Available online at www.sciencedirect.
com
Regulation of the mitochondrial tricarboxylic acid cycle
Adriano Nunes-Nesi1,2, Wagner L Araujo1, Toshihiro Obata3 and
Alisdair R Fernie3
Recent years have seen considerable advances in our
understanding of the particular physiological roles of the
constituent enzymes of the tricarboxylic acid (TCA) cycle.
Despite acquiring a fairly comprehensive overview of the
functional importance of these proteins relatively little is known
concerning how this important pathway is regulated. In this
review we concentrate on the mitochondrial reactions since
this organelle is the only one in which a full cycle can, at least
theoretically, operate. We summarize what is known about the
regulation of the enzymes of the pathway both from historical
kinetic studies as well as discussing more recent transcriptional
and proteomic studies and our enhanced understanding of
subcellular compartmentation within the context of metabolic
regulation.
Addresses
1
Departamento de Biologia Vegetal, Universidade Federal de Vicosa,
36570-000 Vicosa, Minas Gerais, Brazil
2
Max-Planck Partner Group, Departamento de Biologia Vegetal,
Universidade Federal de Vicosa, 36570-000 Vicosa, Minas Gerais, Brazil
3
Max-Planck-Institut fur Molekulare Pflanzenphysiologie, Am
Muhlenberg 1, 14476 Potsdam-Golm, Germany
Corresponding author: Fernie,
Alisdair R (fernie@mpimp-golm.mpg.de)
Current Opinion in Plant Biology 2013, 16:335343
This review comes from a themed issue on Physiology and
metabolism
Edited by John Browse and Edward Farmer
For a complete overview see the Issue and the Editorial
Available online 22nd February 2013
1369-5266/$ see front matter, # 2013 Elsevier Ltd. All rights
reserved.
http://dx.doi.org/10.1016/j.pbi.2013.01.004
Introduction
There is now considerable cumulative evidence that
mitochondrial respiratory function is associated with
proper maintenance of cellular metabolism as a whole.
Reverse molecular genetic approaches have proven
particularly instrumental in establishing the physiological
and metabolic basis for mitochondrial function in living
plant cells [1]. This was particularly true for increasing our
knowledge of mitochondrial metabolism as of fundamen-
tal importance in many other important cellular processes
such as photosynthesis, photorespiration, nitrogen metab-
olism, redox regulation and signalling [24]. That said,
and quite remarkably since it was demonstrated over 50
years ago that exactly the same reactions occur in plant
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cells that were first described by Hans Krebs in pigeon
muscle [5], relatively little is known concerning the
regulation and control of this pathway [1,6!,7!].
The tricarboxylic acid (TCA) cycle is composed by a set of
eight enzymes primarily linking the product of the oxi-
dation of pyruvate and malate (generated in the cytosol) to
CO2 with the generation of NADH for the oxidation by the
mitochondrial respiratory chain [8]. The genomic organ-
ization and the subcellular localization of the enzymes
involved in the Arabidopsis TCA cycle are summarized
in Supplementary Table 1; however those are not
described in detail since firstly it has been expertly
reviewed elsewhere [9] and secondly we intend to con-
centrate here on the mitochondrial reactions since this
organelle is the only one in which a full cycle can, at least
theoretically, operate [10!]. Whilst the presence of organic
acids, particularly TCA cycle intermediates, in all plants is
known to support numerous and diverse functions within
and beyond cellular metabolism, the level of accumulation
of the various organic acids is extremely variable between
species, developmental stages and tissue types [11] pro-
viding further support that the enzymes involved in the
interconversion of these metabolic intermediates are sub-
ject to tight regulatory control. The sequestration of
organic acids into the vacuole and secretion into the rhizo-
sphere additionally represent important mechanisms of
regulating these intermediates; however, these have been
recently reviewed elsewhere [11,12].
Hints to the regulation of the TCA cycle have been
provided by a recent metabolic control analysis which
show that much of the control through this pathway is
resident in fumarase, malate dehydrogenase (MDH) and
2-oxoglutarate dehydrogenase [1], suggesting that these
would be sensitive targets for flux regulation. A lack of
subcellular information concerning the levels of inter-
mediates of the cycle [13] currently precludes us from
being able to assess the potential of the constituent
enzymes to play regulatory roles on the basis of disequi-
librium ratios. However, there is a growing body of
information concerning allosteric and post-translational
regulation of specific enzymes from which mechanisms of
regulation can be inferred (Figure 1). We shall discuss
these studies in two separate sections. In the first we will
describe focused in vitro and in vivo studies investigating
the role of enzyme effectors. The second section will
focus on broad-based studies in which changes in tran-
scripts, protein abundance, post-translational modifi-
cation of proteins or metabolite levels were assessed in
Current Opinion in Plant Biology 2013, 16:335343
336 Physiology and metabolism

