Download Regulation of Primary Metabolism in Response to

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.
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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
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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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
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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. Absolute concentrations of selected primary metabolites used to determine the total 15N label accumulated following
incubation in 15NH4+.
Received February 20, 2015; accepted November 5, 2015; published November
9, 2015.
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