Interaction of cytosolic and plastidic

nitrogen metabolism in plants

Andreas Weber1 and Ulf-Ingo Flugge

Universitat zu Koln, Lehrstuhl Botanik II, Gyrhofstr. 15, D-50931 Koln, Germany

Received 18 July 2001; Accepted 10 December 2001

Abstract

In angiosperms, the assimilation of ammonia result-ing from nitrate reduction and from photorespirationdepends on the operation of the plastidic GS/GOGATcycle. The precursor for ammonia assimilation, 2-oxoglutarate, is synthesized in the mitochondria andin the cytosol. It is imported into the plastid by a2-oxoglutarate/malate translocator (DiT1). In turn,the product of ammonia assimilation, glutamate, isexported from the plastids by a glutamate/malatetranslocator (DiT2). These transport processes linkplastidic and cytosolic nitrogen metabolism and areessential for plant metabolism. DiT1 was purifiedto homogeneity from spinach chloroplast envelopemembranes and identified as a protein with an appar-ent molecular mass of 45 kDa. Peptide sequenceswere obtained from the protein and the correspond-ing cDNA was cloned. The function of the DiT1protein and its substrate specificity were confirmedby expression of the cDNA in yeast cells and func-tional reconstitution of the recombinant protein intoliposomes. Recent advances in the molecular cloningof DiT2 and in the analysis of the in vivo function ofDiT1 by antisense repression in transgenic tobaccoplants will be discussed. In non-green tissues,the reducing equivalents required for glutamateformation by NADH-GOGAT are supplied by theoxidative pentose phosphate pathway. Glucose6-phosphate, the immediate precursor of the oxid-ative pentose phosphate pathway is generated in thecytosol and imported into the plastids by the plastidicglucose 6-phosphate/phosphate translocator.

Key words: Glucose 6-phosphateuphosphate translocator,glutamateumalate translocator, glutamineuglutamate trans-locator, GSuGOGAT, photorespiration, 2-oxoglutarateumalatetranslocator.

Subcellular localization of the main ammoniaassimilation pathway in plants

In green leaves of most C3-type angiosperm plant species,ammonia is assimilated into nitrogen compounds viathe plastidic glutamine synthetaseuglutamate synthase(GSuGOGAT) cycle (Miflin and Lea, 1980) with the plas-tidic GS2 being the predominant isozyme in these tissues(McNally et al., 1983). The plastidic GS2uGOGAT cyclehas to cope with ammonia produced by nitrateunitritereduction and, to a larger extent, with ammonia releasedin the photorespiratory cycle. In several achlorophyllousplant parasites, however, cytosolic GS1 is the onlydetectable isozyme and in several C4-type and CAMplants, the contribution of GS1 to total GS-activity isabout 3580% (McNally et al., 1983).

The predominant role of the GSuGOGAT-system forthe reassimilation of ammonia released during theregeneration of 3-phosphoglycerate in the photorespir-atory pathway was further established by the isolation ofmutants defective in the ferredoxin-dependent glutamatesynthase (Fd-GOGAT) and in GS2. An A. thalianamutant that is defective in Fd-GOGAT activity has beendescribed (Somerville and Ogren, 1980). This mutantis unable to grow under ambient CO2 conditions and isonly viable under high CO2 conditions that suppress

1 To whom correspondence should be addressed. Fax: q49221 4705039. E-mail: andr.weber@uni-koeln.deAbbreviations: GS, glutamine synthetase; GOGAT, glutamine, 2-oxoglutarate aminotransferase; CAM, crassulacean acid metabolism; Fd-GOGAT,

ferredoxin-dependent GOGAT; GUS, b-glucuronidase; DiT1, dicarboxylate translocator 1 (2-oxoglutarateumalate-translocator); DiT2, dicarboxylatetrasnlocator 2 (glutamateumalate-translocator); OPPP, oxidative pentose phosphate pathway; GPT, glucose 6-phosphateuphosphate-translocator;Glc6P, glucose 6-phosphate; NAD(P)H-GOGAT, NAD(P)H-dependent GOGAT; CitT, citrate transporter.