Figure 1

1 Pyruvate 2 Phosphorylation
Redox regulation
CS
Citrate
Citrate
TCA ACO
TCA cycle
cycle Isocitrate

Highly sensitive to
oxidative stress and NO

3 Phosphorylation 4 Phosphorylation
Redox regulation Redox regulation

TCA TCA
cycle Isocitrate cycle
IDH
2-OG light dependent
2-OG SucCoA
2-OGDH regulation

5 6
Phosphorylation Phosphoryation
Redox regulation Redox regulation
Fumarate
TCA TCA
SDH
cycle cycle
Succinate Succinate
SCoAL
light dependent Phytochrome and
SucCoA regulation nitric oxid regulation

7 8 OAA Phosphorylation
Malate MDH Redox regulation
Acetylation
FUM Malate

Fumarate TCA TCA

cycle cycle

Current Opinion in Plant Biology

Current known regulatory mechanisms of the mitochondrial TCA cycle. Each enzymatic step of the TCA cycle is represented having only the enzyme
and its substrate and product whilst specific non-metabolite-mediated regulatory mechanisms are specified. Mechanisms involved in the
transcriptional regulation level for each enzyme are demonstrated in light blue boxes whilst post-transcriptional mechanism are shown in black boxes.
For clarity, cofactors have been omitted the 2-OGDH reaction requires CoA; the CS and SCoA L reactions produce CoA; NAD+ is converted into
NADH by the IDH, 2-OGDH and MDH reactions; ADP is converted into ATP by the SCoA L reaction. CS, citrate synthase (note this actually catalyses
the condensation of acetyl CoA and OAA; pyruvate is just shown to indicate its entrance point); ACO, aconitase; IDH, isocitrate dehydrogenase; 2-
OGDH, 2-oxoglutarate dehydrogenase complex; SCoAL, succinyl CoA ligase; SDH, succinate dehydrogenase; FUM, fumarase; MDH, malate
dehydrogenase; OAA, oxaloacetate; 2-OG, 2-oxoglutarate; TCA cycle, tricarboxylic acid cycle.

response to environmental or experimental perturbation TCA cycle effectors

and from which potential elements of regulation were
identified. Finally we discuss regulatory principles which
have already been described in non-plant systems and
conclude with a perspective for future efforts aimed at
unraveling the regulation of this vital pathway.
In the following paragraphs we will briefly detail the
known metabolite regulators of each enzyme which are
additionally summarized as an overview in Table 1. For
the purpose of the discussion, we have also included the
pyruvate dehydrogenase complex (PDC) reaction which

