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Dicarboxylate Transport (Photorespiration/Malate Shuttle/Glutamine Transport) S

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Dicarboxylate Transport (Photorespiration/Malate Shuttle/Glutamine Transport) S Proc. NatL Acad. Sci. USA Vol. 80, pp. 1290-1294, March 1983 Botany An Arabidopsis thaliana mutant defective in chloroplast dicarboxylate transport (photorespiration/malate shuttle/glutamine transport) S. C. SOMERVILLE*t AND W. L. OGREN*t *Department of Agronomy, University of Illinois, Urbana, Illinois 61801; and tU. S. Department of Agriculture, Agricultural Research Service, Urbana, Illinois 61801 Communicated by Harry Beevers, November 8, 1982 ABSTRACT Reactions of the photorespiratory pathway of C3 ically to CO2 at rates that significantly reduce net CO2 assim- plants are found in three subcellular organelles. Transport pro- ilation (9), and ammoniaaccumulates totoxiclevels (8). Because cesses are, therefore, particularly important for maintaining the glutamate is synthesized in the chloroplast (7) and consumed uninterrupted flow of carbon through this pathway. We describe in the peroxisome, the transfer ofglutamate and 2-oxoglutarate here the isolation and characterization of a photorespiratory mu- between these two organelles is a necessary component ofpho- tant of Arabidopsis thaliana defective in chloroplast dicarboxylate torespiratory nitrogen metabolism. transport. Genetic analysis indicates the defect is due to a simple, Transport systems operating at the peroxisome-bounding recessive, nuclear mutation. Glutamine and inorganic phosphate been characterized. However, studies with transport are unaffected by the mutation. Thus, in contrast to pre- membrane have not vious reports for pea and spinach, glutamine uptake by Arabi- isolated chloroplasts have revealed the presence of a trans- dopsis chloroplasts is mediated by a transporter distinct from the porter, designated the dicarboxylate transporter, which cata- dicarboxylate transporter. Both the inviability and the disruption lyzes the counter-exchange of several dicarboxylic acids across of amino-group metabolism of the mutant under photorespiratory the chloroplast inner membrane (10). These compounds include conditions suggest that the primary function of the dicarboxylate malate, 2-oxoglutarate, aspartate, and glutamate (10). Gluta- transporter in vivo is the transfer of 2-oxoglutarate and glutamate mine is also a reported substrate for this carrier (11, 12). There- across the chloroplast envelope in conjunction with photorespira- fore, the chloroplast dicarboxylate transporter is implicated as tory nitrogen metabolism. The role commonly ascribed to this an important component of the photorespiratory pathway. Be- transporter, conducting malate-aspartate exchanges for the in- cause this transporter is also capable of effecting malate-as- direct export of reducing equivalents from the chloroplast, ap- partate exchanges, ithas been ascribed the role ofmediatingthe pears to be a minor one. indirect export of reducing equivalents to the cytoplasm (13, 14). The constituent reactions of the photorespiratory pathway of The isolation and characterization of a photorespiratory mu- higher plants occur in three organelles, the chloroplast, the mi- tant defective in chloroplast dicarboxylate transport is reported tochondrion, and the peroxisome. Thus, transport processes must here. Biochemical and physiological analyses of the mutant have intervene at several steps of the pathway (Fig. 1) (1, 2). To the proven useful in determining the specificity of this transporter extent that the transport of substrates, products, or cofactors is and in ascertaining its primary in vivo role. limiting, these transport processes may be expected to exert a strong regulatory influence on photorespiratory metabolism. The photorespiratory pathway is initiated by the oxygenation MATERIALS AND METHODS of ribulose bisphosphate by the bifunctional enzyme ribulose- Plant Material andCulture. Both the mutant line CS156, the bisphosphate carboxylase/oxygenase (EC 4.1.1.39) (3, 4). 02 subject of this study, and the previously described line CS113, and CO2 act as competitive substrates for this enzyme, and the a glutamate synthase-deficient mutant (8), were recovered in a degree to which carbon is diverted from the Calvin cycle to the screen for mutants of Arabidopsis thaliana (L.) Heynh. (race photorespiratory pathway is a function of the ratio of these two Columbia) with defects in photorespiratory metabolism (6). The gases in the atmosphere (5). Experimentally, the flux of carbon basis of the mutant selection procedure is that strains with de- through the pathway can be suppressed without adverse effect fects in the photorespiratory pathway cannot survive at atmo- byplacingplants in an atmosphere enriched in CO2 or enhanced spheric levels of CO2 and 02 but grow normally at 