Journal of Experimental Botany, Vol. 53, No. 370,Inorganic Nitrogen Assimilation Special Issue, pp. 865874, April 2002

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photorespiration. Barley mutants defective in GS2have been described previously (Blackwell et al., 1987;Wallsgrove et al., 1987). These mutants developednormally when grown under conditions that minimizedphotorespiration. In air, however, they were unable togrow. The studies using mutants of Arabidopsis andbarley conclusively demonstrated that both GS2 andFd-GOGAT are absolutely necessary for the reassim-ilation of ammonia released during photorespiration inhigher plants.

Fusion of the reporter gene GUS to the promoterregions of the GS1 and GS2 genes from pea and sub-sequent transformation of the reporter constructs intotobacco plants revealed a cell-specific expression patternof the GS2: :GUS fusion in photosynthetic cells, whereasexpression of the GS1: :GUS construct was restrictedto phloem cells. Immunocytochemical studies alsodemonstrated that GS2 was localized to the plastids ofmesophyll cells whereas GS1 could be detected mainly inthe cytosol of phloem companion cells (Carvalho et al.,1992). Since GS1 is also induced by wounding and duringsenescence, differential roles for GS1 (synthesis of aminoacids for long-distance transport) and GS2 (synthesis ofamino acids for protein biosynthesis) have been suggested(Masclaux et al., 2000).

Because the precursor for ammonia assimilation is syn-thesized in the cytosol anduor in the mitochondria (fora recent review on the synthesis of 2-oxoglutarate, seeLancien et al., 2000), plastids rely on a transport systemfor the import of 2-oxoglutarate into the plastids and forthe export of the product of ammonia assimilation,glutamate, from the plastids into the cytosol.

Physiological characterization of plastidicdicarboxylate translocators in C3-plants

Lehner and Heldt first reported on the characterizationof plastidic dicarboxylate transporters in spinachchloroplasts (Lehner and Heldt, 1978). As demonstratedby silicone-oil-filtration-centrifugation, these proteinscatalyse the uptake of 14C-labelled dicarboxylic acids(e.g. 2-oxoglutarate, malate, succinate, aspartate, andglutamate) in an antiport manner. The uptake showedsaturation kinetics and was stimulated by light. Themeasurements of the Km and Ki values for the individualdicarboxylates suggested the existence of more than onetranslocator with overlapping substrate specificities. Wooand Osmond analysed the ammonia and 2-oxoglutarate-dependent O2 evolution in isolated chloroplasts and founda strong stimulation of the (ammonia, 2-oxoglutarate)-dependent O2 evolution by dicarboxylates (e.g. malate,succinate and fumarate) (Woo and Osmond, 1982). Thecalculated maximum rates of ammonia assimilation inisolated chloroplasts were found to be sufficient to cope

with the reassimilation of ammonia generated in thephotorespiratory pathway in vivo. Using a reconstitutedspinach chloroplast system, Anderson and Osmondfound no significant effect of malate on the (glutamine,2-oxoglutarate)-dependent O2 evolution, thereby sup-porting the suggestion that the observed stimulationof (ammonia, 2-oxoglutarate)-dependent O2 evolution ofisolated chloroplasts by malate may be due to a pro-motion of 2-oxoglutarate uptake into chloroplasts bymalate (Anderson and Walker, 1983). However, Lehnerand Heldt had shown that the uptake of 2-oxoglutarateis competitively inhibited by malate (Lehner and Heldt,1978). Thus, the observed malate-stimulation of ammoniaassimilation by isolated chloroplasts would require anentirely newmechanism for the transport of 2-oxoglutarateacross the chloroplast envelope membrane.