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 Regulation of the mitochondrial tricarboxylic acid cycle Nunes-Nesi et al. 337
Table 1
Summary of known metabolite-mediated regulatory characteristics of the TCA cycle enzymes
Enzyme of the pathway Effectors
Pyruvate dehydrogenase Pyruvate, thiamine pyrophosphate
Citrate synthase ADP
Aconitase n.d.
Isocitrate dehydrogenase ADP, NAD+ and Ca2+
2-Oxoglutarate dehydrogenase Thiamine pyrophosphate, AMP,
ADP; NAD+ and Ca2+
Succinyl-CoA ligase 2-Oxoglutarate
Succinate dehydrogenase ATP, ADP, NADH, FAD, QH2
Fumarase n.d.
Malate dehydrogenase n.d.
n.d.: not determined; GABA: g-aminobutyric acid.
is not strictly part of the TCA cycle but is, nevertheless,
intimately associated with it. The PDC is a complex
consisting of three components: pyruvate dehydrogenase
(E1), dihydrolipoyl acetyltransferase (E2) and dihydroli-
poyl dehydrogenase (E3). There are two distinct and
spatially separated forms of the PDC in plant cells;
mitochondrial PDC and plastidial PDC. The mitochon-
drial form of PDC is one of the carbon entry sites into the
TCA cycle, whilst the plastidial PDC provides acetyl-
CoA and NADH for de novo fatty acid biosynthesis [14].
Moreover, the mitochondrial PDC has two associated
regulatory enzymes: pyruvate dehydrogenase kinase
(PDK) and phospho-pyruvate dehydrogenase phospha-
tase [14]. Given the exquisite regulation of this enzyme it
is generally assumed to be a major regulatory point of flux
through the cycle; however, as the discussion below
indicates it is certainly not the only regulatory point
and may not even be the major one. Intriguingly, down
regulation of the inhibitory PDK in Arabidopsis resulted
in plants displaying altered vegetative growth with
reduced accumulation of vegetative tissues, early flower
development and shorter generation time [15], whilst a
mutation of the E2 subunit, conferring an opposite effect
on PDC activity, also resulted in a decreased organ growth
[16]. The apparent contradictory nature of these obser-
vations suggests that further research will be needed to
fully understand the physiological importance of the
phosphorylation of the PDC. What is less contentious
is the fact that the PDC activity interconnects glycolysis,
gluconeogenesis and fatty acid synthesis to the TCA
cycle and is highly regulated by a range of allosteric
effectors [17]. It has long been demonstrated that PDC
as well as TCA cycle dehydrogenases displays product
inhibition in vitro by NADH [14]. Accordingly given that
the in vivo activities of PDC and other TCA cycle
enzymes are responsive to the NADH/NAD+ ratio, this
provides a very sensitive mechanism by which it is
possible to tune balance the rate of pyruvate oxidation
by PDC and the TCA cycle activity with the rate of
oxidative phosphorylation [17]. Because of the fact that
NAD+ is a common co-factor utilized by three TCA cycle
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Inhibitors
NADH, acetyl-CoA, NH4, glyoxylate
ATP, NADH and succinyl-CoA
Citramalate, oxalomalate, NADH, H2O2
ATP and NAD(P)H, succinic semialdehyde, succinate,
glutamate, GABA
NADH
NADH. Malonate, citrate, isocitrate
Oxaloacetate; NADH, nitric oxide
ATP, ADP and AMP; NADH, pyruvate, citrate, 2-oxoglutarate
NADH
enzymes as well as by the PDC, it is reasonable to assume
that mitochondrial NADH/NAD+ ratio has a major impact
on the flux through the TCA cycle. However, it is
important to note both for this enzyme and the other
dehydrogenases of the TCA cycle (discussed below) that
elegant studies from the Mller laboratory have revealed
that the free NADH concentration is kept constant in
plant mitochondria under different metabolic conditions
rendering it important to interpret implications of in vitro
kinetics with caution [18]. In addition to this regulation
the mitochondrial PDC is also regulated by product
inhibition by acetyl CoA [14] and activated by thiamine
pyrophosphate [19!!].
Citrate synthase (CS) is confined to the mitochondrial
matrix, except in tissues converting fatty acids into sugars
in which a peroxisomal isoform is also functional [20].
Interestingly, this peroxisomal isoform is up-regulated
following the antisense repression of the mitochondrial
isoform in tomato, and therefore able to functionally
compensate the absence of the mitochondrial one [21].
Structural and expression characteristics of mitochondrial
isoforms from diverse species have been studied and the
identification of potential inter-domain disulfides in
