236

The oxidative pentose phosphate pathway:

structure and organisation
Nicholas J Kruger
and Antje von Schaeweny
The oxidative pentose phosphate pathway is a major source of
reducing power and metabolic intermediates for biosynthetic
processes. Some, if not all, of the enzymes of the pathway are
found in both the cytosol and plastids, although the precise
distribution of their activities varies. The apparent absence of
sections of the pathway from the cytosol potentially complicates
metabolism. These complications are partly offset, however, by
exchange of intermediates between the cytosol and the plastids
through the activities of a family of plastid phosphate translocators.
Molecular analysis is confirming the widespread presence of
multiple genes encoding each of the enzymes of the oxidative
pentose phosphate pathway. Differential expression of these
isozymes may ensure that the kinetic properties of the activity that
catalyses a specific reaction match the metabolic requirements of
a particular tissue. This hypothesis can be tested thanks to recent
developments in the application of 13 C-steady-state labelling
strategies. These strategies make it possible to quantify flux
through metabolic networks and to discriminate between
pathways of carbohydrate oxidation in the cytosol and plastids.
Addresses


Department of Plant Sciences, University of Oxford, South Parks Road,
Oxford OX1 3RB, UK
e-mail: nick.kruger@plants.ox.ac.uk
y
Institut fur Botanik, Westfalische Wilhelms-Universitat Munster,
Schlossgarten 3, 48149 Munster, Germany
e-mail: schaewen@uni-muenster.de
Correspondence: Nicholas J Kruger

Current Opinion in Plant Biology 2003, 6:236246

This review comes from a themed issue on
Physiology and metabolism
Edited by Alison Smith and Mary Lou Guerinot
1369-5266/03/$ see front matter
2003 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/S1369-5266(03)00039-6

Abbreviations
G6PDH glucose 6-phosphate dehydrogenase
GC
gas chromatography
MS
mass spectroscopy
NMR
nuclear magnetic resonance spectroscopy
oxPPP
oxidative pentose phosphate pathway
6PGDH 6-phosphogluconate dehydrogenase
TA
transaldolase
TK
transketolase
XPT
xylulose phosphate/phosphate translocator

Introduction
Recent years have witnessed a resurgence of interest in
the oxidative pentose phosphate pathway (oxPPP), which
Current Opinion in Plant Biology 2003, 6:236246

has been brought about by an increasing appreciation of

the central role of this pathway in metabolism. In nonphotosynthetic cells, the oxPPP is a major source of
reductant (i.e. NADPH) for biosynthetic processes such
as fatty-acid synthesis and the assimilation of inorganic
nitrogen [1], and maintains the redox potential necessary
to protect against oxidative stress [2]. The reversible nonoxidative section of the pathway is also the source of
carbon skeletons for the synthesis of nucleotides, aromatic amino acids, phenylpropanoids and their derivatives [3]. Although the basic features of the oxPPP are
well-established [4], details of how the pathway operates
in plants and how it influences other processes remain
largely conjecture. This article focuses on the impact of
recent studies on our understanding of the structure and
organisation of the oxPPP in higher plants.

Structure of the oxidative pentose

phosphate pathway
The oxPPP is commonly considered to operate as depicted
in Figure 1a. Varying proportions of fructose 6-phosphate,
and even triose phosphate, are potentially recycled
through the pathway following their conversion to glucose
6-phosphate by glucose-6-phosphate isomerase [5,6].
However, as recently re-emphasised, both transketolase
(TK) and transaldolase (TA) possess broad substrate specificities [7]. This has led to alternative schemes for the
non-oxidative section of the pathway that involve additional metabolites such as octulose 8-phosphate [8]. Support for such schemes is based on the identification of
proposed novel intermediates and the apparent failure of
the conventional pathway to account for the observed
labelling pattern in pathway products. However, both of
these lines of evidence are open to alternative explanations
and the proposed alternative schemes remain strongly
contested [9]. Nevertheless, even if we consider only
recognised pathway intermediates containing up to seven
carbon atoms, it is possible to draw a different but equally
valid pathway, as shown in Figure 1b. This raises the
question of whether it is appropriate to regard the oxPPP
as a rigid formal sequence. The two schemes depicted in
Figure 1 result in the same distribution of carbon atoms
within the pathway intermediates, and so there is no
definitive evidence favouring one scheme over the other.
Indeed, in the absence of any compelling evidence to
suggest that the contributing enzymes are organised in
a way that limits the free diffusion of intermediates
between them, it is difficult to see how they could operate
as a fixed sequence. This view is reinforced by the realisation that varying amounts of ribose 5-phosphate and erythrose 4-phosphate are withdrawn from the pathway for
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Oxidative pentose phosphate pathway Kruger and von Schaewen 237

