Pentosas

The oxidative pentose phosphate pathway:structure and organisationNicholas 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 theactivitiesofa familyofplastidphosphate 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 13C-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.

AddressesDepartment of Plant Sciences, University of Oxford, South Parks Road,Oxford OX1 3RB, UK

e-mail: [email protected] fur Botanik, Westfalische Wilhelms-Universitat Munster,Schlossgarten 3, 48149 Munster, Germany

e-mail: [email protected]

Correspondence: Nicholas J Kruger

Current Opinion in Plant Biology 2003, 6:236246

This review comes from a themed issue onPhysiology 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

AbbreviationsG6PDH glucose 6-phosphate dehydrogenaseGC gas chromatographyMS mass spectroscopyNMR nuclear magnetic resonance spectroscopyoxPPP oxidative pentose phosphate pathway6PGDH 6-phosphogluconate dehydrogenaseTA transaldolaseTK transketolaseXPT xylulose phosphate/phosphate translocator

IntroductionRecent years have witnessed a resurgence of interest inthe oxidative pentose phosphate pathway (oxPPP), which

has been brought about by an increasing appreciation ofthe central role of this pathway in metabolism. In non-photosynthetic cells, the oxPPP is a major source ofreductant (i.e. NADPH) for biosynthetic processes suchas fatty-acid synthesis and the assimilation of inorganicnitrogen [1], and maintains the redox potential necessaryto protect against oxidative stress [2]. The reversible non-oxidative section of the pathway is also the source ofcarbon skeletons for the synthesis of nucleotides, aro-matic amino acids, phenylpropanoids and their deriva-tives [3]. Although the basic features of the oxPPP arewell-established [4], details of how the pathway operatesin plants and how it influences other processes remainlargely conjecture. This article focuses on the impact ofrecent studies on our understanding of the structure andorganisation of the oxPPP in higher plants.

Structure of the oxidative pentosephosphate pathwayThe oxPPP is commonly considered to operate as depictedin Figure 1a. Varying proportions of fructose 6-phosphate,and even triose phosphate, are potentially recycledthrough the pathway following their conversion to glucose6-phosphate by glucose-6-phosphate isomerase [5,6].However, as recently re-emphasised, both transketolase(TK) and transaldolase (TA) possess broad substrate spe-cificities [7]. This has led to alternative schemes for thenon-oxidative section of the pathway that involve addi-tional metabolites such as octulose 8-phosphate [8]. Sup-port for such schemes is based on the identification ofproposed novel intermediates and the apparent failure ofthe conventional pathway to account for the observedlabelling pattern in pathway products. However, both ofthese lines of evidence are open to alternative explanationsand the proposed alternative schemes remain stronglycontested [9]. Nevertheless, even if we consider onlyrecognised pathway intermediates containing up to sevencarbon atoms, it is possible to draw a different but equallyvalid pathway, as shown in Figure 1b. This raises thequestion of whether it is appropriate to regard the oxPPPas a rigid formal sequence. The two schemes depicted inFigure 1 result in the same distribution of carbon atomswithin the pathway intermediates, and so there is nodefinitive evidence favouring one scheme over the other.Indeed, in the absence of any compelling evidence tosuggest that the contributing enzymes are organised ina way that limits the free diffusion of intermediatesbetween them, it is difficult to see how they could operateas a fixed sequence. This view is reinforced by the realisa-tion that varying amounts of ribose 5-phosphate and ery-throse 4-phosphate are withdrawn from the pathway for

236

Current Opinion in Plant Biology 2003, 6:236246 www.current-opinion.com

nucleotide synthesis and phenylpropanoid production,respectively, via the shikimic acid pathway. These con-siderations suggest that the non-oxidative section of theoxPPP should be regarded as a pool of intermediates that isclose to dynamic equilibrium, and that is capable ofadjusting to a new near-equilibrium when any of theintermediates is withdrawn from the pool [4].