higher plant mitochondrial CS suggests paradoxical
differences in redox-sensitivity relative to its animal
counterpart [22]. CS is also regulated by the cellular
NADH/NAD+ ratio, the ATP/ADP ratio and succinyl-
CoA levels whilst being inhibited by ATP, NADH and
succinyl-CoA and activated by ADP [23], suggesting that
its activity is tightly regulated at the metabolite level.
The next step of the TCA cycle is catalyzed by aconitase
which catalyzes the reversible hydration of cis-aconitate to
either citrate or isocitrate. In plants, aconitase has been
characterized in several tissues [24,25]. It has been also
demonstrated that at least in Arabidopsis two isoforms are
present in the mitochondria and one in the cytosol [26!].
Interestingly whilst the number of genes encoding aco-
nitase varies between plant species, the gene products are
often dual targeted to both the mitochondria and the
Current Opinion in Plant Biology 2013, 16:335343
338 Physiology and metabolism
cytosol [25,26!]. Notably the aconitase enzyme is highly
sensitive to oxidative stress [27] since it has highly
oxidation sensitive iron (Fe)sulphur (S) cluster in its
active centre. Studies on citrus fruit vesicles and calli
suggest that Fe availability may also be involved in the
regulation of cytosolic aconitase activity; however the
mitochondrial isoform is not affected by Fe limitation,
suggesting a distinct response of different aconitase
forms to Fe shortage [28]. It has furthermore been
demonstrated that the lack of manganese superoxide
dismutase results in the inhibition of aconitase and the
subsequent enzyme of the cycle, isocitrate dehydrogen-
ase (IDH) [29]. Furthermore, the widely reported plant
metabolite citramalate can act as an endogenous inhibitor
of mitochondrial aconitase [30].
Isocitrate can subsequently be oxidatively decarboxy-
lated to 2-oxoglutarate by either NAD+-dependent or
NADP+-dependent IDH, generating CO2 and NADH or
NADPH, respectively [31]. In higher plants NADP+-
dependent IDH is located to the mitochondria, plastids,
peroxisomes and cytosol [32], whereas NAD+-dependent
IDH is strictly mitochondrial and has been typically
associated with TCA cycle flux [33]. The enzyme iso-
lated from pea leaf mitochondria demonstrates product
inhibition by NADH, and is also inhibited by NADPH
[33]. In addition it has been observed that the latter
inhibition is non-competitive with NAD+ or NADH,
suggesting that NADPH may be an allosteric regulator
since its Ki lies within the physiological range of mito-
chondrial NADPH concentrations [34]. In contrast to the
enzyme from non-plant sources plant NAD+-dependent
IDH is, however, unaffected by adenylates [33]. IDH is
believed to be an important regulatory step of the TCA
cycle and plays an important role in maintaining the 2-
oxoglutarate level and therefore in the regulation of
nitrogen assimilation [35].
The reaction catalyzed by the 2-oxoglutarate dehydro-
genase complex (OGDHC) represents a metabolic
branch point connecting 2-oxoglutarate (and the TCA
cycle) with nitrogen assimilation with 2-oxoglutarate
either being irreversibly degraded by the OGDHC or
providing carbon skeletons for nitrogen assimilation.
OGDHC is allosterically regulated in responses to second
messengers and metabolic indicators, such as Ca2+, ATP/
ADP, NADH/NAD+ and thiamine pyrophosphate [36].
This complex is very similar to the PDC in the context of
its protein organization, cofactor dependency, and mech-
anism of action. Although the OGDHC activity is not
known to be subject to covalent modification, its meta-
bolic regulation is quite complex, likely being regulated
by energy charge, the NADH/NAD+ ratio, as well as by
the levels of both its substrates and products [36].
The subsequent enzyme of the cycle, succinyl-CoA ligase
(ScoAL), is feedback inhibited by intermediates of the
Current Opinion in Plant Biology 2013, 16:335343
pathway of porphyrin biosynthesis as well as being
competitively inhibited by malonate in the reverse
direction but is activated by 2-oxoglutarate and inhib-
ited by both citrate as well as isocitrate and all down-
stream intermediates of the TCA cycle when assayed in
the forward direction [37]. Taken together these data
thus suggest that the reaction catalyzed by ScoAL is
likely co-ordinately regulated with those catalyzed by
other enzymes of the TCA cycle.
Succinate dehydrogenase (SDH), also commonly referred
as complex II, plays a key role in mitochondrial metab-
