Higher plant NADP+-dependent isocitrate dehydrogenases, ammonium assimilation and NADPH production

Review

Higher plant NADP+-dependent isocitrate dehydrogenases,ammonium assimilation and NADPH production

Michael Hodges *, Valerie Flesch, Susana Gálvez, Evelyne Bismuth

Institut de Biotechnologie des Plantes (CNRS, UMR 8618), Bâtiment 630, Université Paris Sud-XI, 91405 Orsay cedex, France

Received 22 November 2002; accepted 20 December 2002

Abstract

The assimilation of ammonium into glutamate is mainly achieved by the GS/GOGAT pathway and requires carbon skeletons in the form of2-oxoglutarate. To date, the exact enzymatic origin of this organic acid for plant ammonium assimilation is unknown. NADP+-dependentisocitrate dehydrogenases can carry out this function and the recent efforts concentrated on evaluating the involvement of different isoforms,distinguished by their subcellular localisation, are analysed. Furthermore, a possible role for these enzymes in the production of NADPH forredox-regulated cell metabolism, such as the recycling of glutathione required in response to oxidative stress will be discussed.

© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: 2-oxoglutarate; Ammonium metabolism; Glutathione; Isoenzyme; NADP+-dependent isocitrate dehydrogenase

1. Ammonium production, the GS/GOGAT cycleand the origin of 2-oxoglutarate

Ammonium is the only reduced inorganic N form avail-able to plants for assimilation into organic compounds likeamino acids [26]. In higher plants, ammonium is generatedmainly by (a) the reduction of nitrate, carried out by thesequential action of cytosolic nitrate reductase (NR) andplastidial nitrite reductase (NiR), (b) the decarboxylation ofglycine to serine by the glycine decarboxylase (GDC), in themitochondria during photorespiration, (c) amino acid deami-nation and nucleic acid catabolism that are especially impor-tant during senescence, (d) the biosynthesis of phenylpro-

panoids and (e) by the nitrogenase activity ofRhizobiabacteria located in legume root nodules (Fig. 1). Ammoniumis assimilated by the concerted action of two enzymes,glutamine synthetase (GS) and glutamate synthase(GOGAT) that form the GS/GOGAT cycle [20]. In higherplant leaves, a specific GS isoform (GS2) and a ferredoxin-dependent GOGAT (Fd-GOGAT), located in mesophyll cellchloroplasts, are responsible for the major part of ammoniumassimilation. The GS/GOGAT pathway transfers ammonium

Abbreviations: AOX, alternative oxidase; DiT1, chloroplastic 2-oxo-glutarate/malate translocator; DiT2, chloroplastic glutamate/malate translo-cator; EST, expressed sequence tag; Fd, ferredoxin; G6PDH, glucose-6-phosphate dehydrogenase; GDC, glycine decarboxylase; GFP, greenfluorescent protein; GOGAT, glutamate synthase; GS1, cytosolic glutaminesynthetase; GS2, chloroplastic glutamine synthetase; ICDH1, cytosolicNADP+-dependent isocitrate dehydrogenase; ICDH2, chloroplasticNADP+-dependent isocitrate dehydrogenase; IDH, NAD+-dependent isoci-trate dehydrogenase; MM, molecular mass; NiR, nitrite reductase; NR,nitrate reductase; OAA, oxaloacetate; PEPc, PEP carboxylase; ROS, reac-tive oxygen species.

* Corresponding author.E-mail address: [email protected] (M. Hodges).

Fig. 1. A scheme showing the assimilation of ammonium by theGS/GOGAT pathway and the production of 2-oxoglutarate by isocitratedehydrogenase. The origin of ammonium in a plant cell is multiple, but it isalmost exclusively assimilated by a GS/GOGAT pathway. In higher plantleaves, this requires ATP, reduced ferredoxin (Fd red) and C-skeletons in theform of 2-oxoglutarate (2OG). This organic acid can be synthesised by anICDH, thus allowing for a net synthesis of glutamate and the production ofCO2 and NAD(P)H.