Flux through the reactions linking the intermediateswithin the pool of pathway metabolites is probably bestconsidered within the framework of elementary modeanalysis. This form of analysis breaks metabolism downinto a series of elementary flux modes, each of whichdefines a minimal set of reactions that allows the meta-bolic system to operate in steady state [10]. Using this

Figure 1

Fructose 6-P

6-P-Gluconate

Ribulose 5-PRibose 5-P

Xylulose 5-P

Sedo-heptulose-7-P

Erythrose-4-P

Triose 3-P

CO2Glucose 6-P

NADPH

NADPH

P-Gluconolactone

Triose 3-P

1 2 3 4 5 6

1 2 3 4 5 6

1 2 3 4 5 6

32 2 3 4 5 6

2 3 4 5

4 5 6

6

2 3 4 5 6

2 3 4 5 6

3 4 5 6

4 5 6

2 3 2 4 5 62 3 3 4 5 6

65

4

3

2

1

8

6

7

Fructose 6-P

6-P-Gluconate

Ribulose 5-P

Sedo-heptulose-7-P

Erythrose-4-P

Triose 3-P

CO2Glucose 6-P

NADPH

NADPH

P-Gluconolactone

1 2 3 4 5 6

1 2 3 4 5 6

1 2 3 4 5 6

32 2 3 4 5 6

4 5 6

2 3 4 5 6

3 4 5 6

2 3 2 4 5 62 3 3 4 5 6

64

5

3

2

1

8

6

7

Ribose 5-P2 3 4 5 6

Xylulose 5-P2 3 4 5 6

(a)

(b)

Current Opinion in Plant Biology

The oxidative pentose phosphate pathway depicted in (a) conventional and (b) alternative formulation. The enzymes ( - ) that catalyse each of thesteps 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).

Oxidative pentose phosphate pathway Kruger and von Schaewen 237

www.current-opinion.com Current Opinion in Plant Biology 2003, 6:236246

approach, five elementary modes have been identifiedwithin a simplified scheme containing the oxPPP andassociated glycolytic reactions [11]. In combination, thesemodes are sufficient to define net flux through each of thecomponent reactions of the system (Table 1). However,the scheme used in this analysis does not allow for thewithdrawal of erythrose 4-phosphate, the broad substratespecificity of TK and TA, or the duplication of oxPPPenzymes in the cytosol and plastids (see below). Each ofthese factors is likely to increase complexity within thesystem, and thus increase the number of elementary fluxmodes required to define the activity of the metabolicnetwork within plant cells.

Compartmentation of the oxidativepentose phosphate pathwayA unique feature of plant metabolism is the extent to whichthe enzymes of carbohydrate oxidation are replicated inthe cytosol and plastids. Both cytosolic and plastidic iso-forms of glucose 6-phosphate dehydrogenase (G6PDH)and 6-phosphogluconate dehydrogenase (6PGDH) havebeen isolated from a sufficiently wide range of photosyn-thetic and non-photosynthetic tissues to suggest that thesubcellular duplication of the oxidative section of theoxPPP is probably ubiquitous in plants [6]. This view issupported by the identification of separate genes encodingdiscrete cytosolic and plastidic isozymes of G6PDH inpotato and tobacco [1214,15] and of 6PGDH in spinachand maize [16,17]. However, the organisation of thereversible non-oxidative section of the pathway is far lessclear. Almost a decade ago, Schnarrenberger et al. [18]suggested that the enzymes catalysing these reactionsmight be restricted to the plastids. Subsequent studies,which were based on the differential centrifugation of cellextracts, confirmed the apparent absence of the enzymes ofthe non-oxidative section of the pathway from the cytosolof spinach and pea leaves, and of pea and maize roots [19].Debnam and Emes [19] found a significant proportion ofthe activity of each of the non-oxidative enzymes outsidethe plastids in tobacco leaves and roots, but more careful

fractionation of tobacco mesophyll protoplasts suggestedthat the vast majority of TK activity is associated withchloroplasts in these cells [20].