Figure 1

(a)

P-Gluconolactone
1 2 3 4 5 6

NADPH

6-P-Gluconate

1 2 3 4 5 6
3

Glucose 6-P

CO2

NADPH

1 2 3 4 5 6

Ribulose 5-P

Ribose 5-P

2 3 4 5 6

2 3 4 5 6
5

Fructose 6-P
6

2 3 2 4 5 6
2 3 3 4 5 6

Triose 3-P

Xylulose 5-P

4 5 6

2 3 4 5 6

Sedoheptulose-7-P

2 3 2 3 4 5 6

Erythrose-4-P
3 4 5 6

Triose 3-P

4 5 6

(b)

P-Gluconolactone
1 2 3 4 5 6

NADPH

6-P-Gluconate

1 2 3 4 5 6
3

Glucose 6-P

CO2

NADPH

1 2 3 4 5 6

Ribulose 5-P

Xylulose 5-P
8

2 3 4 5 6

2 3 4 5 6
4

Fructose 6-P
2 3 2 4 5 6
2 3 3 4 5 6

Triose 3-P

Ribose 5-P

4 5 6

2 3 4 5 6

Sedoheptulose-7-P

2 3 2 3 4 5 6

Erythrose-4-P
3 4 5 6
6
Current Opinion in Plant Biology

The oxidative pentose phosphate pathway depicted in (a) conventional and (b) alternative formulation. The enzymes ( - ) that catalyse each of the
steps are identified in Table 2. The numbers below each compound represent the fate of specific carbon atoms within the glucose 6-phosphate
that enters the pathway through the oxidative steps. Different colours are used to distinguish carbon atoms derived from ribose 5-phosphate (blue) and
xylulose 5-phosphate (green).

nucleotide synthesis and phenylpropanoid production,

respectively, via the shikimic acid pathway. These considerations suggest that the non-oxidative section of the
oxPPP should be regarded as a pool of intermediates that is
close to dynamic equilibrium, and that is capable of
adjusting to a new near-equilibrium when any of the
intermediates is withdrawn from the pool [4].
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Flux through the reactions linking the intermediates

within the pool of pathway metabolites is probably best
considered within the framework of elementary mode
analysis. This form of analysis breaks metabolism down
into a series of elementary flux modes, each of which
defines a minimal set of reactions that allows the metabolic system to operate in steady state [10]. Using this
Current Opinion in Plant Biology 2003, 6:236246

238 Physiology and metabolism

Table 1
Elementary flux modes of the oxidative pentose phosphate pathway.
Flux mode

Net stoichiometry

Comment

3Glc-6-P 8ADP 5Pi 5NAD 6NADP

! 8ATP 5NADH 6NADPH 3CO2 5Pyr
Glc-6-P 2ADP Pi NAD 6NADP ! 2ATP
NADH 6NADPH 3CO2 Pyr
Glc-6-P 12NADP !12NADPH Pi 6CO2

4
5

Glc-6-P 2NADP !2NADPH CO2 Rib-5-Pex

5Glc-6-P ATP ! ADP 6Rib-5-Pex

1
2

No recycling of either fructose 6-P or triose-P through the oxPPP

Complete conversion of fructose 6-P to glucose
6-P and recycling through the oxPPP
Complete recycling of both fructose 6-P and
triose-P through the oxPPP
Formation of Rib-5-P through the oxidative steps of the oxPPP
Production of Rib-5-P through the non-oxidative steps of the oxPPP

The metabolic model used for this analysis assumes that glucose 6-phosphate (Glc-6-P), pyruvate (Pyr, the end-product of glycolysis), ribose
5-phosphate (Rib-5-Pex, withdrawn for nucleotide synthesis) and CO2, together with the cofactors NADP, NADPH, NAD, NADH, ATP, ADP and
Pi, are external metabolites. The steps converting glucose 6-phosphate to ribulose 5-phosphate, converting phosphoenolpyruvate to pyruvate,
and utilising ribose 5-phosphate (Rib-5-Pex) are considered irreversible. A further six elementary flux modes occur if Rib-5-Pex is allowed to enter
the network [11].