The two-translocator model for the transportof dicarboxylic acids and glutamate in plastids

The mechanism of 2-oxoglutarate uptake into chloro-plasts has been investigated further (Woo et al., 1987a;Flugge et al., 1988) and it was shown that the chloroplastenvelope membrane contains at least two distinctdicarboxylate transporters with partially overlapping sub-strate specificities, the 2-oxoglutarateumalate-translocator,(DiT1) and the glutamateumalate-translocator (DiT2).DiT1 was demonstrated to be specific for dicarboxyl-ates (malate, succinate, fumarate, glutarate, and2-oxoglutarate) whereas DiT2, in addition to the sub-strates transported by DiT1, also accepts the amino acidsglutamate and aspartate. A two-translocator model forthe transport of 2-oxoglutarate and glutamate wasproposed (Fig. 1A) that explained the stimulation of2-oxoglutarate transport into isolated chloroplasts and thestimulation of the (ammonia, 2-oxoglutarate)-dependentoxygen evolution in isolated chloroplasts by malate(Woo and Osmond, 1982). According to this model,2-oxoglutarate is imported into the plastid in counter-exchange with malate by DiT1 and is subsequentlyconverted to glutamate by GSuGOGAT. Glutamate isthen exported to the cytosol by DiT2, again in counter-exchange with malate. In summary, this results in acounter-exchange of 2-oxoglutarate with glutamate with-out a net malate transport. By inhibiting DiT2 withhigh concentrations of glutamate, the kinetic propertiesof DiT1 could be analysed in more detail (Yu and Woo,1992a, b). The Km for the uptake of malate into isolatedspinach chloroplasts was found to be 2.7 mM and0.6 mM for DiT1 and DiT2, respectively. The differentaffinities of DiT1 and DiT2 for malate and the othersubstrates provide the kinetic basis of the push and pullmechanism for the transport of dicarboxylates thatwas proposed by the two-translocator model. In potatoleaves, the concentration of malate at the end of the light

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period was found to be 3.2 mM in the cytosol and2.4 mM in the stroma. The concentrations of glutamatein the cytosol and the stroma were determined to be41 mM and 26.4 mM, respectively (Leidreiter et al.,1995). Consequently, the export of glutamate from theplastid stroma to the cytosol occurs against a concentra-tion gradient and is driven by the inverse concentrationgradient for malate. For 2-oxoglutarate, no data on thesubcellular concentration are available. However, it canbe speculated that the concentration of 2-oxoglutaratein the cytosol is in the low micromolar range and evenlower in the stroma (because GSuGOGAT represents astrong sink for 2-oxoglutarate). Therefore, the import of2-oxoglutarate is driven by a concentration gradienttowards the stroma and this, in turn, drives the exportof malate to the cytosol against a concentration gradient.The question arises, why two transporters need to workin concert. Considering the high concentration of glut-amate in the cytosol in comparison with the low concen-tration of 2-oxoglutarate (at least three orders ofmagnitude difference in concentration), it is obvious thatthe transport of 2-oxoglutarate by one single transporterwith affinities for both glutamate and 2-oxoglutarate,would always be out-competed by glutamate and notransport of 2-oxoglutarate would occur. However, thepresence of two transporters with overlapping sub-strate specificities allows the import of 2-oxoglutarateinto the plastid stroma by DiT1 even in the presence ofhigh cytosolic concentrations of glutamate.

The plastidic glutamine translocator andthe three-translocator model

Using a double silicone layer filtering centrifugationsystem, Yu and Woo characterized a specific glutamine

translocator from spinach and oat chloroplasts (Yuand Woo, 1988). The transport of glutamine into thechloroplast was greatly affected by the endogenousdicarboxylate pools of plastids. In glutamine-preloadedchloroplasts, the uptake of glutamine was competit-ively inhibited by glutamate only, but not by otherdicarboxylates that are substrates of DiT2, thus demon-strating that the glutamine transport is mediated by atranslocator different from DiT2. The combined action ofthis putative glutamineuglutamate translocator and bothDiT1 and DiT2 opens the way to export glutamine inexchange for 2-oxoglutarate, with no net glutamate andmalate transport, thereby providing the cytosol withglutamine as a precursor, for example, for biosynthesis ofasparagine (Fig. 1B). The coupling of glutamine exportfrom plastids to the availability of glutamate in thecytosol may act as a control mechanism circumventingglutamine-depletion of the GSuGOGAT-cycle. In sum-mary, it is likely that, in vivo, the transport of dicarb-oxylates, glutamate and glutamine occurs according tothe three-translocator model.