These observations contrast, however, with earlier reportsclaiming that a significant proportion of the activity ofsome or all of the enzymes of the non-oxidative limb ofthe pathway are cytosolic in castor bean endosperm [21],soyabean root nodules [22], and cauliflower buds [23].The current, apparently contradictory, information sug-gests that the distribution of the enzymes of the non-oxidative branch of the pathway is not fixed. The capacityof the cytosol and plastids to catalyse these steps may varybetween species, tissues, developmental stage and envir-onmental conditions. There is an urgent need to clarifyour understanding of the subcellular distribution of theseenzymes, and to establish the extent to which the oxPPPis compartmented in higher plants.

An alternative approach to resolving the compartmenta-tion of the oxPPP is provided by the availability of comp-lete genome sequences. These allow an analysis of themetabolic potential of the organism at the genetic level[24]. The Arabidopsis genome contains multiple copiesof genes that are predicted to encode each of the enzymesof the oxPPP ([25]; see Table 2). The subcellular locationof the products of each of these genes can be inferred fromwhether they encode a transit-peptide sequence that isnecessary to direct the import of the nascent polypeptideinto the plastid. Such analysis suggests that Arabidopsiscontains two cytosolic and four plastidic forms of G6PDH,supporting the previous characterisation of discrete iso-zymes of this enzyme in other species [1214,15]. Simi-larly, the Arabidopsis genome potentially encodes five6-phosphogluconate lactonase enzymes, an activity largelyignored in metabolic studies. At least one of theseenzymes is likely to be plastidic. In apparent contrast,none of the three Arabidopsis isozymes of 6PGDH has anidentifiable transit peptide sequence. However, compar-ison of the Arabidopsis 6PGDH isozymes with related

Table 1

Elementary flux modes of the oxidative pentose phosphate pathway.

Flux mode Net stoichiometry Comment

1 3Glc-6-P 8ADP 5Pi 5NAD 6NADP! 8ATP 5NADH 6NADPH 3CO2 5Pyr

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

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

Complete conversion of fructose 6-P to glucose6-P and recycling through the oxPPP

3 Glc-6-P 12NADP !12NADPH Pi 6CO2 Complete recycling of both fructose 6-P andtriose-P through the oxPPP

4 Glc-6-P 2NADP !2NADPH CO2 Rib-5-Pex Formation of Rib-5-P through the oxidative steps of the oxPPP5 5Glc-6-P ATP ! ADP 6Rib-5-Pex Production of Rib-5-P through the non-oxidative steps of the oxPPPThe 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].

238 Physiology and metabolism


sequences from spinach suggests that two of the Arabi-dopsis genes encode plastidic forms of the enzyme [16].Thus, Arabidopsis has the genetic capacity to catalyse theirreversible oxidative section of the oxPPP in both thecytosol and plastids.

Similar analyses suggest that Arabidopsis contains bothcytosolic and plastidic forms of ribose-5-phosphate iso-merase and ribulose-5-phosphate epimerase. For the lat-ter enzyme, this conclusion has been corroborated by theisolation of cDNA that encodes cytosolic and plastidicisozymes from Arabidopsis, rice and maize [26]. In con-trast, the two remaining enzymes of the non-oxidativesection of the pathway, TK and TA, may be exclusivelyconfined to plastids. Although two genes encode each ofthese enzyme activities, all four genes contain a predictedtransit-peptide sequence, suggesting that none of the fourgene products comprising the two enzymes is cytosolic.This implies that Arabidopsis has the genetic capacityto convert cytosolic ribulose 5-phosphate to ribose5-phosphate and xylulose 5-phosphate, but any further