approach, five elementary modes have been identified

within a simplified scheme containing the oxPPP and
associated glycolytic reactions [11]. In combination, these
modes are sufficient to define net flux through each of the
component reactions of the system (Table 1). However,
the scheme used in this analysis does not allow for the
withdrawal of erythrose 4-phosphate, the broad substrate
specificity of TK and TA, or the duplication of oxPPP
enzymes in the cytosol and plastids (see below). Each of
these factors is likely to increase complexity within the
system, and thus increase the number of elementary flux
modes required to define the activity of the metabolic
network within plant cells.

Compartmentation of the oxidative

pentose phosphate pathway
A unique feature of plant metabolism is the extent to which
the enzymes of carbohydrate oxidation are replicated in
the cytosol and plastids. Both cytosolic and plastidic isoforms of glucose 6-phosphate dehydrogenase (G6PDH)
and 6-phosphogluconate dehydrogenase (6PGDH) have
been isolated from a sufficiently wide range of photosynthetic and non-photosynthetic tissues to suggest that the
subcellular duplication of the oxidative section of the
oxPPP is probably ubiquitous in plants [6]. This view is
supported by the identification of separate genes encoding
discrete cytosolic and plastidic isozymes of G6PDH in
potato and tobacco [1214,15] and of 6PGDH in spinach
and maize [16,17]. However, the organisation of the
reversible non-oxidative section of the pathway is far less
clear. Almost a decade ago, Schnarrenberger et al. [18]
suggested that the enzymes catalysing these reactions
might be restricted to the plastids. Subsequent studies,
which were based on the differential centrifugation of cell
extracts, confirmed the apparent absence of the enzymes of
the non-oxidative section of the pathway from the cytosol
of spinach and pea leaves, and of pea and maize roots [19].
Debnam and Emes [19] found a significant proportion of
the activity of each of the non-oxidative enzymes outside
the plastids in tobacco leaves and roots, but more careful
Current Opinion in Plant Biology 2003, 6:236246

fractionation of tobacco mesophyll protoplasts suggested

that the vast majority of TK activity is associated with
chloroplasts in these cells [20].
These observations contrast, however, with earlier reports
claiming that a significant proportion of the activity of
some or all of the enzymes of the non-oxidative limb of
the pathway are cytosolic in castor bean endosperm [21],
soyabean root nodules [22], and cauliflower buds [23].
The current, apparently contradictory, information suggests that the distribution of the enzymes of the nonoxidative branch of the pathway is not fixed. The capacity
of the cytosol and plastids to catalyse these steps may vary
between species, tissues, developmental stage and environmental conditions. There is an urgent need to clarify
our understanding of the subcellular distribution of these
enzymes, and to establish the extent to which the oxPPP
is compartmented in higher plants.
An alternative approach to resolving the compartmentation of the oxPPP is provided by the availability of complete genome sequences. These allow an analysis of the
metabolic potential of the organism at the genetic level
[24]. The Arabidopsis genome contains multiple copies
of genes that are predicted to encode each of the enzymes
of the oxPPP ([25]; see Table 2). The subcellular location
of the products of each of these genes can be inferred from
whether they encode a transit-peptide sequence that is
necessary to direct the import of the nascent polypeptide
into the plastid. Such analysis suggests that Arabidopsis
contains two cytosolic and four plastidic forms of G6PDH,
supporting the previous characterisation of discrete isozymes of this enzyme in other species [1214,15]. Similarly, the Arabidopsis genome potentially encodes five
6-phosphogluconate lactonase enzymes, an activity largely
ignored in metabolic studies. At least one of these
enzymes is likely to be plastidic. In apparent contrast,
none of the three Arabidopsis isozymes of 6PGDH has an
identifiable transit peptide sequence. However, comparison of the Arabidopsis 6PGDH isozymes with related
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Oxidative pentose phosphate pathway Kruger and von Schaewen 239

Table 2
Summary of Arabidopsis genes likely to encode enzymes of the oxidative pentose phosphate pathway.
Enzyme
1