Plant Physiology and Biochemistry 41 (2003) 577–585

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© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.DOI: 10.1016/S0981-9428(03)00062-7

to glutamate to form glutamine, then the fixed N is trans-ferred to a carbon skeleton in the form of 2-oxoglutarate, thusproducing two molecules of glutamate (Fig. 1). Till date, theexact enzymatic origin of this key organic acid for plantammonium assimilation is still unknown. In the literature,isocitrate dehydrogenases and aspartate aminotransferasesare the two main candidates. Production of 2-oxoglutarate byan isocitrate dehydrogenase allows for net glutamate synthe-sis via the GS/GOGAT cycle, whereas an aspartate ami-notransferase origin leads to the synthesis of aspartate andrequires oxaloacetate (OAA) as a C-skeleton input [24].

Two different isocitrate dehydrogenase activities, depend-ing on cofactor specificity (NAD+ or NADP+), coexist in thecell and both catalyse the oxidative decarboxylation of isoci-trate to form 2-oxoglutarate (Fig. 2). Since this review ispublished in a special edition of Plant Physiology and Bio-chemistry dedicated to Professor Pierre Gadal, attention willbe given to the work carried out over the last two decades atthe UniversitéParis Sud-XI on NADP+-isocitrate dehydroge-nases (ICDH) and their possible role in glutamate synthesis.

2. ICDH enzymes and genes

NADP+-dependent isocitrate dehydrogenases belong to amultiisoenzymatic family, whose members have been lo-cated within the cytosol [5,12], plastids [3,10], mitochondria[14] and peroxisomes [6]. All are homodimeric enzymes, thesubunit MM being ~47 kDa. Their exact physiological func-tion(s) are still not known.

2.1. Abundance, properties and regulation

A comparison of ICDH isoenzyme patterns from 15 plantspecies showed that the cytosolic activity was always pre-dominant in leaf extracts [2]. Indeed, this form is responsiblefor 95% of the total ICDH activity in green tobacco leaves[10], and it is the predominant isoform in tomato fruit [8],

potato [7] and the only detectable form in pine cotyledons[35]. The abundance of cytosolic ICDH has allowed its puri-fication to homogeneity from several plant species[5,7,8,10,35]. On the other hand, the chloroplastic isoformhas been purified only from tobacco cell suspensions [10].This was achieved at Orsay in the early 1990s when it wasdiscovered that green, mixotrophic Nicotiana tabacum cellcultures were enriched in a second ICDH isoform. The pos-sibility to separate two distinct pea ICDH isoforms (ICDH1and ICDH2) by their elution properties, during ion exchangechromatography, had already been determined in 1989 byChen et al. [3]. However, the extremely low ICDH2 activityin pea leaves rendered its purification impractical. Whenapplied to the 45–80% ammonium sulphate precipitated frac-tion from tobacco cell suspensions, two distinct ICDH iso-forms were separated and purified to homogeneity by using aMatrex-Red affinity step and elution with NADP+ and isoci-trate [10]. This allowed for the first detailed comparison ofthe properties of two ICDH isoforms from the same plantspecies. Although the isoenzymes showed differences (e.g.migration on native polyacrylamide gels, elution from theDEAE-Sephacel column, antibody specificity [10]) linked tophysical properties, their kinetic parameters were similar(Table 1). The chloroplastic origin of tobacco ICDH2 wassuggested from the similar elution profile with respect to peachloroplastic ICDH, an absence in etiolated cells and itsappearance during the greening process [10]. This was con-firmed by immunolocalisation by using ICDH2-specific anti-bodies raised against purified native ICDH2 [10]. Till date,plant mitochondrial [7,38] and peroxisomal [6] ICDH iso-forms have not yet been purified to homogeneity althoughthey have been characterised from purified organelles(Table 1).

Unlike bacterial, yeast and animal ICDH enzymes, plantICDH activities are not regulated by allosteric effectors al-though they are inhibited by NADPH. The activity of theEscherichia coli ICDH is controlled by the reversible phos-phorylation of a serine residue; being inactive when phos-phorylated. Although plant ICDHs possess an equivalentresidue [12] there is no substantial evidence to show that they

Fig. 2. The ICDH enzymatic reaction consists of the oxidative decarboxy-lation of isocitrate (a C6 compound) to give the C5 organic acid,2-oxoglutarate. The substrate is an isocitrate-metal ion complex that isinitially oxidised, thus bringing about the reduction of NADP, and thendecarboxylated to 2-oxoglutarate, thus liberating a molecule of CO2. The Hand C atoms used for NAD(P)H and CO2 production are indicated by shadedboxes.