rearrangement of the carbon backbone to regeneratefructose 6-phosphate and triose phosphate must occurwithin plastids. The confidence we can place in suchanalysis depends on two factors. The first consideration isthe reliability with which the subcellular location ofputative gene products can be deduced from the presenceof a potential transit-peptide sequence that is identifiedby TargetP or other search algorithms. Ideally, the pre-dicted location of a specific gene product should beconfirmed experimentally using reporter-gene fusions.Even a gene that encodes a functional transit peptidemay generate a transcript that lacks this region, andtherefore produce a polypeptide that is confined to thecytosol [27]. The second consideration is the possiblefailure to identify distantly related (or unrelated) genesthat encode similar enzymatic activities. The extent ofthis problem is difficult to assess, but it remains a realisticconcern while the functions of up to 30% of the predictedopen-reading frames within the Arabidopsis genome areunknown [25]. More generally, the identification ofcDNA encoding two cytosolic isozymes of TK plus a

Table 2

Summary of Arabidopsis genes likely to encode enzymes of the oxidative pentose phosphate pathway.

Enzyme Gene number TargetP value Predicted location

1 Glucose-6-phosphate 1-dehydrogenase At1g09420 0.895 Plastid

EC 1.1.1.49 At1g24280 0.926 Plastid

At3g27300At5g13110 0.816 Plastid

At5g35790 0.983 Plastid

At5g40760

2 6-Phosphogluconolactonase At1g13700

EC 3.1.1.31 At3g49360


At5g24410

At5g24420

3 6-Phosphogluconate dehydrogenase (decarboxylating) At1g64190 Plastid

EC 1.1.1.44 At3g02360 Plastid

At5g41670

4 Ribose-5-phosphate isomerase At1g71100

EC 5.3.1.6 At2g01290



5 Ribulose-5-phosphate 3-epimerase At1g63290

EC 5.1.3.1 At3g01850


6 Transketolase At2g45290 0.970 Plastid

EC 2.2.1.1 At3g60750 0.960 Plastid

7 Transaldolase At1g12230 0.969 Plastid

EC 2.2.1.2 At5g13420 0.978 Plastid

8 Glucose-6-phosphate isomerase At4g24620 0.949 Plastid

EC 5.3.1.9 At5g42740

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 precedingthe name of each enzyme corresponds to the reaction(s) indicated in Figure 1.



plastidic isoform of the enzyme in the resurrection plantCraterostigma plantagineum [28] demonstrates that there isno reason to assume that the genetic organisation of theoxPPP is the same in all plants.

Interactions between cytosolic andplastidic processesThe possible absence of TK and TA from the cytosol ofsome species raises potential problems in understandingthe fate of pentose phosphates that are generated fromthe oxidative section of the pathway in the cytosol.These problems have been partly alleviated by therecent discovery of a member of the phosphate-translo-cator family of plastid inner-envelope membrane pro-teins that has the capacity to transport pentosephosphates [29]. This translocator is encoded by asingle gene (At5g17630) that is discrete from other sub-classes of phosphate translocator genes within the Ara-bidopsis genome and has homologues in a range of species[29,30]. The substrate specificity of the recombinantArabidopsis protein when reconstituted into liposomessuggests that, in vivo, the translocator preferentiallycatalyses the counter-exchange of xylulose 5-phosphate,

triose phosphate and Pi. On this basis, it has been termeda xylulose phosphate/phosphate translocator (XPT). Thepresence of such an exchange activity in the inner-envelope membrane greatly increases the potential forinteraction between oxPPP reactions in the plastid and inthe cytosol (Figure 2). Specifically, by promoting theexchange of pentose phosphates between the cytosol andthe plastid, the XPT allows the production of NADPHand the provision of biosynthetic precursors to occurindependently of one another in each compartment.In cells that potentially lack cytosolic forms of ribose-5-phosphate isomerase and ribulose-5-phosphate epi-merase as well as TK and TA [19], however, the sourceof ribose 5-phosphate for the synthesis of cytosolicnucleotides remains unresolved. Furthermore, the pre-sence of the XPT translocator does not explain theapparent ability of cytosol and plastids to co-operate inthe provision of NADPH for biosynthesis. Such co-operation has been revealed by studies in which nitritefailed to stimulate flux through the oxPPP in maizemutants that lacked detectable activity of cytosolic6PGDH [31]. Thus, the issues arising from the compart-mentation of the oxPPP are, at best, only partly resolved.