Glucose-6-phosphate 1-dehydrogenase
EC 1.1.1.49

6-Phosphogluconolactonase
EC 3.1.1.31

Gene number

TargetP value

Predicted location

At1g09420
At1g24280
At3g27300
At5g13110
At5g35790
At5g40760

0.895
0.926

Plastid
Plastid

0.816
0.983

Plastid
Plastid

0.964

Plastid

At1g13700
At3g49360
At5g24400
At5g24410
At5g24420

6-Phosphogluconate dehydrogenase (decarboxylating)

EC 1.1.1.44

At1g64190
At3g02360
At5g41670

Ribose-5-phosphate isomerase
EC 5.3.1.6

At1g71100
At2g01290
At3g04790
At5g44520

Plastid
Plastid

0.735
0.829

Plastid
Plastid

Ribulose-5-phosphate 3-epimerase
EC 5.1.3.1

At1g63290
At3g01850
At5g61410

0.925

Plastid

Transketolase
EC 2.2.1.1

At2g45290
At3g60750

0.970
0.960

Plastid
Plastid

Transaldolase
EC 2.2.1.2

At1g12230
At5g13420

0.969
0.978

Plastid
Plastid

Glucose-6-phosphate isomerase
EC 5.3.1.9

At4g24620
At5g42740

0.949

Plastid

The identities of predicted genes within the Arabidopsis genome are based on comparison of the deduced amino-acid sequence with those of
corresponding enzymes from other organisms. The predicted subcellular location of the gene products are derived from analysis of potential
transit peptide sequences using TargetP (http://www.cbs.dtu.dk/services/TargetP/), with the exception of those for 6-phosphogluconate
dehydrogenase, which are assigned on the basis of their similarity to plastidic and cytosolic isozymes from spinach [16]. The number preceding
the name of each enzyme corresponds to the reaction(s) indicated in Figure 1.

sequences from spinach suggests that two of the Arabidopsis genes encode plastidic forms of the enzyme [16].
Thus, Arabidopsis has the genetic capacity to catalyse the
irreversible oxidative section of the oxPPP in both the
cytosol and plastids.
Similar analyses suggest that Arabidopsis contains both
cytosolic and plastidic forms of ribose-5-phosphate isomerase and ribulose-5-phosphate epimerase. For the latter enzyme, this conclusion has been corroborated by the
isolation of cDNA that encodes cytosolic and plastidic
isozymes from Arabidopsis, rice and maize [26]. In contrast, the two remaining enzymes of the non-oxidative
section of the pathway, TK and TA, may be exclusively
confined to plastids. Although two genes encode each of
these enzyme activities, all four genes contain a predicted
transit-peptide sequence, suggesting that none of the four
gene products comprising the two enzymes is cytosolic.
This implies that Arabidopsis has the genetic capacity
to convert cytosolic ribulose 5-phosphate to ribose
5-phosphate and xylulose 5-phosphate, but any further
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rearrangement of the carbon backbone to regenerate

fructose 6-phosphate and triose phosphate must occur
within plastids. The confidence we can place in such
analysis depends on two factors. The first consideration is
the reliability with which the subcellular location of
putative gene products can be deduced from the presence
of a potential transit-peptide sequence that is identified
by TargetP or other search algorithms. Ideally, the predicted location of a specific gene product should be
confirmed experimentally using reporter-gene fusions.
Even a gene that encodes a functional transit peptide
may generate a transcript that lacks this region, and
therefore produce a polypeptide that is confined to the
cytosol [27]. The second consideration is the possible
failure to identify distantly related (or unrelated) genes
that encode similar enzymatic activities. The extent of
this problem is difficult to assess, but it remains a realistic
concern while the functions of up to 30% of the predicted
open-reading frames within the Arabidopsis genome are
unknown [25]. More generally, the identification of
cDNA encoding two cytosolic isozymes of TK plus a
Current Opinion in Plant Biology 2003, 6:236246

240 Physiology and metabolism

plastidic isoform of the enzyme in the resurrection plant

Craterostigma plantagineum [28] demonstrates that there is
no reason to assume that the genetic organisation of the
oxPPP is the same in all plants.