Ammonia assimilation and dicarboxylatetransport in gymnosperms

By contrast to the situation in C3-type angiosperms(McNally et al., 1983), there is increasing evidence thatat least in some gymnosperm species GS1 is the dominantisoform of GS and therefore most of the GS activityis found in the cytosol and not in the plastid stroma(Canton et al., 1993). An immunolocalization study ofGS1 showed that GS1 is localized not only in phloemcells of pine seedlings, but also in mesophyll cells (Garca-Gutierrez et al., 1998). A detailed analysis of GS1

Fig. 1. The two-translocator model for transport of dicarboxylic acids and glutamate (A) and the three-translocator model for the transportof dicarboxylates, glutamate and glutamine (B) into chloroplasts (1, DiT1; 2, DiT2; 3, glutamineuglutamate translocator). Cycling of substratesbetween chloroplast stroma and the cytosol is indicated by broken lines whereas net fluxes are indicated by solid lines.

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expression levels in Scots pine seedlings (Canton et al.,1999) showed that GS1 mRNA levels increased upontransfer of seedlings into the light. These findingssuggested that glutamine synthesis in pine seedlings byglutamine synthetase mainly takes place in the cytosolrather than in the plastid stroma. Since the glutaminerequired for the de novo synthesis of glutamate byplastidic Fd-GOGAT is supplied by cytosolic GS1, anactive glutamateuglutamine exchange able to cope withthe flux of ammonia from nitrate reduction and photo-respiration has to be postulated. Two possible models forthe transport of 2-oxoglutarate, glutamate and glutaminecan be proposed.

Possible models for the transport of 2-oxoglutarate,glutamate and glutamine into gymnosperm plastids

One possible mode of transport could involve DiT2 andthe glutamine translocator working in concert as a doubletranslocator system for 2-oxoglutarate, glutamine andglutamate (Fig. 2A). DiT2 would import 2-oxoglutarateinto the plastid stroma in counter-exchange with anexport of glutamate. Glutamate is converted to glutaminein the cytosol by GS1 and imported into the plastid ina counter-exchange for glutamate. In summary, thiswould result in a net flux of 2-oxoglutarate to glutamatewhereas, in parallel, glutamine and glutamate wouldcycle on DiT2 and the glutamine translocator, respect-ively. In this scenario, the activity of DiT1 would notbe required.

The other possible model would combine DiT1, DiT2and the glutamine translocator to a three-translocator

model (Fig. 2B). In this model, glutamine formed byGS1 in the cytosol is imported into the chloroplast ina 1 : 1 counter-exchange with glutamate. The import of2-oxoglutarate by DiT1 and the export of the secondmolecule of glutamate resulting from Fd-GOGAT actionby DiT2 is coupled to malate counter-exchange in thecascade-like push-and-pull mechanism already describedfor the double translocator system.

In order to check which of the above-described modelsdescribes best the in vivo situation, it will be necessaryto analyse the expression patterns of DiT1 and DiT2in gymnosperms, such as pine seedlings, as well as thetransport characteristics of pine seedling chloroplasts. Itmight be speculated that the three-translocator modelbest describes the situation in vivo.