Figure 2

Sucrose

6-PGlcA

Rib-5-P Xlu-5-P Xlu-5-P

Pi Pi

Rib-5-PXPT

GPT

PPT

Ery-4-P

Glc-6-P

oxPPP

Shikimic acidpathway

Nucleotides

Nucleotides

Plastid

Cytosol

Glc-6-P

NADPH

NADPH

Rbu-5-P Triose-PTriose-P

Pi Pi

Pi Pi

Glc-6-P NADPH

CO2

PEP PEP

CO2

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.



The nature and roles of multiple isoformsof oxidative pentose phosphate pathwayenzymesOne of the surprising outcomes of the sequencing of theArabidopsis genome has been the extent of the duplicationof 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 require-ment for different isozymes to function in the cytosol andplastids. However, this explanation cannot account for themultiplicity of genes encoding enzymes for some oxPPPreactions. Interrogation of cDNA/expressed sequence tag(EST) databases (http://signal.salk.edu/) and the results ofwhole-genomemicroarrayanalyses(http://nasc.nott.ac.uk/)show that all of the known oxPPP genes in Arabidopsis areexpressed to some extent. Studies across a range of speciesindicate that genes encoding individual isozymes may bedifferentially expressed in different tissues, at differentdevelopmental stages, and in response to different growthconditions (especially those that alter demand for NADPHor intermediates of the oxPPP for biosynthesis) [14,16,17,26]. In particular instances, such changes in geneexpression can be linked to changes in the accumulationof specific isoforms of the enzyme that have distinct kineticproperties. This is exemplified by recent studies onG6PDH. All forms of G6PDH are inhibited by high con-centrations of NADPH, and their activity in vivo is prob-ably modulated by the redox balance of the NADPH/NADP pool [4]. In addition, plastidic G6PDH is inacti-vated by a reversible dithioldisulphide interconversion oftwo highly conserved regulatory cysteine residues [32].This interconversion is ferredoxin/thioredoxin-dependentand can be mimicked using dithiothreitol, a non-physio-logical reductant [32].

Two classes of plastidic G6PDH have been distinguishedin higher plants. In potato, the transcripts of one plastidicisozyme (P1) are most prominent in green tissues and,although present in stolons and root tips in the light, arehardly detectable in most non-photosynthetic tissues[13]. In contrast, the second plastidic isozyme (P2) isexpressed throughout the plant, with highest steady-statetranscript levels in stems and roots [14,15]. Althoughboth plastidic activities are inhibited by increasedNADPH/NADP and inactivated by dithiothreitol, theP2 isozyme is strikingly less sensitive than the P1 enzymeto both forms of modulation [14,32]. These kinetic dif-ferences may reflect the differing metabolic requirementsof the cells in which they are expressed. In illuminatedchloroplasts, ferredoxin is reduced directly by compo-nents of Photosystem I and the oxPPP needs to beinactivated in the light so that potentially futile interac-tions with the Calvin cycle are avoided. In contrast, innon-photosynthetic tissues, there is a need to maintainflux through the plastidic oxPPP, even in the face of thehigh stromal NADPH/NADP levels that are required todrive ferredoxin-dependent reactions such as nitrite

reduction and glutamate assimilation [1]. The involve-ment of the P2 form of G6PDH in the provision ofNADPH for ferredoxin-dependent reactions is suggestedby an increase in the expression of genes encoding twoArabidopsis P2 isozymes (At1g24280 and At5g13110; seeTable 2) after transfer from a medium containing ammo-nium to one including nitrate [33]. Similar increases in theexpression of P2 genes upon exposure to nitrate have beenobserved in the roots of tobacco and tomato [15,34].Furthermore, recent studies on barley roots have identi-fied a plastidic isoform of G6PDH that has propertiessimilar to those of P2 and that is induced in response toexposure to ammonium or glutamate [3537]. Theseobservations are complemented by the demonstration thatboth nitrite reduction and glutamate synthesis from glu-tamine and 2-oxoglutarate by plastids isolated from barleyroot depend on a supply of glucose 6-phosphate. Thisresult confirms the ability of the P2 isozyme to supportflux through the plastidic oxPPP in spite of the highNADPH/NADP needed to provide reductant for nitritereduction and glutamate synthesis [37,38].