Interactions between cytosolic and

plastidic processes
The possible absence of TK and TA from the cytosol of
some species raises potential problems in understanding
the fate of pentose phosphates that are generated from
the oxidative section of the pathway in the cytosol.
These problems have been partly alleviated by the
recent discovery of a member of the phosphate-translocator family of plastid inner-envelope membrane proteins that has the capacity to transport pentose
phosphates [29]. This translocator is encoded by a
single gene (At5g17630) that is discrete from other subclasses of phosphate translocator genes within the Arabidopsis genome and has homologues in a range of species
[29,30]. The substrate specificity of the recombinant
Arabidopsis protein when reconstituted into liposomes
suggests that, in vivo, the translocator preferentially
catalyses the counter-exchange of xylulose 5-phosphate,

triose phosphate and Pi. On this basis, it has been termed

a xylulose phosphate/phosphate translocator (XPT). The
presence of such an exchange activity in the innerenvelope membrane greatly increases the potential for
interaction between oxPPP reactions in the plastid and in
the cytosol (Figure 2). Specifically, by promoting the
exchange of pentose phosphates between the cytosol and
the plastid, the XPT allows the production of NADPH
and the provision of biosynthetic precursors to occur
independently of one another in each compartment.
In cells that potentially lack cytosolic forms of ribose5-phosphate isomerase and ribulose-5-phosphate epimerase as well as TK and TA [19], however, the source
of ribose 5-phosphate for the synthesis of cytosolic
nucleotides remains unresolved. Furthermore, the presence of the XPT translocator does not explain the
apparent ability of cytosol and plastids to co-operate in
the provision of NADPH for biosynthesis. Such cooperation has been revealed by studies in which nitrite
failed to stimulate flux through the oxPPP in maize
mutants that lacked detectable activity of cytosolic
6PGDH [31]. Thus, the issues arising from the compartmentation of the oxPPP are, at best, only partly resolved.

Figure 2

Sucrose

Plastid

Glc-6-P
NADPH

Glc-6-P

Glc-6-P

NADPH

6-PGlcA
Pi

Pi

GPT

CO2

NADPH
Rbu-5-P

Triose-P
Pi

Triose-P

oxPPP

Rib-5-P

Pi

XPT

CO2

Nucleotides
Rib-5-P

Nucleotides

Xlu-5-P

Xlu-5-P

PEP

Ery-4-P

PEP
PPT

Pi

Pi

Shikimic acid
pathway

Cytosol
Current Opinion in Plant Biology

Exchange of oxidative pentose phosphate pathway intermediates by the plastid phosphate translocators in Arabidopsis. Glucose 6-phosphate
(Glc-6-P) can enter plastids in exchange for triose phosphate or orthophosphate (Pi) via the Glc-6-P/phosphate translocator (GPT). Exchange of
xylulose 5-phosphate (Xlu-5-P), triose phosphate (Triose-P) and Pi is catalysed by the XPT. In the absence of cytosolic transketolase and
transaldolase, this activity facilitates further metabolism (within plastids) of pentose phosphates that are generated by the oxidative reactions in the
cytosol, as well as the provision of pentose phosphates generated independently of NADPH production for nucleotide synthesis in the cytosol. The
phosphoenolpyruvate/phosphate translocator (PPT) is required for the import of phosphoenolpyruvate into plastids for the biosynthesis of aromatic
acids. The triose phosphate/phosphate translocator, which is expressed only in photosynthetic cells, is omitted for clarity.
Current Opinion in Plant Biology 2003, 6:236246

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Oxidative pentose phosphate pathway Kruger and von Schaewen 241