Role of the plastidic glucose6-phosphate/phosphate translocatorduring ammonia assimilation

Non-green plastids of heterotrophic tissues rely onthe provision of carbohydrates from the cytosol. Theimported carbon is used as a substrate for biosyntheticpathways, for example, for the biosynthesis of starch orfatty acids. Carbon can also be used to drive the oxidativepentose phosphate pathway (OPPP), which is the majorsource of reducing power required for the reduction ofnitrite and the biosynthesis of fatty acids and of aminoacids. The immediate substrate for the OPPP is glucose6-phosphate (Glc6P) that is imported into the plastidsby the Glc6Puphosphate translocator (GPT; Kammerer

Fig. 2. Putative models for the transport processes in pine seedlings during ammonia assimilation by combined action of cytosolic GS1 and plastidicFd-GOGAT. (A) Two-translocator model involving DiT2 (2) and the glutamineuglutamate translocator (3). (B) Three-translocator model involvingDiT1 (1), DiT2 (2) and the glutamineuglutamate translocator (3). Cycling of substrates between chloroplast stroma and the cytosol is indicated bybroken lines whereas net fluxes are indicated by solid lines.

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et al., 1998). The GPT thus links reactions located inthe cytosol, the conversion of sucrose to hexoses andGlc6P, with the metabolism inside the plastid. Contraryto photosynthetic tissues, the main source of ammoniumis not photorespiration but nitrate assimilation. Previousstudies have shown that nitrite reduction and thesubsequent synthesis of glutamate via GSuNAD(P)H-GOGAT is strongly dependent on the provision of theplastids with Glc6P and a functional OPPP that deliversreductants for assimilatory processes in non-greenplastids (Bowsher et al., 1989, 1992; Fig. 3).

Purification and identification of the plastidic2-oxoglutarate/malate translocator

Menzlaff and Flugge reported on the identification ofthe plastidic 2-oxoglutarateumalate translocator as a com-ponent of the plastid envelope membrane (Menzlaff andFlugge, 1993). Detergent solubilized envelope membraneproteins were separated by two successive column chro-matography steps and the activity of DiT1 was monitoredby reconstitution of column fractions into liposomesfollowed by transport measurements. DiT1 transportactivity could be assigned to a protein with an apparentmolecular mass of 45 kDa.

Purified, reconstituted DiT1 accepted malate, suc-cinate, tartrate, glutarate, 2-oxoglutarate, and fumarate,but not the amino acids aspartate and glutamate, ascounter-substrates for the exchange of malate. Thus, thesubstrate specificity of purified, reconstituted DiT1 was inaccordance with the substrate specificity determined forDiT1 in isolated spinach chloroplasts (Woo et al., 1987a, b;Flugge et al., 1988) and therefore in accordance with thefunction of DiT1 proposed in the two-translocator modelfor the transport of dicarboxylates.

cDNA cloning, expression in yeast cells andfunctional characterization of DiT1

The identification of DiT1 as a component of the chloro-plast envelope membrane (Menzlaff and Flugge, 1993)was instrumental for cloning of the corresponding cDNA.Weber et al. developed a preparative purification pro-cedure for the 45 kDa protein based on its solubility inchloroform (Weber et al., 1995). The protein could bepurified by preparative SDSPAGE in amounts sufficientfor subsequent cleavage with the endoproteinase Lys-C.This approach yielded two peptide sequences of DiT1that were used to synthesize corresponding oligonucle-otides and subsequently to screen a spinach leaf cDNAlibrary.

A full-length cDNA was obtained, encoding a highlyhydrophobic protein of 569 amino acids with a predictedmolecular mass of 60.3 kDa and a calculated isoelectricpoint of 9.6. A plastid targeting signal sequence of 90100amino acid residues in length was predicted from theprotein sequence. The in vitro-translated protein wasfound to be imported into intact isolated chloroplasts andprocessed to its mature form.

By contrast to other known translocator proteins ofthe plastid envelope membrane at that time (e.g. the triosephosphateuphosphate translocator; Flugge et al., 1989)that were found to form dimers of two identical sub-units with six a-helices each, the DiT1 protein possesses12 highly hydrophobic membrane-spanning segments.Such a 12 transmembrane helix topology is found inmany bacterial transporters or transporters of the plasmamembrane. Later, a similar topology was also found forother plastidic transporters, for example, the adenylatetranslocator (Neuhaus et al., 1997) and the hexose trans-locator (Weber et al., 2000). Currently, it seems morelikely that the phosphate translocator family is ratherthe exception than the rule in terms of its transmembranetopology (for a recent review on plastid transporterproteins see Neuhaus and Wagner, 2000).