1% CO2, when by transferring plants to an atmosphere enriched in 02(6). Three- photorespiration is suppressed. quarters of the carbon entering the pathway is returned to the Plants were grown according to described methods and con- poolof Calvin-cycle intermediates as phosphoglycerate. The re- ditions (15). For most of this study, a line descended from a maining carbon is lost as CO2 at the glycine decarboxylase step backcross of CS156 to the wild type was used. Experiments were in the mitochondrion. conducted with plants at the rosette stage of development (3- Tightly integrated with the photorespiratory carbon cycle is 4 wk from seeding). Procedures for making genetic crosses and the photorespiratory nitrogen cycle in which ammonia released measuring gas exchange have been described (15). during glycine deamination is refixed by the sequential action Labeling Studies. Plants were labeled with 14CO2 for 10 min, ofglutamine synthetase and glutamate synthase (Fig. 1) (7). The and the distribution of label among products was determined resultant glutamate supports both photorespiratory ammonia by ion-exchange and thin-layer chromatography (15-17). refixation (8) and glyoxylate amination (9). In the absence of Glutamate Synthase Assays. Glutamate synthase was as- adequate glutamate pools, glyoxylate is oxidized nonenzymat- sayed in crude extracts of leaf material after centrifugation at The publication costs of this article were defrayed in partby page charge Abbreviation: Chl, chlorophyll. payment. This article must therefore be hereby marked "advertise- tPresent address: MSU-DOE Plant Research Laboratory, Michigan State ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Univ., East Lansing, MI 48824. 1290 Downloaded by guest on September 29, 2021 Botany: Somerville and Ogren Proc. Natl. Acad. Sci. USA 80 (1983) 1291 Krebs m.M to inhibit C02-dependent 02 evolution (22). Cycy The silicone oil layer filter centrifugation technique was used to measure the transport of radiolabeled compounds into freshly CHHLOROPLAST TP isolated, intact chloroplasts (23). The standard assay medium APG contained, in addition to the standard components, 10-30 tig ADP of Chl and 1-3 ,uCi (3.7-11.1 x 104 Bq) of 3H20. Glutamine L3-dPGA I,-diPGA transport was determined at pH 7.9. The silicone oil layer con- sisted of AR200/AR20 60:40 (wt/wt) (Wacker Chemie, SWS Silicones, Adrian, MI). Chloroplast volumes were determined TP PGA with [14C]sorhitol in parallel experiments under the same con- C02 ditions as the. transport assays (23). The average sorbitol-im- Sucros permeable space was 38 ,ul/mg of Chl and 44 p.l/mg of Chl for P-Glycolate wild-type and mutant chloroplasts, respectively: Transport as- I___________________ I__________ says, commonly 3-, 5-, or 10-sec duration, were performed in I the dark at 50C. For some experiments, chloroplasts were pre- Mdote tMabte Glycerote GlyWcote loaded with the compound to be assayed for uptake by adding a small volume of stock solution to freshly prepared chloroplasts to give a final concentration of 20 mM. After 15 min on ice in OAA OAA OHPyruvate Qlyoxyote the dark, the chloroplasts were collected by centrifugation (270 '4 >342: X g at 40C for 40 sec) and resuspended in medium lacking the -2064 A amg,hb compound. Apparent Km and Vm.x values were estimated from -Asp Sor Gly Asp Scatchard plots of the data presented in the text. Chl was de- PEROXISOE T I termined spectrophotometrically in ethanol (24). J L I x^A RESULTS Ser * Gly Mutant Isolation and Genetic Analysis. In an atmosphere that ,NWCOH suppressed photorespiration (1% C02/99% air), the mutant line THF T NH3 CS156 was capable of normal growth and development. How- ever, in standard atmospheric conditions (N2 containing 0.03% MITOCHONDRION CO2 and 21% 02), the mutant became yellow and lost vigor within NAM NOD 3-4 days. The F1 plants from a CS156 x wild-type cross were healthy in standard atmospheres, suggesting the mutant line carried a recessive, nuclear mutation. In a derivative F2 pop- FIG. 1. Schematicpresentation of thephotorespiratory carbon and ulation, 196 plants exhibited the wild-type phenotype and 55 nitrogen cycles. C1-THF, N5,N'0-methylenetetrahydrofolate; DT, di- carboxylate transporter; OAA, oxaloacetate; 20G, 2-oxoglutarate; PT, were yellow after 4 days in a normal atmosphere. Thus, the mu- phosphate translocator; PGA, phosphoglycerate; RuBP, ribulose bis- tation in line CS 156 responsible for the growth requirement for phosphate; THF, tetrahydrofolate; TP, triose phosphate. high CO2 was inherited as a simple, recessive, nuclear mutation (X2 = 1.276; P > 0.25). The locus defined by this mutant was 30,000 x g and desalting by Sephadex G-25 column chroma- designated dct (dicarboxylate transport). The F1 plants from a tography (8). Protein was determined by a dye-binding assay cross of CS 156 with CS 113, a glutamate synthase-deficient
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