The nature and roles of multiple isoforms

of oxidative pentose phosphate pathway
enzymes
One of the surprising outcomes of the sequencing of the
Arabidopsis genome has been the extent of the duplication
of genes encoding enzymes of central metabolic processes
[25], a trend that is exemplified by the oxPPP (Table 2).
Part of this apparent redundancy may be due to a requirement for different isozymes to function in the cytosol and
plastids. However, this explanation cannot account for the
multiplicity of genes encoding enzymes for some oxPPP
reactions. Interrogation of cDNA/expressed sequence tag
(EST) databases (http://signal.salk.edu/) and the results of
whole-genome microarray analyses(http://nasc.nott.ac.uk/)
show that all of the known oxPPP genes in Arabidopsis are
expressed to some extent. Studies across a range of species
indicate that genes encoding individual isozymes may be
differentially expressed in different tissues, at different
developmental stages, and in response to different growth
conditions (especially those that alter demand for NADPH
or intermediates of the oxPPP for biosynthesis) [14,16,
17,26]. In particular instances, such changes in gene
expression can be linked to changes in the accumulation
of specific isoforms of the enzyme that have distinct kinetic
properties. This is exemplified by recent studies on
G6PDH. All forms of G6PDH are inhibited by high concentrations of NADPH, and their activity in vivo is probably modulated by the redox balance of the NADPH/
NADP pool [4]. In addition, plastidic G6PDH is inactivated by a reversible dithioldisulphide interconversion of
two highly conserved regulatory cysteine residues [32].
This interconversion is ferredoxin/thioredoxin-dependent
and can be mimicked using dithiothreitol, a non-physiological reductant [32].
Two classes of plastidic G6PDH have been distinguished
in higher plants. In potato, the transcripts of one plastidic
isozyme (P1) are most prominent in green tissues and,
although present in stolons and root tips in the light, are
hardly detectable in most non-photosynthetic tissues
[13]. In contrast, the second plastidic isozyme (P2) is
expressed throughout the plant, with highest steady-state
transcript levels in stems and roots [14,15]. Although
both plastidic activities are inhibited by increased
NADPH/NADP and inactivated by dithiothreitol, the
P2 isozyme is strikingly less sensitive than the P1 enzyme
to both forms of modulation [14,32]. These kinetic differences may reflect the differing metabolic requirements
of the cells in which they are expressed. In illuminated
chloroplasts, ferredoxin is reduced directly by components of Photosystem I and the oxPPP needs to be
inactivated in the light so that potentially futile interactions with the Calvin cycle are avoided. In contrast, in
non-photosynthetic tissues, there is a need to maintain
flux through the plastidic oxPPP, even in the face of the
high stromal NADPH/NADP levels that are required to
drive ferredoxin-dependent reactions such as nitrite
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reduction and glutamate assimilation [1]. The involvement of the P2 form of G6PDH in the provision of
NADPH for ferredoxin-dependent reactions is suggested
by an increase in the expression of genes encoding two
Arabidopsis P2 isozymes (At1g24280 and At5g13110; see
Table 2) after transfer from a medium containing ammonium to one including nitrate [33]. Similar increases in the
expression of P2 genes upon exposure to nitrate have been
observed in the roots of tobacco and tomato [15,34].
Furthermore, recent studies on barley roots have identified a plastidic isoform of G6PDH that has properties
similar to those of P2 and that is induced in response to
exposure to ammonium or glutamate [3537]. These
observations are complemented by the demonstration that
both nitrite reduction and glutamate synthesis from glutamine and 2-oxoglutarate by plastids isolated from barley
root depend on a supply of glucose 6-phosphate. This
result confirms the ability of the P2 isozyme to support
flux through the plastidic oxPPP in spite of the high
NADPH/NADP needed to provide reductant for nitrite
reduction and glutamate synthesis [37,38].
The existence of different isozymes of plastidic G6PDH
can be rationalised, but the significance of the multiple
genes that encode other oxPPP enzymes with no obvious
regulatory properties is more difficult to explain. One
possible explanation has been suggested by studies on
transgenic tobacco plants that have decreased TK activity
[20]. The ability of darkened leaves from these plants to
stimulate flux through the oxPPP in response to wounding is compromised. More extensive analysis of photosynthetic carbon metabolism in these plants suggests that
the reduced activity of TK does not directly influence
metabolism. Rather, metabolism is perturbed by the
effects on other metabolic enzymes of changes in the
concentrations of the immediate substrates and products
of TK required to maintain flux through the reactions
catalysed by this enzyme. The relatively large decrease in
phenylpropanoid production in these transgenic plants
demonstrates the sensitivity of other processes to changes
in the levels of oxPPP intermediates. These considerations highlight the need to maintain appropriate levels of
metabolites. In this context, a plausible role for isozymes
may be to allow changes in the steady-state levels of
metabolic intermediates (owing to differences in the
relative affinities of different isozymes for substrates
and products) while maintaining the same net flux
through the pathway.