Table 1Substrate Km values for different NADP-dependent isocitrate dehydrogena-ses. * From purified organelles; ND, not determined

Origin Km (µM) ReferencesIsocitrate NADP Mg2+

CytosolicTobacco 41 7.5 130 [10]Tomato (green) 60 14 45 [8]Tomato (red) 80 16 50 [8]Pine 80 10 ND [35]ChloroplasticTobacco 28 7 120 [10]Mitochondrial*Potato tuber 10.7 5.1 ND [7]Peroxisomal*Young pea leaves 202 ND ND [6]Senescent pea leaves 20 ND ND [6]

578 M. Hodges et al. / Plant Physiology and Biochemistry 41 (2003) 577–585

are regulated by such a mechanism. Attempts to phosphory-late recombinant tobacco ICDH1 and ICDH2 with the E. coliICDH kinase/phosphatase were unsuccessful. However, re-combinant ICDH1 was inactive when the conserved serinewas changed to a negatively charged aspartate residue bysite-directed mutagenesis (M. Hodges et al., unpublishedresults). Furthermore, the activity of purified tobacco ICDH2was increased by 40% when treated with alkaline phos-phatase, while ICDH1 activity was unaffected [9].

2.2. Tobacco ICDH encoding cDNAs and theircorrespondence to different isoenzymes

The biochemical studies to elucidate the physiologicalroles of ICDH carried out by Pierre Gadal’s group at Orsayhad not been conclusive, because the relative ICDH activitiesdid not allow for their purification in sufficient quantitiesfrom the same plant material to carry out comparative stud-ies, and their properties were too similar to distinguish themin crude plant extracts. It was decided to circumvent theseproblems by using a molecular approach, but any advance inthis domain would require the assignment of isolated cDNAsto a specific isoenzyme. At the time, no plant ICDH cDNAswere available in the databases. It was decided to construct atobacco cell suspension cDNA library with the aim of isolat-ing different ICDH encoding cDNAs by using antibodiesraised against purified ICDH proteins. This approach was notfruitful and the first tobacco ICDH cDNAs were finallyisolated by using a soybean cDNA probe provided by Kahn[44]. During this time, three plant ICDH cDNAs isolatedfrom alfalfa [41], soybean [44] and potato [7] became avail-able in the literature. However, the tobacco ICDH cDNAswere the first to be unambiguously assigned to the givenisoenzymes. Previous results suggested that differences inamino acid sequence existed between ICDH1 and ICDH2,and that this could be used to assign a cDNA to an ICDHisoform. It was decided to partially sequence the purified

ICDH1 and ICDH2 proteins and compare them to the de-duced amino acid sequences of the tobacco ICDH cDNAs.This approach led to the assignment of cytosolic ICDH toone of the tobacco cDNAs [12], and this information wassubsequently used to help in identifying the potatocDNA [7].

After rescreening the tobacco cell suspension cDNA li-brary with a homologous probe a new ‘ full length’ cDNA wasobtained that encoded an ICDH sharing only 70–75% aminoacid identity with other known plant ICDH sequences. Fur-thermore, the amino acid sequence deduced from this cDNAhad an N-terminal extension with respect to cytosolic ICDH.The extension of this tobacco protein along with those oforthologous Arabidopsis and potato gene products are givenin Fig. 3A. Common features include two possible transla-tional start sites, a basic N-terminal region (typical of mito-chondrial presequences) and an overall amino acid composi-tion characteristic of chloroplastic transit peptides.Prediction of their subcellular localisation by using Predator(http://www.inra.fr/predotar) suggested that this could de-pend on the start codon (Fig. 3B; mitochondrial or chloro-plastic, first or second Met, respectively). This was interest-ing since dual targeting to mitochondria and chloroplastsexists in plants [36]. To address this possibility, tobacco andArabidopsis plants were transformed with constructs whereeither the entire targeting signal-encoding sequence of thetobacco cDNA or the sequence starting from the 2nd startcodon were inserted in front of a green fluorescent protein(GFP) gene. By optical and confocal microscopy, we showedthat a complete presequence addressed the GFP exclusivelyinto plant mitochondria [14]. However, the ‘second Met’presequence did not allow the GFP to be addressed to plas-tids, as predicted. Although plants contained GFP transcriptsand protein, no differences in green fluorescence were ob-served between transformed and control Arabidopsis plants.Western blot analyses of crude leaf extracts by using com-