The existence of different isozymes of plastidic G6PDHcan be rationalised, but the significance of the multiplegenes that encode other oxPPP enzymes with no obviousregulatory properties is more difficult to explain. Onepossible explanation has been suggested by studies ontransgenic tobacco plants that have decreased TK activity[20]. The ability of darkened leaves from these plants tostimulate flux through the oxPPP in response to wound-ing is compromised. More extensive analysis of photo-synthetic carbon metabolism in these plants suggests thatthe reduced activity of TK does not directly influencemetabolism. Rather, metabolism is perturbed by theeffects on other metabolic enzymes of changes in theconcentrations of the immediate substrates and productsof TK required to maintain flux through the reactionscatalysed by this enzyme. The relatively large decrease inphenylpropanoid production in these transgenic plantsdemonstrates the sensitivity of other processes to changesin the levels of oxPPP intermediates. These considera-tions highlight the need to maintain appropriate levels ofmetabolites. In this context, a plausible role for isozymesmay be to allow changes in the steady-state levels ofmetabolic intermediates (owing to differences in therelative affinities of different isozymes for substratesand products) while maintaining the same net fluxthrough the pathway.

Measurement of flux through the oxidativepentose phosphate pathwayTesting the proposal outlined above and developing ourunderstanding of the regulation of the oxPPP requires theability to determine flux through the component reactionsof this pathway accurately. Traditionally, the activity ofthe oxPPP has been estimated by comparing the releaseof 14CO2 from specifically labelled glucose. (Usually



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

[6-14C]glucose, although other ratios are also informative[5].) Such analyses are often complicated, however, by therearrangement of label within metabolic intermediates inexchange reactions (catalysed by TK and TA) and by therelease of 14CO2 through other metabolic processes (see[5,39] for critical assessment of these problems). Analternative approach takes advantage of the recent demon-stration that plant cells metabolise applied gluconatethrough a route involving 6PGDH [40]. Consequently,release of 14CO2 from [1-

14C]gluconate provides a rapidand convenient method to monitor flux through theoxPPP. However, such estimates of flux are influencedby the rate of gluconate uptake and phosphorylation by thetissue, as well as by dilution of [1-14C]-6-phosphogluco-nate owing to the oxidation of endogenous (unlabelled)glucose 6-phosphate. This approach is therefore unlikelyto provide an accurate quantitative measure of oxPPP flux.More generally, a limitation of all techniques that arebased on measuring 14CO2 release is that they are unableto resolve flux through the oxPPP in the cytosol andplastids [41].

Recently, significant progress has been made in applyingsteady-state labelling techniques involving [13C]glucose,coupled with the detection of metabolic products usingeither nuclear magnetic resonance spectroscopy (NMR)or mass spectroscopy (MS), to resolve flux through thecentral pathways of metabolism in microbial systems[42]. The simplest applications concentrate on quanti-fying the proportion of hexose phosphate that is meta-bolised by the oxPPP relative to that metabolised byglycolysis [43]. However, the power of this approach liesin its potential to quantify exchange fluxes through read-ily reversible reactions as well as net flux through thepathways [42]. A wide range of strategies are beingdeveloped that employ either specifically labelled[13C]glucose or a small proportion of [U-13C6]glucoseand subsequent determination of the relative proportionsof specific groups of 13C-labelled isotopic isomers (knownas isotopomers) of particular metabolites to determineflux through complex networks of metabolic reactions[44,4547]. Although the use of these strategies inplants has been limited, there appear to be no majorpractical problems in applying similar methods to higherplant tissues [48].