Measurement of flux through the oxidative

pentose phosphate pathway
Testing the proposal outlined above and developing our
understanding of the regulation of the oxPPP requires the
ability to determine flux through the component reactions
of this pathway accurately. Traditionally, the activity of
the oxPPP has been estimated by comparing the release
of 14 CO2 from specifically labelled glucose. (Usually
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242 Physiology and metabolism

release of 14 CO2 from [1-14 C]glucose relative to that from

[6-14 C]glucose, although other ratios are also informative
[5].) Such analyses are often complicated, however, by the
rearrangement of label within metabolic intermediates in
exchange reactions (catalysed by TK and TA) and by the
release of 14 CO2 through other metabolic processes (see
[5,39] for critical assessment of these problems). An
alternative approach takes advantage of the recent demonstration that plant cells metabolise applied gluconate
through a route involving 6PGDH [40]. Consequently,
release of 14 CO2 from [1-14 C]gluconate provides a rapid
and convenient method to monitor flux through the
oxPPP. However, such estimates of flux are influenced
by the rate of gluconate uptake and phosphorylation by the
tissue, as well as by dilution of [1-14 C]-6-phosphogluconate owing to the oxidation of endogenous (unlabelled)
glucose 6-phosphate. This approach is therefore unlikely
to provide an accurate quantitative measure of oxPPP flux.
More generally, a limitation of all techniques that are
based on measuring 14 CO2 release is that they are unable
to resolve flux through the oxPPP in the cytosol and
plastids [41].
Recently, significant progress has been made in applying
steady-state labelling techniques involving [13 C]glucose,
coupled with the detection of metabolic products using
either nuclear magnetic resonance spectroscopy (NMR)
or mass spectroscopy (MS), to resolve flux through the
central pathways of metabolism in microbial systems
[42]. The simplest applications concentrate on quantifying the proportion of hexose phosphate that is metabolised by the oxPPP relative to that metabolised by
glycolysis [43]. However, the power of this approach lies
in its potential to quantify exchange fluxes through readily reversible reactions as well as net flux through the
pathways [42]. A wide range of strategies are being
developed that employ either specifically labelled
[13 C]glucose or a small proportion of [U-13 C6 ]glucose
and subsequent determination of the relative proportions
of specific groups of 13 C-labelled isotopic isomers (known
as isotopomers) of particular metabolites to determine
flux through complex networks of metabolic reactions
[44,4547]. Although the use of these strategies in
plants has been limited, there appear to be no major
practical problems in applying similar methods to higher
plant tissues [48].
A potential benefit of steady-state labelling approaches is
that they offer the possibility to resolve intracellular
fluxes in situ. Specifically, the activity of the oxPPP in
the cytosol and plastids is reflected in the labelling
pattern of cytosolic and plastidic pools of hexose phosphate. These patterns can be monitored separately by
measuring the abundance and distribution of label within
the fructosyl and glucosyl moieties of sucrose (synthesised in the cytosol) and in the glucosyl residues of starch
(formed in plastids) [41]. Using this rationale, analysis of
Current Opinion in Plant Biology 2003, 6:236246

the redistribution of label in a carrot cell culture after

growth in either [1-13 C]glucose or [1-13 C]fructose has
shown that the oxPPP operates in both the cytosol and
plastids of these cells [49]. More detailed studies in both
maize root tips and tomato cell cultures have determined
the distribution of flux through a metabolic network using
the 13 C-enrichment of specific carbons in carbohydrates
and amino acids following labelling to isotopic nearsteady-state with [1-13 C]glucose [48,50]. In tomato,
the proportion of carbon flowing through the oxPPP
was remarkably constant, and about 2226% of the glucose metabolised by the cells entered this pathway
throughout the growth cycle of the cell culture [50].
Surprisingly, the redistribution of label in both the maize
and tomato studies suggested that the oxidative steps of
the oxPPP are active only in plastids, whereas TA is active
in both the plastids and the cytosol. Such conclusions
directly contradict the emerging model for the subcellular
organisation of oxPPP that is based on the apparent
distribution of enzyme activities (particularly in maize
roots [19]). However, the [1-13 C]glucose used in these
labelling studies [48,50] is not the most informative
substrate for monitoring oxPPP activity as the isotope is
lost as 13 CO2 during the initial oxidative steps [41]. The
results obtained using this isotope should therefore be
regarded as provisional until the study is repeated using
glucose labelled in a more appropriate position.
A potentially more sensitive strategy involving [1,2-13 C2 ]glucose has recently been applied to isolated embryos
from developing Brassica napus seeds [51]. This
approach is based on the rationale that C-1 of [1,2-13 C2 ]glucose is lost by decarboxylation upon entering the
oxPPP, producing [1-13 C]pentose phosphates. The fructose 6-phosphate regenerated from these [1-13 C]pentose
phosphates will be a mixture of different single 13 C-labelled
isotopomers, whereas that formed directly from glucose
6-phosphate will retain the 13 C13 C bond. The retention
of 13 C2 double-labelling within about 90% of the acetyl
units of C18:1 fatty acid indicates that the vast majority of
the glucose used for lipid synthesis is converted to pyruvate and acetyl-CoA directly through glycolysis, rather
than being metabolised by the oxPPP [51]. Labelling of
amino acids following the metabolism of [U-13 C6 ]glucose
indicates that the precursors of pyruvate and acetyl-CoA
(i.e. glycolytic intermediates) are subjected to extensive
rearrangement through the non-oxidative section of the
oxPPP [51]. Hence, the retention of 13 C13 C bonds
within fatty acids cannot be explained by a form of
metabolic segregation in which the products of the oxPPP
are prevented from entering the glycolytic pathway. In
combination, these considerations suggest that very little
carbohydrate is metabolised by the complete oxPPP in
isolated Brassica napus embryos and that, by implication,
this pathway is unlikely to be the major source of
NADPH for fatty acid synthesis in these cultured
embryos. However, more precise studies using labelling
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Oxidative pentose phosphate pathway Kruger and von Schaewen 243