Surprisingly, no significant homologies were foundto dicarboxylate transporter proteins from mitochondria(Walker and Runswick, 1993; Taniguchi and Sugiyama,1996) although both transporters share similar substratespecificities and functions.

In order to confirm the identity of the DiT1 cDNAwith the proposed function, the cDNA was expressed inyeast cells. Membrane proteins from yeast cells harbour-ing the transgene were isolated and reconstituted intoliposomes. The malate uptake into liposomes contain-ing the recombinant protein was found to exceed thetransport activity of control liposomes by at least twoorders of magnitude. It could also be shown that therecombinant protein possessed substrate specificitiesthat were identical to those of the authentic protein(Weber et al., 1995).

Fig. 3. The role of the plastidic glucose 6-phosphateuphosphate trans-locator (GPT, 4) in supplying the plastidic oxidative pentose phosphatepathway with the precursor glucose 6-phosphate.

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Related sequences to DiT1 in plantsand bacteria

Related sequences in plants

A sequence homology search of the GenBank database(Viridiplantae subsection) was performed to identify genesthat are homologous to DiT1 gene from spinach. Thesehomologues can be grouped into two related, but distinctclasses. The first group contains sequences from A.thaliana, spinach, corn, and tobacco and is closely related

to spinach DiT1 (7085% identity at the protein level).The second group (DiT2-group), containing sequencesfrom A. thaliana, Sorghum bicolor, Flaveria bidentis,tobacco, and spinach shares only about 4555% identityto the spinach DiT1 protein, but 7080% identical aminoacid residues with each other (see phylogenetic tree,Fig. 4). The A. thaliana genome contains three geneshomologous to spinach DiT1, all located on chromo-some 5. The DiT1 homologue is a single copy genewhereas the two other genes fall into the DiT2-group

Fig. 4. Unrooted phylogenetic tree of the dicarboxylate transporter family in plants and eubacteria. The first two letters of the acronyms indicate thespecies (At, Arabidopsis thaliana; Bs, Bacillus subtilis; Cm, Chlamydia muridarum; Cp, Chlamydia pneumoniae; Ct, Chlamydia trachomatis; Ec,Escherichia coli; Fb, Flaveria bidentis; Hi, Haemophilus influenzae; Nt, Nicotiana tabacum; Sa, Staphylococcus aureus; So, Spinacia oleracea; Zm, Zeamays). DiT, dicarboxylate transporter; TST, tartrate succinate transporter; CST, citrate succinate transporter.

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of sequences. The A. thaliana DiT2 genes are arrangedin tandem with a head to tail orientation. No othersignificant homologies were found to other plant proteins.

A cDNA representing DiT2 from spinach has recentlybeen isolated and this cDNA was expressed in yeast.Preliminary experiments showed that the reconstitutedrecombinant DiT2 protein has a substrate specificitycomparable to DiT1 but, in addition to the substratestransported by DiT1, accepts glutamate and aspartate ascounter-substrates for malate. It is therefore very likelythat the DiT2 group of genes encodes the general dicarb-oxylate translocator (glutamateumalate-translocator)of the plastidic double-translocator system for dicarb-oxylates (P Renne, UI Flugge, A Weber, unpublishedresults).