Fig. 3. Analysis of the N-terminal sequences of several plant ICDHs. (A) Comparison of N-terminal sequences deduced from different plant ICDH-encodingcDNAs and (B) the predicted subcellular localisation of the tobacco ICDH (*). Putative START methionines are given in bold and N-terminal basic residues areunderlined. See Fig. 4 for the corresponding accession numbers. Mt, mitochondrial; Chl, chloroplastic.

579M. Hodges et al. / Plant Physiology and Biochemistry 41 (2003) 577–585

mercial antibodies raised against GFP indicated that theexpressed GFP was not processed and still contained theICDH presequence (Flesch et al., unpublished data).

Surprisingly, a comparison of trypsin-digested ICDH2peptide sequences with the deduced amino acid sequence ofthe second tobacco ICDH cDNA indicated that it encoded thechloroplastic isoform [9]. These data appear to be in contra-diction with the GFP experiments, and more work is requiredto determine what is happening in planta.

2.3. ICDH genes and their evolution

In spite of the presence of four plant ICDH isoforms, DNAdatabases indicate the existence of only three nuclear-encoded ICDH genes in higher plants. All Arabidopsis ICDHgenes show a conserved 15 exon/14 intron structure.At1g65930 encodes the cytosolic ICDH, since it shows thehighest identity to tobacco ICDH1 and the absence of anyN-terminal extension. Although no experimental data exist,At1g54340 should encode a peroxisomal ICDH, since thededuced protein sequence contains a C-terminal SRL tripep-tide (typical of a type I peroxisomal targeting (PST1) motif[17]). Therefore,At5g14590 must encode both the mitochon-drial and chloroplastic isoforms. Indeed, this gene is ortholo-gous to the second tobacco gene characterised at Orsay (seeabove). However, experimental data to validate the assump-tion that a single gene encodes two ICDH isoforms and themechanism(s), which regulate the partitioning between twoorganelles are lacking. That a single gene encodes more thanone ICDH isoform would not be exclusive to the plant king-dom. While in yeast there exists three distinct ICDH genes,all three isoforms are encoded by a single gene in the fila-mentous fungus Aspergillus nidulans [43], where two tran-scription start points allow for the production of a mitochon-drial form and a cytosolic/peroxisomal form. In animals, onegene encodes both the cytosolic and peroxisomal ICDHenzymes [15]. How the relative abundance of the cytosolicand peroxisomal ICDHs are controlled is not yet known.Interestingly, the potato and tobacco cytosolic ICDHs show aC-terminal AKA tripeptide that could be interpreted as aPST1 motif.

Protein sequence comparisons show that ICDH isoen-zymes are closely related and have been highly conservedduring evolution. A previous analysis of ICDH sequencesfrom Eukaryotes indicated that cytosolic and mitochondrialisoforms arose from independent gene duplications in ani-mals and fungi [34]. Fig. 4 shows a similar phylogeneticanalysis that includes recent plant ICDH protein sequences.It can be seen that all plant ICDHs are located in a separate‘plant’ group, and that plant mitochondrial ICDH is moreclosely related to other plant isoforms than to animal andfungal mitochondrial ICDH. The same holds true for theperoxisomal and cytosolic isoforms. This confirms the ideathat ICDH isoforms have arisen through independent dupli-cations of an ancestral ICDH gene within each kingdom.