A potential benefit of steady-state labelling approaches isthat they offer the possibility to resolve intracellularfluxes in situ. Specifically, the activity of the oxPPP inthe cytosol and plastids is reflected in the labellingpattern of cytosolic and plastidic pools of hexose phos-phate. These patterns can be monitored separately bymeasuring the abundance and distribution of label withinthe fructosyl and glucosyl moieties of sucrose (synthe-sised in the cytosol) and in the glucosyl residues of starch(formed in plastids) [41]. Using this rationale, analysis of

the redistribution of label in a carrot cell culture aftergrowth in either [1-13C]glucose or [1-13C]fructose hasshown that the oxPPP operates in both the cytosol andplastids of these cells [49]. More detailed studies in bothmaize root tips and tomato cell cultures have determinedthe distribution of flux through a metabolic network usingthe 13C-enrichment of specific carbons in carbohydratesand amino acids following labelling to isotopic near-steady-state with [1-13C]glucose [48,50]. In tomato,the proportion of carbon flowing through the oxPPPwas remarkably constant, and about 2226% of the glu-cose metabolised by the cells entered this pathwaythroughout the growth cycle of the cell culture [50].Surprisingly, the redistribution of label in both the maizeand tomato studies suggested that the oxidative steps ofthe oxPPP are active only in plastids, whereas TA is activein both the plastids and the cytosol. Such conclusionsdirectly contradict the emerging model for the subcellularorganisation of oxPPP that is based on the apparentdistribution of enzyme activities (particularly in maizeroots [19]). However, the [1-13C]glucose used in theselabelling studies [48,50] is not the most informativesubstrate for monitoring oxPPP activity as the isotope islost as 13CO2 during the initial oxidative steps [41]. Theresults obtained using this isotope should therefore beregarded as provisional until the study is repeated usingglucose labelled in a more appropriate position.

A potentially more sensitive strategy involving [1,2-13C2]-glucose has recently been applied to isolated embryosfrom developing Brassica napus seeds [51]. Thisapproach is based on the rationale that C-1 of [1,2-13C2]-glucose is lost by decarboxylation upon entering theoxPPP, producing [1-13C]pentose phosphates. The fruc-tose 6-phosphate regenerated from these [1-13C]pentosephosphateswillbeamixtureofdifferentsingle 13C-labelledisotopomers, whereas that formed directly from glucose6-phosphate will retain the 13C13C bond. The retentionof 13C2 double-labelling within about 90% of the acetylunits of C18:1 fatty acid indicates that the vast majority ofthe glucose used for lipid synthesis is converted to pyr-uvate and acetyl-CoA directly through glycolysis, ratherthan being metabolised by the oxPPP [51]. Labelling ofamino acids following the metabolism of [U-13C6]glucoseindicates that the precursors of pyruvate and acetyl-CoA(i.e. glycolytic intermediates) are subjected to extensiverearrangement through the non-oxidative section of theoxPPP [51]. Hence, the retention of 13C13C bondswithin fatty acids cannot be explained by a form ofmetabolic segregation in which the products of the oxPPPare prevented from entering the glycolytic pathway. Incombination, these considerations suggest that very littlecarbohydrate is metabolised by the complete oxPPP inisolated Brassica napus embryos and that, by implication,this pathway is unlikely to be the major source ofNADPH for fatty acid synthesis in these culturedembryos. However, more precise studies using labelling



strategies that are specifically designed to measure fluxthrough the oxPPP in the cytosol and plastids are requiredto confirm this conclusion.

Similar labelling strategies involving [U-13C6]glucosehave been applied to study metabolism in developingmaize kernels [52,53]. In these studies, analyses of theisotopomer composition of lipids, amino acids, triterpenesand glucosyl units of starch have been used to reconstructthe biosynthetic pathways involved in their formationin vivo. From these measurements, it is possible to inferthe labelling patterns of metabolic intermediates such aserythrose 4-phosphate, ribose 5-phosphate and glucose 6-phosphate. In principle, provided the requirement forlabelling to isotopic steady-state is met [41], such infor-mation could be used to calculate fluxes through thecentral pathways of carbohydrate oxidation. However,such analyses do not appear to have been attempted.