olism both as a member of the electron transport chain
and TCA cycle [38]. The regulation of the enzyme was
investigated in coupled mitochondria by simultaneously
measuring oxygen uptake rates and ubiquinone reduction
levels [39]. This study revealed that the activation state
level of the enzyme is unambiguously reflected in the
kinetic dependence of the succinate oxidation rate upon
the ubiquinone redox poise. Kinetic results indicated that
it is additionally activated by both ATP and ADP [39].
Surprisingly NO, which has been described as an inhibi-
tor of plant respiratory chains owing to its high affinity for
cytochrome c oxidase was recently demonstrated to act
additionally on SDH [40].
The allosteric properties of fumarase from pea (Pisum
sativum L.), revealed inhibition of this enzyme by phys-
iological concentrations of pyruvate, 2-oxoglutarate and
the adenine nucleotides ATP, ADP and AMP, consistent
with this step being an important control point of the
TCA cycle [41]. Accordingly, down regulation of this
enzyme in tomato resulted in a relatively large reduction
in the rate of respiration in comparison to the majority of
other enzymes of the cycle [42].
The cycle is completed by MDH which catalyzes the
reversible oxidation of malate to produce oxaloacetate
(OAA) [43]. Whilst the equilibrium position favors malate
and NAD+ production, the in vivo removal of OAA by CS,
coupled with the removal of NADH by the respiratory
chain, causes the reaction to function in the direction of
malate oxidation in most tissues [44]. Therefore, it is
likely that accumulation of NADH would lead to an
inhibition of the mitochondrial MDH.
To summarize the metabolic regulation of the TCA cycle
is somewhat complex and enzyme specific although sev-
eral of the enzymes are intrinsically coupled with the
redox environment and the NADH/NAD+ ratio. Notably
the extent to which any metabolite mediated regulation
of TCA cycle flux is relevant in vivo is dependent on the
local concentrations of these metabolites. Given this fact
it seems likely that further development of method-
ologies to determine subcellular metabolite concen-
trations [45] as well as a better understanding of the
mitochondrial carrier family of metabolite transporters
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 Regulation of the mitochondrial tricarboxylic acid cycle Nunes-Nesi et al. 339
[46] and indeed transporters of the outer mitochondrial
membrane [47!!] will be crucial facilitators of our com-
prehension of the regulation of flux through the pathway.
Mechanisms of regulation revealed by
profiling studies
By contrast to the directed studies described above, we
will now approach insights achieved from the application
of broad profiling techniques in a more general manner
describing studies based at the transcript, protein and
finally metabolite level. At the transcriptional level it has
been frequently observed that genes associated with the
TCA cycle are down regulated during the night in an
essentially co-ordinated manner. However, a more recent
analysis [48] reveals that genes associated to the TCA
cycle and mitochondrial electron transport chain actually
can be separated into three subgroups depending on their
response to stress. A complete analysis of the expression
changes documented in public microarray databases at
the level of tissue expression and response to stress is
additionally provided in Supplementary Figures 1 and 2,
respectively. Owing to space limitations we will not
comment on all of these suffice to say that there are
significant patterns of similarities and differences across
both tissue type and the application of stress. Of particular
note is the very high up-regulation of the TCA cycle
during osmotic, salt and UV-B stress consistent with
knowledge acquired in other experiments [4951]. Not
visible in these datasets were the above mentioned
responses to light; however, preliminary evidence
suggests that at least in the case of SDH differential
expression is regulated by the phytochrome pathway [52].
The use of proteomics in understanding the TCA cycle
regulation has been two-fold. Firstly, diagnosing changes
in protein abundance and secondly in the identification
and interpretation of post-translational modifications of
the proteins. Interestingly whilst there are several studies
diagnosing the changes in mitochondrial proteome under
stress conditions (reviewed in [53,54]) relatively few