2.4. Isoenzyme expression

By analysing the databases, information concerning theexpression of the different plant ICDH genes can be ob-tained. For instance, in the current MIPS Arabidopsis data-base (http://mips.gsf.de/proj/thal/db/), we find 49 expressedsequence tags (ESTs) for At1g65930, 13 ESTs forAt5g14590 and only 3 ESTs for At1g54340. This reflectswhat might be expected based on the known abundance ofeach isoform (see above). The origin of the different cDNAlibraries containing these ESTs can indicate the organ/tissuelocalisation of each ICDH. Cytosolic ICDH has ESTs inlibraries made from RNA extracted from above ground or-gans, roots, green siliques and from ‘stressed’ material like invitro etiolated liquid culture, NaCl-treated whole plants andin vitro roots infected with nematodes. The ESTs ofAt5g14590 have been found in root, green silique, flower budand inflorescence libraries, while ESTs for the peroxisomalgene have only been reported in root cDNA libraries (normal,nitrate-treated, and liquid culture). Such information are pre-cious, since it is not possible to measure each specific ICDHactivity in a given plant organ. Most experimental data con-cern total measurable ICDH activity. In alfalfa extracts, nod-ules contained the highest ICDH activity, followed by rootsand flower buds, stems and finally leaves [41]. Total ICDHactivity was highest in the roots of soybean and potato [7,44].However, specific 3’ -DNA probes have been used to distin-guish ICDH isoenzyme mRNA. In this way, it was found thattobacco ICDH1 was more abundant in stems, followed byflowers, roots and finally leaves (12). Furthermore, ICDH1transcripts were more abundant in tobacco and potato leaf

Fig. 4. A phylogenetic analysis of ICDH isoenzymes from bacteria, fungi,animals and plants. The dendogram was made using Clustal W. The acces-sion number for each sequence is given. Mt, mitochondrial; Px, peroxiso-mal.


veins when compared to leaf mesophyll cells [7,12]. Thislocalisation was confirmed at the protein level, by Westernblot analyses and immunolocalisation by using ICDH spe-cific antibodies [12]. Immunolocalisation also showed thepreferential accumulation of cytosolic ICDH in the epider-mis and stele parenchyma of nonmycorrhisal and ectomyc-orrhisal lateral roots of Eucalyptus [1].

3. Physiological roles of ICDH isoenzymes

3.1. Nitrogen metabolism

It has been proposed that mitochondrial IDH is the sourceof 2-oxoglutarate for ammonium assimilation [19,23]. How-ever, this remains a hypothesis since IDH appears to have anextremely low measurable in vitro activity and relativelyhigh substrate Km values (Km isocitrate 280–846 µM, KmNAD+ 150–800 µM, see [13]). Of course, the in vivo IDHactivity might be potentially higher than that measured invitro since this enzyme is unstable [28].

On the other hand, the observed low substrate Km valueshave been taken as an argument for the ICDH origin of2-oxoglutarate for plant ammonium assimilation. However,this role for a specific ICDH isoenzyme has not been deter-mined. Although ICDH is found in plastids, the major site ofammonium assimilation, its activity would be too low tosustain the required GOGAT activity. It was proposed byChen and Gadal [4] that cytosolic ICDH could play a majorrole in 2-oxoglutarate production for amino acid synthesis. Inthis hypothesis (see Fig. 5), citrate would leave the TCAcycle to be exported from the mitochondria to the cytosol viaa citrate transporter. Recently, a mitochondrial transportercapable of transporting both tri- and di-carboxylates has beendescribed for Arabidopsis and tobacco [37]. The idea thatcitrate is the major mitochondrial organic acid exported tothe cytosol was experimentally determined by using malate-fed intact mitochondria isolated from photosynthetically ac-tive tissues [16]. The citrate could either be stored in thevacuole or directly used to produce isocitrate via a cytosolicaconitase. The 2-oxoglutarate would be synthesised in the