strategies that are specifically designed to measure flux

through the oxPPP in the cytosol and plastids are required
to confirm this conclusion.
Similar labelling strategies involving [U-13 C6 ]glucose
have been applied to study metabolism in developing
maize kernels [52,53]. In these studies, analyses of the
isotopomer composition of lipids, amino acids, triterpenes
and glucosyl units of starch have been used to reconstruct
the biosynthetic pathways involved in their formation
in vivo. From these measurements, it is possible to infer
the labelling patterns of metabolic intermediates such as
erythrose 4-phosphate, ribose 5-phosphate and glucose 6phosphate. In principle, provided the requirement for
labelling to isotopic steady-state is met [41], such information could be used to calculate fluxes through the
central pathways of carbohydrate oxidation. However,
such analyses do not appear to have been attempted.
These recent studies demonstrate the potential for using
either MS or NMR to obtain detailed information on the
relative isotopomer abundance of a wide range of metabolites following labelling with 13 C-substrates. Despite
the ability to make the relevant measurements, to date,
the application of steady-state labelling strategies to
determine metabolic flux has been relatively underexploited in plants. Such techniques need to be developed if we are to obtain the estimates of flux that will be
necessary to build a quantitative understanding of the
operation and regulation of the oxPPP.

Conclusions
Considering the central importance of the oxPPP in metabolism, our understanding of its organisation and operation
in plant cells is remarkably sketchy. In part, this may be
due to the diversity of processes with which it interacts,
and the flexibility required to meet the varying demands of
such processes for reductant and biosynthetic precursors.
However, prospects for improving our appreciation of the
oxPPP are good. As more genomes are sequenced, we will
develop a better understanding of the genetic capacity of
plant systems to catalyse the reactions involved in the
oxPPP, and have access to a greater range of cognate genes
for comparative analysis [54]. Furthermore, the increasing
availability of whole-genome transcript profiles under
different physiological and developmental conditions will
allow us to determine the relationships between the
expression of specific oxPPP isozymes, and to establish
the contribution of such isozymes to particular physiological processes [55,56]. Hopefully, such comparisons can
be complemented by the development of equally robust
technologies for monitoring changes in the profiles of
a broad range of metabolites. This will enable a full
assessment of the possibly wide-ranging metabolic consequences of perturbations in the expression of specific
oxPPP genes [57]. Finally, conceptual advances in the
way in which groups of enzyme-catalysed reactions are
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treated, coupled with improved techniques for measuring

flux through the oxPPP, should provide the foundation for
a more comprehensive, integrated understanding of the
functioning of this sequence of reactions.

Acknowledgements
Research in the authors laboratories is funded by the Biotechnology and
Biological Sciences Research Council (UK) and Deutsche
Forschungsgemeinschaft (Germany).

References and recommended reading

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Oxidative pentose phosphate pathway Kruger and von Schaewen 245

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46. Maaheimo H, Fiaux J, Cakar ZP, Bailey JE, Sauer U, Szyperski T:


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pathways before its incorporation into starch. In combination with earlier
work [52], this study demonstrates the feasibility of using NMR to obtain
detailed information on the isotopomer composition of metabolites. Such
Current Opinion in Plant Biology 2003, 6:236246

246 Physiology and metabolism

information is required for the calculation of flux through the central

pathways of non-photosynthetic carbon metabolism in plants.
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