changes in abundance of TCA cycle enzymes have been
observed. Namely a decrease of fumarase and accumu-
lation of a mitochondrial MDH and a subunit of PDC
under oxidative stress [55] and the increase of aconitase,
IDH, OGDHC E2 subunit and mitochondrial MDH
under flooding stress [56] were reported. On the other
hand, abundance of TCA cycle enzymes showed dynamic
diurnal changes peaking early in the night, indicating four
diurnal phases of the TCA cycle operation governed by
variation in the capacity of respiratory metabolism [57].
Turning to post-translational modifications a huge num-
ber of potential modifications of proteins are possible;
however, we will limit our discussion here to phosphoryl-
ation, redox modification and protein acetylation since
these are arguably the best understood in terms of the
mitochondrial TCA cycle. Down regulation of PDC
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activity by phosphorylation of E1 subunit has been
reported to be involved in the diurnal regulation of this
enzyme complex. In Escherichia coli phosphorylation of
IDH is also known to play an important role in pathway
regulation [58]. In addition, large scale phosphoproteomic
experiments revealed that many other TCA cycle
enzymes were also phosphorylated in Arabidopsis (e.g.
aconitase, IDH, OGDHC, ScoAL, SDH, and a mitochon-
drial MDH; searched in PhosPhAt 4.0 database [59]). The
results thus render phosphorylation as a candidate mech-
anism for the TCA cycle regulation although the func-
tionality of these phosphorylation events for in vivo
enzyme regulation remains to be tested.
The regulation of photosynthetic enzymes by the
reduction of regulatory disulfide bonds via ferredoxin
(Fdx)/thioredoxin (TRX) system has been well documen-
ted in the plastid; however, plants additionally possess the
extraplastidic NADP/TRX system whereby NADPH
reduces TRX by means of the flavoenzyme NADP/
TRX reductase (NTR) [60]. By contrast to the extensive
studies on the regulatory role of TRX systems in the
plastid and the cytosol, we have scant understanding of
the role of TRX in plant mitochondria. That said, pro-
teomic analyses of TRX binding proteins in Arabidopsis
mitochondria detected seven TCA cycle related enzymes
[61!!], with other proteomic studies on identifying both
cytosolic and mitochondrial MDH as targets of TRX [62].
These results suggest a direct redox regulation of TCA
cycle enzymes via NADP/TRX system as one of the
regulatory mechanisms of this pathway. However, further
studies will be required in order to experimentally assess
the impact of redox regulation on the flux through the
TCA cycle. Lys acetylation of proteins has recently been
demonstrated to play an extensive role in the regulation
of metabolic enzymes [63].
Proteomic studies revealed that all enzymes of TCA cycle
are acetylated in human liver tissue and E. coli and the
levels such as glucose, amino acids and fatty acids, gener-
ally influence the acetylation status of metabolic enzymes
[64,65]. By contrast proteomic analysis of Arabidopsis
detected only a singly acetylated enzyme related to
TCA cycle, NAD+ dependent MDH which is localized
in the cytosol [66]. Further effort should be made here
since as the authors state this most likely merely reflects
that a lower depth of coverage of the Lys-acetylated
proteome was achieved in this study, rather than a lack
of physiologically relevant Lys acetylation [66].
Interrogation of a recent compilation of metabolomics
datasets of plants exposed to abiotic stress reveals that the
cellular level of TCA cycle intermediates respond
dramatically to several stresses [67]. Of particular note
here given changes described above are the changes
following manipulation of the redox status [68], tissue
oxygenation level [69] and of elements of the circadian
Current Opinion in Plant Biology 2013, 16:335343
340 Physiology and metabolism
clock [19!!,70]. Following the manipulation of the thiol-
disulphide status of Arabidopsis leaves by treatment with
DTT there was a decrease in the metabolites aconitate,
isocitrate and 2-oxoglutarate but conversely an increase in
levels of succinate, fumarate and malate [68], consistent
with the proteomic suggestion of direct redox regulation of
the cycle. Similarly, metabolite profiling of waterlogging
induced hypoxia in Lotus japonica revealed the operation of
a bifurcated TCA cycle pathway and an inhibition of the
reaction catalyzed by SDH [69]. Metabolomics on the