Fig. 5. A scheme showing C and N flow between organelles and the possible involvement of I(C)DH in 2-oxoglutarate production for ammonium assimilation.In leaves, ammonium (from nitrate reduction and photorespiration) is assimilated in the chloroplasts by the GS/GOGAT pathway. For net Glu production,GOGAT requires C skeletons in the form of 2OG. Chen and Gadal [4] proposed that citrate is exported from the mitochondria to the cytosol and metabolised to2OG by the action of an aconitase and the cytosolic ICDH. Citrate can be exported by a di/tricarboxylate transporter (DTC [37]). This cytosolic 2OG istransported to the chloroplast by a 2OG/malate translocator (DiT1 [47]). Photorespiratory ammonium re-assimilation by the chloroplastic GS/GOGAT cycle iscoupled to peroxisome metabolism via the cycling of Glu and 2OG between these two organelles. This metabolite shuttling is controlled by a double translocatorsystem composed of DiT1 and DiT2. cPK, cytosolic pyruvate kinase; GDC, glycine decarboxylase; MDH, malate dehydrogenase; PDH, pyruvate dehydroge-nase; RuBP, ribulose bis phosphate.


cytosol by ICDH and imported into chloroplasts via a2-oxoglutarate transporter (DiT1) linked to a glutamatetransporter (DiT2), that require malate cycling as a counter-ion [47]. Till date, there is no direct evidence supporting thisproposition but many observations have been used to arguefor and against it.

3.1.1. For the role of cytosolic ICDH in ammoniumassimilation

For many, the relative abundance of cytosolic ICDH con-solidates the idea that the cytosol is the principal site for2-oxoglutarate production for ammonium assimilation. In-deed, a number of observations link cytosolic ICDH withamino acid metabolism. Tobacco NR mutants that accumu-lated nitrate and 2-oxoglutarate, showed changes in tran-script levels that were interpreted as a functional coordina-tion of ICDH1, GS2 and Fd-GOGAT due to the role ofICDH1 in 2-oxoglutarate production for nitrate assimilation[39]. Diurnal changes in transcript levels and activities ofseveral enzymes, including ICDH1 in wild type and NRtobacco mutants, have also been interpreted within theframework of nitrate assimilation. Cytosolic ICDH transcriptlevels and total ICDH activity were highest at the end of theday/beginning of the night and appeared to be coordinatedwith enzymes (pyruvate kinase and citrate synthase) involvedin C flow to produce 2-oxoglutarate. It was proposed that thisreflected the need to assimilate ammonium and glutamineaccumulated during the day [40]. In pine, a correlation at thetranscript level between ICDH and GS was found duringseed germination and between ICDH, GS and Fd-GOGATduring chloroplast biogenesis. However, this was not ob-served during the advanced stages of cotyledon development,while an inverse relationship was seen along the hypocotyls[35]. The authors concluded that ICDH had other roles, aswell as in GS-GOGAT functioning. A similar conclusion wasmade from ICDH1 expression studies during potato plantdevelopment and in detached potato leaves. The changes inICDH1 transcript levels and total ICDH activity broughtabout by light and mimicked by nitrate and sucrose, asdescribed for other N metabolism genes [42], were indicativeof a role in primary N metabolism [7]. Similarly, inducedsenescence led to an increase in ICDH activity and while leaftotal protein levels decreased, ICDH protein stayed stable[7]. These observations were taken as an indication thatcytosolic ICDH could play an important role in the cycling,redistribution and export of amino acids during senescence.A role in senescence was also suggested following a com-parison of ICDH activity and properties during the ripeningof tomato fruits. A two to threefold increase in ICDH1activity and protein was found that correlated with glutamateaccumulation [8]. The twofold increase in ICDH1 expression(mRNA, protein) and total ICDH activity during the mycor-rhisation of Eucalypt roots with Pisolithus tinctorius wereinterpreted as a role for cytosolic ICDH in glutamate produc-tion linked to glutamine export from the fungal symbiont [1].

3.1.2. Against the role of cytosolic ICDH in ammoniumassimilation

It is well established that most leaf ammonium assimila-tion is carried out by the GS2/Fd-GOGAT cycle located inmesophyll cells. Therefore, the observations showing cyto-solic ICDH to be preferentially located in leaf vascular tis-sues [7,12] are not in agreement with a role in ammoniumassimilation. Interestingly, vascular tissues also contain acytosolic GS1 isoform and an NADH-GOGAT. Therefore, itis possible that cytosolic ICDH might supply 2-oxoglutaratefor other plant metabolic functions involving glutamineand/or glutamate production, perhaps reflecting a potentialrole in amino acid transport events.