These recent studies demonstrate the potential for usingeither MS or NMR to obtain detailed information on therelative isotopomer abundance of a wide range of meta-bolites following labelling with 13C-substrates. Despitethe ability to make the relevant measurements, to date,the application of steady-state labelling strategies todetermine metabolic flux has been relatively under-exploited in plants. Such techniques need to be devel-oped if we are to obtain the estimates of flux that will benecessary to build a quantitative understanding of theoperation and regulation of the oxPPP.

ConclusionsConsidering the central importance of the oxPPP in meta-bolism, our understanding of its organisation and operationin plant cells is remarkably sketchy. In part, this may bedue to the diversity of processes with which it interacts,and the flexibility required to meet the varying demands ofsuch processes for reductant and biosynthetic precursors.However, prospects for improving our appreciation of theoxPPP are good. As more genomes are sequenced, we willdevelop a better understanding of the genetic capacity ofplant systems to catalyse the reactions involved in theoxPPP, and have access to a greater range of cognate genesfor comparative analysis [54]. Furthermore, the increasingavailability of whole-genome transcript profiles underdifferent physiological and developmental conditions willallow us to determine the relationships between theexpression of specific oxPPP isozymes, and to establishthe contribution of such isozymes to particular physiolo-gical processes [55,56]. Hopefully, such comparisons canbe complemented by the development of equally robusttechnologies for monitoring changes in the profiles ofa broad range of metabolites. This will enable a fullassessment of the possibly wide-ranging metabolic con-sequences of perturbations in the expression of specificoxPPP genes [57]. Finally, conceptual advances in theway in which groups of enzyme-catalysed reactions are

treated, coupled with improved techniques for measuringflux through the oxPPP, should provide the foundation fora more comprehensive, integrated understanding of thefunctioning of this sequence of reactions.

AcknowledgementsResearch in the authors laboratories is funded by the Biotechnology andBiological Sciences Research Council (UK) and DeutscheForschungsgemeinschaft (Germany).

References and recommended readingPapers of particular interest, published within the annual period ofreview, have been highlighted as:

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13C6]-glucose or [U-13C12]sucrose. These compositions are used to determinemetabolic flux through the major pathways of carbon metabolism duringembryo development. The work reveals that the cytosolic and plastidicpools of acetyl-CoA have different biogenic origins, and that the latter isderived almost exclusively from glycolysis. More broadly, the studyestablishes the utility of MS-based approaches for the analysis of13C-steady-state labelling in plants. This approach complements thatdeveloped in [52,53] in providing the data required to model fluxthrough metabolic networks.

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13C]acetate. Thedistribution of label is used to deduce the biosynthetic pathways that areactive in developing kernels, and to infer the labelling patterns of meta-bolic intermediates such as erythrose 4-phosphate and ribose 5-phos-phate. The general significance of this work is discussed in [53].

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13C12]sucrose.The distribution of label indicates that applied glucose is extensivelymetabolised via the glycolytic, gluconeogenic and pentose phosphatepathways before its incorporation into starch. In combination with earlierwork [52], this study demonstrates the feasibility of using NMR to obtaindetailed information on the isotopomer composition of metabolites. Such



information is required for the calculation of flux through the centralpathways of non-photosynthetic carbon metabolism in plants.

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The oxidative pentose phosphate pathway: structure and organisationIntroductionStructure of the oxidative pentose phosphate pathwayCompartmentation of the oxidative pentose phosphate pathwayInteractions between cytosolic and plastidic processesThe nature and roles of multiple isoforms of oxidative pentose phosphate pathway enzymesMeasurement of flux through the oxidative pentose phosphate pathwayConclusionsAcknowledgementsReferences and recommended reading

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