The E. coli citrate transporter CitT is related to DiT1

Most Escherichia coli strains are unable to grow on citrateunder oxic conditions. Under anaerobic conditions, how-ever, E. coli is able to grow on citrate in the presence of anoxidizable co-substrate (e.g. glucose or glycerol). Citrateis taken up into the cell and split into acetate andoxaloacetate by citrate lyase (Lutgens and Gottschalk,1980). Oxaloacetate is then reduced to succinate by con-certed reactions of malate dehydrogenase, fumarase andfumarate reductase. The required reducing equivalentsare generated by the oxidation of the co-substrate(e.g. glucose or glycerol). Two enzymes are specificallyrequired for the fermentation of the tricarboxylic acid, acitrate uptake system and citrate lyase. Pos et al. reportedon an open reading frame (designated CitT ) located at13.90 min on the E. coli chromosome between the RNA(RNAse I) and the citrate lyase genes that encodes acitrate carrier (Pos et al., 1998). E. coli cells transformedwith a plasmid expressing CitT were able to grow oncitrate under aerobic conditions. It could be also shownthat CitT catalyses either an homologous exchange of cit-rate or a heterologous exchange with succinate, fumarate,or tartrate (Pos et al., 1998). Since succinate is the end-product of citrate fermentation in E.coli, it is likely thatCitT functions in vivo as a citrateusuccinate antiporter.CitT is a highly hydrophobic protein (487 amino acids,53.1 kDa) with 12 putative transmembrane helices. It isrelated to the 2-oxoglutarateumalate translocator fromspinach chloroplasts (SoDiT) with the amino acid identityto DiT1 from spinach being about 34%.

Proteins related to DiT1 in other bacterial species

Related proteins are also encoded by the genomes ofother bacteria like Heamophilus influenzae, Helicobacterpylori, Staphylococcus aureus, Chlamydia trachomatis,Chlamydia pneumoniae, and Bacillus subtilis. The proteinfrom B. subtilis shows the highest amino acid identity(53%) followed by the proteins from C. trachomatis and

C. pneumoniae with about 48% amino acid identities.DiT2 displays a somewhat lower identity with thebacterial transporters, suggesting that DiT1 is moreclosely related to the common ancestor of the bacterialand plant transporters while DiT2 might have evolvedfrom DiT1 during the evolution of plants. The primaryfunction of DiT1 in early plant cells might have beenthe indirect exchange of redox equivalents between theendosymbiont and the host cell. For example, inChlamydia, 2-oxoglutarate is taken up from the host cellby a DiT1 homologue in exchange for malate. Theoxidation of 2-oxoglutarate to malate leads to thegeneration of reductants and of energy in the form ofGTP. Since no significant homologies are found toproteins from cyanobacteria, it is tempting to speculatethat DiT1 might trace back to the genome of the hostcell of the endosymbiontic event that eventually led tothe evolution of a eukaryotic plant cell. No significanthomologies with proteins from fungi or animals werefound. Together with the bacterial transporters, DiT1 andDiT2 form a class of plant and eubacterial transportersinvolved in the transport of di- and tricarboxylic acids(Fig. 4).

In vivo function of plastidic dicarboxylatetranslocators

Somerville and Ogren isolated an EMS-mutagenizedphotorespiratory mutant of A. thaliana (Somerville andOgren, 1983) that was shown to be defective in theplastidic glutamateumalate-translocator (dct, Somervilleand Somerville, 1985). A corresponding mutant frombarley has also been described (Wallsgrove et al., 1986).

The A. thaliana dicarboxylate translocator mutant dct

The A. thaliana mutant was shown to be not viable underambient CO2-conditions, but grew like the wild typeunder elevated CO2 concentrations that suppress photo-respiration. The phenotype was attributed to the inabilityof the mutant to carry out a functional photorespiratorypathway. A defect in a plastidic dicarboxylate trans-locator would lead to a deficiency in the reassimilation ofammonia resulting from glycine-decarboxylase activityeither because of a lack in the supply of the GSuGOGATcycle with 2-oxoglutarate, the precursor for ammoniaassimilation, or a decreased export capacity for glutamate(or both). In addition, either case would lead to a short-age in glutamate supply for the formation of glycine fromglyoxylate in the peroxisomes, leading to a build-upof toxic photorespiratory intermediates in the mutant.Furthermore, a block in photorespiratory cyclingcauses a depletion of Calvin cycle intermediates. Fromthe physiological characterization, it is difficult to decidewhether the mutant is deficient in the activity of either

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DiT1 or DiT2. Since the complete sequence of theArabidopsis genome is now available, it should be possibleto search for mutations in the three genes related tospinach DiT1 in the Arabidopsis genome in order toidentify the gene defective in dct.

