measurements Respiratory oxygen consumption was measured on excised root pieces (2–6 mm long; total mass approximately 25 mg). Prior to the measurements, excised roots were preincubated in HEPES buffer (100 mM, pH 7.4) for 50 min to reduce woundstress responses. Respiration was determined after transfer of the roots to 1 mL of well-stirred fresh HEPES buffer (100 mM, pH 7.4) in a closed vial at 25°C connected to an OXY-4 multichannel optical oxygen sensor (PreSens). Supplemental Data The following supplemental materials are available. Supplemental Figure S1. Metabolite responses of different species to different hypoxia treatments. Supplemental Figure S2. GC-TOF-MS fumarate signal under all experimental conditions. Supplemental Table S1. GC-TOF-MS relative values and two-way ANOVA for primary metabolites. Supplemental Table S2. Absolute concentrations of selected primary metabolites used to determine the total 13C label accumulated following incubation in [13C]pyruvate. Supplemental Table S3. Absolute concentrations of selected primary metabolites used to determine the total 13C label accumulated following incubation in [13C]Glu. Supplemental Table S4. 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