A further argument against the role of cytosolic ICDH inthe major ammonium assimilatory pathway comes from theanalysis of transgenic plants in which ICDH1 activity wassignificantly and specifically reduced. It was believed that ifcytosolic ICDH was involved in 2-oxoglutarate productionfor GS/GOGAT functioning, then plants exhibiting astrongly reduced activity should show a ‘photorespiratorymutant’ phenotype. In potato, retaining only 8% of their totalICDH activity, no deleterious affects on growth, flowering,tuber yield, photosynthesis, respiration and protein contentwere detected [22]. Such plants showed no noticeable modi-fications in C or N metabolism, and amino acid levels werenot altered. The only significant changes were a 2–2.5-foldincrease in citrate and isocitrate levels and a reduction inhexose levels in the dark. A lack of phenotype under normalgreenhouse conditions was also observed at Orsay with to-bacco plants antisensed by using the tobacco ICDH1 cDNAand exhibiting only 5% of the wild type total ICDH activity(Gálvez et al., unpublished data). Therefore, it appeared thatcytosolic ICDH was not essential for ‘normal’ GS/GOGATcycle functioning, and it was suggested that compensatorypathways perhaps involving IDH could have been induced[22]. Perhaps, the reassimilation of photorespiratory ammo-nium was not aimed at a net synthesis of glutamate and theconstant recycling of 2-oxoglutarate via a glutamate/2-oxoglutarate shuttle between the chloroplast and the peroxi-somes was sufficient (Fig. 5). Indeed, antisensed DiT1 to-bacco plants showed a ‘photorespiratory mutant’ phenotypeand accumulate glyoxylate as well as ammonium. This phe-notype was explained by a limitation in 2-oxoglutarate/glutamate exchange required for Fd-GOGAT activity andperoxisomal photorespiratory glycine synthesis [47].

Further indirect evidence against a role for cytosolicICDH in ammonium assimilation comes from work carriedout at Orsay and Versailles by Lancien et al. [23]. Whenwild-type tobacco plants were submitted to short-termN-starvation followed by nitrate or ammonium re-supply,ICDH1 mRNA levels were not significantly affected. On theother hand, IDH transcripts were induced by the addition ofeither nitrate or ammonium in both the roots and leaves of theN-starved tobacco plants. These modifications were coordi-nated with changes in the RNA levels of other TCA cyclegenes (citrate synthase and aconitase) and N metabolism


genes (NR, GS). These observations were used to argue foran IDH origin of the 2-oxoglutarate required for ammoniumassimilation [19,24].

3.2. NADPH production

As in yeast and animals, plant cytosolic NADPH is be-lieved to be mainly produced by two enzymes of the pentosephosphate pathway, glucose-6-phosphate dehydrogenase(G6PDH) and glyceraldehyde phosphate dehydrogenase, aswell as the malic enzyme (see [33,38]). It appears that such arole for cytosolic ICDH has been overlooked.

In the mitochondria, ICDH is the only major NADP+

reducing enzyme with a low Km for NADP+, although sev-eral NAD+-dependent enzymes (malic enzyme, malate dehy-drogenase, D1-pyrroline-5-carboxylate dehydrogenase andglutamate dehydrogenase) can use NADP+ [32]. The mainsinks for mitochondrial NADPH are reactive oxygen species(ROS) metabolism, NADPH dehydrogenases, folate turn-over and fatty acid biosynthesis [32,33]. Furthermore, alter-native oxidase (AOX) acts as an ‘overflow mechanism’ tohelp prevent over reduction of the electron transfer chain andthus minimises ROS production. Certain keto-acids, likepyruvate and 2-oxoglutarate, can stimulate directly AOXactivity presumably by allosteric mechanisms [29]. Further-more, AOX activity is increased after the reduction of adisulphide bridge that leads to the formation of a nonco-valently linked dimer. It has been found that the addition ofeither citrate or isocitrate to mitochondria enables the forma-tion of this active AOX form [45]. It has been proposed thatNADPH produced by ICDH is used either by a thioredoxin ora glutaredoxin based system to activate AOX [32]. Further-more, NADPH arising from mitochondrial and chloroplasticICDH activity might be important to regenerate glutathioneused in the ascorbate–glutathione cycle and glutathione per-oxidase system (Fig. 6). These two mechanisms are possiblyinvolved in ROS detoxification in both organelles as well asthe cytosol [31].