The barley dicarboxylate transport mutant RPr 79/2

Wallsgrove et al. isolated a barley mutant (RPr 79u2) thatis defective in chloroplast dicarboxylate transport(Wallsgrove et al., 1986). This mutant resembles in manyaspects the Arabidopsis mutant dct, including the labellingpattern of serine, glycine, glycerate, and malate followingfixation of 14CO2, the rapid rise in glutamine content andthe fall in glutamate content in leaves upon transfer fromnon-photorespiratory conditions to air. In contrast todct, the barley mutant does not die in ambient air unlessthe temperature and irradiance are high. The survivalof RPr 79u2 in ambient air might be due to the build-up of2-oxoglutarate levels (up to 10-fold in comparison to thewild type) that might overcome the deficiency in chloro-plast dicarboxylate transport (probably in DiT1) byallowing DiT2 to operate as a 2-oxoglutarateuglutamateexchanger under these conditions.

Knockout lines for plastidic dicarboxylate translocators

The availability of large collections of T-DNA taggedA. thaliana insertion mutants (Krysan et al., 1999;Sussman et al., 2000) enabled a PCR-based reversegenetic screen to be set up to identify mutants that aredefective in either of the three DiT-related genes. Ahomozygous knockout line for A. thaliana DiT2.2 wasisolated (U Kolukisaoglu, B Schulz, A Weber, unpub-lished results) but, surprisingly, the mutant does notdisplay an obvious phenotype. This might be due to aredundancy in gene function with DiT2.1 or to a tissueor cell specific expression pattern of the DiT2.2 genethat does not lead to a visible phenotype in the aerialparts of the plant. As judged from the availability ofA. thaliana ESTs for DiT2.1 and DiT2.2, it can bespeculated that DiT2.1 is expressed at higher levels andmore ubiquitous as compared to DiT2.2. A search for

insertional mutants in DiT1 and DiT2.1 is currentlyunderway.

Antisense repression of DiT1 in transgenictobacco plants

Antisense repression of gene expression has turned outto be a successful approach to study the in vivo func-tion of plant genes. Transgenic tobacco plants showingantisense repression of DiT1 gene expression have beengenerated (AWeber, J Schneidereit, UI Flugge, W Kaiser,unpublished results). These tobacco plants show a distinctphenotype, characterized by bleached veins, altered leafshape, stunted growth and delayed flowering (Fig. 5). Thetransformants possess lower levels of amino acids andprotein, but increased levels of ammonia and glyoxylate,suggesting a defect in the reassimilation of ammoniagenerated in the photorespiratory pathway and in theregeneration of 3-phosphoglyceric acid from glyoxylate.This assumption is supported by the observation thatthe phenotype of DiT1 antisense repression can be atleast partially suppressed by cultivation of the transgenicplants under elevated CO2 levels. In addition to theaspects of the phenotype related to the photorespiratorypathway, profound effects on the accumulation of nitratewere found in the transgenic plants and a striking dis-crepancy between the in vivo activity of nitrate reductaseand the in vivo rate of nitrate reduction.

Taken together, the findings by Somerville andSomerville (Somerville and Somerville, 1985), Wallsgroveet al. (Wallsgrove et al., 1986), and the authors obser-vations on antisense repression of DiT1 in transgenictobacco plants suggest an important role of plastidicdicarboxylate translocators in the co-ordination of theplastidic and the cytosolic C and N-metabolism bycontrolling the flux of 2-oxoglutarate into the plastidstroma and of glutamate to the cytosol.

Acknowledgements

We thank Peter Westhoff and Uta Dressen (University ofDusseldorf, Germany) for supplying us with the cDNAsequences of DiT2 from Flaveria bidentis and Sorghum bicolor.

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