pseudo response regulator prr9 prr7 prr5 triple Ara-
bidopsis mutants [70] and thiamin-pyrophosphate ribos-
witch deficient Arabidopsis [19!!] additionally provide
strong support for the involvement of machinery of the
circadian clock playing a role in the diurnal regulation of
the TCA cycle. In the latter case thiamine biosynthesis was
demonstrated to be under the control of the circadian clock
and rather surprisingly that the supply of thiamine directs
the activity of the thiamine requiring enzymes of the cycle
in a manner which controls its flux.
Inferences from the regulation of the TCA
cycle in non-plant systems and future
perspectives
As alluded to above, a further mode of regulation is that
played by compartmentation whilst studies in plants have
provided a fairly comprehensive view as to how this is
controlled at the organellar level as yet little is known
there in plants at finer sub-compartment resolution. This
is in sharp contrast to mammalian and microbial systems.
Indeed the coining of the term metabolon to define
supermolecular complexes composed of sequential meta-
bolic enzymes was first used in the context of the rat TCA
cycle [71]. In such complexes the active domains of
enzymes are structurally connected in metabolon result-
ing metabolite channeling between the enzymes [72], a
mechanistic process for the direct delivery of a reaction
intermediate from the active site of one enzyme to the
other(s) without prior dissociation into the bulk solvent.
Metabolite channeling offers a means for high metabolic
rates with low bulk concentrations of intermediates and
extremely rapid responses to changes in the metabolic
status of the cell through reversible assembly and disas-
sembly of all or some of the constituent parts [73]. These
features fit very well to those required for TCA cycle
regulation and as stated above the TCA cycle metabolon
was uncovered in mammals and yeast in extensive studies
in the 70s and 90s, whilst interactions of enzymes were
recently reported also in bacteria [74!!]. However, whilst
metabolons have been described for the CalvinBenson
cycle, glycolysis and specific pathways of specialized
metabolism in plants [75,76], to date there is no molecular
evidence showing an interaction of plant TCA cycle
enzymes even in large scale interactome studies [77]. It
is, however, quite possible that this is due to the fact that
the transitory nature of such enzyme complexes renders
them recalcitrant to purification and that preliminary
Current Opinion in Plant Biology 2013, 16:335343
studies have indicated differential association of fumarase
within protein complexes under conditions of oxidative
stress [78]. It seems likely that targeted approaches
designed specifically at the TCA cycle will be required
to better investigate the possibility that the plant cycle like
its mammalian and bacterial counterparts also forms
metabolite channels. To summarize, current understand-
ing of the regulation of the TCA cycle is that it is the result
of an intricate interplay of several levels of regulation
including transcription, protein stability, protein modifi-
cations and allosteric effectors. Proteomics approaches
have clearly suggested that direct redox modulation of
several of the enzymes of the TCA cycle may well
represent a novel mechanism of regulation. It can be
anticipated that further such mechanisms will be uncov-
ered as proteomics technologies mature. Two other devel-
opments that we anticipate having great promise here are
the extension of tools to gain better understanding of
spatial aspects of metabolic regulation [79] and develop-
ment of approaches to provide higher resolution infor-
mation concerning metabolic fluxes through the cycle
[7,80]. We firmly believe that on the application of these
tools we will finally be able to complete the jigsaw allowing
us to address the molecular hierarchy of the metabolic
regulation of the plant mitochondrial TCA cycle.
Acknowledgements
Financial support from the Max-Planck-Society (to WLA, ANN and ARF),
the Deutsche Forschungsgemeinschaft (grant no. DFG-SFB429 to ARF),
and the National Council for Scientific and Technological Development
CNPq-Brazil (grant numbers 478261/2010-1 to ANN and 472787/2011-0 to
WLA) are gratefully acknowledged.
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
! of special interest
!! of outstanding interest
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.pbi.2013.01.004.
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