In peroxisomes, ICDH also serves as an NADPH produc-ing enzyme. This has been described in yeast, where peroxi-somal ICDH produces NADPH for the b oxidation of poly-unsaturated fatty acids carried out by 2,4 dienoyl-CoAreductase [18,46]. Recently, persoxisomal ICDH activity hasbeen investigated in pea during natural senescence. Al-though, senescence did not alter peroxisomal ICDH proteinlevels, the Km for isocitrate was reduced tenfold when com-pared to young leaves (Table 1). It was proposed that ICDHcould have a role in producing NADPH for the ascorbate-glutathione cycle in peroxisomes [6].

Interestingly, recent literature show that ICDH plays a rolein defence mechanisms against oxidative stress in both yeast[30] and mammalian [21,27] cells as ICDH derived NADPHis important for the regeneration of glutathione used in ROSscavenging. This has been shown using mouse cell linestransfected with either mitochondrial [21] or cytosolic/peroxisomal [27] ICDH cDNA. Cells with a reduced ICDHactivity showed increased ROS levels, DNA fragmentation

and lipid peroxidation, and damage to mitochondria becamemore apparent when compared to control cells [21,27]. Onthe other hand, over expression led to reduced ROS-inducibledamage. Although, the activities of other antioxidant en-zymes (including G6PDH) were unchanged, oxidised glu-tathione levels were higher in cells under expressing cytoso-lic ICDH [27]. In rat skeletal muscle, when compared withother NADPH producing enzymes, only the ICDH activitywas inducible with ageing and closely correlated to the mea-sured glutathione peroxidase levels [25]. Furthermore, ayeast double mutant for cytosolic ICDH and G6PDH showeda loss of viability due to the accumulation of intracellularoxidants arising from normal metabolism [30].

Therefore, it seems probable that ICDH may have a simi-lar NADPH-producing role to play in plant defence againstoxidative stress (Fig. 6) as previously suggested [11,19,31,32].

4. Conclusions and perspectives

This review describes our current knowledge concerningNADP+-dependent isocitrate dehydrogenases in higherplants. Arguments for and against an ICDH origin of2-oxoglutarate for plant ammonium assimilation have beendiscussed, and it can be seen that there is no clear answer. Itshould be noted that glutamate auxotrophy is found only in atriple yeast mutant lacking cytosolic ICDH, mitochondrialICDH and IDH [48]. The apparent contradictory observa-tions could reflect an idea that different pathways contributeto the production of 2-oxoglutarate for ammonium assimila-tion depending on specific physiological conditions. It ispossible that the main role for ICDH is not the production of2-oxoglutarate. This is highlighted by the recent work show-ing an important role for ICDH in producing NADPH re-

Fig. 6. Hypothetical roles for ICDH in the production of NADPH used inplant responses to oxidative stress.


quired by glutathione-dependent systems to combat oxida-tive stress in nonplant systems [21,27]. In the near future,such a role will probably be experimentally proven for higherplant ICDH. The identification and characterisation of mu-tants should help determine the role(s) for the different ICDHisoenzymes in plant metabolism. Indeed, a number of puta-tive mutants for each isoform are already available in theT-DNA tagged Arabidopsis mutant libraries. Several are cur-rently under study at Orsay, in an attempt to elucidate ICDHfunction. Furthermore, it will be important to determine theexact cellular localisation of each ICDH within a given plantorgan, to study how expression is modified during plantdevelopment and in response to environmental conditions,and to see whether there is colocalisation and coordinatedexpression with other gene products (involved, for instance,in N metabolism or ROS detoxification).

Acknowledgements

This paper is dedicated to Pierre Gadal, who was respon-sible for developing the work on ICDH at the UniversitéParisXI, Orsay. We thank him for his stimulating support over theyears.

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Higher plant NADP+-dependent isocitrate dehydrogenases, ammonium assimilation and NADPH production